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.

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.

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.

Expression of CCR-5/CXCR-4 in spinal cord of patients with AIDS.

The discovery that chemokines and their receptors (in particular CXCR-4 and CCR-5) play a role in HIV infection challenges traditional views on the pathogenesis of HIV infection in man and identifies new potential targets for therapeutic intervention. Several groups as well as our pilot study have found that increased numbers of CCR-5 positive macrophage/microglia correlate with disease severity in brains of patients with AIDS. Among HIV-related disorders, vacuolar myelopathy (VM) is the most common spinal cord disorder in patients with AIDS. The purpose of this study was to investigate the possible relationship between the expression of CCR-5/CXCR-4 and spinal cord pathology in patients with AIDS. Thirty-four spinal cords (forming two groups: without and with VM) of patients with AIDS and 6 HIV-1-negative controls were investigated by routine histological examination and immunohistochemistry. Elevated expression of CXCR-4 was found in most AIDS cases with/without neuropathological disorders (8/17 and 13/16, respectively). No CCR-5 expression was detected in HIV-1-negative controls. Among 34 cases with AIDS, expression of CCR-5 was detected in 1/16 HIV-1-positive normal spinal cords and 5/18 with VM. Despite the lack of statistical significance between the two groups (P=0.1019), our results suggest that CCR-5/CXCR-4 are present in spinal cord of patients with AIDS and that CCR-5 is more frequently found in association with VM.

Sporadic fatal insomnia: a case study.

A 58-year-old man died after a 27-month illness characterized by insomnia, confirmed by polysomnography. He was homozygous for methionine at codon 129 of the prion gene but had no mutation in the prion gene. Neuropathology showed thalamic and olivary atrophy and no spongiform changes. Paraffin-embedded tissue blotting demonstrated abnormal prion protein in the brain. This is the first case of the sporadic form of fatal familial insomnia with demonstration of the disorder by polysomnography.

Neuronal loss in Onuf's nucleus in three patients with progressive supranuclear palsy.

Disorders of micturition have been reported only sporadically in patients with progressive supranuclear palsy (PSP). We report the results of a clinicopathological study of 3 patients with a definite diagnosis of PSP at various stages of their illness with sphincter abnormalities. Electromyography of the sphincter muscles was performed in all 3 patients and was abnormal in 2. Morphological and morphometric evaluation of Onuf's nucleus in the sacral spinal cord, which is involved in sphincter control, showed severe cell loss, presence of neurofibrillary tangles, neuropil threads, and glial inclusions. We conclude that bladder dysfunction and abnormal sphincter electromyographic results are due to pathological changes in Onuf's nucleus, and we propose that sphincter abnormalities should be included in the list of possible symptoms of PSP.

Detection and localisation of HIV-1 DNA and RNA in fixed adult AIDS brain by polymerase chain reaction/in situ hybridisation technique.

In the brain of patients with AIDS, HIV-1 is localised in a productive form in mononuclear cells. One issue that still needs clarification is whether HIV is localised in cells other than those of mononuclear lineage. Gene amplification by polymerase chain reaction/in situ hybridisation (PCR-IS) could shed light on it. In this study, formalin-fixed, paraffin-embedded brain tissue from ten adult AIDS sufferers was used. Five of them showed evidence of HIV encephalitis (HIVE), five did not show any abnormality. Nested PCR revealed HIV-1 DNA in all HIVE cases and in three of the group without HIVE. HIV-1 DNA and RNA were also detected in situ in seven cases (all seven were also HIV-1 DNA positive in tube). A higher signal was located in the white than in the grey matter. HIV-1 DNA was found in microglia, macrophages, perivascular cells, multinucleated gaint cells (MGC) and in CD68-negative cells. Some of them were identified as endothelial cells, astrocytes and oligodendrocytes. Reverse transcriptase-PCR-IS was positive in macrophages, MGC, endothelial and glial cells. These results confirm infection of endothelial cells and other glial cells and give clues about the route of entry of virus into the central nervous system and the pathogenesis of the disease. This study did not give any convincing evidence supporting an infection of neurons by HIV-1.

