Neuronal loss, demyelination and volume change in the multiple sclerosis neocortex.

AIMS: Indices of brain volume [grey matter, white matter (WM), lesions] are being used as outcomes in clinical trials of patients with multiple sclerosis (MS). We investigated the relationship between cortical volume, the number of neocortical neurons estimated using stereology and demyelination. METHODS: Nine MS and seven control hemispheres were dissected into coronal slices. On sections stained for Giemsa, the cortex was outlined and optical disectors applied using systematic uniform random sampling. Neurons were counted using an oil immersion objective (× 60) following stereological principles. Grey and WM demyelination was outlined on myelin basic protein immunostained sections, and expressed as percentages of cortex and WM respectively. RESULTS: In MS, the mean number of neurons was 14.9 ± 1.9 billion vs. 24.4 ± 2.4 billion in controls (P < 0.011), a 39% difference. The density of neurons was smaller by 28% (P < 0.001) and cortical volume by 26% (P = 0.1). Strong association was detected between number of neurons and cortical volume (P < 0.0001). Demyelination affected 40 ± 13% of the MS neocortex and 9 ± 12% of the WM, however, neither correlated with neuronal loss. Only weak association was detected between number of neurons and WM volume. CONCLUSION: Neocortical neuronal loss in MS is massive and strongly predicted by cortical volume. Cortical volume decline detected in vivo may be similarly indicative of neuronal loss. Lack of association between neuronal density and demyelination suggests these features are partially independent, at least in chronic MS.

Neuronal and glial cerebrospinal fluid protein biomarkers are elevated after West Nile virus infection.

Neurotrophic West Nile virus (WNV) disease is a severe arbovirus infection in which neuronal loss is the likely anatomical substrate for the high morbidity and mortality. We investigated whether cerebrospinal fluid (CSF) protein biomarkers were elevated in vivo and related to disease severity in patients with WNV infection. This exploratory study included 114 patients (24 acute WNV, 77 noninflammatory controls, six peripheral neuropathies, seven aseptic meningoencephalitis). CSF levels of neuronal (neurofilaments, NfH-SMI35) and glial (glial fibrillary acidic protein, GFAP, S100B) biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). Immunocytochemistry was performed in two fatal WNV cases. A significant proportion of patients with WNV had pathological CSF levels for NfH-SMI35 (58%, median concentration 1.01 ng/mL), GFAP (58%, 10 pg/mL), and S100B (90%, 1.29 ng/mL). The results were consistent with postmortem evidence for neuronal death and astrogliosis. Surprisingly, CSF protein biomarker levels were also found to be pathological in a considerable proportion of patients who presented with WNV fever only (100% for GFAP and S100B and 43% for NfH-SMI35). Elevated CSF protein biomarker levels are suggestive of neuronal death and glial pathology in human WNV infection. The results indicate the presence of neuroinvasive disease across the spectrum of WNV disease, including WNV fever.

Neurotrophin-3 administration alters neurotrophin, neurotrophin receptor and nestin mRNA expression in rat dorsal root ganglia following axotomy.

In the months following transection of adult rat peripheral nerve some sensory neurons undergo apoptosis. Two weeks after sciatic nerve transection some neurons in the L4 and L5 dorsal root ganglia begin to show immunoreactivity for nestin, a filament protein expressed by neuronal precursors and immature neurons, which is stimulated by neurotrophin-3 (NT-3) administration. The aim of this study was to examine whether NT-3 administration could be compensating for decreased production of neurotrophins or their receptors after axotomy, and to determine the effect on nestin synthesis. The levels of mRNA in the ipsilateral and contralateral L4 and L5 dorsal root ganglia were analyzed using real-time polymerase chain reaction, 1 day, 1, 2 and 4 weeks after unilateral sciatic nerve transection and NT-3 or vehicle administration via s.c. micro-osmotic pumps. In situ hybridization was used to identify which cells and neurons expressed mRNAs of interest, and the expression of full-length trkC and p75NTR protein was investigated using immunohistochemistry. Systemic NT-3 treatment increased the expression of brain-derived neurotrophic factor, nestin, trkA, trkB and trkC mRNA in ipsilateral ganglia compared with vehicle-treated animals. Some satellite cells surrounding neurons expressed trkA and trkC mRNA and trkC immunoreactivity. NT-3 administration did not affect neurotrophin mRNA levels in the contralateral ganglia, but decreased the expression of trkA mRNA and increased the expression of trkB mRNA and p75NTR mRNA and protein. These data suggest that systemically administered NT-3 may counteract the decrease, or even increase, neurotrophin responsiveness in both ipsi- and contralateral ganglia after nerve injury.

