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. 2015 Mar;63(3):452-65.
doi: 10.1002/glia.22764. Epub 2014 Nov 6.

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival

Michael Porambo  1 , Andre W Phillips, Joel Marx, Kylie Ternes, Edwin Arauz, Mikhail Pletnikov, Mary Ann Wilson, Jeffery D Rothstein, Michael V Johnston, Ali Fatemi
Affiliations

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival

Michael Porambo et al. Glia. 2015 Mar.

Abstract

Objective: Neonatal white matter injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI.

Methods: Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-Day 5 (P5). At P22, intracallosal injections of either enhanced green fluorescent protein (eGFP) + GRPs or saline were performed in control and ligated mice. Neurobehavioral and postmortem studies were performed at 4 and 8 weeks post-transplantation.

Results: GRP survival was comparable at 1 month but significantly lower at 2 months post-transplantation in NWMI mice compared with unligated controls. Surviving cells showed better migration capability in controls; however, the differentiation capacity of transplanted cells was similar in control and NWMI. Saline-treated NWMI mice showed significantly altered response in startle amplitude and prepulse inhibition (PPI) paradigms compared with unligated controls, while these behavioral tests were completely normal in GRP-transplanted animals. Similarly, there was significant increase in hemispheric myelin basic protein density, along with significant decrease in pathologic axonal staining in cell-treated NWMI mice compared with saline-treated NWMI animals.

Interpretation: The reduced long-term survival and migration of transplanted GRPs in an ischemia-induced NWMI model suggests that neonatal ischemia leads to long-lasting detrimental effects on oligodendroglia even months after the initial insult. Despite limited GRP-survival, behavioral, and neuropathological outcomes were improved after GRP-transplantation. Our results suggest that exogenous GRPs improve myelination through trophic effects in addition to differentiation into mature oligodendrocytes.

Keywords: cell therapy; cerebral palsy; ischemia; myelination.

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Conflict of interest statement

