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Cited by 123Lymphatic System and DiseasesNeuroinflammation and Neurodegeneration MechanismsAcute Ischemic Stroke Management

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Brain-to-cervical lymph node signaling after stroke.

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Esposito, Elga, Ahn, Bum Ju, Shi, Jingfei, Nakamura, Yoshihiko, Park, Ji Hyun, Mandeville, Emiri T., Yu, Zhanyang, Chan, Su Jing, Desai, Rakhi, Hayakawa, Ayumi, Ji, Xunming, Lo, Eng H., Hayakawa, Kazuhide·2019·Nature Communications
DOI 10.1038/s41467-019-13324-wPMC PMC6876639
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Abstract

After stroke, peripheral immune cells are activated and these systemic responses may amplify brain damage, but how the injured brain sends out signals to trigger systemic inflammation remains unclear. Here we show that a brain-to-cervical lymph node (CLN) pathway is involved. In rats subjected to focal cerebral ischemia, lymphatic endothelial cells proliferate and macrophages are rapidly activated in CLNs within 24 h, in part via VEGF-C/VEGFR3 signalling. Microarray analyses of isolated lymphatic endothelium from CLNs of ischemic mice confirm the activation of transmembrane tyrosine kinase pathways. Blockade of VEGFR3 reduces lymphatic endothelial activation, decreases pro-inflammatory macrophages, and reduces brain infarction. In vitro, VEGF-C/VEGFR3 signalling in lymphatic endothelial cells enhances inflammatory responses in co-cultured macrophages. Lastly, surgical removal of CLNs in mice significantly reduces infarction after focal cerebral ischemia. These findings suggest that modulating the brain-to-CLN pathway may offer therapeutic opportunities to ameliorate systemic inflammation and brain injury after stroke.

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Fig. 1 Cervical lymph node activation in rats after stroke: a Evans Blue dye (2%, 10 µL) was injected into lateral ventricles in normal male Sprague Dawley (SD) rats. Within 30 min, Evans Blue fluorescence was detected in deep and superficial cervical lymph nodes (CLNs) but not in axillary lymph nodes (ALNs) or inguinal lymph nodes (ILNs). Scale: 500 nm. b–d Male SD rats were subjected to 100 min of transient focal cerebral ischemia. Deep and superficial CLNs were collected and analyzed by immunohistochemistry. We did not count LYVE-1 or Ki67 single-positive cells to exclude potential signal from the macrophage population. b Proliferation of lymphatic endothelial cells were also observed in deep CLNs, but it was not statistically significant. c LYVE-1 positive lymphatic endothelial cells in the area of subcapsular sinus in superficial CLNs rapidly proliferated as early as 3 h until 24 h after focal ischemia (n = 4 biologically independent animals). d At the same time, ILNs were collected as a negative control. Note: Some of Ki67 could be co-expressed with immune cells including macrophages and T cells58. *P < 0.05, **P < 0.01 vs Sham, one-way ANOVA followed by Fisher’s LSD test. Scale: 100 nm. e, f Western blot, following immunoprecipitation, demonstrated that VEGFR3 was phosphorylated in superficial CLNs at 24 h after transient focal ischemia (n = 3). *P < 0.05, unpaired t-test. g PBS (5 µL) or MAZ51 (50 ng/5 µL) was injected into lateral ventricles immediately after reperfusion and FACS analysis was performed at 24 h. h FACS analysis indicated that focal ischemia promoted the proliferation of VEGFR3 positive cells in the superficial CLNs. Intracerebroventricular injection of MAZ51 significantly suppressed the proliferation of VEGFR3-positive cells in CLNs (Sham; n = 3, MCAO; n = 11, MCAO + MAZ51; n = 7 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. i Immunohistochemistry demonstrated that MAZ51 treatment decreased the proliferation of LYVE-1 positive lymphatic endothelium that was observed after ischemia (n = 4). Scale: 100 nm. **P < 0.01, unpaired t-test. All values are mean +/− SD.
