| Objective:Hypoxia-ischemia (HI)-related brain damage, caused by perinatal asphyxia is an important contributor to perinatal mortality and long-term neurological impairments in term and preterm survivors. Based on the establishment of hypoxic-ischemic brain damage (HIBD), the neuron degeneration forms of HIBD in neonatal rats are investigated electron microscopically; using cytidine 5'-diphosphocholine (CDPC) as positive-control drug, the protective effect of the Chinese medicine herbal NiuPoZhiBaoDan (NPZBD) on HIBD is examined, and its neuroprotective mechanism is discussed.Methods: 1. Experimental design: factorial designFactorial design2. Establishment of the neonatal rat model of HIBD and ultrastnicture study on the neuron degeneration forms7 days old Sprague-Dawley rat pups were anaesthetized by placing their head in a jar withcotton containing aether, then the left arteria carotis communis was isolated, double-ligated (5-0 silk) and cut between the ligatures. After surgical procedure, The pups rested with the dam for 1.5 h. Thereafter they were placed in a 2000-ml air-tight incubator and exposed to a humidified 7.7% oxygen with 92.3% nitrogen delivered at 1.5L/min for 60 min. The incubtor was partially submerged in a 47℃ water bath to maintain 36 ℃ constant thermal environment. Finally, the pups were allowed to recover with their mothers. Accoding to their neurological behaviour, TTC staining and pathological examination, we decided whether the model of HIBD in neonatal rats was established successfully.The left cerebral cortex were removed at different recovery time points (4 h, 24 h, 72 h, 1 w), and their ultrastructure were investigated electron microscopically.3. Assessment of the protective effect of NPZBD on the HIBD in neonatal ratsAfter establishment of HIBD model, rats from each litter were randomly divided into the following four groups, ①control group: the rats were ingested normal saline orally, 10ml/kg, bid. ②CDPC group: the rats were injected CDPC intraperitoneally, 4 ml/kg, qd. ③NPZBD group: the rats were ingested Chinese medicine herb NPZBD orally, 55 mg/100 ml/kg, bid. ④combination group: the rats were treated both NPZBD and CDPC. The above mentioned treatments were initiated after reoxygenation, and the whole brain was removed 72 h later. Brain injury was assessed based on the infarct size of left hemisphere measured by TTC staining, and on the weight ratio of left/right hemisphere, and electron microscopically.4. Effect of NPZBD on free radicals in HIBD neonatal ratsAfter establishment of HIBD model in neonatal rats, the grouping and treatments of the rats were the same as those of part 2. But according to time points of brain removal (4 h, 24 h, 72 h after reoxygenation), each group was divided to three sub-groups again. The activity of SOD, the tissue concentration of MDA and NO of left hemisphere were evaluated biochemically.5. Influence of NPZBD on the apoptosis and expression of caspase-3 in HIBD neonatal ratsAfter establishment of HIBD model in neonatal rats, the grouping and treatments of the rats were the same to those of part 2. The brains were removed 72h after reoxygenation. In brain sectionsapoptosis was identified by TUNEL technique, and the immunostaining of caspas-3, cytochrome C were identified by IHC method in the injury cortex. Some left hemispherical cortical tissue were prepared for extracting RNA for determing the content of caspase-3 mRNA and caspase-12 mRNA.6. Infulence of NPZBD on the apoptotic pathway of mitochondrial and endoplasmic reticulum in the HIBD neonatal ratsAfter establishment of HIBD model in neonatal rats, the grouping and treatments of the rats were the same to those of part 2. The protein expression level of cytochrome C on the mitochondrial apoptotic pathway, and the transcription level of caspase-12 on the endoplasmic reticulum apoptotic pathway were checked. The methods were mentioned in part 5.Results:1. Establishment of the neonatal rat model of HIBD and ultrastructure study on the neuron degeneration formsThe pups of HIBD showed rotation to the damage (left) hemisphere and paralysis of contralateral (right) limbs. Contrast to the red contralateral hemisphere, the damaged hemisphere was pale by TTC staining, and revealed a pathological change in cortex and hippocampus light microscopically.The neuron degeneration phenotypes of HIBD