Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Podsumowanie

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Streszczenie

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 °C and positioned in a stereotactic frame with a cervical bend of about 30°. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 µL of blood was injected in the cisterna magna followed by a 30° downward tilt of the animal for 2 minutes. The second infusion of 30 µL of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

Wprowadzenie

Subarachnoid hemorrhage (SAH) accounts for up to 5% of all stroke cases and constitutes a relatively common pathology with an incidence of 7.2 to 9 patients per 100,000 per year, with a mortality rate of 20%-60% depending on the study^1,2,3. In the acute phase, the mortality is attributable to the severity of bleeding, rebleeding, cerebral vasospasm (CVS) and/or medical complications⁴. In survivors, early brain injury (EBI) is associated with parenchymal extension of hemorrhage and abrupt increase in intracranial pressure, which may result in primary cerebral ischemia⁵ and immediate death in about 10%-15% of cases⁶. After the initial "acute" stage of SAH, the prognosis depends on the occurrence of "secondary" or delayed cerebral ischemia (DCI), detected in nearly 40% of patients by cerebral computed tomography, and in up to 80% of patients after magnetic resonance imaging (MRI)^7,8. In addition to the CVS occurring between 4 to 21 days after aneurysm rupture in a majority of SAH patients, DCI⁹ may result from multifactorial diffuse brain lesions secondary to microthrombosis formation, reduced cerebral perfusion, neuroinflammation, and cortical spreading depression (CSD)^10,11,12,13. This affects 30% of SAH survivors and impacts cognitive functions including visual memory, verbal memory, reaction time, and executive, visuospatial and language functions¹⁴ impairing daily life¹⁵. Current standard therapies to prevent CVS and/or the poor cognitive outcomes in SAH patients are based on the blockage of Ca²⁺ signaling and vasoconstriction by using Ca²⁺ channel inhibitors as Nimodipine. However, more recent clinical trials targeting vasoconstriction revealed dissociation between patient’s neurological outcome and prevention of CVS¹⁶, suggesting more complex pathophysiological mechanisms involved in SAH-long-term consequences. Therefore, there is a medical need for greater understanding of the number of pathological events accompanying SAH and the development of valid and standardized animal models to test original therapeutic interventions.

The rupture of an intracranial aneurysm mostly responsible for SAH in humans is likely difficult to mimic in preclinical animal models. Currently, the aneurysm rupture and SAH situation can tentatively be tested by the perforation of the middle cerebral artery (endovascular puncture model) responsible for CVS and sensitivomotor dysfunctions in mice^17,18. Due to the lack of any possible control over the onset of bleeding and the diffusion of blood in this model, other methods have been developed in rodents to generate SAH models without endovascular rupture. More precisely, they consist of the direct administration of arterial blood into the subarachnoid space through a single or a double injection in the magna cisterna¹⁹ or a single injection into the prechiasmatic cistern²⁰. The main advantage of these mouse models without endovascular rupture is the possibility to reproducibly master the surgical procedure and the quality and quantity of the injected blood sample. Another advantage of this model over the model by endovascular perforation in particular is the preservation of the general well-being of the animal. As a matter of fact, this surgery is less invasive and technically less challenging than that required to generate a carotid wall rupture. In this last model, the animal has to be intubated and mechanically ventilated, while a monofilament is inserted in the external carotid artery, and advanced into the internal carotid artery. This likely leads to transient ischemia due to vessel obstruction by the wire path. Consequently, the co-morbidity (moribund state, important pain and death) associated with surgery is less important in double injection model compared with endovascular perforation model. In addition to being a more consistent SAH, the double direct injection method complies with the animal welfare in research and testing (reduced time under anesthesia, pain from tissue disruption in surgery and distress) and leads to a minimum total number of animals used for the protocol study and personnel training.

Moreover, this allows implementation of the same protocol to transgenic mice, leading to an optimized pathological understanding of the SAH and the possibility of comparative testing of potential therapeutic compounds. Here, we present a standardized mouse model of subarachnoid hemorrhage (SAH) by a double daily consecutive injection of autologous arterial blood into the cisterna magna in 6-8 weeks-old male C57Bl/6J mice. The main advantage of this model is the control of the bleeding volume compared with the endovascular perforation model, and the reinforcing of the bleeding event without a drastic increase of intracranial pressure²¹. Recently, the double direct injection of blood into the cisterna magna has been well described on the experimental and physiopathological issues in mice. Indeed, we recently demonstrated CVS of large cerebral arteries (basilar (BA), middle (MCA) and anterior (ACA) cerebral arteries), cerebrovascular fibrin deposition and cell apoptosis from day 3 (D3) to 10 (D10), circulation defects of paravascular cerebrospinal fluid accompanied by altered sensitivomotor and cognitive functions in mice, 10 days post-SAH in this model²². Thus, it makes this model mastered, validated and characterized for short-term and long-lasting events post-SAH. It should be ideally suitable for prospective identification of new targets and for studies on potent and efficient therapeutic strategies against SAH-associated complications.

