Week Of Reperfusion Research Articles

Comparison of salpingectomy and tubal detorsion procedures after experimental ischemia-reperfusion injury in a rat fallopian tube model: biochemical and histopathological evaluation

To compare salpingectomy and detorsion procedures and investigate the biochemical and histopathological changes in the fallopian tubes in the experimentally isolated fallopian tube torsion model in rats. Experimental study. Experimental surgery laboratory in a training and research hospital. Twenty-seven Sprague-Dawley rats in the reproductive period. Group 1, control group (n = 6); group 2, bilateral total salpingectomy group after 4 hours of tubal ischemia (n = 7); group 3: 4 hours of bilateral tubal ischemia plus 1 week of reperfusion (n = 7); and group 4, 4-hour period of bilateral tubal ischemia plus 30 days of reperfusion (n = 7). A 22-gauge catheter was administered before and after surgery using methylene blue through the uterine horn of the rat to evaluate tubal patency. Preoperative and postoperative serum antimüllerian hormone (AMH) levels, histopathological examination of the rat tuba uterine and histopathological damage scores, antioxidant compounds (superoxide dismutase [SOD], catalase, and glutathione peroxidase [GSH-Px]), and oxidative stress end product levels (malondialdehyde [MDA] and 8-hydroxy-2'-deoxyguanosine [8-OHdG]). Although a significant difference was observed in the tissue SOD, GSH-Px, MDA, and 8-OHdG values, no significant difference was observed between the groups in serum samples. The tissue SOD and tissue GSH-Px levels in group 2 significantly decreased, and a significant increase was observed in the tissue MDA and 8-OHdG values in group 2. Among the histopathological parameters, epithelial changes, vascular congestion, and the total fallopian tube mean damage score of 4 showed a significant decrease in group 4. When the methylene blue transitions before and after ischemia-reperfusion injury were compared, the values of the methylene blue transition after ischemia-reperfusion injury in groups 2-4 significantly decreased. When the serum AMH levels were analyzed, the postoperative AMH value in group 2 significantly increased. This study reveals that biochemical and histopathological improvement is observed in the fallopian tube tissues gradually when the detorsion procedure is performed for the necrotized tubal tissue instead of salpingectomy. Although there is restoration of epithelial integrity after reperfusion, tubal passage remains absent. This study was approved by the Local Ethics Committee for Animal Experiments of the Health Sciences University, Istanbul Hamidiye Medicine Faculty (approval number 27.05.2022-9269). The study followed the ethics standards recommended by the Declaration of Helsinki.

Impairments of retinal hemodynamics and oxygen metrics in ocular hypertension-induced ischemia-reperfusion.

Ischemia-reperfusion (I/R) is an established model for retinal neurodegeneration. However, there is limited knowledge of retinal physiological metrics and their relationships to retinal function and morphology in the I/R model. The purpose of the study was to test the hypotheses that retinal hemodynamic and oxygen metrics are impaired and associated with visual dysfunction, retinal thinning, and retinal ganglion cell (RGC) loss due to I/R injury. Intraocular pressure (IOP) was increased in one eye of 10 rats for 90min followed by reperfusion. Fellow eyes served as controls. After one week of reperfusion, multimodal imaging was performed to quantify total retinal blood flow (TRBF) and retinal vascular oxygen contents. Retinal oxygen delivery (DO2) and metabolism (MO2) were calculated. Pattern-evoked electroretinography (PERG) and optical coherence tomography were performed to measure RGC function and retinal thicknesses, respectively. RGCs were counted from retina whole mounts. After one week of reperfusion, TRBF was lower in study eyes than in control eyes (p<0.0003). Similarly, DO2 and MO2 were reduced in study eyes compared to control eyes (p<0.003). PERG amplitude, TRT, IRT, ORT, and RGCs were also lower in study eyes (p≤0.01). DO2 and MO2 were correlated with PERG amplitude, TRT, IRT, and ORT (r≥0.6, p≤0.005). The findings improve knowledge of physiological metrics affected by I/R injury and have the potential for identifying biomarkers of injury and outcomes for evaluating experimental treatments.

Therapeutic Properties of Ayahuasca Components in Ischemia/Reperfusion Injury of the Eye.

