Development of a new cerebral ischemia reperfusion model of Mongolian gerbils and standardized evaluation system

Background:The Mongolian gerbil is an excellent laboratory animal for preparing the cerebral ischemia model due to its inherent deficiency in the circle of Willis.However,the low incidence and unpredictability of symptoms are caused by numerous complex variant types of the circle.Additionally,the lack of an evaluation system for the cer-ebral ischemia/reperfusion(I/R)model of gerbils has shackled the application of this model.Methods:We created a symptom-oriented principle and detailed neurobehavioral scoring criteria.At different time points of reperfusion,we analyzed the alteration in locomotion by rotarod test and grip force score,infarct volume by triphenyltetrazo-lium chloride(TTC)staining,neuron loss using Nissl staining,and histological charac-teristics using hematoxylin-eosin(H&E)straining.Results:With a successful model rate of 56%,32 of the 57 gerbils operated by our method harbored typical features of cerebral I/R injury,and the mortality rate in the male gerbils was significantly higher than that in the female gerbils.The suc-cessfully prepared I/R gerbils demonstrated a significant reduction in motility and grip strength at 1 day after reperfusion;formed obvious infarction;exhibited typi-cal pathological features,such as tissue edema,neuronal atrophy and death,and vacuolated structures;and were partially recovered with the extension of reperfu-sion time.Conclusion:This study developed a new method for the unilateral common carotid artery ligation I/R model of gerbil and established a standardized evaluation system for this model,which could provide a new cerebral I/R model of gerbils with more practical applications.

Neuroprotective effects of rutaecarpine on cerebral ischemia reperfusion injury

Rutaecarpine, an active component of the traditional Chinese medicine Tetradium ruticarpum, has been shown to improve myocardial ischemia repeffusion injury. Because both cardiovascular and cerebrovascular diseases are forms of ischemic vascular disease, they are closely related. We hypothesized that rutaecarpine also has neuroprotective effects on cerebral ischemia reperfusion injury. A cerebral ischemia reperfusion model was established after 84, 252 and 504 pg/kg rutae- carpine were given to mice via intrapedtoneal injection, daily for 7 days. Results of the step through test, 2,3,5-triphenyl tetrazolium chloride dyeing and oxidative stress indicators showed that rutae- carpine could improve learning and memory ability, neurological symptoms and reduce infarction volume and cerebral water content in mice with cerebral ischemia reperfusion injury. Rutaecarpine could significantly decrease the malondialdehyde content and increase the activities of superoxide dismutase and glutathione peroxidase in mouse brain. Therefore, rutaecarpine could improve neu- rological function following injury induced by cerebral ischemia reperfusion, and the mechanism of this improvement may be associated with oxidative stress. These results verify that rutaecarpine has neuroprotective effects on cerebral ischemia reperfusion in mice.

New progress in roles of nitric oxide during hepatic ischemia reperfusion injury

Hepatic ischemia reperfusion injury (HIRI) is a clinical condition which may lead to cellular injury and organ dysfunction. The role of nitric oxide (NO) in HIRI is complicated and inconclusive. NO produced by endothelial nitric oxide synthase (eNOS) activation plays a protective role during early HIRI. But eNOS overexpression and the resulting excessive NO bioavailability can aggravate liver injury. NO induced by inducible nitric oxide synthase (iNOS) may have either a protective or a deleterious effect during the early phase of HIRI, but it may protect the liver during late HIRI. Here, we reviewed the latest findings on the role of NO during HIRI: (1) NO exerts a protective effect against HIRI by increasing NO bioavailability, downregulating p53 gene expression, decreasing inflammatory chemokines, reducing ROS via inhibiting the mitochondrial respiratory chain, activating sGC-GTP-cGMP signal pathway to reduce liver cell apoptosis, and regulating hepatic immune functions; (2) eNOS protects against HIRI by increasing NO levels, several eNOS/NO signal pathways (such as Akt-eNOS/NO, AMPK-eNOS/NO and HIF-1 alpha-eNOS/NO) participating in the anti-HIRI process, and inhibiting over-expression of eNOS also protects against HIRI; and (3) the inhibition of iNOS prevents HIRI. Thus, the adverse effects of NO should be avoided, but its positive effect in the clinical treatment of diseases associated with HIRI should be recognized.

