The overall goal of this present video is to explain the protocol of a double direct injection of blood into the cisterna magna as a model of subarachnoid hemorrhage. The physiology of the subarachnoid hemorrhage is often due to a rupture of an aneurysm. And we first do the extravasation of blood into the compartment between the brain and the tissue that covers the brains.
This compartment is the subarachnoid space. Subarachnoid hemorrhage accounts for up to 7%of all strike cases. Mortality varies between 30 to 50%in population-based studies and subarachnoid hemorrhage survivors commonly experience cognitive sequelae as memory deficits, asthenia, or mood disorders.
The leading cause in the high prevalence of poor outcome is what is cerebral ischemia. Studies made a link between the occurrence of ischemia in the vasoconstriction of the large cerebral arteries, named microscopic vasospasm that can be detected in a majority of patients between the fourth and the fifteenth day after the first bloating event. Because of the inefficiency of the current therapeutic options, there is an obvious medical need for a better understanding of pathological events.
Accompanying subarachnoid hemorrhage and new therapeutic targets should be tested, but this requires varied and standardized animal models. The rupture of intercranial aneurysm mostly responsible of subarachnoid hemorrhage in humans is likely difficult to be making preclinical and human models. In animals, the main difficulties reside in the control incidence of hemorrhage and blood distribution, but recent studies focus on the development of more redicive models of models of subarachnoid hemorrhage.
Here represent a standardized mouse model of two consecutive injections of autologous arterial blood of our two days into the cisterna magna. The main advantages of this model are to reproducibly master a noninvasive surgical procedure, to adapt the quality and quantity of the injected blood, and to reinforce the bleeding event without drastically increase intracranial pressure. Before the beginning of surgery, you have to put another great number of glass capillaries by using a micropipette puller.
The injecting pipette should exhibit an inner diameter of 0.86 millimeter and an outer diameter of 0.5 millimeter. Prepare the artificial cerebral spinal fluid for sham condition, as indicated in the written protocol. You must realize this oxygenated artificial cerebral spinal fluid with a 0.22 micron filter apparatus.
The first step of our subarachnoid hemorrhage model consists in the isolation of the carotid artery along the trachea and the collection of the maximum of blood from carotid artery puncture. After anesthesia by isoflurane, check the lack of reflexes by clamping one or two hind limbs to allow the setting of the experimental procedure. Code a one millimeter syringe with Eprin by using a 26 gauge needle.
This will prevent blood coagulation during the next steps. Weigh each mouse precisely using an electronic balance. In the current study, mice would have body weight within the range of 20 to 25 grams just before surgery.
As previously explained, induced anesthesia of the operated mouse and then shave the neck and the space between each ear by means of a suited electrical clipper. Check mouse is sleeping and mouse head is well blocked. Inject subcutaneously 100 microliters of buprenorphin at 0.1 milligrams per kilo with a 26 gauge needle in the lower back to avoid pain after awakening.
Prevent dry eyes using a protective liquid gel and maintain intraductal temperature of 37 degrees using an auto regulated electric blanket. Before treating the shaved area with an antiseptic solution, all instruments touching the tissue have been sterilized and were handled aseptically. On the first day, cut a one centimeter long incision in the posterior neck, followed by the separation of muscles along the midline, to access the cisterna magna.
Cut the tip of the empty glass pipette with thin scissors. Then adapt to a syringe connected to a flexible silicon connector. Then transfer 60 microliters of blood or artificial cerebral spinal fluid in a 0.5 milliliter tube using a precision micropipette Suck into the glass pipette, the 60 microliters of blood for the subarachnoid hemorrhage condition or 60 microliters of artificial cerebral spinal fluid for the sham condition.
For injection, insert the pipette on the stereotactic frame using, for example, our a ring or a and slowly approach the pipette tip to the membrane at the interface with the cisterna magna. Insert slowly the pipette tip through the atlanto-occipital membrane into the cisterna magna using a micromanipulator of the stereotactic frame. Connect the pipette to the syringe previously filled, ready for pressure induction.
Inject by pressure at a low rate around 10 microliters per minute to avoid acute intracranial hypertension. During injection, monitor closely respiratory rate and rectal temperature. At the end of the injection, carefully take off the pipette using the micromanipulator and visually take care there is no leak during withdrawal.
Achieve homeostasis and carry out two sutures with braided non-absorbable suturing thread. Immediately after surgery, isolate and position the mouse in decline decubitus and cover with a survival blanket in an open box for the duration of recovery. After 24 hours, induce anesthesia again, install the mouse on the stereotactic frame as the day before and remove with care the sutures with micro-scissors.
Prepare as before aseptic of the atlanto-occipital membrane, by applying on the shaved area and aseptic solution with a cotton rod. Inject, as the day before, 30 microliters of blood or artificial cerebral spinal fluid at a low rate. As already mentioned, monitor respiratory rate and rectal temperature.
At the end of the injection, carefully take off the pipette and control the absence of blood leak during withdrawal. Achieve homeostasis and carry out two sutures with a braided absorbable suturing thread. From day one post surgery to the day of sacrifice, the body weight should be daily assessed as a sensitive indicator for general wellbeing.
It showed a significant reduced body weight gain in subarachnoid hemorrhage, compared with sham animals, suggesting a long lasting activity recovery process and prolonged pathological events, post subarachnoid hemorrhage. Why mortality is not in sham condition? Mortality is near of 30%at day seven in subarachnoid hemorrhage condition, with most animals dying on the one or day four after surgery.
Among humane endpoints, a significant weight loss superior of 15%of the initial weight is noted. A hunched-back"posture, slow movements, wounds, abnormal vocalizations or significant aggression are also signs that the animal is suffering. If any of these signs or a combination of signs appears, the tracking of the animal is reinforced within hours that follow its appearance.
If the animal's condition worsens in the hour that's follow or does not improve within 48 hours, it will be considered that a level of intolerable suffering is reached and the killing is carried out. In this example at day one of surgery, it can be observed from a sample brain, blood clots along the large arteries of the Willis polygon in subarachnoid hemorrhage subject. At day five after surgery and before sacrifice, intervascular Indian ink injection allows the detection of macroscopic vasospasm corresponding to vasoconstriction of large cerebellar arteries, Coloration of brain slices with hematoxylin and eosin allows the measurement of cerebral vasospasm.
Here, as illustrated on this photo micrography, you can observe as a vasospasm of the basilar artery. In this model of divert direct injection of blood into the cisterna magna, we have recently demonstrated cerebral vasospasm of large cerebral arteries, cerebral vascular fibril deposition, and cell apoptosis, accompanied by altered sensory, motor, and cognitive functions in mice. Thus, it makes this model validated and characterized for short term and long lasting events, post subarachnoid hemorrhage.
It should be ideally suitable for prospective identification of new targets and for studies on efficient therapeutic strategies against subarachnoid hemorrhage associated complications.
