Isoflurane-induced neuroinflammation and NKCC1/KCC2 dysregulation result in long-term cognitive disorder in neonatal mice.

BACKGROUND: The inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na+-K+-2Cl--1 (NKCC1) and K+-2Cl--2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development. METHODS: We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α were quantified using ELISA. RESULTS: We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1ß levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1ß, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway. CONCLUSIONS: Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.

Protective effect of resveratrol on mitochondrial biogenesis during hyperoxia-induced brain injury in neonatal pups.

BACKGROUND: Neonatal hyperoxic brain injury is caused by exposure to hyperphysiological oxygen content during the period of incomplete development of the oxidative stress defence system, resulting in a large number of reactive oxygen species (ROS) and causing damage to brain tissue. Mitochondrial biogenesis refers to the synthesis of new mitochondria from existing mitochondria, mostly through the PGC-1α/Nrfs/TFAM signalling pathway. Resveratrol (Res), a silencing information regulator 2-related enzyme 1 (Sirt1) agonist, has been shown to upregulate the level of Sirt1 and the expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). We speculate that Res has a protective effect on hyperoxia-induced brain injury through mitochondrial biogenesis. METHODS: Sprague-Dawley (SD) pups were randomly divided into the nonhyperoxia (NN) group, the nonhyperoxia with dimethyl sulfoxide (ND) group, the nonhyperoxia with Res (NR) group, the hyperoxia (HN) group, the hyperoxia with dimethyl sulfoxide (HD) group, and the hyperoxia with Res (HR) group within 12 h after birth. The HN, HD, and HR groups were placed in a high-oxygen environment (80‒85%), and the other three groups were placed in the standard atmosphere. The NR and HR groups were given 60 mg/kg Res every day, the ND and HD groups were given the same dose of dimethyl sulfoxide (DMSO) every day, and the NN and HN groups were given the same dose of normal saline every day. On postnatal day (PN) 1, PN7, and PN14, brain samples were acquired for HE staining to assess pathology, TUNEL to detect apoptosis, and real-time quantitative polymerase chain reaction and immunoblotting to detect the expression levels of Sirt1, PGC-1α, nuclear respiratory factor 1 (Nrf1), nuclear respiratory factor 2 (Nrf2) and mitochondrial transcription factor A (TFAM) in brain tissue. RESULTS: Hyperoxia induced brain tissue injury; increased brain tissue apoptosis; inhibited Sirt1, PGC-1α, Nrf1, Nrf2, TFAM mRNA expression in mitochondria; diminished the ND1 copy number and ND4/ND1 ratio; and decreased Sirt1, PGC-1α, Nrf1, Nrf2, and TFAM protein levels in the brain. In contrast, Res reduced brain injury and attenuated brain tissue apoptosis in neonatal pups and increased the levels of the corresponding indices. CONCLUSION: Res has a protective effect on hyperoxia-induced brain injury in neonatal SD pups by upregulating Sirt1 and stimulating the PGC-1α/Nrfs/TFAM signalling pathway for mitochondrial biogenesis.

Acutely increased aquaporin-4 exhibits more potent protective effects in the cortex against single and repeated isoflurane-induced neurotoxicity in the developing rat brain.

Damage to hippocampus, cerebellum, and cortex associated with cognitive functions due to anesthetic-induced toxicity early in life may cause cognitive decline later. Aquaporin 4 (AQP4), a key protein in waste clearance pathway of brain, is involved in synaptic plasticity and neurocognition. We investigated the effects of single and repeated isoflurane (Iso) anesthesia on AQP4 levels and brain damage. Postnatal-day (P)7 Wistar albino rats were randomly assigned to Iso or Control (C) groups. For single-exposure, pups were exposed to 1.5% Iso in 30% oxygenated-air for 3-h at P7 (Iso1). For repeated-exposure, pups were exposed to Iso for 3 days, 3-h each day, at 1-day intervals (P7 + 9 + 11) starting at P7 (Iso3). C1 and C3 groups received only 30% oxygenated-air. Based on HE-staining and immunoblotting (Bax/Bcl-2, cleaved-caspase3 and PARP1) analyses, Iso exposures caused a higher degree of apoptosis in hippocampus. Anesthesia increased 4-hydroxynonenal (4HNE), oxidative stress marker; the highest ROS accumulation was determined in cerebellum. Increased inflammation (TNF-α, NF-κB) was detected. Multiple Iso-exposures caused more significant damage than single exposure. Moreover, 4HNE and TNF-α contributed synergistically to Iso-induced neurotoxicity. After anesthesia, higher expression of AQP4 was detected in cortex than hippocampus and cerebellum. There was an inverse correlation between increased AQP4 levels and apoptosis/ROS/inflammation. Correlation analysis indicated that AQP4 had a more substantial protective profile against oxidative stress than apoptosis. Remarkably, acutely increased AQP4 against Iso exhibited a more potent neuroprotective effect in cortex, especially frontal cortex. These findings promote further research to understand better the mechanisms underlying anesthesia-induced toxicity in the developing brain.

