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Chronic ethanol exposure during late gestation produces behavioral anomalies in neonatal lambs
Neurotoxicology and Teratology 22 (2000) 205–212
Chronic ethanol exposure during late gestation produces behavioral anomalies in neonatal lambs Jennifer Spear-Smitha, James F. Brienb, Marjorie Grafec, Richard Allricha, James D. Reynoldsd,* a Department of Animal Sciences, Purdue University, West Lafayette, IN, USA Department of Pharmacology and Toxicology, Queen’s University, Kingston, Canada c Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA d Obstetrical Anesthesia Research Laboratory, Division of Women’s Anesthesia, Department of Anesthesiology, Duke University Medical Center, Box 3094, Durham, NC 27710, USA Received 16 July 1999; Accepted 30 August 1999 b
Abstract A total of 45 pregnant ewes were assigned to one of three treatment groups: 1 g ethanol/kg maternal body weight (n ⫽ 18); pair-fed control (n ⫽ 15); and ad lib control (n ⫽ 12). Dosing started at gestational day (GD) 106, and was administered every other day until GD 134. Parturition occurred between GD 144 and 147. Analysis of the placentas indicated that ethanol exposure decreased cotyledon diameter and cotyledon weight compared to the control groups (p ⬍ 0.05). At birth, lambs were given a Vigor Score and then behavior was assessed using a videotape monitoring system for 24 h. Offspring in the ethanol treatment group were significantly less vigorous at birth (p ⬍ 0.05). This finding reversed during the subsequent 24 h such that the ethanol-exposed lambs were significantly more active (p ⫽ 0.001) than the control lambs. Morphometric and histologic examination of the cerebral cortex and hippocampus revealed no differences amongst the three treatment groups. Collectively, the data demonstrate that moderate ethanol exposure during the third-trimester equivalent of gestation can produce placental dysmorphology and postnatal behavioral anomalies in neonatal lambs in the absence of gross neurologic injury. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Behavior; Ethanol; Fetal alcohol syndrome; Placenta; Sheep
1. Introduction Consumption of ethanol during pregnancy can produce a wide spectrum of dose-dependent effects in the developing conceptus, including teratogenesis [1]. The most debilitating manifestation of ethanol teratogenesis would appear to be central nervous system (CNS) dysfunction, which can present as mental deficiency, poor motor coordination, and hyperactivity in postnatal life. Despite being widely recognized as a teratogen, the exact mechanism(s) by which ethanol produces these dysfunctions is not clearly understood [20]. Experimental studies in a variety of animal species have demonstrated that ethanol can produce effects on the conceptus similar to those that occur in the human [9,29]. Many studies have been conducted using the rat as the experimental animal due to its short gestation (about 22 days) and rela* Corresponding author. Tel.: 919-681-6774; Fax: 919-681-7022. E-mail address: [email protected]
tively large litter size. In the rat, a substantial amount of brain development, including the brain growth spurt, occurs postnatally [8]. In contrast, the brain growth spurt in the human is a perinatal event. This is a significant consideration, as the brain growth spurt is a period of rapid neuronal development during which the CNS is very sensitive to drug exposure and pathophysiologic insult [15]. As such, this has required the use of artificial postnatal feeding and rearing programs for neonatal rats to examine the effects of ethanol on the developing CNS during the brain growth spurt [28]. However, postnatal ethanol administration cannot assess any potential contribution of ethanol-induced changes in maternal and/or placental function on CNS development. Other concerns include the different rates of ethanol elimination and, by extension, different patterns of ethanol exposure. The capacity of the neonate to eliminate ethanol by hepatic biotransformation is considerably less compared to the maternal–fetal unit, in which ethanol elimination from the fetus occurs via placental transfer of ethanol into the maternal circulation and subsequent maternal hepatic biotransformation [5].
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In view of this, our laboratories have focused on elucidating the mechanism(s) of ethanol CNS teratogenesis with animal species in which the brain growth spurt occurs prenatally, specifically the sheep and the guinea pig [20]. The use of these species allows for examination of both direct (i.e., target site) and indirect (i.e., maternal, placental, or other fetal sites) effects of maternal ethanol administration on fetal brain development. As such, experiments can be designed and executed that mimic conditions of alcohol consumption in the human, in which virtually all of the offspring’s exposure to ethanol occurs prenatally. Furthermore, with respect to the pregnant sheep, the fetus is of sufficient size to surgically instrument. This allows one to examine the in utero effects of ethanol on the intact functioning fetus. Several studies have been conducted by us and other researchers that investigated the acute fetal effects of maternal ethanol administration in the pregnant sheep and the underlying mechanism(s) of some of these effects, especially in the developing brain [12,21]. However, few studies have investigated the chronic toxicity of ethanol in pregnant sheep, and there have been no published reports demonstrating neurobehavioral teratogenesis in lambs following chronic in utero ethanol exposure. With this in mind, the objective of the present study was to test the hypothesis that chronic in utero exposure to ethanol during the brain growth spurt produces postnatal behavioral deficits and neurological injury in lambs.
2. Method 2.1. Chronic treatment regimen This study was approved by the Purdue University Animal Care and Use Committee. Experiments were performed on 45 mixed-breed ewes (and their offspring) that were bred in-house to produce accurate time-dated pregnancies. Ewes were housed in an open-air barn in experimental groups of four or five in gated pens at the Purdue University Sheep Unit. As a group, they were fed alfalfa haylage daily. The number of fetuses being carried by each ewe was pre determined on gestational day (GD) 30 (term, about 145 days) by an experienced domestic animal ultrasound technician. Ewes were assigned to one of three treatment groups: ethanol, n ⫽ 18; pairfed control, n ⫽ 15; or ad lib control, n ⫽ 12. Each treatment group had two ewes with quadruplets, three ewes with triplets, and six with twins; the remaining ewes were singleton pregnancies. The ewes were weighed weekly to monitor the progression of pregnancy and then dosing commenced on GD 106. The ethanoltreated ewes were administered 1 g ethanol/kg maternal body weight (MBW) by infusing a 40% ethanol/saline v/v solution, warmed to 38⬚C, every other day from GD 106 to 134. The ethanol treatment was divided into four equal doses, and injected directly into the jugular vein at 15 min intervals. This method of ethanol administration was chosen over using indwelling jugular vein catheters because, in a
preliminary study, infection occurred, probably due to the open nature of the housing area. Oral administration of ethanol is not feasible in sheep because they are ruminant animals, which leads to variable drug absorption from the GI tract. The pair-fed and ad lib control animals followed the same infusion schedule, with normal saline substituted for the ethanol solution. To control for any potential nutritional artifact, ethanol-treated ewes were fed a known amount of alfalfa haylage (more than they were expected to consume) after treatment and in the evening of their day off from treatment. The pair-fed control ewes were then given the same amount of haylage that the ethanol-treated animals had consumed the previous day plus an amount of crushed corn isocaloric to the dose of ethanol. The ad lib control ewes had free access to food. All groups had unlimited access to water. One ewe at GD 126 was randomly chosen from the ethanol-treated group to determine maternal blood ethanol concentration (BEC). For this purpose, 3-ml blood samples were taken from the jugular vein opposite to the ethanol infusion site at 10-min intervals from time 0 min (immediately before the first dose) to time 90 min (45 min after the last dose), and then 24 h later. The samples were collected into heparinized tubes and immediately frozen. 