TMEM175 downregulation participates in impairment of the autophagy related lysosomal dynamics following neonatal hypoxic-ischemic brain injury.

The mechanism underlying long-term cognitive impairment caused by neonatal hypoxic-ischemic brain injury (HIBI) remains unclear. Autophagy is a closely related mechanism and may play a role in this process. We aimed to investigate the role of lysosomal transmembrane protein 175 (TMEM175) in the autophagy-lysosome pathway in neonatal rats with HIBI. A neonatal rat model of HIBI was established, hypoxia was induced, followed by left common carotid artery ligation. Expression levels of TMEM175 and the corresponding proteins involved in autophagy flux and the endolysosomal system fusion process were measured. Rats were administered TMEM175 plasmid via intracerebroventricular injection to induce overexpression. Brain damage and cognitive function were then assessed. TMEM175 was downregulated in the hippocampal tissue, and the autophagy-lysosome pathway was impaired following HIBI in neonatal rats. Overexpression of TMEM175 significantly mitigated neuronal injury and improved long-term cognitive and memory function in neonatal rats with HIBI. We found that improvement in the autophagy-lysosome pathway and endolysosomal system homeostasis, which are TMEM175 related, occurred via regulation of lysosomal membrane dynamic fusion. TMEM175 plays a critical role in maintaining the autophagy-lysosome pathway and endolysosomal homeostasis, contributing to the amelioration of neuronal injury and impaired long-term cognitive function following neonatal HIBI.

Wnt Signaling Inhibits High-Density Cell Sheet Culture Induced Mesenchymal Stromal Cell Aging by Targeting Cell Cycle Inhibitor p27.

Mesenchymal stromal cell senescence and apoptosis have been identified as critical molecular hallmarks in aging. In this study, we used stromal cell sheet culture as an in vitro model to study the progressive changes of cellular senescence, apoptosis and underlying mechanism in Wnt3a treated cells. Our results showed fresh bone marrow mesenchymal stromal cells (BMSCs) become senescent and undergo apoptosis with increased inflammatory profile and Reactive Oxygen Species (ROS) in high-density cell sheet cultures. The gene expression level of senescence related proteins and key regulators of apoptosis in cell sheet cultures was significantly increased in older BMSCs at Days 4 and 7 cultures compared with younger cells at Day 1 cultures. More importantly, Wnt signaling activation significantly reduced senescence in cell sheet cultures by direct regulation of cell cycle inhibitor p27. This study not only characterized the cellular and molecular features of aging stromal cells in short-term cell sheet cultures, but also identified the downstream target responsible for Wnt inhibition of cell senescence.

Sevoflurane-Induced Neuroapoptosis in Rat Dentate Gyrus Is Activated by Autophagy Through NF-κB Signaling on the Late-Stage Progenitor Granule Cells.

OBJECTIVE: The mechanisms by which exposure of the late-stage progenitor cells to the anesthesia sevoflurane alters their differentiation are not known. We seek to query whether the effects of sevoflurane on late-stage progenitor cells might be regulated by apoptosis and/or autophagy. METHODS: To address the short-term impact of sevoflurane exposure on granule cell differentiation, we used 5-bromo-2-deoxyuridine (BrdU) to identify the labeled late-stage progenitor granule cells. Male or female rats were exposed to 3% sevoflurane for 4 h when the labeled granule cells were 2 weeks old. Differentiation of the BrdU-labeled granule cells was quantified 4 and 7 days after exposure by double immunofluorescence. The expression of apoptosis and autophagy in hippocampal dentate gyrus (DG) was determined by western blot and immunofluorescence. Western blot for the expression of NF-κB was used to evaluate the mechanism. Morris water maze (MWM) test was performed to detect cognitive function in the rats on postnatal 28-33 days. RESULTS: Exposure to sevoflurane decreased the differentiation of the BrdU-labeled late-stage progenitor granule cells, but increased the expression of caspase-3, autophagy, and phosphorylated-P65 in the hippocampus of juvenile rats and resulted in cognitive deficiency. These damaging effects of sevoflurane could be mitigated by inhibitors of autophagy, apoptosis, and NF-κB. The increased apoptosis could be alleviated by pretreatment with the autophagy inhibitor 3-MA, and the increased autophagy and apoptosis could be reduced by pretreatment with NF-κB inhibitor BAY 11-7085. CONCLUSION: These findings suggest that a single, prolonged sevoflurane exposure could impair the differentiation of late-stage progenitor granule cells in hippocampal DG and cause cognitive deficits possibly via apoptosis activated by autophagy through NF-κB signaling. Our results do not preclude the possibility that the affected differentiation and functional deficits may be caused by depletion of the progenitors pool.

