W hll'l’d’fl 2 THYRC‘ED 3152222522322 222535 AS EN'FLUENSEE EV NiE'FHQfi G? MEAS 222252225227, TYPE QF ANESTHESiA .ANZ} ROUTE 522? THYE’fiXiNfiA 23-24 :NES‘E‘EA? {SN I w {'2 an 2220923 2:02: 22:22: Dawes 02 M. 3. 222222 (‘4 :2 32223 3222332212? Lawrence 302222 2322222222 29269 ' hm ‘- T, ”3'5 - L [B R R Y 2" Michigm State University ABSTRACT THYROID SECRETION RATE AS INFLUENCED BY METHOD OF MEASUREMENT, TYPE OF ANESTHESIA, AND ROUTE OF THYROXINE ADMINISTRATION BY Lawrence John Paulik Three short acting experimental anesthetics (Brevital, Evipal and Metofane) were administered to determine if they significantly effected the thyroid secretion rate (TSR) of rats when compared with a control anesthetic (Nembutal) known not to effect the TSR of these animals. Two methods for measuring TSR were used, the substitution method and the direct output method. The Carworth Farms CFN strain of rat was employed in one substitution experiment and compared with the Sprague Dawley strain used in later substitution eXperi— ments. Evipal was chosen as the anesthetic for use in a sub- sequent infusion experiment. In this infusion eXperiment, a comparison of the substitution method of once daily thyrox- ine injection versus a continuous infusion of thyroxine into unanesthetized, relatively unrestrained rats was performed using the weeks' chronic jugular cannulation procedure, harness, and rotary swivel mechanism. Group I rats were Lawrence John Paulik cannulated, harnessed and received continuous thyroxine in- fusion. Group II (controls) were cannulated, harnessed and received continuous saline infusion with once daily thyrox— ine injection. Group III (controls) were unharnessed, non- cannulated and received once daily thyroxine injection. The results of the anesthesia experiments show that there were no significant differences in the thyroid secre- tion rates of the animals under the eXperimental anesthetics when compared to the control anesthetic within a given method and employing the same strain of rat. The three direct output values "U", "K4", and "I" also show no significant differences between the three anesthetics Evipal, Metofane, and Nembutal. However, in the substitution method, the thy— roid secretion rate of Sprague Dawley rats was significantly higher than the TSR obtained by the direct output method in the same animal strain. The Carworth Farms CFN rats also showed significantly higher TSR values when compared to the Sprague Dawley strain by the substitution method. In the infusion eXperiment, Group I showed higher TSR values when compared to Groups II and III. These results indicate that the higher values of Group I were not due to any stress of the cannulation procedure or confinement in the harness, since these aspects would be reflected in GrOUp II as well, but were due to the greater quantity of infused thyroxine needed to achieve the same inhibitory effects which occur in the once daily injection of thyroxine. THYROID SECRETION RATE AS INFLUENCED BY METHOD OF MEASUREMENT, TYPE OF ANESTHESIA, AND ROUTE OF THYROXINE ADMINISTRATION BY Lawrence John Paulik A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1969 ‘b ‘C\ \‘ ‘ \ S..\ k 1.- \ DEDICATION This thesis is affectionately dedicated to my wife, Barbara E. Paulik. Her de- votion, understanding and artistic talents have made this work possible. Lawrence J. Paulik ii ACKNOWLEDGEMENTS The author wishes to eXpress his sincere appreciation to Dr. E. P. Reineke for his advice and encouragement throughout this work. Dr. Reineke's help was instrumental in the completion of the later stages of this thesis. A sincere thank you to Mrs. Judi Anderson for the iodine determinations and for her constant willingness to be of assistance throughout the various experiments. A final thanks to James R. Stiefel, Associate Profes- sor, for introducing the author to the field of physiology through his excellent course of instruction. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 5 MATERIAL AND METHODS . . . . . . . . . . . . . . . . 8 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 21 SUMMARY OF RESULTS . . . . . . . . . . . . . . . . . 38 CONCLUSION . . . . . . . . . . . . . . . . . . . . . 4O BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 42 iv LIST OF TABLES TABLE 1. Comparing thyroxine concentration by injection and by infusion, assuming a 200 g. rat . . . . . 2. Anesthesia Experiment I utilizing the substitu- tion method to measure thyroid secretion rate per 100 g. Nembutal was used as the control and compared with methohexitol sodium (Brevital) . . 5. Anesthesia Experiment II utilizing the substitu- tion method to measure thyroid secretion rate per 100 g. Nembutal was used as the control and compared with hexobarbitol sodium (Evipal) and methoxyflurane (Metofane). . . . . . . . . . . . 