This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.
Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.
This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida and is in compliance with the “Guide for the Care and Use of Laboratory Animals” (eighth edition, National Academy of Sciences, 2011).
Materials
1. Pre-surgical Steps
2. Pre-cranial Window Preparation
Prior to creating a cranial window, the rat should be prepared for any experiments by implantation of other required hardware and should receive any required surgical procedures. For this protocol, we have previously implanted a guide cannula for endothelin-1 (ET-1) induced MCAO as shown in a companion publication titled “Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat.”
3. Creation of the Cranial Window
After placement of a guide cannula or equipment required for experimentation, a cranial window is created to directly visualize proximal portions of the middle cerebral artery during a stroke procedure.
4. Recording of Cerebral Artery Constriction
To demonstrate how to capture images in real time, a rat that is undergoing ET-1 induced MCAO is used for this protocol.
5. Image Analysis
Vessel diameter can be determined for any part of the visualized MCA. As an example, we will use one branch of the MCA to measure vessel diameter at time-points before and after ET-1 injection. Still frames from the video are captured at 1 min intervals using VLC media player (VideoLAN).
Still images taken from the captured video show that the change in cerebral artery diameter after ET-1 injection can be readily appreciated using this cranial window technique (Figure 2). Within minutes of ET-1 injection, the vessel will begin to constrict. Eventually the vessels will be difficult to visualize and the brain tissue will become pale. After about 20 min the effects of ET-1 will diminish and the vessels will begin to dilate, gradually returning to baseline diameter after about 45 min. In addition to the more obvious vasoconstriction that occurs, the cortical surface becomes paler after ET-1 administration. It is possible to calculate the absolute change in vessel diameter with a calibrated microscope reticle if desired. For comparison between multiple rats we calculate the relative change in vessel diameter that occurs during a procedure. These measurements are performed using ImageJ software (NIH). Then a graph representing the relative changes in vessel diameter over time can then be constructed (Figure 3).
Figure 1. Diagram of the location of temporal craniectomy. This diagram depicts the skeletal anatomy of the rat skull with anterior oriented to the left. The temporalis muscle has its origin along the lateral skull ridge. This muscle must be detached from this ridge and bisected in order to visualize the squamous portion of the temporal bone. An approximately 3-4 mm craniectomy can be performed at this location just posterior to the orbit and superior to the base of the zygomatic process as it reflects off of the temporal bone. The large arrow indicated the location to perform the craniectomy. The 3 small arrows indicate the MCA and its branches. All arteries in this location will be branches of the MCA and arteries can be distinguished from veins by both their non-tortuous appearance and sensitivity to vasoactive compounds.
Figure 2. Cranial window before ET-1 injection, after ET-1 injection, and after reperfusion. Starting at the left, a representative image of MCA branches as viewed through a cranial window is shown. Arteries can be identified by their morphology. The relatively straight MCA enters the field at the lower left and has one major branch point in this image. Other vessels in these pictures are cerebral veins which can be identified by their deeper tone and tortuous appearance. During occlusion arteries will rapidly constrict and the tissue will become pale. Slowly, the artery will dilate and return to baseline diameter.
Figure 3. Representative vessel diameter over time for a single rat. Percent baseline diameter can be calculated over time using the simple formula, current diameter/baseline diameter x 100%. This can be done with any vasoactive compound.
