TGF-beta1 and TGF-beta2 expression after traumatic human spinal cord injury.

STUDY DESIGN: Immunohistochemical investigation in control and lesioned human spinal cords. OBJECTIVES: To assess the spatial and temporal expression patterns of transforming growth factor-beta1 and -beta2 (TGF-beta1 and TGF-beta2) in the human spinal cord after traumatic injury. SETTING: Germany, Aachen, Aachen University Hospital. METHODS: Sections from human spinal cords from 4 control patients and from 14 patients who died at different time points after traumatic spinal cord injury (SCI) were investigated immunohistochemically. RESULTS: In control cases, TGF-beta1 was confined to occasional blood vessels, intravascular monocytes and some motoneurons, whereas TGF-beta2 was only found in intravascular monocytes. After traumatic SCI, TGF-beta1 immunoreactivity was dramatically upregulated by 2 days after injury (the earliest survival time investigated) and was detected within neurons, astrocytes and invading macrophages. The staining was most intense over the first weeks after injury but gradually declined by 1 year. TGF-beta2 immunoreactivity was first detected 24 days after injury. It was located in macrophages and astrocytes and remained elevated for up to 1 year. In white matter tracts undergoing Wallerian degeneration, there was no induction of either isoform. CONCLUSION: The early induction of TGF-beta1 at the point of SCI suggests a role in the acute inflammatory response and formation of the glial scar, while the later induction of TGF-beta2 may indicate a role in the maintenance of the scar. Neither of these TGF-beta isoforms appears to contribute to the astrocytic scar formation in nerve fibre tracts undergoing Wallerian degeneration.

Sequential loss of myelin proteins during Wallerian degeneration in the human spinal cord.

Axons undergo Wallerian degeneration (WD) distal to a point of injury. In the lesioned PNS, WD may be followed by successful axonal regeneration and functional recovery. However, in the lesioned mammalian CNS, there is no significant axonal regeneration. Myelin-associated proteins (MAPs) have been shown to play significant roles in preventing axonal regeneration in the CNS. Since relatively little is known about such events in human CNS pathologies, we performed an immunohistochemical investigation on the temporal changes of four MAPs during WD in post-mortem spinal cords of 22 patients who died 2 days to 30 years after either cerebral infarction or traumatic spinal cord injury. In contrast to experimental studies in rats, the loss of myelin sheaths is greatly delayed in humans and continues slowly over a number of years. However, in agreement with animal data, a sequential loss of myelin proteins was found which was dependent on their location within the myelin sheath. Myelin proteins situated on the peri-axonal membrane were the first to be lost, the time course correlating with the loss of axonal markers. Proteins located within compact myelin or on the outer myelin membrane were still detectable 3 years after injury in degenerating fibre tracts, long after the disappearance of the corresponding axons. The persistence of axon growth-inhibitory proteins such as NOGO-A in degenerating nerve fibre tracts may contribute to the maintenance of an environment that is hostile to axon regeneration, long after the initial injury. The present data highlight the importance of correlating the well documented, lesion-induced changes that take place in controlled laboratory investigations with those that take place in the clinical domain.

Neuropathology: the foundation for new treatments in spinal cord injury.

The first step essential in the search for a cure of human spinal cord injury (SCI) is to appreciate the complexity of the disorder. In this regard, it is not only the loss of ambulation but the sensory and autonomic changes that are equally important in recovery. In addition, there are the serious social emotional psychological and lifestyle effects of SCI which should also be taken into account. It is also true that no two SCI lesions are alike as each is the result of a SCI unique to that individual. Clinically of utmost importance is the segmental level of injury and whether it is complete, incomplete or discomplete (loss of all neurological functions below the injury but with physiological or anatomical continuity of Central nervous system tracts across the lesion). We are not concerned here with primary and secondary prevention or methods designed to limit the severity of the lesion after the event, important as they are, but with the requirements for a cure. Clearly, the greater the number of nerve fibers that can be preserved in the acute stage, the better will be the end result. Our focus at present is on the end-stage lesion with the aim of showing that a cure for SCI will depend upon establishing functionally useful central axonal regeneration and reestablishing physiological reconnections. Existing experimental methods are based on stimulating axonal regeneration by neutralizing inhibitory factors, adding positive trophisms and creating a permissive environment. Better results are obtained by bridging the gap with grafts of peripheral nerves or transplants of Schwann cells and genetically engineered fibroblasts. Recently, the potential for stem cells to enhance this process has created great interest. This is because of the ability of pluripotential cells to differentiate into neural tissue. A cure based on the physiopathology of SCI requires pyramidal, extrapyramidal, sensory, cerebellar and autonomic pathways to be regenerated with their appropriate neurotransmitters restored and reflexes integrated physiologically and in synchrony. In human SCI, there is a very long distance anatomically for axonal regrowth to occur in order to reach their relevant nuclei. This is because of continuing Wallerian degeneration. It also presumes that the target neurons are intact and that there has been no transneuronal degeneration above or below the lesion. Alternatively, in place of regenerated long axons, a multisynaptic pathway may be constructed from stem cells that have developed into neurons. Whether such a pathway would restore useful neurological functions is unknown. At present, the transplant and grafting research teams are exploring these possibilities in experimental animals. Moderate success in gaining axonal regeneration has been reported; however, it must be appreciated that the human lesion differs considerably from that of the experimental animal. In order to be successful, the neuropathology and neurophysiology of human SCI must be taken into account. The purpose of this review is to place the requirements for a cure, using stem cells, within the context of the neuropathology of human SCI.

