Spinal cord injury (SCI) in dogs is a well recognized animal model to study pathogenesis and trea... more Spinal cord injury (SCI) in dogs is a well recognized animal model to study pathogenesis and treatment modalities of the debilitating human disease. To define the contributing role of microglial cell activation to the secondary wave following SCI, microglia from 15 dogs with SCI confirmed by imaging, gross, and histopathological examination were isolated and characterized in terms of morphology, immunophenotype, and function ex vivo by flow cytometry, allowing single cell analysis. The results were compared to region-specific findings obtained from healthy control dogs. Light microscopy revealed a significant enhancement of myelinophagia within the traumatized spinal cord of dogs who had had SCI for ≥5 days. Immunophenotypical characterization revealed increased expression of B7-1, B7-2, MHC II, CD1c, ICAM 1, CD14, CD44, and CD45 emphasizing the enhanced function of microglia as co-stimulators of T cells, in leukocyte adhesion and aggregation, and for lipid or glycolipid presentation. In addition, phagocytosis and reactive oxygen species (ROS) generation were significantly increased in dogs with spinal cord trauma. Regional differences within the spinal cord were observed by demonstrating disparities in microglial immunophenotypes in the traumatized cervical compared to the thoracolumbar spinal cord. In contrast to histopathology, microglia activation analyzed on a single cell basis did not depend upon the time span following SCI.
The purpose of this study was to prove the hypothesis that C-reactive protein (CRP) and nerve gro... more The purpose of this study was to prove the hypothesis that C-reactive protein (CRP) and nerve growth factor (NGF) may be potential biomarkers for lower urinary tract disorders and may be able to distinguish between micturition dysfunctions of different origin in dogs with spinal cord diseases. NGF- and CRP- concentrations were measured in serum and urine samples using specific ELISA-Kits. Results in urine were standardized by urine-creatinine levels. CRP in serum was detectable in 32/76 and in urine samples in 40/76 patients. NGF could be measured in all serum and in 70/76 urine samples. Urinary CRP concentrations were significantly higher in dogs with micturition dysfunction (p = 0.0009) and in dogs with different neurological diseases (p = 0.0020) compared to the control group. However, comparing dogs with spinal cord disorders with and without associated micturition dysfunction no significant difference could be detected for NGF and CRP values in urine or serum samples. Additionally, levels did not decrease significantly, when measured at the time when the dogs regained the ability to urinate properly (urinary NGF p = 0.7962; urinary CRP p = 0.078). Urine samples with bacteria and/or leukocytes had no significant increase in urinary NGF (p = 0.1112) or CRP (p = 0.0534) concentrations, but higher CRP-levels in urine from dogs with cystitis were found compared to dogs without signs of cystitis. From these data we conclude that neither CRP nor NGF in urine or serum can be considered as reliable biomarkers for micturition disorders in dogs with spinal cord disorders in a clinical setting, but their production might be part of the pathogenesis of such disorders. Significantly higher levels of CRP could be found in the urine of dogs with micturition dysfunctions compared to control dogs. This phenomenon could potentially be explained by unspecific extrahepatic CRP production by smooth muscle cells in the dilated bladder.
DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcom... more DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤ 13) and late (≥ 66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage.
Differences in the regulation of surface molecule expression and functional activity of microglia... more Differences in the regulation of surface molecule expression and functional activity of microglia, the resident immune effector elements of the central nervous system (CNS), might give important insights into understanding the predilection sites of some diseases within the CNS. Therefore, canine microglial cells in relation to different topographical regions within the healthy CNS were evaluated ex vivo from the brain, cervical, and thoracolumbar spinal cord using density gradient centrifugation and flow cytometry in a homogenous dog population. Immunophenotypical characterization showed physiological regional differences for B7-1, CD14, CD44, CD1c, CD18, CD11b, and CD11c. Both, phagocytosis and ROS generation revealed differences between the brain, cervical, and thoracolumbar spinal cord. Our results emphasize that microglia displays physiological topographical regional differences within the CNS. The dog seems to be an ideal model to further investigate the role of microglia in focal pathological conditions of the spinal cord.
Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiolog... more Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiological factors contributing to epileptogenesis. Considering the development of combined anti-inflammatory and antioxidant treatment strategies with antiepileptogenic potential, a characterization of the time course of microglial reactive oxygen species generation during epileptogenesis is of major interest. Thus, we isolated microglia cells and analyzed the generation of reactive oxygen species by flow cytometric analysis in an electrical rat post-status epilepticus model. Two days post status epilepticus, a large-sized cell cluster exhibited a pronounced response with excessive production of reactive oxygen species upon stimulation with phorbol-myristate-acetate. Neither in the latency phase nor in the chronic phase with spontaneous seizures a comparable cell population with induction of reactive oxygen species was identified. We were able to demonstrate in the electrical rat post-status-epilepticus model, that microglial ROS generation reaches a peak after the initial insult, is only marginally increased in the latency phase, and returns to control levels during the chronic epileptic phase. The data suggest that a combination of anti-inflammatory and radical scavenging approaches might only be beneficial during a short time window after an epileptogenic brain insult.
