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.

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.

Biomechanical studies in an ovine model of non-accidental head injury.

This paper presents the head kinematics of a novel ovine model of non-accidental head injury (NAHI) that consists only of a naturalistic oscillating insult. Nine, 7-to-10-day-old anesthetized and ventilated lambs were subjected to manual shaking. Two six-axis motion sensors tracked the position of the head and torso, and a triaxial accelerometer measured head acceleration. Animals experienced 10 episodes of shaking over 30 min, and then remained under anesthesia for 6h until killed by perfusion fixation of the brain. Each shaking episode lasted for 20s resulting in about 40 cycles per episode. Each cycle typically consisted of three impulsive events that corresponded to specific phases of the head's motion; the most substantial of these were interactions typically with the lamb's own torso, and these generated accelerations of 30-70 g. Impulsive loading was not considered severe. Other kinematic parameters recorded included estimates of head power transfer, head-torso flexion, and rate of flexion. Several styles of shaking were also identified across episodes and subjects. Axonal injury, neuronal reaction and albumin extravasation were widely distributed in the hemispheric white matter, brainstem and at the craniocervical junction and to a much greater magnitude in lower body weight lambs that died. This is the first biomechanical description of a large animal model of NAHI in which repetitive naturalistic insults were applied, and that reproduced a spectrum of injury associated with NAHI.

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.

Pattern of cerebrospinal immediate early gene c-fos expression in an ovine model of non-accidental head injury.

Expression of the immediate early gene, c-fos, was examined in a large animal model of non-accidental head injury ("shaken baby syndrome"). Lambs were used because they have a relatively large gyrencephalic brain and weak neck muscles resembling a human infant. Neonatal lambs were manually shaken in a manner similar to that believed to occur with most abused human infants, but there was no head impact. The most striking c-fos expression was in meningothelial cells of the cranial cervical spinal cord and, to a lesser degree, in hemispheric, cerebellar, and brainstem meninges. Vascular endothelial cells also frequently showed c-fos immunopositivity in the meninges and hemispheric white matter. It was hypothesised that this c-fos immunoreactivity was due to mechanical stress induced by shaking, with differential movement of different craniospinal components.

Neuropathological changes in a lamb model of non-accidental head injury (the shaken baby syndrome).

Non-accidental head injury (NAHI), also termed the "shaken baby syndrome", is a major cause of death and severe neurological dysfunction in children under three years of age, but it is debated whether shaking alone is sufficient to produce brain injury and mortality or whether an additional head impact is required. In an attempt to resolve this question, we used a lamb model of NAHI since these animals have a relatively large gyrencephalic brain and weak neck muscles resembling those of a human infant. Three anaesthetised lambs of lower body weight than others in the experimental group died unexpectedly after being shaken, proving that shaking alone can be lethal. In these lambs, axonal injury, neuronal reaction and albumin extravasation were widely distributed in the hemispheric white matter, brainstem and at the craniocervical junction, and of much greater magnitude than in higher body weight lambs which did not die. Moreover, in the eyes of these shaken lambs, there was damage to retinal inner nuclear layer neurons, mild, patchy ganglion cell axonal injury, widespread Muller glial reaction, and uveal albumin extravasation. This study proved that shaking of a subset of lambs can result in death, without an additional head impact being required.

Neuroaxonal dystrophy in Australian Merino lambs.

Neuroaxonal dystrophy (NAD) is a morphological abnormality in man and animals that is characterized by the occurrence of numerous axonal swellings (spheroids) in the nervous system. NAD has been described in Suffolk lambs in the USA, Merino lambs in Australia and several breeds of sheep in New Zealand. This paper describes the clinicopathological changes of only the second occurrence of NAD reported in Merino lambs. There were some features (myelin loss, gliosis and visual impairment) in these Australian cases that have not been reported previously in ovine NAD. Application of immunohistochemical markers of axonal transport suggested that disruption of this transport mechanism contributed to spheroid development.

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.

Alzheimer type II astrocytes in the brains of pigs with salt poisoning (water deprivation/intoxication).

The finding of Alzheimer type II astrocytes, in addition to the pathognomonic combination of laminar cerebrocortical necrosis and eosinophil infiltration, in the brains of pigs is reported for the first time in cases of indirect salt poisoning following water deprivation.

Fulminant leucocytoclastic brainstem vasculitis in a patient with otherwise indolent systemic lupus erythematosus.

The spectrum of central nervous system (CNS) vascular pathology in systemic lupus erythematosus (SLE) includes small vessel vasculopathy, thromboembolism, perivascular lymphocytic infiltration and, rarely, overt transmural vasculitis. We present the case of a patient, who experienced three CNS relapses over total disease duration of 26 years, with otherwise indolent disease. The first two relapses were suspicious of vasculitis and the last was proven at autopsy. The short duration between final relapse onset and death in this SLE CNS vasculitis case was, to our knowledge, unique. Histopathological investigation demonstrated multiple confluent areas of haemorrhage in the medulla due to an acute small vessel leucocytoclastic vasculitis.

Selective vulnerability of peripheral nerves in avian riboflavin deficiency demyelinating polyneuropathy.

