Hypoxia-ischemia In Rats Research Articles

Clemastine enhances exercise-induced motor improvement in hypoxic ischemic rats

Neonatal hypoxic ischemia (HI) occurs owing to reduced cerebral oxygen levels and perfusion during the perinatal period. Brain injury after HI triggers neurological manifestations such as motor impairment, and the improvement of impaired brain function remains challenging. Recent studies suggest that cortical myelination plays a role in motor learning, but its involvement in motor improvement after HI injury is not well understood. This study aimed to investigate the impact of myelination on motor improvement following neonatal HI injury. We employed a modified Rice-Vannucci model; the right common carotid artery of postnatal day 7 (P7) Wistar rats was isolated and divided, and the rats were then exposed to hypoxic condition (90 min, 8 % O2). A total of 101 rats (66 males) were divided into four groups: trained-HI (n = 38), trained-Sham (n = 16), untrained-HI (n = 31), and untrained-Sham (n = 16). The trained groups underwent rotarod-based exercise training from P22 to P41 (3 days per week). Structural analysis using magnetic resonance imaging and immunohistochemistry (n = 6 per group) revealed increased fractional anisotropy and myelin density in the primary somatosensory cortex of the trained-HI group. We further evaluated the effect of myelination promotion on rotarod performance by administering clemastine, a myelination-promoting drug, via daily intraperitoneal injections. Clemastine did not enhance motor improvement in untrained-HI rats. However, clemastine-administered trained-HI rats (n = 7) exhibited significantly improved motor performance compared to both saline-administered trained-HI rats (n = 11) and clemastine-administered untrained-HI rats (n = 7). These findings suggest that myelination may be a key mechanism in motor improvement after HI injury and that combining exercise training with clemastine administration could be an effective therapeutic strategy for motor improvement following HI injury.

Neuroprotective Effect of Melatonin in a Neonatal Hypoxia-Ischemia Rat Model Is Regulated by the AMPK/mTOR Pathway.

Melatonin has been shown to be neuroprotective in different animal models of neonatal hypoxic-ischemic brain injury. However, its exact molecular mechanism of action remains unknown. Our aim was to prove melatonin's short- and long-term neuroprotection and investigate its role on the AMPK (AMP-activated protein kinase)/mTOR (mammalian target of rapamycin) pathway following neonatal hypoxic-ischemic brain injury. Seven-day-old Wistar rat pups were exposed to hypoxia-ischemia, followed by melatonin or vehicle treatment. Detailed analysis of the AMPK/mTOR/autophagy pathway, short- and long-term neuroprotection, myelination, and oligodendrogenesis was performed at different time points. At 7 days after hypoxia-ischemia, melatonin-treated animals showed a significant decrease in tissue loss, increased oligodendrogenesis, and myelination. Long-term neurobehavioral results showed significant motor improvement following melatonin treatment. Molecular pathway analysis showed a decrease in the AMPK expression, with a significant increase at mTOR's downstream substrates, and a significant decrease at the autophagy marker levels in the melatonin group compared with the vehicle group. Melatonin treatment reduced brain area loss and promoted oligodendrogenesis with a clear improvement of motor function. We found that melatonin associated neuroprotection is regulated via the AMPK/mTOR/autophagy pathway. Considering the beneficial effects of melatonin and the results of our study, melatonin seems to be an optimal candidate for the treatment of newborns with hypoxic-ischemic brain injury in high- as well as in low- and middle-income countries.

Genistein-3′-sodium sulfonate enhances neurological function in neonatal rats with hypoxia-ischemia during the recovery period

Hypoxic-ischemic (HI) can cause neonatal brain damage leading to disability. Patients with HI experience long-term neurological issues impacting quality of life. Limited clinical treatments are available despite extensive research on HI's molecular mechanisms. Genistein-3′-sodium sulfonate (GSS), a phytoestrogen, has been found to improve acute brain injury in neonatal rats caused by hypoxic-ischemia, but its potential for chronic stage neurological recovery in HI is unknown. HI neonatal rats were treated with 1 mg/kg GSS once a day for 21 days. Then, a series of behavioral experiments was performed to evaluate the learning, memory, cognition, anxiety level and depression-like behaviors of the rats. GSS treatment reduced neuronal loss, enhanced learning, memory and cognitive function while also alleviated anxiety and depression-like behaviors in HI rats during the recovery period. These findings indicated that GSS exerted enhance neurological function in HI rats during the chronic stage, prompting further research on how it works to potentially develop new therapies.

