Stroke is a leading cause of disability and death, but currently available treatments are inadequate¹. Well-established animal models of both ischemic and hemorrhagic stroke serve as the primary method of preclinical evaluation of new stroke treatments, as the complexity of both the nervous system and the pathophysiology of stroke are incompletely understood, and therefore cannot be adequately approximated by other means at this time ¹. Stem cell transplantation has emerged as a promising potential new treatment that may limit the initial brain tissue injury (neuroprotection) or improve recovery through other means (neurorestoration) such as enhancing endogenous repair mechanisms or through cellular replacement ².

Because loss of neurological function after stroke is directly related to loss of cells, and the replacement of the lost cells theoretically could improve recovery, the stroke research community initially explored grafting intact cerebral tissue of animals, followed by suspensions of cultured animal cells, into animal stroke models ³. This rapidly developing area is now evaluating transplantation of numerous animal cell types in stroke models, including neural- and bone marrow-derived cells, many of which have demonstrated graft cell survival and behavioral rescue ². Recently, cultured human cells have been tested in animal stroke models, including multiple types of bone marrow- and tumor-derived cells, adipose cells, umbilical cord blood cells, and neural stem cells (NSCs) from multiple sources ².

Neural stem cells have several potential advantages over other cell types in that they are tissue specific to the central nervous system, and multipotent in their ability to differentiate into the primary cell types lost with stroke. Nonneural cells are not specific to the nervous system, and although they may be able to provide benefit in stroke through other mechanisms, it is unlikely they would be able to participate in large-scale cellular replacement, if that is necessary. Human NSCs behave differently in vitro from those derived from animals, and may therefore also be expected to behave differently in vivo after transplantation as well ^4,5. As cells of human origin will be the NSCs to be further evaluated in human clinical trials, it would seem appropriate that they should be extensively tested in animal stroke models prior to translation to optimize the key variables of the transplantation strategy, and to assess for adverse events.

OBJECTIVE

The purpose of this review is to assess the currently available evidence of human neural stem cell transplantation in animal stroke models, and to evaluate which variables of the transplantation strategy and which mechanistic questions have been adequately defined.

METHODS

Inclusion Criteria

We sought studies transplanting cultured human cells expressing NSC markers in vitro prior to transplantation into an animal stroke model. NSC transplantation needed to occur after the stroke was produced to model the clinical application. We accepted animal models of focal cerebral ischemia, intracerebral hemorrhage, or subarachnoid hemorrhage. We accepted any species and any type of outcome assessment.

Search Strategy

We searched PubMed, Science Citation Index, and Biological Abstracts for published articles matching our inclusion criteria (Appendix 1). Our search was performed in April 2010, and limited to full articles in the English language published prior to January 1, 2010. No other limits were used. This search produced 510 results. We reviewed titles, abstracts, or full articles to determine if our inclusion criteria were met. We found 42 articles that matched our inclusion criteria ^6–47 . The references included in these articles were reviewed for more matching studies but no others were found.

Data Collection

Two reviewers assessed articles for inclusion and extracted data (Appendix 2). A level of evidence for each study was assigned using a modification of the algorithm for determining the level of evidence for an individual study in the Strength of Recommendation Taxonomy (SORT) system ⁴⁸. Our modification for preclinical studies (termed “P-SORT” in Appendix 2) consisted of adding “animal modeling of” to “patient-oriented evidence,” and replacing “bench research” with “in vitro research.” The primary example of “animal modeling of patient-oriented evidence” was assessment of behavioral recovery from stroke. Studies were not considered “High-quality randomized controlled trials” if the article did not mention randomization or blinded assessment of behavioral testing.

RESULTS

Studies

Studies were excluded from this review primarily because they dealt with unrelated topics, were review articles, had cell types not expressing NSC markers in vitro prior to transplantation, or used a model not related to stroke. Not all variables of interest could be determined from the published reports (Appendix 2); only the information that was explicitly stated is presented underneath. No matching studies were found prior to 1998, after which there was an accelerating pace of matching publications (Fig. 1).

Model

Rats were used in 35 studies, mice in five, gerbils in one, and cynomolgus monkeys in one (Fig. 2).

The most common model reported was focal cerebral ischemia that was used in 34 studies, followed by intracerebral hemorrhage in eight studies. No studies of subarachnoid hemorrhage were found.

All studies modeling intracerebral hemorrhage used intracerebral collagenase injection. Several methods were used to produce focal cerebral ischemia, but the most common was transient intraluminal filament middle cerebral artery occlusion (Fig. 3).

Timing

Cells were transplanted at multiple time points after stroke, but the most common was one week, followed by one day, and then one month (Fig. 4).

One study directly compared two time points. Lee et al. compared transplantation at two vs. 24 hours, with both time points having two routes of delivery, intravenous (IV) delivery of 5 million cells vs. intracerebral (IC) delivery of 1 million cells, for four total groups tested in a rat model of IC hemorrhage ³². The primary objective of the study was to assess the effect of NSC transplantation on brain inflammation after stroke, and they reported an association of the two-hour IV group with reduced brain inflammation that was dependent on the spleen. They also reported behavioral rescue with all grafted groups vs. control, and more behavioral rescue for the two-hour IV group than the 24-hour IV group.

Route

The most common route of cell delivery was IC injection (Fig. 5).

Two studies directly compared two transplant routes. The study by Lee et al. is described above in the timing section; they did not report an outcome difference between IV and IC delivery of cells, other than reduced brain inflammation in the two-hour IV group ³². Lappalainen et al. compared IV to intracarotid delivery of cells in a rat ischemic stroke model ³⁴. The primary objective of the study was to assess the ability of SPECT imaging to detect labeled cells accumulating in the brain after transplant, and they reported that the intracarotid group showed a weak label signal in the ischemic hemisphere but the IV group did not, although for both groups the label signal was primarily seen in the viscera instead of brain.

Dose

The dose of transplanted cells in the studies ranged from 5000 to 20 million (Appendix 2). Five studies directly compared two or more doses of transplanted cells. The study by Lee et al. is described above in the timing section; they did not report a difference between doses, although this was confounded by the dose tiers also differing on the route of administration ³². Borlongan et al. reported no difference in outcome between a group given 78×10³ cryopreserved cells vs. a group given 23×10³ fresh cells and, but this was confounded by the cryopreservation difference ⁷. Saporta et al. compared groups given 5, 10, 20, 40, 80, or 160×10³ cells under identical conditions, and reported an association of higher doses with both improved graft cell survival and behavioral rescue ⁹. Daadi et al. compared groups given 50, 200, or 400×10³ cells under identical conditions, and reported an association of higher doses with stronger label signal on imaging ⁴². Stroemer et al. compared groups given 4.5, 45, or 450×10³ cells under identical conditions, and reported an association of higher doses with improved behavioral rescue ⁴⁴.

Immunosupression

Administration of antirejection drugs for the xenotransplantation of human cells into animal models was reported in 21 studies (50%). The most commonly used drug was cyclosporine, although the dosing, schedule, and additional drugs used were all highly variable (Appendix 2). One study directly compared alternative antirejection drug strategies. Borlongan et al. had one group given intraperitoneal cyclosporine 10 mg/kg/day from the transplant day through the six-month survival period vs. an identically treated group not given cyclosporine, and they reported that while behavioral rescue was seen in both groups, it was only sustained in the group given cyclosporine, and that group also showed better graft cell survival ⁷.

Cells

Neural stem cellswere derived from multiple types of tissue, but the most common origin of the cultured cells was fetal brain tissue (Fig. 6). All studies evaluated NSCs of only one origin.

In 22 studies (52%), genetically modified cells were used, most commonly with forms of a myc transgene (Fig. 7). The reported purposes of transgene insertion included immortalizing the graft cell line, labeling the graft cells, or increasing graft cell production of molecules of interest.

Graft

All of the studies that reported performing histology except one found transplanted cells in the brain at the end of the survival period, the duration of which was variable (Appendix 2).

The grafted cells expressed markers of multiple cell types but most commonly neurons (Fig. 8).

Stroke

Five studies found a reduction in stroke volume in transplanted rats but 13 studies did not.

Behavior

All the studies that reported performing behavioral testing found improved behavioral recovery of grafted animals compared to controls on at least one behavioral test (Fig. 9). Only two studies reported negative results on any behavioral test ^21,45.

Adverse Events

No studies reported cell-related adverse events (Appendix 2).

Mechanisms

In vivo evidence suggesting a mechanism of benefit was presented in 15 studies, 12 of which also demonstrated behavioral rescue in the same study.

Two studies demonstrated that graft cells expressed neuronal markers that had formed structures with characteristics of synapses between graft and host cells as well as showing behavioral rescue, suggesting exogenous neuronal replacement as a mechanism of benefit ^14,33. One study did not report behavioral testing, but did demonstrate appropriate electrophysiological responses across graft–host synaptic structures, suggesting the formation of functional graft–host synapses ⁴². Two studies found grafted animals showed increased expression of host neural precursor cell markers, suggesting host neural cell replacement as a potential mechanism of benefit ^44,46.

Five studies showing behavioral rescue also demonstrated a reduction in infarct volume, suggesting neuroprotection as a mechanism of benefit ^{14,22,33,36,47}. The potential mechanisms of neuroprotections in these studies were unclear, but other studies suggested several possibilities. Two studies showing behavioral rescue also demonstrated decreased host apoptosis, one of which found a correlation with increased Bcl-2 expression, and the other found a correlation with increased graft cell glial cell line-derived neurotrophic factor (GDNF) expression, suggesting a role for these factors in decreasing host apoptosis, which could be a mechanism of neuroprotection ^39,40. One study showed reduced brain inflammation in grafted animals through unknown effects dependent on the spleen, suggesting this as a mechanism of neuroprotection ³². One study showed that graft cells expressing astrocyte markers extended processes to blood vessels, suggesting graft effects to repair the blood–brain barrier as a mechanism of neuroprotection ⁴².

If graft cells are augmenting endogenous recovery processes, a proposed component of this likely includes secretion of factors by graft cells that have beneficial effects on host cells. Three studies showed that graft cells engineered to overexpress one factor showed better behavioral rescue than graft cells without this modification, with one study each using genes for brain-derived neurotrophic factor, vascular endothelial growth factor (VEGF), and GDNF ^11,24,39.

Angiogenesis may be necessary to support endogenous restorative processes, and augmenting this process may aid neurorestoration. Two studies found a correlation with increased angiogenesis in grafted animals and behavioral rescue, and another study found the same correlation with VEGF over expressing graft cells compared to graft cells without this modification, suggesting enhancement of angiogenesis, perhaps secondary to increased VEGF secretion, as a mechanism of neurorestoration ^16,24,44.

Reorganization of neuronal pathways in brain white matter may be necessary for recovery by allowing new functional circuitry to form, rerouting around injured areas. One study demonstrated a difference in magnetic resonance imaging between graft and control animals that suggests a role of graft cells in promoting white matter reorganizationthat could contribute to neurorestoration ¹⁹.

DISCUSSION

This review found wide disparities in most of the key variables of neural stem cell transplantation in animal stroke models, but despite this, there was nearly unanimous reporting of graft cell survival in the brain and behavioral rescue, without cell-related adverse events. These data not only support the promise and need for further development of this strategy as a future treatment for stroke patients, but also suggest the possibility of publication bias.

The ratio of studies evaluating NSC transplantation in ischemic (81%) vs. hemorrhagic (19%) stroke models is roughly proportional to the ratio of these stroke types in humans, which seems appropriate. Most of the work to date have used rodents, with only one small uncontrolled study in a nonhuman primate; more data in this area could be helpful.

The specific type of ischemic stroke model used varied widely, which seems to reflect the uncertainty in the experimental stroke research community of which type is optimal for the question of interest ¹. The lack of consensus on the key transplantation variables of timing, route, dose, and antirejection drug regimen suggests that the optimal transplantation strategy also remains unclear at this time.

Debate continues over whether cells derived from nonneural sources such as bone marrow, umbilical cord blood, adipose tissue, or dental pulp truly represent NSCs, or if they express markers associated with NSCs under specific culture conditions without possessing the full multipotency necessary to replace cells of the nervous system ⁴⁹. The lesson of induced pluripotent stem cells, however, is that under appropriate in vitro laboratory conditions, any cell type of the body may be capable of changing into any other. Based on this knowledge, we included studies in this review where NSC markers were expressed even if the exact identity of the pretransplant cells was not entirely clear. These cell types may behave substantially different from NSCs derived from embryonic stem cells or fetal brain tissue, but we did not find evidence of that with this review as every cell type used appeared to be highly successful.

Most of the studies used genetically modified cells for transplant. The introduced transgenes were used to immortalize the cell line, label the cells for histological or image identification, or to make the grafted cells increase production of a molecule of interest. Already with currently available technology, the potential combinations of cell types and transgenes are large, and the expansion of further novel combinations is likely to accelerate as the field gains experience. One could imagine a pipeline of modified cell types awaiting clinical trials, as is currently the case for small molecule drugs, but the complexity and unpredictability of the effects of genetic modification on both the graft cells and host tissue suggest that extensive safety testing should occur with each newly created cell line prior to initial human exposure.

The beneficial mechanisms of NSC transplantation in animal stroke models are unclear, although data supporting several potential contributing factors were found in the reviewed studies. Even though most studies did not report smaller strokes in the grafted groups, all of the studies that reported behavioral testing found behavioral rescue with NSC transplantation. This observation argues against neuroprotection as the primary mechanism of benefit, and instead provides support for neurorestoration, either through enhancement of endogenous repair mechanisms or cellular replacement. Evidence of graft cells replacing lost host cells as part of functional neural circuitry, however, is still limited ⁵⁰.

Six of the included studies evaluated an NSC line derived from an adult teratocarcinoma that led to two small clinical trials where 26 patients had direct IC transplantation of these cells more than six months after their strokes ⁵¹. While too small to detect benefit, these studies reported no cell-related adverse events.

This review has several limitations. The databases searched rely on indexing that could have caused us to miss matching studies. We attempted to compensate for this possibility by ancestral searching of the references of the included articles, on the assumption that the study authors would have the best knowledge of the applicable literature at the time of article preparation. Some articles were written in a way that made it difficult to determine what was done regarding some of the key variables, or the information was simply not stated, so only the information that was clearly stated was included and available for inclusion. A major limitation appears to be the likely existence of publication bias, as we would have expected several studies with negative results by chance alone.

Based on the currently available information reviewed here, further study of this promising potential new stroke treatment is both needed and justified.

Disclosure: The authors declare no conflict of interest and were funded by NIH grant UL1 RR025011.

REFERENCES

1. Durukan A, Tatlisumak T. Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol Biochem Behav. 2007;87:179–197.
2. Bliss T, Guzman R, Daadi M, Steinberg GK. Cell transplantation therapy for stroke. Stroke. 2007;38:817–826.
3. Borlongan C, Koutouzis T, Jorden J, et al. Neural transplantation as an experimental treatment modality for cerebral ischemia. Neurosci Biobehav Rev. 1997;21:79–90.
4. Wright L, Prowse K, Wallace K, Linskens M, Svendsen C. Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro. Exp Cell Res. 2006;312:2107–2120.
5. Hanna J, Cheng A, Saha K, et al. Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse escs. Proc Natl Acad Sci USA. 2010;107(20):9277–9227.
6. Borlongan CV, Tajima Y, Trojanowski JQ, Lee VM, Sanberg PR. Cerebral ischemia and cns transplantation: differential effects of grafted fetal rat striatal cells and human neurons derived from a clonal cell line. Neuroreport. 1998;9:3703–3709.
7. Borlongan CV, Tajima Y, Trojanowski JQ, Lee VM, Sanberg PR. Transplantation of cryopreserved human embryonal carcinoma-derived neurons (nt2n cells) promotes functional recovery in ischemic rats. Exp Neurol. 1998;149:310–321.
8. Borlongan CV, Saporta S, Poulos SG, Othberg A, Sanberg PR. Viability and survival of hnt neurons determine degree of functional recovery in grafted ischemic rats. Neuroreport. 1998;9:2837–2842.
9. Saporta S, Borlongan CV, Sanberg PR. Neural transplantation of human neuroteratocarcinoma (hnt) neurons into ischemic rats. A quantitative dose-response analysis of cell survival and behavioral recovery. Neurosci. 1999;91:519–525.
10. Jeong S-W, Chu K, Jung K-H, Kim SU, Kim M, Roh J-K. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke. 2003;34:2258–2263.
11. Kang S, Lee D, Bae Y, Kim H, Baik S, Jung J. Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats. Exp Neurol. 2003;183:355–366.
12. Chu K, Kim M, Park K-I, et al. Human neural stem cells improve sensorimotor deficits in the adult rat brain with experimental focal ischemia. Brain Res. 2004;1016:145–153.
13. Chu K, Kim M, Chae S-H, et al. Distribution and in situ proliferation patterns of intravenously injected immortalized human neural stem-like cells in rats with focal cerebral ischemia. Neurosci Res. 2004;50:459–465.
14. Ishibashi S, Sakaguchi M, Kuroiwa T, et al. Human neural stem/ progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in mongolian gerbils. J Neurosci Res. 2004;78:215–223.
15. Kelly S, Bliss TM, Shah AK, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci USA. 2004;101:11839–11844.
16. Chu K, Park K-I, Lee S-T, et al. Combined treatment of vascular endothelial growth factor and human neural stem cells in experimental focal cerebral ischemia. Neurosci Res. 2005;53:384–390.
17. Zeng JS, Yu J, Cui CM, et al. Cultured human embryonic neocortical cells survive and grow in infarcted cavities of adult rat brains and interconnect with host brain. Chin Med J (Engl). 2005;118:275–280.
18. Pollock K, Stroemer P, Patel S, et al. A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol. 2006;199:143–155.
19. Jiang Q, Zhang Z, Ding G, et al. Mri detects white matter reorganization after neural progenitor cell treatment of stroke. Neuroimage. 2006;32:1080–1089.
20. Roitberg BZ, Mangubat E, Chen E-Y, et al. Survival and early differentiation of human neural stem cells transplanted in a nonhuman primate model of stroke. J Neurosurg. 2006;105:96–102.
21. Bliss TM, Kelly S, Shah AK, et al. Transplantation of hnt neurons into the ischemic cortex: cell survival and effect on sensorimotor behavior. J Neurosci Res. 2006;83:1004–1014.
22. Kim D, Park S, Lee S, et al. Effect of human embryonic stem cell-derived neuronal precursor cell transplantation into the cerebral infarct model of rat with exercise. Neurosci Res. 2007;58:164–175.
23. Lee H, Kim K, Kim E, et al. Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model. Stem Cells. 2007;25:1204–1212.
24. Lee H, Kim K, Park I, Kim S. Human neural stem cells over-expressing vegf provide neuroprotection, angiogenesis and functional recovery in mouse stroke model. PLoS One. 2007;2:e156.
25. Guzman R, Uchida N, Bliss TM, et al. Long-term monitoring of transplanted human neural stem cells in developmental and pathological contexts with mri. Proc Natl Acad Sci USA. 2007;104:10211–10216.
26. Nagai A, Kim W, Lee H, et al. Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow. PLoS One. 2007;2:e1272.
27. Darsalia V, Kallur T, Kokaia Z. Survival, migration and neuronal differentiation of human fetal striatal and cortical neural stem cells grafted in stroke-damaged rat striatum. Eur J Neurosci. 2007;26:605–614.
28. Hara K, Matsukawa N, Yasuhara T, et al. Transplantation of post-mitotic human neuroteratocarcinoma-overexpressing nurr1 cells provides therapeutic benefits in experimental stroke: in vitro evidence of expedited neuronal differentiation and gdnf secretion. J Neurosci Res. 2007;85:1240– 1251.
29. Kozlowska H, Jablonka J, Janowski M, Jurga M, Kossut M, Domań ska- Janik K. Transplantation of a novel human cord blood-derived neural-like stem cell line in a rat model of cortical infarct. Stem Cells Dev. 2007;16:481–488.
30. Daadi MM, Maag A-L, Steinberg GK. Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model. PLoS One. 2008;3(2):1644.
31. Chang NK, Jeong YY, Park JS, et al. Tracking of neural stem cells in rats with intracerebral hemorrhage by the use of 3t mri. Korean J Radiol. 2008;9:196–204.
32. Lee S-T, Chu K, Jung K-H, et al. Anti-inflammatory mechanism of intravascular neural stem cell transplantation in haemorrhagic stroke. Brain. 2008;131:616–629.
33. Kim S, Yoo S, Park T, et al. Neural induction with neurogenin1 increases the therapeutic effects of mesenchymal stem cells in the ischemic brain. Stem Cells. 2008;26:2217–2228.
34. Lappalainen R, Narkilahti S, Huhtala T, et al. The spect imaging shows the accumulation of neural progenitor cells into internal organs after systemic administration in middle cerebral artery occlusion rats. Neurosci Lett. 2008;440:246–250.
35. Lee TH, Yoon JG. Intracerebral transplantation of human adipose tissue stromal cells after middle cerebral artery occlusion in rats. J Clin Neurosci. 2008;15:907–912.
36. Liu YP, Seckin H, Izci Y, Du ZW, Yan YP, Baskaya MK. Neuroprotective effects of mesenchymal stem cells derived from human embryonic stem cells in transient focal cerebral ischemia in rats. J Cerebr Blood F Met. 2009;29:780–791.
37. Stevanato L, Corteling R, Stroemer P, et al. C-mycertam transgene silencing in a genetically modified human neural stem cell line implanted into mcao rodent brain. BMC Neurosci. 2009;10:86.
38. Lee H, Kim M, Kim H, Kim S. Human neural stem cells genetically modified to overexpress akt1 provide neuroprotection and functional improvement in mouse stroke model. PLoS One. 2009;4:e5586.
39. Lee H, Park I, Kim H, Kim S. Human neural stem cells overexpressing glial cell line-derived neurotrophic factor in experimental cerebral hemorrhage. Gene Ther. 2009;16:1066–1076.
40. Zhang P, Li J, Liu Y, et al. Human neural stem cell transplantation attenuates apoptosis and improves neurological functions after cerebral ischemia in rats. Acta Anaesthesiol Scand. 2009;53:1184–1191.
41. Eve DJ, Musso J, III, Park D-H, et al. Methodological study investigating long term laser doppler measured cerebral blood flow changes in a permanently occluded rat stroke model. J Neurosci Metho. 2009;180:52–56.
42. Daadi MM, Li ZJ, Arac A, et al. Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain. Mol Ther. 2009;17:1282–1291.
43. Song M, Kim Y, Ryu S, Song I, Kim S, Yoon B. Mri tracking of intravenously transplanted human neural stem cells in rat focal ischemia model. Neurosci Res. 2009;64:235–239.
44. Stroemer P, Patel S, Hope A, Oliveira C, Pollock K, Sinden J. The neural stem cell line ctx0e03 promotes behavioral recovery and endogenous neurogenesis after experimental stroke in a dose-dependent fashion. Neurorehabil Neural Repair. 2009;23:895–909.
45. Hicks A, Lappalainen R, Narkilahti S, et al. Transplantation of human embryonic stem cell-derived neural precursor cells and enriched environment after cortical stroke in rats: cell survival and functional recovery. Eur J Neurosci. 2009;29:562–574.
46. Zhang PB, Li J, Liu Y, Chen XL, Kang QY. Transplanted human embryonic neural stem cells survive, migrate, differentiate and increase endogenous nestin expression in adult rat cortical peri-infarction zone. Neuropathol. 2009;29:410–421.
47. Yang KL, Chen MF, Liao CH, Pang CY, Lin PY. A simple and efficient method for generating nurr1-positive neuronal stem cells from human wisdom teeth (tnsc) and the potential of tnsc for stroke therapy. Cytotherapy. 2009;11:606–617.
48. Ebell M, Siwek J, Weiss B, et al. Strength of recommendation taxonomy (sort): a patient-centered approach to grading evidence in the medical literature. Am Fam Physician. 2004;69:548–556.
49. Andres R, Choi R, Steinberg G, Guzman R. Potential of adult neural stem cells in stroke therapy. Regen Med. 2008;3:893–905.
50. Chopp M, Li Y, Zhang Z. Mechanisms underlying improved recovery of neurological function after stroke in the rodent after treatment with neurorestorative cell-based therapies. Stroke. 2009;40:S143–S145.
51. Wechsler L. Clinical trials of stroke therapy. Which cells, which patients? Stroke. 2008;40:s149–s151.

