Role of thyroid hormones in retinotectal axon regeneration in adult zebrafish

Injury to the adult mammalian central nervous system (CNS) leads to detrimental consequences due to its poor regenerative capacity. Thyroid hormones (THs), well known for their key function in CNS development and maturation, have recently emerged as molecules that can influence regeneration. Several studies have found exogenous THs to be beneficial for peripheral nervous system regeneration. Conversely in adult CNS, studies on the role of THs in in vivo regeneration remain scarce. Since the zebrafish optic nerve (ON) is an easily accessible part of CNS and regenerates spontaneously upon trauma, it is widely studied to identify important molecules during CNS regeneration. We therefore investigated the effect of lowering T3 signaling on the regeneration of the ON following crush in adult zebrafish. In addressing the issue we first showed that the reinnervation of the optic tectum (OT) was accelerated when fish were exposed to iopanoic acid (IOP), a compound lowering T3 availability, or to the TH receptor ß antagonist- methylsulfonylnitrobenzoate. At 7 dpi there was a clear increase in the biocytin labeled area in the OT following anterograde tracing of the retinal ganglion cell axons as well as an increased immunostaining of Gap43, a protein expressed in outgrowing axons. Next we showed that the increased reinnervation of OT was attenuated when IOP-treated fish were co-treated with exogenous T3 confirming that increased OT reinnervation was indeed specifically due to decreased T3 signaling.In search for the mechanism behind the observed response, we first looked at the retinal ganglion cells in the retina. ON crush induced very limited cell death and proliferation at the level of the retina in both control and anti-thyroid drug treated groups. While the mRNA expression level of regeneration markers such as gap43, tuba1, socs3b and snap25a changed as expected and klf9 and bdnf did not change following crush, lowering T3 signaling did not influence the pattern. We therefore found no obvious effect of TH inhibition at the level of the retina.We did however find a correlation between the accelerated OT reinnervation and a more rapid resolution of the microglia/MΦs in the ON and the OT of IOP-treated fish. Beside the presence of reactive astrocytes in the injured ON of control and IOP-treted groups, preliminary data also revealed thepresence of reactive astrocytes in the OT of IOP-treated fish at an early time point after injury, which is 4 days after injury. Finally, we could also show an increased myelin staining in the OT of the treated group. Nevertheless these findings need further investigation to fully decipher their relevance.Taken together these data indicate that lowering T3 signaling accelerates retinal ganglion cell axon extension and OT reinnervation following ON crush in adult zebrafish and that an altered inflammatory response may be involved in this process. The next step is trying to further elucidate the underlying molecules and mechanisms and use this knowledge in further research aimed at improving regeneration in the mammalian CNS.Acknowledgement……………………………………………………………i List of abbreviations………………………………………………………..ix RESEARCH FOCUS 1 1. INTRODUCTION 7 1.1. General background on nervous system regeneration 9 1.1.1. Thyroid hormones 10 1.1.2. Thyroid hormone regulation and action 11 1.1.3. Thyroid hormones in nervous system regeneration 15 1.1.4. Thyroid hormones and inflammation 31 1.2. Zebrafish as a model to study CNS regeneration 32 1.2.1. Zebrafish visual system 33 1.2.2. Optic nerve regeneration 37 2. OBJECTIVES 47 3. MATERIALS & METHODS 51 3.1. Zebrafish husbandry 53 3.2. Drug supplementation 53 3.3. Optic nerve crush model 54 3.3.1. Microsurgery 54 3.3.2. Anterograde tracing with biocytin and visualization 55 3.4. Histological analysis 58 3.4.1. BrdU labeling 58 3.4.2. Tissue processing 59 3.4.3. Immunohistochemical (IHC) stainings 59 2.4.4. Standard histochemical stainings 61 3.5. RNA isolation and quantitative RT-PCR 62 3.6. TH extraction and radioimmunoassay 64 3.7. Quantification of microglia/macrophages 64 3.8. Statistics 65 4. RESULTS 67 4.1. Optic Nerve Crush Model Establishment 69 4.1.1. The time course of optic tectum reinnervation 69 4.1.2. Morphological changes of the RGCs in the retina 70 4.1.3. Cell death and cell proliferation in the retina 71 4.2. Effect of IOP treatment 74 4.2.1. Optic tectum reinnervation 74 4.2.2. Cell death and proliferation in the retina 75 4.2.3. mRNA expression of regeneration associated genes and thyroid responsive genes in the retina 75 4.3. Effect of IOP on T3 and T4 levels in the eye and the brain 78 4.4. Effect of IOP pretreatment on the reinnervation of optic tectum 79 4.5. Effect of exogenous T3 on OT reinnervation 79 4.6. Effect of a TRβ antagonist 82 4.6.1. OT reinnervation 82 4.6.2. mRNA expression of regeneration associated genes and thyroid responsive genes in the retina 83 4.7. Effect of low T3 levels on inflammation 83 3.8. Effect of low T3 levels on astrogliosis 86 4.9. Effect of low T3 level on remyelination 92 5. DISCUSSIONS & FUTURE PERSPECTIVES 97 5.1. Effect of lowering T3 signaling on the RGCs at the level of the retina 99 5.2. Effect of lowering T3 signaling on the outgrowth of RGC axons at the level of the optic tectum 101 5.3. Effect of lowering T3 signaling on inflammation at the level of optic nerve and optic tectum 103 5.4. Effect of lowering T3 signaling on astrogliosis at the level of the optic nerve and optic tectum 105 5.5. Effect of lowering T3 signaling on remyelination at the level of the optic nerve and optic tectum 105 5.6. TH regulation of ON regeneration in zebrafish – lessons for mammalian CNS regeneration 106 5.7. Future perspectives 109 SUMMARY 113 Summary 115 Samenvatting 117 REFERENCES 121 CURRICULUM VITAE 147nrpages: 150status: publishe