Mouse Model Neuro-Facial Dysmorphology: Translational & Treatment Studies Progress since September 1, 2007 Feng C. Zhou Charles Goodlett Yun Liang Shiaofen Fang Bruce Anthony

Specific Aims Aim 1. To advance the understanding of sources of variation in abnormal facial development induced by prenatal alcohol exposure as a function of the dose and developmental timing of alcohol exposure in a C57BL/6 mouse model. Aim 2. To determine longitudinally the extent of brain structural and neuro-facial abnormalities as a function of the dose and developmental stage of alcohol exposure. Aim 3. To determine the extent to which the Neurotrophic peptides NAP/SAL will provide long-term protection against alcohol-induced neuro-facial dysmorphology and neurobehavioral deficits.

1. Mouse Model for Dose and Timing of Alcohol Exposure Standardization of C57BL6 mice lines from Halan and Jackson breeder used in Drs. Zhou and Sulik’s laboratories (a) Drinking level—Bruce Anthony (b) Teratogenesis – Scott Parnell

Protocol for Liquid Diet Repeated Deprivation 1. Animals housed in temperature controlled 12-hour reverse light/dark cycle (10am-10pm) 2. Prior to treatment all animals have ad lib chow and water 2 weeks after arrival. 3. From the beginning of treatment all animals housed individually, ages 12-14 weeks Days 1 2 16 20 - 37 E7 E11-E16 P7 and P21 RX 2.4% 4.8 or 3.6 % Alc Chow /Mating 3.6 or 4.8 % alc Isolate or Postnatal surrogate Alcohol groups Pre Treatment Alcohol Pair Fed Control RX 2.4% 4.8 or 3.6 % Alc Chow/Mating 3.6 or 4.8 % Dex . Isolate or Postnatal surrogate Days 1 2 16 20 - 37 E7 E11-E16 P7 and P21 Total Pair Fed Control RX ~2.4% 4.8 or 3.6 % Dex Chow/Mating ~3.6 or 4.8 % . Isolate or Postnatal surrogate Days 1 2 16 20 - 37 E7 E11-E16 P7 and P21 Control RX Chow throughout Mating Chow throughout Isolate or Postnatal surrogate Days 1 20 - 37 E11-E16 P7 and P21

* Statistical Significant N=6 Mating **** *** ** * * Mating Mating Harlan C57Bl/6 Jackson C57Bl/6 Treatment Paradigm 1. Pre treatment 2.4% (x2days) then 4.8 % 14 days. 2. 16 day deprivation and mating 3. E7-E18 4.8% Alc Averages: Harlan pre depreviation 26.6 Avg daily Harlan post deprivation 26.6 Avg daily Jax pre deprivation 22.6 Avg daily Jax post deprivation 22.4 Avg Daily * Statistical Significant N=6 Mating

N=20 pre-treatment/ 10 post treatment * Statistically Significant * * * * * * Mating Mating Harlan C57Bl/6 Jackson C57Bl/6 * * * * * * * * * * * * * * Mating Parnell et al. Paradigm 1. Pre treatment 2.4% (x2days) then 4.8 % 14 days. 2. 4 day deprivation /Max 21 day mating 3. E7-E9 4.8% Alc Averages: Harlan pre deprivation 25.7 +/-0.75 Avg daily Harlan post deprivation 22.3 +/-1 Avg daily Jax pre deprivation 23.8 +/-0.75 Avg daily Jax post deprivation 20.0 +/-1 Avg Daily N=20 pre-treatment/ 10 post treatment * Statistically Significant

2. Facial dysmorph. Analysis A. Microvideo imaging analysis Moving from 2D to 3D --- Shiaofen Fang B. MicroCT imaging analysis ---Yun Liang

Moving from previous 2D to 3D analysis A. Micro-Video Imaging for 3-D Moving from previous 2D to 3D analysis 1. Set of 2D Images 180o at 4o angles 2. Covert each to a binary image to isolate features ==> ==> 3. Project each image onto a 3D volume and carve (remove) non-feature parts ==> 4. 3D model (polygon mesh)

1. 3D geometry and turn-table Collecting Set of 2D Images (180o at 4o angles) M  Ci Axis Image i C1 Image 1

Image Segmentation and 3D Rendering 2. Convert to binary images (2D processing) 3. Automated noise removal and filling eye area (2D) 4. Construct 3D model (polygon mesh) and surface rendering (3D)

Improvements in Microvideo 1. New rotor for Implementation of step motor a. Exact definition of degree changes b. Control of angle progression in capture 2. Images scaled within software a. To ensure proper aspect ratio b. To maintain optimal dimensions software allows 3. Projection algorithm re-done a. Projection done with built-in functions for consistency and quality assurance between users.

B. Micro-CT for Craniofacial Analysis —including facial & skeletal imaging co-registration Evaluate facial and cranial bone dysmorphology in postnatal mice (P7 and P21) using 3D images obtained by a Micro-CT. Identify anthropometric geometries and features to differentiate subjects with alcohol exposure. Correlative studies of facial and skeletal features

Components of MicroCT System Imaging Volume Detector X-Ray Source Opening operture 8cm hole opening Curve arrow --rotation EVS RS-9

Imaging Techniques Scan Resolution Width Length Volume   Scan Resolution Width Length Volume Modes_____(m)_____ (mm)__ (mm)__ Dimensions 1x1 23 36.8 36.8 1600x1600x1600 (~ 2 hours scan over 1000 slices !!) 2x2 46 78.48 41.76 1750x1750x929 4x4 92 78.48 41.76 872x872x464 X-ray output kVp= 50 kVp, I= 1 mA, t= 100 msec , and 276 angular views in a single frame. A total of 8 frames Recon-Resolution Image Quality Radiation Dosage_ 46 m ~ 80-100 HU (noise) 0.8 Gy 276 angular view--- 46 micro is becase merge two slide for analysis Hu ---Hunfiled unit nioce ~ 10% measurment data (it is good enough for bone study) Gy---Geore / kg

MicroCT Study Procedure Subjects: P7 and P21 C57BL/6 mice, Control vs Liquid Alcohol diet treated. Anesthesia: an induction chamber with Isoflurane levels of 1.5 % at a initially flow rate of 0.8-1.2 liter/min followed by a maintenance Isoflurane rate of 0.5 liter/min . CT number Calibration: QA phantom with water, air, acyrilc, bone (SB3) inserts Image Analysis: (1) Semi-automatic: Segmentation of skull with thresholding, followed by manually refining in slice-by-slice way; (2) Volume rendering (shaded-surface-display); (3) Metric measurement with volume/slice relating tool. Analyzed using “MxView” (courtesy of Philips Medical Systems).

Thresholding Sb3 Air water QA Phantom Size of phamtom---1 inch diameter (a model to mimic biological materials. QA quality assurance SB3--for bone meaneral density is about density for skull cortical bone

Relate Slice/Volume

Anthropometric Measurement

Comparative analysis

MicroCT- Surface-Skull fusion

a b c d @ Alcohol Control Figure 3D segmentation of developing craniofacial bone in P1 C57BL6 mice. The threshold is set at 20% of standard cortical bone density (BMD=1073 kg/m3). It is clearly shown that the nasal bone (@) growth among others is retarded in the 25%EDC alcohol-treated group (a, b) as compared with that of two P1 pair-fed control pups (c, d).

There is notable difference in nasal bone development? Compare 3 and 3 animals (FAS vs control)