[30]
THYROID PERFUSION
359
site-receptor reserve model emphasizes the complexity of factors which enter into the determination of Vmax and of apparent dissociation constants for A C T H and its analogs. Specifically, characterization by steroidogenesis alone may be misleading in the delineation of "active centers" and "affinity centers" of the polypeptide hormone.
[30] Perfusion of the Thyroid Gland 1 By JUDAH FOLKMAN and MICHAEL A. GIMBRONE, JR. M a n y methods for perfusing the isolated thyroid gland have been described since Carrel ~ first perfused chicken thyroid. Several of these methods have been tried in our laboratory. However, the best functional and histologic preservation was obtained with the following technique, which has been used to perfuse more than 100 thyroid glands from rabbit and dog. 3-~
PerfusionCircuit Organ Chamber. A sterile circuit is assembled as in Fig. 1. The organ chamber is made from a glass cylinder, 37 mm i.d., 41 mm o.d., and 140 mm long. It has a single outlet of 5 mm o.d., that will fit snugly into gum rubber tubing (1/8 X 3/64 inch). This tubing is used throughout the circuit. The outlet is conical, similar to a glass centrifuge tube. It is etched with volumetric graduations so that flow rate can be measured. A No. 8 silicone rubber stopper is pierced by a short length of glass tube (5 mm o.d.). The arterial cannula is attached to this tube just before the thyroid gland is placed within the chamber. A hypodermic needle is passed through the stopper to serve as an air vent. The arterial cannula is made from glass tubing 5 mm o.d. (Fig. 2). One end is narrowed and beveled so that it will fit easily into the superior thyroid artery. It is 1This work was supported by a grant from the National Cancer Institute (No. 1 RO1 CA 14019-01), a grant from the American Cancer Society (No. DT-2A), and gifts from Alza Corporation, Merck Co., and Mr. Morton Bank. 2A. Carrel and C. A. Lindberg, "The Culture of Organs." Harper (Hoeber), New York, 1938. 3j. Folkman, P. Cole, and S. Zimmerman, Ann. Surg. 164, 491 (1966). 4 M. A. Gimbrone, R. A. Aster, R. S. Cotran, J. Corkery, J. Jandl, and J. Folkman, Nature (London) 222, 33 (1969). J. Folkman, and M. A. Gimbrone, Perfusion of the Thyroid, in "Fourth Karolinska Symposium on Research Methods in Reproductive Endocrinology" (E. Diczfalusy, ed.). Karolinska Inst., Stockholm, 1972.
360
ENDOCRINE AND REPRODUCTIVE TISSUE
[30]
FILTER
STIRRER
FIG. 1. Diagram of perfusion circuit. Scale 1:4.5. Circuit is housed in a plexiglass cabinet which serves as an incubator and which supports the Mettler balance (see inset). convenient to have a variety of cannulas with tips of different sizes when the thyroid gland is being dissected. These should range from 0.5 mm up to 2.0 mm. A cannula tip of about 1.5 mm is usually satisfactory. Three small glass hooks are annealed to the outside of the organ chamber so that it can be suspended from the undersurface of a Mettler balance.
[30]
THYROID PERFUSION
361
Fro. 2. Glass cannula. Gum rubber tubing is held in place by Silastic adhesive and a crimped nylon band (Nylon Tie, SST-1 from Extracorporeal Medical Specialties, Inc. Pennsylvania). The nylon band serves as an anchor point around which silk ligatures can be tightened after they have been tied around the artery following cannulation. Each division = 1 mm.
