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Este artículo se encuentra disponible en la siguiente URL: https://journals.sagepub.com/doi/pdf/10.1177/0300985818817071 This is the pre-peer reviewed version of the following article: Ortega, J., Verdes, J. M., Morrell, E. L., Finnie, J. W., Manavis, J., & Uzal, F. A. (2019). Intramural vascular edema in the brain of goats with "Clostridium perfringens" Type D enterotoxemia. Veterinary Pathology, vol. 56, n. 3 (01 may.), pp. 452?459, which has been published in final form at https://doi.org/10.1177/0300985818817071 Este es el pre-print del siguiente artículo: Ortega, J., Verdes, J. M., Morrell, E. L., Finnie, J. W., Manavis, J., & Uzal, F. A. (2019). Intramural vascular edema in the brain of goats with "Clostridium perfringens" Type D enterotoxemia. Veterinary Pathology, vol. 56, n. 3 (01 may.), pp. 452?459, que se ha publicado de forma definitiva en https://doi.org/10.1177/0300985818817071 Enterotoxemia caused by Clostridium perfringens type D is an important disease of sheep and goats with a worldwide distribution. Cerebral microangiopathy is considered pathognomonic for ovine enterotoxemia and is seen in most cases of the disorder in this species. These lesions are, however, rare and poorly described in goats. In this paper, we describe the vasculocentric brain lesions observed in 44 cases of caprine spontaneous C. perfringens type D enterotoxemia. Only one goat had gross changes in the brain, which consisted of mild cerebellar coning. However, 8/44 (18%) of the cases showed microscopic brain lesions, characterized mainly by intramural vascular proteinaceous edema, a novel and diagnostically significant finding. The precise location of the edema was better observed with PAS and Gomori’s stains. The areas of the brain most frequently affected were cerebral cortex, corpus striatum (basal ganglia) and cerebellar peduncles and both arterioles and venules were involved. Most of the goats of this study showed lesions in the intestinal tract (enteritis, colitis and typhlitis), although pulmonary congestion and edema, hydrothorax, hydropericardium and ascites were also described. This is the largest study to date of neuropathological changes in naturally-occurring cases of caprine type D enterotoxemia, and it describes the main features of the cerebral microangiopathy in this species. Although the intramural edema found, for the first time, in these caprine cases is useful for the diagnosis of enterotoxemia when observed, its absence cannot exclude the disease because, in goats, it is only present in a small number of cases.

Traumatic brain injury constitutes a significant proportion of cases requiring forensic examination, and it encompasses (1) blunt, nonmissile head injury, especially involving motor vehicle accidents, and (2) penetrating, missile injury produced by a range of high- and lower-velocity projectiles. This review examines the complex pathophysiology and biomechanics of both types of neurotrauma and assesses the macroscopic and histologic features of component lesions, which may be used to determine the cause and manner of death resulting from an intentional assault or accident. Estimation of the survival time postinjury by pathologic examination is also important where malicious head injury is suspected, in an attempt to ascertain a time at which the traumatic event might have been committed, thereby evaluating the authenticity of statements made by the alleged perpetrator.

Clostridium perfringens type D causes enterotoxemia in sheep and goats. The disease is mediated by epsilon toxin (ETX), which affects the cerebrovascular endothelium, increasing vascular permeability and leading to cerebral edema. In the present study, we compared the distribution and severity of the cerebrovascular changes induced in lambs by C. perfringens type D strain CN1020, its isogenic etx null mutant, and the ETX-producing complemented mutant. We also applied histochemical and immunohistochemical markers to further characterize the brain lesions induced by ETX. Both ETX-producing strains induced extensive cerebrovascular damage that did not differ significantly between each other in nature, neuroanatomic distribution, or severity. By contrast, lambs inoculated with the etx mutant or sterile, nontoxic culture medium did not develop detectable brain lesions, confirming that the neuropathologic effects observed in these infections are dependent on ETX production. Lambs treated with the wild-type and complemented strains showed perivascular and mural vascular edema, as well as serum albumin extravasation, particularly severe in the cerebral white matter, midbrain, medulla oblongata, and cerebellum. Brains of animals inoculated with the ETX-producing strains showed decreased expression of glial fibrillary acidic protein and increased expression of aquaporin-4 in the end-feet processes of the astrocytes around blood vessels. Early axonal injury was demonstrated with anti–amyloid precursor protein immunohistochemistry. Perivascular accumulation of macrophages/microglia with intracytoplasmic albumin globules was also observed in these animals. This study demonstrates that ETX is responsible for the major cerebrovascular changes in C. perfringens type D–induced disease.

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.

Amyloid precursor protein (APP) immunocytochemistry was used to study axonal and neuronal changes in guinea pig brains exposed to tunicamycin. Substantial axonal injury was found in ischemic-hypoxic foci and more generally, but this injury was not readily appreciable in conventionally stained sections. Neuronal perikaryal APP expression was also widely distributed, possibly as an acute phase response to this neurotoxin.