Fig. 1 Cervical lymph node activation in rats after stroke: a Evans Blue dye (2%, 10 µL) was injected into lateral ventricles in normal male Sprague Dawley (SD) rats. Within 30 min, Evans Blue fluorescence was detected in deep and superficial cervical lymph nodes (CLNs) but not in axillary lymph nodes (ALNs) or inguinal lymph nodes (ILNs). Scale: 500 nm. b–d Male SD rats were subjected to 100 min of transient focal cerebral ischemia. Deep and superficial CLNs were collected and analyzed by immunohistochemistry. We did not count LYVE-1 or Ki67 single-positive cells to exclude potential signal from the macrophage population. b Proliferation of lymphatic endothelial cells were also observed in deep CLNs, but it was not statistically significant. c LYVE-1 positive lymphatic endothelial cells in the area of subcapsular sinus in superficial CLNs rapidly proliferated as early as 3 h until 24 h after focal ischemia (n = 4 biologically independent animals). d At the same time, ILNs were collected as a negative control. Note: Some of Ki67 could be co-expressed with immune cells including macrophages and T cells58. *P < 0.05, **P < 0.01 vs Sham, one-way ANOVA followed by Fisher’s LSD test. Scale: 100 nm. e, f Western blot, following immunoprecipitation, demonstrated that VEGFR3 was phosphorylated in superficial CLNs at 24 h after transient focal ischemia (n = 3). *P < 0.05, unpaired t-test. g PBS (5 µL) or MAZ51 (50 ng/5 µL) was injected into lateral ventricles immediately after reperfusion and FACS analysis was performed at 24 h. h FACS analysis indicated that focal ischemia promoted the proliferation of VEGFR3 positive cells in the superficial CLNs. Intracerebroventricular injection of MAZ51 significantly suppressed the proliferation of VEGFR3-positive cells in CLNs (Sham; n = 3, MCAO; n = 11, MCAO + MAZ51; n = 7 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. i Immunohistochemistry demonstrated that MAZ51 treatment decreased the proliferation of LYVE-1 positive lymphatic endothelium that was observed after ischemia (n = 4). Scale: 100 nm. **P < 0.01, unpaired t-test. All values are mean +/− SD.CC BY · PubMed Central
Fig. 2 Macrophage activation in rat CLNs after stroke: a–c Male SD rats were subjected to 100 min of transient focal cerebral ischemia. Deep and superficial CLNs, and ILNs were collected and analyzed by immunohistochemistry. Lymph node resident CD169 positive macrophages co-labeling with Ki67 were identified as proliferating cells. Immunostaining showed that macrophage proliferation was slightly but not significantly increased in CLNs after focal cerebral ischemia (n = 4 biologically independent animals). Scale: 100 nm. One-way ANOVA followed by Fisher’s LSD test. d Inflammatory factors, including prototypical cytokines such as interleukin-1β (IL-1β) and transforming growth factor beta (TGF-β), were analyzed, by flow cytometry analysis, in CD68 positive macrophages. After stroke, IL-1β positive macrophages were increased in superficial CLNs, but TGF-β positive subsets did not change (Sham; n = 4, MCAO; n = 6 biologically independent animals). *P < 0.05 vs Sham, one-way ANOVA followed by Fisher’s LSD test. e Intracerebroventricular injection of MAZ51 (50 ng/5 µL) significantly reduced IL-1β positive macrophages (n = 4 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. f, g Immunohistochemistry confirmed that CD169 positive macrophages expressed IL-1β after focal cerebral ischemia and MAZ51 treatment reduced IL-1β positive macrophages in subcapsular sinus. Scale: 100 nm. All values are mean +/− SD.
