Additional file 1 of Models of microglia depletion and replenishment elicit protective effects to alleviate vascular and neuronal damage in the diabetic murine retina
Creators: name: Church, Kaira A.
name: Rodriguez, Derek
name: Vanegas, Difernando
name: Gutierrez, Irene L.
name: Cardona, Sandra M.
name: Madrigal, José L. M.
name: Kaur, Tejbeer
name: Cardona, Astrid E.
Publisher: name: figshare
Year: 2023
Subjects: subject: Medicine
subject: Cell Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: 60506 Virology

description: Additional file 1: Fig S1. Characterizing CX3CR1CRE−ER expression modulated by TAM in CX3CreER:R26iTdT mice. (A) Experimental design to confirm that Cre penetrance targets CX3CR1-expressing cells in the retina and brain without affecting peripheral CX3CR1-expressing immune cells. CX3CreER:R26iTdT mice were injected once daily for 5 days with tamoxifen (TAM). One week and 3-weeks after the last TAM injection, flow cytometric analysis was performed on blood leukocytes to track the percentage of TdT+CD11b+CD45Hi leukocytes. At six-weeks post TAM administration, tissues were collected for flow cytometric and immunohistochemical analysis. (B) Gating strategy to identify TdT+CD11b+CD45Hi blood leukocytes. (C) Graphical representation of flow cytometric quantification of TdT+CD11b+CD45Hi blood leukocytes. (D) Gating strategy to identify TdT+CD11b+CD45Lo microglia in brain and spinal cord tissues. E–F, Graphical representation of flow cytometric quantification of TdT+CD11b+CD45Lo microglia (E) and TdT+CD11b+CD45Hi CNS infiltrating leukocytes (F) in brain and spinal cord tissues. Fig S2. Acute DTx treatment in CX3CR1Cre−ER:R26iDTR mice does not induce neurotoxic effects in the non-diabetic CNS. (A) Experimental design to validate the feasibility of depleting CNS-resident microglia without affecting peripheral CX3CR1-expressing immune cells. Four weeks following Cre recombinase induction with TAM (after the 5th TAM injection), CX3CR1Cre−ER:R26iDTR mice were administered 25 ng/g diphtheria toxin (DTx) once daily for 3 days. Tissue collection occurred 24 h after the last DTx injection. Control mice received PBS instead of DTx. (B) Gating strategies to identify CD11b+CD45Hi blood leukocytes. (D) Confocal images of the primary visual cortex for Iba1+ (green), NeuN+ (red) and DAPI+ nuclei (blue). E–F, Quantification of Iba1+ cells/mm3 (E) and NeuN+ cells/mm3 (F) in the primary visual cortex of PBS and DTx treated CX3CR1CreER:R26iDTR mice. (G) Confocal images of the retina for GFAP+ (magenta) and DAPI+ nuclei (blue). (H) Quantification of GFAP+ percent immunoreactive area. Data show mean ± SD, n = 6 to 9 mice per group where each dot represents an individual mouse. **P &lt; 0.01, using Student’s t-test, with Welch’s correction. Fig S3. DTx treatment in diabetic CX3CR1Cre−ER:R26iDTR mice targets CX3CR1+ cells in the CNS and does effect CX3CR1+ cells in the periphery. (A) Gating strategy to identify CD45HiCD11b+SSCHi neutrophils, CD45HiCD11b+SSCLo macrophages, CD45HiCD11b+Ly6CLo tissue resident macrophages, CD45HiCD11b+Ly6CHi inflammatory macrophages, CD45HiCD11b–CD11c+ conventional dendritic cells and CD45HiCD11b+CD11c+ myeloid-derived dendritic cells in blood leukocytes. B-D, Quantification of Bio-Plex Cytokine 23-plex analysis for cytokine and chemokine in serum isolates. (E) Gating strategy to identify CNS resident microglia CD11b+CD45LoP2RY12+Ly6C–, monocyte-derived microglia CD11b+CD45LoP2RY12+Ly6C+ and CD11b+CD45Hi CNS infiltrating leukocytes. F–H, Flow cytometric quantification of CD11b+CD45LoP2RY12+Ly6C– CNS resident microglia (F), CD11b+CD45LoP2RY12+Ly6C+ monocyte-derived microglia (G), and CD11b+CD45Hi CNS infiltrating leukocytes (H). Data show mean ± SD, n = 4 to 6 mice per group where each dot represents an individual mouse. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 ****P &lt; 0.0001 using Student’s t-test, with Welch’s correction. Fig S4. Prolonged DTx exposure and recovery does not alter retinal pathology in the non-diabetic CX3CR1Cre−ER:R26iDTR retina. (A) Experimental design for two-weeks DTx treatment in non-diabetic CX3CR1Cre−ER:R26iDTR mice. Mice received citrate buffer, 2-weeks after TAM administration. At 6–8 weeks post citrate buffer treatment, mice received 3 daily doses of 25 ng/g DTx, followed by 1 dose of 25 ng/g DTx every 48 h for a total of 2-weeks. Tissues were collected after a 2-weeks recovery period. Control non-diabetic mice were administered PBS instead of DTx. (B) Confocal images of Iba1 (green) and NeuN-RBPMS (red). C-D, Quantification of Iba1+ cells/mm3 (C) and NeuN+RBPMS+ cells/mm3 (D). (E) Representation of cellular tracings for transformation index quantification. (F) Transformation index in non-diabetic, PBS and DTx treated mice, n = 98 to 150 