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.

Animal models have played a critical role in elucidating the complex pathogenesis of traumatic brain injury, the major cause of death and disability in young adults in Western countries. This review discusses how different types of animal models are useful for the study of neuropathologic processes in traumatic, blunt, nonmissile head injury.

In a blunt, nonmissile, head impact model of traumatic brain injury in 4-5-week-old Merino lambs, multiple foci of internal granular layer necrosis were found in all 10 impacted animals. This lesion has not previously been reported after human or animal head injury. Temporal lobe impact contusions, predomi- nantly microscopic (8/10) and contralateral contusions (2/10), parenchymal (10/10) and subarachnoid (10/10) hemorrhage, and widely distributed axonal injury were also observed. Although the precise pathogenesis of this focal granule cell necrosis and often attendant red cell change in Purkinje cells was unclear, an ischemic etiology due to trauma-related vascular damage is postulated. 1 its role in pediatric head- injured patients has been less studied. 5 Accordingly, a pre- viously described adult ovine closed head injury model 6 (an impact-generated rotational acceleration of a relatively large gyrencephalic brain in an unconstrained head) was modified to study axonal injury in lambs as an experimental model of pediatric head injury. 4 In this model, all 10 head-impacted lambs showed numerous foci of internal granular layer ne- crosis. This cerebellar lesion was not present in nonimpacted control lambs or in similarly head-injured adult sheep. 6 Anesthetized and ventilated 4-5-week-old Merino lambs were placed in a sphinx position with their heads supported to permit free rotational and lateral movement of the head after impact. A humane stunner (model MKL, Karl Schermer & Co., Karlsruhe, Germany) was aligned with the left temporal fossa, and a constant charge (Schermer No. 11) was used to impact these animals. This mechanical device, approved for humane euthanasia of livestock, 3 uses a charge to propel a captive bolt with an attached, heavy, mushroom-shaped head against the skull. Continuous physiological monitoring for mean arterial blood pressure, intracranial pressure, cerebral blood flow, and blood gases ensured that no hypoxic or hypotensive episodes supervened during the experimental period. Two hours after impact, the brain was perfusion fixed with 4% paraformalde- hyde and then postfixed in formalin for 7 days. Brains were sectioned at 5-mm coronal intervals, embedded in paraffin, and cut and stained with hematoxylin and eosin (HE). Selected small areas of cerebellum were also removed from paraffin blocks, embedded in plastic, cut at 1 m, and stained with toluidine blue. Four control lambs were not impacted but were otherwise subjected to the same experimental protocol. At necropsy, two lambs had fractures of the outer table of the skull, two lambs exhibited depressed fractures, and in all other lambs the inner and outer tables of the skull remained intact. Eight lambs had microscopic impact contusions, and two had contralateral contusions. Microscopically, widely distrib- uted (multifocal) amyloid precursor protein-positive axonal in- jury was found in impacted and contralateral hemispheres and bilaterally in brain stem and cerebellum. Parenchymal and sub- arachnoid hemorrhage was also similarly distributed. Acute multifocal internal granular layer necrosis occurred in all 10 impacted lambs. These necrotic foci were usually small, well circumscribed, and round to ovoid in shape (Fig. 1) or sometimes more extensive and irregular in outline. They were randomly distributed with no discernible predilection for the crests or sulci of the cerebellar folia and often in- volved the full thickness of the granular layer. The granule cell nuclei were markedly condensed, and the cytoplasm ei- ther greatly reduced and paler staining or the hyperchromatic nuclei were disposed in a nonstaining matrix without visible cytoplasm. Necrotic foci had a remarkably similar appear- ance in routinely stained paraffin-embedded sections 2 hours after impact, were well demarcated from adjacent normal compact granular layer (Fig. 1), and sometimes contained hemorrhage. In semithin sections stained with toluidine blue (Fig. 2), damaged granule cell nuclei were condensed with still discernible coarse chromatin clumps or pyknotic, small, and uniformly hyperchromatic. The granule cell cytoplasm was expanded, clear, and poorly delimited. Contiguous Pur- kinje cells showed ischemic (red cell) change with pyknotic nuclei and shrunken, angular, and hypereosinophilic cyto- plasm, exhibited less severe cytoplasmic shrinkage and eo- sinophilia than frankly necrotic neurons, or were apparently unaffected. In plastic-embedded sections, necrotic Purkinje cells were shrunken and darkly stained (Fig. 2), with often fine cytoplasmic vacuolation. There was sometimes edema- tous distension of the layer dissecans, and a few normally inconspicuous Bergmann's glia had swollen, vesicular nuclei and slightly enlarged, clear cytoplasm. Small blood vessels in and immediately adjacent to these necrotic foci in the granular layer were frequently occluded by tightly packed erythrocytes, whereas vessels in the surrounding unaffected parenchyma were normally perfused or contained a few re- sidual unflushed erythrocytes. In all control lambs, the cer- ebellum was microscopically normal and there were no other neuropathologic changes.