Der seltene Befund eines kongenitalen sakrokokzygealen Dermoidsinus Typ IV bei einem 15 Monate al... more Der seltene Befund eines kongenitalen sakrokokzygealen Dermoidsinus Typ IV bei einem 15 Monate alten männlichen Rhodesian Ridgeback wird dargestellt. Die klinischen Symptome umfassten eine Fistelöffnung an der Schwanzwurzel, eine Hyperästhesie im Bereich der Rute und einen beidseitig geringgradig verminderten Flexorreflex der Hintergliedmaßen. Mittels Magnetresonanztomographie konnte die Diagnose gesichert und eine Verbindung des Hautsinus mit dem Duraschlauch bestätigt werden. Zur chirurgischen Resektion des Dermoidsinus waren eine dorsale Laminektomie vom zweiten Schwanzwirbel bis einschließlich des siebten Lendenwirbels sowie eine Durotomie erforderlich. Pathohistologisch wurde die klinische Diagnose Dermoidsinus bestätigt und eine Perineuritis im Bereich kokzygealer Nerven nachgewiesen. Die Wundheilung verlief per primam. 18 Monate nach der Operation ist der Hund ohne neurologische Ausfallserscheinungen und klinisch unauffällig.
Matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs, tissue inhibitors of matrix metalloprote... more Matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs, tissue inhibitors of matrix metalloproteinases), and the membrane-anchored glycoprotein RECK (reversion-inducing cysteine-rich protein with Kazal motifs) contribute to the pathogenesis of many CNS diseases. To assess the potential pathogenetic roles of microglial MMP, TIMP, and RECK generation in extracellular matrix breakdown, opening of the blood brain barrier (BBB) and subsequent recruitment of leukocytes in the CNS, twenty-four dogs suffering from spontaneously occurring different intracranial and extracranial (control group) diseases were examined. Microglia cells were isolated ex vivo by density gradient centrifugation and their expressions of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK were examined via quantitative real-time polymerase chain reaction (qPCR). Zymography on CNS tissues in selected cases was performed to assess differences at the protein level. Dogs were grouped in different disease categories according to histopathological examinations, in groups with or without inflammatory reactions, and in groups with/without contrast enhancement in advanced diagnostic imaging as a function of BBB breakdown. The results showed a significant up-regulation of MMP-9 in dogs with inflammation in the nervous system compared to dogs with non-inflammatory diseases. An increased expression of MMP-9 might lead to a facilitated invasion of white blood cells. Furthermore, down-regulation of MMP-13 was found in dogs with contrast enhancement. Zymographical data reflected MMP-2 qPCR data. In conclusion, differential expression of MMPs and their inhibitors, but not of RECK, which might crucially influence the pathogenesis of a given disease, could be demonstrated in canine microglia. This reflects a further pathway in the microglial repertoire to respond to various disease conditions in the CNS, a characteristic that might be of particular relevance as a target for specific treatments.
Veterinary Clinics of North America: Small Animal Practice, 2010
Discospondylitis and steroid-responsive meningitis-arteritis are the most frequently seen inflamm... more Discospondylitis and steroid-responsive meningitis-arteritis are the most frequently seen inflammatory diseases of the spine. Advanced imaging techniques and the examination of the cerebrospinal fluid are helpful to receive a definitive or presumptive diagnosis. Most inflammatory lesions are of unknown cause, but bacterial, fungal, or parasitic infections may be detected. Specific treatment depends on the cause.
SUMMARY: ››› Microglial cells are the immune effector elements of the central nervous system (CNS... more SUMMARY: ››› Microglial cells are the immune effector elements of the central nervous system (CNS). Every kind of patholo- gical stimulus in the CNS leads to their activation which enables microglial cells to initiate or mediate an adapted immune reaction. Due to the wide range of different rea- ctions performed after their activation, an influence of the pathogenesis of various diseases such as Alzheimer and Parkinson Disease, stroke, depression, and multiple sclero- sis is assumed. Canine distemper virus infection (CDV) is a well-established model for the demyelinating disease multiple sclerosis. By use of flow cytometry a pattern of upregulation of various surface molecules and an enhan- ced phagocytosis could be assessed in dogs infected with CDV. Moreover, a significantly enhanced generation of re- active oxygen species (ROS) could be shown in comparison with control animals and with dogs suffering from other in- tracranial diseases which were examined for comparison of microgl...
