A chronic 1 year assessment of MRI contrast agent-labelled neural stem cell transplants in stroke

Abstract

Non-invasive identification of transplanted neural stem cells in vivo by pre-labelling with contrast agents may play an important role in the translation of cell therapy to the clinic. Understanding the impact of these labels on the cells' ability to repair is therefore vital. In rats with middle cerebral artery occlusion (MCAo), a model of stroke, the transhemispheric migration of MHP36 cells labelled with the bimodal contrast agent GRID was detected on magnetic resonance images (MRI) up to 4 weeks following transplantation. However, compared to MHP36 cells labelled with the red fluorescent dye PKH26, GRID-labelled transplants did not significantly improve behaviour, and performance was akin to non-treated animals. Likewise, the evolution of anatomical damage as assessed by serial, T(2)-weighted MRI over 1 year indicated that GRID-labelled transplants resulted in a slight increase in lesion size compared to MCAo-only animals, whereas the same, PKH26-labelled cells significantly decreased lesion size by 35%. Although GRID labelling allows the in vivo identification of transplanted cells up to 1 month after transplantation, it is likely that some is gradually degraded inside cells. The translation of cellular imaging therefore does not only require the in vitro assessment of contrast agents on cellular functions, but also requires the chronic, in vivo assessment of the label on the stem cells' ability to repair in preclinical models of neurological disease.

Figure 1

Animals with stroke damage show a very significant asymmetry in the removal of adhesive tape from the forepaws compared to controls. Upon transplantation of neural stem cells from the MHP36 cell line, only animals with MHP36 cells labelled with PKH26 showed a significant recovery compared to stroke only animals. Labelling of MHP36 cells with GRID did not result in the same recovery. Both stroke only and the MHP36+GRID groups spontaneously improved their forepaw asymmetry by 26 weeks and no longer exhibited a deficit compared to controls or MHP36+PKH26 transplanted animals. In contrast, the total time of adhesive removal did not exhibit an initial difference between stroke only and control animals. Only 26 weeks following transplantation, when the asymmetry bias disappeared did stroke only animals reveal a significant difference to control and MHP36+PKH26-transplanted animals. MHP36+PKH26 animals showed the same total removal times. MHP36+GRID-transplanted animals performed at an intermediate level between controls and stroke only animals. This suggests that in MHP36+GRID animals, MHP36 cells might have exerted early beneficial effects that might be reflected in later behavioural performance.

Figure 2

(A) shows the evolution of T₂-weighted MR images of animals within the different experimental groups over 52 weeks post-transplantations. Progressively over the 6 weeks post-ischaemia (4 weeks post-grafting), the lesion environment develops into a lesion cavity that is clearly defined on T₂-weighted scans. However, little change in the MRI signature and size of the lesion can be observed beyond this time. (B) Already 1 week post-transplantation, GRID-labelled cells can be detected on T₂-weighted MR images surrounding the vicinity of the lesion (left-facing arrows). Labelled-cells did not constitute the border to the cavity, but were located within intact-appearing tissue. One month following implantation, these GRID-labelled cells were located at the edge of the lesion indicating that GRID-labelled cells did not integrate into tissue undergoing subsequent degeneration. It is likely that this tissue was already severely gliotic and that this inhibits infiltration of stem cells. In 37% of cases, edema (as indicated by a hyperintensity) can be seen at the site of injection (left-facing arrows) and this prevents detection of transplanted cells labelled with GRID which is apparent 1 month following injection when the edema receded. PKH26-labelled cells were not visible on these scans. (C) GRID-labelled cells are detectable by MRI for up to 1 month following injection, but can no longer be detected on images 3 months post-transplantation. The migration pattern that can be observed on these scans indicates that depending on the extent and location of the lesion, transplanted cells either remain at the outer border of the lesion or in case of a subcortical infarct, cells migrate around the cavity and will surround the cavity. It is interesting to note that migration to the lesion always seems to be an extension of the corpus callosum that these cells use to migrate. Grafted cells do not appear to shortcut through the striatum to the site of the lesion indicating a key role of the corpus callosum in cell migration to the site of infarction.

Figure 3

Evolution of anatomical structures. To determine how transplanted cells affect the evolution of brain growth, lesion, striatum and cortex, total volumes prior to transplantation were used as baseline to assess percentage change over time. This removes the intra-animal variability and provides a more powerful assessment of treatment effects. Most significantly here, PKH26-labelled MHP36 cells reduced the size of the lesion by 35% 1 year post-grafting, whereas cells labelled with GRID did not reduce lesion size. Similar effects were seen in the striatum and cortex. Interestingly, no dramatic effects were observed on the asymmetry between ipsi- and contralateral striatum or cortex.

Figure 4

Ex vivo MRI. To provide high-resolution 3 dimensional images of the GRID-transplanted brains, ex vivo MR images were acquired. (A) Even after 1 year the injection tract can still be clearly seen in the damaged brain although in vivo this area was no longer detectable after 3 month. Ex vivo images are less susceptible to intravoxel movement, have a higher resolution to avoid partial volume effects and additional averages can be included to improve detection of smaller clusters of cells compared to in vivo limitations. (B) In the lesion cavity, filamentous elements can be observed on ex vivo images that were not evident on in vivo images. It is possible that these reflect parts of the glial scarring, but their location and distribution strongly suggest that these might be remnants of blood vessels that were reperfused and viable although the surrounding neuropil died away.

Figure 5

Histological assessment of stroke damage and stem cell implantation. (A) Transplanted cells as identified by GRID's red fluorescence can still be seen in the injection tract and the horn of the lateral ventricle (lv) with some of the MHP36 still in corpus callosum. (B) GRID-labelled cells could also still be found in the peri-infarct area. However, far fewer cells could be found here than in previous experiments (Modo et al., 2004a). (C) In the cortex, however, red autofluorescence was also present in normal control animals. (D) This punctate red inclusions had the same appearance than GRID or PKH26 and did not allow a reliable identification of transplanted cells in these areas. (E) Sudan Black staining can remove this autofluorescence by tainting the inclusions black as can be seen here on the brightfield image. However, Sudan Black also covers the fluorescent properties of GRID and PKH26 and therefore still precludes identification of grafted cells.