Conflicts of Interest: None

Figures

Figure 1
Figure 1
In vitro assessment of Glial Restricted Precursors (GRPs): Cells were derived at E13.5 and maintained in GRP culture. Cells were confirmed to be GRPs by their expression of A2B5 (A). As previously reported about 40% of the cells expressed Nestin (B). A week after culturing these immunopanned cells, almost all cells were PDGFR-a positive (C).
Figure 2
Figure 2. Regions of interest for densitometric analysis
In anterior and medial coronal sections (A), the lateral corpus callosum (red), the periventricular corpus callosum (purple), and the mid corpus callosum (yellow) were assessed. In posterior sections (B), in addition to the lateral (red) and medial (yellow) corpus callosum, the fimbriae of the hippocampus (blue) and the internal capsules (green) were also analyzed. Anterior section was 600 microns anterior to injection site (3.9 mm anterior to lambda), and posterior section is was 400 microns posterior to injection site (2.9 mm anterior to lambda).
Figure 3
Figure 3. Patterns of Migration of transplanted GRPs
While in some animals transplanted cells remained mostly limited within the injection track (A), robust migration medially and laterally was observed frequently (B, C). Cell Migration to contralateral hemisphere was observed in controls at 2 months but not in NWMI mice. White oblique arrows point to injection site, vertical gray arrow points at the midsagittal line. Scale bar 100 µm.
Figure 4
Figure 4. GRP cell survival at 1 month and 2 months post-transplantation
At 1 month, both groups showed comparable number of surviving cells (A, B, C). However, at 2 months post-transplantation control mice (D) had significantly more surviving GFP+ cells when compared to NWMI mice (E, F, t(28)=2.33, p = 0.02). Plots (C, F) show total number of cells counted in three sections, the one where the injection tract was most prominent, and the immediate anterior and posterior sections. Error Bar indicates 1SE. Scale bar 100 µm.
Figure 5
Figure 5. Differentiation capacity of transplanted GRPs
A large fraction of surviving GFP+ cells co-expressed CC1, a marker for mature oligodendrocytes, in two control animals (A, B) as well as in two NWMI (C, D) mice. There was no significant difference in the percentage of CC1+/GFP+ cells between controls and NWMI mice (E), unpaired t-test t(16) = 0.32. Little to no GFAP costaining was observed except in a handful cells (arrow, F) suggesting that only a very small fraction of these cells differentiated into astrocytes. Scale bar 20 µm. Error Bar indicates 1SE.
Figure 6
Figure 6. Many transplanted cells Remain as oligodendrocyte progenitor cells (OPCs)
PDGFR-a staining, as an OPC marker, conducted at 2 months post-GRP transplantation showed that a large fraction of transplanted cells remain as progenitor cells. A qualitative assessment of stained sections suggests, that in areas closer to injection tract (A), the fraction of GFP+ cells that were at OPC stage was higher compared to other areas (B).
Figure 7
Figure 7. Effect of GRP transplantation on axonal injury
At 4 weeks post-transplantation, there was a significant difference in degree of pathological axonal staining between the different animal groups; ANOVA for ligated hemisphere F (2, 21) = 9.093, p < 0.01; for contralateral hemisphere F (2, 21) = 3.939, p < 0.05. Saline treated NWMI mice had significantly higher degree of pathological SMI32 staining in both hemispheres as shown in corpus callosum (A–C) and internal capsule (D–F). Treatment with GRPs prevented the increase in SMI32 staining (C,F). Furthermore, we stratified the GRP treated NWMI mice based on survival of transplanted cells (NWMI/GRP+: animals that showed GFP positive cells; NWMI/GRP-: animals without any GFP positive cells). To our great surprise, cell survival did not correlate with outcome (G–H), and whether transplanted cells had survived (NWMI/GRP+) or died (NWMI/GRP-), a comparable ameliorative effects was observed at 4 weeks in GRP treated animals. Scale bar 50 µm. Tukey’s multiple comparison tests p values are shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.
Figure 8
Figure 8. Effect of GRP transplantation on Myelination
At 8 weeks post-transplantation, there were significant differences in hemispheric MBP density values between the different groups; ANOVA for ligated hemisphere F (2, 25) = 4.922, p < 0.05; ANOVA for contralateral hemisphere F (2, 25) = 4.356, p < 0.05. Saline-treated NWMI mice had significantly lower hemispheric MBP staining as shown in the mid corpus callosum (A–C) and the internal capsule (D–F) when compared to controls, while GRP treated NWMI mice showed comparable MBP densities as in controls (C and F). As seen with SMI32 staining, cell survival did not correlate with outcome (G–H), and whether transplanted cells survived (NWMI/GRP+) or died (NWMI/GRP-), animals that had no surviving exogenous cells still showed the same amount of recovery as those that did have surviving cells. This recovery showed a trend that did not gain significance when broken down by cell survival status. Scale bar 50 µm. Tukey’s multiple comparison tests p values are shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.
Figure 9
Figure 9. Effect of GRP transplantation on neurobehavioral outcome
There was a significant difference in the startle response between the animal groups, ANOVA F(2, 87) = 14.057, p < 0.001. The saline treated NWMI group had severely diminished startle response while this deficit was fully reversed by GRP transplantation (A). Prepulse Inhibition (PPI) testing showed again significant difference between the animal group (B); repeated measure ANOVA F(2, 55) = 5.133, p < 0.05. Again the saline treated NWMI group showed reduced amplitudes and diminished habituation to pre-pulse intensity change, while these deficits were fully reversed in the GRP treated group. Tukey’s multiple comparison analysis showed significant differences between control and saline-treated NWMI mice (p <0.05), and between GRP- and saline-treated NWMI mice (p < 0.01), while no significant differences were seen between control and GRP-treated NWMI mice. Post-hoc multiple comparison tests p values shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.
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