Brain Damage In Newborn Rats Research Articles

Neuroprotective Efficacy of Betulinic Acid Hydroxamate, a B55α/PP2A Activator, in Acute Hypoxia-Ischemia-Induced Brain Damage in Newborn Rats.

There is an increasing evidence of the neuroprotective effects of hypoxia inducing factor prolyl-hydroxylase inhibitors (HIF-PHDi) after hypoxic-ischemic (HI) brain damage (HIBD). We studied the neuroprotective effects of betulinic hydroxamate (BAH), a novel B55α/PP2A activator that dephosphorylates and inhibits PHD2 activity, in a rat model of neonatal HIBD. Seven-day-old (P7) Wistar rats were exposed to hypoxia after left carotid artery electrocoagulation and then received vehicle (HI + VEH) or BAH 3mg/kg i.p. 30min post-insult. Brain damage was assessed by magnetic resonance imaging (MRI) and neurobehavioral studies testing motor and cognitive performance at P14 and P37, as well as immunohistochemical studies (TUNEL and myelin basic protein (MBP) signal) at P37. Mechanisms of damage were assessed at P14 determining excitotoxicity (glutamate/N-acetylaspartate ratio by H+-magnetic resonance spectroscopy), oxidative stress (protein nitrosylation by Oxyblot), and inflammation (cytokine and chemokine concentration). BAH reduced brain damage volume and cell death, preventing the development of motor and working memory deficits. BAH showed a robust protective effect on myelination, restoring MBP expression at P37. BAH modulated excitotoxicity, oxidative stress, and inflammation. Most neuroprotective effects were still present despite BAH administration was delayed for 12h, whereas beneficial effects on motor strength at P14 and on cell death and myelination at P37 were preserved even when BAH administration was delayed for 24h. In conclusion, BAH appears as an effective neuroprotective treatment for neonatal HIBD in a manner associated with the modulation of excitotoxicity, oxidative stress, and inflammation, showing a broad therapeutic window.

Olig2 knockdown alleviates hypoxic-ischemic brain damage in newborn rats.

Neuronal damage is an important pathological mechanism in neonatal hypoxic-ischemic brain damage (HIBD). We found in our previous studies that oligodendrocyte transcription factor 2 (Olig2) downregulation was able to increase cell survival in the brain. However, the specific mechanism has yet to be clarified. Sprague-Dawley rats aged 3 d were randomly divided into three groups: the normal control group, the Olig2-RNAi group, and the RNAi-negative control group. The normal control group received no treatment, the Olig2-RNAi group received the Olig2 RNAi adenovirus, and the RNAi-negative control group was given the control adenovirus after the completion of the HIBD model. Infarct lesions and their volumes were observed by triphenyltetrazolium chloride (TTC) staining 3 d after the completion of the adenovirus local injection. The condition of the tissue was characterized by hematoxylin-eosin staining 7 d after the model was established, and cell viability was determined by azure methylene blue staining. Subcellular damage was analyzed by transmission electron microscopy. Rotarod analysis was performed to detect moving behavior ability and an MWM assay was conducted to evaluate the memory. TTC staining showed a smaller brain injury area in the Olig2-RNAi group than in the RNAi-negative control group. Hematoxylin-eosin staining indicated the presence of severe cell injury in the hippocampal region after HIBD, which improved after Olig2 knockdown. Azure methylene blue staining and electron microscopy results suggested that the cells improved after Olig2 knockdown. The rats stayed longer on the rotating rod, and their latency in the water maze test was gradually shortened relative to that of the rats in the Olig2-RNAi negative control group. Olig2 knockdown can promote the repair of hypoxic-ischemic brain damage in newborn rats.

Neuroprotective effects of autophagy inhibition on hippocampal glutamate receptor subunits after hypoxia-ischemia-induced brain damage in newborn rats.

