Effects of thyrotropin-releasing hormone in the fetal lamb

Divon et al.

18. 19. 20. 21.

ration rhythms within several frequency ranges. Automedica 1984;5:77. Harper RM, Walter DO, Leake B, et al. Development of sinus arrhythmia during sleeping and waking states in normal infant~. Sleep 1978; I :33. Hering HE. Uber die Bezeichnung der extracardialen Herznerven zur Steigerung Herzschagzahl bei Muskeltatigheit. Pflugers Arch 1895;60:429. Bainbridge FA. The relationship between respiration and pulse rate. 1 Physiol (Lond) 1920;54: 142. Ekberg DL, Kifle YT, Roberts VL. Phase relationship between normal human respiration and baroreflex responsiveness. 1 Physiol (Lond) 1980;304:489.

December !986 Am J Obstet Gynecol

22. Myers RE, Beard R, Adamson K. Brain swelling in the newborn rhesus monkey following prolonged partial asphyxia. Neurology 1969;19:1012. 23. Laros RK, Wong WS, Weilbron DC, et al. A comparison of methods for quantitating fetal heart rate variability. AM 1 0BSTET GYNECOL 1977;128:381. 24. Sayers BM. Analysis of heart rate variability. Ergonomics 1973;16:17. 25. Divon MY, Zimmer EZ, Platt LD, Paldi E. Human fetal breathing: associated changes in heart rate and beat-tobeat variability. AM1 0BSTET GYNECOL 1985;151:403.

Effects of thyrotropin-releasing hormone in the fetal lamb Jason G. Umans, Ph.D., M.D.,* Hilary R. Umans, B.A., and Hazel H. Szeto, Ph.D., M.D.** New York, New York Thyrotropin-releasing hormone causes neurobehavioral arousal and stimulates breathing in adult, newborn, and preterm experimental animals. Its effects on behavioral state, breathing, blood pressure, and heart rate were studied in the chronically instrumented late term fetal lamb. Fetal intravenous administration of thyrotropin-releasing hormone resulted in behavioral arousal with electrocortical desynchronization, 'increased body and eye movements, rapid and deep breathing movements, and a transient bradycardia followed by prolonged tachycardia, associated with an increase in both systolic and diastolic blood pressure. The effects were similar following intracisternal administration of thyrotropin-releasing hormone. The effects of thyrotropin-releasing hormone on behavior, but not breathing, was abolished in the presence of muscarinic blockade. Thyrotropin-releasing hormone may play a role in the modulation of central regulation of cardiovascular, respiratory, and behavioral activity in the fetus. (AM J OssTET GVNECOL 1986;155:1266-71.)

Key words: Sheep, fetus, thyrotropin-releasing hormone, electrocorticogram, fetal breathing Recently much attention has been directed toward the extrahypothalamic actions of thyrotropin-releasing hormone. The widespread distribution of thyrotropinreleasing hormone in mammalian brain and its apparent involvement in numerous neurophysiologic pro-

From the Department of Pharmacology, Cornell University Medical College. This work was supported in part by Grant R01-DA02475-04 from the Nationallnstitute on Drug Abuse and by Grant S07-RR05396 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National1nstitutes of Health. Presented at the Thirty-third Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, March 19-22, 1986. Reprint requests: Hazel H. Szeto, M.D., Ph.D., Department of Pharmacology, Cornell University Medical College, 1300 York Ave., New York, NY 10021. *Jason G. Umans is the recipient of a Research Fellowship from the Charles H. Revson Foundation. **Hazel H. Szeto is the recipient of a Research Scientist Development Award (K02-DA00100-0l) from the National Institute on Drug Abuse of the Alcohol, Drug Abuse, and Mental Health Administration.

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cesses have led to the concept that thyrotropin-releasing hormone is a ubiquitous neurotransmitter that has been co-opted by the pituitary as a releasing hormone. Intraventricular and/or high-dose parenteral administration of thyrotropin-releasing hormone has been shown to cause behavioral and electrocortical arousal and to antagonize barbiturate-induced narcosis in the adult rabbit,'·5 fowl," and mouse. 7 In addition, it stimulates respiratory activity in the anesthesized adult rat8 and rabbit9 and in the newborn 10 or preterm 11 rabbit. The mechanism of action of thyrotropin-releasing hormone is not understood, but some investigators have demonstrated that the thyrotropin-releasing hormoneinduced arousal, though not the tachypneagenic effect, can be blocked by muscarinic antagonists. In an effort to examine the possible role that thyrotropin-releasing hormone may play in the regulation of neurobehavioral and respiratory activity in the immature animal, we have examined its effects in the fetal lamb. In addition, by studying the effects of thyrotropin-releasing hormone in the presence of atropine, we

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'1 "'1 BP

Fig. 1. Effects of thyrotropin-releasing hormone (TRH) (5.0 mg intravenously) on fetal blood pressure (BP) and heart rate (HR). Top panel shows immediate effect of thyrotropin-releasing hormone; bottom panel shows effect of thyrotropin-releasing hormone 15 to 30 minutes after administration.

