Distinctive waves of innate immune response in the retina in experimental autoimmune encephalomyelitis

Neurodegeneration mediates neurological disability in inflammatory demyelinating diseases of the CNS. The role of innate immune cells in mediating this damage has remained controversial with evidence for destructive and protective effects. This has complicated efforts to develop treatment. The time sequence and dynamic evolution of the opposing functions are especially unclear. Given limits of in vivo monitoring in human diseases such as multiple sclerosis (MS), animal models are warranted to investigate the association and timing of innate immune activation with neurodegeneration. Using noninvasive in vivo retinal imaging of experimental autoimmune encephalitis (EAE) in CX3CR1GFP/+–knock-in mice followed by transcriptional profiling, we are able to show 2 distinct waves separated by a marked reduction in the number of innate immune cells and change in cell morphology. The first wave is characterized by an inflammatory phagocytic phenotype preceding the onset of EAE, whereas the second wave is characterized by a regulatory, antiinflammatory phenotype during the chronic stage. Additionally, the magnitude of the first wave is associated with neuronal loss. Two transcripts identified — growth arrest–specific protein 6 (GAS6) and suppressor of cytokine signaling 3 (SOCS3) — might be promising targets for enhancing protective effects of microglia in the chronic phase after initial injury

Distinctive waves of innate immune response in the retina in experimental autoimmune encephalomyelitis

Neurodegeneration mediates neurological disability in inflammatory demyelinating diseases of the CNS. The role of innate immune cells in mediating this damage has remained controversial with evidence for destructive and protective effects. This has complicated efforts to develop treatment. The time sequence and dynamic evolution of the opposing functions are especially unclear. Given limits of in vivo monitoring in human diseases such as multiple sclerosis (MS), animal models are warranted to investigate the association and timing of innate immune activation with neurodegeneration. Using noninvasive in vivo retinal imaging of experimental autoimmune encephalitis (EAE) in CX3CR1(GFP/+)-knock-in mice followed by transcriptional profiling, we are able to show 2 distinct waves separated by a marked reduction in the number of innate immune cells and change in cell morphology. The first wave is characterized by an inflammatory phagocytic phenotype preceding the onset of EAE, whereas the second wave is characterized by a regulatory, antiinflammatory phenotype during the chronic stage. Additionally, the magnitude of the first wave is associated with neuronal loss. Two transcripts identified - growth arrest-specific protein 6 (GAS6) and suppressor of cytokine signaling 3 (SOCS3) - might be promising targets for enhancing protective effects of microglia in the chronic phase after initial injury

Longitudinal effects of an optic nerve injury on visual behaviour

Une maladie ou un traumatisme du système visuel peut avoir des conséquences à long terme sur la vision. Cependant, des études récentes ont examiné la plasticité cérébrale comme moyen de restaurer une vision fonctionnelle malgré les dommages. Pour mieux comprendre l'implication de la plasticité et de la réorganisation neuronale suite à un déficit, ce mémoire étudie la récupération spontanée des fonctions visuelles par des tests comportementaux chez la souris. L’écrasement partiel du nerf optique (pONC), permettant une vision résiduelle, a été induit selon deux intensités. Les tests comportementaux des fonctions visuelles incluaient le réflexe optomoteur, qui mesure le réflexe du suivi visuel de la souris en réponse à un réseau sinusoïdal, ainsi que le test de la falaise visuelle qui évalue la perception de profondeur. Ces tests ont été effectués une fois avant le pONC, puis à plusieurs moments jusqu'à 28 jours post-opération. La survie des cellules ganglionnaires rétiniennes donnant naissance au nerf optique a ensuite été quantifiée. Les résultats ont montré qu’un pONC de haute intensité entraînait une cécité et une perte de la perception de profondeur, sans amélioration dans les 28 jours suivants, tandis qu’un pONC de faible intensité permettait une récupération partielle des deux paramètres. La perte des cellules rétiniennes était plus forte pour le pONC de haute intensité, surtout dans les régions proximales. Ces résultats montrent une récupération spontanée des fonctions visuelles, à part si le dommage cellulaire est trop important.Disease or trauma to the visual system can cause long-term damage and severe visual deficits. However, recent research has turned to neural plasticity as a means to recover functional vision despite anatomical damage. To understand the involvement of neural plasticity and reorganization following a deficit, the present thesis investigated the spontaneous recovery of visual functions over time using behavioural tests in mice. Specifically, a partial optic nerve crush (pONC) was induced with two injury intensities, while still allowing for residual vision from surviving retinal cells. Behavioural assessments of visual functions included the optomotor reflex test, which measured the mouse’s tracking reflex in response to moving sinusoidal gratings, while the visual cliff test evaluated depth avoidance behaviour by simulating a cliff to observe the animal’s perception of depth. The tests were performed once before the injury, then at multiple time points up to 28 days. Retinal ganglion cell survival was subsequently quantified. Results showed that the high-intensity pONC led to a complete loss of visual acuity and depth avoidance, with no improvement in the following 28 days, whereas the low-intensity pONC showed a partial recovery. There were fewer surviving cells after the high-intensity pONC, especially in proximal regions. These results show evidence of spontaneous recovery of visual functions, but only with a certain amount of cell survival

