Changes in substance P and NK1 receptor immunohistochemistry following human spinal cord injury.

STUDY DESIGN: An immunohistological assessment of substance P (SP), its NK1 receptor and claudin-5 in human spinal cord injury (SCI) tissue. OBJECTIVE: To determine whether SP and NK1 receptor immunoreactivity are altered following human traumatic SCI. SETTING: Australia. SUMMARY OF BACKGROUND DATA: SP has been implicated in the development of neurogenic inflammation and subsequent edema development following both traumatic brain injury and ischemic stroke. In these conditions, inhibition of its NK1 receptor has been shown to be neuroprotective as reflected in a reduction of edema and improved functional outcome. However, the role of SP following human SCI has not yet been assessed. METHODS: Archived human SCI tissue was grouped according to survival times: control (no injury; n=5); immediate (death within an hour of the incident; n=6); 2-5 h (n=3); 3 days (n=5); 1 week (n=3); and 3-4 weeks (n=6). Sections were assessed for SP, its NK1 receptor and claudin-5 using immunohistochemical techniques. RESULTS: Following SCI, dorsal horn SP immunoreactivity demonstrated a profound decrease compared with control tissue, indicating the loss of SP with SCI. A marked increase in perivascular NK1 staining was demonstrated after SCI compared with control levels. No obvious change in claudin-5 immunoreactivity was present immediately following injury, however, by 1 week post-SCI, decreased levels were noted. CONCLUSION: This study demonstrates that severe acute traumatic human SCI results in decreased SP and an immediate increase in NK1 receptor immunoreactivity, suggesting that there is a neurogenic inflammatory component following human SCI.

A Rosenthal fiber encephalomyelopathy resembling Alexander's disease in 3 sheep.

We report an encephalomyelopathy in three 18-month-old Merino sheep with features of adult-onset Alexander's disease (AD), a human primary astrocytic disorder. The signature histologic finding was the presence of numerous hypereosinophilic, intra-astrocytic inclusions (Rosenthal fibers), mainly in perivascular, subpial, and subependymal sites, especially in the caudal brain stem and spinal cord. Although AD usually results from mutations in the glial fibrillary acidic protein (GFAP) gene, no such mutation was detected in these sheep. However, the annual clinical presentation of this disorder in a few sheep in the affected flock is suggestive of a familial pattern of occurrence.

Retinal photoreceptor damage produced in guinea pigs by tunicamycin.

Corynetoxins, members of the tunicamycin group of antibiotics, produce a severe and frequently fatal neurological disorder in ruminant livestock, and guinea pigs are a useful model to study the pathology and pathogenesis of this disease. The aim of this study was to determine whether tunicamycin produced ocular damage in this species, which could have pharmacotherapeutic and diagnostic value. Four 8-week-old guinea pigs were treated with tunicamycin, and two control animals were given the drug vehicle only. Guinea pigs were injected subcutaneously with 400 µg/kg of tunicamycin, in dimethyl sulphoxide, and killed 48 h post-injection. The eyes were then examined by light microscopy. Immunohistochemistry for rhodopsin was also performed. The principal pathological finding was marked retinal photoreceptor damage, which was characterised by disruption and disorganisation of rods, sometimes progressing to necrosis and separation of the outer segment. The cytoplasm of some rods was focally distended by accumulated, proteinaceous material. Rhodopsin immunopositivity in injured rods was markedly diminished and associated with shrinkage and shortening of the injured rod's outer segment. Ocular pathology, in the form of reproducible and extensive retinal photoreceptor damage, was found in guinea pigs given tunicamycin, extending the range of species found to be susceptible to this toxic injury. The guinea pig could prove to be a good animal model to test potential therapeutic interventions, and as brain lesions are often minimal and liver pathology non-specific in intoxicated ruminants, any spontaneously arising ophthalmic injury found in these species could be diagnostically useful.

Characterization of an 'Amyloid Only' Transgenic (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax) Mouse Model of Alzheimer's Disease.