Ischemic eye diseases are major causes of vision impairment. Thus, potential retinoprotective effects of N’N-dimethyltryptamine (DMT) were investigated. To inhibit its rapid breakdown by monoamine-oxidase A (MAO-A) enzyme, DMT was co-administered with harmaline, a β-carboline in the Amazonian Ayahuasca brew. Using ligation, 60 min of ischemia was provoked in eyes of rats, followed by 7 days of reperfusion whilst animals received harmaline alone, DMT + harmaline, or vehicle treatment. After 1 week of reperfusion, electroretinographical (ERG) measurements, histological analysis, and Western blot were performed. Harmaline alone exhibited retinoprotection in ischemia–reperfusion (I/R) which was, surprisingly, counterbalanced by DMT in case of co-administration. As both MAO-A inhibition and DMT increase serotoninergic tone synergistically, communicated to be anti-ischemic, thus, involvement of other pathways was investigated. Based on our experiments, DMT and harmaline exert opposite effects on important ocular proteins such as PARP1, NFκB, MMP9, or HSP70, each having a critical role in a different mechanism of eye-ischemia-related pathologies, e.g., cell death, inflammation, tissue destruction, and oxidative stress. Since DMT is proclaimed to be a promising drug candidate, its potentially undesirable effect on eye-ischemia should be further investigated. Meanwhile, this experiment revealed the potential therapeutic effect of MAO-A inhibitor harmaline in I/R-related eye diseases.

MiRNA-146a Mimic Inhibits NOX4/P38 Signalling to Ameliorate Mouse Myocardial Ischaemia Reperfusion (I/R) Injury.

Evidence suggests that miR-146a is implicated in the pathogenesis of cardiovascular diseases; however, the role of miR-146a in myocardial ischaemia reperfusion (I/R) injury is unclear. The aim of this study was to explore the functional role of miR-146a in myocardial ischaemia reperfusion injury and the underlying mechanism. C57BL/6J mice were subjected to 45 min of ischaemia and 1 week of reperfusion to establish a myocardial I/R injury model. A miR-146a mimic (0.5 mg/kg) was administered intravenously at the beginning of the ischaemia process. Neonatal rat cardiomyocytes were also subjected to hypoxia/reperfusion (H/R). Cells were treated with the miR-146a mimic or antagonist. As a result, the miR-146a mimic attenuated H/R-induced cardiomyocyte injury, as evidenced by increased cell viability and reduced lactate dehydrogenase (LDH) levels. In addition, the miR-146a mimic inhibited oxidative stress in cells suffering from H/R injury. Moreover, the miR-146a antagonist exerted adverse effects in vitro. In mice with myocardial I/R injury, the miR-146a mimic preserved cardiac function and reduced the infarction area and fibrosis. Moreover, the miR-146a mimic decreased the inflammatory response and reactive oxygen species (ROS) accumulation in mouse hearts. Mechanistically, we found that miR-146a directly regulated the transcription of NOX4, which subsequently affected P38 signalling in cardiomyocytes. When we knocked down NOX4, the effects of the miR-146a antagonist in worsening the cell condition were counteracted in in vitro experiments. Taken together, the results suggest that miR-146a protects against myocardial ischaemia reperfusion injury by inhibiting NOX4 signalling. The miR-146a mimic may become a potential therapeutic approach for patients with myocardial ischaemia reperfusion.

Renal Ischemia‐Reperfusion Impairs Whole Kidney Blood Flow Autoregulation in Male and Female Rats

