1. Effects of ethanol on the developing craniofacial region in the chicken model Carissa Temple Advisor: Dr. Lustofin Fetal Alcohol Syndrome (FAS) is the leading cause of preventable mental retardation in the western world (Cudd, 2005, 389). Consuming alcohol while pregnant has been shown to cause growth retardation. Growth retardation may result in craniofacial malformations, retarded overall growth and neurological deficiencies (Snow, Keiver, 2007, 181). Some symptoms consistent with craniofacial malformations are: small head circumference, an underdeveloped jaw, and flattening of nose and other facial features (Cudd, 2005, 392). Using animal models to study FAS has many advantages. However, using the avian model or the chicken model to study FAS may be better than using other animals (Cudd, 2005, 392). The chicken is a good model for several reasons: 1) it has a short gestation period (Cudd, 2005, 392) 2) there is no interference of a placenta or a mother during development, so the exact amount of dosing is known (Becker and Shibley, 1998, 457) 3) it is considered a good model for craniofacial malformations (Cudd, 2005, 392). The stages of development are also well known (Su et. al, 2001, 62). Even though the avian model lacks the soft tissue seen in humans, it does contain similar bone structures to humans (Cudd, 2005, 392). This makes the avian model sufficient to look at craniofacial deformities and allows us to compare these malformations to malformations that occur in humans as a result of FAS (Cudd, 2005, 392). The purpose of this experiment was to determine if different concentration levels of ethanol will cause malformations in the craniofacial region of the developing chick. My hypotheses were: 1) exposing the avian embryo to ethanol will result in craniofacial malformations, similar to that of humans 2) Higher levels of ethanol will cause more significant malformations 3) a lack of growth will be the cause of these malformations. Introduction Measurements of chicken head Figure 1A- Inferior view. Hyoid glossal. Manidibular length and width. Figure 1B- Lateral view. Head height and length. Maxilla and mandibular length. Oral gap and ocular diameter. Figure 1C- Superior view. Nasal length and width. Materials and Methods The effect of ethanol on craniofacial development was determined by using White Leghorn chick eggs. The eggs were separated into 4 even groups of 6. Three of these groups were given a different concentration of ethanol and one group was the control. The different concentrations of ethanol were 8%, 10%, and 12%. Eggs were incubated at 37.5 o C. At hour 18 gestation the eggs were injected (into the egg yolk) with saline or the different concentrations of ethanol (8%, 10% and 12%) in a saline solution. The eggs were injected into the yolk because a fast dispersion of ethanol to the embryo will occur (Su et.al, 2001, 61). The total volume of solution injected into each egg was 250 μl. At day 10 gestation the eggs were candled and the embryos were sacrificed by decapitation. Embryos were harvested at day 10 because this is when facial features of the chick will have completed development (Su et.al, 2001, 62). Two different staining methods were used, however in both methods the heads of the embryos were fixed in 95% EtOH for 3- 5 days (Depew, 2008, 38 & MacLeod, 1980, 299-301). The heads were then skinned and eviscerated. After fixation was completed in the 1 st method, the heads were placed in acetone for 2 days, and then the heads were placed in the staining solution. The first staining solution contained the following: 0.3% Alcian Blue in 70% EtOH, 0.1% Alizarin red S in 95% EtOH, Glacial acetic acid, and EtOH. The specimens were left in the staining solution for 3 days. After the 3 day period the heads were washed in distilled H 2 O. Maceration and clearing of the heads was done by placing them into 1% KOH and distilled water for 12 hours to 2 days, heads were then placed in a 1:1 ratio of ethanol to glycerol for storage (Depew, 2008, 38-39). The first staining method did not warrant clear results so it was necessary to use another method. The second method placed the heads in Alcian Blue for 24 hours. The heads were then placed in 95% EtOH for one day, and then they were placed in 1% KOH overnight. The KOH was drained and the heads were placed in Alizarin Red + 1% KOH for 24 hours and this solution was then drained. The heads were then placed in a 1:1 ratio of ethanol to glycerol for storage (MacLeod, 1980, 299-301). Specimens were then photographed and measured using Infinity Analyze Software. The measurements were then statistically analyzed using an ANOVA method and a Bonferroni Post Hoc test. Results As can be seen in Figures 2, 3, 4, 5, and 6, exposing the embryos to ethanol did cause malformations. However, more malformations occurred in lower concentration groups (8% and 10%) instead of the 12% group (Figures 2,3,4,5 and 6). Also a lack of growth was not seen in all measurements, and in some cases the embryos exposed to ethanol had larger measurements than the control group (Figure 2 and 6). Treatment had no