A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Summary

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Abstract

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Introduction

Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US ¹. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia ^2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist ^4,5.

The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.

Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) ⁶ that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded ^7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter.

The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) ⁹ with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 ¹⁰. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.

Protocol

The use of animals in this protocol has been performed in accordance with procedures approved by the University of California Los Angeles Animal Care and Use Committee.

Note: Begin by identifying the target murine population. In prior studies, only male wild-type C57/Bl6 mice have been used, however various transgenic or knockout mice can also be used. Note that stereotactic coordinates are based on C57/Bl6 anatomy. It is recommended that each user initially verify localization of the stroke to white matter.

1. White Matter Stroke Induction - Medial Angled Approach

1. Begin by preparing a pulled glass pipette using 0.5 mm capillary tube such that the distal diameter is between 15-25 µm ¹¹.
2. Prepare a sterile 10 µl aliquot of L-Nio (N(5)-(1)-iminoethyl-L-ornithine HCl) at 27.4 mg/ml (130 µM) in sterile 0.9% normal saline.
3. Pre-fill the pulled glass pipette with a small volume of L-Nio (2-5 µl) by affixing the glass pipette to tubing connected to a vacuum line. Lay the pipette flat on the bench top and insert the pulled end into the L-Nio solution.
   1. Apply the vacuum until at least 2 mm of the 0.5 mm portion of the pipette is filled. Turn off the vacuum and withdraw the pipette. Place it aside until Step 1.12.
4. Place the mouse in an induction chamber and induce anesthesia of the mouse using standard 32% isoflurane flowed through a vaporizer (5 L/min inhaled with 5 L/min oxygen and 0.5 L/min N₂) for 1 min or until deeply anesthesized. Transfer the mouse to a stereotactic apparatus equipped with a stereotactic microscope. Provide maintenance anesthesia using 32% isoflurane flowed through a vaporizer (2 L/min inhaled with 5 L/min oxygen and 0.5 L/min N₂) and a nose cone. Check depth of anesthesia with a toe pinch. 
5. Adjust the injection arm to 36°.
6. Affix a pulled glass pipette holder to the distal end of a low volume pressure injection system and attach it to the injection arm of the stereotactic setup.
7. Coat the anesthetized animal's whiskers with petroleum jelly and place artificial tear ointment over both eyes. Prepare a sterile surgical field by placing a sterile drape over the animal's head with a 5-10 cm opening over the head. Prepare an aspetic surgical surface by shaving the fur overlying the skull. Clean the scalp with alternating betadine and 70% alcohol swabs.
8. Make a 1.5 cm midline scalp incision with sterile fine scissors to expose the skull surface. Dry the skull with a sterile cotton swab and using a stereotactic microscope at 1-3X magnification, remove any overlying periosteal tissue using a sterile micro point tool.
9. Mark the Bregma as a reference point using a fine point marker.
10. Drill a 2 mm ellipitical craniotomy using a sterile fine stip surgical drill bit beginning posteriorly at the Bregma and extending anteriorly just left of the midline. Remove bone fragments and overlying soft tissue so that the cerebral cortex can be visualized.
11. Keep the surgical field and cortical surface moist by intermittently applying drops of sterile saline.
12. Affix a pulled glass pipette to the injector arm of the stereotactic apparatus. Align the distal end of the pipette with the Bregma and zero the stereotactic coordinates.
13. Advance the pipette to the first anterior/posterior (A/P) and medial/lateral (M/L) coordinates provided in Table 1.
14. Advance the pipette to the cortical surface and zero the dorsal/ventral (D/V) measurement.
15. Slowly pass the pipette into the brain until reaching the first D/V coordinate in Table 1.
16. Using a low volume pressure injection system set at 20 psi for 20 msec pulses, inject 100 nl of L-Nio into the brain and wait 5 min to prevent reflux up the pipette track.
    1. Use a calibrated reticle in the eyepiece of the stereotactic microscope and a magnification of 3X.
    2. Accordingly, displace a total of 0.100 mm³ (0.5 mm length in a 0.5 mm diameter pipette, corresponding to 100 nl) from the pulled glass pipette for each set of coordinates. By using an reticule, measure and standardize since each set up varies depending on the magnification and scales used.
    3. For accurate volume measurement during each injection, approach the angled pipette with the microscope from the side so that the air-fluid meniscus has a sagittal view. The meniscus should appear in the same focal plane of both the inner and outer wall of the pipette.
17. Slowly withdraw the pipette and repeat steps 1.13-1.16.3 at the second and third set of coordinates provided in Table 1.
18. After the final injection, remove the pipette and place enough bone wax to fill the craniotomy site. Approximate the edges of the scalp wound and bind with dermal adhesive.
19. Inject 0.1 ml of 0.5% Marcaine into the wound margins using a sterile 30 G needle to prevent to prevent local pain associated with the scalp incision.
20. Return the animal to housing and supply post-operative antibiotics (0.48 mg/ml trimethoprim-sulfamethoxazole, or 0.5 mg/ml Levofloxacin) in the drinking water for 5 days.

2. White Matter Stroke Induction - Posterior Angled Approach

1. Perform steps 1.1-1.12 as in the medial angled approach protocol, except adjust the injection arm of the stereotactic setup to 45 degrees oriented anterior to posterior.
2. Advance the pipette to the first A/P and M/L coordinates provided in Table 2.
3. Complete remaining steps 1.14-1.20 as in the lateral angled approach protocol.