After peripheral nerve injury, Schwann cells (SC) guarantee for a regeneration-promoting milieu a... more After peripheral nerve injury, Schwann cells (SC) guarantee for a regeneration-promoting milieu and are crucially involved in axonal regeneration. For extended nerve defects, bridging with an autologous nerve transplant is the gold standard therapy. Artificial biohybrid nerve transplants which combine a synthetic conduit with autologous SC genetically modified to express regeneration-promoting proteins may provide an alternative therapy to autotransplantation. The dog seems to be an ideal translational animal model for new treatment strategies. In the present study, utilizing a new transfection protocol, we transplanted enhanced green fluorescent protein (EGFP)-expressing adult canine SC (cSC) into a 5 mm epineural pouch in the sciatic nerve of adult rats (n = 9). The epineurial pouch technique serves as proof of principle to follow the fate of the transplanted cSC for up to 14 days after surgery. Fluorescence microscopy and immunohistochemistry revealed survival and integration of EGFP-expressing cSC into the regenerating host nerve tissue. We demonstrate that transplanted cSC contribute to the formation of bands of Büngner and are located in close vicinity to growth-associated protein-43 (GAP-43) expressing regenerating nerve fibers. This provides first evidence that transplanted genetically modified Schwann cells do successfully integrate into the host tissue where they could actively contribute to the regeneration process.
Tierärztliche Praxis Kleintiere 4/2014 249 Kasuistik Schlüsselwörter Schädel-Hirn-Trauma, zystisc... more Tierärztliche Praxis Kleintiere 4/2014 249 Kasuistik Schlüsselwörter Schädel-Hirn-Trauma, zystische Großhirnläsion, posttraumatische Epi-lepsie, Verhaltensänderung, Magnetresonanztomographie Zusammenfassung Eine 15 Monate alte Große Schweizer Sennenhündin wurde nach ei-nem epileptischen Anfall vorgestellt. Die Besitzerin hatte zudem eine seit Kurzem bestehende Verhaltensänderung des Tieres bemerkt. Auf-grund des progressiven Verlaufs der neurologischen Symptome bei un-auffälligen Blutbefunden erfolgte eine magnetresonanztomographi-sche Untersuchung des Gehirns der Hündin. Hierbei ließ sich eine Por -enzephalie im Lobus parietalis der rechten Großhirnhemisphäre dia -gnostizieren. Das Tier wurde euthanasiert und pathologisch unter-sucht. Aufgrund des vorberichtlichen Hinweises auf ein Schädel-Hirn-Trauma im Welpenalter und der histopathologischen Befunde wird ei-ne traumatische Genese dieser seltenen zystischen Gehirnerkrankung diskutiert. Summary A 15-month-old female Greater Swiss M...
This article outlines the consensus proposal on diagnosis of epilepsy in dogs by the Internationa... more This article outlines the consensus proposal on diagnosis of epilepsy in dogs by the International Veterinary Epilepsy Task Force. The aim of this consensus proposal is to improve consistency in the diagnosis of epilepsy in the clinical and research settings. The diagnostic approach to the patient presenting with a history of suspected epileptic seizures incorporates two fundamental steps: to establish if the events the animal is demonstrating truly represent epileptic seizures and if so, to identify their underlying cause. Differentiation of epileptic seizures from other non-epileptic episodic paroxysmal events can be challenging. Criteria that can be used to make this differentiation are presented in detail and discussed. Criteria for the diagnosis of idiopathic epilepsy (IE) are described in a three-tier system. Tier I confidence level for the diagnosis of IE is based on a history of two or more unprovoked epileptic seizures occurring at least 24 h apart, age at epileptic seizure onset of between six months and six years, unremarkable inter-ictal physical and neurological examination, and no significant abnormalities on minimum data base blood tests and urinalysis. Tier II confidence level for the diagnosis of IE is based on the factors listed in tier I and unremarkable fasting and post-prandial bile acids, magnetic resonance imaging (MRI) of the brain (based on an epilepsy-specific brain MRI protocol) and cerebrospinal fluid (CSF) analysis. Tier III confidence level for the diagnosis of IE is based on the factors listed in tier I and II and identification of electroencephalographic abnormalities characteristic for seizure disorders. The authors recommend performing MRI of the brain and routine CSF analysis, after exclusion of reactive seizures, in dogs with age at epileptic seizure onset <6 months or >6 years, inter-ictal neurological abnormalities consistent with intracranial neurolocalisation, status epilepticus or cluster seizure at epileptic seizure onset, or a previous presumptive diagnosis of IE and drug-resistance with a single antiepileptic drug titrated to the highest tolerable dose. This consensus article represents the basis for a more standardised diagnostic approach to the seizure patient. These recommendations will evolve over time with advances in neuroimaging, electroencephalography, and molecular genetics of canine epilepsy.