Oxygenator. The outer cylinder of the oxygenator is 35 m m o.d., 32 m m i.d. and 150 m m long, with inlet and outlet sidearms of 5 m m o.d. A No. 7 silicone rubber stopper is fitted with a hollow glass core. A thin layer of Silastic 6 cement is applied to this core; the core is then wound with a coil of silicone rubber capillary tubing, 0.025 inch i.d., 0.037 inch o.d., and approximately 30 feet in length. Two No. 23 stainless steel hypodermic needles are driven through the stopper and inserted into the ends of the capillary tubing. The capillary tubes are cemented to the needles and large amounts of Silastic cement are applied to the needle shafts to prevent them from changing position. A mixture of 95% oxygen and 5% carbon dioxide is propelled through one of these needles and escapes from the other. The gas also diffuses through the capillary tubing and oxygenates the perfusate which flows over the coil. The gas is sterilized in this way and air bubbles are eliminated. The space between the coil and the wall of the oxygenator chamber is approximately 2 mm. A third hypodermic needle is passed through the stopper to serve as an air vent. I t is capped with sterile cotton or gauze. A magnetic spinner bar is placed in the bottom of the oxygenator. The bar is turned slowly by a magnetic stirrer to increase efficiency of oxygenation. The outlet of the oxygenator is connected through a three-way stopcock, to a putsatile pump. Pump. A pulsatile p u m p is assembled as shown in Fig. 1. Thin wall silicone tubing 10 m m o.d., 9 m m i.d., is stretched through a l l 0 m m 6Silastic Medical Adhesive, Silicone Type A, Dow Corning Corporation, Midland, Michigan.
362
ENDOCRINE AND REPRODUCTIVE TISSUE
[30]
glass cylinder 14 mm o.d. and 12 mm i.d. The ends of the thin tubing are turned back upon the wall of the glass cylinder and cemented in place. Valves for unidirectional flow are then cemented into each end of the pumping cylinder. These valves are made from silicone tubing 5 mm o.d., 3 mm i.d. and 50 mm long. One end is sealed with Silastic cement and a ~ - i n c h slit is made with a razor in the side wall nearest the sealed end. This end is then inserted into a 60-mm length of glass tubing of 8 mm i.d., the opening of which has been narrowed so that the silicone valve will fit snugly. The valve is cemented in a position that leaves the valve orifice just within the glass tube. The pump chamber is attached to a H a r v a r d small animal respirator 7 with a single length of thick walled T y g o n tubing, ¼ inch i.d. The extra sidearm on the pump chamber is occluded, but can be opened slightly with a screw clamp, should a larger respirator be used with a stroke volume t h a t exceeds the volume of the pump chamber. The outflow of the pump is connected by a short length of gum rubber tubing to a filter. Filter. A filter holder is made of two nylon disks, each 2 inches in diameter and 0.5 inch thick. A shallow well of 0.5 ml is made in each disk. Two layers of unweighted silk mesh are compressed between the two nylon disks. The filter removes cellular debris and platelet aggregates from the circulation. Pressure Transducer. A transducer head is inserted in the circuit between the filter and the arterial cannula. It is connected to an electronic recorder s which measures pressures from 50-150 mm Hg. All components which touch the perfusate are siliconized. 9 The entire circuit is maintained at 35 ° in a plexiglass thermostatic cabinet.
Perfusate
Several types of perfusate may be used including tissue culture solutions such as Eagle's medium with 10% calf serum or hemoglobin solutions2 However, the best thyroid function and the least edema was obtained by Gimbrone in our laboratory, who employed heparinized, autologous platelet-rich plasmaA This perfusate is obtained by plasmaphoresis of fasting animals 2 hours before thyroidectomy. Large (50 lb) dogs with normal erythrocyte sedimentation rates and platelet counts are used as donors. Under light Surital anesthesia, blood is rapidly collected from a femoral artery canHarvard Apparatus Company, Dover, Massachusetts. s Gulton Medical Instruments, Willow Grove, Pennsylvania. Siliclad, Clay-Adams Co., New York.
