Intra vital microscopy of the CRE matric microcirculation is a convenient method to gain insights into ischemia, reperfusion injury of striated Muscle tissue. In a step-by-step Protocol, we depict how to get started with respiration controlled anesthesia under sufficient monitoring via arterial cannulation to keep the animal firmly anesthetized for longer periods of time. We illustrate how to cannulate the jugular vein and then describe the CRE matric preparation as a thin flat sheet for outstanding optical resolution.
We provide a protocol for leukocyte imaging in isch perfusion injury that has been well established in our laboratories. This model can be used to analyze Ocid actation for ischemia. Reperfusion injury research.
Ischemia fusion injury has been implicated in a large area of pathological conditions, such as myocardial infarction, intestinal ischemia, as well as following transplant and cardiovascular surgery. Hyperperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue adjacent to the production of reactive oxygen species, the activation of the complement system and increased microvascular permeability. The activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage During reperfusion.
We here promote intra vital microscopy of the post capillary manual as a convenient method to analyze leukocyte activation in isch reperfusion injury. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate and safely perform the method. Following Approval from ethics committee and aestheticize male rights rats with a body weight of 120 to 180 grams, placed the rat inside the plexiglass box and deliver two to three volume percent iso florin via iso florin vaporizer.
As soon as the appropriate level of anesthesia is achieved, the rat is weighed and shaved on the ventral cervical area. Place the ru dorsa recumbent on a heating pad to maintain body temperature at 37 degrees Celsius and apply iso fluorine at two volume percent using a Silicon mask. The following preparations are best accomplished using a Surgical microscope.
Perform a two centimeter horizontal skin incision in the area of the SRAs stern notch. Mobilize the sali glands laterally. Now you face the ventral neck muscles.
Carefully separate them in the midline and find the trachea. Expose one to two centimeter of the trachea and place a micro forceps from underneath to elevate it. Now SL about halfway through the ventral side of the trachea insert a tube of 14 gauges as a tracheal tube into the lower part of the trachea, which has previously been connected to an animal ventilator.
Respiration can then be volume controlled with a frequency of 35 to 45 breaths per minute. A tidal volume of 4.5 to five M and an ISO fluorine concentration of 1.5 to two volume percent for the carotid cannulation. The right sternal hyoid muscle is separated by blunt dissection.
To locate a carotid artery, separate the vagus nerve carefully from the carotid artery and mount the artery on Anki micro forceps to stop blood flowing from the heart past two pieces of suture beneath the carotid artery in reference to the heart, the more distal suture is tied tightly, whereas the proximal suture is tied loosely around the carotid artery. The application of a second proximal suture prevents bleeding after removing the micro forceps using micro scissors as small cut is made into the carotid artery between the two ligatures. Insert a polyethylene catheter filled with normal saline solution that is connected to a pressure trans user.
Remove the micro forceps and thread the catheter further into the artery. Perform intermittent arterial blood gas analysis using a blood gas analyzer and maintain blood pH within physiological limits for the intravenous application of fluorescent dyes or other drugs of interest. Focus on the area of the left gular vein with the surgical microscope with forceps in each hand, tear the thin fascia to reveal the jugular vein.
Mount the vein on an angled micro forceps to stop the blood flow. Using forceps, two pieces of equal length of suture are passed under the jugular vein in identical fashion to the carotid cannulation, make a small cut into the jugular vein and insert a polyethylene catheter that has been flushed with cell line solution. Thread the catheter toward the heart and tighten the ligator closest to the heart.
Around the vein and the catheter, We use an aluminum Stage four cremasteric imaging that rapidly adopts the desired temperature of the heating pad. The initial incision is made in the skin and external spermatic phy above the scrotum in the very distant end, followed by cautious dilatation of the subdermal space using the fine scissors. As soon as the tissue is exposed, it is moistened with a preheated seline solution.
Connective tissue between the external spermatic fascia and the CRE master muscle is carefully removed to free trimester muscles from the surrounding tissues. Continuous superfusion during dissection hydrates the tissue which facilitate visibility and removal such as the distal end of the CRE master S to hold down and slightly extend the end of the S in inside the distal end of the sac on the ventral aspect and elongate the incision proximally. Using micro scissors carefully categorized bleeding vessels along the incision lines.
Utilizing a thermal Cory, the open cray master muscle lies flat on the aluminum pedestal dose, still connected by a thin ligaments to the EPIs underneath the testicle, including a small artery and vein. Use the ery to seal the vessels and to cut the connective ligaments between discal and cremasteric tissue. Gently push back the isolated testicle into the inguinal canal.
In addition to the first fixation, which are four itches of the tissue and tape the threads cautiously spread the cremasteric tissue radially on the aluminum stage. Place two ends of polyethylene catheter close to the cremaster tissue for the superfusion setup. Draw a line of petroleum gili around the CRE master muscle using a syringe To create a fluid chamber.
Place a square cover slip over the cric tissue and firmly attach the edges to the Vaseline line. The cremasteric tissue is now ready for microscopic Imaging. The basic intra vital setup May vary.
We use an intra vital epi fluorescence microscope equipped with a 470 nanometer LED luminous source for EP lamination. With a water immersion objective, we achieve a magnification of approximately 800 x record observation by means of a high resolution digital camera. For epi fluorescence imaging, the experiment should be executed in a dark room.
For leukocyte labeling, inject rumine six J intravenously via the jugular catheter at concentration of 0.4 milligram per kilogram body weight. Choose a post capillary venue for observation. The size of the vessel should range between 20 to 60 micrometer and blood flow should be sufficient.
Before Ischemia occurs, let the tissue stabilize for 30 minutes. Afterwards, perform a recording of 30 seconds to establish basal values for leukocyte rolling and adherence. Gently place a bemer vessel clip around the very proximal end of the exposed cremasteric tissue by using application forceps.
Remove the vessel clamp after 30 minutes of isch. Subsequent recordings can be made throughout the reperfusion Period. For the quantification of leukocyte rolling, define a virtual line that intersects the vessel vertically, which is consistent in all records.
Count the number of rolling leukocytes that pass the line within 30 seconds manually. For the quantification of adherent leukocytes, define a 200 micrometer vessel section that is consistent. In all records.
Count the number of clearly visible leukocytes that remain static during 30 seconds. Representative results are provided Within the written protocol. Though Isch Reperfusion injury has been implicated in various organ systems, we here describe a method to analyze leukocyte endothelial interaction in reperfusion injury of straight muscle tissue.
Thus, particularly relevant for flip surgery, as it is assumed that post ischemic tissue injury of other organs involves the same inflammatory mechanisms. The described method can be used to gain principle insights into pathological mechanisms in ischemia fusion injury. Applying ischemia to the cremaster muscle can be easily performed and perfectly reflex clinically relevant conditions such as free flap surgery.
Moreover, modifications like cold ischemia or warm ischemia or the chromos teric superfusion with substances that influence inflammation can be easily applied. Intraoral injection may be the method of choice when treatment is needed, hours or days before recording.
