Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models

Summary

A minimally invasive surgical procedure is shown here, which involves placing the laser Doppler probe onto the skull over the distal region of the middle cerebral artery (MCA), a periorbital location suitable for rats and mice, to assess blood flow during transient MCA occlusion.

Abstract

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion is not guaranteed by even the most skilled surgical hands. Errors primarily occur when the filament is not placed at the correct depth and include instances of either no infarction or vessel perforation, which can cause death. Laser Doppler flowmetry (LDF) is a reliable technique that provides real-time feedback on regional cerebral blood flow (CBF) during the MCAO procedure. Here we demonstrate a rapid technique for periorbital placement of a laser Doppler probe for measurement of CBF in both mice and rats. Our rationale was to simplify LDF implementation, encouraging widespread usage for improved surgical reliability. The technique eliminates the need for skull thinning and specialized equipment, with placement at the periorbital region rather than dorsal placement, promoting efficiency and ease of adoption. The protocol described here encompasses presurgical preparations, periorbital Doppler probe placement, and post-operative care. Representative results include visual depictions of procedural elements along with representative LDF tracings illustrating successful MCAO surgeries, with instances of unsuccessful filament placement leading to complications. The protocol illustrates LDF in confirming proper filament placement and offers a simplified procedure compared to alternative methods.

Introduction

The middle cerebral artery occlusion (MCAO) method has been widely used in rodents since introduced to the scientific community for application to rats in 1986¹ with the Longa adaption described in 1990², and adaptations for mice soon following³. Although not described in Longa's publication, the use of laser Doppler flowmetry (LDF) signal to confirm the filament placement was soon described in the literature⁴. LDF employment during the MCAO procedure is prominently featured in the literature but is designated as an optional step in the current Stroke Preclinical Assessment Networks (SPAN) Standard Operative Procedures (SOPs)⁵.

The use of LDF confirms correct filament placement during the MCAO procedure and, therefore, contributes to study design rigor and subsequent results, particularly in experiments designed to explore drug efficacy. LDF use reduces surgical mistakes stemming from incorrect filament placement, which results in the dichotomous situation of either no injury when the filament is not placed far enough or animal deaths due to vessel perforation occurring when the filament is inserted too far. On the other hand, the use of LDF is not associated with infarct size variability commonly observed following the MCAO procedure⁶. The use of LDF in the MCAO procedure may be perceived as difficult and onerous, particularly in rats, because the skull is thicker than in mice and may require skull thinning prior to LDF placement^7,8. Also, dorsal probe placement is often described with some protocols requiring specialized equipment or preparation^7,8,9. With any of these barriers, implementation of the LDF to confirm filament placement may not take place.

In this protocol, we describe the placement of the laser Doppler probe at the skull and over the distal region of the middle cerebral artery-a periorbital placement-for the assessment of blood flow during the MCAO procedure in both mice and rats. Our rationale was to develop a procedure that has multiple advantages over some methods reported in the literature^7,8,9,10 in that it is minimally invasive, fast, and does not require skull thinning or specialized equipment beyond the laser Doppler probe.

Protocol

Adult mice and rats were used to illustrate this protocol (25 g, C57BL/6J, Jackson Laboratories; 250 g, Sprague Dawley, Envigo). Animal handling and experimental procedures were performed with approval and in compliance with the University of Arizona Institutional Animal Care and Use Committee, national laws, and according to the principles of laboratory animal care¹¹. Rats and mice were housed with a 12 h light/dark schedule (7 am-7 pm) with food and water available ad libitum.

1. Presurgical preparation

NOTE: Typically, MCAO surgery in a rodent is performed as a survival surgery, necessitating the use of either an aseptic or tips-only surgical technique, as outlined in an institution-specific IACUC protocol. In this case, the surgeon uses a tips-only technique with sterilized instruments and supplies.

