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 target regions in the white matter³. Additionally, no efficient tools have been developed to date to selectively destroy the planned extent of targeted white matter. Therefore, there has been lack of appropriate models for study of white matter strokes.

Animal stroke models are often used to monitor the progress of motor recovery for development of new rehabilitative and therapeutic methods. It is ideal to utilize an animal model that exhibits a long-term neurological deficit concordant with the anatomical alterations demonstrated in human stroke^4,5. In this regard, rapid recovery of the motor deficit and wide involvement of the brain following infarct lesioning may not be realistic in the pursuit of stroke research. Previous capsular infarct models have been made by the occlusion of the internal carotid or anterior choroidal arteries and diffusion of endothelin-1 (ET-1) into the internal capsule^6-9. Nonetheless, artery occlusion requires careful dissection of arteries, but it produces a wide area of infarct lesion, including the internal capsule, without persistent behavioral deficits. Moreover, ET-1 was not diffused to completely destroy the posterior limb of the internal capsule, and hence less marked or persisting behavioral deficit.

A photothrombotic infarction model has been widely used to generate various types of infarct lesions in the cortex and subcortical structures¹⁰. The technique include intravenous administration followed by focal illumination, which leads to platelet aggregation in the small vessels and generation of infarct lesions¹⁰. Photothrombotic technique has been extensively used to create GMS lesions, whereas it has rarely been used to generate WMS lesions^5,11. For this technique, a combination of Rose Bengal dye and light irradiation has been demonstrated to be useful in destruction of the target structure, causing corresponding functional deficits. The key element of the photothrombotic technique is light irradiation, because it determines the size of infarct lesions. Light irradiation results in different effects on gray matter and white matter, because the scattering of light is more than 4 times higher in white matter compared with gray matter¹²; Accordingly, if the light intensity has a sufficiently low irradiance (<1,140 mW/mm²), one can limit the extension to which photothrombotic lesion affect the extent to the white matter (i.e., internal capsule). For example, light of higher energy can induce infarcts in both gray and white matter, yet lower energy light may induce photothrombosis only in white matter. Furthermore, the penetration of light energy was very limited. Approximately 99% of light energy was lost beyond 1 mm from the source of light¹³. Therefore, it is expected that accurately targeted, lower energy light induces photothrombosis only in the white matter with a minimal encroachment of the neighboring gray matter.

Here, we describe a novel method to create infarct lesions in the forelimb area of the internal capsule in rodents. We describe the method of identification of the forelimb area in the internal capsule, the technology of light irradiation, including the adjustment and delivery of light, and the generation of an infarct lesion. We also describe behavioral testing used to evaluate the completeness of the capsular modeling.

