Neonatal Rat Hippocampus Research Articles

Regulation of CRMP2 by Cdk5 and GSK-3β participates in sevoflurane-induced dendritic development abnormalities and cognitive dysfunction in developing rats

BackgroundGeneral anesthetics such as sevoflurane interfere with dendritic development and synaptogenesis, resulting in cognitive impairment. The collapsin response mediator protein2 (CRMP2) plays important roles in dendritic development and synaptic plasticity and its phosphorylation is regulated by cycline dependent kinase-5 (Cdk5) and glycogen synthase kinase-3β (GSK-3β). Here we investigated whether Cdk5/CRMP2 or GSK-3β/CRMP2 pathway is involved in sevoflurane-induced developmental neurotoxicity. MethodsRats at postnatal day 7 (PND7) were i.p. injected with Cdk5 inhibitor roscovitine, GSK-3β inhibitor SB415286 or saline 20 min. before exposure to 2.8% sevoflurane for 4 h. Western-blotting was applied to measure the expression of Cdk5/CRMP2 and GSK-3β/CRMP2 pathway proteins in the hippocampus 6 h after the sevoflurane exposure. When rats grew to adolescence (from PND25), they were tested for open-field and contextual fear conditioning, and then long term potentiation (LTP) from hippocampal slices was recorded, and morphology of pyramidal neuron was examined by Golgi staining and synaptic plasticity-related proteins expression in hippocampus were measured by western-blotting. In another batch of experiment, siRNA-CRMP2 or vehicle control was injected into hippocampus on PND5. ResultsSevoflurane activated Cdk5/CRMP2 and GSK-3β/CRMP2 pathways in the hippocampus of neonatal rats, reduced dendritic length, branches and the density of dendritic spine in pyramidal neurons. It also reduced the expressions of PSD-95, drebrin and synaptophysin in hippocampus, impaired memory ability of rats and inhibited LTP in hippocampal slices. All the impairment effects by sevoflurane were attenuated by pretreatment with inhibitor of Cdk5 or GSK-3β. Furthermore, rat transfected with siRNA-CRMP2 eliminated the neuroprotective effects of Cdk5 or GSK-3β blocker in neurobehavioral and LTP tests. ConclusionCdk5/CRMP2 and GSK-3β/CRMP2 pathways participate in sevoflurane-induced dendritic development abnormalities and cognitive dysfunction in developing rats.

Dexmedetomidine Alleviates Hypoxia-Induced Synaptic Loss and Cognitive Impairment via Inhibition of Microglial NOX2 Activation in the Hippocampus of Neonatal Rats.

Background Perinatal hypoxia is a universal cause of death and neurological deficits in neonates worldwide. Activation of microglial NADPH oxidase 2 (NOX2) leads to oxidative stress and neuroinflammation, which may contribute to hypoxic damage in the developing brain. Dexmedetomidine has been reported to exert potent neuroprotection in several neurological diseases, but the mechanism remains unclear. We investigated whether dexmedetomidine acts through microglial NOX2 to reduce neonatal hypoxic brain damage. Methods The potential role of microglial NOX2 in dexmedetomidine-mediated alleviation of hypoxic damage was evaluated in cultured BV2 microglia and neonatal rats subjected to hypoxia. In vivo, neonatal rats received dexmedetomidine (25 μg/kg, i.p.) 30 min before or immediately after hypoxia (5% O2, 2 h). Apocynin-mediated NOX inhibition and lentivirus-mediated NOX2 overexpression were applied to further assess the involvement of microglial NOX2 activation. Results Pre- or posttreatment with dexmedetomidine alleviated hypoxia-induced cognitive impairment, restored damaged synapses, and increased postsynaptic density-95 and synaptophysin protein expression following neonatal hypoxia. Importantly, dexmedetomidine treatment suppressed hypoxia-induced microglial NOX2 activation and subsequent oxidative stress and the neuroinflammatory response, as reflected by reduced 4-hydroxynonenal and ROS accumulation, and decreased nuclear NF-κB p65 and proinflammatory cytokine levels in cultured BV2 microglia and the developing hippocampus. In addition, treating primary hippocampal neurons with conditioned medium (CM) from hypoxia-activated BV2 microglia resulted in neuronal damage, which was alleviated by CM from dexmedetomidine-treated microglia. Moreover, the neuroprotective effect of dexmedetomidine was reversed in NOX2-overexpressing BV2 microglia and diminished in apocynin-pretreated neonatal rats. Conclusion Dexmedetomidine targets microglial NOX2 to reduce oxidative stress and neuroinflammation and subsequently protects against hippocampal synaptic loss following neonatal hypoxia.

