Triphenyltetrazolium Chloride Solution Research Articles

Experimental Model of Cerebral Arterial Air Embolism in Conscious Rats.

In this work, an optimal air supply mode was selected to create a model of cerebral arterial air embolism (CAAE) on conscious male Sprague-Dawley rats (n=49). The efficacy of the selected model (administration of 100 μl/kg of air at a rate of 10 μl/min with an infusion pump) was determined by changes in serum biochemical parameters (cholesterol, alkaline phosphatase, inorganic phosphates, AST, and triglycerides), impaired motor functions in the Rotarod test, and visual assessment of the ischemic foci (staining of frontal sections with 1% triphenyltetrazolium chloride solution) at different terms after AAE. The model of AAE created by us confirmed impairment of coordination and motor function in conscious animals and reproduced the lethal consequences of this condition. The obtained results can serve as the basis for drug testing and the development of new approaches to the treatment of ischemic stroke.

The administration of valproic acid exerts a protective effect by reducing the infarct size and the triggering of ventricular fibrillation in an experimental model of chronic myocardial infarction

Abstract Introduction In myocardial infarction there are complications associated with adverse cardiac remodeling that occurs during the fibrotic process, producing an arrhythmogenic substrate that favors the triggering of ventricular arrhythmias such as ventricular fibrillation (VF). These complications also contribute to infarct expansion and mortality. Purpose To analyze, in a model of chronic infarction (5 weeks) in rabbits, the effects of valproic acid (VA), an inhibitor of histone deacetylase enzymes, on the inducibility of ventricular fibrillation and infarct size. Methods 47 New Zealand rabbits were assigned to sham (n=15), control (n=22) and VA-treated (n=10) groups. After midsternal thoracotomy and pericardiotomy, the circumflex coronary artery was occluded (1 hour), followed by reperfusion (control and treated groups). 500 mg/kg of VA were administered every 24 hours for 5 weeks. Then, the animals were sacrificed, their hearts excised and placed in a Langendorff perfusion system. We used a multielectrode recording plate (256 electrodes) and ventricular stimulation electrodes in the left ventricular epicardium. The inducibility of VF was analyzed using ventricular extrastimulus tests (VEST) with two basic stimulation cycle lengths (250 and 150 ms) and up to the three extrastimuli in each cycle. VEST was applied in four peri-infarct stimulation zones. After the electrophysiological study, the circumflex coronary artery was occluded, and the aorta was perfused with thioflavin-S to determine the area at risk (AR). We made sections of the left ventricle (LV) which were incubated in a triphenyltetrazolium chloride solution to determine the infarct size. The images obtained were digitized and manually quantified. The quantification was performed in 3 sections, located below the ligature of the coronary occlusion. Unpaired Student's t-test or chi-square test were used when appropriate (p<0.05). Results Infarct size decreased in the VA-treated group compared to the control group, when expressed as a percentage of AR (40.3±5.3% vs. 69.8±20.4%, respectively) and when expressed as percentage of LV (19.3±7.8% vs. 38.6±16.4% respectively). The myocardium of the sham group did not exhibit areas of necrosis. Regarding VF inducibility, the treated group presented lower arrhythmia inducibility, regardless of the number of extrastimuli and pacing cycle length, triggering the arrhythmia in 40% of cases compared to 59% in the control group (p=0.011). Likewise, more extrastimuli were needed in VA-treated animals to trigger VF than controls (2 (50%) and 3 (50%) extrastimuli vs. 1 (15%), 2 (46%) and 3 (38%) extrastimuli needed, respectively; χ2=16.526; p<0.005). Conclusion VA did exhibit a protective effect, as shown by its ability to limit the extension of the infarct size and to reduce the inducibility of ventricular fibrillation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Carlos III Health Institute and Institute of Health Research (INCLIVA)

Guanxinning Injection Combined With Ischemic Postconditioning Attenuate Myocardial Ischemic Reperfusion Injury in Chronic Renal Failure Rats by Modulating Mitochondrial Dynamics.

