Response of rabbit ear microvasculature to total body cooling as observed through a new implantable window

MICROVASCULAR

RESEARCH

43, 227-234

(1992)

BRIEF COMMUNICATIONS Response

of Rabbit Ear Microvasculature Cooling as Observed through New Implantable Window

F. EDWARD POLLOCK, JR.,*‘? THOMAS L. SMITH,*

to Total Body a

AND L. ANDREW

KoMANt

*Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake University, Medical Center Boulevard, Winston-Salem, North Carolina 27157; and *Department of Orthopaedic Surgery, Bowman Gray School of Medicine, Wake Forest University, Medical Center Boulevard, Winston-Salem, North Carolina 27157 Received

May

Forest

9, 1991

A new implantable window was developed in order to study the native microcirculation of the rabbit ear. The responses of the ear microvasculature to total body cooling were studied before and after implantation of the window. No differences were found between arteriolar and venular vasoconstriction with cooling before and after implantation of the window. Arteriolar constriction was significantly greater than the venular response both before and after placement of the window (P = 0.014). Surface skin temperature of the instrumented and control ears was similar at room temperature (35.2 t 2.6 versus 36.1 * l.o”, respectively; P = 0.280); however, the instrumented ear was slightly warmer during cooling (17.9 ? 1.9 versus 15.2 2 3.1”; P = 0.024), suggesting increased blood flow in the instrumented ear. Details of construction and implantation of the window are described. Q 1992 Academic

Press, Inc.

INTRODUCTION Clark and colleagues described a “preformed tissue” chamber which was developed to facilitate the observation of native vascular beds with an intact nerve supply (Clark et al., 1930). This concept was modified in the current study to examine the microvasculature present in the rabbit ear. A window was designed which is easily constructed and inserted and which allows acute or on-going observations of preexisting vessels to be made. This report details the construction of the window and the surgical procedure for implantation. Finally, a series of rabbits was studied to determine whether window placement in the ear altered normal responses of the vasculature as elicited by exposure to cold thermal stress. MATERIALS

AND METHODS

Chamber Construction and Implantation

The components of the window are easily and inexpensively constructed (Fig. 1). The viewing area of the device is made from a 13-mm Thermonox coverslip 227 0026-2X62/92 $3.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in U.S.A

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Ventral aspect Silastic tubing Polycarbonate cover slip 5-O stainless steel surgical wire

Dorsal aspect 13 FIG. 1. Materials required to implant window

(Miles Scientific; Naperville, IL) into which eight equally spaced anchoring holes have been drilled using a %2 in. drill attached to a hand chuck. A second identical coverslip is prepared for use on the dorsal surface of the ear to discourage edema formation following the implantation surgery; this is accomplished by prevention of swelling and impairment of lymph flow. A H-mm segment of silastic tubing (Dow Corning; Midland, MI) with an i.d. of 0.25 mm is needed to prevent pressure necrosis of the skin flap overlying the window by the stainless wires holding the two coverslips in place. The silastic tubing is cut into 3-mm lengths which are placed over the wire during implantation. Finally, four lengths of 27-guage wire are cut which are used to secure the window in place. All components may be packaged together and gas sterilized using ethylene oxide for subsequent implantation. Surgical Procedure

The rabbit is anesthetized using an intramuscular injection of ketamine (20 mg/kg) and xylazine (8 mg/kg). The animal is also given a subcutaneous injection of a suspension of trimethoprim (40 mg/ml) and sulfadiazine (200 mg/ml) for antiobiotic prophylaxis. When surgical anesthesia is achieved, the dorsal and ventral surfaces of the ear are shaved and disinfected. The ventral surface of the ear is then rinsed with isopropyl alcohol, secured to a small operating platform, and draped as a sterile field. Sterile technique is used throughout the implantation procedure. Using a dissecting microscope, a circle of ventral skin (7-mm diameter) is scored using a sharp skin punch: the skin and underlying loose perichondral tissue are removed and the auricular cartilage is exposed. Minimal bleeding occurs with this procedure; bleeding is controlled, if necessary, with light pressure or topical application of thrombin (Parke-Davis; Morris Plains, NJ). The cartilage within the field is then removed. A thin fibrous layer is interposed between cartilage and the underlying vasculature. This layer protects vessels and sensory and sympathetic nerve fibers supplying the ear; except for nerves and vessels which penetrate to the ventral surface of the ear, this layer should remain intact. Three equally spaced chondrocutaneous flaps are developed which radiate from