Effects of 2,4-dichlorophenoxyacetic acid on the expression of NLRP3 inflammasome and autophagy-related proteins as well as the protective effect of Lycium barbarum polysaccharide in neonatal rats.

The pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) has neurotoxic effects, but its mechanism is not clear. In this study, a 2,4-D (75 mg/kg. b.w) exposure model was established in SD rats with colostrum. Lipopolysaccharide (1 mg/kg b.w) was used as the positive control, and Lycium barbarum polysaccharide (LBP, 50 mg/kg b.w) was used as an intervention factor to explore the neurotoxic effect of 2,4-D and the neuroprotective effect of LBP. Our research results show that 2,4-D causes a decrease in the number of hippocampal CA3 pyramidal cells and pyknosis in nuclei with a triangular or irregular shape and that rats show signs of anxiety or depression. In rat serum, superoxide dismutase, and glutathione peroxidase activity decreased, while malondialdehyde content increased. Protein and mRNA levels of TNFα, IL-6, IL-1ß, IL-18, NLRP3, ASC, caspase-1, IL-1ß, IL-18, and p62 increased, while those of LC3-II/LC3-I and Beclin-1 decreased in hippocampal tissues. In conclusion, 2,4-D increased the oxidative stress level, induced neuroinflammatory response, and decreased the autophagy level in experimental rats. LBP may have upregulated the autophagy level in the body by inhibiting the activation of the NLRP3 inflammasome, thus playing a neuroprotective role.

Long non-coding RNA CRNDE deteriorates intrauterine infection-induced neonatal brain injury.

BACKGROUND: This study aimed to test the hypothesis that long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) could exacerbate brain injury caused by intrauterine infection in neonatal rats. METHODS: Intrauterine infection was induced in pregnant rats by lipopolysaccharide (LPS). After delivery, newborn rats with brain injury caused by intrauterine infection were randomly divided into control, control shRNA, and CRNDE shRNA groups. CRNDE expression in serum and amniotic fluid of pregnant rats and neonatal brain tissues were determined by quantitative real-time PCR (qRT-PCR). Morris water maze (MWM) task was used to test the spatial learning and memory ability. Histological examination and apoptosis detection were performed by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Immunohistochemistry was conducted to evaluate the activation of astrocytes and microglia. RESULTS: LncRNA CRNDE was highly expressed in serum and amniotic fluid of maternal rats and in brain tissues of offspring rats. Furthermore, shRNA-mediated CRNDE downregulation could rescue the spatial learning and memory ability, improve brain histopathological changes and cell death, and inhibit the activation of astrocytes and microglia caused by LPS. CONCLUSION: CRNDE silencing possessed a cerebral protective effect in neonatal rats with brain injury caused by interauterine infection.

Retracted: microRNA-129-5p involved in the neuroprotective effect of dexmedetomidine on hypoxic-ischemic brain injury by targeting COL3A1 through the Wnt/ß-catenin signaling pathway in neonatal rats.

