The overall goal of this procedure is to induce vascular thrombosis and ischemic damage within a given cortical area by means of photo activation of a previously injected light sensitive dye. This is accomplished by first making an incision on the skin in order to expose the skull. After that, a phos sensitive dye, the rose bengal is injected intraperitoneal.
Five minutes later, the target cortical area is exposed to a cold light source for 15 minutes. Rose Bengal circulated into blood vessels. When illuminated rose Bengal is activated and produces cingulate oxygen.
The damage is components of endothelial cell membranes that in turns form thrombo in all the illuminated surface leading To local RA blood flower interruption. At The end of the procedure, The skin is suture to close up the one. The identification of ischemic areas can be quicker accomplished by TT C or C crystal staining.
Hi, I'm v Abes. Hi, my name is Mon Maze. Photo thrombosis is a quick and uninvasive technique for inducing reproducible cortical ischemic lesion in mice.
It's suitable for a number of experimental stroke studies, particularly for the evaluation of cortical plasticity in response to brain injury at either cellular and molecular level. Cheming mod are usually performing rats, which are easier to manipulate on mice. However, mice are less expensive and a large number of congenic strengths is available.
The models that we are using is technically easy to perform, so now we're going to present you how to induce a Romble Deion. In Mice, We rose Bengal in an OV tube disolve sterile cell solution until reaching a final concentration of 15 milligrams per milliliter. Storage it in the dark at room temperature up to two months, and record the mouse body weight.
The dose of Rose Bengal that we are going to use is 10 microliters per gram of body weight. To perform photo thrombosis, you need a stereotactic apparatus and a cold light source. We also suggest to monitor the animal temperature throughout the surgery with a rectal coupled with a heating pad.
For the surgical preparation, you need a pair of forceps, a scalpel, some skin retractors, a pair of scissors, a CI kit, Betadine ethanol, and cotton swab for disinfection and an heating blanket for surgical recovery. Sanitize the surgical area in case of loss of sterility during the surgery, immediately stopped the procedure and started it over again to avoid the risk of infection. Anesthetized mice with isof fluorine vaporized in a mixture of 50%oxygen and 50%the nitrogen monoxide and place the mouse in induction chamber deliver isof fluorine at an initial dose of 3.5%When deep anesthesia is switched, remove the animal from the induction chamber and place it in the stereotaxic frame.
Maintain oph floran dose to 2%throughout the surgery. Use toe pinches in order to ensure the animal is elic sedated. Apply eye ointment in order to prevent the animal side from dry out.
Insert rectal probe coupled to a feedback control heating pad and maintain the mouse temperature at 37 degrees. Shape the mouse Firmly secure the head to the stereotaxic apparatus by inserting the ear person to external mitus. Disinfect the skin of the skull with alternating swipes of 17%Ethanol and Betadine with the scalpel.
Make an incision along the midline in order to fully expose the bone. Position the skin retractors to keep the skull exposed. Retract the periosteum with the scalpel and dry the bone surface using sterile cotton swaps.
Identify the BMA and lamb. Do place a glass micropipet on the bmas reference bone and move it to the coordinates of Your region of interest. Mark the positions with four reference points to ensure The alignment of the optic fiber with the area of interest.
Before illuminating, apply a mask with a hole on the skull surface to the limit the region of interest, and to prevent light from scattering or apply cap directly on the fiber tip for reducing the ture. Using the reference points, position an optic fiber as close as possible on the S skull face, but pay attention not to observe pressure on it. Load the rose Bengal solution in a one milliliter syringe and calculate the volume to be injected according to the body weight.
Considering a dose of 10 microliters per gram proceed to intraperitoneal injection of rose Bengal After five minutes, start illuminating with coal light fibers. 15 minutes later, switch out the fibers And suture the wand. Apply sterile cell solution on the skull surface for rehydrating.
Remove the skin retractors. Close the one using reverse cutting needle and silk or nylon suture thread. Carefully Remove the rectal probe and the ear bars.
Remove the mouse from the stereotaxic apparatus and place it on a pre-war heating blanket until it is fully awake. Then return to its cage. Measurement of the extent of cerebral infarction can be performed by histological staining with RIF, tealium chloride or TTC on fresh brain lysis, which labels in red.
The intact tissue by means of the hydrogenous reaction, the infarcted tissue typically appears pale. The absence of staining is due to the lack of dehydrogenase activity and reveals loss of cell integrity in the damaged areas. Alternatively, the ischemic damage can be revealed by CR violet staining on fixed brain slices.
Many different software can be used for both the measurement and the three dimensional and reconstruction of the infarcted area, Including nma J and neuro elucidate. We've shown you how to Perform photos thrombotic in mice in which any specific cortical area can be targeted with I reproducibility. Furthermore, this model is non-invasive and easy to perform as it does not require colectomy.
However, a series of preliminary experiments should be initially carried out in order to determine the minimum amount of diet to be injected for producing a lesion affecting only the target area or for inducing significant behavioral deficit. Thanks for watching. If you have any question, Please contact us and good luck for your experiment.Goodbye.
