Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Summary

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Abstract

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

Introduction

Until recently, the "gray matter stroke (GMS) models" have been exclusively used to understand the pathophysiology of stroke and to guide the development of new treatments. However, there has been an increasing prevalence of stroke that affects subcortical white matter in elderly individuals, which constitutes 15 - 25% of all strokes^1,2. Numerous studies have been conducted regarding stroke using GMS models, whereas there are few studies that have used white matter stroke (WMS) models. White matter in rodents is substantially less than the white matter in humans or primates. Consequently, it is more difficult to selectively access and destroy the ta....

Protocol

All procedures were conducted according to the institutional guidelines of Gwangju Institute of Science and Technology (GIST), and all procedures were approved by the Institutional Animal Care and Use Committee at GIST.

1. Pre-lesioning Steps

1. Identification of the Forelimb Area in the Internal Capsule using AAV-GFP
   1. House and handle Sprague Dawley rats (~400 g, 11 - 13 weeks) in compliance with institutional and national guidelines.
   2. Sterilize all surgical tools and electrodes using an appropriate sterilizer (Steam or Plasma sterilizer). Use steam sterilizer at 121 °C as setting of 30 min for sterilization and 3....

Results

  The method presented here is intended to create a circumscribed capsular infarct with a persistent motor deficit. Therefore, it is critical to correctly determine the target within the internal capsule in the pre-surgery step. The somatotopic mapping of pyramidal fibers in the internal capsule has not been settled to date. To correctly identify the target within the internal capsule, the forelimb area must be delineated. An injection of AAV-GFP into the forelimb area of the motor cortex.......

Discussion

The capsular infarct model presented here demonstrates a targeted lesion with marked and persistent motor impairment in forelimb function. Previous models of subcortical capsular stroke have demonstrated an insufficient degree of motor impairment and a rapid recovery process^6,8,9 . In this sense, this model resembles the clinical capsular infarct cases which exhibit long-term functional impairment.

The most critical steps in the development of a circumscribed capsular infarct model .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DC Temperature controller	WORLD PRECISION INSTRUMENTS, INC.	ATC1000
Digital Stereotaxic Instruments	STOELTING CO.	51900
Electrical Stimulator	CyberMedic Corp.	EMGFES 2000
Epoxy	Precision Fiber Products, INC.	PFP-353ND1	Mix Ratio: 10(A):1(B-hardener) by weight  Curing Schedule: 1 min @150 °C 2 ~ 5 min @120 °C 5 ~ 10 min @100 °C 15 ~ 30 min @80 °C
Fiber Optic Scribe	THORLABS, INC	S90R
Fiber patch cable	KOREA OPTRON Corp.		Outer diameter: 3 mm Ø200 µm 0.39 NA FC/PC-FC/PC 1 m
Laser Power Supply	CHANGCHUN NEW INDUSTRIES OPTOELECTRONICS TECH. CO., LTD.	MGL-FN-532nm-200mW-14010196
Crimp ring	DAWOOTECH CO.,LTD.		Length: 19 mm Inner diameter: 3 mm Outer diameter: 3.8 mm Material: SUS
Micro4-micro syringe pump controller	WORLD PRECISION INSTRUMENTS, INC	95100
Optical Power Meter	THOLABS, INC	PM100D
Diamond lapping (polishing) sheet	THORLABS, INC	LF3D	Grit : 3 µm
Diamond lapping (polishing) sheet	THORLABS, INC	LF6D	Grit : 6 µm
Rose Bengal	SIGMA-ALDRICH CO. LLC.	330000
Needle for spinal anesthesia with pencil point tip (Spinal needle)	B.BRAUN MELSUNGEN AG	4502027	Size: 27 G Length: 88 mm Needle: 0.40 mm
Waterproof sandpaper	DEERFOS CO.,LTD	CC261	Grit : 1,000 µm
Nanofil 10 µl syringe	WORLD PRECISION INSTRUMENTS, INC	NANOFIL
Nanofil 33 G BVLD needle	WORLD PRECISION INSTRUMENTS, INC	NF33BV-2
AAV-GFP virus	UNC Vector Core	AAV2-CamKIIa-eYFP	2 x 1012 virus molecules/ml
Anti-Green Fluorescent Protein, Rabbit IgG fraction	Life Technologies, INC	A11122	primary antibody (1:200)
Goat Anti-Rabbit IgG (H + L)	Life Technologies, INC	A11034	secondary antibody (1:500)
Ceftezole	GUJU Pharma CO.,LTD.	A27802741	0.1%, 1 ml
Lidocain hydrochloride injection	JEIL PHARMACEUTICAL CO.,LTD.	A04900271	2%, 1 ml
Hand Piece Drill	Seshin
Digital optical power and energy meter	THORLABS, INC	PM100D
Ketoprofen	UNIBIOTech