Appendix A: Search Strategy

PubMed:

Search query: (brain ischemia OR transient ischemic attack OR Stroke OR Middle Cerebral Artery Infarction) AND (stem cell/transplantation OR brain tissue transplantation OR embryonic stem cells/transplantation OR fetal tissue/transplantation OR fetal stem cells/transplantation OR cerebral cortex/transplantation) AND (animal OR mouse OR mice OR murine OR gerbil OR gerbils OR rodentia OR rat OR rats OR heterologous transplantation[MeSH]) AND human

Limits: None

Science Citation Index:

Search query: (neuron* OR neural) AND stem cell* AND transplant* AND (stroke* OR ischemia*) AND (animal OR rat OR rats OR mouse OR mice OR murine OR gerbil) AND human

Limits: None

Refined by Subject Areas: NEUROSCIENCES OR MEDICINE, RESEARCH & EXPERIMENTAL

Biological Abstracts:

Search query: (neuron* OR neural) AND stem cell* AND transplant* AND (stroke* OR ischemia*) AND (animal OR rat OR rats OR mouse OR mice OR murine OR gerbil) AND human

Limits: None

Refined by: Major Concepts NERVOUS SYSTEM

Appendix B

A previously healthy 34-year-old woman, with no remarkable past or family history, presented to the emergency in October 2006 with a 2-week history of bilateral leg weakness and paresthesia. She also complained of clumsiness when walking, fatigue, and severe pain between the shoulders and in both legs. One week later, the symptoms progressed to bilateral leg paralysis and she developed urinary incontinence. Neurological examination revealed bilateral leg hyperreflexia, bilateral extensor plantar responses, and a sensory loss below the T10 level.

A head MRI (Fig. 1) showed five lesions that failed to meet the Barkoff criteria for multiple sclerosis (MS) diagnosis. A spine MRI showed increased T2 signal extending from C7 to T8. The cerebrospinal fluid (CSF) obtained by lumbar puncture showed no cells, normal proteinorrachia, normal glucorrachia, and no oligoclonal bands. A CT of the spine ruled out a massoccupying lesion that could have been compressing the spinal cord. Tests for systemic inflammatory disease, HIV infection, and syphilis were negative while B12 levels were within the normal range. Immunoglobulin levels and protein electrophoresis were also normal. As none of these tests was suggestive of a specific cause, the patient was presumed to have idiopathic transverse myelitis (TM). After a 3-day course of methylprednisolone and intense physiotherapy, her symptoms improved moderately and she was discharged. Three months later, the patient returned to work with mild residual hypoesthesia and numbness below T10.

On May 12, 2008, the patient was readmitted to the ward for worsening of hypoesthesia associated with the return of bilateral leg weakness, a new micturition difficulty, and a girdlelike band of increased sensation extending from the lower chest. There were no visual deficits and the visual evoked potential (VEP) test showed normal patterns. The head MRI performed showed no new lesion. The spine MRI revealed an increased T2 signal extending from T1 to T3 with a regression of the signal documented in 2006. She received another IV methylprednisolone pulse therapy. Subsequently, the paraplegia and sensory disturbances improved moderately during the following 3 months with intensive physiotherapy.

In September 2008 the patient was readmitted for right leg hemiplegia and urinary retention. The spinal MRI showed a new signal extending from T6 to T9 (Fig. 2). Laboratory testing revealed a C-reactive protein (CRP) level at 8 mg/dL, an antinuclear antibodies (ANA) titer of 1/160, markedly low serum complement, negative antiphospholipid antibody, and a persistent 0.37 g/dL proteinuria. The patient was treated with prednisone and methotrexate, and her recovery was similar to that of the previous episodes.

DISCUSSION

Recurring transverse myelopathy (RTM) is uncommon but it can accompany diseases such as MS, systemic lupus erythematosus (SLE) or arterio–venous malformations ^1,2. Acute transverse myelitis (ATM) may be the sole presenting symptom of MS ³, but the possibility of MS is unlikely in our patient and can be excluded by the absence of lesions that fulfill the MRI/Barkoff criteria and of oligoclonal bands on CSF electrophoresis. The immunological (positive ANA test), renal (persistent proteinuria), and inflammatory findings (low CRP levels) during the third episode were suggestive of a connective tissue disease. Even though TM is not among the 11 criteria of SLE, which only consider seizures and psychosis, this is one of the well-known neurological manifestations associated with SLE. During SLE exacerbations, increased formation of immune complexes and the subsequent complement activation often result in hypocomplementemia, explaining the low CRP levels typically found in the disease. Although there was no other clinical evidence of a collagen disease, RTM has already been reported as an isolated manifestation of SLE ¹. Prior to these laboratory findings, an initial diagnosis of idiopathic TM was also considered. In our discussion, we focus on the clinical characteristics and diagnosis criteria of these three common and devastating medical problems: MS, SLE, and TM.

Multiple Sclerosis is the most common inflammatory demyelinating disease of the central nervous system (CNS) in young and middle-aged adults ^3,4. The classic clinical definition of MS is two or more deficits separated in time and space. The diagnosis is based on the presence of typical clinical features, of oligoclonal bands in CSF electrophoresis, together with MRI evidence of specific lesions and abnormal patterns in evoked potentials testings ⁵. Suggestive MRI findings consist of T2-bright areas, representing demyelinative plaques, typically involving the corpus callosum, cortex (juxtacortical U fibers), temporal lobes, brainstem, cerebellum, ventricles, and spinal cord. MRI diagnosis of MS requires lesions to be disseminated in time and space, and to fulfill at least three out of four of the Barkoff criteria (Box 1) ^6,7.

Box 1. The Barkoff criteria: MRI demonstration of space Three of the following four:1. One gadolinium-enhancing or nine T2-hyperintense lesions2. At least one infratentorial lesion3. At least one juxtacortical lesion4. At least three periventricular lesions

The most common symptoms of MS are motor (65–100%) and sensory (48–82%) involvement of trunk and limbs, spasticity (73–100%), and bladder dysfunction (49–93%) because of spinal cord lesions ⁸. Brainstem involvement may produce cranial nerve signs, nystagmus, ataxia, and vertigo. Furthermore, 10–50% of patients with optic neuritis will subsequently develop MS ⁹. MS can present in many ways including as amonosymptomatic disease suggestive of MS, as a disease with insidious progression, or as a disease with typical relapsing–remitting episodes of neurologic deficits followed by gradual, partial recovery from the attack.

Systemic Lupus Erythematosus is a relatively common multisystem disease of autoimmune origin characterized mainly by injury to the skin, joints, kidney, and serosal membranes ¹⁰. The course of the disease is unpredictable but usually involves relapsing–remitting episodes. The clinical presentation of SLE is particularly variable, and at least 4 of the 11 criteria established by the American College of Rheumatology must be present in order to diagnose this disorder. Other features include low blood counts and the presence of an array of autoantibodies, including ANA. The most common CNS manifestations of SLE are cognitive dysfunction, seizures, psychosis, and myelopathy ¹¹. TM is a rare late complication of SLE but it can also occur as the initial presentation ¹². SLE demyelinating syndromes resembling MS are termed lupoid sclerosis and represent a true diagnostic challenge. The most common presentation of lupus-associated TM is a sensory level with spastic lower limb weakness and sphincter disturbance ¹². The main mechanisms underlying CNS manifestations in lupus are ischemia, hemorrhage, white-matter damage, and neuronal dysfunction ^13,14.

Transverse Myelitis may be the presenting manifestation of a variety of potentially life-threatening diseases. A systematic approach to the differential diagnosis of acute TM is therefore necessary (Box 2) and should first aim to eliminate potentially treatable causes of the condition. Acute TM being a monophasic disorder, its recurrences should raise suspicion of MS or other multifocal CNS disease ¹⁵. Idiopathic recurring TM can be distinguished from MS-associated TM on the basis of clinical manifestations of myelopathy, or of findings from MRI or CSF examination. Recurring TM differs from MS in its male preponderance, absence of oligoclonal bands, frequent multiple relapses, and frequent presentation as ATM ¹⁶. As discussed earlier, TM is also a rare complication of SLE, and, although usually a late manifestation of SLE, it can also occur at presentation ¹⁷.

Thus, ATM constitutes a diagnostical challenge, because, even though it is common in MS (90%), it can also be the sole presenting feature of SLE ¹⁴. A clear distinction between the two disorders may be particularly difficult when the clinical manifestations, or the MRI and laboratory findings are incomplete or unspecific.

The recurring neurological symptoms observed in this case could have resulted from several diseases (such as MS, SLE, and TM), hence the importance of a careful clinical and laboratory evaluation and follow-ups.

Box 2. Exclusion Etiologies for Transverse Myelitis ^18–20 1. Compressive myelopathies: tumors, herniated discs, stenosis, abcesses, etc.2. Parainfectiousa. Viral: CNS manifestations of syphilis, Lyme disease, HSV, HTLV-1, VZV, EBV, CMV, HHV-6^*.. Bacterial: Mycoplasma pneumoniae, Lyme borreliosis, syphilis, tuberculosis3. Vascular (spinal cord infarct)a. AVMb. Thrombosis of spinal arteriesc. Vasculitis secondary to heroin abuse4. Postvaccinal5. Paraneoplastic syndrome6. Systemic inflammatory diseases: SLE, antiphospholipid syndrome, Sjögren disease, sarcoidosis7. Multiple sclerosis (brain MRI criteria)8. Delayed radiation myelopathy9. Idiopathic myelopathy

^*SLE, Systemic lupus erythematosus; HTLV-1, human T-cell lymphocytic virus-1; HSV, herpes simplex virus; VZV, varicella zoster virus; EBV, Epstein–Barr virus; CMV, cytomegalovirus; HHV, human herpes virus; AVM, arterio– venous malformation.

Acknowledgments: All authors of this paper have directly and substantially participated in the acquisition of data, conception, and analysis of this case report. All of them have read and approved the final version submitted.

Disclosure: The authors declare no conflicts of interest, no financial interests, and no sources of funding or support related to this case study. The contents of this manuscript have not been previously copyrighted or published, nor are they under consideration for publication elsewhere. The paper will not be copyrighted, submitted, or published elsewhere, while acceptance by the Journal is under consideration.

REFERENCES

1. Yamamoto M. Recurrent transverse myelitis associated with collagen disease. J Neurol. 1986;233:185–187.
2. Campi A, Filippi M, Comi G, Scotti G. Recurrent acute transverse myelopathy associated with anticardiolipin andibodies. Am J Neurology. 1998;19(4):781–786.
3. Berman M, Feldman S, Alter M, Zilber N, Kahana N. Acute transverse myelitis: incidence and etiological considerations. Neurology (NY). 1981;31:966–971.
4. Rosati G. The prevalence of multiple sclerosis in the world: an update. Neurol Sci. 2001;22(2):1590–1874.
5. Blumenfield H. Neuroanatomy Through Clinical Cases. Sinauer, Sunderland, Massachusetts; 2002:241–243.
6. Barkhof F, Filippi M, Miller DH, et al. Comparison of MR imaging criteria at first presentation to predict conversion to clinically definite multiple sclerosis. Brain. 1997;120:2059–2069.
7. McDonald WI, Compston A, Edan G, et al. Recommended diagnosis Criteria for multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis. Ann Neurol. 2001;50:121–127.
8. Rowland LP. Merritt’s Textbook on Neurology. 10th ed. Baltimore, MD: Lippincott Williams and Wilkins; 2000:Table 13.1.
9. Kumar V, Abbas AK, Fausto N, Aster J. Robbins and Cotran Pathologic Basis of Disease. 7th ed. Amsterdam: Elsevier; 2005:1384.
10. Kumar V, Abbas AK, Fausto N, Aster J. Robbins and Cotran Pathologic Basis of Disease. 7th ed. Amsterdam: Elsevier; 2005:227.
11. Devro HH. Systemic lupus erythematosus. In: Fauci AS, Braunwald E, Kasper DL, Hauser SL, Longo DL, Jameson JL, Loscolza J, Harrison’s Principles of Internal Medicine. 17th ed. New York: McGraw-Hill; 2008: 2075– 2082.
12. D’Cruz D, Mellor-Pita S, Joven B, et al. Transverse myelitis as the first manifestation of systemic lupus erythematosus or lupus-like disease: good functional outcome and relevance of antiphospholipid antibodies. J Rheumatol. 2004;31:280–285.
13. Jennekenes F, Kater L. The central nervous system in systemic lupus erythematosus. Part 2. Pathogenetic mechanisms of clinical syndromes: a literature investigation. Rheumatology. 2002;41:619–630.
14. Hughes V, Ferreira S, D’Cruz D. Multiple sclerosis, neuropsychiatric lupus and antiphospholipid syndrome: where do we stand? Rheumatology (Oxford). 2005 Apr 1;44(4):434–442.
15. Tippett DS, Fishman PS, Panitch HS. Relapsing transverse myelitis. Neurology. 1991;41:703–706.
16. Kim KK. Idiopathic recurrent transverse myelitis. Arch Neurol. 2003 Sep;60(9):1290–1294.
17. Lavalle C, Pizarro S, Drenkard C, Sánchez-Guerrero J, Alarcón-Segovia D. Transverse myelitis: a manifestation of systemic lupus erythematosus strongly associated with antiphospholipid antibodies. J Rheumatol. 1990;17:34–37.
18. Krishnan C, Kerr D. Idiopathic transverse myelitis. Arch Neurol. 2005;62:1011–1013.
19. Transverse Myelitis Consortium Working Group. Proposed diagnostic criteria and nosology of acute transverse myelitis. Neurology. 2002;59: 499–505.
20. Berman M, Feldman S, Alter M, et al. Acute transverse myelitis: incidence and etiological considerations. Neurology. 1981;31:96.

GTP cyclohydrolase 1 (GTPCH) is encoded by the GCH1 gene, located on chromosome 14q22.1-q22.2 ¹. Patients heterozygous for GCH1 mutations/deletions may develop the autosomal dominant Segawa syndrome, also called autosomal dominant GTPCH (adGTPCH) deficiency, dopa-responsive dystonia (DRD), and DYT5 dystonia (OMIM 128230) ². GTPCH catalyzes the first and rate-limiting step in the synthesis of tetrahydrobiopterin (BH₄) ³. DRD may be caused by other forms of BH₄ deficiency such as autosomal recessive GTPCH deficiency, 6-pyruvoyl-tetrahydropterin synthase, dihydropteridine reductase, and sepiapterin reductase, but with the exception of the latter, these deficiencies are characterized by neonatal hyperphenylalaninemia ⁴. Further, tyrosine hydroxylase (TH) deficiency, secondary dystonia, and early-onset parkinsonism due to mutations and deletions in the parkin gene may present during childhood with a DRD ^{4, 5}. Segawa syndrome due to adGTPCH deficiency causes a BH₄ deficiency, which leads to decreased dopamine and serotonin biosynthesis, without hyperphenylalaninemia. The disease has an estimated prevalence of 0.5 per million ⁶, although this may be underestimated owing to underdiagnosis and reduced penetrance. The adGTPCH deficiency typically presents insidiously between the ages of 1 and 9 years, average 6 years of age, with a diurnally fluctuating dystonia of one limb spreading to the other extremities after several years, together with subsequent parkinsonian signs developing in some cases ⁷. Clinical features and response to levodopa remained the diagnostic gold standard for this disorder until recently ^{8, 9}. The increasing number of adGTPCH deficiency patients reported as a consequence of the discovering of new mutations and deletions of the GCH1 gene have broadened the phenotype of the disease including exceptions to each of the cardinal points of the diagnosis and many atypical presentations. The highly variable expressivity of DRD, and the fact that the response of adGTPCH deficiency patients to levodopa is less predictable than previously assumed complicate the classic diagnostic approach based on the clinical features and the response to levodopa. Further, DRD is a syndrome with different causes. For these reasons, there is a need to confirm the clinical diagnosis of this treatable disorder through biochemical and/or molecular investigations. Notwithstanding, up to 40% of patients do not have identifiable mutations by sequencing ¹⁰, and the recently incorporated techniques to detect partial and complete deletions of GCH1, the quantitative duplex PCR (qPCR) assay and multiplex ligationdependent probe amplification (MLPA), may only increase the yield of the molecular studies in around 10% ^{11, 12}. Therefore, biochemical studies are many times necessaries.

DOPA-RESPONSIVE DYSTONIA: A WIDE CLINICAL SPECTRUM

Clinical manifestations of DRD are very wide and the phenotypic spectrum of the disease ranges from focal and generalized dystonia causing severe invalidism and parkinsonism to subjective complaints and subtle signs only seen during neurological examination or induced by exercise. The clinical symptoms are characterized by their age dependency ⁷. DRD typically presents in childhood with a diurnally fluctuating postural dystonia of lower limbs causing a gait disorder or abnormal foot posture with asymmetric equinovarus or equinovalgus (there is a preference for the left side) with prominent imbalance and concurrent or subsequent development of parkinsonism (mainly rigidity, reduced facial expression, slow fine finger movements, and bradykinesia) ^{2, 7}. Early motor development usually is normal. In untreated patients, the dystonia progresses with aggravation of dystonic hypertonus and involvement of other limbs quickly or over the next 10–20 years; sometimes, it can cause a loss of ambulation and wheelchair dependency ¹³. A postural tremor of upper limbs, usually asymmetrical, appears during the second decade and this may be the first sign, affecting the four limbs and the neck muscles over the years. Axial dystonia is frequent including laterocollis, torticollis, scoliosis, lumbar hyperlordosis, and, rarely, retrocollis ^{7, 14, 15}. Diurnal fluctuation attenuates with age and the progression of dystonia subsides with age ⁷. This “classic” phenotype is usually described in the majority of individuals. Less frequently, DRD presents in adults with a benign parkinsonism without a previous history of dystonia, manifested with akinesia associated with rigidity (akinetic rigid syndrome) or rest tremor. Akinesia can be expressed as motor slowness (bradykinesia) or as paucity of movement (hypokinesia). These patients may present with isolated parkinsonism or in combination with dystonia. An adult-onset dopa-responsive asymmetric parkinsonism resembling Parkinson's disease has also been reported in carriers of mutations of the GCH1 ^{14, 16}. These adult-onset patients usually lack diurnal fluctuation of the symptoms. When dystonia starts in the second decade (rarely), it usually affects initially the upper limbs. Approximately 40% of carriers of mutations of the GCH1 remain asymptomatic into adulthood or with only very mild symptoms not requiring therapy, although penetrance of the disorder is very variable⁷.

