TERATOLOGY 44:395-404 (1991)
Results of Prenatal Alcohol Exposure on the Dimensions and Binucleation of Cardiac Myocytes in Neonatal and Weanling Rats MARVIN R. ALVAREZ, LINDA C. CLARK, J O ANN MOORE, MARTHA C. MORALES, AND A. MARTIN GERDES Department of Biology, University of South Florida, Tampa, Florida 33620-5150 (M.A.R., L.C.C.)and Department of Anatomy, University of South Florida, Tampa, Florida 336124799 (J.A.M., M.C.M., A.M.G.)
ABSTRACT Sprague-Dawley rats were exposed to ethyl alcohol in utero. The effect of chronic prenatal exposure was examined by giving mature females alcohol in isocaloric liquid diets which served as the sole source of liquid and caloric intake before mating and throughout gestation. Controls consisted of females maintained on laboratory chow or a n isocaloric liquid diet minus alcohol before and during gestation. The offspring were sacrificed a t 21 days of age (weanlings) and the hearts dissociated enzymatically to give purified cardiac myocytes. The effects of daily acute prenatal alcohol exposure were studied by gastric intubation of alcohol to chow-fed females for the duration of pregnancy. The doses used approximated 4 and 5 shots of 80 proof liquor per day by a person weighing 150 lb. These offspring were sacrificed a t 2, 6, and 21 days postnatal and cardiac myocytes prepared as above. Heart weights were determined and cardiac myocytes were analyzed for cell length, volume, cross-sectional area, and percent binucleation. Additionally, nuclear DNA content was measured in all of the 21 day offspring. Statistical analysis of the data showed no significant differences between hearts exposed to prenatal alcohol and nonexposed controls with either regimen with the exception of percent binucleation which was significantly but only slightly higher in the 6-day-old hearts. These findings are discussed in relation to anatomical heart defects found in patients with full fetal alcohol syndrome.
A number of clinical reports in the 1970s correlated chronic maternal alcoholism with a specific pattern of birth defects in children born to such women which is now called fetal alcohol syndrome (FAS) (Jones and Smith, '73; Ferrier et al., '73; Hall and Orenstein, '74; Jones and Smith, '75). Nearly half of affected children have cardiac malformations due to hypoplastic development, causing incomplete anatomical structures such a s ventricular and atrial septa1 defects, hypoplastic left heart, etc. (Sardor et al., '81; Diamond, '89). The authors a r e not aware of any studies on the effect of prenatal ethanol exposure on cardiac myocyte size and binucleation during growth and development in animal models. Yet, one would expect structural anatomical defects to result from morphologically abnormal heart cells and from 0 1991 WILEY-LISS, INC
changes in the rate and timing of cell division. Therefore, the purpose of the present study was to evaluate the Sprague-Dawley rat as a n animal model for the study of effects of prenatal ethanol exposure on cardiomyocyte growth and binucleation. Two approaches for delivery of the alcohol were evaluated: (1)isocaloric liquid diets containing alcohol fed before and during pregnancy and (2) alcohol a t different doses delivered daily during pregnancy by gastric intubation. Enzymatically isolated cardiac myocytes were evaluated in the offspring for cell length, cell volume, cross-sectional area, degree of binucleation, and, in the case of 21day-old offspring, nuclear DNA content. Received January 18, 1991; accepted May 14, 1991. Address reprint requests to Marvin R. Alvarez, Department of Biology, University of South Florida, Tampa, FL 33620-5150.