Prevalence of cerebral vascular amyloid-beta deposition and stroke in an aging Australian population: a postmortem study.

Cerebral amyloid angiopathy (CAA) is a putative risk factor for lobar cerebral haemorrhage and infarction in the elderly. However, the prevalence of stroke in a population with CAA is not known. Amyloid-beta immunohistochemistry was used to assess CAA prevalence as a function of age, and the relationship between CAA and stroke in 100 individuals aged 50-91 years who died unexpectedly and had a Coroner's postmortem. Blocks were taken from several cortical areas and from areas of infarction or haemorrhage. Parenchymal Abeta was first found in the 6th decade, whereas vascular Abeta did not appear until the 7th decade. The prevalence of both vascular and parenchymal Abeta increased with age to a maximum in the 9th decade. The age at onset of vascular Abeta deposition was similar to that in an English study of CAA but a decade later than in Japanese studies. There was no association between the presence of vascular Abeta and cerebral haemorrhage or infarction. The findings indicate differences in the time-course of vascular and parenchymal Abeta deposition with age, as well as racial differences. The lack of association between vascular Abeta and cerebral haemorrhage or infarction indicates that, in the present population, CAA was usually asymptomatic.

Retropulsion of intervertebral discs associated with traumatic hyperextension of the cervical spine and absence of vertebral fracture: an uncommon mechanism of spinal cord injury.

STUDY DESIGN: Case report of a 68-year-old male who sustained cervical trauma following a bodysurfing accident. OBJECTIVE: To describe the pathology of a relatively uncommon mechanism of injury involving extradural cord compression associated with traumatic disc protrusion and herniation, following a cervical hyperextension injury in which there was no vertebral fracture or residual subluxation. SETTING: Department of Neuropathology, Royal Perth Hospital, West Australia. METHOD: Postmortem pathology report. RESULTS: Evidence of multiple ruptures of anterior longitudinal ligament with posterior intervertebral disc herniation and three discrete foci of central cord hemorrhage. CONCLUSION: Observations are consistent with cervical extension injury and an injury vector that involves intense axial loading sufficient to cause multiple disc failures, disc herniation and retropulsion leading to extradural disc compression and cord hemorrhage.

The mRNA of the NR1 subtype of glutamate receptor in Alzheimer's disease.

Glutamate has been implicated in the pathogenesis of Alzheimer's disease (AD). Controversial data exists regarding changes in the N-methyl-D-aspartate (NMDA) receptor complex in AD. We wished to elucidate the hypothesis that the NMDA receptor system is involved in the pathogenesis of AD using a gene expression approach targeting the mRNA of the universal subtype of the NMDA receptor NR1. This was performed using in situ hybridization and antisense 35S-labelled oligonucleotides on brain tissue collected at post-mortem. Relative mRNA expression was measured in standardised optical density units (OD units) using videodensitometry without knowledge of the diagnosis. The study population consisted of AD (n = 6) and neurodegenerative non-Alzheimer controls (non-AD, n = 14). Gene expression was measured in the frontal lobe, superior temporal gyrus and three areas within the hippocampus. We have observed no significant differences in the relative mRNA expression of the NR1 subtype of glutamate receptor in the following regions: frontal lobe AD = 60.7 +/- 14.1 OD units mRNA (x +/- 1SE) vs 52.6 +/- 1 in non-AD (Mann-Whitney test, p = 0.477); the superior temporal gyrus: AD = 53.3 +/- 13.9 vs 38.2 +/- 7 (p = 0.37); the CA1 region: AD = 37.8 +/- 7.75 vs 81.5 +/- 25.7 (p = 0.66); subiculum AD = 46.7 +/- 11.0 vs 105 +/- 43.3 (p = 0.82); parahippocampal gyrus AD = 36.6 +/- 9.3 vs 81.7 +/- 40.6 (p = 0.90). There were trends to a reduction in NR1 mRNA in the hippocampus and increased NR1 within the frontal and superficial temporal gyrus which were not significant. There was variation within and between all patients with and without AD in the magnitude of NR1 expression in all anatomical regions studied. The findings suggest heterogeneity in the involvement of the post-synaptic glutamatergic system in AD.

