Spinal Cord Ependymal Responses to Naturally Occurring Traumatic Spinal Cord Injury in Dogs.

The spinal cord ependymal layer (SEL) is a recent focus in spinal cord injury (SCI) research because of its potential to serve as a source of endogenous neural stem cells. Dogs are an important spontaneous model of SCI; however, there is a paucity of information available in the literature regarding the canine SEL. Here we describe the histologic appearance and immunohistochemical staining patterns of the SEL in normal dogs (n = 4) and dogs with acute SCI caused by intervertebral disk extrusion (n = 7). Immunohistochemical staining for PCNA, Ki-67, caspase 3, E-cadherin, GFAP, and vimentin was employed in both groups. Staining for Ki-67 was absent in the SEL of normal and SCI-affected dogs, indicating possible restricted proliferative capacity of the canine SEL acutely after SCI. GFAP-positive cells were increased after SCI at both at the lesion epicenter and at proximal spinal cord sites (P = .001 and P = .006, respectively), supporting the possibility of astrocytic differentiation within the SEL after SCI. Total E-cadherin staining did not differ between normal and SCI-affected dogs (P = .42 for lesion epicenter, P = .09 at proximal sites) and was restricted to the apical cell surface in normal dogs. After SCI, E-cadherin staining was membrane-circumferential and cytosolic in nature, indicating possible loss of cellular polarity after injury that could drive cell migration from the SEL to injury sites. Enhanced GFAP expression and changes in E-cadherin expression patterns support additional studies to evaluate the canine SEL as a source of endogenous neural precursors that may be modulated for future clinical interventions after SCI.

Quantitative assessment of hsp70, IL-1ß and TNF-α in the spinal cord of dogs with E40K SOD1-associated degenerative myelopathy.

Inflammation is involved in the pathogenesis of many neurodegenerative diseases. Canine degenerative myelopathy (DM) is a progressive adult-onset neurodegenerative disease commonly associated with an E40K missense mutation in the SOD1 gene. DM has many similarities to some familial forms of human amyotrophic lateral sclerosis (ALS) and may serve as an important disease model for therapy development. Pro-inflammatory mediators such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and heat shock protein (hsp) 70 play a role in the pathogenesis of ALS. The focus of the current work was to determine whether an inflammatory phenotype is present in canine DM as defined by IL-1ß, TNF-α, and hsp70 responses in cerebrospinal fluid (CSF) and spinal cord tissue. Concentrations of hsp70, IL-1ß and TNF-α were below the limits of detection by ELISA in the CSF of both normal and DM-affected dogs. Immunohistochemical staining for hsp70 was significantly increased in ependymal cells lining the spinal cord central canal of DM-affected dogs (P = 0.003). This was not associated with increased IL-1ß or TNF-α staining, but was associated with increased CD18 staining in the gray matter of DM-affected dogs. These results suggest that hsp70 in spinal cord tissue is a potential inflammatory signature in canine DM.

Spinal meningeal oligodendrogliomatosis in two boxer dogs.

Two Boxer dogs developed progressive ataxia in association with a neoplastic infiltration of the spinal leptomeninges. In the first dog, the leptomeningeal neoplasm encompassed the entire cord and the ventral aspect of the brainstem and extended bilaterally into the piriform lobes. In the second, the neoplasm surrounded the C1-C3 segments of the spinal cord and the brainstem without involvement of the brain or spinal cord parenchyma. In both dogs, the neoplastic cells had variably distinct cell borders, clear to eosinophilic cytoplasm, and a round to ovoid hyperchromatic nucleus. Neoplastic cells were immunopositive for Olig2 and doublecortin in both dogs and for vimentin in one dog but were immunonegative for glial fibrillary acidic protein, S-100, CD34, E-cadherin, cytokeratin, CD3, and CD20. The morphological and immunohistochemical features of the neoplastic cells were consistent with an oligodendrocyte lineage. This hitherto poorly recognized neoplasm in dogs is analogous to human leptomeningeal oligodendrogliomatosis.