1. Disinfect the surgical work surface with commercial disinfectant and prepare sterile surgical packs of instruments, drapes, gauze, swabs, sutures, and scalpel blades by autoclaving. Maintain sterile conditions during survival surgery according to IACUC-approved techniques, such as covering microscope knobs and draping using food-grade plastic wrap.
2. Use a germinator dry bead sterilizer to re-sterilize surgical instruments between procedures if multiple rodent surgeries are carried out during one session.
3. Place the rat or mouse into an induction chamber and induce anesthesia with 3%-5% isoflurane (1 L/min using combined 80% air and 20% oxygen mixture).
4. Move the animal from the chamber to the bench and nosecone for anesthesia (mouse: 0.5 L/min 80%/20% oxygen/air mixture; rat: 1.5 L/min 80%/20% oxygen/air mixture). Apply ophthalmic ointment to both eyes and carefully shave the space between the ear and eye socket on the side of the MCAO surgery (commonly the right), so as not to cut any skin using clippers. In the rat, when shaved, the whisker dimple will be visible approximately midway between the eye socket and the ear. Continue to shave regions as needed for the MCAO procedure⁹ (not in the scope of this paper).
5. Return the animal to the chamber as needed or move it to a nosecone on the prepared surgical table warmed to 37 °C. Insert a rectal thermometer. The animal should be kept at approximately 36 °C ± 1 °C throughout the procedure and according to the approved IACUC protocol.
6. Apply betadine to a cotton swab and disinfect the skin, spiraling outward from the center of the surgical region. Wipe with an alcohol prep pad. Repeat both steps for a total of three cycles. Confirm proper anesthetization and assess every 15 min during the procedure using the toe pinch reflux.

2. Periorbital Doppler probe placement

1. With small scissors, make a vertical cut (mouse: ~0.5 cm; rat: ~0.7 cm) between the corner of the eye and the fold of the ear, as shown in Figure 2A (mouse) and Figure 3A (rat) to expose super facia and underlying muscle.
   NOTE: Treat for pain preoperatively according to the IACUC protocol approval.
2. Using sharp forceps and curved scissors, carefully cut through the muscle layer until the skull is reached. The skull will be flat and hard to touch with the tip of the forceps. This is the window in which the laser Doppler probe will sit, as visualized in Figure 2B.
3. Use a forceps cautery tool to clot emerging blood, widen the window, and ensure the route to the skull.
4. Once the window is open, blot with cotton swabs to remove excess blood as necessary.
   Place the mouse or rat in the supine position.
5. Use forceps to retract any skin or tissue and place the Doppler probe into the periorbital window so that it abuts the skull perpendicular with no skin or muscle present to impede the laser signal. This region is over the distal MCA as illustrated in Figure 1A,B. Secure the probe placement with laboratory tape. In this protocol, the use of a straight probe is described.
   NOTE: We acknowledge that surgery protocols will vary among laboratories. In the literature (when reported), some grind the skull thin, and others do not⁹. It may be because the skull is thinner in the periorbital region, but this is just speculation. In our experience, direct placement without grinding can be achieved in mice and rats and is demonstrated in this protocol.
6. Monitor the relative blood flow readings on the monitor with appropriate software (Figure 2D, Figure 3C,D). LDF readings will vary. However, the optimal range for detection of blood flow drops is >200 perfusion units (PU).
7. Proceed to the MCAO surgery⁹ (not in the scope of this paper). During the MCAO surgery, test probe placement by briefly tying the common carotid artery, which should cause a sudden drop in blood flow (~50%).

3. Doppler probe removal and post-operative care

1. At the conclusion of the MCAO or whenever blood flow readings are completed, remove the laser Doppler probe from the surgical window. The Doppler probe typically remains in place for the duration of the mouse MCAO surgery because ischemia times are often less than 90 min. On the other hand, in the rat MCAO procedure, the Doppler probe may be removed after the filament is placed and during ischemia (typically > 90 min) and then placed again in the periorbital window to confirm reperfusion by repeating steps 2.6 and 2.7.
   NOTE: A typical LDF continuous tracing from the mouse MCAO may be observed in Figure 4A. Typical LDF tracings from a rat MCAO procedure are illustrated in Figure 4B.
2. Inject IACUC-approved local anesthetic subcutaneously along the prospective incision site.
3. Close the skin with 1-2 sutures using a standard surgical knot with absorbable suture thread or surgical glue, according to approved IACUC protocol. Treat for post-surgical pain according to IACUC protocol approval (e.g. bupivacaine HCL 0.5% & lidocaine 2%, once intra-incision for mice and rats). Animals should not be left unattended until they are able to maintain sternal recumbency, and no animals are returned to the company of other animals until they are fully recovered.

Results

The placement of the laser Doppler probe at the MCA region is visually depicted in Figure 1, offering a pictograph of vasculature and serving as a visual guide from sagittal and dorsal perspectives. Figure 2 summarizes the critical steps for laser Doppler probe placement and outcomes in the mouse. Figure 2A presents an image of an anesthetized and prepared mouse with a dashed marking at the site of the vertical incision necessary for subsequent laser Doppler probe placement. The periorbital window to the skull is illustrated in Figure 2B.