The spatiotemporal pattern of cerebral amyloid deposition, detectable as light microscopically recognizable aggregates in an 'amyloid only' transgenic mouse model of Alzheimer's disease, B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax, is reported for the first time in this strain. Monoclonal and polyclonal antibodies were used to detect amyloid deposition immunohistochemically in brains collected from these mice at 3-12 months of age. Amyloid aggregates (20-200 µm) were first found in serial, whole coronal sections of brain at 4 months of age and these increased progressively, plateauing at 11-12 months. They were most abundant in the cerebral cortices, hippocampus, olfactory bulbs, some white matter tracts and the cerebellar molecular layer; no amyloid aggregates were found in the midbrain, brainstem or spinal cord, or in an equivalent number of brains from wild-type mice. Since the parahippocampal gyrus is severely damaged early in the clinical course of human Alzheimer's disease, amyloid aggregates were also assessed in this brain region and a similar temporal course of amyloid deposition was observed. Moreover, in this gyrus, the amount of aggregated amyloid showed no significant difference between left- and right-sided gyri. However, the polyclonal antibody detected a significantly greater amyloid burden than the monoclonal antibody at 3-10 months of age and the reverse was seen at 11-12 months of age. The pattern of amyloid deposition in the parahippocampal gyrus also resembled that found in the entire brain over time, when the latter was quantified by the colour deconvolution method, suggesting that this gyrus is a good marker for more widely distributed cerebral amyloid deposition. This neuropathological characterization will permit better use of the B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax transgenic mouse strain in future studies of Alzheimer's disease pathogenesis, prevention and treatment.

Increased substance P immunoreactivity and edema formation following reversible ischemic stroke.

Previous results from our laboratory have shown that neurogenic inflammation is associated with edema formation after traumatic brain injury (TBI). This neurogenic inflammation was characterized by increased substance P (SP) immunoreactivity and could be attenuated with administration of SP antagonists with a resultant decrease in edema formation. Few studies have examined whether neurogenic inflammation, as identified by increased SP immunoreactivity, occurs after stroke and its potential role in edema formation. The present study examines SP immunoreactivity and edema formation following stroke. Experimental stroke was induced in halothane anaesthetized male Sprague-Dawley rats using a reversible thread model of middle cerebral artery occlusion. Increased SP immunoreactivity at 24 hours relative to the non-infarcted hemisphere was observed in perivascular, neuronal, and glial tissue, and within the penumbra of the infarcted hemisphere. It was not as apparent in the infarct core. This increased SP immunoreactivity was associated with edema formation. We conclude that neurogenic inflammation, as reflected by increased SP immunoreactivity, occurs following experimental stroke, and that this may be associated with edema formation. As such, inhibition of neurogenic inflammation may represent a novel therapeutic target for the treatment of edema following reversible, ischemic stroke.

Paranodal pathology in Tangier disease with remitting-relapsing multifocal neuropathy.

Pathological studies of a sural nerve biopsy in a man with Tangier disease presenting as a remitting-relapsing multifocal neuropathy showed abnormalities in the paranodal regions, including lipid deposition (65%) and redundant myelin foldings, with various degrees of myelin splitting and vesiculation (43%) forming small tomacula and abnormal myelin terminal loops (4%). The internodal regions were normal in the majority of myelinated fibres. Abnormal lipid storage was also present in the Schwann cells of the majority of unmyelinated fibres (67%). The evidence suggests that the noncompacted myelin region of the paranode is a preferential site for lipid storage in the myelinated Schwann cell, and that the space-occupying effects of the cholesterol esters leads to paranodal malfunction and tomacula formation as the pathological basis for the multifocal relapsing-remitting clinical course.

Temporal Sequence of Autolysis in the Cerebellar Cortex of the Mouse.

This study examined the temporal sequence of post-mortem changes in the cerebellar cortical granular and Purkinje cell layers of mice kept at a constant ambient temperature for up to 4 weeks. Nuclei of granule cell microneurons became pyknotic early after death, increasing progressively until, by 7 days, widespread nuclear lysis resulted in marked cellular depletion of the granular layer. Purkinje cells were relatively unaltered until about 96 h post mortem, at which time there was shrinkage and multivacuolation of the amphophilic cytoplasm, nuclear hyperchromasia and, sometimes, a perinuclear clear space. By 7 days, Purkinje cells had hypereosinophilic cytoplasm and frequent nuclear pyknosis. By 2 weeks after death, Purkinje cells showed homogenization, the cytoplasm being uniformly eosinophilic, progressing to a 'ghost-like' appearance in which the cytoplasm had pale eosinophilic staining with indistinct cell boundaries, and nuclei often absent. The results of this study could assist in differentiating post-mortem autolysis from ante-mortem lesions in the cerebellar cortex and determining the post-mortem interval. Moreover, this information could be useful when interpreting brain lesions in valuable mice found dead unexpectedly during the course of biomedical experiments.