PurposeSalvia miltiorrhiza Bge. (Danshen, DS) and Ligusticum chuanxiong Hort. (Chuanxiong, CX) have been widely used in traditional Chinese medicine to prevent and treat myocardial ischemia and renal insufficiency, and their extracts (Guanxinning injection, GXN) have been reported to exhibit antioxidant, anti-inflammatory, and anti-ischemia-reperfusion injury properties. It is well-established that ischemic postconditioning (IPOC) can protect against myocardial ischemia-reperfusion (I/R) injury in rats with chronic renal failure (CRF). However, little is known on whether GXN combined with IPOC may affect myocardial I/R injury in CRF rats. We sought to observe the effect of GXN combined with IPOC on myocardial I/R injury in CRF rats by quantifying changes in the expression of proteins related to mitochondrial dynamics.Materials and MethodsIn a survey, 90 Wistar rats were randomly divided into 6 groups (15 rats per group): CRF group, I/R group, comorbid group (CRF + I/R), IPOC group, IPOC + GXN group and the sham group. Changes in blood myocardial injury markers, urea, and creatinine were analyzed. Heart tissues were harvested for histomorphometry and western blotting when rats were sacrificed. Myocardial infarction area was measured by Evans blue and Triphenyltetrazolium chloride solution staining. The expressions of mitochondrial fission relative proteins (DRP1 and FIS1) and mitochondrial fusion relative proteins (OPA1 and MFN1) were detected by western blotting.ResultsIPOC could significantly decrease myocardial injury markers and myocardial area of necrosis (AN)/area at risk (AAR) of the comorbid model rats. Further results showed that GXN combined with IPOC could significantly reduce CK-MB levels and myocardial AN/AAR in comorbid model rats compared with the IPOC group. Meanwhile, both IPOC and IPOC + GXN significantly reduced DRP1 levels and increased the MFN1 and OPA1 protein levels in the comorbid model rats. However, compared with the IPOC group, MFN1 and OPA1 protein levels increased significantly in the IPOC + GXN group.ConclusionExtracts of DS and CX combined with IPOC exert a protective effect against myocardial I/R injury in rats with CRF, mediated by increased expression of mitochondrial fusion proteins (MFN1 and OPA1).

Viabilitas dan Vigor Benih Kacang Tanah (Arachis hypogaea L.), Kacang Hijau (Vigna radiata (L.) R. Wilczek), dan Jagung (Zea mays L.) pada Temperatur dan Tekanan Osmotik Berbeda

Viability and vigor are components of seed quality assessment which are influenced by internal and external factors of the seed, i.e., salinity and temperature. Salinity is related to the interaction between osmotic pressure and seed germination. This study used 3 types of seeds, they are peanuts (Arachis hypogaea L.), mung bean (Vigna radiata (L.) R. Wilczek), and corn (Zea mays L.). The purposes of this study were to determine seed viability by using Triphenyl tetrazolium chloride (TTC), to analyze the effects of different osmotic pressures and temperatures on seed viability and vigor. Peanut seed viability test was conducted by using 1% TTC solution. Testing the effect of osmotic pressure on seed viability and vigor was conducted by using various concentrations of NaCl (0.01 M; 0.05 M; 0.5 M; 0.1 M; 1 M). The germination test of the three seeds was conducted by using temperatures of 30°C and 35°C, each with three replications. The test parameters measured included germination, germination rate, germination speed index, vigor index, simultaneous growth, and the speed of seed growth. The results showed that TTC solution was effectively used to determine seed viability. Mung bean had the highest germination rate, LP, IKP, IV, simultaneous growth, and speed of seed growth compared to the other tested seeds in all salinity and temperature treatments. The 30 ⁰C temperature treatment resulted in the best viability and seed vigor for the three tested plants. Treatments of temperature and salinity affect the quality of the three plant seeds which can then be used to determine the quality of peanut, mung bean, and corn seeds.
 