The phenotype of adGTPCH deficiency is much wider than previously assumed. Atypical clinical presentations such as focal dystonia ^{13, 14, 17}; spastic diplegia or cerebral palsy of undetermined etiology by neuroimaging and metabolic studies ^{5, 8, 18–20}; delayed motor development in infancy ^{8, 9, 19, 21–25}; writer's cramp ^{15, 26}; paroxysmal dystonia, including exerciseinduced dystonia; relapsing–remitting dystonia ^{17, 26}; upper-limb tremor ¹⁵; definite signs of cerebellar dysfunction responsive to L-dopa ^{18, 27}, including horizontal gaze-evoked nystagmus, upper and lower limb incoordination, and gait ataxia; infantile hypokinetic rigid syndrome ²³; muscle jerks ^{25, 28}; voice tremor and hypophonia ¹⁴; spasmodic dysphonia ¹⁵; dysphagia; initial hypotonia ¹⁵, and others have considerably expanded the clinical spectrum of Segawa disease in recent years, including the coexistence of psychiatric manifestations such as depression, anxiety, obsessive compulsive disorder, recurrent nonreactive severe mood swings, and sleep disturbances ^{15, 18, 29, 30}.

Abnormal frequent examination findings include supination of the foot (postural dystonia), wrist flexion, focal hand dystonia, poor balance, wide-based gait, failure in tilting response, a gait with lack of the upper limb coordination, action hand tremor, scoliosis, restlessness, limb hypertonus, generalized hyperreflexia (more prominent in childhood, sometimes intermittent), apparent bilateral extensor plantar responses (the “striatal toe”), ankle clonus, slow and clumsy repetitive tasks such as finger-tapping or foot-tapping with progressive decrement in amplitude, clumsiness of diadochokinesis, postural instability, and relative sparing of speech, although dysarthria and spasmodic dysphonia have been reported ¹⁵. A deceleration of the body length appears in childhood together with the neurological symptoms ⁷.

Other signs and symptoms that have been repeatedly reported and should make considering a diagnosis of DRD are a clumsy gait with propensity to fall ²², waddling gait ²⁶, and involuntary movements ²², including blepharospasm ^{14, 15}; restless legs–like symptoms ¹⁵ and oromandibular dystonia ¹⁵; exercise-related worsening of symptoms; pain, stiffness, and cramping of the feet, legs, thighs, hands, and arms worsening toward evening and after exercise ^{30, 31}; and late-presenting mild doparesponsive symptoms of rigidity, frequent falls, and recurrent tendonitis ²⁹.

The disease has a gender-related penetrance with a higher penetrance of symptoms in females as compared with males in many of the families reported ³²; however, in some families the sex penetrance is not significantly different ^{14, 26, 31}. Penetrance in DRD can be higher in families if subtle manifestations of the disorder are considered ^{14, 26, 33}. Many cases appear to be sporadic ²¹. Genotype–phenotype correlations are absent in patients with GCH1 point mutations and deletions ^{10, 17}. A wide phenotypic spectrum of clinical variability has been repeatedly described in individuals belonging to the same family with identical mutations in the GCH1 gene ^{5, 14, 17, 20, 26, 31}. Intrafamilial phenotypic variability has been reported even between monozygotic twins in adGTPCH deficiency ³⁴.

RESPONSE TO LEVODOPA

Classically, it has been reported that patients with adGTPCH deficiency show an excellent and sustained response to small doses of levodopa, in combination with a decarboxylase inhibitor, without any relationship to the longevity of the clinical course and without unfavorable side effects ²¹. A very prompt response is observed in most cases without subsequent on– off phenomena, even in longstanding severely disabled cases ¹⁹. However, sometimes the improvement may initially only be moderate, especially in infants with early-onset DRD and in adults with longstanding symptoms ¹³. A trial of levodopa of at least 3 months of treatment with slowly increasing doses, starting with 1 mg/kg up to 20 mg/kg, is recommended as a reasonable span of time and dose ⁷. Partial or lack of response to levodopa, however, and side effects like drug-induced dyskinesias have also been reported ^{5, 14, 15, 20, 34–37}. Thus, the clinical spectrum of adGTPCH deficiency includes levodopa-induced dyskinesias, introducing the additional diagnostic problem of confusion with patients carrying mutations in the parkin gene. Levodopa treatment frequently causes painful precocious and severe dyskinesias in patients with parkin mutations as opposed to DRD, underlining the importance of biochemical and imaging investigations before treatment is started ¹³. Writer's cramp has been described as a specific levodopa-resistant symptom together with dysphonia and truncal dystonia ¹⁵. Therefore, response to levodopa is less predictable than previously assumed. Further, as DRD is currently considered a syndrome with different causes and a wide clinical spectrum, the classical approach to the diagnosis of adGTPCH deficiency through the identification of the characteristics clinical features and response to levodopa treatment is not advisable, because while this may somewhat improve the movement disorder it can also lead to an inaccurate diagnosis.

MOLECULAR DIAGNOSIS

Molecular genetic testing of GCH1 is available on a clinical basis. To date, there are more than 100 different mutations/deletions associated to the phenotype DRD reported in the database (http://www.biopku.org/BioPKU_DatabasesBIOMDB.asp) curated by N. Blau and Beat Thöny. Most mutations and deletions in GCH1 are unique and up to 40% of patients do not have identifiable mutations in the coding region or the splice sites. Routine mutation analysis of GCH1 detects base changes in the gene; however, this method does not detect heterozygous deletions. The recently developed MLPA has been demonstrated useful for routine deletion analysis of exons 1–3, 5, and 6 of GCH1 but not of exon 4. This exon needs to be investigated separately for a deletion by qPCR ²². Among GCH1 point-mutation-negative patients with a definite diagnosis of DRD, the frequency of GCH1 deletions was 8.7% ¹¹ and 8% ¹². However, the frequency of deletions has not been studied systematically in large patient cohorts. These studies should be incorporated into the routine of the molecular diagnosis of adGTPCH deficiency patients ^{11, 12}. A high mutation rate up to 87% has been reported ¹¹.

It has been speculated that DRD in mutations/deletions–negative patients may be caused by mutations in the noncoding regulatory or intronic regions of GCH1 ^{5, 17}, as well as mutations in other genes having a regulatory effect on GCH1 and mutations in other genes whose products interact with the GTPCH, diminishing its activity ^{1, 17, 38}. The analysis of additional genes in GCH1-negative cases (TH, parkin, SPR gene, the gene encoding sepiapterin reductase) is currently not feasible in a routine setting ¹².

BIOCHEMICAL DIAGNOSIS

The phenylalanine (Phe) loading test is based on stressing the hepatic phenylalanine hydroxylase with a Phe load, which is not converted to tyrosine (Tyr) at a normal rate due to the partial BH4 deficiency in the liver ³⁹. Several reports have demonstrated the usefulness of this test in the diagnosis of this disorder ^39–43. Even though DRD usually presents in childhood, these studies were performed mainly in adult patients; therefore, experience with this test in children with adGTPCH deficiency is very limited. There is no general consensus about the interpretation of the results, although the Phe/Tyr ratio at 4 hours after Phe loading seems the best marker for the diagnosis ⁴². We evaluated the usefulness of Phe loading test in seven pediatric and seven adult patients with adGTPCH deficiency, as described elsewhere ⁴³. Phe/Tyr ratio was increased in all adGTPCH deficiency pediatric patients when compared with controls at 2 and 4 hours after Phe loading. All adult adGTPCH deficiency patients showed an increased Phe/Tyr ratio at any time after Phe load. Only two out of seven pediatric patients showed a ratio higher than 5.25 at 4 h, while six of the seven adult patients showed a higher value. This previously suggested cut-off value was useful for data assessment of adult patients. Regarding pediatric patients, it seems necessary to establish a lower cut-off value. We had to perform the Phe and Tyr quantification in blood spots, since patients were from a different geographical area than that of the laboratory of analysis. Therefore, we could not analyze Phe, Tyr, and pterin concentrations in plasma samples, in contrast to other authors ^39–42. Blood spot analysis of Phe/Tyr after oral Phe load would appear to be reliable for differential diagnosis of this disorder. However, false-negative results have been documented in GCH1 mutation carriers ⁴², and heterozygous phenylketonuric subjects show the same abnormal Phe/Tyr profiles as DRD patients in this test.

The examinations of pterins and neurotransmitter metabolites in CSF require an invasive procedure, but are very specific. Within the central nervous system, the end product for dopamine is homovanillic acid; for serotonin, it is 5-hydroxyindoleacetic acid ⁴⁴. In adGTPCH deficiency patients, low levels of homovanillic acid and reduced levels of neopterin and biopterin in CSF are found. The 5-hydroxyindoleacetic may be normal or reduced as well. Reduced levels of biopterin associated with normal concentration of neopterin can be observed in early-onset parkinsonism. Both pterin metabolites are normal in TH deficiency; neopterin is also normal in sepiapterin reductase deficiency. Viral infections may elevate neopterin in CSF. These studies are available in few laboratories, limiting its usefulness in the clinical practice.

Absolute confirmation for adGTPCH deficiency in cases with normal molecular studies requires enzyme assay in cytokine-stimulated fibroblasts, which seems to be the gold standard laboratory test for the diagnosis of DRD ⁴⁵. Intracellular neopterin and biopterin concentrations and GTPCH activity are measured in this test.

CONCLUSION

We present an algorithm for the diagnostic approach for adGTPCH deficiency in patients with unexplained dystonia, parkinsonism, or spasticity (Figure 1). The primary aim of this diagnostic approach is not to miss the detection of this eminently treatable condition. The correct recognition and differentiation of the motor disturbances remain the basis. Neuroimaging is the initial investigation as it is indispensable to identify secondary causes. Secondary investigations are directed to identify patients in whom motor disturbances could be the presenting symptom of a neurotransmitter disease. Currently, several approaches are possible. The classical approach through a trial of levodopa is less reliable and it may be a source of diagnostic pitfalls. The examinations of pterins and neurotransmitter metabolites in CSF require a lumbar puncture, but are a way to make a reliable diagnosis. The oral Phe loading test may be the most appropriate, as first-line laboratory test, for identifying patients with suspected adGTPCH deficiency. Genetic testing tries to identify the molecular defect although GCH1 DNA sequencing only reveals mutations in about half of adGTPCH deficiency patients ⁴¹. If no mutation is detected, further confirmation of the diagnosis is possible through the measurement of intracellular neopterin and biopterin concentrations and GTPCH activity in cytokine–stimulated fibroblasts; however, these studies are available in very few specialized laboratories worldwide. The secondary genetic testing tries to identify the patient in whom a deletion in the GCH1 gene not identifiable through sequencing causes adGTPCH deficiency. These gene dosage analyses of the GCH1 should be incorporated into the routine of the molecular diagnosis.

In summary, as DRD is a syndrome with different causes and in the recent years a considerable expansion of the clinical spectrum of Segawa disease has emerged, it is advisable to perform an adequate differential diagnosis through a structured series of investigations whose aim is not to miss the detection of this eminently treatable condition and avoid potential pitfalls in the diagnosis. We recommend approaching to a certain diagnosis through a combination of clinical, imaging, as well as biochemical and genetic investigations.

Keywords

Segawa Syndrome, Dopa-Responsive Dystonia, Guanosine Triphosphate Cyclohydrolase I Deficiency, GCH1 Gene

Disclosure: The authors declare no conflict of interest.

REFERENCES

1. Ichinose H, Ohye T, Takahashi E, et al. Hereditary progressive dystonia with marked diurnal fluctuation caused by mutations in the CTP cyclohydrolase I gene. Nat Genet. 1994;8:236–242.
2. Segawa M, Hosaka A, Miyagawa F, Nomura Y, Imai H. Hereditary progressive dystonia with marked diurnal fluctuation. Adv Neurol. 1976;14:215–233.
3. Thöny B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J. 2000;347:1–16.
4. Blau N. Tetrahydrobiopterin deficiencies and movement disorders. In: Fernández-Alvarez E, Arzimanoglou A, Tolosa E, eds. Paediatric Movement Disorders. Progress in Understanding. Montrouge: John Libbey Eurotext; 2005:213–222.
5. Tassin J, Durr A, Bonnet AM, et al. Levodopa-responsive dystonia. GTP cyclohydrolase I or parkin mutations? Brain. 2000;123:1112–1121.
6. Nygard TG. Dopa-responsive dystonia. Delineation of the clinical syndrome and clues to pathogenesis. Adv Neurol. 1993;60:577–585.
7. Segawa M, Nomura Y, Nishiyama N. Autosomal dominant guanosine triphosphate cyclohydrolase I deficiency (Segawa disease). Ann Neurol. 2003;54:S32–S45.
8. Nygaard TG, Waren SP, Levine RA, Naini AB, Chutorian AM. Doparesponsive dystonia simulating cerebral palsy. Pediatr Neurol. 1994;11: 236–240.
9. Bandmann O, Nygaard TG, Surtees R, Marsden CD, Wood NW, Harding AE. Dopa-responsive dystonia in British patients: new mutations of the GTP-cyclohydrolase I gene and evidence for genetic heterogeneity. Hum Mol Genet. 1996;5:403–406.
10. Steinberger D, Korinthenberg R, Topka H, Berghäuser M, Wedde R, Müller U. Dopa-responsive dystonia: mutation analysis of GCH1 and analysis of therapeutic doses of L-dopa. German Dystonia Study Group. Neurology. 2000;55:1735–1737.
11. Hagenah J, Saunders-Pullman R, Hedrich K, et al. High mutation rate in dopa-responsive dystonia: detection with comprehensive GCHI screening. Neurology. 2005;64:908–911.
12. Zirn B, Steinberger D, Troidl C, et al. Frequency of GCH1 deletions in doparesponsive dystonia. J Neurol Neurosurg Psychiatry. 2008;79:183–186.
13. Bandmann O, Wood NW. Dopa-responsive dystonia—the story so far. Neuropediatrics. 2002;33:1–5.
14. López-Laso E, Ochoa-Sepúlveda JJ, Ochoa-Amor JJ, et al. Segawa syndrome due to mutation Q89X in the GCH1 gene: a possible founder effect in Córdoba (southern Spain). J Neurol. 2009;256:1816–1824.
15. Trender-Gerhard I, Sweeney MG, Schwingenschuh P, et al. Autosomaldominant GTPCH1-deficient DRD: clinical characteristics and long-term outcome of 34 patients. J Neurol Neurosurg Psychiatry. 2009;80:839–845.
16. Hjermind LE, Johannsen LG, Blau N, et al. Dopa-responsive dystonia and early-onset Parkinson’s disease in a patient with GTP cyclohydrolase I deficiency? Mov Disord. 2006;21:679–682.
17. Bandmann O, Valente EM, Holmans P, et al. Dopa-responsive dystonia: a clinical and molecular genetic study. Ann Neurol. 1998;44:649–656.
18. Hahn H, Trant MR, Brownstein MJ, Harper RA, Milstien S, Butler IJ. Neurologic and psychiatric manifestations in a family with a mutation in exon 2 of the guanosine triphosphate-cyclohydrolase gene. Arch Neurol. 2001;58:749–755.
19. Cheyette BN, Cheyette SN, Cusmano-Ozog K, Enns GM. Dopa-responsive dystonia presenting as delayed and awkward gait. Pediatr Neurol. 2008;38:273–275.
20. Grimes DA, Barclay CL, Duff J, Furukawa Y, Lang AE. Phenocopies in a large GCH1 mutation positive family with dopa responsive dystonia: confusing the picture? J Neurol Neurosurg Psychiatry. 2002;72:801–804.
21. Nygaard TG, Marsden CD, Fahn S. Dopa-responsive dystonia: long-term treatment response and prognosis. Neurology. 1991;41:174–181.
22. Steinberger D, Trübenbach J, Zirn B, Leube B, Wildhardt G, Müller U. Utility of MLPA in deletion analysis of GCH1 in dopa-responsive dystonia. Neurogenetics. 2007;8:51–55.
23. López-Laso E, Camino R, Mateos ME, et al. Dopa-responsive infantile hypokinetic rigid syndrome due to dominant guanosine triphosphate cyclohydrolase 1 deficiency. J Neurol Sci. 2007;256:90–93.
24. Nitschke M, Steinberger D, Heberlein I, Otto V, Muller U, Vieregge P. Dopa responsive dystonia with Turner’s syndrome: clinical, genetic, and neuropsychological studies in a family with a new mutation in the GTPcyclohydrolase I gene. J Neurol Neurosurg Psychiatry. 1998;64:806–808.
25. Leuzzi V, Carducci Ca, Carducci Cl, Cardona F, Artiola C, Antonozzi I. Autosomal dominant GTP-CH deficiency presenting as a doparesponsive myoclonus-dystonia syndrome. Neurology. 2002;59:1241–1243.
26. Uncini A, De Angelis MV, Di Fulvio P, et al. Wide expressivity variation and high but no gender-related penetrance in two doparesponsive dystonia families with a novel GCH-I mutation. Mov Disord. 2004;19:1139–1145.
27. Chaila EC, McCabe DJ, Delanty N, Costello DJ, Murphy RP. Broadening the phenotype of childhood-onset dopa-responsive dystonia. Arch Neurol. 2006;63:1185–1188.
28. Furukawa Y, Rajput AH. Inherited myoclonus-dystonia: how many causative genes and clinical phenotypes? Neurology. 2002;59:1130–1131.
29. Van Hove JL, Steyaert J, Matthijs G, et al. Expanded motor and psychiatric phenotype in autosomal dominant Segawa syndrome due to GTP cyclohydrolase deficiency. J Neurol Neurosurg Psychiatry. 2006;77:18–23.
30. Venna N, Sims KB, Grant PE. Case records of the Massachusetts General Hospital. Case 26-2006. A 19-year-old woman with difficulty walking. N Engl J Med. 2006;355:831–839.
31. Romstad A, Dupont E, Krag-Olsen B, Østergaard K, Guldberg P, Güttler F. Dopa-responsive dystonia and Tourette syndrome in a large Danish family. Arch Neurol. 2003;60:618–622.
32. Furukawa Y, Lang AE, Trugman JM, et al. Gender-related penetrance and de novo GTP-cyclohydrolase I gene mutations in dopa-responsive dystonia. Neurology. 1998;50:1015–1020.
33. Steinberger D, Weber Y, Korinthenberg R, et al. High penetrance and pronounced variation in expressivity of GCH1 mutations in five families with dopa-responsive dystonia. Ann Neurol. 1998;43:634–639.
34. Grötzsch H, Schnorf H, Morris MA, et al. Phenotypic heterogeneity of dopa-responsive dystonia in monozygotic twins. Neurology. 2004;62: 637–639.
35. De la Fuente-Fernández R. Drug-induced motor complications in doparesponsive dystonia: implications for the pathogenesis of dyskinesias and motor fluctuations. Clin Neuropharmacol. 1999;22:216–221.
36. Robinson R, McCarthy GT, Bandmann O, Dobbie M, Surtees R, Wood NW. GTP cyclohydrolase deficiency; intrafamilial variation in clinical phenotype, including levodopa responsiveness. J Neurol Neurosurg Psychiatry. 1999;66:86–89.
37. Hwang WJ, Calne DB, Tsui JK, de la Fuente-Fernández R. The longterm response to levodopa in dopa-responsive dystonia. Parkinsonism Relat Disord. 2001;8:1–5.
38. Furukawa Y. Genetics and biochemistry of dopa-responsive dystonia: significance of striatal tyrosine hydroxylase protein loss. Adv Neurol. 2003;91:401–410.
39. Hyland K, Fryburg JS, Wilson WG, et al. Oral phenylalanine loading in dopa-responsive dystonia: a possible diagnostic test. Neurology. 1997;48:1290–1297.
40. Hyland K, Nygaard TG, Trugman JM, Swoboda KJ, Arnold LA, Sparagana SP. Oral phenylalanine loading profiles in symptomatic and asymptomatic gene carriers with dopa-responsive dystonia due to dominantly inherited GTP-cyclohydrolase deficiency. J Inherit Metab Dis. 1999;22: 213–215.
41. Bandmann O, Goertz M, Zschocke J, et al. The phenylalanine loading test in the differential diagnosis of dystonia. Neurology. 2003;60:700–702.
42. Saunders-Pullman R, Blau N, Hyland K, et al. Phenylalanine loading as a diagnostic test for DRD: interpreting the utility of the test. Mol Genet Metab. 2004;83:207–212.
43. López-Laso E, Ormazabal A, Camino R, et al. Oral phenylalanine loading test for the diagnosis of dominant guanosine triphosphate cyclohydrolase 1 deficiency. Clin Biochem. 2006;39:893–897.
44. Hyland K. The lumbar puncture for diagnosis of pediatric neurotransmitter diseases. Ann Neurol. 2003;54:S13–S17.
45. Bonafé L, Thöny B, Leimbacher W, Kierat L, Blau N. Diagnosis of doparesponsive dystonia and other tetrahydrobiopterin disorders by the study of biopterin metabolism in fibroblasts. Clin Chem. 2001;47:477–485.