Figure 3 summarizes the laser Doppler probe placement in the rat. An image of an anesthetized and prepared rat with a dashed marking at the site of the vertical incision necessary for subsequent laser Doppler probe placement is shown in Figure 3A. The periorbital window cannot be viewed because it is deeper than the mouse. In this case, the skull can be felt, as mentioned in step 2.3. The laser Doppler probe placed at the periorbital region in the supine rat is shown in Figure 3B.

A typical LDF tracing during the mouse MCAO procedure is shown in Figure 4A. It illustrates a successful induction of ischemia as evidenced by a distinct and immediate drop in relative cerebral blood flow (CBF) when the carotid artery is tied and again when the filament is advanced to the ostium of the MCA. The initiation of reperfusion is shown at the end of the tracing, evidenced by a distinct and immediate increase in relative CBF when the carotid artery is untied and again when the filament is withdrawn from the MCA. A typical LDF reading of ischemia during the rat MCAO surgery is shown in Figure 4B, which shows a successful induction of ischemia followed by probe removal and re-placement for LDF measurement during reperfusion. In rat surgery, ischemia times may be greater than 60 min, and animals may recover during the ischemia period and should be re-anesthetized for reperfusion. In this case, the probe is re-positioned in the periorbital window, and LDF tracings are continued. Ischemia is evidenced in the tracing by a distinct and immediate drop in relative CBF when the carotid artery is tied and the filament advanced to the MCA ostium. Reperfusion is evidenced in the second LDF tracing by a distinct and immediate increase in relative CBF, after which the carotid artery is untied and, again, when the filament is withdrawn from the ostium of the MCA. The periorbital Doppler is positioned over the distal branch of the MCA and, therefore, within the scope of blood distribution of the MCA with an observed decrease in CBF as evidenced by the reduction in blood flow during the MCAO procedure, illustrated in Figure 4.

We show LDF tracings that exemplify failed MCAO surgeries in Figure 5. Figure 5A summarizes an LDF tracing from a rat surgery with a successful carotid occlusion that later became loosened and an unsuccessful filament placement, marked by a slow drop in relative CBF. This LDF pattern is typically associated with a perforated MCA that can be confirmed with necropsy. Another tracing from a mouse MCAO surgery (Figure 5B) illuminates inconclusive carotid artery occlusion that likely contributed to an inability to detect the filament placement at the MCA ostia. Perforation of the MCA was suspected because when the filament was slightly withdrawn, relative CBF exhibited sluggish recovery. These examples highlight the critical role of LDF tracings in identifying successful versus unsuccessful MCAO surgeries and underline the importance of meticulous surgical procedures and measures for reliable experimental outcomes.

Figure 1: Pictograph of approximate laser Doppler probe placement in relation to MCA. Schematic of the laser Doppler probe placement from a (A) sagittal and (B) dorsal view. Created with Biorender.com; KT26JWLYF6. Please click here to view a larger version of this figure.

Figure 2: Illustration of surgery steps for laser Doppler probe placement in the mouse. (A) Image of the location of the vertical incision necessary for laser Doppler probe placement with the mouse in the supine position. (B) Image of the periorbital window and skull prepared by forceps and/or scissors in the mouse. Please click here to view a larger version of this figure.

Figure 3: Illustration of surgery steps for laser Doppler probe placement in the rat. (A) Image of the location of the vertical incision necessary for laser Doppler probe placement with the rat in the supine position. (B) Laser Doppler probe placement at the periorbital region in the supine rat. Please click here to view a larger version of this figure.

Figure 4: Examples of LDF tracings confirming successful MCAO procedure in the mouse and rat. (A) LDF reading of the MCA region during the mouse MCAO procedure. This tracing illustrates confirmation of 1. carotid artery occlusion with tie; 2. filament placement at the MCA; 3. filament removal after 60 min of continuous ischemia; and 4. reperfusion when the carotid artery is untied. Elements a. and b. illustrate what the Doppler tracing looks like when the carotid is untied and tied again during the reading. This technique can be used to confirm the probe placement. (B) LDF readings of the MCA region during the rat MCAO procedure. Like the mouse, this tracing illustrates confirmation of 1. carotid artery occlusion with tie and 2. filament placement at the MCA. In the rat MCAO procedure, the Doppler probe is often removed, and the animal spends the full ischemia time awake and moving. For reperfusion, the LDF is re-established prior to reperfusion: 3. confirms filament removal and 4. confirms that the carotid is untied to complete reperfusion. In both mouse and rat MCAO procedures, successful probe placement is used to visualize correct filament placement and is indicated when the tracing shows two distinct and sudden decreases in LDF (for ischemia) followed by two distinct and sudden increases in LDF (reperfusion). Please click here to view a larger version of this figure.

