RABBIT UTERINE CONT RACTILE ACTIVITY AS MONITORED IN VITRO WITH EXTRALUMINAL CONTRACTILE FORCE TRANSDUCERS Thesis for the Degree of M . S . MIEHIGAN STATE UNIVERSITY CAROL A . ROWLAND 1967 Tum LIBRARY Michigan 5:393 Ummfs‘, $377 ' 'J-gmp-f 800K BINBERY INC. LIBRARY BINDERS * "Imus" ammu- f‘i “'Egafi ~‘.. .1, : amomc av _ L "ME & SUNS' ] ABSTRACT RABBIT UTERINE CONTRACTILE ACTIVITY AS MONITORED IN VITRO WITH EXTRALUMINAL CONTRACTILE FORCE TRANSDUCERS by Carol A. Rowland Although contractile activity patterns of the uterus for the ovarian stages have been established both in vitro and in vivo, no in vitro isometric technique exists for quantification of contractile activity of the longitudinal and circular muscle layers. The purpose of this study is to develop a reliable isometric method for quantification of rabbit uterine in vitro contractile activity. An extraluminal contractile force transducer was waterproofed for in vitro use with consecutive coats of solvent-thinned nitrile rubber solution and subsequent coats of rubber-like epoxy resin. The resulting trans- ducer was smaller in surface area and more convenient for in vitro use than those previously fabricated for in vivo use. The transducers were calibrated following quality control tests for hysteresis, waterproofing and temperature compensation. The ovariectomized, estrogen-treated rabbits were killed and the uterus immediately removed and placed in Carol A. Rowland warmed, oxygenated Ringer-Locke solution for approximately one hour. At this time the extraluminal transducers were affixed to the uterine muscle segment with sutures fas- tened through holes drilled in the transducer shim stock. The transducers were arranged in a number of configura- tions. The segment was suspended in a tissue bath via a Grass transducer. A control segment was similarly sus- pended without attached extraluminal transducers. Re- cordings of contractile activity were obtained on an ink- writing oscillograph. Control records were obtained; then drugs were added to the bath in an effort to differentiate the con- tractile activity of the two muscle layers. Paired com— parison tests were used to determine whether the presence of the transducers on the muscle segment affected their in vitro contractile activity. Analysis of control records showed that extra- luminal transducers were measuring contractile activity of the longitudinal and circular muscle layers separately and simultaneously from the same segment. Oxytocin, va- sopressin, and acetylcholine elicited differential re— sponses from the two muscle layers. The presence of the transducers on the muscle segment did not affect its contractile activity. Longi- tudinally oriented extraluminal transducers detected an average contractile force of 0.57 g./burst while the Carol A. Rowland circularly oriented extraluminal transducer detected an average contractile force of 0.90 g./burst. Average frequency of bursts for both muscle layers was 0.54 bursts/min. Analyzing simultaneous Grass transducer recordings, no statistically significant differences were found between the frequencies or contractile forces of the segments with the extraluminal transducers at- tached and the control segments without extraluminal transducers attached. The longitudinally oriented extraluminal trans- ducer recorded apparent decreased tone when a high ten- sion was set with the attaching sutures. Approved: Date: RABBIT UTERINE CONTRACTILE ACTIVITY AS MONITORED IN VITRO WITH EXTRALUMINAL CONTRACTILE FORCE TRANSDUCERS BY Carol A. Rowland A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1967 ACKNOWLEDGMENTS The author wishes to express sincere appreciation to Dr. David A. Reinke for patience, guidance, assistance and understanding with respect to her endeavors. The author expresses gratitude to the members of her graduate committee, Dr. W. D. Collings and Dr. E. P. Reineke. The author also wishes to acknowledge the fol- lowing: Donna Miller, her mother, for encouragement and technical assistance. David Auerbach for counsel, encouragement and understanding throughout this study. ii TABLE OF CONTENTS ACKNOWLEDGMENTS. . . . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . . . . . Chapter I. INTRODUCTION. . . . . . . . . . . . Review of in vitro methods. . . . Isotonic Recordings . . . . . . Isometric Recordings. . . . . . Statement of the Problem. . . . . II 0 METHODS O O O I O I O O O O O O O O Contractile Force Transducer. . . Transducer Fabrication. . . . . . Transducer Circuitry and Recording Instrument. . . . . . Transducer Calibration. . . . . . Experimental Procedure. . . . . . Drug Concentration Calculations Statistical Analysis of Data. . III 0 RESULTS 0 O O O O O O O O O I O O O Contractile Activity Patterns . . Frequency of Contractile Activity Force of Contractile Activity . . Effects of Drug Stimulation on Contractile Activity . . . . IV. DISCUSSION. 0 O O O O O O O O O O O Contractile Activity Patterns . . Frequency of Contractile Activity Effects of Drug Stimulation . . . v 0 SUMMARY 0 O O O O O O O O O O O O O BIBLIOGRAPHY . . . . . . . . . . . . . . . iii Patterns. Page ii iv 14 15 23 29 29 30 30 4O 45 48 55 55 56 58 61 63 LIST OF TABLES Table Page 1. Comparison of the frequencies of the experimental and control muscle segments. . . . . . . . . . 46 2. Comparison of contractile force developed by the experi- mental and control muscle segments . . . . 47 3. Comparison of contractile force develOped by the longitudinal and circular muscle layers of the same muscle segment. . . . . 49 iv LIST OF FIGURES Figure Page 1. Extraluminal contractile force transducer. . l6 2. Calibration curves . . . . . . . . . . . . . l9 3. Calibration curve for Grass transducer . . . 21 4. Orientations of extraluminal transducers in relation to the Grass FT-03 transducer . . 27 5. Spontaneous contractile activity patterns obtained under the 3VTD configuration. . . 31 6. Spontaneous contractile activity patterns obtained from the 3VTD configuration . . . 33 7. Comparison of contractile activity recorded by a circularly oriented extraluminal transducer and a Grass FT-03 transducer under direction tension . 36 8. Comparison of contractile activity re- corded by a longitudinally oriented extraluminal transducer and a Grass FT-03 transducer under direct tension. . . . . . 38 9. Spontaneous contractile activity pat- terns obtained under indirect tension. . . 41 10. "Mirror image" recording by the longitudi- nally oriented extraluminal transducer . . 43 11. Contractile reSponse to acetylcholine and vasopressin. . . . . . . 50 12. Contractile response to oxytocin . . . . . . 53 CHAPTER ONE INTRODUCTION Early investigators of uterine motility did not maintain a physiological environment; the uterus was ex- posed to air and no attempt was made to maintain body temperature (18). Subsequently, three classes of tech- niques have been devised for studying uterine contractile activity: in vivo, in situ, and in vitro. Luduena (25) advocates use of the in vitro tech- niques for studying properties of mammalian smooth muscle because of the following advantages: 1. The simplicity of the in vitro method provides conditions which permit effective control of variables. 