Nigella sativa relieves the deleterious effects of ischemia reperfusion injury on liver

AIM: To determine whether Nigella sativa prevents hepatic ischemia-reperfusion injury to the liver. METHODS: Thirty rats were divided into three groups as sham (Group 1), control (Group 2), and Nigella sati-va (NS) treatment group (Group 3). All rats underwent hepatic ischemia for 45 min followed by 60 min period of reperfusion. Rats were intraperitoneally infused with only 0.9% saline solution in group 2. Rats in group 3 received NS (0.2 mL/kg) intraperitoneally, before isch-emia and before reperfusion. Blood samples and liver tissues were harvested from the rats, and then the rats were sacrifi ced. Serum aspartate aminotransfera-se (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were determined. Total antioxidant capacity (TAC), catalase (CAT), total oxida-tive status (TOS), oxidative stress index (OSI) and my-eloperoxidase (MPO) in hepatic tissue were measured. Also liver tissue histopathology was evaluated by light microscopy. RESULTS: The levels of liver enzymes in group 3 weresignifi cantly lower than those in the group 2. TAC in liver tissue was significantly higher in group 3 than in group 2. TOS, OSI and MPO in hepatic tissue were signifi cantly lower in group 3 than the group 2. Histo-logical tissue damage was milder in the NS treatment group than that in the control group. CONCLUSION: Our results suggest that Nigella sa-tiva treatment protects the rat liver against to hepatic ischemia-reperfusion injury.

Effects of Lipoxin A4 Pretreatment on Cognitive Function of Aged Rats after Global Cerebral Ischemia Reperfusion

The aim of the present study was to investigate the effect of lipoxin A4 （LXA4） pretreatment on cognitive function of aged rats after global cerebral ischemia reperfusion, and to explore its possible mechanism. Thirty-six aged male Sprague-Dawley rats were randomly divided into three groups （n=12 each）： sham-operation group （S group）, global cerebral ischemia reperfusion group （I/R group） and LXA4-pretreatment group （L group）. The rat model of global cerebral ischemia reperfusion was established by occlusion of the bilateral common carotid artery with hypotension. The cognitive function of rats was determined by a step-down type passive avoidance test and Morris Water Maze test on the third day after reperfusion. Rats were sacrificed after Water Maze test and the pathological changes ofhippocampal CA1 region were observed and the related inflammatory mediators were determined. As compared with S group, the escape latency in I/R group was prolonged from the first day to the fifth day, while that in L group was prolonged from the first day to the third day. The retention time in I/R group and L group in the first quadrant was shortened. The reaction time, frequency of reaction mistake and frequency of escape mistake in I/R group increased, and the latent period shortened. The frequency of escape mistake in L group increased, and the damage in the hippocampal CAI region of I/R group and L group was obvious. The levels of S-10013, TNF-α, IL-1β, IL-10 and NF-κB in I/R group and L group increased. As compared with I/R group, the escape latency in L group was shortened from the first day to the fifth day, and the retention time in the first quadrant prolonged. The reaction time, frequency of reaction mistake and frequency of escape mistake in L group decreased, and the latent period prolonged. The damage in the hippocampal CA1 region of L group was alleviated as well. The levels of S-10013, TNF-α, IL-1β and NF-κB in L group decreased, and those of IL-10 increased. It can be concluded that LXA4 pretreatment can improve the cognitive function in aged rats after global cerebral ischemia reperfusion probably by inhibiting the inflammatory reaction.

