Bilateral Common Carotid Artery Stenosis in Mice: A Model of Chronic Cerebral Hypoperfusion-Induced Vascular Cognitive Impairment

Vascular cognitive impairment (VCI) is a syndrome defined as cognitive decline caused by vascular disease and is associated with various types of dementia. Chronic cerebral hypoperfusion (CCH) is one of the major contributors to VCI. Among the various rodent models used to study CCH-induced VCI, we have found the mouse bilateral common carotid artery stenosis (BCAS) model to be highly suitable. Here, we introduce the BCAS model of C57BL/6J mice generated using microcoils with an internal diameter of 0.18 mm. To produce the mouse BCAS model, the bilateral common carotid arteries are isolated from the adhering tissues and vagus nerves and twined around the microcoils. This model shows cognitive impairment and white matter lesions preceding neuronal dysfunction around postoperative day 28, which is similar to the human clinical picture. Overall, the mouse BCAS model will continue to be useful in studying CCH-induced VCI. Key features • This mouse BCAS model requires approximately 4 weeks to show phenotypes such as cognitive impairment and white matter injury.

This protocol is used in: Sci Adv (2023), DOI: 10.1126/sciadv.adh0102 Vascular cognitive impairment (VCI) is a syndrome defined as cognitive decline caused by vascular disease and is associated with various types of dementia.Chronic cerebral hypoperfusion (CCH) is one of the major contributors to VCI.Among the various rodent models used to study CCH-induced VCI, we have found the mouse bilateral common carotid artery stenosis (BCAS) model to be highly suitable.Here, we introduce the BCAS model of C57BL/6J mice generated using microcoils with an internal diameter of 0.18 mm.To produce the mouse BCAS model, the bilateral common carotid arteries are isolated from the adhering tissues and vagus nerves and twined around the microcoils.This model shows cognitive impairment and white matter lesions preceding neuronal dysfunction around postoperative day 28, which is similar to the human clinical picture.Overall, the mouse BCAS model will continue to be useful in studying CCH-induced VCI.

Background
Vascular cognitive impairment (VCI) refers to cognitive alterations related to vascular disease and is associated with various types of dementia [1].Chronic cerebral hypoperfusion (CCH)-associated small vessel disease is one of the major contributors to VCI [2,3].CCH is elicited by aging and various lifestyle diseases, such as metabolic syndromes, atherosclerosis, hypertension, obesity [4], and hypotension [5].It has also been suggested that CCH induces white matter lesions, which are key characteristics of VCI.In addition, many patients with various types of dementia including VCI have white matter lesions [6].To understand the precise pathological mechanism underlying CCH-induced VCI including white matter lesions, multiple rodent models have been used in many studies.Some widely used rodent models of CCH-induced VCI are the rat bilateral common carotid artery occlusion (BCCAO), mouse unilateral common carotid artery occlusion (UCCAO), rat and mouse 2-vessel gradual occlusion (2-VGO), mouse asymmetric common carotid artery surgery (ACAS), and mouse bilateral common carotid artery stenosis (BCAS) models.However, rodent models have several limitations.First, the visual pathway is damaged and behavioral tests to assess cognitive function are affected in the rat BCCAO and mouse UCCAO models [7,8].Second, the device used in the rat and mouse 2-VGO models and mouse ACAS model is expensive [9].The mouse BCAS model was reported as a CCH model in 2004 by Shibata et al. [10] and is a more suitable

B. Isolation of the common carotid artery (CCA)
1. Place an anesthetized mouse in the supine position with the four limbs spread out.

3.
Separate the submandibular glands laterally to make the trachea visible (Figure 2B).

4.
Move the sternocleidomastoid muscle laterally to make the CCA sheath visible by a handmade wire hook with a weight to open the surgical area (Figure 2C). 5. Carefully isolate the CCA from the adhering tissues and vagus nerve using forceps.Be careful not to damage the CCA and vagus nerve (Figure 2D, Video 1).Note: The CCA and vagus nerve are wrapped in a transparent thin membrane, and the vagus nerve is often seen at a deep depth (dorsal) and on the lateral side of its sheath (Figure 2E).

C. Putting microcoils on the CCAs
1. Gently lift the isolated CCA and put a microcoil under it vertically.Put the CCA in the center of the microcoil (Figure 3A, Video 1). 2. Grab one side of the microcoil and twine the CCA around the other side of the microcoil (Figure 3B, Video 1). 3. Twine the CCA around the other side of the microcoil in the same way as described in step C2 (Figure 3C, Video 1). 4. Remove the wire hook and place the sternocleidomastoid muscle and submandibular glands back. 5. Repeat the procedure described in steps C3 and C4 with the other CCA.The other CCA is on the other side of the trachea at the symmetrical position.As described in step B4, the other CCA sheath is also visible after moving the other sternocleidomastoid muscle laterally.

Data analysis
We measured regional cerebral blood flow (rCBF) at 60 min after surgery with laser Doppler flowmetry [12].BCAS surgery successfully reduced rCBF to ~65 of the baseline, which is similar to the data reported by Shibata et al. [10].In our previous studies, this mouse BCAS model generated using wildtype mice (8-12 weeks old, 20-30 g) showed a decrease in the myelin density by myelin staining and cognitive impairment in a novel object recognition test on postoperative day 28, whereas there were no changes in the number of neuronal cells detected by immunostaining or spatial memory in a novel location recognition test [11,12,14].

Validation of protocol
This protocol or parts of it has been used and validated in the following research articles:  [12].The astrocytic TRPA1 channel mediates an intrinsic protective response to vascular cognitive impairment via LIF production.Sci Adv (Figure 1P-R and supplementary figure 1D).]

General notes and troubleshooting
1. Do not damage CCAs and the vagus nerves.In particular, carefully conduct the procedures during isolation and twining of CCAs. 2. Fully twine CCAs on microcoils.If CCAs are positioned off the end of microcoils, re-twine them or rotate microcoils.3. Conduct the procedures as quickly as possible.If possible, complete this surgery within 10-20 min and take at most 30 min.4. Put microcoils on the lower branch of the carotid artery, not the internal carotid artery or external carotid artery.

Figure 1 .
Figure 1.Surgical equipment and materials.A. Curved suture needle and silk suture.B. Needle holder.C. Micro spring scissors.D. Two straight sharp forceps.E. Curved forceps.F. Handmade wire hook.G. Microcoil with an internal diameter of 0.18 mm.

Figure 2 .
Figure 2. Generation of a ventral cervical skin incision and isolation of the CCA (section B). A. A cervical skin incision was made, and the submandibular glands were visualized.B. The submandibular glands were separated, and the trachea was exposed.C. The surgical area was opened by moving the sternocleidomastoid muscle laterally and the CCA was visible.D. The CCA was isolated from the adhering tissues and vagus nerve with forceps.E. Before isolation of the CCA, the vagus nerve was wrapped in a transparent membrane with the CCA.The dotted line shows the vagus nerve.CCA, common carotid artery.

6 Published: Jul 05, 2024 Figure 3 .Video 1 .D. Completion of surgery 1 . 7 Published: Jul 05, 2024 E. Procedure of the sham operation 1 . 2 .
Figure 3. Placement of a microcoil on the CCA and completion of stenosis (section C). A. A microcoil was put under the isolated CCA.B. The CCA was twined around half of the microcoil.C. The CCA was twined around the other side of the microcoil, and stenosis was completed.CCA, common carotid artery.