Mouse Model Neuro-Facial Dysmorphology: Translational and Treatment Studies Charles Goodlett Bruce Anthony: Sophia Liang Yun: Huisi Ai Shiaofen Fang: Yin Liu Li Shen Leah Wetherill Richard Ward CIFASD 2010 Annual Review

Two alcohol doses– 4.8% or 3.6% V/V. Dose and Stage Two alcohol doses– 4.8% or 3.6% V/V. 2. Two developmental treatment periods –E7-E16 –E7-E11 1 week acclimation Alcohol PF Chow Either 4.8% or 3.6% Alcohol or Par fed Diets From E7-E11 or E7-E17 Mating Alc-Alc Alc-PF PF-PF Chow E7 through E11 or E16 Pregnancy treatment same as pre-pregnant Pre-pregnancy treatment All Pups to Surrogate dams by P1 Diets: (PMI) Purina Test Diet (Micro-stabilized Rodent Liquid Diet, LD101A) with 4.8% and 3.6% alcohol

Mouse Nuerofacial dysmorphology Anthropometic Craniofacial analysis: Embryonic 17 (Mid gestation equivalent): Microvideo images Postnatal day 7 &21 (late gestation, toddler): MicroCT Craniofacial Volume and feature Analysis Embryo: 2D rendered analysis Postnatal 7 & 21: MicroCT Brain volume and neuron analysis Total and Regional volume, neuronal number

Facial Dysmorphology Analysis for E17 Embryos

C57BL/6N E17 Embryo Facial Dysmorphology Analysis Facial morphometric analysis.  P-value of significant treatment main effects (within sub-strain) for each facial measure is indicated. For the paired comparisons, (using Tukey-Kramer procedures) comparisons were made for Alc to Chow, PF to Chow, and Chow to PF groups: Bolded-typed values under columns denote that the overall treatment p-value < the corrected Bonferonni alpha = 0.0033. An asterisk (*) denotes that the Tukey-Kramer pair-wise comparison was significant (p<0.05).  Gray-shaded areas are measurements with significant difference between both control groups and alcohol-exposed group. Results: Significant effects specific to alcohol treatment, (i.e., the Alcohol group differed from each respective control group), were identified for two measures of facial depths (upper facial depth; mid-facial depth) in both sub-strains, Only the B6N strain showed significant effects on two other facial height measures (nasal length; nasal bridge). The two strains showed markedly different patterns of facial alterations comparing the PF and Chow groups. In particular, the B6N sub-strain showed a significantly greater vulnerability to the pair feeding procedure (in terms of smaller facial measures) than did the B6J sub-strain.

C57BL6N Discriminant Analysis of E17 Facial Dysmorphology Discriminant analysis was done with all 15 measurements for correct predictability. Analysis was performed using backward stepwise model for comparison.  Discriminant analysis was performed using individual animal group with comparisons between Alc and Pair-Fed as well as between Alc to all-non-alcohol litters. Percentage of correct predictability is reported for Alc and Non-Alc groups.  “x” denotes the measurement in each column needed in the final classification model. % Reflects the percentage of correct

Silhouette surface extraction Surface alignment A video 3D image volume was formed by stacking 2D video embryo head images taken from a sequence of angle steps Silhouette surface extraction Surface alignment Feature Selection and Analysis Validation CIFASD 2010 Annual Review

Posters and Presentations: Publications: Shiaofen Fang, Ying Liu, Jeffrey Huang, Sophia Vinci-Booher, Bruce Anthony, and Feng Zhou. Alcohol Exposure Analysis of Mouse Embryos Using Multi-angle Facial Image Analysis. Proc. of ACM Symposium on Applied Computing, 2009, 852-856.   Bruce Anthony, Sophia Vinci-Booher, Leah Wetherill, Richard. Ward, Charles Goodlett, Feng C. Zhou. Alcohol Induced Facial Dysmorphology in C57BL/6 Mouse Models of Fetal Alcohol Spectrum Disorder. Alcohol and alcoholism, revision under review. Shiaofen Fang, Ying Liu, Jeffrey Huang, Sophia Vinci-Booher, Bruce Anthony, and Feng Zhou. Surface Feature Analysis using Video Volumes of Mouse Embryos for Fetal Alcohol Syndrome Classification, submitted to International Conference on 3D Data Processing, Visualization, and Transmission (3DPVT). Yun Lian, Huisi Ai, Bruce Anthony, Leah Wetherill, Charles Goodlett, Feng C. Zhou. (2009) Facial Dysmorphology in Craniofacial Bone Formation in C57BL6 after Alcohol Exposure. In preparation. Posters and Presentations: Bruce Anthony, Sophia Vinci-Booher, Leah Wetherill, Richard. Ward, Charles Goodlett, Feng C. Zhou, Research Society on Alcoholism. Poster presentation. San Diego (2009). Alcohol Induced Facial Dysmorphology in C57BL/6 Mouse Models of Fetal Alcohol Spectrum Disorder. Yun Lian, Huisi Ai, Bruce Anthony, Leah Wetherill, Charles Goodlett, Feng C. Zhou. Poster presentation Research Society on Alcoholism San Diego (2009). Micro CT Analysis Facial Dysmorphology in Craniofacial Bone Formation in C57BL6 after Alcohol Exposure. Bruce Anthony. FAS talk. Alcohol Induced Facial Dysmorphology in C57BL/6 Mouse Models of Fetal Alcohol Spectrum Disorder. Research Society on Alcoholism. San Diego (2009). CIFASD 2010 Annual Review CIFASD 2010 Annual Review

