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The effect of ethanol on habituation and the cardiovascular response to stimulation in fetal sheep
European Journal of Obstetrics & Gynecology and Reproductive Biology, 36 (1990) 81-95
87
Elsevier EUROBS 00965
The effect of ethanol on habituation and the cardiovascular response to stimulation in fetal sheep L.R. Leader ‘, F.G. Smith * and E.R. Lumbers * ’ School of Obstetrics & Gynaecology and * School of Physiology & Pharmacology, University of New South Wales, Kensington, Australia
Accepted for publication 27 October 1989
Summary
Fetal cardiovascular (CVS) changes, forelimb movements (FM) and rates of habituation to repeated stimulation, with suffusions of cold saline over the skin, were measured in 12 chronically catheterized fetal sheep aged 130-145 days. Stimulation of the fetuses caused a significant rise in heart rate (HR) (p < 0.01) and blood pressure (BP) (p c 0.001) and FM (p < 0.01). When the ewe was given an intravenous (i.v.) infusion of ethanol which produced fetal ethanol levels of 87 * 1.1 mg/lOO ml, the number of spontaneous FM decreased (p < 0.05). After i.v. ethanol, repeated stimulation of the fetuses still caused an increase in FM (p < 0.01) and a rise in HR (p < 0.05) and BP (p < 0.02) but the fetuses habituated more rapidly (7.25 f 1.28 stimuli) compared to control experiments performed prior to (21.5 f 3.57 stimuli) or after the ethanol (17.75 f 5.83, p < 0.01). Fetal exposure to low concentrations of ethanol does affect the patterns of response and habituation of the developing fetal central nervous system. Habituation; Stimulation; Fetal; Sheep; Ethanol; Cold saline; Cardiovascular
Introduction
The harmful effects of ingestion of large quantities of alcohol during pregnancy are well described [l]. In addition, low doses of maternal ethanol cause suppression of breathing by human fetuses [2,3] and sheep fetuses, who also show altered electrocortical activity for 3 hours after infusion of alcohol [4]. Correspondence: Dr. Leo R. Leader, School of Obstetrics & Gynaecology, University of New South Wales, P.O. Box 1, Kensington NSW, 2033 Australia.
002%2243/90/%03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
88
We have developed a fetal sheep model for studying fetal habituation [5,6]. Fetal sheep respond to stimulation by moving and increasing their heart rate and blood pressure. With repetition of the stimulus, the fetal responses decrease, i.e., it habituates. Habituation is widely regarded as a basic form of learning and there is good evidence that a normal habituation pattern reflects an intact and fully functioning central nervous system [7-91. The patterns of habituation of the human fetus may provide a means of measuring the effects of the intrauterine environment on the fetus, since the pattern of habituation can be altered both in human [lo] and sheep fetuses by decreasing the amount of inspired maternal oxygen [9]. Sedative drugs taken during pregnancy also affect fetal habituation [ll]. We have previously demonstrated that the number of stimuli required for habituation in fetal sheep is similar whether the stimulus is vibroacoustic or cold saline [5,6]. In humans, cold saline injected through the cervix in labour has been shown to cause an increase in fetal heart rate [12]. As alcohol may be one of the most widely ingested drugs during pregnancy, a study was undertaken to examine the effects of a low concentration of alcohol on the habituation patterns of fetal sheep. The stimuli used were repeated suffusions of cold saline through a catheter sutured against the fetal skin. Materials and methods
Studies were performed on 12 sheep aged 130-145 days. Fetuses were surgically prepared 5-7 days before experiments. Ewes were anaesthetised with 1 g sodium thiopentone (Pentothal, Abbott) and maintained with 2% halothane (Fluothane, ICI) in oxygen. Polyvinyl catheters were inserted into the ewes carotid artery and jugular vein. To record electrocortical activity (ECoG), silver ball electrodes were implanted bilaterally onto the fetal parietal dura and held in place by means of teflon screws and acrylic cement. Pairs of electrodes were implanted into the biceps and triceps of each forelimb to record electromyographic activity (EMG), and on the inner and outer canthus of one eye to record electroocular activity (EOG). Polyvinyl catheters (1.0 mm i.d.; 1.5 mm o.d.) were inserted into the fetal carotid artery to measure blood pressure and heart rate, into the fetal trachea to measure fetal breathing activity and into the amniotic cavity to measure intra-amniotic pressure (IAP). A polyvinyl catheter was sutured to the fetal skin against the fetal neck through which the saline was injected. A further catheter was inserted into a fetal jugular vein. All electrodes and catheters were firmly anchored to the fetal back and exteriorized through the maternal flank. After the ewes had recovered, they were housed in metabolic cages and allowed free access to food and water. These experiments were conducted with the approval of the animal ethics committee of the University of New South Wales. Stimulus
Repeated suffusions of 5 ml of cold saline for 3 s through the catheter which had been sutured to the fetal neck were used as the stimuli to elicit habituation. The
89
distal end of the catheter was fenestrated to allow the saline to run across the fetal skin. In a previous series of experiments, a thermistor probe was implanted under the fetal skin on the fetal back in three sheep and no consistent changes in fetal body temperature were observed [6]. More recently, we have measured fetal oesophageal temperature on eight occasions using a temperature probe (Micronta LCD Digital Thermometer) and have shown no change in fetal temperature during stimulation [13] even though in one experiment, the fetus received 40 suffusions of saline before it habituated. Fetal response