Protokół

All procedures were performed under the supervision of H. Castel in accordance with the French Ethical Committee and guidelines of European Parliament Directive 2010/63/EU and the Council for the Protection of Animals Used for Scientific Purposes. This project was approved by the local CENOMEXA and the national ethic committees on animal research and testing. Male C57Bl/6J Rj mice (Janvier), aged 8–12 weeks, were housed under controlled standard environmental conditions: 22 °C ± 1 °C, 12 hours/12 hours light/dark cycle, and water and food available ad libitum.

1. Setup of SAH surgery and preparation for injection

1. Before the beginning of surgery, pull an adequate number of glass capillaries by using a micropipette puller. The injection pipette should exhibit an inner diameter of 0.86 mm and outer diameter of 1.5 mm.
2. Prepare the artificial cerebrospinal fluid (aCSF) for the sham condition.
   1. Prepare a solution with 119 mM NaCl, 2.5 mM KCl, 1 mM NaH₂PO₄, 1.3 mM MgCl₂, 10 mM glucose, 26.2 mM NaHCO₃ in H₂O, pH 7.4.
   2. Gas aCSF with 95% O₂ and 5% CO₂ for 15 min, and then add 2.5 mM CaCl₂.
   3. Sterilize the oxygenated aCSF with a 0.22 µm filter apparatus. The aCSF solution can be stable for 3-4 weeks at 4 °C. If contamination (solution becoming cloudy) or a deposit formation has appeared, discard and make fresh aCSF.
3. Collection of blood from a homologous mouse donor
   1. Isolate the carotid artery along the trachea and collect the maximum amount of blood by puncture of the carotid artery.
   2. In practice, place the mouse into an anesthesia chamber and load the chamber with 5% isoflurane until the animal loses consciousness.
   3. Check the lack of reflexes by clamping one of two hind limbs to allow the setting of the surgical experimental procedure.
   4. Coat a 1 mL syringe with a heparin solution by using a 26 G needle (heparin sodium). This will prevent blood coagulation during the next steps.
   5. Install the animal positioned in dorsal decubitus with legs apart, and the nose in an anesthesia mask (anesthesia maintenance with 2 to 2.5% isoflurane).
   6. Isolate the carotid artery along the trachea by dissecting the omohyoid muscle longitudinally. Once artery isolated, insert the needle towards the heart with the help of the microdissecting hook and forceps and collect the maximum of blood via puncture of the carotid artery (60 µL is needed per SAH mouse).
   7. Sacrifice the anesthetized donor mouse immediately after blood collection by using cervical dislocation.

2. Animal (8-10-week-old C57BL/6J male mice) preparation

1. 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.
2. As previously explained (see steps 1.3.2 and 1.3.3), induce anesthesia of mice to be operated.
3. Shave the neck and the space between the ears with a suitable electric clipper.
4. Install the animal positioned in ventral decubitus with legs apart and the nose in an anesthesia mask (anesthesia maintenance with 2 and 2.5% isoflurane) on a stereotactic frame.
5. Check that the mouse is sleeping and that his head is properly blocked.
6. Subcutaneously inject 100 µL of buprenorphine (0.1 mg/kg) with a 26 G needle in the lower back, to avoid pain after awakening.
7. Prevent dry eyes by using protective liquid gel and maintain an intrarectal temperature of 37 °C by using an auto-regulated electric blanket.
8. Treat the posterior neck shaved area with an antiseptic solution (povidone-iodine or chlorhexidine by using a sterile cotton road).
9. Pre-sterilize all instruments touching the prepared skin/subcutaneous tissue (heating to 200 °C for 2 hours) and handle aseptically.