2.2. Parturition and postnatal behavior assessment At GD 135, 1 day after the treatments were stopped, all groups were moved indoors to straw-bedded pens and provided with haylage ad lib, along with grain supplements to maintain strength for parturition. Once in the lambing area, veterinary technicians were available 24 h/day to monitor the ewes and observe each delivery. Immediately after parturition, a dystocia score was assigned to each ewe based on how much human assistance was required during delivery (1, no assistance; 2, at least one lamb pulled while coming out of the birth canal; 3, at least one lamb pulled from within the uterus; 4, all lambs pulled, ewe down; and 5, Cesarean section). This scoring system has been used since 1982 to assess every ewe that delivers lambs at Purdue University’s Sheep Unit. After parturition was complete, the lambs were weighed and then the ewe and offspring were moved into the behavior assessment area. Each individual ewe and her litter were placed into a 1.3- ⫻ 1.3-m wooden pen with straw bedding and videotaped for 24 h for subsequent behavior analysis. Lamb vigor was assessed 1 h after birth. The Vigor Score was based on each lamb’s ability to stand/nurse/walk (5, very vigorous; 4, moderate vigor; 3, minimal vigor; 2, cannot stand; and 1, dead). Dystocia and lamb vigor scoring was done by the same group of experienced animal technicians who were not aware of the treatment group. In the 2 weeks that followed, lamb deaths were recorded. Behavior was assessed using the Noldus Observer System 3.0 (Noldus Information Technology Inc, Sterling, VA) based on the amount of time the lambs spent in three differ-
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ent activities: active (standing, playing, or moving), resting (laying down whether awake or asleep), and nursing. Videotapes of the 24 h of lamb activity were replayed at a speed 12-fold real time. Again, scorers were unaware of the treatment groups. To further control for any potential bias of the scorers, several tapes were randomly chosen to be assessed by every individual involved in the behavior analysis and then the scores were compared; there was greater than 95% concordance between the scores from the different observers. 2.3. Biochemical and physiologic analyses Maternal blood ethanol concentrations were determined in triplicate by gas-chromatography [18] using a Hewlett Packard 5890 series II Gas Chromatograph with a flame ionization detector and a 6 ft stainless steel chromatograph column packed with factory-conditioned HP Poropak Q 80/ 100 mesh (Hewlett Packard, Wilmington, DE). After heating each airtight, frozen, blood vial for 7 min in a 55⬚C water bath, 1 ml of headspace gas was extracted using an airtight 1.25-ml Hamilton syringe and immediately injected into the column. The ethanol concentration in each sample was calculated by interpolation of the signal peak area to a standard curve constructed from a wide range of ethanol concentrations (10 to 600 mg/dl), using HP Chemostation software. The lower limit of quantitative sensitivity of this method was 10 mg/dl. Placentas were collected after parturition and placental expulsion had occurred. Each lamb develops with its own placenta, so we tried to collect all placentas for analysis. However, placentas from 20 litters were unsuitable for examination because they were damaged during or after delivery (e.g., stepped on by the ewe). The remaining placentas from seven ethanol litters, nine pair-fed litters and four ad lib litters were placed in plastic freezer bags and stored at ⫺20⬚ C. At a later time, the placentas were thawed and surveyed in a “blinded-fashion.” Each placenta was divided into three equal portions, and six cotyledons were randomly selected from each portion. Once the cotyledons were carefully dissected from the placental portion, they were weighed and their diameters measured. Cotyledon weight and cotyledon diameter measurements in each portion were averaged to give a value for the placenta. Results for the placentas were subsequently averaged to give a value for each litter. 2.4. Postnatal histologic analysis Lamb weight gain and survival were monitored in the subsequent weeks. Manpower limitations precluded conducting subsequent behavior assessments. At postnatal day (PD) 90, several lambs were arbitrarily selected for histologic analysis (no lambs were from the same litter). Lambs were euthanized with an overdose of sodium thiopental, and the brains fixed by transcardial perfusion with ice-cold sa-
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line followed by 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4⬚C. Each brain was left in situ overnight, and then carefully removed from the skull, weighed, and photographed. All brains were coded such that the examiners (JDR and MG) had no knowledge of the treatment regimens that were used. Brains were cut into 0.5 cm-thick coronal sections and prepared for standard histopathologic examination and morphometry. The sections, including standard areas of frontal cortex, parietal cortex, and hippocampus were processed and embedded in paraffin. For neuronal morphometry, 20 m-thick sections were cut and stained with cresyl violet. Morphometric analysis was performed using a Nikon Optiphot microscope with a Hitachi HV-C10 CCD color video camera, and Bioscan OPTOMAS 4.10 software on a computer system equipped with an Imaging Technology Vision Plus-AT CFG digitizing card. Brain morphometric analysis involved several measurements. For the frontal and parietal cortex, total cortical width and width of layer one were measured (individual layers two through six could not be accurately distinguished), and cells were counted and classified by size. Only cells that had a distinct nucleus were counted. Small cells were defined as having a mean diameter of greater than 2 m but less than 10 m, medium cells were greater than 10 m but less than 20 m, and large cells had mean diameters greater than 20 m but less than 50 m. These categories were defined by a preliminary evaluation of the slides. In these lambs, essentially all cells in the brain less than 10 m mean diameter were nonneuronal (predominantly glial and endothelial); cells 10–20-m diameter were both glia and small neurons; and cells larger than 20 m diameter were all neurons. All cortical cells in defined 500-m widths of frontal and parietal cortex were counted three times, and a mean value for the three cell sizes were obtained for each brain region (per animal). For the hippocampus, pyramidal cells in both the CA1 region and the CA3 region were counted three times within a 500-m width. The triplicate measurements and counts were then averaged to give a single value per region. Prior to doing the actual measuring and counting, the regions of the frontal cortex, parietal cortex, and hippocampus were identified and marked by MG, a neuropathologist who has extensive experience in sheep histology, to ensure that identical regions were analyzed for all lambs. Brain regions were also examined for evidence of histologic damage. Adjacent sections were cut at a thickness of 6 m and stained with hematoxylin and eosin (H&E) or antibodies to glial fibrillary acidic protein (GFAP; Diaco Corporation, Carpentia, CA). GFAP analysis was conducted using the avidin–biotin–peroxidase technique; staining was visualized with diamino benzidine. In the frontal and parietal cortex, H&E and GFAP staining was used to classify the meninges, cortex, white matter, deep gray matter, and ventricle as normal or abnormal. In the hippocampal sections, the meninges, neocortex, CA1, CA3, dentate gyrus,
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white matter, and ventricle were all evaluated in a similar manner. 2.5. Data presentation and statistical analysis Maternal and fetal/neonatal data are presented as group means ⫾ standard deviations (SD) unless otherwise noted. Both parametric and nonparametric methods were used to statistically analyze the results [30]. Differences in group means for parametric data were determined using one-way analysis of variance (ANOVA). If the analysis produced a significant F-statistic, Students–Newman–Keuls post hoc test was used to determine which group means were statistically different, difference was assumed at p ⬍ 0.05. For the ordinal data (dystocia and vigor scores), differences in group medians were tested for using the Kruskal–Wallis analysis of variance by ranks. With respect to maternal and neonatal death rates, a 2 ⫻ 3 contingency table with 2 analysis was used to assess frequency differences among the three treatment groups.