4. Anesthesia Experiment III utilizing the direct output method to measure thyroid secretion rate per 100 g. Nembutal was used as the control and compared with hexobarbitol sodium (Evipal) and methoxyflurane (Metofane). . . . . . . . . . . . 5. Anesthesia Experiment III direct output method comparing the three values U, K4, and I for the anesthetics Evipal, Metofane, and Nembutal . . . 6. Infusion experiment utilizing the substitution method . . . . . . . . . . . . . . . . . . . . . 7. Calculations for the determination of the per— cent of T4 cleared from plasma, hourly by the liver. . . . . . . . . . . . . . . . . . . . . . Page 20 22 25 26 29 32 LIST OF FIGURES FIGURE Page I. Cannula construction. . . . . . . . . . . . . . 14 II. Swivel mechanism. . . . . . . . . . . . . . . . 16 III. Harness for holding rat . . . . . . . . . . . . 17 IV. Entire assembly with rat attached to harness. Because the swivel turns freely, the rat can move at will without twisting the infusion tubing. . . . . . . . . . . . . . . . . . . . . 18 V. Exogenous thyroxine injection and possible pathways. . . . . . . . . . . . . . . . . . . . 36 VI. Exogenous thyroxine infusion and possible pathways. . . . . . . . . . . . . . . . . . . . 57 vi INTRODUCT I ON Before the introduction of isotopes into the research field, oxygen consumption, goiter prevention, and tadpole metamorphosis were some of the biological methods available to the thyroid physiologist in determining the functions of the thyroid gland. When isotOpes became available, the thyroid physiologist was given the tools which enabled him to more precisely measure thyroid function. Today, 1131 is helping solve the complex mysteries of iodide uptake, hormone synthesis and release, sites of accumulation, rates of degradation, pathways for excretion and absorption, as well as cellular and subcellular mechan- isms for hormone action. The thyroid substitution method has found wide appli_ cation in thyroid research. It is based on the classic negative feedback relationship which exists between the thyroid and the anterior pituitary gland. Thus, graded, daily injections of exogenous thyroxine over a period of time can effectively block the release of thyrotrOpin (TSH) from the pituitary and indicate the level of thyroid secre- tion rate (TSR). Physiologically, the thyroid gland is in a dynamic relationship with the anterior pituitary gland. It is be- lieved that small quantities of hormone are released, minute to minute as peripheral utilization of "free" thyroid hor- mone occurs. Perhaps circadian rhythms (Brody, 1945) could effectively regulate or alter the maximum and minimum quanti— ties of TSH released during specific periods of the day. This would then be reflected in the quantity of thyroxine (T4) released and utilized peripherally. The question arose as to whether small quantities of thyroxine infused over a twenty-four hour period, would be comparable to a once daily injection which is absorbed into the circulation in a matter of a few hours. This is the basis for the following study. LITERATURE REVIEW It has been pointed out by Wiener (1948) that many physiologic processes are regulated by feedback control and he has suggested that much clarification and insight may be gained by the application of servo theory to such functions. The dictionary defines a servo system as one in which the quantity of the output of an apparatus is "fed back" for the control of the system. The pituitary-thyroid axis is an excellent example of a feedback (servo) mechanism. Hoskins (1949) discussed this thyroid-pituitary servo mechanism at length and suggested that in studying this relationship between the pituitary and thyroid, two questions arise: 1) the quantitative re- lationship between the blood thyroxine titer and thyrotropin discharge, and 2) the nature and control of the setting of the pituitary governor. In studying these and other ques~ tions concerning the thyroid and pituitary, researchers have utilized methods that take into account this servo mechanism. Dempsey and Astwood (1945) introduced the goiter pre~ vention method for estimating the daily rate of thyroid hormone secretion. In this method a goitrogen was given to block thyroxine secretion from the thyroid while administer- ing graded doses of thyroxine. The animals were killed and the thyroid glands were excised, weighed and compared with the thyroid glands of control animals. Mixner, Reineke and Turner (1944) studied the effects of thiouracil and thiourea on the thyroid gland of chicks. Reineke, Mixner and Turner (1945) studied metabolism and thyroid weight in the rat by the goiter prevention technique. Schultze and Turner (1945) applied the method in studying the thyroid secretion rate in small experimental animals and fowl, as did Hurst and Turner (1947). The method, however, has several drawbacks. It cannot be used on large animals because of the great expense in sacrificing them to determine thyroid weights. It also gives only an average thyroid secretion rate for a group of animals rather than having the capability of measur- ing each individual animal's TSR. In 1959, Van Middlesworth, Jagiello and Vanderlaan showed that goiter prevention in prOpylthiouracil treated rats was only achieved through daily injections of sodium- 1-thyroxine in amounts which elevated the plasma concentra- tion of protein-bound iodine, two to three times normal. They felt that this clearly raised the possibility that the goiter-prevention method gives erroneously high values for the daily secretion rate of thyroxine by the thyroid gland. Perry (1951) utilized the radioisotOpe, iodinelal, in determining the amount of thyroxine required to suppress turnover of 1131 by the thyroid gland. He showed that when 131 rats are given I and different groups are then given 113: out» graded doses of thyroxine, inhibition of thyroidal put during a 48 hour period is proportional to the dosage of thyroxine administration. Henneman, Griffin and Reineke (1952), and Henneman, Reineke and Griffin (1955), devised a method to determine the daily thyroid secretion rate in intact individual sheep. They injected 1131 into the animals, allowed for Uptake by the thyroid gland and measured the radioactivity of the thy- roid region with a Geiger counter. L-thyroxine was then given to the animals in low concentration, the dosage being increased every three days. The counts were corrected for background and decay and the percentage of the previous cor- rected count was computed. As the quantity of 1-thyroxine injected daily was increased, the percentage of the previous count increased, indicating that the thyroid gland itself was secreting less thyroxine. When the percent of previous count reached the 100% level, this represented the animal's daily thyroid secretion rate. Reineke and Singh (1955) confirmed and extended the method on the intact rat. They observed that in rats there appears to be a small iodine turnover even in those receiving thyroxine, equivalent to their thyroid secretion rate. In normal rats, output is approximately balanced by the uptake. If organic combina~ tion of iodine is blocked by thiouracil, output, even at high thyroxine levels continues at an average rate of 4.2% per day. Thus, rats given thiouracil to prevent further thyroidal combination of 1131 during treatment, yield values higher than those not receiving thiouracil. The substitution method has become well established in studying thyroid function as related to other physiological and environmental factors. Amin, Chai and Reineke (1957) studied the thyroid activity in different strains of mice. An adaptation of the method has been used in determining the TSR of rainbow trout by Hoffert and Fromm (1959). In 1955, Lewis, Reineke and Lodge adapted the technique for thyroid secretion rate in dairy cattle. Lodge, Lewis and Reineke (1957) showed by use of the method that dairy calves were able to recycle iodine and that there were individual and breed differences in iodine assimulation. Flamboe and Reineke (1959) used the substitution method in studying dairy goats and the effects of age, pregnancy, lactation, and seasonal variations on thyroid function. Reineke, Travis and Kifer (1960) measured thyroid parameters in the mink. Recently, Reineke and Lorscheider (1967) found that the thyw 131 output cannot be totally inhibited by thy— roid gland I roxine injections. In thyroids maximally blocked by T4 injection, 1131 is still released at the rate of 1.0 to 1.2 percent per day. They concluded that a more accurate end‘ point for measuring the TSR is the 97.5% of previous count level rather than the 100% level previously used. Nowhere in the literature to date has anyone continu- ously infused thyroxine for the purpose of measuring and comparing the TSR derived by infusion with that derived by the substitution method of once daily thyroxine injection, and indeed, this is why this research work was undertaken. MATERIALS AND METHODS General The experiment was divided into two parts: The first dealt with finding a suitable anesthetic, capable of anes- thetizing the animal for approximately twenty minutes, but not interfering with thyroid secretion rate. The second dealt with chronic infusion of thyroxine into the rat via jugular cannulation. Anesthesia Experiment Perry (1951) originally showed that sodium pentabarbital (Nembutal, Abbott Laboratories, North Chicago, Ill.) does