Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Summary

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Abstract

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide¹. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis^2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke⁴. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research⁵. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Introduction

Stroke is among the principal causes of long-term disability and death worldwide¹, coming second after coronary heart disease. In addition, stroke is the primary cause of long-term disability, underscoring its tremendous socioeconomic impact^6-8. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal⁷.

In the last few decades, data from experimental stroke research has contributed substantially to understanding the complex pathophysiological cascades triggered by ischemia^9,10. Excitotoxicity, apoptosis, peri-infarct depolarization, and inflammation have been identified as the most relevant mediators of cell death following focal cerebral ischemia. Moreover, using animal models of cerebral ischemia, important concepts, diagnostic modalities, and therapeutic approaches have been developed and validated (e.g., "penumbra" and thrombolysis)¹¹.

The availability of experimental stroke models, combined with non-invasive imaging modalities (e.g., magnetic resonance imaging (MRI), computed tomography, or laser speckle contrast analysis), enables the researcher to investigate hyperacute and chronic pathophysiological changes induced by the ischemic insult in a longitudinal manner¹². Along with studying the spatiotemporal profile of the evolving lesion, changes resembling neuronal plasticity can be investigated and correlated to functional outcomes and histological findings. Within the last few years, further methodological advances have been made using the combination of cerebral ischemia models and in vivo microscopy via cranial windows¹³. These new techniques allow investigators to analyze the neurovascular unit at the cellular and molecular level, with great analytic power in the acute, subacute, and chronic phases following focal cerebral ischemia¹⁴. Moreover, in vivo microscopy imaging of microcirculatory dynamics has revealed novel aspects of cerebral microvasculature function and angioarchitecture, with significant pathophysiological relevance^15-17.

In this protocol, we present how to perform a chronic CW preparation lateral to the sagittal sinus and how to surgically induce a distal stroke underneath the window. This mouse model can be applied to sequential imaging of acute, subacute, and chronic changes following focal cerebral ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Protocol

ETHICS STATEMENT: Experiments involving animal subjects were performed in accordance with the guidelines and regulations set forth by Landesamt fuer Gesundheit und Soziales, Berlin, Germany (G0298/13) and the ARRIVE criteria, as applicable. For this study, 10- to 12-week-old male C57Bl/6J mice were used.

1. Lateral Chronic Cranial Window Preparation

1. Perform anesthesia with a subcutaneous injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Test for adequate sedation with a pain stimulus.
2. Sterilize the surgical instruments and surgical field with 70% ethanol.
3. Remove the scalp hair from the neck to the eyes using a rodent shaver.
4. Fix the head in a stereotactic frame.
5. Use dexpanthenol eye ointment on both eyes to avoid dehydration.
6. Clean the surgical area to remove all hairs and sterilize it with 3 layers of 74.1% ethanol and 10% 2-propanolol.
7. Perform a midline incision from the neck to the eyes using a scalpel.
8. Span the skin flap with 4 tenting sutures.
9. Remove the periosteum carefully on the left hemisphere by scraping it off with a scalpel to the point where the temporal muscle begins.
   NOTE: This preparation also serves to create a good adhesion area for the dental glue, which fixes the cover glass.
10. Perform a fronto-parietal craniotomy with a diameter of 4 mm by thinning the skull at the rim of the bone flap using a microdrill. Apply saline solution while drilling to avoid heat injury.
11. Elevate the bone flap with cannulas and remove it using microforceps.
12. Irrigate carefully and extensively with saline solution.
13. Mix the dental glue until it has the proper consistency and is not fluid (i.e., the glue should not produce threads anymore). Place it on the bone around the craniotomy.
14. Place a cover glass with a 6-mm diameter on the prepared glue and fix it with the rest of the dental glue. Be sure that it is watertight. Wait until the glue is dried and hard by testing it with forceps.
    NOTE: An additional irrigation accelerates the curing process.
15. Remove the tenting sutures and close the wound with skin sutures.
16. Pull up the skin of the flank of the mouse. Insert a needle subcutaneously and gently substitute 0.5 ml of sterile saline subcutaneously for fluid balance.
17. After surgery, keep the animals in the heated recovery cage for 90 min. Wait until the animals are fully awake before leaving them unattended. Wait until the animals are completely recovered before returning them to a cage with other animals.
18. Repeat the subcutaneous saline volume substitution after about 12 hr, as described in step 1.16.
19. Always apply analgesia via gavage or directly into the oral cavity after surgery (e.g., paracetamol (10 mg/ml) or other non-steroidal anti-inflammatory drugs).
20. Check the condition of the animals every day after surgery, and always provide mashed animal food on the floor in a petri-dish to make eating simple and to avoid critical weight loss after surgery.
    NOTE: Intravital microscopy can be performed on the first day after cranial window preparation.
21.  Apply isoflurane anesthesia and fix the animal in a head holder. Open the skin suture and clean the window with cotton buds and sterile saline. After 24 hr, the cranial window should be filled with cerebrospinal fluid by that time point, which enables imaging. Perform imaging by using established microscopy protocols¹⁸.