Early entry and widespread cellular involvement of HIV-1 DNA in brains of HIV-1 positive asymptomatic individuals.

There is overwhelming evidence that invasion of the central nervous system (CNS) by HIV-1 takes place at an early stage of the infection. It has been demonstrated that HIV-1 DNA is present in brains of asymptomatic individuals. Evidence of immune activation and increased expression of cytokines suggested that neuropathological changes and neuronal and axonal damage could be the effect of the presence of the virus. The purpose of the study is to ascertain whether target cells for HIV-1 in brain of patients at early stage of the infection are the same as those found in AIDS sufferers or if the distribution seen in AIDS patients results from the late spreading of the infection from cells considered traditionally the reservoir of the virus, i.e. microglial cells. Eighteen brains, all HIV-1 DNA positive, as shown by nested polymerase chain reaction (PCR), were selected among the group of HIV-1 positive asymptomatic cases. In 6 of them, HIV-1 DNA was detected by PCR in situ. Positive cells included astrocytes and endothelial cells, in addition to microglial cells. We conclude that astrocytes and endothelial cells are already infected at an early (asymptomatic) stage of the infection and suggest that they might contribute to the damage of the CNS.

Expression of three oligosaccharide conjugates by neonatal rat dorsal root ganglion neurons: comparison with CGRP and GAP43 immunoreactivity.

Adult dorsal root ganglion neurons express oligosaccharides conjugated to lipids that may be involved in cell-cell recognition, and consequently in the laminar organisation of their central terminations. This paper describes an immunohistochemical study of the developmental expression of 2 lactoseries (LA4 and LD2) and 1 globoseries (SSEA4) oligosaccharide conjugates in rats from embryonic d 19 to postnatal d 60. The expression of calcitonin gene related peptide and the growth associated protein GAP43 was also examined for comparative purposes. We found that these oligosaccharide conjugates begin to be expressed after birth, suggesting that they may be involved in maturation of the central or peripheral terminations, rather than axonal guidance.

The neuropathology of paraneoplastic syndromes.

The term "paraneoplastic neurological syndromes" encompasses a number of uncommon disorders associated with systemic malignancies. In order to be classified a paraneoplastic neurological syndrome, the malignancies must not invade, compress, or metastasize to the nervous system. They can either focally or diffusely involve the central and peripheral nervous system or the neuromuscular junction. This paper reviews the neuropathology of the syndrome. It will first describe the clinical presentation and give an account of the systemic tumors most commonly associated with the various types of disorders. Then it will review the general pathological features that consist of an inflammatory process predominantly affecting the gray matter. Finally, it will describe in detail the main clinico-pathological types, including 1) encephalomyelitis, 2) cortical cerebellar degeneration, 3) peripheral neuropathy, 4) opsoclonus-myoclonus and 5) retinopathy. The Lambert-Eaton myasthenic syndrome will be dealt with separately in another paper in this symposium.

Inhibition of sensory neuron apoptosis and prevention of loss by NT-3 administration following axotomy.

Following permanent transection of their peripheral axons, a proportion of adult rat dorsal root ganglion neurons undergo programmed cell death (apoptosis) over a period of months. The underlying causes of this neuron loss are unclear, but may involve the interruption of the supply of target-derived neurotrophic factors, the replacement of which could prevent this loss from occurring. To investigate whether the administration of neurotrophic factors can prevent the dorsal root ganglion neuron death in adults, a 1 mg/ml solution of ciliary neurotrophic factor or of NT-3 was applied via a silicon reservoir to the proximal stump after unilateral sciatic transection at mid-thigh level. The incidence of apoptotic neurons and neuronal loss in the L4 and L5 ganglia ipsilateral to sciatic nerve transection when compared with the contralateral ganglia was then measured 1 month later. This was assessed by examining serial sections of ganglia for neurons undergoing apoptosis and expressing the total counted as a percentage of the total number of neurons estimated using a stereological neuron counting technique. Our results show that NT-3 administration significantly reduced the incidence of apoptotic neurons and prevented neuron loss, while CNTF had no effect on either parameter.

