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-1β, 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-1β, 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-1β 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-1β, 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.

This study was to explore the mechanism of ferroptosis and hypoxic-ischemic brain damage in neonatal rats. The neonatal rat hypoxic-ischemic brain damage (HIBD) model was established using the Rice-Vannucci method and treated with the ferroptosis inhibitor liproxstatin-1. Cognitive assessment was performed through absentee field experiments to confirm the successful establishment of the model. Brain tissue damage was evaluated by comparing regional cerebral blood flow and quantifying tissue staining. Neuronal cell morphological changes in the rats\' cortical and hippocampal regions were observed using HE and Nissl staining. ELISA was performed to determine GPX4, GSH and ROS expression levels in the rats\' brain tissues, and Western blotting to assess the expression levels of 4-HNE, GPX4, GSS, ACSL4, SLC7A11, SLC3A2, TFRC, FHC, FLC, HIF-1α, and Nrf2 proteins in rat brain tissues. Compared to the Sham group, the HIBD group exhibited a significant decrease in cerebral blood perfusion, reduced brain nerve cells, and disordered cell arrangement. The use of the ferroptosis inhibitor effectively improved brain tissue damage and preserved the shape and structure of nerve cells. The oxidative stress products ROS and 4-HNE in the brain tissue of the HIBD group increased significantly, while the expression of antioxidant indicators GPX4, GSH, SLC7A11, and GSS decreased significantly. Furthermore, the expression of iron metabolism-related proteins TFRC, FHC, and FLC increased significantly, whereas the expression of the ferroptosis-related transcription factors HIF-1α and Nrf2 decreased significantly. Treatment with liproxstatin-1 exhibited therapeutic effects on HIBD and downregulated tissue ferroptosis levels. This study shows the involvement of ferroptosis in hypoxic-ischemic brain damage in neonatal rats through the System Xc--GSH-GPX4 functional axis and iron metabolism pathway, with the HIF-1α and Nrf2 transcription factors identified as the regulators of ferroptosis involved in the HIBD process 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.

White matter injury (WMI) is an important type of preterm brain injury, which may result in severe neurological sequelae and lack of effective treatments. It is ascertained that selective vulnerability of oligodendrocytes is closely related to the WMI in preterm infants. But the alteration of the endogenous oligodendrogenesis over long time after hypoxic-ischemic WMI is still not clearly elucidated.
We adopted an animal model of hypoxic-ischemic WMI in 3-day-old neonatal Sprague-Dawley rats. Immunofluorescence staining and western blotting were used to detect dynamic changes of oligodendrogenesis in the white matter region on postoperative day (POD) 1, 3, 7, 14, 28, 56 and 84.
In the sham group, the oligodendrocyte lineage in the white matter reached a developmental peak from POD 3 to 14. The proliferation and development of oligodendrocyte precursor cells (OPCs) occurred primarily within POD 14. The number of mature oligodendrocytes showed an upward trend and a dynamic change in proliferation over time. While in the WMI group, the oligodendrocyte lineage was upregulated on POD1 and 3 but downregulated on POD 7 and 14. The proliferation of OPCs increased on POD 1 and decreased on POD 3 and 7, with the total number of OPCs significantly reduced from POD 3 to 14. The number of mature oligodendrocytes decreased from POD 3 to 28, and return to the level of the sham group on POD 56 and 84, whereas the MBP expression was still significantly downregulated on POD 56 and 84.
Hypoxia-ischemia can have a long-term dynamic effect on the endogenous oligodendrogenesis of neonatal rat brain white matter. The proliferation of OPCs was promoted on POD 1 but inhibited from POD 3 to 14, which may be an early intervention target to improve oligodendrogenesis. The number of mature oligodendrocytes recover to the normal on POD 56 and 84 but the myelination is still blocked, which suggests it is essential to promote the maturation of oligodendrocyte and its function recovery at the same time within POD 28. Such efforts will provide the opportunity to test new interventions in pre-clinical studies for their promising clinical application.

BACKGROUND: Timing of passaging, passage number, passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells (NSCs) culture. How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.
OBJECTIVE: To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.
METHODS: First, curved tip operating scissors were used to dissect brain tissues from new born rats (2 to 3 d) and the brain tissues were cut into approximately 1 mm3 sections. Filter the single cell suspension through a nylon mesh (200-mesh) and culture the sections in suspensions. Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques. Second, identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation. BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells. Different NSCs specific antibodies (anti-nestin, NF200, NSE and GFAP antibodies) were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.
RESULTS: Brain derived cells from newborn rats (2 to 3 d) proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging. When BrdU was incorporated into the 5th generation of passaged cells, positive BrdU cells and nestin cells were observed by immunofluorescence staining. After induction of dissociation using 5% fetal bovine serum, positive NF200, NSE and GFAP cells were observed by immunofluorescence staining.
CONCLUSIONS: This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.

