System Studies And Technological Advances

Limitations

Discuss about the System Studies and Technological Advances.

In this experiment ischemia can be triggered by the mediators released from the tissues. Receptors specific to the cardiomyocytes like A1 and A3, bradykinin B2, α1-adrenergic, AT1 angiotensin II, muscarinic M2, and δ-opioid receptors can be stimulated by the released mediators. Both single and multiple pathway ischemia protection can be achieved by using freshly isolated cardiomyocytes. Isolated cardiomyocytes experiment would be valuable tool for the evaluation of the mechanistic insight of the ischemia. In comparison with the whole heart experiment, use of cardiomyocytes would be helpful in eliminating endothelial cells and fibroblasts, which would affect analysis. Pure cardiomyocytes would give more robust analysis of desired cell signaling as compared to the whole heart. Visibility using microscope is the most prominent advantage of cardiomyocytes. More number of cells can be made available in freshly isolated cardiomyocytes as compared to the cell cultures.

Limitations: Isolated cardiomyocytes cannot be used for longer time and repeated use of cardiomyocytes is not possible. In the isolation of cardiomyocytes from blood vessel and extracellular matrix, there is the possibility of loss of vital features of pathophysiology of ischemia. Isolated cardiomyocytes are not capable of reproducing ischemia mechanisms similar to the whole heart ¹ .

Methods: Cardiomyocytes will be isolated form isolated heart by enzymatic digestion using calcium-free medium containing collagenase and trypsin. Isolated cardiomyocytes will be centrifuged at very slow speed to get pellet with fluid spread over pellet with fluid volume of one-third volume of the pellet. Supernatant will be covered with oil and will be incubated at 37°C. Preconditioning will be performed by incubation in the absence of glucose. Evaluation of occurrence of ischemia and effect of Bob on the ischemia in cardiomyocytes will be evaluated using Tryptan Blue staining. Four set of isolated cardiomyocytes will be there like normal cardiomyocytes, ischemia induced cardiomyocytes, ischemia induced cardiomyocytes incubated with different concentrations of Bob and cardiomyocytes incubated with Cyclosporin A.   

Cyclosporin A will be used as positive control in this experiment. Cyclosporin A was already evaluated in the cardiomyocytes ischemia experiment and it was proved that Cyclosporin A exhibited its action through cytochrome C which is the mechanism of action Bob. Negative control will be used without any treatment and it will useful in the evaluation of induction of the ischemia in the isolated cardiomyocytes.

Following are the end results of this experiment. Viability of the cells using Tryptan Blue will be useful in the evaluating recovery of cardiomyocytes from ischemia after treatment with Bob. Autofluorescence of NADH in the mitochondrial matrix will be evaluated to assess the redox state of the cardiomyocytes. This redox state will be helpful in inferring the severity of ischemia in negative control, Bob treated cardiomyocytes and Cyclosporin A treated cardiomyocytes.

Methods

Objective: To evaluate effect of Bob on myocardial ischemia in Langendoorf rat heart perfusion.

Rationale: Langendorff isolated heart is a robust model for evaluating ischemia reperfusion injury.

Advantages: Langendoorf rat heart perfusion set up can be used to evaluate real-time pharmacological treatments. It is also useful to get the uniform size of the infracted tissue and also useful in the application of drug directly to the myocardium. In vivo models are useful only in the end point analysis ischemia reperfusion injury, while Langendoorf rat heart perfusion is also useful in the functional analysis of the ischemia reperfusion in real time.     

Limitations: Isolated heart is continuously deteriorating and it can be useful only for short duration. Isolated heart preparations are denerveted and due to this exact pathological ischemia are difficult to achieve.

Methods: Heart will be isolated. Aorta will be cannulated to the reservoir containing oxygenated perfusion fluid. Perfusion fluid will be contained in the sintered glass jacketed reservoir above the isolated heart. Regional ischemia will be induced by perfusing heart with Krebs–Henseleit solution. Perfusion will be continued for 15 minutes and ischemia will be induced for 30 minutes. Apoptotic cells will be identified by the dUTP nick end labelling (TUNEL) using CardioTACS. Heart tissue will be fixed in the 3.7 % formaldehyde for 24 hours and will be  embedded in the paraffin. Mitochondria will be isolated and mitochondrial respiration will be measured using Clarke-type oxygen electrotode. Cyclosporin A will be used in the washing buffer. Skinned cardiac fibers will be prepared and these fibers will be used for measurement of oxygen uptake. Level of cytochrome c will be measured by solubilizing cardiac fibers in Triton X-100. Cytochrome c will be measured using western blotting. Proteins will be identified using primary monoclonal antibodies to cytochrome c using ECL western blotting detection reagents. Caspase activity in the heart homogenate will be measured in sucrose based medium. Cytosolic protein will be incubated with buffer and z-DEVD-pnitroanilide which is a caspase 3substrate. Caspase will be measured using spectrophotometer at 405 nm.