Our study aims to elucidate the mechanisms how microRNA-129-5p (miR-129-5p) involved in the neuroprotective effect of dexmedetomidine (DEX) on hypoxic-ischemic brain injury (HIBI) by targeting the type III procollagen gene (COL3A1) through the Wnt/ß-catenin signaling pathway in neonatal rats. A total of 120 rats were obtained, among which 15 rats were selected as sham group and rest rats as model, DEX, DEX + negative control (DEX + NC), DEX + miR-129-5p mimics, DEX + miR-129-5p inhibitors, DEX + XAV-939, and DEX + miR-129-5p inhibitors + XAV-939 groups. A dual-luciferase reporter assay was performed for the target relationship between miR-129-5p and COL3A1. Weight rate and water content of cerebral hemisphere were detected. Quantitative real-time polymerase chain reaction and Western blot analysis were conducted to detect miR-129-5p expression and expressions of COL3A1, E-cadherin, T-cell factor (TCF)- 4, and ß-catenin. The DEX, DEX + miR-129-5p mimics, DEX + XAV-939 groups had increased weight rate of the cerebral hemisphere, but decreased water content of left cerebral hemisphere, levels of COL3A1, ß-catenin, TCF-4, and E-cadherin in the hippocampus compared with the model and DEX + miR-129-5p inhibitors groups. COL3A1 was verified as the target gene of the miR-129-5p. Compared with the DEX + NC and DEX + miR-129-5p inhibitors + XAV-939 groups, the DEX + XAV-939 and DEX + miR-129-5p mimics groups had elevated weight rate of the cerebral hemisphere, but reduced water content of left cerebral hemisphere, levels of COL3A1, ß-catenin, TCF-4, and E-cadherin in the hippocampus. Our findings demonstrate that miR-129-5p improves the neuroprotective role of DEX in HIBI by targeting COL3A1 through the Wnt/ß-catenin signaling pathway in neonatal rats.

Possible role of mitochondrial K-ATP channel and nitric oxide in protection of the neonatal rat heart.

Cardioprotective effect of ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) in adult hearts is mediated by mitochondrial-K-ATP channels and nitric oxide (NO). During early developmental period, rat hearts exhibit higher resistance to ischemia-reperfusion (I/R) injury and their resistance cannot be further increased by IPC or IPoC. Therefore, we have speculated, whether mechanisms responsible for high resistance of neonatal heart may be similar to those of IPC and IPoC. To test this hypothesis, rat hearts isolated on days 1, 4, 7, and 10 of postnatal life were perfused according to Langendorff. Developed force (DF) of contraction was measured. Hearts were exposed to 40 min of global ischemia followed by reperfusion up to the maximum recovery of DF. IPoC was induced by 5 cycles of 10-s ischemia. Mito-K-ATP blocker (5-HD) was administered 5 min before ischemia and during first 20 min of reperfusion. Another group of hearts was isolated for biochemical analysis of 3-nitrotyrosine, and serum samples were taken to measure nitrate levels. Tolerance to ischemia did not change from day 1 to day 4 but decreased on days 7 and 10. 5-HD had no effect either on neonatal resistance to I/R injury or on cardioprotective effect of IPoC on day 10. Significant difference was found in serum nitrate levels between days 1 and 10 but not in tissue 3-nitrotyrosine content. It can be concluded that while there appears to be significant difference of NO production, mito-K-ATP and ROS probably do not play role in the high neonatal resistance to I/R injury.

Alterations of neuroimmune cell density and pro-inflammatory cytokines in response to thimerosal in prefrontal lobe of male rats.

Evidence suggests that the effect of heavy metals on neuroimmune cells lead to neurogenic inflammatory responses. In this study, immune cells [mast cells (MCs) and microglia] and pro-neuroinflammation cytokines (interleukin-1b and tumor necrosis factor-α) were assessed in the prefrontal lobe of rat brains exposed to thimerosal in different timeframes. A total of 108 neonatal Wistar rats were divided into three groups having three subgroups. The experimental groups received a single dose of thimerosal (300 µg/kg) postnatally at 7, 9, 11, and 15 days. The vehicle groups received similar injections of phosphate-buffered saline in a similar manner. The control groups received nothing. Samples of the prefrontal cortex were collected and prepared for stereological, immunohistochemical, and molecular studies at timeframes of 12 or 48 h (acute phase) and 8 days (subchronic phase) after the last injection. The average density of the microglia and MCs increased significantly in the experimental groups. This increase was more evident in the 48 h group. At 8 days after the last injection, there was a significant decrease in the density of the MCs compared to the 12 and 48 h groups. Alterations in pro-inflammatory cytokines were significant for all timeframes. This increase was more evident in the 48 h group after the last injection. There was a significant decrease in both neuroinflammatory cytokines at 8 days after the last injection. It was found that ethylmercury caused abnormal neurogenic inflammatory reactions and alterations in the neuroimmune cells that remained for a longer period in the brain than in the blood.

