- Количество слайдов: 41
Testing of FASD Therapeutic Agents in Perinatal Mouse Models Feng C. Zhou, Indiana University School of Medicine Charley Goodlett, Indiana University Purdue University • Prenatal: C 57 BL –Chronic Liquid Diet Model Feng C. Zhou, Teresa Powrozek, Youssef Sari, Fang Yuan • Postnatal: C 57 BL–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.
Aim 1. Prenatal Chronic liquid diet model C 57 BL/6 mice Acclimation Treatment At E 7 or E 8 At E 15 -E 18 Young adult Alc PF Body volume Brain weight Chow NAP/SAL Neuronal development Cortical development Alc+NAP/ SAL Analyses Alc: ALCOHOL group fed with liquid diet with 20% or 25% ethanol derived calorie (EDC) and with supplemented minerals, BAC: 60 -120 mg/dl n. PF: Pair-Fed group with isocaloric liquid diet n. Chow: Chow-fed group n NAP/SAL: PF Treated with NAP/SAL 20µg/day, i. p. n Alc+NAP/SAL: ALC treated with NAP or SAL 20µg/day, i. p. n
Neural tube fusion is still an ongoing process at E 11
An Incomplete Neural Tube Fusion (i. NTF) at E 15 Dorsal view Coronal section
An Incomplete Neural Tube Fusion (i. NTF) at E 13 Ventral view Coronal section
Occlusion of Ventral Canal in Brainstem at E 13 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
NAP Treatment Fetal Body Weight Fetal Brain Weight
On Ventrical Enlargement
On Cortical Thinning
E 13 5 -HT Neurons
On Raphe 5 -HT Neurons *=p<0. 05 **=P<0. 01
Summary of Aim 1 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. 2. NAP prevents the fetal alcohol induced deficits on fetal body and brain weight reduction, and cortical thinning, . Continue study / future plan: 1. Study of 5 -HT neurons on other raphe areas 2. Mechanism on ADNF protection 3. Develop behavioral paradigm for midline deficit FAS model
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 3 rd trimester of human gestation. Experimental Paradigm 1. Postnatal alcohol treatment s. c, 2. 5 g/kg twice/day at P 7 2. NAP (2µg/twice/day/pup) is treated at P 7 (concurrent) or P 5 -P 7 (extended) 3. Animal are perfused 6 hrs after last alcohol treatment for caspase 3 immunostaining.
Alcohol Treatment in Postnatal Model – Caspase 3
Striatum Cingulate cortex ALC+NAP Hippocampus
Extended NAP Treatment---Cortex
Extended NAP Treatment---Hippocampus ALC+NAP
Summary of Aim 2 1. NAP fails to prevent the early postnatal alcohol induced apoptosis on limbic neurons in concurrent or extended treatment paradigm. Continue study / future plan: 1. Search additional protective agent(s) for postnatal neuronal apoptosis. 2. 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 E 15.
Crown Rump Length
Measurement of Head Size
Width of Nose
Frequency of Occurrence Treatment Missing Digit Webbed Digit Eye Malformation Total # of Embryos Single Bilateral Chow 0% 0% 8% * 0% 6% 0% 34 Chow +SAL 0% 0% 17 Chow +NAP 0% 0% 0% 4% 0% 25 PF 0% 0% 0% 10% * 0% 0% 30 Alc 10. 3% 0% 3. 4% 13. 8% 3. 4% 20. 7% 10% 0% 29 Alc + Scram 6. 9% 3. 4% 0% 0% 17. 3% 24. 1% 6. 9% 0% 29 Alc + SAL 2. 7% 0% 5. 4% 0% 2. 7% 0% 37 Alc + NAP . Missing Limb 4. 3% 0% 0% 23
3 D 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.
Background • 3 D geometry and turn-table Axis M C 1 Image 1 Ci 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
Image Segmentation and 3 D Rendering • Convert to binary images (2 D processing) • Automated noise removal and filling eye area (2 D) • Construct 3 D model and surface rendering (3 D)
Rotation Axis • Un-calibrated camera position h/2 l/2
Back Projection • The contours of each 2 D image are projected back into the 3 D space.
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: 3 D 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.
Eye Segmentation and Size Measurement
Contour Measurement Circumference: 15. 48 mm Distance: 4. 27 mm Area: 18. 36 mm 2 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
What Geometric 3 -D Analysis of Animal Facial Imaging Can Provide 1. Comparative study to model human facial dysmorphology 2. Longitudial study in shorter time course 3. 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. . . 4. Generating testable new criteria for facial dysmorphology? 5. Translational study in two-way direction 6. Testing potential protecting agents against facial dysmorphology….