Effects of neonatal dexamethasone and CpdA on the expression of genes for apoptosis regulator proteins in the neonatal hippocampus

Glucocorticoids (GC) are crucial regulators of homeostasis and function. Despite its negative side effects, glucocorticoid therapy in neonates is widely used antenatally for accelerating fetal lung maturation in cases of preterm birth. GC action is mediated via glucocorticoid receptors — ligand-activated transcription factors. Cell death and viability in the neonatal brain are regulated by many factors, but the glucocorticoid receptor signalling is high above them. The present work studies the changes in the expression of genes for apoptosis regulators with Bcl-2 homology (BH) domains (Bcl-xL, Bax, Bim, Bok, Bid) in the neonatal rat hippocampus after dexamethasone (DEX) and CpdA administration. CpdA is a dissociative ligand — glucocorticoid receptor modulator — that shifts glucocorticoid receptor (GR) activity toward transrepression. Ligands administration to P2 pups caused different patterns of timeline changes in the expression of the studied genes. We observed the first increase in the mRNA level of the genes which have glucocorticoid response element (GRE) (Bcl-xL, Bim) in their promoter 30 min after DEX administration. Activated GR action on cells in the neonatal hippocampus is complex and long-lasting; it could also contain receptor homo- and hetero-dimerisation. Using rat pheochromocytoma PC12 cells as a test system, we assessed GR-GR and GR-MR (mineralocorticoid receptor) dimerisation with proximity ligation assay (PLA) assay separately in the nucleus and cytoplasm after DEX and CpdA administration. An increase in GR-GR dimers in the cell nucleus was observed only after DEX administration. In the cell cytoplasm, we observed a gradual (DEX more than CpdA) increase in the number of both GR-GR and GR-MR dimers.

Developmental Changes in Early Sharp Waves in the Hippocampus of Neonatal Rats

Synchronized activity the hallmark of neural networks. Early sharp waves (eSPW) form one type of synchronized activity – these are synchronized network discharges of neuronal ensembles seen in the developing hippocampus. Despite the fact that eSPW may play a central role in coordinating neural activity and forming hippocampal functions, little is known of changes in eSPW during early postnatal development. Our experiments on neonatal rat pups using multichannel extracellular electrodes showed that during the first two weeks of postnatal life, along with reductions in the duration of eSPW, there were increases in their frequency and amplitude. We also found an increase in extracellular recorded activity of neurons in the pyramidal layer of the hippocampus. These data lead to the suggestion that the dynamics of changes in eSPW and overall network hippocampal activity are associated with the development of the hippocampal neural network and bottom-up neuromodulator projections.

Early Sharp Wave Synchronization along the Septo-Temporal Axis of the Neonatal Rat Hippocampus

In the neonatal rat hippocampus, the first and predominant pattern of synchronized neuronal network activity is early sharp waves (eSPWs) occurring at a frequency of ~2–4 events per minute. However, how eSPWs are organized longitudinally along the septo-temporal hippocampal axis remains unknown. Using silicone probe recordings from the septal and intermediate segments of the CA1 hippocampus in neonatal rats in vivo we found that eSPWs are highly synchronized longitudinally. The amplitudes of eSPWs in the septal and intermediate segments of the hippocampus were also highly correlated. eSPWs also supported longitudinal synchronization of CA1 multiple unit activity. Spatial-temporal analysis revealed a septal-temporal gradient with more frequent initiation of eSPWs in the septal regions. The speed of eSPW longitudinal propagation attained ~ 250 mm/s. We suggest that longitudinal correlated activity supported by synchronized eSPWs emerges early during postnatal development and may participate in the formation of intrahippocampal connections in the developing hippocampus.