not cause any functional change in thyroid secretion rate. However, Nembutal anesthesia is of too long duration for use with rats restrained in the harness employed in later experi- ments. Therefore, an eXperiment was done to compare the thyroid secretion rate of rats immobilized with three short- acting anesthetics. Nembutal anesthesia was used as the con— trol. The experimental anesthetics used were: 1) Methohexi- tol sodium (Brevital, Eli Lilly and Company, Indianapolis, Ind.), a short acting, intravenously administered anesthetic, 2) Hexobarbitol sodium (Evipal, Winthrop Laboratories, New York, N. Y.) given intraperitoneally, and 5) Methoxyflurane (Metofane, Pitman Moore, Indianapolis, Ind.) an inhalant. The dosages used for each anesthetic were: Brevital--2% solution--0.1 ml/100 g. body wt. intravenously. Evipa --6% solution--0.1 ml/100 gm. body wt. intraperitoneal. Metofane--inhalant given to effect. Nembutal--5% solution--0.1 ml/100 gm. body wt. intraperitoneal. The animals used for the Brevital experiment were female rats from the Carworth Farms CFN strain weighing 180 to 200 grams and maintained on Wayne Lab. BLOX. All other experiments were performed on Sprague Dawley female rats weighing 180 to 200 grams and fed Zinn's Rat Feed. The laboratory changed from the CFN strain to the Sprague Dawley strain after experiencing difficulty in acclimating the CFN rats to a harness mechanism used later in the infusion experiment. Upon recommendation of the breeder, Zinn's Rat Feed was fed to the Sprague Dawley animals after a comparison between the two feeds showed both had similar nutritional and iodine contents. The rat room temperature was maintained at 76.: 20 F and lights were on 14 hours daily. All animals had an adequate water supply. 10 The measurement of TSR in the Brevital experiment was done by the substitution method (Reineke and Singh, 1955). The anesthetic was administered via tail vein injection while the animal was contained in a cylindrical restraining device. The Evipal and Metofane anesthetics were compared with sodium pentabarbital using the substitution method and the direct output method (Reineke and Lorscheider, 1967). Counting Method All animals received subcutaneous injections of 5 to 5 moi of carrier-free sodium iodide (1131). Approximately 72 hours were allowed for I131 fixation by the thyroid and elimination of excess isotOpe by the kidneys and other routes. In_viyg_radioactivity counts were taken with a 2-inch thallium—activated sodium iodide crystal connected to a radiation analyzer-sealer. The rat was positioned over the crystal so maximum counts over the thyroid region were ob- tained by observing a count rate meter. Two to three thyroid counts of one minute duration were then taken on the scaler. A one minute count of the epigastric region was taken to obtain the animal's body background count. Standards of 1/10th the injected dose were counted for one minute. The percent of injected dose over the thyroid region was then calculated from the equations 11 . _ epigastric count general Percent of TthOld count ( 2 + background) injected = 100 dose (Standard count - general background) x 10 The log of percent injected dose was plotted against time. The Substitution Method In the substitution method, thyroid counts were taken on alternate days until an output lepe of usually three points could be plotted. An alkaline saline solution of L-thyroxine (T4), 0.5 ug./100 g. body wt., was injected into the animal after the third count for two successive days. A higher concentration of 1.0 ug. T4/100 g. was injected following the fourth count again for the next two days. The concentration was increased after each count by 0.5 ug. T4/ 100 9. until a leveling off or inhibition of the thyroid I131 output was achieved. Each count was eXpressed as per» cent of the previous count and plotted against T4 concentra~ tion on linear graph paper. Inhibition was considered to occur at the 97.5% level as demonstrated by Reineke and Lorscheider (1967). The T4 dosage corresponding to the 97.5% level then gave the concentration of exogenous thyroxine needed to block TSH release and, therefore, was considered to be equal to the animal's daily endogenous thyroid secre~ tion rate. 12 Direct Output Method The direct output method is based on the fractional daily output of the thyroid gland times the thyroxine equiva- lent of the total iodine released from the thyroid gland. The TSR is calculated as follows: I131 turnover rate constant = X = QLQEQ T3: . _ ' _ mxt _ Fractional turnover - K4 — 1 - e (t — 1 day) where e is the base of the natural logarithms I131 actual turnover rate constant (corrected for the recycling of metabolized iodide to the thyroid) = I =_§-4__ K4 1-U