Immature murine hippocampal neurones do not develop long-term structural changes after a single isoflurane exposure.

BACKGROUND: Studies in developing animals show that a clinically relevant anaesthesia exposure increases neuronal death and alters brain structure. In the hippocampal dentate gyrus, the anaesthetic isoflurane induces selective apoptosis among roughly 10% of 2-week-old hippocampal granule cells in 21-day-old mice. In this work, we queried whether the 90% of granule cells surviving the exposure might be 'injured' and integrate abnormally into the brain. METHODS: The long-term impact of isoflurane exposure on granule cell structure was studied using a transgenic mouse model fate-mapping approach to identify and label immature granule cells. Male and female mice were exposed to isoflurane for 6 h when the fate-mapped granule cells were 2 weeks old. The morphology of the fate-mapped granule cells was quantified 2 months later. RESULTS: The gross structure of the dentate gyrus was not affected by isoflurane treatment, with granule cells present in the correct subregions. Individual isoflurane-exposed granule cells were structurally normal, exhibiting no changes in spine density, spine type, dendrite length, or presynaptic axon terminal structure (P>0.05). Granule cell axon terminals were 13% larger in female mice relative to males; however, this difference was evident regardless of treatment (difference of means=0.955; 95% confidence interval, 0.37-1.5; P=0.010). CONCLUSIONS: A single, prolonged isoflurane exposure did not impair integration of this age-specific cohort of granule cells, regardless of the animal's sex. Nonetheless, although 2-week-old cells were not affected, the results should not be extrapolated to other age cohorts, which may respond differently.

Effects of Sevoflurane Exposure During Mid-Pregnancy on Learning and Memory in Offspring Rats: Beneficial Effects of Maternal Exercise.

Fetal exposure to general anesthetics may pose significant neurocognitive risks but methods to mitigate against these detrimental effects are still to be determined. We set out, therefore, to assess whether single or repeated in utero exposure to sevoflurane triggers long-term cognitive impairments in rat offspring. Since maternal exercise during pregnancy has been shown to improve cognition in offspring, we hypothesized that maternal treadmill exercise during pregnancy would protect against sevoflurane-induced neurotoxicity. In the first experiment, pregnant rats were exposed to 3% sevoflurane for 2 h on gestational (G) day 14, or to sequential exposure for 2 h on G13, G14 and G15. In the second experiment, pregnant rats in the exercise group were forced to run on a treadmill for 60 min/day during the whole pregnancy. The TrkB antagonist ANA-12 was used to investigate whether the brain-derived neurotrophic factor (BDNF)/TrkB/Akt signaling pathway is involved in the neuroprotection afforded by maternal exercise. Our data suggest that repeated, but not single, exposure to sevoflurane caused a reduction in both histone acetylation and BDNF expression in fetal brain tissues and postnatal hippocampus. This was accompanied by decreased numbers of dendritic spines, impaired spatial-dependent learning and memory dysfunction. These effects were mitigated by maternal exercise but the TrkB antagonist ANA-12 abolished the beneficial effects of maternal exercise. Our findings suggest that repeated, but not single, exposure to sevoflurane in pregnant rats during the second trimester caused long-lasting learning and memory dysfunction in the offspring. Maternal exercise ameliorated the postnatal neurocognitive impairment by enhancing histone acetylation and activating downstream BDNF/TrkB/Akt signaling.

Long-term Fate Mapping to Assess the Impact of Postnatal Isoflurane Exposure on Hippocampal Progenitor Cell Productivity.

