Lei-Lei Ma

Both PI3K/Akt and ERK1/2 pathways participate in the protection by dexmedetomidine against transient focal cerebral ischemia/reperfusion injury in rats

Dexmedetomidine (Dex) has been demonstrated to provide neuroprotection against ischemia/reperfusion (I/R) injury. However, the exact mechanism of this protection remains unknown. Here, we explored the neuroprotective effect of Dex in rats exposed to cerebral I/R-induced by middle cerebral artery occlusion (MCAO) and the role of phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and glycogen synthase kinase-3β (GSK-3β) in this protective action. Adult male Sprague-Dawley rats were subjected to MCAO for 90 min followed by reperfusion for 24h and Dex (15 μg/kg, i.v.) was administered immediately after the onset of MCAO. The neurological deficit score, cerebral infarct volume, brain edema, and neuron survival were evaluated at 24h of reperfusion. The effect of Dex on p-Akt, p-ERK1/2 and p-GSK-3β expression in the ischemic hemisphere was assayed by Western blot. Treatment of rats exposed to I/R with Dex caused not only marked reduction in the neurological deficit score, cerebral infarct volume, and brain edema (P <0.01 vs. I/R alone), but also a decrease in neuron death in hippocampal CA1 and cortex (P<0.01 vs. I/R alone). The Dex-induced increment of neuron survival in the ischemic CA1 and cortex was diminished by the PI3K inhibitor LY294002 and the MEK inhibitor U0126. The increasing expressions of p-Akt and p-ERK1/2 induced by Dex in the ischemic hemisphere were markedly inhibited by LY294002 (or wortmannin) and U0126 (or PD98059), respectively. The up-regulation of p-GSK-3β by Dex in the ischemic hemisphere was significantly decreased by both LY294002 (or wortmannin) and U0126 (or PD98059). Our data demonstrated that treatment with Dex reduced cerebral injury in rats exposed to transient focal I/R, and this was mediated by the activation of the PI3K/Akt and ERK1/2 pathways as well the phosphorylation of downstream GSK-3β.

Minocycline Attenuates Cognitive Impairment Induced by Isoflurane Anesthesia in Aged Rats

Postoperative cognitive dysfunction (POCD) is a clinical phenomenon characterized by cognitive deficits in patients after anesthesia and surgery, especially in geriatric surgical patients. Although it has been documented that isoflurane exposure impaired cognitive function in several aged animal models, there are few clinical interventions and treatments available to prevent this disorder. Minocycline has been well established to exert neuroprotective effects in various experimental animal models and neurodegenerative diseases. Therefore, we hypothesized that pretreatment with minocycline attenuates isoflurane-induced cognitive decline in aged rats. In the present study, twenty-month-old rats were administered minocycline or an equal volume of saline by intraperitoneal injection 12 h before exposure to isoflurane. Then the rats were exposed to 1.3% isoflurane for 4 h. Two weeks later, spatial learning and memory of the rats were examined using the Morris Water Maze. We found that pretreatment with minocycline mitigated isoflurane-induced cognitive deficits and suppressed the isoflurane-induced excessive release of IL-1β and caspase-3 in the hippocampal CA1 region at 4 h after isoflurane exposure, as well as the number of TUNEL-positive nuclei. In addition, minocycline treatment also prevented the changes of synaptic ultrastructure in the hippocampal CA1 region induced by isoflurane. In conclusion, pretreatment with minocycline attenuated isoflurane-induced cognitive impairment in aged rats.

Gender-Related Difference of Sevoflurane Postconditioning in Isolated Rat Hearts: Focus on Phosphatidylinositol-3-Kinase/Akt Signaling

