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.

RETRACTED: MicroRNA-29a-3p strengthens the effect of dexmedetomidine on improving neurologic damage in newborn rats with hypoxic-ischemic brain damage by inhibiting HDAC4

Hypoxic-ischemic brain damage (HIBD) is a common brain injury caused by hypoxia or ischemia of the brain. This study aims to investigate the effect of dexmedetomidine (Dex) post-treatment on neurological impairment of newborn rats with HIBD via modulating microRNA-29a-3p (miR-29a-3p) and histone deacetylase 4 (HDAC4). HIBD model of newborn rats was established. Newborn modeled rats were injected with Dex, miR-29a-3p mimic or HDAC4 siRNA to figure their roles in learning and memory abilities, left hemisphere atrophy, brain tissue injury, inflammatory response and apoptosis rate of nerve cells of rats. The expression of miR-29a-3p and HDAC4 in hippocampal tissues of rats were detected, and the potential relationship between miR-29a-3p and HDAC4 was analyzed. Decreased miR-29a-3p and elevated HDAC4 were found in hippocampal tissues of rats with HIBD. In addition, Dex, elevated miR-29a-3p or declined HDAC4 enhanced spatial learning and memory abilities in rats with HIBD. Moreover, Dex, up-regulated miR-29a-3p or declined HDAC4 alleviated brain atrophy, repressed brain tissue injury, retrained the inflammation, repressed the apoptosis of neurons in the hippocampal region of rats with HIBD. HDAC4 was targeted and negatively regulated by miR-29a-3p. The study concludes that miR-29a-3p strengthened the effect of Dex on improving neurologic damage in newborn rats with HIBD by inhibiting HDAC4.

Neuroprotective effect of astaxanthin on newborn rats exposed to prenatal maternal seizures

Maternal epilepsy during pregnancy is associated with an increased incidence of brain damage and cognitive deficits in offspring. Oxidative stress is believed to play a critical role in this process. Astaxanthin, a natural carotenoid and dietary supplement, possesses potent antioxidant properties. This study was designed to investigate whether astaxanthin ameliorates the hippocampal damage in newborn rats induced by maternal epileptic seizures in utero and to explore the underlying mechanisms. Female Sprague-Dawley rats underwent chronic amygdalar kindling. After being fully kindled, all rats were allowed to mate, and electrical stimulation in the amygdala was performed every other day throughout the pregnancy. Astaxanthin was intraperitoneally injected at a dose of 30 mg/kg/d throughout pregnancy. Prenatal astaxanthin administration ameliorated neuronal lesions, decreased oxidative stress and induced the expression of cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus of pups. Astaxanthin also ameliorated placental ischemic damage in epileptic mothers. Based on the results of the present study, we concluded that astaxanthin might serve as a therapeutic agent for preventing brain damage in offspring exposed to prenatal maternal seizures.

RETRACTED: Betulinic acid protects the neuronal damage in new born rats from isoflurane-induced apoptosis in the developing brain by blocking FASL-FAS signaling pathway

Betulinic acid (BA) is a naturally occurring pentacyclic lupane group triterpenoid, has reported to protect cerebral ischemia. Present study evaluates the protective effect of betulinic acid on the isoflurane-induced neuronal damage in neonatal mice. All the mice of 7days age were exposed to isoflurane (2%; 2h) for the duration of 3day. At the end of protocol cognitive function was evaluated by Morris water maze (MWM) test. However count of apoptotic cells were estimated by TUNEL staining. Concentration of oxidative stress parameters [superoxide dismutase (SOD); catalase (CAT) and reduced glutathione (GSH)], cytokines [tumor necrosis factor (TNF-α); interlukin-6 (IL-6) and IL-1β] and expressions of caspase 3, FAS and FASL were estimated in the neuronal cells. Result of the study suggested that treatment with betulinic acid significantly reduces the escape latency and enhances platform crossing time than negative control group. Count of apoptotic cells were also found to be reduced in BA treated group of mice than negative control group. Moreover treatment with BA significantly attenuates the concentration of inflammatory cytokines and oxidative stress in isoflurane induced neonatal mice. However expressions of caspase-3, FAS/FASL was found to be significantly reduced in BA treated group of mice than negative control group. Present study concludes the neuroprotective effect of betulinic acid in isoflurane-induced brain damage in developing brain by attenuating the apoptosis through Fas/FASL pathway inhibition.

