Objective To investigate the expressions of heat shock protein 27 (HSP27), Bcl-2, and Bax proteins of the nerve cells after spinal cord ischemia/reperfusion injury (SCII) in rats and their relationship. Methods Seventy adult male Sprague Dawley rats (weighing, 200-220 g) were randomly divided into the sham operated group (sham group, n=35) and the SCII group (n=35). Only the left renal artery was exposed with no occlusion of the abdominal aorta in the rats of sham group. The left renal artery was exposed with occlusion of the abdominal aorta for 20 minutes in the rats of SCII group. At 4, 8, and 12 hours and at 1, 2, 3, and 5 days, reperfusion treatment was performed in 5 rats respectively, and then the spinal cord tissue was harvested to detect the expressions of HSP27, Bcl-2, and Bax protein of the nerve cells by using immunohistochemistry staining. Results The HSP27 began to express at 4 hours, reached the peak at 3 days, and decreased at 5 days in SCII group; significant differences were found between at 3 and 5 days and at the other time points (P lt; 0.05). The Bcl-2 expression increased at 4 hours, reached the peak at 1 day and maintained a high level at 2 days, and then gradually decreased; significant differences were found between at 1 and 2 days and at the other time points (P lt; 0.05). The Bax expression reached the peak at 12 hours and 3 days, and decreased at 5 days; significant differences were found between at 12 hours and 3 days and at the other time points (P lt; 0.05). A little expression of each protein was observed in sham group at different time points; the expressions of HSP27, Bcl-2, and Bax proteins in SCII group were significantly higher than those in sham group at different time points (P lt; 0.05). Conclusion There may be the time window of self repair after SCII. High expression of HSP27 has an obvious protective effect on the SCII in rat, by promoting the expression of the anti-apoptotic protein Bcl-2 and reducing the expression of the pro-apoptotic protein Bax so as to inhibit spinal cord cell apoptosis.
Objective To study the effect of p38MAPK activity on tumor necrosis factor-α （TNF-α） mRNA and intercellular adhesion molecule 1 （ICAM1） mRNA expressions of isolated rabbit liver during early stage of cold preservation and reperfusion period. Methods Based on the cold preservation and reperfusion model of isolated rabbit liver, the animals were divided into inhibition group （n=12） with 3 μmol/L SB202190 (p38MAPK specificity inhibitor) in perfusate and control group （n=12） with no SB202190 in perfusate. Liver tissue samples were harvested at the time points of before resection, end of cold preservation, and different reperfusion period (10, 30, 60 and 120 min). Protein expression and activity of p38MAPK were detected by Western blot and immunoprecipitation respectively, expression of TNF-α mRNA was detected by RT-PCR, and expression of ICAM1 mRNA was detected by in situ hybridization. Results There was no obvious change of expression of p38MAPK protein in liver tissue both in two groups during the total period （P＞0.05）， and there was no statistically significant difference between two groups （P＞0.05）. At time points of end of cold preservation, 10, 30 and 60 min of reperfusion, the activity of p38MAPK in control group was significantly higher than that at the time points of before resection and 120 min of reperfusion (P＜0.01), and was also significantly higher than that in inhibition group at the same time points (P＜0.01). There was no significant difference in activity of p38MAPK among all time points in inhibition group (P＞0.05). The expressions of TNF-α mRNA and ICAM1 mRNA at the time points of before resection, end of cold preservation, and 10 and 30 min of reperfusion were significantly lower than those in 60 and 120 min of reperfusion in both two groups (P＜0.05, P＜0.01); The expressions of TNF-α mRNA and ICAM1 mRNA in inhibition group were significantly lower than those in control group at the time points of 60 and 120 min of reperfusion (P＜0.01). The activity of p38MAPK of liver tissue during cold preservation and reperfusion period was significantly correlated with the level of TNF-α mRNA and level of ICAM1 mRNA expression （r=0.996, P＜0.01； r=0.985, P＜0.01）. Conclusions These results suggest that p38MAPK pathway may regulate the expressions of TNF-α and ICAM1 at the level of transcription and the activation of p38MAPK can up-regulate TNF-α and ICAM1 expressions, which may be one of the important mechanisms to cause ischemia-reperfusion injury of isolated liver during cold preservation and reperfusion period.