Neuroprotective Effect of Clemastine Improved Oligodendrocyte Proliferation through the MAPK/ERK Pathway in a Neonatal Hypoxia Ischemia Rat Model.

Neonatal hypoxic-ischemic encephalopathy is the most common cause of long-term disability in term neonates, and white matter injury is the primary cause of cerebral palsy. Therapies that focus on the neuroprotection of myelination and oligodendrocyte proliferation could potentially ameliorate long-lasting neurological impairments after hypoxic-ischemic encephalopathy. Clemastine, a histamine H1 antagonist, has been shown to exert neuroprotective effects in multiple sclerosis and spinal cord injury by promoting oligodendrogenesis and re-myelination. In this study, we demonstrated the neuroprotective effects of clemastine in our rat model of neonatal hypoxic-ischemic brain injury. Animals received a single intraperitoneal injection of either vehicle or clemastine (10 mg/kg) for 6 consecutive days. Our results showed a significant reduction in white matter loss after treatment, with a clear effect of clemastine on oligodendrocytes, showing a significant increase in the number of Olig2+ cells. We characterized the MAPK/ERK pathway as a potential mechanistic pathway underlying the neuroprotective effects of clemastine. Altogether, our results demonstrate that clemastine is a potential compound for the treatment of hypoxic-ischemic encephalopathy, with a clear neuroprotective effect on white matter injury by promoting oligodendrogenesis.

Early Neuroprotective Effects of Bovine Lactoferrin Associated with Hypothermia after Neonatal Brain Hypoxia-Ischemia in Rats.

Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) in term newborns is a leading cause of mortality and chronic disability. Hypothermia (HT) is the only clinically available therapeutic intervention; however, its neuroprotective effects are limited. Lactoferrin (LF) is the major whey protein in milk presenting iron-binding, anti-inflammatory and anti-apoptotic properties and has been shown to protect very immature brains against HI damage. We hypothesized that combining early oral administration of LF with whole body hypothermia could enhance neuroprotection in a HIE rat model. Pregnant Wistar rats were fed an LF-supplemented diet (1 mg/kg) or a control diet from (P6). At P7, the male and female pups had the right common carotid artery occluded followed by hypoxia (8% O2 for 60') (HI). Immediately after hypoxia, hypothermia (target temperature of 32.5-33.5 °C) was performed (5 h duration) using Criticool®. The animals were divided according to diet, injury and thermal condition. At P8 (24 h after HI), the brain neurochemical profile was assessed using magnetic resonance spectroscopy (1H-MRS) and a hyperintense T2W signal was used to measure the brain lesions. The mRNA levels of the genes related to glutamatergic excitotoxicity, energy metabolism and inflammation were assessed in the right hippocampus. The cell markers and apoptosis expression were assessed using immunofluorescence in the right hippocampus. HI decreased the energy metabolites and increased lactate. The neuronal-astrocytic coupling impairments observed in the HI groups were reversed mainly by HT. LF had an important effect on astrocyte function, decreasing the levels of the genes related to glutamatergic excitotoxicity and restoring the mRNA levels of the genes related to metabolic support. When combined, LF and HT presented a synergistic effect and prevented lactate accumulation, decreased inflammation and reduced brain damage, pointing out the benefits of combining these therapies. Overall, we showed that through distinct mechanisms lactoferrin can enhance neuroprotection induced by HT following neonatal brain hypoxia-ischemia.

Hypoxia ischemia results in blood brain barrier damage via AKT/GSK-3β/CREB pathway in neonatal rats