 Keywords: germination, salinity, triphenyl tetrazolium chloride, viability, vigour

Comparative evaluation of metformin and liraglutide cardioprotective effect in rats with impaired glucose tolerance

Impaired glucose tolerance (IGT) increases cardiovascular risk and can enlarge myocardial infarction (MI) incidence and severity. There is lack of information about cardioprotective potential of glucose-lowering drugs in IGT. We aimed to evaluate the sustainability of myocardium to ischemia–reperfusion injury in diabetic and IGT rats and to study cardioprotective action of metformin and liraglutide. Type 2 diabetes mellitus (DM) and IGT were modelled in Wistar rats by high-fat diet and streptozotocin + nicotinamide. 4 weeks after rats were divided into 4 groups: DM (without treatment) (n = 4), IGT (without treatment) (n = 4), IGT + MET (metformin 200 mg/kg per os once daily 8 weeks) (n = 4), IGT + LIRA (liraglutide 0.06 mg/kg s.c. once daily for 8 weeks) (n = 4). Control (n = 6) and high-fat diet (n = 8) groups were made for comparison. After 8 weeks ischemia–reperfusion injury in isolated hearts was performed. Hemodynamic parameters were evaluated and MI size was measured by staining of myocardium slices in triphenyltetrazolium chloride solution. Blood glucose level was measured during the study. Both IGT and DM led to similar worsening of hemodynamic parameters during ischemia–reperfusion period, in comparison with control group. MI size in IGT (56.76 (51.58; 69.07) %) and DM (57.26 (45.51; 70.08) %) groups was significantly larger than that in control group (42.98 (33.26; 61.84) %) and did not differ between each other. MI size in high-fat diet group (56.98 (47.11; 62.83) %) was as large as in IGT and DM groups (p > 0.05). MI size in IGT + MET (42.11 (38.08; 71.96) %) and IGT + LIRA (42.50 (31.37; 60.40) %) was smaller than in both DM and IGT groups (p < 0.05 for multiple comparison). Myocardium damage size did not differ in IGT + MET and IGT + LIRA groups (p > 0.05). Only LIRA, but not MET administration to IGT rats led to ischemic contracture reduction. Glycemic control was similarly satisfactory in IGT, IGT + MET, IGT + LIRA groups. Thus, IGT and DM have similarly pronounced negative influence on hemodynamics and MI size in rat transient global ischemia ex vivo. Obesity development also has negative impact on the MI size. Both MET and LIRA have infarct-limiting effect independent on their influence on glucose level. LIRA, but not MET, diminishes ischemic contracture in IGT rats.

A standardised approach for determining heat tolerance in cotton using triphenyl tetrazolium chloride

Improving the heat tolerance of cotton is a major concern for breeding programs. To address this need, a fast and effect way of quantifying thermotolerant phenotypes is required. Triphenyl tetrazolium chloride (TTC) based enzyme viability testing following high-temperature stress can be used as a vegetative heat tolerance phenotype. This is because when live cells encounter a TTC solution, TTC undergoes a chemical reduction producing a visible, insoluble red product called triphenyl formazan, that can be quantified spectrophotometrically. However, existing TTC based cell viability assays cannot easily be deployed at the scale required in a crop improvement program. In this study, a heat stress assay (HSA) based on the use of TTC enzyme viability testing has been refined and improved for efficiency, reliability, and ease of use through four experiments. Sampling factors that may influence assay results, such as leaf age, plant water status, and short-term cold storage, were also investigated. Experiments conducted in this study have successfully downscaled the assay and identified an optimal sampling regime, enabling measurement of large segregating populations for application in breeding programs. The improved HSA methodology is important as it is proposed that long-term improvements in cotton thermotolerance can be achieved through the concurrent selection of superior phenotypes based on the HSA and yield performance in hot environments. Additionally, a new way of interpreting both heat tolerance and heat resistance was developed, differentiating genotypes that perform well at the time of a heat stress event and those that maintain a similar performance level to a non-stressed control.

Rapid microbiological methods (RMMs) for evaluating the activity of cephalosporin antibiotics employing triphenyltetrazolium chloride

Agar diffusion method has been used to evaluate antimicrobial activity since the discovery of penicillin. Nevertheless, little progress has occurred in reducing the time required to determine growth inhibition zones. The aim of this work was to develop, optimize and validate rapid microbiological methods (RMMs) for cephalosporin antibiotics using triphenyltetrazolium chloride (TTC) to reduce the incubation time of the assays. Through a factorial design in which the inoculum suspension, incubation time, and percentage of the TTC solution were varied, it was possible to validate the RMMs for cefazolin, cefuroxime, ceftriaxone, and cefepime. The validated conditions employed 5 mL of MHA medium inoculated with 2% of Staphylococcus aureus suspension, incubation time of 5 h and 30 min at 37 ± 1 °C and the addition of 0.3% TTC solution in 1% agar, cefazolin and cefuroxime in concentrations from 15 to 60 µg mL−1, ceftriaxone and cefepime in concentrations from 20 to 80 µg mL−1. The methods were selective, linear (for standard and sample curves, respectively), accurate, precise, robust and rugged. The results found by the RMMs were statistically similar to those found by conventional microbiological methods, but the advantages of the former decreased the incubation time from 22 h to 5 h and 30 min. Therefore, RMMs can be used in the evaluation and quantification of cephalosporin antibiotics, ensuring their quality, safety, and therapeutic efficacy.