2. The effect of temperature, pH, and electrolyte concentrations on tonus, Spontaneous contractile activity and drug responses can be studied in vitro, but these variables cannot be easily al- tered in vivo. 3. Several segments of a single tissue may be studied simultaneously. This allows accumula- tion of drug information in a shorter period of time and enables selection of the initial drug dosage to be administered to the intact animal. A review of in vitro techniques will show the lack of a reliable isometric method for recording the discrete contractile activity of the different uterine muscle layers. Review of In Vitro Methods Reynolds (34,35) pointed out that many of the dis- crepancies found in the literature regarding the physiology and pharmacology of uterine muscle could be due to the vari- ations in recording technique. Csapo and Corner (16) were the first (1952) to use the isometric technique in vitro in the study of uterine muscle. Prior to this time, isotonic methods had been used. Szent-Gyorgyi and Hadju (45), after investigating the properties of actomyosin, stated that isotonic record- ing may register only the percent of shortening of the muscle strip, while giving no indication of the amount of tension developed. Csapo (14) investigated the effects of temperature on the development of isometric tension and isotonic shortening. He concluded that isotonic shortening of an unloaded uterus is maximal at lower tem- peratures while tension is minimal. A similar effect was obtained by altering the strength of stimulation. Isotonic shortening may be maximal without having maximum muscle activation. Both investigations revealed that the isometric method of recording was a better quantitative estimate of the work the uterine muscle was capable of doing, and gave a better understanding of myometrial function. Isotonic Recordings. Some of the first reliable isotonic recordingstaf rabbit uterine contractile activity occurred in the laboratories of Cushny (18) and Dale (19) in 1906. After anesthetizing the animal with paraldehyde given by stomach tube, cannulas were inserted into the carotid arteries, the jugular vein, and the trachea. A midline abdominal incision was made to the peritoneum. The uterus was exposed after the animal was immersed in physiological saline preheated to body temperature. Two small stitches l" apart were taken in the horn; the threads were connected to levers of an apparatus resembling a myo- cardiograph. The stitches were oriented in either the longitudinal or circular direction. In some pregnant does simultaneous recording of the activity of the two muscle layers was possible by taking four stitches and using a double system of levers. In most preparations the hypo- gastric nerve was severed prior to recording. Contractile activity was recorded with concurrent observation of the motility of the uterus. The contractile activity patterns of rabbits in estrus showed slow, rhythmical contractions which varied in frequency from one contraction every thirty seconds to every three to four minutes. The cir- cular layer contractions were similar to the longitudinal layer contractions. When longitudinal and circular muscle contractions were recorded simultaneously, the contractions were not usually simultaneous. Sometimes the circular layer contracted without the longitudinal layer contrac- ting. If circular and longitudinal contractions did occur simultaneously, the longitudinal was usually masked by the contraction of the circular layer; Cushny stated that the longitudinal muscle showed relaxation on the record. In 1907 Kehrer (24), (according to Csapo and Cor- ner (10)), made pioneer in vitro drug studies on virgin rabbit uteri using the discovery of Rudolf Magnus (26) that strips of cat intestine contracted when put in warm oxygenated saline. Isotonic recordings of the excised myometrium could be made by attaching the muscle to a lever. Later Athias (1) used this method on the guinea pig uteri to demonstrate a definite relationship between the ovaries and uterine motility existed. He ovariec- tomized the animals to show the disappearance of uterine contractile activity; he transplanted ovarian tissue to the castrate and showed the return of contractile activ- ity. In 1923 Corner (9) showed that different in vitro contractile activity patterns existed for each stage of the ovarian cycle in the sow. In estrus the contractions were slower (1.5-2.5 minutes apart) and of greater ampli- tude than after ovulation. Ten days after ovulation the rate was incrased to 6-8 contractions per minute with an amplitude only one-fifth that of estrus contractions. In pregnancy the sow uterus was quiescent. Robson (37,38), using the Trendelenburg method‘ (46), recorded both longitudinal and circular contractile activity of the rabbit myometrium. This method involved recording of the longitudinal muscle contractile activity via an isotonic lever. Changes in intra-uterine pressure represented circular muscle contractile activity. Before suSpending the muscle in the bath, a glass cannula was in- serted into the uterine lumen through a small incision made in the horn. After the cannula was tied in place, warm Ringer-Locke solution was passed through the lumen. The appropriate segment was then tied off, removed, and placed in the bath. In ovariectomized rabbits, Robson (36) found that pretreatment with crystalline ketohy- droxoestrone increased the Spontaneous activity, and reactivity to oxytocin as compared with results ob- tained on nontreated castrates. In 1935 Simeone (43) used cat uteri to demonstrate the effect of initial tension on spontaneous activity. The uterus was exposed by an abdominal midline incision, during Dial anesthesia, and one horn freed by ligation of the ovarian blood vessels and section of the hypogastric nerve. The vaginal end of the uterus was held in place by a dissecting needle while the ovarian end was attached to a light weight writing lever in apposition to a weight pan. The whole horn was then enclosed in a glass cylinder to insure adequate moisture and warmth. The uterus was raised to a vertical position by adjusting the weight in the pan. Care was taken not to stretch the muscle from its resting length which was the Optimum tension for maxi- mum contractile amplitude. Further stretching of the mus- cle rendered it less excitable; an increase in tension caused a decrease in the amplitude of the spontaneous contractions. Bonnycastle and Ferguson (5) used a different ap- proach to the problem of studying the separate muscle layers in the rabbit myometrium. The segment to be stud- ied was separated into its longitudinal and circular com- ponents before being suspended in the bath. The effects of oxytocin (Pitocin) and epinephrine (Adrenalin) on these muscle layers from different levels in the uterus were in- vestigated. Three day prepartum and two day postpartum rabbits were used. These investigators found that in the postpartum rabbit (which is under estrogen dominance ac- cording to Schofield (42)) the longitudinal muscle layer was more sensitive to oxytocin than the circular muscle layer, while the circular muscle layer was more responsive to epinephrine. Isometric Recordings. Csapo and Corner (16) stud- ied ovarian hormones in relation to contraction cycle du- ration and to the "staircase" phenomenon, which had first been observed in cardiac muscle. An isometric method of recording was used for the first time in this in vitro study on contractile activity of the rabbit myometrium, as the isometric method had previously been shown by Szent-Gyorgyi and Hadju to yield more quantitative re- sults (45). Their study involved frog cardiac muscle. The uterine horns were placed in oxygenated Krebs' solu- tion at 0°C. for ten minutes, because at this temperature the uterus reached a state of minimum energy expenditure and established a steady "equilibrium" length 20% shorter than the "resting" length. A muscle segment 25 mm. long was then suspended between two platinum electrodes in a vertical glass tube filled with warm, oxygenated Krebs' solution. The upper hook was attached by a silk thread to the short arm of a lever which was connected to a flat torsion Spring. The long arm of the lever recorded the contraction. This conventional isometric tension recorder allowed less than 6% Shortening of the muscle segment. These authors found that