In this video, we aim to demonstrate two methods of generating int intercerebral hemorrhage. In mice, we will describe a double injection model of autologous whole blood as well as an TAL injection of bacterial collagenase. Both models are commonly used in hemorrhagic stroke research.
Before showing the surgery, we would like to illustrate the procedure starting with the double injection model of autologous whole blood. This schematic represents a coronal brain section 0.2 millimeters anterior of bgma. The lateral ventricle is marked LV CPU stands for ProAm, A part of the corpus striatum, and G identifies the globus palus, both the corpus striatum as well as the globus palus belong to a group of subcortical nuclei known as the basal ganglia.
A 26 gauge Hamilton syringe is positioned to 0.2 millimeters anterior and two millimeters lateral to bgma. The needle is inserted through a cranial bur hole and advanced three millimeters below the dura. Following the insertion, five microliters of autologous whole blood previously collected from the central tail artery are injected inter parenchymal at a rate of two microliters per minute.
The needle is then advanced 0.7 millimeters to the target position of 3.7 millimeters in depth. After a waiting period of five minutes, 25 microliters of autologous blood are injected into the striatum. The needle is left in place for an additional 10 minutes before being slowly withdrawn at a rate of one millimeter per minute.
Coagulated blood of the first injection prevents back leakage along the needle track. In contrast, the collagenase injection model consists of a single intra varietal injection of 0.075 units of bacterial collagenase dissolved in a half microliter of saline. Collagenase is injected at a rate of two microliters per minute.
The needle is slowly withdrawn 10 minutes after the completed injection. Bacterial collagenase disrupts the basal lamina of cerebral vessels causing spontaneous bleeding into the surrounding brain tissue. Subsequent or delayed formation of per hematoma, brain edema aggravates the primary brain injury resulting in worse neuro functional outcomes after intercerebral hemorrhage.
In the following part, we will describe the apparatus used for the induction of intracerebral hemorrhage. In mice, we utilize a small animal stereotaxic instrument, which consists of a U-shaped metal frame and a three-dimensional manipulator arm. The injection pump attached to the horizontal manipulator arm is connected to its electronic control unit.
The nose and tooth bar, as well as the two ear pins demobilize the rodent's head. During surgery, a 26 gauge Hamilton syringe is mounted to the syringe holder of the injection pump. This assembly allows a controlled inter injection of either autologous blood or bacterial collagenase.
In laboratory mice experimental intracerebral hemorrhage is induced in eight week old CD one mice. Before surgery weigh all animals in order to adjust the concentration of anesthesia and eventual treatments According to body weight, a triple beam animal scale can be used for this purpose. To obtain general anesthesia, perform an intraperitoneal co injection of ketamine and xylazine, 100 milligrams per kilogram ketamine, and 10 milligrams per kilogram.
Xylazine allow seven to 10 minutes for the anesthesia to take effect and then shave the animal scalp and apply up thalamic ointment on both eyes. Following that, mount the mouse's head onto the stereotactic frame. Extend its tongue laterally to secure an adequate airway and place the upper incisors into the tooth bar.
Advance the ear pins gently into both ears and fasten them. After feeling the resistance by the skull, secure the nose bar as well. After mounting the animal, disinfect the surgical area using Betadine, followed by 70%ethanol.
This application should be repeated for three cycles of Betadine and ethanol. Make a one centimeter long midline incision of the scalp using a number 10 scalpel blade. Then use cotton tipped applicators to clear away the soft tissue covering the skull.
In order to expose the bgma, mount the Hamilton syringe onto the injector pump and place the needle over bgma with its beveled edge facing the midline. Then move the syringe 0.2 millimeters anterior torema and two millimeters laterally to the right. At these coordinates, make a small cranial bur hole using a variable speed drill with a one millimeter drill bit autologous whole blood for the intercerebral blood injection is collected from the central tail artery, suspend the animal's tail and disinfect its lower surface with 70%ethanol.
Then puncture the tail artery and collect the blood in a capillary tooth. Transfer the blood quickly into a 26 gauge Hamilton syringe. Reattach the now 30 microliters or more of arterial blood containing Hamilton syringe to the injection pump.
Insert the needle through the bur hole just until the beveled edge tip is no longer visible. From this point, advance the needle three millimeters, ventrally, and inject five microliters of autologous blood into the brain at a rate of two microliters per minute. After completion of the first injection, advance the needle 0.7 millimeters further in depth.
After a waiting period of five minutes, inject 25 microliters of blood into the right striatum. In order to perform the collagenous model of intracerebral hemorrhage, fill the Hamilton syringe with 0.075 units of bacterial collagenase dissolved in a half a microliter of saline. Avoid the formation of air bubbles during this process.
Then inject the specified amount of collagenase directly into the right striatum at a rate of two microliters per minute following the injection, the needle is left in place for an additional 10 minutes to prevent backflow of injected blood or collagenase along the needle tract. After 10 minutes, withdraw the needle slowly at a rate of one millimeter per minute, seal the bur hole with bone wax and suture the skin. Lastly, inject 0.05 milligrams per kilogram of buprenorphine subcutaneously and prewarm postoperative fluids.
After completion of surgery, all animals are allowed to recover. Under observation, a self-regulating heating pad is used in order to maintain a physiological body temperature. Immediately after recovery of the anesthesia mice subjected to right Sal injection of autologous blood will show a tendency to circle ipsilateral laterally to the injured hemisphere.
This behavior can also be seen after instal collagenase injection, however, in a delayed manner since collagenase causes a gradual growth of the intracerebral hematoma. Rodents with hemispheric stri attal hemorrhage show impaired sensory motor function compared to healthy animals, these impairments may be evaluated via various behavioral assessments. The corner turn test is commonly employed in preclinical studies investigating intra cerebral hemorrhage in rodents.
To perform the test, mice are allowed to advance into a 30 degree corner consisting of two attached plexiglass walls. To exit the corner, the mouse needs to turn either to the right or to the left side, while healthy animals tend to turn randomly to each side, mice with unilateral brain hemorrhage tend to turn to the side of injury. As a result of the impaired contralateral side only turns involving a full rearing along either wall or recorded.
The following behavior assessment is called the for limb placing test, also known as the rassi elicited for limb placement test. In this test, mice are moved along a firm edge allowing the VI brasse to brush along the surface. Healthy or sham animals as shown here, respond with a placement of the forelimb on the surface mice with right sal hemorrhage, however, show impaired forelimb placements on the contralateral side.
Successful paw placements are recorded out of 10 trials. The figures A and B show the results of previously described behavioral tests. Graph A shows the results of the corner turn test at 24 and 72 hours after surgery.
Mice after right instal injection of autologous blood or bacterial collagenase showed significantly more right turns compared to sham operated animals. Graph B shows the results of the forelimb placing test. Animals with intracerebral hemorrhage showed significantly less left limb placements on the contralateral side.
Compared to sham, no differences were found between blood and collagenous models at given volumes. The amount of brain edema following experimental intercerebral hemorrhage made measured using the wet weight dry weight method. Our results show significantly increased brain water in the ipsilateral cortex and basal ganglia at 24 and 72 hours after ICH.
Histological methods can be used to determine the size, location, and the extent of the brain hemorrhage. Figure A shows a one millimeter thick coronal section of a mouse brain at 24 hours after autologous blood injection. Figure B shows a hemat tolin and eosin stained 10 micrometer thick coronal brain section at 24 hours after Intravital collagenase injection.
