TsI ameliorates myodynamia and motor deficit in HIBD rat
To determine whether TsI can rescue HIBD-induced motor deficits, two different behavioral tests were introduced: grasping test and rotarod test. In grasping test, myodynamia of the right forelimb was significantly decreased compared to that of the left in HIBD rats (HIBD + saline: n = 10, Fig. 1b). Importantly, treatment with TsI (5 mg/kg, i.p.) fully rescued the HIBD-induced myodynamia deficit, as reflected by similar myodynamia in both left and right forelimbs (HIBD + TsI: n = 11, Fig. 1b). Notably, myodynamia was not affected with or without TsI treatment in sham groups (sham: n = 10; sham + TsI: n = 10, Fig. 1b).
In rotarod test, the rats in HIBD group spent much less time on the rod compared with those treated with sham surgery (sham: n = 8; HIBD + saline: n = 8, p < 0.05 vs. sham; Fig. 1c), indicating a significant impairment of motor balance and coordination. Treatment with TsI fully rescued the HIBD-induced motor deficit, as reflected by a dramatic increase in the time spent on the rod (HIBD + TsI: n = 9, p > 0.05 vs. sham, p < 0.05 vs. HIBD + saline; Fig. 1c), whereas TsI treatment have no effect on motor function in the sham group (sham + TsI: n = 8, p > 0.05 vs. sham; Fig. 1c). Taken together, these results indicate that TsI treatment alleviates the deficits of myodynamia and motor function in the neonatal rat after HIBD.
TsI ameliorates spatial learning and memory in HIBD rats
It has been well documented that the impairment of learning and memory is the major sequelae of HIBD in human and a variety of animal models [31, 32]. To identify the effects of TsI treatment on spatial learning and memory deficits induced by hypoxia-ischemia in neonatal rats, the Morris water maze test, a hippocampus-dependent task, was conducted. As shown in Fig. 2, although the escape latency decreased progressively in all groups, the latency in the HIBD group was much longer than that in the sham groups during spatial training period (sham: n = 10; sham + TsI: n = 10, p > 0.05 vs. sham; HIBD + saline: n = 10, p < 0.01 vs. sham; Fig. 2a), indicating an impairment of learning after HIBD. Importantly, daily TsI treatment (5 mg/kg, i.p.) significantly ameliorated the impairment, as reflected by an obvious decrease in the latency to the platform, compared with saline treatment (HIBD + TsI: n = 11, p < 0.05 vs. HIBD + saline; Fig. 2a).
The results from probe test showed that spatial memory retrieval was obviously impaired in HIBD rats since they spent much less time in the quadrant where the hidden platform was previously located (sham: n = 10, 28.8 ± 2.7 s; sham + TsI: n = 10, 26.4 ± 1.8 s, p > 0.05 vs. sham; HIBD + saline: n = 10, 14.5 ± 3.6 s, p < 0.01 vs. sham; Fig. 2b). As expected, TsI treatment significantly increased the time spent in the target quadrant compared with the HIBD group (HIBD + TsI: n = 11, 24.0 ± 2.1 s, p < 0.05 vs. HIBD + saline, p > 0.05 vs. sham; Fig. 2b). Additionally, the results of latency to cross the location of hidden platform (sham: n = 10, 10.2 ± 3.3 s; sham + TsI: n = 10, 12.8 ± 4.8 s, p > 0.05 vs. sham; HIBD + saline: n = 10, 43.8 ± 7.0 s, p < 0.01 vs. sham; HIBD + TsI: n = 11, 15.8 ± 3.4 s, p > 0.05 vs. sham, p < 0.01 vs. HIBD + saline; Fig. 2c) and the number of crossing the location of hidden platform (sham: n = 10, 3.0 ± 0.4; sham + TsI: n = 10, 2.1 ± 0.4, p > 0.05 vs. sham; HIBD + saline: n = 10, 0.8 ± 0.3, p < 0.01 vs. sham; HIBD + TsI: n = 11, 2.5 ± 0.3, p > 0.05 vs. sham, p < 0.01 vs. HIBD + saline; Fig. 2d) further confirmed that spatial memory retrieval was impaired after HIBD, and TsI treatment succeeded in preventing this impairment.
To further determine the therapeutic effects of TsI on HIBD, we treated the rats with TsI daily for 7 days and the first injection was carried out immediately after HIBD. Four weeks after HIBD, the Morris water maze test was performed. As shown in Fig. 3, the protective effects of TsI on spatial learning and memory in HIBD rats were similar to those that found in pretreatment of TsI, as reflected by an obvious decrease in the latency to the platform, compared with saline treatment (HIBD + TsI: n = 7, p < 0.05 vs. HIBD + saline; Fig. 3b) during spatial training period. Although the time spent in the target quadrant among these groups was no significant difference (sham: n = 6, 22.5 ± 2.1 s; sham + TsI: n = 7, 27.1 ± 2.4 s, p > 0.05 vs. sham; HIBD + saline: n = 6, 16.1 ± 1.8 s, p > 0.05 vs. sham; HIBD + TsI: n = 7, 20.9 ± 2.3 s, p > 0.05 vs. sham, p > 0.05 vs. HIBD + saline; Fig. 3c), the latency to cross the location of hidden platform (sham: n = 6, 15.3 ± 4.9 s; sham + TsI: n = 7, 10.1 ± 2.4 s, p > 0.05 vs. sham; HIBD + saline: n = 6, 36.8 ± 7.8 s, p < 0.01 vs. sham; HIBD + TsI: n = 7, 16.8 ± 3.2 s, p > 0.05 vs. sham, p < 0.05 vs. HIBD + saline; Fig. 3d) and the number of crossing the location of hidden platform (sham: n = 6, 2.8 ± 0.4; sham + TsI: n = 7, 2.3 ± 0.4, p > 0.05 vs. sham; HIBD + saline: n = 6, 1.2 ± 0.3, p < 0.01 vs. sham; HIBD + TsI: n = 7, 2.3 ± 0.3, p > 0.05 vs. sham, p < 0.05 vs. HIBD + saline; Fig. 3e) showed that spatial memory retrieval was impaired after HIBD, and TsI treatment succeeded in preventing this impairment.