Experimentally Induced Sepsis Causes Extensive Hypomyelination in the Prefrontal Cortex and Hippocampus in Neonatal Rats.

Neonatal sepsis is associated with cognitive deficit in the later life. Axonal myelination plays a pivotal role in neurotransmission and formation of learning and memory. This study aimed to explore if systemic lipopolysaccharide (LPS) injection would induce hypomyelination in the prefrontal cortex and hippocampus in developing septic neonatal rats. Sprague-Dawley rats (1-day old) were injected with LPS (1mg/kg) intraperitoneally. By electron microscopy, axonal hypomyelination was evident in the subcortical white matter and hippocampus. The expression of myelin proteins including CNPase, MBP, PLP and MAG was downregulated in both areas of the brain at 7, 14 and 28 days after LPS injection. The frequency of MBP and PLP-positive oligodendrocyte was significantly reduced using in situ hybridization in the cerebral cortex and hippocampus at the corresponding time points after LPS injection, whereas the expression of NG2 and PDGFRα was noticeably increased. In tandem with this was reduction of Olig1 and Olig2 expressions which are involved in differentiation/maturation of OPCs. Expression of NFL, NFM, and NFH was significantly downregulated, indicating that axon development was disrupted after LPS injection. Morris Water Maze behavioral test, Open field test, Rotarod test, and Pole test were used to evaluate neurological behaviors of 28 days rats. The rats in the LPS group showed the impairment of motor coordination, balance, memory, and learning ability and represented bradykinesia and anxiety-like behavior. The present results suggest that following systemic LPS injection, differentiation/maturation of OPCs was affected which may be attributed to the inhibition of transcription factors Olig1 and Olig2 expression resulting in impairment to axonal development. It is suggested that this would ultimately lead to axonal hypomyelination in the prefrontal cortex and hippocampus, which may be associated with neurological deficits in later life.

Methylene blue post-treatment improves hypoxia-ischemic recovery in a neonatal rat model

Recent work suggested that methylene blue (MB) has beneficial effects in a variety of neurological disorders, while its role in neonatal hypoxic-ischemic (HI) encephalopathy is still unclear. The current study was designed to investigate the effects of MB on HI-induced brain damage and its underlying mechanisms. The results showed that MB treatment can strongly attenuate HI-induced brain loss and neuronal damage in the cortex and hippocampus of neonatal rats. Further mechanistic analysis suggested that MB treatment was able to significantly reduce blood-brain barrier disruption after HI insult. In addition, MB profoundly inhibited microglia and astrocyte activation and the pro-inflammatory cytokines production in neonatal cortex and hippocampus after HI. Further, MB treatment resulted in dramatic suppression of oxidative damage, as evidenced by robustly decreased DHE and protein carbonyls levels in HI brain. Moreover, MB strongly preserved mitochondrial function by repressing HI-induced mitochondrial fragmentation, and the following neuronal death in cortex and hippocampus. Finally, behavioral tests revealed that MB significantly improved the spatial reference memory and motor coordination of neonatal HI rats. Taken together, these findings demonstrate that the mechanisms behind neuroprotective actions of methylene blue are multifactorial, including suppression of oxidative stress and neuroinflammation, restoration of mitochondrial function, as well as attenuation of blood-brain barrier disruption. Our study might provide further directions for MB as a promising option in neonatal HI encephalopathy therapy.

MicroRNA-9 mediated the protective effect of ferulic acid on hypoxic-ischemic brain damage in neonatal rats.

