Aromatase expression in the cerebellum of the dog infected with canine distemper virus.

Aromatase is the enzyme that catalyzes the biosynthesis of estrogens. It is implicated in neuroprotection.The present study investigated aromatase expression in the cerebellum of dogs infected with canine distemper virus (CDV), a disease characterized by demyelination in the white matter of the cerebellum. The presence of CDV infection was confirmed on the basis of histopathology and immunohistochemical localization of CDV antigen in glial cells of the white matter.The number of aromatase immunoreactive astrocytes were significantly (p < 0.05) higher in CDV-infected dogs compared to control dogs. The results suggest that astrocytes respond to invasion and persistence of CDV by means of increased estrogen production.The results also suggest that the high level of estrogen expression is maintained similarly throughout all stages of the disease since the number of aromatase immunoreactive astrocytes did not vary during the different stages of CDV infection.

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

Canine distemper virus induces apoptosis through caspase-3 and -8 activation in vero cells.

We investigated the signal-transduction pathway of canine distemper virus-Onderstepoort (CDV-Ond) vaccine strain-mediated apoptosis in Vero cells. Canine distemper virus-Onderstepoort at a multiplicity of infection (MOI) of 0.1 induced DNA fragmentation 48 h after infection. Immunofluorescence staining revealed that 57% +/- 4% of the CDV-N-protein-positive cells were terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive, and all TUNEL-positive cells were CDV-N-protein-positive, indicating that CDV-Ond induced apoptosis only in the infected cells. We also found that CDV-Ond infection induced activation of caspase-3 and caspase-8. In the semi-quantitative reverse transcription-polymerase chain reaction assay for apoptosis-related genes, the expression of mRNA of the death receptor, Fas, was also increased in CDV-Ond-infected cells. In contrast, the expressions of Bcl-2 and Bax, regulators for intrinsic apoptotic signaling through the mitochondria, did not change. These results suggest that CDV-Ond induced apoptosis by activating caspase-3, possibly through caspase-8 signaling rather than through p53/Bax-mediated, mitochondrial signaling in the infected cells.

Roles of an extracellular matrix (ECM) receptor and ECM processing enzymes in demyelinating canine distemper encephalitis.

Canine distemper virus (CDV) belongs to the genus Morbillivirus of the Paramyxoviridae family. Due to the central nervous system (CNS) tropism of the virus and associated neuropathological changes, demyelinating canine distemper encephalitis (CDE) represents a relevant model for human demyelinating diseases like multiple sclerosis. The present review decribes the role of CD44 antigen (CD44), the principle cell surface receptor for hyaluronate and extracellular matrix (ECM) processing enzymes (matrix metalloproteinases [MMPs]) and their inhibitors (TIMPs) in the pathogenesis of demyelination. In acute and subacute CDE, a plaque-associated CD44 up-regulation is found that parallels astrocyte activation. Likewise, MMPs and TIMPs are prominently up-regulated in these lesions and are expressed mostly by astrocytes and microglia. In chronic lesions, CD44 expression declines together with the number of glial fibrillary acidic protein (GFAP) positive astrocytes. In addition, in this plaque type, CD44 is expressed on the cell membrane of perivascular mononuclear cells. In this phase, a decrease of MMP and TIMP expressions apart from MMP-11, -12, and -13 is obvious. In summary, CD44 and MMPs might be associated with the onset of demyelination and may interact to initiate ECM disturbances. Ligation of CD44 in the early phase may induce chemokines and cytokines and hence initiate and perpetuate the inflammatory process. In the chronic phase, it is conceivable that a MMP-TIMP imbalance may be the motor for lesion progression with a simultaneous influx of CD44-positive activated immune cells.

Up-regulation of mRNA for matrix metalloproteinases-9 and -14 in advanced lesions of demyelinating canine distemper leukoencephalitis.

Matrix metalloproteinases (MMPs) comprise a family of proteolytic zinc- and calcium-dependent enzymes that are capable of disrupting the blood-brain barrier and mediating the destruction of extracellular matrix and myelin components. MMPs are also involved in facilitating leukocyte migration into inflammatory sites of the central nervous system. To determine the cellular localization and the amount of mRNA for MMP-9, MMP-14 and a tissue inhibitor of metalloproteinases (TIMP-1) in dogs with spontaneous demyelinating distemper encephalitis, formalin-fixed paraffin-embedded cerebella were investigated by in situ hybridization using specific digoxigenin-labeled RNA probes. Additionally, immunohistochemistry was performed to characterize the different types of plaques of demyelinating leukoencephalitis. Furthermore, virus antigen and mRNA were detected by immunohistochemistry and in situ hybridization. Healthy control dogs revealed a weak signal for mRNA for MMP-9, MMP-14, and TIMP-1 in various numbers of neurons, astrocytes, microglial cells and oligodendrocytes. In the cerebella of dogs with distemper, a strong increase of both number and staining intensity of MMP-9, MMP-14, and TIMP-1 mRNA-expressing cells, mainly in subacute inflammatory lesions and chronic plaques, was observed. The number of cells expressing mRNA for MMP-9 and MMP-14 increased about two- to threefold compared to TIMP-1 mRNA-expressing cells, whereas staining intensity of individual cells was similar. In early lesions, especially astrocytes and activated macrophages/microglial cells displayed a positive signal for MMPs and TIMP-1, whereas in older lesions activated microglia/macrophages and infiltrating lymphocytes represented the main source for MMP-9, MMP-14, and TIMP-1 mRNA synthesis as revealed by double-labeling techniques. In summary, the proportionally higher increase of MMP mRNA-expressing cells might indicate an MMP/TIMP imbalance as a cause for lesion initiation and progression in demyelinating canine distemper leukoencephalitis.

