| Objective: To explore upstream pathogenesis of excessive apoptosis in immature brain induced by three kinds anticonvulsants (ACs) with different pharmacological mechanisms-PB, PHT and MK-801- at therapy level, and clarify cognition change in puberal rats after ACs exposure in neonate.Methods: P5 SD rats were divided into 7 groups (n=32): the groups administered with ACs of rescue doses (PB100,PHT100,MK), the groups given conventional-dose ACs (PB50,PHT50,MK0.5) and Control group. After medication once a day for 3 consecutive days, 24 rats were sacrificed for histological observations of brain pathologic changes by HE, Nissl staining. Meanwhile, the following measurements related to apoptosis were detected: TUNEL-positive cells, Protein Bax and Bcl-2 content, Caspase-3 activity, Cytochrome C fluorescence, Oxidant/antioxidant status, Ca2+ concentration. Five weeks after withdrawal, effect of ACs on cognition was assessed by Morris water maze, CaMKâ…¡protein content and Golgi staining with the remaining rats.Results:â‘ No matter what doses were given, all ACs decreased brain weight very significantly (P﹤0.001). And it was still remarkably among intensive-dose groups with negative effect of low body weight excluded.â‘¡HE and Nissl staining revealed a lot of nerve cells degeneration, necrosis, Nissl body reduction, even neurons lost in ACs-group rats, especially of rescue dose.â‘¢The number of TUNEL-positive cells was 2.5 5 times (regular-dose groups) and 3 6 times (rescue-dose groups) as it in Control, their increase difference reached statistical significance (P﹤0.05).â‘£After ACs exposure, content of protein Bax raised significantly, of Bcl-2 slightly droped. Hence, the ratio of Bax to Bcl-2 was increased by 51% 92% in conventional groups and still more in groups of high doses (by 140% 180%).⑤In rats taken conventional-dose ACs, Caspase-3 activity and Cytochromes C fluorescence intensity were 3.73 5.08 times and 3.22 3.56 times normal. Similarly, up to 4.13 6.97 times and 5.29 4.86 times normal were observed in intensive groups, and showed obvious dose dependence.â‘¥Even short-term use, ACs still decreased SOD activity and GSH content, increased MDA content significantly. Such changes were much more obvious in intensive groups than that in conventional groups.⑦Medication with conventional-dose ACs provoked Ca2+ concentration in nerve cell cytoplasm and mitochondria 22% 36% increasing. Moreover, 50% 60% enhancement in cytoplasm, 80% 90% in mitochondria induced by high-dose ACs was observed, and thus be more significant.⑧Histological observation and cognition evaluation in puberty: There were no significant morphology changes in HE, Nissl staining anymore. However, the performances of ACs-group rats in water maze test showed escape latency significantly delayed since the second training day (P﹤0.01 or﹤0.001) and crossing number decreased very remarkably (P﹤0.001). In addition, the dendrites in Golgi staining were significantly decreased, shortened and thinned, protein CaMKâ…¡content strikingly reduced. All these changes were also more obvious in intensive groups.Conlusions:â‘´No matter what dosages use, short-term administration of PB, PHT and MK-801 all can induce nerve cell excessive apoptosis in immature brain, and cause persistent cognitive impairment.⑵The process of excessive apoptosis induced by ACs was mainly via intrinsic apoptotic pathway (mitochondrial apoptosis pathway).⑶Calcium overload and/or oxidative stress occurred to mitochondria, and mitochondrial dysfunction, all may be the potential trigger for apoptosis induced by ACs. Objective: To observe the protective effect of calcium channel blockers (CCBs)- flunarizine (Flu) and amlodipine (Aml)–on immature brain injury caused by PB, PHT, MK-801, and to explore the exact role and position of calcium overload in excessive apoptosis induced by ACs.Methods: P5 SD rats were divided into two groups, given Flu or Aml. Each group was further divided into six subgroups (n=8) and administered with ACs of rescue doses (PB100,PHT100,MK1), with conventional-dose ACs (PB50,PHT50,MK0.5). One hour after administered with Flu 30 mg/kg or Aml 3 mg/kg orally, the rats were intraperitoneal injected with PB, PHT and MK-801 of rescue dose or conventional dose respectively, once a day for 3 consecutive days. The next day, rats were decapitated to detect all indexes within brain as described in PARTâ… .Results:â‘ Significantly different from ACs used alone, there were no differences in body weight and brain weight between CCBs-group and Control rats.