Polyethylene glycols: An effective strategy for limiting liver ischemia reperfusion injury

Liver ischemia-reperfusion injury(IRI) is an inherent feature of liver surgery and liver transplantation in which damage to a hypoxic organ(ischemia) is exacerbated following the return of oxygen delivery(reperfusion). IRI is a major cause of primary nonfunction after transplantation and may lead to graft rejection, regardless of immunological considerations. The immediate response involves the disruption of cellular mitochondrial oxidative phosphorylation and the accumulation of metabolic intermediates during the ischemic period, and oxidative stress during blood flow restoration. Moreover, a complex cascade of inflammatory mediators is generated during reperfusion, contributing to the extension of the damage and finally to organ failure. A variety of pharmacological interventions(antioxidants, anticytokines, etc.) have been proposed to alleviate graft injury but their usefulness is limited by the local and specific action of the drugs and by their potential undesirable toxic effects. Polyethylene glycols(PEGs), which are non-toxic water-soluble compounds approved by the FDA, have been widely used as a vehicle or a base in food, cosmetics and pharmaceuticals, and also as adjuvants for ameliorating drug pharmacokinetics. Some PEGs are also currently used as additives in organ preservation solutions prior to transplantation in order to limit the damage associated with cold ischemia reperfusion. More recently, the administration of PEGs of different molecular weights by intravenous injection has emerged as a new therapeutic tool to protect liver grafts from IRI. In this review, we summarize the current knowledge concerning the use of PEGs as a useful target for limiting liver IRI.

Caffeoylquinic acid derivatives extract of Erigeron multiradiatus alleviated acute myocardial ischemia reperfusion injury in rats through inhibiting NF-kappaB and JNK activations

Erigeron multiradiatus(Lindl.)Benth.,has been used in Tibet folk medicine to treat various inflammatory diseases.The aim of this study was to investigate anti-myocardial ischemia and reperfusion(I/R)injury effect of caffeoylquinic acids derivatives of E.multiradiatus(AE)in vivo and to explain underling mechanism.AE was prepared using the whole plant of E.multiradiatus and contents of 6 caffeoylquinic acid determined through HPLC analysis.Myocardial I/R were induced by left anterior descending coronary artery occlusion for 30 min followed by 24 h of reperfusion in rats.AE administration(10,20 and 40 mg·kg-1)inhibited I/R-induced injury as indicated by decreasing myocardial infarct size,reducing of CK and LDH activities and preventing ST-segment depression in dose-dependent manner.AE decreased cardiac tissue levels of pro-inflammatory factors TNF-αand IL-6 and attenuated leukocytes infiltration.AE was further demonstrated to significantly inhibit I-κB degradation,nuclear translocation of p-65 and phosphorylation of JNK.Our results suggested that cardioprotective effect of AE could be due to suppressing myocardial inflammatory response and blocking NF-κB and JNK activation pathway.Thus,caffeoylquinic acids might be the active compounds in E.multiradiatus on myocardial ischemia and be a potential natural drug for treating myocardial I/R injury.

Pentoxifylline enhances the protective effects of hypertonic saline solution on liver ischemia reperfusion injury through inhibition of oxidative stress

BACKGROUND：Liver ischemia reperfusion（IR）injury triggers a systemic inflammatory response and is the main cause of organ dysfunction and adverse postoperative outcomes after liver surgery.Pentoxifylline（PTX）and hypertonic saline solution（HTS）have been identified to have beneficial effects against IR injury.This study aimed to investigate if the addition of PTX to HTS is superior to HTS alone for the prevention of liver IR injury.METHODS： Male Wistar rats were allocated into three groups. Control rats underwent 60 minutes of partial liver ischemia, HTS rats were treated with 0.4 mL/kg of intravenous 7.5% NaCl 15 minutes before reperfusion, and HPTX group were treated with 7.5% NaC1 plus 25 mg/kg of PTX 15 minutes before reperfusion. Samples were collected after reperfusion for determination of ALT, AST, TNF-α, IL-6, IL-10, mitochondrial respiration, lipid peroxidation, pulmonary permeability and myeloperoxidase. RESULTS： HPTX significantly decreased TNF-α 30 minutes after reperfusion. HPTX and HTS significantly decreased ALT,AST, IL-6, mitochondrial dysfunction and pulmonary myelo- peroxidase 4 hours after reperfusion. Compared with HTS only, HPTX significantly decreased hepatic oxidative stress 4 hours after reperfusion and pulmonary permeability 4 and 12 hours after reperfusion. CONCLUSION： This study showed that PTX added the beneficial effects of HTS on liver IR injury through decreases of hepatic oxidative stress and pulmonary permeability.

