Subretinal Microglia and Donor Photoreceptor Survival in rd1 Mice

Background/Introduction

Inherited retinal diseases lead to irreversible photoreceptor loss.
No effective treatments exist to prevent or slow degeneration.
Photoreceptor cell transplantation is a potential therapy, but survival rates of donor cells are low.
The retina is immune-privileged yet can still cause graft rejection and cell death.
- Improved outcomes are seen when using immune-defective hosts or immune modulation medicines.
Subretinal injections for retinal degeneration are difficult; trans-scleral injections are an alternative that can enhance donor cell survival in rd1 mice.
Microglia are the primary immune cells in the central nervous system, responding to injury or infection.
- In healthy retinas, microglia are found in various layers (ganglion cell, inner plexiform, outer plexiform).
- In degenerating retinas, they migrate to the subretinal space, mediating responses to transplanted grafts and protecting donor photoreceptors.
Goal: To explore the relationship between subretinal microglia and donor photoreceptors in rd1 mice.

Animal Models: Wild-type (C57BL/6J) and rd1 mice (Pde6b mutation) undergo photoreceptor degeneration.
Microglia Manipulation:
- Depletion with PLX5622 (1200 mg/kg) to remove microglia in both WT and rd1 mice.
- Repopulation with AIN-76 to allow microglia to return.
Donor Cell Preparation:
- Photoreceptor precursors collected from P4-P7 neonatal retinas.
- FACS (Fluorescence-Activated Cell Sorting) used to collect tdTomato-labeled photoreceptor precursors.
Transplantation Procedure:
- Utilized trans-scleral injections to avoid immune rejection (as seen in trans-vitreous injections).
- Donor cells injected into the subretinal space, with a sham injection in the contralateral eye.
Post-Transplantation Analysis:
- Methods included immunofluorescence, RPE & microglia isolation, RNA extraction, and cell culture.
- Statistical analysis was performed with significance set at p<0.05.

Figure 1A: Surviving donor photoreceptors detected in rd1 mice; none observed in wild-type mice.
Figure 1B: Quantitative data – 3419 ± 2359 donor cells survived in rd1 mice; 0 in wild-type (p < 0.01).
Figure 1C: Higher fluorescence intensity in surviving donor cells compared to dead cell debris.

Microglia and Macrophage Behavior:
- In wild-type mice: CD68+ macrophages engulfed grafts in the subretinal space.
- In rd1 mice: Microglia surrounded donor cells; macrophages only present in the choroid.
Figure 2A: CD68+ macrophages detected in choroid of rd1 mice irrespective of donor cell survival.
Figure 2B: Microglia and macrophages behavior observed post-trans-scleral vs. trans-vitreous injection showing clear differences in cell interactions around donor cells.
Figure 2C: Increased mRNA levels of chemokines (ccl2, ccl3, cxcl2) in rd1 mice compared to wild-type.
Figure 2D: No significant cytokine level differences between the groups.
Figure 2E: Similar oxidative stress factors in both groups.

PLX5622 treatment (CSF1R inhibitor) eliminated microglia and macrophages.
Figure 3A: Following depletion, donor photoreceptor cells did not survive in rd1 mice (presence of dead cell debris).
Figure 3B: Surviving graft present in repopulated rd1 mice with microglia near donor cells.
Figure 3C: Wild-type mice showed persistence of macrophages, some donor cells survived.
Figure 3D: All donor cells engulfed when both microglia and macrophages were repopulated in rd1 mice.

Gene Expression Analysis:
Figure 4A: In rd1 mice with microglial depletion, lower expression levels of microglial markers compared to those without depletion.
Figure 4B: Upregulated genes related to immune response in rd1 mice without PLX5622.
Figure 4C: Upregulated genes indicating transmembrane transporter activity and synaptic membrane functions in rd1 mice.
Figure 4D: Comparison of axonogenesis-related genes between rd1 and wild-type mice; higher axon development in rd1.
Figure 4E: Genes upregulated in microglial-depleted rd1 mice were related to sensory perception and eye development.
Figure 4F: Comparison showed that PR cells in rd1 mice had a higher survival rate due to microglial presence in the subretinal space.

rd1 mice facilitate graft survival with better outcomes from trans-scleral injections.
Microglia protect donor cells from immune attacks, playing critical roles in survival.
Upregulated genes indicate pathways for axogenesis under microglial influence.
Low surviving donor numbers observed, with high surgical trauma and inflammation risks.

Subretinal microglia significantly help in the survival of donor photoreceptors in rd1 mice.