Journal: bioRxiv

Article Title: Localized calcium accumulations prime synapses for phagocyte removal in cortical neuroinflammation

doi: 10.1101/758193

Figure Lengend Snippet: (A) Orthogonal view of a CX3CR1 GFP cell (magenta) showing a Synapsin-1 inclusion (cyan, left ) colocalized with LAMP-1 staining (white, right ) suggesting removal of synapses by CNS resident microglial cells. (B) Orthogonal view of a CCR2 RFP cell (magenta) showing Synapsin-1 inclusion (cyan), suggesting removal of synapses by blood-derived mononuclear phagocytes. (C) Quantification of reconstructed Synapsin-1 positive spheres and Synapsin-1/LAMP1 double positive spheres inside CX3CR1 GFP cells in healthy control and c-MS d3 mice (n=40 cells from n=4 healthy control and n=5 c-MS d3 mice; mean ± SEM). (D) Left: 3D reconstruction of the cell in A (magenta) and its Synapsin-1 inclusions (cyan) with Imaris software allows studying synapse removal by mononuclear phagocytes. Right: Electron micrograph of a phagocytic cell (violet) in cortical layer 1 at the acute phase of c-MS. Insert : synaptic components (cyan) in a lysosomal compartment of the phagocyte cell. (E) Quantification of reconstructed Synapsin-1 positive spheres and Synapsin-1/LAMP1 double positive spheres inside CCR2 RFP cells in healthy control and c-MS d3 mice (n=0 cells from n=4 healthy control and n=50 cells from n=5 c-MS d3 mice; mean ± SEM). (F, G) Quantification of reconstructed spheres positive for postsynaptic markers ( F: PSD95; G: Homer1) and double positive for LAMP1 inside CX3CR1 GFP cells in healthy control and c-MS d3 mice (n=50 cells from n=5 healthy control and n=5 c-MS d3 mice; mean ± SEM). (H, I) Quantification of reconstructed spheres positive for postsynaptic markers ( H: PSD95; I: Homer1) and double positive for LAMP1 inside CCR2 RFP cells in healthy control and c-MS d3 mice (n=0 cells from n=4 healthy control and n=50 cells from n=5 c-MS d3 mice; mean ± SEM). Scale bars in A and B , 5 μm; D , 2 µm. Mann-Whitney U test in C and E ; Unpaired two-tailed t-test in F-I . **P<0.01, *** P<0.001

Article Snippet: Ten individual CX3CR1 GFP or CCR2 RFP cells per animal were reconstructed automatically by Imaris software in a stack of 5.14 µm.

Techniques: Staining, Derivative Assay, Control, Software, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Localized calcium accumulations prime synapses for phagocyte removal in cortical neuroinflammation