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage (HIBD). However, its regulatory role in HIBD remains unclear and was thus examined here using a rat model. To induce HIBD, the left common carotid artery was ligated in neonatal rats, and the rats were subjected to hypoxia for 2 hours. Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine (10 mM in 10 μL) or the autophagy stimulator rapamycin (1 g/kg) 1 hour before artery ligation. Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR1, but by reduced expression of GluR2. Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury, whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury. Additionally, 3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of GluR1 and downregulation of GluR2 in the hippocampus. By contrast, rapamycin further elevated hippocampal GluR1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD. Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats, at least in part, through normalizing GluR1 and GluR2 expression.

Activation of acetylcholine receptors and microglia in hypoxic-ischemic brain damage in newborn rats

Objective: We previously showed that acetylcholine receptor (AChR) agonist reduced hypoxic-ischemic brain damage in the newborn rats. To further investigated the interaction between hypoxia and chorinergic anti-inflammatory pathway, we examined the effect of AChR antagonist on brain damage and to see the relation between microglial activation and protective effect of AChR agonist. Study design: Seven-day-old Wistar rats were divided into 2 groups, one receiving AChR antagonists to see if they have deleterious effects on hypoxic-ischemic brain damage, and the other receiving AChR agonist, carbachol, to investigate the emergence of microglia in the hippocampus. Rats were subjected to left carotid artery ligation followed by 8% hypoxia. Brains were analyzed histologically and immunohistochemically. Results: Antagonists of AChRs significantly enhanced brain damage in 1-h hypoxia-ischemia. In particular, the nicotinic AChR antagonist showed a marked enhancement of brain damage compared to the saline controls (p<0.01). The hippocampal CA1 was most vulnerable to any AChR antagonists, while the cortex was least vulnerable and only responsive to a higher dose of non-selective nAChR antagonist. Carbachol showed significantly less accumulation of microglia in the hippocampus than the saline controls (p<0.01) in hypoxia–ischemia. Conclusion: An AchR-responsive pathway in the brain plays an important role in modifying perinatal brain damage, in which microglial accumulation may be involved.

The effects of hyperbaric oxygen therapy on hypoxic-ischemic brain damage in newborn rats

Objective To track the effects of hyperbaric oxygen (HBO) therapy on neonatal rats with hypoxic-ischernic brain damage (HIBD).Methods A total of 126 healthy,seven-day-old SD rats were used to model the HIBD and then randomly divided into seven groups of 18:a sham group,a hypoxic-ischemic (HI) group,a 6 h HBO group,a 24 h HBO group,a 48 h HBO group,a 72 h HBO group and a 1 week HBO group.One hour of HBO treatment at 2 atmospheres absolute pressure was administered once daily for a week to the rats in the latter 5groups,starting at 6,24,48,72 hours and 1 week post the HIBD modeling,while no HBO was administered to the sham and HI groups.The effects were assessed in terms of histological changes ( the neuron density and the percentage of neuron apoptosis in the CA1 region of the hippocampus) and nitric oxide (NO),malondialdehyde (MDA) and superoxygen dismustase (SOD) concentrations after 15 days.Results In the 6 h HBO,24 h HBO,48 h HBO and 72 h HBO groups,average neuron density in the CA1 region was significantly higher than in the HI group.But in the 1 week HBO group the average density was not significantly different than in the HI group.In the 6 h HBO,24 h HBO,48 h HBO and 72 h HBO groups the average percentage of neuron cell apoptosis in the CA1 area of the hippocampus was significantly lower than in the HI group,but the 1 week HBO group again showed no significant difference.There were significant differences in average NO,MDA or SOD levels among the 6 h HBO,24 h HBO,48 h HBO,72 h HBO and HI groups,but the 1 week HBO group showed no significantly higher average levels than the HI group.Conclusions The optimal therapeutic window for using HBO to protect against HIBD is within the first week.The best effect can be obtained in the first 6 hours,but after 1 week HBO no longer has a significant effect.The earlier the HBO is administered,the better the effect obtained. Key words: Newborns; Hypoxia; Ischemia; Brain damage; Hyperbaric oxygen; Therapeutic windows