Table I. Criteria for the scoring of the behavioral states indicated by the recordings

Behavioral state

E lectrocortical activity

Eye movements

Dorsal neck electromyographic activity

Arousal Quiet sleep Rapid eye movement sleep

Desynchronized Synchronized Desynchronized

Random Slow/absent Rapid eye movements

Tone, movements Tone, movements Atonia

have sought to confirm the suggestion that some of the effects of this compound may depend on muscarinic cholinergic pathways. Material and methods

Animal preparation. Six unanesthetized, unrestrained pregnant ewes between 121 and 134 days' gestation (term being 145 days) were studied 6 to 18 days after surgery. Details of the surgical procedures were as described previously. 12• 13 Briefly, stainless steel screws were implanted over the fetal parietal cortex to monitor electrocortical activity and over the superior orbital ridge to record eye movements. Stainless steel leads were placed in the dorsal neck muscle to record electromyographic activity. Fetal breathing movements were assessed by stainless steel leads in the diaphragm and by changes in intratracheal pressure by an indwelling saline solution-filled polyvinyl catheter. Polyvinyl catheters were placed in the fetal aorta to monitor blood presure and heart rate and in the maternal and fetal vena cava for thyrotropin-releasing hormone administration. A polyvinyl catheter (0.28 mm inner diameter by 0.64 mm outer diameter, size V1; Bolab Incorporated, Lake Havasu City, Arizona) was chronically implanted in the cisterna magna of four fetuses to allow

direct drug administration into cisternal cerebrospinal fluid. A polyvinyl cuff (1.32 mm outer diameter, size V5) was cemented around the catheter 7 mm from the tip to facilitate suturing the catheter in place. A small hysterotomy was performed, the dorsal aspect of the flexed fetal neck incised, and the skin marsupialized to the uterine wall with Babcock clamps. The nuchal ligaments were divided in the dorsal midline by blunt dissection and the cisternal membranes exposed. A 5-0 silk retention suture was placed in the membrane and the membrane punctured with a 22-gauge hypodermic needle. The saline solution-filled catheter was inserted, secured to the membrane and to the occipital periosteum with silk suture, and the free return of cerebrospinal fluid confirmed after exteriorization of the catheter through the fetal scalp. All catheters and electrodes were exteriorized at a site on the maternal flank. Recording procedures. All physiologic recordings were obtained with the ewe standing or lying quietly in an experimental cart with free access to food and water and in the presence of a companion sheep so as to reduce restlessness. A Gould model 2800 direct pen writing recorder with appropriate amplifiers was used for all studies. Recordings were made with the follow-

1268 Umans, Umans, and Szeto

December 1986 Am J Obstet Gynecol

! .......,...................

FBP

1

FHR

TP

ECoG

~II .1111

EMG-n

EOG

IFF

...

111

••

1 . . . ._,....... ~.

..,

EN!Gd

Fig. 2. Effects of thyrotropin-releasing hormone (5.0 mg intravenously) on the fetus. (FBP, Fetal blood pressure; FHR, fetal heart rate; TP, tracheal pressure; ECoG, electrocorticogram; EMG., dorsal neck electromyogram; EOG, electrooculogram; EMGd, diaphragmatic electromyogram.)

Table II. Effects of thyrotropin-releasing hormone on fetal cardiovascular parameters in the absence and presence of muscarinic blockade Atropine plus thyrotropin-releasing hormone (5 mg) (n = 3)

Thyrotropin-releasing hormone (5 mg) (n = 6) t

Heart rate (bpm) Systolic pressure (mmHg) Diastolic pressure (m Hg)

=0

I

t

= 1 min

I

t

=

15-30 min

t

=0

I

t

=

1 min

171 ::!:: 6 64 ::!:: 2

142 ::!:: 7* 67::!:: 5

248 ::!:: 15* 81 ::!:: 4*

185 ::!:: 5 62 ::!:: 2

205 ::!:: 5*t 77 ::!:: 3*

42::!:: 3

45::!:: 3

50 ::!:: 2*

42 ::!:: 1

57 ± 3*t

I

t

=

15-30 min

285 ::!:: 5* 88 ::!:: 2* 65 ± 3*t

All data expressed as mean ± SE. *Significantly different from control of same group (p < 0.01). tSignificantly different from thyrotropin-releasing hormone alone (p < 0.01).

