A Study Of Neuronal Cell Death And New Born Cell Formation After Hypoxia Ischemia In The Developing Brain

Neonatal hypoxic-ischemix brain damage is one of the most serious diseasethreatening the life and health of newborn infants and leading to disability in nervoussystem of children. The brain response to HI appears as a balance between theactivation of neurodestructive components and endogenous protective system. Themechanism of cell death after HI is still unknown. Recent study show thecaspase-independent apoptosis triggered by apoptosis-inducing factor (AIF) and theprotein nitrosylation induced by excessive nitric oxide (NO) play an important role inneuronal injury. The immunohistochemisty Staining, immunofluorescence staining,western blot, caspase activity were used as parameters to investigate the effect of AIFand nitrosylation in neonatal hypoxia ischemia brain injury.Among the various adaptative responses of the brain to injury, it has beenrecently reported that new neurons can be generated through the proliferation ofprogenitor cells, and thus might help to repair the brain damage. It has beendemonstrated that there is endogenetic neurogenesis in the normal brain. However,there is no report on neurogenesis in developing brain and effect of HI onneurogenesis. We use the Brdu (5-bromo-2-deoxyuriding), the marker of newgenerated cells, to label the new produced cells, combined withimmunohistochemisty staining, immunofluorescence staining, stereological systemand confocal imaging to study the cell proliferation and differentiation in developingbrain after cerebral hypoxia ischemia.Objects: HI group: After HI injury, 1477-day-old newborn Wistar rats were dividedrandomly into 0min, 30min, 1h, 3h, 8h, 14h, 24h, 72h (each time point n=12) groups,BAF treatment group 42 pups (HI 24h n=9, HI 72h n=33), 2-iminobiotin treatment group 9 pups. Normal control group: 60 Wistar rats were divided randomly into 0d,3d, 7d, 8d, 10d, 14d, 21d, 42, adult groups (each time point n=6, except 7d n=12).Forty five 9-day-old C57/BL6 male mice were divided randomly into threegroups. There were 10 mice in each HI group and 5 mice for each normal control. 1521-day-old C57/BL6 male mice were divided into HI group (n=10), normal control(n=5).Methods: 1. The preparation of the hypoxia-ischemia brain injury model: The Wistarrats/C57/BL6 male mice were anesthetized with 1.5%-3.5% halothane and the leftcommon carotid artery was ligated. After 1h recovery, the animals were givenhumidified oxygen (7.7%, 55min for 7-day-old Wistar rat; 10%, 35min for 9-day-oldC57/BL6 mice; 10%, 30min for 21-day-old C57/BL6). 2. Drug administration: (1)BAF was given by intracerebroventicular (ICV) injection 2h and 12h after HI,respectively. The total does of BAF was 5μl (1μl 100nM BAF, 4μl PBS, pH 7.4). (2)2-iminobiotin was given by intraperitoneal injection immediately after HI. The doesis 20mg/kg(10μl/g). (3) Brdu was given by intraperitoneal injection and started theinjection from 1d (group1), 1w(group2), 2w(group3) respectively after HI. The doeswas 50mg/kg/day for seven days. 3. immunohistochemisty staining: To detect AIF,MAP-2, Cyt c, nitrotyrosine, active caspase-3, HPP, Brdu. 4. Immunofluorescentstaining: To detect AIF-HPP-Hoechst 33342, AIF-TUNEL-Hoechst 33342, AIF-Cytc-Hoechst 33342, AIF-COX, AIF-MAP-2, nitrotyrosine-AIF-Hoechst 33342,HPP-nitrotyrosine-Hoechst 33342, caspase-3-nitrotyrosine-Hoechst 33342,Brdu-NeuN, Brdu-APC, Brdu-S100β. 5. Western blot: To detect the proteinexpression of Cyt c, COX, caspase-3, caspase-9, a-tubulin, nitrotyrosine. 6. Thedetermination of caspase activity: To detect the activity of caspase-1, 2, 3, 9. 7. Theconfocal imaging system: To identify the phenotype of the new generated cells. 8.Cell counting: Cell counting was performed in the cortex, striatum, hippocampus,thalamus and Positive cells were counted at 400×magnification. The Brdu positivecells in hippocampus were counted by stereological system. 9. Evaluation of braindamage: Using Micro Image software to measure the MAP-2 negative area. Theinfart volume was calculated by the formula. 10. Statistics: All the data wereexpressed as mean±SD. Unpaired t-test was used when compared two groups.ANOVA with Fisher's post-hoc test was used when comparing more than two groups.Statview software was used to analysis the data. Significance level was assigned at p＜0.05.Results: To study the mechanism of cell death after HI brain damage. 