Role of Recurrent Hypoxia-Ischemia in Preterm White Matter Injury Severity

<div><p>Objective</p><p>Although the spectrum of white matter injury (WMI) in preterm infants is shifting from cystic necrotic lesions to milder forms, the factors that contribute to this changing spectrum are unclear. We hypothesized that recurrent hypoxia-ischemia (rHI) will exacerbate the spectrum of WMI defined by markers of inflammation and molecules related to the extracellular matrix (hyaluronan (HA) and the PH20 hyaluronidase) that regulate maturation of the oligodendrocyte (OL) lineage after WMI.</p><p>Methods</p><p>We employed a preterm fetal sheep model of <i>in utero</i> moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. The response to rHI was compared against corresponding early or later single episodes of HI. An ordinal rating scale of WMI was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microglial activation. Late oligodendrocyte progenitors (preOLs) were quantified by stereology. Analysis of hyaluronan and the hyaluronidase PH20 defined the progressive response of the extracellular matrix to WMI.</p><p>Results</p><p>rHI resulted in a more severe spectrum of WMI with a greater burden of necrosis, but an expanded population of preOLs that displayed reduced susceptibility to cell death. WMI from single episodes of HI or rHI was accompanied by elevated HA levels and increased labeling for PH20. Expression of PH20 in fetal ovine WMI was confirmed by RT-PCR and RNA-sequencing.</p><p>Conclusions</p><p>rHI is associated with an increased risk for more severe WMI with necrosis, but reduced risk for preOL degeneration compared to single episodes of HI. Expansion of the preOL pool may be linked to elevated hyaluronan and PH20.</p></div

Spectrum of WMI in the four experimental conditions, as assessed by analysis of GFAP and Iba-1 staining using quantitation of area fractions stained for each marker.

<p>(A) Astrogliosis, measured by GFAP area fraction (AF), is increased following rHI (Kruskal-Wallis H = 13.96, p = 0.003, on 3df; mean ± SD Control: 0.20±0.06; Late HI: 0.25±0.05; Early HI: 0.33±0.05; rHI: 0.37±0.04; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **rHI vs. Control, p = 0.002; *Early HI vs. Control, p = 0.018; *rHI vs. Late HI, p = 0.021). (B) Iba-1 AF revealed similar patterns to Iba-1 pathology in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112800#pone-0112800-g003" target="_blank">Fig. 3</a> (Kruskal-Wallis H = 11.7, p = 0.008; mean ± SD Control: 0.06±0.05; Late HI: 0.08±0.06; Early HI: 0.18±0.13; rHI: 0.33±0.170; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **rHI vs. Control p = 0.01; *rHI vs. Late HI p = 0.020). (C) Iba-1 and GFAP AFs are significantly associated over a broad spectrum of WMI (***Spearman’s Rank Correlation: ρ = 0.91, p&lt;0.0001).</p

The pool of premyelinating OL lineage cells is significantly increased following rHI; PreOLs are less susceptible to acute degeneration following rHI than following late HI.

<p>(A–B) Representative confocal images of premyelinating OL lineage cells in the PVWM labeled with the O4 monoclonal antibody (arrowheads) in controls (A) and after rHI (B). Scale bars 20 µm. (C) Unbiased stereological counts of O4-labeled cells show an expansion of the preOL pool in the rHI group relative to control and early HI (Kruskal-Wallis: H = 7.7, p = 0.053, on 3 df; mean ± SD Control: 16,854±4,752 cells/mm³; Early HI: 16,430±7,182 cells/mm³; Late HI: 22,855±3,210 cells/mm³; rHI: 29,917±11,254 cells/mm³; CE range: 0.048–0.154). (D) Stereology further shows a decreased rate of preOL death following rHI relative to late HI (Kruskal-Wallis: H = 7.5, p = 0.057, on 3 df; mean ± SD Control: 0.9±0.4%; Early HI: 1.2±0.9%; Late HI: 5.0±2.4%; rHI: 1.7±1.8%; Bonferroni-corrected post-hoc Mann-Whitney U-tests: *Late HI vs. Control, p = 0.037; CE range for pyknotic counts: 0.21–0.95). (E) There is a greater density of activated caspase 3-labeled cells following a single late HI episode (Kruskal-Wallis H = 10.5, p = 0.015, on 3df; mean ± SD, Control: 1.62±0.76 Cells/mm²; Late HI: 6.30±5.40; Early HI: 0.92±0.21 cells/mm²; rHI: 1.73±1.33 cells/mm²; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **Early HI vs. Late HI, p = 0.001). (F) Representative confocal images of a degenerating O4-positive cell with a fragmented pyknotic nucleus in a late HI case. (F1) O4, (F2) Hoechst 33342 nuclear stain, (F3) Merge. Scale bar: 5 µm.</p

HA is present in fetal ovine white matter and displays a persistent increase for several weeks after HI.

<p>Representative confocal image of staining for HA with an HA binding protein (HABP) in frontal white matter (pseudocolors: green: HABP; red: Hoechst 33342-labeled nuclei). (A) Control: One-week post-insult. (B) Early HI. (C) Late HI. (D) rHI. (E) Control: 24-hours post-insult. (F) HI: 24-hours post-insult. (G) Control: Two weeks post-insult. (H) HI: Two weeks post-insult. (I) Control: Four weeks post-insult. (J) HI: Four weeks post-insult. Scale bars 20 µm.</p

Spectrum of WMI in the four experimental conditions, as assessed by analysis of H &amp; E staining using ordinal rating scores.

