Table 1.
Antibodies.
Table 2.
Summary of triple immunolabeling cell quantifications.
Figure 1.
Representative images of compressive SCI in rats at 12 days post-injury.
(A) Images of thoracic spinal cord cross sections at various rostral and caudal distances to the injury center were counter-stained with Luxol Fast Blue (LFB) and hematoxylin/eosin (H&E). We found the presence of an intact central canal (CC) at around 2.5 mm away from the injury center in all rats. (B) Magnified area in the box in (A) show the ependymal layer that lines the central canal. (C-D) Confocal images of the central canal immunostained for vimentin.
Figure 2.
ChABC efficiently degrades CSPG in the injured spinal cord.
(A, B) Confocal images of horizontal and cross sections of vehicle treated spinal cords 12 days after SCI shows the intense expression of CSPGs (red) at the lesion site. (C, D) Treatment with ChABC at 4 days post-injury for one week substantially reduced CSPGs deposits in the injured spinal cord. (E-G) Successful degradation of CSPGs was further confirmed by pronounced appearance of chondroitin- 4-sulfate (C4S) in ChABC treated rats, which marks the tetrasaccharride linker region, or stub, that results from ChABC enzymatic action. Inset image (E) represents the enlarged box in F that shows a magnified area of C4S immunoreactivity in ChABC treated-injured rats. (H, I) Degradation of CSPGs by ChABC was confirmed by slot blotting. Quantification of CSPGs revealed a significant reduction in CSPG level after ChABC treatment compared to the vehicle treated injured rats. This was supported by dramatic appearance of C4S immunoreactivity in slot blotting in ChABC treated injured rats with no apparent immunoreactivity in vehicle group. (*p<0.05, n = 3/group).
Figure 3.
Increased number of proliferating cells after ChABC and GF treatments.
(A–B) Representative cross sections of a subacutely injured spinal cord at 12 days after SCI. At approximately 2.5 mm rostral point to the injury center, there was a significant number of BrdU labelled cells within the injured spinal cord particularly in the dorsal column. (B) Higher magnification of the boxed area in (A) shows the presence of BrdU labelled cells (red). (C–G) BrdU immunohistochemistry showed rare proliferative activity inside the spinal cord under normal condition (C). Our results showed an increase in the number of BrdU-labeled cells (evidence of proliferation) after SCI under baseline condition (D). Interestingly, GF and/or ChABC treatments were able to enhance cell proliferation in the areas distant to the injury center (E–G). The effect was significant for ChABC+GF treated group in the rostral point and for all treatment groups in the zone caudal to the lesion epicentre compared to the vehicle group (H). *p<0.05, n = 6/group.
Figure 4.
ChABC and GFs enhance the proliferation of NPCs after SCI.
(A–B) Confocal images showing ependymal region in the central canal (CC) of the spinal cord. After SCI (B), there was a significant increase in the expression of nestin (red) in the ependymal/subependymal cells compared to the un-injured spinal cord with no apparent nestin immunostaining (A). SCI also induces the proliferation of ependymal cells evident by BrdU labelling (green). (C–G) Representative confocal images of the ependymal region of a rat treated with ChABC+GFs. Triple labelling for Nestin/GFAP/BrdU was used to identify proliferating/activating NPCs marked as BrdU+/nestin+/GFAP-. This immunostaining combination was used to distinguish nestin+ NPCs from nestin positive reactive astrocytes after SCI (Table 2). Normal astrocytes (GFAP+) and reactive astrocytes were mostly confined in the subependymal layer whereas Nestin+/GFAP- cells resided in the ependymal layer (C–G). We found BrdU+/nestin+/GFAP- that were migrating away from the ependymal layer to reside in parenchyma (arrows in G) Our quantitative confocal immunohistochemistry revealed a significant number of BrdU+/nestin+/GFAP- (in all treatment groups compared to the vehicle-injured group particularly around the central canal at both 3 mm rostral and caudal points to the injury center (H-I, *p<0.05, n = 6/group). Note: Our immunolabeling showed the absence of GFAP expression in ependymal cell. Although GFAP positive astrocytes were closely surrounding the ependymal layer and sending their process into the region, no GFAP expressing cell was observed inside the ependymal layer.