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.

Neuroprotective Effect of Erythropoietin on Phenylhydrazine-Induced Hemolytic Hyperbilirubinemia in Neonatal Rats.

Neonatal unconjugated hyperbilirubinemia might cause severe bilirubin neurotoxicity in especially hemolytic conditions. The study aimed to elucidate the potential neuroprotective effects of erythropoietin (EPO) in hemolysis-induced hyperbilirubinemia. In newborn rats, hyperbilirubinemia secondary to hemolysis was induced by injecting with phenylhydrazine hydrochloride (PHZ) and rats were injected with either vehicle or EPO. At 54th hour of the PHZ injection, rats were decapitated. Serum levels of TNF-α, IL-1β, IL-10, brain-derived neurotrophic factor (BDNF) and S100-B and brain malondialdehyde, glutathione levels and myeloperoxidase activities were measured. TUNEL staining and NF-κB expression were evaluated. As compared to control pups, in vehicle-treated PHZ group, TNF-α and IL-1β levels, malondialdehyde level and myeloperoxidase activity were increased with concomitant decreases in IL-10 and glutathione. All EPO regimens reversed PHZ-induced alterations in IL-10, TNF-α, malondialdehyde and glutathione levels. Three-day-treatment abolished increases in myeloperoxidase activity and IL-1β levels, while BDNF and S100-B were elevated. Increased TUNEL (+) cells and NF-κB expressions in the brain of PHZ group were reduced in the 3-day-treated group. EPO exerted anti-inflammatory effects on PHZ-induced neural damage in newborn rats, while the neuroprotection was more obvious when the treatments were repeated successively. The results suggest that EPO treatment may have a therapeutic potential in supporting neuroplasticity in the hyperbilirubinemic neonates.

N-methyl-D-aspartate receptor activation mediates lung fibroblast proliferation and differentiation in hyperoxia-induced chronic lung disease in newborn rats.

BackgroundPrevious studies have suggested that endogenous glutamate and its N-methyl-D-aspartate receptors (NMDARs) play important roles in hyperoxia-induced acute lung injury in newborn rats. We hypothesized that NMDAR activation also participates in the development of chronic lung injury after withdrawal of hyperoxic conditions.MethodsIn order to rule out the anti-inflammatory effects of NMDAR inhibitor on acute lung injury, the efficacy of MK-801 was evaluated in vivo using newborn Sprague-Dawley rats treated starting 4 days after cessation of hyperoxia exposure (on postnatal day 8). The role of NMDAR activation in hyperoxia-induced lung fibroblast proliferation and differentiation was examined in vitro using primary cells derived from the lungs of 8-day-old Sprague-Dawley rats exposed to hyperoxic conditions.ResultsHyperoxia for 3 days induced acute lung injury in newborn rats. The acute injury almost completely disappeared 4 days after cessation of hyperoxia exposure. However, pulmonary fibrosis, impaired alveolarization, and decreased pulmonary compliance were observed on postnatal days 15 and 22. MK-801 treatment during the recovery period was found to alleviate the chronic damage induced by hyperoxia. Four NMDAR 2 s were found to be upregulated in the lung fibroblasts of newborn rats exposed to hyperoxia. In addition, the proliferation and upregulation of alpha-smooth muscle actin and (pro) collagen I in lung fibroblasts were detected in hyperoxia-exposed rats. MK-801 inhibited these changes.ConclusionsNMDAR activation mediated lung fibroblast proliferation and differentiation and played a role in the development of hyperoxia-induced chronic lung damage in newborn rats.