Fig. 2 Macrophage activation in rat CLNs after stroke: a–c Male SD rats were subjected to 100 min of transient focal cerebral ischemia. Deep and superficial CLNs, and ILNs were collected and analyzed by immunohistochemistry. Lymph node resident CD169 positive macrophages co-labeling with Ki67 were identified as proliferating cells. Immunostaining showed that macrophage proliferation was slightly but not significantly increased in CLNs after focal cerebral ischemia (n = 4 biologically independent animals). Scale: 100 nm. One-way ANOVA followed by Fisher’s LSD test. d Inflammatory factors, including prototypical cytokines such as interleukin-1β (IL-1β) and transforming growth factor beta (TGF-β), were analyzed, by flow cytometry analysis, in CD68 positive macrophages. After stroke, IL-1β positive macrophages were increased in superficial CLNs, but TGF-β positive subsets did not change (Sham; n = 4, MCAO; n = 6 biologically independent animals). *P < 0.05 vs Sham, one-way ANOVA followed by Fisher’s LSD test. e Intracerebroventricular injection of MAZ51 (50 ng/5 µL) significantly reduced IL-1β positive macrophages (n = 4 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. f, g Immunohistochemistry confirmed that CD169 positive macrophages expressed IL-1β after focal cerebral ischemia and MAZ51 treatment reduced IL-1β positive macrophages in subcapsular sinus. Scale: 100 nm. All values are mean +/− SD.CC BY · PubMed Central
Fig. 3 Cervical lymphatic activation in mice after stroke: a Male C57BL6 mice were subjected to transient 60 min of focal ischemia. Note: the artery occlusion time was titrated to achieve equivalent levels of injury in mice and rats. Sixty min of transient middle cerebral artery occlusion in mice result in the same infarct volume as 100 min of occlusion in rats, i.e. approximately 30% of the ipsilateral hemisphere. Western blot demonstrated that LYVE-1 expression was significantly upregulated in CLNs. LYVE-1 upregulation was robust in superficial CLNs compared to deep CLNs after focal ischemia. There were no significant changes in axillary lymph nodes or spleen after focal ischemia (n = 6 biologically independent animals). *P < 0.05 vs Sham resulted from Fisher’s LSD test. b, c Immunohistochemistry using LYVE-1 antibody (Sham; n = 13, MCAO; n = 6 biologically independent animals) and Podoplanin antibody (Sham; n = 7, MCAO; n = 7 biologically independent animals) confirmed that lymphatic endothelial intensity was increased in superficial CLNs at 24 h after focal cerebral ischemia. Scale: 100 nm. *P < 0.05, **P < 0.01, unpaired t-test. Images were taken in the area of subcapsular sinus. d Experimental design for microarray using lymphatic endothelium isolated from CLNs after focal ischemia. Male C57BL6 mice were subjected to transient focal ischemia. At 3 h after reperfusion, LYVE-1 positive lymphatic vessels were collected from CLNs pooled from 4 mice, and RNAs were extracted for microarray analysis. e Focal ischemia upregulated genes in lymphatic endothelium (red in heat maps, n = 3, total animal number = 12). f Gene Set Enrichment Analysis (GSEA) suggested that differentially expressed genes were largely related to matrix and transmembrane receptor protein tyrosine kinase pathways. g Volcano plot showed some upregulated genes after cerebral ischemia including Fkbp5, Hdc, Sesn1, Lyve1, Cyp2b10, Dccp2, Flt1, Ccl28, IL1b, Kdr, and Flt4. All values are mean +/− SD.
Fig. 3 Cervical lymphatic activation in mice after stroke: a Male C57BL6 mice were subjected to transient 60 min of focal ischemia. Note: the artery occlusion time was titrated to achieve equivalent levels of injury in mice and rats. Sixty min of transient middle cerebral artery occlusion in mice result in the same infarct volume as 100 min of occlusion in rats, i.e. approximately 30% of the ipsilateral hemisphere. Western blot demonstrated that LYVE-1 expression was significantly upregulated in CLNs. LYVE-1 upregulation was robust in superficial CLNs compared to deep CLNs after focal ischemia. There were no significant changes in axillary lymph nodes or spleen after focal ischemia (n = 6 biologically independent animals). *P < 0.05 vs Sham resulted from Fisher’s LSD test. b, c Immunohistochemistry using LYVE-1 antibody (Sham; n = 13, MCAO; n = 6 biologically independent animals) and Podoplanin antibody (Sham; n = 7, MCAO; n = 7 biologically independent animals) confirmed that lymphatic endothelial intensity was increased in superficial CLNs at 24 h after focal cerebral ischemia. Scale: 100 nm. *P < 0.05, **P < 0.01, unpaired t-test. Images were taken in the area of subcapsular sinus. d Experimental design for microarray using lymphatic endothelium isolated from CLNs after focal ischemia. Male C57BL6 mice were subjected to transient focal ischemia. At 3 h after reperfusion, LYVE-1 positive lymphatic vessels were collected from CLNs pooled from 4 mice, and RNAs were extracted for microarray analysis. e Focal ischemia upregulated genes in lymphatic endothelium (red in heat maps, n = 3, total animal number = 12). f Gene Set Enrichment Analysis (GSEA) suggested that differentially expressed genes were largely related to matrix and transmembrane receptor protein tyrosine kinase pathways. g Volcano plot showed some upregulated genes after cerebral ischemia including Fkbp5, Hdc, Sesn1, Lyve1, Cyp2b10, Dccp2, Flt1, Ccl28, IL1b, Kdr, and Flt4. All values are mean +/− SD.CC BY · PubMed Central