396
M.R. ALVAREZ ET AL
TABLE 1 . Liquid diet composition % Alcohol-
Diet LC LDA HDA
derived calories 0.0 17.0 34.0
Ethanol (95% v/v), 5.25 cal/ml % cal vil00 ml 0.0 0.0 3.0 16.9 6.0 33.8
Sustacal 1.01 caliml vilO0 ml % cal 61.0 66.1 61.0 66.1 61.0 66.1
MATERIALS AND METHODS
Experimental protocol for liquid diets Virgin Sprague-Dawley rats were housed one to a cage and initially given laboratory chow and water ad libitum. Those to be placed later on liquid diets containing alcohol were brought to their final alcohol levels gradually to prevent shock from sudden exposure. All of the rats to be placed on liquid diets were started on the diet without alcohol (control liquid diet) and weighed weekly. When weight had stabilized (about two weeks) some of the animals were placed on the low alcohol liquid diet and some left on the control liquid diet. Again when the animals’ weight stabilized, some of the animals which had been on the low alcohol liquid diet were placed on the high alcohol liquid diet. Thus, after several weeks, the initial group was divided into four dietary regimens; laboratory chow and water (chow controls) (CC), liquid diet without alcohol (liquid diet controls) (LC), liquid diet with low alcohol content (LDA), and liquid diet with high alcohol content (HDA). The liquid diets were made isocaloric by adding appropriate amounts of sucrose. The diets consisted of chocolate-flavored Sustacal@with added Vitamin Fortification Mixture (0.26 gi100 ml). The composition of the diets is given in Table 1. Each rat was served 75 ml of liquid diet diluted with 125 ml water daily. All showed weight gains while they were on liquid diets. Vaginal smears were made beginning a week or two before mating. When a smear pattern indicative of fertility was noted, the females were placed in cages with males which had never been exposed to alcohol. The animals were given laboratory chow until mating was achieved. Successful mating was judged by the presence of a vaginal plug or vaginal sperm. The female was a t this time returned to her cage and returned to her respective premating diet and continued on i t until parturition. At that time the
Sucrose (45% wiv) 1.81caliml v/100 ml % cal 17.4 33.8 8.7 16.9 0.0 0.0
Water added, v/100 ml
Final calorie contentiloo ml
21.6 27.3 33.0
93.0 93.0 93.0
litter size was adjusted to eight pups per dam and the dam was placed on the next lower alcohol content each day so that by a maximum of 3 days postpartum she was on chow and water. The pups were sacrificed a t 21 days after birth (weaning age) for morphometric analysis of the hearts and measurement of nuclear DNA content. Blood alcohol levels were determined in parallel series of nonpregnant rats acclimated to the high alcohol diet. The animals were anesthetized with intraperitoneal injections of sodium pentobarbital and the blood drawn by cardiac puncture. The enzymatic assay was done using a n Ethyl Alcohol Kit (Sigma).
Experimental protocol for force-fed alcohol Virgin Sprague-Dawley rats were mated and the presence of a vaginal plug or vaginal sperm were taken a s a n indication of successful mating, a t which time the males were removed. The pregnant females were administered alcohol once a day throughout pregnancy a s a 25% (wiv) solution in tap water. The alcohol solutions were administered to the esophagus with a 20-gauge feeding needle and syringe without anesthesia. The animals showed disequilibrium wit.hin a few minutes and visibly recovered within 3 hr. One set received daily doses of 4 g alcohol per kg body weight (4gfkg) and a second set 5 gikg. Some of the pups from dams receiving 5 glkg were sacrificed a t 2 and 6 days after birth. Pups from dams receiving both doses were sacrificed at 21 days. Litters to be analyzed a t 21 days after birth were randomly culled to 8 pups per dam. Myocyte isolation procedure Animals were anesthetized (45 mgikg, sodium pentobarbital, ip) and the hearts quickly removed, blotted, and weighed. The hearts were cannulated in the aorta and attached to a Langendorff apparatus for retrograde coronary perfusion. In the case of the pups sacrificed at 21 days after birth,
PRENATAL ALCOHOL EFFECTS ON CARDIAC MYOCYTES
isolated myocytes were prepared by a calcium-tolerant procedure (Montini et al., '81). This technique produced a high yield of elongated myocytes which could be used either for morphometry or for nuclear isolation. Myocytes from hearts of pups sacrificed a t 2 or 6 days after birth were prepared by retrograde coronary perfusion with collagenase dissolved in calcium-free Joklik media (pH 7.2) as described by Bishop and Drummond ('79). The Bishop and Drummond method was used for the smaller (2and 6-day-old) hearts so that morphometric data could be obtained from individual hearts from a given litter. The calcium-tolerant method would have required pooling myocytes from several hearts in order to obtain enough elongated myocytes to allow nuclear isolation. Myocytes isolated by the Bishop and Drummond method became rapidly damaged following isolation thus requiring immediate fixation. This does not allow enough time for nuclear isolation. After perfusion, both ventricles were separated from the atria and minced in the isolation media. The cells were passed through a 2 5 0 - ~ mnylon mesh and the samples centrifuged twice through a sucrose gradient to remove nonmyocyte cells. The portion of the purified myocyte preparation to be used for morphometry was fixed in 2% glutaraldehyde in 0.08 M phosphate buffer. The remaining portion was suspended in phosphate buffer, pH 7.4 and used for nuclear isolation.