Repetitive and profound insulin-induced hypoglycemia results in brain damage in newborn rats: an approach to establish an animal model of brain injury induced by neonatal hypoglycemia

The human neonate is at a higher risk for hypoglycemia-induced neuronal injury than other pediatric and adult patients. Repetitive and profound neonatal hypoglycemia can result in severe neurologic sequelae, of which the mechanisms was not elucidated by hitherto. Moreover, no reliable animal model of brain injury induced by neonatal hypoglycemia is available in order to carry out more research. Therefore, we tried to induce neonatal hypoglycemia in newborn rats by fasting and insulin injection, and then examined the neuronal degeneration after repetitive hypoglycemic insults by Fluoro-Jade B (FJB) staining. Experimental animals were randomly divided into four groups: insulin-treated rats with short hypoglycemia, insulin-treated rats with prolonged hypoglycemia, fasted rats, and control rats. Insulin injection and fasting both could induce consistent hypoglycemia in newborn rats. But from FJB staining results, only in insulin-treated rats with prolonged hypoglycemia could extensive neurodegeneration be detected. We can conclude that FJB staining is a useful method of marking neuronal degeneration in neonatal rats following hypoglycemic brain damage. Repetitive and profound neonatal hypoglycemia can result in extensive neurodegeneration, and it seems that neurons of the cortex, dentate gyrus of the hippocampus, the thalamus, and the hypothalamus are more vulnerable to hypoglycemic insult in newborn rats. Repetitive and profound insulin-induced hypoglycemia in newborn rats can establish a reliable animal model of brain injury resulting from neonatal hypoglycemia.

Characterization of the Neuroprotective Effect of the Cannabinoid Agonist WIN-55212 in an In Vitro Model of Hypoxic-Ischemic Brain Damage in Newborn Rats

Brain slices from 7-d-old Wistar rats were exposed to oxygen-glucose deprivation (OGD) for 30 min. OGD slices were incubated with vehicle or with the CB1/CB2 cannabinoid agonist WIN55212 (50 microM), the CB1 agonist arachidonyl-2-chloroethylamide (ACEA) (50 microM), or the CB2 agonist JW133 (50 microM), alone or combined with the CB1 and CB2 receptor antagonist SR 141716 (50 microM) or SR 144528 (50 microM), respectively. Neuronal damage was assessed by histologic analysis and spectrophotometric determination of lactate dehydrogenase (LDH) efflux into the incubation medium. Additionally, medium glutamate levels were determined by high-performance liquid chromatography (HPLC) and those of tumor necrosis factor alpha (TNF-alpha) by enzyme-linked immunosorbent assay. Finally, inducible nitric oxide synthase (iNOS) and CB1/CB2 receptor expression were determined in slices homogenate by Western blot. Both CB1 and CB2 receptors were expressed in slices. OGD increased CB1 expression, cellular damage, LDH efflux, glutamate and TNF-alpha release, and inducible nitric oxide synthase (iNOS) expression; WIN55212 inhibited all these actions. SR141716 and SR144528 inhibited the effect of R(+)-WIN-55212-2 (WIN), as well as the reduction of LDH efflux by ACEA and JW133, respectively. In conclusion, WIN55212 afforded robust neuroprotection in the forebrain slices exposed to OGD, by acting on glutamatergic excitotoxicity, TNF-alpha release, and iNOS expression; this neuroprotective effect seemed to be mediated by CB1 and CB2 receptors.