where U = zero time uptake Hormonal iodine released = output rate K4 x iodine content of thyroid T4 activity, T4 equivalent = K4 x total thyroid iodine (mg.) x 1.53 x 1.52, the factor 1.52 is used to allow for the proportional contribu_ tion of triiodothyronine (T3) presumed to be released from the thyroid. Thyroid Substitution by_Intravenous Infusion Cannula Construction (1) (3) (4) (5) The cannulas used for infusion (Weeks and Davis, 1964) were constructed of 26 gauge needle tubing (Small Parts, Inc., Biscayne Annex, Miami, Fla.), silicone rubber tubing 15 and polyethylene as indicated in Figure I. The ridges were formed on the polyethylene tubing by passing a wire stylus through the lumen (Heatley and Weeks, 1964). Rotation of the tubing on the stylus wire in presence of a jet of hot air, together with linear compression formed the ridges. The flared pieces were made by holding the tubing near an open flame. A silicone adhesive (Clear Sealerm-Dow Corning Corporation, Midland, Michigan) was used to give the conm necting joint flexibility. Cannula Implantation The surgery was performed on 180 to 200 g. female rats, anesthetized with Metofane. The hair was shaved from the dorsal side of the neck region and over the right jugular area on the ventral side. An incision was made just to the left of the midline in the dorsal neck region. A transverse incision was also made over the jugular region. Approxim mately 5 mm. of the jugular vein was exposed by blunt dim section. A curved hemostat was inserted subcutaneously into the incision in the dorsal neck region and worked posteriorly around the right foreleg to the ventral skin incision. The cannula was then clamped (polyethylene side) with the hem0m stat and pulled back through the dorsal incision. A syringe containing 0.85% NaCl was attached to the polyethylene end of the cannula. The vein was punctured with a 25 gauge needle and the silicone rubber portion of the cannula was 14 .coauosnumcoo MHDGGMU .H wusmflm mwam anon pm omam>mn .EE OH numsma .mmsmm mm ,mcflnsa mawmmz Hmmpm mmmacflmpm mono nuOQ um comma“ as mungumcmq om .m.m u HM mono Umumamcoc Eoum .EE wilmwmoflm m .pcm pmumam .EE obllnumcmq IIIIII EOHM .EE Nilmmoflm d manned. Hmnnsm mcooHHHm n MMMM .EE Owaalnumcmq ON .m.m n g w%mM 05mmflu m>fluuwccoo Ho maomsa maflpcsouusm ou pmuozocm mucflom mcflnzusm GHm> HmHDmDfl Quad pmpummcfl mafinsp mo COHAHom 0000000 .0900000 000000000 000 000 < m>Hmm£Um COUHHHm wmpflm Hmmm Amman paw Umumam 15 inserted into the vein. The suturing points were then attached to surrounding muscle tissue. The connective tis- sue on the ventral side was reunited over the cannula with absorbable sutures, and wound clips were used to close both incisions. The animal was allowed a week to recover from the Operation before it was acclimated an additional week in the harness. The Harness and Pump The harness used in this method allows the rat to pivot freely because of a swivel mechanism (Lehigh Valley Elec- tronics, Fogelsville, Pa.)(see Figures II through IV). When the rat is securely placed in the harness it cannot get at the cannula, yet is not restrained from moving freely around the cage. The thyroxine solution was infused into the animal by using a Sage constant speed syringe pump (Model 254~7, Sage Instruments, Inc., White Plains, New York) modified to push four syringes simultaneously. One c.c. tuberculin syringes were used. The flow rates were varied by changing the gear ratio. A minimum flow rate of 0.25 ml/day up to a maximum flow rate of 0.57 ml/day could thus be achieved. 16 Tubing inlet *-————) E (a ,‘ho Swivel ———> l‘~ ,2 I. v Tubing outlet -————-9 (_ .Spring extending to the harness Figure II. Swivel mechanism. 17 Spring attached to swivel Figure III. Harness for holding the rat. 18 Figure IV. Entire assembly with rat attached to harness. Because the swivel turns freely, the rat can move at will without twisting the infusion tubing. 19 Once Daily Injection vs. Chronic Infusion of T4 The eXperiment was performed on 5 groups of 4 rats each. The first group contained cannulated rats in harnesses, receiving thyroxine by infusion. The second group contained cannulated rats in harnesses, receiving 0.85% saline by in— fusion, but subcutaneous thyroxine injections. The third group contained non—cannulated, unharnessed rats receiving subcutaneous thyroxine injections. Calculating the Amount of Té Infused per 100 Grams of Rat First pump speed equals 0.25 ml/day ‘Thyroxine concentration equals 6 ug/ml 0.46 ml/48 hours of infusion x 6 ug/ml = 2.76 ug/48 hr. infusion Average weight of rat between the 5rd and 4th count equals 209 g. 2.76 ug/2.09 g. = 1.52 ug T4/100 g./48 hr. 0.66 ug T4/100 g./24 hr. infusion All new solutions were completely flushed through the polyethylene tubing from the pump to the junction point where the polyethylene tubing meets the cannula on the rat before a new infusion was begun. Table 1 20 . Comparing thyroxine concentration by injection and by infusion, assuming a 200 g. rat. LA Thyroxine dosage and concentration by infusion expressed as micrograms. T4/100 9-/24 hr. Concentration T4/ml Days_post 1131 0.7 pg. 6 ug. 9-11 1.05 pg. 6 pg. 12—14 1.80 ug. 6 pg. 15-17 2.28 ug. 8 ug. 18-20 2.70 pg. 9 pg. 21-25 5.50 pg. 11 pg. 24-26 Graded thyroxine dosage by injection. pg, 100 0.5 1.0 1.5 2.0 2.5 5.0 g rat 229. 