3. SAH induction

1. On the first day (D-1)
   1. Cut a 1 cm incision with thin scissors in the posterior neck, followed by the separation of muscles along the midline to access the cisterna magna.
   2. Cut the tip of the empty glass pipette with thin scissors. Then, adapt to a syringe connected to a flexible silicone connector.
   3. Transfer 60 µL of blood or aCSF (for SAH or sham condition, respectively) in a 0.5 mL tube using a precision micropipette.
   4. Suck into the glass pipette the 60 µL of blood for the SAH condition or 60 µL of aCSF for the sham condition.
   5. For injection, install the pipette on the stereotactic frame using a ring or blue-tack and slowly bring the pipette tip to the membrane at the interface with the cisterna magna.
   6. Slowly insert the pipette tip through the atlanto-occipital membrane into the cisterna magna, using a micro-manipulator of the stereotactic frame.
   7. Connect the pipette previously filled with blood or aCSF to the syringe ready for pressure induction.
   8. Inject by pressing the plunger at a low rate around 10 µL/min, to avoid acute intracranial pressure.
   9. During injection, closely monitor the respiratory rate and the rectal temperature.
   10. At the end of the injection, carefully take off the pipette via the micro-manipulator and visually ensure that there is no leak during withdrawal.
   11. Achieve hemostasis using an absorbable hemostat and run two sutures with braided non-absorbable suturing thread.
   12. Immediately after surgery, isolate and position the mouse in decline decubitus and cover it with a survival blanket in an open box for the duration of recovery.
2. Second day of induction (D0)
   1. After 24 hours, induce anesthesia (see steps 1.3.2 and 1.3.3). Subcutaneously inject 100 µL of buprenorphine again (0.1 mg/kg) and prevent dry eyes by using protective liquid gel (see steps 2.7 and 2.8).
   2. Install the animal on the stereotactic frame as the day before.
   3. Carefully remove the sutures with microscissors.
   4. Prepare the atlanto-occipital membrane as before and apply antiseptic preparation on the shaved area of the neck with a sterile cotton rod.
   5. Inject 30 µL of blood or aCSF at a low rate (see steps 3.1.2 to 3.1.8). Monitor the respiratory rate and the rectal temperature.
   6. At the end of the injection, carefully take off the pipette and control the absence of blood leak during withdrawal.
   7. Achieve hemostasis and run two sutures with braided absorbable suturing thread.

4. Postoperative follow-up and end of the experiment

1. Immediately after surgery, isolate and position the mouse in decline decubitus with a survival blanket on its back in an open box during recovery.
2. Weigh and carefully observe daily the behavior of each mouse until sacrifice (e.g., D7 post-surgery).
3. Among humane endpoints, a significant weight loss (>15% of the weight) is classically noticed. A "hunched back" posture, slow movements, prostration, abnormal vocalizations of hurt and/or significant aggressive behavior are also important signs of animal suffering. If any of these signs or a combination of signs appears, the monitoring of the animal is reinforced within hours of their appearance. If the animal's welfare worsens or does not improve within 48 hours, it will be considered that a level of intolerable suffering is reached, and euthanasia is carried out.
4. At the time of choice, sacrifice anesthetized mice by decapitation, and harvest brains for further analyses.
5. Perform euthanasia (decapitation) after isoflurane anesthesia (5%).

Wyniki

Experimental timeline, procedure, follow-up and mortality
Figure 1A and Figure 1B summarize the SAH model protocol by double intracisternal injection of blood. Briefly, on the first day of SAH induction (D-1), 60 µL of blood withdrawn from a homologous mouse or 60 µL of artificial cerebrospinal fluid (aCSF) were injected into the cisterna magna in SAH or sham conditions, respectively. The next day (D0), 30 µL of blood withdr...

Dyskusje

Despite the intensity of the research in the field of SAH and the development of therapeutic strategies such as endovascular and pharmacological treatment options increasing over the past twenty years, mortality remains high within the first week of hospital admission and reaches about 50% during the following 6 months^24,25. This current preclinical model by daily double injection of homologous arterial blood into the cisterna magna has been reco...

Materiały

Name	Company	Catalog Number	Comments
absorbable hemostat	Ethicon	Surgicel
absorbable suturing thread	Ethicon	Vicryl 5.0
auto-regulated electric blanket	Harvard Apparatus	50-7087-F
bluetack for capillary fixation	UHU	Patafix
electronic balance	Denver Instrument	MXX-2001
glass capillaries	Harvard Apparatus	GC150F-15	inner diameter 0.86 mm outer diameter 1.5 mm
isoflurane vaporizer	Phymep	V100
micropipette puller	Sutter Instrument Company	P-97
needle 26 G	BD microbalance	300300
non absorbable suturing thread	Peters surgical	Filapeau 4.0
stereotaxic frame	David Kopf instruments	Model 902
surgical equipment	Kent scientific	clamp, microscissors, thin scissors
syringe 20 mL TERUMO	Thermofisher	11866071