Different Astrocytic Activation between Adult Gekko japonicus and Rats during Wound Healing In Vitro

Abstract

Glial scar formation is a major obstacle to regeneration after spinal cord injury. Moreover, it has been shown that the astrocytic response to injury differs between species. Gekko japonicas is a type of reptile and it shows differential glial activation compared to that of rats. The purpose of the present study was to compare the proliferation and migration of astrocytes in the spinal cords of geckos and rats after injury in vitro. Spinal cord homogenate stimulation and scratch wound models were used to induce astrocytic activation in adult and embryonic rats, as well as in adult geckos. Our results indicated that astrocytes from the adult rat were likely activated by mechanical stimulation, even though they showed lower proliferation abilities than the astrocytes from the gecko under normal conditions. Furthermore, a transcriptome analysis revealed that the differentially expressed genes in astrocytes from adult rats and those from geckos were enriched in pathways involved in proliferation and the response to stimuli. This implies that intrinsic discrepancies in gene expression patterns might contribute to the differential activation of astrocytes between species.

Fig 1. Gliosis after spinal cord injury is different in rats and Gekko japonicus.

Nissl staining (A, B) and glial fibrillary acidic protein (GFAP) immunehistochemical analysis (C, D) of adult geckos (A, C) and adult rats (B, D) after spinal cord transection (n = 6 per group). GFAP staining is shown in panels C and D. GFAP expression in activated astroglia was observed at 1 week post-injury in both adult geckos and rats. In geckos, the GFAP level was weakened at 4 weeks post-injury, while GFAP in adult rats was still strong 4 weeks post-injury.

Fig 2. Spinal cord homogenates (SCHs) stimulated the proliferation of rat astrocytes, but not gecko astrocytes.

Different concentrations of SCHs from adult rats stimulated the proliferation of cultured adult rat astrocytes (A-Rat AS), but had no effect on the proliferation of cultured adult gecko astrocytes (A-Gecko AS) (A). SCHs from Gekko japonicus stimulated the proliferation of A-Rat AS, but had no effect on A-Gecko AS (B). The optical density (OD) value was measured, and data are represented as the mean ± the standard deviation (SD). Data were normalized, as described in the results. **p < 0.01 vs. control; *p < 0.05 vs. control; ns = no significant change vs. the control.

Fig 3. Astrocytic responses from adult and embryonic rats and geckos after in vitro scratch wound.

The responses in adult rat astrocytes (A-Rat AS) were different from that of astrocytes from embryonic rats (E18-Rat AS) and adult geckos (A-Gecko AS). (A) Representative images of wound healing combined with BrdU assays at 4, 24, and 48 h. A-Gecko AS showed obvious delays in covering the wound and decreased proliferation abilities compared with A-Rat AS. For E18-Rat AS, the capacity for proliferation and migration was between that of adult rats and geckos. (B) Graph showing the percentage of cell migration in the cleaned space at 4, 24, and 48 h after the scratch wounding. (C) Graph showing the percentage of BrdU-positive cells that migrated into the cleaned space at 4, 24, and 48 h after the scratch wounding. Data are represented as the mean ± SD. **p < 0.01, *p < 0.05 vs. A-Rat AS. (D, E) the expression of GFAP and CSPG after scratch injury. GFAP expression in astrocytes from the adult rat, embryonic rat, and adult gecko increased following injury. The extent of the increase was highest in A-Rat AS, and lowest in A-Gecko AS. In addition, GFAP expression ceased to increase after 24 h in E18-Rat AS and A-Gecko AS, while GFAP expression remained high in A-Rat AS. CSPG expression was observed in all three groups, but did not show obvious variations after injury.

Fig 4. Under normal conditions, astrocytes from geckos showed poor migration abilities compared to astrocytes from rats.

A–C show representative transwell images of cresyl violet staining in astrocytes from the adult rat (A-Rat AS) (A), embryonic rat E18-Rat AS (B), and adult gecko (A-Gecko AS) (C). (D) Graph showing transferred cells of three kinds of astrocytes, data are represented as the mean ± SD, compared to A-Rat AS, **p<0.01.

Fig 5. The gene ontology (GO) term enrichment analysis of differentially expressed genes (DEGs).

DEFs between astrocytes from adult geckos, embryonic rats, and adult rats were examined. 5230 DEGs between the astrocytes from adult and embryonic rats were identified. Among these DEGs, 1585 genes showed higher or lower expression (more than 2-fold differences) in astrocytes from the adult rat versus astrocytes from the ebryonic rat and adult gecko. Pie charts show the GO term and its relation to various biological processes, molecular functions, and cellular components.

Fig 6. Relative fibroblast growth factor receptor 1 (FGFR1) and FGFR2 mRNA expression.

The expression of FGFR1 and FGFR2 in cultured adult rat astrocytes (A-Rat AS), embryonic rat astrocytes (E18-Rat AS), and adult gecko astrocytes (A-Gecko AS) was examined. The EF1α gene served as an internal control. Graphic representation of the real-time PCR results in different astrocytes; **p < 0.01.