BACKGROUND: Exposure to isoflurane increases apoptosis among postnatally generated hippocampal dentate granule cells. These neurons play important roles in cognition and behavior, so their permanent loss could explain deficits after surgical procedures. METHODS: To determine whether developmental anesthesia exposure leads to persistent deficits in granule cell numbers, a genetic fate-mapping approach to label a cohort of postnatally generated granule cells in Gli1-CreER::GFP bitransgenic mice was utilized. Green fluorescent protein (GFP) expression was induced on postnatal day 7 (P7) to fate map progenitor cells, and mice were exposed to 6 h of 1.5% isoflurane or room air 2 weeks later (P21). Brain structure was assessed immediately after anesthesia exposure (n = 7 controls and 8 anesthesia-treated mice) or after a 60-day recovery (n = 8 controls and 8 anesthesia-treated mice). A final group of C57BL/6 mice was exposed to isoflurane at P21 and examined using neurogenesis and cell death markers after a 14-day recovery (n = 10 controls and 16 anesthesia-treated mice). RESULTS: Isoflurane significantly increased apoptosis immediately after exposure, leading to cell death among 11% of GFP-labeled cells. Sixty days after isoflurane exposure, the number of GFP-expressing granule cells in treated animals was indistinguishable from control animals. Rates of neurogenesis were equivalent among groups at both 2 weeks and 2 months after treatment. CONCLUSIONS: These findings suggest that the dentate gyrus can restore normal neuron numbers after a single, developmental exposure to isoflurane. The authors' results do not preclude the possibility that the affected population may exhibit more subtle structural or functional deficits. Nonetheless, the dentate appears to exhibit greater resiliency relative to nonneurogenic brain regions, which exhibit permanent neuron loss after isoflurane exposure.

Overexpression of hSNF2H in glioma promotes cell proliferation, invasion, and chemoresistance through its interaction with Rsf-1.

hSNF2H partners with Rsf-1 to compose the Rsf complex to regulate gene expression. Recent studies indicated that hSNF2H was overexpressed in several human cancers. However, its expression pattern and biological mechanism in glioma remain unexplored. In this study, we found that hSNF2H was overexpressed in 32 % of glioma specimens. hSNF2H overexpression correlated with advanced tumor grade (p = 0.0338) and Rsf-1 positivity in glioma tissues (p = 0.016). Small interfering RNA (siRNA) knockdown was performed in A172 and U87 cell lines. MTT, colony formation assay, and cell cycle analysis showed that knockdown of hSNF2H inhibited cell proliferation, colony formation ability, and cell cycle transition. Matrigel invasion assay showed that hSNF2H depletion inhibited invasive ability of glioma cells. In addition, we demonstrated that hSNF2H depletion decreased temozolomide resistance of A172 and U87 cell lines and increased temozolomide induced apoptosis. Furthermore, hSNF2H depletion decreased cyclin D1, cyclin E, p-Rb, MMP2, cIAP1, Bcl-2 expression, and phosphorylation of IκBα and p65, suggesting hSNF2H regulates apoptosis through NF-κB pathway. Immunoprecipitation showed that hSNF2H could interact with Rsf-1 in both cell lines. To validate the involvement of Rsf-1, we checked the change of its downstream targets in Rsf-1 depleted cells. In Rsf-1 depleted cells, changes of cyclin E, Bcl-2, and p-IκBα were not significant using hSNF2H siRNA treatment. In conclusion, our study demonstrated that hSNF2H was overexpressed in human gliomas and contributed to glioma proliferation, invasion, and chemoresistance through regulation of cyclin E and NF-κB pathway, which is dependent on its interaction with Rsf-1.

Hydrogen-Rich Saline Attenuates Lipopolysaccharide-Induced Heart Dysfunction by Restoring Fatty Acid Oxidation in Rats by Mitigating C-Jun N-Terminal Kinase Activation.

Sepsis is common in intensive care units (ICU) and is associated with high mortality. Cardiac dysfunction complicating sepsis is one of the most important causes of this mortality. This dysfunction is due to myocardial inflammation and reduced production of energy by the heart. A number of studies have shown that hydrogen-rich saline (HRS) has a beneficial effect on sepsis. Therefore, we tested whether HRS prevents cardiac dysfunction by increasing cardiac energy. Four groups of rats received intraperitoneal injections of one of the following solutions: normal saline (NS), HRS, lipopolysaccharide (LPS), and LPS plus HRS. Cardiac function was measured by echocardiography 8 h after the injections. Gene and protein expression related to fatty acid oxidation (FAO) were measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. The injection of LPS compromised heart function through decreased fractional shortening (FS) and increased left ventricular diameter (LVD). The addition of HRS increased FS, palmitate triphosphate, and the ratio of phosphocreatinine (PCr) to adenosine triphosphate (ATP) as well as decreasing LVD. The LPS challenge reduced the expression of genes related to FAO, including perioxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), perioxisome proliferator-activated receptor alpha (PPARα), Estrogen-related receptor alpha (ERRα), and their downstream targets, in mRNA and protein level, which were attenuated by HRS. However, HRS had little effect on glucose metabolism. Furthermore, HRS inhibited c-Jun N-terminal kinase (JNK) activation in the rat heart. Inhibition of JNK by HRS showed beneficial effects on LPS-challenged rats, at least in part, by restoring cardiac FAO.