397
Nuclear DNA content determined by flow cytometry Unifixed isolated myocytes from 21-dayold hearts were divided into aliquots and allowed to settle a t 37°C. Excess fluid was removed and the cells resuspended in 10 drops of 0.01 M Hepes buffer for 5 min, shaking gently every 2 min. Two to 3 drops of lysis buffer (Engelmann and Gerrity, '88) were added. After 10 min (shaking every 2 min) the solution was microfuged for 3 sec and the nuclei resuspended in DMSO and stored at - 70°C. Isolated nuclei were thawed and stained with DAPI. Flow DNA measurements of the isolated myocyte nuclei were done with a mercury arc flow cytometer (Alvarez and Stone, '88). DAPI fluorescence was quantitated using a BG-1 exciter filter, a TK 405 dichroic mirror, and a n LP 395 barrier filter. Nuclear concentration, injection rate and sheath flow were adjusted to give a sample flow rate of 200 nuclei/sec. The data are displayed a s frequency distribution histograms. Statistical analyses Two statistical analyses were applied to each set of morphometric data: a one-way analysis of variance (ANOVA) and Scheffe's multiple contrast test. RESULTS
Liquid diets Rats on the liquid diets showed consistent weight gains indicating that caloric conMyocyte morphometry sumption was adequate and that they were The volume of fixed myocytes was deter- ingesting alcohol. Most of the individuals mined with a Coulter Channelyzer using a consumed the total amount they were 70-km aperture for cells from 2- and 6-day- served daily, yet, blood alcohol levels were old pups and a 100-Fm aperture for cells found to be variable a t any given sample from 21-day weanlings. Because the myo- time. For example, in one set consisting of cytes are elongated, a shape factor of 1.05 12 animals on HDA, cardiac blood alcohol based on a cell lengthlwidth ratio of 1:7 was levels were found to range from 35 to 160 applied (Hurley, '70; Gerdes et al.; '86). The mgidl. Since the liquid diet was the only source of nutrition o r liquid, the high degree median cell volume was used. Cell length (longest length parallel to the of variation which was observed appeared to long axis) was measured directly with a be dependent on the time elapsed since the phase-contrast microscope. The mean was last drink to the time of sampling. The rate of alcohol loss from the blood was calculated from a sample size of 40 myocytes. Myocyte cross-sectional area was de- determined in a series of animals intubated termined by the ratio of cell volume/cell with 4 g/kg alcohol in water followed by length. The number of nuclei per myocyte blood alcohol determinations a t intervals. was counted in cells stained with 4,6-diami- The results are shown in Table 2. These data show that blood alcohol peaks dino-2-phenylindole-2 HCl (DAPI) using a n at 2 h r after ingestion followed by a deepiillumination fluorescence microscope.
398
M.R. ALVAREZ ET AL. 2000
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Fig. 1. Nuclear DNA content of cardiac myocytes from 21-day-old rats exposed in utero to alcohol in liquid diets. A, CC; B, LC; C , LDH; D, HDA.