Effects of hypothermia during propofol anesthesia on learning and memory ability and hippocampal apoptosis in neonatal rats.

OBJECTIVE: At present, the harm of hypothermia to the central nervous system has received a great attention from scholars. The present study aimed to investigate the effects of hypothermia on learning and memory abilities and hippocampal apoptosis in neonatal rats and the role of p-ERK and p-CREB in anesthesia. METHODS: In this study, 60 Sprague Dawley newborn rats (age 7-day-old) were randomly divided into 3 groups (n = 20), including Control Group (Group C), Anesthesia Group (Group A), and Anesthesia Hypothermia Group (Group AH). Group C was intraperitoneally injected with 0.1 ml saline, and rectal temperature was maintained in the range of 38-39 °C; Group A was intraperitoneally injected with 25 mg/kg of propofol (0.1 ml), the 1/2 initial dose was added per each period of 20 min, anesthesia was maintained for 2 h, and rectal temperature was kept in the range of 38-39 °C. The anesthesia mode and duration of Group AH were as same as Group A, room temperature was set to 23 °C, which caused body's temperature naturally dropped down. After the anesthesia recovered, each group randomly involved five rats for analyzing by Western blot to detect the expression level of p-ERK and p-CREB, and other five rates were also analyzed by flow cytometry assay to detect hippocampal apoptosis rate. The remaining 10 rats in each group were kept up to 30 days for conducting the Morris water maze test, five rats were tested for detecting the expression level of p-ERK and p-CREB, as well as hippocampal apoptosis rate in each group. RESULTS: Compared with Group C and Group A, the rectal temperature of Group AH was decreased significantly (P < 0.05); At the age of 7 days, compared with Group C and Group A, apoptosis rate of hippocampal tissue in Group AH was increased (P < 0.05), the expression level of p-ERK and p-CREB proteins in Group AH was significantly reduced (P < 0.05), and there were no significant differences between Group C and Group A. At the age of 36 days, there were no significant differences in the results of behavioral test, apoptotic rates, and expression level of the proteins. CONCLUSION: Our findings suggest that hypothermia during anesthesia can increase the apoptosis rate in the hippocampus of neonatal rats, whose mechanism may be related to the downward adjustment of p-ERK and p-CREB. However, it has no obvious influence on the long-term learning and memory abilities.

Leptin and adiponectin supplementation modifies mesenteric lymph node lymphocyte composition and functionality in suckling rats.

At birth, when immune responses are insufficient, there begins the development of the defence capability against pathogens. Leptin and adiponectin, adipokines that are present in breast milk, have been shown to play a role in the regulation of immune responses. We report here, for the first time, the influence of in vivo adipokine supplementation on the intestinal immune system in early life. Suckling Wistar rats were daily supplemented with leptin (0·7 µg/kg per d, n 36) or adiponectin (35 µg/kg per d, n 36) during the suckling period. The lymphocyte composition, proliferation and cytokine secretion from mesenteric lymph node lymphocytes (on days 14 and 21), as well as intestinal IgA and IgM concentration (day 21), were evaluated. At day 14, leptin supplementation significantly increased the TCRαß + cell proportion in mesenteric lymph nodes, in particular owing to an increase in the TCRαß + CD8+ cell population. Moreover, the leptin or adiponectin supplementation promoted the early development CD8+ cells, with adiponectin being the only adipokine capable of enhancing the lymphoproliferative ability at the end of the suckling period. Although leptin decreased intestinal IgA concentration, it had a trophic effect on the intestine in early life. Supplementation of both adipokines modulated the cytokine profile during (day 14) and at the end (day 21) of the suckling period. These results suggest that leptin and adiponectin during suckling play a role in the development of mucosal immunity in early life.

