Canine model of ischemic stroke with permanent middle cerebral artery occlusion: clinical features,magnetic resonance imaging,histopathology, and immunohistochemistry

The purpose of this study was to identify time-related changes in clinical, MRI, histopathologic, and immunohistochemical findings associated with ischemic stroke in dogs. Additionally, the association of cerebrospinal fluid (CSF) and tissue levels of interleukin (IL)-6 with clinical prognosis was assessed. Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO) in nine healthy experimental dogs. The dogs were divided into three groups according to survival time and duration of the experimental period: group A (survived only 1 day), group B (1-week experimental period), and group C (2-week experimental period). Neurologic status was evaluated daily. Magnetic resonance imaging (MRI) was performed according to a predetermined schedule. Concentration of IL-6 in CSF was measured serially after ischemic stroke. Postmortem examination was performed for all experimental dogs. During histopathological examination, variable degrees of cavitation and necrosis due to neuronal cytopathic effects, such as pyknotic nuclei and cytoplasmic shrinkage, were observed on the affected side of the cerebral cortex in all dogs. Immunohistochemistry specific for IL-6 showed increased expression in the ischemic lesions. CSF IL-6 concentrations and ischemic lesion volumes 1 day after ischemic stroke were significantly higher in group A compared to groups B and C.     

Parvovirus-induced encephalitis in a juvenile raccoon

A juvenile raccoon was euthanized because of severe neurologic signs. At postmortem examination, no significant gross lesions were present. Histologic evaluation demonstrated nonsuppurative encephalitis in thalamus, brainstem, and hippocampus, cerebellar Purkinje cell loss, as well as poliomyelitis and demyelination of the spinal cord. Parvovirus antigen–specific immunohistochemistry revealed immunopositive neurons in the brainstem, cerebral cortex, and hippocampus. A few Purkinje cells were also immunopositive. DNA extracted from formalin-fixed, paraffin-embedded brain tissue (thalamus, hippocampus, cerebral cortex) yielded a positive signal using PCR targeting both feline and canine parvovirus. Sequencing analyses from a fragment of the NS1 gene and a portion of the VP2 gene confirmed the presence of DNA of a recent canine parvovirus variant (CPV-2a–like virus) in the cerebellum. Our case provides evidence that a recent canine parvovirus (CPV) strain (Carnivore protoparvovirus 1) can infect cerebral and diencephalic neurons and cause encephalitis in an otherwise healthy raccoon. Parvovirus-induced encephalitis is a differential diagnosis of rabies and canine distemper in raccoons with neurologic signs.     

Computed tomographic findings of ceroid lipofuscinosis in a dog.

A two-year and seven-month-old, castrated male border collie was presented for a two-month history of progressive neurological signs including blindness, ataxia, dementia, and partial seizures. A complete blood count, serum biochemical profile, urinalysis, thoracic radiographs, and cerebrospinal fluid analysis were within reference ranges. Computed tomography (CT) of the brain showed dilatation of the ventricles and atrophy of the cerebral cortex. A central nervous system (CNS) storage disease was suspected, and the dog was euthanized due to a poor prognosis. Light and electron microscopic examination revealed neuronal degeneration with pigment accumulation in neurons of the CNS, in ganglia of the peripheral nervous system, and in several non-nervous tissues. Ceroid lipofuscinosis was diagnosed based on the microscopic and ultrastructural lesions detected. This is the second report of CT findings in a canine clinical patient with ceroid lipofuscinosis.     

Lesions in the central nervous system associated with perinatal lamb mortality

OBJECTIVE: To identify and describe the occurrence of neurological lesions that could have an effect on lamb mortality. PROCEDURE: The central nervous system was investigated macroscopically (n = 92) and microscopically (n = 72) in lambs dying in the perinatal period during 3 years in flocks of adult Corriedale ewes. The central nervous system was removed intact and samples of cerebral cortex, basal ganglia, thalamus, hippocampus, mesencephalon, cerebellar cortex, medulla oblongata, and cervical spinal cord were scored microscopically for the severity of neuronal dead, cytotoxic and perivascular oedema, and haemorrhage. RESULTS: Neurologic findings between birth and 6 days included haemorrhages in meninges, brain congestion and oedema, neuronal ischemic necrosis, intraparenchymal haemorrhages in medulla oblongata and cervical spinal cord, parasagittal cerebral necrosis, and periventricular leukomalacia. No significant lesions were found in anteparturient deaths or in those aged between 7 and 16 days. Oedema was more severe in the brain than in other regions of the central nervous system. Ischaemic neurons first appeared 24 hours post partum, increased linearly in number between 48 hours and 5 days post partum, and had a laminar distribution in the cerebral cortex, indicating a hypoxic-ischemic encephalopathy. Haemorrhages were most severe in the gray matter of medulla oblongata and cervical spinal cord, suggesting trauma due to instability of atlantoaxialis joint. CONCLUSION: Lesions in the central nervous system can explain most deaths at birth and within 6 days of birth. The lesions were hypoxic-ischemic and appeared to be related to birth injury.     