estrogen dominated uteri ex- hibited a "positive" staircase (a frequency of l/min. electrical stimulation after a resting period of ten minutes caused an increase in the contractile amplitude develOped without tone change), while progesterone domi- nated uteri exhibited a "negative" staircase (electrical stimulation at a frequency of l/min. or greater than l/min. caused a decrease in contractile amplitude de- velOped without a change in tone). Estrogen dominance increased while progesterone dominance decreased the du- ration of the contraction-relaxation cycle (the time from beginning of contraction to the end of relaxation) when compared to the contraction-relaxation cycle of the castrate rabbit's uterus. In 1953 Csapo and Goodall (17), using the same in vitro isometric technique, determined that the muscle was most capable of developing maximum contraction ampli- tude at the "resting" length. In 1954 Csapo (l4) dis- cussed the adjustment of the muscle to obtain this "resting" length. The muscle was first put in Kreb's solution at 0°C. where it obtained its equilibrium length which was approximately 20% shorter than the "resting" length. It was then suspended between two hooks in Krebs' solution at body temperature, where it elongated after ten minutes to a length 25% longer than the equilibrium length. If the distance between the two hooks was increased, the muscle strip lengthened; it adjusted to the new length by an initial increase in the resting tension followed by a decrease (indicated by a slight rise in the tracing above the baseline and then a return to the baseline or below). If this limit of muscle adjustment was overstepped, the tracing did not return to the baseline. Subsequent in- crease in stretch of the muscle segment brought a still greater increase in amplitude above the baseline. The point at which the muscle fiber could not adjust to the new length without an accompanying increase in tension was designated as the "resting" length. This is the pres- ent standard for adjustment of "resting" tension in in vitro work, although more recent investigators have omitted putting the muscle in 0°C. bathing solution be- fore starting (20,21,28). In 1965 Miller and Marshall (28) used the tech- nique first developed by Varagic (47) for an isolated hypogastric nerve-uterine muscle preparation for studying the effects of stimulation on the rabbit myometrium. The animal was "bled out" to facilitate identification of the nerves. The nerves were tied 5—7 mm. below the bifurca- tion of the abdominal aorta.‘ The bladder was then removed to make the dissection easier. The uterus and nerves, along with a piece of colon, were removed intact. The nerves were then carefully isolated. Miller and Marshall used only a segment of either horn while Varagic immersed the whole uterus in the bath. The segment, including the cervix, was tied by the cervical end to a hook in the or- gan bath. The other end was attached to a Grass force displacement transducer, FT-03, which measured isometric tension deve10ped by the segment. The nerves were threaded through insulated platinum electrodes connected to a Grass stimulator. They concluded that the rabbit uterus con- tains both a-excitatory and B-inhibitory adrenergic re- ceptors with the former predominating in the estrogen— dominated rabbit. Diamond and Brody (20,21) investigated the rela- tionship between in vitro contraction of rat uteri, the 10 biochemical changes occurring in the muscle, and the ef- fect of catecholamines on its contractile activity. They used an in vitro isometric technique, but allowed the mus- cle to equilibrate in a warm, oxygenated bathing solution, rather than a 0° solution. Resting tension was adjusted according to the method of Csapo (14). They concluded that the estrogen-primed rat uterus contains both a-ex- citatory and B-inhibitory receptors with the latter pre- dominating. Statement of the Problem Although accepted contractile activity patterns for the ovarian stages have been established both in vitro (4,9,10,16,18,28) and in vivo (2,8,32,33,41,42), this re- view has Shown that no in vitro isometric technique exists for the quantification of the contractile activity of the uterine circular and longitudinal muscle layers. The extraluminal contractile force transducer has been developed and used in vivo for recording contractile activity of the smooth muscle layers in the gut and uterus (2,3,8,23,30,31,39)._ This transducer is unidirectional (it measures only contractile activity of that muscle layer with which its longitudinal axis is aligned). In yizg in the uterus (2) and in the gut (3), this transducer with an attached electrode has been used to measure elec- trical and contractile activity; correlations have been made from the data obtained. 11 The transducer was modified by fabricating without the silicone rubber protection used in vivo. The resulting transducer was smaller in surface area and allowed conven- ient in vitro use. A combination of the Grass force dis- placement transducer with the extraluminal contractile force transducers measuring the contractile activity of the muscle layers on the same segment would allow a dis- crete monitoring of the separate muscle layers simultane- ously and an analysis of the contractile activity which is measured by the Grass transducer. The purpose of this thesis is to develOp an in zitrg isometric method for quantification of rabbit uter- ine muscle layer contractile activity. This will be at- tempted using the extraluminal contractile force trans- ducer. CHAPTER TWO METHODS Contractile Force Transducer The extraluminal contractile force transducer moni- tors the contractile activity of that portion of the muscle layer which is beneath the two ends of the transducer. The transducer consists of two resistance strain gages bonded one to each side of an arched metal shim stock. The prin- ciple of this method of contractile activity measurement is based on the detection of linear deformation of-the strain gage grid, which may result from muscle contraction or relaxation, either active or passive. The force of this contractile activity is detected by a change in the elec- trical resistance of the strain gage grid. Discrete activ- ity of the muscle layer may be measured by orienting the longitudinal axis of the grid parallel to the muscle fibers of the muscle layer to be monitored. A detailed description of the transducer fabrica- tion used for in vivo recording, along with a list of mate- rials required, has been given (23,30). A description of the modification of this fabrication for in vitro work follows. 12 13 Transducer Fabrication Figure 1 shows a schematic drawing of the trans- ducer. The 2.5 X 6.0 mm. shim stock was cut from 2-1/2% beryllium c0pperl alloy Sheets, in the "quarter hard" con- dition. The shim stock was 0.15 mm. thick. A cylindrical mold was used to press the shim stock into an arch of 6 mm. diameter. The curved shim stocks were then heat cured for four hours at 600°F. After removal from the oven, the heat treated shim stocks were immediately placed in room-temperature distilled water. They were inspected, polished with Silicon carbide paper,2 and cleaned with "metal conditioner"2 and isoprOpyl alcohol to improve the surface for bonding. The square cor- ners were rounded off with a file to eliminate the poss- bility of trauma to the muscle. A jeweler's drill (approxi- mately 0.75 mm. diameter, #65) was used to drill one small hole in each end, to permit the transducer to be sewn di- rectly on the muscle. Two strain gages3 were bonded, one to each side of the shim stock, using a filled epoxy cement.4 1Compliments of the Beryllium Corp., Reading, Pa. 2Materials described may be obtained from William T. Bean, 18915 Grand River Ave., Detroit, Michigan. 