Aneurysmal subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke due to rupture of a cerebral aneurysm, accounts for 2–5% of all new strokes, but results in 22–25% of cerebrovascular deaths ¹. Nearly one-third of patients require lifelong care ^{2, 3}. Worldwide incidence is 10.5/100 000 personyears ². The incidence of this disease has remained stable over the past three decades, but the case-fatality index has improved significantly from 50% to 60% to approximately 30–35% ^4–6. Reasons for improved survival likely include earlier diagnosis of brain aneurysms, alternatives to surgery for ruptured aneurysms, endovascular therapy for cerebral vasospasm, and dedicated neurointensive care units for the care of patients with SAH. A thorough understanding of SAH and its complications, as well as a familiarity with modern multimodality neuromonitoring technology facilitate optimal patient care. This article reviews strategies for the optimal management of SAH in the intensive care setting.

EMERGENCY MANAGEMENT

Emergency management of SAH patients focuses on prompt and accurate diagnosis as well as hemodynamic and neurologic stabilization and support. SAH should be suspected in patients who present with sudden onset of a severe headache. Associated symptoms include nausea, vomiting, neck pain, photophobia, and altered consciousness. Physical examination may reveal retinal hemorrhages, cranial nerve palsies, weakness, altered consciousness, or a normal neurological exam. Hypertension, tobacco use, and heavy alcohol consumption have been consistently identified as independent risk factors for SAH ⁷. Other risk factors include female gender, advanced age, family history of SAH, and cocaine use ⁸. Particular racial and ethnic groups have a higher risk of aneurysmal SAH, as do those with certain genetic syndromes such as Ehlers–Danlos type IV or autosomal dominant polycystic kidney disease ^9–11. The risk of cerebral aneurysm rupture depends heavily on the size and location of aneurysm; large aneurysms and those in the posterior circulation have a higher tendency to bleed ¹².

Initial signs and symptoms may predict outcome. Decreased level of consciousness portends a poor outcome in SAH. Several clinical grading scales are used to determine candidacy for intervention and to predict response to treatment ¹³. The most commonly used clinical grading scales are the Hunt and Hess and the World Federation of Neurological Surgeons (WFNS) scales. The Hunt and Hess score is based on level of consciousness and the presence of neurological signs, while the WFNS scale combines focal neurological findings with the Glasgow Coma Scale (Table 1). Radiological scales, such as the Fisher classification (Table 2), assess quantity of blood in the subarachnoid space and presence of intraventricular hemorrhage and correlate with risk for delayed ischemic deficits and overall outcome ^{14, 15}.

A computed tomography (CT) scan of the brain should be urgently obtained for all patients with a suspected SAH (Figure 1). Sensitivity of head CT for SAH decreases over time due to rapid clearance of blood. Head CT scans have a sensitivity of 93% at 24 hours, 70% at 72 hours, and 50% by day 7 ¹⁶. Lumbar puncture should be performed in patients with a high clinical suspicion of SAH and a negative head CT. Cerebrospinal fluid (CSF) cell counts should be obtained in both tubes 1 and 4 to avoid erroneous diagnosis of SAH. For example, if the number of red blood cells decreases to zero in the fourth tube then traumatic needle insertion may explain the presence of red blood cells in tube 1. The presence of CSF xanthochromia at least 12 hours after symptom onset corroborates a diagnosis of SAH. The combination of CT and LP can exclude SAH in 100% of cases ¹⁷. Catheter angiography or CT angiography may be employed to assess further for the presence of cerebral aneurysms (Figure 2). One study suggests that CT followed by CT angiography (CTA) reliably excludes aneurysmal SAH with >99% posttest probability ¹⁸.

The initial goals of treatment are to stabilize the patient's airway, breathing, and circulation. Serial assessments are required as patients may rapidly deteriorate in the hours after presentation. Placement of an endotracheal tube with rapid sequence intubation should be performed in those patients who are unable to protect their airway or in those with respiratory compromise. Once cardiopulmonary stability has been achieved, the patient must be admitted to an intensive care unit (ICU). Transferring the patient to a specialized neuroscience ICU in a tertiaryor quaternary-care setting with dedicated vascular neurosurgeons, endovascular specialists, and neurointensivists should be considered.

INTENSIVE CARE MANAGEMENT

The ICU management of aneurysmal SAH can be divided into two phases, the early phase, prior to treatment of the aneurysm, and the late phase, after aneurysm treatment. The early ICU pretreatment phase focuses on the prevention of rebleeding. The later ICU posttreatment phase is centered on treatment of DCI and other medical and neurological complications.

Early ICU Management

Aneurysmal Rebleeding

Aneurysmal rebleeding is the most threatening complication in the early management period, with the greatest risk occurring in the first 24 hours ^{19, 20}. Therefore, definitive treatment of the aneurysm should be performed early. The mainstay of therapy to secure a ruptured aneurysm is either surgical clipping or endovascular embolization, usually with detachable coils. The International Subarachnoid Aneurysm Trial (ISAT) randomized patients to either surgical clipping or endovascular coiling ²⁰. Although endovascular treatment was associated with a slightly higher rate of SAH recurrence, it was also found be less morbid than surgical clipping. There was no significant difference in mortality between the treatment groups.

Until procedural exclusion of the aneurysm is performed, medical measures are employed to minimize the risk of rebleeding. Blood pressure reduction is recommended; one study showed that the incidence of prehospital rebleeding was greater among patients whose systolic blood pressure was greater than 160 mmHg ²¹. However, treatment of acute hypertension has never been proven to reduce the risk of rebleeding. Commonly used medications include nicardipine, labetalol, and esmolol. Caution must be exercised when lowering blood pressure in patients with either chronic hypertension or elevated intracranial pressure as aggressive measures may precipitate cerebral ischemia. Secondary measures to prevent rebleeding may include bed rest, pain control, and relief of anxiety. Prolonged use of antifibrinolytic agents, such as tranexamic acid and epsilon-aminocaproic acid, may reduce the risk of rebleeding but their benefit is mitigated by an increased risk of cerebral infarction ²². Further study is needed to determine whether a short course of antifibrinolytic therapy reduces the rate of early rebleeding without incurring an increased risk of stroke.

Hydrocephalus

Hydrocephalus occurs in 20–30% of patients with SAH ^23–25. Both communicating and non-communicating hydrocephalus may occur; the latter occurs more frequently due to the presence of intraventricular blood. It can occur immediately at the time of aneurysmal rupture, or may develop later in the course of the disease. In addition to intraventricular hemorrhage, risk factors for the development of hydrocephalus include premorbid diagnosis of hypertension, posterior aneurysm location, and high Hunt and Hess grade ²⁴. The presence of hydrocephalus is associated with poor outcome ²⁴. When hydrocephalus is associated with a diminished level of consciousness, a ventriculostomy should be placed. CSF diversion often leads to an improved level of arousal. However, when hydrocephalus occurs with lesser symptoms, the clinical significance and therapeutic management are less clear. In patients with a ventriculostomy prior to aneurysm treatment, CSF should not be drained rapidly or frequently as this may change in transmural pressure across the aneurysm and may increase the likelihood of rerupture. It is reasonable to maintain the collection chamber open at 20 cm above the tragus.

Seizures

The risk and impact of seizures after SAH is not well defined. Shaking and other abnormal movements may occur at the time of hemorrhage; however, it is unclear whether this represents true epileptic phenomena. In a retrospective single-center observational study of over 500 SAH patients, the incidence of seizures was 5%, but the incidence was double in patients who eventually died ²⁶. In a case series of selected patients undergoing continuous electroencephalography (cEEG) monitoring, nonconvulsive status epilepticus was observed in 19% of comatose patients an average of 18 days after the ictus ²⁷.

Although the risk of seizures may be high in the SAH population, the use of prophylactic antiepileptic drugs (AEDs) remains controversial. Cumulative exposure to phenytoin is associated with poor long-term cognitive outcome ²⁸; however, the incidence of nonconvulsive status in the comatose patient is high. It may be reasonable to treat with prophylactic AEDs prior to treatment of the aneurysm, as a seizure-related surge in blood pressure may result in rebleeding. Once the aneurysm is secured, discontinuation of prophylactic AEDs may be reasonable, especially if cEEG monitoring is available.

Cardiac Arrhythmias

Cardiac arrhythmias, including supraventricular and ventricular tachycardias, and sinoatrial and atrioventricular blocks, occur in 90–100% of patients ²⁹ and are generally not associated with coronary artery disease. Of these, 5% are lifethreatening ³⁰. The frequency and severity of arrhythmias are greatest in the first 48 hours after the hemorrhage. These arrhythmias are often associated with prolongation of the QT segment and hypokalemia. Six to 22% of patients have STsegment elevations or depressions ^{30, 31}. In a meta-analysis, Q waves, ST-segment depression and T wave abnormalities were significantly associated with increased mortality ³¹. STsegment depression increased the relative risk of both poor outcome and delayed cerebral ischemia (DCI) by a factor of 2.4. Cardiac arrhythmias should be treated according to standard ACLS protocols.

Late ICU Management

Diagnosis of Cerebral Vasospasm and Delayed Cerebral Ischemia

After successful treatment of the ruptured aneurysm, patients remain at risk for potentially devastating cerebral ischemia. This risk is particularly great within the first 2 weeks posttreatment. The terminology used to describe the clinical deterioration from cerebral ischemia occurring mainly between days 4 and 14 is varied; it has been called “delayed neurological ischemic deficit”, “delayed neurological deficit”, “delayed ischemic deficit”, “secondary cerebral ischemia”, “clinical vasospasm”, and “symptomatic vasospasm” ³². In an effort to standardize research definitions and clinical strategies, the use of the term “delayed cerebral ischemia” has been suggested by a multidisciplinary research group ³². This group recommends that the term “vasospasm” be reserved for radiographic (digital subtraction angiography [DSA], CTA, magnetic resonance angiography [MRA]) evidence of arterial narrowing.

Narrowing of the large arteries at the base of the brain demonstrated by catheter angiography is referred to as “angiographic vasospasm” (Figure 3). It is associated with DCI, presumably from reduced cerebral perfusion. Although angiographic vasospasm and DCI are associated, not all angiographic vasospasm results in DCI and not all DCIs result from vasospasm. Angiographic vasospasm is seen in 21–70% of patients, with a typical onset 3–5 days after hemorrhage, maximal narrowing at 6–8 days, and resolution over 2–4 weeks ^{33, 34}. Thickness of clot in the basilar cisterns correlates with risk of developing angiographic vasospasm; however, the pathophysiology is poorly understood ³⁵. In about one-half of cases, angiographic vasospasm is manifested by the occurrence of DCI. DCI may resolve spontaneously, or may progress to irreversible injury. DCI accounts for most morbidity and 50% of mortality in patients who survive the initial hemorrhage.

Vasospasm may not be the sole cause of DCI; microvascular dysfunction due to in situ thromboemboembolism from a local hypercoagulable and inflammatory state has been proposed as a complementary mechanism ³⁶. Alternatively, cortical spreading depression (CSD) and cortical spreading ischemia (CSI) may play a role ³⁷. CSD describes a wave of neuronal depolarization and edema, instigated by an influx of cations, which overwhelms ATP-dependent sodium and calcium pump function. CSI describes the resultant ischemia from prolonged CSD coupled with inadequate perfusion ³⁷. Diagnosis of CSD and the measurement of the negative DC potential shift currently require invasive technologies.

A variety of strategies are employed to screen for DCI. All patients should be examined serially for subtle changes in the neurological exam. Spontaneous hypertension may herald cerebral ischemia as it may represent a compensation for increased cerebrovascular resistance and should not be treated. DCI may occur without clear clinical manifestations, especially in the comatose patient.

Transcranial Doppler ultrasound (TCD) is a widely used screening method for DCI. It is a safe, portable, noninvasive tool that may be performed repeatedly, even continuously, at the patient's bedside. However, the sensitivity and specificity of this diagnostic study remains controversial. The chief limitation of TCD is that it measures cerebral blood flow (CBF) velocities in limited segments of large intracranial vessels. TCD values are most accurate for the middle cerebral artery, but tend to be poor for other arteries ^38–40. TCD is operator-dependent and in 15% patients, readings are unobtainable due to poor sonographic windows from relative hyperostosis of the temporal bones. A variety of factors, such as vessel anatomy, age, intracranial pressure, mean arterial blood pressure, hematocrit, arterial CO2 content, collateral flow patterns, and response to therapeutic interventions, influence flow velocities ⁴¹. The ratio of the velocities in the middle cerebral artery and the ipsilateral extracranial carotid artery, or Lindegaard ratio, differentiates elevated blood flow velocities due to vasospasm from other causes ⁴². Lindegaard ratios of 5–6 indicate severe vasospasm ⁴³. Despite the variable sensitivity of TCD, the American Academy of Neurology Expert committee supports its use since it can identify severe vasospasm with fairly high reliability ⁴¹.

Continuous electroencephalography is emerging as a promising means of detecting cerebral ischemia ⁴⁴. Ischemia produces characteristic changes in brain electrical activity with increased levels of delta and theta waveforms. Computer algorithms analyze these waveforms and generate simplified graphic representations indicating trends of ischemia in real time. Continuous EEG provides information about both the location and degree of ischemia. In one series, a loss of relative alpha variability was 100% sensitive for the detection of angiographic vasospasm ⁴⁵. Changes on continuous EEG may be evident prior to clinical deterioration ⁴⁶. Its noninvasive nature, high degree of sensitivity, and the fact that evidence of ischemia precedes clinical change make cEEG an appealing diagnostic monitor. However, further study is needed to define the optimal use of cEEG in SAH patients. Trained electroencephalographers are required to confirm the bedside impression of ischemia.

The gold standard for detection of arterial narrowing is catheter-based cerebral DSA. It is an invasive test that requires significant resources and entails some risk. Angiographic vasospasm may be evident in patients who never manifest clinical symptoms; in these patients, treatment may not be warranted. Neurological complications of catheter angiography include thrombotic and embolic stroke, arterial perforation and dissection, and intracranial hemorrhage. Other complications include those related to sedatives and anesthesia, puncture site complications, and contrast nephropathy and allergic reactions. The overall complication rate is 1–2% ⁴⁷.

Noninvasive angiographic techniques including CTA and magnetic resonance angiography (MRA) are being used more commonly in lieu of catheter angiography for the diagnosis of vasospasm ^{48, 49}. CTA has a high sensitivity and high negative predictive value for severe vasospasm, but it is less useful for detecting mild to moderate vasospasm ⁵⁰. Its diagnostic utility may be increased with the addition of CT perfusion, but the requisite contrast load, and thus the risk for nephrotoxicity, also increases. Clip and coil artifact, patient transport, and prolonged study time make MRA a less viable option for detection of vasospasm.

The use of novel diagnostic modalities for the detection of DCI is becoming more widespread. Xenon CT and single photon emission computed tomography (SPECT) provide quantitative global and regional blood flow information ^51–53. Xenon can be inhaled as a gas or administered intravenously. An advantage of xenon CT is that it can be performed at the patient's bedside; however, there is a risk of apnea with xenon gas in the nonintubated patient. Near-infrared spectroscopy (NIRS) can be used to noninvasively estimate cerebral blood flow (CBF) through the intact skull. This method uses near-infrared light to measure the absorption spectra of oxyhemoglobin and deoxyhemoglobin in the frontal cerebral cortex. It is rapid, safe, and relatively inexpensive. Cerebral microdialysis (CMD) is a technique in which a thin tube with a semipermeable membrane is placed into brain tissue allowing free diffusion of water and solutes between the interstitial fluid and perfusate. Markers of brain tissue ischemia, such as lactate, pyruvate, and glutamate, as well as markers of cell injury, such as glycerol, are quantified and trended ^{54, 55}. Important factors affecting recovery of these substances in vivo include the length and permeability of the membrane, the diffusion coefficient and molecular weight of the substance of interest, and the perfusion flow rate. Cerebral ischemia may cause a decrease in the partial pressure of oxygen in the interstitium (PbtO2). PbtO2 can be monitored continuously via a Clark-type electrode placed directly into brain parenchyma and may enhance diagnosis of DCI ^{56, 57}.

Treatment of Cerebral Vasospasm and DCI

The goal for the management of DCI is to reduce the risk of brain infarction. Once the aneurysm has been treated, measures are taken to ensure adequate cerebral perfusion. Intracranial hypertension is treated aggressively and blood pressure parameters are liberalized. Systemic and metabolic insults such as fever, acidosis, electrolyte imbalance, hypoglycemia, and hypoxia should be avoided and treated promptly if they do occur. Infection, sepsis, and the systemic inflammatory immune response syndrome (SIRS) have all been shown to increase the risk of vasospasm and DCI ^58–60. Efforts should be made to maintain blood sugars within a normal range. Although multiple pilot clinical trials suggested that intravenous magnesium improved outcome in SAH, two recent Phase 3 randomized placebo-controlled trials demonstrated conflicting results regarding the utility of intravenous magnesium sulfate ^61–63. Optimal hemoglobin concentration is unknown. Both anemia and red blood cell transfusion are associated with harm in the SAH population ^{64, 65}. Studies are underway to determine the optimal transfusion threshold.

Hemodynamic augmentation to improve CBF is the mainstay of DCI treatment, although its efficacy has been studied with only one randomized controlled trial. The main components of this therapy include induced hypertension, induced hypervolemia, and hemodilution (“triple H therapy”). Triple H therapy has been used to treat DCI but should not be used prophylactically ^{66, 67}. Avoidance of hypovolemia and hypotension is clearly supported by the literature, likely because of compromised CBF ^{68, 69}. However, support for aggressive hypervolemia is less strong. Induced hypertension using vasopressors for DCI has been shown to be efficacious by a number of studies ^70–72. Hemodilution is the least well-studied component of this therapy. It is thought to result in rheological improvements in CBF; however, it may come at a cost to cerebral oxygen delivery. The authors recommend euvolemic-induced hypertension for augmentation of CBF in patients with DCI.

The only medication that is known to reduce the risk of poor outcome after SAH is oral nimodipine, an L-type dihydropyridine calcium channel blocker ⁷³. Administration of this drug reduces the incidence of cerebral infarction and poor outcome at 3 months compared to placebo ⁷³. The mechanism by which nimodipine exerts its beneficial effects is unclear; it does not appear to reverse angiographic vasospasm ⁷³. Interestingly, intravenous nicardipine, another L-type calcium channel blocker, reduces the incidence of angiographic vasospasm but does not improve outcome ⁷⁴. Administration of 60 mg of nimodipine orally every 4 hours for 21 days is warranted unless contraindicated.

Catheter-based intra-arterial administration of vasodilators, such as papaverine and calcium channel blockers, has been shown to reverse angiographic vasospasm and result in clinical improvement ^75–77. Intracranial pressures should be monitored during administration of these medications, as vasodilators can increase ICP through an increase in blood volume ^78–80. Anecdotal reports suggest that endovascular balloon angioplasty effectively reverses vasospasm and may be a more durable treatment in large proximal vessels. One study suggests that the odds of being discharged home are higher at centers that routinely perform angioplasty for angiographic vasospasm ⁸¹. However, there are no data from randomized controlled trials on the efficacy of this intervention. The combination of intra-arterial vasodilators and balloon angioplasty has been reported in the literature; it is unknown whether combination therapy is superior to either single therapy ^{82, 83}. Current American Stroke Association guidelines for the management of acute SAH state that cerebral angioplasty may be reasonable after, or together with, or in place of tripleH therapy depending on clinical scenario (Class IIb, Level of evidence B) ³².

Many novel approaches to the treatment of cerebral vasospasm and DCI are currently being evaluated. Statins upregulate endothelial nitric oxide synthase (eNOS) to improve vasomotor reactivity, increase CBF and mitigate the development of a cerebral vasculopathy. Early administration of high-dose statin therapy in SAH patients has been shown to reduce the incidence of arterial vasospasm, DCI, and mortality in small Phase II studies ^{84, 85}. However, a recent meta-analysis of its efficacy demonstrated no benefit ⁸⁶. The results of a large-scale Phase III study are expected to provide more data about efficacy.

Free radical scavengers and endothelin-receptor antagonists appear to reduce radiographic vasospasm and DCI but have not been shown to consistently improve outcomes ^87–89. Cisternal fibrinolysis with urokinase has been shown to reduce vasospasm and improve outcome, but further study is needed ⁹⁰. Treatment with high-dose methylprednisolone to reduce the inflammatory component of vasospasm did not reduce the incidence of vasospasm, but did improve functional outcome at 1 year ⁹¹.

Hyponatremia

Hyponatremia occurs in 10–30% of SAH patients and may be associated with poor outcome ^92–94. Risk factors for hyponatremia include hydrocephalus, anterior communicating artery aneurysms, and poor clinical grade ^{94, 95}. Although controversial, cerebral salt wasting (CSW) is thought to be the cause of hyponatremia after SAH. It is a hypovolemic hyponatremia resulting from renal sodium loss. CSW is distinguished from the syndrome of inappropriate antidiuretic hormone (SIADH) by determination of intravascular volume status, which is difficult in practice. Regardless of the etiology, the treatment of hyponatremia in SAH patients is administration of salt. Most cases can be treated by enteral sodium chloride and intravenous isotonic crystalloid. Fluid restriction should be avoided as volume contraction has been linked to vasospasm ^{96, 97}. Fludrocortisone may help correct the hyponatremia and minimize the need for volume resuscitation ⁹⁶.

