Name: double direct injection of blood into the cisterna magna as a model of subarachnoid hemorrhage
Uploaded: 2020-08-30T14:00:00
Description: Watch this Scientific Journal Video about Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage at JoVE.com

We thank the PRIMACEN platform (Normandie Rouen University, France) for imaging equipment and Mr. Arnaud Arabo, Mrs Julie Maucotel and Mrs Martine Dubois, for animal housing and care. We thank Mrs. Celeste Nicola for lending her voice to the videotaping of the protocol. This work was supported by Seinari Normandy maturation program, Fondation AVC under the aegis of the FRM, Normandie Rouen University and Inserm. The Normandy Region and the European Union (3R project). Europe gets involved in Normandy with European Regional Development Fund (ERDF).

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&#8211;12 weeks, were housed under controlled standard environmental conditions: 22 &#176;C &#177; 1 &#176;C, 12 hours/12 hou...

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 months24,25. This current preclinical model by daily double injection of homologous arterial blood into the cisterna magna has been reco...

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 study1,2,3. 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 &#34;acute&#34; stage of SAH, the prognosis depends on the occurrence of &#34;secondary&#34; 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&#8217;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 mice17,18. 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.

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double direct injection of blood into the cisterna magna as a model of subarachnoid hemorrhage

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 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. 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 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L 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.

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 &#181;L of blood withdrawn from a homologous mouse or 60 &#181;L of artificial cerebrospinal fluid (aCSF) were injected into the cisterna magna in SAH or sham conditions, respectively. The next day (D0), 30 &#181;L of blood withdr...

Watch this Scientific Journal Video about Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage at JoVE.com

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

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.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

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