ing band widths: electrocorticogram, 0.3 to 30 Hz; electrooculogram, I to 10 Hz; electromyogram, 30 to 1000Hz. Experimental protocol. Control recordings of all fetal physiologic parameters were made for 3 hours before thyrotropin-releasing hormone administration. Thyrotropin-releasing hormone (0.5 to 5.0 mg; Sigma Chemical Company) was administered to the fetus by the intravenous or intracisternal routes (0.1 ml volume). All physiologic parameters were recorded continuously for at least 4 hours afterwards. In addition, three lambs that had received thyrotropin-releasing

hormone (5 mg intravenously) received the same dose on a second occasion during the continuous infusion of atropine (5 11g/min). This dose of atropine resulted in a continuous 30 bpm tachycardia before thyrotropinreleasing hormone administration and has been shown to antagonize the effects of cholinergic agonists in this preparation (Hinman DJ, Szeto HH. Cholinergic influences on behavioral states and breathing movements in the fetus. Unpublished observations). Data analysis. All recordings were divided into !minute epochs and scored as arousal, quiet sleep, or rapid eye movement sleep by visual analysis with use

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min

Fig. 3. Effects of thyrotropin-releas ing hormone (5.0 mg intravenously) on fetal blood pressure (BP) and heart rate (HR) in the presence of muscarinic blockade with atropine. Top panel shows immediate effect of thyrotropin-releas ing hormone. Note absence of the initial transient bradycardia. Bottom panel shows effects of thyrotropin-releas ing hormone J5·to 30 minutes after administration.

EOG

,

min

Fig. 4. Effects of thyrotropin-releas ing hormone (5.0 mg intravenously) on the fetus in the presence of muscarinic blockade with atropine. Note continuous deep breathing movements associated with synchronized electrocorticogram. (FBP, Fetal blood pressure; FHR, fetal heart rate; ECoG, electrocorticogram; EMG., dorsal neck electromyogram; EOG, electrooculogram; EMGd, diaphragmatic electromyogram.)

of the criteria given in Table I. Student's t test was used for analysis of data. Results

During the control period all fetuses spent 5% to 15% of total recording time in a state of arousal with the remainder spent alternating between quiet sleep and

rapid eye movement sleep. Fetal breathing movements averaged 57% ± 4% of total recording time and were observed only during arousal and rapid eye movement sleep. These results are in agreement with those previously reported.' 2· ' 3 Thyrotropin-rel easing hormone in intravenous doses of 0.5 and 1.0 mg did not result in significant

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December 1986 Am J Obstet Gynecol

Table III. Effects of thyrotropin-releasing hormone on fetal behavioral states and breathing movements

in the absence and presence of muscarinic blockade Thyrotropin-releasing hormone (n = 6)

Incidence (%) Arousal Quiet sleep Rapid eye movement sleep Breathing

Atropine plus thyrotropin-releasing hormone (n = 3) After administration

After administration

Before administration

6 ± 2 38 ± 3 56 ± 5

65 ± 6* 30 ± 6 5 ± 3*

9 ± 3 43 ± 3

14 ± lOt 81 ± lO*t

48 ± 5

5 ± 4*

58± 4

74 ± lO

48 ± 6

63 ± 20

Before administration

I

I

All data expressed as mean ± SE. *Significantly different from control of same group (p < 0.01). tSignificantly different from thyrotropin-releasing hormone alone (p < 0.01).

changes in any of the fetal parameters (n = 6). Doses of 2.5 and 5.0 mg (n = 7) resulted in a transient mild bradycardia within 1 to 2 minutes followed by a prolonged tachycardia and an increase in both systolic and diastolic blood pressure (Fig. 1). These doses were also associated with immediate desynchronization of the electrocorticortical activity, rapid and deep breathing movements, and increased muscle tone and movements (Fig. 2). Although the breathing movements induced by thyrotropin-releasing hormone were initially associated with a state of arousal, they were observed to persist for a period of time after return of electrocorticogram synchrony. This is significantly different from the normal fetus in which electrocorticogram synchrony is associated with apnea, and breathing movements are only present during rapid eye movement sleep or arousaL When administered by the intracisternal route (n = 4), the effects were similar to those observed intravenously except that there was a longer latency to the onset of effect (3 to 6 minutes) and the initial bradycardiac response was not observed. There was also a tendency towards greater potency, with effects observed at 1.0 mg of thyrotropin-releasing hormone being comparable to those observed with 5.0 mg of thyrotropin-releasing hormone intravenously. When thyrotropin-releasing hormone (5.0 mg intravenously) was administered during the steady-state infusion of atropine (n = 3), the effects were similar to those observed in the absence of muscarinic blockade, except for the notable absence of the initial transient bradycardia (Fig. 3). Rapid, deep breathing movements were present, but they were associated with a synchronized rather than desynchronized electrocorticogram (Fig. 4). A comparison of the effects of 5.0 mg ofthyrotropin-releasing hormone alone and in the presence of atropine on cardiovascular parameters, and behavioral and breathing activity can be seen in Table II and Table III, respectively. None of the above effects were observed following

the administration of thyrotropin-releasing hormone (5.0 mg intravenously) to the mother (n = 2), indicating a direct effect of thyrotropin-releasing hormone on the fetus.