Einfluss der Experimentellen Autoimmunen Encephalomyelitis auf elektrische und chemische Synapsen der murinen Retina

Die Multiple Sklerose gehört zu den chronisch-degenerativen Erkrankungen des ZNS. Im Rahmen der neuropathologischen Krankheitsprozesse kommt es durch das patienteneigene Immunsystem zur zentralen Inflammation und Autoantikörperbildung. Folge ist eine fokale Degeneration der neuronalen Myelinscheiden an multiplen Stellen im ZNS (REALE, SANCHEZ-RAMON, 2017). In der Pathogenese der MS spielen intra- und interzelluläre Kanalproteine (Panx1 und verschiedene Connexin-Subtypen) eine bedeutende Rolle. Sie sind beispielsweise bei der Aktivierung zytotoxischer autoreaktiver T-Lymphozyten, bei der Vermittlung der Entzündungsreaktion an den Myelinscheiden und bei einer Fortleitung pro- inflammatorischer Botenstoffe in weitere Zellen beteiligt (ADAMSON, LEITINGER, 2014; MAIER et al., 2004; RIPPS, 2002; THOMPSON, 2015; WONG et al., 2014). Rund ein Drittel der Patienten mit MS entwickelt als Frühsymptom eine Neuritis des Nervus opticus, verschiedenartige Sehstörungen bis hin zum völligen Ausfall des Gesichtsfeldes unter Beteiligung der Retina (HEROLD et al., 2015; LARABEE et al., 2016; SYC et al., 2012). Die genauen Pathomechanismen dieser Mitbeteiligung des visuellen Systems sowie das Ausmaß der retinalen Läsion bei der MS sind aktuell jedoch noch nicht im Detail bekannt. Es wurde daher in dieser Arbeit in der ersten Hypothese postuliert, dass die durch die zentrale Inflammation induzierte axonale Schädigung im N. opticus die Apoptose retinaler Ganglienzellen zur Folge hat sowie sich eine Reduktion synaptischer Verbindungen innerhalb der Retina zeigt. Experimentell wurde in dieser Arbeit mit dem Tiermodell der Experimentellen Autoimmunen Encephalomyelitis (EAE) gearbeitet. Im zweiten Teil dieser Arbeit wurde ein möglicher neuroprotektiver Effekt des Pannexin1 Inhibitors Probenecid (PBN) auf die retinalen Neurone der Versuchstiere untersucht. Hypothese des zweiten Teils war, dass eine PBN-Intervention mit 100mg/kg Körpergewicht ab Tag eins nach EAE-Induktion eine Prävention der neuronalen Schädigung im Auge zur Folge hat. Es wurde angenommen, dass retinale Strukturproteine von elektrischen und chemischen Synapsen in der präventiv mit PBN behandelten Gruppe im Vergleich zur EAE- Gruppe in höherer Dichte immunologisch nachweisbar sein würden und weniger Zellen in eine EAE-induzierte Apoptose eintreten. In beiden Teilen dieser Arbeit wurde die Zahl der Ganglienzellen über den Nachweis des Ganglienzell-spezifischen Proteins RBPMS sowie der programmierte Zelltod über das Apoptose-assoziierte Protein Cleaved-Caspase-3 bestimmt. Connexin 36, das Gap junction Protein elektrischer Synapsen der Retina, sowie RIBEYE, exprimiert in den chemischen Ribbon-Synapsen der Retina, wurden analysiert, um die synaptische Verschaltung in der Retina bei EAE und unter Therapie zu untersuchen. Im ersten Teil dieser Arbeit konnte gezeigt werden, dass die Induktion der EAE im Tiermodell sowohl zu klinisch messbaren Paralysen der Versuchstiere führte als auch degenerative I Veränderungen mit signifikant verringerter Expression der untersuchten Proteine an neuronalen Zellen verschiedener Schichten der Retina stattfanden. Im zweiten Teil der Arbeit konnte gezeigt werden, dass sich klinisch die Motorik der Versuchstiere signifikant unter präventiver PBN-Gabe verbesserte. Zudem traten unter Therapie mit PBN signifikant weniger Ganglienzellen in die Apoptose ein. Trotz PBN-Gabe kam es jedoch zum Verlust synaptischer Proteine im Vergleich zur Kontrollgruppe. Zusammengenommen legen die Ergebnisse dieser Arbeit nahe, dass nicht nur der optische Nerv und die Ganglienzellen im Rahmen des Krankheitsgeschehens der MS degenerieren, sondern auch synaptische Bestandteile weiterer retinaler Schichten geschädigt werden. Zudem konnte gezeigt werden, dass präventiv verabreichtes PBN einen protektiven Effekt auf die Versuchstiere mit einem besseren motorischen Ergebnis unter EAE und einem