The role of laser induced retinal injury on visual spatial memory and its rescue by subretinal transplantation of human umbilical cord blood derived lineage negative stem Cells in mice

Abstract

In this study we aim to evaluate the efficacy of lineage negative stem cells derived from hUCB in reversing the retinal injury and visual memory by subretinal transplantation in mice model. We established a mouse model at different degrees of laser-induced retinal injury by laser photocoagulator. The disruption in the RPE- Bruch s membrane interface, resulting in retinal degeneration, was validated by Fundus Fluorescein Angiography (FFA) and histological characterisation. Lin-ve stem cells derived from human umbilical cord blood were isolated by the MACS apparatus. Through flow cytometry, isolated Lin-ve stem cells were characterized for surface markers CD45, 34, and 117 and the number of CD34 cells was significantly high. In the laser-injured mouse model, 50,000 Lin-ve stem cells were transplanted after 24h of laser injury. Subretinal transplantation was done at the RPE-retina junction adjacent to the laser shots. The animals were sacrificed after one month to analyze the time-dependent effect of the stem cells. Neurobehavioural tests were done in which it was found that 2 laser + stem cell mouse models have rescued memory loss in comparison to 2 laser injury models. Similar results were found in a passive avoidance test, mice spent more time in the light region in 2 laser + stem cells, 4 laser + stem cells and 8 laser + stem cell groups to avoid electric shock in darker regions. The retinal s amples were analysed for gene expression through IHC and Real-time PCR for retinal, neurotropic, proliferative and apoptotic markers. The data have shown that milder injury (2 laser) rescued faster than severe injury (8 laser injury). The stem cell may be able to proliferate at the target site and may have shown neuroprotective function after an increase in BDNF and CNTF expression. Based on this data, we propose that the Lin-ve stem cells tend to get localised in the RPE, and were able to reverse the injury by neuroprotective effect in mild injury.