J. Physiol. (1975), 244, pp. 353-364 With 1 plate and 10 text-figures Printed in Great Britain

353

ELECTRICAL ACTIVITY OF GRAFTED MYOMETRIUM AND ITS RECORDING BY RADIO-TELEMETRY IN UNRESTRAINED RABBITS

BY GORM WAGNER From the Institute of Medical Physiology B, University of Copenhagen, Denmark

(Received 6 May 1974) SUMMARY

1. The electrical activity in myometrial grafts in fifty-one female rabbits with ear chambers fitted with pick-up electrodes has been monitored. An FM telemetry system has made long-term studies possible in the freely moving animal. 2. Two distinct patterns of electrical activity were observed either arising spontaneously or provoked by oxytocin. These were the 'long. duration burst' containing about 50-150 spikes, and the 'short-duration burst' containing 1-10 spikes. 3. The spike frequency within the long-duration bursts varied between 0 9 and 1-3 spikes sec-1 and was fairly constant during the first half of the burst. The frequency of the bursts in the long series of short-duration bursts was about 2-3 min-. 4. When activity from grafts in two chambers in the same animal was monitored for long periods, spontaneous activity occurred at random throughout the day in each graft, indicating that the spontaneous activity was probably myogenic. INTRODUCTION

The aim of this work was to study the electrical activity in myometrial tissue grafted into ear chambers in unrestrained rabbits by means of radio-telemetry. It was hoped that by studying grafted myometrium rather than myometrium in situ information could be obtained regarding the various factors regulating the activity of the myometrium. The graft in the ear of the rabbit is readily accessible and allows repeated study of a single group of cells for long periods (Cliff, Martin & Michael, 1963). So far no studies of the spontaneous electrical activity of the myometrium have been undertaken in the unrestrained rabbit.

GORM WAGNER

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METHODS

A transparent ear chamber (Text-fig. 1) was inserted in fifty-one female rabbits. Platinium electrodes protruded from the base of the chamber into the connective tissue that grew into the chamber. This tissue served as a medium for the graft, which was placed as close as possible to the pick-up electrode (P1. 1). The grafts were taken from the upper third of one of the uterine horns. The size varied from 0 5 to 1 mm3. In order to keep the blood perfusion of the ear constant and to avoid nervous influence upon the graft, ipsilateral cervical sympathectomy was performed at the time of grafting. In some cases it was done a few days before the start of recording of electrical activity.

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Text-fig. 1. Cross-section of the chamber. A: upper celluloid cover plate. B: nickel-plated copper ring. C: filter-paper ring. D: mica plate. E: upper acryl part of chamber. F: acryl base through which the platinum electrodes were led. G: lower celluloid cover plate. The new tissue will grow between E and F.

Radio-telemetry transmission systems The platinum pick-up electrodes were connected to the input of the radio transmitter. Four different transmitters have been used through the years and three different receivers. For recording of signals either an oscilloscope, a tape recorder, or a paper recorder was used, or all three together (Text-fig. 2). The impedance of each electrode pair was measured in situ. A constant a.c. field was applied across the ear and the potential difference was measured between the electrodes which were loaded with a resistance of known value. At a frequency of 7-5 Hz the impedance was 170-190 kfl, at 15 Hz about 100-110 knt, and at 30 Hz about 50-70 kQ. The transmitters and receivers All transmitters have been built in the electronic workshop of the Institute. They are based on the principle of direct frequency modulation (FM), using the tuning coil of the oscillator as the transmitting antenna. The relevant data of the four transmitters (B-2-B-5) used in connexion with the receivers (M-2-M-4) are given in Table 1. A 5 mVpp input to the transmitter provides a frequency deviation of + 100 kHz. The maximal allowable signal is 10 mVpp. The receivers were equipped with