Lymphocyte contamination of laryngoscope blades--a possible vector for transmission of variant Creutzfeldt-Jakob disease.

Variant Creutzfeldt-Jakob disease (vCJD) is associated with extensive prion infection of lymphoreticular tissues during the prolonged asymptomatic incubation period. Instruments exposed to infected tissues of preclinically infected individuals during medical or surgical procedures represent a potential risk of iatrogenic transmission of vCJD prions. We assessed the frequency of contamination with lymphoid tissue of single-use laryngoscope blades used for tracheal intubation for general anaesthesia. Using a cyto-centrifugation technique, lymphocytes were detected from 30% of laryngoscope blades studied. As prions resist routine sterilisation procedures, the use of non-disposable laryngoscope blades poses a risk of transmitting vCJD from patient to patient. The use of such instruments should be abandoned and disposable alternatives used.

Brain biopsy in dementia.

Brain biopsy has an uncertain role in the diagnosis of dementia. Here we report a retrospective analysis of 90 consecutive cerebral biopsies undertaken for the investigation of dementia in adults at a tertiary referral centre between 1989 and 2003. In most cases (90%), biopsy consisted of a right frontal full thickness resection of cortex, white matter and overlying leptomeninges. Fifty-seven per cent of biopsies were diagnostic: the most frequent diagnoses were Alzheimer's disease (18%), Creutzfeldt-Jakob disease (12%) and inflammatory disorders (9%). Other diagnoses in individual patients included Pick's disease, corticobasal degeneration and other tauopathies, Lewy body dementia, multiple sclerosis, Whipple's disease, progressive multifocal leucoencephalopathy, cerebral autosomal dominant arteriopathy with subcortical ischaemic leucoencephalopathy, vasculopathies and paraneoplastic encephalopathy. The most frequent biopsy finding in the non-diagnostic group and for the series as a whole (37%) was non-specific gliosis variably affecting both cortex and white matter. Complications (11%) included seizures, intracranial and wound infections, and intracranial haemorrhage; there were no deaths or lasting neurological sequelae attributable to the procedure. No trends in diagnostic yield or complication rate over the course of the series were identified. Information obtained at biopsy determined treatment in 11%. A raised cerebrospinal fluid cell count was the only robust predictor of a potentially treatable (inflammatory) process at biopsy. The constellation of behavioural change, raised CSF protein and matched oligoclonal bands in CSF and serum was associated with non-specific gliosis at biopsy. This series underlines the value of cerebral biopsy in the diagnosis of dementia, and suggests that certain clinical and laboratory features may be useful in guiding the decision to proceed to brain biopsy where a treatable disease cannot be excluded by other means.

Young onset limb spasticity with PSP-like brain and spinal cord NFT-tau pathology.

A 30-year-old white man presented with a sporadic form of gradually progressive spastic gait and, later, supranuclear vertical and horizontal gaze palsy, mild cognitive impairment, loss of postural reflexes, and falls. DNA analysis revealed H1/H1 haplotype without tau gene (exons 9 to 13) mutation. Eight years later, postmortem revealed a tauopathy similar to progressive supranuclear palsy. Unusual aspects were early age at onset, neurofibrillary tangle, and tau involvement of the cord.

The spectrum of antineuronal autoantibodies in a series of neurological patients.

