Maternal Insult and Embryonic Neurodevelopmental Damage

1. Maternal Insult and Embryonic Neurodevelopmental Damage Fetal hypoxia Hava Golan, Mahmoud Huleihel, Ira Kashtuzki-Philip, Maya Gerstein, Revital Mane, Miriam Stilman, Tamar Levav, Vered Kaplan Dept of Developmental Genetics & Zlotowski center for Neuroscience Energy failure Transcriptional and translational regulation Cell death Regeneration Immediate response Delayed response

2. Study Design P 1 4h pretreatment Saline or Mg 2h hypoxia or air 9% O2 3% CO2 P 14 P 180 E17 E18 E20 P1 P7 P14 P21 P120-P180 • Animals: Pregnant, Jackson Black C-57 mice at gestation day 17 were assigned to one of four groups: • Saline injections (i.p) in room air exposure (S+A), • Saline injections (i.p) with hypoxia chamber exposure (S+H), • Mg injections (i.p) in room air exposure (Mg+A), • Mginjections (i.p) with hypoxia chamber exposure (Mg+H). Mg administration: 270 mg/kg followed by 27 mg/kg every 20 minutes for 4 hours. A second loading dose of 270 mg/kg was given at the end of the 4-hour period. Control mice were injected with saline following the same volume and schedule.

3. IS MAGNESIUM-SULFATE LOAD A PROPHLACTIC TREATMENT FOR PRENATAL HYPOXIA? S+H Mg+H Behavior Motor impaired partial protection Learning impaired short and long term Sort-term normal long-term not Brain morphology Cerebellum low PC density PC density normal thinner granular layer not prevented Cortex low cell density L 2-3,5-6 L5-6 protected Hippocampus no effect thinner CA1 layer GABAergic neurons Cortex PV and CB decreased L2-3, 5-6 L2-3 partial protected Hippocampus PV in CA3 decreased not prevented Inhibitory synaptogenesis Cortex not affected increased in all layers Hippocapus VGAT in CA3 & hilus decreased not prevented Glutamatergic synaptogenesis Cerebellum reduced NR2AB density in PC at P1 normal NR2AB density Cortex no effect no effect Hippocampusreduced NR2AB in all regions at P1 normal NR2AB density

4. Behavior - Exploration in open field First minute Fifth minute S+A S+H Mg+H

5. % of mice failed Motor Function Wire Clinging Footprint Pattern * Stride distance (mm) % of mice failed % of Mice Stopped Vertical Pole N=11-15 animals in each group Golan et al., 2004

6. Morphology - Cerebellum Cerebellum Clb 3 S+H Mg+H S+A * ** * Purkinje cells density (cell/100mm) Granular Layer Width (mm) Golan et al., 2004 N=3-4 animals in each group, 3-4 section per animal

7. SH - Control MgH - Control 634 57 9 291 12 48 250 196 66 P-value < 0.01 SH - Control MgH - Control SH - Control MgH - Control P-value < 0.05 P-value < 0.01 SH - Control MgH - Control 2h 1912 1108 594 P-value < 0.05 24h

8. Differential expression of genes Number of genes Time after hypoxia (hours) Number of genes folds change > 1.3, P<0.05

9. Spearman Rank Correlation With centered data, Pairwise centroid linkage Pattern 1 Pattern 2 Pattern 3

10. Pattern 1

11. Pattern 2

12. Pattern 3