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Summary

Abstract

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Published: February 22nd, 2013

DOI:

10.3791/50015

1Department of Physiology and Functional Genomics, University of Florida , 2Department of Neurosurgery, McKnight Brain Institute, University of Florida , 3Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

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. Animals: Eight-week-old, male, Sprague Dawley rats (Charles River Farms, Wilmington, MA, USA) weighing 250-300 g at the time of surgery.
  2. Anesthesia
    1. Inhalation anesthesia system (VetEquip In.......

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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 add.......

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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 .......

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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).

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Name Company Catalog Number Comments
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

  1. Levasseur, J. E., Wei, E. P., Raper, A. J., Kontos, A. A., Patterson, J. L. Detailed description of a cranial window technique for acute and chronic experiments. Stroke. 6, 308-317 (1975).
  2. Baumbach, G. L., Dobrin, P. B., Hart, M. N., Heistad, D. D. Mechanics of cerebral arterioles in hypertensive rats. Circ. Res. 62, 56-64 (1988).
  3. Regrigny, O., et al. Effects of melatonin on rat pial arteriolar diameter in vivo. Br. J. Pharmacol. 127, 1666-1670 (1999).
  4. Mecca, A. P., O'Connor, T. E., Katovich, M. J., Sumners, C. Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion. Exp. Physiol. 94, 937-946 (2009).
  5. Mecca, A. P., et al. Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke. Exp. Physiol. 96, 1084-1096 (2011).

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