The aim of the present study is to identify the range of neurological disorders expressing antineuronal antibodies, evaluate the number of different patterns of reactivity that can be detected, and analyse the contribution of these studies to the identification of subgroups of patients. The records of 882 patients were reviewed and their sera and cerebrospinal fluids tested for antineuronal antibodies. Patients were initially divided into four groups according to suspected clinical diagnosis. Autoantibodies were detected by immunohistochemistry, Western blot of gradient-separated neuronal and recombinant proteins and by RIA. Cerebellar degeneration and sensory neuropathies were the most common neurological disorders in which paraneoplastic-related anti-neuronal antibodies were detected. However, in addition to PCA1/anti-Yo and ANNA1/anti-Hu antibodies, we found other reactivities in six patients with cerebellar degeneration: anti-GAD in three females and atypical in the other cases. The widest range of different anti-neuronal antibodies was detected in patients with peripheral sensory neuropathy. Few patients with Stiff-Person syndrome, temporal lobe epilepsy and myoclonus harboured anti-GAD antibodies. Atypical antibodies were detected in single cases with motor neuron disorder and multiple system atrophy. No anti-neuronal antibodies were detected in patients with neurological complications of connective tissue disorders other than Sjögren's syndrome, or in neurological diseases other than motor neuron disease and multiple system atrophy. Our study shows that the spectrum of neurological disorders in which anti-neuronal antibodies can be detected is wider than previously thought. In addition, we found patterns of neuronal staining and Western blot reactivity that differed from those so far reported. This may permit identification of subgroups of patients in whom strategies directed at removing and/or suppressing antibody production could be of some benefit.

Parallel changes in bladder suburothelial vanilloid receptor TRPV1 and pan-neuronal marker PGP9.5 immunoreactivity in patients with neurogenic detrusor overactivity after intravesical resiniferatoxin treatment.

OBJECTIVE: To compare PGP9.5 and transient receptor potential vanilloid receptor (TRPV1) suburothelial immunoreactivity between controls and patients with spinal neurogenic detrusor overactivity (NDO) before and after treatment with intravesical resiniferatoxin, as suburothelial PGP9.5-staining nerve fibres decrease in patients with spinal NDO who respond to intravesical capsaicin, and TRPV1 is present on these suburothelial nerve fibres in normal and overactive human urinary bladder. PATIENTS AND METHODS: Patients with refractory NDO were enrolled in a prospective, randomized, parallel-group, double-blind, placebo-controlled trial using escalating doses of resiniferatoxin to a maximum of 1 micro mol/L. Flexible cystoscopic bladder biopsies obtained at baseline, 4 weeks after each instillation and at the time of maximum clinical response were compared with biopsies taken from control subjects. Frozen sections were incubated with rabbit antibodies to TRPV1 and PGP9.5, and assessed using standard immunohistochemical methods. PGP9.5 nerve density was analysed using a nerve-counting graticule by an observer unaware of sample origin. Another two independent observers unaware of each other's results used a random grading scale to evaluate TRPV1 nerve fibre density and intensity. The immunohistochemistry results were compared with histology findings (haematoxylin-eosin), and the Mann-Whitney test used to assess any differences (P < 0.05 significant) and the Pearson test for correlation. RESULTS: There were eight controls and 20 patients with spinal NDO, 14 (five clinical responders and nine not) who received the maximum dose of resiniferatoxin. There were more PGP9.5 and TRPV1 nerve fibres in patients with NDO than in controls (P = 0.007 and 0.002, respectively). Immunoreactivity before resiniferatoxin was similar in both groups for both PGP9.5 and TRPV1. In responders there were fewer PGP9.5 and TRPV1-positive fibres after treatment (P = 0.008 for each) but no change in those not responding. Changes after treatment for TRPV1 correlated well with those for PGP9.5 (r = 0.88, P < 0.001). CONCLUSIONS: The decrease of PGP9.5 and TRPV1 immunoreactive nerve fibres in responders to resiniferatoxin (to levels in control tissues) suggests that the increased numbers of nerve fibres in patients with NDO are mainly of sensory origin and express TRPV1. As baseline nerve fibre values were similar in responders and nonresponders, an additional factor may account for the difference in treatment outcome.

