Journal: CNS Neuroscience & Therapeutics

Article Title: Mast Cell–Microglia Crosstalk Drives Aberrant Synaptic Pruning Through PAR ‐2/ MAPK / NF ‐ κB Signaling in Neonatal Hypoxic–Ischemic Encephalopathy

doi: 10.1002/cns.71189

Figure Lengend Snippet: HI insult increases synaptic complement labeling, microglia associated CD68 and CX3CR1 signals, and engulfment of synaptic material. (A) Representative immunofluorescence images showing C1q (yellow), NeuN (red), Iba1 (green), and DAPI (blue) in the hippocampus of Sham and HI rats, together with enlarged views and single‐channel images. (B) Representative immunofluorescence images showing C3 (yellow), NeuN (red), Iba1 (green), and DAPI (blue), together with enlarged views and single‐channel images. (C–E) Fluorescence‐intensity surface plots of C1q, quantification of C1q fluorescence intensity, and quantification of C1q + /NeuN + cells, respectively. (F–H) Corresponding analyses for C3. (I) Representative immunofluorescence images showing Iba1 (green), CD68 (red), and DAPI (blue), together with enlarged views and single‐channel images. (J) Representative immunofluorescence images showing Iba1 (green), CX3CR1 (red), and DAPI (blue), together with enlarged views and single‐channel images. (K–M) Fluorescence‐intensity surface plots of CD68, quantification of CD68 fluorescence intensity, and quantification of CD68 + /Iba1 + cells, respectively. (N–P) Corresponding analyses for CX3CR1. Scale bar = 100 μm in panel A, B, I, J. (Q) Representative confocal images of Iba1 + microglia (red), PSD95 (green), and DAPI (blue) in the hippocampus, together with 3D reconstructions. Scale bars = 10 μm. (R) Representative transmission electron micrographs and pseudo‐colored images of hippocampal synaptic structures. Scale bar = 1 μm (S, T). Quantification of PSD95 volume and PSD95 puncta internalized within Iba1 + microglia. (U, V) Quantification of postsynaptic density length and synaptic vesicle number per synaptosome. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4 animals per group.

Article Snippet: To obtain direct evidence for microglia‐dependent synaptic engulfment, we performed 3D reconstruction of immunofluorescence labeled Iba1 + microglia and the postsynaptic marker PSD95 using Imaris software.

Techniques: Labeling, Immunofluorescence, Fluorescence, Confocal, Transmission Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Mast Cell–Microglia Crosstalk Drives Aberrant Synaptic Pruning Through PAR ‐2/ MAPK / NF ‐ κB Signaling in Neonatal Hypoxic–Ischemic Encephalopathy

doi: 10.1002/cns.71189

Figure Lengend Snippet: Microglia‐targeted F2rl1 silencing attenuates HI‐associated PAR‐2 upregulation, downstream MAPK/NF‐κB signaling activation, and excessive microglial synaptic engulfment. (A) Schematic illustration of the experimental design. Neonatal rats received stereotaxic hippocampal injection of AAV9‐CX3CR1‐shNC or AAV9‐CX3CR1‐shF2rl1 on postnatal Day 1 (P1). HI injury or sham surgery was performed on P7, and hippocampal tissues were collected on P8 for subsequent analyses. (B) Schematic representation of the injection site and a representative coronal brain section showing the AAV associated reporter signal in the hippocampus. (C) Relative F2rl1 mRNA expression in hippocampus determined by quantitative real‐time PCR. (D) Representative immunoblots of PAR‐2, p‐p38 MAPK, p38 MAPK, p‐NF‐κB p65, NF‐κB p65, CD68, PSD95, SYP, and β‐actin in hippocampal tissue. (E) Quantification of PAR‐2, p38 MAPK, NF‐κB p65, CD68, PSD95, and SYP normalized to β‐actin. (F, G) Quantification of p‐p38 MAPK/p38 MAPK and p‐NF‐κB p65/NF‐κB p65 ratios, respectively. (H, I) Representative confocal images of Iba1 + microglia (red), PSD95 (green), and DAPI (blue) in the hippocampus, together with 3D reconstructions. Scale bars = 10 μm. Quantification of PSD95 puncta internalized within Iba1 + microglia was performed to evaluate microglia‐mediated synaptic engulfment. n = 4 animals per group. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To obtain direct evidence for microglia‐dependent synaptic engulfment, we performed 3D reconstruction of immunofluorescence labeled Iba1 + microglia and the postsynaptic marker PSD95 using Imaris software.

