Il1-β Involvement in Cognitive Impairment after Sepsis

Sepsis is defined as the host's reaction to infection and characterised by a systemic inflammatory response with important clinical implications. Central nervous system dysfunction secondary to sepsis is associated with local generation of pro- and anti-inflammatory cytokines, impaired cerebral microcirculation, an imbalance of neurotransmitters, apoptosis and cognitive impairment. It's known that the IL-1β is one of the first cytokines to be altered. Thus, the objective of this study was to evaluate the role of IL-1β in cognitive parameters in brain tissue through the use of an IL-1β (IL-1ra) receptor antagonist up to 10 days and to assess blood–brain barrier permeability, cytokine levels, oxidative parameters and energetic metabolism up to 24 h, after sepsis induction. To this aim, we used sham-operated Wistar rats or submitted to the cecal ligation and perforation (CLP) procedure. Immediately after, the animals received one dose of 10 μg of IL-1ra. After 24 h, the rats were killed and were evaluated for biochemical parameters in the pre-frontal cortex, hippocampus and striatum. After 10 days, the animals were submitted to the habituation to the open field and step-down inhibitory avoidance task. We observed that the use of IL-1ra reverted the increase of blood–brain barrier permeability in the pre-frontal cortex, hippocampus and striatum; the increase of IL-1β, IL1-6 and TNF-α levels in the pre-frontal cortex and striatum; the decrease of complex I activity in the pre-frontal, hippocampus and striatum; the increase of oxidative parameters in pre-frontal cortex, hippocampus and striatum; and cognitive impairment. In conclusion, the results observed in this study reinforce the role of acute brain inflammatory response, in particular, the IL1β response, in the cognitive impairment associated with sepsis.     

Kinetics of Apoptosis and Expression of Apoptosis-Related Proteins in Rat CA3 Hippocampus Cells After Experimental Diffuse Brain Injury

The present study examined kinetics of apoptosis and expression of apoptosis-related proteins Bcl-2, Bax, and caspase-3 in the CA3 hippocampus cells after diffuse brain injury (DBI) induced experimentally in rats. Percentage of apoptotic cells and expressions of above proteins were examined by flow cytometry and immunohistochemistry. Substantial neuronal apoptosis was documented in the CA3 hippocampus cells after DBI (22.26 ± 2.97 % at 72 h after DBI vs. 2.92 ± 0.88 % in sham-operated animals). Expression of Bc1-2 decreased, while expression of Bax and caspase-3 increased after DBI, with caspase-3 expression peaking after that of Bax (72 vs. 48 h, respectively). Further, the Bc1-2/Bax expression ratio decreased prior to increase of caspase-3 expression. In conclusion, cell apoptosis and altered expressions of Bcl-2, Bax, and caspase-3 are present in the CA3 region of hippocampus after experimental DBI. Changes in the Bc1-2/Bax expression ratio may facilitate activation of caspase-3 and aggravate neuronal apoptosis after brain injury.     

Mitochondrial respiratory chain and creatine kinase activities in rat brain after sepsis induced by cecal ligation and perforation

The mechanisms responsible to the development of brain dysfunction during sepsis are not well understood. The objective of this study is to evaluate mitochondrial respiratory chain and creatine kinase activities in the brain after cecal ligation and perforation (CLP) in rats. We performed a prospective, controlled experiment in male Wistar rats. Rats were subjected to CLP (sepsis group) with saline resuscitation (at 50mL/kg immediately and 12h after cecal ligation and perforation) or sham operation (control group). Several times (0, 6, 12, 24, 48 and 96h) after CLP six rats were killed by decapitation, and brain structures (cerebellum, hippocampus, striatum and cortex) were isolated. Mitochondrial respiratory chain and creatine kinase activity were then measured. It was observed that animals submitted to CLP presented decreased mitochondrial respiratory chain activity in complex I, but not in complex II, III and IV, 24, 48 and 96h in all analyzed structures. Activity of succinate dehydrogenase was decreased in 48 and 96h in all analyzed structures. Creatine kinase activity increased after CLP in cerebellum, hippocampus and cortex (after 0h) and striatum (after 6h). Sepsis associated brain injury may include dysfunction in the mitochondrial respiratory chain activity.     