BACKGROUND Previous studies have reported that female gender confers cardioprotection against ischemia/reperfusion (I/R) injury, partly because estrogen activates phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. We have previously proven that cardioprotection of sevoflurane postconditioning is mediated by PI3K/Akt pathway in male rats. The purpose of the present study was to determine whether the cardioprotection of sevoflurane postconditioning is influenced by gender, and the role of PI3K/Akt pathway in such gender difference. MATERIALS AND METHODS Isolated hearts from 2-mo-old male and female SD rats were subjected to ischemia for 40 min and reperfusion for 2 h in the Langendorff apparatus, and were randomly assigned to the following groups: no ischemia/reperfusion (CON), ischemia/reperfusion (I/R), I/R+sevoflurane postconditioning (I/R+SPC), I/R+100 nM wortmannin (I/R+WOR), and I/R+SPC+WOR. Postconditioning was performed with administration of 3.0% sevoflurane at the first 10 min of reperfusion. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and myocardial lactate dehydrogenase (LDH) release were measured. Infarct size was detected by riphenyltetrazolium chloride staining. The protein expression of total Akt (t-Akt) and phosphorylated Akt (Ser(473)) (p-Akt) were determined by Western blot. RESULTS The I/R group showed lower LVDP and higher LVEDP than CON group in the same gender during reperfusion period. The LDH release and infarct size were smaller in the female I/R group (P < 0.05 versus male I/R group). Sevoflurane postconditioning markedly improved left ventricular function and decreased LDH, infarct size in the male I/R+SPC group (P < 0.05 versus male I/R group) but not in the female I/R+SPC group. Wortmannin abolished the cardioprotection of sevoflurane postconditioning in the male I/R+SPC+Wort group (P < 0.05 versus male I/R+SPC group), and markedly increased the infarct size and LVEDP and decreased LVDP in female rats. The t-Akt protein expression was no significant difference in all groups. The ratio of p-Akt/t-Akt expression in the male CON group was a little lower than that in the female CON group, but there was no statistical significance. In male rats, the ratio of p-Akt/t-Akt was no difference between CON and I/R group, but it was higher in I/R+SPC group than that in I/R group (P < 0.05). In female rats, the level of p-Akt was markedly increased by I/R, which was markedly higher than that in male I/R group (P < 0.05). However, p-Akt was not different between I/R and I/R+SPC groups. Wortmannin decreased the p-Akt expression in both male and female rats. CONCLUSIONS It is concluded that female rat hearts showed greater resistance to I/R injury, and sevoflurane postconditioning developed cardioprotection in male rats but not in female rats. The PI3K/Akt pathway may be involved in the cardioprotection by both sevoflurane postconditioning and gender.

Hypercholesterolemia blocked sevoflurane-induced cardioprotection against ischemia–reperfusion injury by alteration of the MG53/RISK/GSK3β signaling

BACKGROUND Recent studies have demonstrated that volatile anesthetic preconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. METHODS Normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane during three 5 min periods with and without PI3K antagonist wortmannin (10 μg/kg, Wort) or the ERK inhibitor PD 98059 (1 mg/kg, PD). The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined. RESULTS Two hundred and six rats were analyzed in the study. In the healthy rats, sevoflurane significantly reduced infarct size by 42%, a phenomenon completely reversed by wortmannin and PD98059 and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β. In the hypercholesterolemic rats, sevoflurane failed to reduce infarct size and increase the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. CONCLUSIONS Hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.

Endoplasmic reticulum stress pathway mediates isoflurane-induced neuroapoptosis and cognitive impairments in aged rats.

Postoperative cognitive dysfunction (POCD) is increasingly being recognized as an important clinical syndrome. Although it has been documented that volatile anesthetics induce neuronal apoptosis and cognitive deficits in several aged animal models, the underlying mechanisms are not well understood. Endoplasmic reticulum stress (ERS) is considered as an initial or early response of cells under stress and linked to neuronal death in various neurodegenerative diseases. The study was designed to explore the possible role of ERS pathway in isoflurane-induced neuroapoptosis and cognitive impairments. In the present study, twenty-month-old rats were exposed to 1.3% isoflurane for 4h. Two weeks later, the rats were subjected to behavioral study. Protein and mRNA expressions of ERS markers were evaluated. Meanwhile, hippocampal neuronal apoptosis was also detected. We found that isoflurane triggered ERS as evidenced by increased phosphorylation of eukaryotic initiation factor (EIF) 2α, and increased expression of 78-kDa glucose-regulated protein (GRP78), activating transcription factor (ATF) 4 and C/EBP homologous protein (CHOP). Furthermore, the level of apoptosis in the hippocampus was significantly up-regulated after isoflurane exposure, and salubrinal (ERS inhibitor) treatment attenuated the increase. More importantly, cognitive impairments caused by isoflurane were also effectively alleviated by salubrinal pretreatment. These results indicate that ERS-mediated apoptotic pathway is involved in isoflurane neurotoxicity in aged rats. Inhibition of ERS overactivation contributes to the relief of isoflurane-induced neurohistopathologic changes.

Hypercholesterolemic Myocardium Is Vulnerable to Ischemia-Reperfusion Injury and Refractory to Sevoflurane-Induced Protection

Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia. Therefore, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. In the present study, normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane for 5 min or 3 cycles of 10-s ischemia/10-s reperfusion. The hemodynamic parameters, including left ventricular developed pressure, left ventricular end-diastolic pressure and heart rate, were continuously monitored. The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined. We found that both sevoflurane and ischemic postconditioning significantly improved heart pump function, reduced infarct size and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β in the healthy rats. In the hypercholesterolemic rats, neither sevoflurane nor ischemic postconditioning improved left ventricular hemodynamics, reduced infarct size and increased the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. In conclusions, hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.