Previous studies have showed that the permeability of blood brain barrier (BBB) increased after hypoxia ischemia (HI). The current research uncovered the mechanism of altered BBB permeability after hypoxic-ischemic brain damage (HIBD) through AKT/GSK-3β/CREB signaling pathway in neonatal rats. Firstly, Magnetic resonance imaging (MRI) combined with hematoxylin-eosin (H&E) staining was used to assess brain injury. Initial findings showed abnormal signals in T2-weighted imaging (T2WI) and diffusion weighted imaging (DWI). Changes also happened in the morphology of nerve cells. Subsequently, we found that BBB damage is manifested as leakage of immunoglobulin G (IgG) and destruction of BBB-related proteins and ultrastructure. Meanwhile, the levels of matrix metalloproteinase-9 (MMP-9) significantly increased at 24 h after HIBD compared to a series of time points. Additionally, immunohistochemical (IHC) staining combined with Western blot (WB) was used to verify the function of the AKT/GSK-3β/CREB signaling pathway in BBB damage after HI in neonatal rats. Results showed that less Claudin-5, ZO-1, p-AKT, p-GSK-3β and p-CREB, along with more MMP-9 protein expression were visible on the damaged side of the cerebral cortex in the HIBD group in contrast to the sham and HIBD + SC79 groups. Together, our findings demonstrated that HI in neonatal rats might upregulate the levels of MMP-9 protein and downregulate the levels of Claudin-5 and ZO-1 by inhibiting the AKT/GSK-3β/CREB pathway, thus disrupting the BBB, which in turn aggravates brain damage after HI in neonatal rats.

Effects of prenatal mobile phone radiation exposure on MMP9 expression: Implications for inflammation, oxidative stress, and sensory-motor impairment after neonatal hypoxia- ischemia in rats

ObjectiveNon-ionizing radiofrequency radiation, which finds application in various sectors such as industry, commerce, medicine, and particularly in mobile phone technology, has emerged as a topic of significant concern during pregnancy. The aim of this study was to investigate the effect of cell phone radio-frequency (RF) radiation during pregnancy on the Matrix metalloproteinase 2 (MMP-2) and (MMP-9) 9 expressions after neonatal hypoxia-ischemia (HI) in rats. Materials and methodsTwo groups were formed by randomly assigning female Wistar rats: Group 1 consisted of female rats that were not exposed to RF radiation during pregnancy, while Group 2 comprised female rats that were exposed to RF radiation during pregnancy. After delivery, male offspring were divided into four groups including: (a) SHAM, (b) Exposure (EXP), (c) hypoxia-ischemia (HI), (d) HI/Exposure (HI/EXP). Seven days after HI induction, neurobehavioral tests were performed, and then brain tissue was taken from the skull to measure MMP-2 and MMP-9 expressions, inflammation, oxidative stress, infarct volume and cerebral edema. ResultsMMP-9 mRNA expression in the HI/EXP group was significantly higher than the HI, SHAM and EXP groups. MMP-2 mRNA expression levels in the HI group were significantly higher than Sham and the EXP groups.TNF-α and Total oxidant capacity (TOC) levels in the HI/EXP group were significantly higher than HI, EXP and SHAM groups. Total antioxidant capacity (TAC) level in the HI/EXP group were significantly lower than HI, EXP and SHAM groups. Cerebral edema and infarct volume in the HI/EXP group were significantly greater than the HI group. Sensory-motor function was significantly weaker in HI/EXP as compared HI group. ConclusionOur findings indicate that during pregnancy, exposure to mobile phone RF radiation intensifies damage from HI in rat pups by elevating MMP-9 activity.

Neuroserpin As an Adjuvant Therapy for Hypothermia on Brain Injury in Neonatal Hypoxic-Ischemic Rats.

