Models of microglia depletion and replenishment elicit protective effects to alleviate vascular and neuronal damage in the diabetic murine retina

Abstract

Microglia, the resident phagocytes of the retina, are believed to influence the development of retinopathy, but their exact contributions to vascular integrity and neuronal loss are unknown. Therefore, utilizing two models of microglia depletion, we aimed to deplete and repopulate microglia to clarify the contribution of microglia to neuronal loss and vascular damage in the diabetic retina in an STZ-induced model of hyperglycemia. Here, we report that 2 weeks exposure to diphtheria toxin (DTx) in diabetic CX3CR1CreER:R26iDTR transgenic mice induced a 62% increase in Iba1+ microglia associated with an increase in TUJ1+ axonal density and prevention of NeuN+RBPMS+ neuronal loss. Conversely, diabetic PBS controls exhibited robust TUJ1+ axonal and NeuN+RBPMS+ neuronal loss compared to non-diabetic controls. A 2-week recovery period from DTx was associated with a 40% reduction in angiogenesis and an 85% reduction in fibrinogen deposition into the diabetic retina in comparison to diabetic PBS-treated controls. Analysis of microglia morphology and marker expression revealed that following a 2-week recovery period microglia displayed a P2RY12+Ly6C– phenotype and high transformation index (TI) values complimented by a ramified-surveillant morphology closely resembling non-diabetic controls. In contrast, diabetic PBS-treated control mice displayed P2RY12+Ly6C+ microglia, with a 50% reduction in TI values with an amoeboid morphology. To validate these observations were due to microglia depletion, we used PLX-5622 to assess vascular and neuronal damage in the retinas of diabetic mice. Confocal microscopy revealed that PLX-5622 also induced an increase in TUJ1+ axonal density and prevented fibrinogen extravasation into the diabetic retina. mRNAseq gene expression analysis in retinal isolates revealed that PLX-5622-induced microglia depletion and repopulation induced a downregulation in genes associated with microglial activation and phagocytosis, B2m, Cx3cr1, and Trem2, and complement-associated synaptic pruning, C1qa, C1qb, and C1qc. Although the levels of microglia depletion induced with DTx in the CX3CR1CreER:R26iDTR model and those induced with the CSF-1R antagonists are distinct, our results suggest that microglia depletion and replenishment is neuroprotective by inducing the proliferation of a homeostatic microglia pool that supports neuronal and vascular integrity.

Pharmacological depletion of microglia alleviates neuronal and vascular damage in the diabetic CX3CR1-WT retina but not in CX3CR1-KO or hCX3CR1I249/M280-expressing retina

“…In addition to a heightened proinflammatory response in diabetic CX3CR1 -KO mice, previous studies showed that CX3CR1 -KO microglia display a gene signature of premature ageing, with an increased expression of proinflammatory genes under naïve conditions that is further amplified with LPS treatment ( 45 ). We recently reported that transient microglia depletion and repopulation utilizing the genetic CX3CR1 CreER :R26 iDTR model of microglia depletion prevented NeuN + RBPMS + retinal ganglion cell and TUJ1 + axonal loss and alleviated vascular damage in the diabetic retina ( 26 ). This study revealed that in mice that retain functional CX3CR1-FKN signaling, microglia can be reprogrammed to be protective in the diabetic retina ( 26 ).…”

“…We recently reported that transient microglia depletion and repopulation utilizing the genetic CX3CR1 CreER :R26 iDTR model of microglia depletion prevented NeuN + RBPMS + retinal ganglion cell and TUJ1 + axonal loss and alleviated vascular damage in the diabetic retina ( 26 ). This study revealed that in mice that retain functional CX3CR1-FKN signaling, microglia can be reprogrammed to be protective in the diabetic retina ( 26 ). However, CSF-1R antagonism in diabetic CX3CR1 -KO and hCX3CR1 I249/M280 mice did not alleviate TUJ1 + axonal loss nor vascular damage in contrast to CX3CR1 -WT mice ( Figures 4 , 5 ).…”

“…Acute LPS treatment can cause hypoglycemia and induce large variation in circulating glucose levels in diabetic mice ( 37 , 38 ). Therefore, to avoid large fluctuations in glucose levels and to prevent masking hyperglycemia, 10 weeks diabetic mice, ( Figures 3 – 5 and Supplementary Figures 2 – 6 ) received one daily injection of 0.08mg/kg LPS for 2 consecutive days prior to euthanasia as previously characterized ( 26 ).…”

“…Microglial depletion models are used to interrogate the role of microglia-mediated inflammation in disease initiation and progression ( 22 – 25 ). Utilizing the genetic model, CX3CR1 CreER :R26 iDTR , in which microglia become susceptible to the effects of diphtheria toxin (DTx), we recently showed that depletion and repopulation of microglia correlated with decreased neuronal cell loss and vascular damage in the diabetic retina ( 26 ). We also reported changes in the retinal transcriptome with reduced expression of complement-associated synaptic pruning and microglial activation genes ( 26 ).…”

“…Utilizing the genetic model, CX3CR1 CreER :R26 iDTR , in which microglia become susceptible to the effects of diphtheria toxin (DTx), we recently showed that depletion and repopulation of microglia correlated with decreased neuronal cell loss and vascular damage in the diabetic retina ( 26 ). We also reported changes in the retinal transcriptome with reduced expression of complement-associated synaptic pruning and microglial activation genes ( 26 ). The CX3CR1-FKN signaling axis has also been shown to regulate microglial repopulation following ablation in CNS tissues, and delayed microglial repopulation is associated with lower CX3CR1 expression ( 27 , 28 ).…”