The expression of TRPM7 is up-regulated after SCI
In the event of cerebral ischemia and hypoxia, ionic imbalances, including calcium overload inside the neuronal cells, are the established cellular and molecular mechanisms for ischemia and hypoxic neuronal cell death followed brain damage [31]. An in vivo study also showed that the expression of TRPM7 was increased in hippocampal neurons through PI-3K signal pathway after middle cerebral artery occlusion model (MCAO) [32].
To test the hypothesis that TRPM7 channels may contribute to biological events following SCI, we first examined the profile of TRPM7 expression in injured rat spinal cords. TRPM7 expression was markedly up-regulated after SCI (Fig. 1A and B). RT-PCR and Western blot analysis revealed that TPRM7 expression was increased and peaked at 7 d after SCI. TRPM7 was also mainly localized in neurons in the GM and oligodendrocytes in the WM, in the uninjured rat spinal cord (Fig. 1C, Sham). By double-immunofluorescence analysis, TRPM7 was colocalized in neurons (NeuN) and oligodendrocytes (CC1), respectively (Fig. 1D, Sham). Interestingly, TRPM7 expression was increased in the blood vessels at or near the lesion site in the injured spinal cord (Fig. 1C, 7d). Consistent with these results, this up-regulation of TRPM7 expression was not observed in the blood vessels of uninjured spinal cords (Fig. 1D, Sham) and majority of TRPM7-positive blood vessels in the injured spinal cord were also positive for the endothelial cell marker RECA1 (Fig. 1D, 7d). These results indicate that TRPM7 is up-regulated in the blood vessels after SCI.
CAR inhibits BSCB disruption and preserves tight junction (TJ) integrity after injury
It is well known that SCI causes in the breakdown of the BSCB [33], and blocking BSCB disruption enhances functional recovery by alleviating the infiltration of blood cells including neutrophils and macrophages followed inflammatory responses [21]. To determine whether ion influx through TRPM7 affects the BSCB disruption, we evaluated the effect of CAR, a TRPM7 inhibitor, on BSCB disruption at 1 d after injury by Evans blue assay. Compared with sham group, the amount of Evans blue dye extravasation was markedly increased at 1d after SCI, implying BSCB leakage (Fig. 2A). Furthermore, CAR administration significantly reduced the amount of Evans blue dye extravasation at 1 d after SCI as compared with the vehicle group (Veh 72.69 ± 5.05 vs. CAR 15.61 ± 2.51 µg/g tissue, p < 0.05). Next, we examined the effect of CAR on the TJ protein levels of ZO-1 and occludin at 4 h and 5 d after injury. As shown in Fig. 2B, CAR significantly attenuated the decrease in TJ protein levels after injury compared with the vehicle control (ZO-1, Veh 0.68 ± 0.04 vs. CAR 0.94 ± 0.04; occludin, Veh 0.62 ± 0.03 vs. CAR 0.94 ± 0.07, p < 0.05). In addition, double immunofluorescence staining for ZO-1 and RECA-1 showed that the fragmentation of capillary blood vessel was increased after SCI and ZO-1 immunoreactivity was decreased upon injury, compared with the sham control, whereas CAR treatment attenuated the fragmentation of the capillary blood vessels and ZO-1 loss (Fig. 2C), indicating that TRPM7 affected TJ integrity and BSCB disruption upon SCI.
TRPM7 is also up-regulated and regulates the integrity of TJ in OGD-induced bEnd.3 cells
To understand more about the molecular mechanism underlying TRPM7-mediated regulation in injured blood vessels, an in vitro OGD/reperfusion model with a bEnd.3 mouse brain microvessel endothelial cell line was used. Under the optimized condition of OGD/reperfusion injury to induce the maximal expression of the TRPM7 gene in bEnd.3 cells (data not shown), the expression of the TRPM7 mRNA and protein was increased in the bEnd.3 cells subjected to 6 h of OGD treatment followed by 0.5 h, 1 h, and 3 h of reperfusion (Fig. 3A and B). By Western blot analysis with anti-ZO-1 and occludin antibodies, the expression of the TJ proteins, ZO-1 and occludin, was decreased in the OGD/reperfusion injury-induced bEnd.3 cells (Fig. 3C, +OGD). However, CAR substantially attenuated the decrease in ZO-1 and occludin expression in the bEnd.3 cells after OGD/reperfusion injury (Fig. 3C, +OGD/CAR) (ZO-1, +OGD 0.6 ± 0.03 vs. +OGD/CAR 1.0 ± 0.04; occludin, +OGD 0.7 ± 0.07 vs. +OGD/CAR 0.9 ± 0.04, p < 0.05). Consistently, trans-endothelial electrical resistance (TEER) was decreased in the OGD/reperfusion injury-induced bEnd.3 cells compared with the untreated control (Fig. 3D, +OGD). In parallel with these results, the decrease of TEER by OGD/reperfusion injury was significantly inhibited by CAR treatment (Fig. 3D, +OGD/CAR) (+ OGD 35 ± 6 vs. +OGD/CAR 72 ± 7, p < 0.05). These results indicate that TRPM7 affects TJ integrity by increasing the degradation of TJ molecules.
