Brain Research Bulletin,
Vol. 3, pp. 571-574. Printedin the U.S.A.
BRIEF COMMUNICATION Stereotaxic Implant Microinjection and Temperature Recording System for Rabbits WILSON
A. SUAREZ
AND WILLIAM
Southern Illinois University, School of Medicine, (Received
S. HUNTER
Carbondale,
IL 62901
1 March 1978)
SUAREZ, W. A. AND W. S. HUNTER. Stereotaxic implant, microinjection and temperature recording system for ruhbits. BRAIN RES. BULL. 3(S) 571-574, 1978.-Modification of standard rabbit stereotaxic apparatus to allow adjustment of the tooth bar in the vertical plane may be accomplished by simple machining of a standard laboratory clamp and aluminum rod. The modification greatly increases speed of head positioning for implant surgery. Machining the chrome and brass luer hub of standard stainless steel hypodermic needles to the described configurations produces improved guide tubes for brain temperature recording and cannulae for brain microinjections. Data from hypothalamic injection of prostaglandin E, and records of resulting changes in brain temperature support the effectiveness of the methods described. Rabbit stereotaxic
apparatus
Brain temperature
methods
RECENTLY, there has been increasing use of the rabbit for studies of body temperature regulation; particularly of fever [ 1,3]. Often chronic brain implants are made for measurement of hypothalamic temperature and for microinjection of various substances into the ventricles, or other brain regions [ 1,5]. Because rabbits are easily excited, and their body temperature is quite labile, injection and measurements must be made with minimal disturbance and handling. This report describes a modification of stereotaxic apparatus, successfully employed in our laboratory to increase the speed and reliability of chronic brain implants in rabbits. In addition, we will describe a method for long term (i.e., several days) continuous monitoring of rabbit brain temperature in the normothermic and febrile states. We feel that the ready availability and comparative low cost (discounting the time required for fabrication) of the system components make it of particular interest and usefulness. DESIGN AND CONSTRUCTION
Stereotaxic
Frame Modification
Because stereotaxic methods for rabbits usually do not include the use of ear bars (zygomatic clamps are used instead), antero-posterior or dorsoventral coordinates relative to the interaural line cannot be used, nor can the midsaggital point of the interaural line be used for dorsoventral coordinates. In order to use zygomatic clamps, coordinates relative to bregma are used, and the head is positioned so that lambda is 1.5 mm lower than bregma [4]. With stereotaxic apparatus similar to that used for the present report (David Kopf Instruments frame No. 1504 with rabbit adaptor), attaining the desired lambda-bregma relationship can be tedious and time
Copyright
o 1978 ANKHO
International
Rabbit brain implant
consuming. Delay in head positioning results in increased chance of surgical complications. Figure 1 A-C, illustrates a simple modification of the rabbit adaptor which allows quick and accurate lambda-bregma head positioning by means of a tooth bar adjustable in the vertical plane. To make the tooth bar vertically adjustable, an aluminum school clamp (Labline 7050-l) or aluminum block can be easily machined to attach to the swivel base of the head holder (Fig. 1, A). The tooth bar is attached to a 10 cm length of 13 mm dia. aluminum rod (Fig. 1, B) which can be slid up or down inside a hole of matching diameter in the school clamp, and held in position by a thumb screw pushing against a nylon, teflon, or polyethylene cylinder between the end of the thumb screw and the aluminum rod (Fig. 1, C). Using this modification, the rabbit’s head can be placed in the frame in the normal manner, probes set at the lambda and bregma positions, and the tooth bar raised or lowered until labmda is 1.5 cm below bregma. Instead of setting separate probes at lambda and bregma, the planilabe device ([2], David Kopf No. 1244) can be used. Implant
Cunnulae
and Guide Tubes
Cannulae for microinjection, and guide tubes for thermocouple measurement of hypothalamic temperature, were made by machining the conventional chrome and brass luer hub of appropriately sized hypodermic needles to the contiguration shown in Fig. 2. The microinjection cannula is kept clean and patent by a trocar (0.3 mm stainless steel wire) soldered in a slot cut in the end of a 2.5 mm long, 2.14 mm dia. screw which fits threads cut in the brass hub of the cannula (Fig. 2, A). The screw, and wire trocar are assembled so that the trocar tip
Inc.-0361-9230/78/050571-04$0090/O
572
SUAREZ AND HUNTER
FIG. 1. Photograph
of rabbit skull with implant
in modified
reaches exactly to the end of the cannula when screwed in place. Injection is made by a 30 ga hypodermic needle cut to extend 0.5 mm beyond the tip of the 23 ga cannula. The injection needle is machined to the configuration shown in Fig. 2B, and connected with an oil filled microsyringe and length of PE 20 polyethylethene tubing. A 1 cm section of 21 ga stainless steel tubing is soldered in the brass hub to allow direct connection of the 30 ga needle and PE 20 tubing. Paraffin oil or silicone fluid may be used as a fluid piston to drive a small volume of injectate filling the 30 ga needle [S]. The guide tube for recording hypothalamic temperature is closed at the distal end by peening and burnishing until closed as completely as possible. The remaining perforation is sealed with solder and the end of the cannula coated with a thin layer of biologically inert insulation (epoxylite) to avoid possible adverse tissue reaction by the solder. Alternatively. the peened, burnished end can be sealed by inert gas plasma arc,welding. The luer hub of the needle is machined to an outside diameter of approximately 3.5 mm, with an inside diameter of 2 mm and the inside threaded to accept a
head holder (see text for explanation
of lettered
parts).
