Abbreviated Exposure to Hypoxia Is Sufficient to Induce CNS Dysmyelination, Modulate Spinal Motor Neuron Composition, and Impair Motor Development in Neonatal Mice

<div><p>Neonatal white matter injury (nWMI) is an increasingly common cause of cerebral palsy that results predominantly from hypoxic injury to progenitor cells including those of the oligodendrocyte lineage. Existing mouse models of nWMI utilize prolonged periods of hypoxia during the neonatal period, require complex cross-fostering and exhibit poor growth and high mortality rates. Abnormal CNS myelin composition serves as the major explanation for persistent neuro-motor deficits. Here we developed a simplified model of nWMI with low mortality rates and improved growth without cross-fostering. Neonatal mice are exposed to low oxygen from postnatal day (P) 3 to P7, which roughly corresponds to the period of human brain development between gestational weeks 32 and 36. CNS hypomyelination is detectable for 2–3 weeks post injury and strongly correlates with levels of body and brain weight loss. Immediately following hypoxia treatment, cell death was evident in multiple brain regions, most notably in superficial and deep cortical layers as well as the subventricular zone progenitor compartment. PDGFαR, Nkx2.2, and Olig2 positive oligodendrocyte progenitor cell were significantly reduced until postnatal day 27. In addition to CNS dysmyelination we identified a novel pathological marker for adult hypoxic animals that strongly correlates with life-long neuro-motor deficits. Mice reared under hypoxia reveal an abnormal spinal neuron composition with increased small and medium diameter axons and decreased large diameter axons in thoracic lateral and anterior funiculi. Differences were particularly pronounced in white matter motor tracts left and right of the anterior median fissure. Our findings suggest that 4 days of exposure to hypoxia are sufficient to induce experimental nWMI in CD1 mice, thus providing a model to test new therapeutics. Pathological hallmarks of this model include early cell death, decreased OPCs and hypomyelination in early postnatal life, followed by dysmyelination, abnormal spinal neuron composition, and neuro-motor deficits in adulthood.</p></div

Long hypoxia vs abbreviated hypoxia.

<p>Long hypoxia vs abbreviated hypoxia.</p

Abbreviated hypoxia is sufficient to induce persistent motor-deficits in mice.

<p>Mice reared under hypoxia (P3 <b>→</b> P7) or room air were tested at weaning age (P21), early adulthood and adulthood (P43, P80) for motor coordination and strength (D-F), strength (A-C), balance and endurance (G-I), front limb grip strength (L), body composition (E, F), global nocturnal activity in groups of three mice per box (J, K) and global nocturnal activity of single mice per box (I, J). A-C: hanging wire-single test at P21 (A), P43 (B), P80 (C); D-F: hanging wire-mesh test at P21 (D), P43 (E), P80 (F); G-I: Rotarod test at P21 (G), P43 (H), P80 (I). J, K: Global nocturnal activity of grouped mice (3 per activity box) showing horizontal hourly beambreaks (J) and vertical hourly beambreaks (K) for 5 nights from P91-P96. L: Grip strength meter test at P90. N per test and time-point >34 animals for hanging wire tests, Rotarod and grouped nocturnal activity; N = 14 per group for grip strength meter test with *** equals p < 0.001; ** equals p < 0.01; * equals p < 0.05.</p

Behavioral motor testing—Male mice.

<p>Behavioral motor testing—Male mice.</p

Comparison of growth and survival in models of neonatal hypoxic injury.

