1. Testing of FASD Therapeutic Agents in Perinatal Mouse Models
Feng C. Zhou, Indiana University School of Medicine
Charley Goodlett, Indiana University Purdue University
Prenatal: C57BL –Chronic Liquid Diet Model
Feng C. Zhou, Teresa Powrozek, Youssef Sari, Fang Yuan
Postnatal: C57BL–Bing Exposure Model
Feng C. Zhou, Teresa Powrozek, Bruce Anthony
Charley Goodlett
Mark Stanton, Lygine Calizo

2. Specific Aims
Specific Aim 1. Prenatal model: Identify the neuroprotective and /or neurotrophic effect of NAP/SAL against alcohol neurotoxicity to (1) midline deficit, (2) raphe-serotonin (5-HT) neurons
Specific Aim 2. Postnatal model: Identify the neuroprotective and neurotrophic effect of NAP/SAL on an alcohol induced apoptotic injury of the excitatory hippocampal neurons.
Publications
Zhou FC, Sari Y, Powrozek TA, Spong CY.A neuroprotective peptide antagonizes fetal alcohol exposure-compromised brain growth.J Mol Neurosci. 2004;24(2):189-99
Goodlett CR, Horn KH, Zhou FC. Alcohol teratogenesis: mechanisms of damage and strategies for intervention. Exp Biol Med (Maywood) (6):
Zhou FC, West JR, Blake CA. General discussion at the fetal alcohol syndrome symposium.
Exp Biol Med (Maywood) Jun;230(6):

3. Aim1. Prenatal Chronic liquid diet model
Acclimation
C57BL/6 mice
Treatment
Chow PF
Alc
NAP/SAL
Alc+NAP/SAL
At E7 or E8
At E15-E18
Young adult
Body volume Neuronal development
Brain volume Cortical development
Brain weight
Analyses
Alc: ALCOHOL group fed with liquid diet with 20% or 25% ethanol derived
calorie (EDC) and with supplemented minerals, BAC:60-120mg/dl
PF: Pair-Fed group with isocaloric liquid diet
Chow: Chow-fed group
NAP/SAL: PF Treated with NAP/SAL 20µg/day, i.p.
Alc+NAP/SAL: ALC treated with NAP or SAL 20µg/day, i.p.

4. Neural tube fusion is still an ongoing process at E11

5. An Incomplete Neural Tube Fusion (iNTF) at E15
Dorsal view
Coronal section

6. An Incomplete Neural Tube Fusion (iNTF) at E13
Ventral view
Coronal section

7. Occlusion of Ventral Canal in Brainstem at E13
opening

8. SAL treatment
SAL prevents the fetal alcohol induced deficits on fetal body and brain weight reduction, midline opening, ventrical enlargement, and cortical thinning.--
Zhou et al, J Mol Neurosci. 2004;24(2):189-99
Effect of SAL on fetal alcohol-compromised body and brain weight. Fetal alcohol exposure reduced both body and brain weight at E15. The SAL-treatment (20ug/0.2ml/dam/once daily, i.p.) through out the period of alcohol exposure protected the embryos from alcohol-induced reduction with greater effect on brain weight than body weight. Values are expressed as mean  standard error of the mean; significance of group comparisons used ANOVA and post hoc tests (Fisher’s PLSD). **, * =p<0.01 and p<0.05, respectively. No significant difference was found between Chow and PF groups.

9. NAP Treatment
Fetal Body Weight
Fetal Brain Weight
Fig. 2. Prenatal alcohol treatment decreases the embryonic body weights at E15. The NAP treatment protects against the alcohol induced reduction. The fetal body weight at E15 was increased in the ALC/NAP group from that of the level of the ALC group (**:P<0.01; *: P<0.05), but did not reach that of PF and Chow control groups.

10. On Ventrical Enlargement
Fig. 5. Alcohol induced enlargement of lateral ventricle in ALC as compared with that of PF and Chow groups at E15 was prevented by administration of NAP (ALC/NAP; **:P<0.01).