Progressive multifocal leukoencephalopathy: unusual MRI findings and prolonged survival in a pregnant woman.

We report a biopsy-proven case of progressive multifocal leukoencephalopathy (PML) in a pregnant woman without obvious underlying immune disorder. MRI showed lesion enhancement and deep gray matter involvement, which are uncommon imaging patterns in PML. The clinical course in this case is also rather atypical, as the patient is alive 16 months after disease onset. The combination of these unusual characteristics in PML is unknown and indicates that the criteria for this disease may need to be redefined.

Generation of labeled probes by PCR.

Nucleic acid probes are an important tool in molecular diagnosis. To facilitate the detection of hybridized probes, they are labeled with a reporter molecule, which is usually a radioisotope. For diagnostic techniques carried out in a clinical laboratory, radioisotopes are hazardous and, thus, recently there is a move to use nonisotopic labels, such as biotin and digoxigenin. Nonisotopic probes also have the advantage of much better stability over time compared with isotopic probes that have limited half-lives.

Axonal damage revealed by accumulation of beta-APP in HIV-positive individuals without AIDS.

The presence of neuropsychological disturbances in HIV-positive, pre-symptomatic individuals is a controversial issue. Neuroimaging studies have not shown brain atrophy or hyperintensity in the white matter, whereas proton magnetic resonance spectroscopy has revealed some abnormality of cerebral biochemistry. Using an antibody to beta-amyloid precursor protein (beta-APP), we previously demonstrated frequent and widespread axonal changes in the brains of AIDS patients. In this study, we extended the use of beta-APP to asymptomatic patients in order to establish a possible morphological correlation with neuropsychological disorders. Brain samples from 29 patients were examined. Results showed bundles of beta-APP-positive axons in 8/29 cases (27%). The changes, seen in both superficial and deep white matter, were either focal or diffuse, could not be visualized by silver or ubiquitin stains, and did not coexist with any change in distribution or morphology of astrocytes and microglial cells. We conclude that in HIV-positive asymptomatic individuals, axonal changes: (a) may be related to the state of immune activation with consequent presence of toxic substances, including cytokines, observed in these patients; (b) may represent mild changes that could undergo repair, unless other pathological events, such as the supervening of the AIDS stage and the specific encephalitis, make them permanent.

Accumulation of beta-amyloid precursor protein in HIV encephalitis: relationship with neuropsychological abnormalities.

The pathogenesis of neuropsychological abnormalities in patients with human immunodeficiency virus type 1 (HIV-1) encephalitis is obscure because neurons are not the target of infection and severe neuronal loss occurs only late during the disease. Moreover, there is evidence indicating that HIV dementia is not a homogeneous entity and could partially reverse after treatment with zidovudine. The finding that impaired axonal flow, evidenced by beta-amyloid precursor protein immunoreactivity, could contribute to the neuropsychological deficits prompted the present study. Brains of patients with full-blown acquired immunodeficiency syndrome (AIDS) were studied and findings compared with those of normal and abnormal control subjects. The presence of HIV-1 DNA was investigated by nested polymerase chain reaction; axonal abnormalities were detected by beta-amyloid precursor protein, ubiquitin immunohistochemistry, and silver staining. Accumulation of beta-amyloid precursor protein was observed in all the HIV encephalitis brains studied; the appearance of the immunostaining varied from globular structures to bundles of parallel formations. In 2 AIDS brains without pathological abnormalities, only the latter pattern was detected. The brains with trauma were strongly reactive with beta-amyloid precursor protein antibody and the different reactivity within them correlated with posttrauma survival, only globular structures being detected in the older cases. No correlation was found between the different pattern of beta-amyloid precursor protein reactivity and dementia in AIDS patients. These results show that widespread axonal injury is a constant feature in AIDS brains and suggest that it could play a role in the pathogenesis of the neuropsychological abnormalities of these patients.

Circular forms of unintegrated human immunodeficiency virus type 1 DNA and high levels of viral protein expression: association with dementia and multinucleated giant cells in the brains of patients with AIDS.

