Role Of MTⅠ+Ⅱ In Delayed Protective Effects Of Sevoflurane Preconditioning In Hippocampal Neurons Of Rats

PART I IDENTIFICATION OF GENES DIFFERENTIALLY REGULATED IN RAT LIVER BRAIN AND HEART FOLLOWING SEVOFLURANE EXPOSUREObjective To study the effect of sevoflurane exposure on gene expression profiles of rat liver brain and heart by DNA Microarray. Methods Identification genes that were differentially expressed in adult rat liver, brain and heart following a clinically relevant exposure to the inhaled anesthetic sevoflurane by DNA microarray. In order to verify results from our microarray, three of the highly regulated genes from the liver array were selected for verification by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Results Based on our selection criteria, all of the tissues tested showed differential gene expression in response to a90min dose of2.0%sevoflurane. Of the differential genes,34of the4937genes on the arrays were differentially regulated in the same direction in at least2tissues. There was good agreement between microarray and qRT-PCR differential expression levels, suggesting that array values reflect a reasonable approximation of differential expression levels under the conditions we used. Conculsion We identified a number of genes that were differentially expressed in rat liver brain and heart following a clinical dose of sevoflurane. Consistent with our prediction, there were a number of genes that were differentially regulated in a number of tissues. PART II A ROLE FOR METALLOTHIONEINS-Ⅰ+II IN SEVOFLURANE PRECONDITIONING OF PRIMARY MURINE NEURONAL CULTURESObjective To study protective characteristics of delayed sevoflurane preconditioning in dissociated neuronal culture and the role that MT-Ⅰ+Ⅱ play in conferring APC-mediated protection. Methods1) Primary neuronal cultures;2) Sevoflurane Preconditioning;3) Oxygen-Glucose Deprivation;4) Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) of MT-Ⅰ+Ⅱ RNA levels;5) Lactate Dehydrogenase assay for determination of cellular toxicity;6) Microtubule associated protein-2(MAP2)/Glia fibrillary associated protein (GFAP) colorimetric assays for determination of cellular toxicity;7) MAP2, GFAP, MT-Ⅰ+Ⅱ immunofluorescence microscopy for determination of MT Localization.;8) siRNA Knockdown of MT-Ⅰ+Ⅱ;9) MT-Ⅰ+Ⅱ protein transfection. Results1) A3hr pretreatment with1.5%v/v sevoflurane (a clinically relevant exposure) provided protection that was significant (two-tailed Student’s t-test; p<0.01) compared to paired, nonpreconditioned controls for all time points. In detail, at24hrs the protection amounted to11.7±8.2%(n=22), increasing to peak protection of37.5±4.3%at72hrs (n=22). The effect persisted and was still robust at96hrs (31.9±5.2%; n=29).2)3hrs OGD was toxic to both neuronal and glial populations, neurons were particularly sensitive to this treatment with survival rates of only39.6±7.8%vs80.2±10.5%(n=16) for glial cells. Neurons were also more responsive to sevoflurane preconditioning showing recovery to a85.3±9.2%(n=16) survival rate i.e.～125%increase in survival. To a lesser extent glial cells were also protected with a relative increase in survival rate of only～22%. For neuronal populations, a2×2ANOVA analysis showed significant main effects of Oxygen-Glueose Deprivation (OGD) and of sevoflurane preconditioning (APC) with a significant interaction between these variables (p<0.01). This interaction indicates the effectiveness of APC in counteracting OGD-mediated cell death and is supported by a Student’s t-test that confirmed a significant (p<0.01) difference between ’OGD alone’ and ’APC+OGD’ conditions. For glial cells, the2×2ANOVA analysis revealed significant main effects of OGD and of sevoflurane preconditioning (APC)(p<0.05) however there was no significant interaction between these variables (p=0.53). The lack of a significant interaction can be interpreted to mean that sevoflurane preconditioning is not effective in protecting glia against OGD. However, a Student’s t-test comparing the’OGD alone’and’APC+OGD’conditions revealed a significant (p<0.01) difference between these groups suggesting that APC was effective but that the level of OGD toxicity was not as pronounced in this cell population.3) Cultures transfection by exogenous MT-Ⅰ+Ⅱ protein prior to a3hr OGD challenge and then assessed toxicity by LDH. Compared to transfection with a similarly-sized control protein (oxidized insulin13-chain), MT protein transfection conferred protection against3hr OGD, rising from3.1±1.4%protection at1.6μM MT (not significant; n=10) to significant protection of34.8±9.2%(p<0.001; n=10) with3.2μM MT.4) Both MT-Ⅰ and MT-Ⅱ were rapidly upregulated with increases in mRNA levels observed as early as3hrs. For both metallothioneins-Ⅰ+Ⅱ, mRNA levels peaked at-12hrs with levels that were2.2±0.8and2.9±1.1fold the non-preconditioned cultures for MT I