The Decrease on Na+, K+-ATPase Activity in the Cortex, but not in Hippocampus, is Reverted by Antioxidants in an Animal Model of Sepsis

In the present study, we investigated whether sepsis induced by cecal ligation and puncture (CLP) modifies Na⁺, K⁺-ATPase activity, mRNA expression, and cerebral edema in hippocampus and cerebral cortex of rats and if antioxidant (ATX) treatment prevented the alterations induced by sepsis. Rats were subjected to CLP and were divided into three groups: sham; CLP??rats were subjected to CLP without any further treatment; and ATX?CCLP plus administration of N-acetylcysteine plus deferoxamine. Several times (6, 12, and 24) after CLP or sham operation, the rats were killed and hippocampus and cerebral cortex were isolated. Na⁺, K⁺-ATPase activity was inhibited in the hippocampus 24?h after sepsis, and ATX treatment was not able to prevent this inhibition. The Na⁺, K⁺-ATPase activity also was inhibited in cerebral cortex 6, 12, and 24?h after sepsis. No differences on Na⁺, K⁺-ATPase catalytic subunit mRNA levels were found in the hippocampus and cerebral cortex after sepsis. ATX treatment prevents Na⁺, K⁺-ATPase inhibition only in the cerebral cortex. Na⁺, K⁺-ATPase inhibition was not associated to increase brain water content. In conclusion, the present study demonstrated that sepsis induced by CLP inhibits Na⁺, K⁺-ATPase activity in a mechanism dependent on oxidative stress, but this is not associated to increase brain water content.     

Lithium Treatment Prevents Apoptosis in Neonatal Rat Hippocampus Resulting from Sevoflurane Exposure

We aimed to observe the therapeutic effects of lithium on inhalational anesthetic sevoflurane-induced apoptosis in immature brain hippocampus. From postnatal day 5 (P5) to P28, male Sprague–Dawley pups were intraperitoneally injected with lithium chloride or 0.9 % sodium chloride. On P7 after the injection, pups were exposed to 2.3 % sevoflurane or air for 6 h. Brain tissues were harvested 12 h and 3 weeks after exposure. Cleaved caspase-3, nNOS protein, GSK-3β,p-GSK-3β were assessed by Western blot, and histopathological changes were assessed using Nissl stain and TUNEL stain. From P28, we used the eight-arm radial maze test and step-through test to evaluate the influence of sevoflurane exposure on the learning and memory of juvenile rats. The results showed that neonatal sevoflurane exposure induced caspase-3 activation and histopathological changes in hippocampus can be attenuated by lithium chloride. Sevoflurane increased GSK-3β activity while pretreatment of lithium decreased GSK-3β activity. Moreover, sevoflurane showed possibly slight but temporal influence on the spatial learning and the memory of juvenile rats, and chronic use of lithium chloride might have the therapeutic effect. Our current study suggests that lithium attenuates sevoflurane induced neonatal hippocampual damage by GSK-3β pathway and might improve learning and memory deficits in rats after neonatal exposure.     

Axonal Accumulation of Lysosomal-Associated Membrane Protein 1 (LAMP1) Accompanying Alterations of Autophagy Dynamics in the Rat Hippocampus Upon Seizure-Induced Injury

