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.

Non-coding VMA21 deletions cause X-linked myopathy with excessive autophagy.

X-linked Myopathy with Excessive Autophagy (XMEA) affects proximal muscles of the lower extremities and follows a progressive course reminiscent of muscular dystrophy. It is caused by mutations in VMA21 whose protein product assembles lysosomes' proton pumps. All XMEA mutations to date have been single-nucleotide substitutions that reduce VMA21 expression, which leads to modest lysosomal pH increase, the first step in the disease's pathogenesis. We now report a new class of XMEA mutations. We identified two VMA21 non-coding microdeletions, one intronic (c.54-16_54-8del), the other in the 3'UTR (c.*13_*104del). Both resulted in a relatively more severe (early ambulation loss), diffuse (extra-ocular and upper extremity involvement), and early (neonatal) onset disease compared to previously reported patients. Our cases highlight the importance of including non-coding regions of VMA21 in genetic testing panels of dystrophies and myopathies. Specific diagnosis of XMEA will be particularly important as therapies aimed at correcting the modest rise in lysosomal pH at the root of this disease are developed.

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.

Mortality and its predominant causes in a large cohort of patients with biopsy-determined inflammatory myositis.

BACKGROUND: There is a paucity of literature on the patterns and predictors of mortality in idiopathic inflammatory myopathies (IIM). AIMS: To determine the patterns and predictors of mortality in a South Australian cohort of patients with biopsy-proven IIM. METHODS: The living/deceased status (and for deceased patients the causes of death) of patients with histologically determined IIM was determined from the Births, Deaths and Marriages Registry. Standardised mortality ratios (SMR) were generated compared with the age/gender matched South Australian population. The effect of presence/absence of the components of the Bohan and Peter criteria on risk ratios (RR) for mortality was determined. The effect of comorbidities and autoantibodies on mortality was investigated. RESULTS: The SMR for mortality in IIM was 1.75 and was significantly increased in all disease subgroups, being highest in patients with dermatomyositis (2.40). Dominant causes of death were cardiovascular disease (31%), infections (22%) and malignancy (11%). Risk factors for death were age at time of biopsy (hazard ratio 1.05), ischaemic heart disease (RR 2.97, P < 0.0001), proximal weakness at diagnosis (RR 1.8, P= 0.03), definite diagnosis of IIM per the Bohan and Peter criteria (RR 2.14, P < 0.0001), and the absence of autoantibodies (RR 1.9, P < 0.001). CONCLUSIONS: Patients with IIM are at 75% increased risk for mortality, and cardiovascular diseases account for the commonest causes of death. This study suggests a thorough cardiovascular evaluation of these patients is indicated, and raises the possibility that targeted interventions such as the use of aspirin or statins may improve outcomes in IIM.

A novel mitochondrial DNA deletion producing progressive external ophthalmoplegia associated with multiple sclerosis.

We report a previously undescribed 7676 base pair mitochondrial (mt)DNA deletion involving genes of complex I, complex IV subunits 2 and 3 (cytochrome oxidase [Cox] II, III), adenosine triphosphatase 8 and 6, cytochrome b and 8 transfer (t)RNA genes producing myopathy and progressive external ophthalmoplegia (PEO) in a 44-year-old right-handed Caucasian man with features of multiple sclerosis (MS). We performed complete mtDNA sequencing and deletion analysis, spectrophotometric analysis of muscle and platelet respiratory chain activity, measurement of platelet mitochondrial membrane potential with the potentiometric dye JC-1 and magnetic resonance spectroscopy (MRS) and MRI studies of normal-appearing and lesional cerebral tissue. The deletion resulted in significant respiratory chain deficiency in muscle and blood and abnormalities of the platelet mitochondrial membrane potential. However, cerebrospinal fluid analysis, magnetic resonance spectroscopy and MRI features suggested inflammatory central nervous system demyelination rather than a primary respiratory chain disorder. We conclude that this novel mtDNA deletion causing myopathy and PEO is associated with severe muscle and platelet cellular energetic abnormalities. Furthermore, clinical and paraclinical features of multiple sclerosis were found. The potential pathomechanistic interaction between mtDNA variation and multiple sclerosis is reviewed.

Clinical heterogeneity and prognostic features of South Australian patients with anti-synthetase autoantibodies.