Neonatal hypoxic-ischemic brain damage (HIBD) is prone to cognitive and memory impairments, and there is no effective clinical treatment until now. Ferulic acid (FA) is found within members of the genus Angelica, reportedly shows protective effects on neuronal damage. However, the protective effects of FA on HIBD remains unclear. In this study, using the Morris water maze task, we herein found that the impairment of spatial memory formation in adult rats exposed to HIBD was significantly reversed by FA treatment and the administration of LNA-miR-9. The expression of miRNA-9 was detected by RT-PCR analyses, and the results shown that miRNA-9 was significantly increased in the hippocampus of neonatal rats following HIBD and in the PC12 cells following hypoxic-ischemic injury, while FA and LNA-miR-9 both inhibited the expression of miRNA-9, suggesting that the therapeutic effect of FA was mainly attributed to the inhibition of miRNA-9 expression. Indeed, the silencing of miR-9 by LNA-miR-9 or FA similarly attenuated neuronal damage and cerebral atrophy in the rat hippocampus after HIBD, which was consistent with the restored expression levels of brain-derived neurotrophic factor (BDNF). Therefore, our findings indicate that FA treatment may protect against neuronal death through the inhibition of miRNA-9 induction in the rat hippocampus following hypoxic-ischemic damage.

Odor-mediated contextual learning induced memory consolidation and hippocampus development in neonate rat.

The hippocampus in neonatal rats is not fully developed after birth, and the effect of odor-aversion learning on memory consolidation in the immature hippocampus is not well understood. Therefore, the aim of the present study was to explore the effects of odor-aversion learning in neonatal rats on memory consolidation and neurodevelopment in the immature hippocampus. The effect of hippocampal-induced learning was measured at two different developmental stages using the Y-maze and c-Fos protein levels. Furthermore, hippocampal cell proliferation and growth-associated protein 43 (GAP-43) expression were evaluated at different developmental stages, namely, postnatal day 7 (PN7) and PN24, after odor-aversion learning. Both PN7 and PN24 rats avoided conditioned odor stimuli after odor-aversion learning. PN7 and PN24 rats in the odor-averse learning groups exhibited high c-Fos protein levels. PN7 rats exhibited high cell proliferation rates and GAP-43 protein levels after odor-aversion learning. These results showed that the immature hippocampus can participated in odor-aversion learning, which may induce cell proliferation and axonal development.

Effects of neural stem cells in combination with nerve neurotrophic factor 3 gene modified olfactory ensheathing cell co-transplants on functional recovery after traumatic brain injury in the adult rats

Objective To observe the effects of neural stem cells (NSCs) combined with neurotrophin-3 gene modified olfactory ensheathing cells (OECs-NT-3) co-transplants on functional recovery after traumatic brain injury in the adult rats. Methods NSCs and OECs were cultured from hippocampus and olfactory bulb of neonatal rats and then identified. OECs-NT-3 was constructed by lentiviral vector containing NT-3 gene. Male SD rats weighing (200±20) g were randomly divided into sham group, model group, OECs-NT-3 transplantation group, NSCs transplantation group and OECs-NT-3 combined NSCs transplantation group. The rat brain injury model was established. The corresponding cells were transplanted. At day 1, 7, 14 and 28 after transplantation, the modified neurological severity score (mNSS) was used to evaluate neurological function, and brain tissues were taken at each time point for paraffin sections. Hematoxylin-eosin staining was performed. Cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) method. Immunohistochemical staining of BrdU and p75 was done. Results At day 28 of transplantation, the mNSS scores in rats in the combined group (4.00±1.05) were significantly different from those in the model group (8.50±1.43), the OECs-NT-3 group (5.50±1.35) and the NSCs group (6.00±1.49) (t=-7.997, -2.764, -3.464, P 0.05). Hematoxylin and eosin (HE) staining revealed that the cell density in the sham group, model group, OECs-NT-3 group, NSCs group and the combined group was (220±22), (299±47), (347±35), (390±72) and (523±55) cells, respectively. As compared with the model group, OECs-NT-3 group and NSCs group, the cell density in the combined group was significantly increased (t=7.246, 5.996, 3.373, P<0.05), and the tissue structure was relatively regular. The p75 and BrdU immunohistochemistry showed that the two kinds of transplanted cells could survive continuously and were widely distributed around the lesion. At day 7 after transplantation, the apoptotic index in the sham group, model group, OECs-NT-3 group, NSCs group and combined group was (1.67±0.17)%, (47.49±7.92)%, (31.22±3.97)%, (34.48±4.69)% and (18.17±3.13)% respectively. The apoptosis rate in the combined transplantation group was significantly lower than that in the model group, OECs-NT-3 group and NSCs group (t=5.963, 4.470, 5.012, P<0.05). Conclusion Transplantation of NSCs combined with OECs-NT-3 can alleviate neuronal apoptosis after brain injury in rats, repair tissue defects, and promote the recovery of nerve function. Key words: Neural stem cell; Neurotrophin-3 gene; Gene modify; Olfactory ensheathing cells; Combined transplantation; Traumatic brain injury