ObjectiveTo investigate the effect of power-assisted intravascular shunt in replantation of amputated limbs of rabbits. MethodsEighty rabbits weighing 1.8-2.5 kg (male or female) were selected to establ ish the model of circular amputation at the hind groin, only femoral arteries and veins were completely preserved. After the femoral artery was clamped in 60 rabbits, the rabbits underwent power-assisted intravascular shunt with high-flow rate (group A, n=20), powerassisted intravascular shunt with low-flow rate (group B, n=20), and no power-assisted intravascular shunt (group C, n=20) to reconstruct blood supply; the femoral artery was not clamped in another 20 rabbits of sham group (group D). Before and after intravascular shunt (1, 3, 6, and 12 hours), the malondialdehyde (MDA), lactate dehydrogenase (LDH), and creatine kinase (CK) of the serum were determined. The myeloperoxidase (MPO), MDA, and wet to dry weight ratio (W/D ratio) of the gastrocnemius muscle were measured, and the thrombogenesis and survival rate of limb were observed. ResultsBefore intravascular shunt, MDA, LDH, and CK of the serum and MPO, MDA, and W/D ratio of the muscle showed no significant difference among 4 groups (P>0.05). At each time point after intravascular shunt, no significant difference was found in all indexes between groups A and D (P>0.05); the indexes of groups B and C were significantly higher than those of groups A and D (P<0.05); the values were the highest in group C (P<0.05), and reached the peak at 12 hours. All limbs of group A survived with low thrombosis rate, and less limbs could survive with high thrombosis rate in group C. ConclusionThe power-assisted intravascular shunt with high-flow rate can effective ensure the blood supply of the amputated limbs of rabbits with lower limb injury and higher survival rate of amputated limbs after replantation.
ObjectiveTo explore performances of functional magnetic resonance imaging (MRI) in evaluation of hepatic warm ischemia-reperfusion injury.MethodThe relative references about the principle of functional MRI and its application in the assessment of hepatic warm ischemia-reperfusion injury were reviewed and summarized.ResultsThe main functional MRI techniques for the assessment of hepatic warm ischemia-reperfusion injury included the diffusion weighted imaging (DWI), intravoxel incoherent motion (IVIM), diffusion tensor imaging (DTI), blood oxygen level dependent (BOLD), dynamic contrast enhancement MRI (DCE-MRI), and T2 mapping, etc.. These techniques mainly used in the animal model with hepatic warm ischemia-reperfusion injury currently.ConclusionsFrom current results of researches of animal models, functional MRI is a non-invasive tool to accurately and quantitatively evaluate microscopic information changes of liver tissue in vivo. It can provide a useful information on further understanding of mechanism and prognosis of hepatic warm ischemia-reperfusion injury. With development of donation after cardiac death, functional MRI will play a more important role in evaluation of hepatic warm ischemia-reperfusion injury.