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automatic frequency control (AFC). For detailed technical descriptions, see Dj0rup, Nielsen & Wagner (1970) and Nielsen & Wagner (1972). Receiving antennae. Two types were used: one was a A/2 dipole; the other was a single loop around the animal's cage. The oscilloscope was a two-channel Tektronix 502 A, and the frequency range 1-5 Hz-1 MHz. The input impedance on the differential amplifier was 2 x 1 MKI, each shunted by 47 pF. The tape recorder used was an FM-7-channel (Philips Ana-log 7) with frequency range from d.c. to 312 Hz at lowest speed (23-75 mm sec-1) and from d.c. to 10 kHz at highest speed (380 mm sec-1); 12 hr of continuous recording is possible at lowest speed for each channel. Two different pen-recordere were used: (1) a 2-channel Dynograph (Beckmann) with thermal rectilinear recording system, and frequency range 5-3-35 Hz at full scale (15% deflexion at 150 Hz); (2) a Brush Recorder 220, rectilinear, with 2 analogue channels and frequency range d.c. -40 Hz at full scale (20 % deflexion at 100 Hz).

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Text-fig. 2. Block diagram showing the transmission system. The FM transmitter is placed upon the rabbit. The modulated high-frequency signals are demodulated in the receiver. From the receiver the signals are fed into one channel of a two-channel oscilloscope for direct observation, a paper recorder for screening purposes and a tape recorder. Experimental procedure The transmitter was carefully fixed with adhesive tape upon a plastic cap cut into the appropriate shape for the particular rabbit. After the animal had had the cap fitted it was placed in an acryl cage in the laboratory. Usually no recordings were made for a day or two in order to be certain that the animal had got used to its new situation. The noise level of the transmitters was low, and because of the localization of the graft no electrical interference from the myocardium or from striated muscle occurred. No disturbances occurred from other radio transmissions. A severe disadvantage using the present transmitters has been the noise occurring when the animal was restless and/or moved strongly. This often made parts of records useless.

RADIO-TELEMETRY AND MYOMETRIAL GRAFTS

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Careful fixation of the transmitter and twisting of the leads from the ear chamber to the transmitter eliminated or reduced the noise. When ix.v. injections of synthetic oxytocin were given the animal was sitting in a 'pillory' box or held down on a specially designed table with the transmitter lying beside the animal. In both situations it was neither anaesthetized nor sedated. Observations could be made for several hours in this way. I min

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Text-fig. 3. The two distinct types of electrical activity in myometrial grafts. A, one long-duration burst following iv. injection of 50mi-u. oxytocin ( ~) on day 15 of pregnancy. B, one series of short-duration bursts following i.v. injection of 50 in-u. oxytocin (4)in oestrus. Same animal (B) 18 days before A. RESULTS

Electrical activity, usually in the form of bursts of spikes, either occurred spontaneously or followed the injection of oxytocin. In the many instances in which the graft was observed microscopically simultaneous with electric recording a burst of spikes was invariably accompanied by visible contraction and blanching of the graft. Single spikes rarely occurred and were not associated with blanching. When a physiological dose of oxytocin (e.g. 50 in-u.) was injected the response might either be a single, continuous burst of spikes of long duration or a series of bursts of spikes of short duration. These two different patterns will be described in the following as 'long-duration burst' and 'short-duration burst' (Text-fig. 3).

Long-duration burst A burst of long duration contained a varying number of spikes (from about 50 to 150) and lasted from about 30 to 120 seconds. Usually the burst was not followed by another within a period of 5-10 min. A sample record of 20 hr continuous registration is given in Text-fig. 4. In this case spontaneous long-duration burst activity is seen to occur 2-5 times per hour. The shortest period between two bursts is 14h min i 17Y

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Text-fig. 5. Spontaneous long-duration burst containing 122 spikes from myometrial graft in freely moving rabbit on day 26 of pregnancy. a Shows the total burst, b, c and d are parts of the same burst played back from tape at different paper speeds. Same part of the burst is seen to the left of the vertical line at all speeds. Time scale only valid for d. B-2/M-3 transmission system. Record from same animal (rabbit K9) as in Text-fig. 4, but 6 days later.