Effect of sevoflurane inhalation on electrophysiology and ultrastructure of hippocampus in neonatal rats

ObjectiveThe hippocampal neuron electrophysiology and structure might be the basis of postoperative cognitive function‐related diseases after anesthesia. Here, we examined whether sevoflurane inhalation affected the electrophysiological and ultrastructural morphology of the hippocampal neurons.MethodsThe rats were randomly divided into the air/oxygen and Sevoflurane group (SEV group). The air/oxygen group inhaled carrier gas (1 L/min O2 + 1 L/min air) for 2 hours; The SEV group inhaled 3.4% sevoflurane &amp; carrier gas for 2 hours. After that, the hippocampal electrophysiology signals were observed 1 week after anesthesia. Then, Transmission Electron Microscope was used to observe the neuron cell morphology, structures, and related synaptic morphology in hippocampi.ResultsCompared with the air/oxygen group, the numbers of spontaneous discharge and the values of maximum current significantly decreased in the SEV group, and the neuron cytoplasm edema and organelles also were damaged.ConclusionThe results showed that sevoflurane could weaken the electrophysiological signals of hippocampal neurons and damage the ultrastructure of neurons.

Sirtuin 2 Inhibition Attenuates Sevoflurane-Induced Learning and Memory Deficits in Developing Rats via Modulating Microglial Activation.

Sevoflurane is a widely used inhalational anesthetic in pediatric medicine that has been reported to have deleterious effects on the developing brain. Strategies to mitigate these detrimental effects are lacking. Sirtuin 2 (SIRT2) is a member of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases involved in a wide range of pathophysiological processes. SIRT2 inhibition has emerged as a promising treatment for an array of neurological disorders. However, the direct effects of SIRT2 on anesthesia-induced damage to the immature brain are unclear. Neonatal rats were exposed to 3% sevoflurane or 30% oxygen for 2h daily with or without SIRT2 inhibitor AK7 pretreatment from postnatal day 7 (P7) to P9. One cohort of rats were euthanized 6, 12, and/or 24h after the last gas exposure, and brain tissues were harvested for biochemical analysis and/or immunohistochemical examination. Cognitive functions were evaluated using the open field and Morris water maze tests on P25 and P28-32, respectively. SIRT2 was significantly up-regulated in neonatal rat hippocampus at 6 and 12h post-anesthesia. Pretreatment with SIRT2 inhibitor AK7 reversed sevoflurane-induced hippocampus-dependent cognitive impairments. Furthermore, AK7 administration mitigated sevoflurane-induced neuroinflammation and microglial activation. Concomitantly, AK7 inhibited pro-inflammatory/M1-related markers and increased anti-inflammatory/M2-related markers in microglia. AK7 might prevent sevoflurane-induced neuroinflammation by switching microglia from the M1 to M2 phenotype. Downregulation of SIRT2 may be a novel therapeutic target for alleviating anesthesia-induced developmental neurotoxicity.

The expression of glucose transporters and mitochondrial division and fusion proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity

Purpose: Evidence has shown that propofol may cause widespread apoptotic neurodegeneration. Hypoxic preconditioning has been demonstrated to provide neuroprotection and brain recovery from both acute and chronic neurodegeneration in several cellular and animal models. However, the mechanism has not been well elucidated. Therefore, the present study was designed to investigate the expression of glucose transporters (GLUT1 and GLUT3) and mitochondrial division and fusion (Drp1 and Mfn2) proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.Methods: Propofol (100 mg/kg) was given to 7-day-old Sprague-Dawley rats; in some rats, hypoxic preconditioning was administered before intraperitoneal propofol injection by subjecting rats to five cycles of 10 min of hypoxia (8% O2) and 10 min of normoxia (21% O2). Then, the rats were allowed to breathe room air for 2 h. Neuronal mitochondrial morphology was observed by transmission electron microscopy. ATP content was detected using an ATP assay kit. The expression levels of GLUT1, GLUT3, pDrp1, Drp1 and Mfn2 were detected by Western blot, and the expression levels of GLUT1 and GLUT3 were further examined by immunohistochemistry.Results: Propofol damaged mitochondria, and decreased ATP content and GLUT3 and pDrp1 protein expression. However, our results suggested that hypoxic preconditioning could attenuate propofol neurotoxicity by reducing mitochondrial damage and increasing ATP content and pDrp1, GLUT1 and GLUT3 protein expression.Conclusion: Hypoxic preconditioning reduced propofol-induced damage in the hippocampus of neonatal rats by attenuating the increase in mitochondrial division and decrease in GLUT3 expression.