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 30 min for dry.
   3. Anesthetize the animal with a mixture of ketamine hydrochloride (100 mg/kg) and xylazine (7 mg/kg) via an intramuscular injection. Check the depth of anesthesia by paw pinching. Maintain the body temperature at 37.5 ± 0.5 °C via a heating pad under the body of the animal.
   4. Place the animal in a stereotactic frame using an ear bar and mouth holder.
   5. Clean and disinfect the surgical site with 70% alcohol and povidone iodine solution. Infiltrate 2% lidocaine hydrochloride under the scalp in the intended skull incision area to reduce the intraoperative pain.
   6. Apply vet ophthalmic ointment to prevent drying of the eyes. Place a sterile drape over the animal to the operative sites. Maintain all procedures in sterile conditions.
   7. Perform a midline skull incision of 2 cm using a scalpel and retract the skin bilaterally with wire retractors. Dry the skull with cotton swabs and 30% hydrogen peroxide.
   8. Make a hole using a hand piece drill over the forelimb area of motor cortex (AP: +2.5 from bregma, ML: ±2.5 from the midline) and clear the tract with micro-currette for virus-injection.
   9. Thaw the AAV-GFP (2 x 10¹² virus molecules/ml) on ice and load 1 µl of the virus in a 10 µl syringe. Place the syringe on the stereotactic frame.
   10. Move the needle to the pre-made hole and lower the needle 1 mm deep into the dura.
   11. Inject the virus slowly (0.1 µl/min) using a high precision micropump and leave the needle in place for an additional 10 min to allow the virus to diffuse out.
   12. After cleaning the operative site with saline irrigation, secure the wound with 3-0 nylon suture; release the rat from the stereotactic frame and transfer it to a recovery chamber. Administer  ketoprofen (2 mg/kg) via an intramuscular injection  for postoperative pain control.
   13. Maintain the body temperature (37 °C) with heating pad and administer second generation cephems-class antibiotics (0.1%, 1 ml) via an intramuscular injection and 2% lidocaine hydrochloride via subcutaneous injection as necessary.  Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Single house the animal until full recovery.
   14. Following 2 - 3 week recovery, deeply anesthetize the rat with an overdose of ketamine hydrochloride (300 mg/kg) via an intramuscular injection in the hood. Confirm the death of animal by lack of toe pinching response, pulse, and breathing. Place the rat supine in the hood.
   15. Open the abdominal cavity via a 'Y'-shaped incision to open the chest cavity. Tightly clamp the descending aorta with a hemostat and rupture the right atrium of the rat heart for blood drainage. Initiate perfusion into the left ventricle of the heart with cold 1% paraformaldehyde for 5 min (10 ml/min) followed by 4% paraformaldehyde for 30 min (10 ml/min).
   16. Remove the rat head from carcass using a pair of scissors. Make a midline incision from the neck to the nose and remove the neck muscles using scissor or rongeur so that skull is exposed. Gently dissect the skull bones and duras out from the brain.
   17. Extract the brain and place the rat brain in a 50 ml conical tube filled with 4% paraformaldehyde overnight. The next day, wash the brain with 1x PBS 3 times and place it in a 30% sucrose solution.
   18. After the brain completely sinks to the bottom of the 30% sucrose solution, place the brain in the cryomold with OCT compound at -20 °C in cryotome. Slice the brain in coronal plane at a thickness of 40 μm and an interval of 200 μm.
   19. Perform GFP immunohistochemistry staining using the slide method¹⁴. Apply primary antibody (1:200 of Anti-Green Fluorescent Protein, Rabbit IgG fraction) to brain slices overnight at 4 °C. On 2^nd day, wash with 1% Phosphate Buffered Saline with Tween-20 (PBST) solution 3 times and apply the secondary antibody (1:500 of Goat Anti-Rabbit IgG (H + L)) for 1 hr. Rinse the slide with 1% PBST 3 times. Place the cover glass on the brain slice.
   20. Using a fluorescent microscope (excitation wavelength 470 nm, emission wavelength 525 nm, magnification 5X), observe the AAV-GFP transduced axons in the internal capsule. Compare the locations of transduced axons with the Rat Brain Atlas¹⁵ to determine the stereotactic coordinates of the transduced axons
2. Pre-lesioning Adjustment of the Light Intensity Appropriate for Capsular Infarct Modeling
   1. Construction of the Optical Neural Interface
      1. Cut an appropriate length (4 cm) of a 27 gauge spinal needle with a stylet inside using a cutting drill.
         NOTE: Cutting may compress and crush the spinal needle tip; remove the stylet and polish the spinal needle tip to remove the crushed portion of the spinal needle and maintain the inner caliber of the spinal needle.
      2. Strip an appropriate length (10 cm) of the jacket of the optical fiber (125 µm with a 62.5 µm core) of one side patch cord.
      3. Insert the unjacked optical fiber into the metal tube (external diameter: 3.8 mm, internal diameter: 3.3 mm and length: 17 mm), which is then clamped around the fiber. The metal tube is helpful to fill the space between the optical fiber and the hub of the spinal needle. Clamp the lower 1/2 of the metal tube with a presser twice.
      4. Apply the heat-curable epoxy on the optical fiber and insert the optical fiber into the spinal needle. Apply an additional epoxy to the empty space in the hub. Cure the epoxy for 20 min at 100 °C for stable fixation.
      5. Cleave the optical fiber that protrudes out of the spinal needle and polish the optical fiber at the tip of the spinal needle using diamond lapping (polishing) sheets.
      6. Connect the FC/PC connector part of patch cord to the coupler of green laser system and measure the light intensity from the tip of the optical fiber using digital optical power and energy meter.