We found a dramatic upregulation in the expression of LC3 in the hippocampus of rats upon status epilepticus (SE). However, the enhancement in LC3 expression might be caused by a reduction in lysosomal activity or by alterations in autophagosome-lysosome fusion leading to a cytosolic vesicular retention. In order to dissect this aspect, we monitored the spatial and temporal expression of LC3 and LAMP1 in the hippocampus of rats with SE. The Western blot analysis showed that the expression of LAMP1 was slightly increased in hippocampal cells at 6, 24, and 48 h post-SE. However, immunofluorescence analysis showed dramatic spatial changes in LAMP1 distribution within the hippocampus. LAMP1 in controls was localised only in cytosol as dot like staining, however at 24 h post-SE LAMP1 was not only highly expressed, but accumulated in mossy fibers of dentate gyrus. In parallel, we found few scattered LC3-positive-dots in neurites of dentate gyrus which co-localise with LAMP1-positive structures. We conclude that SE not only increased autophagosomal abundance, but also lysosomal activities and a massive accumulation of LAMP1 in axons of dentate gyrus. This could support the hypothesis that the marked increased autophagosomal abundance in cytosol reflects an increase in the autophagic activity more than an inhibition of autophagosomal clearance. Although LAMP1 may have contributed to cell damage in the selective vulnerable hippocampal CA1-subfield, it is also possible that lysosomal/autophagic mechanisms in mossy fibers were compensatory and reflected an attempt to survive the epileptic insult by breaking down non-essential components.     

Treadmill exercise suppresses ischemia-induced increment in apoptosis and cell proliferation in hippocampal dentate gyrus of gerbils

Cerebral ischemia resulting from transient or permanent occlusion of cerebral arteries leads to neuronal cell death and eventually causes neurological impairments. In the present study, the effects of treadmill exercise on apoptosis and cell proliferation in the hippocampal dentate gyrus following transient global ischemia in gerbils were investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry for caspase-3 and 5-bromo-2'-deoxyuridine (BrdU). It was shown that apoptotic cell death and cell proliferation in the hippocampal dentate gyrus were significantly increased following transient global ischemia in gerbils and that treadmill exercise suppressed the ischemia-induced increase in apoptosis and cell proliferation in the dentate gyrus. The present results suggest that treadmill exercise may protect cells from apoptotic death and aid in recovery from the central nervous system sequelae following stroke.     

Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated

Recent studies from our laboratory demonstrated that mucosal lymphoid tissue such as Peyer's patch cells and lamina propria (LP) B lymphocytes from mice shows evidence of increased apoptosis after sepsis that is associated with localized inflammation/activation. The mechanism for this is poorly understood. Endotoxin as well as Fas/Fas ligand (FasL) have been shown to augment lymphocyte apoptosis; however, their contribution to the increase of apoptosis in LP B-cells during sepsis is not known. To study this, sepsis was induced by cecal ligation and puncture (CLP) in endotoxin-tolerant C3H/HeJ or FasL-deficient C3H/HeJ-FasL(gld) (FasL(-)) mice and LP lymphocytes were isolated 24 h later. Phenotypic, apoptotic, and functional indexes were assessed. The number of LP B cells decreased markedly in C3H/HeJ mice but not in FasL-deficient animals at 24 h after CLP. This was associated with comparable alteration in apoptosis and Fas antigen expression in the B cells of these mice. Septic LP lymphocytes also showed increased IgA production, which was absent in the FasL-deficient CLP mice. Furthermore, Fas ligand deficiency appeared to improve survival of septic challenge. These data suggest that the increase in B cell apoptosis in septic animals is partially due to a Fas/FasL-mediated process but not endotoxin.     

Hippocampal enlargement in <Emphasis Type="Italic">Bassoon</Emphasis>-mutant mice is associated with enhanced neurogenesis,reduced apoptosis,and abnormal BDNF levels