3Catalogue #SK-09-031DE-350. Micro-measurements, Inc., Romulus, Michigan. 4Tatnall GA—5 epoxy cement. Instruments Division, Budd Co., Phoenixville, Pa. 14 Bonding pressure of one pound was applied via neg'ator clamps5 while the unit was being heat cured at 220°F. for two hours. A three-conductor lead wire system was used to compensate for the heat-induced lead wire resistance changes. The lead wire was Teflon-insulated, with seven strands per conductor.5 The bared ends of the lead wires were soldered with Special strain gage solder5 to the four soldering tabs of the strain gage, as shown in Fig- ure 1. After soldering, the solder joints were cleaned with rosin solvents and the whole transducer was coated with three coats of Gagekote #25 (drying time of 15 min- utes per coat), followed by two coats of Gagekote #55 (drying time of 6 hours per coat). Needles were placed in the holes in the shim stock to prevent filling the holes with the water-proofing materials. The combination of these two materials completely waterproofed the trans- ducer for immersion in the in vitro bath. Transducer Circuitry and Recording Instrument The two-gage transducer was incorporated into a double active arm Wheatstone half-bridge circuit. The "free" end of the lead wire was soldered to a nine pin— position Cannon plug (MDl-9SL1),6 which connected the 5Materials described may be obtained from William T. Bean, 18915 Grand River Ave., Detroit 23, Michigan. 6ITT Cannon Electric, Inc., Los Angeles, California. 15 two resistance elements located within the transducer to two external resistors located outside and adjacent to the strain gage coupler of the ink-writing oscillograph7 used in this study. Sensitivities of 0.05 and 0.02 millivolts per centimeter pen deflection were used in recording con- tractile activity via the extraluminal contractile force transducers. Sensitivities of 0.5, 0.2 and 0.1 millivolts per centimeter pen deflection were used in recording con- tractile activity via the Grass force displacement trans- ducer. A paper speed of 12.5 mm./min., 25.0 mm./min., 0.1 cm./sec. or 0.5 cm./sec. was used to record contractile activity. Transducer Calibration The transducers were calibrated to determine the contractile force deve10ped by each muscle layer. Initially calibration of the circularly oriented transducer was done according to the method of Reinke (30). This consisted of taping the transducer to a supporting rod to secure it in a horizontal position. A MersileneCmO suture9 was fastened to the transducer and passed through a small hole located 7Type R Dynograph, Beckman Instruments, Inc., Spinco Division, Schiller Park, Illinois. 8Model FT-03 without springs. Grass Instrument Co., Quincy, Mass. 9Mersilene 000 suture, Ethicon #R-552, Ethicon, Inc., Somerville, N. J. 16 .HO>MH odomse maamcfiosuflmcoa on“ no mufl>flpom mafluomuucoo mcflpuoowu How com: on o» moonwmcmuu we» no cowumucmfluo mufiz coma one .mla .Hmmma maomSE “masouflo mcu mo wufl>fluom oafluomnucoo mcflpuoomu How poms on on noospmcmuu may no coflumucmauo onwz coma one .mna .xooum Eflnm 50mm mo moflm m>mocoo on“ ou pmccon ma comm cflmnum Hmnuoad .mcfimooumnmumz ou Hoaum cowumucmwuo oHH3lpmmH com muoogpmcmuu on» no coflumuumsaafi oaumEEmHmmao m Hmospmcmuu money mawuomuucoo HmcHESHmuuxm .H musmflm 18 in each end, under the transducer and over a pulley on the Opposite Side. Weights were hung from the sutures to Simu- late contractions of the muscle. A different method of calibration of the longi- tudinally oriented transducer was necessary due to the orientation of its lead wire. The longitudinally oriented transducer was secured in a horizontal position by clamp- ing the non-lead wire end of the transducer in a vise. Contraction was simulated by hanging weights from a Mer- silene 000 suture passed through the hole and fastened to the lead wire end of the transducer, a modification of the calibration procedure.of Jacoby (23). A calibration curve obtained by Jacoby's method for the circularly oriented transducer showed that a lesser degree of deflection was developed per gram by this method than by Reinke's method. The orientation of the lead wires for the longitudinally oriented extralumi- nal transducers did not allow it to be calibrated accord- ing to Reinke's method.' Calibration curves obtained us- ing Jacoby's method were used to compare contractile force deve10ped by the muscle layers. These curves are shown in Figure 2—A and B. The Grass FT-03 transducers were calibrated by suspending weights from the muscle attachment point. A calibration curve obtained in this manner is Shown in Figure 3. Figure 2. l9 Calibration curves A. B. A typical calibration curve for a circularly oriented transducer. By knowing the de- flection of the pen in mm., the grams of force developed by a particular contraction can be read directly from this graph. A typical calibration curve for a longi- tudinally oriented transducer. By knowing the deflection of the pen for a contraction recorded by this transducer, the force of contraction can be calculated from this graph. a. Sensitivity at 0.05 mv./cm. b. Sensitivity at 0.1 mv./cm. c. Sensitivity at 0.2 mv.cm. Units--millimeters. A0 20 20 O PEN . DEFLECTION IO . b UNITS " . C o I 2 3 4 5 LOAD (GRAMS) a. 2. . :: O PEN DEFLECTION l0 .. .. b UNITS " " 0:234'5 I AAHIflBAIID\ 21 Figure 3. Calibration curve for Grass transducer A typical calibration curve for a Grass FT-03 transducer. By knowing the deflection of the pen in mm., the grams of force deve10ped by a particular contraction can be read directly from this graph. 40 so PEN DEFLECTION 2° umrs ' IO 0.| vacm. 22 0.2 mv./cm. 0.5 vacm. IO I5 20 25 LOAD(GRAMS) 23 The air around the transducers was warmed to 37-38°C. by a heat lamp and stabilized at that temperature for the entire calibration period, to eliminate the difference that might have occurred between calibration at room temperature and bath temperature. The stability and waterproofing of the transducers was tested by completing the bridge curcuit to the Dyno- graph recorder and immersing them in water warmed to 37°C. for one-half hour., If any deviation from the baseline oc- curred, the transducers were discarded. They were tested for hysteresis during the calibration period, by rapid addition and removal of a weight. If the pen deflection did not return to its initial baseline position, the transducer was not used. Experimental Procedure It is well known that rabbits are induced ovula- tors; they may ovulate due to some stimulus other than coitus (mechanical stimulation to the cervix, play with other doe». This produces a pseudopregnant condition in which progesterone is the dominant hormone and the uterus shows a contractile activity pattern which differs from the contractile activity pattern of the estrous state. It was difficult to ascertain the ovarian state of the rabbit by external examination. In an effort to standardize experimental conditions 16 of the 24 rabbits tested were ovariectomized, then pretreated with 100 pg. 24 of estradiol cypionatelo daily for five days prior to use. This treatment has been shown to simulate the estrous stage of the rabbit (17,22,41). The rabbits were killed by a blow on the head. The uterine horns were immediately removed and placed in Ringer-Locke solution at 37°C. The composition of the medium was as follows: NaCl, 9.0 g.; KCl, 0.42 g.; CaClz, 0.24 9.; MgCl 0.20 g.; NaHCO3, 0.50 g.; and d-glucose, 2, 0.50 g. (q.s. to 1000 ml. with distilled water). The solution was gassed with a mixture of 95% oxygen and 5% carbon dioxide.r The muscle was allowed to equilibrate under these conditions; during this time it relaxed and lengthened. It was necessary to allow the muscle to equilibrate for 45-60 minutes before sewing on the trans- ducers; if they were sewn on before complete equilibra- tion had occured, the muscle wrinkled under the trans- ducer and reduced the ability of the transducer to regis- ter contractile activity. Mersilene 000 suture was used to sew the trans- ducers on the muscle. The tissue was kept immersed in Ringer-Locke solution during most of the procedure; care was taken to handle the tissue as little as possible. Two sutures were used to secure each transducer on the muscle. After the extraluminal transducers were sewn 10Upjohn Company, Kalamazoo, Michigan. 