Neurogenic Stunned Myocardium and Cardiomyopathy

A reversible cardiomyopathy occurs in 10–28% of SAH patients and is likely related to excessive norepinephrine release from sympathetic nerve terminals in the heart ⁹⁸. Myocardial perfusion is normal and coronary artery disease is absent. A variety of wall motion abnormalities may be seen, none of which respect coronary arterial distributions. Reported risk factors include female sex, younger age, smaller body surface area, anterior aneurysm location, higher clinical SAH grade, and prior cocaine or amphetamine use ^{98, 99}.

Presenting signs of neurogenic stunned myocardium can range from sinus tachycardia to frank cardiogenic shock. STsegment elevation is the most common ECG abnormality. Nonspecific T-wave changes and a new bundle branch block may also be observed. Troponin I elevation correlates with neurological, not cardiac, injury ¹⁰⁰. Echocardiography may demonstrate hypokinesis or akinesis of the apical segment of the left ventricle with preservation of function at the base ^101–103. This pattern produces apical ballooning or the socalled Tako-Tsubo pattern. However, more common after SAH is hypokinesis of the mid-segment (apical-sparing) or hypokinesis of the base with preserved function at the apex (inverted Tako-Tsubo pattern) ^{101, 103, 104}.

Treatment of neurogenic stunned myocardium is largely supportive as this condition is transient and reverses within days ^{105, 106}. Administration of intravenous crystalloids may suffice if symptoms are mild. Vasopressors and/or inotropes may be used for more significant cardiac dysfunction. It may be beneficial to choose a noncatecholamine inotrope, such as milrinone, as catecholamines are thought to cause the inciting injury. Dynamic left ventricular outflow obstruction should be excluded prior to using inotropes. Judicious use of diuretics is imperative given the association between volume depletion and poor outcome after SAH. Vasopressors, inotropes, and intra-aortic balloon counterpulsation should be employed as necessary when cardiogenic shock is present. Rare complications of neurogenic stunned myocardium include left ventricular free-wall rupture and formation of intracardiac thrombus.

CONCLUSIONS

Critical to the management of patients with SAH is the ability to make a prompt and accurate diagnosis, to anticipate the natural history and complications of the disease, and to prevent further neurological injury. Aneurysmal rebleeding, hydrocephalus, seizures, vasospasm, and delayed neurological ischemic deficits represent major threats. Systemic complications, such as hypotension, hyponatremia, and cardiac dysfunction, can exacerbate neurological deficits and cause further damage. Early identification and treatment of these disturbances improves outcome. Technological advances may improve the detection of ischemia, and help to mitigate secondary injury.

Keywords

Aneurysm, subarachnoid hemorrhage, delayed cerebral ischemia, cerebral vasospasm, stroke, cerebral infarction, ICU management, hypertension

Disclosure: The authors declare no conflict of interest.

REFERENCES

1. King JT, Jr. Epidemiology of aneurysmal subarachnoid hemorrhage. Neuroimaging Clin N Am. 1997 Nov;7(4):659–668.
2. Linn FH, Rinkel GJ, Algra A, van Gijn J. Incidence of subarachnoid hemorrhage: role of region, year, and rate of computed tomography: a meta-analysis. Stroke. 1996 Apr;27(4):625–629.
3. Hop JW, Rinkel GJ, Algra A, van Gijn J. Case-fatality rates and functional outcome after subarachnoid hemorrhage: a systematic review. Stroke. 1997 Mar;28(3):660–664.
4. Nieuwkamp DJ, Setz LE, Algra A, Linn FH, de Rooij NK, Rinkel GJ. Changes in case fatality of aneurysmal subarachnoid haemorrhage over time, according to age, sex, and region: a meta-analysis. Lancet Neurol. 2009 Jul;8(7):635–642.
5. Stegmayr B, Eriksson M, Asplund K. Declining mortality from subarachnoid hemorrhage: changes in incidence and case fatality from 1985 through 2000. Stroke. 2004 Sep;35(9):2059–2063.
6. Ingall TJ, Whisnant JP, Wiebers DO, O’Fallon WM. Has there been a decline in subarachnoid hemorrhage mortality? Stroke. 1989 Jun;20(6):718–724.
7. Teunissen LL, Rinkel GJ, Algra A, van Gijn J. Risk factors for subarachnoid hemorrhage: a systematic review. Stroke. 1996 Mar;27(3):544–549.
8. Bederson JB, Connolly ES, Jr., Batjer HH, et al. Guidelines for the management of aneurysmal subarachnoid hemorrhage: a statement for healthcare professionals from a special writing group of the Stroke Council, American Heart Association. Stroke. 2009 Mar;40(3):994–1025.
9. Ruggieri PM, Poulos N, Masaryk TJ, et al. Occult intracranial aneurysms in polycystic kidney disease: screening with MR angiography. Radiology. 1994 Apr;191(1):33–39.
10. Lozano AM, Leblanc R. Cerebral aneurysms and polycystic kidney disease: a critical review. Can J Neurol Sci. 1992 May;19(2):222–227.
11. Kato T, Hattori H, Yorifuji T, Tashiro Y, Nakahata T. Intracranial aneurysms in Ehlers-Danlos syndrome type IV in early childhood. Pediatr Neurol. 2001 Oct;25(4):336–339.
12. Wiebers DO, Whisnant JP, Huston J, 3rd, et al. Unruptured intracranial aneurysms: natural history, clinical outcome, and risks of surgical and endovascular treatment. Lancet. 2003 Jul 12;362(9378):103–110.
13. Dupont SA, Wijdicks EF, Manno EM, Lanzino G, Rabinstein AA. Prediction of angiographic vasospasm after aneurysmal subarachnoid hemorrhage: value of the Hijdra sum scoring system. Neurocrit Care. 2009;11(2):172–176.
14. Frontera JA, Claassen J, Schmidt JM, et al. Prediction of symptomatic vasospasm after subarachnoid hemorrhage: the modified Fisher scale. Neurosurgery. 2006 Jul;59(1):21–27; discussion 7.
15. Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery. 1980 Jan;6(1):1–9.
16. Sames TA, Storrow AB, Finkelstein JA, Magoon MR. Sensitivity of newgeneration computed tomography in subarachnoid hemorrhage. Acad Emerg Med. 1996 Jan;3(1):16–20.
17. Perry JJ, Spacek A, Forbes M, et al. Is the combination of negative computed tomography result and negative lumbar puncture result sufficient to rule out subarachnoid hemorrhage? Ann Emerg Med. 2008 Jun;51(6):707–713.
18. McCormack RF, Hutson A. Can computed tomography angiography of the brain replace lumbar puncture in the evaluation of acute-onset headache after a negative noncontrast cranial computed tomography scan? Acad Emerg Med. Apr;17(4):444–451.
19. Sundt TM, Jr., Whisnant JP. Subarachnoid hemorrhage from intracranial aneurysms. Surgical management and natural history of disease. N Engl J Med. 1978 Jul 20;299(3):116–122.
20. Molyneux A, Kerr R, Stratton I, et al. International Subarachnoid Aneurysm Trial (ISAT) of neurosurgical clipping versus endovascular coiling in 2143 patients with ruptured intracranial aneurysms: a randomized trial. J Stroke Cerebrovasc Dis. 2002 Nov–Dec;11(6):304–314.
21. Ohkuma H, Tsurutani H, Suzuki S. Incidence and significance of early aneurysmal rebleeding before neurosurgical or neurological management. Stroke. 2001 May;32(5):1176–1180.
22. Roos Y, Rinkel G, Vermeulen M, Algra A, van Gijn J. Antifibrinolytic therapy for aneurysmal subarachnoid hemorrhage: a major update of a cochrane review. Stroke. 2003 Sep;34(9):2308–2309.
23. Suarez-Rivera O. Acute hydrocephalus after subarachnoid hemorrhage. Surg Neurol. 1998 May;49(5):563–565.
24. Mehta V, Holness RO, Connolly K, Walling S, Hall R. Acute hydrocephalus following aneurysmal subarachnoid hemorrhage. Can J Neurol Sci. 1996 Feb;23(1):40–45.
25. Sheehan JP, Polin RS, Sheehan JM, Baskaya MK, Kassell NF. Factors associated with hydrocephalus after aneurysmal subarachnoid hemorrhage. Neurosurgery. 1999 Nov;45(5):1120–1127; discussion 7–8.
26. Wartenberg KE, Schmidt JM, Claassen J, et al. Impact of medical complications on outcome after subarachnoid hemorrhage. Crit Care Med. 2006 Mar;34(3):617–623; quiz 24.
27. Dennis LJ, Claassen J, Hirsch LJ, Emerson RG, Connolly ES, Mayer SA. Nonconvulsive status epilepticus after subarachnoid hemorrhage. Neurosurgery. 2002 Nov;51(5):1136–1143; discussion 44.
28. Naidech AM, Kreiter KT, Janjua N, et al. Phenytoin exposure is associated with functional and cognitive disability after subarachnoid hemorrhage. Stroke. 2005 Mar;36(3):583–587.
29. Kopelnik A, Zaroff JG. Neurocardiogenic injury in neurovascular disorders. Crit Care Clin. 2006 Oct;22(4):733–752; abstract ix–x.
30. Solenski NJ, Haley EC, Jr., Kassell NF, et al. Medical complications of aneurysmal subarachnoid hemorrhage: a report of the multicenter, cooperative aneurysm study. Participants of the Multicenter Cooperative Aneurysm Study. Crit Care Med. 1995 Jun;23(6):1007–1017.
31. van der Bilt IA, Hasan D, Vandertop WP, et al. Impact of cardiac complications on outcome after aneurysmal subarachnoid hemorrhage: a meta-analysis. Neurology. 2009 Feb;72(7):635–642.
32. Vergouwen MD, Vermeulen M, van Gijn J, et al. Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group. Stroke. 2010 Oct;41(10):2391–2395.
33. Heros RC, Zervas NT, Varsos V. Cerebral vasospasm after subarachnoid hemorrhage: an update. Ann Neurol. 1983 Dec;14(6):599–608.
34. Kistler JP, Crowell RM, Davis KR, et al. The relation of cerebral vasospasm to the extent and location of subarachnoid blood visualized by CT scan: a prospective study. Neurology. 1983 Apr;33(4):424–436.
35. Fisher CM, Roberson GH, Ojemann RG. Cerebral vasospasm with ruptured saccular aneurysm—the clinical manifestations. Neurosurgery. 1977 Nov–Dec;1(3):245–248.
36. Stein SC, Levine JM, Nagpal S, LeRoux PD. Vasospasm as the sole cause of cerebral ischemia: how strong is the evidence? Neurosurg Focus. 2006;21(3):E2.
37. Dreier JP, Major S, Manning A, et al. Cortical spreading ischaemia is a novel process involved in ischaemic damage in patients with aneurysmal subarachnoid haemorrhage. Brain. 2009 Jul;132(Pt 7):1866–1881.
38. Vora YY, Suarez-Almazor M, Steinke DE, Martin ML, Findlay JM. Role of transcranial Doppler monitoring in the diagnosis of cerebral vasospasm after subarachnoid hemorrhage. Neurosurgery. 1999 Jun;44(6):1237–1247; discussion 47–48.
39. Grosset DG, Straiton J, McDonald I, Cockburn M, Bullock R. Use of transcranial Doppler sonography to predict development of a delayed ischemic deficit after subarachnoid hemorrhage. J Neurosurg. 1993 Feb;78(2):183–187.
40. Sloan MA, Haley EC, Jr., Kassell NF, et al. Sensitivity and specificity of transcranial Doppler ultrasonography in the diagnosis of vasospasm following subarachnoid hemorrhage. Neurology. 1989 Nov;39(11): 1514–1518.
41. Sloan MA, Alexandrov AV, Tegeler CH, et al. Assessment: transcranial Doppler ultrasonography: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. Neurology. 2004 May 11;62(9):1468–1481.
42. Lindegaard KF, Nornes H, Bakke SJ, Sorteberg W, Nakstad P. Cerebral vasospasm diagnosis by means of angiography and blood velocity measurements. Acta Neurochir (Wien). 1989;100(1–2):12–24.
43. Mizuno M, Nakajima S, Sampei T, et al. Serial transcranial Doppler flow velocity and cerebral blood flow measurements for evaluation of cerebral vasospasm after subarachnoid hemorrhage. Neurol Med Chir (Tokyo). 1994 Mar;34(3):164–171.
44. Claassen J, Hirsch LJ, Kreiter KT, et al. Quantitative continuous EEG for detecting delayed cerebral ischemia in patients with poorgrade subarachnoid hemorrhage. Clin Neurophysiol. 2004 Dec;115(12):2699–2710.
45. Vespa PM, Nuwer MR, Juhasz C, et al. Early detection of vasospasm after acute subarachnoid hemorrhage using continuous EEG ICU monitoring. Electroencephalogr Clin Neurophysiol. 1997 Dec;103(6):607–615.
46. Labar DR, Fisch BJ, Pedley TA, Fink ME, Solomon RA. Quantitative EEG monitoring for patients with subarachnoid hemorrhage. Electroencephalogr Clin Neurophysiol. 1991 May;78(5):325–332.
47. Cloft HJ, Joseph GJ, Dion JE. Risk of cerebral angiography in patients with subarachnoid hemorrhage, cerebral aneurysm, and arteriovenous malformation: a meta-analysis. Stroke. 1999 Feb;30(2):317–320.
48. Otawara Y, Ogasawara K, Ogawa A, Sasaki M, Takahashi K. Evaluation of vasospasm after subarachnoid hemorrhage by use of multislice computed tomographic angiography. Neurosurgery. 2002 Oct;51(4):939– 942; discussion 42–43.
49. Westerlaan HE, van der Vliet AM, Hew JM, Metzemaekers JD, Mooij JJ, Oudkerk M. Magnetic resonance angiography in the selection of patients suitable for neurosurgical intervention of ruptured intracranial aneurysms. Neuroradiology. 2004 Nov;46(11):867–875.
50. Anderson GB, Ashforth R, Steinke DE, Findlay JM. CT angiography for the detection of cerebral vasospasm in patients with acute subarachnoid hemorrhage. AJNR Am J Neuroradiol. 2000 Jun–Jul;21(6):1011–1015.
51. Chieregato A, Battaglia R, Sabia G, et al. A diagnostic flowchart, including TCD, Xe-CT and angiography, to improve the diagnosis of vasospasm critically affecting cerebral blood flow in patients with subarachnoid haemorrhage, sedated and ventilated. Acta Neurochir Suppl. 2008;104:251–253.
52. Egge A, Sjoholm H, Waterloo K, Solberg T, Ingebrigtsen T, Romner B. Serial single-photon emission computed tomographic and transcranial doppler measurements for evaluation of vasospasm after aneurysmal subarachnoid hemorrhage. Neurosurgery. 2005 Aug;57(2):237–242; discussion 42.
53. Hillman J, Sturnegk P, Yonas H, et al. Bedside monitoring of CBF with xenon-CT and a mobile scanner: a novel method in neurointensive care. Br J Neurosurg. 2005 Oct;19(5):395–401.
54. Unterberg AW, Sakowitz OW, Sarrafzadeh AS, Benndorf G, Lanksch WR. Role of bedside microdialysis in the diagnosis of cerebral vasospasm following aneurysmal subarachnoid hemorrhage. J Neurosurg. 2001 May;94(5):740–749.
55. Nilsson OG, Brandt L, Ungerstedt U, Saveland H. Bedside detection of brain ischemia using intracerebral microdialysis: subarachnoid hemorrhage and delayed ischemic deterioration. Neurosurgery. 1999 Nov;45(5):1176–1184; discussion 84–85.
56. Rose JC, Neill TA, Hemphill JC, 3rd. Continuous monitoring of the microcirculation in neurocritical care: an update on brain tissue oxygenation. Curr Opin Crit Care. 2006 Apr;12(2):97–102.
57. Bader MK. Recognizing and treating ischemic insults to the brain: the role of brain tissue oxygen monitoring. Crit Care Nurs Clin North Am. 2006 Jun;18(2):243–256, xi.
58. Tam AK, Ilodigwe D, Mocco J, et al. Impact of systemic inflammatory response syndrome on vasospasm, cerebral infarction, and outcome after subarachnoid hemorrhage: exploratory analysis of CONSCIOUS-1 database. Neurocrit Care. 2010 Oct;13(2):182–189.
59. Dhar R, Diringer MN. The burden of the systemic inflammatory response predicts vasospasm and outcome after subarachnoid hemorrhage. Neurocrit Care. 2008;8(3):404–412.
60. Yoshimoto Y, Tanaka Y, Hoya K. Acute systemic inflammatory response syndrome in subarachnoid hemorrhage. Stroke. 2001 Sep;32(9): 1989–1993.
61. Westermaier T, Stetter C, Vince GH, et al. Prophylactic intravenous magnesium sulfate for treatment of aneurysmal subarachnoid hemorrhage: a randomized, placebo-controlled, clinical study. Crit Care Med. 2010 May;38(5):1284–1290.
62. Wong GK, Poon WS, Chan MT, et al. Intravenous magnesium sulphate for aneurysmal subarachnoid hemorrhage (IMASH): a randomized, double-blinded, placebo-controlled, multicenter phase III trial. Stroke. 2010 May;41(5):921–926.
63. Zhao XD, Zhou YT, Zhang X, Zhuang Z, Shi JX. A meta analysis of treating subarachnoid hemorrhage with magnesium sulfate. J Clin Neurosci. 2009 Nov;16(11):1394–1397.
64. Smith MJ, Le Roux PD, Elliott JP, Winn HR. Blood transfusion and increased risk for vasospasm and poor outcome after subarachnoid hemorrhage. J Neurosurg. 2004 Jul;101(1):1–7.
65. Kramer AH, Gurka MJ, Nathan B, Dumont AS, Kassell NF, Bleck TP. Complications associated with anemia and blood transfusion in patients with aneurysmal subarachnoid hemorrhage. Crit Care Med. 2008 Jul;36(7):2070–2075.
66. Shimoda M, Oda S, Tsugane R, Sato O. Intracranial complications of hypervolemic therapy in patients with a delayed ischemic deficit attributed to vasospasm. J Neurosurg. 1993 Mar;78(3):423–429.
67. Medlock MD, Dulebohn SC, Elwood PW. Prophylactic hypervolemia without calcium channel blockers in early aneurysm surgery. Neurosurgery. 1992 Jan;30(1):12–16.
68. Egge A, Waterloo K, Sjoholm H, Solberg T, Ingebrigtsen T, Romner B. Prophylactic hyperdynamic postoperative fluid therapy after aneurysmal subarachnoid hemorrhage: a clinical, prospective, randomized, controlled study. Neurosurgery. 2001 Sep;49(3):593–605; discussion 6.
69. Lennihan L, Mayer SA, Fink ME, et al. Effect of hypervolemic therapy on cerebral blood flow after subarachnoid hemorrhage: a randomized controlled trial. Stroke. 2000 Feb;31(2):383–391.
70. Darby JM, Yonas H, Marks EC, Durham S, Snyder RW, Nemoto EM. Acute cerebral blood flow response to dopamine-induced hypertension after subarachnoid hemorrhage. J Neurosurg. 1994 May;80(5):857–864.
71. Muizelaar JP, Becker DP. Induced hypertension for the treatment of cerebral ischemia after subarachnoid hemorrhage. Direct effect on cerebral blood flow. Surg Neurol. 1986 Apr;25(4):317–325.
72. Kosnik EJ, Hunt WE. Postoperative hypertension in the management of patients with intracranial arterial aneurysms. J Neurosurg. 1976 Aug;45(2):148–154.
73. Allen GS, Ahn HS, Preziosi TJ, et al. Cerebral arterial spasm—a controlled trial of nimodipine in patients with subarachnoid hemorrhage. N Engl J Med. 1983 Mar 17;308(11):619–624.
74. Haley EC, Jr., Kassell NF, Torner JC, Truskowski LL, Germanson TP. A randomized trial of two doses of nicardipine in aneurysmal subarachnoid hemorrhage. A report of the Cooperative Aneurysm Study. J Neurosurg. 1994 May;80(5):788–796.
75. Biondi A, Ricciardi GK, Puybasset L, et al. Intra-arterial nimodipine for the treatment of symptomatic cerebral vasospasm after aneurysmal subarachnoid hemorrhage: preliminary results. AJNR Am J Neuroradiol. 2004 Jun–Jul;25(6):1067–1076.
76. Badjatia N, Topcuoglu MA, Pryor JC, et al. Preliminary experience with intra-arterial nicardipine as a treatment for cerebral vasospasm. AJNR Am J Neuroradiol. 2004 May;25(5):819–826.
77. Feng L, Fitzsimmons BF, Young WL, et al. Intraarterially administered verapamil as adjunct therapy for cerebral vasospasm: safety and 2-year experience. AJNR Am J Neuroradiol. 2002 Sep;23(8):1284–1290.
78. McAuliffe W, Townsend M, Eskridge JM, Newell DW, Grady MS, Winn HR. Intracranial pressure changes induced during papaverine infusion for treatment of vasospasm. J Neurosurg. 1995 Sep;83(3): 430–434.
79. Cross DT, 3rd, Moran CJ, Angtuaco EE, Milburn JM, Diringer MN, Dacey RG, Jr. Intracranial pressure monitoring during intraarterial papaverine infusion for cerebral vasospasm. AJNR Am J Neuroradiol. 1998 Aug;19(7):1319–1323.
80. Milburn JM, Moran CJ, Cross DT, 3rd, Diringer MN, Pilgram TK, Dacey RG, Jr. Increase in diameters of vasospastic intracranial arteries by intraarterial papaverine administration. J Neurosurg. 1998 Jan;88(1): 38–42.
81. Khatri R, Tariq N, Vazquez G, Suri MF, Ezzeddine MA, Qureshi AI. Outcomes after nontraumatic subarachnoid hemorrhage at hospitals offering angioplasty for cerebral vasospasm: a national level analysis in the United States. Neurocrit Care. [Epub Sep 14].
82. Coyne TJ, Montanera WJ, Macdonald RL, Wallace MC. Percutaneous transluminal angioplasty for cerebral vasospasm after subarachnoid hemorrhage. Can J Surg. 1994 Oct;37(5):391–396.
83. Elliott JP, Newell DW, Lam DJ, et al. Comparison of balloon angioplasty and papaverine infusion for the treatment of vasospasm following aneurysmal subarachnoid hemorrhage. J Neurosurg. 1998 Feb;88(2): 277–284.
84. Lynch JR, Wang H, McGirt MJ, et al. Simvastatin reduces vasospasm after aneurysmal subarachnoid hemorrhage: results of a pilot randomized clinical trial. Stroke. 2005 Sep;36(9):2024–2026.
85. Tseng MY, Czosnyka M, Richards H, Pickard JD, Kirkpatrick PJ. Effects of acute treatment with pravastatin on cerebral vasospasm, autoregulation, and delayed ischemic deficits after aneurysmal subarachnoid hemorrhage: a phase II randomized placebo-controlled trial. Stroke. 2005 Aug;36(8):1627–1632.
86. Vergouwen MD, de Haan RJ, Vermeulen M, Roos YB. Effect of statin treatment on vasospasm, delayed cerebral ischemia, and functional outcome in patients with aneurysmal subarachnoid hemorrhage: a systematic review and meta-analysis update. Stroke. 2010 Jan,41(1): e47–e52.
87. Kramer A, Fletcher J. Do endothelin-receptor antagonists prevent delayed neurological deficits and poor outcomes after aneurysmal subarachnoid hemorrhage?: a meta-analysis. Stroke. 2009 Oct;40(10):3403–3406.
88. Sillberg VA, Wells GA, Perry JJ. Do statins improve outcomes and reduce the incidence of vasospasm after aneurysmal subarachnoid hemorrhage: a meta-analysis. Stroke. 2008 Sep;39(9):2622–2626.
89. Macdonald RL, Kassell NF, Mayer S, et al. Clazosentan to overcome neurological ischemia and infarction occurring after subarachnoid hemorrhage (CONSCIOUS-1): randomized, double-blind, placebocontrolled phase 2 dose-finding trial. Stroke. 2008 Nov;39(11):3015– 3021.
90. Kinouchi H, Ogasawara K, Shimizu H, Mizoi K, Yoshimoto T. Prevention of symptomatic vasospasm after aneurysmal subarachnoid hemorrhage by intraoperative cisternal fibrinolysis using tissue-type plasminogen activator combined with continuous cisternal drainage. Neurol Med Chir (Tokyo). 2004 Nov;44(11):569–575; discussion 76–77.
91. Gomis P, Graftieaux JP, Sercombe R, Hettler D, Scherpereel B, Rousseaux P. Randomized, double-blind, placebo-controlled, pilot trial of highdose methylprednisolone in aneurysmal subarachnoid hemorrhage. J Neurosurg. 1990 Mar;112(3):681–688.
92. Hasan D, Wijdicks EF, Vermeulen M. Hyponatremia is associated with cerebral ischemia in patients with aneurysmal subarachnoid hemorrhage. Ann Neurol. 1990 Jan;27(1):106–108.
93. Hasan D, Vermeulen M, Wijdicks EF, Hijdra A, van Gijn J. Management problems in acute hydrocephalus after subarachnoid hemorrhage. Stroke. 1989 Jun;20(6):747–753.
94. Qureshi AI, Suri MF, Sung GY, et al. Prognostic significance of hypernatremia and hyponatremia among patients with aneurysmal subarachnoid hemorrhage, Neurosurgery. 2002 Apr;50(4):749–755; discussion 55–56.
95. Sayama T, Inamura T, Matsushima T, Inoha S, Inoue T, Fukui M. High incidence of hyponatremia in patients with ruptured anterior communicating artery aneurysms. Neurol Res. 2000 Mar;22(2):151–155.
96. Mori T, Katayama Y, Kawamata T, Hirayama T. Improved efficiency of hypervolemic therapy with inhibition of natriuresis by fludrocortisone in patients with aneurysmal subarachnoid hemorrhage. J Neurosurg. 1999 Dec;91(6):947–952.
97. Wijdicks EF, Vermeulen M, Hijdra A, van Gijn J. Hyponatremia and cerebral infarction in patients with ruptured intracranial aneurysms: is fluid restriction harmful? Ann Neurol. 1985 Feb;17(2):137–140.
98. Banki NM, Kopelnik A, Dae MW, et al. Acute neurocardiogenic injury after subarachnoid hemorrhage. Circulation. 2005 Nov;112(21): 3314–3319.
99. Kothavale A, Banki NM, Kopelnik A, et al. Predictors of left ventricular regional wall motion abnormalities after subarachnoid hemorrhage. Neurocrit Care. 2006;4(3):199–205.
100. Levine JM. Critical care management of subarachnoid hemorrhage. Curr Neurol Neurosci Rep. 2008 Nov;8(6):518–525.
101. Cardin C, Roncalli J, Lairez O, et al. Subarachnoid haemorrhage associated with midventricular Tako-Tsubo syndrome. Int J Cardiol. 2009 [Epub Apr 8].
102. D’Aloia A, Vizzardi E, Faggiano P, Fiorina C, Cas LD. Intracranial bleeding mimicking an extensive acute myocardial infarction with reversible apical ballooning and systolic left ventricular dysfunction. A case report. Monaldi Arch Chest Dis. 2007 Mar;68(1):44–47.
103. Lee VH, Oh JK, Mulvagh SL, Wijdicks EF. Mechanisms in neurogenic stress cardiomyopathy after aneurysmal subarachnoid hemorrhage. Neurocrit Care. 2006;5(3):243–249.
104. Hakeem A, Marks AD, Bhatti S, Chang SM. When the worst headache becomes the worst heartache! Stroke. 2007 Dec;38(12):3292–3295.
105. Deibert E, Barzilai B, Braverman AC, et al. Clinical significance of elevated troponin I levels in patients with nontraumatic subarachnoid hemorrhage. J Neurosurg. 2003 Apr;98(4):741–746.
106. Deibert E, Aiyagari V, Diringer MN. Reversible left ventricular dysfunction associated with raised troponin I after subarachnoid haemorrhage does not preclude successful heart transplantation. Heart. 2000 Aug;84(2):205–207.