BRIEF

IIII1

Stainless surgical

steel wire

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COMMUNICATIONS

i

--)

-

Y

Silastic

tubing

.Ii

Dorsal

skin

-,

1; ./II 1

Polycarbonate

Ventral

slip

skin

Perichondral ”

cover

connective

Auricular

cartilage

Vascular

bed

tissue

FIG. 2. Cross-sectional drawing of window in place in the rabbit ear.

the exposed vasculature approximately 10 mm. The skin and cartilage of the flap are raised as a unit to preserve the ventral blood supply of the tissue. Vessels and nerves beneath the flaps are protected by the fibrous layer as the cartilage is elevated. A coverslip is then rinsed with normal saline and carefully placed over the fibrous layer and vascular bed. The anchoring holes are positioned over avascular areas to avoid hemorrhage when the window is secured. Two to 3 mm of ventral skin and cartilage are trimmed from the original opening, allowing the flaps to be reopposed with the skin edges covering all of the anchoring holes; the ventral skin opening then measures approximately 10 mm. The radial incisions used to create the flaps are repaired with 5-O Vicryl (Ethicon; Sommerville, NJ). The window is then secured permanently. The ventral surface of the ear is transilluminated so that the position of the anchoring holes can be ascertained. A 25gauge needle is inserted through the ventral chondrocutaneous flap, the anchoring hole of the window, and the dorsal skin to act as a cannula through which a segment of wire is threaded. The needle is then withdrawn, leaving the wire protruding through both dorsal and ventral surfaces of the ear. A 3-mm segment of silastic tubing is cut and threaded over the wire projecting from the ventral surface of the ear. The ventral portion of the wire is then passed through a cannula placed in the flap, an adjacent anchoring hole, and the dorsal skin. The cannula is withdrawn leaving both ends of the wire protruding from the dorsal surface of the ear. This procedure is repeated three more times so that all eight of the anchoring holes are filled by four lengths of wire. The wires are then inserted through the anchoring holes of a second coverslip so that this coverslip is resting against the dorsal surface of the ear. Wires are twisted together holding the coverslip securely against the dorsal skin. The pressure exerted by the wires should be adjusted so that the circulation beneath the window is not compromised; blood flow is checked periodically throughout this step of the procedure. The implantation procedure is now complete. A cross-sectional drawing of the window in place in the ear is shown in Fig. 2. Windows have been successfully implanted in 28 rabbits; 6 rabbits were not available for chronic study because of perisurgical complications (hemorrhage, broken wires, and infection). The window is well tolerated by the animal and may be left in place for several months during which observations can be made.

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Following completion of the surgery, blood flow, vasomotion, and other hemodynamic events may be observed at once. However, quantitative observations should be delayed for 1 week to allow the acute inflammatory response which occurs following surgery to subside. Experimental