Role of NF-kappaB in Liver Ischemia Reperfusion Injury of Rats

To determine the role of NF-kappaB in ischemia reperfusion (I/R) injury of the rat liver, rats underwent partial hepatic ischemia and reperfusion. The left and median lobes of the liver were subjected to ischemia for 90 min followed by reperfusion for defined times. NF-kappaB activity was analyzed by electrophoretic mobility shift assay (EMSA). Semiquantitative reverse-transcriptase polymerase chain reaction was used to analyze TNF-α and ICAM-1 mRNA levels. Results showed during liver I/R injury, NF-kappaB activation was induced in a time dependent manner. NF-kappaB was activated within 1h and 2h after the initiation of reperfusion and decreased after 4 h. Messenger RNA expression of TNF-α and ICAM-1 were increased after the reperfusion of 2 h. It was concluded that during hepatic I/R injury, NF-kappaB was activated and could bind to special sequence in the promoters of budget genes, which can up-regulate the expression of TNF-α and ICAM-1 mRNA to result in ischemia reperfusion injury of the rat liver.

Role of Nuclear Factor kappaB in Intestine Injury Induced by Hepatic Ischemia Reperfusion

Summary: The role of nuclear factor kappaB in intestine injury induced by hepatic ischemia reperfusion was investigated. Eighteen male Wistar rats were divided into 3 groups randomly: sham operation group (group A), hepatic ischemia reperfusion group (group B) and hepatic ischemia reperfusion plus pyrrolidine dithiocarbamate (PDTC) group (group C). The rats in group A were only subjected to laparotomy, those in group B underwent partial hepatic ischemia reperfusion (ischemia for 1 h and reperfusion for 2 h) and those in group C underwent the same procedure as that of group B but received PDTC 200 mg/kg i.v. before and after ischemia. After reperfusion, tissues of jejunum and venous blood were obtained for measurement of TNF-α, MDA and MPO. The levels of TNF-α in jejunum and venous blood, the levels of MPO in jejunum in group B were significantly higher than those in group A and group C (P<0.05). There was no significant different in the levels of MDA between group B and group C. The severity of histological intestinal injury in group B and group C was similar. Hepatic ischemia reperfusion caused intestine injury, NF-kappaB may play an important role in this course and the targeting of upstream components of the inflammatory response, such as NF-kappaB, may have important therapeutic applications.

Fructose 1,6-diphosphate alleviates myocardial ischemia reperfusion injury in rats through JAK2/STAT3 pathway

Objective: To study the effect of fructose 1,6-diphosphate(FDP) on myocardial ischemia reperfusion injury in rats and its molecular mechanism.Methods: Male SPF SD rats were selected as experimental animals and randomly divided into four groups.Sham group received sham operation, I/R group were made into myocardial ischemia reperfusion injury models, FDP group were made into myocardial ischemia reperfusion injury models and then were given FDP intervention, and FDP+AG490 group were made into myocardial ischemia reperfusion injury models and then were given FDP and JAK2 inhibitor AG490 intervention.Results: CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of I/R group were significantly higher than those of Sham group whereas Bcl-2, p-JAK2 and p-STAT3 protein expression in myocardial tissues were significantly lower than those of Sham group; CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of FDP group were significantly lower than those of I/R group whereas Bcl-2, p-JAK2 and p-STAT3 protein expression in myocardial tissue were significantly higher than those of I/R group; CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of FDP+AG490 group were significantly higher than those of FDP group whereas Bcl-2 protein expression in myocardial tissue was significantly lower than that of FDP group.Conclusion: FDP could reduce the myocardial ischemia reperfusion injury in rats by activating the JAK2/STAT3 pathway.

