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Journal of the Anatomical Society of India

Skeletal Anomalies In Fetal Alcohol Syndrome: A Study On Developing Mice Embryos

Author(s): Mishra, S.R., Sahai, A., Srivastava, A.K., Agrawal, A.K., Singh, P.J., Mishra, R.K.

Vol. 52, No. 1 (2003-01 - 2003-12)

Department of Anatomy, King George's Medical College, Lucknow, INDIA

Abstract

Present study has been envisaged to elicit in mice: the possible teratogenic effect of ethanol on the skeleton of developing mice embryos; dose effect relationship by choosing two doses; and to establish the time effect relationship when a particular dose is given during total gestational period or during major organogenesis period. Skeletal anomalies met in the study were in the form of unossified skull bones, maxillary hypoplasia, nasal bone hypoplasia, scoliosis, and many types of rib anomalies.

Key words: Fetal alcohol syndrome, Teratogenic effect of alcohol, Alizarin red-S stain.

Introduction:

In the modern industrial society alcohol has an essential place. Its expanding use in industries, agriculture, medicine and other fields of daily life has played a major role in achieving the quality and life expectancy enjoyed today. With the advent of new drugs and environmental chemicals, and their potential toxic effect on the human reproduction (Thalidomide disaster in 1960's) it was felt that this ubiquitous drug generlly accepted as safe may be contributing to infants death and morbidity (Green, 1974.)

Jones & Smith (1973) described specific abnormalities in infants of alcoholic mothers and named the symptom complex as Fetal Alcohol Syndrome (FAS). A wide variety of variable pregnancy outcome have been reported after controlled human studies on this aspect which includes brain malformation, cerebral palsy, intrauterine growth retardation, decreased birth weight etc. (Clarren et al, 1976; Jone et al, 1974, Ouelletle et al, 1977; Olegard et al, 1979; Little 1977 ; Kaminiski et al; (1976). The adverse effects of alcohol on pregnant mice have also been reported by, Guy & Suches ten (1986), Parson & Sojitra (1995). But Schwelz et al (1978) did not find any teratogenic effect of 15% ethanol in pregnant mice, rat and rabbit when administered during organogenesis period. Persaud & Kennedy (1983) stated that alcohol is non teratogenic in rats when consumed moderately during early gestation.

Scanning through the literature showed that not much work has been done on the teratogenic effect of ethanol on the developing mice embryos in India. Hence the present study has been undertaken to find out the teratogenic effect of ethanol on skeletal system of mice embryos.

Material and Method:

The study was done on sexually mature Swiss albino mice. The mice were divided into three groups as follows : (Table-I)

Table I: Plan of the work

Groups Subgroups No. of pregnant mice taken for study Material treated with Days of gestation when treated Dose per day
I Control group I-A 6 Distilled water 0-18 0.5ml/mice
  I-B 6 Distilled water 6-15 0.5ml/mice
II Low-dose group II-A 6 25% Ethanol 0-18 2.9gm/kg/mice
  II-B 6 25% Ethanol 6-15 2.9gm/kg/mice
III High-dose group III-A 6 25% Ethanol 0-18 5.8gm/kg/mice
  III-B 6 25% Ethanol 6-15 5.8gm/kg/mice

Group I (Control group): The pregnant mice of this group were kept under similar and identical experimental conditions as treatment group. Each mouse received 0.5 ml of distilled water orally on the scheduled dosage days.

Group II (Low dose treatment group): Each pregnant mouse of this group was administered 25% ethanol v/v at the dose level of 2.9 gm/kg body weight/day by oral route.

Group III (High dose treatment group): Each pregnant mouse of this group was administered 25% ethanol v/v at the dose level of 5.8 gm/kg body weight/day by oral route.

Each group was further divided into two subgroups A and B, each consisting of 6 pregnant mice. Subgroup-A received the dose on day 0-18 of gestation (total gestation period), while subgroup B recieved the same dose on day. 6-15 of gestation (major organogenesis period).

Technique of skeletal study:

Skeletal study was done by the technique described by Dawson in 1926. The specimens were stored in 70% ethanol, and then eviscerated by puncturing the adbomen.

The specimens were placed in 2% aqueous potassium hydroxide until the flesh was sufficiently clear to render the skeleton visible. The solution was changed whenever it became discolored. The specimens were then placed in Mall's solution. To this solution Alizarin red-S was added to color it deep purplish red. The solution was gently agitated a few times per hour. This solution was left until the bones were stained the desired color. Specimens were then again passed through Mall's solution for the removal of any overstaining and then passed through 25%, 50%, 80% glycerol and finally preserved permanently in 90% glycerol solution. 1 or 2 crystals of thymol were added as preservative.

A total of 189 fetuses were obtained (by caesarian section on 19th day) from 29 dams out of 36 dams. One mouse died without giving any litter and remaining 6 exhibited total resorption. 93 fetuses out of 189 were randomly selected for skeletal study. Rest 96 fetuses were preserved for histological studies.