Fetal movements (measured as changes in the integrated EMG) which occurred either during the stimulus or within 2.5 s of it were considered a response. If the fetus was moving at the time that the stimulus was due, it was withheld until the movement ceased. The stimulus was repeated approximately every 20 s until habituation occurred. This was defined as a lack of a movement response to 5 successive stimuli [14,15,5,6]. The habituation pattern of an individual fetus was determined in control experiments and then after an infusion of ethanol to the ewe by counting the number of stimuli needed to produce habituation. Data recording
Data were simultaneously recorded on an eight channel Devices recorder and an Apple IIe microcomputer with an Isaacs Cyborg interface using specially designed software. The software averages the integrated EMG recording for 4.5 s preceding the stimulus, 3 s during and 2.5 s after the stimulus. A response was recorded when the mean fluctuation of the integrated EMG recording during or within 2.5 s of the stimulus increased to twice the mean amplitude of the fluctuation in the 4.5 s preceding the stimulus. This is similar to the criteria used by Natale et al. [16]. The computer analysis also allowed the precise temporal relationship between the stimulus and the response to be measured [6]. The unintegrated EMG recordings were simultaneously displayed on a two channel oscilloscope sweeping at 1 cm/s and the fetal responses noted. The fetal ECoG and EOG were also displayed on an oscilloscope to assist in determining fetal state. Data collected during the experiments were stored on floppy disks and later analyzed. The number of stimuli required for habituation as assessed by the observation of the oscilloscope and paper records was compared to the computer analysis of the number of stimuli required for habituation. There was a highly significant correlation (p < 0.0001, r = 0.99) between the two methods of assessing habituation. Experimental
protocol
Two experimental protocols were carried out at least once in each sheep. These were a control and an ethanol experiment. Two to 5 days after a control experiment, six sheep were tested for habituation after an intravenous (i.v.) ethanol infusion into the ewe. A further control experiment was performed 2 to 5 days later. Four other sheep were given i.v. ethanol before a control experiment to ensure that the results obtained were not due to the
90
order of experiments or the gestational age; then a further control experiment was performed 2 to 5 days later. Control experiment Control observations were made for 10 to 20 min prior to stimulating the fetus. During this time, fetal blood pressure (corrected for amniotic pressure), heart rate and amniotic pressures were recorded. The fetal state (high or low voltage) was determined by observing the ECoG, EOG and tracheal pressure recordings. The number of spontaneous fetal movements were also recorded. Only movements that occurred 10s or more apart were considered separate. The duration of the observation period was recorded and the number of fetal movements expressed per min. The fetus was then stimulated and the number of stimuli delivered before habituation occurred were counted. Fetal and maternal arterial samples were taken for pH, p0, and pC0, analysis before and after each experiment. Ethanol experiments Control observations were made for 10 min, then 1 gm/kg of maternal body weight of a 40% (v/v) solution of ethanol was injected i.v. over 10 min into the ewe. No observations were made for the next 10 min. Fetal HR, BP and FM were recorded for a further 10 min after which the fetus was stimulated until it habituated. Fetal and maternal arterial samples (1 ml) were taken simultaneously before the infusion of ethanol, 20 min later (before the 2nd control period), 30 min later (preceding the onset of stimulation) and then when the fetus had habituated. Plasma ethanol levels were measured using an enzymatic method (Behring kit). The coefficient of variation for this assay is less than 1%. Glucose concentrations were measured in plasma using a glucose hexokinase method on a Sentrifichem analyser (Roche). The coefficient of variation for this method is less than 2%. Data analysis Thirty three experiments were conducted in 12 sheep. Results are reported as means and standard errors of the mean (SEM). Data were analyzed by means of the Student’s t-test (paired or unpaired as appropriate) or by means of a one way analysis of variance followed by the Tukey test to establish differences between population means [17]. Results
Ethanol, glucose concentrations and blood gases The maternal and fetal blood ethanol levels are shown in Table levels in Table II. The fetal ethanol levels were 92 f 8.4 mg/lOO the period of stimulation and had fallen to 76 f 5.0 mg/lOO ml by habituated. Neither the fetal arterial blood glucose (1.95 f 0.15 mmol/l) levels (3.31 f 0.29 mrnol/l) changed during the experiment.
I and the glucose ml at the start of the time the fetus nor the maternal
91 TABLE
I
Fetal and maternal
Fetal Maternal
ethanol
levels (mg/lOO
ml) in IO fetal sheep. Data are expressed
as means f SE
Pre-ethanol control
Post-ethanol control
Prestim
Post-habit
0 0
92+8.4 84f7.1
86.3 + 6.3 71.1 k4.2
76k5.0 55f7.1
Prestim, pre-stimulation. Post-habit, post-habituation.
TABLE
II
Fetal and maternal
Fetal Maternal
glucose
levels (mmol/l)
in 10 fetal sheep. Data are expressed
as means f SE
Pre-ethanol control
Post-ethanol control
Prestim
Post-habit
1.95 f 0.15 3.31 kO.29
1.81+0.14 2.77 + 0.25
1.73 * 0.12 2.69 + 0.21
1.85 kO.14 2.63 * 0.25
Prestim, pre-stimulation. Post habit, post-habituation.