SUMO-1 marks subdomains within glial cytoplasmic inclusions of multiple system atrophy.

Conjugation of the small ubiquitin-like modifier, SUMO-1, to target proteins is linked to the regulation of multiple cellular pathways, including nucleocytoplasmic trafficking, cell cycle progression, the ubiquitin-proteasome system and apoptosis. Recently, the accumulation of SUMOylated proteins in pathological neuronal intranuclear aggregates has been found in several neurodegenerative diseases. The aim of our study was to examine SUMO-1 in the alpha-synucleinopathy diseases, Multiple System Atrophy (MSA) and Dementia with Lewy Bodies (DLB). We conducted anti-SUMO-1 immunostaining of fixed brain tissue sections and smears of unfixed brain tissue homogenates of DLB and MSA cases. We found that oligodendroglial cytoplasmic inclusions, the alpha-synuclein-positive cytoplasmic aggregates that characterize MSA, exhibit robust punctate SUMO-1 immunostaining, marking discrete submicron-sized subdomains within the inclusion bodies. Lewy bodies in smears of DLB tissue homogenates showed similar SUMO-1-positive structures, although these were not detected in fixed tissue. In cell culture experiments, we found that the nuclear and perinuclear accumulation of SUMO-1 aggregates could be induced in glioma cells by chemical inhibition of proteasomal protein degradation.

Alpha B-crystallin is a major component of glial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of alpha-synuclein filaments. AlphaB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of alphaB-crystallin in GCIs in MSA brains. We also examined the influence of alphaB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed alphaB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that alphaB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for alphaB-crystallin. Proteasome-inhibited cells transfected with GFP-tagged alpha-synuclein resulted in ubiquitin- and alphaB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that alphaB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

Age-related loss of dorsal vagal neurons in Parkinson's disease.

Older patients who die with Parkinson's disease (PD) have fewer pigmented neurons in the locus coeruleus and fewer substance P-containing neurons in mesopontine tegmental nuclei. We analyzed two other medullary nuclei, the dorsal vagal nucleus and the hypoglossal nucleus, in eight PD patients and six normal controls by counting neurons in serial Nissl stained sections to determine the relationship between age at death and cell loss in these nuclei. PD-related neurodegenerative changes (Lewy bodies and neuronal loss) were present only in the dorsal vagal nucleus (13,637 +/- 1,323 neurons in PD, 24,885 +/- 1,157 in normal controls). Cells in the intermediate rostrocaudal part of the nucleus were most severely affected. There was a significant correlation between loss of vagal neurons and age at death in PD patients. No age-related cell loss was present in the dorsal vagal nucleus of normal brains, or in the hypoglossal nucleus in either PD or normal brains. These results confirm that age-related cell death depends on whether or not there is coexistent PD.

Topography of axonal injury as defined by amyloid precursor protein and the sector scoring method in mild and severe closed head injury.

Axonal injury (AI), as defined by amyloid precursor protein (APP) positive axonal swellings, was recorded on a series of line diagrams of standard brain sections divided into 116 sectors to provide an Axonal Injury Sector Score (AISS) ranging from 0 to 116. This sector scoring method of recording axonal damage and providing a topographic overview of AI was applied to a series of 6 mild head injury cases [Glasgow Coma Scale (GCS) 13-15] and six severe head injury cases (GCS 3-8). The AISS ranged from 4 to 107 overall and varied from 4 to 88 in the mildly injured group and 76 to 107 in the severe head injury group, supporting the concept that there is a spectrum of AI in traumatic head injury and that the AISS is a measure of the extent of AI. APP immunostaining demonstrated positive axonal swellings 1.75 h after head injury and analysis of the pattern of AI in the mild and severe head injury groups showed that axons were more vulnerable than blood vessels and that the axons in the corpus callosum and fornices were the most vulnerable of all.