Hyperoxia with 100% Oxygen following Hypoxia-Ischemia Increases Brain Damage in Newborn Rats

Objective: To describe the effect of reoxygenation with 100% O₂ as compared to the effect of room air in newborn rat brains after asphyxia. Methods: Experimental asphyxia (carotid artery ligation followed by hypoxic exposure with 8% O₂ for 2 h) was performed on 7-day-old rats. After hypoxia-ischemia the rats were reoxygenated with either 100% O₂ (hyperoxia group) or 21% O₂ (room air group) for 24 h and then returned to the dam. The rats were killed 1 week after the experiment to study the cerebral cortex and hippocampus. Results: Rats reoxygenated with 100% O₂ post-asphyxia showed more frequency of cortical damage (10 of 24 rats) than those reoxy genated with room air (3 of 24 rats) (χ² test, p = 0.02). Conclusion: We consider that hyperoxia with 100% oxygen after hypoxia-ischemia can cause more damage in the cerebral cortex than room air in newborn rats.

Cerebral blood flow distribution and hypoxic-ischemic brain damage in newborn rats.

Our purpose was to assess the cerebral blood flow distribution and resulting grade of hypoxicischemic brain damage in newborn rats. Seven-day-old Wistar rats (n = 75) underwent left common carotid artery ligation followed by 2 hours hypoxia (8% oxygen in nitrogen) at 33 degrees C. The control animals were exposed to hypoxia without ligation (n = 8). Colored microspheres of 15 microm in diameter were administered into the left cardiac ventricle percutaneously at the end of hypoxia. They were killed 24 hours after induced injury. Brain sections 2 mm in thickness were removed for microtubule-associated protein 2 (MAP-2) staining, and remaining parts were separated into left and right hemispheres for counting the microspheres. The blood flow distribution to the ligated side was expressed as the difference from the non-ligated control side. Severity of MAP-2 disappearance was ranked as normal, mild or severe. In the control rats, there was no loss of MAP-2 staining. The blood flow equally distributed into both cerebral hemispheres. The cerebral blood flow distribution on the side of carotid artery ligation decreased by 44.7 +/- 21.9% in the mildly damaged group and 65.8 +/- 16.8% in the severely damaged group. The greater the percentage difference of blood flow distribution from the non-ligated side, the more severe the brain damage.

Long-term magnesium sulfate treatment as protection against hypoxic-ischemic brain injury in seven-day-old rats

Objective: Our purpose was to study the effects of long-term treatment with magnesium sulfate on hypoxic-ischemic brain damage in newborn rats. Study Design: Seven-day-old rat pups (n = 120) were exposed to unilateral carotid artery ligation and 2 hours of hypoxia (8% oxygen in 92% nitrogen). Neuronal loss was evaluated in 4 groups. The loading dose group (n = 25) received a bolus injection of 270 mg/kg magnesium sulfate intraperitoneally. The maintenance group (n = 26) received 3 days of continuous infusion of magnesium sulfate with a micro-osmotic pump at a rate of 72 mg/kg per hour. The loading dose–plus–maintenance group (n = 23) received both. The control group (n = 46) did not receive magnesium. Seven days after the injury the pups were killed and the brains were removed for analysis. Severity of neuronal loss was evaluated in the cerebral cortex and hippocampus and compared among groups by χ2 test. Results: Cyst formation resulting from necrosis was significantly decreased in the maintenance group (15%) and loading dose–plus–maintenance group (9%) but not in the loading dose group (52%) relative to the control group (46%). Severity of neuronal loss in the cerebral cortex and the hippocampus was also significantly improved in the maintenance group and the loading dose–plus–maintenance group but not in the loading dose group. Mortality rates were not different among the groups. Magnesium ion concentrations at 24 hours were significantly decreased from the preinfusion value of 0.49 ± 0.01 mmol/L in the control group (0.34 ± 0.03 mmol/L) but remained within normal ranges in the maintenance group (0.46 ± 0.02 mmol/L with a pump infusing 72 mg/kg per hour and 0.56 ± 0.05 mmol/L with a pump infusing 24 mg/kg hour). There was an inverse relationship between the maintenance dose and neuronal loss but not between the maintenance dose and mortality rate. Conclusion: Long-term magnesium administration had neuroprotective effects against hypoxia-ischemia in newborn rats. (Am J Obstet Gynecol 2001;184:185-90.)