Polioencephalomalacia in the dog

Disturbance of cerebral blood flow from causes such as meningitis, thromboembolic disease and atherosclerosis was considered an important factor in the pathogenesis of polioencephalomalacia in 25 dogs. In dogs with polioencephalomalacia of undetermined cause, the distribution of lesions in neocortex and paleocortex suggested a change of neuronal metabolism secondary to cerebral anoxia/ischemia. Five dogs with canine distemper infection had bilateral necrosis of the hippocampus and pyriform cortex. Convulsions, central visual impairment and hemiparesis were the most prominent neurologic signs.     

Magnetic resonance imaging findings of hepatic encephalopathy in a dog with a portosystemic shunt

A 6-year-old ShihTzu presented with tonic-clonic cluster seizure. T2-weighted magnetic resonance (MR) images showed bilateral diffuse hyperintense lesions at the cerebral cortex with enlarged sulci. Computed tomography revealed a portosystemic shunt (PSS) and azygos continuation. Based on the clinical signs, blood examinations and diagnostic images, the dog was diagnosed with hepatic encephalopathy secondary to PSS. The neurologic signs were gradually improved after medical therapy for hyperammonemia. This is the first report of hyperintensity of the cerebral cortex on T2-weighted MR images associated with acute hepatic encephalopathy in a dog.     

Polioencephalomalacia in captive harbour seals (Phoca vitulina)

In a colony of 11 harbour seals (Phoca vitulina Linné 1758) two episodes of central nervous disorders occurred within 2 years causing fatalities in seven adult animals. Clinical signs comprised dyspnoea, anorexia, apathy, incoordination and lateral recumbency. Vitamin B complex therapy was successful once. Pathomorphological examination of seven carcasses revealed acute and subacute malacia of the cerebellar grey matter. Additional acute malacic lesions located in the cerebral cortices and basal ganglia were observed. Mesencephalic nuclei were less severely affected and displayed acute changes. Despite intense search for environmental toxins and infectious agents, the cause of the fatalities remained undetermined. However, the type and pattern of the lesions are most suggestive of a thiamine deficiency.     

Necrotizing meningoencephalitis in five Chihuahua dogs

An acute to chronic idiopathic necrotizing meningoencephalitis was diagnosed in 5 Chihuahua dogs aged between 1.5 and 10 years. Presenting neurologic signs included seizures, blindness, mentation changes, and postural deficits occurring from 5 days to 5.5 months prior to presentation. Cerebrospinal fluid analyses from 2 of 3 dogs sampled were consistent with an inflammatory disease. Magnetic resonance imaging of the brain of 2 dogs demonstrated multifocal loss or collapse of cortical gray/white matter demarcation hypointense on T1-weighted images, with T2-weighted hyperintensity and slight postcontrast enhancement. Multifocal asymmetrical areas of necrosis or collapse in both gray and white matter of the cerebral hemispheres was seen grossly in 4 brains. Microscopically in all dogs, there was a severe, asymmetrical, intensely cellular, nonsuppurative meningoencephalitis usually with cystic necrosis in subcortical white matter. There were no lesions in the mesencephalon or metencephalon except in 1 dog. Immunophenotyping defined populations of CD3, CD11d, CD18, CD20, CD45, CD45 RA, and CD79a immunoreactive inflammatory cells varying in density and location but common to acute and chronic lesions. In fresh frozen lesions, both CD1b,c and CD11c immunoreactive dendritic antigen-presenting cells were also identified. Immunoreactivity for canine distemper viral (CDV) antigen was negative in all dogs. The clinical signs, distribution pattern, and histologic type of lesions bear close similarities to necrotizing meningoencephalitis as described in series of both Pug and Maltese breed dogs and less commonly in other breeds.     

