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

2. 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.

3. 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

4. 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 weeks
Days E7
E11-E P7 and P21
RX % 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% or 3.6 %
Alc
Chow/Mating or 4.8 %
Dex .
Isolate or Postnatal surrogate
Days E7
E11-E 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 E7
E11-E P7 and P21
Control RX
Chow throughout
Mating Chow throughout
Isolate or Postnatal surrogate
Days 1
E11-E P7 and P21

5. * 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.
day deprivation and mating
3. E7-E18 4.8% Alc
Averages:
Harlan pre depreviation Avg daily
Harlan post deprivation Avg daily
Jax pre deprivation Avg daily
Jax post deprivation Avg Daily
* Statistical Significant N=6
Mating

6. 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 /-0.75 Avg daily
Harlan post deprivation /-1 Avg daily
Jax pre deprivation / Avg daily
Jax post deprivation /-1 Avg Daily
N=20 pre-treatment/ 10 post treatment
* Statistically Significant

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

8. 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)

9. 1. 3D geometry and turn-table
Collecting Set of 2D Images (180o at 4o angles) M  Ci
Axis
Image i C1
Image 1

10. 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)

11. 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.

12. 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

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

14. Imaging Techniques Scan Resolution Width Length Volume
Scan Resolution Width Length Volume
Modes_____(m)_____ (mm)__ (mm)__ Dimensions
1x x1600x1600
(~ 2 hours scan over 1000 slices !!)
2x x1750x929
4x x872x464
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 ~ HU (noise) 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

15. 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 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).

16. 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

17. Relate Slice/Volume

18. Anthropometric Measurement

19. Comparative analysis

20. MicroCT- Surface-Skull fusion

21. 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).

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