2.5x2.14 mm screw (Fig. 2C). A 1.0x3.0 mm slot is cut in the side of the hub so that a short section of I.5 mm OD silicone rubber tubing can be pushed into it. The thermocouple is slid through the silicone tubing extending from the side slot, and advanced to the closed end of the cannula. With the thermocouple in place, the screw (Fig. 21)) can be tightened down to squeeze the silicone tubing enough to hold the thermocouple wires firmly in place without damaging them. To secure the implants, dental acrylic (Fig. 2E) is molded around the extracranial parts of the cannula, guide tube and 3 stainless steel screws tapped into the skull. Figure 3 presents recording of febrile change in hypothalamic temperature of a rabbit after remote injection of a 1 microliter solution containing 1 microgram PGE,, using the system described here. DISCUSSION
Modification of the rabbit head holder (David Kopf) so that the tooth bar is adjustable in the vertical plane reduces
RABBIT STERBOTAXI~
573
IMPLANT
-
lmm
FIG, 2. (A) diagram of cannula; (B) microinjection needle; (C, D) thermocouple guide tube assembly: and (E) dental acrylic matrix for head mount.
FIG. 3. Recording of brain temperature following hypothalamic microinjection of PGE,, using thermocouple guide tube and microinjection cannula.
the time necessary to establish the proper head position for stereotaxic implants in rabbits. If used in conjunction with the Kopf Model 1244 alignment device [2j, skufi alignment is further facilitated. Also, methods described above have been successfully used to remotely administer mic~injections of PGE, into the
anterior hypothalamus of confined but un~st~~n~ rabbits, and to continuously record hypothahtmic temperature for periods of hours or days. The cannula and guide tube design is potentially applicable in a wide variety of species in situations where implantation of parallel guide tubes does not require their centers to be less than 3 mm apart.
SUAREZ
ACKNOWLEDGEMENTS
The authors would like to express their thanks to Mr. Jeffrey Parks for his technical assistance, and to Dr. David Bliss for his review and suggestions during preparation of the manuscript. Pros-
AND HUNTER
taglandin E, was generously supplied by Dr. John Pike, Upjohn Laboratories, Kalamazoo, Michigan, USA. Photography and aid in figure preparation was provided by Southern Illinois University, Carbondale, Biomedical Communications.
REFERENCES 1. Cranston, W. I., C. W. Duff, R. F. Hellon, D. Mitchell and Y. Townsend. Evidence that brain prostaglandin synthesis is not essential in fever. .I. Physid. 259: 23%249, 1976. 2. Crawford, I. L., J. I. Kennedy and J. M. Lipton. A simple “planilabe” for rapid establishment of the stereotaxic horizontal zero plane in rabbits. Bruin Res. Bull. 2: 397-398, 1977. 3. Milton, A. S. and S. Wendiandt. Effect on body temperature of prost~andins of the A, E and F series on injection into the third ventricle of unanesthetized cats and rabbits. f. Physid. 218: 325-336, 1971.
4. Sawyer,
C., J. W. Everett and J. D. Green. The rabbit diencephalon in stereotaxic coordinates. J. camp. Neural. 101: 801-824,
1954.
5. Stitt, J. T. Prostaglandin 232: 163-179, 1973.
E fever induced in rabbits. J.