and MT Ⅱ, respectively. Compared to untreated paired controls, MTI mRNA fold changes were significant (two-tailed Student’s t-test; p<0.05) at6and12hrs and MTII mRNA fold changes were significant at3,6and12hrs.5) MT-Ⅰ+Ⅱ mRNA levels were assessed under basal and under MT stimulating conditions (addition of20μM ZnC12). Though we were able achieve only partial knockdown of either gene, MT-Ⅰ and MT-Ⅱ mRNA levels were diminished in a dose-dependent manner by MT-siRNA transfection. Zinc stimulation and siRNA knockdown both showed greater effect on MT-Ⅱ than on MT-Ⅰ. We assessed the effect of sham knockdown (ShamKD) or metallothionein knockdown on sevoflurane-mediated protection against3hr OGD using an LDH assay, sevoflurane-preconditioned ShamKD cultures demonstrated substantial protection19.4±3.5%, similar to our findings for WT C57cultures. ANOVA analysis of the siRNA data revealed main effects of metallothionein knockdown (p<0.05) and of APC (p<0.01). However, there was no significant interaction found between these variables (p=0.564). This implies that the partial knockdown of the metallothioneins was not effective in reducing the protective influence of APC significantly. However. metallothionein-Ⅰ+Ⅱ knockdown conditions without preconditioning showed substantially increased OGD-mediated toxicity compared to ShamKD. This suggests that MT-Ⅰ+II below typical basal levels renders cells particularly vulnerable to OGD insults.6) Preconditioned background (129)-derived cultures showed significant protection (two tailed Student’s t-test; p<0.001) against OGD toxicity (22.5±3.4%; n=16) as compared to paired non-preconditioned samples.7) In C57-and129-derived cells the metallothionein antibody colocalized strongly with MAP2labeled cell bodies indicating strong neuronal localization. GFAP-labeled cells showed metallothionein co-localization to a lesser extent (data not shown). Neither MT knockout-derived cultures nor129wild type (WT) cultures coincubated with10μM horse MT-Ⅰ+Ⅱ protein demonstrated measurable MT labeling, consistent with MT-Ⅰ+Ⅱ specific labeling. Conculsion1.)establish a time-course for metallothionein Ⅰ+Ⅱ expression in response to sevoflurane treatment,2.) show that both neurons and glia are protected during delayed APC and3.) show that neurons were both more susceptible to oxygen-glucose deprivation toxicity and showed greater protection from preconditioning.4.) establish, through the use of knockdown and knockout studies, that metallothioneins are integrally involved in sevoflurane-mediated delayed protection and5.) demonstrate through direct transfection of exogenous metallothionein protein that metallothioneins likely act as end effectors of protection against ischemic injury. PART Ⅲ THE MECHANISM OF METALLOTHIONEIN I+Ⅱ AS OXIDATIVE STRESS SENSOR IN SEVOFLURANE PRECONDITIONING PROTECTIONObjective Investigated a possible role for metallothioneins as sensors of oxidative stress following preconditioning with the inhaled anesthetic sevoflurane, the reactive nitrogen species nitric oxide and the oxygen radical superoxide. Methods1) Primary neuronal cultures;2) Sevoflurane Preconditioning;3) Oxygen-Glucose Deprivation (OGD);4) Lactate Dehydrogenase assay for determination of cellular toxicity;5) Isolation of nuclear proteins for determination of MTF-1translocation;6) Western Determination of MTF-1levels. Results1) SNAP preconditioned cultures demonstrated significant protection against OGD (38.2±2.9%; n=28) compared to non-preconditioned cells in WT cultures (p<0.001; n=28). Cells treated with the same concentration of SNAP that had been heat degraded (37°,24hrs) were not significantly protected7.9±4.1%(n=14) and were significantly less protected than the SNAP preconditioned cultures (p<0.01; n=14). Paraqu at preconditioned cultures showed significant protection (23.8±6.2%; n=14) compared to untreated controls. SNAP, decomposed SNAP and paraquat showed only small differences from basal control LDH levels (75.2±2.6%, p=0.18, n=6),(82±3.1%; p=0.26, n=6) and (65.5±1.1%; p<0.05; n=6) of control levels respectively.; p=0.18and p<0.05respectively; n=6). Following2hrs on from the initiation of preconditioning, SNAP and paraquat treatments resulted in increased nuclear MTF-1levels (by74±18%and224±85%above untreated control levels respectively; n=3,). Degraded SNAP showed minimal MTF-1translocation compared to non-degraded SNAP (16±5.8%above untreated control; n=3). Conculsion1.) blocking NO or.O2production during APC decreased both protection and nuclear localized MTF-1.2.) The NO and.02generators alone were sufficient to precondition mixed neuronal cultures and that this too, corresponded with increased nuclear localization of MTF-1.3.) Finally, knockout of metallothionein Ⅰ+Ⅱ genes not only eliminated protection induced by NO and.O2but also diminished preconditioning-mediated increases in nuclear MTF-1levels.