Phase-dependent expression of matrix metalloproteinases and their inhibitors in demyelinating canine distemper encephalitis.

Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent enzymes that cleave molecules of the extracellular matrix, and thus are able to open the blood-brain-barrier and affect myelin. Their inhibitors (TIMPs) are important candidates for the therapy of demyelinating diseases. To establish an immunohistochemical profile of MMP and TIMP expression in plaque variants in dogs with spontaneous demyelinating distemper encephalitis, paraffin-embedded cerebella were studied employing the avidin-biotin-peroxidase complex method with a panel of nine polyclonal (anti-MMP-1, -3, -7, -9, -12, -13, -14, -TIMP-1, and -2) and two monoclonal antibodies (anti-latent MMP-2, and -MMP-11). All MMPs and TIMPs were prominently up-regulated in acute and subacute non-inflammatory lesions, and double-labeling techniques showed that they were mainly expressed by astrocytes and brain macrophages/microglia. In subacute inflammatory and chronic plaques, a moderate to strong decrease of MMP and TIMP expression compared to acute lesions was observed. In these phases MMP-11, -12, and -13 were still moderately present. In addition to astro- and microglia, invading perivascular mononuclear cells were positive for MMPs and TIMPs. In summary, there seems to be a phase-dependent expression of MMPs and TIMPs in demyelinating canine distemper encephalitis, and an MMP-TIMP imbalance might account for the lesion progression in this disease.

Distribution of 3beta-hydroxysteroid dehydrogenase in the cerebellum in canine distemper virus infection.

The cerebella of eight dogs naturally infected with canine distemper virus (CDV) and two normal dogs were examined immunohistochemically for glial fibrillary acidic protein (GFAP) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The clinical diagnosis of canine distemper was confirmed histopathologically and by the immunohistochemical demonstration of CDV antigen. In all dogs (healthy and infected), the Purkinje cells of the cerebellum were immunolabelled for 3beta-HSD activity. In infected dogs, 3beta-HSD labelling was prominent in astrocytes (particularly in areas of astrocytosis) whereas in healthy dogs such immunolabelling was weak. Double immunolabelling demonstrated that all GFAP-positive cells (especially in demyelinating areas) were also positive for 3beta-HSD. The results suggest that 3beta-HSD expression by astrocytes is associated with demyelination in CDV infection.

Inhibition of tyrosine hydroxylase expression within the substantia nigra of mice infected with canine distemper virus.

Experimental infection of mouse brain with a neuroadapted strain of canine distemper virus (CDV) leads to early acute encephalitis, followed by late neurological diseases such as motor pathologies (paralysis and turning behavior) or obesity syndrome. We have previously shown that, during the early stage of infection, CDV replicates transiently in selective structures of the brain including the substantia nigra, a structure known to play a critical role in motor control. In this study we demonstrate that CDV replication in the substantia nigra induces an early decrease in transcript level of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. The CDV infection of neuroblastoma cell culture, constitutively expressing TH, results in downregulation of TH transcription in the absence of cell death. In the few surviving mice with motor deficiencies, a pronounced decrease in TH expression is associated with a loss of dopaminergic cell bodies in the absence of any viral transcripts and proteins, suggesting that the initial CDV infection was sufficient to trigger irreversible neurodegenerative processes.

Plasmalogenase is elevated in early demyelinating lesions.

Plasmalogenase catalyzes the hydrolysis of ethanolamine plasmalogens to long-chain aldehydes and 2-acyl-sn-glycero-3-phosphoethanolamines. During development, plasmalogenase activity parallels myelination. The enzyme is most concentrated within oligodendroglial cells and is absent from myelin. The normal function of plasmalogenase in white matter may be related to its specificity for plasmalogens that contain most of the thromboxane and prostaglandin precursors. Plasmalogenase activities are elevated in demyelinating CNS tissues including canine white matter with lesions due to distemper virus. Elevated plasmalogenase activity precedes cellular invasion and lysosomal activation as indicated by beta-glucuronidase, acid proteinase and neutral proteinase activities. The elevation of plasmalogenase activity was 4.9-fold greater than normal in an early demyelinating lesion caused by the Snyder-Hill strain of distemper virus. Phospholipases acting on phosphatidyl ethanolamine were not activated in this tissue and have activities much lower than plasmalogenase in control tissues. Plasmalogenase activities are also elevated after intracerebral injections of complement-dependent anti-myelin antibody and after ischemia. Plasmalogenase acting on the oligodendrocyte plasma membrane may be responsible for necrosis of the oligodendrocyte that results in demyelination.