CONTROL
ALCOHOL
POST ALCOHOL CONTROL
Fig. 1. This demonstrates the effect of ethanol on habituation rates compared to control experiments performed either prior to or after ethanol data are prescribed as means k SE and represent paired samples in the same animal. * * * p -C 0.01.
92 TABLE
III
The fetal cardiovascular changes that occur in response Data are presented as means f SE
Before stim During stim
to stimulation
in control
experiments
Heart rate beats/mm
Systolic BP rnmHg
Diastolic BP mmHg
166.1 f 6.1 189.9 f 9.8 *
59.4* 1.9 67.9f2.0 **
41.2kl.7 46.9k1.9
(n =19).
**
* p < 0.01. ** p
Fetal and maternal arterial PO,, pCOz and pH were 19.45 f 0.77 mmHg, 40.2 f0.87 mmHg, 7.35 f 0.02 and 94.56 f 1.9 mmHg, 27.55 + 0.79 mmHg and 7.45 * 0.01, respectively and did not change during the experiment. Fetal movements
During the control period preceding stimulation there were 1.56 & 0.12 (n = 33) fetal movements/mm The number of fetal movements in those fetuses who remained in low voltage (LV) throughout the control period did not differ significantly from the fetal activity of those who remained in high voltage (HV). (LV 1.56 k 0.19 moves/min, n = 15; HV 1.18 f 0.15 moves/mm, n = 9). Nine fetuses were excluded from the state analysis as they changed their ECoG voltage during the control period. During stimulation, the number of fetal movements increased to 2.05 f 0.17 moves/mm (p = 0.01). After the ethanol infusion, the number of spontaneous fetal movements was significantly reduced (before 1.75 + 0.18 moves/mm; after 0.89 f 0.22 moves/mm, p < 0.05, n = 10). Each fetus that received alcohol acted as its own control. The ECoG state of the fetus had no significant effect on the amount of fetal activity (LV 0.94 + 0.16 moves/min, n = 4; HV 0.85 f 0.37 moves/mm, n = 6). During stimulation there was a significant increase in the number of fetal movements (2.42 + 0.34 moves/min, p < 0.01). This is similar to the increase in fetal movements seen during stimulation in the control experiments.
TABLE
IV
The fetal cardiovascular changes that occur in response. to stimulation Data are presented as means * SE
1. Pre-ethanol 2. Post-ethanol 3. During stim
after the ethanol
infusion
Heart rate beats/mm
Systolic BP mmHg
Diastolic BP rnmHe.
164.3k4.9 173.9k 5.2 202.9 f 9.4 *
61.9k 3.0 64.1 f 2.7 72.8 f 5.4 * *
41.4 f 2.6 43.3 f 3.1 49.5 rt 4.2 * *
2 vs. 3: * p < 0.05 * * p < 0.02. n, number of experiments.
(n = 8).
93
Habituation patterns The number of stimuli required to cause habituation
are shown in Fig. 1. When tested after an ethanol infusion, the fetuses habituated significantly faster (7.25 f 1.28, n = 10) compared to the control period which preceded alcohol (21.5 + 3.57, n = 6, p < 0.01). There was no difference in the number of stimuli required to cause habituation when the control experiment was performed first or second (i.e., after the alcohol experiment 21.5 f 3.57 vs. 17.75 f 5.83, n = 14). Both in control and ethanol experiments, there was no correlation between the number of stimuli required for habituation and the gestational age of the fetus. Cardiovascular changes
Repeated stimulation of the fetus was associated with a significant increase in heart rates and blood pressures (Table III). Following the infusion of ethanol prior to stimulation, there was a small non-significant increase in the fetal heart rate and blood pressure (Table IV). Fetal stimulation was once again associated with a significant increase in fetal heart rate (p < 0.05) and blood pressure (p < 0.02, n = 8). In two fetuses, the arterial catheter became obstructed after the control experiments preventing recording of CVS data. Discussion
The fall in spontaneous fetal movements seen after an i.v. infusion of ethanol to the ewe was not due to a change in the fetal ECoG activity because the amount of spontaneous fetal movements in LV is similar to those observed in HV. In the present study, there was no obvious change in ECoG activity after the ethanol. By contrast to the reduction in spontaneous fetal movements, ethanol had no effect on the ability of the fetus to respond to stimulation in that the number of fetal movements and CVS changes during stimulation were similar in both ethanol and control experiments (Tables III and IV). Infusion of ethanol in the ewe was associated with a small nonsignificant rise in fetal heart rate and blood pressure. This observation is similar to that of Ayromlooi et al. [18]. The stimulus of cold saline against the fetal skin is a powerful stimulus and elicits a very positive response by the fetus. However, using the same stimulus, Leader et al. [6] have shown that hypoxia reduced the amount of spontaneous fetal activity and fetal stimulation failed to cause an increase in fetal movements activity [6] or heart rate and blood pressure [13]. Fetal sheep stimulated during an intravenous infusion of noradrenaline did not show an increase in fetal movements and habituated more rapidly [6]. Ethanol altered the fetal habituation pattern, i.e., they habituated more rapidly after ethanol (p < 0.01). This effect was not due to the order of the experiments because in four sheep, the ethanol experiments were done before control experiments. It is also not due to the effects of gestational age of the fetus as there was no correlation between fetal age and the number of stimuli needed for habituation.