Early migration of Sarcocystis neurona in ponies fed sporocysts.

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM), a neurologic disease of the horse. In the present work, the kinetics of S. neurona invasion is determined in the equine model. Six ponies were orally inoculated with 250 x 10(6) S. neurona sporocysts via nasogastric intubation and killed on days 1, 2, 3, 5, 7, and 9 postinoculation (PI). At necropsy, tissue samples were examined for S. neurona infection. The parasite was isolated from the mesenteric lymph nodes at 1, 2, and 7 days PI; the liver at 2, 5, and 7 days PI; and the lungs at 5, 7, and 9 days PI by bioassays in interferon gamma gene knock out mice (KO) and from cell culture. Microscopic lesions consistent with an EPM infection were observed in brain and spinal cord of ponies killed 7 and 9 days PI. Results suggest that S. neurona disseminates quickly in tissue of naive ponies.

An equine protozoal myeloencephalitis challenge model testing a second transport after inoculation with Sarcocystis neurona sporocysts.

Previous challenge studies performed at Ohio State University involved a transport-stress model where the study animals were dosed with Sarcocystis neurona sporocysts on the day of arrival. This study was to test a second transportation of horses after oral inoculation with S. neurona sporocysts. Horses were assigned randomly to groups: group 1, transported 4 days after inoculation (DAI); group 2, at 11 DAI; group 3, at 18 DAI; and group 4, horses were not transported a second time (controls). An overall neurologic score was determined on the basis of a standard numbering system used by veterinarians. All scores are out of 5, which is the most severely affected animal. The mean score for the group 1 horses was 2.42; group 2 horses was 2.5; group 3 horses was 2.75; and group 4 horses was 3.25. Because the group 4 horses did not have a second transport, they were compared with all other groups. Statistically different scores were present between group 4 and groups 1 and 2. There was no difference in the time of seroconversion between groups. There was a difference between the time of onset of first clinical signs between groups 1 and 4. This difference was likely because of the different examination days. Differences in housing and handling were likely the reason for the differences in severity of clinical signs. This model results in consistent, significant clinical signs in all horses at approximately the same time period after inoculation but was most severe in horses that did not experience a second transport.

Experimental infection of ponies with Sarcocystis fayeri and differentiation from Sarcocystis neurona infections in horses.

Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in the Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses, whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In this study, 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 1 x 10(5) to 1 x 10(7) sporocysts of S. fayeri obtained from dogs that were fed naturally infected horse muscles. All ponies remained asymptomatic until the termination of the experiment, day 79 postinoculation (PI). All serum samples collected were negative for antibodies to S. neurona using the Western blot at the initial screening, just before inoculation with S. fayeri (day 2) and weekly until day 79 PI. Cerebrospinal fluid samples from each pony were negative for S. neurona antibodies. Using the S. neurona agglutination test, antibodies to S. neurona were not detected in 1:25 dilution of sera from any samples, except that from pony no. 4 on day 28; this pony had received 1 X 10(7) sporocysts. Using indirect immunofluorescence antibody tests (IFATs), 7 serum samples were found to be positive for S. neurona antibodies from 1:25 to 1:400 dilutions. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies, with heaviest infections in the tongue. All sarcocysts examined histologically appeared to contain only microcytes. Ultrastructurally, S. fayeri sarcocysts could be differentiated from S. neurona sarcocysts by the microtubules (mt) in villar protrusions on sarcocyst walls; in S. fayeri the mt extended from the villar tips to the pellicle of zoites, whereas in S. neurona the mt were restricted to the middle of the cyst wall. Results indicate that horses with S. fayeri infections may be misdiagnosed as being S. neurona infected using IFAT, and further research is needed on the serologic diagnosis of S. neurona infections.

Intrinsic thermal resistance of the canine brain.