Protective Effects of Epigallocatechin-3-gallate on Intestinal Ischemia Reperfusion Injury through Enhanced Activation of PI3K/Akt Pathway in Rats

Inflammation plays a critical role in intestinal ischemia reperfusion injury（IRI）. Epigallocatechin-3-gallate（EGCG） has been demonstrated to possess anti-inflammatory effect. This study examined the effect of EGCG on intestinal IRI and explored the possible mechanisms. Male Wistar rats were randomly divided into three groups： sham-operated group（Sham）, IRI control group（IRI） and IRI-EGCG group（EGCG）. Rats in IRI-EGCG group were administered dissolved EGCG in drinking water（0.4 mg/m L） for 14 days prior to IRI induction. A rat model of intestinal IRI was established by ligating the superior mesenteric artery（SMA） for 30 min, followed by reperfusion for 1 h. Intestinal histology, pro-inflammatory cytokines and mediators were examined and the effect of EGCG on PI3K/Akt signalling was assessed. EGCG significantly alleviated the pathological changes of the intestine and suppressed the IRI-induced up-regulation of TNF-α, IL-1 and IL-6 m RNA and protein expression in the serum and intestine. The mechanism might be that EGCG enhanced the activation of PI3K/Akt signalling pathway. In conclusion, the administration of EGCG can significantly mitigate the acute intestinal IRI in rats by enhancing the activation of PI3K/Akt signalling pathway to suppress inflammatory response and might be a promising alternative for the prevention or treatment of intestinal IRI in the clinical practice.

Effects of “Nourishing Liver and Kidney” Acupuncture Therapy on Expression of Brain Derived Neurotrophic Factor and Synaptophysin after Cerebral Ischemia Reperfusion in Rats

The aim of the present study was to investigate the effect of ＂nourishing liver and kidney＂ acupuncture therapy on motor and cognitive deficits,and the underlying mechanism following cerebral ischemia-reperfusion（I/R） via increasing the expression of brain derived neurotrophic factor（BDNF） and synaptophysin（SYN） in the hippocampus.Healthy adult male SD rats were randomly divided into sham operation group（n=51）,model group（n=51）,acupuncture group（n=51） and acupuncture control group（n=51）.The middle cerebral I/R model was established.Acupunctures were performed in the acupuncture group and acupuncture control group at acupoints of Taixi（K103）,Taichong（ST09） of both sides,for 30 min once daily every morning.The animals in the sham operation group and model group were conventionally fed in the cage,without any intervention therapy.The rats of each group were assessed with modified neurological severity scores（m NSS）.The expression of BDNF and SYN in the hippocampus was detected by immunohistochemical SP method and the synaptic structure in hippocampus area was assessed morphologically and quantitatively at the 3rd,7th and 14 th day.The Morris water Maze（MWM） test was used to evaluate the rats＇ learning and memory abilities on the 15 th day after acupuncture.The animals in the acupuncture control group and sham operation group presented no neurological deficit.In the acupuncture group,the nerve functional recovery was significantly better than that in the model group at the 7th and 14 th day after modeling.The average MWM escape latency in the acupuncture group was shorter than that in the model group at the 3rd,4th and 5th day.The number of crossings of the platform quadrant in the acupuncture group was significantly more than that in the model group.At the each time point,the expression levels of BDNF and SYN in the hippocampal regions increased significantly in the model group as compared with the sham operation group and the acupuncture control group.In the acupuncture group,the expression levels of BDNF at the 7th and 14 th day increased more significantly than those in the model group.In the acupuncture group,the expression levels of SYN at the each time point increased more significantly than those in the model group.The post-synaptic density（PSD） was significantly increased and the synapse cleft width was narrowed in the acupuncture group as compared with other groups.The synaptic curvatures were improved obviously in the acupuncture group in contrast to the model group.It was concluded that the ＂nourishing liver and kidney＂ acupuncture therapy has positive effects on behavioral recovery,as well as learning and memory abilities,probably by promoting the expression of BDNF and SYN,and synaptic structure reconstruction in the ipsilateral hippocampus after I/R in rats.The ＂nourishing liver and kidney＂ acupuncture therapy can promote the functional recovery in rats after cerebral ischemia injury.