Expression of Neurotrophic Factor 3 in the Hippocampus of Neonatal Rats after Administration of Dexamethasone

Abstract—Elevated levels of glucocorticoids in the perinatal period of ontogeny may provoke further development of neuropathology, whose mechanisms can involve apoptosis of brain cells caused by impaired expression of neurotrophins, including the practically unstudied neurotrophic factor 3 (NT3). In order to clarify this issue, we explored the effect of glucocorticoid dexamethasone (DEX) on the NT3 and the key apoptotic protease caspase-3, mRNA levels as well as immature (proNT3) and mature (matNT3) forms of the investigated neurotrophin in the hippocampus of 3–4-day-old rats 6 or 24 hours after DEX administration. In 6 hours but not in 24 hours DEX increased the NT3 mRNA levels in the whole hippocampus, as well as the proNT3 and matNT3 proteins contents the CA1–3 fields and the dentate gyrus of the structure. In this case, the expression of apoptogenic proNT3 temporarily predominated over matNT3; however, this was not accompanied by an increase in the caspase-3 mRNA level.

Effects of propofol on the expression of B-cell lymphoma-2 assaciated protein and B-cell lymphoma-2 in the hippocampus of neonatal rats

Objective To investigate the effects of propofol on the expression of B-cell lymphoma-2 assaciated protein (Bax) and B-cell lymphoma-2(Bcl-2) in the hippocampus of neonatal rats. Methods A total of 112 male newborn Sprague Dawley (SD) rats aging 7 d were randomly divided into 8 groups (n=14): control group (Con1 group, intraperitoneally given normal saline for 5 d), and P50, P100 and P150 groups which received 50, 100 mg/kg and 150 mg/kg propofol once per day for 5 d, respectively; and control 2 group (Con2 group, intraperitoneallyl receiving normal saline for 5 d, and appendectomy was performed under propofol anesthesia for the last day) and SP50, SP100 and SP150 groups which received 50, 100 mg/kg and 150 mg/kg propofol for 5 d respectively, and appendectomy was performed under propofol anesthesia for the last day). Two months after the experiments, the level of Bcl-2 (inhibitor of apoptosis proteins) and Bax (the proapoptotic protein) were detected using immunohistochemical methods. Results Compared with the Con1 group, the levels of Bax in the P50, P100 and P150 groups increased, while the amounts of Bcl-2 gradually decreased in the P100 and P150 groups(P<0.05). Compared with the P50 group, the levels of Bax increased (P<0.05), while the amounts of Bcl-2 decreased in the P100 and P150 groups. Compared with the Con2 group, the level of Bax in the SP50, SP100 and SP150 groups gradually increased (P<0.05), while the amount of Bcl-2 in the SP50 and SP150 groups gradually decreased (P<0.05). Compared with the SP50 group, the level of Bax in the SP100 and SP150 groups increased (P<0.05), while the amounts of Bcl-2 was decreased in the SP150 group (P<0.05). Conclusions Repeated administration of propofol can inhibit the activation of Bcl-2 and enhance the expression of Bax. It can promote the apoptosis of hippocampal neurons of newborn rats in a dose-dependent manor. Key words: Propofol; Neonatal rat; Hippocampus; Apoptosis

Synaptic structure and alterations in the hippocampus in neonatal rats exposed to lipopolysaccharide

