DSC examination of intestinal tissue following warm ischemia and reperfusion injury

The fact that small bowel is extremely sensitive to ischemia/reperfusion (I/R) injury had encouraged us to compare the conventional histology and differential scanning calorimetry (DSC) methods in intestinal structural changes following experimental warm I/R models. Our histological findings showed that longer warm I/R period caused more severe damage in structure of mucosa and crypts, but there were no changes in the muscular layer. According to our DSC data (transition temperature, calorimetric enthalpy) suggest that the thermal destruction of mucosa, muscular layer and total intestinal wall following I/R injury revealed significant differences compared to normal bowel structure.     

Ischemic postconditioning protects cardiomyocytes against ischemia/reperfusion injury by inducing MIP2

Cardiomyocytes can resist ischemia/reperfusion (I/R) injury through ischemic postconditioning (IPoC) which is repetitive ischemia induced during the onset of reperfusion. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a member of the WD-40 family proteins, we previously showed that MIP2 was up-regulated during ischemic preconditioning (IPC). As IPC and IPoC engaged similar molecular mechanisms in cardioprotection, this study aimed to elucidate whether MIP2 was up-regulated during IPoC and contributed to IPoC-mediated protection against I/R injury. The experiment was conducted on two models, an in vivo open chest rat coronary artery occlusion model and an in vitro model with H9c2 myogenic cells. In both models, 3 groups were constituted and randomly designated as the sham, I/R and IPoC/hypoxia postconditioning (HPoC) groups. In the IPoC group, after 45 min of ischemia, hearts were allowed three cycles of reperfusion/ischemia phases (each of 30 s duration) followed by reperfusion. In the HPoC group, after 6 h of hypoxia, H9c2 cells were subjected to three cycles of 10 minute reoxygenation and 10 minute hypoxia followed by reoxygenation. IPoC significantly reduced the infarct size, plasma level of Lactate dehydrogenase and creatine kinase MB in rats. 12 h after the reperfusion, MIP2 mRNA levels in the IPoC group were 10 folds that of the sham group and 1.4 folds that of the I/R group. Increased expression of MIP2 mRNA and attenuation of apoptosis were similarly observed in the HPoC group in the in vitro model. These effects were blunted by transfection with MIP2 siRNA in the H9c2 cells. This study demonstrated that IPoC induced protection was associated with increased expression of MIP2. Both MIP2 overexpression and MIP2 suppression can influence the IPoC induced protection.     

TRIM45 causes neuronal damage by aggravating microglia-mediated neuroinflammation upon cerebral ischemia and reperfusion injury

Excessive and unresolved neuroinflammation is a key component of the pathological cascade in brain injuries such as ischemic stroke. Tripartite motif-containing 45 (TRIM45) is a ubiquitin E3 ligase involved in various critical biological processes. However, the role of TRIM45 in cerebral ischemia remains unknown. Here, we found that the TRIM45 protein was highly expressed in the peri-infarct areas of mice subjected to cerebral ischemia and reperfusion injury induced by middle cerebral artery occlusion. This study systemically evaluated the putative role of TRIM45 in the regulation of neuroinflammation during ischemic injury and the potential underlying mechanisms. We found that TRIM45 knockdown significantly decreased proinflammatory cytokine and chemokine production in primary cultured microglia challenged with oxygen-glucose deprivation and reoxygenation (OGD/R) treatment. Mechanistically, we demonstrated that TRIM45 constitutively interacted with TAB2 and consequently facilitated the Lys-63-linked polyubiquitination of TAB2, leading to the formation of the TAB1–TAK1–TAB2 complex and activation of TAK1, which was ultimately followed by activation of the nuclear factor-kappa B (NF-κB) signaling pathway. In an in vitro coculture Transwell system, downregulation of TRIM45 expression also inhibited the OGD/R-induced activation of microglia and alleviated neuronal apoptosis. More importantly, microglia-specific knockdown of TRIM45 in mice significantly reduced the infarct size, mitigated neurological deficit scores, and improved cognitive function after ischemic stroke. Taken together, our study reveals that the TRIM45–TAB2 axis is a crucial checkpoint that controls NF-κB signaling in microglia during cerebral ischemia and reperfusion injury. Therefore, targeting TRIM45 may be an attractive therapeutic strategy.Subject terms: Cell death in the nervous system, Stroke     

养阴通脑颗粒中黄酮对脑缺血再灌注损伤保护作用的实验研究

为了进一步探讨养阴通脑颗粒中有效成分治疗缺血性脑血管病的作用及其作用机理.我们开展了黄酮对大鼠脑缺血再灌注损伤保护作用研究.现将其对脑缺血再灌注损伤大鼠抗凝、纤溶等指标的影响报告如下.     