94
The finding of more rapid habituation could be due to the release of fetal catecholamines by ethanol. A recent study in fetal sheep found that an iv. fetal infusion of noradrenaline is associated with more rapid habituation [6]. We have previously demonstrated faster habituation in some ‘high risk’ human fetuses [15]. The change in fetal habituation rates was not due to alterations in fetal state in that the rates of habituation were similar in those fetuses who were in LV compared to those in HV. Using vibroacoustic stimulation [5] and cold saline stimulation [6], we have shown that the rates of habituation do not differ significantly in HV and LV. On the other hand, the alteration in habituation may be due to a direct effect of ethanol on fetal brain function. Ethanol has been shown to decrease cerebral oxygen consumption, as well as decrease blood flow to the cerebral hemispheres [19]. From the study of fetal cerebral oxidative metabolism [19], it has been suggested that ethanol may cause effects for up to 24 hours after ingestion. In the present study, the effects of ethanol were no longer present when control experiments were done 3-5 days later. Although ethanol depressed spontaneous fetal movement activity, it did not alter fetal .responsiveness to stimulation but the fetus habituated more rapidly. This suggests that fetal movement and cardiovascular responses to stimulation are independent of each other and that habituation may be a more sensitive measure of fetal CNS function. This study presents further evidence that even low concentrations of ethanol effect the fetal CNs and may alter cognitive function. Acknowledgements
The authors wish to thank Dr. D. Naidoo, Department of Biochemistry, St. Vincents Hospital, Sydney for performing the ethanol and glucose assays and Professor M.J. Bennett for his assistance with the manuscript. References 1 Barrisom IG, Waterson El, Murray-Lyon IM. Adverse effects of alcohol in pregnancy. Br J Addict 1985;80:11-22. 2 Fox HE, Steinbrecher M, Pessel D, Inglis J, Medvid L, Angel E. Maternal ethanol ingestion and the occurrence of human fetal breathing movements. Am J Obstet Gynecol 1978;132:354. 3 McLeod W, Brien 3, Loomis C, Carmichael L, Probert C, Patrick J. Effect of maternal ethanol ingestion on fetal breathing movements, gross body movements, and heart rate at 37 to 40 weeks’ gestational age. Am J Obstet Gynecol 1983;145:251. 4 Patrick J, Richardson B, Hasen G, Clarke D, Wlodek M, Bousquet J, Brien J. Effects of maternal ethanol infusion on fetal cardiovascular and brain activity in lambs. Am J Obstet Gynecol 1985;151:859-867. 5 Leader LR, Stevens AD, Lumbers ER. Measurement of fetal responses to vibroacoustic stimuli: habituation in fetal sheep. Biol Neonate 1988;53:73-85. 6 Leader LR, Smith FG, Lumbers ER, Stevens AD. The effect of hypoxia and catecholamines on the habituation rates of chronically catheterised ovine fetuses. Biol Neonate 1989;56:218-227. 7 Lewis M. Individual differences in the measurement of early cognitive growth. In: Hellmuth, ed. Exceptional infant: two studies in abnormalities, 1971:172-210.
95 8 Leader LR, Baillie P, Martin B, Molten0 C, Wynchank S. Fetal responses to vibrotactile stimulation: a possible predictor of fetal and neonatal outcome. Aust NZ J Obstet Gynaecol 1984;24:251-256. 9 Madison LS, Adubato SA, Madison JK, Nelson RM, Anderson JC, Erickson J, Kuss LM, Goodlin RC. Fetal response decrement: true habituation? Dev Behav Ped 1986;17:14-20. 10 Leader LR, Baillie P. The changes in fetal habituation patterns due to a decrease in inspired maternal oxygen. Br J Obstet Gynaecol 1988;95:664-668. 11 Leader LR, Bennett MJ. Fetal Habituation. In Fetal and Neonatal Neurology and Neurosurgery. Levene MI. Bennett MJ, Punt J. London: Churchill Livingston, 1988. 12 Timor-Tritsch IE. The effect of external stimuli on fetal behaviour. Eur J Obstet Gynecol Reprod Biol 1986;21:321-329. 13 Leader LR, Smith FG, Lumbers ER, Stevens AD. The effects of alcohol on fetal habituation. Presented at the meeting of The Society for the Study of Fetal Physiology, Cairns, 1988. 14 Brackbill Y, Kane J, Mamriello RL, Adamson D. Obstetric premeditation and infant outcome. Am J Obstet Gynecol 1974;118:377-384. 15 Leader LR, Baillie P, Martin B. Vermeulen E. Fetal habituation in high-risk pregnancies. Br J Obstet Gynaecol 1982;89:441-446. 16 Natale R, Clewlow F, Dawes GS. Measurement of fetal forelimb movements in the lamb in utero. Am J Obstet Gynecol 1981;140:545-551. 17 Zar JH. Biostatistical Analysis, 2nd Edn. Prentice-Hall International Editions, 1984. 18 Ayromlooi J, Tobias M, Berg PD. Desiderio D. Effects of ethanol on the circulation and acid-base balance of pregnant sheep. Obstet Gynecol 1979;54:624-630. 19 Richardson B, Patrick J, Homan J, Carmichael L, Brien J. Cerebral oxidative metabolism in fetal sheep with multiple-dose ethanol infusion. Am J Obstet Gynecol 1987;157:1496-1502.