Synaptic structure integrity plays a key role in learning and memory. Previous studies have shown that there is cognitive dysfunction in septic neonates in later life. In this study, intraperitoneal injection of lipopolysaccharide (LPS) in the developing rats was used as a sepsis model to determine whether hippocampal synapses would be affected. Expression of synaptophysin (Syn), synaptosomal associated protein of 25 kD (SNAP-25), and N-methyl d-aspartate receptor (NMDAR) in the hippocampus in septic brain were significantly reduced. Consistent with this, the number of dendritic spines associated with the pyramidal neurons in the CA1 region of hippocampus at 28d after LPS administration was decreased. Additionally, the number of synapse and synaptic vesicles were reduced and appeared swollen. The number of neurons in the CA1 and CA3 of hippocampus at 14, and 28d after LPS injection remained unchanged. Coupled with the above was upregulated expression of interleukin-1β (IL-1β), IL-1 receptor 1 (IL-R1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) at 1-3d after LPS injection. IL-1β expression was specifically detected in activated microglia. The plasma corticosterone (CORT) concentration in the LPS treatment rats was increased; but the glucocorticoid receptor (GR) expression in the hippocampus was decreased. We conclude that LPS injection in neonatal rats can cause synaptic disruption in the hippocampus which may be attributed to inflammatory response due to excess production of proinflammatory cytokines e.g., IL-1β derived from activated microglia. The significance of increased plasma CORT concentration and decreased GR expression in the hippocampus is discussed.

Potential role of the cAMP/PKA/CREB signalling pathway in hypoxic preconditioning and effect on propofol‑induced neurotoxicity in the hippocampus of neonatal rats.

Hypoxic preconditioning (HPC) is neuroprotective against ischaemic brain injury; however, the roles of potential anti-apoptotic signals in this process have not been assessed. To elucidate the molecular mechanisms involved in HPC-induced neuroprotection, the effects of HPC on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signalling pathway and apoptosis in Sprague-Dawley pups (postnatal day 7) treated with propofol were investigated. Western blot and histological analyses demonstrated that HPC exerts multiple effects on the hippocampus, including the upregulation of cAMP and phosphorylation of CREB. These effects were partially blocked by intracerebroventricular injection of the protein kinase antagonist H89 (5 µmol/5 µl). Notably, the level of cleaved caspase-3 was significantly downregulated by treatment with the cAMP agonist Sp-cAMP (20 nmol/5 µl). The results indicate that propofol increased the level of cleaved caspase-3 and Bax by suppressing the activity of cAMP-dependent proteins and Bcl-2; thus, HPC prevents propofol from triggering apoptosis via the cAMP/PKA/CREB signalling pathway.

Both GSK-3β/CRMP2 and CDK5/CRMP2 Pathways Participate in the Protection of Dexmedetomidine Against Propofol-Induced Learning and Memory Impairment in Neonatal Rats.

Dexmedetomidine has been reported to ameliorate propofol-induced neurotoxicity in neonatal animals. However, the underlying mechanism is still undetermined. Glycogen synthase kinase-3β (GSK-3β), cycline-dependent kinase-5 (CDK5), and Rho-kinase (RhoA) pathways play critical roles in neuronal development. The present study is to investigate whether GSK-3β, CDK5, and RhoA pathways are involved in the neuroprotection of dexmedetomidine. Seven-day-old (P7) Sprague Dawley rats were anesthetized with propofol for 6 h. Dexmedetomidine at various concentrations were administered before propofol exposure. Neuroapoptosis, the neuronal proliferation, and the level of neurotransmitter in the hippocampus were evaluated. The effects of GSK-3β inhibitor SB415286, CDK5 inhibitor roscovitine, or RhoA inhibitor Y276321 on propofol-induced neurotoxicity were assessed. Propofol-induced apoptosis in the hippocampal neurons and astrocytes, inhibited neuronal proliferation in the dentate gyrus region, down-regulated the level of γ-aminobutyric acid and glutamate in the hippocampus, and impaired long-term cognitive function. These harmful effects were reduced by pretreatment with 50 μg·kg-1 dexmedetomidine. Moreover, propofol-activated GSK-3β and CDK5 pathways, but not RhoA pathway, by reducing the phosphorylation of GSK-3β (ser 9), increasing the expression of CDK5 activator P25 and increasing the phosphorylation of their target sites on collapsin response mediator protein 2 (CRMP2) shortly after exposure. These effects were reversed by pretreatment with 50 μg·kg-1 dexmedetomidine. Furthermore, SB415286 and roscovitine, not Y276321, attenuated the propofol-induced neuroapoptosis, brain cell proliferation inhibition, γ-aminobutyric acid and glutamate downregulation, and learning and memory dysfunction. Our results indicate that dexmedetomidine reduces propofol-induced neurotoxicity and neurocognitive impairment via inhibiting activation of GSK-3β/CRMP2 and CDK5/CRMP2 pathways in the hippocampus of neonatal rats.