The Effects of Short‐Term Versus Long‐Term Exercise on Reducing Infarct Size After Ischemia‐Reperfusion in Male and Female Rat Hearts

Ischemic heart disease is the leading cause of death in both males and females worldwide, with myocardial infarction due to ischemia‐reperfusion (IR) injury being the most common manifestation. Exercise is known to protect the myocardium against IR injury, but the volume of exercise sufficient to confer cardioprotection is not well understood. A single bout of exercise has been shown to confer protection against IR injury, but it is not known whether longer term exercise training (weeks) adds additional cardioprotective benefit. In addition, it is unclear to what extent sex differences contribute to cardioprotection. To address these questions, we studied female and male Sprague‐Dawley rats (3 mo of age) randomly assigned to a 5 day exercise training (EX5d) group (n=20), an 8 week exercise training (EX8w) group (n=20), or a sedentary (Sed) control group (n=10). EX5d rats ran for 5 consecutive days, while EX8w animals ran 5 days a week for 8 weeks. The speed was gradually increased until animals were running 21–23 m/min for 60 min. Maximal running capacity improved in all groups as a result of training. EX5d animals improved maximal distance run by 31.9 ± 9% (pre vs post) while EX8w animals improved by 118.5 ± 15.6% (p&lt;0.01, EX5d vs EX8w). At least 24 h after the last training session, animals were anesthetized and the left anterior descending coronary artery was occluded via suture to induce regional ischemia for 45 min, followed by 24 h of reperfusion. After 24 hours, occlusion was reestablished and 0.5% Evans blue dye was injected into the aorta and perfused through the heart. Hearts were then sectioned, placed in a 0.1% triphenyltetrazolium chloride solution and incubated at 37° C for 10 minutes, then flash frozen in OCT. Infarct areas were quantified using ImageJ software, and infarct values calculated as a percentage of the zone at risk (ZAR). Exercise training led to a significant reduction in infarct size, with Sed, 5d, and 8w animals having infarct sizes of 37.2 ± 3, 27.5 ± 2, and 21.4 ± 1% of the ZAR, respectively (p&lt;0.01, Sed vs EX5d and Sed vs EX8w). Hearts from Sed females had significantly smaller infarcts than those of Sed males (41.8 ± 3 and 32.6 ± 1% of the ZAR, respectively). This sex difference persisted in response to exercise training, with females exhibiting smaller infarct sizes following 5 days of exercise training compared to males (25.1 ± 1 and 29.9 ± 1% of the ZAR, in females and males respectively (p&lt;0.05)), as well as 8 weeks of training (19.4 ± 1 and 23.5 ± 1% of the ZAR, respectively (p&lt;0.05)). When comparing within‐sex differences in cardioprotection, 8 weeks of exercise did not lead to significant infarct size reduction compared to 5 days of exercise in males (23.5 vs 29.9% of the ZAR). However, hearts from EX8w females had significantly smaller infarct sizes when compared to EX5d females (19.4 vs 25.1% of the ZAR, respectively; p&lt;0.01). These results show that 1) long‐term exercise training can augment infarct sparing against IR injury when compared to short duration exercise, and 2) this effect is sex‐dependent.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diagnosis of Early Myocardial Infarction by Histochemical Staining of Heart on Autopsy