Figure 5: Examples of laser Doppler flowmetry tracings that indicate unsuccessful MCAO procedures in the mouse and rat. (A) An example of an LDF tracing that indicates an inconclusive filament placement in the rat. While the carotid was tied (1.), became loose (2.), and then tied again (3.), there is an indistinct and slow drop, rather than a sudden drop in LDF when the filament was at the MCA Ostia. This tracing may indicate a perforated MCA, and the animal may not survive the first 24 h of reperfusion; filament perforation can be confirmed with necropsy. (B) An example of an LDF tracing that indicates an inconclusive filament placement in the mouse. In this tracing, evidence of carotid tie is inconclusive (1.), followed by an indistinct drop in LDF with filament placement at the MCA ostia (2.). In addition, when the filament was slightly withdrawn, the relative cerebral blood flow increased sluggishly (3.). Please click here to view a larger version of this figure.

Discussion

The MCAO is the gold-standard procedure for modeling cerebral artery occlusion and reperfusion in rodents and has been the cornerstone of preclinical stroke research, enabling the induction of focal ischemia in rodents to mimic human stroke pathophysiology. It is an exacting surgical procedure with significant inter- and even intra-surgeon variability. While there is no evidence that the application of LDF reduces variability, it may improve scientific rigor and study outcomes in some designs. This is accomplished because LDF validates filament placement and provides real-time information on CBF during surgery, confirming whether occlusion of the MCA has occurred. Approximately 75% of mouse studies describe using a laser Doppler probe^12,13, whereas this was not a reported parameter in a meta-analysis of rat studies¹⁴. An often-used alternative to LDF is to measure the filament insertion (e.g., 18-20 mm from the common carotid artery bifurcation in rats) using marked filaments or to measure the remaining filament protruding from the insertion point (e.g., 10 mm)¹⁰. However, this alternative is inferior to LDF because the exact depth of filament insertion may vary slightly depending on factors such as the size and weight of the rat, as well as the specific protocol being used in the experiment. Such differences are essential considerations for the reproducibility of the MCAO surgery, given the risk of vessel perforation or incomplete occlusion-both negative surgical outcomes.

Although LDF is a method to validate filament placement and is supported by SPAN, it is not a required step in the published SPAN-SOP⁵. The described protocol overcomes barriers such as the use of special equipment and difficult probe placement on dorsal sections of the skull when the main procedure is carried out on a supine-positioned animal; these factors often extend surgical times. In contrast, this protocol uses a single, straight laser Doppler probe and monitor, periorbital placement location, and no skull thinning. When combined, these steps result in a greatly simplified and shortened (often less than 5 min) procedure-a significant improvement from similar but alternative methods described in the literature^7,8,9,10. This protocol may also be adapted by surgeons as required. Troubleshooting of the described techniques may necessitate and include probe repositioning, especially if a decrease in blood flow is not observed in step 2.8.

The use of LDF is limited to confirming filament placement and has not been associated with infarct size variability, which can be >20% in mouse and rat methods^12,14. Infarct variability has been a long-standing problem in preclinical stroke research. Whereas this increased variability may mimic the diversity of infarct sizes observed in the human condition¹³, when not properly accounted for, observed variability may result in studies that are not properly powered. While there is not a single factor predicting method variability, confirming placement is an important factor for drug efficacy studies. The protocol described here, while possibly already employed in mouse MCAO procedures, may be underutilized in rats, given the more frequent descriptions of dorsal placement in the literature⁹. We believe that this description of a simplified laser Doppler probe placement contributes to refining preclinical stroke research methodologies, aiming for increased experimental precision and translational relevance.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
curved spring scissors	Castroviejo	1501710
forceps #5	Fine science tools	11250-20
forceps #5/45	Fine science tools	1151-35
Forcepts Cautery tool	Conmed	M18019-01
Laboratory tape	Fisherbrand Labeling Tape	15-950
Laser Doppler Monitor	Moore Instruments	MOORVMS-LDF
LDF software	Perisoft for Windows or moorSOFT	NA
Mouse clippers	Philips Norelco	MG7910
Periflux System 4000, probe 407	Perimed	equipment no longer available
plastic wrap	Glad	press n seal
Rat clippers	oster	A5 or similar
Small rodent anesthesia	JD Medical	custom order
small scissors	excelta	362 Sissors or similar
Temperature monitor system with probe	Physitemp	TCAT-2AC Controller