87
Elsevier EUROBS 00965
The effect of ethanol on habituation and the cardiovascular response to stimulation in fetal sheep L.R. Leader ‘, F.G. Smith * and E.R. Lumbers * ’ School of Obstetrics & Gynaecology and * School of Physiology & Pharmacology, University of New South Wales, Kensington, Australia
Accepted for publication 27 October 1989
Summary
Fetal cardiovascular (CVS) changes, forelimb movements (FM) and rates of habituation to repeated stimulation, with suffusions of cold saline over the skin, were measured in 12 chronically catheterized fetal sheep aged 130-145 days. Stimulation of the fetuses caused a significant rise in heart rate (HR) (p < 0.01) and blood pressure (BP) (p c 0.001) and FM (p < 0.01). When the ewe was given an intravenous (i.v.) infusion of ethanol which produced fetal ethanol levels of 87 * 1.1 mg/lOO ml, the number of spontaneous FM decreased (p < 0.05). After i.v. ethanol, repeated stimulation of the fetuses still caused an increase in FM (p < 0.01) and a rise in HR (p < 0.05) and BP (p < 0.02) but the fetuses habituated more rapidly (7.25 f 1.28 stimuli) compared to control experiments performed prior to (21.5 f 3.57 stimuli) or after the ethanol (17.75 f 5.83, p < 0.01). Fetal exposure to low concentrations of ethanol does affect the patterns of response and habituation of the developing fetal central nervous system. Habituation; Stimulation; Fetal; Sheep; Ethanol; Cold saline; Cardiovascular
Introduction
The harmful effects of ingestion of large quantities of alcohol during pregnancy are well described [l]. In addition, low doses of maternal ethanol cause suppression of breathing by human fetuses [2,3] and sheep fetuses, who also show altered electrocortical activity for 3 hours after infusion of alcohol [4]. Correspondence: Dr. Leo R. Leader, School of Obstetrics & Gynaecology, University of New South Wales, P.O. Box 1, Kensington NSW, 2033 Australia.
002%2243/90/%03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
88
We have developed a fetal sheep model for studying fetal habituation [5,6]. Fetal sheep respond to stimulation by moving and increasing their heart rate and blood pressure. With repetition of the stimulus, the fetal responses decrease, i.e., it habituates. Habituation is widely regarded as a basic form of learning and there is good evidence that a normal habituation pattern reflects an intact and fully functioning central nervous system [7-91. The patterns of habituation of the human fetus may provide a means of measuring the effects of the intrauterine environment on the fetus, since the pattern of habituation can be altered both in human [lo] and sheep fetuses by decreasing the amount of inspired maternal oxygen [9]. Sedative drugs taken during pregnancy also affect fetal habituation [ll]. We have previously demonstrated that the number of stimuli required for habituation in fetal sheep is similar whether the stimulus is vibroacoustic or cold saline [5,6]. In humans, cold saline injected through the cervix in labour has been shown to cause an increase in fetal heart rate [12]. As alcohol may be one of the most widely ingested drugs during pregnancy, a study was undertaken to examine the effects of a low concentration of alcohol on the habituation patterns of fetal sheep. The stimuli used were repeated suffusions of cold saline through a catheter sutured against the fetal skin. Materials and methods
Studies were performed on 12 sheep aged 130-145 days. Fetuses were surgically prepared 5-7 days before experiments. Ewes were anaesthetised with 1 g sodium thiopentone (Pentothal, Abbott) and maintained with 2% halothane (Fluothane, ICI) in oxygen. Polyvinyl catheters were inserted into the ewes carotid artery and jugular vein. To record electrocortical activity (ECoG), silver ball electrodes were implanted bilaterally onto the fetal parietal dura and held in place by means of teflon screws and acrylic cement. Pairs of electrodes were implanted into the biceps and triceps of each forelimb to record electromyographic activity (EMG), and on the inner and outer canthus of one eye to record electroocular activity (EOG). Polyvinyl catheters (1.0 mm i.d.; 1.5 mm o.d.) were inserted into the fetal carotid artery to measure blood pressure and heart rate, into the fetal trachea to measure fetal breathing activity and into the amniotic cavity to measure intra-amniotic pressure (IAP). A polyvinyl catheter was sutured to the fetal skin against the fetal neck through which the saline was injected. A further catheter was inserted into a fetal jugular vein. All electrodes and catheters were firmly anchored to the fetal back and exteriorized through the maternal flank. After the ewes had recovered, they were housed in metabolic cages and allowed free access to food and water. These experiments were conducted with the approval of the animal ethics committee of the University of New South Wales. Stimulus
Repeated suffusions of 5 ml of cold saline for 3 s through the catheter which had been sutured to the fetal neck were used as the stimuli to elicit habituation. The
89
distal end of the catheter was fenestrated to allow the saline to run across the fetal skin. In a previous series of experiments, a thermistor probe was implanted under the fetal skin on the fetal back in three sheep and no consistent changes in fetal body temperature were observed [6]. More recently, we have measured fetal oesophageal temperature on eight occasions using a temperature probe (Micronta LCD Digital Thermometer) and have shown no change in fetal temperature during stimulation [13] even though in one experiment, the fetus received 40 suffusions of saline before it habituated. Fetal response
