To comparatively evaluate molecular and morphological changes in a simplified environment devoid of peripheral blood supply, organotypic slice cultures of the canine spinal cord have been established as an in vitro model for canine SCI.
Organotypic slice cultures of CNS tissue can be regarded as an intermediate model between dissociated cell cultures and animal experiments (Holopainen, 2005;
Huuskonen et al., 2005). Spinal cord slices of different species have been cultured previously (Casha et al., 2005; Stavridis et al., 2005; Jeong et al., 2011; Imbschweiler et al., 2012).
Spinal cord slices have been established as an in vitro model for SCI by several investigators (Krassioukov et al., 2002; Pan et al., 2002; Casha et al., 2005; Jeong et al., 2011). In the present study, organotypic spinal cord slices of young adult dogs were cultivated for 0, 3, and 9 days, respectively, roughly reflecting the groups of the in vivo study. In this design, slices immediately embedded after the initial chopping process (0 days) were regarded as the control tissue in which secondary injury processes can be assumed to be minimal. The cultivation of canine spinal cord slices was associated with immune processes highly reflecting the findings in naturally traumatized dogs and thus validating the chosen in vitro approach. Slices incubated for 9 days exhibited a similar up-regulation of MHC class II on phagocytic cells. In fact, it is known that microglial cells preserve their ability to transform to an amoeboid
morphology, form multinucleated giant cells, and enhance their expression of MHC class II when exposed to membranes of neuronal or glial origin in vitro (Beyer et al., 2000). In contrast, phagocytosis of latex beads induced an amoeboid morphology but did not cause an increase in the expression of immunologically relevant molecules (Beyer et al., 2000). As early as 1996 it was demonstrated that microglial cells exhibit an activated morphology after 3 days in vitro in cultured hippocampal slices (Hailer et al., 1996). Moreover, treatment with IFN-γ has shown to induce MHC class II-expression on microglia in hippocampal slices of neonatal rats (Neumann et al., 1996).
The spontaneous expression of MHC class II on phagocytic cells in the present study might reflect a tissue response to the trauma that is caused by the production of slices and thus substantiates organotypic spinal cord slice cultures as an in vitro model for secondary injury processes during SCI.
Interestingly, the cultivation of slices was associated with a prominent up-regulation of inflammatory cytokines (TNF, IL-6, IL-8, TGF-β) during the entire culturing period and a comparatively transient up-regulation of IL-10 on day 3 in culture. Compared to the situation in naturally traumatized dogs there is thus a similar expression of inflammatory cytokines. However, in slice cultures it is prolonged. This might implicate that intrinsic regulation mechanisms in vivo might counteract a prolonged expression of potentially harmful cytokines in the subacute disease phase during naturally occurring SCI. Despite such differences in the cytokine orchestration, the cultivation of canine spinal cord slices clearly demonstrates that resident CNS cells are capable to produce the investigated cytokines. Similarly, acute slices of adult rats exhibit an immediate induction of the pro-inflammatory cytokines IL-1α, IL-1β, TNF, and IL-6, consistent with a local tissue source (Pan et al., 2002). Spinal cord slice cultures are devoid of the peripheral blood supply, thus, molecular and morphological immune processes are vastly restricted to endogenous CNS cells (Koshinaga et al., 2000). Collectively, the results obtained in cultured spinal cord slices are similar to those derived from dogs with IVDD related SCI. This substantiates the hypothesis that resident CNS cells, and here predominantly microglial cells, play a pivotal, though temporally dependent role during SCI.
With regard to axonal pathology, there was a similar swelling of axons in cultivated spinal cord slices as detected by hematoxylin and eosin (HE) staining. However, immunohistochemically, compared to the in vivo situation axonal damage was exclusively characterized by axonal expression of n-NF whereas β-APP and GAP-43 were not detected. This difference might result from the fact that the kind of primary injury is different in slice cultures and IVDD: the production of slices leads to immediate axotomy and separates the axon from its perikaryon. This might explain why there were neither hints for axonal regeneration nor accumulation of proteins that need to undergo axonal transport mechanisms. The axonal expression of n-NF however demonstrates that changes in the phosphorylation of axonal cytoskeletal intermediate filaments may additionally be a process that occurs locally in axon fragments disconnected from their neuronal perikaryon.