Magnetic resonance imaging findings in histologically confirmed Pug dog encephalitis

The purpose of the study was to describe magnetic resonance (MR) imaging features of histologically confirmed necrotizing encephalitis in four Pugs and to compare those findings with MR imaging characteristics of necrotizing encephalitis in other breeds. All dogs had the following common findings: lesions restricted to the forebrain, both cerebral hemispheres diffusely but asymmetrically affected, lesions affected gray and white matter resulting in loss of distinction between both, most severe lesions in occipital and parietal lobes, lesions were irregularly T2-hyperintense and T1-isointense to slightly T1-hypointense, and no cavitation. There were various degrees of contrast enhancement of brain and leptomeninges. Asymmetry of lateral ventricles and midline shift was seen in one dog each. Two dogs had brain herniation, which may have contributed to the progression of neurologic signs. Hyperintensity on T2-weighted and fluid attenuated inversion recovery images in the hippocampus and piriform lobe was consistent with excitotoxic edema, whereas similar imaging features in other forebrain areas corresponded to areas of inflammation or liquefaction on histopathology. In comparison with necrotizing encephalitis in other canine breeds, Pug dog encephalitis has some unique MR imaging features. Therefore, these characteristics cannot be applied to other breeds, nor should imaging features of necrotizing encephalitis of other canine breeds be used for interpretation of MR images in Pug dogs.     

Do canine parvoviruses affect canine neurons? An immunohistochemical study

In cats (most of which died from panleukopenia), cerebral neurons have recently been shown to be susceptible to canine parvovirus infection. In addition to positive immunostaining and distinct in situ hybridization signals, signs of neurodegeneration were identified by histopathology, mainly in the diencephalic area. Similar histological lesions of the diencephalic regions in dogs have also attracted attention; therefore, an immunohistochemical study was initiated to determine the possible infection of canine neurons with canine parvoviruses. The study was carried out on formalin-fixed and paraffin-embedded brain tissue, with and without signs of neurodegeneration, from 40 dogs, most of them dying from parvovirus enteritis. Immunohistochemistry, using polyclonal antiserum against canine parvoviruses, was negative in all 40 cases, suggesting that, unlike cats, canine parvoviruses do not seem capable of infecting canine neurons.     

Multifocal oligodendroglioma in three dogs

This report describes the clinical, histopathologic, and imaging findings of multifocal oligodendrogliomas from three canine patients. Clinical history varied but included seizure activity and behavior changes. Neurologic examination abnormalities included ataxia, proprioceptive deficits, cranial nerve deficits, and changes in mentation. MRI in one patient revealed multifocal brain lesions; however, the MRI was normal in another one of the patients. Histopathologic evaluation identified multifocal neoplastic infiltrates in all three patients involving the cerebral cortex, brainstem, and spinal cord, with leptomeningeal extension in two of the three patients. All three patients were euthanized due to progression of their neurologic condition and/or complications due to aspiration pneumonia. Oligodendrogliomas should be considered a differential diagnosis for patients with multifocal brain disease.     

Increased expression of vascular endothelial growth factor in neo-vascularized canine brain tissue

This retrospective study was done to characterize the levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 (HIF-1α) in dog brains with neo-vascularization in the cerebral cortex of frontal, temporal, and parietal lobe by using immunohistochemistry (IHC) and Western blot. In neo-vascularized (NV) brains, we analyzed the number and area of blood vessels and the expression of VEGF and HIF-1α. The IHC results showed that the number and area of blood vessels, as assessed by immunolabeling for von Willebrand factor, was higher in the NV brain than in the control brain. The Western blot results showed that the level of VEGF was increased, predominantly in NV brain of the cerebral cortex relative to the clinically normal cerebral cortex, whereas the expression of HIF-1α in NV brains was not different from the control brains. Our study showed that dilatation of vessels and development of new vessels in the cerebral cortex were observed in cases of canine CNS disease and found increased expression of VEGF in canine brains with neo-vascularization.     

MAGNETIC RESONANCE IMAGING AND PATHOLOGIC FINDINGS ASSOCIATED WITH NECROTIZING ENCEPHALITIS IN TWO YORKSHIRE TERRIERS

Two young adult Yorkshire terriers had neurologic signs consistent with forebrain and brainstem involvement or forebrain involvement alone. On magnetic resonance imaging studies there were asymmetric bilateral lesions mainly in the cerebral cortex, and in the diencephalon. These areas were hyperintense on T2-weighted and FLAIR images, but hypointense or isointense on T1-weighted images. Lesions had a varying degree of contrast enhancement. Areas which were isointense on T1-weighted images had no contrast enhancement or only foci of contrast enhancement. Lesions with hypointensity in T1-weighted images had no enhancement or more frequently ring-like enhancement around the lesion. Necrotizing encephalitis was confirmed pathohistologically in both dogs. The degree of contrast enhancement appeared to be related to the degree of lymphohistiocytic inflammation on histologic examination.     