Axonal injury in falls.

Amyloid precursor protein (APP) immunocytochemistry was used as a marker for axonal injury (AI) in a series of 16 cases of head trauma associated with fatal falls. Nine cases were falls from not more than the person's own height (falls from < or = own height) and seven cases were falls from a distance greater than the person's own height (falls from > own height). AI was recorded on a series of line diagrams of standard brain sections divided into 116 sectors. AI around focal lesions (infarcts, hemorrhages, contusions) was distinguished from nonfocal axonal injury that was distant from any focal area of damage. The percentage of sectors showing focal AI provided the Focal Axonal Injury Score (FAIS) and the percentage showing nonfocal AI the Non-Focal Axonal Injury Score (NFAIS). The FAIS is a measure of secondary AI and the NFAIS of diffuse axonal injury (DAI). The percentage of sectors involved with AI (focal and nonfocal) provided the cumulative Axonal Injury Score (AIS). A semiquantitative grading system was also used to assess the severity of axonal injury in each sector and the sum of the grades from all sectors was expressed as a percentage to provide the Axonal Injury Severity Score (AISS). Widespread AI was present in all cases irrespective of the height of the fall. AI was present in the midbrain (94%), pons (94%), corpus callosum (100%), central grey matter (100%), and cerebral hemispheric white matter (94%). AIS ranged from 10 to 94 in falls from < or = own height (mean 73) and from 38 to 92 in falls from > own height (mean 82). AISS ranged from 6 to 95 in falls from < or = own height (mean 65) and 28 to 95 in falls from > own height (mean 72). There was no statistically significant difference in AIS or AISS between the two groups. The extent and severity of AI cannot be predicted from biomechanical data, such as the height of the fall, as the total AI in a given case is a variable mixture of Nonfocal AI (DAI) and Focal AI arising by secondary mechanisms, and APP immunostaining is unable to distinguish primary from secondary AI. However, the combination of the Hypoxic-Ischemic Score (HIS) defined as the percentage of sectors showing any hypoxic-ischemic damage ranging from neuronal "red cell change" to infarction in conjunction with the FAIS and NFAIS provided a measure of the relative contribution of primary and secondary AI in a given brain.

Upregulation of neuronal amyloid precursor protein (APP) and APP mRNA following magnesium sulphate (MgSO4) therapy in traumatic brain injury.

The aim of this study was to assess and quantitate topographically the effects of posttraumatic intravenous magnesium sulphate (MgSO4) on neuronal perikaryal APP antigen and messenger RNA (mRNA) expression in sheep brains 2 h after a controlled focal head impact. The percentage brain area with APP immunoreactive neuronal perikarya was 71, 56, 27.5 and 5.5%, respectively, in MgSO4-treated head-injured animals, head-injured animals without any treatment, MgSO4 treated nonimpacted animals, and nontreated nonimpacted control sheep. Although there was no statistically significant difference in APP immunoreactive neuronal perikarya in the MgSO4-treated HI group (mean 71%) compared to the HI group without any treatment (mean 56%), northern analysis showed that there was a 2.3-+/-0.2-fold increase in APP mRNA in the thalamus of treated impacted animals compared to untreated impacted animals (p < 0.005). However, MgSO4 treated nonimpacted control animals also showed a 1.6-+/-0.1-fold increase in APP mRNA compared to untreated nonimpacted controls (p < 0.005). MgSO4 therapy results in upregulation of neuronal APP mRNA and APP expression that is quantitatively greater following a focal head impact.

A head impact model of early axonal injury in the sheep.