Similar to naturally traumatized dogs the cultivation of slices was accompanied by a temporally distinct expression of MMP-2 and MMP-9 mRNA. As it is true for the detected cytokines this underlines the hypothesis that local tissue cells may function as the main source of such molecules. Here, activated microglial cells might be the main candidate as these cells are known to be capable of the expression of both gelatinases (Stein et al., 2011, 2012). In this respect, a differential expression of MMPs including MMP-2 and MMP-9 was recently demonstrated in isolated microglial cells from dogs suffering from spontaneously occurring different intracranial and extracranial diseases (Stein et al., 2011).
Conclusively the obtained results substantiate that organotypic spinal cord slice cultures represent a valuable simplified in vitro model to study the local molecular and morphological response to CNS tissue injury and stress.
7 Chapter 7: Summary
Ingo Spitzbarth - A morphological and molecular investigation on the spatio-temporal development of spinal cord lesions following spinal injury in dogs
Spinal cord injury (SCI) represents a common and devastating traumatic neurological disease that requires the development of novel therapeutic strategies such as cell transplantation techniques. Most data on secondary injury processes during SCI are based on well established experimental rodent models; however, knowledge of the underlying pathogenetical processes during naturally occurring SCI is strikingly sparse. Here, the impact of the immune response including the cytokine orchestration during SCI on the local tissue integrity is discussed controversially. In addition, axonal damage is a central hallmark of SCI and is one of the major causes for the clinical disability of affected patients. Data on the spatio-temporal development of immune processes and axonal alterations during clinical SCI are insufficient. Dogs represent a species that is commonly affected by naturally occurring SCI, most often due to intervertebral disk disease (IVDD) and have recently gained increasing importance as a translational animal model for human SCI. The spontaneity of canine SCI may bridge the gap between results obtained in rodent models and clinical SCI.
The aims of the present study were (i) to comparatively review the current understanding of the cytokine orchestration during spontaneous canine central nervous system (CNS) diseases followed by (ii) an investigation of the spatio-temporal development of immunophenotypical changes, the local cytokine orchestration, and axonal pathology in dogs suffering from naturally occurring SCI, and (iii) to comparatively evaluate the results including the cytokine response in an in vitro model for SCI using cultivated organotypic spinal cord slices devoid of peripheral blood supply. A multidirectional approach using histology, transmission electron microscopy, immunohistochemistry, and reverse transcriptase quantitative polymerase chain reaction was applied. Spinal cord tissue of dogs with acute SCI
(n=9), subacute SCI (n=8), and neurologically healthy control animals (n=10) was investigated.
A time-dependent increase of major histocompatibility complex (MHC) class II-expression was noted on gitter cells during subacute canine SCI. In parallel, a significant up-regulation of the pro-inflammatory cytokines interleukin (IL)-6, IL-8 and a trend of up-regulation of tumor necrosis factor during the acute phase of naturally occurring SCI was detected. In contrast, expression of neuroprotective cytokines remained unchanged (IL-10) or showed a delayed up-regulation (transforming growth factor-β). The hypothesis that resident cells play a pivotal role during the immune response of SCI was verified using organotypic spinal cord slices which displayed a similar activation of MHC class II positive microglia with a distinct but prolonged up-regulation of inflammatory cytokines. Moreover, the local cytokine orchestration during canine SCI showed high similarities to other canine CNS diseases such as canine distemper leukoencephalitis, suggesting that a derailment of the molecular immune response might be a key feature during canine CNS injury, independent from the disease entity.
Axonal pathology during naturally occurring canine SCI was characterized by an enhanced axonal expression of non-phosphorylated neurofilament (n-NF), and β-amyloid precursor protein (β-APP) that exhibited a significant time-dependent spatial progression in segments distal to the lesion epicenter during subacute SCI.