Riboflavin (vitamin B2) deficiency in young chickens produces a demyelinating peripheral neuropathy. In this study, day-old broiler meat chickens were fed a riboflavin-deficient diet (1.8 mg/kg) and killed on posthatch days 6, 11, 16, 21, and 31, while control chickens were given a conventional diet containing 5.0 mg/kg riboflavin. Pathologic changes were found in sciatic, cervical, and lumbar spinal nerves of riboflavin-deficient chickens from day 11 onwards, characterized by endoneurial oedema, hypertrophic Schwann cells, tomacula (redundant myelin swellings), demyelination/remyelination, lipid deposition, and fibroblastic onion bulb formation. Similar changes were also found in large and medium intramuscular nerves, although they were less severe in the latter. However, by contrast, ventral and dorsal spinal nerve roots, distal intramuscular nerves, and subcutaneous nerves were normal at all time points examined. These findings demonstrate, for the first time, that riboflavin deficiency in young, rapidly growing chickens produces selective injury to peripheral nerve trunks, with relative sparing of spinal nerve roots and distal nerve branches to muscle and skin. These novel findings suggest that the response of Schwann cells in peripheral nerves with riboflavin deficiency differs because either there are subsets of these cells in, or there is variability in access of nutrients to, different sites within the nerves.

Aquaporin-4 in acute cerebral edema produced by Clostridium perfringens type D epsilon toxin.

Sheep, particularly lambs, with high circulating levels of Clostridium perfringens type D epsilon toxin develop severe neurologic signs and often die suddenly. On microscopic examination, in the brain, there is microvascular endothelial injury and diffuse vasogenic edema. The aquaporin (AQP) family of membrane water-channel proteins, especially AQP-4, is important in the regulation of water balance in the brain and facilitates reabsorption of excess fluid. In rats given epsilon toxin, generalized cerebral edema was demonstrated by marked albumin extravasation and was correlated with widespread upregulation of AQP-4 in astrocytes. These results suggest that AQP-4 has a role in the clearance of edema fluid from brains damaged by this clostridial toxin.

Novel fibroblastic onion bulbs in a demyelinating avian peripheral neuropathy produced by riboflavin deficiency.

The finding of novel fibroblastic onion bulb-like structures in peripheral nerves is reported for the first time in avian riboflavin deficiency. Day old broiler meat chickens were fed a riboflavin deficient diet (1.8 mg/kg) and were killed on postnatal days 6, 11, 16, 21 and 31, whereas control chickens were fed a conventional diet containing 5.0 mg/kg riboflavin. The fibroblastic onion bulb-like structures were found in sciatic and brachial nerves from day 11 onwards and consisted of long cytoplasmic processes of hypertrophied fibroblasts surrounding demyelinated, remyelinated and normally myelinated axons. The fibroblast cytoplasmic processes often enveloped more than one nerve fibre to produce a unique compound-like onion bulb structure. These onion bulb-like structures occurred early in the course of segmental demyelination at the same time as tomacula formation and became increasingly more prominent in the later stages of demyelination and remyelination. The molecular basis of formation of these unique structures requires further study as to the basis of the attraction of the fibroblast processes to nerve fibres associated with myelinating Schwann cells. The model may also be useful in investigating the role of endoneurial fibroblasts in endoneurial fibrosis as the early fibroblastic response in the onion bulbs is distinct from the more usual fibroblastic deposition of collagen in end-stage peripheral nerve disease.

Avian riboflavin deficiency: an acquired tomaculous neuropathy.

The finding of tomacula, focal areas of sausage-shaped hypermyelination in peripheral nerves, is reported for the first time in avian riboflavin deficiency. Day-old, meat-type chickens were fed a riboflavin-deficient diet (1.8 mg/kg) and were killed on postnatal days 6, 11, 16, and 21, while control chickens were fed a conventional diet containing 5.0 mg/kg riboflavin. Tomacula were found in sciatic and brachial nerves from day 11 onward, became more frequent and prominent with increasing time, and preceded the onset of segmental demyelination.

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.

Early paranodal myelin swellings (tomacula) in an avian riboflavin deficiency model of demyelinating neuropathy.

INTRODUCTION: Disruption of the complex architectural and molecular organization of the paranodal region of myelinated peripheral nerve fiber may initiate the evolving time dependent process of segmental demyelination. In support of this notion was the finding of focal paranodal myelin swellings (tomacula) due to redundant folding of myelin sheaths, early in the time course of an avian riboflavin deficiency model of demyelinating neuropathy. METHODS: Newborn broiler meat chickens were maintained either on a routine diet containing 5.0 mg/kg riboflavin (control group) or a riboflavin-deficient diet containing 1.8 mg/kg riboflavin. Riboflavin concentrations in the liver were measured at postnatal day 11. Peripheral nerves were morphologically examined at days 6, 11, 16 and 21 using light and electron microscopy and teased nerve fiber techniques. RESULTS: Riboflavin-deficient chickens showed signs of a neuropathy from days 8 and pathological examination of peripheral nerves revealed a demyelinating neuropathy with paranodal tomacula formation starting on day 11. Paranodal tomacula consisted of redundant myelin infoldings or outfoldings, increased in size and frequency after day 11. After day 16, the paranodal swellings showed prominent degenerative changes accompanied by an increased frequency of myelinated fibers showing demyelination. CONCLUSION: Tomacula due to redundant myelin folds are generally considered a remyelination phenomenon, yet in this avian riboflavin deficiency model of demyelination, the paranodal tomacula occurred early in the course of demyelination.

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.