Evidence of intervertebral disk degeneration (IVDD) is extremely common in dogs, and its prevalen... more Evidence of intervertebral disk degeneration (IVDD) is extremely common in dogs, and its prevalence increases with age. It has many important consequences because degeneration of the intervertebral disks often is a prelude to disk herniation, which can injure the spinal cord, spinal nerves, or both. This review summarizes the advances in diagnosis and treatment of IVDD that have been made since the 1950s when the first detailed description of the degenerative changes was published. It also discusses new approaches to treatment of the associated spinal cord injury and new methods by which to classify injury severity that are currently under development.
Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiolog... more Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiological factors contributing to epileptogenesis. Considering the development of combined anti-inflammatory and antioxidant treatment strategies with antiepileptogenic potential, a characterization of the time course of microglial reactive oxygen species generation during epileptogenesis is of major interest. Thus, we isolated microglia cells and analyzed the generation of reactive oxygen species by flow cytometric analysis in an electrical rat post-status epilepticus model. Two days post status epilepticus, a large-sized cell cluster exhibited a pronounced response with excessive production of reactive oxygen species upon stimulation with phorbol-myristate-acetate. Neither in the latency phase nor in the chronic phase with spontaneous seizures a comparable cell population with induction of reactive oxygen species was identified. We were able to demonstrate in the electrical rat post-status-epilepticus model, that microglial ROS generation reaches a peak after the initial insult, is only marginally increased in the latency phase, and returns to control levels during the chronic epileptic phase. The data suggest that a combination of anti-inflammatory and radical scavenging approaches might only be beneficial during a short time window after an epileptogenic brain insult.
A key common feature all but three known mammalian genera is the strict seven cervical vertebrae ... more A key common feature all but three known mammalian genera is the strict seven cervical vertebrae blueprint, suggesting the involvement of strong conserving selection forces during mammalian radiation. This is further supported by reports indicating that children with cervical ribs die before they reach reproductive age. Hypotheses were put up, associating cervical ribs (homeotic transformations) to embryonal cancer (e.g., neuroblastoma) or ascribing the constraint in cervical vertebral count to the development of the mammalian diaphragm. Here, we describe a spontaneous mutation c.196A > G in the Bos taurus T gene (also known as brachyury) associated with a cervical vertebral homeotic transformation that violates the fundamental mammalian cervical blueprint, but does not preclude reproduction of the affected individual. Genome-wide mapping, haplotype tracking within a large pedigree, resequencing of target genome regions, and bioinformatic analyses unambiguously confirmed the muta...
A 15-month-old female Greater Swiss Mountain Dog was presented after an epileptic episode. In add... more A 15-month-old female Greater Swiss Mountain Dog was presented after an epileptic episode. In addition, the owner had noticed a recent marked change in the animal's behaviour. Because of the progressive nature of the neurological signs, a magnetic resonance imaging scan of the brain was performed and porencephaly in the parietal lobe of the right hemisphere was diagnosed. The dog was euthanized and submitted for pathology. Because of the histopathological findings and the history of a craniocerebral injury whilst a puppy, a traumatic genesis of this rare cystic lesion is discussed.
Veterinary immunology and immunopathology, Jan 15, 2013
Canine distemper virus (CDV) infection causes immunosuppression and demyelinating leukoencephalit... more Canine distemper virus (CDV) infection causes immunosuppression and demyelinating leukoencephalitis in dogs. In viral diseases, an ambiguous function of regulatory T cells (Treg), with both beneficial effects by reducing immunopathology and detrimental effects by inhibiting antiviral immunity, has been described. However, the role of Treg in the pathogenesis of canine distemper remains unknown. In order to determine the effect of CDV upon immune homeostasis, the amount of Foxp3(+) Treg in spleen and brain of naturally infected dogs has been determined by immunohistochemistry. In addition, splenic cytokine expression has been quantified by reverse transcriptase polymerase chain reaction. Splenic depletion of Foxp3(+) Treg was associated with an increased mRNA-expression of tumor necrosis factor and decreased transcription of interleukin-2 in the acute disease phase, indicative of disturbed immunological counter regulation in peripheral lymphoid organs. In the brain, a lack of Foxp3(+...