[Effects of different doses of dexmedetomidine on the recovery quality from general anesthesia undergoing thyroidectomy].

OBJECTIVE: To investigate effects of two doses of dexmedetomidine (Dex) on the recovery quality from general anesthesia undergoing thyroidectomy. METHODS: A prospective randomized controlled double-blind trial was conducted in 90 patients admitted to Shengjing Hospital of China Medical University who were scheduled for thyroidectomy. They were randomly divided into three groups: group D0.4 received Dex 0.4 µg/kg intravenously, group D0.8 received Dex 0.8 µg/kg intravenously, and control group with same volume of normal saline. There were 30 patients in each group, and all the patients received the above drug or saline 30 minutes after intubation. The heart rate (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded before Dex administration, at the end of surgery, time of eye opening and extubation, and 10 minutes after extubation. Time of eye opening after surgery and time of extubation after eye opening were recorded. End-tidal partial pressure of carbon dioxide (PETCO2) before and after operation as well as restlessness score (RS), Ramsay sedative score, and visual analogue pain scale (VAS) scores at 10 minutes after extubation were recorded. Events during extubation including cough, nausea and vomiting, respiratory depression were also recorded. RESULTS: (1) Compared with control group, in D0.4 and D0.8 groups incidence of lowering of HR, SBP and DBP was lower at time of eye opening and extubation, and 10 minutes after extubation. Compared with D0.4 group, D0.8 group had lower HR at the time of extubation and 10 minutes after extubation, SBP was lower at the time of eye opening and extubation, and lower DBP at the time of eye opening. (2) D0.4 and D0.8 groups showed lower RS and VAS scores than those of control group, Ramsay sedative score in groups D0.4 and D0.8 was higher than that in control group (RS: 1.40±0.51, 1.20±0.42 vs. 1.90±0.56; VAS: 1.50±0.52, 0.80±0.63 vs. 2.50±0.52; Ramsay: 2.10±0.56, 2.40±0.51 vs. 1.60±0.51, P<0.05 or P<0.01), and VAS score in group D0.8 was lower than that in D0.4 group (P<0.01). The time of eye opening and extubation were longer in group D0.8 as compared with those in control and D0.4 groups (12.50±1.08 minutes vs. 10.50±1.58 minutes, 10.40±1.26 minutes; 15.00±0.94 minutes vs. 13.00±1.63 minutes, 12.80±1.13 minutes, P<0.05 or P<0.01), but there was no significant difference between the latter two groups. No significant difference in PETCO2 was found among three groups before and after surgery. (3) The incidence rate of cough, nausea and vomiting in control group (16.7%, 13.3%) were significantly higher than those in groups D0.4 (3.3%, 0) and D0.8 (0, 0). There was no respiratory depression in the three groups. CONCLUSIONS: Adjunctive infusion of Dex 0.4 µg/kg at 30 minutes after anesthesia induction was recommended as it may result in more steady hemodynamics, with shorter recovery time and extubation time after thyroidectomy.

Isoflurane post-conditioning protects primary cultures of cortical neurons against oxygen and glucose deprivation injury via upregulation of Slit2/Robo1.

Different mechanisms have been suggested to contribute to isoflurane-mediated neuroprotection. Previous studies have suggested that the protein Slit can abrogate neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation (OGD) and reperfusion (OGD/R). We hypothesized that isoflurane increases the expression of Slit and its receptor Robo when cortical neurons are exposed to OGD/R. To test this hypothesis, we exposed primary cortical neurons to OGD for 90 min and reperfusion for 24h and investigated how isoflurane post-conditioning affected cell survival and expression of Slit2 and receptors Robo1 and Robo4. Cell survival increased after administration of isoflurane, as assessed by the lactate dehydrogenase assay, trypan blue analysis, and propidium iodide staining. Western blot analysis showed that cleaved caspase-3 was increased after OGD/R(P<0.01) but reduced by isoflurane post-conditioning. Real-time PCR and Western blot analysis showed that the expression levels of Slit2 and Robo1, but not Robo4, were increased after OGD/R (P<0.5) and increased even further by isoflurane post-conditioning (P<0.01). Our results suggest that isoflurane post-conditioning markedly attenuates apoptosis and necrosis of cortical neurons exposed to OGD/R possibly in part via elevation of Slit2 and Robo1 expression. These findings provide a novel explanation for the pleiotropic effects of isoflurane that could benefit the central nervous system.