Axonal injury (AI), one of the principal determinants of clinical outcome after head injury, may evolve over several hours after injury, raising the future possibility of therapeutic intervention during this period. A new head impact model of AI in sheep was developed to examine pathological and physiological changes in the brain resulting from a graded traumatic insult. In this preliminary study 10 anesthetized and ventilated Merino ewes were used. Head injury was produced by impact from a humane stunner to the temporal region of an unrestrained head. Eight sheep were studied for 1, 2, 4, or 6 h after impact. Two sham animals (no impact, 6 h survival) were also examined. Arterial blood pressure, intracranial pressure, and cerebral blood flow were monitored continuously. A physiological index of injury severity was calculated by weighting the percentage shift from preinjury values for each monitored parameter over the first hour after injury. Immunostaining with amyloid precursor protein (APP) was used as a marker of axonal damage and the distribution of APP positive axons was recorded according to a sector scoring method (APPS). Widespread AI was identified in 7 of the 8 impacted animals, around cerebral contusions and in hemispheric white matter, central gray matter, brain stem, and cerebellum, and was detected as early as 1 h after injury. The degree of axonal injury (APPS) correlated well with an index of physiological response to injury (r = 0.83, p = 0.005).

Widespread axonal injury in gunshot wounds to the head using amyloid precursor protein as a marker.

In order to determine whether axonal injury (AI) is a factor in cases of penetrating head injury, the brains of 14 patients who died shortly after sustaining a fatal gunshot wound (GSW) to the head were examined, and the presence of AI determined using immunohistochemical staining for amyloid precursor protein (APP). The distribution of AI was mapped throughout the cerebral hemispheres and brain stem. AI was present in all cases in a diffuse distribution distant to the missle track with severe involvement of the brain stem in all cases. There was no axonal APP immunoreactivity in the direct region of the missle track at the point of primary axotomy. The APP-positive AI in these cases is likely to be a mixture of primary and secondary AI as APP immunostaining is unable to distinguish primary AI due to mechanical deformation from AI secondary to hypoxic-ischemic damage.

Cerebral malakoplakia.

A case of cerebral malakoplakia is described in an 18-year-old woman who died as the consequence of a postpartum stroke. The malakoplakic lesion occurred in areas of cerebral infarction. The features of this unique case are compared with the small number of previously reported cases of cerebral malakoplakia which occurred in much younger patients and against a background of herpes simplex infection.

A novel method for correlating internal and external structure of individual myelinated nerve fibres.

A new method is described that enables longitudinal and cross sections of an individual nerve fibre to be cut at multiple specified sites along the fibre by the use of a unique marker system. In this way the internal structure of the fibre can be correlated with the external appearance. Individual myelinated nerve fibres are teased apart in epoxy resin and mounted onto a carbon-coated slide, and after orientation and marking of specific sites of interest are snap frozen to liberate the orientated and marked fibres for embedding on the surface of an epoxy resin block for subsequent longitudinal or transverse sectioning. This method is particularly useful for the correlative study of the myelin-axon relationships.

Galanin-containing fibers innervate substance P-containing neurons in the pedunculopontine tegmental nucleus in humans.

Galanin is a putative peptide transmitter distributed widely in the central nervous system. Galanin shows potent inhibitory effects on neuronal activity and learning behaviors induced or mediated by acetylcholine. Here, we demonstrate that in humans there are abundant galanin-containing fibers in the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus and the oral pontine reticular nucleus. These neurons contain both acetylcholine and substance P (SP). Using a double-immunostaining method, we show that galanin-containing terminals from pericellular baskets around many SP-positive neurons in these nuclei. Our results suggest galanin may influence brain functions via inputs to the ascending mesopontine acetylcholine-containing nuclei.

Loss of brainstem serotonin- and substance P-containing neurons in Parkinson's disease.

Using postmortem immunohistochemical analysis, we have identified degeneration of several different neuronal cell groups in the brainstem of patients dying with idiopathic Parkinson's disease. We report the first chemically identified loss of presumed serotonin neurons in the median raphe nucleus of the pons and of substance P-containing preganglionic neurons in the dorsal motor vagal nucleus. This evidence is concordant with other evidence that the primary neuropathological process is not confined either to a single pathway or to neurons containing a particular transmitter. Rather it appears that Parkinson's disease affects several classes of neurons in localized areas of the brainstem.

Polymyositis presenting with respiratory failure.

A 36-year-old woman presented with a 3-month history of increasing dyspnoea, culminating in respiratory failure due to paralysis of respiratory muscles. She required assisted ventilation. Subsequently, mild limb and neck weakness was noted. Muscle biopsy of the right vastus lateralis showed polymyositis. The patient died suddenly and the postmortem revealed polymyositis with predominant involvement of the diaphragm and intercostal muscles.