TABLE 2. Rate of change in blood alcohol with time Elapsed time from Blood alcohol intubation (hr) (mg/dl) 0.5 150 155 230 160 120 70
crease to near zero by 5 h r after ingestion. Thus, the blood alcohol present in a given animal at any point in time is greatly influenced by the time elapsed since drinking. Observation of the rats on the alcohol containing diets showed that they tended to drink for short periods and that the time between drinks was variable. The DNA content of cardiac myocytes from 21-day-old weanlings is compared among the four dietary regimens in Figure
1. These histograms show the fluorescence intensity distribution of isolated DAPIstained nuclei on the abscissa as channel number; the ordinate gives the number of signals in a given channel. The individual histograms are A, CC; B, LC; C, LDA; D, HDA. Note that all four histograms show modal peaks in the same position. These peaks are interpreted to represent the Go/G1 population of the cell cycle. The negligible number of signals detected to the right of the modal peaks signifies a n absence of nuclei in the S and G , + M phases of the cell cycle. The small signals in the post-G,,/G, peak probably represent artifact consisting of a small number of nuclei forming doublets, triplets, etc. No evidence of polyploidy is seen. Figure 2 shows a comparison of heart weight and three morphometric parameters of the cardiac myocytes; cell length, volume, and cross-sectional area for the four prena-
PRENATAL ALCOHOL EFFECTS ON CARDIAC MYOCYTES
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Fig. 2. Comparison of heart weights and cardiac myocyte dimensions from 21-day-old rats exposed in utero to alcohol in liquid diets. Data were statistically analyzed by one-way analysis of variance (ANOVA) and Scheffe's multiple contrast test. Myocyte dimensions
were measured in 40 cellsiheart, n = number of hearts. Heart weight, n = 12, 16, 8, 12, left-to-right; myocyte cross-sectional area, n = 3 , 4 , 4 , 3 ;myocyte length, n = 3, 4, 2, 3; myocyte volume, n = 4, 8, 4, 6.
tal alcohol regimens measured in the 21day-old offspring. No significant differences were found among treatments for these parameters nor between treated and controls. Thus, it appears that chronic prenatal exposure to alcohol does not alter heart weight, myocyte dimensions, or nuclear DNA content in weanling rats.
peaks, interpreted as nuclei in the G,/G, phase of the cell cycle, were seen. No evidence of polyploidy or nuclei in S or G2 M phases of the cell cycle was observed. Heart weight, myocyte length, volume, cross-sectional area, and percent binucleation were measured in 2- and 6-day-old pups exposed to 5 g alcohol/kg maternal body weight. Because of the small sizes of the hearts, the yield of elongated myocytes was not large enough to allow isolation of nuclei in sufficient number to obtain reliable measurements of nuclear DNA by flow cytometry. Instead, percent binucleation in DAPI-stained myocytes was used as a n index of nuclear proliferation. The results are shown in Figures 4 (2 day old) and 5 (6 day old). No significant differences in heart weight, myocyte length, volume, or cross-sectional area were detected between treated and control pups, either at 2 or 6 days after birth. However, the percent of binucleated myocytes was greater in the alco-
Alcohol intubation The effects of daily maternal ingestions of alcohol on the offspring were evaluated at weaning age. Figure 3 shows the mean heart weight and the dimensions of cardiac myocytes in weanlings (21-day-old) exposed to acute prenatal alcohol. Again, no significant differences among treated and control sets were found for these morphometric parameters. Measurements of nuclear DNA content from 21-day-old offspring were compared among controls, 4 g/kg and 5 glkg exposures. As in the liquid diet study, single
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Fig. 3. Comparison of heart weights and cardiomyocyte dimensions from 21-day-old rats exposed to alcohol in utero by gastric intubation. Statistical analysis done by one-way ANOVA and Scheffe’s multiple con-
trast test. Myocyte dimensions were measured in 40 cellslheart, n = number of hearts. Heart weight, n = 8, 12, 14; myocyte length, n = 8, 12, 12; myocyte crosssectional area, n = 7, 12, 12.
hol-exposed hearts than in controls. The difference between treated and control myocytes from 2-day hearts was not significant. However, in the 6-day old hearts the difference in binucleation between controls and alcohol-exposed myocytes was small but significantly (P
Results of Prenatal Alcohol Exposure on the Dimensions and Binucleation of Cardiac Myocytes in Neonatal and Weanling Rats MARVIN R. ALVAREZ, LINDA C. CLARK, J O ANN MOORE, MARTHA C. MORALES, AND A. MARTIN GERDES Department of Biology, University of South Florida, Tampa, Florida 33620-5150 (M.A.R., L.C.C.)and Department of Anatomy, University of South Florida, Tampa, Florida 336124799 (J.A.M., M.C.M., A.M.G.)