Ca2+-PKCα-ERK1/2 signaling pathway is involved in the suppressive effect of propofol on proliferation of neural stem cells from the neonatal rat hippocampus

Neonatal exposure to propofol induces persistent behavioral abnormalities in adulthood. In addition to triggering the apoptosis of neurons in the developing brain, anesthetics may contribute to the development of cognitive deficits by interfering neurogenesis. Given the importance of neural stem cell (NSC) proliferation in neurogenesis, the effect of propofol on NSC proliferation and the mechanisms underlying this effect were investigated. Hippocampal NSC proliferation from neonatal rats was examined using 5-bromo-2′-deoxyuridine incorporation assays in vitro. The [Ca2+]i was analyzed using flow cytometry. The activations of protein kinase C (PKC)-α and extracellular signal-regulated kinases 1/2 (ERK1/2) were measured by western blot. Our results showed that propofol significantly inhibited NSC proliferation in vitro. [Ca2+]i and activations of PKCα and ERK1/2 in NSCs were markedly suppressed by propofol (5, 10, 20, 40 and 80 μM). Ca2+ channel blocker verapamil, PKCα inhibitor chelerythrine and ERK1/2 kinase inhibitor PD98059 exerted their maximal effects on NSC function at concentrations of 20, 10 and 20 μM, respectively. Propofol (20 μM) could not produce further additional suppression effects when used in combination with verapamil (20 μM), chelerythrine (10 μM) or PD98059 (20 μM). In addition, phorbol-12-myristate-13-acetate (PMA, a activator of PKC) markedly attenuated the suppressive effects of propofol on ERK1/2 phosphorylation and NSC proliferation. The inhibition effects on PKCα activation, ERK1/2 phosphorylation and NSC proliferation induced by propofol were significantly improved by BayK8644 (a calcium channel agonist). These results indicate that propofol can inhibits hippocampal NSC proliferation by suppressing the Ca2+-PKCα-ERK1/2 signaling pathway.

Sevoflurane induces temporary spatial working memory deficits and synaptic ultrastructure impairments in the hippocampus of neonatal rats.

Exposure to volatile anesthetics in neonatal rats could induce neurotoxicity, learning deficits and abnormal social behaviors. The aim of this study is to investigate the potential neurotoxicity induced by sevoflurane. Postnatal day 7 (P7) Sprague-Dawley (SD) rats were continuously exposed to 2% sevoflurane plus 40% oxygen/air for 2 h. We used Morris water maze (MWM) to examine subsequent neurobehavioral performance. Transmission electron microscopy (TEM) was used to observe the histopathological changes in the hippocampus. Neonatal exposure to 2% sevoflurane for 2 hours impaired short-term spatial working memory but not reference memory at P25. It induced synaptic ultrastructure impairments in the CA3 region of hippocampal, including fewer numbers of synapses, thinner thickness of postsynaptic dense, broader synaptic cleft width and smaller synaptic curvature. Our results also showed that all synaptic ultrastructure impairments and neurocognitive deficits had almost completely recovered at P53. We showed that a single sevoflurane exposure to neonatal rats led to temporary spatial working memory deficits. It might be associated with synaptic ultrastructure impairments in the CA3 region of the hippocampus, including fewer numbers of synapses, thinner thickness of PSD and broader synaptic cleft width. Fortunately, all the neurotoxicity and neurocognitive deficits were reversible.