3. Results A total of 45 ewes were treated in this study. All animals tolerated the jugular vein injections well, with no overt evidence that this method of chronic drug administration produced more than minimal discomfort or distress. Following IV administration, all of the ethanol-treated ewes exhibited signs of moderate ethanol intoxication (e.g., balance impairment, unsteady gait). In the ewe tested at GD 126, peak maternal BEC was 120 mg/dl, and occurred 5 min after the fourth ethanol injection; 24 h later, the BEC was too low to quantify (i.e., ⬍10 mg/dl). The pregnancy and neonatal outcome data are presented in Table 1. Three ewes died during the period of ethanol treatment, another two ethanol-treated ewes died following spontaneous abortion after the ethanol treatments had ceased (i.e., after GD 134). No maternal deaths occurred in the pair-fed or ad lib control groups. Among animals that delivered, the length of gestation and the average litter sizes
were similar. There was no difference in birth weight between the ethanol-exposed lambs and the pair-fed or ad lib control lambs. Lamb postnatal death occurred in all three treatments with a similar incidence between groups (2 ⫽ 1.75; 2crit ⫽ 5.99). Postpartum analysis of the placentas indicated that maternal ethanol exposure decreased cotyledon diameter and cotyledon weight compared to the two control groups (p ⬍ 0.05). A total of 87 lambs were delivered to 40 ewes. Dystocia and lamb vigor scores for the offspring of the ethanoltreated and pair-fed and ad lib control ewes are presented in Fig. 1. Dystocia scores were similar amongst the three groups. In contrast, ethanol-exposed lambs were significantly less vigorous compared to offspring of the two control groups (p ⬍ 0.05). In three instances (one litter in each treatment group), lambs were unable to suckle on their own and were force-fed colostrum within a few hours of birth. This was a normal and expected behavior; however, those animals were not utilized in any subsequent analyses due to the interruption in their behavior patterns. The 24-h postnatal cumulative behavior data, divided into time spent active (moving, playing and standing), resting (including sleeping), or nursing, are presented in Figure 2. Ethanol-exposed lambs were more active (p ⫽ 0.001) than the pair-fed or ad lib control lambs. On PD 90, several ethanol-exposed (n ⫽ 4), pair fed control (n ⫽ 5), and ad lib control lambs (n ⫽ 4) were selected for brain histologic evaluation. Gross examination of the individual brains revealed no evidence of overt anomalies. Likewise, there was no evidence of ethanol-induced microencephaly, as brain weights were similar between treatment groups. A summary of the histologic morphometry data for the frontal and parietal regions of the cerebral cortex is presented in Table 2. All three groups were similar with respect to cortical widths and cell numbers. Similarly, within the CA1 and CA3 regions of the hippocampus, no morphometric differences were observed (data not shown). The presence or absence of histologic injury in these three brain regions was scored following H&E and GFAP staining. In a like fashion to the morphometry results, there was
Table 1 Pregnancy and neonatal outcome data Treatment Parameter
Ethanol (n ⫽ 18)
Pair fed (n ⫽ 15)
Ad lib (n ⫽ 12)
Maternal death Spontaneous abortion Length of gestation (days) Average litter size Average birthweight (kg) Cotyledon diameter (mm) Cotyledon weight (g) Neonatal death (before PD 14)
3 2 145 ⫾ 1 (n ⫽ 13) 2 (range 1–3) 5.1 (n ⫽ 32) 27.1 ⫾ 1.9* (n ⫽ 7 placentas) 2.4 ⫾ 0.5* (n ⫽ 7 placentas) 7 of 32 (21%)
0 0 146 ⫾ 1 2 (range 1–4) 5.2 (n ⫽ 27) 32.6 ⫾ 1.7 (n ⫽ 9 placentas) 3.5 ⫾ 0.4 (n ⫽ 9 placentas) 3 of 27 (11%)
0 0 146 ⫾ 1 2 (range 1–4) 5.2 (n ⫽ 28) 34.5 ⫾ 1.6 (n ⫽ 4 placentas) 3.4 ⫾ 0.4 (n ⫽ 4 placentas) 3 of 28 (11%)
The data for maternal death, spontaneous abortion and neonatal death are reported as number of occurrences; other data are reported as mean ⫾ SD. *Cotyledon diameter and cotyledon weight in placentas from the ethanol-treated animals were significantly less compared to cotyledons from both the pair-fed and ad lib control animals (p ⬍ 0.05).
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Fig. 1. (A) Dystocia scores for the ethanol-treated (n ⫽ 13), pair-fed (n ⫽ 15), and ad lib (n ⫽ 12) control ewes. Individual values are given by the black squares; the median dystocia score ⫾ first and third quartile range is marked by the line. A score was assessed to each ewe based on how much human assistance was required during delivery: 1, no assistance; 2, at least one lamb pulled while coming out birth canal; 3, at least one lamb pulled from within the uterus; 4, all lambs pulled, ewe down; and 5, Cesarean section. (B) Vigor scores (median ⫾ first and third quartile range) for litters born to ethanol-treated (n ⫽ 13), pair-fed (n ⫽ 15), and ad lib (n ⫽ 12) control ewes. The average litter score was based on each lamb’s ability to stand/nurse/walk: 5, very vigorous; 4, moderate vigor; 3, minimal vigor; 2, cannot stand; and 1, dead. Ethanol-exposed lambs were significantly less vigorous compared to offspring of the two control groups (p ⬍ 0.05).
no evidence of ethanol-induced histologic injury in any of the brain regions examined (data not shown). 4. Discussion The pregnant sheep has been used by us and other investigators to examine the acute effects of maternal ethanol exposure on a variety of different biochemical and biophysical parameters in the intact functioning fetus [12,21,22]. While informative, it is difficult to extrapolate the results from such acute experiments to the chronic situation, as few studies have investigated the effects of chronic ethanol exposure in pregnant sheep. Of the three studies we are aware of, two focussed on ethanol-induced fetal growth restriction, and did not allow the ewes to deliver [19,24]. The third involved first-trimester ethanol exposure (GD 31–GD 52, i.e., prior
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Fig. 2. The 24-h postnatal cumulative behavior data for ethanol-exposed (n ⫽ 12 litters), pair-fed (n ⫽ 14 litters), and ad lib (n ⫽ 11 litters) control lambs. The data are presented as group means (⫾SD) and divided up into percent time spent active (moving, playing, and standing), resting (including sleeping) or nursing. Ethanol-exposed lambs were significantly more active (p ⫽ 0.001) than the pairfed or ad lib control lambs.
to the brain growth spurt), and focussed on the newborn cerebrovascular response to carbon dioxide exposure or hypoxemia [13]. To date, there have been no published reports demonstrating neurobehavioral teratogenicity in lambs following chronic in utero ethanol exposure. With this in mind, the objective of the present study was to test the hypothesis that chronic in utero exposure to ethanol during the third-trimester equivalent produces postnatal behavioral deficits and neurological injury in lambs. Fifteen treatments were administered on an alternating day basis from GD 106 to GD 134. The ethanol-treated ewes received 1 g ethanol/kg MBW/alternating day, which produced a peak maternal blood ethanol concentration (and assumed peak fetal blood ethanol concentration [5]) of 120 mg/dl. This blood ethanol concentration is associated with a fully recognizable level of human alcohol intoxication [23], and is comparable to the maternal sheep blood ethanol concentrations reported in previous studies using a similar dosing regimen [21]. Five of the ethanol-treated ewes died prior to parturition. Although the exact causes of death were not determined, multiple fetuses appear to play a role; the two ewes carrying four fetuses died, and the other three ewes were carrying twins or triplets. As such, it appears prudent to suggest that future chronic ethanol experiments should involve the use of singleton pregnant ewes.
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Table 2 Summary of cerebral cortical histologic morphometry data Treatment Parameter Brain weight (g) Frontal cortex width (m) Layer I Total Cell Counts 2 ⬍ small ⬍ 10 m 10 ⬍ medium ⬍ 20 m 20 ⬍ large ⬍ 50 m Parietal cortex width (m) Layer I Total Cell Counts 2 ⬍ small ⬍ 10 m 10 ⬍ medium ⬍ 20 m 20 ⬍ large ⬍ 50 m
Ethanol (n ⫽ 4)
Pairfed (n ⫽ 5)
Ad lib (n ⫽ 4)
105.9 ⫾ 7.4
105.8 ⫾ 1.7
110.5 ⫾ 9.1
373 ⫾ 154 1738 ⫾ 160
338 ⫾ 91 1675 ⫾ 273
371 ⫾ 42 1681 ⫾ 254
1266 ⫾ 269 421 ⫾ 139 63 ⫾ 44
1039 ⫾ 253 335 ⫾ 40 49 ⫾ 73
1148 ⫾ 276 283 ⫾ 90 51 ⫾ 45
299 ⫾ 70 1516 ⫾ 288
338 ⫾ 112 1514 ⫾ 156
317 ⫾ 31 1696 ⫾ 300
1139 ⫾ 215 425 ⫾ 54 38 ⫾ 44
1043 ⫾ 115 395 ⫾ 102 13 ⫾ 22
1360 ⫾ 456 446 ⫾ 129 26 ⫾ 30
Brain weight and morphometric data from randomly selected lambs perfusion fixed for histologic analysis at postnatal day 90. Cells in the frontal and parietal cortex were counted and classified as small, medium, or large based on mean diameter. There were no differences amongst the three treatment groups.