A new PRNP mutation (G131V) associated with Gerstmann-Sträussler-Scheinker disease.

BACKGROUND: Gerstmann-Sträussler-Scheinker disease is a rare form of prion disease. OBJECTIVE: To determine the prion mutation in a 51-year-old man without a family history of neurologic disease who died from Gerstmann-Sträussler-Scheinker disease. PATIENT AND METHODS: The patient was a 51-year-old man who died after a 9-year illness characterized by dementia and eventually ataxia. Neuropathologic studies were performed, the results of which revealed abundant prion protein-immunopositive amyloid plaques in the cerebellum without spongiform degeneration. RESULTS: Genetic analysis of the prion protein gene showed a novel mutation at codon 131 that caused a valine-for-glycine substitution (G131V) and homozygosity at codon 129 (129M). Proteinase K-resistant prion protein was detected by Western blot analysis. CONCLUSIONS: This is the first mutation described in the short, antiparallel beta-sheet domain of the prion protein. This report highlights the importance of genetic analysis of patients with atypical dementia even in the absence of a family history.

Severe gamma-sarcoglycanopathy caused by a novel missense mutation and a large deletion.

We report two siblings with a relatively severe limb-girdle muscular dystrophy. The elder sister presented at 8 years of age with inability to climb and abnormal gait. At 12 years she was barely ambulant. Her sister followed a similar course. Serum creatine kinase was 8500-10000 IU (N 25-200) in the elder sister and 17000-19000 IU in the younger sister. Muscle biopsy of the elder sister at 8 years showed chronic myopathic changes with loss of muscle fibres, active necrosis and regeneration. Immunocytochemistry demonstrated normal spectrin and dystrophin, reduced alpha-sarcoglycan and absent gamma-sarcoglycan--indicating a gamma-sarcoglycanopathy. Haplotype analysis for the markers D13S115, D13S232, D13S292, D13S787, D13S1243 and D13S283 internal to and flanking the gamma-sarcoglycan gene showed the affected sisters shared haplotypes, indicating it was possible they were suffering from a gamma-sarcoglycanopathy. Non-inheritance of paternal alleles for D13S232, D13S292 and D13S1243 suggested the inheritance of a deletion, which was confirmed by FISH, using a genomic probe from the gamma-sarcoglycan gene. The gamma-sarcoglycan cDNA was amplified by reverse transcriptase PCR from the muscle biopsy of the elder sister and sequenced. A missense mutation changing codon 69 from GGC glycine to CGC arginine was identified. HhaI digestion of exon 3 genomic PCR products showed the two affected sisters were hemizygous for the mutation, while the mother and grandmother were heterozygotes. The mutation, identified by SSCP analysis, was not observed in 116 unrelated, unaffected individuals. Previously, only two other missense mutations, the Cys283Tyr missense mutation in Gypsies and the Leu193Ser mutation in a Dutch family, have been described in the gamma-sarcoglycan gene. The fact that the affected individuals in the current and Gypsy families are gamma-sarcoglycan negative may indicate that codons 69 and 283 are important in gamma-sarcoglycan function.

Amyloid precursor protein gene isoforms in Alzheimer's disease and other neurodegenerative disorders.