INTRODUCTION : The incidence of Sudden Unexpected Death is increasing globally. Cardio vascular disease is the most common cause for sudden death. Eighty percent of sudden cardiac death is due to coronary arterial disease36. In 25% of cases death occurs abruptly and unexpectedly within first hour of onset of clinical symptom. Most often, the identification of early change of Myocardial infarction becomes difficult during postmortem examination, as gross change of infarct will not be apparent for 24 to 48 hrs following myocardial ischemic damage Histochemical staining techniques using azo dyes are based on the fact that ischemic myocardial cells lose their membrane integrity and release their enzyme contents in to the blood, resulting in a marked decrease or total depletion of these enzymes in the ischemic areas of the myocardium. Enzyme depleted infarct myocardium remain unstained, which is the principle of this study. AIMS AND OBJECTIVES : 1. To diagnose early Myocardial Infarction by Histochemical staining of Myocardium using Triphenyl Tetrazolium Chloride (TTC) while performing autopsy, in the absence of appreciable macroscopic changes in the myocardial tissue. 2. To confirm those identified areas of early myocardial infarction by Histopathological Examination. 3. To determine the diagnostic validity of the Histochemical Staining (TTC) of heart in gross detection of early Myocardial Infarction. MATERIALS AND METHODS: Slices from 18 hearts of suggested or suspected sudden cardiac death were incubated in 1% Triphenyl Tetrazolium Chloride Solution for 20 min. after adjusting the pH of the solution using phosphate buffer. The heart slices were examined for unstained area. Normal myocardium was stained whereas infarct areas remain unstained. Both stained and unstained areas were subjected to Histopathological examination for early Myocardial Infarction. OBSERVATION AND RESULTS: Of 18 hearts stained with TTC solution, 11 hearts showed positive TTC result, 5 hearts showed negative result, 1 heart showed false positive result and 1 heart showed false negative result. CONCLUSION : Histochemical staining using 1% TTC solution is a reliable method in detection of early myocardial infarction. Though this method has diagnostic validity of 88.88%, it could not identify death due to arrhythmias.

Corrigendum: Engineered Substrate-Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics.

In the middle panel of Figure 1 c of this Communication, a wrong image for the ψDrp1-treated heart was included. The correct image is shown below. This change does not alter the overall data or the conclusion of the study. Cardioprotective activity of the peptides as measured in whole hearts subjected to simulated myocardial infarction, ex vivo. […] b,c) Triphenyltetrazolium chloride solution (TTC) staining (red indicates live tissue and white indicated dead tissue; n=6/hearts per treatment), […]

Development, Optimization, and Validation of a Microplate Bioassay for Relative Potency Determination of Linezolid Using a Design Space Concept, and its Measurement Uncertainty.

The aim of this study was to develop, optimize, and validate a microplate bioassay for relative potency determination of linezolid in pharmaceutical samples using quality-by-design and design space approaches. In addition, a procedure is described for estimating relative potency uncertainty based on microbiological response variability. The influence of culture media composition was studied using a factorial design and a central composite design was adopted to study the influence of inoculum proportion and triphenyltetrazolium chloride in microbial growth. The microplate bioassay was optimized regarding the responses of low, medium, and high doses of linezolid, negative and positive controls, and the slope, intercept, and correlation coefficient of dose-response curves. According to optimization results, design space ranges were established using: (a) low (1.0 μg/mL), medium (2.0 μg/mL), and high (4.0 μg/mL) doses of pharmaceutical samples and linezolid chemical reference substance; (b) Staphylococcus aureus ATCC 653 in an inoculum proportion of 10%; (c) antibiotic No. 3 culture medium pH 7.0±0.1; (d) 6 h incubation at 37.0±0.1ºC; and (e) addition of 50 μL of 0.5% (w/v) triphenyltetrazolium chloride solution. The microplate bioassay was linear (r2=0.992), specific, precise (repeatability RSD=2.3% and intermediate precision RSD=4.3%), accurate (mean recovery=101.4%), and robust. The overall measurement uncertainty was reasonable considering the increased variability inherent in microbiological response. Final uncertainty was comparable with those obtained with other microbiological assays, as well as chemical methods.