Thirty-one histologically abnormal brains from patients with AIDS were studied in order to establish the relationship between multinucleated giant cells, viral protein expression, the various forms of human immunodeficiency virus type 1 (HIV-1) DNA, and clinical evidence of dementia. Unintegrated HIV-1 DNA of 2 to 8 kb was found in 22 of the 31 brains. Multinucleated giant cells without any other pathology were found in 14 cases; unintegrated 1-long terminal repeat (1-LTR) circular forms of HIV-1 DNA and strongly positive immunohistochemistry for gp41 and p24 were found in most of these brains. Most of these patients had a clinical diagnosis of HIV-1-associated dementia and cerebral atrophy. In all the other brains studied, 1-LTR circles were absent and immunohistochemistry for gp41 and p24 was usually negative. Very few of these patients had a clinical diagnosis of dementia. Sequence comparison of the LTR region from integrated HIV-1 DNA with that from unintegrated 1-LTR circular forms of HIV-1 DNA in 12 cases showed no significant differences. A further comparison of these brain-derived LTR sequences with LTR sequences derived directly from lymphoid tissue also showed strong sequence conservation. The V3 loop of the virus from the brain was sequenced in 6 cases and had a non-syncytium inducing-macrophage-tropic genotype. Our results show that (i) although unintegrated HIV-1 DNA was present in most brains from patients with AIDS, molecular evidence of high levels of viral replication was associated with the presence of multinucleated giant cells and dementia, and that (ii) the HIV-1 LTR is not a determinant of neurotropism. These observations suggest that replication of HIV-1 and not just the presence of HIV-1 DNA within giant cells makes the important contribution to central nervous system damage.

Folliculo-stellate cells of human pituitary adenomas: immunohistochemical study of the monocyte/macrophage phenotype expression.

Folliculo-stellate cells (FS) represent a small percentage of anterior pituitary elements of still undetermined embryological origin. They are sparse among endocrine pituitary cells and are characterized by the lack of secretory granules and by the presence of few branching processes inserted between hormone-secreting cells. Although FS cell role is still under discussion, recent reports showed that they produce monocyte-derived cytokines able to influence the hormone production and modulate the immunoendocrine connections. In this study we applied three monocyte-macrophage markers (HAM56, KP1, HLA-DR) to 15 pituitary adenomas in order to ascertain whether FS cells belong to the macrophage lineage. In this case FS cells could be considered the resident macrophages of the pituitary. FS cells were identified according to the reactivity to S-100, GFAP and vimentin. We confirm that S-100 represents the most useful marker for these cells that were detected scattered between tumor cells in more than half of the adenomas. GFAP stained only a percentage of FS cells, while vimentin recognized in addition to stellate cells endothelia, perivascular and infiltrating macrophages. We were unable to detect the expression of the macrophage markers on S-100 and GFAP reactive cells. Indeed, HAM56, KP1 and HLA-DR-positive cells were mostly round, small size and located in the perivascular and septal positions where FS cells were never detected. Lack of expression of monocyte-macrophage lineage markers by FS cells in pituitary adenomas suggests their preferential neuroectodermal origin. However, further studies on normal human pituitary will be needed before ruling out a possible role for FS cells as resident pituitary macrophages.

Early HIV-1 infection of the central nervous system.