18F-annexin V apoptosis imaging for detection of myocardium ischemia and reperfusion injury in a rat model

¹⁸F-annexin V was labeled with ¹⁸F-SFB, after size-column exclusion chromatography, the radiochemical purity exceeded 95 %. High focal uptake of ¹⁸F-annexin V was visualized in myocardium ischemia–reperfusion injury (RI) area of rat models, which was consistent with autoradiography, the standard uptake values (SUV) of RI was 7.93 ± 3.85, whereas remote viable myocardium was 1.02 ± 0.19 (p < 0.001). Histological staining and flow cytometry confirmed ongoing apoptosis in RI. Activated caspase-3, cells in sub-G0 phase, apoptotic cells measuring with FITC-annexin V in RI was 23.04 ± 2.25, 4.62 ± 1.96, 8.84 ± 1.25 respectively, whereas activated caspase-3 in remote viable myocardium was only 3.49 ± 1.83 (p < 0.001). Therefore, ¹⁸F-annexin V can be serve as a candidate for the early detection of myocardial ischemia and injury.     

Ginsenoside Rb1 attenuates intestinal ischemia reperfusion induced renal injury by activating Nrf2/ARE pathway

Intestinal ischemia reperfusion (IIR) is a serious clinical condition associated with simultaneous multiple organ dysfunction. The aim of this study was to investigate the effects of ginsenoside Rb1 on IIR induced renal injury in mice. An intestinal ischemia reperfusion mouse model was established by superior mesenteric artery (SMA) occlusion for 45 min, followed by reperfusion for 2 h. IIR induced renal injury characterized by increase of BUN, Cr and NGAL in serum, MDA levels and decrease of SOD levels in the renal tissues. Ginsenoside Rb1 (30, 60 mg/kg) given intraperitoneally before reperfusion attennuated renal injury, which was associated with decrease of BUN, Cr and NGAL in serum, MDA levels and increase of SOD levels in the renal tissues. Furthermore, the immunohistochemistry and Western blot data showed that ginsenoside Rb1 dramatically reversed IIR induced renal injury, associated with upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in renal tissues. Our data suggests that ginsenoside Rb1 attenuates acute renal injury induced by intestinal ischemia reperfusion by activating the Nrf2/ARE pathway.     

The role of molecular chaperonins in warm ischemia and reperfusion injury in the steatotic liver: A proteomic study

ABSTRACT: BACKGROUND: The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software. RESULTS: The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R. CONCLUSION: Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.     

Effect of phalloidin on the skeletal muscle ADP-actin filaments

The effect of phalloidin on the thermal stability of skeletal actin filaments polymerized from ADP-binding monomers was investigated with the method of differential scanning calorimetry. Phalloidin shifted the melting temperature of the ADP-F-actin from 59.1±1.0 to 80.0±1.2°C. The stabilizing effect of phalloidin propagated cooperatively along the filament. The cooperativity factor according to the applied model was 1.07±0.11. With these measurements it was possible to demonstrate that the binding of phalloidin has lower influence on the adjacent protomers in ADP- (k=1) than in ATP-actin filaments (k=3).     

Influence of pectins on preparation characteristics of lactoferrin bioadhesive tablets

For the treatment of chronic inflammation in the oral cavity, we attempted to develop bioadhesive tablets of bovine lactoferrin (B-LF). Pectin was used as a bioadhesive polymer, and the influence of the degree of esterification and the molecular weight of pectins on the characteristics of B-LF tablets were investigated. Concerning bioadhesive force, a tendency increasing the value according to increase of the esterification of the pectin was confirmed. Sustained release of B-LF from the tablets was observed as the esterification increased, and a possibility for prediction of the time required to release 50% of B-LF by using the equation given by the degree of esterification and the logarithm of the molecular weight was suggested. Pectin cross-linked with Ca(2+) (Ca-PC) was also used for the preparation of the B-LF tablets. Prolonged release of B-LF from the tablets was observed as the Ca(2+) in Ca-PC increased. Our findings suggest that pectin with a high degree of esterification is suitable as a bioadhesive polymer since high bioadhesive force and sustained release are shown. Furthermore, a possibility that the B-LF release could be controlled by adjusting the Ca(2+) concentration in Ca-PC was suggested.     

Purification of rabbit skeletal muscle troponin C

Troponin C binds to phenyl-Sepharose in the presence of Ca2+ and can be eluted with EDTA. This property was used as an essential step in the purification of this protein from rabbit skeletal muscle. Troponin C was extracted with 6M urea from extensively washed ground muscle. The protein was bound to and eluted from DEAE-Sephadex, fractionated by size on Sephadex G75, and in a final step purified from UV-absorbing non-protein impurities on phenyl-Sepharose. The total yield of electrophoretically pure protein was 60 mg per 100 g of muscle, which is considerably higher than that previously obtained.     