Immunohistochemical and histopathological study of experimental rabies infection in mice.

An immunohistochemical and histopathological study using the ABC technique was carried out to examine time-sequential virus spread in the central nervous system (CNS) of mice after inoculation with the CVS strain of fixed rabies virus by different routes; intracerebral (ic), intraocular (io), intranasal (in), intramuscular (im) and subcutaneous (sc). Only the ic and io inoculations caused fatal infections, so that detailed analysis was conducted on mice inoculated by these two routes. In ic-inoculated mice, viral antigens were detected mainly in neurons in the cerebral cortex and in the pyramidal cells and granular cells of the hippocampus. After io inoculation, viral antigen was first detected in the trigeminal nerve ganglia, following which it spreads to the cerebral cortex and cerebellum. In the hippocampus only a few cells were viral antigen-positive at the early stage after io inoculation. There were no inflammatory lesions or Negri bodies in the CNS of mice infected by either route. This suggests that clinical signs such as ataxia or depression leading to death may be due to the direct effect of the virus on the functions of neural cells, but not to inflammatory reactions. The ABC method will be useful for the early diagnosis of suspected patients or animals to have the disease when conventional histopathological and immunofluorescent antibody techniques can not detect lesions or viral antigens.     

基于网络药理学研究丹参-川芎抗脑缺血再灌注损伤作用机制及试验验证

【目的】 通过网络药理学研究丹参-川芎药对治疗缺血性脑卒中的有效性成分及相应基因靶标，探讨其作用机制。【方法】 应用中药系统药理学计数平台（TCMSP）、GeneCards、OMIM数据库，筛选出丹参-川芎药对治疗缺血性脑卒中的潜在靶点并进行GO功能和KEGG通路富集分析。分别设置假手术组、模型组、丹参-川芎低、中、高剂量组，采用预防和治疗给药方式对线栓制备的脑缺血再灌注模型大鼠进行干预，采用Zea-longa评分法对脑缺血再灌注24 h模型大鼠进行神经功能评分，苏木素-伊红（HE）染色法观察脑组织病理学变化进行初步验证。【结果】 本研究共筛选到丹参-川芎药对主要有效活性成分共72个，其中川芎7个，丹参65个，缺血性脑卒中的靶点基因1 972个，有效活性成分与缺血性脑卒中共同作用靶标94个，包括IL6、IL10、TNF、MMP9、VEGFA、CASP3等；GO功能和KEGG通路富集结果提示，丹参-川芎药对治疗缺血性脑卒中与PI3K-Akt、cGMP-PKG、VEGF等多个信号通路有关。动物试验结果表明，丹参-川芎可减轻脑组织病理改变，减轻神经功能缺损，改善脑缺血再灌注损伤。【结论】 丹参-川芎药对可能是通过PI3K/Akt信号通路改善脑缺血再灌注损伤，发挥抗细胞凋亡的作用。     

Development of lesions and tissue distribution of parasite in lambs orally infected with sporulated oocysts of Toxoplasma gondii

The host-pathogen interaction is as a key feature during the formation of tissue cysts of Toxoplasma gondii within intermediate hosts. In this study, we investigated whether oral infection of lambs with T. gondii oocysts may be used as an experimental model in sheep to study this interaction, with the main objective being to detect the presence and distribution of lesions and parasite within different organs at different time points after oral infection. Lambs were infected with 5 × 10(3) and 5 × 10(5) sporulated T. gondii oocysts and culled at 2, 3, 5 and 6 weeks post-infection (WPI). During the infection, rectal temperature of the animals and serological antibodies against T. gondii were monitored. The presence of inflammatory lesions and parasite were evaluated through histological and immunohistochemical methods at different organs (brain, liver, lung, heart and lymph nodes). The lambs showed no clinical signs other than fever, and lesions appeared mainly in the brain, characterized by glial foci and perivascular cuffs, and in the heart, denoted by foci of interstitial myositis. Tissue cysts and tachyzoite-like structures were observed at all time points studied in the brain, where together with the glial foci they appeared mainly in the cerebral cortex of the forebrain and in the midbrain, but also in the heart, lung and lymph nodes. This study shows that oral infection with sporulated oocysts in lambs may provide a model for investigating the host-parasite interaction in situ during the development of tissue cysts.     