For all groups, dosing was stopped at GD 134, and each ewe was allowed to deliver without further manipulation. Parturition occurred between GD 145 and GD 146, with the chronic ethanol treatment having no apparent effect on the length of gestation. Likewise, ethanol did not have a significant effect on delivery itself, as there was no difference in the dystocia scores between the three treatment groups. Ethanol did affect placental morphology. Specifically, cotyledon diameter and cotyledon weight were significantly less (p ⬍ 0.05) in the intact placentas obtained from the ethanoltreated ewes compared with the cotyledons of the pair-fed and ad lib control animals. Although a decrease in cotyledon size has not been previously reported, other placental effects of ethanol exposure have been observed. In humans, chronic ethanol exposure can decrease placental weight, and has been associated with placental pathology including abnormal cord insertion, chorioamnionitis, villitis, and other placental lesions [3]. Changes in human placental ultrastructure have also been reported [2]. Alterations in placental morphology have been proposed to contribute to the growth restriction that can be produced following chronic in utero ethanol exposure. However, in the present study there was no evidence of ethanol-induced growth restriction, as neonatal lamb weights were similar between the three treatment groups. As such, it appears that an adequate level of oxygen and nutrient exchange is maintained between the ewe and fetus during moderate ethanol exposure despite alterations in placental morphology. Following delivery, the viability of each lamb was assessed using a five-point lamb vigor scale. This vigor score has been used by the Purdue University Sheep Unit for over 15 years to evaluate lamb viability and is analogous to the Apgar score used to assess human neonatal physical/neurologic status during the first few minutes of life. Ethanolexposed lambs were significantly less (p ⬍ 0.05) vigorous 1
h after birth compared to the pair-fed and ad lib control lambs. A similar effect has been observed in human neonates. Maternal consumption of more than 120 g ethanol/ week is associated with decreased 1 and 5 min Apgar scores [27]. Furthermore, the ethanol-exposed lambs exhibited the same languid behavior and low levels of arousal during early postnatal life that have been observed in ethanolexposed human infants [25]. The vigor score and the Apgar score, in broad terms, are measures of how successful the offspring is (are) in adapting to the extrauterine environment. The nature of the score suggests that ethanol sensitizes the lamb to novel physiologic stress, such as parturition, although the mechanism behind this sensitivity remains to be determined. Certainly, the decrease in lamb vigor did not result from a direct depressant effect of ethanol. The treatments were stopped well in advance of parturition (⭓10 days) to ensure that any observed differences in dystocia or postnatal lamb behavior were not due to ethanol being present in the maternal, fetal, or neonatal circulation. The pharmacokinetics of ethanol elimination in the near-term pregnant ewe are such that the 1 g ethanol/kg MBW dose would be completely metabolized within 24 h [5]. Indeed, 24 h after ethanol administration on GD 126, the maternal blood ethanol concentration was too low to be quantitated (i.e., less than 10 mg/dl; data not shown). Despite the decreased vigor observed during the first hour of postnatal life, the ethanol-exposed lambs “recovered,” and exhibited an increase in active behavior during the subsequent 24 h of videotape monitoring. This postnatal hyperactivity is consistent with the results from other chronic ethanol studies with different animal species, including the guinea pig [6] and the rat [4]. It is also consistent with the behavioral anomalies reported for human infants following in utero ethanol exposure. An assessment study
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conducted on the third day of life demonstrated that children of mothers who continuously drank during gestation exhibited increased crawling and limb movements, and were more often awake and active than children whose mothers did not drink or stopped drinking early on in pregnancy [7]. Another early behavioral measure reported to be affected by prenatal ethanol exposure is habituation [25]. Habituation is a measurement of an infant’s ability to “tune out” environmental stimuli after a few repetitions. A decrease in habituation is indicative of an attention deficit and hyperactivity, again indicating the association between prenatal ethanol exposure and increased activity during early postnatal life. Of specific relevance to the present study is that the reduced habituation was observed during the first day of postnatal life, between 9 and 27 h after birth. Furthermore, poor habituation was correlated with levels of maternal alcohol consumption (35 to 70 g ethanol/day) that resembled the ethanol regimen employed in this sheep study. Collectively, the results demonstrate that moderate ethanol exposure during the third trimester equivalent produces early postnatal behavioral anomalies in lambs analogous to those reported for humans following similar magnitudes of ethanol exposure. Although we were unable to assess behavior at subsequent postnatal time points, it is interesting to note that the human infants initially observed with similar behavior anomalies at PD 1 continued to exhibit behavior dysfunction, including hyperactivity, as they grew older [26]. These similarities appear to justify the continued use of pregnant sheep to investigate mechanisms of ethanol teratogenesis. In the instrumented pregnant sheep, acute maternal exposure to 1 g ethanol/kg MBW has been reported to decrease cerebral oxidative metabolism [22], impair placental uptake of amino acids [11], and alter fetal cerebral cortical prostaglandin E concentration [21]. It remains to be determined if such effects contribute to the observed behavioral dysfunction in the present study. The early postnatal behavioral deficits occurred in the absence of structural brain injury at PD 90. Exposure to ethanol did not produce microencephaly, as the brain weights were similar between the three treatment groups and were comparable to previously published lamb brain-weight data [16]. Similarly, there was no evidence of any gross malformations found upon visual inspection of the lamb brains. Morphometric analysis indicated that the cerebral cortex and hippocampus of the ethanol-exposed lambs were indistinguishable from the pair-fed and ad lib control lambs. Likewise, scoring for the presence of neuronal injury following H&E and GFAP staining revealed no differences in the brain regions examined among the three treatment groups. A number of experimental animal studies have demonstrated that ethanol can produce permanent gross and subtle neurologic injury, depending on the amount and type of developmental exposure. The present study employed a modest ethanol dosage regimen so the histologic methods for examination of the lamb brains were chosen to optimize the
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detection of subtle abnormalities. For instance, reactive gliosis, shown by an increase in GFAP staining, can persist for weeks to months after insult, making this a useful technique to identify prior neurologic injury [17]. Despite this, no injury was detected. Behavior was examined on PD 1 while the histologic analysis was conducted on PD 90. As such, it is possible that the neurologic anomalies responsible for the altered behavior resolved during the subsequent postnatal period. Indeed, resolution of prenatal ethanol-induced neurologic damage (e.g., decreased GFAP staining; restoration of hippocampal morphometry) has been reported [10,14]. For now, we are not in a position to determine if histologic injury was present earlier in postnatal life. Further histologic studies are ongoing in an attempt to ascertain if moderate ethanol exposure produces specific degrees of neurologic impairment (e.g., change in receptor populations) that could account for the postnatal hyperactivity. In conclusion, this is the first study to report neurobehavioral deficits in lambs following chronic prenatal ethanol exposure during the third-trimester equivalent of pregnancy. The data demonstrate that, in pregnant sheep, chronic moderate ethanol exposure (1 g/kg MBW) during this period of gestation, which includes the brain growth spurt, can produce postnatal behavioral anomalies in the offspring (and placental dysmorphology) that resemble those reported in humans following in utero exposure to ethanol. Although the exact mechanism(s) to account for these effects remains to be determined, the results do support the continuing use of pregnant sheep to investigate mechanisms of ethanol teratogenesis.
References [1] E.L. Abel, Fetal Alcohol Syndrome: From Mechanism to Prevention, CRC Press, Boca Raton, 1996. [2] K.S. Amankwah, R.C. Kaufmann, Ultrastructure of human placenta: effects of maternal drinking, Gynecol Obstet Invest 18 (1984) 311–316. [3] V.J. Baldwin, P.M. MacLeod, K. Bernirschke, Placental findings in alcohol abuse during pregnancy, Birth Defects 18 (1982) 89–94. [4] N.W. Bond, Fetal alcohol exposure and hyperactivity in rats: The role of the neurotransmitter systems involved in arousal and inhibition, in: J.R. West, (Ed.), Alcohol and Brain Development, Oxford University Press, New York, 1986, p. 45. [5] J.F. Brien, D.W. Clarke, Disposition and fetal effects of ethanol during pregnancy, in: S. Kacew, S. Lock (Ed.), Toxicologic and Pharmacologic Principles in Pediatrics, Hemisphere Publishing Corporation, New York, 1988, pp. 199–222. [6] M.C. Catlin, A. Abdollah, J.F. Brien, Dose-dependent effects of prenatal ethanol exposure in the guinea pig, Alcohol 10 (1993) 109–115. [7] C.D. Coles, I. Smith, P.M. Fernhoff, A. Falek, Neonatal neurobehavioral characteristics as correlates of maternal alcohol use during gestation, Alcohol Clin Exp Res 9 (1985) 454–460. [8] J. Dobbing, J. Sands, Comparative aspects of the brain growth spurt, Early Hum Dev 3 (1979) 79–83. [9] C.D. Driscoll, A.P. Streissguth, E.P. Riley, Prenatal alcohol exposure: Comparability of effects in humans and animal models, Neurotoxicol Teratol 12 (1990) 231–237. [10] I. Ferrer, F. Galofre, D. Lopez-Tejero, M. Llobera, Morphological recovery of hippocampal pyramidal neurons in the adult rat exposed in utero to ethanol, Toxicology 48 (1988) 191–197.