229 - 229 - 229 - 229 - 229 - Daysgpost 1131 injection 9-11 12—14 15-17 18-20 21-25 24-26 RESULTS AND DISCUSSION Anesthesia is a necessity when taking thyroid radio- activity counts on small animals. However, the anesthetic must not alter thyroid secretion rate. Anesthesia Experi— ments I through III were performed to find a suitable anes— thetic for the harnessed rats. The control anesthetic, Nembutal, was compared with experimental anesthetics by two different methods (substitution and direct output) for measurement of TSR. Table 2 shows the results of the first experiment. Nembutal was compared with Brevital (intravenous injection) by the substitution method. The average thyroid secretion rate of the rats under Brevital anesthesia was 1.94.: 0.159 pg? of thyroxine per 100 g. of rat with a range of 1.15 ug. to 5.10 ug./100§L Nembutal gave an average TSR of 2.14.: 0.220 ug./100 g. T4 with a range of 1.50 to 5.58 ug./100 g. The student "t" test (Goldstein, 1964) was performed on the data and showed the difference to be non-significant (P.> 0.10). In Anesthesia EXperiment II (Table 5), Nembutal was compared with Evipal (subcutaneous injection) and Metofane (veterinary inhalant) by the substitution method. The average *Standard error of the mean. 22 Table 2. Anesthesia Experiment I utilizing the substitu- tion method to measure thyroid secretion rate per 100 g. Nembutal used as the control and compared with methohexitol sodium (Brevital). Brevital Nembutal Rat No. TSR/100 g./day Rat No. TSR/100 g./day 1 5.100 1 5.575 2 2.415 2 5.125 5 2.150 5 5.050 4 2.125 4 2.165 5 1.950 5 2.100 6 1.875 6 2.050 7 1.850 7 1.850 8 1.825 8 1.575 9 1.475 9 1.525 10 1.450 10 1.425 11 1.125 11 1.500 Average TSR/100 g./day* 1.94 i.0.159 (11) Average TSR/100 g./day 2.14 i.0.220 (11) *- Mean 4 SE (N) 25 Table 5. Anesthesia Experiment II utilizing the substitution method to measure thyroid secretion rate per 100 g. Nembutal was used as the control and compared with hexobarbitol sodium (Evipal) and methoxyflurane (Metofane). Rat Evipal Metofane Nembutal Number TSR/100 g./day TSR/100 g. TSR/100 g./day 1 2.800 2.900 2.125 2 1.950 1.850 1.875 5 1.875 1.700 1.875 4 1.800 1.600 1.825 5 1.775 1.525 1.675 6 1.575 1.550 1.675 7 1.550 1.225 1.625 8 1.550 1.100 1.500 9 1.475 1.000 1.525 10 1.225 Average TSR/100 g2 1.76 .2; 0.154(10) 1.58 _+_ 0.189(9) 1.72 1 0.078(9) * Mean : S.E. (N). 24 TSR for Evipal, Metofane, and Nembutal was 1.76 :_0.154 ug./ 100 g., 1.58 i_0.189 ug./100 g., and 1.72 i 0.078 Hg./100 g., respectively. Evipal showed a range of 1.25 ug. to 2.80 ug./100 g. Metofane showed 1.00 ug. to 2.90 ug. T4/100 g. and Nembutal gave a range of 1.55 Hg. to 2.15 Mg. T4/100 g. The student "t" test showed a non—significant difference (P > 0.10) between the three groups. In experiment I, female rats from the Carworth Farms CFN strain were used whereas all other experiments were per- formed on Sprague Dawley female rats. Inasmuch as there were no differences between groups within either strain, a single mean was calculated for each strain of rat. The mean TSR's of the CFN and Sprague Dawley strains, respectively, were 2.04 1.0.154 ug./100 g., and 1.69 i 0.080 ug./100 g. This is a significant difference P2< 0.05 level. In eXperiment III (Table 4), Nembutal was again compared with Evipal and Metofane using the direct output method. Brevital was not further tested because it would not have been possible to give intravenous injections to harnessed rats via tail vein. Table 4 gives an average TSR for Evipal of 1.15 i.0.096 ug. T4 100 g. rat with a range of 0.656 ug. to 1.472%} T4/100 g. Metofane showed a TSR average of 1.16 i 0.129 ug./100 g. with a range of 0.775 ug. to 1.72 ug T4/100 g. The Nembutal control averaged 1.51 1.0.121 ug./100 g. with a range of 0.850 ug. to 1.76 ug. T4/100 g. The student "t" test showed a non-significant difference P.