Differential expression of the amyloid precursor protein gene (APP) may be important in the development of amyloidosis in Alzheimer's disease (AD) and experimentally in the brain's response to injury. Controversial data suggests that APP isoforms containing the Kunitz protease inhibitor isoform (APP KPI+) are over expressed in the brains of patients with AD when compared to the non-Kunitz protease inhibitor containing isoforms (APP KPI-). We have investigated this hypothesis using a quantitative analysis of gene expression on brain tissue collected at post-mortem. In situ hybridization has been used with synthetic oligonucleotide probes labelled with 35S to detect the two principal splice variants of APP: APP 695 (KPI-) and APP 751 (KPI+). A prospective brain bank of frozen brain specimens has been established and includes pathologically proven AD (n=15) and other neurodegenerative disorders as controls (n=18). The controls consist of frontal lobe atrophy (n=4), Huntington's disease (n=5), Parkinson's disease (n=4), motor neuron disease (n=2), multi-infarct dementia (n=1), multisystem atrophy (n=1), and subacute sclerosing panencephalitis (n=1). We have observed no significant differences in the expression of APP 695 KPI- mRNA in frontal lobe: 17.49+/-3.26 optical density (OD) units of mRNA expression in AD vs. 16.13+/-1.76 OD units mRNA in controls (P=0.80, linear regression); or temporal lobe: 14.73+/-2.96 in AD vs. 16.49+/-2.15 in controls (P=0.55). No significant differences have been found in APP 751 KPI+ in frontal lobe: 12.86+/-2.98 in AD vs. 13.70+/-2.88 in controls (P=0.97); and temporal lobe: 13.31+/-4.93 in AD vs. 11.07+/-1.99 in controls (P=0. 65). Analysis of the ratios of APP 751 KPI+ OD units of mRNA to APP 695 KPI- mRNA revealed a trend to an increased ratio which did not reach statistical significance: frontal lobe APP 751 KPI+/APP 695 KPI- 1.92+/-1.04 in AD vs. 0.86+/-0.17 in controls (P=0.54); temporal lobe 2.54+/-1.59 in AD vs. 0.96+/-0.11 controls (P=0.34). Our data has not revealed differential expression of APP mRNA isoforms in AD and supports the hypothesis that post-translational events in APP metabolism are important in amyloidogenesis and the pathogenesis of AD.

Butyrycholinesterase K variant and Alzheimer's disease.

This study attempted to corroborate findings on the association between butyrylcholinesterase K variant and Alzheimer's disease. This was performed on an autopsy-confirmed series of patients with Alzheimer's disease and controls. The butyrylcholinesterase K variant was found to be of increased allele frequency in patients with sporadic Alzheimer's disease. When related to APOE epsilon4 typing the association was specific but not sensitive for the diagnosis of Alzheimer's disease.

Novel mutation in the myelin protein zero gene in a family with intermediate hereditary motor and sensory neuropathy.

OBJECTIVES: To determine the molecular basis for autosomal dominant intermediate hereditary motor and sensory neuropathy (HMSN) in a four generation family. The gene defects in families with intermediate HMSN are not known, but it has been suggested that most have X linked HMSN. METHODS: All participating family members were examined clinically. Genomic DNA was obtained from 10 affected and seven unaffected members. Linkage analysis for the known HMSN loci was first performed. Mutations in the peripheral myelin protein zero gene (PMP0) were sought in two affected members, using one unaffected member for comparison, by amplification of the six exons of the gene followed by single strand conformation polymorphism (SSCP) analysis, dideoxy fingerprinting (ddF), and sequencing. Subsequently, the mutation was screened for in all affected and unaffected members in the family using Alu I digestion and in 100 unrelated control subjects using "snap back" SSCP analysis. Sequencing of cDNA from a sural nerve biopsy from an affected member was also performed. RESULTS: The clinical phenotype was of variable severity, with motor nerve conduction velocities in the intermediate range. Linkage to PMP0 was demonstrated. Analysis of genomic DNA and cDNA for PMP0 identified a novel codon 35 GAC to TAC mutation. The mutation produces an inferred amino acid change of aspartate to tyrosine at codon six of the processed protein (Asp6Tyr) in the extracellular domain and was present in all affected family members but not in 100 unrelated controls. CONCLUSIONS: The present findings further extend the range of phenotypes associated with PMP0 mutations and indicate that families with "intermediate" HMSN need not necessarily be X-linked as previously suggested.

The contribution of molecular genetics in the diagnosis and management of neuromuscular disorders.