Scutellarin Reduces Cerebral Ischemia Reperfusion Injury Involving in Vascular Endothelium Protection and PKG Signal

Flavonoid glycoside scutellarin(SCU)has been widely applied in the treatment of cerebral ischemic diseases in China.In this article,we conducted research on the working mechanisms of SCU in hypoxia reoxygenation(HR)injury of isolated cerebral basilar artery(BA)and erebral ischemia reperfusion(CIR)injury in rat models.In isolated rat BA rings,HR causes endothelial dysfunction(ED)and acetylcholine(ACh)induces endothelium-dependent vasodilation.The myography result showed that SCU(100μM)was able to significantly improve the endothelium-dependent vasodilation induced by Ach.However,SCU did not affect the ACh-induced relaxation in normal BA.Further studies suggested that SCU(10-1000μM)dose-dependently induced relaxation in isolated BA rings which were significantly blocked by the cGMP dependent protein kinase(PKG)inhibitor Rp-8-Br-cGMPs(PKGI-rp,4μM).Pre-incubation with SCU(500μM)reversed the impairment of endothelium-dependent vasodilation induced by HR,but the reversing effect was blocked if PKGI-rp(4μM)was added.The brain slice staining test in rats’model of middle cerebral artery occlusion(MCAO)induced CIR proved that the administration of SCU(45,90 mg/kg,iv)significantly reduced the area of cerebral infarction.The Western blot assay result showed that SCU(45 mg/kg,iv)increased brain PKG activity and PKG protein level after CIR surgery.In conclusion,our findings suggested that SCU possesses the ability of protecting brain cells against CIR injury through vascular endothelium protection and PKG signal.

Apoptosis of motor neurons in the spinal cord after ischemia reperfusion injury delayed paraplegia in rabbits

Objective To clarify the pathologic change of the motor neuron on spinal cord ischemia reperfusion injury delayed paraplegia.Methods The infrarenal aorta of White New Zealand rabbits(n=24) was occluded for 26 minutes using two bulldog clamps.Rabbits were killed after 8,24,72,or 168 hours(n=6 per group),respectively.The clamps was placed but never clamped in sham-operated rabbits(n=24).The lumbar segment of the spinal cord(L5 to L7) was used for morphological studies,including hematoxylin and eosin staining,the expression of bcl-2 and bax proteins in spinal cord was detected with immunohistochemistry.The apoptotic neurons in spinal cord were measured with terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling of DNA fragments(TUNEL) staining.Results Delayed paraplegia occurred in all rabbits of ischemia reperfusion group at 16-24 hours,but not in sham groups.Motor neurons were selectively lost at 7 days after transient ischemia.After ischemia,the positive expression of bcl-2 protein were in the sham controls but decreased significantly as compared with that of the IR group(P<0.01),especially in 72 hours reperfusion.The positive expression of bax protein were also in the sham controls, but increased in the IR group,especially in 72 hours reperfusion;In addition, TUNEL study demonstrated that no cells were positively labeled until 24 hours after ischemia,but nuclei of some motor neurons were positively labeled at peak after ischemia reperfusion at 72 hours.Conclusion Spinal cord ischemia in rabbits induces morphological and biochemical changes suggestive of apoptosis.These data raise the possibility that apoptosis contributes to neuronal cell death after spinal cord ischemia reperfusion.

