After Intracerebral Hemorrhage, Oligodendrocyte Precursors Proliferate and Differentiate Inside White-Matter Tracts in the Rat Striatum

As expected, there was no hemorrhage in control, saline-injected rats, while a hemorrhage developed when collagenase was injected into the striatum (Fig. 1). At 1 day, collagenase injection produced a large hematoma that was confined to the striatum, and in both coronal (Fig. 1a) and sagittal (Fig. 1b) sections, cell damage was apparent, as shown by reduced cresyl violet staining of Nissl bodies. While a hematoma was evident throughout the 28-day period, the area of diminished cresyl violet staining decreased with time and the blood clot was gradually resorbed. In addition, the lateral ventricles expanded (ventriculomegaly), which is a common outcome after ICH and is due to impaired drainage of cerebrospinal fluid (CSF) [21] or tissue loss [22]. Others, using the same ICH model, have observed similar changes to the size of the hematoma and ventricles for 28 days and longer [22, 23]. The darker staining at 7 and 14 days is due to chromogenic iron that is released from ruptured erythrocytes [24, 25]. In order to quantify changes in the spatial distribution of oligodendrocyte-lineage cells, we used coronal sections and examined three regions: the peri-hematoma, surrounding striatum, and contralateral striatum. Approximate sample locations are illustrated by the boxes in Fig. 1. Then, sagittal sections were used to quantify cells inside versus outside white-matter bundles in the peri-hematoma region. The sample boxes were slightly larger to visualize more bundles and a larger proportion of each bundle.

To examine the progression of damage to axons and myelin in a qualitative manner, we used coronal (Fig. 2a) and sagittal (Fig. 2b) sections. Myelinated axon tracts in the striatum arise from excitatory cortical neurons [26]. They were visualized using an antibody against MBP, and axons were labeled with a pan-NF antibody that recognizes the three major neurofilament subunits: light, medium, and heavy chain. The coronal sections show approximately circular myelinated axon tracts extending in the rostral-caudal axis (Fig. 2a). In the saline-injected (control) striatum, bundles are filled with densely packed myelinated axons, as indicated by the abundant overlap of the MBP and pan-NF stains (Fig. 2a). At high magnification, the two stains do not precisely co-localize (also see [27]) because myelin wraps around each neurofilament-containing axon. In sagittal sections from saline-injected animals, there were dense longitudinal tracts of myelinated axons throughout the striatum (Fig. 2b). The sagittal orientation was especially useful for assessing damage to white-matter bundles close to the hematoma.

We previously showed a progressive, dramatic decrease in staining for normal MBP and a concomitant increase in damaged MBP in the peri-hematoma region from 1 to 7 days after ICH [16‐18]. In coronal (Fig. 2a) and sagittal sections (Fig. 2b), despite the normal variation in diameter of striatal white-matter tracts, a similar increase in myelin damage is clearly evident over the first week. There was progressive bundle swelling and fragmentation. Interestingly, at 7 days, myelin and axon staining were not well organized into dense bundles, and the high-magnification coronal image shows diffuse neurofilament staining inside fragmented bundles. Beyond the first week, the bundles of myelinated axons appeared denser, less swollen, and less fragmented (i.e., day 14 was similar to day 3). The high-magnification coronal image at 28 days clearly shows that the axons were more aligned into dense myelinated tracts. From the sagittal images, it appears that some bundles were thinner at 14 and 28 days than in the saline control. The improved appearance of the myelinated axon tracts after the first week, and presence of both axons and myelin in the peri-hematoma region throughout the first month after ICH, raises the prospect for re-myelination. Thus, we next determined if oligodendrocyte-lineage cells survived and differentiated into mature cells that are capable of myelin production.

We quantified temporal changes in the density of oligodendrocyte-lineage cells in the peri-hematoma region, surrounding striatum, and undamaged contralateral striatum from 1 to 28 days. All oligodendrocyte-lineage cells, regardless of maturation state, express the transcription factor, Olig2 [28], and, as expected, we found that Olig2 co-localized with the nuclear marker, DAPI (Fig. 3a). It was previously shown that saline injection does not cause damage [15, 25, 29, 30]. As expected, saline-injected controls showed the same density of oligodendrocyte-lineage cells at 1, 3, and 7 days; hence, ICH samples were compared with the 1-day saline control. After ICH, the density of Olig2⁺ cells increased from 1 to 7 days in the peri-hematoma region, at which time they were ∼11-fold higher than controls (Fig. 3b) and formed a dense band around the hematoma (Fig. 3a; inset to right). Their numbers were markedly decreased at 14 and 28 days. In the surrounding striatum further from the hematoma, and in the contralateral striatum, the density of Olig2⁺ cells was elevated at 1, 3, 7, and 14 days and then decreased to the control level at 28 days.

We examined sagittal sections from the peri-hematoma region to ask whether the oligodendrocyte-lineage cells increased inside myelinated white-matter tracts, and in the neuron-rich neuropil, which contains many cell bodies of medium spiny neurons that are damaged after ICH [16‐18, 31]. The bundles of myelinated axons were swollen and MBP was degraded at 7 days, as previously shown. As shown in Fig. 2, many myelinated bundles had re-appeared by 14 and 28 days (Fig. 4a). Although Olig2⁺ cells were present both inside and outside the bundles, they preferentially accumulated inside the white-matter tracts (Fig. 4b). Again, their density peaked at 7 days and then decreased. Their density also apparently increased outside the bundles (∼2-fold higher at 7 days) but this did not reach statistical significance.