Potentially suggestive of axonal regeneration attempts, significant axonal expression of growth-associated protein (GAP)-43 during acute and subacute SCI was noted, verified by immunoelectron microscopy. In organotypic slices axonal pathology was restricted to enhanced expression of n-NF suggestive of a local axonal phenomenon.
MBP immunoreactivity was decreased exclusively in dogs with subacute naturally occurring SCI. Ultrastructurally, axons of traumatized dogs predominantly displayed degenerative axoplasmic alterations including neurofilament and dense body accumulations. Axonopathy was paralleld by dysregulated mRNA-expression of matrix metalloproteinase (MMP)-2 and MMP-9 with a prominent up-regulation of MMP-9 during acute SCI, correlating with axonal β-APP-expression.
The results demonstrate that naturally occurring canine SCI is characterized by an early pro-inflammatory microenvironment paralleled by degenerative axonal alterations such as inadequate neurofilament phosphorylation and disturbances in fast axonal transport mechanisms. Moreover, there are significant parallels in the local cytokine orchestration compared to other canine CNS diseases such as canine distemper leukoencephalitis. The acute axonal damage seems to trigger delayed myelin loss with subsequent dominating responses of microglial cells in the subacute phase of clinical SCI. The results support the hypothesis, that phagocytic microglial cells play an essential, however time-dependent role in the initiation and regulation of inflammatory events in the traumatized spinal cord. Axonal damage exhibits a significant spatio-temporal progression with increasing pathology in segments distant to the epicenter during the advanced disease phase. Moreover, there are hints for spontaneous regenerative axonal changes. The results validate the canine species for translational research on SCI and suggest that future therapies should concentrate on a modulation of the pro-inflammatory microenvironment, the microglia/macrophage response, and axonopathy adjunctive to cell transplantation techniques.
8 Chapter 8: Zusammenfassung
Ingo Spitzbarth - Eine morphologische und molekulare Untersuchung der zeitlich-räumlichen Entwicklung kaniner Rückenmarksverletzungen
Rückenmarksverletzungen stellen sowohl in der Veterinärmedizin als auch in der Humanmedizin eine vergleichsweise häufige und gefürchtete neurologische Erkrankung dar, für die die Entwicklung neuer Therapieansätze wie die Transplantation regenerationsfördernder Zellen notwendig ist.
Das bisherige Wissen über pathologische Prozesse im traumatisierten Rückenmark basiert weitgehend auf hoch standardisierten Nagetiermodellen, während relativ wenig über die zeitliche Entwicklung von Veränderungen im natürlich traumatisierten Rückenmark bekannt ist. Hunde sind durch die hohe Prävalenz von Bandscheibenvorfällen häufig von spontanen Rückenmarksverletzungen betroffen und eignen sich daher als translationales Tiermodell für die humane Erkrankung. Die Spontaneität kaniner Rückenmarksverletzungen stellt hierbei den entscheidenden Unterschied zu Nagetiermodellen dar und macht die Spezies Hund so zu einem Brückenmodell zwischen Laborversuchen und der klinisch relevanten Erkrankung.
Die Ziele der vorliegenden Studie bestanden in i) einer zusammenfassenden Analyse der bisherigen Daten über die Zytokinexpression bei verschiedenen spontanen Erkrankungen des zentralen Nervensystems (ZNS) des Hundes, gefolgt von einer ii) Untersuchung der zeitlich-räumlichen Entwicklung von Immunprozessen, der lokalen Zytokinexpression und von axonalen Veränderungen im Rückenmarksgewebe von Hunden mit Bandscheibenvorfall-assoziierten Rückenmarksverletzungen und iii) der vergleichenden Evaluation derartiger Veränderungen in organotypischen Schnittkulturen vom kaninen Rückenmark als reduktionistisches in vitro Modell für Rückenmarkstraumata. Für die in vivo Studie wurde Rückenmarksgewebe von Kontrolltieren (n = 10), von Hunden mit akuter Rückenmarksverletzung (n=9; 1-4 Tage nach Bandscheibenvorfall) und Rückenmarksgewebe von Hunden mit subakuter Rückenmarksverletzung (n=8; 5-10 Tage nach Bandscheibenvorfall)
untersucht. Die organotypischen Kulturen kaniner Rückenmarksschnitte von 6 Monate alten Hunden (n=6) wurden für 0, 3 und 9 Tage kultiviert.