Fetal movements (measured as changes in the integrated EMG) which occurred either during the stimulus or within 2.5 s of it were considered a response. If the fetus was moving at the time that the stimulus was due, it was withheld until the movement ceased. The stimulus was repeated approximately every 20 s until habituation occurred. This was defined as a lack of a movement response to 5 successive stimuli [14,15,5,6]. The habituation pattern of an individual fetus was determined in control experiments and then after an infusion of ethanol to the ewe by counting the number of stimuli needed to produce habituation. Data recording
Data were simultaneously recorded on an eight channel Devices recorder and an Apple IIe microcomputer with an Isaacs Cyborg interface using specially designed software. The software averages the integrated EMG recording for 4.5 s preceding the stimulus, 3 s during and 2.5 s after the stimulus. A response was recorded when the mean fluctuation of the integrated EMG recording during or within 2.5 s of the stimulus increased to twice the mean amplitude of the fluctuation in the 4.5 s preceding the stimulus. This is similar to the criteria used by Natale et al. [16]. The computer analysis also allowed the precise temporal relationship between the stimulus and the response to be measured [6]. The unintegrated EMG recordings were simultaneously displayed on a two channel oscilloscope sweeping at 1 cm/s and the fetal responses noted. The fetal ECoG and EOG were also displayed on an oscilloscope to assist in determining fetal state. Data collected during the experiments were stored on floppy disks and later analyzed. The number of stimuli required for habituation as assessed by the observation of the oscilloscope and paper records was compared to the computer analysis of the number of stimuli required for habituation. There was a highly significant correlation (p < 0.0001, r = 0.99) between the two methods of assessing habituation. Experimental
protocol
Two experimental protocols were carried out at least once in each sheep. These were a control and an ethanol experiment. Two to 5 days after a control experiment, six sheep were tested for habituation after an intravenous (i.v.) ethanol infusion into the ewe. A further control experiment was performed 2 to 5 days later. Four other sheep were given i.v. ethanol before a control experiment to ensure that the results obtained were not due to the
90
order of experiments or the gestational age; then a further control experiment was performed 2 to 5 days later. Control experiment Control observations were made for 10 to 20 min prior to stimulating the fetus. During this time, fetal blood pressure (corrected for amniotic pressure), heart rate and amniotic pressures were recorded. The fetal state (high or low voltage) was determined by observing the ECoG, EOG and tracheal pressure recordings. The number of spontaneous fetal movements were also recorded. Only movements that occurred 10s or more apart were considered separate. The duration of the observation period was recorded and the number of fetal movements expressed per min. The fetus was then stimulated and the number of stimuli delivered before habituation occurred were counted. Fetal and maternal arterial samples were taken for pH, p0, and pC0, analysis before and after each experiment. Ethanol experiments Control observations were made for 10 min, then 1 gm/kg of maternal body weight of a 40% (v/v) solution of ethanol was injected i.v. over 10 min into the ewe. No observations were made for the next 10 min. Fetal HR, BP and FM were recorded for a further 10 min after which the fetus was stimulated until it habituated. Fetal and maternal arterial samples (1 ml) were taken simultaneously before the infusion of ethanol, 20 min later (before the 2nd control period), 30 min later (preceding the onset of stimulation) and then when the fetus had habituated. Plasma ethanol levels were measured using an enzymatic method (Behring kit). The coefficient of variation for this assay is less than 1%. Glucose concentrations were measured in plasma using a glucose hexokinase method on a Sentrifichem analyser (Roche). The coefficient of variation for this method is less than 2%. Data analysis Thirty three experiments were conducted in 12 sheep. Results are reported as means and standard errors of the mean (SEM). Data were analyzed by means of the Student’s t-test (paired or unpaired as appropriate) or by means of a one way analysis of variance followed by the Tukey test to establish differences between population means [17]. Results
Ethanol, glucose concentrations and blood gases The maternal and fetal blood ethanol levels are shown in Table levels in Table II. The fetal ethanol levels were 92 f 8.4 mg/lOO the period of stimulation and had fallen to 76 f 5.0 mg/lOO ml by habituated. Neither the fetal arterial blood glucose (1.95 f 0.15 mmol/l) levels (3.31 f 0.29 mrnol/l) changed during the experiment.
I and the glucose ml at the start of the time the fetus nor the maternal
91 TABLE
I
Fetal and maternal
Fetal Maternal
ethanol
levels (mg/lOO
ml) in IO fetal sheep. Data are expressed
as means f SE
Pre-ethanol control
Post-ethanol control
Prestim
Post-habit
0 0
92+8.4 84f7.1
86.3 + 6.3 71.1 k4.2
76k5.0 55f7.1
Prestim, pre-stimulation. Post-habit, post-habituation.
TABLE
II
Fetal and maternal
Fetal Maternal
glucose
levels (mmol/l)
in 10 fetal sheep. Data are expressed
as means f SE
Pre-ethanol control
Post-ethanol control
Prestim
Post-habit
1.95 f 0.15 3.31 kO.29
1.81+0.14 2.77 + 0.25
1.73 * 0.12 2.69 + 0.21
1.85 kO.14 2.63 * 0.25
Prestim, pre-stimulation. Post habit, post-habituation.