In principle, the increased density of Olig2⁺ cells observed in the peri-hematoma region could be due to proliferation, migration, or both. To address whether these cells were proliferating within the damaged striatum, we labeled Ki67, a nuclear protein present in all active phases of the cell cycle but not in the G0 phase [32]. Because Olig2 and DAPI co-localize in the nucleus (Fig. 3), we could readily identify proliferating oligodendrocyte-lineage cells as those double-labeled for Olig2 and Ki67. In general, many proliferating cells (Ki67⁺) were present in the peri-hematoma region at 3 and 7 days (Fig. 5a). The higher-magnification images (Fig. 5b) show examples of proliferating (Ki67⁺) Olig2⁺ cells, some oligodendrocyte-lineage cells not in active cell-cycle phases (Ki67⁻Olig2⁺), and some proliferating cells that were not of oligodendrocyte lineage (Ki67⁺Olig2⁻). Proliferating Olig2⁺ cells were then quantified in two ways: as the density of cells double-labeled for Ki67 and as the percentage of Olig2⁺ cells that had re-entered the cell cycle. In the peri-hematoma region, their density increased ∼16-fold between 1 and 3 days and then progressively declined by 14 days to the level seen at 1 day (Fig. 5c). Of note, increased proliferation of Olig2⁺ cells preceded their maximal increase in density at 7 days (Fig. 3). At 3 days, 55 ± 5 % of the Olig2⁺ cells expressed Ki67, while only 9 ± 1 % did so at 7 days (Fig. 5d). In the surrounding striatum, their proliferation showed a similar time course but much lower density, and the proportion of proliferating Olig2⁺ cells was only 18 ± 2 % at 3 days. No proliferating Olig2⁺ cells were detected in the striatum of saline-injected control rats or the contralateral striatum of ICH animals (not shown). Next, we addressed whether proliferating Olig2⁺ cells in the peri-hematoma were likely to have simply migrated from the SVZ, a site of oligodendrogenesis throughout life [33]. While proliferating cells were present in the SVZ at each time point, almost none of them were Olig2⁺ (Fig. 5a, b). Of those few that were double-labeled, their numbers did not change between 1 and 28 days (not shown); thus, proliferation of Olig2⁺ cells at the damage site is apparently important for their increased density.

As shown above, the density of oligodendrocyte-lineage cells increased dramatically and peaked at 7 days, at which time many were inside the damaged white-matter bundles (Figs. 3 and 4). However, in order to generate and repair myelin, they must undergo maturation [34]; therefore, we next quantified their maturation states. Immature oligodendrocyte precursor cells (OPCs) were identified as cells with Olig2⁺ nuclei that also stained for NG2 chondroitin sulfate proteoglycan [35]. It was necessary to double-label the cells (see high-magnification image in Fig. 6a) because activated microglia and macrophages can also express NG2 [36]. In the undamaged contralateral striatum, there was no change in the density of OPCs (NG2⁺Olig2⁺) compared with saline controls (Fig. 6a, b). In contrast, in the peri-hematoma region, their density increased dramatically at 3 and 7 days (∼8-fold at 7 days) and then decreased, reaching control levels by 28 days. Sagittal sections showed a similar time-dependent increase in OPCs, preferentially inside the white-matter bundles (Fig. 6c, d). In the surrounding striatum, there was a much smaller increase, with statistical significance only at 3 days (Fig. 6b).

To quantify the density of mature oligodendrocytes, cells were double-labeled for Olig2 and CC-1. CC-1 labels adenomatous polyposis coli (APC), a tumor suppressor protein that is present in the cell body of mature oligodendrocytes and thought to regulate their adhesive properties [37]. Double-labeling for CC-1 and Olig2 is illustrated in the high-magnification inset in Fig. 7a. In the peri-hematoma region, the density of mature oligodendrocytes increased with time, reached a peak at 7 days that was ∼7-fold higher than that of saline controls (Fig. 7a, b), and then declined. This time-dependent profile was similar to that of total oligodendrocyte-lineage cells (Fig. 3b) and OPCs (Fig. 6b), but the density of mature cells was higher than that of OPCs. In the surrounding striatum, mature oligodendrocytes increased at 7 and 14 days and then decreased to control levels at 28 days. In the undamaged contralateral striatum, the density of mature oligodendrocytes was increased by 2–3-fold as early as 1 day and remained elevated for at least 14 days (Fig. 7a, b). Because there was no increase in immature OPCs (Fig. 6b) or proliferating Olig2⁺ cells, this suggests that the cells migrated into the striatum from other regions soon after ICH. Finally, mature oligodendrocytes increased preferentially inside the white-matter bundles (Fig. 7c, d), as was shown for total oligodendrocyte-lineage cells (Fig. 4) and OPCs (Fig. 6). However, the greatest increases in mature cells were at 7 and 14 days (Fig. 7) compared with 3 and 7 days for OPCs (Fig. 6). Together with the proliferation data (Fig. 5), these observations provide evidence for local replenishment of both immature OPCs and mature oligodendrocytes in the peri-hematoma region, and preferentially inside white-matter tracts.