There is a consensus of opinion that central nervous system (CNS) involvement takes place in a large proportion of patients with the acquired immune deficiency syndrome (AIDS). However, uncertainty still remains about how often and how early the CNS is infected during the early asymptomatic stage as some researchers still believe that low copy of human immunodeficiency virus type 1 (HIV-1) identified in the brains using polymerase chain reaction (PCR) represents HIV harboured in the infected cells trapped in cerebral blood vessels. In this review, the neurological abnormalities in HIV-1 positive pre-AIDS individuals are discussed from three points of view: neuropsychiatric and neurophysiological, involvement of cerebrospinal fluid (CSF) and brain pathology. In particular, our investigations of the brains of asymptomatic individuals have demonstrated that HIV-1 DNA was present in about half (17/36) of brains studied (copy numbers of HIV-1 DNA were detected and the possibility of contamination from the blood was calculated and excluded). Astro- (34/36) and micro- (31/36) gliosis and meningitis (11/36) were found. Immune activation, revealed by elevated expression of major histocompatibility complex (MHC) class II antigens, was previously demonstrated in the brains of patients with AIDS and was also present before the development of AIDS. Furthermore, demonstration of highly expressed cytokines (tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, 4, 6) possibly explains the neuropathological changes and neuronal damage (confirmed by the demonstration of apoptotic neurons by in situ end labelling) seen in these brains. We conclude that HIV-1 is present in the brains of HIV-1 infected individuals at early stages of the infection and that HIV-1 induces brain damage in a direct as well as indirect way. This is a worrying conclusion which makes it mandatory to reconsider the time at which treatment must be applied in HIV-1 infection.

Detection of paraneoplastic anti-neuronal autoantibodies on paraffin-embedded tissues.

Anti-neuron-specific autoantibodies are widely recognised as useful, though non-specific, diagnostic markers of paraneoplastic neurological disorders. However, controversies on the best way to detect these autoantibodies have recently arisen, and the use of different procedures for their detection by different laboratories has made results difficult to compare. The aim of this study was to adapt the existing immunohistochemical techniques used for the detection of anit-neuron autoantibodies to improve their visualisation and to facilitate a wide application of these procedures. Sera and cerebrospinal fluid (CSF) were obtained from 15 patients known to carry paraneoplastic anti-neuronal autoantibodies; in addition, one serum with "atypical" anti-neuron autoantibody and 18 control sera were studied. Paraformaldehyde-fixed, paraffin-embedded rat nervous tissue and formalin-fixed, paraffin-embedded human nervous tissue treated in a microwave oven were used as substrate; the reactions were developed by immunoperoxidase methods. At the dilutions used for diagnostic purposes, all the sera and CSFs showed staining whose intensity and specificity was comparable to that obtained using frozen tissue; the end-point dilutions were, however, reduced. The atypical pattern of staining of one serum was confirmed and better emphasised using these procedures; all control sera and CSFs were negative. The morphology was improved by the use of paraffin-embedded tissues; moreover, the results obtained are permanent because of peroxidase staining, which makes it possible to use them as standards for further investigations and for comparison between different laboratories. The convenience of using paraffin-embedded material could facilitate a wide application of these procedures in clinical neurology.

HIV-1 DNA in brains in AIDS and pre-AIDS: correlation with the stage of disease.

Seventeen asymptomatic individuals positive for human immunodeficiency virus type 1 (HIV-1) and 16 patients with acquired immunodeficiency syndrome (AIDS), all with polymerase chain reaction evidence of HIV-1 DNA, were selected for quantitative analysis to correlate the levels of HIV-1 DNA in brain tissue with the stage of infection. The AIDS patients either were clinically asymptomatic or presented various abnormalities. Neuropathological lesions were assessed by morphological and immunohistochemical methods. To determine the level of HIV-1 DNA, semiquantitative nested polymerase chain reaction was applied using a digoxigenin-labeled primer and chemiluminescence. Serial dilutions of standard HIV DNA were run in parallel with brain DNA samples. Among the 16 AIDS brains studied, 9 showed changes characteristic of HIV encephalitis/leukoencephalopathy while 1 showed focal pontine leukoencephalopathy and 6 showed no obvious neuropathological lesions. Abnormalities in pre-AIDS individuals included meningitis, microgliosis, and astrogliosis. Copy numbers of HIV-1 DNA in the brains of AIDS patients were higher than those in asymptomatic individuals (median, 135 vs 45 copies/150,000 cells). However, there was some degree of overlapping between the two groups, with some AIDS patients showing low figures while 3 asymptomatic individuals had high copy numbers. This suggests that the use of HIV-1 DNA load in the central nervous system as an indicator of progression of the disease should be restricted to large series and not single patients.