included classic apoptosis, hybrid form, classic necrosis, lipid degeneration, and dark cell.2. Assessment of the protective effect of NPZBD on the HIBD in neonatal rats 2.1 Influence of NPZBD on brain infarct size in HD3D neonatal ratsBoth NPZBD and CDPC could reduce the size of infarct (F=26.212, P=0.000; F=12.951, P=0.001; respectively); In comparison with control group (31.87± 5.55), NPZBD group (22.76± 2.70) and CDPC group (24.67±3.79) reduced the size of infarct significantly (p=0.000, both).There was interaction between the two drugs (F=4.631, P=0.038) which showed synergetic effect; Compared with NPZBD group and NPZBD group, the infarct size of combination group (20.95 + 3.19) failed by 7.95 %, 14.96 % respectively.22 Inflence of NPZBD on the weight ratio of the damage / contralateral hemisphereBoth NPZBD and CDPC could enhanced the weight ratio of left/right hemisphere (F=9.747, P=0.004; F=11.853, P=0.002; respectively); In comparison with control group (0.85 ±0.05), NPZBD group (0.93 ±0.04) and CDPC group (0.92 ±0.03) enhanced the weight ratio significantly (P=0.000; P=0.001, respectively), increased by 9.41 %, 8.23 % respectively.There was interaction between the two drugs (F=4.345, P=0.045) which showed synergetic effect; Compared with NPZBD group and NPZBD group, the weight ratio of combination group (0.94 ± 0.03) increased by 1.07 %, 2.17 % respectively.23 Influence of NPZBD on the ultrastructure of neuron in HIBD neonatal ratsUltrastructurally, the two drugs either used alone or combined, both NPZBD and CDPC could protect organelle especially mitochondria and endoplasmic reticulum from hypoxic-ishemic injury.3. Effect of NPZBD on free radicals in HIBD neonatal rats3.1 Dynamic change of SOD, MDA, and NO of brain tissue in HEBD neonatal rats4 h, 24 h after reoxygenation, the activity of SOD was weak; but the level of MDA increased greatly, and showed a peak 24 h after reoxygenation. Incomparison with 4 h after reoxygenation (75.97±13.01), the level of MDA 24 h (90.81 ± 16.96) increased significantly (P=0.045); As the age growing, the activity of SOD increased, and the activity of SOD 72 h after reoxygenation (197.74 ±56.23) was higher than those of 4 h (147.32 ±22.66), and 24 h (155.26 ±20.29) significantly (p=0.014, 0.041, respectively). As the activity of SOD increased, the level of MDA decreased, and the level of MDA of 72 h after reoxygenation (67.34 ± 10.36) was lower than that of 24 h (90.81 ± 16.96) significantly (P=0.003).4 h, 24 h, 72h after oxygenation, the level of NO in the left brain tissue showed a peak at 4 h, and then decreased a little, and reached apeak again at 72 h (135.69±31.98) which was higher than those of 4 h (97.41 ±35.83), 24 h (78.19±19.79) significantly (P=0.044, P=0.005, respectively).32 Influence of NPZBD on the level of SOD, MDA, NO in HIBD neonatal rats4h, 24 h after oxygenation, both NPZBD and CDPC could enhance the activity of SOD (P<0.05, both) and decrease the level of MDA. There was a significant difference between control group and NPZBD group, CDPC group (P<0.05, both). There was interaction between the two drugs (P<0.05, both) which showed synergetic effect. 72 h after oxygenation, neither NPZBD norCDPC could show the influence on the activity of SOD (P>0.05), and no interaction between them (P>0.05). But at the time, the two drugs could decreased the level of MDA(P<0.05), and has interaction (P<0.05) which showed synergetic effect.4 h, 24 h, 72 h after oxygenation, NPZBD could decreased the level of NO (F=13.923, P= 0.001; F=17.536, P=0.000;F=44.318, P=0.000; respectively), but CDPC couldn't (F=2.026, P=0.164; F=1.680, P=0.204; F=3.500, p=0.073; respectively); and the two drugs has no interaction (F=0.818, P=0.373; F=1.298, P=0.263; F=2.047, P=0.164; respectively).4. Influence of NPZBD on the apoptosis and expression of caspase-3 in HIBD neonatal rats4.1 Influence of NPZBD on the apoptosis in HIBD neonatal rats72 h after reoxyegnation, counting the number of TUNEL-posivite cells per high power visual field in the damaged area of left hemisphere, both NPZBD and CDPC could inhibit excessive apoptosis (F=40.036, P=0.000; F=34.004, P=0.001, respectively). The average number of posivite cells of control group (38.80 ± 5.76) was higher than those of NPZBD group (21.80±3.11) and CDPC group (23.20±3.03) significantly (P=0.000, both). There was interaction between