Spinal cord injury (SCI) in dogs is a well recognized animal model to study pathogenesis and trea... more Spinal cord injury (SCI) in dogs is a well recognized animal model to study pathogenesis and treatment modalities of the debilitating human disease. To define the contributing role of microglial cell activation to the secondary wave following SCI, microglia from 15 dogs with SCI confirmed by imaging, gross, and histopathological examination were isolated and characterized in terms of morphology, immunophenotype, and function ex vivo by flow cytometry, allowing single cell analysis. The results were compared to region-specific findings obtained from healthy control dogs. Light microscopy revealed a significant enhancement of myelinophagia within the traumatized spinal cord of dogs who had had SCI for ≥5 days. Immunophenotypical characterization revealed increased expression of B7-1, B7-2, MHC II, CD1c, ICAM 1, CD14, CD44, and CD45 emphasizing the enhanced function of microglia as co-stimulators of T cells, in leukocyte adhesion and aggregation, and for lipid or glycolipid presentation. In addition, phagocytosis and reactive oxygen species (ROS) generation were significantly increased in dogs with spinal cord trauma. Regional differences within the spinal cord were observed by demonstrating disparities in microglial immunophenotypes in the traumatized cervical compared to the thoracolumbar spinal cord. In contrast to histopathology, microglia activation analyzed on a single cell basis did not depend upon the time span following SCI.
The purpose of this study was to prove the hypothesis that C-reactive protein (CRP) and nerve gro... more The purpose of this study was to prove the hypothesis that C-reactive protein (CRP) and nerve growth factor (NGF) may be potential biomarkers for lower urinary tract disorders and may be able to distinguish between micturition dysfunctions of different origin in dogs with spinal cord diseases. NGF- and CRP- concentrations were measured in serum and urine samples using specific ELISA-Kits. Results in urine were standardized by urine-creatinine levels. CRP in serum was detectable in 32/76 and in urine samples in 40/76 patients. NGF could be measured in all serum and in 70/76 urine samples. Urinary CRP concentrations were significantly higher in dogs with micturition dysfunction (p = 0.0009) and in dogs with different neurological diseases (p = 0.0020) compared to the control group. However, comparing dogs with spinal cord disorders with and without associated micturition dysfunction no significant difference could be detected for NGF and CRP values in urine or serum samples. Additionally, levels did not decrease significantly, when measured at the time when the dogs regained the ability to urinate properly (urinary NGF p = 0.7962; urinary CRP p = 0.078). Urine samples with bacteria and/or leukocytes had no significant increase in urinary NGF (p = 0.1112) or CRP (p = 0.0534) concentrations, but higher CRP-levels in urine from dogs with cystitis were found compared to dogs without signs of cystitis. From these data we conclude that neither CRP nor NGF in urine or serum can be considered as reliable biomarkers for micturition disorders in dogs with spinal cord disorders in a clinical setting, but their production might be part of the pathogenesis of such disorders. Significantly higher levels of CRP could be found in the urine of dogs with micturition dysfunctions compared to control dogs. This phenomenon could potentially be explained by unspecific extrahepatic CRP production by smooth muscle cells in the dilated bladder.
DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcom... more DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤ 13) and late (≥ 66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage.
Differences in the regulation of surface molecule expression and functional activity of microglia... more Differences in the regulation of surface molecule expression and functional activity of microglia, the resident immune effector elements of the central nervous system (CNS), might give important insights into understanding the predilection sites of some diseases within the CNS. Therefore, canine microglial cells in relation to different topographical regions within the healthy CNS were evaluated ex vivo from the brain, cervical, and thoracolumbar spinal cord using density gradient centrifugation and flow cytometry in a homogenous dog population. Immunophenotypical characterization showed physiological regional differences for B7-1, CD14, CD44, CD1c, CD18, CD11b, and CD11c. Both, phagocytosis and ROS generation revealed differences between the brain, cervical, and thoracolumbar spinal cord. Our results emphasize that microglia displays physiological topographical regional differences within the CNS. The dog seems to be an ideal model to further investigate the role of microglia in focal pathological conditions of the spinal cord.
Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiolog... more Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiological factors contributing to epileptogenesis. Considering the development of combined anti-inflammatory and antioxidant treatment strategies with antiepileptogenic potential, a characterization of the time course of microglial reactive oxygen species generation during epileptogenesis is of major interest. Thus, we isolated microglia cells and analyzed the generation of reactive oxygen species by flow cytometric analysis in an electrical rat post-status epilepticus model. Two days post status epilepticus, a large-sized cell cluster exhibited a pronounced response with excessive production of reactive oxygen species upon stimulation with phorbol-myristate-acetate. Neither in the latency phase nor in the chronic phase with spontaneous seizures a comparable cell population with induction of reactive oxygen species was identified. We were able to demonstrate in the electrical rat post-status-epilepticus model, that microglial ROS generation reaches a peak after the initial insult, is only marginally increased in the latency phase, and returns to control levels during the chronic epileptic phase. The data suggest that a combination of anti-inflammatory and radical scavenging approaches might only be beneficial during a short time window after an epileptogenic brain insult.