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[11] S.E. Fisher, M. Atkinson, I. Holzman, R. David, D.H. Van Thiel, Effect of ethanol upon placental uptake of amino acids, Prog Biochem Pharmacol 18 (1981) 216–223. [12] C.A. Gleason, K.J. Hothkiss, Cerebral responses to acute maternal alcohol intoxication in immature fetal sheep, Pediatr Res 31 (1992) 645–648. [13] C.A. Gleason, H. Iida, K.J. Hotchkiss, F.J. Northingon, R.J. Traystman, Newborn cerebrovascular responses after first trimester moderate maternal ethanol exposure, Pediat Res 42 (1997) 39–45. [14] C.R. Goodlett, J.T. Leo, J.P.O. Callaghan, J.C. Mahoney, J.R. West, Transient cortical astrogliosis induced by alcohol exposure during the neonatal brain growth spurt in rats, Dev Brain Res 72 (1993) 85–97. [15] J.W. McDonald, M.V. Johnston, Physiological and pathophysiological roles of excitatory amino acids during central nervous system development, Brain Res Rev 15 (1990) 41–70. [16] G.H. McIntosh, K.I. Baghurst, B.J. Potter, B.S. Hetzel, Foetal brain development in the sheep, Neuropathol Appl Neurobiol 5 (1979) 103–114. [17] C.K. Petito, S. Morgello, J.C. Felix, M.L. Lesser, The two patterns of reactive astrocytosis in postischemic rat brain, J Cereb Blood Flow Metab 10 (1990) 850–859. [18] L.A. Pohorecky, J. Brick, A new method for the determination of ethanol levels in rodents, Pharmacol Biochem Behav 16 (1982) 693–696. [19] B.J. Potter, G.B. Belling, M.T. Mano, B.S. Hetzel, Experimental production of growth retardation in the sheep fetus after exposure to ethanol, Med J Aust 2 (1980) 191–193. [20] J.D. Reynolds, J.F. Brien, Ethanol neurobehavioural teratogenesis and the role of L-glutamate in the fetal hippocampus, Can J Physiol Pharmacol 73 (1995) 1209–1223. [21] J.D. Reynolds, D.H. Penning, K.A. Kimura, F. Dexter, J.L. Hender-
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Chronic ethanol exposure during late gestation produces behavioral anomalies in neonatal lambs Jennifer Spear-Smitha, James F. Brienb, Marjorie Grafec, Richard Allricha, James D. Reynoldsd,* a Department of Animal Sciences, Purdue University, West Lafayette, IN, USA Department of Pharmacology and Toxicology, Queen’s University, Kingston, Canada c Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA d Obstetrical Anesthesia Research Laboratory, Division of Women’s Anesthesia, Department of Anesthesiology, Duke University Medical Center, Box 3094, Durham, NC 27710, USA Received 16 July 1999; Accepted 30 August 1999 b
Abstract A total of 45 pregnant ewes were assigned to one of three treatment groups: 1 g ethanol/kg maternal body weight (n ⫽ 18); pair-fed control (n ⫽ 15); and ad lib control (n ⫽ 12). Dosing started at gestational day (GD) 106, and was administered every other day until GD 134. Parturition occurred between GD 144 and 147. Analysis of the placentas indicated that ethanol exposure decreased cotyledon diameter and cotyledon weight compared to the control groups (p ⬍ 0.05). At birth, lambs were given a Vigor Score and then behavior was assessed using a videotape monitoring system for 24 h. Offspring in the ethanol treatment group were significantly less vigorous at birth (p ⬍ 0.05). This finding reversed during the subsequent 24 h such that the ethanol-exposed lambs were significantly more active (p ⫽ 0.001) than the control lambs. Morphometric and histologic examination of the cerebral cortex and hippocampus revealed no differences amongst the three treatment groups. Collectively, the data demonstrate that moderate ethanol exposure during the third-trimester equivalent of gestation can produce placental dysmorphology and postnatal behavioral anomalies in neonatal lambs in the absence of gross neurologic injury. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Behavior; Ethanol; Fetal alcohol syndrome; Placenta; Sheep
1. Introduction Consumption of ethanol during pregnancy can produce a wide spectrum of dose-dependent effects in the developing conceptus, including teratogenesis [1]. The most debilitating manifestation of ethanol teratogenesis would appear to be central nervous system (CNS) dysfunction, which can present as mental deficiency, poor motor coordination, and hyperactivity in postnatal life. Despite being widely recognized as a teratogen, the exact mechanism(s) by which ethanol produces these dysfunctions is not clearly understood [20]. Experimental studies in a variety of animal species have demonstrated that ethanol can produce effects on the conceptus similar to those that occur in the human [9,29]. Many studies have been conducted using the rat as the experimental animal due to its short gestation (about 22 days) and rela* Corresponding author. Tel.: 919-681-6774; Fax: 919-681-7022. E-mail address: [email protected]
tively large litter size. In the rat, a substantial amount of brain development, including the brain growth spurt, occurs postnatally [8]. In contrast, the brain growth spurt in the human is a perinatal event. This is a significant consideration, as the brain growth spurt is a period of rapid neuronal development during which the CNS is very sensitive to drug exposure and pathophysiologic insult [15]. As such, this has required the use of artificial postnatal feeding and rearing programs for neonatal rats to examine the effects of ethanol on the developing CNS during the brain growth spurt [28]. However, postnatal ethanol administration cannot assess any potential contribution of ethanol-induced changes in maternal and/or placental function on CNS development. Other concerns include the different rates of ethanol elimination and, by extension, different patterns of ethanol exposure. The capacity of the neonate to eliminate ethanol by hepatic biotransformation is considerably less compared to the maternal–fetal unit, in which ethanol elimination from the fetus occurs via placental transfer of ethanol into the maternal circulation and subsequent maternal hepatic biotransformation [5].
0892-0362/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 9 9 ) 0 0 0 5 9 - 8
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In view of this, our laboratories have focused on elucidating the mechanism(s) of ethanol CNS teratogenesis with animal species in which the brain growth spurt occurs prenatally, specifically the sheep and the guinea pig [20]. The use of these species allows for examination of both direct (i.e., target site) and indirect (i.e., maternal, placental, or other fetal sites) effects of maternal ethanol administration on fetal brain development. As such, experiments can be designed and executed that mimic conditions of alcohol consumption in the human, in which virtually all of the offspring’s exposure to ethanol occurs prenatally. Furthermore, with respect to the pregnant sheep, the fetus is of sufficient size to surgically instrument. This allows one to examine the in utero effects of ethanol on the intact functioning fetus. Several studies have been conducted by us and other researchers that investigated the acute fetal effects of maternal ethanol administration in the pregnant sheep and the underlying mechanism(s) of some of these effects, especially in the developing brain [12,21]. However, few studies have investigated the chronic toxicity of ethanol in pregnant sheep, and there have been no published reports demonstrating neurobehavioral teratogenesis in lambs following chronic in utero ethanol exposure. With this in mind, the objective of the present study was to test the hypothesis that chronic in utero exposure to ethanol during the brain growth spurt produces postnatal behavioral deficits and neurological injury in lambs.