> 0.10 for the three groups. In Table 5, the values of "U", the percent 25 Table 4. Anesthesia Experiment III utilizing the direct out- put method to measure thyroid secretion rate per 100 g. Nembutal was used as the control and com- pared with hexobarbitol sodium (Evipal) and methoxyflurane (Metofane). *Rat Evipal Metofane Nembutal Number TSR/100g./day TSR/100g./day TSR/100g./day 1 1.470 1.725 1.757 2 1.585 1.578 1.414 5 1.515 1.557 1.590 4 1.271 1.064 1.278 5 1.266 0.976 1.196 6 1.262 0.859 0.850 7 0.877 0.775 8 0.841 9 0.656 Average * TSR/100g. 1.15 i,0.096(9) 1.16 i_0.129(7) 1.51 : 0.121(6) Mean i_S.E. (N) sz .m.m + cmmz* Lee .2 Ame Ame Ape Ape Ase Ame Ame Ame mnm.N + 26 SodH 3:. H 08;” H 30.8.. MNNAH mom; H moo. H 304 H me.>H >0mo.o mm.afi oao.mfi ommo.o aw.m mNH.Nfi mmoa.o mm.oa *mmsam> mmmum>¢ omm.m wmmo.o mm.dfi mmo.fifi mdmo.o om.> mmo.«fi $580.0 m4.0fi 00m.> Hamfi.o m>.m oom.ma wmwo.o mw.m mm¢.dd Nmmo.o «m.m mmm.om wo~o.o mm.m ma¢.m meow.o m>.mfi me.mfi wmmo.o ma.m mdo.aa mmmo.o no.0H omw.wfi memo.o do.> oom.m mwmfi.o ma.oa www.mfi mmmo.o mw.m mao.afi mmdd.o mm.oa omm.ma mmmO.o >m.ma oom.¢fi ddmo.o om.0d oom.>m oemo.o md.m wam.>a memo.o ma.> www.mfi mmfid.o am.mfi mmo.mm Hmmo.o oa.mfi www.mfi wmmfi.o >m.ma m>¢.afi memfi.o oo.wfi AmsvH «M D ASEVH ex : AmsvH «m D amusnfimz mamm0pmz Hmmfl>m .HMHDQEmz Cam mammoumz .mefl>m mUHuwnumwcm on» How H cam .2X .5 mmsHm> mwnnu mzu mafiummEOO .Uonumfi usmuso uomufln .HHH ucwEflnmmxm MHmmnumwcd .m manna 27 of thyroidal 1131 uptake extrapolated to zero time, "K4", the fractional I131 output rate per day corrected for re- cycling of metabolized 1131, and "I", the total iodine present in the thyroid gland were compared for each of the three anesthetics (Evipal, Metofane, and Nembutal) used in the direct output experiment. All three parameters, ”U", "K4", and "I" gave non—significant differences between groups using the student "t" test (P.> 0.10). The average "U" values for Evipal, Metofane, and Nembutal were 10.29_: 1.041, 9.81.: 1.225, and 11.56 i 2.428 respectively. This shows that the experimental anesthetics did not alter or radically change the uptake of I131 by the thyroid gland when compared to the control anesthetic. The "K4" values were 0.1025 1.0.008 for Evipal, 0.0950 1.0.015 for Metofane, and 0.08007 : 0.011 for Nembutal. Here again, the experi- mental anesthetic values show non-significant differences from the control anesthetic indicating no gross altera~ tions in the animals' fractional output or release of I131 from the thyroid gland. The values for total iodine ("I“) present in the thyroid gland were 12.425,i 1.505 pg. for Evipal, 15.010.i 1.510 ug. for Metofane, and 17.469 1.2.875 ug. for Nembutal. The non—significance of the experimental values when compared to the control, show no adverse changes in the iodine content of the thyroid gland. All of these values indicate no physiological change in the normal func- tion of the thyroid gland due to repeated use of these experimental anesthetics. 28 The thyroid secretion rate obtained by the substitu~ tion method in Sprague Dawley rats was consistently higher than the TSR obtained by the direct output method in the same strain of rat. Since there were no significant dif- ferences between either method due to the anesthetics used, the values for each method were combined. The overall average TSR's were 1.69 1.0.080 ug./100 g. by the substitu- tion method and 1.20 i_0.064 ug./100 g. by the direct output method. The difference was highly significant by the stun dent "t" test (P‘< 0.01). Singh and Reineke (1968), measur~ ing TSR in the chicken, obtained values by the direct output method that were one-half as large as those obtained using other methods for measuring TSR. Evipal was chosen as the anesthesia for the infusion experiments. It is short acting, has minimal side effects, and, as the tables indicate, does not effect the TSR. Thyroid secretion rates of rats receiving thyroxine by continuous infusion are compared in Table 6 with two control grOUps in which the T4 was injected once daily. Group I had a mean TSR of 2.84.: 0.175 Mg. T4/100 g. which was sig~ nificantly higher than either the mean TSR value of 2.12_i 0.248 g. T4/100 g. Group II ( P <.0.05) or the mean TSR value of 1.86 i,0.147 ug. T4/100 g. for Group III (P < 0.01). Group I ranged from 1.70 ug. to 5.45 ug. T4/100 9. Groups II and III had a range of 1.40 ug. to 5.95 ug. T4/100 g. and 1.18 Hg. to 5.28 ug. T4/100 g., respectively. The student "t" test gave a non-significant difference (P £u msocmmoxm mo mwmzfipmm mHQHmmom .