Der seltene Befund eines kongenitalen sakrokokzygealen Dermoidsinus Typ IV bei einem 15 Monate al... more Der seltene Befund eines kongenitalen sakrokokzygealen Dermoidsinus Typ IV bei einem 15 Monate alten männlichen Rhodesian Ridgeback wird dargestellt. Die klinischen Symptome umfassten eine Fistelöffnung an der Schwanzwurzel, eine Hyperästhesie im Bereich der Rute und einen beidseitig geringgradig verminderten Flexorreflex der Hintergliedmaßen. Mittels Magnetresonanztomographie konnte die Diagnose gesichert und eine Verbindung des Hautsinus mit dem Duraschlauch bestätigt werden. Zur chirurgischen Resektion des Dermoidsinus waren eine dorsale Laminektomie vom zweiten Schwanzwirbel bis einschließlich des siebten Lendenwirbels sowie eine Durotomie erforderlich. Pathohistologisch wurde die klinische Diagnose Dermoidsinus bestätigt und eine Perineuritis im Bereich kokzygealer Nerven nachgewiesen. Die Wundheilung verlief per primam. 18 Monate nach der Operation ist der Hund ohne neurologische Ausfallserscheinungen und klinisch unauffällig.
Matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs, tissue inhibitors of matrix metalloprote... more Matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs, tissue inhibitors of matrix metalloproteinases), and the membrane-anchored glycoprotein RECK (reversion-inducing cysteine-rich protein with Kazal motifs) contribute to the pathogenesis of many CNS diseases. To assess the potential pathogenetic roles of microglial MMP, TIMP, and RECK generation in extracellular matrix breakdown, opening of the blood brain barrier (BBB) and subsequent recruitment of leukocytes in the CNS, twenty-four dogs suffering from spontaneously occurring different intracranial and extracranial (control group) diseases were examined. Microglia cells were isolated ex vivo by density gradient centrifugation and their expressions of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK were examined via quantitative real-time polymerase chain reaction (qPCR). Zymography on CNS tissues in selected cases was performed to assess differences at the protein level. Dogs were grouped in different disease categories according to histopathological examinations, in groups with or without inflammatory reactions, and in groups with/without contrast enhancement in advanced diagnostic imaging as a function of BBB breakdown. The results showed a significant up-regulation of MMP-9 in dogs with inflammation in the nervous system compared to dogs with non-inflammatory diseases. An increased expression of MMP-9 might lead to a facilitated invasion of white blood cells. Furthermore, down-regulation of MMP-13 was found in dogs with contrast enhancement. Zymographical data reflected MMP-2 qPCR data. In conclusion, differential expression of MMPs and their inhibitors, but not of RECK, which might crucially influence the pathogenesis of a given disease, could be demonstrated in canine microglia. This reflects a further pathway in the microglial repertoire to respond to various disease conditions in the CNS, a characteristic that might be of particular relevance as a target for specific treatments.
Veterinary Clinics of North America: Small Animal Practice, 2010
Discospondylitis and steroid-responsive meningitis-arteritis are the most frequently seen inflamm... more Discospondylitis and steroid-responsive meningitis-arteritis are the most frequently seen inflammatory diseases of the spine. Advanced imaging techniques and the examination of the cerebrospinal fluid are helpful to receive a definitive or presumptive diagnosis. Most inflammatory lesions are of unknown cause, but bacterial, fungal, or parasitic infections may be detected. Specific treatment depends on the cause.
SUMMARY: ››› Microglial cells are the immune effector elements of the central nervous system (CNS... more SUMMARY: ››› Microglial cells are the immune effector elements of the central nervous system (CNS). Every kind of patholo- gical stimulus in the CNS leads to their activation which enables microglial cells to initiate or mediate an adapted immune reaction. Due to the wide range of different rea- ctions performed after their activation, an influence of the pathogenesis of various diseases such as Alzheimer and Parkinson Disease, stroke, depression, and multiple sclero- sis is assumed. Canine distemper virus infection (CDV) is a well-established model for the demyelinating disease multiple sclerosis. By use of flow cytometry a pattern of upregulation of various surface molecules and an enhan- ced phagocytosis could be assessed in dogs infected with CDV. Moreover, a significantly enhanced generation of re- active oxygen species (ROS) could be shown in comparison with control animals and with dogs suffering from other in- tracranial diseases which were examined for comparison of microgl...