In summary, this cranial window preparation technique is very versatile as it can be altered to meet the needs of many experiments with minor modifications4, 5. For example, we have successfully monitored cerebral blood flow in specific MCA branches using laser doppler flowmetry to focus directly on a cerebral artery visualized through a cranial window (Mecca AP 2009 and 2011). In addition, a similar preparation with the dura incised can be used with topical administration of vasoactive compounds to create an in vivo vascular reactivity bath3. Several factors should be taken into consideration when preparing a cranial window in order to decrease the failure rate for this technique. Many of these factors are related to obtaining good visualization of cerebral arteries. First, care must be taken when creating the craniectomy so that the dura or blood vessels overlying it are not disrupted with the drill bit. This is best accomplished by frequent washes with sterile saline to clear debris and cool the skull. Second, the bone fragment should be lifted gently when it is removed. If the fragment does not pull away easily, then the drill bit should be used to cut away more bone. Lastly, small amounts of blood or CSF can easily alter the appearance of the cranial window during this procedure. The craniectomy performed provides an opening in the skull that is larger than required for visualization. Therefore, it is easy to place several absorbent sponges in the dependent portion of the surgical site to prevent fluid from accumulating. These sponges can be changed as needed if care is used not to obstruct the window with surgical tools.
The authors have nothing to disclose.
This work was supported by grants from the American Heart Association Greater Southeast Affiliate (09GRNT2060421), the American Medical Association, and from the University of Florida Clinical and Translational Science Institute. Adam Mecca is a NIH/NINDS, NRSA predoctoral fellow (F30 NS-060335). Robert Regenhardt received predoctoral fellowship support from the University of Florida Multidisciplinary Training Program in Hypertension (T32 HL-083810).
Name of the reagent | Company | Catalogue number | Comments (optional) |
Inhalation anesthesia system | VetEquip Inc., Pleasanton, CA, USA | 901806 | |
Isoflurane anesthetic | Baxter Pharmaceutics, Deerfield, IL, USA | 1001936060 | |
Small animal stereotaxic system | David Kopf Instruments, Tujunga, CA, USA | 900 | |
Non-rupture ear bars, rat | David Kopf Instruments, Tujunga, CA, USA | 957 | |
Rat gas anesthesia head holder | David Kopf Instruments, Tujunga, CA, USA | 1929 | |
BAT-12 microprobe thermometer | World Precision Instruments, Inc., Sarasota, FL, USA | BAT-12 | |
T/PUMP, Thermal blanket | Gaymar Industries, Inc., Orchard Park, NY, USA | T/PUMP, TP600 | |
Metzenbaum Scissors | World Precision Instruments, Inc., Sarasota, FL, USA | 501254 | |
Iris forceps | World Precision Instruments, Inc., Sarasota, FL, USA | 15915 | |
Bulldog clamp retractors | World Precision Instruments, Inc., Sarasota, FL, USA | 14119-G | |
10 μl syringe 26-gaugue | World Precision Instruments, Inc., Sarasota, FL, USA | SGE010RNS | |
Bovie, high temperature cautery kit | World Precision Instruments, Inc., Sarasota, FL, USA | 500392 | |
Rat tooth forceps 0.12 | Stotz | E1811 | |
Micromotor drill | Stoelting, Wood Dale, IL, USA | 51449 | |
0.8 mm round drill bur | Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA | RS-6280C-1 | |
STORZ Bonn suturing forceps | Bausch and Lomb, Inc., Rochester, NY, USA | ||
Nylon Suture, size 3.0 | Oasis, Mettawa, IL, USA | MV-663 | |
Cotton swabs | Fisher Scientific, Pittsburg, PA, USA | 22-029-488 | |
Puralube eye ointment | Fisher Scientific, Pittsburg, PA, USA | NC0138063 | |
Electric hair clippers | Oster, Providence, RI, USA | 78005-301 | |
ET-1 diluted to 80 μM concentration in PBS | American Peptide, Sunnyvale, CA, USA | 88-1-10A | |
Chlorhexidine, 2% | Agrilabs, St. Joseph, MO, USA | 1040, Rev. 6-06, NAC No.: 10580322 | |
Surgical microscope | Seiler Instrument and Manufacturing, St. Louis, MO, USA | Evolution xR6 | |
Sony Handycam | Sony, Minato, Tokyo, Japan | HDR-SR12 | |
Fiber optic illuminator | TechniQuip Corp., Livermore, CA, USA | FO1–150 | |
VLC media Player | (Paris, France) | ||
Image J software | U.S. National Institutes of Health, Bethesda, MA, USA |