Propofol increases expression of basic fibroblast growth factor after transient cerebral ischemia in rats.

Anesthetics such as propofol can provide neuroprotective effects against cerebral ischemia. However, the underlying mechanism of this beneficial effect is not clear. Therefore, we subjected male Sprague-Dawley rats to 2 h of middle cerebral artery occlusion and investigated how post-ischemic administration of propofol affected neurologic outcome and the expression of basic fibroblast growth factor (bFGF). After 2 h of ischemia, just before reperfusion, the animals were randomly assigned to receive either propofol (20 mg kg(-1) h(-1)) or vehicle (10 % intralipid, 2 ml kg(-1) h(-1)) intravenously for 4 h. Neurologic scores, infarct volume, and brain water content were measured at different time points after reperfusion. mRNA level of bFGF was measured by real-time PCR, and the protein expression level of bFGF was analyzed by immunohistochemistry and Western blot. At 6, 24, 72 h, and 7 days of reperfusion, infarct volume was significantly reduced in the propofol-treated group compared to that in the vehicle-treated group (all P < 0.05). Propofol post-treatment also attenuated brain water content at 24 and 72 h and reduced neurologic deficit score at 72 h and 7 days of reperfusion (all P < 0.05). Additionally, in the peri-infarct area, bFGF mRNA and protein expression were elevated at 6, 24, and 72 h of reperfusion compared to that in the vehicle-treated group (all P < 0.05). These results show that post-ischemic administration of propofol provides neural protection from cerebral ischemia-reperfusion injury. This protection may be related to an early increase in the expression of bFGF.

[The comparison of the vasoactive effects of dopamine on isolated rabbit pulmonary and systemic arteries after incubation with lipopolysaccharide].

OBJECTIVE: To compare the vasoactive effects of dopamine (DOPA) of different concentrations on isolated rabbit pulmonary and systemic arteries after incubation with lipopolysaccharide (LPS). METHODS: Six white male rabbits were used. Thirty-six pulmonary arterial rings and 36 systemic arterial rings were prepared. The 36 pulmonary arterial rings were divided into six groups to determine the effect of different concentrations of DOPA (4x10(-5) , 8x10(-5), 16x10(-5) micromol/L) on the tension of the normal pulmonary artery (PN-DOPA4, PN-DOPA8, PN-DOPA16 groups, respectively), and the tension of the pulmonary artery rings after being incubated with LPS (PL-DOPA4, PL-DOPA8, PL-DOPA16 groups, respectively). The 36 systemic arterial rings were also divided into six groups as the pulmonary arterial rings, including normal groups (SN-DOPA4, SN-DOPA8 , SN-DOPA16) and LPS groups (SL-DOPA4, SL-DOPA8 , SL-DOPA16). RESULTS: (1) DOPA relaxed the arterial rings in PN-DOPA4 and SN-DOPA4 groups, while it produced contraction in PN-DOPA8, PN-DOPA16, SN-DOPA8 and SN-DOPA16 groups, and the contraction was more marked with the increase in concentration of DOPA. (2) After preincubation with LPS, the relaxation property of DOPA in PL-DOPA4 and SL-DOPA4 groups was observed to be reversed to contraction [(22.60+/-6.68)% vs. -(2.25+/-0.58)%, (3.80+/-0.52)% vs. -(3.65+/-0.75)%, P<0.05 and P<0.01]; the contraction response of DOPA in PL-DOPA8 group decreased compared with PN-DOPA8 group by (14.52+/-0.59)% (P<0.05), while increased by (25.90+/-1.75)% in SL-DOPA8 group compared with SN-DOPA8 group (P<0.05), and no response was observed in PL-DOPA16 and SL-DOPA16 groups. (3)After preincubation with LPS, changes in pulmonary arterial tension (PL/PN) in DOPA4 group were more obvious than those in systemic arterial tension (SL/SN, -10.90+/-5.06 vs. -1.00+/-0.24, P<0.05), while the SL/SN in DOPA8 group were more obvious (1.80+/-0.35 vs. 0.48+/-0.17, P<0.01). CONCLUSION: DOPA in low concentrations had the function of relaxation on the pulmonary arterial and systemic arterial rings. After the arterial rings are preincubated with LPS, the relaxation response of DOPA of low concentrations is changed to be vaso-contraction, and the changes in pulmonary arterial rings are most marked. DOPA of different concentrations all produce contraction effect on LPS-preincubated arterial rings.