Mice mutant for the presynaptic protein Bassoon develop epileptic seizures and an altered pattern of neuronal activity that is accompanied by abnormal enlargement of several brain structures, with the strongest size increase in hippocampus and cortex. Using manganese-enhanced magnetic resonance imaging, an abnormal brain enlargement was found, which is first detected in the hippocampus 1 month after birth and amounts to an almost 40% size increase of this structure after 3 months. Stereological quantification of cell numbers revealed that enlargement of the dentate gyrus and the hippocampus proper is associated with larger numbers of principal neurons and of astrocytes. In search for the underlying mechanisms, an approximately 3-fold higher proportion of proliferation and survival of new-born cells in the dentate gyrus was found to go hand in hand with similarly larger numbers of doublecortin-positive cells and reduced numbers of apoptotic cells in the dentate gyrus and the hippocampus proper. Enlargement of the hippocampus and of other forebrain structures was accompanied by increased levels of brain-derived neurotrophic factor (BDNF). These data show that hippocampal overgrowth in Bassoon-mutant mice arises from a dysregulation of neurogenesis and apoptosis that might be associated with unbalanced BDNF levels.     

Irradiation-induced progenitor cell death in the developing brain is resistant to erythropoietin treatment and caspase inhibition

One hemisphere of postnatal day 8 (P8) rats or P10 mice was irradiated with a single dose of 4-12 Gy, and animals were killed from 2 h to 8 weeks after irradiation (IR). In the subventricular zone (SVZ) and the granular cell layer (GCL) of the dentate gyrus, harboring neural and other progenitor cells, nitrosylation and p53 peaked 2-12 h after IR, followed by markers for active caspase-3, apoptosis-inducing factor and TUNEL (6-24 h). Ki67-positive (proliferating) cells had disappeared by 12 h and partly reappeared by 7 days post-IR. The SVZ and GCL areas decreased approximately 50% 7 days after IR. The development of white matter was hampered, resulting in 50-70% less myelin basic protein staining. Pretreatment with erythropoietin did not confer protection against IR. Caspase inhibition by overexpression of XIAP prevented caspase-9 and caspase-3 activation but not cell death, presumably because of increased caspase-independent cell death.     

Single-Prolonged Stress Induces Endoplasmic Reticulum - Dependent Apoptosis in the Hippocampus in a Rat Model of Post-Traumatic Stress Disorder

Background

Our previous research indicated that apoptosis induced atrophy in the hippocampus of post-traumatic stress disorder (PTSD) rats. Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of several disorder diseases. The aim of this study was to investigate whether endoplasmic reticulum-related pathway is involved in single-prolonged stress (SPS) induces apoptosis in the hippocampus of PTSD rats by examining the expression levels of three important indicators in the ER-related apoptotic pathway: Glucose-regulated protein (GRP) 78, caspase-12 and Ca²⁺/CaM/CaMkinaseIIα (CaMkIIα).

Methods

Wistar rats were sacrificed at 1, 4 and 7 days after SPS. SPS is a reliable animal model of PTSD. The apoptotic cells in the hippocampus were assessed by TUNEL method and transmission electron microscopy (TEM). Free intracellular Ca²⁺ concentration was measured. GRP78 expression was examined by immunohistochemistry, western blotting and RT-PCR. mRNA of caspase-12 and CaM/CaMkIIα were determined by RT-PCR.

Results

Our results showed that apoptotic cells were increased in the SPS rats. TEM analysis revealed characteristic morphological changes of apoptosis in these cells. We observed that GRP78 was significantly up-regulated during early PTSD, and then recovered at 7 days after SPS. By RT-PCR, we observed that the change in caspase-12 expression level was similar to that in GRP78. Moreover, the free intracellular Ca²⁺ concentration was significantly higher at 1 day after SPS and decreased in 7 days. CaM expression increased significantly, while CaMKIIα expression decreased significantly in the hippocampus at 1 day after SPS.

Conclusion

SPS induced change in the expression levels of GRP78, caspase-12 and Ca²⁺/CaM/CaMkIIα in the hippocampus of PTSD rats indicated that the endoplasmic reticulum pathway may be involved in PTSD-induced apoptosis.     

