Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Summary

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Abstract

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) ¹. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex ², is the most common site of human stroke ³, and ischemia within its territory can result in extensive dysfunction or death ^1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.

Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Introduction

In order to induce ischemic conditions that effectively mimic human ischemic stroke, several animal stroke models are widely employed, with varying volumes of infarct resulting. In the photothrombotic model, the brain is irradiated through the intact skull using laser illumination after intravenous injection of a photosensitive substance (such as rose-bengal), resulting in photochemical coagulation, blockage of the irradiated vessels, and ischemia within the surrounding tissue ^6,7. Photothrombosis can result in very small, isolated regions of infarct and is typically used as a means of modeling "mini-strokes", or "micro-strokes".

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Protocol

1. Getting Started: Required Surgical Instruments

See Figure 4

1. Dental drill (Kavo Dental Equipment, Model: UMXL-TM), 2-bit drill, and 3-bit drill
2. Two ~30 gauge hypodermic needles
3. Serrated tweezers, curved tip optional (can be helpful but not essential)
4. Two fine tip tweezers
5. wire cutters
6. Suture thread
7. Micro scissors

2. Creating the Surgical Window

1. Anesthesia: Procedures are in compliance with NIH guidelines and have been approved by UC Irvine Animal Care and Use Committee. Experimental subjects are 295-400 g....

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Results

Successful occlusion of a vessel can be confirmed using laser speckle imaging (LSI) among other blood flow imaging techniques. Blood flow in the major cortical branches of MCA should drop to ~25% of baseline or less following occlusion depending on the level of noise in the recording system and sensitivity of the technique. See Figure 3 for a representative LSI image of a segment of a cortical branch of MCA before and after MCA occlusion. When the described occlusion technique is applied.......

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Discussion

This protocol was developed in order to induce ischemia within the rodent cortex, and to do so with minimal peripheral impact to experimental subjects. The double occlusion and transection method allows for visual confirmation that the vessel has been permanently occluded, and may be performed without excessive invasion or tissue damage, and with a high survival rate. This occlusion protocol may be applied to any cortical vessel that can be accessed via craniotomy in order to induce ischemia within a specific cortical do.......

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Materials

Name	Company	Catalog Number	Comments
Extra Fine Graefe Forceps - 0.5 mm Tips Slight Curve (1)	Fine Science Tools	11151-10
Ceramic Coated Dumont #5 Forceps (2)	Fine Science Tools	11252-50
Extra Fine Bonn Scissors, straight (1)	Fine Science Tools	14084-08
Round 3/8 (16 mm) Suture Needles	Fine Science Tools	12050-02
6-0 Braided Silk Suture	Fine Science Tools	NC9071061 Harvard Apparatus No.:510461
30 gauge needle, ½" length	Fine Science Tools	NC9867376 No.:ZT-5-030-5-L/COL