11. On Cortical Thinning
Fig. 4. The cortical thickness was reduced in ALC as compared with PF and Chow groups. Treatment with L-NAP along the alcohol treatment time course significantly increased medial frontal and cingulate cortical thickness as compared to that of alcohol-treated . Treatment with L-NAP increased the thickness of the medial frontal and cingulate cortices from the level of the ALC to levels comparable to PF and Chow control groups. (**:P<0.01; *P<0.05).

12. E13 5-HT Neurons

13. On Raphe 5-HT Neurons
peptide of Activity dependent neurotrophic factor protects serotonin neurons in rostral raphe against Fetal alcohol exposure in midgestation brain
*=p<0.05
**=P<0.01

14. Summary of Aim 1
SAL prevents the fetal alcohol induced deficits on fetal body and brain weight reduction, midline opening, ventrical enlargement, and cortical thinning, and median raphe 5-HT neurons reduction.
NAP prevents the the fetal alcohol induced deficits on fetal body and brain weight reduction, and cortical thinning,.
Continue study / future plan:
Study of 5-HT neurons on other raphe areas
Mechanism on ADNF protection
Develop behavioral paradigm for midline deficit FAS model

15. Experimental Paradigm
Specific Aim 1. Prenatal model: Identify the neuroprotective and / or neurotrophic effect of NAP/SAL against alcohol neurotoxicity to (1) midline deficit, (2) raphe-serotonin (5-HT) neurons
Specific Aim 2. Identify the neuroprotective and neurotrophic effect of NAP/SAL on an alcohol induced apoptotic injury of the excitatory hippocampal neurons.
This aim tests the hypothesis that NAP or SAL will protect the limbic hippocampal system with binge ethanol exposure at its vulnerable neonatal stage equivalent to 3rd trimester of human gestation.
Experimental Paradigm
Postnatal alcohol treatment s.c, 2.5g/kg twice/day at P7
NAP (2µg/twice/day/pup) is treated at P7 (concurrent) or P5-P7 (extended)
Animal are perfused 6 hrs after last alcohol treatment for caspase 3 immunostaining.

16. Alcohol Treatment in Postnatal Model – Caspase 3
Postnatal alcohol treatment s.c, 2.5g/kg twice/day at P7, animal are perfused 6 hrs after last alcohol treatment for caspase 3 immunostaining

17. ALC+NAP Cingulate cortex Striatum Hippocampus
ALC ( s.c, 2.5g/kg twice/day)+ NAP (2ug/twice/day/pup) at P7
Hippocampus

18. Extended NAP Treatment---Cortex
One day Alcohol, three days NAP treatment

19. Extended NAP Treatment---Hippocampus
ALC
ALC+NAP

20. Summary of Aim 2
NAP fails to prevent the the early postnatal alcohol induced apoptosis on limbic neurons in concurrent or extended treatment paradigm.
Continue study / future plan:
Search additional protective agent(s) for postnatal neuronal apoptosis.
Develop behavioral paradigm for postnatal alcohol exposure FAS model on limbic deficits

21. A Test of ADNF on
Facial Dysmorphology

22. SAL antagonizes the alcohol exposure reduced head size at E15.
Figure 3. Effect of SAL on fetal alcohol-induced head size reduction. The fetal alcohol exposure in the liquid diet model (with 25% ethanol-derived calories from E7 to E15) retarded the growth of embryos as compared to Chow and PF. The embryos have a smaller head size with corresponding lower body and brain weight, and smaller brain dimension. The reduction in head size is attenuated by the SAL treatment (ALC+ SAL , i.p., 20ug/0.2ml/daily during alcohol treatment). The Chow with SAL treatment (Chow+SAL) however did not create an abnormally large embryo. Scale bars: a,b,c,d,e=1mm.