ObjectiveTo investigate the protective effect and the regulation mechanism of oxaloacetate (OAA) on myocardial ischemia reperfusion injury in rats. MethodsSixty rats, weight ranged from 200 to 250 grams, were randomly divided into 6 groups:a negative control group, a sham operation control group, a model control group, an OAA pretreatment myocardial ischemia-reperfusion model group (three subgroups:15 mg/kg, 60 mg/kg, 240 mg/kg). We established the model of myocardial ischemia reperfusion of rats and recorded the internal pressure of left ventricle (LVSP), the maximal rate of left ventricular pressure change (±dp/dtmax) and left ventricular end diastolic pressure (LVEDP). We restored reperfusion 180 minutes after ligating the left anterior descending coronary artery 30 minutes and determinated cardiac troponin Ⅰ (cTn-I), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). We took out heart tissues, stained it and calculated the infarcted size. We used the Western blot to detect the expression of NF-E2 related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1) and heme oxygenase-1 (HO-1). ResultsCompared with the sham operation group, heart function indexes in the negative control group had no significant difference (P>0.05). But in the model control group there was a decrease (P<0.05) And the serum levels of LDH, cTn-I, and myocardial infarcted size were significantly increased (P<0.01). Compared with the model control group, heart function indexes in the OAA pretreatment groups improved, the serum LDH, cTn-I activity, and infarct size decreased (P<0.05), SOD and GSH-Px activity increased (P<0.05). And these results were statistically different (P<0.01) in the high dose OAA pretreatment groups. Compared with the model control group, the expression of Keap1 in the OAA pretreatment group was down-regulated (P<0.001) while total Nrf2, nucleus Nrf2 and its downstream HO-1 was up-regulated (P<0.001), which suggested that OAA enhanced antioxidant capacity by (at least in part) Keap1-Nrf2 pathway, resulting in reducing myocardial damage and protecting myocardium after acute myocardial ischemia reperfusion injury. ConclusionOxaloacetate can provide protective effects on myocardial ischemia reperfusion injury through down-regulating the expression of Keap1 and up-regulating the expression of Nrf2 and its downstream peroxiredoxins to improve antioxidant capacity.
OBJECTIVE: To observe the changes of heme oxygenase-1 (HO-1) expression in the skeletal muscle after ischemia-reperfusion of hind limb in rats. METHODS: A model of hind limb ischemia was made by clamping femoral artery with a microvascular clip. Soleus muscle was obtained from the animals received sham operation, 4 h ischemia without reperfusion and 2 h, 4 h, 8 h, 16 h, 24 h reperfusion after 4 h ischemia. Soleus histology and malondialdehyde (MDA) content were measured. The levels of HO-1 mRNA and protein were measured in different time by Northern blotting, Western blotting and immunohistochemistry technique. RESULTS: After ischemia-reperfusion of limb, HO-1 mRNA increased at the 2nd hour, reached a peak at the 8th hour, and returned toward baseline at the 24th hour. The change of protein level was essentially in agreement with that of mRNA. Immunohistochemical results showed that HO-1 expressed primarily in skeletal muscle cytoplasma. There were no positive signals of mRNA and protein in sham group and in ischemia group. After limb reperfusion, MDA contents in the soleus muscle increased significantly when compared with that in the sham group (P lt; 0.05). MDA content of the 8th after reperfusion decreased significantly when compared with that of the 4 h after reperfusion (P lt; 0.05). CONCLUSION: Ischemia-reperfusion can induce HO-1 expression in skeletal muscle in rats, which may provide protection for injured tissue.
Objective To investigate the effect of N-acetylserotonin (NAS) on the retinal microglia polarization in retinal ischemia-reperfusion injury (RIRI) rats and explore its mechanism via nucleotide-bound oligomeric domain 1 (NOD1)/receptor interacting protein 2 (Rip2) pathway. MethodsHealthy male Sprague Dawley rats were randomly divided into Sham (n=21), RIRI (n=21) and NAS (injected intraperitoneally 30 min before and after modeling with NAS, 10 mg/kg, n=18) groups, using random number table. And the right eye was used experimental eye. The RIRI model of rats in RIRI group and NAS group was established by anterior chamber high intraocular pressure method. Rats in NAS group were intraperitoneally injected with 10 mg/kg NAS before and 30 min after modeling, respectively. The retinal morphology and the number of retinal ganglion cell (RGC) in each group were detected by hematoxylin-eosin staining and immunohistochemical staining. The effect of NAS on polarization of retinal microglia was detected by immunofluorescence staining. Transcriptome sequencing technology was used to screen out the differentially expressed genes between Sham and RIRI groups. Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to examine the differentially expressed genes. Immunohistochemical staining, Western blot and RT-PCR were used to investigate the effect of NAS on the expression of NOD1 and Rip2 protein and mRNA in retinal tissue and microglia of rats. General linear regression analysis was performed to determine the correlation between the number difference of NOD1+ cells and the number difference of M1 and M2 microglia in retinal tissues of rats in NAS group and RIRI group. ResultsA large number of RGC were observed in the retina of rats in Sham group. 