The overall goal of this procedure is to generate left intravital brain hematoma formation in a mouse. This is accomplished by first anesthetizing the mouse and mounting the head in a stereotactic frame. The next steps of the procedure are to expose the skull and drill a bur hole.
Then the left striatum is injected with collagenase or blood and the wound is closed up. Ultimately worsened. Neurobehavioral function can be detected by rotor rod testing, and the hematoma formation can be detected by magnetic resonance imaging and hemat toxicant and eosin Staining.
Intra varietal collagenase or autologous blood injection can help answer key questions in the field of intra cerebral hemorrhage, such as the effects of hematoma expansion, cerebral edema formation, and secondary injury mechanisms. These techniques allow in vivo testing of promising therapeutic interventions aimed at secondary injury mechanisms after intra cerebral hemorrhage. At the same time that discovery of these targets occur and transgenic systems are created.
Generally individuals new to this method may initially struggle with mouse intubations drilling craniotomies, intra TAL injections without leakage, and appropriate post-surgical care. Visual demonstration of these models is critical as highly reproducible injury is essential to in vivo study of pathophysiology and interventions. There are many critical steps that require experience to avoid error and subsequent production of dissimilar injuries.
Demonstrating the collagenase injection procedure will be bayle. A research scientist in the multidisciplinary neuroprotection laboratories demonstrating the autologous blood injection procedure will be Washington Shang, an assistant professor and the Department of Anesthesiology at Duke University. Intubation is necessary to control ventilation and subsequent effects on cerebral blood flow.
Once anesthetized and unresponsive to a toe pinch, use a 30 millimeter 20 gauge intravenous catheter to intubate the trachea. Ventilate the lungs at 105 breaths per minute with a tidal volume of point 75 milliliters. Next, shave the scalp back at the surgical bench.
Secure the head in a stereotaxic frame level. It coronal and Sally. Use an underbody circulating waterbed to maintain the mouse's body temperature.
Also apply ophthalmic ointment and monitor the mouse's internal temperature. Then wipe down the exposed scalp with three alternations of Betadine and 70%ethanol. Be sure to have prepared in advance Type four s clostridial collagenase in normal saline at a concentration of 0.0 75 units per 0.4 microliters.
Begin by making a one centimeter midline scalp incision. Then expose the bgma by wiping the periosteum laterally with a sterile cotton tipped applicator. Next, using a water cooled drill, open a one millimeter bur hole, 2.2 millimeters to the left of the bgma.
Next, rotate the collagenase vial five times to ensure it is well mixed. Then attach a 0.5 microliter syringe with a 25 gauge needle to the stereotactic frame and wash it out with 0.5 microliters of collagenase solution four times. Then draw up 0.5 microliters of collagenase solution into the syringe.
Align the needle tip to the hole and prime it expel. Point one microliters of collagenase and wipe the needle bevel clean with a razor. Now advance the needle three millimeters into the cortex and wait 30 seconds.
Then over the next 90 seconds, eject the remaining collagenase after the injection, decrease the isof fluorine to the ventilator to 1%and leave everything still for five minutes. Then withdraw the needle slowly. Once the needle is fully withdrawn, apply a few drops of point 25%bupivacaine into the surgical wound.
Then suture the skin. Turn off the isof fluorine and release the mouse from the frame. Once breathing, return it to a clean cage with three axis to food and water.
First, prepare the animal for surgery as previously described up to having drilled the hole to access the brain. Then draw 50 microliters of saline into a 30 gauge 50 microliter syringe. Attach a 70 centimeter length of polyethylene tube to the syringe and expel the saline into the tube to push out all air from the tube.
Now, carefully pull the plunger out one millimeter to create an air bubble at the distal end of the assembly. This will prevent s saline and blood from mixing when it is used on the mouse's tail. Use 70%ethanol to wipe clean the area with the central artery can be accessed.
Then cut the artery with a razor from one half to one centimeter from the tail tip. Collect 40 microliters of blood into the assembled syringe and tube after blood collection. Hemostatic control generally occurs through pressure application alone.
However, when necessary electrocautery may be utilized, then attach the syringe to an injection pump and at the other end, connect the metal cannula portion of a 27 gauge needle to the tube and secure the needle to the frame. Prime the needle by expelling two microliters of blood and wipe the bevel clean with a razor. Align the needle with the hole and insert it three millimeters into the cortex.
Then slowly inject 35 microliters of the blood at two microliters per minute. Once the injection is complete, reduce the isof fluorine flow to 1%Wait 10 minutes and complete the surgery as described in the previous section. In the evening after the surgery, inject a half milliliter of saline subcutaneously into the back of the mouse's neck to ensure overnight hydration over the first seven postoperative days.
Provide fresh softened food and water directly in the cage so the animal can more easily stay nourished. Monitor the mouse's weight wound healing and general character during this week. If recovery takes longer than a week, then manually remove the sutures under light.Anesthesia.
Differences are seen in hematoma formation between intravital autologous blood and collagenase injections. Intra varietal injections of 35 microliters of autologous blood do not produce hematomas as large as mice injected with 0.0 75 units of type four s clostridial collagenase ipsilateral turning is demonstrated immediately after wake up for autologous blood injected mice, and within two to four hours after collagenase injection as hematoma expansion occurs. If this is an observed, the injury may be insignificant on the first post-injury day.
Mice in both models should demonstrate significant neurological deficits which begin to return to baseline over the first seven days after injection at 24 hours after injection ipsilateral hemispheres show stable hematoma volumes. Furthermore, brain water content should be expected to be about 79.8%in collagenase injected mice and about 79.3%in autologous blood injected Mice Once mastered inal injection can be done in approximately 20 to 30 minutes for collagenase injection and 50 to 60 minutes for blood injection if it is performed properly. While attempting these procedures, it's important to remember to perform each step precisely with meticulous controlled motions following intra varietal injection.
Other methods like neurobehavioral testing and complimentary immunohistochemistry can be performed in order to answer additional questions. Regarding neuroinflammation, neuronal injury and recovery. Don't forget that working with mice, needles, clostridial, collagenase, and blood can be hazardous.
Universal precautions should be observed and care should always be taken for sanitary elimination of all waste. After performing these procedures.