Cyclosporin A will be used as positive control in this experiment. Heart will be preperfused with Cyclosporine A for about 15 minutes. Cyclosporine A protects heart from ischemia by preventing loss of cytochrome c. Use of Cyclosporine A is valid in this experiment because, Bob also exhibits similar mechanism of action. In this experiment, negative control will be without any type of treatment. This negative control will be useful in the evaluation of induction of ischemia and also evaluation of treatment effect of Bob and cyclosporine A.

Langendorff rat heart perfusion set up to allow ischemia-reperfusion

Cyclosporine A and Bob will be reducing level of cytochrome c release from the ischemic isolated rat heart. Pretreatment of heart with Cyclosporine A and Bob will be resulting in the no decrease in the respiratory rate of mitochondria which will be measured using membrane potential. Also, dependence of cytochrome release on caspase will be evaluated in this experiment.  Cyclospprine A and Bob will be inhibiting ischemia-induced caspase activation.

Objective: To evaluate effect of Bob on transient coronary artery ligation model of ischemia-reperfusion.

Rationale: Transient coronary artery ligation model exhibits exact ischemia pathology.

Advantages: Availability of experimental rat makes this model more feasible. Possibility of genetic modification in rat leads to the development of more specific model. Possibility of reperfusion makes this model make more clinically relevant because reperfusion always occurs in clinical setting prior to treatment. Ischemic period can be effectively controlled in this model based on the requirement of the intervention.

Limitations: Variability of infract size is more in this rat surgical model. Variation of the infract size is mainly depends on the location of ligation, hence it is very important to select same location for all the animals in the study. Range of infract size is between 20 – 90 % of the entire left ventricle and as a result this model requires large number of animals in a study. Variability due to modification in the surgical procedure has also been reported from different laboratories. Also, there is reported variability due to different time of analysis. Despite the benefits of reperfusion, there is the possibility of damage of part of the myocardium ^8-9.  

Methods: Rats will be approved from the Institutional Ethical Committee. Rats will be housed and procedure will be carried out in the aseptic condition. Rats will be anesthetized with suitable anesthetic agent. Chest and neck region of the rat will be shaved and will be placed in supine position under a stereomicroscope. Transient coronary artery ligation will be performed by making incision in the skin, chest muscle will be separated and thoracotomy will be performed in the fourth intercostals space. Pericardium will be removed and left coronary artery will ligated  at 1 mm distal end of the tip of left auricle with the help of 7-0 polypropylene suture. Ligated artery will be released after 30 minutes of ischemia. Rats survived after surgery will be assigned to different treatment groups like curaglutide and Bob. Sham control group will performed with the similar surgical procedure without ligation of the coronary artery. Drugs will be administered to the animals intraperitoneally at different time points after reperfusion like 0, 5, 15 and 30 minutes. Sham control animals will be treated with normal saline. An additional set of animals will be utilized for the evaluation of caspase 3 activity.

Transient coronary artery ligation

Each sample will be homogenized with RIPA buffer containing protease inhibitor and will be centrifuged. Fluorescence of the protein in treatment groups will be measured and will be compared with protein of the sham treated group to evaluate activity of caspase – 3 activity. Tunnel assay will be performed to measure apoptotic rate. After the antigen retrieval it will be incubated with monoclonal antibody against actin and it will be stained with 6-diamidino-2-phenylinidole (DAPI) ^10-12.   

Curaglutide will be used as positive control as it already proved its efficacy in the transient coronary artery ligation model. Negative control will be considered as the group with surgery without any type of treatment.

Results obtained will be decrease in the caspase 3 activity and reduction in the infract size. Other hemodynamic parameters will also be evaluated like left ventricular systolic pressure and heart rate.

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