the two drugs (F=5.248, P=0.037) which showed synergetic effect, and the average number of posivite cells of combination group (15.00±4.16) was lower than those of NPZBD group and CDPC group, decreased by 31.19 %, 35.34 % respectively.42 Influence of NPZBD on the transcription and protein expression of Caspase-3The immunostaining intensity of Caspase-3 was examined by HIC method. Both NPZBD and CDPC could decreased the immunostaining intensity of Caspase-3 (F=12.376, P=0.002; F=10.008, P=0.004; respectively), and the PU value of NPZBD group (14.96±3.61) and CDPC group (15.41 ± 3.14) were lower than that of control group (22.38 ± 4.48) significantly (P=0.000; 0.001 respectively). There was interaction between the two drugs (F=5.091, P=0.033) which showed synergetic effect. Compared with NPZBD group and CDPC group, the PU value of combination group (13.79±2.27) failed by 7.82 %, 10.51 % respectively.The content of caspase-3 mRNA in the damage hemiphere cortex was examined by the RT-PCR technique. Both NPZBD and CDPC could downregulated the content of caspase-3 mRNA(F=20.511, P=0.000; F=31.074, P=0.000; respectively), and the content of NPZBD group (59.74± 8.15) and CDPC group (53.86±9.27) were lower than that of control group (97.25 ± 18.84) significantly (P=0.000, both), failed by 38.57 %, 44.62 % respectively. There was interaction between the two drugs (F=4.561, P=0.049) which showed synergetic effect. Compared with NPZBD group and CDPC group, the content of combination group (40.39±11.24) failed by 32.39 %, 33.35 % respectively.5. Infulence of NPZBD on the apoptotic pathway of mitochondrial and endoplasmic reticulum in the HIBD neonatal rats5.1 Infulence of NPZBD on the cytochrome C of the mitochondrial apoptotic pathway in HEBD neonatal rats72 h after reoxygenation, counting the number of cytochrome C-posivite cells per high power visual field in the damaed area of left hemispere. Both NPZBD and CDPC could decreased the expression of Cyt C (F=22.839, P=0.000; F=18.087, P=0.001; respectively), and the average number of positive cells of NPZBD group (10.80 ± 2.39) and CDPC group (11.40±1.82) were lower than that of control group (18.20±3.56) significantly (P=0.000, both). There was interaction between the two drugs (F=5.000, p=0.041) which showed synergetic effect. In comparison with NPZBD group and CDPC group, the average number of positive cells of combination group (8.5 ± 1.29) failed by 21.3 %, 25.44 % respectively.52 Influence of NPZBD on the transcription of caspase-12 of the endoplasmic reticulum apoptotic pathwayThe content of caspase-12 mRNA in the damaged hemisphere cortex was examined by RT-PCR technique. Both NPZBD and CDPC could downregulate the content of caspase-12 mRNA (F=41.309, P=0.001; F=6.266, P=O.O24; respectively); but there was no interaction between the two drugs (F=0.559, P=0.466).Conclusions:1. There is a variety of neuron degeneration of HIBD in neonatal rats.2. Decreasing the size of brain infarct, and enhancing the weight ratio of thedamage/contralateral (left/right) hemisphere, and protecting the ultrastructure, NPZBD shows protective effect on the HIBD in the neonatal rats. Combined with CDPC, they show synergetic effect.3. Oxygen free radicals play important role during the early phase of reoxygenation, whereas NO injures the brain during the early phase and delay phase of reoxygenation; NPZBD may enhance the activity of SOD, and decrease the content of free radicals including NO, and elongate the therapeutic window; CDPC may decrease the level of lipid peroxidation, but it has no effect on NO level; Combined with CDPC, they only showed synergetic effect on elimination of oxygen free radicals.4 An excessive apoptosis of brain cells contributes to the brain injury during the reoxgenation phase. Similar to CDPC, NPZBD restrains the excessive brain cell apoptosis, which is related to downregulate the transcription and protein expression of caspase-3. Combined with CDPC, they show synergetic effect on the influence of Caspase-3.5 NPZBD may downregulate the protein level of cytochrome C, which is a upstream factor of mitochondrial apoptotic pathway, and the transcription level of caspase-12, located in upsteam of endoplasmic reticulum apoptotic pathway. Combined with CDPC, they only show the synergetic effect on the Cyt C.
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