Profiling of Inflammatory Mediators During Regeneration in Adult Zebrafish Retina

Abstract

Retinal injuries, caused due to various reasons, are great burden and it often leads to serious consequences due to permanent tissue damage and life-long impairments. The human CNS possesses very low regenerative capacity and there are no therapies available to cure disorders that impact the normal function of the CNS. Retinal injury is accompanied by strong neuroinflammation, including accumulation of immune cells, activation of microglia (immune cells of the CNS), secretion of various inflammatory-mediators (pro-inflammatory and anti-inflammatory cytokines in case of mammals secreted by two distinct M1, M2 activated microglial subtypes). In case of humans, neuroinflammation persist over years contributing to chronic scar formation and is hence regarded as a major determental process that is considered as the cause of low regenerative capacity. In contrast, zebrafish is known to successfully regenerate injured tissues including retina after the lesion. Importantly, the neuroinflammatory response is required for the injury induced MG proliferation, and regenerative neurogenesis in the zebrafish. However, identification of specific inflammatory-mediators involved in this process remained obscure. Hence, this study was involved in the identification of the molecular and cellular composition of the neuroinflammatory response upon light lesion in the adult zebrafish retina. Additionally, the role of NF-kB signalling in case of inflammation has been known for many years. Hence, some preliminary experiments were performed to decipher the role of NF-kB signalling in regeneration during the proliferation of MG and how this signalling pathway impacts MG proliferation in the presence and absence of microglia. In order to understand the changes in gene expression pattern of entire zebrafish retina in response to the light lesion, the temporal profiling of inflammatory-mediator expression was investigated. Initially, a detailed temporal expression pattern-based analysis of the known inflammatory mediators within the retina was performed using RT-qPCR. Instead of a distinct M1-M2 cytokine profile, expression of pro- and anti-inflammatory cytokines occurs largely simultaneously. Furthermore, the chromogenic in-situ results revealed that the expression of two inflammatory-mediators (il11a, il11b respectively) were observed in the INL at early time points, followed by their expression in the ONL at later time points. To understand the role of NF-kB signalling in the proliferation of MG, FISH was performed using the transgenic reporter line Tg(NF-κB:GFP) for NF-KB. This was then combined with the antibody staining for the PCNA (to check proliferating cells), and Zrf-1(to label MG). Results suggest that there is no co-localization between proliferating MG and NF-kB positive MG, showing that the NF-kB is not involved in proliferation of MG cells and might have some other role in the regeneration. Apart from this, another preliminary study to examine the effect of modulating the macrophage/microglia injury response to investigate their contribution to the reactive proliferation and regeneration after photoreceptor ablation. In order to get the overall idea III about how the presence of microglia in the adult zebrafish retina affects the NF-kB signalling, another transgenic line irf8-/-;Tg(NF-kB:GFP), in which the microglia/monocytes are depleted, for NF-kB was used. FISH was performed to combine GFP in-situ hybridization with the immunostaining of PCNA (to check proliferating cells), and Zrf-1(to label MG). These results were then compared to the FISH for irf8+/-;Tg(NF-kB:GFP). The preliminary from this study suggests that the NF-kB activation did not show much difference at 24hpl, but it is significantly downregulated in 44hpl in homozygous (irf8-/-) compared to heterozygous (irf8+/-) fish. In case of proliferation, we see that proliferation peaks at 44hpl in heterozygous (irf8+/-) fish. But the peak of proliferation for homozygous(irf8-/-) fish is at 24hpl. From this, we hypothesize that the absence of microglia shifts the proliferation peak to earlier time points and this early proliferation could be a reason for low NF-kB activation at 44hpl in homozygous (irf8-/-) compared to the heterozygous (irf8+/-) fish. Taken together, this study demonstrates the power of using zebrafish as a model to better understand how neuroinflammation can facilitate retina regeneration. This work, in combination with the earlier understanding from the work of the previous lab member (Viktoria Bosak) on the telencephalon regeneration in zebrafish shows that the changes in the expression profile of different inflammatory-mediators occur very rapidly upon injury in zebrafish. These findings indicate that in zebrafish there is a substantial temporal overlap of expression of different inflammatory-mediators at very early time points after injury. This could on the one hand be a mechanism by which zebrafish are able to quickly resolve inflammatory reactions upon injury. This study also revealed that the proliferation of MG is independent of NF-kB signalling. But this was the preliminary study and further experiments can be done to confirm the hypothesis. Collectively, this knowledge in combination with the earlier understanding on zebrafish regeneration, will be crucial for the identification of novel therapeutic targets and the development of future immune-modulating therapies with the potential to ultimately improve retina regeneration in humans.