effect on the following measurements: head length, maxilla length, mandible length and nasal length. P = 0.535, P = 1.00, P = 1.00, and P = 0.876 respectively. * * Conclusions In conclusion, my hypotheses were not supported. The malformations that occurred in the treatment groups do not resemble the malformations that are seen in humans with FAS. It is common to see a wide, elongated face in humans with FAS, and this did not occur in my treatment groups. More malformations tended to occur in the treatment groups exposed to 8% and 10% ethanol when compared to the 12% exposure group. Finally, a lack of growth did seem to cause some of the malformations (Figures 3, 4 and 5), but some measurements were larger in the treatment groups than they were in the control group (Figures 2, and 6). My results do not reflect the results that have been seen in other research. In previous research it has been common to see a larger nasal width and a larger head height in the groups exposed to ethanol. This would give an appearance of midfacial flattening which is a common symptom of FAS (Su et.al., 2001, 62). This did not occur in my treatment groups. A probable cause of these results is the genetics of the chickens. Even though all of the chicken eggs were from White Leghorn chickens, the genetic background of each individual chicken may be highly different. This would make each embryo react to the ethanol in different ways (Su et.al., 2001, 62). Also, very small group sizes were used and this may have caused the results that were seen. Literature Cited Becker Bupp SR., Shibley IA Jr.1998. Teratogenicity of Ethanol in Different Chicken Strains. Alcohol and Alcoholism Vol. 33 No. 5: 457-464. Chaudhuri DJ. 2004. Effect of a single dose of ethanol on developing skeletal muscle of chick embryos. Alcohol Vol. 34: 279-283. Cudd, T. 2005. Animal Model Systems for the Study of Alcohol Teratology. Society for Experimental Biology and Medicine. 389-393. Depew M. 2008. Analysis of Skeletal Ontogenesis through Differential Staining of Bone and Cartilage. Methods in Molecular Biology Vol. 461: 37-45. MacLeod, MJ. 1980. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratol. 22:299-301. Snow ME., Keiver K. 2007. Prenatal ethanol exposure disrupts the histological stages of fetal bone development. Bone Vol. 41 No. 2: 181-187, 7p. Su B, Debelak K, Tessmer L, Cartwright M, Smith S. 2001. Genetic Influences on Craniofacial Outcome in an Avain Model of Prenatal Alcohol Exposure. Alcoholism: Clinical and Experimental Research Vol. 25 No. 1: 60-68. I would like to express my appreciation and thankfulness to: Dr. Lustofin, my capstone advisor for all of her support, feedback, and help; Dr. Brown, for all of his feedback and support; Dr. Spilatro for providing the saline for my experiment; Dr. Klein for helping me with my statistics; the Biology Department for funding my experiment; my fellow capstone students for all of the support and feedback. Acknowledgements Figure 2: Average mandibular width measurements Figure 3. Average nasal width measurements Figure 4. Average head height measurements Results Average hyoid glossal measurements are shown in Figure 6. An ANOVA analysis of these averages did not give any overall significant results (Figure 6). As can be seen in Figure 6, when a Bonferroni Post Hoc test was performed to analyze each group individually, it was discovered that the 8% group was significantly larger than then 10%, 12% and control groups (p< 0.0001). Figure 6. Average Hyoid Glossal measurements Average head measurements in each group are shown in Figure 4. As can be seen there were no overall trends exhibited from the ANOVA analysis (Figure 4). When the groups were compared individually using a Bonferroni Post Hoc test, the average head height of the 8% group was significantly smaller than the average head heights of the control, 10% and 12% groups (Figure 4). P value < 0.05 Average Ocular Diameter results can be seen in Figure 5. When the average ocular diameter results were compared using an ANOVA analysis, no overall significant results occurred. However, when a Bonferroni Post Hoc test was used to compare the groups individually, the 10% group had a significantly smaller ocular diameter average than the control, 8% and 12% groups (Figure 5). P value < 0.0001 Figure 5. Average Ocular Diameter measurements. Average nasal width measurements for each group can be seen in Figure 3. An ANOVA analysis was performed and no overall significant differences occurred (Figure 3). However, when a Bonferroni Post Hoc test was performed the 8% and 10% groups were significantly smaller than the control and 12% groups when they were compared individually (Figure 3). P value of < 0.05. Average mandibular width measurements in each group are shown in Figure 2. An ANOVA analysis did not indicate any overall significant differences for all of the groups. However, when the groups were compared individually using a Bonferroni Post Hoc test, it was discovered that the 8% and the 10% groups were significantly larger than the control and 12% groups (Figure 2). P value of < 0.0001. *