25 on the muscle, a segment approximately 20 mm. long was cut and it, with the attached transducers, was suSpended ver- tically in a double-walled, preheated bath which contained Ringer-Locke solution, which was continually being gassed with 95% oxygen and 5% carbon dioxide mixture. The seg- ment was connected to a glass rod via a platinum hook on one end, and the other end was connected with thread to a Grass force displacement transducer (FT-O3, without springs). The transducer arrangement was as follows: 1. A muscle segment approximately 20 mm. long, with two extraluminal contractile force transducers attached, was connected to a Grass force displace- ment transducer.. The extraluminal transducers were arranged in a vertical-inverted "T" formation on the antimesometrial side of the segment and in a direct line with the tension exerted on the seg- ment by the attaChment of the Grass force dis- placement transducer. The longitudinally oriented extraluminal transducer was positioned above and perpendicular to the circularly oriented extra- luminal transducer. Another segment, cut from the opposite horn at approximately the same level, was suspended in a second bath and connected only to a Grass transducer as a control segment without extraluminal transducers attached. This arrange- ment shall be referred to as 3VTD (3 transducers in a vertically-inverted "T" formation in direct line with the tension exerted on the muscle seg- ment by the Grass force displacment transducer). Refer to Figure 4-A. Another group of recordings was obtained with the following transducer arrangements: 2. Two adjacent muscle segments, 15 mm. long, were suspended in separate baths.- One extraluminal transducer measured the contractile activity of the circular muscle layer of one segment, while a Grass FT-03 transducer measured contractile 26 activity of the entire segment. A second extra- luminal transducer measured the contractile activity of the longitudinal muscle layer in the second seg- ment, while a second Grass FT-03 transducer measured contractile activity of this entire segment. This arrangement shall be referred to as 2D (2 trans- ducers attached to one muscle segment; the extra- luminal transducer in a direct line with the ten- sion exerted on the muscle segment by the Grass FT-03 transducer). 3. To determine whether contractile activity of the muscle layers differed at the same level in the segment, the extraluminal transducers were placed in a horizontal "T" formation, with the circularly oriented transducer on the mesometrial side of the segment and the longitudinally oriented transducer on the antimesometrial Side. The Grass FT-03 transducer was attached at a point midway between the two extraluminal transducers. A control seg- ment was set up as in #1 above. This arrangement shall be referred to as 3HTI (3 transducers at- tached to one segment; the extraluminal transducers in a horizontal "T" formation and not in a direct line with the tension exerted on the segment by the attachment of the Grass FT-03 transducer). Refer to Figure 4-B. Resting tension, as determined by recording from the Grass transducer, was adjusted according to the method of Csapo as explained on page 8 of this thesis. A slight increment in tension of the muscle under the extraluminal transducer was set at the time the transducer was sewn on. The distance between the attachment sutures was 3.5 mm. Spontaneous contractile activity usually occurred within one hour after suspension of the uterine segment in the in vitro bath. Numerous control records were obtained; then drugs were used to show differential responses of the outer longitudinal and inner circular muscle layers, known to be present in rabbit uteri (ll,27,29,33). 27 Figure 4. Orientation of extraluminal transducers in relation to the Grass FT-03 transducer A. Orientation of the extraluminal transducer in a vertically-inverted "T" formation in a direct line with the resting tension exerted on the muscle segment by the Grass FT-03 transducer. This configuration Shall be designated as 3VTD (3 transduc- ers; two extraluminal transducers in a vertically-inverted "T" under direct ten- sion). Orientation of the extraluminal transducers in a horizontal "T" formation when the resting tension exerted on the muscle seg- ment by the Grass FT-03 transducer is not in a direct line with the transducers. This configuration shall be designated as 3HTI (3 transducers; two extraluminal transducers in a horizontal "T" and not under direct tension). G--Grass FT-03 transducer thread connection. C--circularly oriented extraluminal trans- ducer. L--Longitudinally oriented extraluminal transducer. 28 E D S I.— C G \ a; v‘ .. . . as... ....... .i -.\.. .....11/:1.-Ih/ G L C m A ‘bwflaflbmoflV "II. S Aflvgf - \ O > I‘ll, O ”I II I I I I I I I I ‘l ‘. MESO METRIAL .(‘llI-l.‘llllllllllll"l.1 inll!‘ Ill-'1‘ 11". III-Illa... II... IIIIIII...II 29 Drug Concentration Calculations Effective drug dosages are reported in terms of molarity of final bath concentration. Initially the drug dose administered to the bath was in terms of micrograms (ug.) or units/total bath capacity. A typical final bath concentration calculation is that for acetylcholine (ACh). Bath capacity 80 ml. Initial drug dose 40159. Molecular Weight of Acetylcholine 163 g. A one molar solution contains one gram molecular weight/liter. 1 molar ACh = 163 g./l. Given: 40 INL ACh/80 ml. = 0.5 pg./ml. bath concentrations. Then: 0.5 pg. ACh/ml. = 0.5 mg./l. = 5 x 10'4 g./1. The final bath concentration in molarity is calculated in the following manner: 163 g.ACh/l. = 5 x 10‘4 g. ACh/l. 1 molar x molar 5 x 10"4 -6 x molar = 9: = 3.0 x 10 molar. 163 g. Statistical Analysis of Data In an effort to detect a difference between con- tractile activity of the experimental and control muscle segments, a paired comparison11 was made of data obtained. 11Frederick E. Croxton, Elementary Statistics with Applications in Medicine and the Biological Sciences,_ Dover Publications, Inc., New York, 1953. CHAP TE R TH RE E RESULTS Characteristic activity patterns for both the longitudinal and circular muscle layers, recorded sepa- rately and/or Simultaneously in vitro, were obtained for the estrogen dominated rabbit myometrium using the extra- luminal contractile force transducers. Correlations be- tween the contractile activity recorded by these trans- ducers and the contractile activity recorded by the Grass FT-03 transducer were made. Contractile Activity Patterns Two types of contractile activity records were ob- tained using the three transducer arrangements.