2. Method 2.1. Chronic treatment regimen This study was approved by the Purdue University Animal Care and Use Committee. Experiments were performed on 45 mixed-breed ewes (and their offspring) that were bred in-house to produce accurate time-dated pregnancies. Ewes were housed in an open-air barn in experimental groups of four or five in gated pens at the Purdue University Sheep Unit. As a group, they were fed alfalfa haylage daily. The number of fetuses being carried by each ewe was pre determined on gestational day (GD) 30 (term, about 145 days) by an experienced domestic animal ultrasound technician. Ewes were assigned to one of three treatment groups: ethanol, n ⫽ 18; pairfed control, n ⫽ 15; or ad lib control, n ⫽ 12. Each treatment group had two ewes with quadruplets, three ewes with triplets, and six with twins; the remaining ewes were singleton pregnancies. The ewes were weighed weekly to monitor the progression of pregnancy and then dosing commenced on GD 106. The ethanoltreated ewes were administered 1 g ethanol/kg maternal body weight (MBW) by infusing a 40% ethanol/saline v/v solution, warmed to 38⬚C, every other day from GD 106 to 134. The ethanol treatment was divided into four equal doses, and injected directly into the jugular vein at 15 min intervals. This method of ethanol administration was chosen over using indwelling jugular vein catheters because, in a
preliminary study, infection occurred, probably due to the open nature of the housing area. Oral administration of ethanol is not feasible in sheep because they are ruminant animals, which leads to variable drug absorption from the GI tract. The pair-fed and ad lib control animals followed the same infusion schedule, with normal saline substituted for the ethanol solution. To control for any potential nutritional artifact, ethanol-treated ewes were fed a known amount of alfalfa haylage (more than they were expected to consume) after treatment and in the evening of their day off from treatment. The pair-fed control ewes were then given the same amount of haylage that the ethanol-treated animals had consumed the previous day plus an amount of crushed corn isocaloric to the dose of ethanol. The ad lib control ewes had free access to food. All groups had unlimited access to water. One ewe at GD 126 was randomly chosen from the ethanol-treated group to determine maternal blood ethanol concentration (BEC). For this purpose, 3-ml blood samples were taken from the jugular vein opposite to the ethanol infusion site at 10-min intervals from time 0 min (immediately before the first dose) to time 90 min (45 min after the last dose), and then 24 h later. The samples were collected into heparinized tubes and immediately frozen. 2.2. Parturition and postnatal behavior assessment At GD 135, 1 day after the treatments were stopped, all groups were moved indoors to straw-bedded pens and provided with haylage ad lib, along with grain supplements to maintain strength for parturition. Once in the lambing area, veterinary technicians were available 24 h/day to monitor the ewes and observe each delivery. Immediately after parturition, a dystocia score was assigned to each ewe based on how much human assistance was required during delivery (1, no assistance; 2, at least one lamb pulled while coming out of the birth canal; 3, at least one lamb pulled from within the uterus; 4, all lambs pulled, ewe down; and 5, Cesarean section). This scoring system has been used since 1982 to assess every ewe that delivers lambs at Purdue University’s Sheep Unit. After parturition was complete, the lambs were weighed and then the ewe and offspring were moved into the behavior assessment area. Each individual ewe and her litter were placed into a 1.3- ⫻ 1.3-m wooden pen with straw bedding and videotaped for 24 h for subsequent behavior analysis. Lamb vigor was assessed 1 h after birth. The Vigor Score was based on each lamb’s ability to stand/nurse/walk (5, very vigorous; 4, moderate vigor; 3, minimal vigor; 2, cannot stand; and 1, dead). Dystocia and lamb vigor scoring was done by the same group of experienced animal technicians who were not aware of the treatment group. In the 2 weeks that followed, lamb deaths were recorded. Behavior was assessed using the Noldus Observer System 3.0 (Noldus Information Technology Inc, Sterling, VA) based on the amount of time the lambs spent in three differ-
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ent activities: active (standing, playing, or moving), resting (laying down whether awake or asleep), and nursing. Videotapes of the 24 h of lamb activity were replayed at a speed 12-fold real time. Again, scorers were unaware of the treatment groups. To further control for any potential bias of the scorers, several tapes were randomly chosen to be assessed by every individual involved in the behavior analysis and then the scores were compared; there was greater than 95% concordance between the scores from the different observers. 2.3. Biochemical and physiologic analyses Maternal blood ethanol concentrations were determined in triplicate by gas-chromatography [18] using a Hewlett Packard 5890 series II Gas Chromatograph with a flame ionization detector and a 6 ft stainless steel chromatograph column packed with factory-conditioned HP Poropak Q 80/ 100 mesh (Hewlett Packard, Wilmington, DE). After heating each airtight, frozen, blood vial for 7 min in a 55⬚C water bath, 1 ml of headspace gas was extracted using an airtight 1.25-ml Hamilton syringe and immediately injected into the column. The ethanol concentration in each sample was calculated by interpolation of the signal peak area to a standard curve constructed from a wide range of ethanol concentrations (10 to 600 mg/dl), using HP Chemostation software. The lower limit of quantitative sensitivity of this method was 10 mg/dl. Placentas were collected after parturition and placental expulsion had occurred. Each lamb develops with its own placenta, so we tried to collect all placentas for analysis. However, placentas from 20 litters were unsuitable for examination because they were damaged during or after delivery (e.g., stepped on by the ewe). The remaining placentas from seven ethanol litters, nine pair-fed litters and four ad lib litters were placed in plastic freezer bags and stored at ⫺20⬚ C. At a later time, the placentas were thawed and surveyed in a “blinded-fashion.” Each placenta was divided into three equal portions, and six cotyledons were randomly selected from each portion. Once the cotyledons were carefully dissected from the placental portion, they were weighed and their diameters measured. Cotyledon weight and cotyledon diameter measurements in each portion were averaged to give a value for the placenta. Results for the placentas were subsequently averaged to give a value for each litter. 2.4. Postnatal histologic analysis Lamb weight gain and survival were monitored in the subsequent weeks. Manpower limitations precluded conducting subsequent behavior assessments. At postnatal day (PD) 90, several lambs were arbitrarily selected for histologic analysis (no lambs were from the same litter). Lambs were euthanized with an overdose of sodium thiopental, and the brains fixed by transcardial perfusion with ice-cold sa-
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line followed by 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4⬚C. Each brain was left in situ overnight, and then carefully removed from the skull, weighed, and photographed. All brains were coded such that the examiners (JDR and MG) had no knowledge of the treatment regimens that were used. Brains were cut into 0.5 cm-thick coronal sections and prepared for standard histopathologic examination and morphometry. The sections, including standard areas of frontal cortex, parietal cortex, and hippocampus were processed and embedded in paraffin. For neuronal morphometry, 20 m-thick sections were cut and stained with cresyl violet. Morphometric analysis was performed using a Nikon Optiphot microscope with a Hitachi HV-C10 CCD color video camera, and Bioscan OPTOMAS 4.10 software on a computer system equipped with an Imaging Technology Vision Plus-AT CFG digitizing card. Brain morphometric analysis involved several measurements. For the frontal and parietal cortex, total cortical width and width of layer one were measured (individual layers two through six could not be accurately distinguished), and cells were counted and classified by size. Only cells that had a distinct nucleus were counted. Small cells were defined as having a mean diameter of greater than 2 m but less than 10 m, medium cells were greater than 10 m but less than 20 m, and large cells had mean diameters greater than 20 m but less than 50 m. These categories were defined by a preliminary evaluation of the slides. In these lambs, essentially all cells in the brain less than 10 m mean diameter were nonneuronal (predominantly glial and endothelial); cells 10–20-m diameter were both glia and small neurons; and cells larger than 20 m diameter were all neurons. All cortical cells in defined 500-m widths of frontal and parietal cortex were counted three times, and a mean value for the three cell sizes were obtained for each brain region (per animal). For the hippocampus, pyramidal cells in both the CA1 region and the CA3 region were counted three times within a 500-m width. The triplicate measurements and counts were then averaged to give a single value per region. Prior to doing the actual measuring and counting, the regions of the frontal cortex, parietal cortex, and hippocampus were identified and marked by MG, a neuropathologist who has extensive experience in sheep histology, to ensure that identical regions were analyzed for all lambs. Brain regions were also examined for evidence of histologic damage. Adjacent sections were cut at a thickness of 6 m and stained with hematoxylin and eosin (H&E) or antibodies to glial fibrillary acidic protein (GFAP; Diaco Corporation, Carpentia, CA). GFAP analysis was conducted using the avidin–biotin–peroxidase technique; staining was visualized with diamino benzidine. In the frontal and parietal cortex, H&E and GFAP staining was used to classify the meninges, cortex, white matter, deep gray matter, and ventricle as normal or abnormal. In the hippocampal sections, the meninges, neocortex, CA1, CA3, dentate gyrus,
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white matter, and ventricle were all evaluated in a similar manner. 2.5. Data presentation and statistical analysis Maternal and fetal/neonatal data are presented as group means ⫾ standard deviations (SD) unless otherwise noted. Both parametric and nonparametric methods were used to statistically analyze the results [30]. Differences in group means for parametric data were determined using one-way analysis of variance (ANOVA). If the analysis produced a significant F-statistic, Students–Newman–Keuls post hoc test was used to determine which group means were statistically different, difference was assumed at p ⬍ 0.05. For the ordinal data (dystocia and vigor scores), differences in group medians were tested for using the Kruskal–Wallis analysis of variance by ranks. With respect to maternal and neonatal death rates, a 2 ⫻ 3 contingency table with 2 analysis was used to assess frequency differences among the three treatment groups.
