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 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

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 system and temperature controllers with heating lamp and rectal probe. Prepare hypoxia gas with 2% isoflurane in 7.5% O₂ balanced by 92.5% N₂.
4. One hour before surgery, mice are analgesized by subcutaneous injection of a slow-release Meloxicam (4.0 mg/kg).

2. Transient Cerebral Hypoxia-ischemia (Figure 1B)

1. Anesthetize 10-13 week old male C57BL/6 mice weighing 22 to 30 g in the anesthesia induction chamber with 3% isoflurane until the animal is unresponsive to foot squeeze, and then remove the hair on right neck using an electronic shaver.
2. Place mice on the surgical bed connected with 2% isoflurane in medical air at flow rate of 2 L/min. Secure forelimbs stretched out along neck roll at sides using medical tape.
3. Clean the surgical site for incision with betadine followed by alcohol and then cotton swabs.
4. Under dissecting microscope, make a 0.5 cm right-cervical incision using a straight forceps and a micro scissors about 0.2 cm lateral from midline skin.
5. Use a pair of fine serrated forceps to pull apart the fascia and tissue to expose the right common carotid artery (RCCA). Carefully separate the RCCA from the vagal nerve using a pair of fine smooth forceps.
6. Live knot two precut 5-0 silk suture (releasable) on the RCCA, and then sew up the skin using 4-0 Nylon monofilament suture (Figure 1C).
7. Apply eye ointment on both eyes to prevent dryness.
8. Quickly transfer the mice to hypoxia system and put nose and mouth in face-mask with 2% isoflurane in 7.5% O₂ at flow rate of   0.5-1 L/min for 30 min.
   1. During hypoxia, use temperature controllers with heating lamps to control the rectal temperature at 37.5 ± 0.5 °C. Monitor the respiratory rate at 80-120 breaths/min. The maintenance of the body temperature above 37 °C during the hypoxia is important to create consistent brain infarction. Low respiratory rate usually happens after 20 min hypoxia. Remove the face-mask and allow normal air supply if the respiratory rate drops below to 40. This takes 1-2 min and does not count into the 30 min hypoxia duration.
9. After hypoxia, transfer mice to a surgical bed and release the two sutures from RCCA. Close the wound using tissue glue, and then return mice to the cage. Exclude the animals if both of two live knots are unexpectedly released after hypoxia.
10. Monitor the mice for 5-10 min to recover from hypoxia and anesthesia. Place the wetted food in the cage and return it to animal care facility.
    Note: Animals showing mild to severe circling behavior at 24 hr after tHI are correlated with brain infraction. Most animals with seizure symptoms die before the 24 hr timepoint after tHI.

3. Laser Speckle Contrast Imaging

Note: Although this is not an essential procedure of the tHI model, a two-dimensional laser speckle contrast imaging system¹⁶ can be used to characterize the changes of cerebral blood flow (CBF) during or after transient hypoxia-ischemia. To document the alterations of CBF under tHI, record immediately after the step 2.6. Alternatively, to compare CBF recovery after tHI insult, these procedures can be performed following the step 2.10.

1. Place an anesthetized mouse in the prone position and perform a 1 cm-long midline incision on the scalp with the skull exposed but unopened.
2. Monitor CBF in both cerebral hemispheres under a blood flow imager according to manufacturer’s protocol and start recording the cerebral blood flow immediately after the CCAO surgery (step 2.6). Continue for 50 min.
3. Show CBF image with arbitrary units in a 16-color palette and analyze in real-time the selected regions using the MoorFLPI software following the manufacturer’s instructions (Figure 2).
4. After recording the CBF image, close the scalp with tissue glue and return the animal to the cage.

4. tPA Administration

1. Inject animals at the tail vein with the solvent or 10 mg/kg recombinant tPA (220-300 μl of 1 mg/m tPA) at 0.5, 1, or 4 hr after tCCAo plus hypoxia (Figure 4).

5. Brain Damage Detection with Several Different Options

Note: To collect brain samples, euthanize the mice at 1, 4 or 24 hr after tHI.

1. Perform quantification of infarct volume by in vivo 2,3,5-Triphenyltetrazolium chloride (TTC) method at 24 hrs after tHI insult as previous described.¹⁷
   1. Intraperitoneally inject animals with 1.4 M mannitol solution (~0.1 ml/g body weight) 30 min before transcardial perfusion. Transcardial perfuse mice with PBS followed by 10 ml of 2% TTC.
   2. Remove the brain of animals with surgical instruments after 10 min and place into 4% paraformaldehyde for fixation overnight and section into 1 mm thickness with a vibratome.
   3. Snap a series of four sectioned brain slides by digital microscope and quantify the infarct volume as the ratio of the infarcted area (white area in the right side) to the area of the undamaged, contralateral hemisphere using the ImageJ software.
2. Alternatively, perform thrombosis formation by immunofluorescence at 1 hr after tHI insult.
   1. Freeze the fixed brain in O.C.T. compound and section the brains at 12 μm thickness using a cryostat.
   2. Incubate the brain slide with rabbit anti-fibrinogen antibody (1:100) following by goat anti-rabbit Alexa Fluro 488 dye (1:200) to observe the fluorescence on a fluorescent microscope.
3. Alternatively, perform vessel obstruction by tail vein injection of 100 µl 2% Evans blue dye at 4 hr after tHI insult.
   1. Euthanize the mice and quickly cut head to remove brains into 4% paraformaldehyde after Evans blue injection. Note: It takes 5-10 min for Evans blue circulation with blue color of both fore- and hind limbs.
   2. Section fixed brains at 100 μm thickness using a sliding microtome and observe the fluorescence using a 680 nm emission filter on a fluorescent microscope.

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