Protocol

Eight New Zealand White male rabbits weighing between 3.5 and 4.0 kg were used to examine the effects of total body cold stress on the thermoregulatory vessels of the ear. The protocol was used for each rabbit and was approved by the Animal Care and Use Committee of Bowman Gray School of Medicine. On Day 0, each rabbit was transported to an animal surgery laboratory and anesthetized using intramuscular injections of ketamine (30 mg/kg) and xylazine (8 mg/kg). Both ears were then shaved and coated with a commercial depilatory (Nair; Carter Products; New York, NY). Once the hair was removed, the ears were observed under a dissecting microscope (Wild M650; Heerbruug, Switzerland) using a cold fiber-optic transilluminating light source. Vessel beds were marked for later observation using a permanent ink marker. The rabbit was then allowed to recover fully without further intervention. On Day 1, the rabbit was again brought to the laboratory and placed in a restraining box; each animal had been conditioned to the box prior to the beginning of the experiment by repetitive restraints. The temperature of the room was maintained between 22 and 23”. Reusable thermistor probes (YSI Model 427; Yellow Springs Instrument Co.; Yellow Springs, OH) were attached to the dorsal surface of each ear with tape; each probe was placed 5 cm from the tip over the central neurovascular pedicle. A rectal probe was also placed to measure core temperature. A probe was also used to monitor ambient room temperature. All probes were connected to a multichannel analog monitor (YSI Model 46; Yellow Springs Instrument Co.); the display for each channel was read directly from the monitor during the experiment and recorded on data sheets. One ear was placed in a restraint which was specifically constructed to hold the ear in a vertical orientation. Mineral oil was applied to the dorsal surface, and a large glass slide was used to stabilize the microvasculature. Small plastic blocks equal to the thickness of the ear (3 mm) had been applied to the edge of the ear restraint to ensure that no pressure was applied to the ear surface by the glass slide. The previously marked vessel bed was transilluminated using a fiber-optic light source and then photographed through a dissecting microscope (Wild M650; Heerbruug) using a 35-mm camera with extension rings (Nikon F3; Tokyo, Japan). The same procedure was performed on the opposite ear. The restrained animal, monitoring equipment, microscope, and camera were then transported to a refrigerated room (average temperature 6”) and allowed to remain undisturbed for 30 min. Each ear was photographed at a uniform magnification, and the animal and equipment were returned to the lab for a 1-hr recovery period. No anesthesia was used during these procedures, nor did the rabbit exhibit signs of discomfort. The rabbit was next anesthetized, and a window was placed in a randomly selected ear using the previously described surgical procedure. After the vessels were checked to verify normal blood flow, the animal was allowed to recover without further manipulation.

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Unlimited access to food and water was available for the next 7 days. The window and surrounding skin were observed daily for signs of hemorrhage, loosening of the components, or infection. On Day 8, the rabbit was returned to the laboratory to undergo monitoring and photographic sessions of each ear as previously described. Enlargements (11 x 14 in.) were made of the photographs of all ears producing a final magnification of x 225. Vessels in the enlargement were systematically mapped so that diameters could be measured at each branch point. Vessels were also divided into arterioles and venules based on characteristic appearance and geography. Diameters to be measured were labeled and numbered so that repeated measures could be performed on the same vessel in both warm and cold environments. The enlargements were randomized so that blinded measurements could be made. Measurements were made on a precalibrated digital analysis pad and a custom software program. Diameters were measured in micrometers and were individually recorded on data sheets for later analysis. In order to compare the response of a wide range of vessel sizes, the reactivity of a vessel was calculated by dividing the diameter obtained after cooling by the warm diameter and multiplying the result by 100. This value was calculated for both arterioles and venules. Temperature and diameter measurements were compared prior to and after window placement. Statistical analysis was performed by means of Student’s paired and unpaired t tests; significance was determined at P < 0.05. RESULTS Prior to window placement, average core temperature was 39.3” in the warm environment and 38.9” in the cold. Since this difference was not significantly different, the data were pooled and compared to core temperature following window placement. Average core temperature in warm and cold environments after window placement was 39.5” and 38.9”, respectively; again, no significant difference was detected. Surface ear temperature with an ambient temperature of 23” showed no difference between ears either before or after surgery. Ears at all data points showed highly significant decreases in temperature when rabbits were placed in the cold room. However, the average temperature of the instrumented ear during cold stress was significantly warmer than either the average ear temperature before surgery or the temperature of the uninstrumented ear under the same conditions. Arteriolar reactivity was no different between right and left ears prior to surgery (JJ = 0.257). Th ese results were therefore pooled and compared to those for instrumented and uninstrumented ears. No significant differences were noted between the three data points. Venular reactivity showed a similar pattern of response. However, venular constriction during cold was less than arteriolar constriction at all three time points; this difference reached statistical significance in ears before surgery and in ears containing the window (Table 1). DISCUSSION The microvasculature of the rabbit ear has been used extensively as a model for the physiologic response of vessels in the human digit (Grant and Bland, 1929-

232

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COMMUNICATIONS TABLE

1

ARTER~OLAR AND VENULAR REACTIVITY TO COOLING BEFORE SURGERY AND IN INSTRUMENTED AND UINSTRUMENTED EARS AFTER WINDOW IMPLANTATION (PERCENTAGE OF CONTROL 2 STANDARD DEVIATION)

Arterioles Venules

Ears before surgery

Uninstrumented ears

58.9 f 18.1 76.5 k 15X*

68.4 2 22.6 76.2 _’ 32.0

Instrumented ears 60.4 k 24.5 83.5 + 13.5*

* P < 0.05 when venules are compared to arterioles for each ear.