Abstract 427: Comparison of RF Renal Denervation Systems: Multiple Bipolar Electrodes With Blood Flow Control vs. Single Monopolar Electrode

Introduction: Boston Scientific’s Vessix TM Over-the-Wire (OTW) balloon catheter uses bipolar radiofrequency (RF) energy and blood flow control to deliver significantly less energy than monopolar RF renal denervation systems. We hypothesize that the greater energy used in monopolar RF systems increases the risk for non-target tissue injury and inconsistent lumenal lesion formation. Methods: A computational model was used to simulate the clinical settings and control algorithms of a monopolar single point catheter and bipolar balloon catheter. The electrical field was solved to calculate the Joule heating in tissue and a transient thermal model was solved for the temperature change for the duration of RF energy delivery. In a swine model, RF treated renal arteries and select psoas muscles were rinsed and submerged in a beaker containing TTC (Triphenyl Tetrazolium Chloride) solution to aide in identification of thermal lesions. Non-viable tissue does not metabolize the stain and remains white, in contrast to red stained viable tissue. Results: The computational model predicted that the monopolar RF lesion varied due to electrode-tissue contact conditions. Lesion depth was dependent on the electrical conductivity of the artery wall and structures near the electrode. In contrast, the Vessix bipolar balloon produced consistent lesion pattern and depth due to fixed balloon apposition and blood flow control, focal energy delivery, and the temperature control algorithm. TTC stained renal arteries treated with the bipolar balloon device had accurate &amp; consistent electrode placement. The single electrode monopolar device had inconsistent tip placement, with lesion depths decreasing with decreasing treatment time. Non-target lesions (psoas muscle and bowel) were observed with the monopolar device. Psoas muscle thermal lesion depth decreased from 1.5mm with 120s ablation time (67% incidence) to 0.3mm with 30s ablation time (17% incidence) with a single electrode unipolar device (N=16). Non-target lesions were not observed with the bipolar balloon device (N=13). Conclusions: The BSC bipolar balloon renal denervation system had increased accuracy of electrode placement in the renal artery with no observations of non-target thermal injury.

Viability of barley seeds by the tetrazolium test

The tetrazolium test is used to control seed quality of various plant species since it allows a rapid evaluation of viability. Freshly harvested barley seeds show dormancy that can make the germination test ineffective for an immediate evaluation. Therefore, the development of more efficient methods, such as the tetrazolium test, is necessary. The objective of this research work was to study various procedures for performing the tetrazolium test on barley seeds. Five lots of cv. BRS 195 barley seeds were used and subjected to the following treatments: two different methods of seed preconditioning (direct immersion in H2O and between sheets of moistened paper towels); two types of preparation for staining (longitudinal cross-section of the seed through the embryo with immersion of one half in a 2,3,5 triphenyl tetrazolium chloride solution or placing both halves on top of filter paper moistened with the tetrazolium salt solution); two methods of staining (on top of filter paper and direct immersion in the tetrazolium salt solution). Three concentrations of the tetrazolium salt solution (0.1%, 0.5%, and 1.0%) were used. It was concluded that the tetrazolium test on barley seeds may be accomplished with preconditioning by direct immersion in H2O and staining by immersing in a 0.1% or 0.5% concentration of tetrazolium salt solution or staining on top of filter paper moistened with such solution at a 1.0% concentration.

Protein Kinase C δ (δPKC)-Annexin V Interaction: A REQUIRED STEP IN δPKC TRANSLOCATION AND FUNCTION

Protein Kinase C δ (δPKC)-Annexin V Interaction: A REQUIRED STEP IN δPKC TRANSLOCATION AND FUNCTION

Protein Kinase A-mediated Phosphorylation Modulates Cytochrome c Oxidase Function and Augments Hypoxia and Myocardial Ischemia-related Injury

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.

Intraperitoneal injection induces a delayed preconditioning-like effect in mice

We hypothesized that intraperitoneal injections of anaesthetics or fluid per se might evoke a delayed preconditioning-like response in mice hearts isolated and Langendorff perfused 24 h later. To test this, mice were given opioid anaesthesia by intraperitoneal injections or sham treated and the hearts were harvested and subjected to global ischaemia and reperfusion 24 h later in series 1. In series 2, mice were subjected to intraperitoneal injection of Ringer, sham needle prick procedure, or no intervention 24 h before heart isolation. In series 3, intraperitoneal Ringer injection 24 h earlier was compared with the effects of classic preconditioning or no pretreatment of the isolated heart or no treatment. Heart function was measured in all series. At the end of reperfusion, hearts in series 1 and 2 were frozen and infarct size was estimated by triphenyltetrazolium chloride solution. In series 3, separate hearts were frozen for immunoblotting to detect phosphorylation of mitogen-activated protein (MAP) kinases. Cardiac activation of nuclear factor kappa B (NFkappaB) was measured using a NFkappaB luciferase firefly reporter mouse. The ischaemia-induced impairment of left ventricular function was attenuated by opioid anaesthesia injected 24 h earlier, which also reduced infarct size. Injection of fluid, but not the sham needle prick procedure, reduced infarct size. The functional protection afforded by classic preconditioning and Ringer pretreatment was comparable. Neither cardiac MAP kinases nor NFkappaB were influenced by the interventions. In conclusion, this study demonstrates a delayed preconditioning-like effect of the heart caused by intraperitoneal administration of opioid anaesthetics and of fluid only in the mouse. The mechanism of protection remains to be determined.