3. Retrograde Neuronal Labeling

1. Prepare a sterile 10 µl aliquot of L-Nio at 54.8 mg/ml in 0.9% normal saline.
2. Prepare a sterile aliquot of 20% Fluororuby (or 20% biotinylated dextran amine or 2% Fluorogold) in 0.9% normal saline.
3. Dilute together 1:1 for final concentrations of 27.4 mg/ml L-Nio and 10% Fluororuby.
4. Perform the stroke protocol as above in steps 1.3-1.23.
5. Visualize the natively fluorescent tracer in tissue section by perfusion fixation, cryosectioning and microscopy as previously described⁸.

4. Tissue Processing for Immunofluorescence

1. At an appropriate post-stroke interval ranging from 3 hr to 14 days after stroke, euthanize mice via isoflurane overdose or local IACUC approved procedure.
2. Open the thoracic cavity using angled scissors and insert a 23 G butterfly needle into the left ventricle.
3. Place a small cut in the right atrium using fine scissors to allow an outflow track for the perfusion fluid.
4. Transcardially perfuse with 30-40 ml of cold phosphate-buffered saline followed by 30-40 ml of cold 4% paraformaldehyde at a rate of 10 ml/min at RT.
5. Decapitate the mouse and remove the brain using sterile scissors to open the skull posteriorly and then gently remove the overlying skull with a spatula, place the brain into cold 4% PFA for 24 hr, and then transfer to 30% sucrose in PBS for 48 hr.
6. Prepare forty micron floating sections using a cryostat and perform antibody processing as previously described ^6-8. In this study, use the following antibodies: rabbit anti-neurofilament 200 (1:500 dilution); rabbit anti-vimentin (1:500); goat anti-GFAP (1:500); rabbit anti-Iba-1 (1:1,000).

5. Tissue Processing for Protein or RNA Analysis

1. At an appropriate post-stroke interval ranging from 3 hr to 14 days after stroke, euthanize via isoflurane overdose or local IACUC approved procedure.
2. Decapitate the mouse and remove the brain using sterile scissors to open the skull posteriorly and then gently remove the overlying skull with a spatula.
3. Insert a sterile 4 mm spatula at the front of the brain to sever the olfactory bulb and optic nerves. Gently lift the brain out of the calvarium and place into ice-cold dissection buffer (1x Hank's Balanced Salt Solution, 25 mM HEPES-KOH, pH 7.4, 35 mM glucose, 4 mM sodium bicarbonate, and 0.01 mg/ml cyclohexamide).
4. Using a brain block and sterile new razor blades, prepare 2-3 mm slabs containing the stroke and place into cold dissection buffer.
5. Under a dissecting microscope, identify the white matter underlying motor cortex in the injected hemisphere. At longer post-stroke intervals, the region may be visually identified by focal necrosis and myelin pallor.
   Note: At earlier post-stroke intervals, injection of L-Nio mixed with 1 µl of 10% Fast Green can allow visual identification of the stroke (Figure 4A).
6. Under guidance of a dissecting microscope and using a fresh scalpel, carefully dissect the region of white matter containing the stroke, identified by either Fast Green labeling or tissue loss. Remove overlying cortex and underlying striatum as desired.

Results

Using the model presented, the white matter underlying forelimb sensorimotor cortex can reliably be targeted. This chemically induced stroke model produces focal axonal and myelin loss, astrocytosis, and microgliosis (Figure 1), as is typically seen in human lacunar infarcts. By using three injections, a clinically useful model is established with early impairment on forelimb motor tasks ⁷ and a small but significant portion of brain tissue experiences ischemia...

Discussion

A number of prior models of subcortical stroke have been described including focal injections of endothelin-1 into the internal capsule, subcortical white matter and striatum in the rat ^12-14 and mouse ^6,15. More recent models of small focal strokes have utilized cholesterol microemboli injection in the carotid artery ¹⁶ and photothrombotic occlusion of a single penetrating arteriole ¹⁷. Each of these models has both advantages and disadvantages ⁵. The presently desc...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-N5-(1-Iminoethyl)ornithine, Dihydrochloride	Calbiochem	400600-20MG
Isoflurane	Phoenix Pharmaceutical, Inc.	NDC 57319-559-06
Capillary tubes	World Precision Instruments	50-821-807
Picospritzer	Parker Instrumentation	Picospritzer II
Stereotactic setup	Kent Scientific	KSC51725
Pipette puller	KOPF	Model 720
Stereomicroscope SZ51	Olympus	88-124
Fine scissors	Fine Scientific Tools	14084-08
Forceps	Harvard Apparatus	PY2 72-8547
Curved Forceps	Harvard Apparatus	PY2 72-8598
Blunt dissection tool	Fine Scientific Tools	10066-15
Drill	Dremel	8220-1/28
Drill bits	Fine Scientific Tools	19007-05
Vetbond	3M	1469SB
Marcaine	HOSPIRA	NDC 0409-1610-50
Trimethoprim-Sulfamethaxole	STI Pharmacy	NDC 54879-007-16
Fluororuby	Fluorochrome Inc	30 mg
Paraformaldehyde	Fisher	O4042-500
Sucrose	Fisher	BP220-10
Cryostat	Leica CM3050 S	14047033518
Glass slides	Fisher	12-544-7
Fast Green	Sigma	F7252-5G
Dissection microscope	Nikon	SMZ1500
23 G butterfly needle	Fisher	14-840-35
10x Hank's Balanced Salt Solution	Life Technologies	14065056
1 M HEPES-KOH, pH 7.4	Affymetrix	16924
D-Glucose	Sigma	G8270
Sodium bicarbonate	Sigma	S5761
Cyclohexamide	Sigma	01810