Neuroprotective effects of Tacrolimus (FK-506) and Cyclosporin (CsA) in oxidative injury

Abstract

The detrimental effects of hypoxic damage to central nervous system lead to energy depletion, free radical formation, lipid peroxidation (LPO), and increased calcium. We hypothesized that in vitro tacrolimus (FK-506) and cyclosporine A (CsA) could be protective against hypoxic damage in spinal cord. Dorsal columns were isolated from the spinal cord of adult rats and injured by exposure to hypoxic condition for 1 h, and treated with FK-506 (0.1 μM) and CsA (0.1 μM). After injury, reperfusion was carried out for 2 h. Tissues were collected, processed for biochemical assays, and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Spinal cord hypoxia caused a significant decrease (P < 0.001) in mitochondrial ATP (30.64%) and tissue reduced glutathione (GSH) (60.14%) content. Conversely, a significant increase (P < 0.001) in tissue LPO level (57.77%) and myeloperoxidase (MPO) activity (461.24%) was observed in hypoxic group. Mitochondrial swelling was also significantly increased in hypoxic group (90.0%). Treatment with either FK-506 or CsA showed that significant neuroprotective effects (P < 0.05-0.01) were measured in various parameters in hypoxic groups. FK-506 and CsA treatment showed increase in ATP by 11.19% and 16.14% while GSH content increased by 66.46% and 77.32%, respectively. Conversely, LPO content decreased by 18.97% and 24.06% and MPO level by 42.86% and 18.66% after FK-506 and CsA treatment. Calcium uptake was also decreased in mitochondria as exhibited by the increase in absorbance by 11.19% after FK-506 treatment. TTC staining also showed increased viability after FK-506 and CsA treatment. In conclusion, present study demonstrates the neuroprotective effect of FK-506 and CsA treatment against spinal cord hypoxia induced damage is mediated via their antioxidant actions.

Figure 1

ATP content in various groups. Values are expressed as pmol ATP per mg protein (mean ± SE, n = 6). Significant difference ^§P < 0.001 when compared with sham; *P < 0.05, **P < 0.01 when compared with hypoxic group. Hypoxia resulted in significant decrease (P < 0.001) in ATP content, which was significantly increased (P < 0.05–0.01) by the treatment of FK-506 and cyclosporine A (CsA) in hypoxic group. Both the drugs showed same ATP-restoring effect. There was no difference between both the groups.

Figure 2

Calcium uptake in spinal cord mitochondria. Mitochondria (25 mg/reaction) were deenergized via incubation in isotonic buffer at room temperature for 3 min. After incubation, calcium (100 μM) was added to the reaction mixture and absorbance was measured at 540 nm for 5 min at every 1-min time interval. Results are shown as percent change in absorbance corresponding to the absorbance of deenergized mitochondria.

Figure 3

Rate of lipid peroxidation (LPO) in different groups. Values are expressed as mean ± SE (n = 6). Significant difference ^§P < 0.001 when compared with sham; *P < 0.01, **P < 0.001 when compared with hypoxic group. Hypoxia resulted in significant increase (P < 0.001) in LPO, which was significantly decreased by the treatment of FK-506 and CsA in hypoxic group. Both the drugs showed the same LPO-lowering effect.

Figure 4

Reduced glutathione (GSH) content in various groups. Values are expressed as mean ± SE (n = 6). Significant difference ^§P < 0.001 when compared with sham; *P < 0.01 when compared with hypoxic group. Hypoxia resulted in significant decrease (P < 0.001) in GSH content, which was significantly increased (P < 0.01) by the treatment of FK-506 and CsA in hypoxic group. There is no difference between drugs group.

Figure 5

Myeloperoxidase (MPO) activity in various groups. Values are expressed as mean ± SE (n = 6). Significant difference ^§P < 0.001 when compared with sham; *P < 0.01, **P < 0.001 when compared with hypoxic group and ^#P < 0.01 when a comparison was made between both the drugs. Hypoxia resulted in significant increase (P < 0.001) in MPO activity, which was significantly decreased by the treatment of FK-506 and CsA in hypoxic group. FK-506 showed significantly (P < 0.01) more MPO lowering effect than CsA.

Figure 6

Neuroprotective effect of FK-506 and CsA in spinal cord hypoxia. (A) Representative photographs of spinal cord sections stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). White patches show the infraction. (B) Infract (%) measurement in different groups. There is significant increase in infract size in hypoxic group as compared to sham (P < 0.001). Treatment of FK-506 and CsA significantly reduces the infract size (P < 0.05) as compared to hypoxic group.