Renal autoregulation is critical in maintaining a stable renal blood flow (RBF) and glomerular filtration rate over a wide range of renal perfusion pressure. Our recent in vitro work demonstrated that accumulating reactive oxygen species (ROS) contribute to loss of renal autoregulatory capability in ischemia reperfusion (IR)‐induced acute kidney injury (IR‐AKI) in male rats. Evidence shows that sex differences might contribute to differential injury in IR‐AKI. Therefore, we hypothesized that renal IR leads to impaired RBF autoregulation which may be associated with ROS accumulation and inflammation in male and female rats. IR was induced by occluding both renal arteries for 60 minutes followed by 24 hours or 1 week of reperfusion. Plasma creatinine was markedly increased in IR rats but more so in males than females at 24 hours (3.8±0.2 vs 1.9±0.4 mg/dl, P&lt;0.05, n=7/group). Plasma creatinine was still elevated in IR males one week post‐IR (1.3±0.1 vs 1.1±0.0 mg/dl in sham, P&lt;0.05, n=13–14/group). Whole kidney autoregulation was assessed in vivo. IR significantly lowered baseline RBF in males (5.9±0.6 vs. 9.1±0.5 ml/min/gkw in sham, P&lt;0.05, n=6/group) but not in females (8.3±0.6 vs. 10.3±0.9 ml/min/gkw in sham, P&gt;0.05, n=5–8/group) 24 hours post‐IR. RBF was lower in IR males compared to IR females (P&lt;0.05). RBF decreased further in IR rats at week 1 (4.1±1.0 ml/min/gkw in males and 4.5±0.2 ml/min/gkw in females, n=2–3). RBF remained constant during stepwise reduction of renal perfusion pressure from 120 to 100 mmHg in both sham males and females at 24 hours and 1 week, indicating intact autoregulation as reflected by autoregulatory index (ARI) between 0.1±0.0 and 0.2±0.1. IR led to impaired RBF autoregulation in males at 24 hours and 1 week, and in females at 24 hours as indicated by ARI between 0.8±0.1 and 1.1±0.2. Interestingly, autoregulation partially recovered in females at week 1 as indicated by ARI of 0.5. Preglomerular microvessels (PGMV) were collected for mRNA measurement (n=4–7 rats/each group). IR increased mRNA expression of p67phox in males at 24 hours (7.3±1.5 vs. 1.1±0.2 in sham, P&lt;0.05) and 1 week (2.2±0.3 vs. 1.0±0.1 in sham, P&lt;0.05). In contrast, IR elevated p67phox mRNA expression in females at 24 hours only (3.9±0.4 vs. 1.5±0.2 in sham, P&lt;0.05) but to a lesser degree compared to IR males (P&lt;0.05). The p47phox mRNA expression was elevated in IR males at 1 week (2±0.3 vs. 1.0±0.1 in sham, P&lt;0.05) but remained unchanged in IR females. IR also markedly increased mRNA expression of MCP‐1 in both male and female rats at 24 hours and 1 week (P&lt;0.05 vs respective sham). TGF‐β mRNA expression tends to increase in IR males at 24 hours and reached statistical significance at week 1 (2.5±0.2 vs 1.1±0.1 in sham, P&lt;0.05). TGF‐β was significantly elevated in IR females though still lower compared to IR males at week 1 (1.7±0.2 vs 2.5±0.2, P&lt;0.05). In conclusion, these results suggest that IR impairs RBF autoregulation in male and female rats which might be associated with activation of NOX, MCP‐1 and TGF‐β pathways in PGMV. Female rats have less kidney injury which might be attributed to less NOX expression in PGMV.Support or Funding InformationThis study was supported by grants from NIH (DK106500) to ZG, and NIH (DK044628 and HL095499) to EWI.

Characterization of Klotho/FGF23 signaling in cardiorenal syndrome‐induced cardiac hypertrophy

IntroductionIn diseases that generate an inflammatory situation, such as cardiorenal syndrome, different clinical conditions promote cardiac and renal dysfunction. There are type 3 and 4 which are characterized by renal insults that subject the heart to dysfunctions; Among the changes that renal injury can lead, we can mention the modulation in Klotho levels, a gene that is negatively regulated by fibroblast growth factor 23 (FGF23). The objective of this study is to evaluate the participation of this axis in cardiac hypertrophy induced by renal ischemiaMethodsC57BL/6 male mice were subjected to surgical occlusion of left renal pedicle for 60 min followed by reperfusion in acute term (24, 48 and 72h), short term (for 8 and 15 days) and long term (4,6 and 8 weeks). Total heart RNA was extracted for gene expression of Klotho, FGF23 and hypertrophy markers using real‐time PCR.ResultsThe renal injury was confirmed by decreasing of left kidney weight and urea/creatinine levels starting at the 15th day of reperfusion. Cardiac hypertrophy was confirmed by increased a‐actin levels at day 15 and 4 weeks of renal reperfusion; however, heart weight has maintained increased until the 8th week of reperfusion. Concerning the FGF23/Klotho axis, both genes were modulated during the cardiorenal syndrome type 3. FGF23 gene expression was mostly increased in heart tissue after 8 days of reperfusion, having its peak during the 4th week. In renal tissue, FGF23 did not present a pattern, although it shows a peak in the first 24 hours, in the 8th day and 15th day (the same timepoints which the heart tissue is affect which electrophysiological and immunological changes). By the other side, Klotho gene expression was mostly reduced in renal tissue after 48h of reperfusion, evidencing even more the renal injury (once some authors consider Klotho is a renal injury biomarker). In heart tissue, klotho expression is reduced since the first 24h, but this is attenuated around the 4th week of reperfusion.ConclusionWe found that cardiac FGF23/Klotho axis dynamics are affected by renal I/R indicating a possible interaction between these two responses during cardiorenal syndrome.Support or Funding InformationFinancial Support: FAPESP 2015/19107‐5 and 2018/03089‐6.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential effects of intestinal ischemia and reperfusion in rat enteric neurons and glial cells expressing P2X2 receptors.

Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. The objective was to analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of the rat ileum after different periods of reperfusion. The superior mesentery artery was occluded for 45 minutes, and animals were euthanized after 24 hours and 1 week of reperfusion. Immunohistochemical analyses were performed with antibodies against the P2X2 receptor in combination with antibodies against nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, calretinin, the pan-neuronal marker anti-HuC/D, or S100β (glial marker). Dual immunolabeling demonstrated that approximately 100% of NOS-, ChAT-, calbindin-, and calretinin-immunoreactive neurons in all groups expressed the P2X2 receptor. Following I/R, the neuronal density decreased in the P2X2 receptor-, ChAT-, calretinin-, and HuC/D-immunoreactive neurons at 24 hours and 1 week following injury compared to the densities in the control and sham groups. The calbindin-immunoreactive neuron density was not reduced in any of the groups. The density of enteric glial cells increased by 40% in the I/R group compared to the density in the sham groups. We also observed increases of 12%, 16%, and 23% in the neuronal cell body profile areas of the NOS-, ChAT-, and calbindin-immunoreactive neurons, respectively, at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% in the I/R group at 24 hours. This work demonstrates that I/R is associated with a significant loss of different classes of neurons in the myenteric plexus accompanied by morphological changes and an increased density of enteric glial cells; all of these effects may underlie conditions related to intestinal motility disorder.

Abstract 333: Deficiency of DJ-1 Leads to Increased Injury following Myocardial Ischemia by Enhancing Apoptosis and Oxidative Stress

Background: DJ-1 is a ubiquitously expressed protein that has typically been associated with the development of early onset Parkinson’s disease. Recent data suggests that it also plays a role in the cellular response to stress. Although much is known about DJ-1 in the brain, very little has been investigated in the heart. Here, we tested the hypothesis that a deficiency in DJ-1 would enhance myocardial ischemia-reperfusion (MI/R) injury. Methods and Results: Wild-type (WT) control and DJ-1 knockout (DJ-1 KO) mice were subjected to 45 min of left coronary artery ischemia followed by 24 hrs of reperfusion. The deficiency of DJ-1 significantly increased myocardial infarct size relative to both the area-at-risk and entire left ventricle, as well as increased circulating troponin-I levels (Panels A-B). Echocardiography and hemodynamic analysis at 1 week of reperfusion revealed that DJ-1 KO mice experienced greater left ventricular dilatation and hypertrophy, displayed exacerbated left ventricular dysfunction, and displayed worse contractility and relaxation when compared to WT controls. In an effort to evaluate the signaling mechanism responsible for the increased injury in DJ-1 KO mice, additional WT and KO animals were subjected to 45 min of ischemia followed by 4 hrs of reperfusion. DJ-1 KO hearts were found to display higher levels of oxidative stress, greater caspase-3 activity (Panel C), enhanced phosphorylation of Jnk, and enhanced activation of the mitochondrial fission protein, dynamin-related protein 1 (Drp1). Conclusions: These findings provide important information that DJ-1 plays a protective role in heart against acute MI/R injury.

Evaluation of Early Regenerative Processes in a Preclinical Pig Model of Acute Kidney Injury

Renal failure due to ischemic injury is a common denominator of various clinical situations in critically ill patients. This study was designed to characterize the TPSO/Cholesterol synthesis and cell division pathways in response to different levels of ischemia. Porcine kidneys were subjected to either 60 min-warm ischemia (WI) or auto-transplanted after cold storage for 24 h at 4°C (CS), or both conditions (WI+CS), pathway activation and function were evaluated at 3 h, 3 and 7 days after reperfusion. CS combined to WI affects renal functions indicating a high degree of injury. During the first week of reperfusion, renal levels of free and esterified cholesterol, major cellular components, increased in CS group with an attenuated production when WI was associated. CS and WI+CS groups exhibited an elevated expression of cell cycle induction markers such as PCNA and stathmin. TSPO expression was highest in groups with the lowest injury, and correlated with kidney outcome, revealing its potential for diagnosis.

Neuroprotective effect of grafting GDNF gene-modified neural stem cells on cerebral ischemia in rats