Quantitative assessment of infarct size in vivo by myocardial contrast echocardiography in a murine acute myocardial infarction model

Background: In vivo measurement of the infarct size in a small animal model is still challenging. The purpose of this study was to evaluate the feasibility of quantitative assessment of infarct size by myocardial contrast echocardiography (MCE) in the acute myocardial infarction (AMI) model of the rat. Methods: In 32 Sprague–Dawley rats with AMI, we measured total myocardial area (TMA) and infarct area (IA) of the rats by MCE study (MCE method). They were compared with those of postmortem heart measured by planimetry after histochemical staining with triphenyl tetrazolium chloride solution (TTC method). Simple TTC staining was done in 13 rats (Group 1). To reduce the postmortem change, continuous aortic and left ventricular (LV) pressure was loaded during TTC staining in 19 rats (Group 2). Results: The TMA, IA, and IA/TMA ratio measured by the MCE method were 38.4±3.4 mm 2, 18.3±0.8 mm 2, and 0.37±0.02 in Group 1, and 43.7±1.8 mm 2, 15.8±1.1 mm 2, and 0.37±0.02 Group 2, respectively. Those measured by the TTC method were 66.1±2.2 mm 2, 29.3±1.1 mm 2, and 0.44±0.01 in Group 1, and 65.9±2.5 mm 2, 26.5±1.7 mm 2, and 0.40±0.02 in Group 2, and 65.9±2.5 mm 2, 26.5±1.7 mm 2, and 0.44±0.02 in Group 2, respectively. Compared with the TTC method, the MCE method underestimated the TMA and IA in both groups ( p<0.001). There was no difference in TMA and IA between the two groups in both methods. IA/TMA ratio showed significant correlation between the two methods in both groups ( r=0.85, p<0.001). Conclusion: The IA/TMA ratio measured by the MCE method may be useful for in vivo estimation of the myocardial infarct size in the AMI model of the rat.

The Cardioprotective Effect of Ischemic Preconditioning : Role of Adenosine and Protein Kinase C

Background:Brief episodes of coronary blood flow interruption, ischemic preconditioning(IP), following a prolonged ischemia induces myocardial tolerance to ischemia and improves myocardial function during reperfusion by undefined mechanism. Recently, it has been suggested that the signal transduction pathway of the cardiomyocyte itself may involve in this protection. The aims of the present study were:(1) to examine the effect of adenosine in early phase of IP, (2) to define the relationship between the adenosine and protein kinase C (PKC). Methods and Results:Hearts isolated from New Zealand White rabbit (1.2-1.5kg body weight, n=78) were perfused with Tyrode solution by non-recirculating Langendorff technique. After stabilization of baseline hemodynamics, the hearts were subjected to receiving 45min global ischemia (I) and 120min reperfusion (R) with or without IP. IP was induced by a single dose of 5min I and 10min R. A part of the IP hearts, calphostin C (200nmol/L), a PKC inhibitor, was administered 5min before IP and sustained during IP regimen. Left ventricular function and coronary flow were monitored. Infarct size was determined by staining with 1% triphenyltetrazolium chloride solution and computerized planimetry. Adenosine concentration in the coronary flow was determined by HPLC. Myocardial cytosolic and membrane PKC activities were measured by P-γ-ATP incorporation into PKC specific peptide. Expression of PKCe and PKCd was determined by SDSPAG-e and Western blot. IP enhanced improvement of functional recovery (p<0.05, in the left ventricular developed and end-