2. Distal MCAo

NOTE: The MCAo procedure should be performed about 5 d after CW preparation. This minimizes interference from the immune reaction caused by the CW preparation with the stroke-induced immune reaction.

Figure 1. Overview of Distal MCAo. A. This is a nice overview about the vessels before the op. B. The vessels after first bipolar contact. C. The vessels after second bipolar contact. D. The overview about the vessels, which are completely closed now. E. Final overview with lower magnification. Please click here to view a larger version of this figure.

1. Anaesthetize the mice using an anesthesia mask and an appropriate anesthetic regime, in consultation with veterinary staff (e.g., induction with 1.5 - 2% isoflurane and maintenance with 1.0 - 1.5% isoflurane in 2/3 N₂O and 1/3 O₂ via a vaporizer).
2. Shave and remove the hair and disinfect the skin and surrounding fur with an appropriate agent (e.g., 70% ethyl alcohol), and dry it afterwards.
3. Maintain the body temperature of the mice at 36.5 °C ± 0.5 °C during surgery. A feedback controlled heating pad, heated according to the rectal temperature of the mouse, is highly recommended.
4. Place the animal in the lateral position. Fix the nose in the anesthesia mask and adjust the isoflurane concentration to 1.0 - 1.5%.
5. Apply wet ointment (dexpanthenol) to both eyes.
6. Use the skin incision made for the CW preparation.
7. Gently separate the skin and identify the temporal muscle underneath.
8. Adjust the energy of the high-frequency generator (5 - 7 W) and use the bipolar mode.
9. Use the electrocoagulation forceps and carefully remove the temporal muscle from the skull, creating a muscle flap, without totally removing the muscle.
10. Identify the MCA below the transparent skull in the rostral part of the temporal area, dorsal to the retro-orbital sinus. If the MCA bifurcation cannot be identified, try to identify the vessel most rostral.
11. Thin the skull above the MCA branch with a microdrill while continuously irrigating to avoid heat damage.
12. Lift the bone with cannulas and remove it with microforceps.
13. Decrease the energy of the high-frequency generator to 3 - 5 W.
14. Approach the artery from above and gently touch it with the bipolar forceps on both sides without lifting the vessel.
15. Coagulate the artery proximally and distally to the vessel bifurcation.
16. Wait for 30 sec, and then touch the artery gently to ensure that the blood flow is permanently interrupted. Repeat the electrocoagulation if a recanalization is observed.
17. Fix the temporal muscle with 1 or 2 stitches at the muscle insertion to cover the bone defect, if possible.
18. Suture the wound and place the animal into the heated recovery box. In general, animals recovery quickly after volatile anesthesia.
19. For volume substitution, apply 0.5 ml of sterile saline subcutaneously, as described in step 1.16.
20. After surgery, allow the animals to stay in the heated recovery cage for 90 min. Wait until the animals are conscious before leaving them unattended. Only return them to a cage with other animals when they are fully recovered.
21. Repeat the volume substitution, as explained in step 1.16, after 12 hr.
22. Apply postoperative analgesia via drinking water (e.g., paracetamol (10 mg/ml) or other non-steroidal anti-inflammatory drugs).
23. Check the medical condition of the animals every day after surgery. Provide mashed animal food in a petri-dish on the floor to simplify eating and to minimize postoperative weight loss.

3. Sham Treatment

1. Perform all procedures identically to steps 1 and 2, described above-including CW preparation-except do not coagulate the exposed middle cerebral artery.

Results

The timeline and representative results are shown in Figures 2 and 3. The cranial window preparation, with a small cranial window lateral to the superior sagittal sinus (Figure 2 B, C, D) results in a very low mortality and morbidity rate when performed by an experienced surgeon. All of the 10 animals survived, and all chronic CW could be used for high-quality imaging, even 28 days after surgery. There was no problem with wound infections...

Discussion

Stroke is among the principal causes of long-term disability and death worldwide¹. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal⁷. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. In fact, models inducing transient or permanent MCAo mimic one of the most common types of focal cerebral ischemia in patients⁴. Besides proximal occlusion of the MCA, the...

Materials

Name	Company	Catalog Number	Comments
Binocular surgical microscope	Zeiss	Stemi 2000 C
Light source for microscope	Zeiss	CL 6000 LED
Heating pad with rectal probe	FST	21061-10
Stereotactic frame	Kopf	Model 930
Anaethesia system for isoflurane	Draeger
Isoflurane	Abott
Dumont forceps #5	FST	11251-10
Dumont forceps #7	FST	11271-30
Bipolar Forceps	Erbe	20195-501
Bipolar Forceps	Erbe                              20195-022
Microdrill	FST                              18000-17
Needle holder	FST	12010-14
5-0 silk suture	Feuerstein, Suprama
7-0 silk suture	Feuerstein,Suprama
8-0 silk suture	Feuerstein, Suprama
Veterinary Recovery Chamber	Peco Services	V1200