Mitigation of Hypoxic‐Ischemic Damage in Newborn Rats by Misoprostol, a Prostaglandin E2 Receptor Agonist

Hypoxic‐Ischemic (HI) episodes occur in approximately 50% of preterm infants and result in significant morbidity and mortality. HI activates the nuclear transcription factor HIF1α, which in turn up‐regulates a variety of antioxidant response element‐driven genes including Bnip3, whose protein product has a pivotal role in hypoxia‐induced apoptosis and/or necrosis. Increased Bnip3 expression has been implicated in necrotizing enterocolistis (NEC), an inflammatory bowel disease of premature infants. Previously our laboratory has shown that Misoprostol, a prostaglandin E2 receptor agonist, mitigates the effects of Bnip3 in an enterocyte cell model. We hypothesized that misoprostol regulates intestinal Bnip3 activity in a rat model of HI. Dams and their pups were exposed to environmental hypoxia (10% O2) or normoxia (21% O2) from post‐natal days (PND) 3–10. Experimental animals were injected subcutaneously with 0.1mg/kg Misoprostol each day between PND 3–10, while control animals received saline injections over the same period. PND10 pups were sacrificed, and transverse colon regions were isolated and analyzed for Bnip3 protein expression via western blotting. HI induced a 17‐fold increase in Bnip3 protein expression (p<0.001); in contrast, Misoprostol induced a 72% decrease in protein expression (p<0.05) in hypoxic rats. To explore this effect mechanistically, we examined mitochondrial permeability transition pore (mPTP) opening in HEK293 cells. As expected, Bnip3 transfection increased mPTP opening, whereas Misoprostol inhibited mPTP opening in cells transfected with Bnip3. Furthermore, cotransfection with both Bnip3 and protein kinase A (PKA) mitigated Bnip3‐induced cell death in 3T3 fibroblasts. Finally, we present data showing that PKA phosphorylates Bnip3 protein. We suggest that misoprostol may attenuate hypoxia‐induced cell death via cAMP/PKA signaling pathways.Support or Funding InformationAthabasca UniversityNatural Sciences and Engineering Research Council of CanadaChildren's Hospital Research Institute of Manitoba

Investigation of the effect of the efficiency of noise at different intensities on the DNA of the newborns.

Hearing loss can occur in newborns exposed to high-level noise; noise exposure can cause more physiological stress and can lead to DNA damage. This study was designed to determine DNA damage in newborn rats exposed to sound at different concentrations. For this purpose, 28 newborn (3-6 days old) rats were divided into four groups of 7 rats in each group (Control and Groups of 40 decibel (dB), 70 dB, and 110 dB]. In the experimental groups, 40 dB, 70 dB, and 110 dB (7.5-15 kHz) of sound was applied to the experimental groups for 30 min a day for 7 days. DNA damage levels in the serums obtained from this study were determined by the enzyme-linked immunosorbent assay (ELISA) method. According to this, it was determined that DNA damage in the group exposed to 110 dB showed a statistically significant increase (P < 0.05) compared to the compared to the control, 40 dB, and 70 dB groups. Related to the subject, it was concluded that DNA damage may occur in newborns exposed to 110 dB or higher sound in neonatal units, wards, and home environments with newborn babies. Mothers should be warned about this situation and noise should be kept under 110 dB volume in the environments with the newborns.

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.

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.

Detection of clastogenic and aneugenic damage in newborn rats

The last 25 years have seen an ever-growing use of the erythrocyte micronucleus test for measuring damage to mammalian chromosomes in vivo. In addition, staining micronuclei with antikinetochore antibodies from CREST serum discriminates aneugenic from clastogenic damage. The use of the micronucleus test in rats, however, has been problematic because the spleen of adult rats efficiently removes micronucleated erythrocytes from the blood. In the present study, we have treated 5-day-old rats with either X-rays (a clastogen) or vinblastine (an aneugen) and measured micronuclei in erythrocytes from the blood and liver. Each treatment increased the frequency of micronuclei in both tissues, with the percentages of CREST-staining micronuclei reflecting the mechanism of micronucleus induction by the two agents. The results indicate that performing the micronucleus assay in the liver and peripheral blood of 5-day-old rats may be a useful approach for detecting the in vivo genotoxicity of chemical and physical agents.

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.

Spinal dorsal horn cell receptive field size is increased in adult rats following neonatal hindpaw skin injury.