The apoptosis of peripheral blood lymphocytes promoted by hyperbaric oxygen treatment contributes to attenuate the severity of early stage acute pancreatitis in rats

The aim of this study was to investigate the immunoregulatory effects of hyperbaric oxygen (HBO) via promoting the apoptosis of peripheral blood lymphocytes (PBLs) to attenuate the severity of early stage acute pancreatitis (AP) in rats. Additionally, the persistence of the HBO treatment effects was evaluated. One hundred and twenty male Wistar rats were randomized into four groups: sham, AP, AP + normobaric oxygen (NBO), and AP + HBO. Each group consisted of 30 rats. Four hours after the induction of AP, the 30 rats in the AP + NBO group were given normobaric oxygen treatment with 100 % oxygen at 1 atm for 90 min. The 30 rats in the AP + HBO group received 100 % oxygen at 2.5 atm for 90 min, with a compression/decompression time of 15 min. The 30 rats in the AP group remained untreated. At 6, 12, and 24 h after the induction of AP, surviving rats from each group were sacrificed, and the blood and tissue samples were collected for the following measurements: the partial pressure of oxygen (PaO₂) and oxygen saturation (SaO₂) of the arterial blood, the levels of serum amylase, lipase, interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-10 (IL-10), hepatocyte growth factor (HGF), and reactive oxygen species (ROS), and the mitochondrial membrane potential (?Ψm) of the PBLs. The expression levels of procaspase-3, caspase-3, procaspase-9, and caspase-9 were also evaluated in the PBLs. Additionally, the apoptosis of PBLs was assessed, and the pancreatic tissues were subjected to a histopathological analysis by pathological grading and scoring. The histopathology of the lung, liver, kidney, duodenum, and heart was also analyzed at 12 h after the induction of AP. Significant differences were found at 6 and 12 h after AP induction. The HBO treatment significantly elevated the PaO₂ and SaO₂ levels, and the ROS levels in the PBLs. Additionally, HBO downregulated the levels of amylase and lipase. The HBO treatment also reduced the ?Ψm levels, upregulated the expression of caspase-3 and caspase-9, and increased the apoptosis rate of the PBLs. Moreover, the HBO treatment decreased the serum concentrations of IL-2, IFN-γ and HGF, and reduced the pathological scores of the pancreatic tissue. The histopathological changes of the lung, liver, kidney, duodenum, and heart were also improved. A significant elevation of IL-10 occurred only at the 12-h time point. However, no obvious differences were found at the 24-h time point. This study demonstrated that the HBO treatment can promote the apoptosis of PBLs via a mitochondrial-dependent pathway and inhibit the inflammatory response. These immunoregulatory effects may play an important therapeutic role in attenuating the severity of early stage AP. The repeated administration of HBO or the use of HBO in combination with other approaches may further improve outcomes.     

Electroconvulsive shock causes neuronal death in the mouse hippocampus: correlation of neurodegeneration and convulsive activity

Relations between seizures induced by repeated electroshock (ES) and structural changes in the hippocampus were investigated in Balb/C mice. Brain sections of the animals 2 or 7 days after the last ES were stained for Nissl or TUNEL (apoptotic nuclei). Direct measurement of caspase-3 activity (a key enzyme of apoptosis) in brain regions was performed immediately after the last ES. Statistically significant, albeit moderate cell loss was demonstrated in the CA1 field and dentate gyrus, but not in the CA3 field of the hippocampus. The number of neurons in these fields inversely correlated with seizure severity. No apoptotic nuclei could be revealed either in hippocampus or in other brain regions. Caspase-3 in the hippocampus decreased after ES. The data obtained support the results from other groups showing prominent functional changes in neurons induced by repeated ES and extend this concept directly testifying for a moderate (within 10%), albeit statistically significant neuronal death in selected hippocampal fields. The inverse correlation of cell number with severity of seizures suggest that these are seizures inducing neuronal death.     

Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway

Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.     