Upregulated expression of S100A8 in mice brain after focal cerebral ischemia reperfusion

BACKGROUND:Recent studies have showed that S100A8 has been implicated in the pathobiology of inflammatory disorders,and that cerebral ischemia reperfusion(l/R) rapidly activates inflammation responses via Toll-like receptor 4(TLR4).This study aimed to explore the expression of S100A8 and the relationship between S100A8 and TLR4 in focal cerebral ischemia reperfusion injury.METHODS:C3H/HeJ mice(n=30) and C3H/HeN mice(n=30) were divided randomly into a C3H/HeJ model group(n=18),a C3H/HeJ control group(n=12),a C3H/HeN model group(n=18),and a C3H/HeN control group(n=12).Middle cerebral artery l/R model in mice was produced using a thread embolism method.The brains of the mice were collected after ischemia for 1 hour and reperfusion for 12 hours.Stroke outcome was evaluated by determination of infarct volume and assessment of neurological impairment scores.Brain injury after cerebral l/R was observed by an optical microscope after TTC and HE dyeing.The immunofluorescence technique and real time PCR were used to test the expression level of S100A8 in brain damage.RESULTS:Compared with C3H/HeN mice,TLR4-deficient mice(C3H/HeJ) had lower infarct volumes and better outcomes in neurological tests.The levels of S100A8 increased sharply in the brains of mice after l/R injury.In addition,mice that lacked TLR4(C3H/HeJ) had lower expression of l/R-induced S100A8 than C3H/HeN mice in the model group,indicating that a close relationship might exist between the levels of S100A8 and TLR4.CONCLUSION:S100A8 interaction with TLR4 might be involved in brain damage and in inflammation triggered by l/R injury.

Effects of phycocyanin on apoptosis and expression of superoxide dismutase in cerebral ischemia reperfusion injury

BACKGROUND ： The application of exogenous antioxidant is always the focus in the prevention and treatment of cerebral ischemia. Phycocyanin has the effects against oxidation and inflammation, but its role in the pathophysiological process of cerebral ischemia reperfusion injury still needs further investigation. OBJECTIVE： To observe the effects of phycocyanin on the expression of superoxide dismutase （SOD） apoptosis and form of the nerve cells in rats after cerebral ischemia reperfusion injury. DESIGN： A randomized control animal experiment SETTING ： Institute of Cerebrovascular Disease, Medical School Hospital of Qingdao University MATERIALS： Fifty-two healthy adult male Wistar rats of clean degree, weighing 220-260 g, were used. Phycocyanin was provided by the Institute of Oceanology, Chinese Academy of Sciences. METHODS： The experiments were carried out in Shangdong Key Laboratory for Prevention and Treatment of Brain Diseases from May to December 2005. ① All the rats were divided into three groups according to the method of random number table： sham-operated group （n=4）, control group （n=24） and treatment group （n=24）. Models of middle cerebral artery occlusion/reperfusion （MCAO/R） were established by the introduction of thread through external and internal carotid arteries in the control group and treatment group. After 1-hour ischemia and 2-hour reperfusion, rats in the treatment group were administrated with gastric perfusion of phy- cocyanin suspension （0.1 mg/g）, and those in the control group were given saline of the same volume, and no treatment was given to the rats in the sham-operated group. ②The samples were removed and observed at ischemia for 1 hour and reperfusion for 6 and 12 hours and 1, 3, 7 and 14 days respectively in the control group and treatment group, 4 rats for each time point, and those were removed at 1 day postoperatively in the sham-operated group. Forms of the nerve cells were observed with toluidine blue staining. Apoptosis after cerebral ischemia reperfusion was detected with TUNEL technique. SOD expression was detected with immunohistochemical technique.③ The intergroup difference was compared with the ttest. MAIN OUTCOME MEASURES： The apoptosis of the nerve cells and SOD expression were mainly observed in each group. RESULTS： Finally, 52 rats were involved in the analysis of results. ① Number of apoptotic cells： In the sham-operated group, a few apoptotic cells could be observed in brain tissue. The apoptotic cells at each time point in the control group and treatment group were obviously more than those in the sham-operated group （P 〈 0.05）. In the treatment group, the numbers of apoptotic cells at 12 hours, 1 and 3 days after reperfusion were significantly fewer than those in the control group, and those at 6 hours, 7 and 14 days were similar to those in the control group. ② Number of SOD positive cells： In the sham-operated group, there was weak expression of SOD in brain tissue, and the positive cells were extremely few, the positive cells at each time point were significantly more in the control group and treatment group than in the sham-operated group （P 〈 0.05）. In the treatment group, the numbers of positive cells at 6 and 12 hours, 1 and 3 days after reperfusion were significantly fewer than those in the control group, and those at 7-14 days were similar to those in the control group. ③ Cellular forms： In the control group, the karyopyknosis occurred in the nerve cells, which were irregularly distributed, nucleolus disappeared, and some scattered cell fragments were observed. The forms of the nerve cells in the treatment group were generally normal. CONCLUSION ： Phycocyanin plays a neuroprotective role in cerebral ischemia reperfusion injury by activating the SOD expression and inhibiting apoptosis.