doi: 10.1101/758193

Figure Lengend Snippet: (A) Schematic diagram showing treatment with CSF-1R inhibitor or vehicle in the c-MS model. (B) Representative images of deconvoluted apical dendritic stretches from layer V pyramidal neurons showing spines in c-MS d3 mice treated with vehicle ( left ) or CSF-1R inhibitor ( right ) for 4 days. (C) Spine density of apical dendritic stretches from layer V pyramidal neurons in c-MS d3 mice treated with vehicle or CSF-1R inhibitor (n=65 and n=48 neurons from n=8 and n=7 mice; mean ± SEM). The dotted line shows the average level of spine density in healthy control mice based on . (D, E) FACS analysis of cell populations in brains of c-MS model mice. (D) After separation of debris and doublets, live cells were sorted using CD45 and CD11b staining as gating strategies to identify mononuclear phagocytic cells. (E) CD45 high CD11b high cells were further gated into two populations depending on the presence of CCR2-RFP positivity. (F-H) Volcano plot based on differentially expressed genes between CSF-1R inhibitor treatment vs. vehicle in CD45 int CD11b int cells (F) , CD45 high CD11b high CCR2 positive cells (G) and CD45 high CD11b high CCR2 negative cells (H) . (I) Heatmap of treatment-affected genes associated with Apoe signaling as measured in transcriptomic analysis of CD45 high CD11b high CCR2 negative cells (vehicle vs treatment, n=5 and n=6 mice respectively) (J) Genes predicted by Ingenuity Pathway Analysis to be directly regulated by CSF1 signaling (p-value cutoff of 0.05 and fold change cutoff of −2) in CD45 high CD11b high CCR2 negative cells (vehicle vs treatment, n=5 and n=6 mice respectively) (K) Quantification of CX3CR1 GFP cells (CNS resident microglial cells) in cortical sections of c-MS d3 mice treated with vehicle or CSF-1R inhibitor (n=6 mice per group; mean ± SEM). (L) Normalized LAMP1 positive volume and (M) number of Synapsin-1/LAMP1 double positive spheres per CX3CR1 GFP cells in c-MS d3 mice treated with vehicle or CSF-1R inhibitor (n=5 mice per group; mean ± SEM). (N) Quantification of Iba-1 positive CX3CR1 GFP negative cells (infiltrating mononuclear cells) in c-MS d3 mice treated with vehicle or CSF-1R inhibitor (n=6 mice per group; mean ± SEM). (O) Normalized LAMP1 positive volume and (P) number of Synapsin-1/LAMP1 double positive spheres per CCR2 RFP cells in c-MS d3 mice treated with vehicle or CSF-1R inhibitor (vehicle vs treatment, n=4 vs n=5 respectively; mean ± SEM). Scale bar in B , 2 µm. Unpaired two-tailed t-test in C, K-P. *P<0.05 **P<0.01, *** P<0.001.

Article Snippet: Ten individual CX3CR1 GFP or CCR2 RFP cells per animal were reconstructed automatically by Imaris software in a stack of 5.14 µm.

Techniques: Control, Staining, Two Tailed Test

Journal: Biomedical Optics Express

Article Title: Histology correlated adaptive optics polarisation sensitive optical coherence tomography

doi: 10.1364/BOE.590238

Figure Lengend Snippet: Top row: log-scale reflectance and DOPU of albino mouse retina. Second row: log-scale reflectance and DOPU of C57BL/6 mouse retina. Third row: log-scale reflectance and DOPU of Cx3cr1 GFP Ccr2 RFP mouse retina. Retinal layers as indicated on reflectance image in top row: retinal nerve fibre layer (RNFL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL); external limiting membrane (ELM); photoreceptor outer segments (OS); photoreceptor inner segments (IS); retinal pigment epithelium (RPE); choroid.

Article Snippet: Cx3cr1 GFP Ccr2 RFP transgenic mice (Jackson Laboratory, strain no. 032127; RRID: IMSR_JAX:032127) were used to generate time-course images.

Techniques: Membrane

Journal: Scientific Reports

Article Title: Macrophages recruited by implanted fibrin gels promote regeneration of injured lymphatic vessels

doi: 10.1038/s41598-026-39167-2

Figure Lengend Snippet: Fibrin gel implantation following lymphatic injury results in macrophage and neutrophil recruitment. ( a ) Percentage of macrophage infiltration on day 10 after lymphatic injury in the absence and presence of fibrin gel measured by flow cytometry. ( b ) Representative confocal images of the tissue surrounding uninjured and injured collecting lymphatic vessels with gel implantation. The data show that two weeks post-gel implantation, CX3CR1(+) and CCR2(+) cells accumulate around the regenerating vessel, where lectin-647 (white) indicates saphenous lymphatic vessels (see also Supplementary Fig. ). ( c , d ) The top panel illustrates the percentage of various cell types within the CD45 + population. The bottom panel shows the percentage of CCR5 + cells within each identified population. Each group comprised 4 mice, with tissue dissection and flow cytometry conducted 10 days following injury and gel implantation. ANOVA and post-hoc Tukey HSD tests were utilized to determine statistical significance. P-values are presented for statistically significant groups.

Article Snippet: PROX1-eGFP (kind gift of Taija Mäkinen, Uppsala University, and Young-Kwon Hong, University of Southern California), CX3CR1 GFP/+ and CX3CR1 GFP/+ CCR2 RFP/+ reporter mice (C57BL/6, JAX), double mutant CX3CR1 GFP/RFP CCR2 RFP/RFP mice (C57BL/6, JAX), and CCL5-KO mice (C57BL/6, JAX) were housed in MGH Center for Comparative Medicine facilities.