Neonatal rat age, sex and strain modify acute antioxidant response to ozone.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US and its impact continues to increase in women. Oxidant insults during critical periods of early life appear to increase risk of COPD through-out the life course. To better understand susceptibility to early life exposure to oxidant air pollutants we used Fisher (F344), Sprague-Dawley (SD) and Wistar (WIS) male and female neonatal rat pups to assess: (A) if strain (i.e. genetics), sex, or stage of early life development affected baseline lung antioxidant or redox enzyme levels and (B) if these same factors modulated antioxidant responsiveness to acute ozone exposure (1 ppm × 2 h) on post-natal day (PND) 14, 21, or 28. In air-exposed pups from PND14-28, some parameters were unchanged (e.g. uric acid), some decreased (e.g. superoxide dismutase), while others increased (e.g. glutathione recycling enzymes) especially post-weaning. Lung total glutathione levels decreased in F344 and SD pups, but were relatively unchanged in WIS pups. Post-ozone exposure, data suggest that: (1) the youngest (PND14) pups were the most adversely affected; (2) neonatal SD and WIS pups, especially females, were more prone to ozone effects than males of the same age and (3) F344 neonates (females and males) were less susceptible to oxidative lung insult, not unlike F344 adults. Differences in antioxidant levels and responsiveness between sexes and strains and at different periods of development may provide a basis for assessing later life health outcomes - with implications for humans with analogous genetic or dietary-based lung antioxidant deficits.

Role of histone acetylation in long-term neurobehavioral effects of neonatal Exposure to sevoflurane in rats.

Human studies, and especially laboratory studies, provide evidence that early life exposure to general anesthesia may affect neurocognitive development via largely unknown mechanisms. We explored whether hippocampal histone acetylation had a role in neurodevelopmental effects of sevoflurane administered to neonatal rats. Male Sprague-Dawley rats were exposed to 3% sevoflurane or were subjected to maternal separation only for 2h daily at postnatal days 6, 7, and 8. The histone deacetylase inhibitor, sodium butyrate (250mg/kg, intraperitoneally), or saline was administered starting 2h prior to anesthesia or maternal separation and continued daily until the end of behavioral tests, which were performed between postnatal days 33 and 50. Upon completion of the behavioral tests, the brain tissues were harvested for further analysis. Rats neonatally exposed to sevoflurane exhibited decreased freezing time in the fear conditioning contextual test and increased escape latency, decreased time in target quadrant, and number of platform crossings in the Morris water maze test. The sevoflurane-exposed rats had lower hippocampal density of dendritic spines, reduced levels of the brain-derived neurotrophic factor, c-fos protein, microtubule-associated protein 2, synapsin1, postsynaptic density protein 95, pCREB/CREB, CREB binding protein, and acetylated histones H3 and H4, and increased levels of histone deacetylases 3 and 8. These neurobehavioral abnormalities were normalized in the sevoflurane-exposed rats treated with sodium butyrate. Our findings provide evidence that neonatal exposure to sevoflurane induces neurobehavioral abnormalities and long-lasting alterations in histone acetylation; normalization of histone acetylation may alleviate the neurodevelopmental side effects of the anesthetic.

Sevoflurane induces cognitive impairments via the MiR-27b/LIMK1-signaling pathway in developing rats.

Exposure to sevoflurane in neonatal rats could induce learning deficits and abnormal social behaviors, but the specific molecular mechanism is unknown. Postnatal day-7 SD rats were treated with 3% sevoflurane plus 30% oxygen/air or 30% oxygen/air. As the rats grew, the Morris water maze (MWM) and fear conditioning tests were performed to evaluate cognitive function, while the expression of LIMK1 was analyzed by western blot. Luciferase reporter assay was performed to investigate the interaction between LIMK1 and miR-27b. The expression of miR-27b was measured by real-time polymerase chain reaction (PCR) after exposure to sevoflurane. Once the miR-27b inhibitor was transfected into the neurons, the expression of LIMK1 was analyzed by real-time PCR and western blot. Exposure to sevoflurane in neonatal rats induced memory and learning impairments according to the MWM and fear conditioning tests. Sevoflurane increased the expression of miR-27b and reduced the expression of LIMK1 in the brain tissues of rats compared to the control group. The results of the luciferase reporter assay showed that LIMK1 was a direct target of miR-27b. In the primary neurons, the inhibition of miR-27b could reverse the down-regulating effects of sevoflurane on LIMK1 expression. We suggest that sevoflurane-induced learning and memory impairments in rats might be mediated via the miR-27b-LIMK1-signaling pathway.