<p>A: Experimental timeline showing rearing strategies of neonatal CD1 mice under abbreviated hypoxia (red) (P3 → P7) followed by normoxia (gray), or long-duration hypoxia (green) (P3 <b>→</b> P12). B: Body weight comparison under abbreviated or long hypoxia (P3 <b>→</b> P7 or P3 <b>→</b> P12) (normoxia: n = 102; 10d hypoxia: n = 20; 4d hypoxia: n = 102). C: Survival rate of neonatal CD1 mice at P12 under abbreviated (red bar, n = 102) or long hypoxia (green bar), n = 20. D, E: Body (D) and (E) brain weight development of neonatal mice after hypoxia (P3 <b>→</b> P7) or normoxia (P3, n = 73 normoxic, 78 hypoxic; P7, n = 108 normoxic, 119 hypoxic; P13, n = 84 normoxic, 96 hypoxic; P27, n = 25 normoxic, 34 hypoxic; P43, n = 25 normoxic, 34 hypoxic; P80, n = 24 normoxic, 23 hypoxic mice). Litter sizes in A-E were 12 neonatal mice per dam. F-I: Body weight (F-H) and body weight ratios (I) in cross-fostered (F, G) and non-cross-fostered (H) normoxic and hypoxic (P3 <b>→</b> P7) CD1 and C57/Bl6 mice. Litter sizes in F were 6 neonatal mice per dam (non-cross-fostered CD1 mice: P7, n = 23 normoxic, 18 hypoxic; P13, n = 18 normoxic, 12 hypoxic mice; cross-fostered CD1 mice by C57/bl6 dam: n = 12 normoxic and 12 hypoxic mice for P7, P13 and P27; cross-fostered C57/Bl6 mice by CD1 dam: n = 12 normoxic and 12 hypoxic mice for P7, P13 and P27). Data are shown as mean ± std.-dev. *** p < 0.001; ** p < 0.01; * p < 0.05.</p

Fold-change in gene transcripts at P13, as detected by RT-PCR.

<p>Fold-change in gene transcripts at P13, as detected by RT-PCR.</p

Abbreviated hypoxia (P3 → P7) does not increase levels of OPCs but causes substantial apoptosis throughout cortical layers, hippocampus and SVZ.

<p>A: Immunohistochemistry and stereologic analysis of mouse cerebra at P13 using OL markers Olig-2 (OPCs, immature OLs, mature OLs), MBP (mature OLs) and NKX2.2 (OPCs). B: Immunohistochemical staining of level matched hypoxic and control cerebra at P7 showing anti-CC3 (red) and nuclear marker DAPI (green arrows indicate specific regions SVZ, DG and CA1/3 field; yellow arrows mark levels of high apoptotic intensity in hypoxic mice). C: Representative Western blots using total brain homogenates from hypoxic and control CD1 mice at P7 using apoptosis marker CC3 and β-actin as a loading control. Densitometric analysis of Western blots from 3 independent experiments showing brain levels of CC3 at P7 in hypoxic and control mice with *** equals p < 0.001; ** equals p < 0.01; * equals p < 0.05.. SVZ, subventricular zone; DG, dentate gyrus; CA1-3, hippocampal CA fields; Ctx, cortex, I-VI = cortical layers 1–6. (n = 6 hypoxic + 6 normoxic animals for immunohistochemistry and Western blotting (each)).</p

Abbreviated hypoxia changes axonal composition in spinal cords and causes dysmyelination of spinal axons.

<p>A, B: 6 month old hypoxic mice showed strong motor deficits in hanging wire tests (A) without having different body weights (B) (n = 4 normoxic + 6 hypoxic animals). C: Spinal cord thick sections from anterior and lateral funiculi (thoracic regions) in hypoxic and normoxic mice 6 month after the hypoxic insult (4 control + 4 hypoxic mice). D: Automated axon counts of spinal cord thick sections from C. showing abnormal axonal compositions in spinal anterior funiculi with increased small and medium diameter axons and decreased numbers of large diameter axons relative to normoxic controls (small = 1–4 μm; medium = 4–10 μm; large = > 10 μm) (4 control + 4 hypoxic mice). E: Electron microscopy of spinal cord sections left and right of the anterior median fissure (thoracic spinal cord) in hypoxic and control animals (magnification: 8kx). Higher g-ratios (thinner myelin sheaths) and loosely wrapped myelin around axons was prominent by Electron microscopy in thoracic spinal motor neurons (anterior funiculi) (4 control + 4 hypoxic mice, 100 axons per animal). F. Chi-Square analysis of the g-ratio/axon diameter relationship in hypoxic and normoxic animals. G: Scatter plots of g-ratio vs. axon diameter in control (black) and hypoxic animals (red) with best fitting curves, with *** equals p < 0.001; ** equals p < 0.01; * equals p < 0.05.</p