longitudinal 3D-craniofacial dysmorphology: microCT study in the C57BL/6J mouse Yun Liang1, Huisi Ai1, Li Shen1, Bruce Anthony2, Richard Ward3, Leah Wetherill4, Feng C. Zhou2 Dept of Radiology1, Dept of Anatomy and Cell Biology2, Dept of Anthropology3, Dept of Medical and Molecular Genetics4, IU School of Medicine, Indianapolis, IN CIFASD 2010 Annual Review

Components of MicroCTSystem X-Ray Source Imaging Volume Detector CT System - EVS RS-9 (GE Healthcare, London, Ontario N6G 4X8, Canada) http://www.gehealthcare.com/usen/fun_img/pcimaging/products/microctscanner.html. CIFASD 2010 Annual Review

Imaging Techniques Scanner volume coverage: 36.8 mm x 36.8 mm x 78 mm Scan time: ~ 45 minutes Recon-resolution: 46 m Image Quality : 80-100 HU (noise) X-ray Dosage: ~ 0.8 Gy X-ray CT provides detailed bone anatomic information http://www.imtekinc.com CIFASD 2010 Annual Review

Scan Procedure CT number Calibration: QA phantom with water, air, acrylic, bone (SB3) inserts Air water Sb3 QA Phantom CIFASD 2010 Annual Review CIFASD 2010 Annual Review

Image Analysis Methods Bone Data Analysis: Part I- 2D craniofacial anthropometric Part II- individual bone volumes segmented and measured Image analysis tool: All image analyses are performed with software tool “MxView” (courtesy of Philips Medical Systems) and “Amiral” (Commercial Image Analysis). CIFASD 2010 Annual Review

Table 1. Cranium morphometric measurement and landmarks Part I Table 1. Cranium morphometric measurement and landmarks Type Number Measurement Definition of Landmarks Width 1 Minimal Frontal Width Measure distance from junction of Parietal and Frontal bone suture at the top most inner ridge of the orbital surface 2 Bizygomat Width* Top view, the distance from the left to right central auditory canal parallel to a line perpendicular to Frankfort Horizontal 3 Bitragal Width Measure a straight line distance perpendicular to Frankfort horizontal that begins at the upper most margin of the Tympanic Bulla. 4 Bigonial Width Measure widest distance between the lateral aspects of mandible 5 Inner Canthal Width Measure the shortest distance between the inner ridge of the orbital surface 6 Outer Canthal Width* (NA) Measure the longest distance to the inner edge of the outer orbital socket 7 Philtrum Length* Distance from the lowest border of the nasal cavity to the bottom of incisive bone Depth 8 Upper Facial Depth (L/R) Distance from the central point of the tympanic bulla (central ear canal) to the nasion. 9 Mid Facial Depth (L/R) (central ear canal) to the forward upper tip of the incisive bone at the junction of the nasal and incisive bones. 10 Lower Facial Depth (L/R) (central ear canal) to the forward tip of the mandible. Length 11 Nasal Length Distance from the Nasion to the forward upper tip of the incisive bone at the junction of the nasal and incisive bones. 12 Nasal Bridge Length (Shown in Figure 3i below) at the junction of the nasal and incisive bones New 14 Inner Orbital Width Front view, the minimum separation (width) between orbital bones. 15 Head Circumference The outer perimeter of skull enclosure in the axial plane at the level of “nasion” 16 Bony Mandible Shown in Figure o). Bony mandible length is the distance between the gonia and the tip of the menton. CIFASD 2010 Annual Review

Perform anthropometric measurement with landmarks MicroCT of P21 c57BL mouse facial bone with embryonic analogue division ad landmark measurements. CIFASD 2010 Annual Review CIFASD 2010 Annual Review