CAR inhibits the infiltration of neutrophils and macrophages and the expression of inflammatory factors after SCI
BSCB disruption and blood cell infiltration after SCI are known to mediate inflammatory responses, thereby contributing to the secondary injury cascade by releasing inflammatory mediators such as Il-6, Tnf-α, Cox-2, and iNos [20, 34, 35]. Furthermore, the early increase in chemokines such as Mcp-1, Mip-1β, Gro-α, and Mip-2α following SCI is considered to cause neutrophil and macrophage infiltration, thus facilitating inflammatory responses [36–39]. Since CAR prevented BSCB disruption following, the effect of CAR treatment on blood cell infiltration was examined by immunofluorescence staining and Western blot with neutrophil and macrophage cell markers, MPO and ED-1 antibodies. Immunofluorescence staining showed that numerous MPO-positive cells (after 1 d) and ED-1-positive cells (after 5 d) were observed in the dorsal column of the injured spinal cord. However, CAR treatment attenuated the infiltration of these cells compared with the infiltration into the vehicle control (Fig. 4A and B). Additionally, relative fluorescence intensity analysis revealed that CAR treatment significantly reduced the infiltration of blood cells compared with that of the vehicle control (MPO, Veh 1.0 ± 0.07 vs. CAR 0.6 ± 0.05; ED-1, Veh 1.0 ± 0.02 vs. CAR 0.5 ± 0.08, p < 0.05). By Western blot and quantitative analyses, the level of ED-1 was markedly increased after SCI and was significantly attenuated by CAR treatment (Fig. 4C) (Veh 1.0 ± 0.07 vs. CAR 0.6 ± 0.06, p < 0.05). These findings suggest that the inhibition of TRPM7 reduced the infiltration of blood cells by preventing BSCB disruption after SCI.
Next, RT-PCR and Western blot tests were used to look at the effect of CAR on the expression of inflammatory mediators and chemokines after SCI. The results revealed that the increases CAR inhibited the increases in Tnf-α, IL-1β (at 2 h), IL-6, Cox-2, and iNos (at 6 h) mRNA levels after SCI (Fig. 4D and E). Furthermore, CAR suppressed the increases in the mRNA levels of Mcp-1, Mip-1α, Mip-1β, Gro-α (at 2 h), and Mip-2α (at 6 h) following injury (Fig. 4F and G). CAR also decreased COX-2 and iNOS protein levels at 1 d after injury as compared to vehicle control (Fig. 4H).
CAR inhibits the apoptotic cell death of neurons and oligodendrocytes
TRPM7 is known to play an important role in neuronal cell death in various neurodegenerative disease models, including SCI [10, 12, 18]. Furthermore, after BSCB damage, infiltrated blood cells such as neutrophils and macrophages are known to produce inflammatory mediators such as cytokines and chemokines, which contribute to cell death [40–42]. Thus, based on our results showing that TRPM7 is involved in the BCSB disruption after SCI, we next examined whether CAR inhibits apoptotic cell death by attenuating inflammatory responses following BSCB disruption. As previously mentioned, a massive loss of VMN was observed in the lesion area following injury [30], when compared to the vehicle control, CAR treatment reduced VMN loss both rostral and caudal to the lesion epicenter (Fig. 5A). The cleaved caspase-3-positive cells in the WM 5 days after SCI were CC1-positive oligodendrocytes, according to double immunofluorescence staining. Immunofluorescence with cleaved anti-caspase-3 antibody showed that CAR treatment significantly decreased the number of active caspase-3-positive cells in the WM at 5 d after injury as opposed to the vehicle control (Fig. 5B and C) (Veh 221 ± 16 vs. CAR 141 ± 11; p < 0.05). By Western blot, CAR treatment significantly reduced the levels of cleaved caspase-3 at 1 d and 5 d after injury compared with the vehicle control (Fig. 5D and E) (1 d, Veh 4.1 ± 0.4 vs. CAR 2.2 ± 0.4; 5 d, Veh 4.9 ± 0.4 vs. CAR 2.0 ± 0.3 p < 0.05).