[301
THYROID PERFUSION
363
nula into a plastic Fenwall bag TM containing sufficient heparin so that the final concentration will be 2.5 mg/ml. An average volume of 600 ml is collected, representing about one-third of the donor's blood volume. An equivalent amount of Ringers lactate solution is then infused intravenously. Immediately after separation of the platelet-rich plasma by centrifugation, the packed red cells are reinfused into the donor animal. Platelet-rich plasma is prepared by slow centrifugation of blood (1000 rpm = 253 g) for 10 minutes at 20 ° in a bucket-head International Centrifuge. Sterile techniques are used throughout, hemolysis and foaming are avoided, and the plasma is allowed to come into contact with only nonwettable plastic or siliconized surfaces. Platelet counts by phase microscopy routinely vary between 150 and 200 X 103/mm 3 when plasma is prepared in this way. About 70 ml is needed to prime the perfusion circuit. The remaining plasma is stored at room temperature in plastic bags for later use.
Thyroid Gland A careful anatomical dissection is carried out in the donor dog. A thyroid lobe attached to its respective superior thyroid artery pedicle is removed. Both thyroid lobes may be removed if two perfusion circuits are available. All veins are allowed to bleed freely, but tiny arterial branches severed during the dissection, are ligated so that normal arterial pressures can be maintained within the gland. Immediately preceding removal, the donor animal is systemically heparinized (2 mg/kg). A glass cannula is then inserted into the superior thyroid artery or into the carotid artery near the branch of the superior thyroid artery, and fixed in position with silk ligatures. The gland is placed in the organ chamber and the eannula attached to the glass inlet tube. The initial venous effluents are contaminated with blood and are discarded before they enter the oxygenator. After the venous effluent has become clear (about 10-15 minutes), it is allowed to enter the oxygenator and recireulate.
Management of Perfusion The stroke volume is initially adjusted to maintain a systolic pressure of 100-120 mm Hg, but thereafter, both rate and stroke volume are kept constant so that changes in perfusion pressure and flow rate will reflect changes of vascular resistance within the perfused lobe. The flow rate is measured periodically by occluding the outlet of the organ chamber and measuring the time required for 1 ml to accumulate in the conical 1oFenwall Transfer Pack, Code No. TA-1.
364
ENDOCRINE AND REPRODUCTIVE TISSUE
[31]
end of the chamber. During technically satisfactory perfusions, flow rates range between 0.75 and 3.00 ml per gram of tissue per minute. Oxygen tensions of perfusate sampled distal to the filter, average 180 mm Hg (pO2) and the pH of venous effluents remains within the range 7.30 to 7.55. The oxygenator chamber is drained and refilled with 50 ml fresh plasma every 12 hours. Perfusions can be continued for 5-7 days.
Studies of Function and Morphology during Perfusion Donor animals were injected with 400 ~Ci of 12~I (as NaI) 48 hours prior to thyroidectomy. This allowed the monitoring of TCA-precipitable and butanol-extractable 12~I in the perfusate. In other perfusions, 1~I was injected in vitro and the percent of iodine incorporated into triiodothyronine was studied. Mean vascular resistance was estimated as the ratio of effluent flow rate to mean systolic pressure. The increase in mean vascular resistance , expressed as a percentage of initial, averaged 26% after 5 hours of perfusion. After 5 hours thyroid glands perfused with platelet-rich plasma gained an average of 15% in overall weight. All other types of perfusate caused significantly more rapid weight gain. For example when plateletpoor plasma was used, the average weight gain was 41% at 5 hours. Serial histological studies were carried out by both light and electron microscopy. In general, the capillary endothelial cells showed normal preservation up to 5 hours and beyond, the thyroid follicles up to 24 hours in the central portion of the gland and up to 3-5 days in the most peripheral portion of the gland. The best histological preservation was observed in the parathyroid glands which had a normal appearance even after 1 week of perfusion. In other experiments 3,~ tumors implanted into these glands during perfusion, grew well up to diameters of 1-2 mm and then remained alive but dormant and unvascularized.
[31] T h e i n V i t r o Perfused Pancreas By GEROLDM. GRODSKYand Rt~DOLPHE. FANSKA A technique for removing rat pancreas and maintaining it in a perfusion apparatus was briefly described by Anderson and Long in 1947.I Subsequently the technique was modified and used for studying the influ1E. Anderson and J. A. Long, Endocrinology 40, 92 (1947).