3. Results A total of 45 ewes were treated in this study. All animals tolerated the jugular vein injections well, with no overt evidence that this method of chronic drug administration produced more than minimal discomfort or distress. Following IV administration, all of the ethanol-treated ewes exhibited signs of moderate ethanol intoxication (e.g., balance impairment, unsteady gait). In the ewe tested at GD 126, peak maternal BEC was 120 mg/dl, and occurred 5 min after the fourth ethanol injection; 24 h later, the BEC was too low to quantify (i.e., ⬍10 mg/dl). The pregnancy and neonatal outcome data are presented in Table 1. Three ewes died during the period of ethanol treatment, another two ethanol-treated ewes died following spontaneous abortion after the ethanol treatments had ceased (i.e., after GD 134). No maternal deaths occurred in the pair-fed or ad lib control groups. Among animals that delivered, the length of gestation and the average litter sizes
were similar. There was no difference in birth weight between the ethanol-exposed lambs and the pair-fed or ad lib control lambs. Lamb postnatal death occurred in all three treatments with a similar incidence between groups (2 ⫽ 1.75; 2crit ⫽ 5.99). Postpartum analysis of the placentas indicated that maternal ethanol exposure decreased cotyledon diameter and cotyledon weight compared to the two control groups (p ⬍ 0.05). A total of 87 lambs were delivered to 40 ewes. Dystocia and lamb vigor scores for the offspring of the ethanoltreated and pair-fed and ad lib control ewes are presented in Fig. 1. Dystocia scores were similar amongst the three groups. In contrast, ethanol-exposed lambs were significantly less vigorous compared to offspring of the two control groups (p ⬍ 0.05). In three instances (one litter in each treatment group), lambs were unable to suckle on their own and were force-fed colostrum within a few hours of birth. This was a normal and expected behavior; however, those animals were not utilized in any subsequent analyses due to the interruption in their behavior patterns. The 24-h postnatal cumulative behavior data, divided into time spent active (moving, playing and standing), resting (including sleeping), or nursing, are presented in Figure 2. Ethanol-exposed lambs were more active (p ⫽ 0.001) than the pair-fed or ad lib control lambs. On PD 90, several ethanol-exposed (n ⫽ 4), pair fed control (n ⫽ 5), and ad lib control lambs (n ⫽ 4) were selected for brain histologic evaluation. Gross examination of the individual brains revealed no evidence of overt anomalies. Likewise, there was no evidence of ethanol-induced microencephaly, as brain weights were similar between treatment groups. A summary of the histologic morphometry data for the frontal and parietal regions of the cerebral cortex is presented in Table 2. All three groups were similar with respect to cortical widths and cell numbers. Similarly, within the CA1 and CA3 regions of the hippocampus, no morphometric differences were observed (data not shown). The presence or absence of histologic injury in these three brain regions was scored following H&E and GFAP staining. In a like fashion to the morphometry results, there was
Table 1 Pregnancy and neonatal outcome data Treatment Parameter
Ethanol (n ⫽ 18)
Pair fed (n ⫽ 15)
Ad lib (n ⫽ 12)
Maternal death Spontaneous abortion Length of gestation (days) Average litter size Average birthweight (kg) Cotyledon diameter (mm) Cotyledon weight (g) Neonatal death (before PD 14)
3 2 145 ⫾ 1 (n ⫽ 13) 2 (range 1–3) 5.1 (n ⫽ 32) 27.1 ⫾ 1.9* (n ⫽ 7 placentas) 2.4 ⫾ 0.5* (n ⫽ 7 placentas) 7 of 32 (21%)
0 0 146 ⫾ 1 2 (range 1–4) 5.2 (n ⫽ 27) 32.6 ⫾ 1.7 (n ⫽ 9 placentas) 3.5 ⫾ 0.4 (n ⫽ 9 placentas) 3 of 27 (11%)
0 0 146 ⫾ 1 2 (range 1–4) 5.2 (n ⫽ 28) 34.5 ⫾ 1.6 (n ⫽ 4 placentas) 3.4 ⫾ 0.4 (n ⫽ 4 placentas) 3 of 28 (11%)
The data for maternal death, spontaneous abortion and neonatal death are reported as number of occurrences; other data are reported as mean ⫾ SD. *Cotyledon diameter and cotyledon weight in placentas from the ethanol-treated animals were significantly less compared to cotyledons from both the pair-fed and ad lib control animals (p ⬍ 0.05).
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Fig. 1. (A) Dystocia scores for the ethanol-treated (n ⫽ 13), pair-fed (n ⫽ 15), and ad lib (n ⫽ 12) control ewes. Individual values are given by the black squares; the median dystocia score ⫾ first and third quartile range is marked by the line. A score was assessed to each ewe based on how much human assistance was required during delivery: 1, no assistance; 2, at least one lamb pulled while coming out birth canal; 3, at least one lamb pulled from within the uterus; 4, all lambs pulled, ewe down; and 5, Cesarean section. (B) Vigor scores (median ⫾ first and third quartile range) for litters born to ethanol-treated (n ⫽ 13), pair-fed (n ⫽ 15), and ad lib (n ⫽ 12) control ewes. The average litter score was based on each lamb’s ability to stand/nurse/walk: 5, very vigorous; 4, moderate vigor; 3, minimal vigor; 2, cannot stand; and 1, dead. Ethanol-exposed lambs were significantly less vigorous compared to offspring of the two control groups (p ⬍ 0.05).
no evidence of ethanol-induced histologic injury in any of the brain regions examined (data not shown). 4. Discussion The pregnant sheep has been used by us and other investigators to examine the acute effects of maternal ethanol exposure on a variety of different biochemical and biophysical parameters in the intact functioning fetus [12,21,22]. While informative, it is difficult to extrapolate the results from such acute experiments to the chronic situation, as few studies have investigated the effects of chronic ethanol exposure in pregnant sheep. Of the three studies we are aware of, two focussed on ethanol-induced fetal growth restriction, and did not allow the ewes to deliver [19,24]. The third involved first-trimester ethanol exposure (GD 31–GD 52, i.e., prior
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Fig. 2. The 24-h postnatal cumulative behavior data for ethanol-exposed (n ⫽ 12 litters), pair-fed (n ⫽ 14 litters), and ad lib (n ⫽ 11 litters) control lambs. The data are presented as group means (⫾SD) and divided up into percent time spent active (moving, playing, and standing), resting (including sleeping) or nursing. Ethanol-exposed lambs were significantly more active (p ⫽ 0.001) than the pairfed or ad lib control lambs.
to the brain growth spurt), and focussed on the newborn cerebrovascular response to carbon dioxide exposure or hypoxemia [13]. To date, there have been no published reports demonstrating neurobehavioral teratogenicity in lambs following chronic in utero ethanol exposure. With this in mind, the objective of the present study was to test the hypothesis that chronic in utero exposure to ethanol during the third-trimester equivalent produces postnatal behavioral deficits and neurological injury in lambs. Fifteen treatments were administered on an alternating day basis from GD 106 to GD 134. The ethanol-treated ewes received 1 g ethanol/kg MBW/alternating day, which produced a peak maternal blood ethanol concentration (and assumed peak fetal blood ethanol concentration [5]) of 120 mg/dl. This blood ethanol concentration is associated with a fully recognizable level of human alcohol intoxication [23], and is comparable to the maternal sheep blood ethanol concentrations reported in previous studies using a similar dosing regimen [21]. Five of the ethanol-treated ewes died prior to parturition. Although the exact causes of death were not determined, multiple fetuses appear to play a role; the two ewes carrying four fetuses died, and the other three ewes were carrying twins or triplets. As such, it appears prudent to suggest that future chronic ethanol experiments should involve the use of singleton pregnant ewes.