Fig. 4 Cervical lymphatic inflammation through VEGFR3 tyrosine kinase in mice after stroke: a Male C57BL6 mice were subjected to transient focal ischemia and, immediately after reperfusion the vehicle (PBS 10 µL) or MAZ51 (3 ng/10 µL) were injected into the nasal cavity. b MAZ51 treatment suppressed tyrosine phosphorylation in superficial CLN lymphatic endothelium at 72 h after focal ischemia (Sham; n = 4, MCAO; n = 9, MCAO + MAZ51; n = 8 biologically independent animals). *P < 0.05, one-way ANOVA followed by Fisher’s LSD test. c FACS analysis demonstrated that MAZ51 treatment significantly decreased pro-inflammatory TNF-α positive macrophages in superficial CLNs; no clear changes were noticed in TGF-β positive macrophages. *P < 0.05, one-way ANOVA followed by Fisher’s LSD test. d–f. Immunostaining revealed that CD169 positive macrophages increased TNF-α, while MAZ51 treatment decreased pro-inflammatory macrophages. Scale: 100 nm. g–h Confocal microscopy analysis demonstrated that TNF-α was highly co-localized with CD169 positive macrophages in the subcapsular sinus of superficial CLNs. MAZ51 treatment decreased the co-localization. TNF-α expression was not observed in ILN macrophages. Scale: 100 nm. i–l Western blot confirmed that TNF-α, IL-1β and CCL28 were significantly increased in post-stroke superficial CLNs. MAZ51 treatment reduced cytokine/chemokine expression (n = 4 biologically independent animals). ILNs weakly responded to focal cerebral ischemia as to TNF-α, IL-1β or CCL28 expression (n = 3 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. All values are mean +/− SD.
Fig. 4 Cervical lymphatic inflammation through VEGFR3 tyrosine kinase in mice after stroke: a Male C57BL6 mice were subjected to transient focal ischemia and, immediately after reperfusion the vehicle (PBS 10 µL) or MAZ51 (3 ng/10 µL) were injected into the nasal cavity. b MAZ51 treatment suppressed tyrosine phosphorylation in superficial CLN lymphatic endothelium at 72 h after focal ischemia (Sham; n = 4, MCAO; n = 9, MCAO + MAZ51; n = 8 biologically independent animals). *P < 0.05, one-way ANOVA followed by Fisher’s LSD test. c FACS analysis demonstrated that MAZ51 treatment significantly decreased pro-inflammatory TNF-α positive macrophages in superficial CLNs; no clear changes were noticed in TGF-β positive macrophages. *P < 0.05, one-way ANOVA followed by Fisher’s LSD test. d–f. Immunostaining revealed that CD169 positive macrophages increased TNF-α, while MAZ51 treatment decreased pro-inflammatory macrophages. Scale: 100 nm. g–h Confocal microscopy analysis demonstrated that TNF-α was highly co-localized with CD169 positive macrophages in the subcapsular sinus of superficial CLNs. MAZ51 treatment decreased the co-localization. TNF-α expression was not observed in ILN macrophages. Scale: 100 nm. i–l Western blot confirmed that TNF-α, IL-1β and CCL28 were significantly increased in post-stroke superficial CLNs. MAZ51 treatment reduced cytokine/chemokine expression (n = 4 biologically independent animals). ILNs weakly responded to focal cerebral ischemia as to TNF-α, IL-1β or CCL28 expression (n = 3 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. All values are mean +/− SD.CC BY · PubMed Central
Fig. 5 Activated lymphatic endothelium upregulates pro-inflammatory macrophages in vitro: a LYVE-1 antibody-conjugated magnetic beads were used for lymphatic endothelial isolation using CLNs isolated from 4 mice. b VEGFR3 was activated by adding VEGF-C (10 ng/ml) to mouse CLN lymphatic endothelial cells for 24 h, then cells were co-cultured with mouse peritoneal macrophages for another 24 h and analyzed by immunostaining. c Immunostaining showed that the expression of iNOS (M1-like) was upregulated, but CD206 (M2-like) expression did not change. Scale: 50 nm. d–f Western blot confirmed that iNOS and IL-1β were upregulated, but TGF-β did not change. *P < 0.05, unpaired t-test. g–i VEGF-C (10 ng/mL) was directly added to macrophages for 24 h. Western blot confirmed that VEGF-C itself did not change macrophage iNOS, IL-1β, and TGF-β expressions (n = 6 biologically independent cells, n = 3 biologically independent experiments), unpaired t-test. All values are mean +/− SD.