Taken together, these results suggest that systemic administration of TsI either pretreatment or immediately after HIBD is able to prevent the HIBD-induced impairment of spatial learning and memory.
TsI reduces HIBD-induced neuron loss
The number of NeuN-immunoreactive neurons was examined to confirm the neuroprotective effects of TsI on pyramidal neurons after hypoxic-ischemic insult in the CA1 area of hippocampus. The results showed that the number of NeuN-immunoreactive neurons in the CA1 area dramatically decreased in the HIBD group compared with the sham group, and administration of TsI significantly suppressed the decrease of NeuN-immunoreactive neurons (sham: n = 4; sham + TsI: n = 4, 108.0 ± 3.5% sham, p > 0.05 vs. sham; HIBD + saline: n = 6, 63.5 ± 3.2% sham, p < 0.01 vs. sham; HIBD + TsI: n = 5, 96.9 ± 8.1% sham, p > 0.05 vs. sham, p < 0.01 vs. HIBD + saline; Fig. 4).
TsI reverses the decreased antioxidant and the increased pro-oxidant in HIBD rats
It has been well known that oxidative injury plays an important role in pathogenesis of HIE [6], and TsI has been shown to exert antioxidant action in various experimental models both in vitro [33, 34] and in vivo [35, 36]. We therefore wanted to determine whether the reduction of neuron loss in HIBD rats treated with TsI could be attributed to its suppressing effect on oxidative stress. As shown in Fig. 5, a significant decrease in the production of antioxidants including T-AOC, GSH, CAT and T-SOD was observed in HIBD group. However, compared with saline treatment, TsI dramatically rescued the T-AOC (sham: n = 11, sham + TsI: n = 11, 99.5 ± 2.3% sham, p > 0.05 vs. sham; HIBD + saline: n = 12, 86.9 ± 2.1% sham, p < 0.05 vs. sham; HIBD + TsI: n = 13, 99.4 ± 2.5% sham, p > 0.05 vs. sham, p < 0.05 vs. HIBD + saline; Fig. 5a), GSH (sham: n = 8, sham + TsI: n = 8, 110.2 ± 13.3% sham, p > 0.05 vs. sham; HIBD + saline: n = 8, 55.8 ± 5.2% sham, p < 0.01 vs. sham; HIBD + TsI: n = 8, 90.1 ± 6.3% sham, p > 0.05 vs. sham, p < 0.01 vs. HIBD + saline; Fig. 5b), CAT (sham: n = 9, sham + TsI: n = 10, 105.1 ± 9.8% sham, p > 0.05 vs. sham; HIBD + saline: n = 10, 74.4 ± 3.7% sham, p < 0.01 vs. sham; HIBD + TsI: n = 12, 94.0 ± 3.5% sham, p > 0.05 vs. sham, p < 0.05 vs. HIBD + saline; Fig. 5c) and T-SOD (sham: n = 8, sham + TsI: n = 7, 101.8 ± 3.1% sham, p > 0.05 vs. sham; HIBD + saline: n = 8, 85.4 ± 5.2% sham, p < 0.01 vs. sham; HIBD + TsI: n = 7, 107.0 ± 4.6% sham, p > 0.05 vs. sham, p < 0.01 vs. HIBD + saline; Fig. 5d) activity following HIBD in the brain.
Meanwhile, as shown in Fig. 6, a significant increase in the production of pro-oxidants including H2O2, TNOS and iNOS was observed in HIBD group. As expected, TsI dramatically suppressed the increase of H2O2 (sham: n = 10, sham + TsI: n = 10, 90.9 ± 7.9% sham, p > 0.05 vs. sham; HIBD + saline: n = 12, 132.7 ± 10.5% sham, p < 0.01 vs. sham; HIBD + TsI: n = 12, 109.0 ± 6.8% sham, p > 0.05 vs. sham, p < 0.05 vs. HIBD + saline; Fig. 6a), TNOS (sham: n = 11, sham + TsI: n = 9, 99.4 ± 3.6% sham, p > 0.05 vs. sham; HIBD + saline: n = 11, 133.0 ± 3.7% sham, p < 0.01 vs. sham; HIBD + TsI: n = 13, 108.3 ± 5.6% sham, p > 0.05 vs. sham, p < 0.05 vs. HIBD + saline; Fig. 6b) and iNOS (sham: n = 10, sham + TsI: n = 10, 99.4 ± 16.3% sham, p > 0.05 vs. sham; HIBD + saline: n = 12, 140.3 ± 8.4% sham, p < 0.01 vs. sham; HIBD + TsI: n = 14, 102.7 ± 6.3% sham, p > 0.05 vs. sham, P < 0.01 vs. HIBD + saline; Fig. 6c) following HIBD in the brain, compared with saline treatment. These results indicate a powerful antioxidative capacity of TsI in HIBD rats.