Previous studies indicated the beneficial effects of glial cell line-derived neurotrophic factor (GDNF) and transplanted neural stem cells (NSCs) on stroke. Here, we explored whether transplantation of neural stem cells (NSCs) modified by GDNF gene provides a better therapeutic effect than native NSCs after stroke. Primary rat NSCs were transfected with GDNF plasmid (GDNF/NSCs, labeled by green fluorescent protein from AdEasy-1, GFP). Adult rats were subjected to two-hour middle cerebral artery occlusion and reperfusion, followed by infusion of NSCs (labeled with5-bromo-2′-deoxyuridine before infusion, BrdU), GDNF/NSCs and saline at 3 days after reperfusion (NSCs group, GDNF/NSCs group, control group), respectively. All rats were sacrificed at 1, 2, 3, 5, and 7 weeks after reperfusion. Modified Neurological Severity Scores (mNSS) test and H and E staining were respectively performed to evaluate neurological function and lesion volume. Immunohistochemistry was used to identify implanted cells and observe the expressions of Synaptophysin (Syp) and postsynaptic density-95 (PSD-95) and caspase-3. TdT-mediated dUTP–biotin nick-end labeling (TUNEL) was employed to observe apoptotic cells. Western blotting was used to detect brain-derived neurotrophic factor (BDNF) and NT-3 protein expression. Significant recovery of mNSS was found in GDNF/NSCs rats at 2 and 3 weeks after reperfusion compared with NSCs rats. Lesion volume in the NSCs and GDNF/NSCs groups was reduced significantly compared with control group. The number of NSCs in the GDNF/NSCs group was significantly increased in comparison with NSCs group. Moreover, Syp-immunoreactive product at 2 and 3 weeks after reperfusion and PSD-95 immunoreactive product in the GDNF/NSCs group were significantly increased compared with NSCs group. In contrast, caspase-3 positive cells and TUNEL-positive cells in the GDNF/NSCs group were significantly decreased compared with NSCs group. Significant increase of BDNF protein in the GDNF/NSCs and NSCs groups was observed compared to the control group at different time points of reperfusion, and GDNF/NSCs grafting significantly increased BDNF protein expression compared to NSCs grafting. In addition, significant increase of NT-3 protein in GDNF/NSCs and NSCs groups was detected only at 1 week of reperfusion compared to control group. The results demonstrate that grafting NSCs modified by GDNF gene provides better neuroprotection for stroke than NSCs grafting alone.

Ischemia/Reperfusion Effects on Bladder Muscle and Mucosa Cell Contractile Regulatory Proteins

Objectives: Ischemia/reperfusion (I/R) has been shown to be the major etiologic factor in animal models in a variety of bladder dysfunctions. Herein we investigate the direct effect of I/R on rabbit bladder contractile regulatory proteins.Methods: Male rabbit bladders were subjected to IR. The contractile responses were recorded and the protein levels of rho‐kinase (ROK), caldesmon (CaD) and myosin light chain kinase (MLCK) were analyzed in both bladder muscle and mucosa.Results: For the mucosa layer, ROK was unchanged after ischemia, but increased significantly after 1 week of reperfusion. MLCK increased after ischemia and then significantly increased after 1 and 2 weeks of reperfusion. In the muscle layer, ROK increased at 2 h of reperfusion, decreased significantly following 1 week of reperfusion, then returned to control level by 2 weeks of reperfusion. MLCK expression significantly decreased as early as ischemia alone and did not recover after reperfusion. For CaD expression in the mucosa layer, both CaD isoforms significantly increased at 1 week of reperfusion, then progressively decreased at 2 weeks of reperfusion. In the muscle layer, both CaD isoforms had different expressions. In the muscle layer, smooth muscle caldesmon (h‐CaD) decreased at ischemia alone and at 2 h of reperfusion, but significantly increased at 2 weeks of reperfusion; whereas non‐muscle caldesmon (l‐CaD) significantly decreased at 2 weeks of reperfusion.Conclusion: As bladder muscle and mucosa have markedly different purposes, it is not surprising that they respond differently to I/R. Bladder mucosa and muscle have different kinds of alternations on contractile regulatory proteins.

Improved Survival of Murine Island Skin Flaps by Prevention of Reperfusion Injury

Studies have demonstrated that blocking a single specificity of self-reactive immunoglobulin M with a 12-amino acid peptide mimic of the antigen of immunoglobulin M can attenuate murine intestinal and skeletal muscle injury following ischemia and reperfusion. The aim of this study was to ascertain whether peptide (P8) had protective effects in an axial island skin flap model, where tissue loss is attributed to ischemia-reperfusion injury. Dorsal lateral thoracic artery island skin flaps (3.5 x 1.5 cm) were elevated in 82 male C57BL/6 mice and rendered ischemic for 10 hours by placing a 7-mm microclamp on the vascular pedicle followed by 7 days of reperfusion. Group I (n = 7), the sham group, had no clamp placed. Group II (n = 21) had clamp placement but no other treatment. Thirty minutes before clamp placement, group III (n = 18) received 0.25 cc of saline intravenously, group IV (n = 18) received 25 microg/0.25 cc P8 peptide, and group V (n = 7) received 25 microg/0.25 cc random 12-mer peptide. Animals in group VI (n = 11) underwent two cycles of 20 minutes of ischemic preconditioning before 10 hours of ischemia. After 1 week of reperfusion, percent necrosis was measured and results were compared using analysis of variance and an unpaired t test. In animals treated with P8 peptide, flap necrosis was 14.61 +/- 2.77 percent. This represents a statistically significant, 56 percent reduction in flap necrosis compared with controls (p < 0.001). These data demonstrate that prevention of ischemia-reperfusion injury with P8 peptide produces a significant reduction in necrosis of treated flaps.