We aimed to assess the effects of neuroserpin and its combination with hypothermia on hypoxic-ischemic (HI) brain injury in neonatal rats. Neuroserpin is an axon-secreted serine protease inhibitor and is important for brain development, neuronal survival, and synaptic plasticity. Male Wistar-Albino rats on postnatal day 7 (P7) were randomly divided into five groups: sham group (n = 10), (HI; n = 10), hypoxic-ischemic hypothermia (HIH; n = 10), hypoxic-ischemic neuroserpin (HIN; n = 10), and hypoxic-ischemic neuroserpin-hypothermia (HINH; n = 10). The P7 rat brain's maturation is similar to a late preterm human brain at 34 to 36 weeks of gestation. HI was induced in rats on P7 as previously described. A single dose of 0.2 µM neuroserpin (HINH and HIN) or an equivalent volume of phosphate-buffered saline (sham, HIH, and HI) was administered intraventricularly by a Hamilton syringe immediately after hypoxia. In the follow-up, pups were subjected to systemic hypothermia or normothermia for 2 hours. Euthanasia was performed for histopathological evaluation on P10. Apoptosis was detected by caspase-3 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and was counted in the hippocampus. In comparison to the HI group, the TUNEL-positive and caspase-3-positive neurons in the sham, HIN, HIH, and HINH groups were considerably lower (13.4 ± 1.0 vs. 1.9 ± 0.9, 6.0 ± 0.9, 5.3 ± 1.6, and 4.0 ± 1.1; p < 0.001) and (13.5 ± 1.7 vs. 1.2 ± 0.7, 9.1 ± 2.7, 4.8 ± 1.0, and 3.9 ± 1.6; p < 0.001). HIN, HIH, and HINH, compared to the sham group, showed more TUNEL-positive and caspase-3-positive neurons (6.0 ± 0.9, 5.3 ± 1.6, 4.0 ± 1.1 vs. 1.9 ± 0.9 and 9.1 ± 2.7, 4.8 ± 1.0, 3.9 ± 1.6 vs. 1.2 ± 0.7; p < 0.001). The HINH group (synergistic effect) had significantly fewer TUNEL-positive neurons and caspase-3-positive neurons than the HIN group (4.0 ± 1.1 vs. 6.0 ± 0.9 and 3.9 ± 1.6 vs. 9.1 ± 2.7; p < 0.001). Our study showed that both neuroserpin alone and as an adjuvant treatment for hypothermia may have a neuroprotective effect on brain injury. · Neuroserpin decreased brain injury.. · Neuroserpin showed a synergistic effect when used as an adjuvant treatment for hypothermia.. · The neuroprotective effect of neuroserpine was related to its antiapoptotic properties..

Transcranial Doppler Ultrasonography detection on cerebral infarction and blood vessels to evaluate hypoxic ischemic encephalopathy modeling

BackgroundThis study aimed to observe changes of rats' brain infarction and blood vessels during neonatal hypoxic ischemic encephalopathy (NHIE) modeling by Transcranial Doppler Ultrasonography (TCD) so as to assess the feasibility of TCD in evaluating NHIE modeling. MethodsPostnatal 7-days (d)-old Sprague Dawley (SD) rats were divided into the Sham group, hypoxic-ischemic (HI) group, and hypoxia (H) group. Rats in the HI group and H group were subjected to hypoxia-1 hour (h), 1.5 h and 2.5 h, respectively. Evaluation on brain lesion was made based on Zea-Longa scores, hematoxylin-eosin (HE) staining and Nissl staining. The brain infarction and blood vessels of rats were monitored and analyzed under TCD. Correlation analysis was applied to reveal the connection between hypoxic duration and infarct size detected by TCD or Nissl staining. ResultsIn H and HI modeling, longer duration of hypoxia was associated with higher Zea-Longa scores and more severe nerve damage. On the 1 d after modeling, necrosis was found in SD rats' brain indicated by HE and Nissl staining, which was aggravated as hypoxic duration prolonged. Alteration of brain structures and blood vessels of SD rats was displayed in Sham, HI and H rats under TCD. TCD images for coronal section revealed that brain infarct was detected at the cortex and there was marked cerebrovascular back-flow of HI rats regardless of hypoxic duration. On the 7 d after modeling, similar infarct was detected under TCD at the cortex of HI rats in hypoxia-1 h, 1.5 h and 2.5 h groups, whereas the morphological changes were deteriorated with longer hypoxic time. Correlation analysis revealed positive correlation of hypoxic duration with infarct size detected by histological detection and TCD. ConclusionsTCD dynamically monitored cerebral infarction after NHIE modeling, which will be potentially served as a useful auxiliary method for future animal experimental modeling evaluation in the case of less animal sacrifice.

Systemic administration of clinical-grade multilineage-differentiating stress-enduring cells ameliorates hypoxic–ischemic brain injury in neonatal rats

Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative pluripotent stem cells present in the bone marrow, peripheral blood, and organ connective tissues. We assessed the homing and therapeutic effects of systemically administered nafimestrocel, a clinical-grade human Muse cell-based product, without immunosuppressants in a neonatal hypoxic–ischemic (HI) rat model. HI injury was induced on postnatal day 7 (P7) and was confirmed by T2-weighted magnetic resonance imaging on P10. HI rats received a single dose nafimestrocel (1 × 106 cells/body) or Hank’s balanced salt solution (vehicle group) intravenously at either three days (on P10; M3 group) or seven days (on P14; M7 group) after HI insult. Radioisotope experiment demonstrated the homing of chromium-51-labeled nafimestrocel to the both cerebral hemispheres. The cylinder test (M3 and M7 groups) and open-field test (M7 group) showed significant amelioration of paralysis and hyperactivity at five weeks of age compared with those in the vehicle group. Nafimestrocel did not cause adverse events such as death or pathological changes in the lung at ten weeks in the both groups. Nafimestrocel attenuated the production of tumor necrosis factor-α and inducible nitric oxide synthase from activated cultured microglia in vitro. These results demonstrate the potential therapeutic benefits and safety of nafimestrocel.