MAGNETIC RESONANCE IMAGING OF INTRACRANIAL INFLAMMATORY CONDITIONS IN DOGS: SENSITIVITY OF SUBTRACTION IMAGES VERSUS PRE‐ AND POST‐GADOLINIUM T1‐WEIGHTED IMAGE PAIRS

Ante mortem diagnosis of canine meningoencephalitis is usually based on the results of neurologic examination, cerebrospinal fluid analysis and magnetic resonance (MR) imaging. It has been hypothesized that subtraction MR imaging may increase the sensitivity of MR for intracranial inflammatory lesions compared to conventional post‐gadolinium T1‐weighted imaging. Sensitivity of pre‐ and post‐gadolinium (C‐/C+) image pairs and dynamic subtraction (DS) images was compared in a retrospective diagnostic accuracy study of 52 dogs with inflammatory cerebrospinal fluid and 67 dogs with idiopathic epilepsy. Series of transverse C‐/C+ and DS images were reviewed independently for signs of abnormal enhancement affecting the pachymeninges, leptomeninges or intra‐axial structures. Sensitivity of C‐/C+ image pairs and DS images was 48% (95% CI: 35–61%) and 65% (95% CI: 52–77%), respectively (P = 0.01). Intra‐axial lesions were observed more frequently than meningeal lesions in both C‐/C+ (43% vs. 31%) and DS images (61% vs. 22%). The difference in sensitivities of C‐/C+ and DS series was entirely due to increased sensitivity of DS images for intra‐axial lesions. Eight (12%) dogs with epilepsy had evidence of intra‐axial gadolinium accumulation affecting the cerebral cortex in DS images. This finding may represent a false‐positive result or a true sign of pathology, possibly associated with a leaky blood–brain barrier in areas of the brain affected by neovascularization secondary to repeated seizures. Results suggest that DS imaging has higher sensitivity than comparison of pre‐ and post‐gadolinium image pairs for inflammatory intra‐axial lesions.     

Necrotizing encephalitis in Yorkshire terriers

During the past six years these authors have observed a distinctive multifocal non-suppurative necrotizing encephalitis in Yorkshire terriers. Histopathological findings were different from those in canine encephalitides of known and unknown causes. Clinically, the Yorkshire terriers presented primarily brain stem signs or evidence of cerebral involvement, including seizures. The course of the disease was mostly chronic and progressive. Protozoal, bacterial and mycotic organisms were not found on histopathological examinations. The morphology of the lesions was strongly suggestive of a viral aetiology. Immunocytochemistry as well as in situ hybridisation failed to provide evidence for canine distemper virus infection. Likewise, canine herpesvirus was not detected by immunocytochemistry. Other known canine encephalitides could be excluded on clinical and morphological grounds; however, certain similarities may exist to pug dog encephalitis.     

Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits

ABSTRACT: Atypical bovine spongiform encephalopathy (BSE) has recently been identified in Europe, North America, and Japan. It is classified as H-type and L-type BSE according to the molecular mass of the disease-associated prion protein (PrPSc). To investigate the topographical distribution and deposition patterns of immunolabeled PrPSc, H-type BSE isolate was inoculated intracerebrally into cattle. H-type BSE was successfully transmitted to 3 calves, with incubation periods between 500 and 600 days. Moderate to severe spongiform changes were detected in the cerebral and cerebellar cortices, basal ganglia, thalamus, and brainstem. H-type BSE was characterized by the presence of PrP-immunopositive amyloid plaques in the white matter of the cerebrum, basal ganglia, and thalamus. Moreover, intraglial-type immunolabeled PrPSc was prominent throughout the brain. Stellate-type immunolabeled PrPSc was conspicuous in the gray matter of the cerebral cortex, basal ganglia, and thalamus, but not in the brainstem. In addition, PrPSc accumulation was detected in the peripheral nervous tissues, such as trigeminal ganglia, dorsal root ganglia, optic nerve, retina, and neurohypophysis. Cattle are susceptible to H-type BSE with a shorter incubation period, showing distinct and distinguishable phenotypes of PrPSc accumulation.