ABSTRACT Sprague-Dawley rats were exposed to ethyl alcohol in utero. The effect of chronic prenatal exposure was examined by giving mature females alcohol in isocaloric liquid diets which served as the sole source of liquid and caloric intake before mating and throughout gestation. Controls consisted of females maintained on laboratory chow or a n isocaloric liquid diet minus alcohol before and during gestation. The offspring were sacrificed a t 21 days of age (weanlings) and the hearts dissociated enzymatically to give purified cardiac myocytes. The effects of daily acute prenatal alcohol exposure were studied by gastric intubation of alcohol to chow-fed females for the duration of pregnancy. The doses used approximated 4 and 5 shots of 80 proof liquor per day by a person weighing 150 lb. These offspring were sacrificed a t 2, 6, and 21 days postnatal and cardiac myocytes prepared as above. Heart weights were determined and cardiac myocytes were analyzed for cell length, volume, cross-sectional area, and percent binucleation. Additionally, nuclear DNA content was measured in all of the 21 day offspring. Statistical analysis of the data showed no significant differences between hearts exposed to prenatal alcohol and nonexposed controls with either regimen with the exception of percent binucleation which was significantly but only slightly higher in the 6-day-old hearts. These findings are discussed in relation to anatomical heart defects found in patients with full fetal alcohol syndrome.
A number of clinical reports in the 1970s correlated chronic maternal alcoholism with a specific pattern of birth defects in children born to such women which is now called fetal alcohol syndrome (FAS) (Jones and Smith, '73; Ferrier et al., '73; Hall and Orenstein, '74; Jones and Smith, '75). Nearly half of affected children have cardiac malformations due to hypoplastic development, causing incomplete anatomical structures such a s ventricular and atrial septa1 defects, hypoplastic left heart, etc. (Sardor et al., '81; Diamond, '89). The authors a r e not aware of any studies on the effect of prenatal ethanol exposure on cardiac myocyte size and binucleation during growth and development in animal models. Yet, one would expect structural anatomical defects to result from morphologically abnormal heart cells and from 0 1991 WILEY-LISS, INC
changes in the rate and timing of cell division. Therefore, the purpose of the present study was to evaluate the Sprague-Dawley rat as a n animal model for the study of effects of prenatal ethanol exposure on cardiomyocyte growth and binucleation. Two approaches for delivery of the alcohol were evaluated: (1)isocaloric liquid diets containing alcohol fed before and during pregnancy and (2) alcohol a t different doses delivered daily during pregnancy by gastric intubation. Enzymatically isolated cardiac myocytes were evaluated in the offspring for cell length, cell volume, cross-sectional area, degree of binucleation, and, in the case of 21day-old offspring, nuclear DNA content. Received January 18, 1991; accepted May 14, 1991. Address reprint requests to Marvin R. Alvarez, Department of Biology, University of South Florida, Tampa, FL 33620-5150.