Fig. 5 Activated lymphatic endothelium upregulates pro-inflammatory macrophages in vitro: a LYVE-1 antibody-conjugated magnetic beads were used for lymphatic endothelial isolation using CLNs isolated from 4 mice. b VEGFR3 was activated by adding VEGF-C (10 ng/ml) to mouse CLN lymphatic endothelial cells for 24 h, then cells were co-cultured with mouse peritoneal macrophages for another 24 h and analyzed by immunostaining. c Immunostaining showed that the expression of iNOS (M1-like) was upregulated, but CD206 (M2-like) expression did not change. Scale: 50 nm. d–f Western blot confirmed that iNOS and IL-1β were upregulated, but TGF-β did not change. *P < 0.05, unpaired t-test. g–i VEGF-C (10 ng/mL) was directly added to macrophages for 24 h. Western blot confirmed that VEGF-C itself did not change macrophage iNOS, IL-1β, and TGF-β expressions (n = 6 biologically independent cells, n = 3 biologically independent experiments), unpaired t-test. All values are mean +/− SD.CC BY · PubMed Central
Fig. 6 Effects of blocking cervical lymphatic activation in brain infarct formation after stroke: a, b Male C57BL6 mice were subjected to transient focal ischemia. Neuronal staining with NeuN antibody and basal lamina staining with collagen IV (Col IV) antibody or endothelial staining with vWF antibody were conducted to provide visual landmarks for immune cell infiltration including monocytes/macrophages, neutrophils or T-cells. Immunohistochemistry confirmed that monocytes/macrophages and neutrophils were accumulated in the ischemic area and peri-infarct cortex. A few T-cells were infiltrated in the same area at 72 h after stroke. Scale: 100 nm. c The percentage of IgG leaked area after MCAO was assessed. MAZ51 treatment did not influence IgG leakage after focal ischemia (n = 4 biologically independent animals). unpaired t-test. d, e CD16/32 antibody was used to identify pro-inflammatory macrophage phenotype. At 72 h after focal ischemia, pro-inflammatory macrophages were accumulated in ipsilateral cortex (d) and striatum (e). Intranasal treatment with MAZ51 (3 ng/10 µL) significantly reduced pro-inflammatory macrophages (n = 4 biologically independent animals). Scale: 100 nm. **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. f MAZ51 (3 ng/10 µL) significantly decreased cerebral infarction volumes at 72 h (MCAO; n = 8, MCAO + MAZ51; n = 9 biologically independent animals). Vehicle: 100.1 +/− 23.8 mm3, MAZ51: 71.2 +/− 19.7 mm3. *P < 0.05, unpaired t-test. All values are mean +/− SD.
Fig. 6 Effects of blocking cervical lymphatic activation in brain infarct formation after stroke: a, b Male C57BL6 mice were subjected to transient focal ischemia. Neuronal staining with NeuN antibody and basal lamina staining with collagen IV (Col IV) antibody or endothelial staining with vWF antibody were conducted to provide visual landmarks for immune cell infiltration including monocytes/macrophages, neutrophils or T-cells. Immunohistochemistry confirmed that monocytes/macrophages and neutrophils were accumulated in the ischemic area and peri-infarct cortex. A few T-cells were infiltrated in the same area at 72 h after stroke. Scale: 100 nm. c The percentage of IgG leaked area after MCAO was assessed. MAZ51 treatment did not influence IgG leakage after focal ischemia (n = 4 biologically independent animals). unpaired t-test. d, e CD16/32 antibody was used to identify pro-inflammatory macrophage phenotype. At 72 h after focal ischemia, pro-inflammatory macrophages were accumulated in ipsilateral cortex (d) and striatum (e). Intranasal treatment with MAZ51 (3 ng/10 µL) significantly reduced pro-inflammatory macrophages (n = 4 biologically independent animals). Scale: 100 nm. **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. f MAZ51 (3 ng/10 µL) significantly decreased cerebral infarction volumes at 72 h (MCAO; n = 8, MCAO + MAZ51; n = 9 biologically independent animals). Vehicle: 100.1 +/− 23.8 mm3, MAZ51: 71.2 +/− 19.7 mm3. *P < 0.05, unpaired t-test. All values are mean +/− SD.CC BY · PubMed Central
Fig. 7 Superficial cervical node lymphadenectomy reduces brain damage after stroke: a Superficial CLNs were surgically removed from C57BL6 mice, then mice were subjected to transient 60 min focal cerebral ischemia. b, c Flow cytometry analysis demonstrated that accumulations of neutrophils and F4/80 positive monocytes/macrophages in both systemic circulation (b) and injured brain (c) were significantly reduced by superficial CLN removal (Control; n = 3, CLN removal; n = 4 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. d. Percentage of IgG leaked area in the ipsilateral hemisphere was assessed. CLN removal did not significantly influence IgG leakage after focal ischemia (n = 4 biologically independent animals). unpaired t-test. e CLN lymphadenectomy significantly decreased pro-inflammatory macrophages in ipsilateral cortex and striatum (n = 4 biologically independent animals), unpaired t-test. Scale: 100 nm. f CLN lymphadenectomy significantly reduced infarct volume at 72 h post-stroke (Control; n = 10, CLN removal; n = 11 biologically independent animals). Control: 99.9 + /− 22.3 mm3, CLN removal: 73.4 + /− 22.7 mm3. *P < 0.05, unpaired t-test. g Inguinal lymph nodes (ILN) lymphadenectomy did not decrease infarct volume at 72 h post-stroke (Control; n = 7, ILN removal; n = 6 biologically independent animals). Control: 90.2 +/− 19.1 mm3, ILN removal: 89.9 +/− 9.6 mm3, unpaired t-test. All values are mean +/− SD. h Schematic of the proposed brain-to-cervical lymph node pathway in the regulation of inflammatory response via VEGFR3 signaling after focal cerebral ischemia.