geringeren Ganglienzellschaden im Vergleich zur nicht behandelten Gruppe hat, es aber dennoch zur verringerten Expression synaptischer Proteine kommt.Summary Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). In the process of the neuropathological genesis of this disease, the patient’s own immune system initiates a central inflammation and generates autoantibodies, causing focal degeneration of neuronal myelin sheaths in multiple areas within the CNS (REALE, SANCHEZ-RAMON, 2017). The pathogenesis of MS is based mainly on intra- and intercellular protein channels (Panx1 and multiple subtypes of connexin-channels). These channels e.g. play an important role in the activation of cytotoxic T-lymphocytes, in passing of inflammatory reactions at the myeline sheath and for the transport of pro-inflammatory substances to bystanding cells (ADAMSON, LEITINGER, 2014; MAIER et al., 2004; RIPPS, 2002; THOMPSON, 2015; WONG et al., 2014). Approximately one third of MS-patients develop as initial symptom a neuromyelitis optica, i.e. a wide range of visual disorders up to a complete failure of the visual perceptive field including damage of the retina (HEROLD et al., 2015; LARABEE et al., 2016; SYC et al., 2012). It is still not certain how those pathomechanisms involving the visual system precisely work and the extent of retinal lesions are currently still unknown. Therefore, the first hypothesis of this thesis postulates that axonal damage of the optical nerve, caused by central inflammation, results in an apoptosis of retinal ganglion cells as well as in a reduction of retinal synaptic structures of multiple retinal layers. The research work for this thesis is based on the animal experimental autoimmune encephalomyelitis (EAE) model. The second part of this thesis deals with research examining a potential neuro-protective effect of Panx1 inhibitor PBN on retinal neurons of laboratory mice. This part is based on a hypothesis saying that a PBN intervention of 100mg/kg body weight injected at day one after an EAE induction can prevent neuronal damage in the animal`s eye. It was assumed that an immunological staining would prove a higher density of structural retinal molecules of electrical and chemical synapses within a preventively treated PBN group, when compared with EAE-groups, and that a smaller number of cells would enter an EAE-induced apoptosis. In both parts of this thesis retinal ganglion cells were counted using the ganglion cell specific protein RBPMS. Apoptosis was analyzed using the apoptosis associated protein cleaved caspase-3. In order to examine retinal synaptic connections under EAE and under preventive treatment, connexin 36, the gap junction protein of retinal electric synapses, as well as RIBEYE, expressed in retinal chemical ribbon synapses, were analyzed. In the first part of this thesis it could be demonstrated that an induction of EAE in an animal model leads to detectable paralyses of the animals as well as to degenerations with a significantly decreased expression of analyzed proteins in multiple layers of the retina. The second part of this thesis could prove that motoric results of animals preventively treated with III PBN increased significantly. Furthermore, under PBN-treatment a significant number of retinal ganglion cells were prevented from entering apoptosis. But yet, despite PBN treatment, synaptic proteins decreased in expression in comparison with the control group. To sum up the results of this thesis, it may be presumed that during the pathogenesis of MS, lesions are not limited to the optic nerve and the retinal ganglion cells but degeneration also takes place in multiple retinal synaptic components. Additionally, it could be demonstrated that preventively applicated PBN has a neuro-protective effect on laboratory mice with a better motoric outcome under EAE induction, in comparison with groups without treatment. But still, depletion of expressed retinal synaptic proteins could not be prevented by this treatment