The Long-Term Neuroprotective Effect of the Endocannabinoid 2-AG and Modulation of the SGZ's Neurogenic Response after Neonatal Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) often causes hypoxic-ischemic encephalopathy (HIE), a neurological condition that can lead to overall disability in newborns. The only treatment available for affected neonates is therapeutic hypothermia; however, cooling is not always effective to prevent the deleterious effects of HI, so compounds such as cannabinoids are currently under research as new therapies. Modulating the endocannabinoid system (ECS) may reduce brain damage and/or stimulate cell proliferation at the neurogenic niches. Further, the long-term effects of cannabinoid treatment are not so clear. Here, we studied the middle- and long-term effects of 2-AG, the most abundant endocannabinoid in the perinatal period after HI in neonatal rats. At middle-term (postnatal day 14), 2-AG reduced brain injury and increased SGZ's cell proliferation and the number of neuroblasts. At post-natal day 90, the treatment with the endocannabinoid showed global and local protection, suggesting long-lasting neuroprotective effects of 2-AG after neonatal HI in rats.

Transcranial Doppler Ultrasonography detection on cerebrovascular flow for evaluating neonatal hypoxic-ischemic encephalopathy modeling.

This study aimed to investigate the feasibility of Transcranial Doppler Ultrasonography (TCD) in evaluating neonatal hypoxic-ischemic encephalopathy (NHIE) modeling through monitoring the alteration of cerebrovascular flow in neonatal hypoxic-ischemic (HI) rats. Postnatal 7-day-old Sprague Dawley (SD) rats were divided into the control group, HI group, and hypoxia (H) group. TCD was applied to assess the changes of cerebral blood vessels, cerebrovascular flow velocity, and heart rate (HR) in sagittal and coronal sections at 1, 2, 3, and 7 days after the operation. For accuracy, cerebral infarct of rats was examined by 2,3,5-Triphenyl tetrazolium chloride (TTC) staining and Nissl staining to simultaneously verify the establishment of NHIE modeling. Coronal and sagittal TCD scans revealed obvious alteration of cerebrovascular flow in main cerebral vessels. Obvious cerebrovascular back-flow was observed in anterior cerebral artery (ACA), basilar artery (BA), middle cerebral artery (MCA) of HI rats, along with accelerated cerebrovascular flows in the left internal carotid artery (ICA-L) and BA, decreased flows in right internal carotid artery (ICA-R) relative to those in the H and control groups. The alterations of cerebral blood flows in neonatal HI rats indicated successful ligation of right common carotid artery. Besides, TTC staining further validated the cerebral infarct was indeed caused due to ligation-induced insufficient blood supply. Damage to nervous tissues was also revealed by Nissl staining. Cerebral blood flow assessment by TCD in neonatal HI rats contributed to cerebrovascular abnormalities observed in a real-time and non-invasive way. The present study elicits the potentials to utilize TCD as an effective means for monitoring the progression of injury as well as NHIE modeling. The abnormal appearance of cerebral blood flow is also beneficial to the early warning and effective detection in clinical practice.

Effect of type 1 diabetes during pregnancy and lactation on neonatal hypoxia-ischemia injury and apoptotic gene expression.

