Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

Summary

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Abstract

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.

We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.

This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

Introduction

Animal models of cerebral venous hypertension have proved to be a key tool in the understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas^1-7. The most widely used is the rat model created through an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV), which provokes a consistent cerebral venous hypertension (CVH) in the rat^1,8-10. Equivalent mice models, by opening the possibility of using different transgenic mice strains, would allow further study on not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases. Furthermore, the experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.¹¹ However, previous attempts to construct these mice models have demonstrated the difficulties with patency of the fistula due to the diminutive anatomy^5,12. Here, we describe our step-by-step protocol for a successful anastomosis of the murine CCA and EJV that translates into a long-term patent fistula and a durable venous hypertension in the mouse.

Protocol

1. Preparing the Mouse

1. Induce general anesthesia in the mouse with isoflurane gas. Inject 0.15 ml of intraperitoneal brupenorphine for pain management. Before proceeding, check if the anesthesia level is satisfactory by pricking the mouse's paws.
2. Put the mouse in dorsal recumbency with the four limbs fixed by adhesive tape. Remove the hair of the neck and the upper chest with scissors. Via subcutaneous injection, administer 0.2-0.4 ml of 0.9% saline to keep the mouse hydrated during the surgical procedure.
3. Prepare the operative field following a strict sterile method. The area of the skin incision should be cleaned with 90% alcohol.

2. Dissecting the Common Carotid Artery and the External Jugular Vein

1. Make a horizontal midline cervical incision across the lower neck area of the mouse. After deepening the wound, elevate the salivary glands and cervical soft tissue by using a traction suture (Figure 1). Expose the right external jugular vein (EJV) lateral to the sternocleidomastoid muscle (SCM). This step should be performed under the microscope, since excessive traction over the vein might damage it and induce its thrombosis.
2. Carefully dissect the right EJV along its course from the clavicle to the skull base. Usually electric bipolar forceps, coagulate and divide any branches to prepare an adequate length for the later temporary clip placement and anastomosis.
3. Lateral to the trachea and medial to the SCM, explore the common carotid artery (CCA). It should be carefully exposed from the clavicle to just beyond its bifurcation into the external and internal carotid arteries. During this step, attention should be given again to avoid excessive traction to the EJV that could later compromise the patency of the anastomosis.

3. Preparing the Anastomosis

1. Ligate the CCA with 10-0 Nylon just proximal to its bifurcation. Next, apply a temporary clip over the proximal CCA as close to the clavicle as possible.
2. Once the flow has been interrupted, transect the artery just under the bifurcation ligature and irrigate it with saline to wash out any remaining blood inside the lumen. Avoid bipolar coagulation in this step, since thermal injury to the artery wall could put the future anastomosis in jeopardy.
3. In order to improve the visibility of the edges of the EJV, the medial wall is marked with a blue marking pen along the course of the planned venotomy. Once the EJV is marked, use a 10-0 suture to ligate the distal end as caudal as possible and apply a temporary vascular clip on the proximal end as cranial as possible.
4. With a fine 30 G needle and 0.5 ml syringe, make an initial aperture over the marked area of the EJV and immediately irrigate the lumen with saline to avoid thrombus formation. Next, extend the venotomy with the microscissors until the length is about 2-3 times the diameter of the CCA. Avoid violent stretching and pay attention to keep a sharp and neat cutting edge.
5. Approximate the end of the CCA to the EJV. Make a side-cut incision in the donor end of the CCA to adjust the diameter to the length to the venotomy size (Figure 2).

4. End-to-side Anastomosis

1. Use a 11-0 monofilament nylon suture for the CCA-to-EJV end-to-side anastomosis. Suturing the medial wall of the anastomosis in a craneo-caudal direction is the initial step. Every stitch should be placed from outside-in the venous wall first (Figure 3) and from inside-out the arterial wall (Figure 4) next. This will keep the knot on the outside surface of the anastomotic vessels at all times (Figure 5). Either interrupted or continuous sutures can be used, but all continuous sutures should be tightened as the final step.
2. Once the medial wall has been sutured, repeat the procedure from caudal to cranial with the lateral wall. Now every stitch should be placed from outside-in the arterial wall first and from inside-out the venous wall next. Saline irrigation will help keep the lumen of the anastomosis visible at all times during the procedure.
3. After finishing all the steps of the anastomosis, remove the temporary clip from the vein first and from the artery next. The arterial blood will flow into the EJV with no or little oozing from the anastomosis (see videos VH model and VH model²). The minimal bleeding should stop without using cotton compression over the anastomosis. This must be avoided in order to prevent thrombosis of the delicate vein.
4. Once pulsatile flow through the anastomosis is confirmed and no apparent bleeding is observed, irrigate the surgical field with saline and close the cervical incision with a 6-0 nylon suture. Finally, administer 0.15 ml more of intraperitoneal brupenorphine for postoperative pain management and 0.2-0.4 ml of subcutaneous 0.9% saline to replenish any blood loss during the surgery.

Results

A successful outcome of the model is a patent arteriovenous fistula that induces venous hypertension in the murine brain. To validate the model we initially measured the intracranial venous pressure in the sagital sinus of the mice at 2, 3 and 4 weeks after surgery. 6 different mice were assigned to every time group. The sinus pressure was 8.8 ± 1.2 mmHg in the group measured two weeks after the surgery. In the 6 mice measured 3 weeks after surgery, the sinus pressure was 4.7 ± 1.4 mmHg. Finally, the 6 mice mea...

Discussion

Sustained cerebral venous hypertension has been closely related with more severe clinical manifestations and poor prognosis in patients with dural AVFs and brain AVMs³. These effects of CVH have been widely studied in rat models^1,2,8. An equivalent model in the mouse would allow the use of genetically modified animals which would ultimately allow the analysis of molecular pathways involved on the pathogenesis of venous hypertension and its relationship with dural AVF and brain AVM.

Materials

Name	Company	Catalog Number	Comments
10-0 Sterile Microsuture	Arosurgical Ic.	VT5A010Q10
11-0 Sterile Microsuture	Arosurgical Ic	VT4A00N07
DUROTIP Scissors	Aesculap	BC210R
Micro-Adson Tissue Forceps	Aesculap	BD510R
Microscissors	Aesculap	OC496R
Micro Forceps #5 Jewelers	Aesculap	BD331R
Angled Jewelers Forceps	Aesculap	BD329R
Micro Suture Forceps	Aesculap	BD338R
DUROGRIP Needle Holder	Aesculap	BM009R