A Modified Model for Rat Sciatic Nerve Ischemia/Reperfusion Injury

Sir: A number of different models of unilateral ischemia/reperfusion injury in rat sciatic nerve have been developed. In one method described by Mitsui et al.,1 the abdominal aorta, the right iliac and femoral arteries, and all identifiable collateral vessels supplying the right sciatic-tibial nerve are ligated for 3 hours and then reperfused for different time intervals. In another model introduced by Saray and his colleagues,2 only the femoral artery and vein–-just distal to the inguinal ligament–-are clamped for 3 hours, followed by reperfusion. The first method is time-consuming and requires advanced surgical techniques and instruments, and the second one, albeit easy to perform, does not produce severe injury.3 The aim of this study was to develop a practical model producing serious neurologic deficits that was still technically feasible. For this purpose, clamping of the femoral artery as well as the ipsilateral common iliac artery was used to induce sciatic nerve ischemia/reperfusion. For this pilot study, 12 Sprague–Dawley male rats weighting 150 to 200 g were randomly divided into two groups: the sham-operated group and the ischemia/reperfusion group. All animals were anesthetized with ketamine (50 mg/kg) and xylazine (4 mg/kg) and subjected to laparotomy. In the ischemia/reperfusion group, the right common iliac artery and the femoral artery–-just distal to the inguinal ligament–-were clamped for 3 hours using two Yasargil aneurysm clips providing 125 g (1.24 N) of force. By using a thermal pad, the deep rectal temperature was monitored by a rectal probe inserted 5 cm into the rectum and maintained at 36.5°C ± 1°C. All procedures were carried out for the sham-operated group, but arterial clamping was excluded. After 1 week of reperfusion, each animal's behavioral score was assessed based on gait, grasp, paw position, and pinch sensitivity; the score for each index was based on a scale of 0 (no function) to 3.0 (normal function), except for pinch sensitivity, the score for which ranged from 0 to 2.1 Then, the sciatic nerve was fixed in situ for 30 minutes using 4% formaldehyde in phosphate buffer (pH adjusted to 7.4), and then trifurcation of the sciatic nerve was removed, the nerve was embedded in paraffin, and sections were stained with hematoxylin and eosin and trichrome Gomori for light microscopy studies. The median behavioral scores were 5 and 11 in the ischemia/reperfusion and sham groups, respectively, and were significantly different (p < 0.05, Mann-Whitney U test). The pathologic markers for ischemia/reperfusion, including epineurial and endoneurial edema and demyelination in trichrome Gomori staining, were observed in the ischemia/reperfusion group but were absent in the sham-operated group (Figs. 1 and 2).Fig. 1.: Transverse section of the rat sciatic nerve after 7 days of reperfusion shows epineurial and endoneurial edema (hematoxylin and eosin stain).Fig. 2.: Transverse section of the rat sciatic nerve after 7 days of reperfusion shows epineurial and endoneurial edema and demyelination (trichrome Gomori stain).Our results show that common iliac artery and femoral artery clamping induces ischemia/reperfusion injury in the rat sciatic nerve. This method offers the obvious advantage of producing an easily inducible, moderate to severe neurologic deficit compared with current methods. However, a more comprehensive study containing several groups of different reperfusion intervals and quantification of pathological changes is needed to make the method practical for use in experimental studies. Mohammad R. Rasouli, M.D. School of Medicine Mohsen Nouri, M.D. School of Medicine Reza Rahimian, Ph.D. Department of Pharmacology Fahimeh Asadi Amoli, M.D. Trauma and Surgery Research Center Vafa Rahimi-Movaghar, M.D. Trauma and Surgery Research Center Ahmad R. Dehpour, Ph.D. Department of Pharmacology School of Medicine Medical Sciences/Tehran University Tehran, Iran

Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of myocardial infarction

Transplantation of stem cells may improve regional perfusion and post-infarct ventricular function, but the optimal dose and efficacy of cell delivery via the intravenous route has not been determined. This study tested the hypothesis that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) enhances regional perfusion and improves ventricular function after myocardial infarction. In a closed-chest pig model, the LAD coronary artery was occluded for 75 min by angioplasty balloon inflation followed by 12 weeks of reperfusion. After 15 min of reperfusion, pigs randomly received 1 of 4 treatments: (1) Vehicle (Control, n = 10); (2) 1 x 10(6) MSCs/kg (1 mill, n = 7); (3) 3 x 10(6) MSCs/kg (3 mill, n = 8) and (4) 10 x 10(6) MSCs/kg (10 mill, n = 8). Angiogenesis was demonstrated by immunohistochemical staining, myocardial blood flow (steady state and vasodilator reserve) was measured using 15 microm neutron-activated microspheres, and cardiac function was determined by contrast left ventriculography (ejection fraction) and pressure-volume relationships. After 12 week of reperfusion, von Willebrand Factor-positive vessels and tissue vascular endothelial growth factor (VEGF) expression in the scar zone was significantly greater in all MSCs-treated animals relative to Control. Steady state myocardial blood flow in the scar tissue was comparable among groups. However, adenosine recruited vasodilator reserve in the scar zone induced by intracoronary adenosine was significantly higher in the MSC-treated animals compared to Control. Furthermore, preload-recruitable stroke work and systolic performance were significantly greater compared to Control. In conclusion, these data demonstrate that intravenous delivery of MSCs during early reperfusion augments vasculogenesis, enhances regional perfusion, and improves post-infarct ventricular function. The results suggest that intravenous infusion of MSCs is an effective modality for the treatment of ischemia/reperfusion induced myocardial injury.