â‘¡HE, Nissl staining revealed no significant morphological changes in rats after CCBs and conventional-dose ACs exposure. Although there were still some abnormal nerve cells in rats given rescue-dose ACs and CCBs, but their number was less and distribution was more scattered than rats without CCBs.â‘¢Histological observation and cognition testing in puberty: Five weeks after discontinuation, HE, Nissl staining did not show morphological changes anymore. Similarly, no matter Flu / Aml combined with what dose ACs, their escape latencies in water maze test were significantly shortened (P﹤0.05 or﹤0.01), crossing numbers increased remarkably (P﹤0.01 or﹤0.001), dendrites in Golgi staining more decrease, shorter and thinner, as well as protein CaMKâ…¡content strikingly increased (P﹤0.05 or﹤0.01) compared with rats without CCBs use.â‘£CCBs significantly reduced Caspase-3 activity, Cytochrome C fluorescence intensity, and number of TUNEL-positive cells in rats taken ACs by over 50%, 38% 60%, and 60% respectively as compared with single ACs administration. These decreases all had very significantly difference.⑤As for CCBs and ACs combination, content of Bax protein were significantly decreased, of Bcl-2 protein was remarkably increased. So the ratio of Bax to Bcl-2 returned to normal range in Flu + conventional-dosage ACs groups, and individually reduced by 41.4% 44.6% and 35% 37.8% in ACs rats with Flu / Aml than that in ACs rats without CCBs.â‘¥No matter Flu or Aml use, these two CCBs strikingly improved the inhibi tion of SOD activity, GSH reduction and MDA excessive increase due to ACs administration remarkably.⑦CCBs (Flu and Aml) all significantly reduced calcium overload induced by ACs not only in cytoplasm but in mitochondrial (P﹤ 0.001), and much more obvious in mitochondrial.⑧Each measurement showed Flu was more effective than Aml in protection of immature brain injury caused by ACs. Especially in water maze performances, Bax/Bcl-2 ratio, caspase-3 activity, Cytochromes C fluorescence, their differences all reached statistically significance.Conclusions:â‘´CCBs - Flu (T-type) and Aml (L-type), both can effectively prevent PB, PHT and MK-801 - associated immature brain damages. Each measurements showed Flu was much more effective than Aml.⑵The significant improvement of calcium overload and oxidative-stress status within mitochondrial, the recovery of mitochondrial function, all are the important basis of molecular biology for preventive effect of CCBs (Flu and Aml) on excessive apoptosis induced by ACs.⑶After combination with Flu/Aml, the reduction of Ca2+ concentration within mitochondrial was much more notable than it within cytoclasm, indicate that calcium overload elimination within mitochondrial should be the key link in starting CCBs against ACs induction of excessive apoptosis Objective: To observe the protective effect of antioxidants - Edaravone (Eda) and Melatonin (Mel) - on immature brain injury induced by PB, PHT, MK-801, and to explore the specific position of oxidative stress reaction in mechanism of ACs-associated brain damage.Methods: P5 SD rats given Eda or Mel were divided into two groups. Each group was further divided into six subgroups (n=8), administered with ACs of rescue doses (PB100,PHT100,MK1) or conventional doses (PB50,PHT50,MK0.5). Five minutes after injected with Eda 3 mg/kg or Mel 5 mg/kg intraperitoneally, the rats were respectively administered with PB, PHT or MK-801 of rescue dose or conventional dose for 3 consecutive days. The next day, rats were decapitated to detect all indexes within brain as described in PARTâ… .Results:â‘ With antioxidants, all ACs still decreased body weight, brain weight significantly, even more serious in Mel groups. However, no remarkable loss in brain weight was noted by excluding negative effect of body weight drop.â‘¡Compared with ACs used alone, HE and Nissl staining revealed much more scattered and fewer normal nerve cells in Eda or Mel groups.â‘¢Histological observation and cognition testing in puberty: Consistent with no morphological changes in HE, Nissl staining after five weeks withdrawal, the escape latency was significantly shortened since second training day, crossing number remarkably increased, dendrites in Golgi staining significantly increased, lengthened and coarsened, as well as protein CAMKâ…¡content strikingly increased in rats after antioxidants exposure than that in rats with single ACs use.