It is true that the recent advances in molecular genetics have generated a medical revolution. This is especially true for the inherited neuromuscular disorders. There have been many spectacular recent discoveries with new genes being found and their protein products identified. One of the most remarkable aspects of this progress is the nexus which has developed between the basic discovery and its clinical application. As soon as a new genetic mutation is reported, the information may be used immediately to establish the molecular diagnosis for that disorder in any part of the world which has a DNA laboratory. This is done by using primers derived from the published DNA sequences using the polymerase chain reaction (PCR). This development is of immense value for the clinician as it provides an exact molecular diagnosis often with prognostic information and the test results can be used for genetic counselling and prenatal diagnosis. One of the unexpected outcomes of this work has been the surprising variation which has been shown to exist between genotype and phenotype. Previously, one mutation was believed to be responsible for one clinical disorder. However, it is now known that one genotype may be responsible for a variety of phenotypes and vice versa. In the field of neuromuscular disorders the most notable advances have occurred for Duchenne muscular dystrophy and the related dystrophinopathies and for the group of limb girdle muscular dystrophies, especially the subgroup of sarcoglycanopathies. Other areas are the congenital myopathies, the 'channel-opathies' and the mitochondrial cytopathies. In this review the most commonly used molecular genetic and immunocytochemical methods using antibodies to the protein product are outlined together with the principles of their application in the neuromuscular clinic. Included are the provisos and pitfalls which need to be kept in mind in the interpretation of DNA results for each patient.

A review of the neuropathology of human spinal cord injury with emphasis on special features.

A judicious understanding of the basic neuropathology of spinal cord injuries (SCI) is essential knowledge for the clinician responsible for SCI management. An appreciation of the nature of human SCI is also necessary for the neuroscientist searching for a cure. The neuropathology of human SCI described here is derived from the study of 564 cases of spinal cord trauma held in a tissue bank and database of the Department of Neuropathology, Royal Perth Hospital in Australia. The main features of SCI neuropathology are reviewed and special aspects such as early axonal lesions, traumatic demyelination-remyelination, and quantification of white matter tracts are reported in more detail. One of the remarkable outcomes of this work is the finding that the majority of SCI patients have a proportion of spinal cord white matter maintained across the level of the lesion, an observation that has important therapeutic implications.

Dorsal root ganglion injuries in 109 blunt trauma fatalities.

In an autopsy study of 180 cervical spines, 109 were from victims of fatal blunt injury. A search was made for injuries to the dorsal root ganglia. The whole cervical spines, from the skull base to T1, were formalin fixed, deep frozen and sagittally sectioned on a specially adapted band saw in 2.5 mm thick slices. In 15 of the 109 fatally injured individuals, 44 examples of interstitial haemorrhage into a dorsal root ganglion (DRG) were found. This was sometimes accompanied by neural tissue disruption, visible only on histological study. The intraneural DRG haemorrhage was found in 13.8 per cent of all the injured individuals, but this prevalence rose to 34.5 per cent when only those (29) individuals surviving the injury between 2 h and 7 days were considered. The possible relevance of such injuries, in survivors of injury, to acute and chronic pain syndromes is discussed.

Dominantly inherited proximal myotonic myopathy and leukoencephalopathy in a family with an incidental CLCN1 mutation.

A two generation family of Greek origin with mild myotonia, predominantly proximal muscle weakness, and cataracts compatible with the syndrome of proximal myotonic myopathy, is reported. In addition, brain MRI showed a diffuse leukoencephalopathy in the propositus. Molecular genetic studies showed the R894X mutation in exon 23 of the muscle chloride channel gene in the propositus but in only one of her two clinically affected offspring, indicating that it is not the mutation causing disease in this family.

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Age-related congophilic inclusions in the brains of apolipoprotein E-deficient mice.

The hippocampal region of apolipoprotein E-deficient mice of varying ages was examined for any morphological changes by light and electron microscopy. Unusual periodic acid-Schiff-positive granules were seen in the hippocampal area of these animals as early as the fourth week of life and their numbers increased gradually with age. These granules were never found in control C57BL/6J (B6) mice before six months-of-age and their numbers were invariable low. They were strongly congophilic when stained with a modified Congo Red technique and reacted with a monoclonal antibody specific to amino acids 17-24 and 35-43 of the beta-amyloid peptide. The immunostaining of these granules with the beta-amyloid peptide was lost after specific adsorption with the appropriate synthetic peptide. These granules were identified ultrastructurally as non-membrane-bound fibrillogranular material in the cytoplasm of protoplasmic astrocytes. The data indicate that an amyloid-like protein accumulates in the protoplasmic astrocytes of the hippocampus of apolipoprotein E-deficient mice, especially in the brains of old animals.