24 h after modeling, compared with Sham group, the inner retinal thickness of rats in RIRI group was significantly increased and the number of RGC was significantly decreased. The thickness of inner retina in NAS group was significantly thinner and the number of RGC was significantly increased. Compared with Sham group, the number of retinal microglia of M1 and M2 in RIRI group was significantly increased. Compared with RIRI group, the number of M1 microglia decreased significantly and the number of M2 microglia increased significantly in NAS group. There was statistical significance in the number of M1 and M2 microglia in the retina of the three groups (P＜0.05). Transcriptome sequencing results showed that retinal NOD1 and Rip2 were important differential genes 24 h after modeling. The mRNA and protein relative expressions of NOD1 and Rip2 in retina of RIRI group were significantly higher than those of Sham group, with statistical significance (P＜0.05). The number of NOD1+ and Rip2+ cells and the relative expression of mRNA and protein in retinal microglia in RIRI group were significantly higher than those in Sham group, and NAS group was also significantly higher than that in Sham group, but lower than that in RIRI group, with statistical significance (P＜0.05). The number of Iba-1+/NOD1+ and Iba-1+/Rip2+ cells in retinal microglia in RIRI group was significantly increased compared with that in Sham group, and the number of Iba-1+/Rip2+ cells in NAS group was significantly decreased compared with that in RIRI group, but still significantly higher than that in Sham group, with statistical significance (P＜0.05). Correlation analysis results showed that the difference of retinal NOD1+ and Rip2+ cells in NAS group and RIRI group was positively correlated with that of M1 microglia (r=0.851, 0.895), and negatively correlated with that of M2 microglia (r=?0.797, ?0.819). The differences were statistically significant (P＜0.05). ConclusionNAS can regulate the microglial polarization from M1 to M2 phenotype, the mechanism is correlated with the NOD1/Rip2 pathway.
Objective To study the influence of ischemia-reperfusion on the expression of the hyperpolarization activated cycl icnucleotide gated cation channel 4 (HCN4) and to discuss the mechanism of functional disturbance of sinoatrial node tissue (SANT) after ischemia reperfusion injury (IRI). Methods Eighty five healthy adult rabbits, weighing 2-3 kg, were randomly divided into 3 groups: control group [a suture passed under the root section of right coronary artery (RCA) without l igation, n=5], experimental group A (occluding the root section of RCA for 30 minutes, then loosening the root 2,4, 8 and 16 hours, n=10), experimental group B (occluding the root section of RCA for 1 hour, then loosening the root 2, 4,8 and 16 hours, n=10). At the end of the reperfusion, the SANT was cut off to do histopathological, transmission electronmicroscopical and immunohistochemical examinations and semi-quantitative analysis. Results The result of HE stainingshowed that patho-injure of sinoatrial node cell (SANC) happened in experimental groups A and B after 2 hours of reperfusion, the longer the reperfusion time was, the more serious patho-injure of SANC was after 4 and 8 hours of reperfusion, SANC reached peak of damage after 8 to 16 hours of reperfusion; patho-injure of SANC was more serious in experimental group B than in experimental group A at the same reperfusion time. Immunohistochemical staining showed that the expression of HCN4 located in cellular membrane and cytoplasm in the central area of SANC and gradually decreased from the center to borderl ine. The integral absorbance values of HCN4 expression in the control group (397.40 ± 34.11) was significantly higher than those in the experimental group A (306.20 ± 35.77, 216.60 ± 18.59, 155.40 ± 19.11 and 135.00 ± 12.30) and in the experimental group B (253.70 ± 35.66, 138.70 ± 13.28, 79.10 ± 9.60 and 69.20 ± 8.42) after 2, 4, 8 and 16 hours of reperfusion (P lt; 0.05). With reperfusion time, the expression of HCN4 of SANC decreased, which was lowest after 8 hours of reperfusion; showing significant difference among 2, 4 and 8 hours after reperfusion (P lt; 0.05) and no significant difference between 8 and 16 hours after reperfusion (P gt; 0.05). At the same reperfusion time, the expression of HCN4 was higher in the experimental group A than in the experimental group B. The result of transmission electron microscope showed that ultramicrostructure of SANC was damaged after reperfusion in experimental groups A and B. The longer the reperfusion time was, the more serious ultramicrostructure damage of SANC was, and reached the peak of damage after 8 hours of reperfusion. Ultramicrostructure of SANC was not different between 8 and 16 hours of reperfusion. At the same reperfusion time, the ultramicrostructure damage of SANC was moreserious in experimental group B than in experimental group A. Conclusion IRI is harmful to the morphous and structure ofSANC, and effects the expression of HCN4 of SANC, which is concerned with functional disturbance and arrhythmia.