Perinatal hypoxic-ischemic (HI) brain injury leads to mortality and morbidity in neonates and children. There are no effective and practical methods to attenuate this brain injury. This study determined whether desflurane, a volatile anesthetic with limited effect on the cardiovascular system, protected against HI-induced brain damage and the role of transient receptor potential ankyrin 1 (TRPA1), a mediator for simulated ischemia-induced myelin damage, in this protection. Seven-day-old male and female Sprague-Dawley rats had brain HI. They were exposed to 4.8%, 7.6% or 11.4% desflurane immediately or 4.8% desflurane at 0.5, 1 or 2 h after the HI. Brain tissue loss was evaluated 7 days later. Neurological functions and brain structures of rats with HI and 4.8% desflurane post-treatment were evaluated 4 weeks after the HI. TRPA1 expression was determined by Western blotting. HC-030031, a TRPA1 inhibitor, was used to determine the role of TRPA1 in the HI-induced brain injury. HI induced brain tissue and neuronal loss, which was attenuated by all tested concentrations of desflurane. Desflurane post-treatment also improved motor function, learning and memory in rats with brain HI. Brain HI increased the expression of TRPA1 and this increase was inhibited by desflurane. TRPA1 inhibition reduced HI-induced brain tissue loss and impairment of learning and memory. However, the combination of TRPA1 inhibition and desflurane post-treatment did not preserve brain tissues, learning and memory better than TRPA1 inhibition or desflurane post-treatment alone. Our results suggest that desflurane post-treatment induces neuroprotection against neonatal HI. This effect may be mediated by inhibiting TRPA1.

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.
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.
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.
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.

Sevoflurane anesthesia induces neurocognitive impairment and pyroptosis in the developing brain. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was involved in neuronal apoptosis, oxidative stress and inflammation during ischemic stroke. The role of PHLDA1 in sevoflurane-induced pyroptosis in developing rats was investigated. Firstly, neonatal rats at day 7 was exposed to 2.0% sevoflurane for 6 h to induce neurotoxicity. Pathological analysis showed that sevoflurane anesthesia induced hippocampal injury and reduced the number of neurons. The expression of PHLDA1 was elevated in hippocampus of sevoflurane-treated rats. Secondly, sevoflurane anesthesia-treated neonatal rats were injected with adeno-associated virus serotype (AAV) to mediate knockdown of PHLDA1. Injection with AAV-shPHLDA1 ameliorated sevoflurane-induced hippocampal injury and neurocognitive impairment in rats. Moreover, knockdown of PHLDA1 increased the number of neurons in sevoflurane-treated rats. Silence of PHLDA1 suppressed neuronal apoptosis, and inhibited pyroptosis in sevoflurane-treated rats. Thirdly, PHLDA1 was also elevated in sevoflurane-treated primary neuronal cells. Loss of PHLDA1 also enhanced cell viability and suppressed pyroptosis of sevoflurane-treated primary neuronal cells. Lastly, silence of PHLDA1 reduced protein expression of TRAF6 and p-Rac1 in sevoflurane-treated rats and neuronal cells. Over-expression of TRAF6 attenuated PHLDA1 silence-induced increase of cell viability and decreased pyroptosis in neuronal cells. In conclusion, loss of PHLDA1 protected against sevoflurane-induced pyroptosis in developing rats through inhibition of TRAF6-mediated activation of Rac1.

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.

Ketamine is a widely-used anesthetic in the field of pediatrics and obstetrics. Multiple studies have revealed that ketamine causes neurotoxicity in developing animals. However, further studies are needed to determine whether clinical doses of ketamine (20 mg/kg) are able to cause kidney damage in developing animals. Herein, we investigated the effects of continuous ketamine exposure on kidney injury and pyroptosis in seven-day-old rats. Serum renal function indicators, renal histopathological analysis, pyroptosis, as well as oxidative stress indicators, were tested. Additionally, the NLRP3 inhibitor MCC950 and the Caspase-1 inhibitor VX765 were used to evaluate the role of the NLRP3/Caspase-1 axis in ketamine-induced kidney injury among developing rats. Our findings indicate that ketamine exposure causes renal histopathological injury, increased the levels of blood urea nitrogen (BUN) and creatinine (Cre), and led to upregulation in the levels of pyroptosis. Furthermore, we found that ketamine induced an increase in levels of reactive oxygen species (ROS) and malonaldehyde (MDA), as well as a decrease in the content of glutathione (GSH) and catalase (CAT) in the kidneys of neonatal rats. Moreover, targeting NLRP3 and caspase-1 with MCC950 or VX765 improved pyroptosis and reduced renal damage after continuous ketamine exposure. In conclusion, this study suggested that continued exposure to ketamine caused kidney damage among neonatal rats and that the NLRP3/Caspase-1 axis-related pyroptosis may be involved in this process.