Hyperthermia above a critical threshold results in multisystemic changes that include neurological manifestations of heat stroke. It is unknown if the latter represents an intrinsic thermal sensitivity of the CNS or whether injury is secondary to physiological responses of non-CNS origin. To address this issue, the present work examined functional, structural, and biochemical changes in the CNS of dogs subjected to a thermal dosage immediately below that which induces disseminated intravascular coagulation with secondary multiple organ injury. The experimental approach is previously reported, inducing a 42.5 degrees C, 90 min, whole body hyperthermia while preventing other physiological responses to treatment, including respiratory alkalosis and significant reductions in mean arterial pressure. Functional analyses included neurologic examinations and brainstem auditory evoked potential recordings in the post-treatment interval in both hyperthermic and euthermic control populations. Biochemical and structural analyses examined the expression of 70-kDa heat shock proteins, cytokines, markers of astroglial and microglial injury/activation, evidence of vascular endothelial damage, and evidence of neuronal and axonal injury in brain between 0.5 h and 8 days from the end of the treatment. The only significant change associated with treatment was induction of the major inducible 70-kDa heat shock protein, this being most prominent in the cerebellum with maximal expression at 6 h and a return to baseline by 8 days.Collectively, from these results we suggest that the canine brain is intrinsically resistant to sublethal hyperthermia such that when CNS lesions occur, they do so in the presence of other physiological derangements.

Life cycle of Sarcocystis neurona in its natural intermediate host, the raccoon, Procyon lotor.

Sarcocystis neurona causes encephalomyelitis in many species of mammals and is the most important cause of neurologic disease in the horse. Its complete life cycle is unknown, particularly its development and localization in the intermediate host. Recently, the raccoon (Procyon lotor) was recognized as a natural intermediate host of S. neurona. In the present study, migration and development of S. neurona was studied in 10 raccoons that were fed S. neurona sporocysts from experimentally infected opossums; 4 raccoons served as controls. Raccoons were examined at necropsy 1, 3, 5, 7, 10, 14, 15, 22, 37, and 77 days after feeding on sporocysts (DAFS). Tissue sections of most of the organs were studied histologically and reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical test. Parasitemia was demonstrated in peripheral blood of raccoons 3 and 5 DAFS. Individual zoites were seen in histologic sections of intestines of raccoons euthanized 1, 3, and 5 DAFS. Schizonts and merozoites were seen in many tissues 7 to 22 DAFS, particularly in the brain. Sarcocysts were seen in raccoons killed 22 DAFS. Sarcocysts at 22 DAFS were immature and seen only in skeletal muscle. Mature sarcocysts were seen in all skeletal samples, particularly in the tongue of the raccoon 77 DAFS; these sarcocysts were infective to laboratory-raised opossums. This is the first report of the complete development of S. neurona schizonts and sarcocysts in a natural intermediate host.

Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and experimental induction of neurologic disease in raccoons.

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses in the Americas and Sarcocystis neurona is the most common etiologic agent. The distribution of S. neurona infections follows the geographical distributions of its definitive hosts, opossums (Didelphis virginiana, Didelphis albiventris). Recently, cats and skunks were reported as experimental and armadillos as natural intermediate hosts of S. neurona. In the present report, raccoons (Procyon lotor) were identified as a natural intermediate host of S. neurona. Two laboratory-raised opossums were found to shed S. neurona-like sporocysts after ingesting tongues of naturally-infected raccoons. Interferon-gamma gene knockout (KO) mice fed raccoon-opossum-derived sporocysts developed neurologic signs. S. neurona was identified immunohistochemically in tissues of KO mice fed sporocysts and the parasite was isolated in cell cultures inoculated with infected KO mouse tissues. The DNA obtained from the tongue of a naturally-infected raccoon, brains of KO mice that had neurological signs, and from the organisms recovered in cell cultures inoculated with brains of neurologic KO mice, corresponded to that of S. neurona. Two raccoons fed mature S. neurona sarcocysts did not shed sporocysts in their feces, indicating raccoons are not likely to be its definitive host. Two raccoons fed sporocysts from opossum feces developed clinical illness and S. neurona-associated encephalomyelitis was found in raccoons killed 14 and 22 days after feeding sporocysts; schizonts and merozoites were seen in encephalitic lesions.