Objective To investigate the effects of adenosine 2A receptor (A2AR) activation on oxidative stress in small-forsize liver transplantation. Methods A rat orthotopic liver transplantation model was performed using 40% graft, 18 recipients were given intravenously saline (control group), CGS21680 (A2AR agonist, CGS21680 group) or ZM241385 (A2AR antagonist, CGS21680+ZM241385 group) randomly. Aspartate aminotransferase (AST), enzymatic antioxidants 〔superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase (GSH-Px)〕, non-enzymatic antioxidants 〔ascorbic acid (AA); glutathione (GSH); α-tocopherol (TOC)〕 and lipid oxidant metabolites malondialdehyde (MDA) were measured and analyzed at 6 h after reperfusion. Results Compared with the control group and CGS21680+ZM241385 group, A2AR activation increased the activities of SOD and GSHPx (Plt;0.05), reduced the productions of AST and MDA (Plt;0.05), increased the levels of AA, GSH and TOC (Plt;0.05) in CGS21680 group. But there was no significant change in CAT activity (Pgt;0.05) among 3 groups. Conclusions A2AR activation improves the antioxidant enzyme activities, promotes the production of antioxidants, and slowes down the increase in MDA level, depresses of the increase in AST activity. A2AR activation suppresses oxidative damage and increases the antioxidant capacity which in turn minimizes their harmful effects of ischemia-reperfusion in small-for-size liver transplantation.
ObjectiveTo investigate the effect of post-conditioning with fospropofol disodium on hepatic ischemiareperfusion (I/R) and its possible mechanism in rats. MethodsForty-eight Sprague-Dawley rats were randomly divided into four groups, including sham group (S), control group (C), propofol group (P) and fospropofol disodium group (F). According to the different periods after reperfusion, each group was further divided into 2-hour and 4-hour reperfusion subgroups respectively (n=6 in each subgroup), named S2h, C2h, P2h, and F2h subgroups and S4h, C4h, P4h, and F4h subgroups. The livers of rats were reperfused after hepatic ischemia for one hour. In the beginning of reperfusion, normal saline was infused intravenously in group S and group C continuously, propofol was infused intravenously in group P continuously, fospropofol disodium was infused continuously in group F. The blood was sampled at the end of ischemia and reperfusion for assay of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The bcl-2 and bax protein contents in liver tissue were detected by immunohistochemical analysis, and liver samples were stained with hematoxylin-eosine for histological observation and damage degree evaluation by counting the proportion of necrosis cells. ResultsThe activity of ALT and AST, the rate of necrosis cells and the amount of bcl-2 and bax protein after reperfusion in group C, group P and group F were higher than those in group S at matched reperfusion time points (P<0.05). The activity of ALT and AST, the proportion of necrosis cells and bax protein contents decreased in group P and group F, compared with group C at the same reperfusion time points, while the contents of bcl-2 protein were significantly increased (P<0.05). ConclusionFospropofol disodium can alleviate hepatic injury induced by ischemia-reperfusion in rats, in which the bcl-2 and bax protein may play important roles.