THYROID PERFUSION
359
site-receptor reserve model emphasizes the complexity of factors which enter into the determination of Vmax and of apparent dissociation constants for A C T H and its analogs. Specifically, characterization by steroidogenesis alone may be misleading in the delineation of "active centers" and "affinity centers" of the polypeptide hormone.
[30] Perfusion of the Thyroid Gland 1 By JUDAH FOLKMAN and MICHAEL A. GIMBRONE, JR. M a n y methods for perfusing the isolated thyroid gland have been described since Carrel ~ first perfused chicken thyroid. Several of these methods have been tried in our laboratory. However, the best functional and histologic preservation was obtained with the following technique, which has been used to perfuse more than 100 thyroid glands from rabbit and dog. 3-~
PerfusionCircuit Organ Chamber. A sterile circuit is assembled as in Fig. 1. The organ chamber is made from a glass cylinder, 37 mm i.d., 41 mm o.d., and 140 mm long. It has a single outlet of 5 mm o.d., that will fit snugly into gum rubber tubing (1/8 X 3/64 inch). This tubing is used throughout the circuit. The outlet is conical, similar to a glass centrifuge tube. It is etched with volumetric graduations so that flow rate can be measured. A No. 8 silicone rubber stopper is pierced by a short length of glass tube (5 mm o.d.). The arterial cannula is attached to this tube just before the thyroid gland is placed within the chamber. A hypodermic needle is passed through the stopper to serve as an air vent. The arterial cannula is made from glass tubing 5 mm o.d. (Fig. 2). One end is narrowed and beveled so that it will fit easily into the superior thyroid artery. It is 1This work was supported by a grant from the National Cancer Institute (No. 1 RO1 CA 14019-01), a grant from the American Cancer Society (No. DT-2A), and gifts from Alza Corporation, Merck Co., and Mr. Morton Bank. 2A. Carrel and C. A. Lindberg, "The Culture of Organs." Harper (Hoeber), New York, 1938. 3j. Folkman, P. Cole, and S. Zimmerman, Ann. Surg. 164, 491 (1966). 4 M. A. Gimbrone, R. A. Aster, R. S. Cotran, J. Corkery, J. Jandl, and J. Folkman, Nature (London) 222, 33 (1969). J. Folkman, and M. A. Gimbrone, Perfusion of the Thyroid, in "Fourth Karolinska Symposium on Research Methods in Reproductive Endocrinology" (E. Diczfalusy, ed.). Karolinska Inst., Stockholm, 1972.
360
ENDOCRINE AND REPRODUCTIVE TISSUE
[30]
FILTER
STIRRER
FIG. 1. Diagram of perfusion circuit. Scale 1:4.5. Circuit is housed in a plexiglass cabinet which serves as an incubator and which supports the Mettler balance (see inset). convenient to have a variety of cannulas with tips of different sizes when the thyroid gland is being dissected. These should range from 0.5 mm up to 2.0 mm. A cannula tip of about 1.5 mm is usually satisfactory. Three small glass hooks are annealed to the outside of the organ chamber so that it can be suspended from the undersurface of a Mettler balance.
[30]
THYROID PERFUSION
361
Fro. 2. Glass cannula. Gum rubber tubing is held in place by Silastic adhesive and a crimped nylon band (Nylon Tie, SST-1 from Extracorporeal Medical Specialties, Inc. Pennsylvania). The nylon band serves as an anchor point around which silk ligatures can be tightened after they have been tied around the artery following cannulation. Each division = 1 mm.