RADIO-TELEMETRY AND MYOMETRIAL GRAFTS 359 deflexions. Ten minutes after this recording a single injection of 100 m-u. oxytocin was given i.v. and a similar burst containing 92 spikes was observed. In all animals long-duration bursts of spontaneous origin could be reproduced by i.v. injection of oxytocin. Usually the threshold dose was of the order of 50-100 m-u. Oddly, no increase in response was observed on increasing the dose of oxytocin. Spikes/sec

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50 75 100 sec Text-fig. 6. Frequency of spikes as a function of the burst duration in long-duration bursts from grafts in three different animals. A, spontaneous burst 11 days post partum. B, response to i.v. injection of 100 m-u. oxytocin on day 26 of pregnancy. C, response to stretch 8 days after

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The spike frequency within the burst tends to be fairly constant in the first 20-30 sec of the burst and then falls off through the rest of the period. Text-fig. 6 shows the spike frequency as a function of the duration of the burst. The spike frequency was calculated as the reciprocal of the time interval between the onset of the spikes. The frequency range in the period with greatest constancy varied from 0 9 to 1x3 spikes/sec. Within the long-duration burst the single spike potentials, being bior triphasic, had a duration of 80-120 msec in general - while the characteristic spike of the short-duration burst (see below) would be biphasic and have a shorter duration, i.e. 45-70 msec. Short-duration burst Short-duration (i.e. from about 1 to 15 sees) bursts often occurred repetitively. The number of such bursts appearing was increased by I7-2

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RADIO-TELEMETRY AND MYOMETRIAL GRAFTS 361 oxytocin in a dose-dependent manner. Text-fig. 7 shows the response to increasing doses of intravenously injected oxytocin in an ovariectomized, oestrogen-treated rabbit. Text-fig. 8 shows the burst frequency as a function of the duration of the series of bursts. The period with greatest constancy is characterized by 2-3 bursts/min. If a single burst contains more spikes than the preceding one the interval immediately after was increased (Text-fig. 8C). Before pregnancy occurs and around the time of parturition the short-duration burst was most often the sole electrical manifestation. Long-duration bursts were typically the only electrical activity of the graft during pregnancy. If the same animal was followed during different endocrine states a lowering of the measured amplitude (peak to peak value) invariably followed the change from short- to long-duration burst. An increase in amplitude always followed the return to short-duration burst activity. The amplitude of the spikes of the long-duration burst was in the order of 01-05 of that of the short-duration burst in the same graft. Cutting the ipsilateral cervical sympathetic nerve had no effect upon the electrical pattern of the graft.

Position of graft in relation to pick-up electrodes PI. 1 is a photomicrograph showing the relation between a graft and a pick-up electrode. In a series of animals two grafts were placed in the same chamber, each upon its 'own' electrode. The chance of obtaining successful grafts and recordings was thus increased. In some cases both grafts took, and spike activity could be recorded from both grafts via the same pair of electrodes. Text-fig. 9 is a drawing of two grafts and their positions relative to the electrodes. Both grafts were functioning spontaneously, as well as when oxytocin was given. When leads were taken from electrodes A and C two distinct groups of spikes were observed, one with a large amplitude (average 1-5 mV) and one with a smaller (average 03 mV). The frequency of the bursts in the two groups was different. Contraction of the graft at A (Text-fig. 9) was observed concomitantly with the bursts with the lowest frequency and containing the large spikes. Contractions of the graft at B (Text-fig. 9) were concomitant with the bursts with the highest frequency and containing the small spikes. Textfig. 10 is a record of the electrical activity in the two grafts evoked by I.v. injection of oxytocin. During the series of bursts the leads were shifted from electrodes A-C to electrodes B-C. Simultaneously the contractions in the two grafts were observed. The amplitude of the spikes from graft A dropped from 1-5 mV to about 0-05 mV while the amplitude from graft B

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362 GORM WAGNER increased from 0-3 to 0-7 mV. When the leads again were taken from the electrodes A-C the original pattern was obtained. In this particular animal it was observed that the spontaneous activity always appeared in both grafts simultaneously. However, it was possible to activate only one graft at a time by electrical stimulation.