Hypoxia-ischemia (HI) is one of the major disorders that causes a variety of abnormalities and infant mortality. Type 1 diabetes is one of the most common worldwide metabolic disorders, and its prevalence has become one of the public health concerns of the 21st century. This study aims to evaluate the effect of type 1 diabetes during pregnancy and lactation on vulnerability to neonatal HI in rats. Female Wistar rats (weighing 200 ± 220 g) were randomly assigned into two groups: Group 1 (rats that received 0.5mL/day of normal saline solution) and Group 2 (type 1 diabetes was induced in rats on the second day of pregnancy with a single dose of intraperitoneal injection of alloxan monohydrate [150 mg/kg]). After delivery, offspring were divided into four groups: (a) Control (Co), (b) Diabetic (DI), (c) Hypoxia-ischemia (HI), (d) HI + Diabetic (HI + DI). Seven days after HI induction, neurobehavioral tests were performed, and then cerebral edema, infarct volume, inflammatory factors, Bax-Bcl2 expression, and oxidative stress were measured. The BAX level in the DI + HI (p = 0.0355) group was significantly higher than the HI group. The Bcl-2 expression levels in the HI (p = 0.0027) and DI + HI (p < 0.0001) groups were significantly lower than the DI group. Total antioxidant capacity (TAC) levels in the DI + HI (p < 0.0001) group were significantly lower than HI and CO groups. TNF-α, CRP, and total oxidant status (TOS) levels in the DI + HI (p < 0.0001) group were significantly higher than the HI group. Infarct volume and cerebral edema in the DI + HI (p < 0.0001) group were significantly higher than the HI group. According to the results, type 1 diabetes during pregnancy and lactation increased the destructive effects of HI injury in pups. It also decreased the expression of the anti-apoptotic Bcl-2 and increased the expression BAX apoptosis factor gene in pups.

Inter-Alpha Inhibitor Proteins Modify the Microvasculature after Exposure to Hypoxia-Ischemia and Hypoxia in Neonatal Rats.

Microvasculature develops during early brain development. Hypoxia-ischemia (HI) and hypoxia (H) predispose to brain injury in neonates. Inter-alpha inhibitor proteins (IAIPs) attenuate injury to the neonatal brain after exposure to HI. However, the effects of IAIPs on the brain microvasculature after exposure to HI have not been examined in neonates. Postnatal day-7 rats were exposed to sham treatment or right carotid artery ligation and 8% oxygen for 90 min. HI comprises hypoxia (H) and ischemia to the right hemisphere (HI-right) and hypoxia to the whole body, including the left hemisphere (H-left). Human IAIPs (hIAIPs, 30 mg/kg) or placebo were injected immediately, 24 and 48 h after HI/H. The brains were analyzed 72 h after HI/H to determine the effects of hIAIPs on the microvasculature by laminin immunohistochemistry and calculation of (1) the percentage area stained by laminin, (2) cumulative microvessel length, and (3) density of tunneling nanotubes (TNTs), which are sensitive indicators of the earliest phases of neo-vascularization/collateralization. hIAIPs mainly affected the percent of the laminin-stained area after HI/H, cumulative vessel length after H but not HI, and TNT density in females but not males. hIAIPs modify the effects of HI/H on the microvasculature after brain injury in neonatal rats and exhibit sex-related differential effects. Our findings suggest that treatment with hIAIPs after exposure to H and HI in neonatal rats affects the laminin content of the vessel basal lamina and angiogenic responses in a sex-related fashion.

Maternal treadmill exercise ameliorates impairment of neurological outcome, caspase-1 and NLRP3 gene expression alteration in neonatal hypoxia-ischemia rats.

Neonatal hypoxia-ischemia (HI) is one of the most important causes of neurological disorders in children. Various studies suggest that maternal exercise during pregnancy has a beneficial impact on the health status of offspring infants. In this study, the effect of maternal treadmill exercise during pregnancy on neurological and molecular changes induced by HI in newborn rats was investigated. In this experiment, 24 pregnant female rats were divided into two groups; the first group was subjected to treadmill exercise for six weeks. The treadmill exercise program was initiated by running for 17 min at 5-10 m/min at 0 inclination in the first week, followed by running for 21 min at 5-25 m/min at 5° inclination in the second week, running for 25 min at 5-30 m/min at 10° inclination in the third and fourth weeks, running for 25 min at 5-15 m/min at 10° inclination in the fifth and sixth weeks. The second group was left untreated and did not perform the exercise. Newborn rats were assigned to four groups; (1) control, (2) control+exercise, (3) HI, and (4) HI+exercise. HI was developed in the offspring on the 8th postnatal day. One week following the induction of HI, the Garcia test was carried out. The histological morphology of neonates was assessed, and the expression levels of caspase-1 and NLRP3 were evaluated. The data showed that maternal exercise during pregnancy significantly improved neural cell death (P<0.001) and the Garcia score (P<0.05), while it attenuated the expression levels of caspase-1 (P<0.001) and NLRP3 (P<0.05) genes in newborn rats induced by HI. These results demonstrated that maternal treadmill exercise during pregnancy could reverse the neurological deficits, as well as the expression levels of caspase-1 and NLRP3 genes, which occur in neonatal hypoxia-ischemia.