Observations:

The percentage of skeletal anomalies was rather higher among the fetuses of subgroup I-A (16.67%) than I-B (8%). In both the subgroups the skeletal anomalies were in the form of unossified temporal bone (I/I-A), increased intercostal space (2/I-A), unossified frontal bone (I/I-B) and rudimentary rib (I/I-B). (Table-II).

In subgroup II-A, 4 fetuses (36.36%) presented with skeletal anomalies which included one fetus each of unossified occipital bone, maxillary hypoplasia, nasal bone hypoplasia, increased intercostal space between 8th and 9th rib and wavy rib. In subgroup II-B two fetuses (10.53%) exhibited skeletal anomalies which included one fetus each of maxillary hypoplasia and scoliosis. (Table-II)

Subgroup III-A exhibited 75% skeletal anomalies, which included unossified occipital bone, wide open cranial suture lines, wide open fontanellae (figure-4), scoliosis, curling of tail (figure-1), maxillary hypoplasia. increased intercostal space, and rudimentary ribs. Multiple anomalies were also recorded. In subgroup II-B, 4 (25%) out of 16 fetuses exhibited skeletal anomalies, which included one unossified occipital bone, one scoliosis, one increased intercostal space and one wavy rib.

The percentage of skeletal anomalies in subgroup III-A (75%) and III-B (25%) were much higher compared to control group (i.e. 16.67% in I-A and 8% in I.B). (Table-II).

Table-II: Various skeletal anomalies met among the fetuses of various groups

Groups No. of pregnant mice No. of fetuses with skeletal anomalies
Examined Affected Description
I-A 6 18 3(16.67%) Unossified temporal bone, increased intercostal space
I-B 6 25 2(8.0%) Unossified frontal bone, rudimentary ribs
II-A 6 11 4 (36.36%) Unossified occipital bone, maxillary hypoplasia, nasal bone hypoplasia, increased intercostal space
II-B 6 19 2 (10.53%) Scoliosis
III-A 6 8 6(75.0%) Unossified occipital bone, maxillary hypoplasia, scoliosis, wide and increased intercostal spaces, rudimentary ribs
III-B 6 16 4 (25.0%) Unossified occipital bone, scoliosis, increased intercostal space, wavy ribs

Discussion:

Present work was undertaken to study the effect of prenatal exposure of alcohol on developing skeleton of mice embryos. The skeletal anomalies met in this study were in the form of unossified temporal (figure-2), frontal and occipital bones (figure3). As revealed by subgroup I-A, I-B and II-A, maxillary hypoplasia, nasal bone hypoplasia, scoliosis, large cases of increased intercostal spaces (figure1) and other rib anomalies (figure-3) are met in the present study. Alizarin stain failed to reach upto tail region, but curling of tail seen during gross examination in 9 fetuses (1 in subgroup I-A, 2 each in subgroups I-B, II-A, II-B, 1 in subgroup III-A and 2 in subgroup III-B) is indicative of vertebral column anomaly.

Delayed occiput ossification was found in our study as also reported by Chernoff in 1977. Delayed skeletal growth was reported by Stucky & Berry in 1984. Maxillary hypoplasia was noted by Webster et al (1983) and Rasmussen & Christansen, (1979). In this study maxillary hypoplasia exhibited as either a flat midface or a narrow snout that can be correlated with the typical human FAS features i.e. a flat midface associated with maxillary hypoplasia, a short ruptured nose and a diminished or absent philtrum with a long gap between the thinned upper lip (Clarren et al, 1978.)

The present study reported significant increase in the incidence of malformation of face and limbs skeletons in group II-A, II-B, III-A & III-B (Table-II) in which alcohol was administered either during total gestation (II-A and III-A) or during major organogenesis period i.e. 6-15 days (II-B and III-B) which shows time and dose dependency in almost accordance with the reports of other workers. Similarly investigation in inbred and outbreed mice (Webster et al. 1980) have shown that acute alcohol exposure during the organogenesis period is highly teratogenic causing facial, brain and limb anomalies.

Hence we can conclude that alcohol consumption during pregnancy has adverse effect on the developing skeleton of the fetuses.

References:

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  3. Dawson A.B. (1926): A note on the staining of the skeleton of cleared specimen with Alizarin Red-S. Stain Technology. 1 : 123-124.
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Figure-1

Alizarin preparation of fetuses showing increased intercostal space, and curled tail, suggestive of vertebral column deformity.

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Figure-2

Alizarin preparation of fetus showing unossified temporal bone.

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Figure-3

Alizarin preparation of fetus showing unossified occipital bone and wavy ribs.

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Figure-4

(Left to Right) Normal fetus; Fetus with wide open sutures; and Fetus with wide anterior fontanel.

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