High field MRI correlates of myelin content and axonal density in multiple sclerosis--a post-mortem study of the spinal cord.

Different MRI techniques are used to investigate multiple sclerosis (MS) in vivo. The pathological specificity of these techniques is poorly understood, in particular their relationship to demyelination and axonal loss. The aim of this study was to evaluate the pathological substrate of high field MRI in post-mortem (PM) spinal cord (SC) of patients with MS. MRI was performed in PMSCs of four MS patients and a healthy subject on a 7 Tesla machine. Quantitative MRI maps (PD; T2; T1; magnetization transfer ratio, MTR; diffusion weighted imaging) were obtained. After scanning, the myelin content and the axonal density of the specimens were evaluated neuropathologically using quantitative techniques. Myelin content and axonal density correlated strongly with MTR, T1, PD, and diffusion anisotropy, but only moderately with T2 and weakly with the apparent diffusion coefficient. Quantitative MR measures provide a promising tool to evaluate components of MS pathology that are clinically meaningful. Further studies are warranted to investigate the potential of new quantitative MR measures to enable a distinction between axonal loss and demyelination and between demyelinated and remyelinated lesions.

CDKN2A, CDKN2B and p14ARF are frequently and differentially methylated in ependymal tumours.

Ependymal tumours are histologically and clinically varied lesions. Numerical abnormalities of chromosome 9 are frequently associated with these tumours. Nevertheless, the three important tumour suppressor genes located in this chromosome, CDKN2A, CDKN2B and p14 ARF, have not been reported to be commonly altered in them. We studied promoter methylation of these genes, an important mechanism associated with gene silencing in a series of 152 ependymal tumours of WHO grades I to III. Methylation status of the CDKN2A, CDKN2B and p14 ARF promoters was assessed by methylation-specific polymerase chain reaction and the genetic results were correlated to clinicopathological features. We observed promoter methylation for CDKN2A in 21% (26/123) of tumours, for CDKN2B in 32% (23/71) and p14 ARF in 21% (23/108). For all three genes, posterior fossa ependymomas were less frequently methylated in paediatric patients than in adults. For CDKN2B, extracranial tumours were more frequently methylated than intracranial ones. For CDKN2B and p14 ARF, methylation was more frequent in low-grade tumours; the reverse was observed for CDKN2A. CDKN2A, CDKN2B and p14 ARF promoters were methylated in 21-32% of the tumours. Frequencies of methylation varied according to clinicopathological features. This suggests a role for these genes in ependymoma tumorigenesis.

Stereotactic co-registration of magnetic resonance imaging and histopathology in post-mortem multiple sclerosis brain.

A number of groups have examined the pathological substrate of signal changes on magnetic resonance imaging (MRI) in post-mortem (PM) brain of patients with multiple sclerosis (MS). Such studies will benefit from using a standardized method to reliably co-register regions of interest on MRI and tissue specimens. We investigated the usefulness of a stereotactic navigation system for this purpose. We also addressed the sensitivity of different standard MRI sequences with regard to lesion conspicuity in PM MS brain. Post-mortem brains of eight patients with MS were studied. Formalin-fixed coronal slices were placed in the head frame of a stereotactic system. Proton density-, T2-weighted and fast fluid-attenuated inversion recovery (FLAIR) scans were obtained and visually matched with scans that had been previously obtained on the same, but fresh, specimens. Guided by the stereotactic target points, the dissection of the fixed specimens was performed. After processing the blocks for embedding in paraffin, sections were stained with haematoxylin-eosin and Luxol fast blue. T2-weighted MRI of fixed brain revealed 24 areas suspected to be MS lesions, all of which were confirmed histologically. Three of these lesions were not visible on macroscopic inspection. There were 14 additional hyperintensities on T2-weighted or FLAIR MRI of the fresh specimens, five of which did not correlate to MS lesions histologically. Stereotactic navigation is a useful approach to co-register MRI and histopathology in PM brain of MS patients and may improve the precision of MRI-guided sampling of tissue specimens. Standard T2-weighted MRI appeared to be the single most useful approach for lesion detection in fresh and fixed specimens.