Diagnostic validity of electroencephalography in equine intracranial disorders.

Electroencephalography (EEG) is a valuable diagnostic test to identify functional disturbances in brain activity. The purpose of this study was to assess the validity of EEG as a diagnostic indicator of intracranial diseases in horses. The validity of EEG was estimated by comparing clinical, clinicopathologic, and histopathologic findings to EEG findings in 20 horses examined for seizures. collapse, or abnormal behavior between 1984 and 1997. A bipolar left-to-right, back-to-front montage and a bipolar circular montage were recorded from sedated (4) and anesthetized (16) horses. Visual and semiquantitative masked analysis of EEG recording Ist was validated on 10 horses presented for problems other than intracranial diseases. EEG pattern was normal in 7 of the 20 clinically affected horses. Abnormal EEG patterns included high-voltage slow waves and discrete paroxysmal activity with or without generalized activity in 13 horses. Histopathologic diagnoses in 10 horses included meningoencephalitis, neuronal necrosis, congenital anomalies. cerebral edema. and abscess. All of these horses had abnormal EEG patterns (sensitivity, 100%) with a positive neuroanatomic correlation in 7 animals. Localization of histopathologic and EEG abnormalities did not correlate in 15% of the horses (3/20). The cause of neurologic signs could not be explained at postmortem examination in 10 animals and the EEG pattern was normal in 7 of these horses (specificity, 70%). In conclusion, equine EEG was a sensitive tool in the diagnosis of intracranial disorders.

Utilization of stress in the development of an equine model for equine protozoal myeloencephalitis.

Neurologic disease in horses caused by Sarcocystis neurona is difficult to diagnose, treat, or prevent, due to the lack of knowledge about the pathogenesis of the disease. This in turn is confounded by the lack of a reliable equine model of equine protozoal myeloencephalitis (EPM). Epidemiologic studies have implicated stress as a risk factor for this disease, thus, the role of transport stress was evaluated for incorporation into an equine model for EPM. Sporocysts from feral opossums were bioassayed in interferon-gamma gene knockout (KO) mice to determine minimum number of viable S. neurona sporocysts in the inoculum. A minimum of 80,000 viable S. neurona sporocysts were fed to each of the nine horses. A total of 12 S. neurona antibody negative horses were divided into four groups (1-4). Three horses (group 1) were fed sporocysts on the day of arrival at the study site, three horses were fed sporocysts 14 days after acclimatization (group 2), three horses were given sporocysts and dexamethasone 14 days after acclimatization (group 3) and three horses were controls (group 4). All horses fed sporocysts in the study developed antibodies to S. neurona in serum and cerebrospinal fluid (CSF) and developed clinical signs of neurologic disease. The most severe clinical signs were in horses in group 1 subjected to transport stress. The least severe neurologic signs were in horses treated with dexamethasone (group 3). Clinical signs improved in four horses from two treatment groups by the time of euthanasia (group 1, day 44; group 3, day 47). Post-mortem examinations, and tissues that were collected for light microscopy, immunohistochemistry, tissue cultures, and bioassay in KO mice, revealed no direct evidence of S. neurona infection. However, there were lesions compatible with S. neurona infection in horses. The results of this investigation suggest that stress can play a role in the pathogenesis of EPM. There is also evidence to suggest that horses in nature may clear the organism routinely, which may explain the relatively high number of normal horses with CSF antibodies to S. neurona compared to the prevalence of EPM.

Alterations in hemostasis associated with hyperthermia in a canine model.