396
M.R. ALVAREZ ET AL
TABLE 1 . Liquid diet composition % Alcohol-
Diet LC LDA HDA
derived calories 0.0 17.0 34.0
Ethanol (95% v/v), 5.25 cal/ml % cal vil00 ml 0.0 0.0 3.0 16.9 6.0 33.8
Sustacal 1.01 caliml vilO0 ml % cal 61.0 66.1 61.0 66.1 61.0 66.1
MATERIALS AND METHODS
Experimental protocol for liquid diets Virgin Sprague-Dawley rats were housed one to a cage and initially given laboratory chow and water ad libitum. Those to be placed later on liquid diets containing alcohol were brought to their final alcohol levels gradually to prevent shock from sudden exposure. All of the rats to be placed on liquid diets were started on the diet without alcohol (control liquid diet) and weighed weekly. When weight had stabilized (about two weeks) some of the animals were placed on the low alcohol liquid diet and some left on the control liquid diet. Again when the animals’ weight stabilized, some of the animals which had been on the low alcohol liquid diet were placed on the high alcohol liquid diet. Thus, after several weeks, the initial group was divided into four dietary regimens; laboratory chow and water (chow controls) (CC), liquid diet without alcohol (liquid diet controls) (LC), liquid diet with low alcohol content (LDA), and liquid diet with high alcohol content (HDA). The liquid diets were made isocaloric by adding appropriate amounts of sucrose. The diets consisted of chocolate-flavored Sustacal@with added Vitamin Fortification Mixture (0.26 gi100 ml). The composition of the diets is given in Table 1. Each rat was served 75 ml of liquid diet diluted with 125 ml water daily. All showed weight gains while they were on liquid diets. Vaginal smears were made beginning a week or two before mating. When a smear pattern indicative of fertility was noted, the females were placed in cages with males which had never been exposed to alcohol. The animals were given laboratory chow until mating was achieved. Successful mating was judged by the presence of a vaginal plug or vaginal sperm. The female was a t this time returned to her cage and returned to her respective premating diet and continued on i t until parturition. At that time the
Sucrose (45% wiv) 1.81caliml v/100 ml % cal 17.4 33.8 8.7 16.9 0.0 0.0
Water added, v/100 ml
Final calorie contentiloo ml
21.6 27.3 33.0
93.0 93.0 93.0
litter size was adjusted to eight pups per dam and the dam was placed on the next lower alcohol content each day so that by a maximum of 3 days postpartum she was on chow and water. The pups were sacrificed a t 21 days after birth (weaning age) for morphometric analysis of the hearts and measurement of nuclear DNA content. Blood alcohol levels were determined in parallel series of nonpregnant rats acclimated to the high alcohol diet. The animals were anesthetized with intraperitoneal injections of sodium pentobarbital and the blood drawn by cardiac puncture. The enzymatic assay was done using a n Ethyl Alcohol Kit (Sigma).
Experimental protocol for force-fed alcohol Virgin Sprague-Dawley rats were mated and the presence of a vaginal plug or vaginal sperm were taken a s a n indication of successful mating, a t which time the males were removed. The pregnant females were administered alcohol once a day throughout pregnancy a s a 25% (wiv) solution in tap water. The alcohol solutions were administered to the esophagus with a 20-gauge feeding needle and syringe without anesthesia. The animals showed disequilibrium wit.hin a few minutes and visibly recovered within 3 hr. One set received daily doses of 4 g alcohol per kg body weight (4gfkg) and a second set 5 gikg. Some of the pups from dams receiving 5 glkg were sacrificed a t 2 and 6 days after birth. Pups from dams receiving both doses were sacrificed at 21 days. Litters to be analyzed a t 21 days after birth were randomly culled to 8 pups per dam. Myocyte isolation procedure Animals were anesthetized (45 mgikg, sodium pentobarbital, ip) and the hearts quickly removed, blotted, and weighed. The hearts were cannulated in the aorta and attached to a Langendorff apparatus for retrograde coronary perfusion. In the case of the pups sacrificed at 21 days after birth,
PRENATAL ALCOHOL EFFECTS ON CARDIAC MYOCYTES
isolated myocytes were prepared by a calcium-tolerant procedure (Montini et al., '81). This technique produced a high yield of elongated myocytes which could be used either for morphometry or for nuclear isolation. Myocytes from hearts of pups sacrificed a t 2 or 6 days after birth were prepared by retrograde coronary perfusion with collagenase dissolved in calcium-free Joklik media (pH 7.2) as described by Bishop and Drummond ('79). The Bishop and Drummond method was used for the smaller (2and 6-day-old) hearts so that morphometric data could be obtained from individual hearts from a given litter. The calcium-tolerant method would have required pooling myocytes from several hearts in order to obtain enough elongated myocytes to allow nuclear isolation. Myocytes isolated by the Bishop and Drummond method became rapidly damaged following isolation thus requiring immediate fixation. This does not allow enough time for nuclear isolation. After perfusion, both ventricles were separated from the atria and minced in the isolation media. The cells were passed through a 2 5 0 - ~ mnylon mesh and the samples centrifuged twice through a sucrose gradient to remove nonmyocyte cells. The portion of the purified myocyte preparation to be used for morphometry was fixed in 2% glutaraldehyde in 0.08 M phosphate buffer. The remaining portion was suspended in phosphate buffer, pH 7.4 and used for nuclear isolation.