Oxygenator. The outer cylinder of the oxygenator is 35 m m o.d., 32 m m i.d. and 150 m m long, with inlet and outlet sidearms of 5 m m o.d. A No. 7 silicone rubber stopper is fitted with a hollow glass core. A thin layer of Silastic 6 cement is applied to this core; the core is then wound with a coil of silicone rubber capillary tubing, 0.025 inch i.d., 0.037 inch o.d., and approximately 30 feet in length. Two No. 23 stainless steel hypodermic needles are driven through the stopper and inserted into the ends of the capillary tubing. The capillary tubes are cemented to the needles and large amounts of Silastic cement are applied to the needle shafts to prevent them from changing position. A mixture of 95% oxygen and 5% carbon dioxide is propelled through one of these needles and escapes from the other. The gas also diffuses through the capillary tubing and oxygenates the perfusate which flows over the coil. The gas is sterilized in this way and air bubbles are eliminated. The space between the coil and the wall of the oxygenator chamber is approximately 2 mm. A third hypodermic needle is passed through the stopper to serve as an air vent. I t is capped with sterile cotton or gauze. A magnetic spinner bar is placed in the bottom of the oxygenator. The bar is turned slowly by a magnetic stirrer to increase efficiency of oxygenation. The outlet of the oxygenator is connected through a three-way stopcock, to a putsatile pump. Pump. A pulsatile p u m p is assembled as shown in Fig. 1. Thin wall silicone tubing 10 m m o.d., 9 m m i.d., is stretched through a l l 0 m m 6Silastic Medical Adhesive, Silicone Type A, Dow Corning Corporation, Midland, Michigan.
362
ENDOCRINE AND REPRODUCTIVE TISSUE
[30]
glass cylinder 14 mm o.d. and 12 mm i.d. The ends of the thin tubing are turned back upon the wall of the glass cylinder and cemented in place. Valves for unidirectional flow are then cemented into each end of the pumping cylinder. These valves are made from silicone tubing 5 mm o.d., 3 mm i.d. and 50 mm long. One end is sealed with Silastic cement and a ~ - i n c h slit is made with a razor in the side wall nearest the sealed end. This end is then inserted into a 60-mm length of glass tubing of 8 mm i.d., the opening of which has been narrowed so that the silicone valve will fit snugly. The valve is cemented in a position that leaves the valve orifice just within the glass tube. The pump chamber is attached to a H a r v a r d small animal respirator 7 with a single length of thick walled T y g o n tubing, ¼ inch i.d. The extra sidearm on the pump chamber is occluded, but can be opened slightly with a screw clamp, should a larger respirator be used with a stroke volume t h a t exceeds the volume of the pump chamber. The outflow of the pump is connected by a short length of gum rubber tubing to a filter. Filter. A filter holder is made of two nylon disks, each 2 inches in diameter and 0.5 inch thick. A shallow well of 0.5 ml is made in each disk. Two layers of unweighted silk mesh are compressed between the two nylon disks. The filter removes cellular debris and platelet aggregates from the circulation. Pressure Transducer. A transducer head is inserted in the circuit between the filter and the arterial cannula. It is connected to an electronic recorder s which measures pressures from 50-150 mm Hg. All components which touch the perfusate are siliconized. 9 The entire circuit is maintained at 35 ° in a plexiglass thermostatic cabinet.
Perfusate
Several types of perfusate may be used including tissue culture solutions such as Eagle's medium with 10% calf serum or hemoglobin solutions2 However, the best thyroid function and the least edema was obtained by Gimbrone in our laboratory, who employed heparinized, autologous platelet-rich plasmaA This perfusate is obtained by plasmaphoresis of fasting animals 2 hours before thyroidectomy. Large (50 lb) dogs with normal erythrocyte sedimentation rates and platelet counts are used as donors. Under light Surital anesthesia, blood is rapidly collected from a femoral artery canHarvard Apparatus Company, Dover, Massachusetts. s Gulton Medical Instruments, Willow Grove, Pennsylvania. Siliclad, Clay-Adams Co., New York.