After peripheral nerve injury, Schwann cells (SC) guarantee for a regeneration-promoting milieu a... more After peripheral nerve injury, Schwann cells (SC) guarantee for a regeneration-promoting milieu and are crucially involved in axonal regeneration. For extended nerve defects, bridging with an autologous nerve transplant is the gold standard therapy. Artificial biohybrid nerve transplants which combine a synthetic conduit with autologous SC genetically modified to express regeneration-promoting proteins may provide an alternative therapy to autotransplantation. The dog seems to be an ideal translational animal model for new treatment strategies. In the present study, utilizing a new transfection protocol, we transplanted enhanced green fluorescent protein (EGFP)-expressing adult canine SC (cSC) into a 5 mm epineural pouch in the sciatic nerve of adult rats (n = 9). The epineurial pouch technique serves as proof of principle to follow the fate of the transplanted cSC for up to 14 days after surgery. Fluorescence microscopy and immunohistochemistry revealed survival and integration of EGFP-expressing cSC into the regenerating host nerve tissue. We demonstrate that transplanted cSC contribute to the formation of bands of Büngner and are located in close vicinity to growth-associated protein-43 (GAP-43) expressing regenerating nerve fibers. This provides first evidence that transplanted genetically modified Schwann cells do successfully integrate into the host tissue where they could actively contribute to the regeneration process.
Tierärztliche Praxis Kleintiere 4/2014 249 Kasuistik Schlüsselwörter Schädel-Hirn-Trauma, zystisc... more Tierärztliche Praxis Kleintiere 4/2014 249 Kasuistik Schlüsselwörter Schädel-Hirn-Trauma, zystische Großhirnläsion, posttraumatische Epi-lepsie, Verhaltensänderung, Magnetresonanztomographie Zusammenfassung Eine 15 Monate alte Große Schweizer Sennenhündin wurde nach ei-nem epileptischen Anfall vorgestellt. Die Besitzerin hatte zudem eine seit Kurzem bestehende Verhaltensänderung des Tieres bemerkt. Auf-grund des progressiven Verlaufs der neurologischen Symptome bei un-auffälligen Blutbefunden erfolgte eine magnetresonanztomographi-sche Untersuchung des Gehirns der Hündin. Hierbei ließ sich eine Por -enzephalie im Lobus parietalis der rechten Großhirnhemisphäre dia -gnostizieren. Das Tier wurde euthanasiert und pathologisch unter-sucht. Aufgrund des vorberichtlichen Hinweises auf ein Schädel-Hirn-Trauma im Welpenalter und der histopathologischen Befunde wird ei-ne traumatische Genese dieser seltenen zystischen Gehirnerkrankung diskutiert. Summary A 15-month-old female Greater Swiss M...
This article outlines the consensus proposal on diagnosis of epilepsy in dogs by the Internationa... more This article outlines the consensus proposal on diagnosis of epilepsy in dogs by the International Veterinary Epilepsy Task Force. The aim of this consensus proposal is to improve consistency in the diagnosis of epilepsy in the clinical and research settings. The diagnostic approach to the patient presenting with a history of suspected epileptic seizures incorporates two fundamental steps: to establish if the events the animal is demonstrating truly represent epileptic seizures and if so, to identify their underlying cause. Differentiation of epileptic seizures from other non-epileptic episodic paroxysmal events can be challenging. Criteria that can be used to make this differentiation are presented in detail and discussed. Criteria for the diagnosis of idiopathic epilepsy (IE) are described in a three-tier system. Tier I confidence level for the diagnosis of IE is based on a history of two or more unprovoked epileptic seizures occurring at least 24 h apart, age at epileptic seizure onset of between six months and six years, unremarkable inter-ictal physical and neurological examination, and no significant abnormalities on minimum data base blood tests and urinalysis. Tier II confidence level for the diagnosis of IE is based on the factors listed in tier I and unremarkable fasting and post-prandial bile acids, magnetic resonance imaging (MRI) of the brain (based on an epilepsy-specific brain MRI protocol) and cerebrospinal fluid (CSF) analysis. Tier III confidence level for the diagnosis of IE is based on the factors listed in tier I and II and identification of electroencephalographic abnormalities characteristic for seizure disorders. The authors recommend performing MRI of the brain and routine CSF analysis, after exclusion of reactive seizures, in dogs with age at epileptic seizure onset <6 months or >6 years, inter-ictal neurological abnormalities consistent with intracranial neurolocalisation, status epilepticus or cluster seizure at epileptic seizure onset, or a previous presumptive diagnosis of IE and drug-resistance with a single antiepileptic drug titrated to the highest tolerable dose. This consensus article represents the basis for a more standardised diagnostic approach to the seizure patient. These recommendations will evolve over time with advances in neuroimaging, electroencephalography, and molecular genetics of canine epilepsy.