Comment The intravenous administration of thyrotropin-releasing hormone to the unanesthesized fetal Iamb causes electrocortical and behavioral arousal, consistent with prior observations in other systems.'·' Although the increase in the incidence of fetal breathing movements is apparently related to the effect of thyrotropinreleasing hormone on behavioral state, the occurrence of episodes of breathing movements during quiet sleep after thyrotropin-releasing hormone administration suggests a dissociation of its effects on fetal behavior and breathing movements. This dissociation has previously only been demonstrated in this system by inhibitors of prostaglandin synthesis. 14 In contrast to the results obtained in anesthesized rats, 8 the behavioral and breathing effects were accompanied by significant cardiovascular responses, including moderate hypertension and a biphasic effect on fetal heart rate. The enhanced potency of thyrotropin-releasing hormone after intracisternal administration suggests that its effects are mediated predominantly within the central nervous system, while the delayed onset after central administration suggests that its effects are mediated more rostrally in the neuroaxis, Although the simultaneous blood pressure data suggest that the initial thyrotropin-releasing hormone-induced bradycardia is not the result of a baroreflex, the blockade of the initial effect by atropine suggests that it is mediated by another cholinergic mechanism. The similar lack of an initial bradycardia after administration of intracisternal thyrotropin-releasing hormone suggests that the atropine-sensitive bradycardic effect may be mediated exclusively at peripheral muscarinic sites. As demonstrated by Horita eta!! in the adult rabbit,

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our results show that atropine selectively antagonizes the arousal but not the tachypneagenic effect of thyrotropin-releasing hormone in the fetal lamb. These results suggest that the respiratory effect of thyrotropin-releasing hormone is independent of its effects on behavioral activity in the fetal lamb. In summary these results suggest that thyrotropinreleasing hormone affects cardiovascular, respiratory, and behavioral activity in the fetal lamb. However, the amount of hormone required to produce these effects suggests that it is unlikely to be of major physiologic significance. REFERENCES 1. Carino MA, Smith JR, Weick BG, Horita A. Effects of thyrotropin-releasing hormone microinjected into various brain areas of conscious and pentobarbital-pretreated rabbits. Life Sci 1976;19:1687-92. 2. Horita A, Carino MA, Smith JR. Effects of TRH on the central nervous system of the rabbit. Pharmacal Biochem Behav 1976;5:111-6. 3. Andry DK, Horita A. Thyrotropin-releasing hormone: physiological concomitants of behavioral excitation. Pharniacol Biochem Behav 1977;6:55-9. 4. Beale JS, White RP, Huang SP. EEG and blood pressure effects of TRH in rabbits. Neuropharmacology 1977; 16:499-506. 5. Maurelli M, Tartara A, Machioni E, Savoldi F. The spec-

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trum of actions of thyrotropin-releasing hormone on the CNS: a polygraphic study in rabbits. Neurosci Lett 1984; 46:85-90. Nistico G, Rotiroti D, DeSarro A, StephansonJD. Behavioural, electrocortical and body temperature effects after intracerebral infusion of TRH in fowls. Eur J Pharmacal 1978;50:253-60. Breese GR, CottJM, Cooper BR, Prange AJ, Lipton MA, Plotnikoff NP. Effects of thyrotropin-releasing hormone (TRH) on the actions of pentobarbital and other centrally acting drugs. J Pharmacal Exp Ther 1975;193:11-22. Hedner J, Hedner T, Jonason J, Lundberg D. Central respiratory stimulant effect of thyrotropin releasing hormone in the rat. Neurosci Lett 1981;25:317-20. Homma I, Oouchi M, Ichikawa S. Facilitation of inspiration by intracerebroventricu1ar injection of thyrotropinreleasing hormone in rabbits. Neurosci Lett 1984;44: 265-70. Yamamoto Y, Lagercrantz H, von Euler C. Effects of substance P and TRH on ventilation and pattern of breathing in newborn rabbits. Acta Physiol Scand 1981;113:541-3. Hedner T, Hedner J,JonasonJ, LUndberg D. Respiratory effects of TRH in preterm rabbits. Pediatr Res 1982; 16:543-8. Szeto HH. Effects of narcotic drugs on fetal behavioral activity: acute methadone exposure. AMJ 0BSTET GYNECOL 1983;146:211-8. Umans JG, Szeto HH. Effects of opiates on fetal behavioral activity in utero. Life Sci 1983;33:639-42. Kitterman JA, Liggins GC, Clements JA, Tooley WH. Stimulation of breathing movements in fetal sheep by inhibitors of prostaglandin synthesis. J Dev Physiol 1979; 1: 453-66.