Intranasal IGF-1 Reduced Rat Pup Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is the most devastating neurological problem of premature infants. Current treatment strategies are ineffective and brain injury is unpreventable. Insulin-like growth factor 1 (IGF-1) is an endogenous protein shown to have multiple neuroprotective properties. We therefore hypothesized that IGF-1 would reduce brain injury after GMH. Neonatal rats (P7 age) received stereotactic collagenase into the right ganglionic eminence. The following groups were studied: (1) sham, (2) GMH + vehicle, (3) GMH + intranasal IGF-1. Three days later, the animals were evaluated using the righting-reflex (early neurobehavior), Evans blue dye leakage (blood-brain barrier (BBB) permeability), brain water content (edema), and hemoglobin assay (extent of bleeding). Three weeks later, juvenile rats were tested using a water maze (delayed neurobehavior), and then were sacrificed on day 28 for assessment of hydrocephalus (ventricular size). Intranasal IGF-1 treated animals had improved neurological function, and amelioration of BBB permeability, edema, and re-bleeding. IGF-1 may play a part in protective brain signaling following GMH, and our observed protective effect may offer new promise for treatment targeting this vulnerable patient population.

PAR-1, -4, and the mTOR Pathway Following Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is the most common cause of neurological complications of prematurity and has lasting implications. PAR-1 and PAR-4 receptors are involved with upstream signaling pathways following brain hemorrhage in adult models of stroke, of which the mammalian target of rapamycin (mTOR) is a potential downstream mediator. Therefore, we hypothesized a role for PAR-1, -4/ mTOR signaling following GMH brain injury. Postnatal day 7 Sprague-Dawley rats were subjected to GMH through stereotactic infusion of collagenase into the right ganglionic eminence. Rodents were euthanized at 72 h (short term), or 4 weeks (long term). Short-term mTOR expression was evaluated by Western blot in the context of PAR-1 (SCH-79797) and PAR-4 (P4pal10) inhibition. Pups in the long-term group were administered the selective mTOR inhibitor (rapamycin) with neurobehavioral and brain pathological examinations performed at 4 weeks. Pharmacological PAR-1, -4 antagonism normalized the increased mTOR expression following GMH. Early inhibition of mTOR by rapamycin improved long-term outcomes in rats. Mammalian-TOR signaling plays an important role in brain injury following neonatal GMH, possibly involving upstream PAR-1, -4 mechanisms.

Osteopontin-Rac1 on Blood-Brain Barrier Stability Following Rodent Neonatal Hypoxia-Ischemia.

Osteopontin (OPN) is a neuroprotective molecule that is upregulated following rodent neonatal hypoxic-ischemic (nHI) brain injury. Because Rac1 is a regulator of blood-brain barrier (BBB) stability, we hypothesized a role for this in OPN signaling. nHI was induced by unilateral ligation of the right carotid artery followed by hypoxia (8 % oxygen for 2 h) in P10 Sprague-Dawley rat pups. Intranasal (iN) OPN was administered at 1 h post-nHI. Groups consisted of: (1) Sham, (2) Vehicle, (3) OPN, and (4) OPN + Rac1 inhibitor (NSC23766). Evans blue dye extravasation (BBB permeability) was quantified 24 h post-nHI, and brain edema at 48 h. Increased BBB permeability and brain edema following nHI was ameliorated in the OPN treatment group. However, those rat pups receiving OPN co-treatment with the Rac1 inhibitor experienced no improvement compared with vehicle. OPN protects the BBB following nHI, and this was reversed by Rac1 inhibitor (NSC23766).

Remote Ischemic Postconditioning (RIPC) After GMH in Rodents.

Germinal matrix hemorrhage (GMH) is the most common and devastating neurological injury of premature infants, and current treatment approaches are ineffective. Remote ischemic postconditioning (RIPC) is a method by which brief limb ischemic stimuli protect the injured brain. We hypothesized that RIPC can improve outcomes following GMH in rats. Neonatal rats (P7) were subjected to either stereotactic ganglionic eminence collagenase infusion or sham surgery. Groups were as follows: sham (n = 0), GMH non-RIPC (n = 10), GMH + 1 week RIPC (n = 10), GMH + 2 weeks RIPC (n = 10). Neurobehavior analysis at the fourth week consisted of Morris water maze (MWM) and rotarod (RR). This was followed by euthanasia for histopathology on day 28. Both 1- and 2-week RIPC showed significant improvement in FF and RR motor testing compared with untreated animals (i.e., GMH without RIPC). RIPC treatment also improved cognition (MWM) and attenuated neuropathological ventricular enlargement (hydrocephalus) in juvenile animals following GMH. RIPC is a safe and noninvasive approach that improved sensorimotor and neuropathological outcomes following GMH in rats. Further studies are needed to evaluate for mechanisms of neuroprotection.