Techniques: Activation Assay, Injection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Tissue, Confocal

Journal: CNS Neuroscience & Therapeutics

Article Title: Mast Cell–Microglia Crosstalk Drives Aberrant Synaptic Pruning Through PAR ‐2/ MAPK / NF ‐ κB Signaling in Neonatal Hypoxic–Ischemic Encephalopathy

doi: 10.1002/cns.71189

Figure Lengend Snippet: Tryptase inhibition with APC366 is associated with reduced microglial PAR‐2 signal, attenuated MAPK/NF‐κB activation, and reduced synaptic engulfment after HI. (A) Representative immunofluorescence images showing Iba1 (green), PAR‐2 (red), and DAPI (blue) of Sham+Veh, HI+Veh, and HI+APC366 rats, together with enlarged views and single‐channel images. Scale bar = 100 μm. (B, C) Fluorescence‐intensity surface plots of PAR‐2 and quantification of PAR‐2 fluorescence intensity, respectively. (D) Quantification of Iba1 + /PAR‐2 + cells per field of view. (E) Representative immunoblots of tryptase, PAR‐2, p‐p38 MAPK, p38 MAPK, p‐NF‐κB p65, NF‐κB p65, and β‐actin in hippocampal tissue from Sham+Veh, HI+Veh, and HI+APC366 rats. (F) Quantification of tryptase, PAR‐2, p38 MAPK, and NF‐κB p65 normalized to β‐actin. (G, H) Quantification of p‐p38 MAPK/p38 MAPK and p‐NF‐κB p65/NF‐κB p65 ratios, respectively. (I) Representative immunoblots of CD68, PSD95, SYP, and β‐actin. (J) Quantification of CD68, PSD95, and SYP normalized to β‐actin. (K) Representative confocal images of Iba1 + microglia (red) and PSD95 (green), together with merged images and 3D reconstructions. Scale bars = 10 μm. (L, M) Quantification of PSD95 puncta engulfed by Iba1 + microglia and PSD95 volume within Iba1 + microglia, respectively. Data are presented as mean ± SEM. n = 4 animals per group.

Article Snippet: To obtain direct evidence for microglia‐dependent synaptic engulfment, we performed 3D reconstruction of immunofluorescence labeled Iba1 + microglia and the postsynaptic marker PSD95 using Imaris software.

Techniques: Inhibition, Activation Assay, Immunofluorescence, Fluorescence, Western Blot, Tissue, Confocal

Journal: CNS Neuroscience & Therapeutics

Article Title: Mast Cell–Microglia Crosstalk Drives Aberrant Synaptic Pruning Through PAR ‐2/ MAPK / NF ‐ κB Signaling in Neonatal Hypoxic–Ischemic Encephalopathy

doi: 10.1002/cns.71189

Figure Lengend Snippet: APC366 restores dendritic spine density, synaptic ultrastructure, and spatial memory performance in HI rats. (A) Representative images of neuronal morphology (left: Neuronal shell analysis, right: Dendritic spine imaging) from Sham+Veh, HI+Veh, and HI+APC366 groups. Scale bars = 5 μm. (B) Sholl analysis showing the number of dendritic intersections as a function of distance from the soma. (C) Quantification of dendritic spine density per 10 μm dendrite in each group. (D) Representative transmission electron micrographs (upper) and pseudo color illustrations (lower) displaying postsynaptic densities (PSD) and synaptic vesicles in hippocampal CA1 regions. Scale bars = 500 nm. (E) Quantification of PSD length in hippocampal synapses. (F) Quantification of synaptic vesicle number per presynaptic terminal. (G) Probe test traces and navigation test swim paths in the Morris water maze for each experimental group. (H) Escape latency curves during training days in the Morris water maze task. (I) Quantification of platform area crossings during the probe test. (J) Percentage of time spent in the target quadrant during the probe test. (K) Swimming speed measurement among groups in the Morris water maze. (L) Representative heatmaps of swim patterns in the Y‐maze novel arm test. (M) Quantification of the percentage of time spent in the novel arm during the Y‐maze test. (N) Quantification of the number of entries into the novel arm in the Y‐maze. Data are presented as mean ± SEM. n = 4 animals per group for histological and biochemical analyses and n = 8 animals per group for behavioral tests.

Article Snippet: To obtain direct evidence for microglia‐dependent synaptic engulfment, we performed 3D reconstruction of immunofluorescence labeled Iba1 + microglia and the postsynaptic marker PSD95 using Imaris software.

Techniques: Morphology, Analysis, Imaging, Transmission Assay