> musmflm mmomm zH smog nzm ome¢obezoo qum 2H 49 ome¢opezoo oz¢ mmmm Vw mzHemmezH A49 wqmzez .qum 2H mmmmmmozH oneoame mqmoqom qoz¢ebm mo moz«m¢mqo AonaeqnomHo .mmmmmmozH onsmmezmozoo «a wee qmsmomv oooum oezH.wvmmmeHneHe.2v. Mooqm aooqm some 49 mmamqo mm>Hq ommmommmmm 29 meme umoneoznm oneommm szqua 09 nzpom we mm>02mm wAQHmam mmoz mm>HA + 4 szqu¢ 09 waxes: ozwwm «a mm>Hq 2H mnemoem szDmummmm Aomev zHADmoqo oansz mszomwme omeampeam .mqowo nooum nooqm oezH onsomnzH mszomwme nmmmomm< wqumHN .. mpomzaesomnm mpozmooxm 57 .COHmswcH unflxoumzu msocmmOXm mo mwm3£umm manflmmom .H> musmflm mmomm ZH BmOA .ATIIII mZHBmMBZH A WQHm A mm>HA Mm QflBdODbZOO \\\N\ 99 mo mqm>mq son mm>Hq 2H moamosm .MHHHHHHHHHHHHHHHHHHHU\\ mszeomm oooqm % oansz ZHZqufimmm mzom EBHS OmB Omsmmbfidm MAEOAm ”MQUNU QOOAm I, ZOHmDmZH BOQm SUMMARY OF RESULTS Three short acting experimental anesthetics (Brevital, Evipal, and Metofane) were administered to determine if they significantly effected the thyroid secretion rate of rats when compared with a control anesthetic (Nembutal) known not to effect the TSR of these animals. The experi— mental anesthetics were compared to the control anesthetic by two separate methods for measuring the thyroid secretion rate; namely, the direct output method and the subsitution method. The Carworth Farms CFN strain of rat was employed in one substitution experiment and compared with the Sprague Dawley strain used in a later substitution eXperiment. Evipal was chosen as the best anesthetic for use in the subsequent infusion experiment. In this infusion eXperiment, the substitution method employing once daily thyroxine in~ jections was compared to a continuous infusion of thyroxine into unanesthetized, relatively unrestrained laboratory rats. The weeks' chronic jugular cannulation procedure, harness, and rotary swivel mechanism was employed to achieve the eXperimental results. Two sets of control animals were used. One group was cannulated, harnessed, and received continuous 58 59 saline infusion with once daily thyroxine injection. The other control group was unharnessed, non—cannulated and received once daily thyroxine injections. The experimental group of rats were cannulated, harnessed, and received continuous thyroxine infusion. The two sets of controls were compared to each other to determine if any stress due to the Weeks‘ procedure was pre- sent and also compared to the experimental rats to observe any significant difference between the continuous infusion of thyroxine versus the once daily thyroxine injection. CONCLUSION g 1) The results of the anesthesia experiments I, II, and III show that there is no significant difference in the thyroid secretion rate of the animals under the eXperi- mental anesthetics (Evipal, Metofane and Brevital) when compared to the control anesthetic (Nembutal) Within a given method and employing the same strain of rat. 2) The thyroid secretion rate obtained by the substitu- tion method in Sprague Dawley rats was significantly higher than the thyroid secretion rate obtained by the direct out~ put method in the same strain of rat. The overall average thyroid secretion rates were 1.69 3.0.080 ug. T4/100 g./day by the substitution method and 1.20 1.0.064 ug. T4/100 g./ day by the direct output method. The difference was highly significant (P >v0.01). 5) The three direct output parameters "U", "K4” and “I" showed non—significant differences (P >-0.10) for the three anesthetics, Evipal, Metofane, and Nembutal by the student "t" test. 4) The Carworth Farms CFN strain of rat gave a mean thyroid secretion rate of 2.04 i 0.154 ug. T4/100 g./day for the substitution method. The Sprague Dawley rats gave 40 41 a mean TSR of 1.69 1 0.80 ug. T4/100 g./day for the same method. A significant difference (P >>0.05) in the thyroid secretion rate exists between these two strains. 5) In the infusion experiment Group II and Group III controls had mean TSR values of 2.12 1.0.248 ug. T4/100 g./ day and 1.86 1.0.147 ug. T4/100 g./day, respectively. The student ”t" test gave a non-significant difference (P'< 0.10) between the two control groups. The Group I experimental animals had a mean TSR of 2.84.1 0.175 ug. T4/100 g./day which was significantly higher than either the mean TSR value for Group II (P <10.05) or the mean TSR value for Group III (P'< 0.01). The results therefore showed that the higher TSR values of Group I were not due to any stress of the cannulation procedure or confinement in the harness, since these aspects would be reflected in Group II as well, but were due to the significantly greater quantity of in- fused thyroxine needed to achieve the same inhibitory effects which occur in the once daily injection of thyroxine. BIBLIOGRAPHY Albert, A. and F. R. Keating, 1952. The Role of the Gastro- intestinal Tract, Including Liver, in the Metabolism of Radiothyroxine. Endocrinology, 51:427—445. Amin, A., C. K. Chai, and E. P. Reineke, 1957. Differences in Thyroid Activity of Several Strains of Mice and F1 Hybrids. Am. J. Physiol., 191:54-56. Best, C. and N. Taylor, 1950. Physiological Basis of Medical Practice. Baltimore, The Williams and Wilkins Company, p. 18. Brody, S., 1945. 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