Pl7.vsiol.
Vol. 3, pp. 571-574. Printedin the U.S.A.
BRIEF COMMUNICATION Stereotaxic Implant Microinjection and Temperature Recording System for Rabbits WILSON
A. SUAREZ
AND WILLIAM
Southern Illinois University, School of Medicine, (Received
S. HUNTER
Carbondale,
IL 62901
1 March 1978)
SUAREZ, W. A. AND W. S. HUNTER. Stereotaxic implant, microinjection and temperature recording system for ruhbits. BRAIN RES. BULL. 3(S) 571-574, 1978.-Modification of standard rabbit stereotaxic apparatus to allow adjustment of the tooth bar in the vertical plane may be accomplished by simple machining of a standard laboratory clamp and aluminum rod. The modification greatly increases speed of head positioning for implant surgery. Machining the chrome and brass luer hub of standard stainless steel hypodermic needles to the described configurations produces improved guide tubes for brain temperature recording and cannulae for brain microinjections. Data from hypothalamic injection of prostaglandin E, and records of resulting changes in brain temperature support the effectiveness of the methods described. Rabbit stereotaxic
apparatus
Brain temperature
methods
RECENTLY, there has been increasing use of the rabbit for studies of body temperature regulation; particularly of fever [ 1,3]. Often chronic brain implants are made for measurement of hypothalamic temperature and for microinjection of various substances into the ventricles, or other brain regions [ 1,5]. Because rabbits are easily excited, and their body temperature is quite labile, injection and measurements must be made with minimal disturbance and handling. This report describes a modification of stereotaxic apparatus, successfully employed in our laboratory to increase the speed and reliability of chronic brain implants in rabbits. In addition, we will describe a method for long term (i.e., several days) continuous monitoring of rabbit brain temperature in the normothermic and febrile states. We feel that the ready availability and comparative low cost (discounting the time required for fabrication) of the system components make it of particular interest and usefulness. DESIGN AND CONSTRUCTION
Stereotaxic
Frame Modification
Because stereotaxic methods for rabbits usually do not include the use of ear bars (zygomatic clamps are used instead), antero-posterior or dorsoventral coordinates relative to the interaural line cannot be used, nor can the midsaggital point of the interaural line be used for dorsoventral coordinates. In order to use zygomatic clamps, coordinates relative to bregma are used, and the head is positioned so that lambda is 1.5 mm lower than bregma [4]. With stereotaxic apparatus similar to that used for the present report (David Kopf Instruments frame No. 1504 with rabbit adaptor), attaining the desired lambda-bregma relationship can be tedious and time
Copyright
o 1978 ANKHO
International
Rabbit brain implant
consuming. Delay in head positioning results in increased chance of surgical complications. Figure 1 A-C, illustrates a simple modification of the rabbit adaptor which allows quick and accurate lambda-bregma head positioning by means of a tooth bar adjustable in the vertical plane. To make the tooth bar vertically adjustable, an aluminum school clamp (Labline 7050-l) or aluminum block can be easily machined to attach to the swivel base of the head holder (Fig. 1, A). The tooth bar is attached to a 10 cm length of 13 mm dia. aluminum rod (Fig. 1, B) which can be slid up or down inside a hole of matching diameter in the school clamp, and held in position by a thumb screw pushing against a nylon, teflon, or polyethylene cylinder between the end of the thumb screw and the aluminum rod (Fig. 1, C). Using this modification, the rabbit’s head can be placed in the frame in the normal manner, probes set at the lambda and bregma positions, and the tooth bar raised or lowered until labmda is 1.5 cm below bregma. Instead of setting separate probes at lambda and bregma, the planilabe device ([2], David Kopf No. 1244) can be used. Implant
Cunnulae
and Guide Tubes
Cannulae for microinjection, and guide tubes for thermocouple measurement of hypothalamic temperature, were made by machining the conventional chrome and brass luer hub of appropriately sized hypodermic needles to the contiguration shown in Fig. 2. The microinjection cannula is kept clean and patent by a trocar (0.3 mm stainless steel wire) soldered in a slot cut in the end of a 2.5 mm long, 2.14 mm dia. screw which fits threads cut in the brass hub of the cannula (Fig. 2, A). The screw, and wire trocar are assembled so that the trocar tip
Inc.-0361-9230/78/050571-04$0090/O
572
SUAREZ AND HUNTER
FIG. 1. Photograph
of rabbit skull with implant
in modified
reaches exactly to the end of the cannula when screwed in place. Injection is made by a 30 ga hypodermic needle cut to extend 0.5 mm beyond the tip of the 23 ga cannula. The injection needle is machined to the configuration shown in Fig. 2B, and connected with an oil filled microsyringe and length of PE 20 polyethylethene tubing. A 1 cm section of 21 ga stainless steel tubing is soldered in the brass hub to allow direct connection of the 30 ga needle and PE 20 tubing. Paraffin oil or silicone fluid may be used as a fluid piston to drive a small volume of injectate filling the 30 ga needle [S]. The guide tube for recording hypothalamic temperature is closed at the distal end by peening and burnishing until closed as completely as possible. The remaining perforation is sealed with solder and the end of the cannula coated with a thin layer of biologically inert insulation (epoxylite) to avoid possible adverse tissue reaction by the solder. Alternatively. the peened, burnished end can be sealed by inert gas plasma arc,welding. The luer hub of the needle is machined to an outside diameter of approximately 3.5 mm, with an inside diameter of 2 mm and the inside threaded to accept a
head holder (see text for explanation
of lettered
parts).