Effect and mechanism of adrenomedullin on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury

Objective To investigate the effect and mechanism of adrenomedullin ( AM ) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group,IRI group, empty plasmid group and AM group. One week after re-

Molecular mechanisms of liver ischemia reperfusion injury:Insights from transgenic knockout models

Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation,as well as expanding the potential pool of usable donor grafts.The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes,increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis.Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury.IRI involves a complex interplay between neutrophils,natural killer T-cells cells,CD4+ T cell subtypes,cytokines,nitric oxide synthases,haem oxygenase-1,survival kinases such as the signal transducer and activator of transcription,Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways.Transgenic animals,particularly genetic knockout models,have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies.Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein.This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.

Protective effect of hyperoside on cardiac ischemia reperfusion injury through inhibition of ER stress and activation of Nrf2 signaling

Objective:To study the protective effect of hyperoside(Hyp) on cardiac ischemia reperfusion injury and its potential mechanism.Methods:Rats were divided into two groups for the evaluation,the Hyp(50 uM Hyp;n=8) and the control group(n=8).Rat hearts were isolated and perfused with Krebs-Henseleit buffer(KHB) for 30 min.After being inhibited with cardioplegic solution,they were stored for 4 h in B21 solution at 4℃.Afterwards,rat hearts were perfused with KHB again for 45 min.In this period.Hyp was added into solutions of cardioplegia for storage and KHB.Parameters of cardiac functions,including heart rate,the systolic pressure of the left ventricle,the end-diastolic pressure of the left ventricle,the developed pressure of the left ventricle,the left-ventricular systolic pressure and the peak rise rate of the pressure of the left ventricle were recorded.The levels of adenosine triphosphate(ATP),the content of malondialdehyde and apoptotic cells were determined to evaluate the protective effect of Hyp on hearts suffered from ischemia reperfusion injury.Moreover,cultured cardiac myocytes were subjected to the process simulating ischemia/reperfusion.What were analyzed included the endoplasmic reticulum(ER) stress hallmarks expressions,such as binding immunoglobulin protein and C/EBP homologous protein,using the western blot and real-time PCR.Besides,the NF-E2-related factor 2(Nrf2) expression was measured to explore the potential mechanism.Results:Compared with the control group,the Hyp group had better cardiac functional parameters and higher ATP levels;pretreatment of Hyp greatly relieved the apoptosis of myocyte,decreased oxidative stress as well as ER stress and activated the signaling pathway of anti-oxidative Nrf2 to a further extent.Conclusions:Hyp plays an important role in preserving cardiac function by improving ATP levels of tissue,easing oxidative injury of myocardium and reducing apoptosis following IRI dramatically,while the ER stress inhibition and the downstream Nrf2 signaling activation may contribute to the effects of protection.