microglia per group where each dot represents an individual microglia cell and bars show mean ± SD. (G) Confocal images of TUJ1 (turquoise) and GFAP (magenta). H-I, Quantification of percent immunoreactive area of TUJ1+ (H) and GFAP+ (I). (J) Confocal images of CD31 (red) and fibrinogen (white). K-L, Quantification of percent immunoreactive area of CD31+ (K) and fibrinogen+ (L). Data show mean ± SD, n = 6 to 9 mice per group where each dot represents an individual mouse (C, D, H, I, K, L). Data are mean ± SD, n = 120 to 180 microglia per group where each dot represents an individual microglia cell from n = 4–10 mice (E). *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 ****P &lt; 0.0001 using Student’s t-test, with Welch’s correction. Fig S5. The effects of two-weeks DTx recovery on the CNS in CX3CR1Cre−ER:R26iDTR mice. (A) Experimental design for two-weeks DTx treatment in non-diabetic and diabetic CX3CR1Cre−ER:R26iDTR mice. Diabetes was induced via streptozotocin (STZ) in CX3CR1CreER:R26iDTR mice 2-weeks after the last TAM injection. Control mice received citrate buffer as a vehicle control. 6–8 weeks after the last STZ (or citrate buffer control) injection, mice received 3 daily doses of 25 ng/g DTx, followed by 1 dose of 25 ng/g DTx every 48 h for a total of 2-weeks. Tissues were collected after a 2-weeks recovery period following the last DTx injection. Control non-diabetic and diabetic mice were administered PBS instead of DTx. (B) Gating strategy to identify CNS resident microglia CD11b+CD45LoP2RY12+Ly6C–, monocyte-derived microglia CD11b+CD45LoP2RY12+Ly6C+ and CD11b+CD45Hi CNS infiltrating leukocytes. C-E, Flow cytometric quantification of CD11b+CD45LoP2RY12+Ly6C– CNS resident microglia (C), CD11b+CD45LoP2RY12+Ly6C+ monocyte-derived microglia (D), and CD11b+CD45Hi CNS infiltrating leukocytes (E). Data show mean ± SD, n = 4 to 6 mice per group where each dot represents an individual mouse. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 ****P &lt; 0.0001 using Student’s t-test, with Welch’s correction. Fig S6. Differences in microglia depletion efficiencies in the CX3CR1CreER:R26iDTR model compared to PLX-5622 model. (A) Experimental design to compare microglia depletion efficiency in CX3CR1CreER:R26iDTR model and PLX-5622 model of microglia depletion. Diabetic CX3CR1CreER:R26iDTR mice were DTx treated for 2-weeks and in diabetic CX3CR1-WT mice were PLX-5622 treated for 2-weeks. Control diabetic CX3CR1CreER:R26iDTR mice were given PBS and control diabetic CX3CR1-WT mice remained on normal chow. Tissues were collected immediately after the 2-weeks treatment regimen. (B) Flow cytometric quantification of CD11b+CD45LoZombie– microglia. C-D, Quantification of the number of Iba1+ cells/mm3 (C) and transformation index, where n = 98 to 150 microglia per group where each dot represents an individual microglia cell and bars show mean ± SD (D). Data show mean ± SD, n = 4 to 10 mice per group where each dot represents an individual mouse (B, C). Data are mean ± SD, n = 52 to 150 microglia per group where each dot represents an individual microglia cell from n = 4–10 mice (D). *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 ****P &lt; 0.0001 using Student’s t-test, with Welch’s correction. Fig S7. Transcriptional profile changes associated with the glial limitans. (A) Experimental design to pharmacologically deplete and repopulate microglia using PLX-5622 for retinal mRNAseq analysis. Six weeks following STZ-induced diabetes, CX3CR1-WT mice were fed PLX-5622 for two weeks, followed by a 2-weeks recovery period. Diabetic control mice were fed normal chow. B-G, Graphical analysis of the fold change in gene expression for Gfap (B), Claudin-1 (C), Claudin-4 (D), F11r (E), Adamts13 (F) and Csf3 (G). Fig S8. Principal component analysis plots of diabetic samples and non-diabetic samples. Principal component analysis plots to show biological replicate clustering by sample type for non-diabetic n = 6 (green) versus 6-weeks diabetic n = 5 (blue) (A), non-diabetic n = 6 (green) versus 8-weeks diabetic normal chow n = 5 (blue) (B), non-diabetic n = 6 (green) versus 8-weeks diabetic PLX-5622 chow treated n = 4 (blue) (C), non-diabetic n = 6 (green) versus 10-weeks diabetic normal chow recovery n = 5 (blue) (D) and non-diabetic n = 6 (green) versus 10-weeks diabetic PLX-5622 chow recovery n = 5 (blue) (E). Fig S9. Principal component analysis plots of diabetic normal chow versus diabetic PLX-5622 chow samples. Principal component analysis plots to show biological replicate clustering by sample type for 8-weeks diabetic PLX-5622 chow treated n = 4 (green) versus 8-weeks diabetic normal chow n = 5 (blue) (A) and 10-weeks diabetic PLX-5622 chow recovery n = 5 (green) versus 10-weeks diabetic n = 5 (blue) (B).