1931; Harada, 1971; Kluger et al., 1971; Mohler and Heath, 1981). The two structures have vessels of similar size and architecture (Stirrat and Seaber, 1979; Tsai, 1975). Both organs have very minimal metabolic requirements, thereby allowing them to regulate heat loss through changes in blood flow (Burton, 1939; Gonzalez et al., 1971). Finally, each serves as a sensor and effector of thermoregulation (Gordon and Heath, 1983; Kluger et al., 1971). The impetus for this project was the development of a device which could be implanted over the native vessels of the rabbit ear to study microcirculatory anatomy, function, and response to manipulation. Rabbit ear chambers have been successfully employed for the last 70 years to study blood vessel morphology and function. Nims and Irwin have reviewed the different types of chambers which have been described, noting that most devices require ingrowth of neovasculature (Nims and Irwin, 1973). The angiogenesis which occurs within the chambers is largely composed of venous channels which have developed in an environment of granulation tissue (Zawicki et al., 1981). In order to study preformed vessels, Clark and colleagues described a chamber consisting of multiple components fixed in place with small bolts and nuts; the authors note that the chamber is useful for studies of blood flow, vessel wall structure, and arteriolar contraction (Clark et al., 1930). Experience with this device is limited (Branemark and Lindstrom, 1963). However, similar devices have been used to study the function and morphology of native vascular beds in other animal models with good success (Greenblatt and Shubik, 1967; Smith et al., 1985; Zarem and Dimitrievich, 1970; Zweifach and Lipowsky, 1977). The components of the window described in this study are simple to make, and implantation of the device requires approximately 1 hr of surgical time. Observations of vascular architecture, vessel morphology, and blood flow characteristics may be performed immediately after the window has been placed, compared with devices designed for vessel ingrowth which require 2 to 4 weeks before a mature vascular bed can be studied (Leaf and Zarem, 1970; Wood et al., 1966). The acute inflammatory response which accompanies the surgical procedure is resolved by 72 hr, making possible accurate observations of vasomotion, network perfusion, and vascular response to physiologic thermal stress. Eight rabbits underwent total body cold stress to determine whether vascular behavior was altered by implantation of the window. Surface ear temperature was unchanged before and after implantation when measured at an ambient temperature of 23”, indicating that blood flow under those environmental conditions was not influenced by the presence of the window. However, the average ear

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233

temperature during cold stress of the instrumented ear was 2.5“ warmer than that of either the preoperative or uninstrumented ear. This result indicates that increased blood flow was present in the instrumented ear, but also suggests the same qualitative response to cooling as that of the control ears. The increased temperature may reflect mild chronic inflammation causing increased blood flow through hyperemia (Grant and Bland, 1933) or angiogenesis (Zawicki et al., 1981). The reactivity of vessel diameters to cooling showed similar behavior in both instrumented and control ears. This reaction demonstrates that both the sympathetic and the local vasoconstrictor mechanisms which have been described in detail in the rabbit ear by Grant and Bland function normally with the window in place (Grant and Bland, 1933). Arteriolar constriction is known to be more intense than venular constriction in the human digit and in the rabbit ear (Grant and Bland, 1933; Roddie, 1983). This phenomenon represents the mechanism for venular pooling which is thought to contribute to thermoregulation in proximal areas of the organism (Grant and Pearson, 1938; Hirata et al., 1989). SUMMARY The window provides a simple and effective means for studying normal and pathologic behavior of native microvascular beds in the rabbit ear. It allows repeated visualization of networks of preformed arterioles, venules, and anastomotic channels which cannot otherwise be observed. The procedure for implantation is uncomplicated and requires relatively little time to complete. The window is well tolerated and may be used to examine parameters of blood flow and vessel behavior in thermoregulatory vascular beds. REFERENCES BRANEMARK, P. I., AND LINDSTROM, J. (1963). A modified rabbit’s ear chamber: High-power highresolution studies in regenerated and pre-formed tissues. Anat. Rec. 145,533-540. BURTON, A. C. (1939). The range and variability of the blood flow in the human fingers and the vasomotor regulation of body temperature. Am. J. Physiol. 127, 437-453. CLARKE, E. R., KIRBY-SMITH, H. T., REX, R. O., AND WILLIAMS, R. G. (1930). Recent modifications in the method of studying living cells and tissues in transparent chambers inserted in the rabbit’s ear. Anat. Rec. 47, 187-211. GONZALEZ, R. R., KLUGER, M. J., AND HARDY, J. D. (1971). Partitional calorimetry of the New Zealand White rabbit at temperatures of 5-35°C. J. Appl. Physiol. 31, 728-734. GORWN, C. J., AND HEATH, J. E. (1983). Reassessment of the neural control of body temperature: Importance of oscillating neural and motor components. Comp. Biochem. Physiol. 74A, 479-489. GRANT, R. T., AND BLAND, E. F. (1929-1931). Observations on arteriovenous anastamoses in human skin and in the bird’s foot with special reference to the reaction to cold. Heart 15, 385-411. GRANT, R. T., AND BLAND, E. F. (1933). Observations on the vessels and nerves of the rabbit’s ear with special reference to the reaction of cold. Heart 16, 69-101. GRANT, R. T., AND PEARSON, R. S. B. (1938). The blood circulation in the human limb: Observations on the difference between the proximal and distal parts and remarks on the regulation of body temperature. Clin. Sci. 3, 119-139. GREENBLATT, M., AND SHUBIK, P. (1967). Hamster cheek pouch chamber. Cancer Bull. 19, 65-81. HARADA, E. (1971). A characteristic pattern of fluctuation of skin temperature of the rabbit’s ear in response to alteration of the environmental temperature. J. Physiol. Sot. Jupan 33, 303-316. HIRATA, K., NAGASAKA, T., AND NODA, Y. (1989). Venous return from distal regions affects heat loss from the arms and legs during exercise-induced thermal loads. Eur. J. Appl. Physiol. 58, 865-872.

