Fig 1.
Surgery for the CCI mouse model.
(a) In the injury group, the skull was removed, and a rounded metal tip was used to directly impact the brain. (b) For the sham group, resection of the external table and upper part of the internal table of the skull was performed using a drill. (c) The rounded metal tip was pushed into the bone flaps manually to transmit the pressure to the brain via the bone flaps in the sham group.
Fig 2.
Isolation of meningeal lymphatic endothelial cells (LECs) via flow cytometry.
Representative dot and contour plots showing the gating strategy used to isolate LECs via flow cytometry from the meninges of mice with traumatic brain injury and the sham mice. The cell count graph shows the number of cells, which were gated by CD45−CD31+PDPN+, labeled with the anti-LYVE1 antibody.
Table 1.
Sequences of the primers used for RT-PCR.
Fig 3.
Count of viable LYVE1 positive meningeal lymphatic endothelial cells (LECs) via flow cytometry.
The count of viable, CD45-CD31+PDPN+, LYVE1-positive meningeal LECs was compared between the injury and sham groups 3 days after injury via flow cytometry. (*p = 0.0122 between the injury and sham groups, n = 5 for each group).
Table 2.
Average number of each cell gated by flow cytometry.
Fig 4.
Principal component analysis (PCA) and volcano plots of the microarray data.
(a) PCA shows clustering of samples. (b) The volcano plots illustrate the number of significantly differentially expressed transcripts (fold change ≥ 2 and p ≤ 0.05). Significantly downregulated transcripts are represented by green dot plots and significantly upregulated transcripts by red dot plots. Several transcripts with altered expression are indicated. (n = 3 for each group).
Fig 5.
Pathways and gene ontology (GO) analyses using g:Profiler.
Pathway and GO analyses of significantly differentially expressed genes (fold change ≥ 2 and p ≤ 0.05) are shown. The numbers identified in Fig 4 are further described in Table 3. (MF: molecular functions. BP: biological processes. CC: cellular components, REAC: Reactome pathway. KEGG: Kyoto Encyclopedia of Genes and Genomes pathway. n = 3 for each group).
Table 3.
Important terms obtained in gene ontology (GO) and pathway analyses via g:Profiler for 657 genes in 1242 transcriptswith ≥ 2-folds changes and P-values ≤ 0.05.
Fig 6.
Interaction degree of the protein–protein interaction (PPI) network assessed via Cytoscape using the Search Tool for the Retrieval of Interacting Genes (STRING) plug-in.
The interaction degree of the PPI network is shown based on significantly differentially expressed genes (fold change ≥ 2 and p ≤ 0.05). Near red color indicates upregulation, and near black color indicates downregulation; the larger the circle, the larger the value of interaction degree. (n = 3 for each group).
Fig 7.
Module of molecular complex detection (MCODE) via the protein–protein interaction (PPI) network using Cytoscape and the Search Tool for the Retrieval of Interacting Genes (STRING) plug-in and MCODE tool.
The module of MCODE from significantly differentially expressed genes (fold change ≥ 2 and p ≤ 0.05) is shown. (a–n) Modules from 1 to 14. Near red color indicates upregulation, and near black color indicates downregulation; the larger the circle, the larger the value of interaction degree. (n = 3 for each group).
Fig 8.
Lymphangiogenesis pathway gene of the protein–protein interaction (PPI) network obtained using Cytoscape with the Search Tool for the Retrieval of Interacting Genes (STRING) plug-in.
(a) The PPI network for all lymphangiogenesis pathway genes is shown. The PPI network of genes regulating or regulated by (b) Vegfc, (c) Flt4, and (d) Nrp2. Near red color indicates upregulation, and near black color indicates downregulation; the larger the circle, the larger the value of interaction degree. (n = 3 for each group).
Fig 9.
Microarray, RT-PCR and ELISA data for meningeal LECs from mice with injury and sham mice 3 days after injury.
(a) The mRNA expression levels of Nrp2 (left Y-axis), Flt4 (right Y-axis), and Lyve1 (right Y-axis) were determined using microarrays. The mRNA expression levels of (b) Nrp2, (c) Flt4, and (d) Lyve1 were normalized to those of that of Hprt, Ppia, and S100b using RT-PCR. The protein expression levels of (e) NRP2 and (f) FLT4 in meningeal LECs, as determined using ELISA, were compared between the injury group and the sham group 3 days after injury. (*p < 0.05 between injury and sham group, n = 3 for each group, as assessed using microarrays, n = 5 for each group, as assessed using RT-PCR and ELISA).
Table 4.
Interaction degree of the protein-protein interaction network for the lymphangiogenesis pathway gene set.
(n = 3 for each group).