Evidence of intervertebral disk degeneration (IVDD) is extremely common in dogs, and its prevalen... more Evidence of intervertebral disk degeneration (IVDD) is extremely common in dogs, and its prevalence increases with age. It has many important consequences because degeneration of the intervertebral disks often is a prelude to disk herniation, which can injure the spinal cord, spinal nerves, or both. This review summarizes the advances in diagnosis and treatment of IVDD that have been made since the 1950s when the first detailed description of the degenerative changes was published. It also discusses new approaches to treatment of the associated spinal cord injury and new methods by which to classify injury severity that are currently under development.
Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiolog... more Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiological factors contributing to epileptogenesis. Considering the development of combined anti-inflammatory and antioxidant treatment strategies with antiepileptogenic potential, a characterization of the time course of microglial reactive oxygen species generation during epileptogenesis is of major interest. Thus, we isolated microglia cells and analyzed the generation of reactive oxygen species by flow cytometric analysis in an electrical rat post-status epilepticus model. Two days post status epilepticus, a large-sized cell cluster exhibited a pronounced response with excessive production of reactive oxygen species upon stimulation with phorbol-myristate-acetate. Neither in the latency phase nor in the chronic phase with spontaneous seizures a comparable cell population with induction of reactive oxygen species was identified. We were able to demonstrate in the electrical rat post-status-epilepticus model, that microglial ROS generation reaches a peak after the initial insult, is only marginally increased in the latency phase, and returns to control levels during the chronic epileptic phase. The data suggest that a combination of anti-inflammatory and radical scavenging approaches might only be beneficial during a short time window after an epileptogenic brain insult.
A key common feature all but three known mammalian genera is the strict seven cervical vertebrae ... more A key common feature all but three known mammalian genera is the strict seven cervical vertebrae blueprint, suggesting the involvement of strong conserving selection forces during mammalian radiation. This is further supported by reports indicating that children with cervical ribs die before they reach reproductive age. Hypotheses were put up, associating cervical ribs (homeotic transformations) to embryonal cancer (e.g., neuroblastoma) or ascribing the constraint in cervical vertebral count to the development of the mammalian diaphragm. Here, we describe a spontaneous mutation c.196A > G in the Bos taurus T gene (also known as brachyury) associated with a cervical vertebral homeotic transformation that violates the fundamental mammalian cervical blueprint, but does not preclude reproduction of the affected individual. Genome-wide mapping, haplotype tracking within a large pedigree, resequencing of target genome regions, and bioinformatic analyses unambiguously confirmed the muta...
A 15-month-old female Greater Swiss Mountain Dog was presented after an epileptic episode. In add... more A 15-month-old female Greater Swiss Mountain Dog was presented after an epileptic episode. In addition, the owner had noticed a recent marked change in the animal's behaviour. Because of the progressive nature of the neurological signs, a magnetic resonance imaging scan of the brain was performed and porencephaly in the parietal lobe of the right hemisphere was diagnosed. The dog was euthanized and submitted for pathology. Because of the histopathological findings and the history of a craniocerebral injury whilst a puppy, a traumatic genesis of this rare cystic lesion is discussed.
Veterinary immunology and immunopathology, Jan 15, 2013
Canine distemper virus (CDV) infection causes immunosuppression and demyelinating leukoencephalit... more Canine distemper virus (CDV) infection causes immunosuppression and demyelinating leukoencephalitis in dogs. In viral diseases, an ambiguous function of regulatory T cells (Treg), with both beneficial effects by reducing immunopathology and detrimental effects by inhibiting antiviral immunity, has been described. However, the role of Treg in the pathogenesis of canine distemper remains unknown. In order to determine the effect of CDV upon immune homeostasis, the amount of Foxp3(+) Treg in spleen and brain of naturally infected dogs has been determined by immunohistochemistry. In addition, splenic cytokine expression has been quantified by reverse transcriptase polymerase chain reaction. Splenic depletion of Foxp3(+) Treg was associated with an increased mRNA-expression of tumor necrosis factor and decreased transcription of interleukin-2 in the acute disease phase, indicative of disturbed immunological counter regulation in peripheral lymphoid organs. In the brain, a lack of Foxp3(+...
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