Synthesis and protective effect of scutellarein on focal cerebral ischemia/reperfusion in rats

Scutellarein, the main metabolite of scutellarin in vivo, has relatively better solubility, bioavailability and bio-activity than scutellarin. However, compared with scutellarin, it is very difficult to obtain scutellarein from Nature. Therefore, the present study focused on establishing an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin. Neurological deficit score and cerebral infarction volume with the administration of scutellarein were then used to compare its neuroprotective effects on focal cerebral ischemia/reperfusion in rats induced by middle cerebral artery occlusion (MCAO) with those of scutellarin. The results showed that scutellarein had better protective effect on focal cerebral ischemia/reperfusion than scutellarin, which laid the foundation for further research and development of scutellarein as a promising candidate for ischemic cerebro-vascular disease.     

Electrophoretic analysis of electrically trained skeletal muscle.

Sheep latissimus dorsi muscle was electrically trained, thereby inducing fast-to-slow fibre-type transformation. Using a combination of one- and two-dimensional gel electrophoresis techniques with computer analysis, we have analysed altered expression of contractile protein isoforms at protein and mRNA level over a time course of electrical training extending to 5 months. Myosin heavy chain and regulatory myosin light chain analysis showed predominant expression of their slow isoforms (86% and 92%, respectively) after 3 months of training. At the same time point, however, tropomyosin analysis revealed that the slow isoform of the alpha-subunit accounted for 64% of the total alpha expression. Troponin T isoform switching proceeded more slowly over the same time course than tropomyosin and the thick filament proteins studied. Troponin T analysis revealed 5 fast and 2 slow isoforms in the sheep, of which the second slow isoform only became clearly visible after 5 months' training. At this time point the two slow isoforms were more predominant than their fast counterparts. This suggests that a wide heterogeneity of fast and slow isoform combinations are possible in the thin filament of skeletal muscle.     

Infrared (810 nm) low-level laser therapy in experimental model of strain-induced skeletal muscle injury in rats: effects on functional outcomes

Muscle strains are among the most prevalent causes for athletes' absence from sport activities. Low-level laser therapy (LLLT) has recently emerged as a potential contender to nonsteroidal anti-inflammatory drugs in muscle strain treatment. In this work we investigated effects of LLLT and diclofenac on functional outcomes in the acute stage after muscle strain injury in rats. Muscle strain was induced by overloading the tibialis anterior muscle of rats during anesthesia. The injured groups received either no treatment, or a single treatment with diclofenac 30 min prior to injury, or LLLT (810 nm, 100 mW) with doses of 1, 3, 6 or 9 J, at 1 h after injury. Functional outcome measures included a walking index and assessment of electrically induced muscle performance. All treatments (except 9 J LLLT) significantly improved the walking index 12 h postinjury compared with the untreated group. The 3 J group also showed a significantly better walking index than the drug group. All treatments significantly improved muscle performance at 6 and 12 h. LLLT dose of 3 J was as effective as the pharmacological agent in improving functional outcomes in the early phase after a muscle strain injury in rats.     

Spatiotemporally controlled contraction of micropatterned skeletal muscle cells on a hydrogel sheet

We have developed gel sheet-supported C(2)C(12) myotube micropatterns and combined them with a microelectrode array chip to afford a skeletal muscle cell-based bioassay system. Myotube line patterns cultured on a glass substrate were transferred with 100% efficiency to the surface of fibrin gel sheets. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT). We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell.     

Cyclophosphamide treatment evoked side effects on skeletal muscle monitored by DSC

Journal of Thermal Analysis and Calorimetry - Polyneuropathy is defined as a simultaneous malfunction of several peripheral nerves, which could be a side effect of cancer therapy as well. Many...     

Influence of internal structure on kinetics of drug release from wax matrix tablets

To examine the influence of the internal structure of a wax matrix tablet on in vitro drug release, the release rates of several tablets consisting of various proportions of drug and wax were compared with the water penetration rates from the compressed and lateral surfaces of the tablets. The penetration rates from the lateral surface were found to be much faster than those from the compressed surface in all cases. A theoretical equation involving a two-dissolving-direction was derived on the basis of the boundary retreating concept. The retreating rate constants deduced from the dissolution results were well coincident with the values directly determined by the needle penetration method, suggesting good applicability of the proposed equation. The results suggest that the tortuosity of the water channels created in a tablet during dissolution is generally smaller in the horizontal direction than that in the vertical direction. This would be caused by the drug particles or granules being elongated in the horizontal direction by compression.     