Minimum Frontal Width Upper Facial Depth P7 P21 P7 P21 Nasal Length CIFASD 2010 Annual Review

Sample ALC-ALC ALC-PF PF-PF CHOW P7 29 35 P21 26 28 40

Statistical Analysis for P7 Table 2. Craniofacial morphometric analysis.  P-value of significant treatment main effects for each morphometric measure is indicated. The yellow shaded values under the first column denote that the overall treatment p-value < the corrected Bonferonni alpha=0.0038. Yellow- shaded areas in the rest of columns denote that the Tukey-Kramer pair- wise comparison was significant (p<0.05).  CIFASD 2010 Annual Review

Dysmophology Classification for P7 CIFASD 2010 Annual Review

Dysmophology Classification for P21 CIFASD 2010 Annual Review

Future Work Perform longitudinal comparison for P7 and P21 at 4.8% alcohol; Continue examination for 3.6% alcohol liquid diet; Correlate facial (video) facial and craniofacial dysmorphology. CIFASD 2010 Annual Review

Part II. 3D Bone Analysis The bone growth in differentiated anthropometry regions are evaluated. The development of ossification centers and sutures are being evaluated. The Shape analysis of regions and areas are planned. CIFASD 2010 Annual Review

Procrustes Analysis Definition: A means of comparing two arbitrary multidimensional configurations by removing the transnational and rotational differences between them such that they best fit. Procedures: Using anthropometric landmarks to match the overall sizes of the objects, and to identify and compare the regional/global differences (in shape, size,…). CIFASD 2010 Annual Review

Landmarks Landmarks Index Name 1 forward center tip of nasal bone fctn 2 nasion n 3l, 3r 3,4 inner canthal width icw 4l, 4r 5,6 minimal frontal width mfw 5l, 5r 7,8 bigonial width bgo 6l, 6r 9,10 bitragal width btr 7 11 forward tip of mandible ftm 8l, 8r 12,13 nasal length nl 9l, 9r 14,15 interior point of orbital socket in 10l, 10r 16,17 exterior point of orbital socket ex 11l, 11r 18,19 bony mandible man 12 20 bregma breg CIFASD 2010 Annual Review

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CIFASD 2010 Annual Review (1) (2) (3) (4) (11) (8) (9) (10) (5) (6) (7) CIFASD 2010 Annual Review

Bone Segmentation: P21 Examples Frontal Occipital Parietal Jaw CHOW ALC-ALC ALC-PF

Sample ALC-ALC ALC-PF CHOW P7 19 10 32 P21 20 36

Bone Volumes vs. Centroid Size The Centroid is defined as the geometric center of all landmarks; The Centroid_size is defined as the square root of the summed squared distances between the landmarks and the centroid. CIFASD 2010 Annual Review

General Linear Model (GLM) on P21

GLM on P21 without Covariates Tests of Between-Subjects Effects Source Dependent Variable df F Sig. Frontal 2 10.715 .000 Parietal 25.773 Occipital 19.507 Jaw 4.264 .018 Centroid_Size 16.464 Pairwise Comparisons (Sig.) ALC-ALC vs ALC-PF ALC-ALC vs CHOW ALC-PF vs CHOW Frontal .003 .000 .296 Parietal .889 Occipital .042 Jaw .110 .005 .281 Centroid_Size .712

GLM on P21 with Centroid_Size as Covariate Tests of Between-Subjects Effects Source Dependent Variable df F Sig. Corrected Model Frontal 3 47.157 .000 Parietal 52.713 Occipital 45.181 Jaw 21.676 Group 2 .705 .498 7.004 .002 5.413 .006 1.178 .314 Pairwise Comparisons (Sig.) ALC-ALC vs ALC-PF ALC-ALC vs CHOW ALC-PF vs CHOW Frontal .723 .553 .248 Parietal .001 .595 Occipital .472 .006 .016 Jaw .146 .512 .275

Summary ALC-ALC vs CHOW: easy to distinguish P21: 7 out of 9 measures can tell the difference P7: 5 out of 9 measures can tell the difference ALC-ALC vs ALC-PF: easy to distinguish P21: 5 out of 9 measures can tell the difference P7: 2 out of 9 measures can tell the difference ALC-PF vs CHOW: very hard to distinguish Only Occipital at P21 can tell the difference (p=0.42, p=0.16)

Summary Interesting measures Centroid_Size at both P7 and P21 stages can distinguish ALC_ALC from ALC_PF and CHOW, but not between ALC_PF and CHOW. Occipital at P21 can distinguish all three groups from one another

CIFASD 2010 Annual Review