Techniques: Flow Cytometry, Dissection

Journal: Scientific Reports

Article Title: Macrophages recruited by implanted fibrin gels promote regeneration of injured lymphatic vessels

doi: 10.1038/s41598-026-39167-2

Figure Lengend Snippet: Macrophage and neutrophil depletion abolishes lymphatic regeneration after fibrin gel implantation. ( a ) Representative epifluorescence and confocal images of mouse saphenous tissue treated with control and clodronate liposomes after injury showing clodronate liposomes abolished lymphatic endothelial sprouting (PROX1-GFP+) within the fibrin gel. Mice treated with control liposomes exhibit lymphatic sprouting and reconnection within the gel. The fibrin gel was loaded with 10% v/v clodronate or control liposomes prior to polymerization in vivo. The white circle indicates the approximate injury site, and the white arrows show the direction of lymphatic flow. ( b ) Longitudinal near-infrared imaging was used to assess lymphatic pumping and regeneration after macrophage depletion by clodronate liposomes ( n = 4) and control liposomes ( n = 4) using CX3CR1GFP/+ mice. Representative NIR images of the popliteal collecting lymphatic vessels before surgery, two and four weeks after fibrin gel implantation. The red and blue arrows indicate lymphatics distal and proximal to the injury site, respectively. ( c ) Analysis of functional NIR imaging reveals a lack of lymphatic regeneration and loss of lymphatic pumping (packet frequency) after treatment by clodronate liposomes proximal to the injury site. ( d ) After gel implantation and treatment with clodronate liposomes in CX3CR1 GFP/+ mice, injured collecting lymphatic vessels did not adequately regenerate to re-connect the injured vessels (failure of proximal filling). In contrast, CX3CR1 GFP/+ mice treated with control liposomes successfully reconnected injured vessels (success of proximal filling two-sided Fisher’s exact test p < 0.05). ( e ) Confocal imaging was used to evaluate the depletion success in CX3CR1 GFP/+ mice. Quantification of CX3CR1 + cells shows that the number of cells significantly decreased following treatment by clodronate liposomes ( n = 3). Statistically significant differences between presurgical values and values on days 14, and 28 following injury were tested via ANOVA with Tukey’s post hoc tests (* p < 0.05, ** p < 0.01). An unpaired t-test was used to test for significant differences between the control liposomes and clodronate liposome groups at each time (* p < 0.05, ** p < 0.01).

Article Snippet: PROX1-eGFP (kind gift of Taija Mäkinen, Uppsala University, and Young-Kwon Hong, University of Southern California), CX3CR1 GFP/+ and CX3CR1 GFP/+ CCR2 RFP/+ reporter mice (C57BL/6, JAX), double mutant CX3CR1 GFP/RFP CCR2 RFP/RFP mice (C57BL/6, JAX), and CCL5-KO mice (C57BL/6, JAX) were housed in MGH Center for Comparative Medicine facilities.

Techniques: Control, Liposomes, In Vivo, Imaging, Functional Assay

Journal: Scientific Reports

Article Title: Macrophages recruited by implanted fibrin gels promote regeneration of injured lymphatic vessels