ROLE OF CARDIOMYOCYTE APOPTOSIS IN TRAUMA-INDUCED SENSITIZATION TO MI INJURY

Previous epidemiological studies have demonstrated that trauma exposure is linked to adverse health outcomes. Moreover, recent clinical observation suggests that trauma patients who later develop myocardial ischemia (MI) have a much higher mortality rate than age-matched controls. These results highly suggest that traumatic injury may increase the risk for cardiovascular diseases. We have recently demonstrated that nonfatal mechanical trauma results in significant cardiomyocyte apoptosis. The current study was designed to determine whether trauma-induced cardiomyocyte apoptosis may "prime" the heart to be more susceptible to MI injury. Adult male Swiss Webster mice were anesthetized and subjected to nonfatal trauma (Noble-Collip drum, 200 revolutions at 40 rpm). Sham traumatic mice were subjected to the same number of revolutions without trauma. One week after trauma, mice were subjected to 30 min MI followed by 1 week of reperfusion (R). Cardiac function assessed by echocardiography before ischemia was normal, and there was no difference between trauma mice and non-trauma mice. However, ischemic cardiac dysfunction assessed by both non-invasive and invasive techniques was significantly aggravated in those animals subjected to a nonfatal trauma (P < 0.01). Moreover, infarct size (fraction of the infarcted left ventricle as the percentage of length of circumference) was significantly enlarged in those animals exposed to a mild trauma 1 week before (74 ± 4.6% vs. 56 ± 3.9% in sham trauma plus MI, n = 10/group, P < 0.01). Treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor (4 mg/kg, i.p.), given immediately after trauma, blocked trauma-induced apoptosis and abolished traumainduced sensitization to MI/R injury. These results suggest that traumatic injury might be a "silent" risk factor for ischemic MI and that cardiomyocyte apoptosis contributes to trauma-induced sensitization to MI/R injury.Figure

Expression of HSP 70 and its mRNAS during ischemia-reperfusion in the rat bladder

HSP 70 is an important protein that repairs damaged tissue after injury. In the present study, we investigated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder dome. Male Wistar rats, 8 weeks old, were divided into six groups: controls, 30-min ischemia, 30-min ischemia and 30-, 60-minute, 1- and 7-day reperfusion, groups A, B, C, D, E, and F, respectively. In functional studies, contractile responses to carbachol were measured in these groups. The expression of HSP 70-1/2 mRNAs was quantified using a real-time PCR method, and that of HSP 70 proteins was measured using ELISA in the bladders. In the functional study, Emax values of carbachol to bladders in the A, B, C, D, E and F groups were 9.3 ± 1.3, 7.9 ± 1.7, 4.3 ± 0.8, 4.2 ± 0.7, 4.5 ± 0.6, and 8.1 ± 1.2 g/mm 2, respectively. In the control group, the expression of HSP 70-1/2 mRNA was detected, and the expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1/2 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70 proteins was increased shortly after the expression of their mRNAs. The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels. Our data indicate that contractile responses of the bladder were decreased by ischemia reperfusion, and that expression of HSP 70 and its mRNAs appeared to increase after a short period of the insult.

Reperfusion Injury of the Rat Bladder is Worse Than Ischemia

Previous studies have demonstrated that in vivo and in vitro ischemia of the bladder results in decreased contractile responses. However, to our knowledge the effect of reperfusion following ischemia of the bladder is not known. Adult male rats were subjected to bilateral bladder ischemia and varying periods of reperfusion. In vivo ischemia was created for 4 hours by reversibly clamping the 2 vesical arteries for 4 hours. Reperfusion was produced by removing the clamps and allowing the animals to recover for 1 day, 1 week or 1 month after surgery. Following recovery bladders strips were studied using field stimulation (FS), carbachol and KCl. The maximal contractile response and rate of response generated were recorded digitally and analyzed. The maximal responses to FS, carbachol and adenosine triphosphate (ATP) were not decreased by 4-hour ischemia alone, whereas the response to KCl was decreased significantly. The contractile responses to FS and KCl were significantly decreased after 1 day and 1 week of reperfusion. Responses after 1 month of reperfusion were increased significantly compared with responses after 1 week of reperfusion. The responses to ATP were not affected by ischemia or reperfusion. The contractile response to KCl was significantly more sensitive to ischemia than the responses to carbachol, ATP or FS, whereas the contractile response to FS was significantly more sensitive to reperfusion than the other forms of stimulation. This study demonstrates clearly that injury by reperfusion following ischemia is more detrimental than the effects of ischemia alone and FS contraction is the most sensitive form of stimulation to reperfusion damage. This study also demonstrates the ability of the bladder to recover from ischemic and reperfusion injuries.