â‘£No matter antioxidants (Eda or Mel) combined with whatever dose ACs, their protein Bax content was increased by 43.4% 62.7%, Bcl-2 content slightly increased, thus the ratio of Bax to Bcl-2 very significantly decreased as compared with only ACs use.⑤In antioxidants-ACs rats, Caspase-3 activity and cytochromes C fluorescence intensity dropped from 2.7 5.0 and 1.3 2.9 times normal (single ACs use) to 0.57 1.4 and 0.4 1.3 times normal. Similarly, TUNEL-positive cells were also decreased by 51.4% 59.6%.â‘¥After antioxidants exposure, the significantly inhibited SOD, GSH and over increased MDA due to ACs administration all returned to normal range.⑦Different from calcium channel blockers used, there was no marked dcrease of calcium concentration within cytoplasm and mitochondrial after antioxidants administration.⑧All measurements showed no efficacy difference between such two antioxidants in protection against immature brain injury induced by ACs.Conclusions:â‘´With Eda or Mel, oxidative stress indicators (SOD, GSH and MDA etc) in each ACs group all back to normal range, fully shows the prominent oxidative resistance of such two drugs.⑵Based on significant anti-oxidative action, Eda and Mel all show the remarkable efficacy against apoptosis in immature brain excessively and widely caused by ACs.⑶Combined with ACs and antioxidants, the overoxidation status and mitochondrial dysfunction but not calcium overload within cytoplasm and mitochondrial normalized, suggest mitochondrial overoxidation and dysfunction may be another source independent of calcium overload in starting apoptosis.Objective: To observe the protective efficacy of mitochondrial nutrients (MN) on immature brain injury induced by PB, PHT, MK-801 at therapy level.Methods: P5 SD rats given MN (including B vitamins, Vitamin E, Vitamin C, L- carnitine, Coenzyme Q10) were divided into six groups, and administered with ACs of rescue doses (PB100 , PHT100,MK1) or conventional doses (PB50,PHT50,MK0.5). One hour after administered with MN orally, the rats were intraperitoneally injected with PB, PHT and MK-801 of rescue dose or conventional dose respectively for three consecutive days. The next day, rats were decapitated to detect all indexes within brain as described in PARTâ… .Results:â‘ Significantly different from ACs used alone, there were no changes in body weight and brain weight in rats after MN exposure anymore.â‘¡As compared with ACs used alone, HE and Nissl staining revealed much more scattered and fewer nerve cells in MN groups.â‘¢Histological observation and cognition testing in puberty: Consistent with no morphological changes in HE, Nissl staining after five weeks withdrawal, the escape latency was significantly shortened since second training day, crossing number remarkably increased, dendrites in Golgi staining significantly increased, lengthened and coarsened, as well as protein CAMKâ…¡content strikingly increased in rats after antioxidants exposure than that in rats with single ACs use.â‘£After MN combination, protein Bax and Bcl-2 contents in conventional-group rats were all return to normal. Even though there was still some changes in rescue-group rats, but their Bax content was decreased by 54.1% 63.2%, Bcl-2 increased by 60.4% 65.0%, and thus the ratio of Bax to Bcl-2 further dropped by 67.3%～73.4% as compared with single ACs use.⑤No matter which kind ACs used, MN significantly reduced Caspase-3 activity and Cytochromes C fluorescence intensity by 91.6% 93.3% and 74.5% 77.9%, TUNEL-positive cells by 72% 74%.â‘¥Being similar to antioxidants, MN caused the changed SOD activity, GSH and MDA contents by ACs returned to normal range.⑦Significantly different from calcium channel blockers use, no marked drop in Ca2+ concentration within cytoplasm and mitochondrial after MN administration.Conclusions:â‘´MN can effectively prevent immature brain against ACs-induced excessive apoptosis, and is more predominant than single calcium channel blocker or antioxidant used alone.⑵MN can not only improve overoxidation to mitochondrial, but show outstanding performance in significantly reducing mitochondrial cytochromes C spillover and Caspase-3 activation, maintaining the integrity of molecular structure and function of mitochondria.⑶MN composed of varied nutrients, and thus may be the possible reason for being more effective than single calcium channel blocker or antioxidant. Based on this, it is necessary to further explore more effective and targeted mitochondrial nutrition cocktails for neonate...
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