Polymyositis masquerading as mitochondrial toxicity.

A 66 year old HIV infected male heavy smoker presented with arthralgia, myalgia, and weight loss which was originally ascribed to nucleoside induced mitochondrial toxicity. Despite withdrawal of antiretroviral therapy a proximal myopathy developed. Further investigation excluded malignancy. Polymyositis was diagnosed on muscle biopsy. The patient recovered completely with oral prednisolone. This case highlights the importance of muscle biopsy in HIV infected patients whose myopathy persists despite withdrawal of antiretroviral therapy and the need for thorough investigation of non-specific symptoms in HIV infected patients who are receiving antiretroviral therapy.

Tumour necrosis and microvascular proliferation are associated with 9p deletion and CDKN2A alterations in 1p/19q-deleted oligodendrogliomas.

A subset of oligodendrogliomas and oligoastrocytomas has been associated with 1p/19q deletion. Subsequently, this genetic alteration was linked to chemosensitivity and classic histology of oligodendrogliomas. Tumoural progression includes deletions of 9p, 10q and alterations of CDKN2A. However, these (epi)genetic changes have not been associated with specific histological features. In a series of 45 gliomas including oligodendrogliomas, oligoastrocytomas and astrocytomas, deletions of chromosomal regions implied in these tumours (1p, 9p, 10, 17p13, 19q and 22) were looked for by microsatellite analysis. Tumours that were deleted for 1p and 19q were selected. Subsequently, presence of deletions in the other studied regions, (epi)genetic changes in p14ARF, CDKN2A and CDKN2B, as well as histological features, were associated to these tumours. 1p/19q deletion was observed in 22 tumours. Twenty-one of them presented regions of classic histology of oligodendroglioma. A deletion of 9p was found in eight of them, always in association with tumour necrosis and/or microvascular proliferation. In addition, (epi)genetic alterations of CDKN2A were observed in 71% of these tumours. Presence of regions of classic histology of oligodendroglioma in a tumour sample is predictive of 1p/19q deletions. Necrosis and/or microvascular proliferation are signs of an additional 9p deletion. Finally, as CDKN2A (epi)genetic alterations were found in 71% of the 1p/19q/9p-deleted oligodendrogliomas, CDKN2A may have a role in oligodendroglioma-associated microvascular proliferation.

Sudden and unexpected death in epilepsy (SUDEP): evidence of acute neuronal injury using HSP-70 and c-Jun immunohistochemistry.

Post-mortem and neuropathological examination in sudden and unexpected death in epilepsy (SUDEP) shows no specific lesions and the exact cause and mechanism of death in these cases remains undetermined. There is clinical evidence to support the fact that SUDEP is a seizure-mediated event, and patients with poorly controlled seizures are at higher risk. We aimed to identify any evidence of acute neuronal injury in SUDEP cases at post-mortem to support that a recent seizure had occurred. We analysed the distribution and frequency of heat shock protein (HSP)-70 and c-Jun immunopositive neurones in the hippocampus in 18 SUDEP cases and 22 control cases, both markers being nonspecific but early and reliable indicators of acute neuronal injury. Post-mortem control groups included patients with epilepsy with cause of death other than SUDEP (including status epilepticus and accidental death), and patients with sudden cardiac death without an epilepsy history. An additional surgical control group included patients with refractory epilepsy and hippocampal sclerosis who had undergone temporal lobectomy. Semiquantitative analysis of the distribution of HSP-70 staining showed significantly more SUDEP cases with positively labelled neurones in hippocampal subfields compared to epilepsy and cardiac post-mortem controls (P < 0.001) but not compared to the epilepsy surgical controls (P = 0.4). No significant difference in immunostaining patterns between groups was seen in the parahippocampal gyrus with HSP-70 or with c-Jun in either the hippocampus or parahippocampal gyrus regions. The detection of HSP-70 positive neurones in the hippocampus in SUDEP is supportive of ante-mortem neuronal injury including a recent seizure prior to death.

Profile of adult rat sensory neuron loss, apoptosis and replacement after sciatic nerve crush.