397
Nuclear DNA content determined by flow cytometry Unifixed isolated myocytes from 21-dayold hearts were divided into aliquots and allowed to settle a t 37°C. Excess fluid was removed and the cells resuspended in 10 drops of 0.01 M Hepes buffer for 5 min, shaking gently every 2 min. Two to 3 drops of lysis buffer (Engelmann and Gerrity, '88) were added. After 10 min (shaking every 2 min) the solution was microfuged for 3 sec and the nuclei resuspended in DMSO and stored at - 70°C. Isolated nuclei were thawed and stained with DAPI. Flow DNA measurements of the isolated myocyte nuclei were done with a mercury arc flow cytometer (Alvarez and Stone, '88). DAPI fluorescence was quantitated using a BG-1 exciter filter, a TK 405 dichroic mirror, and a n LP 395 barrier filter. Nuclear concentration, injection rate and sheath flow were adjusted to give a sample flow rate of 200 nuclei/sec. The data are displayed a s frequency distribution histograms. Statistical analyses Two statistical analyses were applied to each set of morphometric data: a one-way analysis of variance (ANOVA) and Scheffe's multiple contrast test. RESULTS
Liquid diets Rats on the liquid diets showed consistent weight gains indicating that caloric conMyocyte morphometry sumption was adequate and that they were The volume of fixed myocytes was deter- ingesting alcohol. Most of the individuals mined with a Coulter Channelyzer using a consumed the total amount they were 70-km aperture for cells from 2- and 6-day- served daily, yet, blood alcohol levels were old pups and a 100-Fm aperture for cells found to be variable a t any given sample from 21-day weanlings. Because the myo- time. For example, in one set consisting of cytes are elongated, a shape factor of 1.05 12 animals on HDA, cardiac blood alcohol based on a cell lengthlwidth ratio of 1:7 was levels were found to range from 35 to 160 applied (Hurley, '70; Gerdes et al.; '86). The mgidl. Since the liquid diet was the only source of nutrition o r liquid, the high degree median cell volume was used. Cell length (longest length parallel to the of variation which was observed appeared to long axis) was measured directly with a be dependent on the time elapsed since the phase-contrast microscope. The mean was last drink to the time of sampling. The rate of alcohol loss from the blood was calculated from a sample size of 40 myocytes. Myocyte cross-sectional area was de- determined in a series of animals intubated termined by the ratio of cell volume/cell with 4 g/kg alcohol in water followed by length. The number of nuclei per myocyte blood alcohol determinations a t intervals. was counted in cells stained with 4,6-diami- The results are shown in Table 2. These data show that blood alcohol peaks dino-2-phenylindole-2 HCl (DAPI) using a n at 2 h r after ingestion followed by a deepiillumination fluorescence microscope.
398
M.R. ALVAREZ ET AL. 2000
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Fig. 1. Nuclear DNA content of cardiac myocytes from 21-day-old rats exposed in utero to alcohol in liquid diets. A, CC; B, LC; C , LDH; D, HDA.
