Fig 2: The location and expression of CTRP1 in the brain analyzed by immunofluorescence (n = 3). (A) The expression of CTRP1 in neuron in the cortex, NeuN was used to label neuron. CTRP1 expression was observed by fluorescence microscope and is shown by green fluorescence. NeuN expression is shown by red fluorescence. The nuclei were stained with DAPI and is shown by blue fluorescence. (B) The expression of CTRP1 in astroglia in the cortex. GFAP was used to label astroglia. CTRP1 expression is shown by red fluorescence. GFAP expression is shown by green fluorescence. (C) The expression of CTRP1 in microglia in the cortex. IBA1 was used to label microglia. CTRP1 expression is shown by green fluorescence. IBA1 expression is shown by red fluorescence. (D) The intensity of CTRP1 and NeuN in the cortex. The representative images were acquired under × 400 magnification, scale bars = 50 µm. ****p < 0.0001 vs. sham group.

Fig 3: Immunofluorescence characterization of neurons (magnification, ×200). (A) Bright-field micrographs of neurons on the culture plate surface. (B) NeuN, (C) DAPI and (D) combined NeuN and DAPI staining of neurons on the culture plate surface. NeuN, neuronal nuclei.

Fig 4: Expression and source of TSP2 in surgical specimens. a Representative Western blot bands (upper panel) showing TSP2 protein expression in the control group (control, n = 5), LGG group (n = 13), and HGG group (n = 15). Normalized densitometric analyses of Western blots (middle panel) showed a higher level of the TSP2 protein in the LGG (P = 0.028) and HGG groups (P < 0.001) than in the Ctrl group, and a higher level was detected in the HGG group than in the LGG group (P < 0.001). Spearman’s correlation analysis (lower panel) between the WHO grade of glioma and the expression of the TSP2 protein (r = 0.826, P < 0.001). b Representative IHC staining of surgical specimens from the center of LGGs/HGGs and the cerebral cortex of patients with traumatic injuries (control). The magnified view of each image shows the distribution of the TSP2 protein (bar: 100 µm). The average optical density values indicate higher expression of TSP2 in the LGG (P < 0.001) and HGG groups (P < 0.001) than in the Ctrl group and higher expression in the HGG group than in the LGG group (P < 0.001). c Representative IF staining for TSP2 (red) and GFAP (green) in the Ctrl group, LGG group, and HGG group (bar: 100 µm). The enlarged view of each image from left to right shows the TSP2 protein, GFAP ( +) cells, and the colocalization of TSP2 and GFAP. d Representative IF staining for TSP2 (red), NeuN (green), and DAPI (blue) in the Ctrl group, LGG group, and HGG group (bar: 100 µm). The enlarged view of each image from left to right shows the TSP2 protein, neurons, nuclei, and the colocalization of TSP2 and NeuN

Fig 5: Recombinant WNT3A promotes regeneration of injured brain tissue of controlled cortical impact (CCI) mice. (A) The effect of WNT3A on neuronal regeneration was assessed using in vivo CCI mouse model. Cortical impact on motor and somatosensory cortex was generated and the mice were given PBS or 50 ng WNT3A intranasally during 0–3 dpi. Injured brain tissues were fixed, cryosectioned on 4 dpi, and subjected to immunofluorescence staining with anti-TUJ1 (green), anti-GFAP (red) antibodies, and DAPI (blue). White dashed lines depict the border of CCI sites. The regions of interest (ROIs) are indicated in the boxed regions. Scale bar, 1 mm. Enlarged images of the ROIs are shown at the bottom. Scale bar, 0.5 mm. (B) The number of NeuN+ cells and (C) the relative intensity of GFAP of CCI cryosections from sham group (n = 10), PBS-treated (n = 12) and WNT3A-treated (n = 8) CCI animals were quantified as described in the Materials and Methods. GFAP-immunostained cryosection on the left panel in (C) defines 0.05 mm2-ROIs within white boxes. Dashed lines indicate the borders of the impact site. Data are presented as mean ± SEM. ** p = 0.01, *** p = 0.001, **** p = 0.0001, analyzed by one-way ANOVA followed by Tukey’s test.