Stroke-induced progenitor cell proliferation in adult spontaneously hypertensive rat brain: effect of exogenous IGF-1 and GDNF.

Progenitor cells in the dentate gyrus of hippocampus (DG) and the subventricular zone of lateral ventricles (SVZ) generate new neurons throughout the life of mammals. Cerebral ischemia increases this basal progenitor cell proliferation. The present study evaluated the time frame of proliferation, length of survival and the phenotypes of the new cells formed after transient middle cerebral artery occlusion (MCAO) in adult spontaneously hypertensive rats. Compared to sham controls, ischemic rats showed a significantly higher number of newly proliferated cells (as defined by BrdU immunostaining) in both the DG (by fourfold, p < 0.05) and the SVZ (by twofold, p < 0.05). DG showed increased proliferation only in the first week of reperfusion and 49% of the cells formed in this period survived to the end of third week. Whereas, SVZ showed a continuous proliferation up to 3 weeks after MCAO, but the cells formed survived for less than a week. In both DG and SVZ, at the end of the first week of reperfusion, majority of the BrdU-positive (BrdU+) cells were immature neurons (DCX positive). In the DG, 28% of the cells formed in the first week after MCAO mature into neurons (NeuN positive). The ischemic cortex and striatum showed several BrdU+ cells which were ED-1 positive microglia/macrophages. At 1 week of reperfusion, MCAO-induced progenitor cell proliferation in the ipsilateral DG was significantly increased by i.c.v. infusion of IGF-1 (by 127 +/- 14%, p < 0.05) and GDNF (by 91 +/- 5%, p < 0.05), compared to vehicle. In the growth factor treated rats subjected to transient MCAO, several BrdU+ cells formed in the first week survived up to the third week.

Morphologic changes and lipid peroxidation in renal tissues of young rats following intestinal ischemia-reperfusion.

Intestinal ischemia-reperfusion (IIR) is a complex phenomenon causing local and remote tissue destruction, and even multiple-organ failure. To examine the hypothesis that IIR affects renal function, 21-day-old male Sprague-Dawley rats underwent 45 min superior mesenteric artery occlusion and control rats were subjected to a sham laparotomy. After 2 and 24 h and 1 week of reperfusion, blood was sampled for urea and the kidneys were harvested for lipid peroxidation and histologic examination. Malondialdehyde (MDA) levels as an indicator of lipid peroxidation were significantly increased in renal tissue after 2 h of reperfusion, and this finding was in accordance with serum urea levels (SU) and endothelial injury. However, at 24 h of reperfusion MDA and SU had returned to normal. These data were supported by electron-microscopic studies suggesting reversibility of the changes. It is concluded that IIR leads to renal injury and that free radicals may be responsible for this injury.

Ultrastructural Changes of the Neuromuscular Junction in Reperfusion Injury

Ischemia followed by reperfusion of skeletal muscle frequently takes place in trauma surgery. Anoxia followed by reoxygenation leads to reperfusion injury, which damages the involved tissues. However, no information is available about how the neuromuscular junction is affected by ischemia-reperfusion. Tourniquet ischemia of the left hind limb was applied in the anesthetized rat for 2 h. Reperfusion lasted for 2 and 24 h and for 1, 2 and 4 weeks. The extensor digitorum longus and the soleus muscles from both legs were prepared for electron-microscopic analyses. Morphological changes of the neuromuscular junction were investigated. In all cases only the nerve endings (terminals) were affected. The postsynaptic structures were not affected. Changes can be grouped in two categories: degeneration and recovery. Degeneration consisted of the loss of synaptic vesicles, disruption of the presynaptic membrane, degeneration of the mitochondria and the development of vacuoles. It was most severe at 24 h and was still present at 4 weeks of reperfusion. Recovery started at 1 week of reperfusion and lasted at least for 4 weeks. It consisted of the slow reappearance of synaptic vesicles and mitochondria, and restoration of the presynaptic membrane with active zones. Ultrastructure of the skeletal muscle fibers did not show pathological changes. The recovery of the structures may be regulated by the Schwann cells and also by the postsynaptic membrane which is not affected by 2 h of ischemia followed by reperfusion.