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Some experiments therefore have been done with one ear chamber placed in each of the ears of the rabbit, thus spacing the grafts sufficiently to exclude interference between the two. Three such animals with two chambers have been studied during long periods through pregnancy and the puerperium. The records showed that each graft commonly exhibited electrical activity not synchronous with the other; synchronous activity was much less usual. Both long-duration bursts and single spikes were found to appear asynchronously.

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DISCUSSION

The radio-telemetry system used in this work has made it possible to follow the spontaneous electrical activity occurring in grafted myometrium in the freely moving animal. If no electrical activity is recorded for long periods the problem arises whether to ascribe this to a silent tissue or to technical faults in the transmission system. An unfavourable position of the graft in relation to the electrode may also play a role in such instances. As was demonstrated in the chamber with two grafts the amplitudes of the recorded potentials are not predictable from a simple evaluation of the distance to the pick-up electrodes. Electrical activity recorded in vivo directly from the myometrium in situ in the restrained animal has shown a characteristic relation between the type of electrical activity and the intra-uterine pressure (Csapo & Takeda, 1965). These authors found that when the electrical activity was of short duration in a given area, the pressure increase was larger and shorter than when the electrical activity was prolonged. The present findings of two distinct types of electrical activity thus suggest that the types of electrical pattern previously described also occur in the grafted myometrium. Pointing in the same direction is the increase of spike amplitude at the same time as short-duration activity occurs. In long-term studies of electrical activity in myometrium in situ in restrained rabbits Kao (1959) found an increase of amplitude when monitoring the same group of cells at the end of pregnancy. The graft was not found to be influenced by the sympathetic nervous system and no mechanical factors (endometrial swelling or conceptus) could influence the electrical activity. When therefore electrical patterns found in grafted myometrium are similar to or identical with those found in the organ in situ, a predominantly humoral regulation of the electrical activity of the myometrium is the most reasonable assumption. The records obtained when two grafts were placed in two ear chambers in the same rabbit showed that spontaneous activity (long-duration bursts) occur randomly during the 24 hr for each of the grafts. This phenomenon indicates a 'true' spontaneous (myogenic) activity in grafted myometrium. The help of Dr J. Rostgaard, Institute of Medical Anatomy C, University of Copenhagen, with the histological work is gratefully acknowledged. REFERENCES

CLIFF, W. J., MARTIN, J. D. & MICHAEL, C. A. (1963). Use of ear chamber with uterine muscle transplant to differentiate myometrial and vascular activity. Nature, Lond. 199, 399-400.

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CsAPo, A. I. & TAKEDA, H. (1965). Effect of progesterone on the electrical activity and intrauterine pressure of pregnant and parturient rabbits. Am. J. Obatet. Gynec. 91, 221-321. DJ0RUP, A. NIELSEN, J. F. & WAGNER, G. (1970). Implantable FM-telemetry transmitter for long-term registration of biopotentials. Dan. med. Bull. 17, 136-137. KAo, C. Y. (1959). Long term observations of spontaneous electrical activity of the uterine smooth muscle. Am. J. Physiol. 196, 343-350. NIELSEN, J. F. & WAGNER, G. (1972). Implantable FM-telemetry transmitters for registration of biopotentials. In Biotelemetry, pp. 360-364. Leiden: Meander N.V. EXPLANATION OF PLATE

PLATE 1

Photomicrograph of myometrial graft (arrows) in connective tissue. The hole to the right of the graft has been produced by the platinum electrode protruding from the base of the ear chamber. The amplitude of the spikes from this graft (rabbit in oestrus) was in the order of 0 8 mVp