Neuroprotective effect of lipopolysaccharides in a dual-hit rat pup model of preterm hypoxia–ischemia

The combination of lipopolysaccharide (LPS) and hypoxia–ischemia (HI) has been used to model the brain injury sustained by sick pre-term infants in order to study the pathological conditions of diffuse white matter injury, which is a major cause of preterm morbidity. Prior studies have shown that the timing and dose of LPS administration will determine whether the injury is reduced or exacerbated. Here we show that administering a single injection of LPS (0.1 mg/kg) to postnatal-day-2 rat pups 14 h before inducing HI effectively protects the brain from HI-associated damage. We show that the LPS-treated HI rat pups have significantly less histopathology compared to the saline-treated HI rat pups. Apoptotic deaths were dramatically curtailed in both the neocortex and white matter when evaluated at 2 days of recovery. Microglial activation was reduced when the percentage of CD68+/Iba1+ cells was quantified in the neocortex of the LPS-treated vs the saline-treated HI rat pups. One mechanism through which LPS pre-treatment appears to be preventing injury is through the AKT-endothelial nitric oxide synthase (eNOS) pathway as LPS induced an increase in both the expression and phosphorylation of eNOS. Altogether these data show that the neocortex, as well as the white matter sustain damage after HI at this timepoint in forebrain development and that acutely activating the immune system can protect the brain from brain injury.

Hypothermia Does Not Boost the Neuroprotection Promoted by Umbilical Cord Blood Cells in a Neonatal Hypoxia-Ischemia Rat Model

Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of death and long-term disability in the perinatal period. Currently, therapeutic hypothermia is the standard of care for this condition with modest efficacy and strict enrollment criteria. Therapy with umbilical cord blood cells (UCBC) has come forward as a strong candidate for the treatment of neonatal HIE, but no preclinical studies have yet compared the action of UCBC combined with hypothermia (HT) with the action of each therapy by itself. Thus, to evaluate the potential of each therapeutic approach, a hypoxic-ischemic brain lesion was induced in postnatal day ten rat pups; two hours later, HT was applied for 4 h; and 24, 48, and 72 h post-injury, UCBC were administered intravenously. The neonatal hypoxic-ischemic injury led to a brain lesion involving about 48% of the left hemisphere that was not improved by HT (36%) or UCBC alone (28%), but only with the combined therapies (25%; p = 0.0294). Moreover, a decrease in glial reactivity and improved functional outcomes were observed in both groups treated with UCBC. Overall, these results support UCBC as a successful therapeutic approach for HIE, even when treatment with therapeutic hypothermia is not possible.

Protective effect of isoflurane preconditioning on neurological function in rats with HIE.

Hypoxic-ischemic encephalopathy (HIE) is an important cause of neonatal death and disability, which can lead to long-term neurological and motor dysfunction. Currently, inhalation anesthetics are widely used in surgery, and some studies have found that isoflurane (ISO) may have a positive effect on neuroprotection. In this paper, we investigated whether ISO pretreatment has a neuroprotective effect on the neurological function of HIE rats. Here, 7-day-old neonatal rats were randomly divided into a sham group, a hypoxic-ischemic (HI) group, and an ISO pretreatment (pretreatment) group. The pretreatment group was pretreated with 2% ISO for 1 h, followed by the HI group to establish an HI animal model. The HI‑induced neurological injury was evaluated by Zea‑Longa scores and triphenyltetrazolium (TTC) staining. Neuronal number and histomorphological changes were observed with Nissl staining and Hematoxylin-eosin (HE) staining. In addition, motor learning memory function was evaluated by the Morris water maze (MWM), the Y-maze, and the rotarod tests. HI induced severe neurological dysfunction, brain infarction, and cell apoptosis as well as obvious neuron loss in neonatal rats. In the MWM, the rats in the pretreatment group showed a decrease in escape latency (p = 0.042), indicating that pretreatment with ISO could improve the learning ability of HI rats. The results of Nissl staining showed that in the HI group, there was an irregular arrangement of neurons and nuclear fixation; however, the cell damage was significantly reduced and the total number of neurons was increased after ISO pretreatment (p < 0.001). In conclusion, ISO pretreatment improved cognitive function and attenuated HI-induced reduction of Nissl-positive cells and spatial memory impairment, suggesting that pretreatment with ISO before HI modeling could reduce neuronal cell death in the hippocampus after HI.