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Table 2 Summary of cerebral cortical histologic morphometry data Treatment Parameter Brain weight (g) Frontal cortex width (m) Layer I Total Cell Counts 2 ⬍ small ⬍ 10 m 10 ⬍ medium ⬍ 20 m 20 ⬍ large ⬍ 50 m Parietal cortex width (m) Layer I Total Cell Counts 2 ⬍ small ⬍ 10 m 10 ⬍ medium ⬍ 20 m 20 ⬍ large ⬍ 50 m
Ethanol (n ⫽ 4)
Pairfed (n ⫽ 5)
Ad lib (n ⫽ 4)
105.9 ⫾ 7.4
105.8 ⫾ 1.7
110.5 ⫾ 9.1
373 ⫾ 154 1738 ⫾ 160
338 ⫾ 91 1675 ⫾ 273
371 ⫾ 42 1681 ⫾ 254
1266 ⫾ 269 421 ⫾ 139 63 ⫾ 44
1039 ⫾ 253 335 ⫾ 40 49 ⫾ 73
1148 ⫾ 276 283 ⫾ 90 51 ⫾ 45
299 ⫾ 70 1516 ⫾ 288
338 ⫾ 112 1514 ⫾ 156
317 ⫾ 31 1696 ⫾ 300
1139 ⫾ 215 425 ⫾ 54 38 ⫾ 44
1043 ⫾ 115 395 ⫾ 102 13 ⫾ 22
1360 ⫾ 456 446 ⫾ 129 26 ⫾ 30
Brain weight and morphometric data from randomly selected lambs perfusion fixed for histologic analysis at postnatal day 90. Cells in the frontal and parietal cortex were counted and classified as small, medium, or large based on mean diameter. There were no differences amongst the three treatment groups.
For all groups, dosing was stopped at GD 134, and each ewe was allowed to deliver without further manipulation. Parturition occurred between GD 145 and GD 146, with the chronic ethanol treatment having no apparent effect on the length of gestation. Likewise, ethanol did not have a significant effect on delivery itself, as there was no difference in the dystocia scores between the three treatment groups. Ethanol did affect placental morphology. Specifically, cotyledon diameter and cotyledon weight were significantly less (p ⬍ 0.05) in the intact placentas obtained from the ethanoltreated ewes compared with the cotyledons of the pair-fed and ad lib control animals. Although a decrease in cotyledon size has not been previously reported, other placental effects of ethanol exposure have been observed. In humans, chronic ethanol exposure can decrease placental weight, and has been associated with placental pathology including abnormal cord insertion, chorioamnionitis, villitis, and other placental lesions [3]. Changes in human placental ultrastructure have also been reported [2]. Alterations in placental morphology have been proposed to contribute to the growth restriction that can be produced following chronic in utero ethanol exposure. However, in the present study there was no evidence of ethanol-induced growth restriction, as neonatal lamb weights were similar between the three treatment groups. As such, it appears that an adequate level of oxygen and nutrient exchange is maintained between the ewe and fetus during moderate ethanol exposure despite alterations in placental morphology. Following delivery, the viability of each lamb was assessed using a five-point lamb vigor scale. This vigor score has been used by the Purdue University Sheep Unit for over 15 years to evaluate lamb viability and is analogous to the Apgar score used to assess human neonatal physical/neurologic status during the first few minutes of life. Ethanolexposed lambs were significantly less (p ⬍ 0.05) vigorous 1
h after birth compared to the pair-fed and ad lib control lambs. A similar effect has been observed in human neonates. Maternal consumption of more than 120 g ethanol/ week is associated with decreased 1 and 5 min Apgar scores [27]. Furthermore, the ethanol-exposed lambs exhibited the same languid behavior and low levels of arousal during early postnatal life that have been observed in ethanolexposed human infants [25]. The vigor score and the Apgar score, in broad terms, are measures of how successful the offspring is (are) in adapting to the extrauterine environment. The nature of the score suggests that ethanol sensitizes the lamb to novel physiologic stress, such as parturition, although the mechanism behind this sensitivity remains to be determined. Certainly, the decrease in lamb vigor did not result from a direct depressant effect of ethanol. The treatments were stopped well in advance of parturition (⭓10 days) to ensure that any observed differences in dystocia or postnatal lamb behavior were not due to ethanol being present in the maternal, fetal, or neonatal circulation. The pharmacokinetics of ethanol elimination in the near-term pregnant ewe are such that the 1 g ethanol/kg MBW dose would be completely metabolized within 24 h [5]. Indeed, 24 h after ethanol administration on GD 126, the maternal blood ethanol concentration was too low to be quantitated (i.e., less than 10 mg/dl; data not shown). Despite the decreased vigor observed during the first hour of postnatal life, the ethanol-exposed lambs “recovered,” and exhibited an increase in active behavior during the subsequent 24 h of videotape monitoring. This postnatal hyperactivity is consistent with the results from other chronic ethanol studies with different animal species, including the guinea pig [6] and the rat [4]. It is also consistent with the behavioral anomalies reported for human infants following in utero ethanol exposure. An assessment study
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conducted on the third day of life demonstrated that children of mothers who continuously drank during gestation exhibited increased crawling and limb movements, and were more often awake and active than children whose mothers did not drink or stopped drinking early on in pregnancy [7]. Another early behavioral measure reported to be affected by prenatal ethanol exposure is habituation [25]. Habituation is a measurement of an infant’s ability to “tune out” environmental stimuli after a few repetitions. A decrease in habituation is indicative of an attention deficit and hyperactivity, again indicating the association between prenatal ethanol exposure and increased activity during early postnatal life. Of specific relevance to the present study is that the reduced habituation was observed during the first day of postnatal life, between 9 and 27 h after birth. Furthermore, poor habituation was correlated with levels of maternal alcohol consumption (35 to 70 g ethanol/day) that resembled the ethanol regimen employed in this sheep study. Collectively, the results demonstrate that moderate ethanol exposure during the third trimester equivalent produces early postnatal behavioral anomalies in lambs analogous to those reported for humans following similar magnitudes of ethanol exposure. Although we were unable to assess behavior at subsequent postnatal time points, it is interesting to note that the human infants initially observed with similar behavior anomalies at PD 1 continued to exhibit behavior dysfunction, including hyperactivity, as they grew older [26]. These similarities appear to justify the continued use of pregnant sheep to investigate mechanisms of ethanol teratogenesis. In the instrumented pregnant sheep, acute maternal exposure to 1 g ethanol/kg MBW has been reported to decrease cerebral oxidative metabolism [22], impair placental uptake of amino acids [11], and alter fetal cerebral cortical prostaglandin E concentration [21]. It remains to be determined if such effects contribute to the observed behavioral dysfunction in the present study. The early postnatal behavioral deficits occurred in the absence of structural brain injury at PD 90. Exposure to ethanol did not produce microencephaly, as the brain weights were similar between the three treatment groups and were comparable to previously published lamb brain-weight data [16]. Similarly, there was no evidence of any gross malformations found upon visual inspection of the lamb brains. Morphometric analysis indicated that the cerebral cortex and hippocampus of the ethanol-exposed lambs were indistinguishable from the pair-fed and ad lib control lambs. Likewise, scoring for the presence of neuronal injury following H&E and GFAP staining revealed no differences in the brain regions examined among the three treatment groups. A number of experimental animal studies have demonstrated that ethanol can produce permanent gross and subtle neurologic injury, depending on the amount and type of developmental exposure. The present study employed a modest ethanol dosage regimen so the histologic methods for examination of the lamb brains were chosen to optimize the
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detection of subtle abnormalities. For instance, reactive gliosis, shown by an increase in GFAP staining, can persist for weeks to months after insult, making this a useful technique to identify prior neurologic injury [17]. Despite this, no injury was detected. Behavior was examined on PD 1 while the histologic analysis was conducted on PD 90. As such, it is possible that the neurologic anomalies responsible for the altered behavior resolved during the subsequent postnatal period. Indeed, resolution of prenatal ethanol-induced neurologic damage (e.g., decreased GFAP staining; restoration of hippocampal morphometry) has been reported [10,14]. For now, we are not in a position to determine if histologic injury was present earlier in postnatal life. Further histologic studies are ongoing in an attempt to ascertain if moderate ethanol exposure produces specific degrees of neurologic impairment (e.g., change in receptor populations) that could account for the postnatal hyperactivity. In conclusion, this is the first study to report neurobehavioral deficits in lambs following chronic prenatal ethanol exposure during the third-trimester equivalent of pregnancy. The data demonstrate that, in pregnant sheep, chronic moderate ethanol exposure (1 g/kg MBW) during this period of gestation, which includes the brain growth spurt, can produce postnatal behavioral anomalies in the offspring (and placental dysmorphology) that resemble those reported in humans following in utero exposure to ethanol. Although the exact mechanism(s) to account for these effects remains to be determined, the results do support the continuing use of pregnant sheep to investigate mechanisms of ethanol teratogenesis.
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