Figure 5.
ChABC and GFs reduce astrocyte differentiation and promote the formation of new oligodendrocytes after SCI.
(A–C) Representative confocal images showing BrdU labelled astrocytes that were newly generated after treatments in the injured spinal cord. (D–E) Our cell quantification for BrdU+/GFAP+ profiles (arrows in C) showed a significant decrease in the number of new astrocytes after treatment with ChABC and ChABC+GFs compared to the vehicle group in the 3 mm rostral point to the injury center. We also found a significant reduction in astrocyte proliferation in all treatment groups at 3 mm caudal distance to the injury center relative to the vehicle group (D, E, *p<0.05, n = 6/group). (F–H) Representative confocal images show triple labelling for BrdU/Olig2/GFAP (arrows). To quantify BrdU labelled cells with oligodendrocyte phenotype, we only quantified Olig2+/GFAP− to exclude Olig2 expressing reactive astrocytes. Interestingly, there was a significant increase in the number of newly generated oligodendrocytes (arrows) in GFs and ChABC+GF in comparison to the vehicle group at both rostral and caudal points to SCI center (*p<0.05, n = 6/group).
Figure 6.
ChABC and GF treatments enhance the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) after SCI.
(A–D) Representative confocal images showing triple immunostaining for NG2, Olig2 and BrdU in injured spinal cord. To accurately identify OPCs among BrdU+ cells, we used double labelling for Olig2 and NG2 (Table 2). (E–F) Our quantification for BrdU+/Olig2+/NG2+ (arrows in D) revealed a significant increase in the number of OPCs after ChABC+GF treatments relative to vehicle (approximately a three-fold increase in 3 mm rostral point and a four-fold increase in 3 mm caudal point). (G–J) We also used BrdU/Olig2/APC immunostaining combination to mark and quantify mature oligodendrocytes after ChABC and GF treatments. Our analysis showed a positive trend for an increase in the number of newly generated mature oligodendrocytes under ChABC and/or GF treatments compared to vehicle at both 3 mm rostral and caudal points to the injury center (K–L). However, this increased level did not reach a statistically significant level. *p<0.05, n = 6/group.
Figure 7.
ChABC and GF treatments attenuate the proliferation of microglia/macrophages and promote the generation of new endothelial cells after SCI.
(A–D) Representative confocal images of BrdU+/NG2+ macrophages/microglia marked with Iba-1 in the injured spinal cord (arrows). (E–F) Under baseline SCI condition, macrophages/microglia comprised about 25% and 17% of BrdU+/NG2+ cells in rostral and caudal points to the injury center, respectively. After treatment with ChABC and/or GFs, we found a reduction in the number of BrdU+/NG2+/IbA-1+ cells that was statistically significant for ChABC and ChABC+GFs treatment groups relative to the vehicle group. (G–J) Representative confocal images show newly generated endothelial cells marked by Reca-1 and NG2 among BrdU+ cells. Reca-1 positive endothelial cells comprised a subpopulation of proliferating NG2+ cells after SCI (J). (K–L) Quantification of BrdU+/NG2+/Reca-1+ cells showed a significant number of newly generated endothelial cells after treatment with ChABC and/or GFs at both rostral and caudal points to the injury center compared to the vehicle group. *p<0.05, n = 6/group.
Figure 8.
ChABC and GF treatments reduce the injury-induced astrogliosis and inflammation at the lesion site.
(A–F) Cross sections of the spinal cord at the lesion site from vehicle and ChABC+GF treated animals show immunostaining for astrocytes (marked by GFAP) and macrophages/microglia (marked by OX42). Confocal images clearly show an overall reduction in the expression of GFAP and OX42 particularly in the surrounding paranchymal region in ChABC+GFs treated spinal cords relative to the vehicle group. Our immunoblotting analyses indicated a significant reduction in GFAP and Iba-1(macrophages/microglia) at the lesion site in ChABC+GF and ChABC and ChABC+GF treatments, respectively. *p<0.05, n = 4/group.