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-....

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 eye....

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

List of materials used in this article

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