doi: 10.1038/s41598-026-39167-2

Figure Lengend Snippet: Fibrin gel implantation leads to increased tissue CCL5 after lymphatic vessel injury and loss of CCL5 inhibits the recovery of lymphatic filling proximal to the injury site. ( a ) A comparative analysis of the levels of cytokines and growth factors after gel implantation demonstrating the upregulation of CCL5 and MMP9 ( n = 3 mice pooled in each group). Data generated by Proteome Profiler Cytokine Array (R&D systems, Minneapolis, MN) according to the manufacturer’s protocol. The values indicate fold changes in different groups relative to the values in the sham group. ( b ) Mice lacking CCL5 (CCL5-/-) cytokine are less likely than control mice to regain filling of vessels proximal to the injury site (proximal filling) subsequent to gel implantation 4-week post-injury ( p = 0.0498, two-sided Fisher’s exact test) while deficiency of CX3CR1 and CCR2 are not associated with the success/failure of proximal filling recovery following gel implantation ( p = 0.5105, two-sided Fisher’s exact test). ( c ) A comparison of lymphatic pumping frequencies via NIR technique proximal and distal to the injury site among CCL5-KO, CX3CR1(GFP/GFP), CCR2(-/-), and double-deficient CX3CR1(GFP/GFP)-CCR2(RFP/RFP) mice following gel implantation. To test the association between categorical variables (success/ failure of proximal filling after gel implantation), the two-sided Fisher’s exact test was used ( p < 0.05). One-way ANOVA analysis with the Tukey post hoc test was used to test the significant difference between different groups after gel implantation (* p < 0.05, ** p < 0.01, *** p < 0.001). All data were presented as mean (± s.e.m.).

Article Snippet: PROX1-eGFP (kind gift of Taija Mäkinen, Uppsala University, and Young-Kwon Hong, University of Southern California), CX3CR1 GFP/+ and CX3CR1 GFP/+ CCR2 RFP/+ reporter mice (C57BL/6, JAX), double mutant CX3CR1 GFP/RFP CCR2 RFP/RFP mice (C57BL/6, JAX), and CCL5-KO mice (C57BL/6, JAX) were housed in MGH Center for Comparative Medicine facilities.

Techniques: Generated, Control, Comparison

Journal: Biomedical Optics Express

Article Title: Histology correlated adaptive optics polarisation sensitive optical coherence tomography

doi: 10.1364/BOE.590238

Figure Lengend Snippet: Top row: log-scale reflectance and DOPU of albino mouse retina. Second row: log-scale reflectance and DOPU of C57BL/6 mouse retina. Third row: log-scale reflectance and DOPU of Cx3cr1 GFP Ccr2 RFP mouse retina. Retinal layers as indicated on reflectance image in top row: retinal nerve fibre layer (RNFL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL); external limiting membrane (ELM); photoreceptor outer segments (OS); photoreceptor inner segments (IS); retinal pigment epithelium (RPE); choroid.

Article Snippet: Cx3cr1 GFP Ccr2 RFP mice are derived from a C57BL/6 genetic background.C57BL/6J mice (Charles River, strain no. 027; RRID: IMSR_CRL:027) and albino (Charles River, strain number 022, Crl:CD1(ICR)) mice were used for validation.

Techniques: Membrane

Journal: bioRxiv

Article Title: Meningeal neutrophil infiltration drives inflammation-exacerbated pediatric stroke through IL-36γ signaling

doi: 10.64898/2026.04.03.716365

Figure Lengend Snippet: a, Expression of neutrophil activation markers (ICAM1, iNOS, CD62L and CXCR2) in the ipsilateral hemisphere was assessed by flow cytometry at 24 h post-stroke in PT and LPS/PT groups (N = 7 per group). Data were presented as mean ± S.E.M. ****p < 0.0001 compared with the PT group by unpaired t-test. b-c, Mice were treated with a neutrophil-depleting anti-Ly6G antibody (clone 1A8) or an isotype control antibody beginning one day prior to stroke induction and continuing daily for three consecutive days (P15–P17). Brain sections (1mm sections) were collected 3 days post-stroke and stained with TTC. Infarct volumes were quantified in mm3. (N> 10 per group). *p < 0.05 by one-way ANOVA. d, CCR2–RFP knock-in mice, including homozygous CCR2-deficient (CCR2RFP/RFP) and heterozygous CCR2-sufficient (CCR2RFP/+) littermates, were subjected to PT or LPS/PT. Brain sections were harvested 3 days post-stroke and infarct volumes were quantified by TTC staining and expressed as a percentage of the contralateral hemisphere (N > 5 per group). ns, Not significant, p>0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA.

Article Snippet: C57BL/6J, CCR2 RFP/RFP (JAX#017586), and GFAP-Cre (JAX#012886) mice were purchased from The Jackson Laboratory.

Techniques: Expressing, Activation Assay, Flow Cytometry, Control, Staining, Knock-In