Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Summary

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Abstract

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

Introduction

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States¹. Approximately 87% of all strokes are ischemic, while a majority of the remaining 13% are hemorrhagic². An ischemic stroke is defined as the blockage of blood flow in an artery to the surrounding tissue. This occlusion results in oxygen deprivation and subsequent necrosis that often leads to permanent disability in surviving patients. While there has been a decrease in the mortality rate of stroke³, its prevalence is expected to increase to 3.4 million people by 2030⁴. This ....

Protocol

The experiments were conducted in accordance with IUCAC at Duke University and University of California Los Angeles. 8 to 12-week-old male C57Bl/6J mice were used in this study. The animals were housed under controlled temperature (22 ± 2 °C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum.Analgesia and sedation protocols are described as approved by the IUCAC but might differ from protocols used in other laboratories.

Animals may be prematu.......

Discussion

Here we demonstrate an easily reproducible, minimally invasive, permanent stroke model and describe how to inject a biomaterial into the infarct five days after stroke. The use of the photothrombotic dye Rose Bengal and a 520 nm collimated laser connected to the stereotaxic device gives us the ability to position the stroke at the motor cortex of the mouse with enhanced precision. Five days after stroke, the location of the infarct is visible by eye at the center of irradiation, 2.0 mm medio-lateral to the bregma. Hydrog.......

Materials

Name	Company	Catalog Number	Comments
10% Normal Goat Serum	VWR	100504-028	For blocking buffer
2-ply alcohol pre pad, Sterile, Medium	Medline	MDS090735
25uL Hamilton Syringe 702RN, no needle	Fishcer Scientific	14824663	Syringes used to inject biomaterials
25uL Positive displacement pipette	Gilson	M-25
2x Beam Expander, 400-650nm	Thorlabs	GBE02-A	Laser beam expander
Adjustable Stage Platform	Kopf Instruments	901
Anti-Glucose Transporter GLUT1 antibody, rabbit	Abcam	ab113435
Anti-Iba1 Antibody, goat	abcam	ab5076
BD Vacutainer Safety-Lok Blood Collection Sets. 25G, 12"	Medsupply	367294	For perfusions
BKF12- Matte Black Aluminum Foil	Thorlabs	BKF12	To cover anything that is reflective when using laser.
Bone Iris Mini Scissors - 3-1/2"	Sklar surgical instruments	64-2035
C57BL/6 Mice	Jackson Laboratory	000664	8-12 weeks of age
Cage Assembly Rob	Thorlabs	ER3-P4	3" Long, diameter 6mm, 4 pack - for attaching laser to sterotax
Carbon Steel Burrs -0.5mm Diameter	Fine Science Tools	19007-05	For creating burr hole
Chromium(III) potassium sulfate dodecahydrate	VWR	EM1.01036.0250
Compact Controller for pigtailed lasers	Thorlabs	CLD1010LP
Cotton Swabs	VWR	89031-288
CP 25 pipette tips	Gilson	F148012
Donkey anti-goat IgG H&L (488)	abcam	ab150129
Donkey Anti-rabbit IgG H&L (647)	abcam	ab150075
Donkey Anti-rat IgG H&L (555)	abcam	ab150154
EMS DPX Mountant	Elecron Microscopy Sciences	13512	Mounting solution for slides
EMS Gelatin Powder Type A 300 Bloom	Electron Microscopy Sciences	16564	For gelatin coating slides
EMS Paraformaldehyde, Granular	VWR	100504-162	For making 45 PFA
ESD Worstation kit	Elmstat	WSKK5324SB	Need for setting up the laser
Fiber Bench Wall Plate, unthreaded	Thorlabs	HCA3	Need for connecting laser to Kopf shaft
FiberPort	Thorlabs	PAF-X-5-A	FC/APC& APC, f=4.6mm, 350-700nm, diameter 0.75mm
Fine Scissors - straight/sharp-blunt/10cm	Fine Science Tools	14028-10
GFAP Antibody, rat	Thermo Fisher Scientific	13-0300
Heating Plate	Kopf Instruments	HP-4M
ImmEdge Hydrophobic Barrier Pen	Vector Laboratories	H-4000	For staining slides
IMPAC 6-Integrated Multi Patient Anesthesia Center	VetEquip	901808
Iodine Prep Pads	Medx Supple	MED MDS093917H
Jewlers Forceps #5	GFS chemicals	46085
Laser Safety Glasses	Thorlabs	LG10B	Amber Lenses, 35% Visible light (googles versions available too)
M27-1084 Powerful LED Dual Goose-neck	United Scope	LED-11C
Medical USP Grade Oxygen	Airgas	OX USP250
Miltex Adson Dressing Forceps, Disecting-grade	Intefra Miltex	V96-118
Mini Cord/Cordless Small Animal Trimmer	Harvard aparatus	72-6110
Mini-pump variable flow	Thomas Scientific	70730-064	Pump for perfusions
Mouse Brain Matrices, Coronal Slices, 1mm	Kent Scientific	RBMA-200c	For TTC slices
Mouse Gas Anethesia Head Holder	Kopf Instruments	923-B
Nanojet Control Box	Chemyx	10050
Nanojet pump header	Chemxy	10051	Attach to stereotaxic device for injecting biomaterial
Needle RN 30G PT STY 3, 0.5 inch	Fishcer Scientific	NC9459562
Non-rupture ear bars 60º	Kopf Instruments	922
PBS buffer pH 7.4	VWR	97062 338
Pigtaled laser 520 nm, 100mW, 5G Pin	Thorlabs	LP520-MF100
Positive charge glass slides	Hareta	AHS90-WH
Power engergy meter	Thorlabs	PM100D	Used to measure your mW laser output
Puralube Vet Ointment	Dr. Foster Smith	9N-76855
Rectal Probe Mouse	kopf Instruments	Ret-3-ISO
Rose Bengal Dye 95%	Sigma-Aldrich	330000-5G
Shaft Modified 8-32 threaded hole 1/2" depth	Kopf Instruments	1770-02	For connecting laser to sterotaxic device
Slim photodiode power sensor	Thorlabs	S130VC	Used with power energy meter
SM1-Threaed 30 mm Cage Plate 0.35" thick 2 Retaining	Thorlabs	CP02	For connecting laser expander
Sol-M U-100 Insuline syringe with 1/2 unit markings 0.5 mL	VWR	10002-726	To inject rose bengal
StainTray Slide Staining System	Simport Scientific	M920-2	For staining slides
Sterotaxic device	Kopf Instruments	940	Small Animal Stereotaxic Instrument
Student Adson Forceps -1x2 teeth	Fine Science Tools	91127-12
Student Fine Forceps - straight/broad Shanks	Fine Science Tools	91113-10
Temperature Controller	Kopf Instruments	TCAT-2LV
Tissue-Tek OCT compound	VWR	25608-930
Triton X-100	VWR	97063-864
Upper Bracket Clamp	Kopf Instruments	1770-c	For connecting laser to sterotaxic device
Vetbond Tissue Adhessive 3mL	Santa Cruz Biotechnology	sc-361931
Vogue Professional My Manicurist	Bargin Source	6400	For Burrs
VWR Bead Sterilizers	VWR	75999-328
Tissue Tek OCT compound	Sakura	4583	For tissue embeding