Cyclooxygenase-2 Inhibition Provides Lasting Protection Following Germinal Matrix Hemorrhage in Premature Infant Rats.

Germinal matrix hemorrhage (GMH) is a major cause of brain damage in prematurity and has long-lasting neurological implications. The development of brain inflammation contributes to brain injury, leading to a lifetime of neurologic deficits. PAR-1 and 4 receptors are involved with inflammatory pathways after brain hemorrhage in adult models of stroke, of which cyclooxygenase-2 (COX-2) is a potential mediator. We therefore hypothesized a role for PAR-1, 4/ COX-2 signaling following GMH. Postnatal day 7 Sprague-Dawley rats were subjected to GMH induction, which entailed stereotactic collagenase infusion into the ganglionic eminence. Animals were euthanized at two time points: 72 h (short-term) or 4 weeks (long-term). Short-term COX-2 expression was evaluated in the context of PAR-1 (SCH-79797) and PAR-4 (P4pal10) inhibition. Pups in the long-term group were administered the selective COX-2 inhibitor (NS-398); and the neurobehavioral and pathological examinations were performed 4 weeks later. Pharmacological PAR-1, 4 antagonism normalized COX-2 expression following GMH and reduced hydrocephalus. Early inhibition of COX-2 by NS-398 improved long-term neurobehavioral outcomes. COX-2 signaling plays an important role in brain injury following neonatal GMH, possibly through upstream PAR-1, 4 receptor mechanisms.

Intranasal Osteopontin for Rodent Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is the most common and devastating neurological problem of premature infants. Current treatment is largely ineffective and GMH has been nonpreventable. Osteopontin (OPN) is an endogenous protein that has been shown to be neuroprotective, however, it has not been tested in GMH. P7 neonatal rats were subjected to stereotactic ganglionic eminence collagenase infusion. Groups were as follows: (1) sham, (2) GMH + vehicle, (3) GMH + intranasal OPN. Seventy-two hours later, the animals were evaluated using righting reflex, blood-brain barrier (BBB) permeability by Evans blue dye leakage, brain water content, and hemoglobin assay. Intranasal OPN improved outcomes after GMH by attenuation of brain swelling, BBB function, re-bleeding, and neurological outcomes. OPN may play an important role in enhancing neuroprotective brain signaling following GMH. These observed effects may offer novel possibilities for therapy in this patient population.

Amikacin induced renal damage and the role of the antioxidants on neonatal rats.

Amikacin (AK) is frequently used on the treatment of Gram-negative infections on neonates, but its usage is restricted because of nephrotoxicity. In this study, on neonatal rats, we aimed to investigate the effects of erythropoietin and vitamin E on AK induced nephrotoxicity. A total of 35 newborn Wistar Albino rats were divided into four groups: (1) injected with saline (serum physiological was administered to placebo controls), (2) injected with AK (1200 mg/kg), (3) injected with AK + vitamin E (150 mg/kg), (4) injected with AK + erythropoietin (EPO) (300 IU/kg/day). In renal tissue, AK levels were significantly high in all groups except the control. Tissue malondialdehyde (MDA) and nitric oxide (NO) levels were statistically higher in AK -treated group than the control. MDA and NO levels were significantly decreased with the administration of vitamin E and EPO. Glutathione peroxidase (GPX) levels were statistically low in AK group compared with the controls. The levels of GPX, in vitamin E group, were increased significantly. However, superoxide dismutase and catalase levels were not significantly different in none of the groups. Insulin-like growth factor-1 values in AK, EPO and vitamin E groups were significantly higher than the control group. Histomorphological changes such as tubular epithelial necrosis were seen in AK treated group. Histopathological improvements observed with EPO and vitamin E administration. AK nephrotoxicity is related to oxidative stress and is supported with biochemical and histopathological findings. Vitamin E and EPO, as antioxidants, can be useful renoprotective agents for ameliorating AK induced nephrotoxicity in neonates.