Local tissue damage in newborn rats can lead to changes in skin sensitivity that last into adulthood and this is likely to be due to plasticity of developing peripheral and central sensory connections. This study examines the functional connections of dorsal horn neurons in young and adult rats that have undergone local skin damage at birth. Newborn rat pups were halothane anaesthetised and received either a unilateral subcutaneous plantar injection of 1 % lambda-carrageenan or a unilateral plantar foot injury made by removal of 2 mm x 2 mm of skin. At 3 weeks, (postnatal day (P) 19-23) and 6 weeks (P40-44) in vivo extracellular recordings of single dorsal horn cells with plantar cutaneous receptive fields were made under urethane anaesthesia (2 g kg-1) and responses to mechanical and electrical stimulation of the skin were assessed. Following neonatal carrageenan inflammation, dorsal horn neuron properties and receptive field sizes at 3 weeks were the same as those of controls. In contrast, following neonatal skin injury, dorsal horn cell receptive field sizes were significantly greater than those of controls at 3 weeks (2.5-fold) and at 6 weeks (2.2-fold). Mechanical thresholds, mechanical response magnitudes and evoked responses to single and repeated A and C fibre stimulation remained unaffected. These results show that early skin injury can cause prolonged changes in central sensory connections that persist into adult life, long after the skin has healed. Enlarged dorsal horn neuron receptive field sizes provide a physiological mechanism for the persistent behavioural hypersensitivity that follows neonatal skin injury in rats and for the prolonged sensory changes reported in human infants after early pain and injury.

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.)

Modelling the prolonged effects of neonatal pain

This chapter discusses the prolonged effects of neonatal pain. A substantial inflammatory response lasting for 2 weeks follows from a single injection of 2% carageenan into one hindpaw within 24 h of birth. This causes no lasting effect on behavioral sensory thresholds or inflammatory pain responses in adulthood. It is important to emphasize, however, that electrophysiological and neuroanatomical analysis may reveal changes in sensory connections that are not evident in reflex behavioral tests and that more research is required in this area. In addition, the reapplication of 2% carageenan or complete Freunds adjuvant (CFA) in rats, when they had reached maturity, caused normal inflammatory, hyperalgesic, and allodynic responses that did not differ from controls. Repetitive painful experiences, prolonged tissue or nerve damage in newborn rats can lead to long-lasting neurobehavioral sequelae not observed when the same stimuli are applied to adults.

The role of nitric oxide and metalloproteinases in the pathogenesis of hyperoxia-induced lung injury in newborn rats.

The effects of nitric oxide (NO) and metalloproteinases (MMP-2 and MMP-9) in the pathogenesis of hyperoxia-induced lung damage in newborn rats were examined. Three-day-old rat pups were subjected to hyperoxia (> or = 95% O2) or room air for 7 and 14 days. Some animals were treated with NG-L-nitro-L-arginine methyl ester (L-NAME, 10 mg kg(-1), s.c., daily). Histology, morphometry, oedema, Ca2+-dependent and -independent NO synthase (NOS) activities, expression of NOS isoforms and the activities of MMP-2 and MMP-9 were measured in lungs of hyperoxic and control animals. Exposure of rats to hyperoxia for 7 days resulted in alveolar sac injury characterized by the presence of cellular debris, red cell extravasation and inflammatory infiltration with mononuclear cells. Lung water content, epithelial, smooth muscle layers and total airway thickness was similar to controls. In contrast, exposure of rats to hyperoxia for 14 days resulted in lung oedema, inflammation and epithelial proliferation. Hyperoxia caused a decrease in Ca2+-dependent NOS activity, an effect that was associated with increased expression of eNOS protein. In control rats, Ca2+-dependent NOS activity and expression of eNOS were reduced at 14 days. Hyperoxia caused 10 fold increase in the activity of Ca2+-independent NOS that remained significantly elevated after 14 days of exposure to hyperoxia. The activity of this enzyme was unchanged in control rats. In lungs of hyperoxic rats, the immunoblot showed time-dependent, biphasic expression (peak at 7 days) of iNOS. The profile of expression of iNOS in control rats was similar. The activities of MMPs were increased in lungs of hyperoxic animals. The L-NAME treatment of hyperoxic animals reduced lung oedema and epithelial proliferation, but enhanced the activities of MMPs. L-NAME exerted no significant effects in control rats. It is concluded that increased generation of NO contributes to the pathogenesis of hyperoxia-induced lung damage in newborn rats.