‘ The record obtained depended on whether the extraluminal force trans- ducers were in the 3VTD or the 3HTI configuration. The first type of contractile activity pattern re- corded by the extraluminal transducers and the Grass FT-03 transducer occurred when the 3VTD and the 2D configurations were used. Two typical examples of Spontaneous contractile activity patterns obtained from a 3VTD transducer configu- ration are shown in Figures 5 and 6. The results obtained when the extraluminal trans- ducers were aligned with the tension exerted on the muscle 30 31 .cHE\.EE m.mallpmmmm momma .Hmosomcmuu moIBm mmmuw on» we popuoomu muw>fluom mafluomHDSOOIIO .Hoospmcmuu Hmcfladamnuxm poucmwuo maamcflpsuflmcoH ha omouooon Mufi>wuom mHHDOMHHSOOIIA .Hmospmcmup HmcHEsHmupxm pmucmfluo mammasonflo >9 popuooou >DH>Huom mafluomuucoollu .c0flumusmwmcoo QB>m m CH pmucmfluo mums mucosomcmnu may sows3 cw .mcflouoomn m mo mcwomuu was» SH c303m ma >ua>wuom mawpomuucoo msomcmucomm coflumusmflmcoo oe>m map Hops: omcflmuno mcumuumm >ua>wuom mawuomnucoo msomcmucomm .m musmflm 33 .Hmospmcmnu melhm mmmuw m an pmusmmme muH>Huom mHSUUMHDGOOIIO .uwospmcmup Hmcwfidamuuxm omucmfino adamcflpsuamcoH m an UmHSmmmE >DH>fluom oHHDOMHDGOOIIA .umUSUmcmnu Hmcflasamuuxm omucmfluo haumasouflo m an Umusmmme mufl>fiuom mafiuomuucoollu .omm\.Eo H.ollommmm ummmm .Hmospmcmuu HmcHEsHmHuxm poucmfluo adamcwpsuflmcoH on» an oopuoomn mcwmn soaps Ixmaou usmnmamw mzonm mcHoHoomu M NO mcwomuu mane coaumusmflmcoo QB>m mnu Eoum pmcfimuno mcumuumm mufl>fiuom maauomuucoo msomcmucomm .m musmflm 35 segment by the Grass FT-03 transducer may be summarized as follows: 1. The Grass FT-03 transducer and the transversely oriented extraluminal contractile force trans- ducer registered contractions simultaneously, while the longitudinally oriented extraluminal contractile force transducer showed superimposed contractions on an apparent decrease in tone. 2. A contraction (superimposed on decreased tone) re- corded by the longitudinally oriented extraluminal transducer was also recorded (without decreased tone) by the Grass FT-03 transducer, while the transversely oriented extraluminal transducer was recording an initial apparent relaxation followed by a contraction of the circular muscle. 3. The Grass FT-03 transducer, the transversely oriented extraluminal transducer, and the longi- tudinally oriented extraluminal transducer often registered contraction Simultaneously. Recordings from the transversely oriented extra- luminal transducer and the Grass FT-03 transducer (Figure 7) which were attached to the same muscle segment showed the following results: 1. The Grass FT-03 transducer recorded Simultaneous contractions with the transversely oriented extra- luminal transducer. 2. The two transducers often recorded contraction peaks which did not coincide. Recordings from the longitudinally oriented extra- luminal transducer and the Grass FT-03 transducer (Figure 8) which monitored contractile activity of the same muscle seg- ment showed the following results: 1. The Grass FT-03 transducer recorded simultaneous contractions with the longitudinally oriented extraluminal contractile force transducer. Figure 7. 36 Comparison of contractile activity recorded by a circularly oriented extraluminal trans- ducer and a Grass FT-03 transducer under direct tension These tracings Show a recording of the contrac— tile activity of a muscle segment recorded by the circularly oriented extraluminal trans- ducer and a Grass FT-03 transducer. The transducer arrangement was the ZD configura- tion. Paper Speed--upper record, 25 mm./min.; lower record, 5 mm./sec. C--contractile activity recorded by the cir- cularly oriented extraluminal transducer. G--contractile activity recorded by the Grass FT-03 transducer. —--~-,— —- h‘ 37 ' 0.l MIN ' 38 .Hoospmcmuu mouBm mmmuo onu we popuooou huw>fluoo oafluoonucooIIw .Hoosomconu Hocflfidaouuxo poucofluo aaaocfiosu Iflmcoa on» >3 popuooon wufl>wuoo oafipooHDGOOIIA .oom\.Eo H.OIIUoomm Homom .cofluouomflmcoo Om on» SH pomcouuo oHo3 muoosomcouu one .uoosomcouu melam mmouw m can Hoospmcouu Hocflfidaouuxo poacofluo maaocflpsuflmcoH onu an popuooou ucosbom oHomSE o no >ua>fluoo oafluoonucoo on» mo mafipuooon o m3onm mcfloouu mane coflmcou Doouflp Hops: nonopmcouu melam momuo m was nooso Imcouu Hocasdaouuxo poucofluo waaochSDAmcoH m we popuooon >ua>fluoo oaauomuucoo mo GOmAHMQEou .m ousmflm 39 3 HI/W 2:2 3.0 H J). .. JI- 4O 2. The Grass FT-03 transducer showed a contraction peak while the longitudinally oriented extraluminal transducer showed an initial contraction peak, and then a slight relaxation followed by another con- traction peak. The second contractile activity pattern recorded by the extraluminal transducers and the Grass FT-03 trans- ducer occurred when the 3HTI configuration was used. Tracings of recordings from this configuration are shown in Figures 9 and 10. These two figures Show the following results: 1. The characteristics of this contractile activity of the circular muscle layer does not differ from that obtained by the 3VTD or 2D configurations. 2. The contractile activity of the longitudinal mus- cle layer differs greatly from that obtained when the 3VTD or 2D configuration was used. a. The contractile activity of the longitudinal muscle Shows a decreased tone with superimposed contractions. Close examination shows that this contractile activity approximates a mirror image of the contractile activity recorded by the Grass FT-03 transducer. b. Figure 10 (the polarity of the recording pen for the longitudinally oriented extraluminal transducer was reversed) shows that the con- tractile activity recorded by the longitudi- nally oriented extraluminal transducer is a mirror image of the contractile activity re- corded by the Grass FT-03 transducer. Super- imposed on this passive relaxation are con- tractions of the longitudinal muscle located under the extraluminal transducer. Frequency of Contractile Activity The frequency of bursts of contractile activity (number of bursts/minute) for both muscle layers in the isolated estrogen primed rabbit myometrium did not differ 41 .uoosomnouu mouem moonw on» an oopuooou >HH>Huoo oHHuoouunooIIw .HoUSpmnmuu HmnHEsHmuuxo pounoHHo wHHonHosu IHmnHH onn an popuooon >HH>Huom oHHuUMHHCOOIIA .uoospmnmuu HonHEsHouuxo pounoHHo >HHoHsouHo on» an poonooou muH>Huoo oHHuooHHGOOIIO - .oom\.Eo H.¢Iuooomm uomom .noHuousmHmnoo Hemm onu nH pomnouuo ouo3 muoospmnouu one .Hoospmnouu pounoHHo mHHMHso IMHO on» mo noHuHmom onu mo omoHpuomoH ponHouno ouo3 Hoon oHomse HoasouHo onu scum monHUHooou uoHHEHm .oon HoHHuoEomoEHuno onu Eoum mcHoHoo now was Hoospmconu pounoHHo mHHoansuHmnoH onn oHHn3 unoemom oHomSE onu Ho opHm HoHHuoEOmoE onu Eoum mnHouooou mo3 Hoosomnouu monem moouw one .mnHoonu mHnu nH nsonm mH Hoospmnouu momma on» an popuooou >DH>Huoo oHHuomuunoo on» mo woodponouu HonHEdeuuxo pounoHHo >HHmannuHmnoH onu mn mnHoHoooH =omoEH HOHHHE= ouoEonummo one nOHmnou pooHHan moons ponHou Ino mnuouuom mquHuom oHHuoouunoo msoonounomm .m ousmHm 43 .Hoosomnonu melee mmouo on» an popuooon wuH>Huoo oHHuooHuGOOIIO .AthHoHom pomuo>ouv Hoozpmnouu HonHESHoHuxo pounoHuo wHHonHUSH IHmGOH onu an oopuooou >UH>Huom oHHuoouunooIIq .nHE\.EEmNIIUoomm Homom .Hemm mo3 SOHuonsmHmnoo nonopmnoue .oUHm HoHHHoEOmoEHunm on» Sony muH>Huoo oHHuooHunoo mnHouoooH mo3 Houdpmnouu melee mmouw onu oHHn3 .oon HoHHuoEOmoE on» no Hoon oHomSE HonHonHmnoH onu mo >HH>Hu loo oHHuooHunoo mnHHoanOE mo3 Hooaomnouu Hon IHEsHopro one .GOHuoouunoo monoOchH mnHooHu uo3oH onu CH nom onu mo noHuoonop phosmn no oHHn3 .noHuoonoH mouooHonH mnHooHu momma onu nH com on» Ho COHuoonop ouo3ms an .oomuo> Ion noon won HoOSUmnoHu HonHEsHmnuxo pounoHHo SaamcnosuHmaoH on» an cmuouHcos suH>Huom mHH» Ioouunoo onu mnHUHoooH nom onu mo muHHoHom one uoonpmnonp HonHEDHoHuxo pounoHHo MHHonHosuHmnoH on» an manHooou =omoEH HOHHH2= .OH ousmHm A): e 3 is .37 . 