Hypercholesterolemia abrogates sevoflurane-induced delayed preconditioning against myocardial infarct in rats by alteration of nitric oxide synthase signaling.

ABSTRACT The aim of the current study was to determine whether hypercholesterolemia affects the delayed sevoflurane preconditioning against myocardial ischemia-reperfusion (IR) injury and, if so, the underlying mechanism. Male Sprague-Dawley rats fed 2% cholesterol-enriched chow for 8 weeks were subjected to sevoflurane preconditioning (2.4% vol/vol, 1 h) 24 h before myocardial ischemia was induced by occluding the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. The hemodynamic parameters left ventricular developed pressure, left ventricular end-diastolic pressure, and maximal rise/fall rate of left ventricular pressure were continuously monitored, and myocardial infarct size was determined at the end of reperfusion. The protein expression of myocardial nitric oxide synthase (NOS), Bcl-2, and Bad was assessed before ischemia. We found that the left ventricular hemodynamic parameters during the whole IR procedure and the myocardial infarct size did not significantly differ between the normocholesterolemic and hypercholesterolemic control groups. The hemodynamic parameters were all markedly improved during the reperfusion period, and the myocardial infarct size was significantly reduced by delayed sevoflurane preconditioning in normocholesterolemic rats, but all of these improvements were reversed by N-(3-(aminomethyl)benzyl) acetamidine (1400W, 1 mg/kg; i.v., 10 min before ischemia), a selective inducible NOS (iNOS) inhibitor, and 5-hydroxy decanoate sodium (5 mg/kg, i.v., 10 min before ischemia), a mitochondrial ATP-dependent K+ channel blocker. Such cardiac improvement induced by delayed sevoflurane preconditioning did not occur in hypercholesterolemic rats and was not exacerbated by 1400W or 5-hydroxy decanoate sodium. The expression of myocardial iNOS was markedly enhanced by delayed sevoflurane preconditioning in normocholesterolemic, but not in hypercholesterolemic rats. The expression of endothelial NOS and Bad did not differ among all groups. The expression of myocardial phosphorylated endothelial NOS, Bcl-2, and phosphorylated Bad in normocholesterolemic rats was not affected by delayed sevoflurane preconditioning but was decreased in the hypercholesterolemic control group, and this was not reversed by sevoflurane, compared with the normocholesterolemic control group. Taken together, these results indicate that sevoflurane preconditioning exerts delayed cardioprotection against IR injury in normocholesterolemic rats, which is blocked by hypercholesterolemia potentially via interference with the iNOS/mitochondrial ATP-dependent K+ channel pathway.

Hypertrophied myocardium is refractory to sevoflurane-induced protection with alteration of reperfusion injury salvage kinase/glycogen synthase kinase 3β signals.

ABSTRACT Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired by ventricular hypertrophy, we investigate whether anesthetic-induced cardiac protection was maintained in rat hearts with ventricular hypertrophy. Transverse aortic constriction operation was performed on male Sprague-Dawley rats to induce left ventricular (LV) hypertrophy, then sham-operated or hypertrophied rat hearts were subjected to 40 min of global ischemia and 2 h of reperfusion. The isolated hearts received 3% sevoflurane for 10 min or six cycles of 10-s ischemia/10-s reperfusion after reperfusion. The hemodynamics, infarct size, PTEN (phosphatase and tensin homolog deleted on chromosome ten), phosphorylated Akt, phosphorylated extracellular regulated protein kinase (ERK) 1/2, and phosphorylated glycogen synthase kinase 3&bgr; (GSK3&bgr;) were determined. We found the myocardial expression of PTEN, phosphorylated Akt, ERK1/2, and phosphorylated GSK3&bgr; did not significantly differ between sham-operated and transverse aortic constriction–control groups. Both sevoflurane and ischemic postconditioning significantly improved LV hemodynamics, reduced infarct size, and increased the phosphorylation of Akt, ERK1/2, and their downstream target of GSK3&bgr; in the sham-operated rat hearts. In contrast, neither sevoflurane nor ischemic postconditioning improved LV hemodynamic, reduced infarct size, and increased the phosphorylated Akt, ERK1/2, and GSK3&bgr; in hypertrophied myocardium. All the results above indicate that ventricular hypertrophy abrogated sevoflurane-induced cardioprotection against ischemia-reperfusion injury by alteration of RISK/GSK3&bgr; signals.