CONTROL
ALCOHOL
POST ALCOHOL CONTROL
Fig. 1. This demonstrates the effect of ethanol on habituation rates compared to control experiments performed either prior to or after ethanol data are prescribed as means k SE and represent paired samples in the same animal. * * * p -C 0.01.
92 TABLE
III
The fetal cardiovascular changes that occur in response Data are presented as means f SE
Before stim During stim
to stimulation
in control
experiments
Heart rate beats/mm
Systolic BP rnmHg
Diastolic BP mmHg
166.1 f 6.1 189.9 f 9.8 *
59.4* 1.9 67.9f2.0 **
41.2kl.7 46.9k1.9
(n =19).
**
* p < 0.01. ** p
Fetal and maternal arterial PO,, pCOz and pH were 19.45 f 0.77 mmHg, 40.2 f0.87 mmHg, 7.35 f 0.02 and 94.56 f 1.9 mmHg, 27.55 + 0.79 mmHg and 7.45 * 0.01, respectively and did not change during the experiment. Fetal movements
During the control period preceding stimulation there were 1.56 & 0.12 (n = 33) fetal movements/mm The number of fetal movements in those fetuses who remained in low voltage (LV) throughout the control period did not differ significantly from the fetal activity of those who remained in high voltage (HV). (LV 1.56 k 0.19 moves/min, n = 15; HV 1.18 f 0.15 moves/mm, n = 9). Nine fetuses were excluded from the state analysis as they changed their ECoG voltage during the control period. During stimulation, the number of fetal movements increased to 2.05 f 0.17 moves/mm (p = 0.01). After the ethanol infusion, the number of spontaneous fetal movements was significantly reduced (before 1.75 + 0.18 moves/mm; after 0.89 f 0.22 moves/mm, p < 0.05, n = 10). Each fetus that received alcohol acted as its own control. The ECoG state of the fetus had no significant effect on the amount of fetal activity (LV 0.94 + 0.16 moves/min, n = 4; HV 0.85 f 0.37 moves/mm, n = 6). During stimulation there was a significant increase in the number of fetal movements (2.42 + 0.34 moves/min, p < 0.01). This is similar to the increase in fetal movements seen during stimulation in the control experiments.
TABLE
IV
The fetal cardiovascular changes that occur in response. to stimulation Data are presented as means * SE
1. Pre-ethanol 2. Post-ethanol 3. During stim
after the ethanol
infusion
Heart rate beats/mm
Systolic BP mmHg
Diastolic BP rnmHe.
164.3k4.9 173.9k 5.2 202.9 f 9.4 *
61.9k 3.0 64.1 f 2.7 72.8 f 5.4 * *
41.4 f 2.6 43.3 f 3.1 49.5 rt 4.2 * *
2 vs. 3: * p < 0.05 * * p < 0.02. n, number of experiments.
(n = 8).
93
Habituation patterns The number of stimuli required to cause habituation
are shown in Fig. 1. When tested after an ethanol infusion, the fetuses habituated significantly faster (7.25 f 1.28, n = 10) compared to the control period which preceded alcohol (21.5 + 3.57, n = 6, p < 0.01). There was no difference in the number of stimuli required to cause habituation when the control experiment was performed first or second (i.e., after the alcohol experiment 21.5 f 3.57 vs. 17.75 f 5.83, n = 14). Both in control and ethanol experiments, there was no correlation between the number of stimuli required for habituation and the gestational age of the fetus. Cardiovascular changes
Repeated stimulation of the fetus was associated with a significant increase in heart rates and blood pressures (Table III). Following the infusion of ethanol prior to stimulation, there was a small non-significant increase in the fetal heart rate and blood pressure (Table IV). Fetal stimulation was once again associated with a significant increase in fetal heart rate (p < 0.05) and blood pressure (p < 0.02, n = 8). In two fetuses, the arterial catheter became obstructed after the control experiments preventing recording of CVS data. Discussion
The fall in spontaneous fetal movements seen after an i.v. infusion of ethanol to the ewe was not due to a change in the fetal ECoG activity because the amount of spontaneous fetal movements in LV is similar to those observed in HV. In the present study, there was no obvious change in ECoG activity after the ethanol. By contrast to the reduction in spontaneous fetal movements, ethanol had no effect on the ability of the fetus to respond to stimulation in that the number of fetal movements and CVS changes during stimulation were similar in both ethanol and control experiments (Tables III and IV). Infusion of ethanol in the ewe was associated with a small nonsignificant rise in fetal heart rate and blood pressure. This observation is similar to that of Ayromlooi et al. [18]. The stimulus of cold saline against the fetal skin is a powerful stimulus and elicits a very positive response by the fetus. However, using the same stimulus, Leader et al. [6] have shown that hypoxia reduced the amount of spontaneous fetal activity and fetal stimulation failed to cause an increase in fetal movements activity [6] or heart rate and blood pressure [13]. Fetal sheep stimulated during an intravenous infusion of noradrenaline did not show an increase in fetal movements and habituated more rapidly [6]. Ethanol altered the fetal habituation pattern, i.e., they habituated more rapidly after ethanol (p < 0.01). This effect was not due to the order of the experiments because in four sheep, the ethanol experiments were done before control experiments. It is also not due to the effects of gestational age of the fetus as there was no correlation between fetal age and the number of stimuli needed for habituation.