[301
THYROID PERFUSION
363
nula into a plastic Fenwall bag TM containing sufficient heparin so that the final concentration will be 2.5 mg/ml. An average volume of 600 ml is collected, representing about one-third of the donor's blood volume. An equivalent amount of Ringers lactate solution is then infused intravenously. Immediately after separation of the platelet-rich plasma by centrifugation, the packed red cells are reinfused into the donor animal. Platelet-rich plasma is prepared by slow centrifugation of blood (1000 rpm = 253 g) for 10 minutes at 20 ° in a bucket-head International Centrifuge. Sterile techniques are used throughout, hemolysis and foaming are avoided, and the plasma is allowed to come into contact with only nonwettable plastic or siliconized surfaces. Platelet counts by phase microscopy routinely vary between 150 and 200 X 103/mm 3 when plasma is prepared in this way. About 70 ml is needed to prime the perfusion circuit. The remaining plasma is stored at room temperature in plastic bags for later use.
Thyroid Gland A careful anatomical dissection is carried out in the donor dog. A thyroid lobe attached to its respective superior thyroid artery pedicle is removed. Both thyroid lobes may be removed if two perfusion circuits are available. All veins are allowed to bleed freely, but tiny arterial branches severed during the dissection, are ligated so that normal arterial pressures can be maintained within the gland. Immediately preceding removal, the donor animal is systemically heparinized (2 mg/kg). A glass cannula is then inserted into the superior thyroid artery or into the carotid artery near the branch of the superior thyroid artery, and fixed in position with silk ligatures. The gland is placed in the organ chamber and the eannula attached to the glass inlet tube. The initial venous effluents are contaminated with blood and are discarded before they enter the oxygenator. After the venous effluent has become clear (about 10-15 minutes), it is allowed to enter the oxygenator and recireulate.
Management of Perfusion The stroke volume is initially adjusted to maintain a systolic pressure of 100-120 mm Hg, but thereafter, both rate and stroke volume are kept constant so that changes in perfusion pressure and flow rate will reflect changes of vascular resistance within the perfused lobe. The flow rate is measured periodically by occluding the outlet of the organ chamber and measuring the time required for 1 ml to accumulate in the conical 1oFenwall Transfer Pack, Code No. TA-1.
364
ENDOCRINE AND REPRODUCTIVE TISSUE
[31]
end of the chamber. During technically satisfactory perfusions, flow rates range between 0.75 and 3.00 ml per gram of tissue per minute. Oxygen tensions of perfusate sampled distal to the filter, average 180 mm Hg (pO2) and the pH of venous effluents remains within the range 7.30 to 7.55. The oxygenator chamber is drained and refilled with 50 ml fresh plasma every 12 hours. Perfusions can be continued for 5-7 days.
Studies of Function and Morphology during Perfusion Donor animals were injected with 400 ~Ci of 12~I (as NaI) 48 hours prior to thyroidectomy. This allowed the monitoring of TCA-precipitable and butanol-extractable 12~I in the perfusate. In other perfusions, 1~I was injected in vitro and the percent of iodine incorporated into triiodothyronine was studied. Mean vascular resistance was estimated as the ratio of effluent flow rate to mean systolic pressure. The increase in mean vascular resistance , expressed as a percentage of initial, averaged 26% after 5 hours of perfusion. After 5 hours thyroid glands perfused with platelet-rich plasma gained an average of 15% in overall weight. All other types of perfusate caused significantly more rapid weight gain. For example when plateletpoor plasma was used, the average weight gain was 41% at 5 hours. Serial histological studies were carried out by both light and electron microscopy. In general, the capillary endothelial cells showed normal preservation up to 5 hours and beyond, the thyroid follicles up to 24 hours in the central portion of the gland and up to 3-5 days in the most peripheral portion of the gland. The best histological preservation was observed in the parathyroid glands which had a normal appearance even after 1 week of perfusion. In other experiments 3,~ tumors implanted into these glands during perfusion, grew well up to diameters of 1-2 mm and then remained alive but dormant and unvascularized.
[31] T h e i n V i t r o Perfused Pancreas By GEROLDM. GRODSKYand Rt~DOLPHE. FANSKA A technique for removing rat pancreas and maintaining it in a perfusion apparatus was briefly described by Anderson and Long in 1947.I Subsequently the technique was modified and used for studying the influ1E. Anderson and J. A. Long, Endocrinology 40, 92 (1947).