1005-78 Myocardial Preconditioning by Laser Irradiation

We previously demonstrated that transmural laser channels made after coronary occlusion did not acutely enhance blood flow or reduce infarct size. In contrast, other studies (which did not measure blood flow) reported increased survival and/or reduction of necrosis when laser channels were made prior to coronary occlusion. To reconcile this apparent paradox, we speculated that laser channels made prior to occlusion might protect the heart via a mechanism independent of blood flow. To test this hypothesis, we randomized 12 rats to either; (1) control: 1 hour of coronary artery occlusion followed by 5 hours of reperiusion. or (2) laser: 6 non-transmural channels were made in the anterior wall of the left ventricle using a holmium:YAG laser coupled to a 400 micron diameter optic fiber. Two minutes after the last channel was made, the left coronary artery was occluded for 1 hour and then reperiused for 5 hours. We monitored lead I of the ECG and noted the incidence of ventricular tachycardia (VT) and fibrillation (VF). At the end of the protocol. the artery was briefly reoccluded and we determined the area at risk (AR) by injection of pigment into the circulation. Heart slices were incubated in triphenyltetrazolium chloride solution to delineate the area of necrosis (AN), which was expressed as a percent of area at risk (AN/AR). [data given as mean ± SEM]Empty CellARAN/ARVT/vFControl49 ± 361 ± 95Laser45 ± 535 ± 4*0**p &lt; 0.03 versus control p &lt; 0.03 versus control Although laser channels were associated with thermally-induced necrosis, total infarct size was smaller in laser-treated hearts. Protection of muscle was most prominent in heart slices containing laser channels. but also occurred in slices without channels. Laser treatment also significantly reduced the incidence of VT and VF. In conclusion, non-transmural laser channels “preconditioned” the heart against subsequent ischemia. Although the mechanism of protection is unknown, it cannot be attributed to increased blood flow to the tissue because the channels did not connect to the ventricular cavity.

Blood flow-dependent uptake of indium-111 monoclonal antimyosin antibody in canine acute myocardial infarction

Objectives. The relation of myocardial blood flow and indium-111 (111In) antimyosin antibody uptake was studied by inducing myocardial infarction in 18 dogs, 8 with closed chest left anterior descending artery balloon occlusion for 3 h followed by reperfusion (group A) and 10 dogs with open chest left anterior descending artery ligation (without reperfusion, group B).Background. The relation of antimyosin uptake to myocardial injury has been documented. However, its relation to tracer delivery by myocardial blood flow has not been studied and has been assumed to be independent.Methods. Indium-111 antimyosin antibody, 2 mCi, was injected 20 min after reperfusion and 3 h after coronary artery ligation in groups A and B, respectively. Regional blood flows were determined by radiolabeled microspheres during occlusion and 24 h later in both groups. On day 2, dogs were killed after risk zone delineation with gentian violet. The heart was excised and stained with triphenyltetrazolium chloride solution and graded for increasing severity of tissue injury based on extent of staining. Microsphere activity and 111In antimyosin activity were measured in control tissue (grade 1), noninfarct tissue at risk (grade 2), mixed tissue (grade 3), infarct tissue (grade 4) and hemorrhagic infarct tissue (grade 5, present only in group A dogs). count activity was normalized to that of the mean value in control tissue (grade 1) and expressed as a ratio of activity.Results. Indium-111 astimyosin activity was high in triphenyltetrazolium chloride grade 4 tissue in both groups but was attenuated in grade 4 tissue in group B dogs (10.6 ± 5.1 vs. 5.0 ± 4.5; p < 0.05 group A vs. group B), which had lower blood flow on day 2 (0.51 ± 0.36 vs. 0.23 vs. 0.22; p < 0.01). Normalizing 111In antimyosin activity for blood flow on day 2 resulted in equivalent 111In antimyosin uptake for infarct tissue (32.6 ± 21.6 vs. 36.6 ± 29.8 for group A vs. group B; p = NS).Conclusions. Thus, 111In antimyosin uptake is a specific marker of necrotic tissue with a high signal ratio in reperfused tissue. However, its uptake is dependent on residual blood flow in the infarct territory. Indium-111 antimyosin could potentially serve as a suitable tracer for infarct sizing if myocardial blood flow in the same region were factored simultaneously.