23. Crown Rump Length
Crown Rump Length

24. Measurement of Head Size

25. Width of Nose

26. Eye Distance
Eye Distance

27. ALCs

28. Frequency of Occurrence
Treatment
Missing Limb
Missing Digit
Webbed Digit
Eye
Malformation
Total # of Embryos
Single
Bilateral
Chow 0%
8% * 6% 3% 34
Chow +SAL 17
Chow +NAP 4% 25 PF
10% * 30
Alc
10.3%
3.4%
13.8%
20.7%
10% 29
Alc + Scram
6.9%
17.3%
24.1%
Alc + SAL
2.7%
5.4% 37
Alc + NAP
4.3% 23 .
Table 1: The frequency of malformations following prenatal alcohol exposure and treatment with the ADNF protective peptides. The number of abnormalities seen including missing limbs, missing digits, undifferentiated digits and malformations of the eye were higher in the alcohol group usually occurring unilaterally, though in the case of webbing of the digits the results were generally bilateral. These effects were not prevented with use of a scrambled peptide of the SAL sequence but were prevented with both the use ADNF peptides NAP and SAL

29. 3D Model Construction of Mouse Embryos from Video Imaging
Feng C. Zhou
Jeff Huang
Shiaofen Fang
Indiana University School of Medicine
IU school of Science

30. Microscopic Video image and 3-D Modeling
3-D model construction in computer vision and photogrammetry allow for virtual 3-D vector measurement.
With limitation of laser image acquisition of small objects, we adopted a method of turn-table image sequences by moving an embryo around single axis under microscope digital vedio camera, and the multi-angle 2-D images can be acquired.
A 3-D mouse model is generated from a sequence of 2-D images.
(53 images depending on video clips.
vidio with eye, hair will be
3-D medel construction -->Geometry process

31. Background 3D geometry and turn-table Axis M C1  Image 1 Ci Image i
Using angle (video data) instead of depth (by laser data) to generate a 3-D information
6 or more degree, it does not to be same angle
Image i

32. Methodology Segmentation Remove background information
Silhouette extraction
Determine rotation center
Calibrated camera position
Un-calibrated camera position
Volume Carving/Back projection
Texture Mapping
For video to making into depth and 3-D mesh image
After getting mesh image (with 3-D data pionts), the processes for measurement of parameters are the same.

33. Image Segmentation and 3D Rendering
Convert to binary images (2D processing)
Automated noise removal and filling eye area (2D)
Construct 3D model and surface rendering (3D)

34. Rotation Axis
Un-calibrated camera position
h/2
l/2

35. Back Projection
The contours of each 2D image are projected back into the 3D space.
Is to do the carving to produce 3-D object. The end product is become a 3-D object

36. Volume Carving
The object is represented as a volume and is initially estimated as a cube.
The volume is rotated by an angle of  around the rotation axis. The volume is projected into each of the corresponding silhouettes and sections that fall outside the silhouettes are discarded or “carved off”.
Iterative call of the volume carving program to generate a volume model:
Iterative call--do multiple carving
3D polygon mesh model with rotation

37. Texture Mapping For visualization purpose
The average intensities (R,G,B) were calculated for the volume during volume carving. The output intensities represent the colors for the final volume.
Just for visual purpose

38. Eye Segmentation and Size Measurement
Rapid form will be used for measurement of 2-D as well as 3-D
2-D any image, for 3-D the mesh image will used .

39. Contour Measurement Circumference: 15.48mm Distance: 4.27mm
Area: 18.36mm2
Centroid: 6.49, 5.61, 4.40

40. Binocular Distance Measurement
Point 1: , ,
Point 2: 9.035, ,
Distance: mm
Point 1: , ,
Point 2: 9.035, ,
Distance: mm

41. What Geometric 3-D Analysis of Animal Facial Imaging Can Provide
Comparative study to model human facial dysmorphology
Longitudial study in shorter time course
Testing correlation between facial dysmorphology parameters (e g. Palpebral fissure, with head circumference, and brain volume) in controlled experimental paradigms (stages and patterns of exposure, BAC, gender...
Generating testable new criteria for facial dysmorphology?
Translational study in two-way direction
Testing potential protecting agents against facial dysmorphology….