Natürlich auftretende Rückenmarkstraumata sowie die Kultivierung kaniner Rückenmarksschnitte gingen mit einer prominenten axonalen Schwellung einher, der eine von major histocompatibility complex (MHC)-II exprimierenden Mikroglia/Makrophagen dominierte Immunantwort in der subakuten Phase bzw. nach 9 Tagen in Kultur folgte. Mittels reverser Transkriptase quantitativer Polymerase (RTqPCR) Kettenreaktion zeigte sich eine Aufregulation pro-inflammatorischer Zytokine (Interleukin [IL]-6, IL-8) sowie ein statistischer Trend einer Aufregulation von Tumor Nekrose Faktor (TNF) während des akuten Stadiums natürlicher Rückenmarksverletzungen. Potentiell neuroprotektiv wirkende Zytokine zeigten keine (IL-10) bzw. eine verspätete Aufregulation im subakuten Stadium (transforming growth factor [TGF]-β). In Schnittkulturen wurde eine ähnliche, aber im Vergleich zur in vivo Situation verlängerte Expression inflammatorischer Zytokine beobachtet. Die molekularen Veränderungen in der Zytokinexpression weisen darüber hinaus Ähnlichkeiten zu anderen kaninen ZNS-Erkrankungen wie der demyelinisierenden, nervösen Staupe auf. Dies impliziert, dass eine Dysregulation der molekularen Immunantwort eine Schlüsselrolle bei kaninen ZNS-Erkrankungen, unabhängig von der Krankheitsentität, spielt.
Immunhistologisch stellte sich bei natürlich traumatisierten Tieren die axonale Pathologie durch eine früh einsetzende axonale Expression von nicht-phosphoryliertem Neurofilament (n-NF) sowie β-Amyloid Vorläuferprotein (β-APP) dar. Darüber hinaus ergaben sich Hinweise auf spontane axonale Regenerationsversuche (Expression von growth-associated protein [GAP]-43), die immunelektronenmikroskopisch verifiziert wurden. In der subakuten Phase kam es ausgehend vom Epizentrum der Läsion zu einer progressiven Ausdehnung der axonalen Veränderungen in kranio-kaudaler Richtung. In Schnittkulturen beschränkten sich die axonalen Veränderungen auf eine Expression von n-NF. Die axonalen Veränderungen wurden von einer Dysregulation von Matrix Metalloproteinase (MMP)-9 und MMP-2 begleitet, deren zeitlich abhängige Aufregulation auch in Schnittkulturen nachvollzogen werden konnte. Darüber hinaus
korrelierte die mittels RTqPCR bestimmte Kopienzahl von MMP-9 mit der axonalen β-APP-Expression.
Die Ergebnisse zeigen, dass kanine Rückenmarksverletzungen durch ein früh einsetzendes pro-inflammatorisches Mikromilieu und prominente Axonpathologie gekennzeichnet sind, die möglicherweise als Auslöser für eine im subakuten Stadium folgende Ausdehnung morphologischer Alterationen und für die durch residente Zellen (Mikroglia/Makrophagen) dominierte Immunantwort fungieren. Die pro-inflammatorisch dominierte, früh einsetzende Zytokinexpression weist darüber hinaus Parallelen zu anderen kaninen ZNS-Erkrankungen, wie beispielsweise der nervösen Staupe, auf. Zusätzlich fanden sich jedoch auch Hinweise auf spontane axonale Regerationsversuche. Die Ergebnisse validieren die Spezies Hund als geeignetes translationales Forschungsmodell und verdeutlichen, dass zukünftige Therapieansätze insbesondere auf die Modulation des pro-inflammatorischen Mikromilieus, der Mikroglia/Makrophagen-Antwort sowie auf Axonopathien abzielen sollten, um spätere Zelltransplantationsversuche zu optimieren.
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