Most patients suffering from Parkinson's disease (PD) in the advanced stage are poorly managed with medications alone. Neurosurgical interventions including lesioning and deep brain stimulation (DBS) provide effective choices but with the risk of adverse events mainly caused by the surgical procedure. DBS for PD was widely accepted in the late 1990s. Compared to other neurosurgical methods, DBS is attractive because of its adjustability and reversibility. Most DBS treatments target the subthalamic nucleus (STN) because the STN plays an important role in the modulation of the cortico-striato-pallido-thalamocortical (CSPTC) motor pathways in PD. The increasing activity of STN results in excessive inhibitory outflow from the basal ganglia to the thalamus and brain stem, which is associated with the motor symptoms in PD patients. By modulating the STN neural activity, the CSPTC pathway can be restored.

Positron emission tomography (PET) can show the functional network of CSPTC pathways. In previous [¹⁸F]fluorodeoxyglucose(FDG)-PET studies, Eidelberg and colleagues have found that PD is associated with the presence of a specific abnormal metabolic brain network ^{1, 2}. This Parkinson's disease-related pattern (PDRP) is characterized by lentiform, thalamic, and brain stem hypermetabolism covarying with metabolic reductions in the lateral premotor and supplementary motor area (SMA). STN lesioning was found to produce a marked and sustained reduction in network activity in advanced PD patients with changes in network activity highly correlated with clinical effects. STN DBS can also modulate the activity of STN. Effective electrical STN stimulation significantly increases regional cerebral blood flow (rCBF) of the cingulate cortex and, especially the dorsolateral prefrontal cortex (DLPFC), when compared to the internal segment of the globus pallidus (GPi) DBS stimulation ³. Ceballos-Baumann et al. reported that rCBF increases in activation of rostral SMA, DLPFC, and premotor cortex ipsilateral to stimulation were observed when stimulation was on during contralateral movement ⁴. The activated regions were similar to those of pallidotomy. The difference was, during both rest and movement period, decreases in rCBF in primary motor cortex were induced by STN stimulation. This was probably caused by the stimulation that conversely activated the projection from the cerebral cortex to STN. The STN stimulation also induced the increase of thalamic rCBF, which indicates that the decreased output of STN leads to the removing of the thalamic inhibition. The use of glucose metabolism for the assessment of the impact of bilateral electrical STN stimulation on the basal ganglia circuit was so far less reported.

The purpose of this study is to evaluate the effect of bilateral STN DBS on medically intractable advanced PD patients and investigate the mechanism of bilateral STN DBS with the metabolic network modulation.

MATERIALS AND METHODS

Subjects

We studied five patients with advanced-stage (Hoehn and Yahr Stage IV) PD (four men and one woman; age [mean±SD] 61.6±3.9 years; disease duration 7.3±2.8 years). The clinical characteristics of these patients are shown in Table 1. All patients received a daily L-dopa usage ranging from 700 to 1650 mg/day (mean 1030±397.8 mg/day). All patients underwent bilateral STN DBS implantation at the Huashan Hospital, Fudan University in Shanghai, China. Installation of the Leksell sterotaxic frame was under local anesthesia. The STN was localized by GE 1.5T high-resolution MRI combined with Schaltenbrand–Bailey atlas. Deep brain stimulation electrodes (Medtronic 3387 or 3389, Medtronic, USA) were implanted in bilateral STN. Under supportive intravenous anesthesia, subclavicular electrodes were implanted synchronously (four cases) or asynchronously (one case). The stimulation parameters (stimulation sites, frequency, length of pulse, and voltage) were adjusted by the programmed computer 1 week later to achieve the best control of the symptom without side effects. Written informed consent was obtained from all participants.

FDG-PET Scans

Patients were scanned approximately 6 months (mean 6.2±0.02 months) following STN DBS implantation. In a fasting state, each of the five patients underwent FDG-PET scans in the ON and OFF DBS conditions, defined by the presence or absence of stimulation for at least 24 hours prior to the PET imaging. The ON and OFF scanning sessions were performed on separate days with the order randomized across patients. The PET scans were performed with an ECAT EXACT HR+ tomography (CTI, Knoxville, USA) with 3D mode. This scanner acquires 63 slices with an interslice spacing of 2.43 mm. The PET scanner had a total axial view of 15.5 cm and no interplane dead space, ensuring coverage of the brain from the vertex to the lower cerebellum. PET scan lasting for 10 minutes started 30 minutes after an intravenous bolus injection of 5 mCi of ¹⁸F-FDG and a 10-minute transmission scan was performed for attenuation correction. Hanning filters were used, giving transaxial and axial cut-off frequency of 0.5. No arterial blood sampling was performed. All studies were performed with the subject's eye open in a dimly lit room with minimal auditory stimulation.

Data Analysis

The PET image data were analyzed with statistical parametric mapping (SPM2, Wellcome Department of Cognitive Neurology, London, UK) implemented in Matlab 6.5.1 (Mathworks Inc, Sherborn, MA, US). The scans from each subject were realigned, and the aligned PET images were then normalized into Montreal Neurological Institute (MNI) space involving linear and nonlinear three-dimensional transformations. The normalized PET images were filtered with a Gaussian kernel of 10 mm (FWHM) in the x, y, and z axes.

Effects of Bilateral STN DBS on Brain Metabolism

The effects of the treatment condition were estimated according to the general linear model at each voxel. Mean signal changes over the whole brain were removed by proportional scaling. To find changes in brain metabolism with bilateral STN stimulation, the estimates were compared by paired t-tests with statistical significance of p<0.01 at cluster level over the entire volume in the brain analyzed. All results are presented as SPM maps, showing significant changes in brain metabolism with bilateral STN DBS.

Effects of Bilateral STN DBS on Parkinson's Disease Network Activity

The mean effects of stimulation on brain metabolism evidence with SPM2 do not take into account the functional connectivity that exists between multiple brain function regions constituting a whole distributed neural network. It has previously been shown that regional covariance analysis can be used to identify interesting changes in network activity following localized sterotaxic interventions ^5–8. In this study, we assessed changes in the activity of the PDRP during stimulation. This was accomplished by quantifying individual subjects scores of PDRP in the ON and OFF states. Automated voxel-based network computations (software from Center for Neuroscience, Institute for Medical Research, North ShoreLong Island Health System) were performed on a whole-brain basis. Network activity changes of ON–OFF were computed for each subject. Clinical improvement was measured objectively by the change in motor UPDRS ratings between stimulation conditions (ie, [ON–OFF]/OFF).

RESULTS

Effects of Bilateral STN Stimulation on Clinical Symptoms

Bilateral STN DBS significantly improved the clinical symptoms of five advanced-stage PD patients. UPDRS motion scores in “ON” and “OFF” conditions were 20±3.4 and 46.6±11.1, respectively (Table 1). Clinical improvement with the change in motor UPDRS ratings between stimulation conditions was 56.6±3.6%. The tremor was completely relieved, muscular tension was close to normal, and gait and posture were significantly improved. All five patients who used L-dopa as the only medication for PD reduced their daily dosage by approximately 30.7% (from 1030±397.8 mg/day to 700±273.9 mg/day) after the surgery.

Effects of Bilateral STN Stimulation on Brain Metabolism

Besides the significant improvement in clinical symptoms of these PD patients, bilateral STN DBS also markedly changed the brain metabolism (Figure 1): decreased metabolism of bilateral parietal cortex, the cerebellum, internal segment of the GPi, as well as the thalamus; and increased metabolism of the midbrain and pons.

Effects of Bilateral STN Stimulation on Metabolic Network Activity

To assess the effect of STN DBS on metabolic network activity, we quantified the changes in whole-brain PDRP expression that were induced by this intervention in each patient. Network activity declined on individual subject basis. For the group as a whole, the PDRP score in “ON” and “OFF” condition were 2.12±15.24 and 4.93±13.01, respectively. Patients with a decreased score in PDRP were considered as effective DBS treatment, the p value between ON and OFF condition was 0.03 (p<0.05) using one-tailed t-test. Although there is no significant correlation between PDRP and UPDRS scores, the clinical symptoms of PD patients were improved with the reduction of the PDRP score.

DISCUSSION

The traditional surgical treatments of PD, such as sterotaxic pallidotomy, can cause permanent complications such as defect of visual field, hemiplegia, dysarthria, and dysphagia. Other cognitive impairments are also commonly seen. The destruction of the specific site can usually be operated only once and is irreversible. Although DBS may worsen the symptom like verbal fluency and dysarthria ^{9, 10}, it remains an effective treatment for advanced PD patients for its nondestructive, reversible feature for carefully selected patients compared to best medical treatment ¹¹. Both GPi and STN stimulation can significantly improve the clinical symptoms of PD patients; improvement rate of tremor was 80%, improvement rate of bradykinesia and rigidity was over 60%, and improvement rate of motion abnormality and improvement rate of posture and gait disorder was between 40% and 50% ¹².

Three-month and 6-month follow-up of our five PD cases showed that the dose of the dopaminergic drug was significantly decreased. Compared to the “OFF” condition before operation, electrical stimulation can markedly improve the motor symptoms of PD patients by 50.5% at 3 months and 54.5% at 6 months, respectively.

With the wide application of DBS in surgical treatment, researchers are increasingly interested in functional mechanism of DBS. FDG-PET can detect the regional cerebral glucose metabolism of the PD patients. It can not only detect the degree of degeneration of dopaminergic system-related substantia nigra–corpus striatum, but also detect the cerebral metabolic changes in regions distal to the focus. Using spatial covariance analysis of FDG-PET scan, Eidelberg et al. found a PD-related metabolic pattern whose expression was correlated with the severity of the disease ². Treatment with levodopa drugs improves the clinical symptoms by reducing the high metabolism of GPi ¹³. The GPi DBS reduced expression of an abnormal PDRP network correlating significantly with clinical improvements in UPDRS motor ratings ¹⁴.

Destructive operation of GPi and STN reduces the high metabolism of lentiform nucleus, thalamus, and pons cerebelli and improves the clinical symptoms at the same time. A recent research of Eidelberg showed that STN DBS can reduce the glucose metabolism of lentiform nucleus and midbrain along with the reduction (downregulation) of PDRP level ⁵. But the PET scans of the CBF from DBS-treated PD patients confirmed that, differing from unilateral electrical STN stimulation, bilateral STN DBS could specifically increase the rCBF of GPi ¹⁵. Microdialysis of animal research also confirmed that electrical STN stimulation can increase the amount of the GABA transmitters in corpus striatum and the excitatory glutamate transmitter in GPi ¹⁶.

In our research, we found that bilateral electrical STN stimulation decreased the metabolism in the GPi, the cerebellum, and the thalamus. These findings were consistent with the abnormal metabolism in the previous study ¹⁷, and may be caused by the reductions in the synaptic activity of STN projections to the interconnected brain regions, similar to the impact of dopaminergic therapy ⁵. In parkinsonism, STN is overexcited, which leads to the increase of excitatory output from STN to GPi/SNr (Substantia Nigra pars reticulata). Upper brain stem is extremely important in initiating and controlling motion, midline movement, and maintaining the muscle tension. While receiving the inhibitive GABA transmitters from GPi/SNr, it also receives the excitive glutamic transmitters from STN. Increasing the release of GABA transmitter will suppress the activeness of the pedunculopontine nucleus (PPN), and thus worsen the symptoms of akinesia. The increasing release of glutamic transmitters can facilitate the activeness of PPN, which improves the symptoms of akinesia and abnormal muscle tension ¹⁸.

Bilateral STN DBS increases the metabolism of midbrain and pons, and thus improves all motion symptoms of PD patients. This might be related to the increasing of release of the excitive glutamic transmitters.

In this study, we also quantified PDRP, a previously validated abnormal disease-related metabolic covariance pattern, to assess the effects of bilateral STN DBS. We found that STN DBS stimulation reduced PDRP network activity and that network change correlated significantly with the clinical improvement in advanced PD patients. Patients with a relatively high PDRP score suppression were found to have the better clinical improvement with the intervention, in accordance with the previous results of Eidelberg and his colleagues.

In summary, bilateral STN DBS achieves its therapeutic results in PD patients by reducing the abnormal disease-related metabolic network. These findings suggest that DBS functions by inhibiting the activeness of local neurons at target point and exciting the axons distal to the target point, that is, DBS is more likely to function by regulating the entire neuronetwork rather than merely exciting or inhibiting certain nucleus.

Keywords

PET, Parkinson's disease, deep brain stimulation

Disclosure: Dr Chuantao Zuo is the principal investigator for this report.

REFERENCES

1. Eidelberg D, Moller JR, Dhawan V, et al. The metabolic topography of parkinsonism. J Cere Blood Flow Metab. 1994;14:783–801.
2. Eidelberg D, Moller JR, Ishikawa T, et al. Assessment of disease severity in parkinsonism with fluorine-18-fluorodeoxyglucose and PET. J Nucl Med. 1995;36:378–383.
3. Limousin P, Greene J, Pollak P, et al. Changes in cerebra l activity pattern due to subthalamic nucleus or internal pallidum stimulation in Parkinson’s disease. Ann Neurol. 1997;42:283–291.
4. Ceballos-Baumann AO, Bartenstein P, Boecker H, et al. A positron emission tomographic study of subthalamic nucleus stimulation in Parkinson’s disease: enhanced movement-related activity of motor association cortex and decrease motor cortex resting activity. Arch Neurol. 1999;56:997–1003.
5. Asanuma K, Tang C, Ma Y, et al. Network modulation in the treatment of Parkinson’s disease. Brain. 2006;129:2667–2678.
6. Eidelberg D, Moeller JR, Ishikawa T, et al. Regional metabolic correlates of surgical outcome following unilateral pallidotomy for Parkinson’s disease. Ann Neurol. 1996;39:450–459.
7. Su PC, Ma Y, Fukuda M, et al. Metabolic changes following subthalamotomy for advanced Parkinson’s disease. Ann Neurol. 2001;50:514–520.
8. Trost M, Su S, Su P, et al. Network modulation by the subthalamic nucleus in the treatment of Parkinson’s disease. Neuroimage. 2006;31:301– 307.
9. Marconi R, Landi A, Valzania F. Subthalamic nucleus stimulation in Parkinson’s disease. Neurol Sci. 2008;29(Suppl. 5):S389–S391.
10. Pahwa R, Lyons KE, Wilkinson SB, et al. Long-term evaluation of deep brain stimulation of the thalamus. J Neurosurg. 2006;104(4):506–512.
11. Witt K, Daniels C, Reiff J, et al. Neuropsychological and psychiatric changes after deep brain stimulation for Parkinson’s disease: a randomized, multicentre study. Lancet Neurol. 2008;7(7):565–567.
12. Limousin P, Krack P, Pollak P, et al. Electrical stimulation of the subthalamic nucleus in advanced Parkinson’s disease. New Engl J of Med. 1998;339(16):1105–1111.
13. Feigin A, Fukuda M, Dhawan V, et al. Metabolic correlates of levodopa response in Parkinson’s disease. Neurology. 2001;57(11):2083–2088.
14. Fukuda M, Mentis MJ, Ma Y, et al. Networks mediating the clinical effects of pallidal brain stimulation for Parkinson’s disease: a PET study of resting-state glucose metabolism. Brain. 2001;124:1601–1609.
15. Strafella AP, Dagher A, Sadikot AF. Cerebral blood flow changes induced by subthalamic stimulation in Parkinson’s disease. Neurology. 2003;60(2):1039–1042.
16. Windels F, Bruet N, Poupard A, et al. Effects of high frequency stimulation of subthalamic nucleus on extracellular glutamate and GABA in substantia nigra and globus pallidus in the normal rat. Eur J Neurosci. 2000;12:4141–4146.
17. Eckert T, Barnes A, Dhawan V, et al. FDG PET in the differential diagnosis of parkinsonian disorders. Neuroimage. 2005;26:912–921.
18. Parent A, Cicchetti F. The current model of basal ganglia organization under scrutiny. Mov Disord. 1998;13:199–202.

