A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Summary

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Abstract

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 ± 0.5 °C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Introduction

Thrombolysis and recanalization is the most effective therapy of acute ischemic stroke in clinical practice¹. Yet, the majority of preclinical neuroprotection research was performed in a transient mechanic obstruction model (intraluminal suture middle cerebral artery occlusion) that produces rapid recovery of cerebral blood flow upon removal of the vascular occlusion and shows little to no benefits by tPA thrombolysis. It has been suggested that the dubious choice of stroke models contributed, at least in part, to the difficulty in translating neuroprotective therapy to patients^2,3. Hence, there is an increasing call for employing tPA-responsive ....

Protocol

This protocol is approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University and follows the National Institutes of Health Guideline for Care and Use of Laboratory Animals.

1. Setup

1. Prepare the surgical bed on warming pad connected with heat pump at 37 °C for at least 15 min before the surgery. Place a neck roll using the barrel of 3 ml syringe on the surgical bed. Prepare the anesthesia gas with 2% isoflurane in medical air.
2. Prepare autoclaved forceps, scissors, micro needle holders, hemostat, cotton swabs and sutures. Prepare tissue glue and eye ointment.
3. Set up the hypoxia ....

Results

Two-dimensional laser speckle contrast imaging (LSCI)¹⁶ was used to compare the alterations of cerebral blood flow (CBF) by 30 min transient unilateral carotid occlusion (tCCAO), 30 min exposure to hypoxia (7.5% oxygen), and 30 min unilateral carotid ligation under hypoxia (tHI). This experiment revealed that tCCAO under normoxia suppressed the CBF on the carotid ligated hemisphere to ~50% of the baseline value, which quickly recovered to above 85% after release of the carotid occlusion (R in Figure 2A.......

Discussion

Stroke is a major health issue of growing significance for any society with an aging population. Globally, stroke is the second-leading cause of death with an estimated 5.9 million fatal events in 2010, equivalent to 11.1% of all deaths¹⁸. Stroke is also the third-leading cause of disability-adjusted life years (DALYs) lost globally in 2010, rising from the fifth position in 1990¹⁹. These epidemiological data highlight the need of more effective therapies of acute (ischemic) stroke. However, despite.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
adult male mice	Charles River	C57BL/6	10-13 weeks old (22-30 g)
Mobile Laboratory Animal Anesthesia System	VetEquip	901807	anesthesia
Medical air (Compressed) air tank	Airgas	UN1002	anesthesia
Isoflurane	Piramal Healthcare	NDC 66794-013-25	anesthesia
Multi-Station Lab Animal AnesthesiaSystem	Surgivet	V703501	hypoxia system
7.5% O2 balanced by 92.5% N2 tank	Airgas	UN1956	hypoxia system
Temperature Controller with heating lamp	Cole Parmer	EW-89000-10	temperature controllers
Rectal probe	Cole Parmer	NCI-00141PG	temperature controllers
Dissecting microscope	Olympus	SZ40	surgical setup
Heat pump with warming pad	Gaymar	TP700	surgical setup
Fine curved forceps (serrated)	FST	11370-31	surgical instrument
Fine curved forceps (smooth)	FST	11373-12	surgical instrument
micro scissors	FST	15000-03	surgical instrument
micro needle holders	FST	12060-01	surgical instrument
Halsted-Mosquito hemostats	FST	13008-12	surgical instrument
5-0 silk suture	Harvard Apparatus	624143	surgical supplies
4-0 Nylon monofilament suture	LOOK	766B	surgical supplies
Tissue glue	Abbott Laboratories	NC9855218	surgical supplies
Puralube Vet ointment	Fisher	NC0138063	eye dryness prevention
MoorFLPI-2 blood flow imager	Moor	780-nm laser source	Laser Speckle Contrast Imaging
Mannitol	Sigma	M4125	in vivo TTC
2,3,5-triphenyltetrazolium chloride (TTC)	Sigma	T8877	in vivo TTC
Vibratome	Stoelting	51425	brain section for in vivo TTC
Digital microscope	Dino-Lite	AM2111	whole-brain imaging
O.C.T compound	Sakura Finetek	4583
goat anti-rabbit Alexa Fluro 488	Invitrogen	A11008	Immunohistochemistry
Cryostat	Vibratome	ultrapro 5000	brain section for IHC
Evans blue	Sigma	E2129	Detecting vascular perfusion
Microtome	Electron Microscopy Sciences	5000	brain section for histology
Avertin (2, 2, 2-Tribromoethanol)	Sigma	T48402	euthanasia
Fluorescent microscope	Olympus	DP73
Meloxicam SR	ZooPharm		NSAID analgesia