Next, by TUNEL staining of spinal tissue at 1 d and 5 d after SCI, TUNEL-positive cells were also observed within the lesion site in the GM at 1 d and outside the lesion area (WM) at 5 d. Consistent with our previous reports [24, 43], the majority of TUNEL-positive cells in the GM at 1 d were identified as neurons. TUNEL-positive cells in the WM at 5 d were also observed outside of the lesion area, extending the entire length of the section (20 mm) and were known as oligodendrocytes. As shown in Fig. 5F and G, CAR treatment resulted in a significant reduction in the number of TUNEL-positive cells when compared with the vehicle control in the GM at 1 d and the WM at 5 d (1 d, Veh 405 ± 36 vs. CAR 262 ± 4; 5 d, Veh 325 ± 12.2 vs. CAR 185 ± 8.3, p < 0.05). As a result, our findings show that inhibiting TRPM7 with CAR prevents apoptotic cell death in neurons and oligodendrocytes following injury.
CAR increases functional recovery after SCI
To assess the effect of CAR on functional recovery, CAR (50 mg/kg, i.p) was administered immediately and 8 hours after injury and then once daily for 7 days. Functional recovery was assessed 28 d after injury using BBB scale, inclined plane test, grid, and footprint analysis. As shown in Fig. 6A, CAR significantly improved hindlimb locomotor function from 14 d to 35 d after injury compared with the vehicle group (At 28 d, CAR 11.8 ± 1.1 vs. Veh 8.8 ± 0.9, p < 0.05). Furthermore, the ability to control and accurately position the hindlimbs was checked on a horizontal grid at 28 d after injury. From 14 to 28 days after damage, the angle of the incline was significantly higher in the CAR-treated rats than in the vehicle group (Fig. 6B) (At 28 d, CAR 66.3 ± 3.1 vs. Veh 55 ± 2.7 %, p < 0.05). As shown in Fig. 6C, the number of mistakes (footfalls on the grid walk) was significantly smaller in the CAR-treated group than in the vehicle group (At 28 d, CAR 43 ± 1.9 vs. Veh 73.6 ± 4.2, p < 0.05). Finally, footprint examination indicated that fairly consistent forelimb-hindlimb coordination was observed in both the vehicle-treated and CAR-treated rats at 35 d after SCI, but CAR-treated group was very little toe dragging, compared to inconsistent dorsal stepping and extensive dragging in the vehicle rats, as revealed by ink streaks extending from both hindlimbs (Fig. 6D).
CAR decreases lesion volume and inhibits the loss of axon and myelin after SCI
We performed a histological study of the spinal cord tissues from the animals used in the behavioral tests to confirm the correlation between the behavioral results and histological results such as axon loss, myelin loss, and lesion volume. To determine whether CAR retains axons after injury, immunostaining with anti-NF200 and anti-5-HT antibodies was used to detect the remaining axons, and the density of the preserved axons was measured as described in the Materials and Methods section. In the sham control, NF200-positive axons were dense and axonal packing was uniform in both the ventral and dorsolateral funiculi (Fig. 7A and B, Sham). However, the axon density was significantly decreased in the injured tissue (Fig. 7A and B, Veh). The number of NF200-positive axons was significantly higher in the CAR-treated group than in the vehicle control group in both the ventral and dorsolateral funiculi (Fig. 7A and B) (rostral 2 mm, ventral funiculus, CAR 61 ± 5.3 vs. Veh 26 ± 5.4 %; dorsolateral funiculus, CAR 57 ± 5.3 vs. Veh 29 ± 6.0 %, P < 0.05). Furthermore, the density of the 5-HT serotonergic axons in the ventral horn was higher in the CAR-treated group than in the vehicle control (Fig. 7C). These findings imply that CAR treatment reduces axon loss following SCI.
Next, the extent of myelin loss after injury was assessed by Luxol fast blue staining. At 35 d after injury, extensive myelin loss near the lesion area was evident in the vehicle-treated group but not in the sham control (Fig. 7D, Veh); however, CAR treatment attenuated myelin loss (Fig. 7D, CAR). Serial longitudinal sections were stained with Cresyl violet and the lesion volume was assessed to evaluate the tissue loss after SCI. Figure 7E reveals that the overall lesion volume was significantly reduced in the CAR-treated group compared to the vehicle-treated group at 35 d after injury (Fig. 7E) (CAR 4.5 ± 0.3 vs. Veh 7.6 ± 0.9 mm3, P < 0.05).