In 1931, Dr. Lucy Wills demonstrated that yeast extract was effective against the tropical macrocytic anemia seen in late pregnancy in India.¹ In the subsequent decade, the compound responsible for this effect, folate, was isolated. Folate is a water-soluble B vitamin, found particularly in such foods as leafy green vegetables, citrus fruits, and legumes. Folic acid is the synthetic form of the vitamin, which is given as a supplement or added to fortified foods. The value of preconceptual dietary supplementation with folate in women of child bearing age in reducing major congenital malformations (MCMs) and neural tube defects (NTDs) has been demonstrated in a number of large studies. This has led to explicit guidelines from the American Academy of Neurology (ANN) and the European Registration of Congenital Anomalies and Twins (Eurocat) regarding such supplementation and fortification,^{2, 3} and has to led to folic acid fortification of food in many countries as a public health measure for the reduction of NTD occurrence rates.⁴

The role and dosing of folate in women with epilepsy (WWE) who are taking antiepileptic medication (AEDs) is less clear. Some AEDs can theoretically interfere with folate metabolism, thus presumably increasing the risk of both MCM and NTD occurrence. Despite the lack of convincing evidence for a protective effect of folate supplementation in WWE, guidance from organizations such as the American Epilepsy Society (AES)² and the National Institute for Clinical Excellence⁵ in the UK have strongly recommended it.

In the course of this article, we will review some of the basic mechanisms of folic acid metabolism, the role of folate in the development of the neural tube, and the evidence underlying the recommendations for folic acid supplementation in women of child bearing age and in WWE. We will examine the evidence that these recommendations have been shown to affect the outcomes of pregnancy in WWE and whether folic acid in itself is associated with any adverse outcomes in the doses recommended.

METABOLISM

In adults (aged 19 or older) the recommended daily allowance (RDA) of folate in the diet in is 400 µg per day, rising to 600 µg in pregnancy, and 500 µg during lactation.⁶ There is a difference in bioavailability of folate and the synthetic preparation folic acid,⁷ such that the equivalent RDA of folic acid is 240 µg per day, rising to 360 µg in pregnancy.

Dietary folate is initially hydrolyzed from the polyglutamate forms to monoglutamate forms in the intestinal wall. Absorption occurs through the jejunum and much of the monoglutamate is taken up from the portal circulation into the liver.⁸ In the liver, monoglutamyl folate is first reduced by dihydrofolate reductase to a dihydrofolate compound and then reduced further to tetrahydrofolate (THF). Subsequent to the formation of THF, 5,10-methylene-THF is formed by the addition of methylene groups from one of serine, glycine, or formaldehyde (see Figure 1). The 5,10-methylene-THF is subsequently converted into 5-methyl-THF through the action of methylene tetrahydrofolate reductase (MTHFR). The 5-methyl-THF serves to replenish the methylated form of vitamin B12, which is a cofactor for methionine synthase. This is the enzyme that converts homocysteine to methionine, which is in turn converted to S-adenosyl-L-methionine (SAM), the principal methyl donor for DNA methyltransferases.

NEURAL TUBE DEVELOPMENT

Folic acid has received the most attention in the prevention of NTDs and MCMs. However, to appreciate the importance of folate in this process, it is important to review the normal process of neural tube formation in the development of the central nervous system.

In the development of the embryo, the primitive streak develops by embryonic day 13, elongates until day 16 and then regresses until day 28. During this regression, primitive notochordal cells ingress to form the notochordal canal by approximately day 16. This structure is further modified over subsequent days and, by day 25, has become the true notochord.

Under the influence of soluble growth factors secreted by the developing notochord, ectodermal cells are induced to form neuroectoderm, which then forms the thick and flat neural plate above the notochord. As this develops, the process of primary neurulation begins, usually at embryonic day 17. This process is responsible for forming brain and the majority of the spinal cord. As a result of the expanding epidermis, the neural plate folds, giving rise to the neural groove as a midline furrow (see Figure 2) and the neural folds elevated laterally. The process continues with the formation of the paired lateral hinge points, and pressure on these hinge points causes convergence of the neural folds toward the midline and fusion of the neural folds. Fusion of the neural folds begins near the cervicomedullary junction at about embryonic day 20 and then proceeds both cranially and caudally. The last two points of closure are the cranial neuropore at day 24 postovulation and the caudal neuropore at day 26.

The process of secondary neurulation begins after this and forms the spinal cord caudal to S2, but is less organized than primary neurulation. In this process, a cell population at the caudal extremity of the primitive streak differentiates and forms a neuroepithelium surrounding a central cavity. The neural tube formed by this process then fuses with the primary neural tube more rostrally.

The role of folic acid in this process is speculative and whether folic acid is any more pivotal than any other neurochemical remains unclear. Nevertheless, the high rate of neural cell division that occurs during the highly structured primary neurulation phase suggests that methylation, via folate metabolism, of proteins and lipids is crucial. It is likely therefore that interruptions in this process, whether due to genetic or environmental factors, are an important cause of NTDs. The epidemiological and scientific evidence is reviewed below.

FOLIC ACID SUPPLEMENTATION AND MCMs/NTDs

The spectrum of NTDs is considerable, and some—such as anencephaly—are incompatible with life. The NTDs are a considerable cause of neonatal mortality and morbidity worldwide, with an estimated incidence of more than 300,000 new cases per year.⁹ Over 95% of these cases are a first occurrence.^{10, 11}

There are numerous possible mechanisms for NTD formation in humans (Figure 3). Genetic factors clearly play a significant role in some cases. The recurrence risk of NTD in siblings of index cases is 2%–5%—approximately 50 times more than in the general population.¹² Some studies have suggested that 70% of the variance in NTD prevalence may be due to genetic factors,¹³ but twin studies of NTD frequency have suggested a lower contribution.¹⁴

Most NTDs show sporadic occurrence and this suggests a multifactorial polygenic or oligogenic pattern of inheritance. In the studies carried out thus far on candidate genes causative of NTDs, the most robust findings are the two genetic polymorphisms in the MTHFR gene, C677T and A1298C.^{15, 16}

However, it is clear that genetic factors alone are not responsible for NTD formation. As an example, mice whose genetics have been engineered to disrupt methionine synthesis (MTHFR-null mice) do not develop spinal defects.¹⁷ Similarly, the increased rate of NTD formation with the polymorphisms specified above is not seen in all populations.¹⁸ It is likely that there is a genetic predisposition to NTD formation that then requires an environmental factor for this to manifest. This is suggested by studies in mice, where folate deficiency causes NTDs only in the setting of a genetic predisposition.

Environmental factors associated with increased NTD occurrence include season of conception,¹⁹ socioeconomic class,²⁰ maternal diabetes,²¹ maternal age,²² maternal alcohol abuse,²³ and maternal use of certain medications (in particular, sodium valproate).²⁴ Deficiency states, such as folate or vitamin B12 deficiency, are also associated with NTD occurrence.^{25, 26} More recently, maternal smoking and exposure to smoke have been associated with an increased rate of NTDs.²⁷

Of the environmental factors mentioned above, folate deficiency has been extensively investigated. Although the mechanisms by which folate deficiency leads to NTD formation are not fully understood, it is clear that in the normal process of cell formation, methylation is needed for synthesis of membrane phospholipids and myelin basic protein.^{28, 29} It has been demonstrated that folate deficiency is associated with global DNA hypomethylation³⁰ and an increased rate of malformations.³¹ The reasons why the developing nervous system is particularly vulnerable to the effects of folate deficiency compared to other tissues are not fully understood. Precursor cells for both the neural crest and neuroepithelial cells have a higher level of folate receptor expression than others.³² Given this and the rapid growth and differentiation seen during gastrulation and neural tube formation, these features may explain why folate deficiency seems to be particularly detrimental to the nervous system.

The beneficial role of periconceptual folic acid supplementation in the prevention of NTDs has been demonstrated in a number of trials.^33–35 A recent meta-analysis of the published data³⁵ estimated that folic acid supplementation can give a reduction in the first occurrence of NTD of 62%. The estimated effect of folic acid fortification of food products in the prevention of NTD is 46%. Data from randomized trials suggest that folic acid supplementation is effective in 70% of cases in the prevention of recurrent NTD.

However, there are difficulties in the interpretation of studies on the protective effects of folic acid supplementation. The dose of folic acid used in studies varies, from 0.36 mg to 5 mg per day.³⁶ The designs of these studies are often quite different, ie, randomized controlled trials³⁵ versus observational studies,³⁷ and direct comparisons can be difficult. Overall, however, the benefits of folic acid supplementation are felt to be clear-cut for the general population of women of child bearing age,⁴ to the extent that many countries have initiated fortification of foods with folic acid as a public health measure.³⁸ Such fortification has been justified on the basis of the benefits of folic acid supplementation and the limited impacts of public health campaigns in ensuring preconceptual folic acid use. Previous studies of public health³⁹ and mass media campaigns⁴⁰ have shown that these methods are not successful in increasing folic acid use in women in the long term.

It should be noted that the benefit of folic acid supplementation may be limited in the prevention of NTDs. In one recent observational study after the introduction of folic acid fortification in some European countries, the rate of NTD occurrence fell to 7 or 8 cases at birth or abortion per 10000 births, suggesting a “floor effect” for the potential benefits seen.⁴¹

Folic acid supplementation has also been shown to cause a reduction in MCMs beyond NTDs. It has been shown that folic acid supplementation can reduce the rate of congenital heart defects, limb defects, and anomalies of the urinary tract.⁴² There is also a growing body of evidence that folic acid supplementation may also be associated with a lower rate of certain childhood malignancies,^43–45 although the evidence for this is not as robust as other findings.

PREGNANCY IN WWE

While there appears to be at least some robust evidence in favor of folic acid as a periconceptual supplement in women in the general population, to what extent this can be applied to WWE is unclear. It has been recognized for many years that epilepsy and its treatment have significant implications for both maternal and fetal health. A PubMed search for publications relating to maternal epilepsy and congenital malformations reveals over 500 citations. Initial reports go back to the 1960s noting the link to anticonvulsant treatment and malfomations.^{46, 47} There are many reports in the literature of complications associated with epilepsy in pregnancy including increased rates of caesarean section, preterm delivery, and low birth rate.^{48, 49} However, based on more recent data from a number of pregnancy registries, it is now accepted that the vast majority of WWE will have uncomplicated pregnancies.^50–52

The issue of NTDs/MCMs in pregnancy in WWE is one that is complicated by a number of factors. The frequency of MCM in pregnancies of women without epilepsy varies, but has been reported in studies as approximately 1.6–2.1%.^{53, 48} In WWE not taking AEDs, the rate of MCM is estimated to be slightly higher (2.8%) although clearly the numbers used for these estimates is considerably lower.⁵⁴ The effect of AED therapy on the rate of NTDs or MCMs is also variable and depends greatly on the medications used. Rates as low as 0% with levetiracetam monotherapy have been reported (but again with very small numbers),⁵⁵ compared to as high as 17.1% with sodium valproate monotherapy.^{24, 56} Polypharmacy is associated with a higher rate of MCMs than monotherapy.⁵⁷ The mechanisms of NTD formation by AEDs are still largely unknown. Some of the AEDs, such as phenytoin, carbamazepine and primidone, are metabolized by the CYP450 system in the liver. These enzyme-inducing drugs can interfere with the action of MTHFR,⁵⁸ thus impairing folate metabolism and action. However, this clearly cannot be the principal mechanism, as sodium valproate is not an enzyme-inducer and has the greatest rate of teratogenic complications.

There are risks seen in relation to not taking AEDs during pregnancy. It has been shown that even complex partial seizures can induce fetal bradycardia.⁵⁹ Trauma related to seizures and status epilepticus appear to be major causes of excess maternal mortality in WWE.⁶⁰ There are also reports of excess fetal loss in WWE not taking AEDs.⁶¹ Given the dangers of uncontrolled seizures to both the mother and the fetus, all guidelines recommend the continuation of AED therapy during pregnancy.

Generally, WWE are seen as being a high risk group for the occurrence of MCMs and NTDs in pregnancy. The guidelines from the various national and international federations reflect this, as they recommend the use of folic acid in all cases.^{2, 5} Many authors have expressed the opinion that the higher doses of folic acid available (4–5 mg) should be used, instead of the 0.4 mg dose that is recommended for lower risk groups.^{62, 63} However, the evidence to support this is lacking.

There has been concern that the adverse effects of AEDs in causing MCMs are not amenable to prevention by folic acid supplementation. In a paper this year, those WWE who were on high-dose (5 mg) preconceptual folic acid did not have a lower rate of MCMs than those who were not.⁶⁴ The lack of effectiveness of folic acid has also been shown in results of trials from the UK pregnancy registry and from other sources.⁶⁵ In contrast to this, the findings from other registries have suggested that the use of folic acid reduced but did not eliminate the rate of MCMs in pregnancy in WWE.⁶⁶ It has also been reported that the use of folic acid has been associated with a lower rate of spontaneous abortions, especially in those on sodium valproate.⁶⁷

In short, it can be said that folic acid supplementation should be used in WWE, based more on the data from studies in the general population rather than on clearly demonstrated efficacy in trials on populations of WWE. It is unclear why the effects are not as clear-cut in WWE. It is recognized that the mechanism of teratogenicity of AEDs is not limited to interference with folate metabolism, and this may explain why the results of high-dose folic acid as a protective agent are somewhat disappointing. Nevertheless, the use of high-dose folic acid continues to be recommended as such doses may maximize any potential benefit. However, it is worth reviewing the question of the potential harm associated with high-dose folic acid.

POTENTIAL ADVERSE EFFECTS OF FOLIC ACID SUPPLEMENTATION

Previously, the principal concern with the use of folic acid has been the potential masking of vitamin B12 deficiency, leading to a delay in diagnosis. Folic acid supplementation can correct the laboratory abnormalities of vitamin B12 deficiency, allowing the adverse effects of the deficiency to progress undetected until much later.

Folic acid has been studied in areas other than the prevention of NTDs. Given the effects it has on lowering homocysteine levels, it has been proposed that it may be of use in the prevention of vascular events such as stroke and myocardial infarction,⁶⁸ and it has been proposed as a protective agent against a number of cancers.⁶⁹ Some of the observational studies and trials to assess this potential use have raised concerns about the use of folic acid, especially in supraphysiological doses.

Epidemiological studies in cancer prevention have suggested that higher folate status is associated with a decreased risk of a number of cancers including those of the colorectum, pancreas, and stomach.⁶⁹ Studies on folate status and colorectal cancer suggest a 20%–40% reduction in adenoma or cancer rate in those with the highest status.^{70, 71} Small trials have also shown that folic acid supplementation can decrease adenoma recurrence rate after resection.⁷² However, other trials have not shown any significant effect.⁷³

The concern is whether the use of folic acid, especially in supraphysiological doses, could be associated with increasing the risk of malignancy. Epidemiological studies from the United States have shown an increase in the incidence of colorectal cancer after the introduction of folic acid dietary fortification.⁷⁴ The results of the Aspirin/Folate Polyp Prevention Study seemed to suggest a potential tumor-promoting effect of folic acid supplementation. In this trial, the use of folic acid seemed to be associated with an increased risk of developing advanced lesions with malignant potential and of developing any other cancer.⁷⁵ Animal studies of this question would seem to offer an explanation of such results. In animal models, folate deficiency in normal colorectal mucosa seems to promote tumor genesis but in established neoplasms, folic acid supplementation seems to promote tumor progression.⁶⁹

In studies of cancers outside of the colorectum, a prospective study showed that increased folate plasma concentrations were associated with an increased risk of premenopausal breast cancer (although the result was not felt to be statistically significant),⁷⁶ and a prospective cohort study showed an increased risk of postmenopausal breast cancer in those whose folate consumption was highest.⁷⁷ However, other studies have generally shown a protective effect on folate intake against breast cancer.⁷⁸ In two prospective cohort studies, multivitamin use (containing folic acid) was associated with a nonsignificant increased risk of pancreatic cancer.⁷⁹ How much these results are due to the dose of folic acid, or on the use folic acid supplements rather than dietary folate, is unclear, and it should be noted that in many cases the results did not reach statistical significance.

CONCLUSION

There is no doubt about the benefit of folic acid use in the population as a whole. Neurologists should be aware of this and should be more active in the use or prescription of folic acid for WWE. It has been shown in a number of studies that the guidelines for prepregnancy counseling and the use of folic acid are not well adhered to⁸⁰—by both neurologists and other specialists. However, specifically on the issue of dosing, the evidence available does not support the use of high-dose folic acid in those women who do not have either a personal or family history of NTD or MCM occurrence. Furthermore, in some patients, the use of folic acid may not be as risk free as is commonly supposed. From our current reading of the available evidence, the following points should be considered:

1. Standard dose (0.4 mg) of folic acid use should be encouraged in all WWE of child bearing potential.

2. The evidence to support the use of high-dose (ie, 5 mg) folic acid in WWE who do not have a personal or family history of NTD/MCM occurrence is not strong.

3. The evidence that folic acid supplementation will protect WWE from MCMs and NTDs is poor. However, given the potential interference in the metabolism of folate by some AEDs, high-dose folic acid (5 mg) should be prescribed in WWE but only in those without a personal history of malignancy.

4. Folic acid should be used in those with a history of malignancy only with caution, after a full discussion of the possible benefits and risks, and with regular review.

The rate of NTD occurrence can be lessened, but not eliminated by the use of folic acid, a fact that patients should be made aware.