43.12% illj JR: 45 for any one segment. A burst is defined as an increase in contractile amplitude consisting of one or more contractions which may be superimposed on a tone increase. The range of frequencies of bursts for all rabbits tested was from 0.18 to l.08/min., with an average frequency of 0.54 bursts/minute. In those experiments which were designed to deter- mine whether the presence of the extraluminal transducers affected frequency, no statistically significant difference in frequencies was found between the segments with attached transducers and the control segments. Data used in this comparison are shown in Table 1. Force of Contractile Activity The average force developed by the uterine muscle layers during spontaneous activity was determined for all segments with transducers arranged in the 3VTD or 3HTI con- figuration. The average contractile force, per burst as measured by the Grass FT-03 transducers, was 12.8 9. To determine whether the presence of the extraluminal trans- ducers affected contractile force developed, a paired com- parison was made between the force developed by the muscle segment with the attached extraluminal transducers and the control muscle segment without extraluminal transducers attached. No statistically Significant difference was found, as shown in Table 2. A paired comparison between the average contractile force per burst developed by the circular and longitudinal 46 Table 1. Comparison of the frequencies* of the experi- mental** and control muscle segments Rabbit Cogtrol Experimental X D-=S D2 1 2 l 2 1 0.28 0.18 0.10 0.0100 2 0.42 0.42 0.00 0.0000 3 0.48 0.25 0.23 0.0529 4 1.0 1.08 -0.08 0.0064 5 0.83 0.33 0.50 0.2500 6 0.22 0.21 0.01 0.0001 7 1.0 0.44 0.66 0.4356 8 0.71 0.90 -0.19 0.0361 Total 4.94 3.81 (1.23 0.7911 *Bursts/minute. **The transducers were arranged in the 3VTD or 3HTI configuration. XD = 0.1537; CD = 0.2932; OED = 0.1036 T = 1.48 p >0.10--At this level these differences in fre- quencies are not statistically significant. 47 Table 2. Comparison of contractile force* deve10ped by the experimental** and control muscle segments as monitored by Grass FT-03 transducers :- L Rabbit Cogtrol Experimental X D-=x D2 . l 2 l 2 1 9.5 18.0 -8.5 72.25 2 18.4 18.0 0.4 0.16 3 20.0 9.5 10.5 110.25 4 11.0 15.0 -4.0 16.0 5 5.5 6.2 -0.7 0.49 6 16.0 7.0 9.0 81.00 Total 80.4 73.7 6.7 280.05 *Grams; the summation of maximum amplitude of contractile activity of bursts divided by number of bursts. **Transducers were arranged in the 3VTD or 3HTI configuration. XD = 1.11; CD = 7.38; OED = 3.01 T = 0.368 p > 0.05--At this level these differences in contractile force are not significant. 48 muscle layers indicated that there was no statistically Sig- nificant difference (see Table 3). Effects of Drug Stimulation on Contractile Activity To establish further the differentiation between the contractile activity of the longitudinal and circular muscle layers, drugs were added to the in vitro baths. Re- sults from the longitudinal and circular muscle layers of the same segment were compared at the same time. Acetylcholine chloride, in a final bath concentration of 3.0 X 10.6 molar of base, caused a sustained increase in tone in the circular muscle layer which surpassed any tone measured in the control activity. The contractile amplitude was greater than any amplitude recorded in the control bursts. The longitudinal muscle did not Show an increase in tone. There appeared to be numerous contractions super- imposed on the decreased tone; the amplitude of these mul- tiple contractions did not appear to differ from the con; trol. A tracing from a recording of this drug's action on the muscle is shown in Figure ll-A. I Contractile activity, monitored by both the Grass FT-03 and the circularly oriented extraluminal transducers, in response to vaSOpressin in a bath concentration of 6.2 x 10'2 units/ml., was similar to the responses de- scribed previously to acetylcholine administration, al- though vasopressin allowed less complete relaxation ini- tially. This response was recorded from the same muscle .Illlll 'lllllac'l"! llul.ln1 1' III-Ill l.‘ llll." (Ilf I. I f I I III 49 Table 3. Comparison of contractile force* deve10ped by the longitudinal and circular muscle layers of the the same muscle segment as monitored by extra- luminal transducers Circular Longitudinal Rabbit muscle muscle D = D2 X - X X X l 2 l 2 l 0.50 1.0 -0.50 0.2500 2 0.76 0.24 0.52 0.2704 3 1.50 1.25 0.25 0.0625 4 1.40 0.25 1.15 1.3225 5 0.35 0.10 0.25 0.0625 Total 4.51 2.84 1.67 1.9679 *Grams; the summation of the maximum amplitude of contractile activity of bursts divided by number of bursts. XD = 0.335; CD = 0.373; OED = 0.166 T = 2.01 p > 0.10--At this level these differences in contractile force are not Significant. Figure 11. 50 Contractile responses to acetylcholine and vaSOpressin A. This tracing of a recording shows the contractile responses of the muscle layers to acetylcholine (3.0 X 10‘6 molar bath concentration). The circu- lar muscle responded with a sustained increase in tone and greater contractile amplitude than any control contractile amplitude. The longitudinal muscle ap- peared to Show an increased frequency of contractions superimposed on de- creased tone. B. This tendency from the same recording as the tracing described above shows the contractile response of the muscle layers to vasopressin (6.2 X 10"2 units/ml. bath concentration). The longitudinal muscle Shows an increase in tone with increased frequency of contractile activity. C--contractile activity as measured by the circularly oriented extraluminal trans- ducer. L--contractile activity as measured by the longitudinally oriented extraluminal transducer. G--contractile activity as measured by the Grass FT-03 transducer. Arrow indicates drug delivery. 52 segment as the previous description for response to acetyl- choline. However, the contractile response of the longi- tudinal muscle layer was markedly different from that re- sponse recorded after the administration of acetylcholine. The longitudinally oriented extraluminal transducer meas- ured contractile activity which coincided with that re- corded by the Grass FT-03 and the extraluminal transducer. A tracing of this recording is shown in Figure ll-B. Oxytocin in a bath concentration of 6.2 X 10-3 units/ml. elicited a similar response as that recorded from the longitudinal muscle after acetylcholine admin- istration. Following initial tone increase in the cir- cular muscle, more rhythmic, higher amplitude contractions were recorded as Opposed to the control bursts recorded from this muscle layer. A tracing of the response of the muscle segment to this drug is shown in Figure 12. Figure 12. 53 Contractile response to oxytocin This tracing of a recording shows the con- tractile response of the muscle layers to oxytocin (bath concentration of 6.2 X 10"2 units/ml.). The circular muscle layer shows a sustained increase in tone followed by higher amplitude, more rhythmic concentrations than obtained in the control bursts. The longitudinal muscle layer shows a response similar to that obtained from the addition of acetylcholine to the bath--increased fre- quency of contractions superimposed on de- creased tone. C--contractile activity as measured by the circularly oriented extraluminal trans- ducer. L--contractile activity as measured by the longitudinally oriented extraluminal trans- ducer. G--contracti1e activity as measured by the Grass FT-O3 transducer. Arrow indicates drug delivery. 