Timing of appearance of late oligodendrocyte progenitors coincides with enhanced susceptibility of preterm rabbit cerebral white matter to hypoxia-ischemia

Abstract

Emerging evidence supports that premature infants are susceptible to both cerebral white and gray matter injury. In a fetal rabbit model of placental insufficiency, preterm rabbits at embryonic day 22 (E22) exhibited histologic evidence of gray matter injury but minimal white matter injury after global hypoxia-ischemia (H-I). We hypothesized that the dissociation between susceptibility to gray and white matter injury at E22 was related to the timing of appearance of late oligodendrocyte progenitors (preOLs) that are particularly vulnerable in preterm human white matter lesions. During normal rabbit oligodendrocyte (OL) lineage progression, early OL progenitors predominated at E22. PreOL density increased between E24 and E25 in major forebrain white matter tracts. After H-I at E22 and E25, we observed a similar magnitude of cerebral H-I, assessed by cortical microvascular blood flow, and gray matter injury, assessed by caspase activation. However, the increased preOL density at E25 was accompanied by a significant increase in acute white matter injury after H-I that coincided with enhanced preOL degeneration. At E29, significant white matter atrophy developed after H-I at E25 but not E22. Thus, the timing of appearance of preOLs coincided with onset of a developmental window of enhanced white but not gray matter susceptibility to H-I.

Figure 1

Cell degeneration in cerebral white matter of the embryonic day 22 (E22) rabbit was uniformly very low as determined by TUNEL staining (A, C, E). Regional boundaries were defined by fluorescent nuclear counterstain with Hoechst 33324 (B, D, F). Degenerating cells were enriched in the cortical plate (CxP), Putamen (Pu), Piriform cortex (Pi), and the caudate neuroepithelium in (E). TUNEL-positive cells were rarely seen in cerebral white matter tracts such as the intermediate zone (subcortical white matter; ICx), internal capsule (IC), or external capsule (EC). LV, lateral ventricle; M, marginal zone (cortical layer 1). Scale bars: 500 μm.

Figure 2

The overall density of degenerating cells, derived from the average from four major forebrain white matter tracts (corpus callosum, corona radiata, internal capsule, and external capsule), was very low at 24 h after hypoxia-ischemia (H-I) at E22, as determined by three complementary methods to assess cell death: O4-staining, Hoechst 33324-staining and TUNEL staining. ^*P<0.001, independent samples t-tests. See ‘Materials and methods' for details.

Figure 3

Numerous early oligodendrocyte (OL) progenitors are restricted to the basal ganglionic eminence at E22. (A) Olig2-positive OL progenitors (red, arrowheads) are distinct from O4-positive preOLs (green, arrows). (B) NG2-positive OL progenitors (green, arrowheads) are distinct from isolectin-B4-positive microglia (red, arrows). (C) Olig2-positive OL progenitors were highly enriched in the putamen (Pu). Arrowheads delineate the region of the external capsule (EC) shown in (D). Ctx, cerebral cortex; IC, internal capsule. (E, F) OL progenitors were intercalated among the axon bundles of the EC and often had a pseudo-unipolar morphology (arrows in F). Scale bars: A, 50 μm; B, 100 μm; C, 500 μm; D, 200 μm; E, F, 100 μm.

Figure 4

OL lineage progression in four major rabbit cerebral white matter tracts between E22 and E32 at the levels of the mid-septal nuclei and the rostral hippocampal formation. PreOL density (cells/mm²) was coordinately upregulated at E24 to E25; ^*P<0.001, independent samples t-tests. Data presented as mean±s.e.m. Arrowheads indicate the time of birth at E32.

Figure 5

The preOL-enriched cerebral white matter is highly susceptible to injury 24 h after H-I at E25. (A) Numerous O4 antibody-labeled preOLs are seen in periventricular white matter. Arrows in (A) delineate a region shown in (B) with extensive preOL degeneration in the corpus callosum and more lateral corona radiata. Note the clusters of intact-appearing preOLs at the edges of lesion. LV, lateral ventricle. (C) A typical lesion showing numerous pyknotic preOLs at various stages of degeneration (arrows). Intact-appearing preOLs are seen at lower right (arrowheads). (D) Density of degenerating cells in the corpus callosum and corona radiata was significantly increased in the H-I animals relative to control animals as determined by O4 staining, Hoechst 33324 staining, and TUNEL staining; ^*P<0.001, independent samples t-tests. Scale bars: A, B, 500 μm; C, 200 μm.

Figure 6

Cortical neuronal degeneration, as assessed by staining for activated caspase-3 (AC-3), was similar at E23 (A) and E26 (B) at 24 h after the onset of H-I. (A, B) Low power montages of the distribution of AC-3 stained cell bodies in the cerebral cortex at the level of the septal nuclei that have been pseudo-colored in blue (parasagittal cortex) and red (ventrolateral/piriform cortex). The insets in (A) and (B) show details of the AC-3 staining pattern in deeper cortical layers. The bar and whisker plots show a similar density of AC-3-labeled cells in the parasagittal (A) and piriform (B) lesions at E23 versus E26. (C, D) Neurons in cortical layers 4 and 6 were particularly susceptible to AC-3-mediated degeneration. In (C), the typical distribution of AC-3 staining in cortical layers 1 to 6 and the subplate (SP) is shown from an animal at E26. The merged image in (D) shows triple immunohistochemical staining for AC-3 (red) and the neuronal-specific markers NeuN (green) and Ctip2 (blue). Note that the pattern of AC-3 staining in layer 4 overlapped with Ctip2, which was particularly enriched in this layer.

Figure 7

(A) White matter injury at E29 after H-I at E22 or E25 was determined for the three major forebrain white matter tracts (corpus callosum (CC), corona radiata (CR), and internal capsule (IC)); ^*P<0.05, ANOVA, Tukey post hoc comparison. (B, C) Total number of preOLs (B) and immature OLs (C) in each of the three white matter tracts at E29. Statistical analysis was performed for the E22 and E25 H-I groups relative to E29 control animals (^*P<0.05, ANOVA, Tukey post hoc comparison for weighted means). Weighted means for each white matter tract were calculated by multiplication of raw total cell counts with a correction factor obtained from all groups.