1. Thechanges of apoptosis-related protein in developing brain: The total levels of AIF werevirtually unchanged duing normal brain development from postnatal day to adult. Themitochondrial marker cytochrome c oxidase (COX) displayed an increase, and so didcytochrome c and caspase-9. Caspase-3 decreased as the brain growth spurt leveledout. 2. AIF and cytochrome c translocation after HI: AIF was translocated to nucleiimmediately following HI and reached peak at 8h post-HI. The AIF positive cellsstaining grew increasingly stronger and more condensed during reperfusion,eventually outlining only pyknotic nuclei. The redistribution of AIF is earlier thanthat of cytochrome c. 3. AIF nuclei translocation precedes the DNA damage. 4. TheAIF redistribution was no changed after BAF treatment which indicated AIFmediated caspase-independent cell death passway. 5. Nitrotyrosine immunoreactivity:The time course of nitrotysine immuneoreactivity in ipsilateral hemisphere (cortex,striatum, hippocampuss and thalamus) was detected already 30min post-HI andincreased peak at 3h and decreased afterwards. In the cortex, there appeared to be asecond increase at 72h post-HI. The nitrotyrosine immuneoreactivity was particularlystrong in the subependymal layer, where stem cells and progenitors reside. 6.Nitrotyrosine formation preceded AIF translocation to nuclei and caspase-3 activation.7. Nitrotyrosine formation preceded DNA damage and the positive cells located inMAP-2 negative area (infarct area). 8. The nitrotyrosine formation and the activationof caspase-3 were decreased after 2-iminobiotin (the inhibitor of nNOS and iNOS)treatment, but the AIF nuclear translocation was not altered.Cell proliferation and differentiation in developing brain after cerebral HI. 1.Cell proliferation in the cortex and striatum after HI in the developing brain: Thenumber of Brdu labeled cells decreased significantly in both cortex and striatum withbrain development. HI insult increased Brdu positive cells significantly in theipsilateral cortex and striatum at early recovery of the immature brain. In the juvenile,large amount of new born cells was only seen in the ipsilateral striatum. 2. Celldifferentiation in the cortex and striatum after HI in the developing brain: A smallportion of Brdu and NeuN double labeled cells could be detected in the cortex at veryearly recovery in the immature brain and in the striatum of all the groups. Themajority of Brdu labeled cells were neuroglia. The number of the neuroglia cells decreased dramatically with brain maturation. HI insult stimulated to produce a largenumber of neuroglia cells in the ipsilateral cortex and striatum of immature brain atearly recovery after HI. In the juvenile striatum, Iba1 and S-100βpositive cellsincreased 50 and 8 folds after HI, but the number of APC positive cells was nosignificant change. 3. Compare the cell proliferation in neonatal (P9) and juvenile(P21) brain after HI insult: The basal level of Brdu labeled cells and neurogenesis wasmuch higher in the immature brain than that of juvenile. HI insult increased Brdulabeled cells dramatically in the whole juvenile hippocampus, but not in the immaturedentate gyrus (DG). 4. Compare the cell differentiation in neonatal (P9) and juvenile(P21) brain after HI insult: The neurogenesis was increased significantly in thejuvenile compared with the age matched control; however, there was no significantlyincrease in the immature brain. The absolute number of newly generated neuron wasno different between immature and juvenile. More microglia and oligodendrocytewere differentiated in the juvenile compared to immature.Conclusions: 1. AIF translocation is an early marker of DNA damage in neonatalcerebral HI and AIF-mediated cell death may play an important role in HI inducedneuronal loss in the immature brain. 2. Nitrotyrosine is an early marker of neuronaldamage after neonatal HI brain injury. The inhibition of nNOS and iNOS show theeffect of neuronal protection. 3. Cell proliferation, differentiation and survive wasbrain regions, developmental stages and injury time courses related. 4. Thedevelopment related differences in the neurogenesis and gliagenesis respond to HIinjury may underline the differences in tissue restoration in developing brain.