Changes in hippocampal neurons and memory function during the developmental stage of newborn rats with hypoxic-ischemic encephalopathy

BACKGROUND: Under the normal circumstance, there exist some synapses with inactive functions in central nervous system (CNS), but these functions are activated following nerve injury. At the early stage of brain injury, the abnormal functions of brain are varied, and they have very strong plasticity and are corrected easily. OBJECTIVE: To observe the changes of neuronal morphology in hippocampal CA1 region and memory function in newborn rats with hypoxic-ischemic encephalopathy(HIE) from ischemia 6 hours to adult. DESIGN: Completely randomized grouping, controlled experiment. SETTING: Taian Health Center for Women and Children; Taishan Medical College. MATERIALS: Altogether 120 seven-day-old Wistar rats, of clean grade, were provided by the Experimental Animal Center, Shandong University of Traditional Chinese Medicine. Synaptophysin (SYN) polyclonal antibody was provided by Maixin Biological Company, Fuzhou. METHODS: This experiment was carried out in the Laboratory of Morphology, Taishan Medical College between October 2000 and December 2003. ① The newborn rats were randomly divided into 2 groups: model group and control group, 60 rats in each group. Five rats were chosen from each group at postoperative 6 hours, 24 hours, 72 hours, 7 days, 2 weeks and 3 weeks separately for immunohistochemical staining. Fifteen newborn rats were chosen from each group at postoperative 4 weeks and 2 months separately for testing memory ability (After test, 5 rats from each group were sacrificed and used for immunohistochemical staining)② The right common carotid artery of newborn rats of model group was ligated under the anesthetized status. After two hours of incubation, the rats were placed for 2 hours in a container filled with nitrogen oxygen atmosphere containing 0.08 volume fraction of oxygen, thus, HIE models were created; As for the newborn rats in the control group, only blood vessels were isolated, and they were not ligated and hypoxia-treated. ③ Thalamencephal tissue sections of newborn rats of two groups were performed DAB developing and haematoxylin slight staining. Cells with normal nucleous in 250 μm-long granular layer which started from hippocampal CA1 region were counted with image analysis system under high-fold optical microscope (×600), and the thickness of granular layer was measured. The absorbance (A) of positive reactant of SYN in immunohistochemically-stained CA1 region was measured. Learning and memory ability were measured with step through test 3 times successively. ④ t test and paired t test were used for comparing intergroup and intragroup difference of measurement data respectively, and Chi-square for comparing the difference of enumeration data. MAIN OUTCOME MEASURES: Comparison of cytological changes in hippocampal CA1 region and memory ability at different postoperative time points between two groups. RESULTS: Totally 120 newborn rats were involved in the result analysis. ① Cell morphological changes in hippocampal CA1 region: In the control group, with aging, perikaryon, nucleus and nucleolus in cortex of parietal lobe were significantly increased, Nissl body was compacted, the amount of neurons was declined, but the A of SYN positive reactant was relatively increased. In the model group, at postoperative each time point, neurons were seriously shrunk and dark-stained, nucleus was contracted, chromatin was condensed, nucleolus was unclear, even cells disappeared, especially the cells in 6 hours and 24 hours groups. The amount of neurons with normal morphology in hippocampal CA1 region and granular layer thickness in the model group at postoperative each time point were significantly less or smaller than those in the control group at postoperative 6 hours respectively (t =3.002-1.254, P < 0.01). The A value of SYN positive reactant at postoperative 2, 3 and 4 weeks was significantly higher than that at previous time point (t =2.011-2.716,P < 0.05-0.01). ② Test results of learning and memory ability: In the first test, there was no significant difference in the ratio of rats which kept memory ability between two groups (P > 0.05); In the third test, the ratio of rats which kept memory ability in the model group was significantly lower than that in the control group at postoperative 4 weeks and 2 months[53%(8/15),100%(15/15);60%(9/15),93%(14/15),χ 2=2.863,2.901,P < 0.01]. CONCLUSION: The destroyed hippocampal structure induces the decrease of learning and memory ability of developmental rats. Early interference can increase the quality of neurons and also promote functional development of the nervous system.