Differential regulation of apoptosis in the corpus luteum of pregnancy and newly formed corpus luteum after parturition in rats

Apoptosis contributes to luteal regression in many species. In the postpartum rat, there are two different types of corpora lutea (CL) in the ovary: CL of pregnancy (CLP) and newly formed CL (NCL). To investigate the regulation of apoptosis in the two different types of CL during luteal regression, apoptosis and caspase-3 activity were examined in the CL obtained on Days 7, 15, and 21 of pregnancy and Days 0, 1, 3, 5, 7, and 9 postpartum. Furthermore, the effect of lactation on apoptosis in the CL was examined in two groups of postpartum rats: lactating rats that nurse more than 10 pups, and nonlactating rats that nurse no pups. Apoptotic cells were detected after Day 21 of pregnancy. In the CLP, remarkable increases in the number of apoptotic cells on Days 5 and 9 postpartum were observed in nonlactating rats (P < 0.01), but not in lactating rats. Changes in caspase-3 activity in the CLP were not consistent with those in number of apoptotic cells. In the NCL, an increase in apoptosis was found only on Day 5 postpartum in nonlactating rats (P < 0.01), but not in lactating rats. Changes in caspase-3 activity in the NCL were consistent with those in number of apoptotic cells. In conclusion, apoptosis is, at least in part, involved in luteal regression after parturition, and lactation appears to inhibit apoptosis. This study also suggests the presence of a caspase-3-independent mechanism for apoptosis in CLP regression in the rat.     

Effects of Copper on Hemocyte Apoptosis,ROS Production,and Gene Expression in White Shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

Copper, a common chemical contaminant in aquatic environment, is known to be toxic to aquatic life at high concentrations. In the present study, we evaluated the apoptotic cell ratio and ROS production in hemocytes of the white shrimp Litopenaeus vannamei exposed to 1 or 5 mg L^?1 Cu for 0, 3, 6, 12, 24, and 48 h. The expression changes of antioxidant biomarker genes, i.e., copper-zinc superoxide dismutase (Cu-Zn SOD) and catalase (CAT), apoptosis-related genes, i.e., caspase-3 and inhibitor of apoptosis protein (IAP), and a specific biomarker gene of heavy metal pollution, i.e., metallothionein (MT), were also determined in hemocytes. Significant increases in ROS production were observed in both treatment groups at each time points. The apoptotic cell ratios were significantly increased at 6–48 h among shrimp exposed to 1 mg L^?1 Cu and at each time points in 5 mg L^?1 Cu group. These results indicated that Cu would induce oxidative stress and apoptosis in the hemocyte of L. vannamei. Quantitative real-time PCR analysis revealed that the relative expression levels of Cu-Zn SOD, CAT, caspase-3, IAP, and MT were upregulated in a dose-dependent and time-dependent manner, suggesting the involvement of these genes in stress response against Cu exposure.     

Upregulation of myocardial syntaxin1A is associated with an early stage of polymicrobial sepsis

This study was designed to test whether increased sympathetic stimulation during polymicrobial sepsis modulates the profile of the syntaxin1A and norepinephrine transporter (NET) in the heart. Sepsis of mild and severe intensity was induced in male Sprague-Dawley rats (275–350 g) using the cecal inoculum (CI) and cecal ligation and puncture (CLP) methods, respectively. The heart samples were isolated from sham, 1, 3, and 7 day post-sepsis in the CI model and at 2 and 20 h post-sepsis in the CLP model. In the CI model, the plasma concentration of norepinephrine (NE) significantly increased at 1, 3, and 7 days post-CI compared to the sham group. The myocardial syntaxin1A mRNA and protein expression also significantly increased at 1 day post-CI compared to the sham group. However, the sepsis-induced increase in syntaxin1A returned to the baseline values at 3 and 7 days post-CI. The expressions of myocardial NET mRNA and protein were not altered at 1 day post-CI but significantly decreased at 3 days post-CI compared to the sham and 1 day post-CI groups. The immunohistochemical analyses revealed an increased localization of NET and syntaxin1A in the heart tissue sections of the 1 day post-CI group. In the CLP model of severe sepsis, the myocardial syntaxin1A mRNA protein expressions significantly increased at 2 h post-CLP, but remained unchanged at 20 h post-CLP compared to the sham group. In contrast, the myocardial expressions of NET mRNA and protein significantly decreased at both 2 and 20 h post-CLP compared to the sham group. It appears that during severe sepsis (CLP model), both the upregulation of syntaxin1A and the downregulation of NET contribute to increased concentrations of NE during the early and late stages of sepsis.     