Use of hyperthermia in the treatment of cancer and viral infection has received renewed interest. However, the in vivo relationship between hyperthermia and direct versus indirect effects upon hemostasis are incompletely defined, although we do know that disseminated intravascular coagulation (DIC) is a common sequel to heat stroke. The purpose of the present study was to more precisely define the relationship between hyperthermia and derangements of hemostasis, thereby providing a guideline for the development of safe hyperthermia treatment regimens. The present investigation examined the in vivo effects of high-grade whole-body hyperthermia (WBH) (42.5 degrees C, 90 min) on hemostasis in a canine model. Induction of hyperthermia via extracorporeal circulation of heated blood (ECC-WBH) caused thrombocytopenia, increased plasma fibrin degradation products (FDPs), prolonged clotting times, increased serum liver enzymes, and evidence of spontaneous bleeding. However, when WBH was induced by peritoneal lavage (PL-WBH), transient thrombocytopenia was the only significant alteration. Temporal correlation between hemostatic alterations and elevations in serum alanine aminotransferase (ALT) levels in the ECC-WBH treatment group suggested that liver injury is responsible, at least in part, for the coagulopathy associated with high-grade hyperthermia and that in the absence of liver injury, identical degrees of hyperthermia cause only incidental decreases in platelet numbers.

Whole body hyperthermia: effects upon canine immune and hemostatic functions.

In this study, the effects of induced whole body hyperthermia (WBH; 42.3 degrees C for 90 min) on peripheral blood mononuclear cell (PBMC) phenotype distribution, in vitro blastogenic responsiveness and selected parameters of hemostasis were determined in dogs. Hyperthermia was induced by heating venous blood during extracorporeal circulation (EC); induction of WBH by peritoneal lavage (PL) and perfusion without heating (i.e. euthermic EC and euthermic PL) were used as controls. Whole body hyperthermia was associated with lymphopenia and thrombocytopenia that persisted throughout the eight-day post-treatment observation interval. The lymphopenia was selective in that CD5-positive T lymphocytes were more sensitive than were sIg-positive B cells and, within the T-cell compartment, suppressor (CD8-positive) cells were more sensitive to hyperthermic stress than helper (CD4 positive) lymphocytes. Lymphopenia was also observed in EC and PL euthermic controls, although that lymphopenia was transient and nonselective. Persistent suppression of T-cell phytomitogen-induced blastogenesis was induced by WBH in contrast to transient suppression in euthermic controls. For all treatment groups, lymphopenia and suppressed blastogenesis were correlated to elevated plasma cortisol levels. Induction of WBH by EC resulted in coagulopathy characterized by thrombocytopenia, increased plasma fibrin degradation products, prolonged clotting times, and evidence of spontaneous bleeding. These hemostatic alterations were correlated temporally to increased serum levels of liver enzymes. However, there was no evidence of hepatic injury when WBH was induced by PL, where transient thrombocytopenia was the only significant hemostatic alteration. These results indicate that 42.3 degrees C whole body hyperthermia can be well-tolerated and, although associated with suppression of general indexes of immunocompetence, is not associated with opportunistic infections.

The effects of extracorporeal whole body hyperthermia on the functional and phenotypic features of canine peripheral blood mononuclear cells (PBMC).

In this study the effect of transient 42.3 degrees C whole body hyperthermia (WBH) on the distribution of PBMC phenotypes and in vitro blastogenic responsiveness was determined in dogs. Hyperthermia (n = 6) was induced by heating venous blood during extracorporeal circulation (venous perfusion WBH); perfused non-heated dogs (n = 4) were used as controls. Both euthermic and hyperthermic perfusion produced transient lymphopenia which normalized in controls after perfusion but persisted in hyperthermic animals throughout the 8-day post-perfusion observation interval. The transient lymphopenia in control dogs was non-selective. In contrast, WBH-associated lymphopenia was selective, in that CD5+ T lymphocytes were more sensitive to hyperthermia than sIg+ B cells and, within the T cell compartment, suppressor (CD8+) cells were more sensitive to hyperthermic stress than helper (CD4+) lymphocytes. Functional analyses showed that WBH caused persistent suppression of PBMC blastogenesis in response to T cell phytomitogens. Increased plasma cortisol levels were correlated to peak lymphopenia and hyporesponsiveness to phytomitogens. Despite these alterations, high grade WBH was well tolerated and there was no evidence of opportunistic infection.