Human–rat integrated microRNAs profiling identified a new neonatal cerebral hypoxic–ischemic pathway melatonin‐sensitive

Neonatal encephalopathy (NE) is a pathological condition affecting long‐term neurodevelopmental outcomes. Hypothermia is the only therapeutic option, but does not always improve outcomes; hence, researchers continue to hunt for pharmaceutical compounds. Melatonin treatment has benefitted neonates with hypoxic–ischemic (HI) brain injury. However, unlike animal models that enable the study of the brain and the pathophysiologic cascade, only blood is available from human subjects. Therefore, due to the unavailability of neonatal brain tissue, assumptions about the pathophysiology in pathways and cascades are made in human subjects with NE. We analyzed animal and human specimens to improve our understanding of the pathophysiology in human neonates. A neonate with NE who underwent hypothermia and enrolled in a melatonin pharmacokinetic study was compared to HI rats treated/untreated with melatonin. MicroRNA (miRNA) analyses provided profiles of the neonate's plasma, rat plasma, and rat brain cortexes. We compared these profiles through a bioinformatics tool, identifying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways common to HI brain injury and melatonin treatment. After evaluating the resulting pathways and the literature, to validate the method, the key proteins expressed in HI brain injury were investigated using cerebral cortexes. The upregulated miRNAs in human neonate and rat plasma helped identify two KEGG pathways, glioma and long‐term potentiation, common to HI injury and melatonin treatment. A unified neonatal cerebral melatonin‐sensitive HI pathway was designed and validated by assessing the expression of protein kinase Cα (PKCα), phospho (p)‐Akt, and p‐ERK proteins in rat brain cortexes. PKCα increased in HI‐injured rats and further increased with melatonin. p‐Akt and p‐ERK returned phosphorylated to their basal level with melatonin treatment after HI injury. The bioinformatics analyses validated by key protein expression identified pathways common to HI brain injury and melatonin treatment. This approach helped complete pathways in neonates with NE by integrating information from animal models of HI brain injury.

Mitigated Oxidative Stress and Cognitive Impairments in Transient Global Ischemia using Niosomal Selegiline-NBP delivery.

Stroke is the most common reason for adult disabilities and the second ground for death worldwide. Our previous study revealed that selegiline serves as an alternative candidate in transient hypoxia-ischemia. However, aggressive and restless behavior was observed in stroke-induced rats receiving 4 mg/kg selegiline. In comparison, 1 mg/kg selegiline could induce negligible therapeutic effects on mitochondrial dysfunction and histopathological changes. Therefore, we designed oral noisome-based selegiline attached to 4-(4-nitrobenzyl) pyridine to improve transient global ischemia by attenuating cognitive impairments, oxidative stress, and histopathological injury. The investigation was performed in transient hypoxia-ischemia-induced rats by oral administration of nanoformulation containing selegiline (0.25-1 mg/kg) for 4 weeks (3 times a week). Novel object recognition (NOR) was considered to evaluate their cognitive dysfunction. Oxidative stress parameters and brain histopathological assessments were determined following the scarification of rats. Outstandingly, our data demonstrated slower selegiline release from niosomes relative to free drug, which was also in a controlled manner. Our data confirmed significant improvement in cognitive behavior in the NOR test, an increase in glutathione level and total antioxidant power, a decline in MDA and protein carbonyl level, as well as a decreased number of dead cells in histopathological assessment after being exposed to (0.5-1 mg/kg) selegiline-NBP nanoformulation. These data manifested that the selegiline-NBP nanoformulation (0.5-1 mg/kg) could significantly reduce oxidative damage, cognitive dysfunction, and histopathological damage compared to transient hypoxia-ischemia rats, which is 20 times lower than the therapeutic dose in humans. Therefore, the proposed nanoformulation would be capable as an alternative candidate without side effects in stroke.