<p>(A) Neuropathological scoring of H &amp; E staining in frontal white matter (Kruskal-Wallis H = 8.57, p = 0.036 on 3df; mean ± SD Control: 0.20±0.45; Late HI: 0.28±0.44; Early HI: 0.80±0.84; rHI: 2.0±1.3). (B) Neuropathological scoring of H &amp; E stained parietal white matter (Kruskal-Wallis H = 10.5, p = 0.015; mean ± SD Control: 0.60±0.89; Late HI: 1.00±0.00; Early HI: 1.4±0.89; rHI: 2.3±0.82; Bonferroni-corrected post-hoc Mann-Whitney U-test: *rHI vs. Late HI, p = 0.036). (C–F) Representative images of H &amp; E staining from (C) Control, (D) Early HI, (E) Late HI, (F) rHI frontal white matter. Panel scale bars: 200 µm; inset scale bars: 20 µm.</p

Schematic timeline of the ten day protocol for rHI studies showing the assignment of animals from twin pairs to the four experimental conditions.

<p>Animals recovered from surgery for 3 days before the initial exposure to hypoxemia with or without concurrent ischemia (black arrowheads indicate the timing of ischemia). The first gray shaded bar indicates that all groups sustained a 30-minute period of maternal hypoxemia at this time. Note that the twin control for the early HI group and the late HI twin of the rHI group did not sustain ischemia at this time. Thereafter, the early HI group and their twin controls survived for 7 days (i.e., 10 days after surgery). The rHI cases were exposed to a second episode of HI six days after the first HI episode. The corresponding twins for the rHI group were the late HI animals. The rHI group and the late HI cases were both exposed to an insult that involved maternal hypoxemia (gray shaded bar) and ischemia (arrowheads) at 24-hours before brain collection (i.e., 7 days after the initial insult and 10 days after surgery). Hence, the animals in all four groups survived for 10 days after surgery.</p

More severe WMI is associated with more severe gray matter injury.

<p>(A–D) Photomicrographs from four animals that sustained rHI that illustrate Iba1 staining used to define progressively more severe frontal cortical injury scores of 0 (A), 1 (B), 2 (C) and 3 (D), as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112800#s2" target="_blank">Materials and Methods</a>. Note the variable cortical injury in the animals in B, C and D all of which sustained moderate-to-severe WMI. Note also the focal collections of activated microglia in B (arrowheads); the more diffuse distribution of activated microglia in C and the infiltrates of macrophages in a focal cystic necrotic cortical lesion in D. Scale bars A–D: 250 µm. (E, F) Neuropathological scoring of Iba-1 staining in frontal cortex (E; Kruskal-Wallis: H = 5.38, p = 0.146 on 3df; Mean ± SD Control: 0.00±0.00; Early HI: 0.80±1.10; Late HI: 0.67±0.82; rHI: 1.50±1.38) and thalamus (F; Kruskal-Wallis H = 7.32, p = 0.062 on 3df; Mean ± SD Control: 0.00±0.00; Early HI: 1.40±1.34; Late HI: 1.33±1.21; rHI: 2.00±1.26, Bonferroni-corrected post-hoc Mann-Whitney U-test: rHI vs. Control: p = 0.059). (G–I) Diffuse WMI was accompanied by a paucity of axonal degeneration as defined by staining for β-amyloid precursor protein (β-APP). (G) No degenerating axons were observed in control animals. (H) Representative image of the low levels of axonal degeneration observed in more severe diffuse WMI in an animal from the rHI group. This image from a rHI lesion shows rare degenerating axons (arrowheads) and within the inset box. (I) Detail of the inset shows the elevated staining for β-APP in several dystrophic-appearing axons (arrowheads) in this apparent small focus of necrosis. Scale bars (G–H) 100 µm; (I) 25 µm.</p

Spectrum of WMI in the four experimental conditions, as assessed by analysis of Iba-1 staining using ordinal rating scores.

<p>(A) Neuropathological scoring of Iba-1 staining in frontal white matter (Kruskal-Wallis H = 10.41, p = 0.015 on 3df; mean ± SD Control: 0.80±0.45; Late HI: 0.83±0.41; Early HI: 1.20±1.10; rHI: 2.50±0.84; Bonferroni-corrected post-hoc Mann-Whitney U-tests: *rHI vs. Control, p = 0.016; **rHI vs. Late HI, p = 0.01). (B) Neuropathological scoring of Iba-1 staining in parietal white matter (Kruskal-Wallis: H = 10.02, p = 0.018, on 3df, mean ± SD Control: 1.4±0.55; Late HI: 1.0±0.00; Early HI: 1.8±0.84; rHI: 2.5±0.84; Bonferroni-corrected post-hoc Mann-Whitney U-tests: *rHI vs. Late HI, p = 0.033). (C–F) Representative images of Iba-1 staining from (C) Control, (D) Early HI, (E) Late HI, (F) rHI frontal white matter. Panel scale bars: 200 µm, inset scale bars: 20 µm.</p

The total pool of OL lineage cells, defined by staining for Olig2, is increased in response to early HI.

<p>(A) Cell counts of Olig2 in the frontal PVWM for the four experimental conditions (Kruskal-Wallis H = 8.4, p = 0.038, on 3df; mean ± SD Control: 117.2±18.9 cells/mm²; Late HI: 105.1±23.0 cells/mm²; Early HI: 154.6±15.5 cells/mm²; rHI: 151.5±48.5 cells/mm²; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **Early HI vs. Late HI: p = 0.008). (B) GFAP area fraction and Olig2 density are significantly associated over a broad spectrum of WMI (**Spearman’s Rank Correlation ρ = 0.63, p = 0.005). (C–F) Representative Olig2 images. (C) Control, (D) Early HI, (E) Late HI, (F) rHI. Panel scale bars: 100 µm. Inset scale bars: 10 µm.</p