Constitutive overexpression of the major inducible 70 kDa heat shock protein mediates large plaque formation by measles virus.

Induction of the cellular stress response elevates cytoplasmic levels of heat shock proteins (HSPs) belonging to multiple families. When infected with canine distemper virus or measles virus (MV), cells containing elevated HSPs support increased viral gene expression and cytopathic effect. The present work tests the hypothesis that increases in the major inducible 70 kDa HSP (hsp72) are sufficient to mediate the effect of stress response induction on infection phenotype. Human astrocytoma cells (U373) were stably transfected with the human hsp72 gene under control of the beta-actin promoter. Constitutive overexpression of hsp72 was demonstrated in multiple clones by Western blot analysis of cytoplasmic total protein. Southern blot analysis of cell DNA confirmed the recovery of genetically distinct clones. Infection of these clonal populations with MV resulted in increased viral transcript production relative to infected control cell lines. Increased transcript production was associated with increased viral membrane glycoprotein expression and cytopathic effect (i.e., mean plaque area). Increases in cytopathic effect were due to the emergence of a large plaque phenotype from a small plaque-purified inoculum, mimicking the effect of cellular stress response induction upon viral infection phenotype. Large plaque phenotypic variants reported in the literature are associated with enhanced neurovirulence, a fact that highlights the potential significance of physiologic elevations in hsp72 (e.g., fever-induced) that accompany in vivo viral infection.

Results of postmortem examination of psittacine birds with cardiac disease: 26 cases (1991-1995).

OBJECTIVE: To characterize prevalence and type of cardiac disease evident in psittacine birds during postmortem examination. DESIGN: Retrospective study. ANIMALS: 26 psittacine birds with gross and histologic evidence of cardiac disease. PROCEDURE: Records of postmortem examinations of psittacine birds necropsied during a 4-year period were reviewed. Data on gross and histologic evidence of cardiac disease were analyzed. Birds identified included those in which congestive heart failure (CHF) was considered the primary cause of death and those in which substantial cardiac disease was evident, despite a lack of postmortem findings supportive of CHF. RESULTS: Of 269 psittacine birds necropsied, 26 (9.7%) had evidence of cardiac disease. In 15 (58%) birds with cardiac disease, changes consistent with CHF were evident and were sufficiently severe as to be considered the cause of death. The remaining 11 birds had cardiac lesions secondary to other systemic diseases; cardiac lesions were considered to be an incidental finding in these birds, and CHF was not evident. Of the 15 birds with CHF, 10 had evidence of right ventricular or biventricular failure, whereas only 5 had evidence of left ventricular failure. CLINICAL IMPLICATIONS: Prevalence of cardiac disease in the psittacine birds reported here was similar to that seen clinically in other companion animals. The high incidence of right ventricular or biventricular heart failure in psittacine birds was similar to that for poultry in which lesions of right-sided heart failure predominate.

Phosphorylation of canine distemper virus P protein by protein kinase C-zeta and casein kinase II.

Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-zeta) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-zeta has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-zeta has only minor activity. Selective inhibition of PKC-zeta activity within CV1 cells eliminated permissiveness to CDV replication, indicating an in vivo role for PKC-zeta in the virus replication cycle. The broad tissue expression of PKC-zeta parallels the pantropic nature of CDV infections, suggesting that PKC-zeta activity is a determinant of cellular permissiveness to CDV replication.