Hypercholesterolemia Abrogates Remote Ischemic Preconditioning-Induced Cardioprotection: Role of Reperfusion Injury Salvage Kinase Signals.

ABSTRACT Remote ischemic preconditioning (RIPC) is one of the most powerful intrinsic cardioprotective strategies discovered so far and experimental data indicate that comorbidity may interfere with the protection by RIPC. Therefore, we investigate whether RIPC-induced cardioprotection was intact in hypercholesterolemic rat hearts exposed to ischemia reperfusion in vivo. Normal or hypercholesterolemic rat hearts were exposed to 30 min of ischemia and 2 h of reperfusion, with or without RIPC, PI3K inhibitor wortmannin, MEK-ERK1/2 inhibitor PD98059, GSK3&bgr; inhibitor SB216763. Infarct size, apoptosis, MG53, PI3K-p85, p-Akt, p-ERK1/2, p-GSK3&bgr;, and cleaved Caspase-3 were determined. RIPC reduced infarct size, limited cardiomyocyte apoptosis following IR that was blocked by wortmannin but not PD98059. RIPC triggered unique cardioprotective signaling including MG53, phosphorylation of Akt, and glycogen synthase kinase-3ß (GSK3&bgr;) in concert with reduced proapoptotic active caspase-3. In contrast, RIPC failed to reduce myocardial necrosis and apoptosis as well as to increase the phosphorylated Akt and GSK3&bgr; in hypercholestorolemic myocardium. Importantly, we found that inhibition of GSK with SB216763 reduced myocardial infarct size in healthy and hypercholesterolemic hearts, but no additional cardioprotective effect was achieved when combined with RIPC. Our results suggest that acute GSK3&bgr; inhibition may provide a novel therapeutic strategy for hypercholesterolemic patients during acute myocardial infarction, whereas RIPC is less effective due to signaling events that adversely affect GSK3&bgr;.

Ventricular hypertrophy abrogates intralipid-induced cardioprotection by alteration of reperfusion injury salvage kinase/glycogen synthase kinase 3β signal.

ABSTRACT Recent studies have demonstrated that intralipid (ILP) conferred myocardial protection against ischemia-reperfusion (IR) injury through activation of reperfusion injury salvage kinase (RISK) pathway. As RISK signal has been shown to be impaired in hypertrophied myocardium, we investigated whether ILP-induced cardiac protection was maintained in hypertrophied rat hearts. Transverse aortic constriction was performed on male Sprague-Dawley rats to induce left ventricular hypertrophy, then sham-operated or hypertrophied rat hearts were isolated and perfused retrogradely by the Langendorff for 30 min (equilibration) followed by 40 min of ischemia and then 120 min of reperfusion. The isolated hearts received 15-min episode of 1% ILP separated by 15 min of washout or three episodes of 5-min ischemia followed by 5-min reperfusion before ischemia. The hemodynamics, infarct size, apoptosis, phosphorylated protein kinase B (p-Akt), phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated glycogen synthase kinase 3&bgr; (GSK3&bgr;), Bcl-2, phosphorylated Bad, and Bax were determined. We found that ILP significantly improved left ventricular hemodynamics and reduced infarct size and the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)–positive cells in the sham-operated rat hearts exposed to IR. However, such myocardial infarct–sparing effect of ILP was completely blocked by phosphatidylinositol-3-kinase inhibitor wortmannin, but only partially by mitogen-activated protein kinase kinase inhibitor PD98059 in sham-operated hearts. Intralipd upregulated the phosphorylation of Akt, extracellular regulated protein kinase 1/2 (ERK1/2), and their downstream target of GSK3&bgr; and antiapoptotic Bcl-2 expression in healthy rat hearts. Nonetheless, ILP failed to improve left ventricular hemodynamics and reduced infarct size and apoptosis and increase the phosphorylated Akt, ERK1/2, GSK3&bgr;, and antiapoptotic Bcl-2 in hypertrophied myocardium. In contrast, ischemic preconditioning increased the phosphorylation of Akt, ERK1/2 and GSK3&bgr;, improved heart pump function, and reduced myocardial necrosis in sham-operated hearts, a phenomenon partially attenuated by ventricular hypertrophy. Interestingly, GSK inhibitor SB216763 conferred cardioprotection against IR injury in sham-operated hearts, but failed to exert cardioprotection in hypertrophied myocardium. Our results indicated that ventricular hypertrophy abrogated ILP-induced cardioprotection against IR injury by alteration of RISK/GSK3&bgr; signal.