Enhancement of matrix metalloproteinases 2 and 9 accompanied with neurogenesis following collagen glycosaminoglycan matrix implantation after surgical brain injury

Abstract

Surgical brain injury may result in irreversible neurological deficits. Our previous report showed that partial regeneration of a traumatic brain lesion is achieved by implantation of collagen glycosaminoglycan (CGM). Matrix metalloproteinases (MMPs) may play an important role in neurogenesis but there is currently a lack of studies displaying the relationship between the stimulation of MMPs and neurogenesis after collagen glycosaminoglycan implantation following surgical brain trauma. The present study was carried out to further examine the expression of MMP2 and MMP9 after implantation of collagen glycosaminoglycan (CGM) following surgical brain trauma. Using the animal model of surgically induced brain lesion, we implanted CGM into the surgical trauma. Rats were thus divided into three groups: (1) sham operation group: craniotomy only; (2) lesion (L) group: craniotomy + surgical trauma lesion; (3) lesion + CGM (L + CGM) group: CGM implanted following craniotomy and surgical trauma lesion. Cells positive for SOX2 (marker of proliferating neural progenitor cells) and matrix metalloproteinases (MMP2 and MMP9) in the lesion boundary zone were assayed and analyzed by immunofluorescence and ELISA commercial kits, respectively. Our results demonstrated that following implantation of CGM after surgical brain trauma, significant increases in MMP2⁺/SOX2⁺ cells and MMP9⁺/SOX2⁺ cells were seen within the lesion boundary zone in the L + CGM group. Tissue protein concentrations of MMP2 and MMP9 also increased after CGM scaffold implantation. These findings suggest that implantation of a CGM scaffold alone after surgical brain trauma can enhance the expression of MMP2 and MMP9 accompanied by neurogenesis.

Keywords: collagen glycosaminoglycan; matrix metalloproteinases; neural regeneration; neurogenesis; surgical brain trauma.

Figure 1

Proliferating neural progenitor cells (SOX2⁺) with MMP2 immunoreactivity in the LBZ of rats on day 21 following implantation of CGM. (A–C) Representative microphotographs of double immunofluorescence staining of SOX and MMP2 in representative brain sections from L + CGM group rats on day 21 following surgical brain trauma. (A) Image of immunoreactivity of SOX2 (green; marker of proliferating neural progenitor cells), and (B) MMP2 (red) positive cells and (C) merged image in the LBZ of rats in the L + CGM group. (D) Numbers of MMP2⁺/SOX2⁺ cells in the LBZ from the brain sections of sham (SHAM), L and L + CGM groups on days 7, 14, 21 and 28 after surgery. Data are expressed as the mean ± SD. ***P < 0.001, vs. SHAM group; +P < 0.05, ++P < 0.01 and +++P < 0.001, vs. L group (one-way analysis of variance with the Bonferonni correction). MMP2: Matrix metalloproteinase-2; CGM: collagen glycosaminoglycan matrix; LBZ: lesion boundary zone; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 2

Proliferating neural progenitor cells (SOX2⁺) with MMP9 immunoreactivity in the LBZ of surgical brain lesion on day 14 following implantation of CGM. (A–C) Representative microphotographs of double immunofluorescence staining of representative brain sections from L + CGM group rats on day 14 following surgical brain trauma. (A) Image of SOX2⁺ (green; marker of proliferating neural progenitor cells), and (B) MMP9⁺ (red) cells, and (C) merged image in LBZ of rats in the L + CGM group. (D) Numbers of MMP9⁺/SOX2⁺ cells in the LBZ from the brain sections of rats in the sham (SHAM), L and L + CGM groups on days 7, 14, 21 and 28 after surgery. Data are expressed as the mean ± SD. ***P < 0.001, vs. SHAM group; ++P < 0.01, +++P < 0.001, vs. L group (one-way analysis of variance with the Bonferonni correction). MMP9: Matrix metalloproteinase-9; CGM: collagen glycosaminoglycan matrix; LBZ: lesion boundary zone; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 3

Merged image of GFAP⁺ cells and MMP2⁺ cells in the lesion boundary zone of surgical brain lesion following implantation of CGM. (A–C) Representative microphotographs of double immunofluorescence staining of representative brain sections from the L + CGM group rats on day 14 following surgical brain trauma. Merged image of GFAP⁺ (green; astrocyte marker), and MMP2⁺ (red) cells in the sham (SHAM; A), L (B) and L + CGM (C) groups. GFAP: Glial fibrillary acidic protein; MMP2: matrix metalloproteinase-2; CGM: collagen glycosaminoglycan matrix.

Figure 4

Merged image of GFAP⁺ cells and MMP9⁺ cells in the lesion boundary zone of surgical brain lesion following implantation of CGM. (A, B) Representative microphotographs of double immunofluorescence staining for GFAP (green; astrocyte marker)/MMP9 (red) in representative brain sections of rats from the L + CGM group on day 14 following surgical brain trauma. B is a magnified image from the box in A. GFAP: Glial fibrillary acidic protein; DAPI: 4′,6-diamidino-2-phenylindole; MMP9: matrix metalloproteinase-9; CGM: collagen glycosaminoglycan matrix.

Figure 5

Increased SOX2-, MMP2-, and MMP9-immunoreactive cells in the LBZ of surgical brain lesion rats following implantation of CGM. (A) Representative microphotographs of SOX2-immunoreactive cells (immunofluorescence staining: green) in the LBZ of representative brain sections from Sham, L and L + CGM rats on day 14 following surgical brain trauma. (B–D) Numbers of Sox2- (B), MMP2- (C), and MMP9-immunoreactive (D) cells in the LBZ of brain sections of rats in the sham, L and L + CG groups on days 7, 14, 21 and 28 after surgery. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001, vs. SHAM group; +P < 0.05, ++P < 0.01 and +++P < 0.001, vs. L group (one-way analysis of variance with the Bonferonni correction). MMP2: Matrix metalloproteinase-2; MMP9: matrix metalloproteinase-9; LBZ: lesion boundary zone; CGM: collagen glycosaminoglycan matrix.

Figure 6

Increase of tissue concentrations of MMP2 and MMP9 in the lesion boundary zone in the surgical brain lesion rats following implantation of CGM. MMP2 and MMP9 protein concentrations as measured by ELISA in the surgical brain lesion rats from the sham (SHAM), L and L + CGM groups. Data are expressed as the mean ± SD. ***P < 0.001, vs. SHAM group; +++P < 0.001, vs. L group (one-way analysis of variance with the Bonferonni correction). MMP2: Matrix metalloproteinase-2; MMP9: matrix metalloproteinase-9; CGM: collagen glycosaminoglycan matrix.