Chinese Herbal Medicine Xingnaojing Injection(醒脑静注射液) for Hypoxic Ischemic Encephalopathy in Newborns:A Systematic Review and Meta-Analysis

Objectives： To evaluate the efficacy and safety of Chinese herbal medicine Xingnaojing Injection（醒脑静注射液） for newborns with hypoxic ischemic encephalopathy（HIE）. Methods： Literature was identified by searching the Pub Med, EMBASE, Cochrane Library, Cochrane Central, and four Chinese literature databases from the establishment of database to October in 2013. Relevant reference lists were also screened. Two reviewers independently evaluated the methodological quality of included studies. We also conducted the meta-analysis. Results： Thirteen trials involving 1,169 patients were included. There was no trial reported death or disability at the end of follow-up period. Meta-analysis of 4 trials（n=371） showed that there was no significant difference in the reduction of mortality [risk ratios（RR）=0.48, 95% confidence intervals（CI, 0.21, 1.13）, P=0.09] between the Xingnaojing and control groups. Meta-analysis of 5 trials（n=359） showed that there was significant difference in reducing the major neurodevelopmental disability [RR=0.36, 95% CI（0.19, 0.66）, P=0.001]. Meta-analysis of 6 trials（n=447） showed that there was a significant difference in the author self-defined symptom improvement [RR=1.25, 95% CI（1.14, 1.37）, P〈0.01]. No fatal side-effects were reported. Conclusion： Based on the limited evidence, the routine use of Xingnaojing Injection for treatment of HIE in newborns is not recommended. Further well-conducted trials are justified.

Clinical Manifestations and Amplitude-integrated Encephalogram in Neonates with Early-onset Epileptic Encephalopathy

Background： The patients with early-onset epileptic encephalopathy （EOEE） suffer from neurodevelopmental delay. The aim of this study was to analyze the clinical manifestations and amplitude-integrated encephalogram （aEEG） characteristics of infants with EOEE with onset within the neonatal period, to make early diagnosis to improve the prognosis. Methods： One-hundred and twenty-eight patients with neonatal seizure were enrolled and followed up till 1 year old. Sixty-six neonates evolved into EOEE were as the EOEE group, the other 62 were as the non-EOEE （nEOEE） group. Then we compared the clinical and aEEG characteristics between the two groups to analyze the manifestations in neonates with EOEE. Results： Compared to the nEOEE group, the incidence of daily seizure attacks, more than two types of convulsions, more than two antiepileptic drugs （AEDs） application, severely abnormal aEEG background, absence of cyclicity, and more than two seizures detection were significantly higher in the EOEE group （P 〈 0.05） （97% vs. 54.8%; 30.3% vs. 14.5%; 97.0% vs. 25.4%; 39.4% vs. 3.2%; 57.6% vs. 9.7%; and 56% vs. 3.2%, respectively）. Severely abnormal background pattern （odds ratio [OR] = 0.081, 95% confidence interval [CI]：0.009-0.729, P = 0.025） and more than two seizures detection by aEEG （OR = 0.158, 95% CI： 0.043-0.576, P = 0.005） were the independent risk factors for the evolvement into EOEE. The upper and lower margins of active sleep （AS） and quiet sleep （QS） were significantly higher in EOEE group than those of the control group （P 〈 0.05） （34.3 ± 13.6 vs. 21.3 ± 6.4; 9.9 ± 3.7 vs. 6.7 ± 2.2; 41.2 ± 15.1 vs. 30.4 ± 11.4;and 11.9 ± 4.4 vs. 9.4 ± 4.0; unit： μV, respectively）. AS upper margin was demonstrated a higher diagnostic specificity and sensitivity for EOEE than another three parameters according to the receiver operating characteristic curves; the area under the curve was 0.827. Conclusions： The clinical characteristics of the neonatal seizure which will evolve into EOEE were more than two AEDs application, high seizure frequency （daily attack）, and more than two types of the seizure. Significant high voltage, severely abnormal background, absence of cyclicity, and more than two seizures detected on aEEG were the meaningful indicators to the prediction of EOEE.