Effect of Ginkgo biloba extract 50 on immunity and antioxidant enzyme activities in ischemia reperfusion rats

The aim of the study was to investigate the effect of Ginkgo biloba extract 50 (GBE50), a well-known natural antioxidant, against immunity and antioxidant enzyme activities in ischemia reperfusion (IR) rats. Rats were then divided into six groups fed for 15 days with the same diet: three groups (IV, V, VI) were treated by different doses of GBE50 suspension [20, 40, or 60 mg/kg body weight by oral gavage every day at a fixed time (10.00 a.m.)] (equal to 5, 10 and 20 times, respectively, the maximum recommended human dose), and three groups (I, II, III) were untreated. At the end of the experiment, rats' hearts were subjected to 30 min of ischemia followed by 90 min of reperfusion. Results showed that IR significantly enhanced heart rate, S-T height, myocardium (myeloperoxidase) MPO activity and blood interleukin-8 (IL-8), tumor necrosis factor Alpha (TNF-a), interleukin-1β (IL-1β) levels, blood aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatinine kinase (CK) activities, reduced myocardium sodium-potassium adenosine triphosphatase (Na(+)-K(+)-ATPase), calcium-magnesium adenosine triphosphatase (Ca(2+)-Mg(2+)-ATPase) activities and antioxidant enzyme activities in IR group (III) compared to sham control group (II). Pretreatment of GBE50 markedly significantly reduced heart rate, S-T height, myocardium MPO activity and blood IL-8, TNF-a, IL-1β levels, blood AST, LDH, and CK activities, enhanced myocardium Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase activities and antioxidant enzyme activities in IR group (II) compared to IR group (III). The results suggested that the GBE50 may reduce the oxidative stress in the reperfused myocardium, and increased immunity and antioxidant activities in IR rats.     

Effect of laser phototherapy on wound healing following cerebral ischemia by cryogenic injury

Laser phototherapy emerges as an alternative or auxiliary therapy for acute ischemic stroke, traumatic brain injury, degenerative brain disease, spinal cord injury, and peripheral nerve regeneration, but its effects are still controversial. We have previously found that laser phototherapy immunomodulates the response to focal brain damage. Following direct cortical cryogenic injury the effects of laser phototherapy on inflammation and repair was assessed after cryogenic injury (CI) to the central nervous system (CNS) of rats. The laser phototherapy was carried out with a 780 nm AlGaAs diode laser. The irradiation parameters were: power of 40 mW, beam area of 0.04 cm(2), energy density of 3 J/cm(2) (3s) in two points (0.12 J per point). Two irradiations were performed at 3 h-intervals, in contact mode. Rats (20 non-irradiated - controls and 20 irradiated) were used. The wound healing in the CNS was followed in 6 h, 1, 7 and 14 days after the last irradiation. The size of the lesions, the neuron cell viability percentages and the amount of positive GFAP labeling were statistically compared by ANOVA complemented by Tukey's test (p<0.05). The distribution of lymphocytes, leukocytes and macrophages were also analyzed. CI created focal lesions in the cortex represented by necrosis, edema, hemorrhage and inflammatory infiltrate. The most striking findings were: lased lesions showed smaller tissue loss than control lesions in 6 h. During the first 24 h the amount of viable neurons was significantly higher in the lased group. There was a remarkable increase in the amount of GFAP in the control group by 14 days. Moreover, the lesions of irradiated animals had fewer leukocytes and lymphocytes in the first 24 h than controls. Considering the experimental conditions of this study it was concluded that laser phototherapy exerts its effect in wound healing following CI by controlling the brain damage, preventing neuron death and severe astrogliosis that could indicate the possibility of a better clinical outcome.     

Preparation and analysis of heart and skeletal muscle specimens with LAMMA

Summary A technique is described to obtain shockfrozen tissue specimens from a beating heart in situ using liquid propane as cooling agent. The method allows to take up to 8 samples from the ischemic area of the left ventricle of a pig heart following acute coronary artery occlusion.A freeze-drying apparatus was developed in which, under a vacuum of 10^–6 mm Hg, the specimens remain for 10 days with the temperature slowly rising from initially –100 C to room temperature. Freezing damages in the tissues samples due to the formation of ice crystals either during shock-freezing or freeze-drying are usually below the spatial resolution of the microscope system employed in the LAMMA instrument. Already after a few minutes of ischemia, a considerable decrease of the intracellular K/Na relation was measured. LAMMA spectra from skeletal muscle were taken to check for possible ion redistribution which had eventually occurred during the various steps of the preparation procedure employed.Supported by Deutsche Forschungsgemeinschaft, SFB 68, A 10