54 CHAPTER FOUR DISCUSSION Contractile ActiviteratternS Contractile activity patterns for the estrous stage of the ovarian cycle have been described for many Species (4,9,18,34). This contractile activity is generally de- scribed as rhythmic, Slow, and of greater amplitude than contractile activity found in a different stage in the cycle. Reynolds (35) describes the motility of the intact uterus during estrus as being initiated by a pacemaker lo- cated in the isthmus of the Fallopian tube. These contrac- tions begin in the circular fibers at intervals of 7-15 seconds; they cause first a lengthening of the tube, fol- lowed by a shortening which is due to contraction of the longitudinal fibers. These contraction waves spread to the utero-tubal junction where only every third or fourth contraction continues into the horn. The above description pertains to the rabbit Fal- lopian tube. The results obtained in the present in vitro study monitoring contractile activity of the circular and longitudinal muscle layers of the rabbit uterine horn sup- port the view that the two muscle layers contract Simul- taneously. Contraction of the circular fibers was recorded 55 56 by the circularly oriented extraluminal transducer and may have been recorded by the Grass FT-03 transducer. A Simul- taneous contraction of the longitudinal fibers was recorded by the Grass FT-03 transducer and the longitudinally ori- ented extraluminal transducer, if the tension set at the time of attaching the extraluminal transducer was minimal. If the tension set in attaching the extraluminal transducer was great, it recorded an apparent decrease in tone when the adjacent muscle contracted. Figure 6 illustrates the longitudinal muscle contractile activity with setting ten- sion high, while Figure 8 shows it with setting tension low. With regard to smooth muscle layer contractile activ- ity in the gastrointestinal tract, Reinke, gt_§l. (30) state that in burst activity in the small intestine, the longitudinal muscle contracted before the circular muscle layer. Frequency of Contractile Activiteratterns Previous investigators have characterized estrous contractile activity as exhibiting one contraction every minute up to one contraction every four or five minutes (9,18,28,34). This quantification, when converted to fre- quency, gives an average frequency for estrous contractile activity which approximates that obtained from this inves- tigation (0.55 bursts/minute). This rate of Spontaneous activity has been attributed to the myogenic property of 57 uterine muscle fibers, which is modified by the dominant hormone, ionic concentrations, amount of actomyosin, and presence of calcium (17). The characteristic of this myogenic prOperty is related to the resting membrane po- tential, which is under the control of hormones (17). Estrogen has been Shown to control the synthesis of the contractile protein, actomyosin, and to raise the resting membrane potential to the critical level, that level at which rhythmic, Spontaneous contractile activity occurs (12,13,15,17). The role of the autonomic nervous system in the generation of spontaneous contractile activity has been considered (34,44). The presence of intrinsic innervation in the myometrium was debated; positive identification of its presence has been made (29), although exact morphology and extent is unknown. Recent in vivo and in vitro experi- ments on dogs, which involved nerve sections, electrical and pharmacological nerve stimulation, and acetycholine depletion were designed to discover other possible mecha- nisms which could contribute to spontaneous contractile activity (44). The investigators concluded from the re- sults of their experiments that the cholinergic neuro- hormone, acetylcholine, was reSponsible for the generation and rhythmicity of contractile activity. It is possible that the intrinsic nervous system is acting in conjunction with the myogenic prOperty of uterine smooth muscle cells in vitro. 58 This study showed that bursts of contractile activity from the longitudinal and circular muscle layers occur simul- taneously. It is not known whether the cells in the longi- tudinal muscle layer and the circular muscle layer are alike. It is possible that the outer longitudinal muscle of the uterus is analogous to that in the small intestine and that it generates a BER (slow wave) which controls the rate at which the circular muscle layer contracts. Effects of Drug Stimulation A recent hypothesis set forth by Shabanah, Toth, and Maughan (44), suggests that the intrinsic nerve supply in the "autonomous" (excised) uterus consisting of post- ganglionic parasympathetic fibers and their endogenous acetylcholine, sets the pace for spontaneous contractility by acting on the longitudinal muscle fiber membranes. In this study acetylcholine appeared to differentially affect the longitudinal and circular muscle layers. The increase in frequency of contraction recorded from the high tension longitudinally oriented transducer following acetylcholine administration was likely due to increased tension in the muscle beneath this transducer resulting from: (1) contrac- tion of adjacent longitudinal muscle above and below the extraluminal transducer, and (2) the direction action of acetylcholine on the muscle beneath the extraluminal trans- ducer. The recorded apparent tone decrease was likely due 59 to the predominance of the adjacent longitudinal muscle contractile activity. Bortoff (6) used the cat small in- testine in vitro to show that longitudinal muscle was 9 molar) stimulated to contract by small doses (2 X 10- of acetylcholine while the circular muscle did not con- tract. Higher concentrations elicited contractions from both muscles. Burnstock and Holman (7) state that drugs which change the pattern of contractile activity in iso- lated preparations are probably acting directly on the smooth muscle membrane, which would indicate a mecha- nism for its action on the longitudinal muscle. Oxytocin elicited a response similar to that recorded after acetyl- choline. Burnstock and Holman (7) state that oxytocin has an action similar to acetylcholine on smooth muscle. In contrast to the longitudinal muscle response to acetylcholine, the vaSOpressin frequency and amplitude stimulation was superimposed on a sustained increase in tone. The in vitro method deve10ped for this study and the results obtained by it indicate that the extraluminal contractile force transducer is a reliable isometric in vitro method if adequate attention is given to the amount of tension applied at the time of attachment of the transducer to the muscle. The influence of this set- ting tension is more apparent in the longitudinal muscle layer due to the greater amount of adjacent muscle tissue. 60 Thus there is a possibility that the longitudinally ori- ented extraluminal contractile force transducer may record an apparent decrease in tone. CHAPTER FIVE SUMMARY The extraluminal contractile force transducer has been modified for use in vitro by changing the orientation of the lead wires to facilitate immersion in the bath, and by fabricating with waterproofing materials which reduced the size of the transducer from that previously used in vivo. 1. Although a contraction of the circular muscle layer occurs in the vertically oriented segment, the con- tribution made by the circular muscle to the con- traction recorded by the Grass FT-03 transducer cannot be quantified. The contribution of the longitudinal muscle layer as recorded by the longitudinally oriented extra- luminal transducer to the contraction recorded by the Grass FT-03 transducer depended on the degree of tension set at the time of attachment. If the force of contractile activity of the longitudinal muscle adjacent to the extraluminal transducer at each end was greater than the contractile force of the longitudinal fibers under the transducer, the longitudinally oriented transducer recorded ap- parent decrease in tone. 6' 62 The presence of the extraluminal transducers on the muscle segment did not affect frequency or force of contractile activity, as shown statistically by paired comparisons. The extraluminal transducers allowed recording of the contractile activity of the longitudinal and circular muscle layers separately and simultaneously as shown by the addition of drugs to the in vitro bath. 10. 11. BIBLIOGRAPHY Athias, M. Effets de la castration sur les movements automatiques de uterus chez 1e cogaye. J. Physiol. Pathol. gen. 18:731, 1919. Bass, P., and Callantine, M. R. Simultaneous record- ing of electrical and mechanical activity of the uterus in the unanesthetized animal. Nature. 203:1367, 1964. 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