2.5x2.14 mm screw (Fig. 2C). A 1.0x3.0 mm slot is cut in the side of the hub so that a short section of I.5 mm OD silicone rubber tubing can be pushed into it. The thermocouple is slid through the silicone tubing extending from the side slot, and advanced to the closed end of the cannula. With the thermocouple in place, the screw (Fig. 21)) can be tightened down to squeeze the silicone tubing enough to hold the thermocouple wires firmly in place without damaging them. To secure the implants, dental acrylic (Fig. 2E) is molded around the extracranial parts of the cannula, guide tube and 3 stainless steel screws tapped into the skull. Figure 3 presents recording of febrile change in hypothalamic temperature of a rabbit after remote injection of a 1 microliter solution containing 1 microgram PGE,, using the system described here. DISCUSSION
Modification of the rabbit head holder (David Kopf) so that the tooth bar is adjustable in the vertical plane reduces
RABBIT STERBOTAXI~
573
IMPLANT
-
lmm
FIG, 2. (A) diagram of cannula; (B) microinjection needle; (C, D) thermocouple guide tube assembly: and (E) dental acrylic matrix for head mount.
FIG. 3. Recording of brain temperature following hypothalamic microinjection of PGE,, using thermocouple guide tube and microinjection cannula.
the time necessary to establish the proper head position for stereotaxic implants in rabbits. If used in conjunction with the Kopf Model 1244 alignment device [2j, skufi alignment is further facilitated. Also, methods described above have been successfully used to remotely administer mic~injections of PGE, into the
anterior hypothalamus of confined but un~st~~n~ rabbits, and to continuously record hypothahtmic temperature for periods of hours or days. The cannula and guide tube design is potentially applicable in a wide variety of species in situations where implantation of parallel guide tubes does not require their centers to be less than 3 mm apart.
SUAREZ
ACKNOWLEDGEMENTS
The authors would like to express their thanks to Mr. Jeffrey Parks for his technical assistance, and to Dr. David Bliss for his review and suggestions during preparation of the manuscript. Pros-
AND HUNTER
taglandin E, was generously supplied by Dr. John Pike, Upjohn Laboratories, Kalamazoo, Michigan, USA. Photography and aid in figure preparation was provided by Southern Illinois University, Carbondale, Biomedical Communications.
REFERENCES 1. Cranston, W. I., C. W. Duff, R. F. Hellon, D. Mitchell and Y. Townsend. Evidence that brain prostaglandin synthesis is not essential in fever. .I. Physid. 259: 23%249, 1976. 2. Crawford, I. L., J. I. Kennedy and J. M. Lipton. A simple “planilabe” for rapid establishment of the stereotaxic horizontal zero plane in rabbits. Bruin Res. Bull. 2: 397-398, 1977. 3. Milton, A. S. and S. Wendiandt. Effect on body temperature of prost~andins of the A, E and F series on injection into the third ventricle of unanesthetized cats and rabbits. f. Physid. 218: 325-336, 1971.
4. Sawyer,
C., J. W. Everett and J. D. Green. The rabbit diencephalon in stereotaxic coordinates. J. camp. Neural. 101: 801-824,
1954.
5. Stitt, J. T. Prostaglandin 232: 163-179, 1973.
E fever induced in rabbits. J.
Pl7.vsiol.