Rapamycin Protects Sepsis-Induced Cognitive Impairment in Mouse Hippocampus by Enhancing Autophagy

The purpose of this study is to test the hypothesis that the mammalian target of rapamycin (mTOR) signaling pathway might mediate neuroprotection in a mouse model of septic encephalopathy and also to identify the role of autophagy. Mice were subjected to cecal ligation and puncture (CLP) or a sham operation, and all 50 mice were randomly assigned to five groups: sham, CLP+ saline, CLP+ rapamycin (1, 5, 10 mg/kg) groups. Two weeks after the operation, Morris water maze was conducted for behavioral test; Nissl staining was used for observing glia infiltration; immunohistochemical staining and biochemical measures in hippocampi were performed to detect mTOR targets and autophagy indicators. Immunochemistry revealed significant loss of neurons and increased glia infiltration in hippocampus after CLP operation. Inhibition of mTOR by rapamycin rescued cognitive deficits caused by sepsis (p < 0.05). Rapamycin did not affect total mTOR targets, while phosphorylated mTOR targets (p-mTOR-Ser2448, p-p70S6k-Thr389, p-AKT-S473) decreased (p < 0.05) and autophagy indicators (LC3-II, Atg5, Atg7) were increased, and P62 was decreased in rapamycin-treated CLP mice compared with the untreated (p < 0.05) in hippocampus. Rapamycin improves learning after sepsis through enhancing autophagy and may be a potentially effective therapeutic agent for the treatment of sepsis-induced cognitive impairment.     

杏仁核注射红藻氨酸诱导大鼠边缘叶癫痫发作时海马中Smac和XIAP蛋白的表达

应用红藻氨酸(kainic acid,KA)诱导的大鼠边缘叶癫痫发作模型,检测第二个线粒体源的半胱天冬蛋白酶激活物,直接与凋亡抑制蛋白结合的低等电点蛋白(second mitochondrial activator of caspases／direct inhibitor of apoptosis protein-binding protein of low isoelectric point[PI],Smac／DIABLO)和X染色体连锁的凋亡抑制蛋白(X-chromosome-linked inhibitor of apoptosis protein,XIAP)在癫痫大鼠海马神经元表达。单侧杏仁核内注射KA诱导癫痫发作,1h后用安定终止发作,然后分别用TUNEL染色和cresyl violet染色观察海马神经元存活和凋亡的变化,用免疫荧光和Western blot检测海马Smac／DIABLO、XIAP和半胱天冬蛋白酶-9(caspase-9)的表达。结果表明,发作终止2h时KA注射同侧海马CA3区细胞浆内Smac／DIABLO蛋白表达增加,4h时caspase-9出现裂解片断,8h时出现TUNEL阳性细胞,24h时达高峰。脑室内注射caspase-9抑制剂z-LEHD-fluoromethyl ketone(z-LEHD-fmk)可减少TUNEL阳性细胞,增加存活神经元。发作后KA注射同侧海马CA3区神经元caspase-9免疫反应性增强,Smac／DIABLO和XIAP弥散于整个神经元内。对侧海马未检测到TUNEL阳性细胞及Smac／DIABLO和XIAP蛋白的上述变化。以上结果提示,癫痫发作可诱导Smac／DIABLO蛋白从线粒体向细胞浆的移位、XIAP亚细胞分布改变和caspase-9的激活,Smac／DIABLO、XIAP和caspase-9可能参与了癫痫神经元损伤的病理生理机制,caspase-9可能是潜在的治疗靶点。