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

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). 2005 230(6):394-406. Zhou FC, West JR, Blake CA. General discussion at the fetal alcohol syndrome symposium. Exp Biol Med (Maywood). 2005 Jun;230(6):407-12.

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.

Neural tube fusion is still an ongoing process at E11

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

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

Occlusion of Ventral Canal in Brainstem at E13 opening

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.

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.

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).

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).

E13 5-HT Neurons

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

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

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.

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

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

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

Extended NAP Treatment---Hippocampus ALC ALC+NAP

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

A Test of ADNF on Facial Dysmorphology

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.

Crown Rump Length Crown Rump Length

Measurement of Head Size

Width of Nose

Eye Distance Eye Distance

ALCs

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

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

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

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

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.

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)

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

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

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

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

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 .

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

Binocular Distance Measurement Point 1: 7.724, 7.722, 4.800 Point 2: 9.035, 4.956, 4.800 Distance: 3.062 mm Point 1: 7.724, 7.722, 4.800 Point 2: 9.035, 4.956, 4.800 Distance: 3.062 mm

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….