Exploring experimental autoimmune optic neuritis using multimodal imaging

BACKGROUND Neuro-axonal injury is a key contributor to non-reversible long-term disability in multiple sclerosis (MS). However, the underlying mechanisms are not yet fully understood. Visual impairment is common among MS patients, in which episodes of optic neuritis (ON) are often followed by structural retinal damage and sustained functional impairment. Alterations in the optic nerve and retina have also been described in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Thus, investigating structural anterior visual pathway damage may constitute a unique model for assessing mechanisms and temporal sequence of neurodegeneration in MS. We used a multimodal imaging approach utilizing optical coherence tomography (OCT) and diffusion tensor imaging (DTI) to explore the mechanisms and temporal dynamics of visual pathway damage in the animal model of MS. METHODS 7 EAE-MOG and 5 healthy female C57BL/6J mice were used in this study. Ganglion cell complex (GCC) thickness was derived from an OCT volume scan centred over the optic nerve head, while the structure of the optic nerve and tracts was assessed from DTI and co-registered T2-weighted sequences performed on a 7T MRI scanner. Data was acquired at baseline, disease onset, peak of disease and recovery. Linear mixed effect models were used to account for intra-subject, inter-eye dependencies, group and time point. Correlation analyses assessed the relationship between GCC thickness and DTI parameters. Immunofluorescence staining of retina and optic nerve sections was used to assess distribution of marker proteins for microglia and neurodegeneration (nerve filaments). RESULTS In EAE mice, a significant increase in GCC thickness was observed at disease onset (p < 0.001) followed by a decrease at recovery (p < 0.001) compared to controls. The EAE group had significant GCC thinning at recovery compared to all other time points (p < 0.001 for each). Signal increase on T2-weighted images around the optic nerves indicative of inflammation was seen in most of the EAE mice but in none of the controls. A significant decrease in axial diffusivity (AD) and increase in radial diffusivity (RD) values in EAE optic nerves (AD: p = 0.02, RD: p = 0.01) and tract (AD: p = 0.02, RD: p = 0.006) was observed compared to controls. GCC at recovery was positively correlated with AD (optic nerve: rho = 0.74, p = 0.04, optic tract: rho = 0.74, p = 0.04) and negatively correlated with RD (optic nerve: rho = -0.80, p = 0.02, optic tract: rho = -0.75, p = 0.04). Immunofluorescence analysis indicated the presence of activated microglia in the retina and optic nerves in addition to astrocytosis and axonal degeneration in the optic nerve of EAE mice. CONCLUSION OCT detected GCC changes in EAE may resemble what is observed in MS-related acute ON: an initial phase of swelling (indicative of inflammatory edema) followed by a decrease in thickness over time (representative of neuro-axonal degeneration). In line with OCT findings, DTI of the visual pathway identifies EAE induced pathology (decreased AD, and increased RD). Immunofluorescence analysis provides support for inflammatory pathology and axonal degeneration. OCT together with DTI can detect retinal and optic nerve damage and elucidate to the temporal sequence of neurodegeneration in this rodent model of MS in vivo