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M. J., GONZALES, R. R., MITCHELL, J. W., AND HARDY, J. D. (1971). The rabbit ear as a temperature sensor. Life. Sci. 10, 895-899. LEAF, N., AND ZAREM, H. A. (1970). Construction and use of a miniaturized rabbit ear chamber. KLUGER,

Microvas. MOHLER,

Res. 2, 72-85. F. S., AND HEATH,

J. E. (1988). Oscillating heat flow from rabbit’s pinna. Am. J. Physiol.

255,R464-R469. NIMS, J. C., AND IRWIN, J. W. (1973). Chamber techniques to study the microvasculature. Microvas. Res. 5, 105-118. RODDIE, I. C. (1983). Circulation to skin and adipose tissue. In “Handbook of Physiology” (S. R. Geiger, Ed.), Sect. 2, Vol. III, Part 1, pp. 285-317. American Physiological Society, Bethesda, MD. SMITH, T. L.. LYNCH, C. D., KHRAIBI, A. A., LEVITT, M., AND HUTCHINS, P. M. (1985). A pial window for microvascular observations in unanesthetized rats. Microvas. Rex 29, 251. STIRRAT, C. R., AND SEABER, A. V. (1979). The microsurgery laboratory. In “American Academy of Orthopaedic Surgeons: Symposium on Microsurgery: Practical Use in Orthopaedics” (J. R. Urbaniak, Ed.), pp. 12-39. Mosby, St. Louis, MO. TSAI, T. M. (1975). Experimental and clinical application of microvascular surgery. Ann. Surg. 181, 169-177. WOOD, S., LEWIS, R., MULHOLLAND, J. H., AND KNAACK, J. (1966). Assembly, insertion and use of a modified rabbit ear chamber. Bull. Johns Hopkins Hosp. 119, l-15. ZAREM, H. A., AND DIMITRIEVICH, G. S. (1970). In vivo observations of the effects of Imuran on the microvasculature within full thickness mouse allografts. Plast. Reconstr. Surg. 45, 51-57. ZAWICKI, D. F., JAIN, R. K., SCHMID-SCHOENBEIN,G. W., AND CHIEN, S. (1981). Dynamics of neovascularization in normal tissue. Microvas. Res. 21, 27-47. ZWEIFACH, B. W., AND LIPOWSKY, H. H. (1977). Quantitative studies of microcirculatory structure and function. III. Microvascular hemodynamics of cat mesentary and rabbit omentum. Circ. Res.

41. 380-390.