Following permanent transection of the adult rat sciatic nerve, sensory neuron apoptosis in the contributing L4 and L5 dorsal root ganglia can be observed for at least 6 months afterwards. To establish the profile of any sensory neuron apoptosis and loss over time when axonal regeneration is allowed, serial sections of L4 and L5 ganglia were examined and the neurons counted using a stereological technique 1, 2 and 3 months after crushing the right sciatic nerve at mid-thigh level. Our results show that an identical degree of sensory neuron loss and apoptosis occurs 1 month after crush as at 1 month after permanent transection. However, at 3 months no neurons undergoing apoptosis could be observed and no significant loss could be detected in the ipsilateral ganglia when compared to unoperated controls. One explanation was a neuronal replacement mechanism, which was investigated by administering bromodeoxyuridine to rats for 1 month after sciatic nerve transection or crush, prior to detection using immunohistochemistry on sections of their ganglia after 2 months. The presence of bromodeoxyuridine in the nuclei of occasional cells that would be counted as neurons on the basis of size and morphology indicates that a process of apparent neurogenesis may underlie the profile of sensory neuron loss after axotomy.

Bilateral isolated hippocampal malformation in temporal lobe epilepsy.

Hippocampal malformations in patients with epilepsy usually are reported in the context of widespread cortical malformations. Isolated hippocampal malformations are more rarely identified in MRI studies with little documentation of their pathologic appearance. Postmortem examination revealed abnormal position and complex convolutional malformations isolated to the hippocampal formation in an adult with temporal lobe epilepsy in whom MRI demonstrated bilateral hippocampal abnormalities.

Progressive myoclonic ataxia and JC virus encephalitis in an AIDS patient.

A case of progressive myoclonic ataxia in an AIDS patient is described, which evolved over a 13 month period. The ataxia persisted as the only clinical finding for several months before the appearance of a severe tetraparesis and cachexia. Throughout the clinical progression, magnetic resonance imaging (MRI) revealed the presence of bilateral, progressive, isolated, and symmetrical lesions involving the red nuclei, subthalami, thalami, lenticular nuclei, and primary motor cortices. Neuropathological examination, supplemented by in situ hybridisation for JC virus DNA, confirmed that the lesions were those of progressive multifocal leucoencephalopathy (PML). The exceptional clinical presentation of PML in this case is the first report of progressive myoclonic ataxia caused by PML. The selective nature of the lesions confirms the role of the dentato-rubral-thalamo-cortical tract in the pathogenesis of progressive myoclonic ataxia. The atypical MRI findings further emphasise the need for expanded diagnostic criteria for PML in AIDS patients and support the use of more aggressive diagnostic methods as new treatments become available.

Evidence for pathological involvement of the spinal cord in motor neuron disease-inclusion dementia.

Fronto-temporal dementia (FTD), characterised pathologically by ubiquitinated inclusions in the hippocampal dentate fascia and fronto-temporal cortex, may develop in association with motor neuron disease (MND). However, FTD with identical pathological hallmarks may occur in isolation and has been termed motor neuron disease-inclusion dementia (MND-ID). We studied the pathology of three cases of MND-ID including the spinal cord, which has not previously been examined in this condition. The ages of the patients at death were 53, 70 and 68 years and the onset of FTD 10, 15 and 9 years before death. Neuropathological findings in all cases included micro-vacuolation in cortical layer II and ubiquitinated intraneuronal inclusions in fronto-temporal cortex and hippocampal dentate fascia. One case showed unusual basophilic, ubiquitinated neuronal cytoplasmic inclusions in the brain stem. Qualitative assessment of the spinal cord was normal in two cases, while the third showed mild pallor of the lateral cortico-spinal tracts and ubiquitinated inclusions in motor neurons typical of MND. Morphometry did not reveal any significant loss or decrease in size of anterior horn motor neurons. The results of this study are consistent with the hypothesis that the pathological changes of pure MND and MND-ID form a spectrum and demonstrate that pathological involvement of the motor system may occur in MND-ID without clinical evidence of MND.