En trombotisk hjerneslag modell basert på Transient Cerebral Hypoksi-iskemi

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 forskning har tålt mange tilbakeslag i å oversette nevro terapi i klinisk praksis. I motsetning til den virkelige verden terapi (Pa trombolyse) produserer sjelden fordeler i mekaniske okklusjon-baserte eksperimentelle modeller, som dominerer preklinisk hjerneslag forskning. Denne fordelingen mellom benk og nattbord antyder behovet for å ansette Pa-responsive modeller i preklinisk hjerneslag forskning. For dette formål, er en enkel og tPA-reaktive trombotisk slag modell oppfunnet og beskrevet her. Denne modellen består av transient okklusjon av den ensidige arteria carotis communis og levering av 7,5% oksygen gjennom en ansiktsmaske i voksen mus i 30 minutter, under opprettholdelse av dyret rektal temperatur på 37,5 ± 0,5 ° C. Selv reversibel ligering av den ensidige carotid arterie eller hypoksi hver trykkes cerebral blodstrøm bare forbigående, er kombinasjonen av begge fornærmelser forårsaket varig reperfusjon underskudd, fibrin og blodplateavleiring, og store INFARct i midten cerebral arterie-leverte territorium. Viktigere, haleveneinjeksjon av rekombinant tPA ved 0,5, 1, eller fire timer etter Thi (10 mg / kg) gitt tidsavhengig reduksjon av dødelighet og infarktstørrelsen. Denne nye slagmodell, er enkel og kan standardiseres tvers laboratorier for å sammenligne eksperimentelle resultater. Videre induserer det trombose uten craniectomy eller innføre forhånds dannet emboli. Gitt disse unike fordeler, er thi modellen et nyttig tillegg til repertoaret av preklinisk hjerneslag forskning.

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 thromboembolic stroke models in preclinical research, but such models also have technical problems (see Discussion)^4-7. Here we describe a new thrombotic stroke model based on unilateral transient hypoxic-ischemic (tHI) insult and its responses to intravenous tPA therapy⁸.

The tHI stroke model was developed based on the Levine procedure (permanent ligation of the unilateral common carotid artery followed by exposure to transient hypoxia in a chamber) that was invented for experiments with adult rats in 1960⁹. The original Levine procedure faded into obscurity because it only produced variable brain damage, but the same insult caused consistent neuropathology in rodent pups when it was re-introduced by Robert Vannucci and his colleagues as a model of neonatal hypoxic-ischemic encephalopathy (HIE) in 1981¹⁰. In recent years, some investigators re-adapted the Levine-Vannucci model to adult mice by adjusting the temperature in the hypoxic chamber¹¹. It is plausible that the inconsistent brain lesions in the original Levine procedure may arise from fluctuating body temperatures of adult rodents in the hypoxic chamber. To test this hypothesis, we modified the Levine procedure by administering hypoxic gas through a facemask, while maintaining the rodent core temperature at 37 °C on the surgical table¹². As expected, stringent body temperature control greatly increased the reproducibility of HI-induced brain pathology. The HI insult also triggers coagulation, autophagy, and gray- and white-matter injury¹³. Other investigators have also used the HI model to investigate post-stroke inflammatory responses¹⁴.

A unique feature of the HI stroke model is that it closely follows the Virchow’s triad of thrombus formation, including the stasis of blood flow, endothelial injury (e.g. due to HI-induced oxidative stress), and hypercoagulability (HI-induced platelet activation) (Figure 1A)¹⁵. As such, the HI model may capture some pathophysiological mechanisms relevant to real-world ischemic stroke. With this idea in mind, we further refined the HI model with reversible ligation of the unilateral common carotid artery (therefore to create a transient HI insult), and tested its responses to tPA thrombolysis with or without Edaravone. Edaravone is a free radical scavenger already approved in Japan to treat ischemic stroke within 24 hr of onset⁹. Our experiments showed that as brief as 30 min transient HI triggers thrombotic infarction, and that combined tPA-Edaravone treatment confers synergistic benefits⁸. Here we describe detailed surgical procedures and methodological considerations of the tHI model, which can be used to optimize reperfusion treatments of acute ischemic stroke.

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 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. Prepare autoc…

Representative 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

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-braina 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