Fig. 7 Superficial cervical node lymphadenectomy reduces brain damage after stroke: a Superficial CLNs were surgically removed from C57BL6 mice, then mice were subjected to transient 60 min focal cerebral ischemia. b, c Flow cytometry analysis demonstrated that accumulations of neutrophils and F4/80 positive monocytes/macrophages in both systemic circulation (b) and injured brain (c) were significantly reduced by superficial CLN removal (Control; n = 3, CLN removal; n = 4 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. d. Percentage of IgG leaked area in the ipsilateral hemisphere was assessed. CLN removal did not significantly influence IgG leakage after focal ischemia (n = 4 biologically independent animals). unpaired t-test. e CLN lymphadenectomy significantly decreased pro-inflammatory macrophages in ipsilateral cortex and striatum (n = 4 biologically independent animals), unpaired t-test. Scale: 100 nm. f CLN lymphadenectomy significantly reduced infarct volume at 72 h post-stroke (Control; n = 10, CLN removal; n = 11 biologically independent animals). Control: 99.9 + /− 22.3 mm3, CLN removal: 73.4 + /− 22.7 mm3. *P < 0.05, unpaired t-test. g Inguinal lymph nodes (ILN) lymphadenectomy did not decrease infarct volume at 72 h post-stroke (Control; n = 7, ILN removal; n = 6 biologically independent animals). Control: 90.2 +/− 19.1 mm3, ILN removal: 89.9 +/− 9.6 mm3, unpaired t-test. All values are mean +/− SD. h Schematic of the proposed brain-to-cervical lymph node pathway in the regulation of inflammatory response via VEGFR3 signaling after focal cerebral ischemia.CC BY · PubMed Central

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Authors
Esposito, E., Ahn, B. J., Shi, J., Nakamura, Y., Park, J. H., Mandeville, E. T., Yu, Z., Chan, S. J., Desai, R., Hayakawa, A., Ji, X., Lo, E. H., & Hayakawa, K.
Affiliations
  • Harvard University (US)
  • Capital Medical University (CN)
Corresponding author
  • Lo, Eng H.
  • Hayakawa, Kazuhide
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-11-22
Volume
10
Issue
1
Pages
5306-5306
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-13324-w
PMC
PMC6876639
OpenAlex
W2990744279
Cited by
123
References
70
Citations by year
2026: 13 · 2025: 20 · 2024: 18 · 2023: 16 · 2022: 27 · 2021: 24 · 2020: 5
FWCI
5.85
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • National University of Singapore (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke
Topics
  • Health Sciences › Medicine › Oncology › Lymphatic System and Diseases
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Acute Ischemic Stroke Management
Keywords
stroke, cervical lymph nodes, focal cerebral ischemia, systemic inflammation, lymphatic endothelial cells, macrophage activation, infarct size, lymphatic drainage
MeSH terms
Animals, Brain, Brain Ischemia, Endothelium, Lymphatic, Inflammation, Lymph Nodes, Macrophages, Neck, Brain Infarction, Stroke, Vascular Endothelial Growth Factor Receptor-3, Vascular Endothelial Growth Factor C, Lymphangiogenesis, Endothelial Cells, Cell Proliferation, Mice, Rats

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