Abstract
Purpose:
Multiple factors play important roles in the development of glaucoma, with retinal ganglion cell death (RGC) as a major hallmark of the disease. In the central nervous system, microglia is activated in response to neurodegenerative disease (e.g. Alzheimer's disease) as well as in glaucoma. Thus, it is suggested that inflammation can contribute to disease progression. The purpose of the present project was to study the temporal aspects of microglial activation, cytokine-release profile and RGC death after lipopolysaccharide (LPS)-induced inflammation in the mouse retina.
Methods:
An organotypic culture system of the postnatal mouse retina was used. The optic nerve (ON) axotomy, and the explantation process results in RGC loss and causes microglia activation. LPS (100 ng/ml) was added to cultures at 48 hours after dissection, and at 24 h, 48 h and 5 days retinas were fixed. Microglia activation is assessed by 1) numbers of proliferating microglia (Ki-67 immunostaining), 2) morphological changes using Iba-1 and ED1-immunostaining and 3) cytokine release. Conditioned media were collected and a multiplex immunoassay is used to determine cytokine levels. Degeneration in the ganglion cell layer is examined by quantification of number of nuclei (DAPI- staining), apoptotic cells (TUNEL) staining) and RGCs (NeuN- staining).
Results:
We demonstrate a 50% significant loss of NeuN-positive RGCs by apoptosis within 48 hrs after axotomy of the ON. Thereafter the numbers of DAPI- as well as NeuN-positive cells is stable up to 14 days. Initial results show enhanced microglial activation after LPS-treatment judged by a higher number of ED1-positive cells in the inner retina at all time-points studied, suggesting more activated microglia, but no significant difference in RGC numbers. Further analysis is on-going.
Conclusions:
Since inflammation in the central nervous system may have a critical role in the neurodegenerative process in many diseases, including glaucoma, appropriate models of neuroinflammation are necessary for understanding the disease progression. Here we propose a controlled system for studying the effects of microglial activation on the degeneration in the retina, and especially the RGCs, using an organotypic mouse retina model.
Keywords: 557 inflammation •
694 retinal culture •
691 retina: proximal (bipolar, amacrine, and ganglion cells)