AIM: To determine the clinical, serological and prognostic features of patients with autoantibodies against three aminoacyl-transfer RNA synthetases (ARS), namely Jo-1 (histidyl-tRNA synthetase), PL-7 (threonyl-tRNA synthetase) and PL-12 (alanyl-tRNA synthetase) in South Australia. METHODS: Patients with autoantibodies against ARS detected by line immunoassay (anti-Jo1, anti-PL7, anti-PL12) or enzyme-linked immunosorbent assay (anti-Jo1) were identified from existing laboratory databases for the period 1994-2009. Demographic, clinical and serological data were obtained by retrospective review of patients' medical records and laboratory databases. RESULTS: Forty-two patients with autoantibodies were identified (anti-Jo1 = 37, anti-PL7 = 4, anti-PL12 = 1). Females were more commonly affected than males (M : F = 12:30). Twenty-one patients had polymyositis (anti-Jo1 = 17, anti-PL7 = 4), seven dermatomyositis (anti-Jo1 = 6, anti-PL12 = 1), 10 overlap syndrome (anti-Jo1 = 10; lupus = 4, scleroderma = 3, Sjögren's syndrome = 2 and rheumatoid arthritis = 2) and four had interstitial lung disease (ILD) only (anti-Jo1 = 4). ILD was present in 69%, polyarthritis in 59% and positive anti-nuclear antibody (ANA) in 43% of patients. Concurrence of autoantibodies against Ro-52 with Jo-1 was seen in 12 patients. The mean follow-up period was 8.3 years (95% CI 5.8-10.8) with 12 deaths. Poor prognostic indicators were age of onset >60 years (P= 0.001), cancer (P= 0.002), negative ANA (P= 0.006) and negative autoantibodies to extractable nuclear antigens (P= 0.02). CONCLUSION: Patients with autoantibodies against ARS present with varied clinical features and occasionally with isolated lung involvement (amyopathic ILD). Older age of onset, malignancy and negative immunologic tests are predictors of poor prognosis. Concurrence of autoantibodies against Jo-1 and Ro-52 may reflect a coupling effect during generation of autoimmunity.

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.

Localized hypertrophic neuropathy: a case report and review of the literature.

The case is presented of a 71-year-old man with a 6-year history of symptoms suggestive of carpal tunnel syndrome, which did not improve despite two surgical procedures. On further investigation, a fusiform enlargement of the median nerve was found above the elbow, which was found on biopsy to be localized hypertrophic neuropathy (LHN). This case is the first to be described affecting the median nerve. The literature regarding LHN is reviewed, with discussion of the differential diagnoses and possible etiology of this rare lesion.

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.

Idiopathic inflammatory myopathies.

The idiopathic inflammatory myopathies are a group of systemic autoimmune syndromes characterized by striated muscle inflammation. Here, we discuss the clinical features of this group of conditions and review the recent developments in the understanding of the pathogenesis and immunogenetics of the idiopathic inflammatory myopathies. The role of myositis-specific autoantibodies and their clinical significance and an overview of management are also provided.

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.

The spatiotemporal pattern of somal and axonal pathology after perikaryal excitotoxic injury to retinal ganglion cells: a histological and morphometric study.

OBJECTIVE: To describe the spatiotemporal pattern of somal and axonal pathologic changes after perikaryal excitotoxic injury to retinal ganglion cells in-vivo. METHODS: 40 male Sprague-Dawley rats were killed at 0 h, 24 h, 72 h and 7 days after injecting 20 nM N-methyl-D-aspartate (NMDA) into the vitreous chamber of left eye. Saline-injected right eyes served as control. After perfusion fixation, the eyes and retrobulbar optic nerves from half of the animals in each group were embedded in paraffin and tissues from the other half embedded in resin. Paraffin-embedded eyes and resin-embedded proximal (intraorbital) and distal (intracranial) optic nerve segments were evaluated by light microscopy. Light microscopic photographs of proximal and distal optic nerve segments were compared using the following parameters: axon counts, axonal swellings and myelin changes. RESULTS: Retinas showed cell loss in ganglion cell layer (GCL) and reduction in inner retinal thickness at 72 h after NMDA injection (p<0.05), with changes becoming more advanced after 7 days (p<0.001). The cell count in GCL correlated strongly with the axonal counts (R=0.929, p<0.001). Axon loss, axon swellings and myelin damage were seen in both proximal and distal segments of optic nerves 72 h post-NMDA exposure (p<0.05), with changes increasing further at 7 days (p<0.001). Pathological changes were more prominent in the distal segments (p<0.05). CONCLUSION: Excitotoxic perikaryal injury causes an axonopathy, which is synchronous with the somal degeneration and which is most prominent in the distal portions of the axon, consistent with "dying-back like neuropathy".