TABLE 2. Rate of change in blood alcohol with time Elapsed time from Blood alcohol intubation (hr) (mg/dl) 0.5 150 155 230 160 120 70
crease to near zero by 5 h r after ingestion. Thus, the blood alcohol present in a given animal at any point in time is greatly influenced by the time elapsed since drinking. Observation of the rats on the alcohol containing diets showed that they tended to drink for short periods and that the time between drinks was variable. The DNA content of cardiac myocytes from 21-day-old weanlings is compared among the four dietary regimens in Figure
1. These histograms show the fluorescence intensity distribution of isolated DAPIstained nuclei on the abscissa as channel number; the ordinate gives the number of signals in a given channel. The individual histograms are A, CC; B, LC; C, LDA; D, HDA. Note that all four histograms show modal peaks in the same position. These peaks are interpreted to represent the Go/G1 population of the cell cycle. The negligible number of signals detected to the right of the modal peaks signifies a n absence of nuclei in the S and G , + M phases of the cell cycle. The small signals in the post-G,,/G, peak probably represent artifact consisting of a small number of nuclei forming doublets, triplets, etc. No evidence of polyploidy is seen. Figure 2 shows a comparison of heart weight and three morphometric parameters of the cardiac myocytes; cell length, volume, and cross-sectional area for the four prena-
PRENATAL ALCOHOL EFFECTS ON CARDIAC MYOCYTES
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Fig. 2. Comparison of heart weights and cardiac myocyte dimensions from 21-day-old rats exposed in utero to alcohol in liquid diets. Data were statistically analyzed by one-way analysis of variance (ANOVA) and Scheffe's multiple contrast test. Myocyte dimensions
were measured in 40 cellsiheart, n = number of hearts. Heart weight, n = 12, 16, 8, 12, left-to-right; myocyte cross-sectional area, n = 3 , 4 , 4 , 3 ;myocyte length, n = 3, 4, 2, 3; myocyte volume, n = 4, 8, 4, 6.
tal alcohol regimens measured in the 21day-old offspring. No significant differences were found among treatments for these parameters nor between treated and controls. Thus, it appears that chronic prenatal exposure to alcohol does not alter heart weight, myocyte dimensions, or nuclear DNA content in weanling rats.
peaks, interpreted as nuclei in the G,/G, phase of the cell cycle, were seen. No evidence of polyploidy or nuclei in S or G2 M phases of the cell cycle was observed. Heart weight, myocyte length, volume, cross-sectional area, and percent binucleation were measured in 2- and 6-day-old pups exposed to 5 g alcohol/kg maternal body weight. Because of the small sizes of the hearts, the yield of elongated myocytes was not large enough to allow isolation of nuclei in sufficient number to obtain reliable measurements of nuclear DNA by flow cytometry. Instead, percent binucleation in DAPI-stained myocytes was used as a n index of nuclear proliferation. The results are shown in Figures 4 (2 day old) and 5 (6 day old). No significant differences in heart weight, myocyte length, volume, or cross-sectional area were detected between treated and control pups, either at 2 or 6 days after birth. However, the percent of binucleated myocytes was greater in the alco-
Alcohol intubation The effects of daily maternal ingestions of alcohol on the offspring were evaluated at weaning age. Figure 3 shows the mean heart weight and the dimensions of cardiac myocytes in weanlings (21-day-old) exposed to acute prenatal alcohol. Again, no significant differences among treated and control sets were found for these morphometric parameters. Measurements of nuclear DNA content from 21-day-old offspring were compared among controls, 4 g/kg and 5 glkg exposures. As in the liquid diet study, single
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Fig. 3. Comparison of heart weights and cardiomyocyte dimensions from 21-day-old rats exposed to alcohol in utero by gastric intubation. Statistical analysis done by one-way ANOVA and Scheffe’s multiple con-
trast test. Myocyte dimensions were measured in 40 cellslheart, n = number of hearts. Heart weight, n = 8, 12, 14; myocyte length, n = 8, 12, 12; myocyte crosssectional area, n = 7, 12, 12.
hol-exposed hearts than in controls. The difference between treated and control myocytes from 2-day hearts was not significant. However, in the 6-day old hearts the difference in binucleation between controls and alcohol-exposed myocytes was small but significantly (P