Keywords

folic acid, women with epilepsy, supplementation, major congenital malformations

Disclosures: The authors have no disclosures or conflicts of interest and funding

REFERENCES

1. Wills L. Treatment of pernicious anaemia of pregnancy and tropical anaemia with special reference to yeast extract curative agent. Br Med J. 1931;1:1059–1064.
2. Harden C, Pennell P, Koppel B, et al. Practice parameter update: management issues for women with epilepsy. Focus on pregnancy (an evidence based review): vitamin K, folic acid, blood levels and breastfeeding: report of the Quality Standards Subcommittee and Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology and the American Epilepsy Society. Neurology. 2009;73:142–149.
3. Meijer W, de Walle H. Differences in folic-acid policy and the prevalence of neural-tube defects in Europe; recommendations for food fortification in a EUROCAT report. Ned Tijdschr Geneeskd. 2005;149(46):2561–2564.
4. Vidailhet M, Bocquet A, Bresson J, et al. Folic acid and prevention of neural tube closure defects: the question is not solved yet. Arch Pediatr. 2008;15(7):1223–1231.
5. The epilepsies; the diagnosis and management of the epilepsies in adults and children. Epilepsy NICE guideline October 2004. www.nice.org.uk
6. Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin and Choline. Washington, DC: National Academy Press; 1998.
7. Suitor C, Bailey L. Dietary folate equivalents: interpretation and application. J Am Diet Assoc. 2000;100:88–94.
8. Lucock M. Folic acid: nutritional biochemistry, molecular biology and role in disease processes. Mol Gent Metab. 2000;71:121–138.
9. Botto L, Moore C, Khoury M, Erickson J. Neural tube defects. N Engl J Med. 1999;341:1509–1519.
10. Wald N. Folic acid and the prevention of neural tube defects. Ann NY Acad Sci. 1993;678:112–129.
11. Blom H, Shaw G, den Heijer M, Finnell R. Neural tube defects and folate: case far from closed. Nat Rev Neurosci. 2006;7(9):724–731.
12. Rampersaud E, Melvin E, Speer M. Nonsyndromic neural tube defects: genetic basis and genetic investigations. In: Wyszynski D, ed. Neural Tube Defects: from Origin to Treatment. Oxford: Oxford University Press; 2006:165– 175.
13. Leck I. Causation of neural tube defects: clues from epidemiology. Br Med Bull. 1974;30:158–163.
14. Elwood JM, Elwood JH Epidemiology of Anencephalus and Spina Bifida. Oxford: Oxford University Press; 1980.
15. Botto L, Yang Q. 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review. Am J Epidemiol. 2000; 151(9):862–877.
16. De Marco P, Calevo M, Moroni A, et al. Study of MTHFR and MS polymorphisms as risk factors for NTD in the Italian population. J Hum Genet. 2002;47(6):319–324.
17. Copp A, Greene N, Murdoch J. The genetic basis of mammalian neurulation. Nat Rev Genet. 2003;4:784–793.
18. Gutiérrez Revilla J, Pérez Hernández F, Calvo Martín M, Tamparillas Salvador M, Gracia Romero J. C677T and A1298C MTHFR polymorphisms in the etiology of neural tube defects in Spanish population. Med Clin (Barc). 2003;120(12):441–445.
19. Fedrick J. Anencephalus in the Oxford Record Linkage Study area. Dev Med Child Neurol. 1976;18(5):643–656.
20. Rosano A, Del Bufalo E, Burgio E. Socioeconomic status and risk of congenital malformations. Epidemiol Prev. 2008;32(1):21–26.
21. Dheen S, Tay S, Boran J, et al. Recent studies on neural tube defects in embryos of diabetic pregnancy: an overview. Curr Med Chem. 2009;16(18):2345–2354.
22. Vieira A, Castillo Taucher S. Maternal age and neural tube defects: evidence for a greater effect in spina bifida than in anencephaly. Rev Med Chil. 2005;133(1):62–70.
23. Grewal J, Carmichael S, Ma C, Lammer E, Shaw G. Maternal periconceptual smoking and alcohol consumption and risk for select congenital anomalies. Birth Defects Res A Clin Mol Teratol. 2008;82(7):519– 526.
24. Jentink J, Loane MA, Dolk H, et al; EUROCAT Antiepileptic Study Working Group. Valproic acid monotherapy in pregnancy and major congenital malformations. N Engl J Med. 2010;362:2185–2193.
25. Kirke P, Molloy A, Daly L, Burke H, Weir D, Scott J. Maternal plasma folate and vitamin B12 are independent risk factors for neural tube defects. Quart J Med. 1993;86:703–708.
26. Steen M, Boddie A, Fisher A. Neural tube defects are associated with low concentrations of cobalamin (vitamin B12) in amniotic fluid. Am J Hum Genet. 1997;61(4):A166.
27. Suarez L, Felkner M, Brender J, Canfield M, Hendricks K. Maternal exposures to cigarette smoke, alcohol and street drugs and neural tube defect occurrence in offspring. Matern Child Health J. 2008;12(3):394–401.
28. Chen Z, Karaplis A, Ackerman S, et al. Mice deficient in methyltetrahydrofolate reductase exhibit hyperhomocysteinaemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet. 2001;10:433–443.
29. Biniszkiewicz D, Girbnau J, Ramsahoye B, et al. Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting and embryonic lethality. Mol Cell Biol. 2002;22:2124–2135.
30. Bohnsack B, Hirschi K. Nutrient regulation of cell cycle progression. Annu Rev Nutr. 2004;24:433–453.
31. Boot M, Steegers-Theunissen R, Poelmann R, Iperen L, Lindemans J, Gittenberger-de Groot A. Folic acid and homocysteine affect neural crest and neuroepithelial cell outgrowth and differentiation in vitro. Dev Dyn. 2003;227:301–308.
32. Rampersaud G, Kauwell G, Hutson A, Cerda J, Bailey L. Genomic DNA methylation decreases in response to moderate folate depletion in elderly women. Am J Clin Nutr. 2000;72:998–1003.
33. MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet. 1991;338:131–137.
34. Czeizel A, Dudas I. Prevention of the first occurrence of neural tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992;327:1832–1835.
35. Czeizel A, Dudas I, Metneki J. Pregnancy outcomes in a randomised controlled trial of periconceptional multivitamin supplementation. Final report. Arch Gynecol Obstet. 1994;255:131–139.
36. Blencowe H, Cousens S, Modell B, Lawn J. Folic acid to reduce neonatal mortality from neural tube disorders. Int J Epidem. 2010;39:i110–i121.
37. Ray J, Meier C, Vermeulen M, Boss S, Wyatt P, Cole D. Association of neural tube defects and folic acid fortification in Canada. Lancet. 2002;360:2047–2048.
38. Eicholzer M, Tönz O, Zimmermann R. Folic acid: a public health challenge. Lancet. 2006;367:1352–1361.
39. De Jong-Van den Berg L, Hernandez-Diaz S, Werler M, Louik C, Mitchell A. Trends and predictors of folic acid awareness and periconceptual use in pregnant women. Am J Obstet Gynecol. 2005;192:121–128.
40. De Walle H, van der Pal K, de Jong-van den Berg L, et al. Effect of mass media campaign to reduce socioeconomic differences in women’s awareness and behaviour concerning use of folic acid: cross sectional study. BMJ. 1999;319:291–292.
41. Heseker H, Mason J, Selhub J, Rosenberg I, Jacques P. Not all cases of neural tube defect can be prevented by increasing the intake of folic acid. Br J Nutr. 2009;102(2):173–180.
42. Goh Y, Koren G. Folic acid in pregnancy and fetal outcomes. J Obstet Gynaecol. 2008;28:3–13.
43. French A, Grant R, Weitzman S, et al. Folic acid fortification is associated with a decline in neuroblastoma. Clin Pharmacol Ther. 2003;74:288–294.
44. Bunin G, Kuijten R, Buckley J, Rorke L, Meadows A. Relationship between maternal diet and subsequent primitive neuroectodermal brain tumours in young children. N Engl J Med. 1993;329:536–541.
45. Schuz J, Weihkopf T, Kaatsch P. Medication use during pregnancy and the risk of childhood cancer in the offspring. Eur J Paediat. 2007;166:433– 441.
46. Mueller-Kueppers M. On the problem of fetal damage during pregnancy caused by intake of anticonvulsants. Acta Paedopsychiatr. 1963;30:401–405.
47. Massey K. Teratogenic effects of diphenylhydantoin sodium. J Oral Ther Pharmacol. 1966;2(5):380–385.
48. Olafsson E, Hallgrimsson J, Hauser W, Ludvigsson P, Gudmundsson G. Pregnancies of women with epilepsy: a population based study in Iceland. Epilepsia. 1998;39:887–892.
49. Laskowska M, Leszczynska-Gorlezak B, Oleszczuk J. Pregnancy in women with epilepsy. Gynaecol Obstet Invest. 2001;51:99–102.
50. Fairgrieve S, Jackson M, Jonas P, et al. Population based, prospective study of the care of women with epilepsy in pregnancy. Br Med J. 2000; 321:674–675.
51. EURAP. Seizure control and treatment in pregnancy: observations from the EURAP Epilepsy and Pregnancy Registry. Neurology. 2006;66:354–360.
52. Meador K, Baker G, Finnell R, et al. In utero antiepileptic drug exposure: fetal death and malformations. Neurology. 2006;67:407–412.
53. Holmes LB, Harvey EA, Coull BA, et al. The teratogenicity of anticonvulsant drugs. N Engl J Med. 2001;344:1132–1138.
54. Artama M, Auvinen A, Raudaskoski T, Isojarvi I, Isojarvi J. Antiepileptic drug use of women with epilepsy and congenital malformations in offspring. Neurology. 2005;64:1874–1878.
55. Hunt S, Craig J, Russell A, et al. Levetiracetam in pregnancy: preliminary experience from the UK Epilepsy and Pregnancy Register. Neurology. 2006;67:1876–1879.
56. Vaida F, Eadie M. Maternal valproate dosage and foetal malformations. Acta Neurol Scand. 2005;112:137–143.
57. Pennell P. The importance of monotherapy in pregnancy. Neurology. 2003;60(suppl 4):S31–S38.
58. Dansky L, Rosenblatt D, Andermann E. Mechanisms of teratogenesis: folic acid and antiepileptic therapy. Neurology. 1992;42(suppl 5):S32–S42.
59. Nei M, Daly S, Liporace J. A maternal complex partial seizure in labor can affect fetal heart rate. Neurology. 1998;51:904–906.
60. Barrett C, Richens A. Epilepsy and pregnancy: report of an Epilepsy Research Foundation Workshop. Epilepsy Res. 2003;52:147–187.
61. De Bree A, Verschuren W, Kromhout D, Kluijtmans A, Blom H. Homocysteine determinants and the evidence to what extent homocysteine determines the risk of coronary heart disease. Pharmacol Rev. 2002;54:599–618.
62. Centres for Disease Control. Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. MMWR Recomm Rep. 1992;41:1–7.
63. Kluger B, Meador K. Teratogenicity of antiepileptic medications. Semin Neurol. 2008;28(3):328–335.
64. Mawer G, Briggs M, Baker G, et al. Pregnancy with epilepsy: obstetric and neonatal outcome of a controlled study. Seizure. 2010;19:112–119.
65. Morrow J, Hunt S, Russell A, et al. Folic acid use and major congenital malformations in offspring of women with epilepsy: a prospective study from the UK Epilepsy and Pregnancy Register. J Neurol Neurosurg Psychiatry. 2009;80:506–511.
66. Kjaer D, Horyath-Puhó E, Christensen J, et al. Antiepileptic drug use, folic acid supplementation, and congenital abnormalities: a population-based case-control study. BJOG. 2008;115(1):98–103.
67. Pittschieler S, Brezinka C, Jahn B, et al. Spontaneous abortion and the prophylactic effect of folic acid supplementation in epileptic women undergoing antiepileptic therapy. J Neurol. 2008;255(12):1926–1931.
68. Kim Y. Folate and colorectal cancer: an evidence-based critical review. Mol Nutr Food Res. 2007;51:267–292.
69. Kim D, Smith-Warner S, Hunter D. Pooled analysis of prospective cohort studies on folate and colorectal cancer. Pooling project of diet and cancer investigators. Am J Epidemiol. 2001;153:S118.
70. Sanjoaquin M, Allen N, Couto E, Roddam A, Key T. Folate intake and colorectal cancer risk: a meta-analytical approach. Int J Cancer. 2005;113:825–828.
71. Jaszewski R, Misra S, Tobi M, et al. Folic acid supplementation inhibits recurrence of colorectal adenomas: a randomised chemoprevention trial. World J Gastroenterol. 2008;14:4492–4498.
72. Logan R, Grainge M, Shepherd V, Armitage N, Muir K. Aspirin and folic acid for the prevention of recurrent colorectal adenomas. Gastroenterology. 2008;134:29–38.
73. Mason J, Dickstein A, Jacques P, et al. A temporal association between folic acid fortification and an increase in colorectal cancer rates may be illuminating important biological principles: a hypothesis. Cancer Epidemiol Biomarkers Prev. 2007;16:1325–1329.
74. Cole B, Baron J, Sandler R, et al. Folic acid for the prevention of colorectal adenomas: a randomised clinical trial. JAMA. 2007;297:2351–2359.
75. Lin J, Lee I, Cook N, et al. Plasma folate, vitamin B6, vitamin B12 and risk of breast cancer in women. Am J Clin Nutr. 2008;87(3):734–743.
76. Stolzenberg-Solomon R, Chang S, Leitzmann M, et al. Folate intake, alcohol use and postmenopausal breast cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Am J Clin Nutr. 2006;83(4):895–904.
77. Ericson U, Sonestedt E, Gullberg B, Olsson H, Wirfält E. High folate intake is associated with lower breast cancer incidence in postmenopausal women in the Malmo Diet and Cancer Cohort. Am J Clin Nutr. 2007;86(2):434–443.
78. Skinner H, Michaud D, Giovannucci E, et al. A prospective study of folate intake and the risk of pancreatic cancer in men and women. Am J Epidemiol. 2004;160(3):248–258.
79. Kampman M, Johansen S, Stenvold H, Acharya G. Management of women with epilepsy: are guidelines being followed? Results from casenote reviews and a patient questionnaire. Epilepsia. 2005;46(8):1286–1292.
80. Schwarz E, Postlethwaite D, Hung YY, Armstrong M. Documentation of contraception and pregnancy when prescribing potentially teratogenic medications for reproductive-age women. Ann Intern Med. 2007;147:370– 376.

The most frequent causes of solitary transient lesions in the splenium of the corpus callosum (SCC) are viral encephalitis, antiepileptic drug toxicity/withdrawal, and hypoglycemic encephalopathy.^{1, 2} Influenza, adenovirus, and mumps have been found in some patients but in most encephalitic cases the causative germ remained obscure. Other etiologies include rapid correction of hyponatremia resulting in osmotic myelinolysis, dural sinus thrombosis, multiple sclerosis,³ bacterial meningoencephalitis⁴ malnutrition,⁵ as well as systemic lupus erythematosus.⁶ In most of these latter conditions, however, the splenial lesion is not isolated. The clinical course is usually mild with good prognosis.^{1, 2, 7–9} Accompanying symptoms include disorientation, drowsiness, cognitive and memory impairments,⁴ hallucinations,⁷ headache,¹ confusion,¹⁰ but detailed clinical description of callosal disconnection in patients with reversible lesions has only rarely been reported.¹¹ Routine neuropsychological examination can miss these signs, which can often be detected only by targeted tests.

We report a 64-year-old male patient who presented with fever, agitated logorrhea, and deficiency of interhemispheric transfer, associated with an isolated lesion of the SCC. Methodology included neurological and detailed neuropsychological examinations, radiological investigations, cerebrospinal fluid (CSF), and routine laboratory analyses. Neuropsychological examination was oriented to evaluate interhemispheric transfer.

CASE REPORT

A 64-year-old right-handed male patient was admitted for agitation, fever and flu-like symptoms as well as difficulty in using his both hands simultaneously. He only took his regular antihypertensive drug and paracetamol.

The neurological examination was unremarkable, apart from some psychomotor agitation and logorrhea. He was well oriented in time and space and had no hallucinations or delusions, no aphasia or apraxia. Cranial nerves, motor, sensory, and cerebellar functions were found to be normal. A detailed neuropsychological examination, however, revealed important asymmetry of dichotic audition (ie, left auditive extinction): the patient completely ignored words presented to his left ear. He was unable to perform simultaneous bimanual tapping in a synchronized manner as well as to copy words that were presented in the left visual hemifield (dyscopia). Besides these signs of impaired interhemispheric transfer he had a moderate deficit of visuo-spatial memory, but the rest of the neuropsychological evaluation was normal.

No drugs other than paracetamol were given to the patient during this period. The CSF analysis showed mild pleocytosis (19/mm³), 0.57 g/l proteins, and two oligoclonal bands in the CSF, the same as in the serum. At the brain magnetic resonance imaging (MRI), the only abnormality was an oval-shaped hyperintense lesion in T2 and FLAIR sequences in the splenium of the corpus callosum, with restricted diffusion and no gadolinium enhancement (Figure 1a).

Tests for various infections (Lyme disease, syphilis, cytomegalovirus (CMV), ebstein barr virus (EBV), Herpes simplex 1-2, HHV-6, mumps, measles, rubella, varicella, HIV, influenza A and B, and adenovirus), systemic autoimmune, or thyroid disorders were negative. Medical history did not reveal excessive alcohol use and the patient had never taken antiepileptic drugs. Serum electrolytes, glucose, renal and hepatic functions, and vitamin B levels were normal. Without any treatment, the patient's neuropsychological deficits and the corpus callosum lesion disappeared within one week (Figure 1d,e).

DISCUSSION

The diagnosis of encephalitis was supported by the acute onset of brain dysfunction and the inflammatory changes of the CSF. The typical MRI finding in transient SCC, as in our case, is a hyperintense oval lesion in the splenium of the corpus callosum in T2 and FLAIR sequences with restricted diffusion and low apparent diffusion coefficient (ADC), which normalizes after a few days or weeks. The corpus callosum connects the cerebral hemispheres and it is the major component of interhemispheric communication. A complete callosotomy results in a callosal disconnection syndrome (ie, “split brain”): left ideomotor apraxia, left auditive extinction, left tactile anomia, left hemi-alexia and dysgraphia, right constructive apraxia, and intermanual conflict such as alien hand phenomenon.¹²

Depending on the localization and extension of the lesion, incomplete syndrome can be encountered. For example, posterior lesions of the corpus callosum result in motor ignoring of the left hand, impairment of posture, dyscopia from the left visual hemifield, dysgraphia and left tactile agnosia.^{12, 13} Our patient had intermanual conflict and dyscopia, which were also reported in posterior corpus callosum lesions from ruptured arterio-venous malformations.¹² A middle corpus callosum infarct may cause left ideomotor apraxia, intermanual conflict, left auditive extinction on dichotic listening but no left tactile or visual field anomia.¹⁴ In anterior lesions, left ideomotor apraxia, dysgraphia, and right construction apraxia have been observed.¹⁵

Concerning the patomechanism of SCC, osmotic myelinolysis or myelin swelling is hypothesized.⁷ In our case, we suspect a parainfectious origin. The splenial lesion was probably due to intramyelinic rather than cytotoxic edema, which explains its rapid and complete reversibility. For the same reason, a parainfectious immune-mediated mechanism seems more likely than a direct viral invasion.

In five patients, Conti et al³ detected low mean ADC values within the splenial lesions, and they postulated that this may be related to vasogenic edema. Prilipko et al² hypothesized a reversible cytotoxic edema in a case of SCC, induced by abrupt changes of serum antiepileptic drug concentrations and an increased vasopressin (AVP) secretion. In one report, interleukin-6 and tumor necrosis factor-alpha were markedly elevated in serum and CSF samples from two patients with influenza encephalopathy who died after a rapid fulminate course.¹⁶ On the contrary, in a 12-year-old Japanese girl, with influenza virus induced SCC, the serum and CSF analysis did not reveal any increase of this cytokines.⁸ A postmortem examination of one fatal case revealed vasogenic brain edema with generalized vasculopathy, which suggests that impairment of vascular endothelial cells might play a central role in the pathophysiology of this disease.¹⁶

Our case underlines the spontaneously benign nature of isolated SCC with full recovery of the clinical symptoms and of the MRI abnormalities. Targeted neuropsychological evaluation is needed to detect callosal dysfunction. This typical neuroradiological picture should orient the search for potential etiologies and prevent useless diagnostic or therapeutic procedures.

Keywords

Encephalitis, corpus callosum, splenium

Acknowledgement: In honor of the late Dr. E. Mayer, who conducted the neuropsychological examination.

Disclosure:The authors declare no conflict of interest.

REFERENCES

1. Maeda M, Tsukahara H, Terada H, et al. Reversible splenial lesion with restricted diffusion in a wide spectrum of diseases and conditions. J Neuroradiol. 2006;33(4):229–236.
2. Prilipko O, Delavelle J, Lazeyras F, Seeck M. Reversible cytotoxic edema in the splenium of the corpus callosum related to antiepileptic treatment: report of two cases and literature review. Epilepsia. 2005;46(10):1633– 1636.
3. Conti M, Salis A, Urigo C, Canalis L, Frau S, Canalis CG. Transient focal lesion in the corpus callosum: MR imaging with an attempt to clinicalpsychopathological explanation and review of the literature. Radiol Med. 2007;112:921–935.
4. Takanashi J, Hirasawa K, Tada H. Reversible restricted diffusion of entire corpus callosum. J Neurol Sci. 2006;247:101–104.
5. Kosugi T, Isoda H, Imai M, Sakahara H. Reversible focal splenial lesion of the corpus callosum on MR images in a patient with malnutrition. Magn Reson Med Sci. 2004;3(4):211–214.
6. Appenzeller S, Faria A, Marini R, Lavras Costallat LT, Cendes F. Focal transient lesions of the corpus callosum in systematic lupus erythematosus. Clin Rheumatol. 2006;25:568–571.
7. Takanashi J, Barkovich A, Yamaguchi K, Kohno Y. Influenza-associated encephalitis/encephalopathy with a reversible lesion in the splenium of the corpus callosum: a case report and literature review. AJNR. 2004;25:798–802.
8. Matsubara K, Kodera M, Nigami H, Yura K, Fukaya T. Reversible splenial lesion in influenza virus encephalopathy. Pediatr Neurol. 2007;37:431–434.
9. Yaguchi M, Yaguchi H, Itoh T, Okamoto K. Encephalopathy with isolated reversible splenial lesion of the corpus callosum. Intern Med. 2005;44:1291–1294.
10. Tada H, Takanashi J, Barkovich AJ, et al. Clinically mild encephalitis/ encephalopathy with a reversible splenial lesion. Neurology. 2004;63:1854–1858.
11. Gallucci M, Limbucci N, Paonessa A, Caranci F. Reversible focal splenial lesions. Neuroradiology. 2007;49:541–544.
12. Buklina BS. The corpus callosum, interhemisphere interactions, and the function of the right hemisphere of the brain. Neurosci Behav Physiol. 2005;35(5):473–480.
13. Balsamo M, Trojano L, Giamundo A, Grossi D. Left hand tactile agnosia after posterior callosal lesion. Cortex. 2008;44(8):1030–1036.
14. Servan J, Verstichel P, Elghozi D, Duclos H. Interhemispheric disconnection syndrome caused by partial infarction of the corpus callosum: neuropsychological study and MRI. Rev Neurol (Paris). 1996;152(3):165– 173.
15. Giroud M, Dumas R. Clinical and topographical range of callosal infarction: a clinical and radiological correlation study. JNNP. 1995; 59:238–242.
16. Togashi T, Matsuzono Y, Narita M, Morishima T. Influenza-associated acute encephalopathy in Japanese children in 1994–2002. Virus Res. 2004;103:75–78.