94
The finding of more rapid habituation could be due to the release of fetal catecholamines by ethanol. A recent study in fetal sheep found that an iv. fetal infusion of noradrenaline is associated with more rapid habituation [6]. We have previously demonstrated faster habituation in some ‘high risk’ human fetuses [15]. The change in fetal habituation rates was not due to alterations in fetal state in that the rates of habituation were similar in those fetuses who were in LV compared to those in HV. Using vibroacoustic stimulation [5] and cold saline stimulation [6], we have shown that the rates of habituation do not differ significantly in HV and LV. On the other hand, the alteration in habituation may be due to a direct effect of ethanol on fetal brain function. Ethanol has been shown to decrease cerebral oxygen consumption, as well as decrease blood flow to the cerebral hemispheres [19]. From the study of fetal cerebral oxidative metabolism [19], it has been suggested that ethanol may cause effects for up to 24 hours after ingestion. In the present study, the effects of ethanol were no longer present when control experiments were done 3-5 days later. Although ethanol depressed spontaneous fetal movement activity, it did not alter fetal .responsiveness to stimulation but the fetus habituated more rapidly. This suggests that fetal movement and cardiovascular responses to stimulation are independent of each other and that habituation may be a more sensitive measure of fetal CNS function. This study presents further evidence that even low concentrations of ethanol effect the fetal CNs and may alter cognitive function. Acknowledgements
The authors wish to thank Dr. D. Naidoo, Department of Biochemistry, St. Vincents Hospital, Sydney for performing the ethanol and glucose assays and Professor M.J. Bennett for his assistance with the manuscript. References 1 Barrisom IG, Waterson El, Murray-Lyon IM. Adverse effects of alcohol in pregnancy. Br J Addict 1985;80:11-22. 2 Fox HE, Steinbrecher M, Pessel D, Inglis J, Medvid L, Angel E. Maternal ethanol ingestion and the occurrence of human fetal breathing movements. Am J Obstet Gynecol 1978;132:354. 3 McLeod W, Brien 3, Loomis C, Carmichael L, Probert C, Patrick J. Effect of maternal ethanol ingestion on fetal breathing movements, gross body movements, and heart rate at 37 to 40 weeks’ gestational age. Am J Obstet Gynecol 1983;145:251. 4 Patrick J, Richardson B, Hasen G, Clarke D, Wlodek M, Bousquet J, Brien J. Effects of maternal ethanol infusion on fetal cardiovascular and brain activity in lambs. Am J Obstet Gynecol 1985;151:859-867. 5 Leader LR, Stevens AD, Lumbers ER. Measurement of fetal responses to vibroacoustic stimuli: habituation in fetal sheep. Biol Neonate 1988;53:73-85. 6 Leader LR, Smith FG, Lumbers ER, Stevens AD. The effect of hypoxia and catecholamines on the habituation rates of chronically catheterised ovine fetuses. Biol Neonate 1989;56:218-227. 7 Lewis M. Individual differences in the measurement of early cognitive growth. In: Hellmuth, ed. Exceptional infant: two studies in abnormalities, 1971:172-210.
95 8 Leader LR, Baillie P, Martin B, Molten0 C, Wynchank S. Fetal responses to vibrotactile stimulation: a possible predictor of fetal and neonatal outcome. Aust NZ J Obstet Gynaecol 1984;24:251-256. 9 Madison LS, Adubato SA, Madison JK, Nelson RM, Anderson JC, Erickson J, Kuss LM, Goodlin RC. Fetal response decrement: true habituation? Dev Behav Ped 1986;17:14-20. 10 Leader LR, Baillie P. The changes in fetal habituation patterns due to a decrease in inspired maternal oxygen. Br J Obstet Gynaecol 1988;95:664-668. 11 Leader LR, Bennett MJ. Fetal Habituation. In Fetal and Neonatal Neurology and Neurosurgery. Levene MI. Bennett MJ, Punt J. London: Churchill Livingston, 1988. 12 Timor-Tritsch IE. The effect of external stimuli on fetal behaviour. Eur J Obstet Gynecol Reprod Biol 1986;21:321-329. 13 Leader LR, Smith FG, Lumbers ER, Stevens AD. The effects of alcohol on fetal habituation. Presented at the meeting of The Society for the Study of Fetal Physiology, Cairns, 1988. 14 Brackbill Y, Kane J, Mamriello RL, Adamson D. Obstetric premeditation and infant outcome. Am J Obstet Gynecol 1974;118:377-384. 15 Leader LR, Baillie P, Martin B. Vermeulen E. Fetal habituation in high-risk pregnancies. Br J Obstet Gynaecol 1982;89:441-446. 16 Natale R, Clewlow F, Dawes GS. Measurement of fetal forelimb movements in the lamb in utero. Am J Obstet Gynecol 1981;140:545-551. 17 Zar JH. Biostatistical Analysis, 2nd Edn. Prentice-Hall International Editions, 1984. 18 Ayromlooi J, Tobias M, Berg PD. Desiderio D. Effects of ethanol on the circulation and acid-base balance of pregnant sheep. Obstet Gynecol 1979;54:624-630. 19 Richardson B, Patrick J, Homan J, Carmichael L, Brien J. Cerebral oxidative metabolism in fetal sheep with multiple-dose ethanol infusion. Am J Obstet Gynecol 1987;157:1496-1502.