2. Photothrombotic Infarct Lesioning in the Internal Capsule

1. 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 30 min for dry.
2. Anesthetize the animal (~400 g, 11 - 13 weeks) with a mixture of ketamine hydrochloride (100 mg/kg) and xylazine (7 mg/kg) via an intramuscular injection. Check the depth of anesthesia by paw pinching. Maintain the body temperature at 37.5 ± 0.5 °C via a heating pad under the body of the animal.
3. Place the animal in a stereotactic frame using an ear bar and mouth holder.
4. Clean and disinfect the surgical site with 70% alcohol and povidone iodine solution. Infiltrate 2% lidocaine hydrochloride under the scalp in the intended skull incision area to reduce the intraoperative pain. Apply vet ophthalmic ointment to prevent drying of the eyes.
5. Apply a sterile drape over the animal and expose the operative sites. Maintain all procedures in sterile conditions.
6. Perform a midline skull incision of 2 cm and retract the skin bilaterally with wire retractors. Dry the skull with cotton swaps and hydrogen peroxide.
7. Adjust the height of the nose clamp until the bregma and lambda are aligned at the same level. CRITICAL STEP: This alignment is critical to correctly approach a deeper structure, such as in performing an infarct lesion in the internal capsule in the main experiment.
8. Make a hole (diameter: 2 mm; AP: -2.04 from bregma; ML: ±3.0 from the midline) using a drill to induce photothrombosis.
9. Polish and clean the optical fiber tip of the optical interface. Fix the ONI to the stereotaxic frame without bending. Check the tip of the ONI and wipe it out clearly before and after the insertion of the optical interface.
10. Measure the laser intensity from the tip of the optical fiber prior to the insertion of the optical interface to the target site of the rat brain. Adjust the laser intensity to 3.5 mW, as confirmed by pre-surgery steps, at the tip of the optical fiber.
11. Insert the ONI into the target area of the internal capsule (-7.8 mm confirmed from pre-surgery step) through the drill hole.
12. Maintain the body temperature at 37.5 ± 0.5 °C during photothrombosis. A lower body temperature may not produce the expected extent of infarction. Inject Rose Bengal (2 ml/kg) through the tail vein.
13. Turn on the 532 nm green laser for 90 sec 1 min after the Rose Bengal injection. After irradiation, gently remove the ONI from the brain. After cleaning the operative site, secure the wound secure the wound with 3-0 nylon suture; release the rat from the stereotactic frame and transfer it to a recovery chamber.
14. For sham-operated group (SOG), perform an identical lesion-making procedure, except for injection of saline (0.2 ml/100 g) instead of Rose-Bengal.
15. Maintain the body temperature (37 °C) with heating pad after surgery and administer antibiotics (second generation cephalosporin, 0.1%, 1 ml) via an intramuscular injection. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return post-surgical animals to the cage occupied by other animals until fully recovered.
    NOTE: Preliminary experiment was performed in the same procedures to find the optimal light intensity from 1 mW to 5 mW and the procedure may be required to acquire the satisfactory extent of lesion in different condition.

3. Evaluation of Capsular Infarct Lesioning

1. Behavioral Testing and Animal Grouping
   1. Perform single pellet reaching tasks as described by Whishaw et al.¹⁴ to evaluate the motor deficit of the forelimb every day for 1 week after the stroke modeling. Perform a single pellet reaching task (SPRT) in  food-restricted animals (90% of control body weight) using clear Plexiglas (30 x 15 x 35 cm height) with a 1 cm wide slit and a food shelf in the front of the middle of the front wall.
   2. Place a pellet on the food shelf obliquely contralateral to the preferred forelimb. Administer 20 pellets per session for 3 weeks.
      NOTE: A successful number of SPRTs is defined as a reach in which the animal grasps a food pellet and puts it into the mouth without dropping it.
   3. Calculate the score as a percentage of successful reaches, which is defined by the following formula:
      
      NOTE: We divide animals into 3 groups: the sham-operated group (SOG), moderate recovery group (MRG), and poor recovery group (PRG). If a post-stroke SPRT score >50%, we classify the rats as the MRG, which indicates the presence of a substantial lesion, but not complete destruction of the target. If the post-stroke SPRT score is <50% compared with the pre-stroke SPRT score, we classify the group as PRG, which indicates complete lesioning in the target.
2. Neurohistological Confirmation of Infarct Lesioning
   1. Perform cardiac perfusion with 4% paraformaldehyde as previously described. After the brain completely sinks in the 30% sucrose solution, perform coronal sectioning at a thickness of 10 µm and an interval of 200 µm using a microtome or cryotome⁴.
   2. Stain with H&E, Nissl, Luxol fast blue-PAS, Neurofilament protein-L or Glial fibrillary acid protein staining and observe the histological findings to determine the optimal light intensity that can cover the entire breadth of the internal capsule in the target area to observe staining^4,17.
   3. Using ImageJ software, measure the volume of the photothrombotic infarct area of the internal capsule on the brain slides.
      1. To measure the volume of infarct area, launch the 'ImageJ' software. To open the files to be stacked, select 'Image to Stacks' ('Image' → 'Stacks' → 'Image to Stacks'). Edit file name and select 'Set the Scale' ('Analyze' → 'Set the Scale') to edit scale.
      2. In 'Plugins', select 'Measure Stacks' to calculate volume or area of images. Insert the distance interval of 2 images into 'Slice Spacing'. Make a drawing of the ROI (Region Of Interest) of all images and click 'Measure'.
         NOTE: The software 'ImageJ' automatically calculates the area and volume of each image and total volume of them.

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