Método de intervención

The work presented in this thesis opens up different paths for further investigation regarding cellular MRI in SCI research.

One of the questions regarding cell therapy for SCI treatment is optimal delivery method. Different routes have been used such as intra-arterial,7 intravenous,8 lumbar puncture,9 and direct transplantation.10 However, there is no clear picture of which, if any, would produce optimal results. We have shown here how cellular MRI can be used to better understand the fate of administered stem cells. From the moment of transplantation we were able to verify cell delivery to the target, which as de Vries et al. have reported, may be of crucial importance for therapy success.11 _{Thus, the techniques here developed could}

be applied to compare different transplantation sites and techniques. We were also able to observe distribution to other tissues, which might help get a better understanding of the homing of the cells and whether that is required for the trophic and immunomodulatory effects that stem cells can provide. Especially after the report of increased pain and spasticity by Kishk et al.,12 upon cell transplantation, being able to monitor cells over time at a high resolution could give us a clearer picture of their distribution. Things to investigate might include if cells are going to the sensory tracts, for example, and if this is

an undesirable target to home to. As one of the differences among the early clinical studies has been dose difference that would be certainly another thing to compare in the future with longitudinal studies as shown here.

One of the limitations in this study was establishing the long-term fate of the cells, as we could not distinguish viable and non-viable cells or iron transfer several weeks after the transplant. Recent studies have investigated the ability to differentiate between live and dead cells based on changes in relaxation times, and contrast, when iron goes from being compartmentalized to free in solution.13, 14 _{These techniques may be useful for}

monitoring cell death in transplantation studies, however, they have yet to be validated in vivo. An alternative exciting new development that could be incorporated in cell tracking in SCI models is the use of MRI reporter genes. A recent study used embryonic stem cells transduced with a reporter gene that can be targeted with an iron oxide conjugated antibody to provide information on cell viability, by providing differences in contrast from viable cells, even as they divided and differentiated, compared to apoptotic cells.15 Another study used ferritin as a reporter gene by transducing stem cells to overexpress ferritin and then transplanted them into a myocardial infarction model where the authors reported the possibility of tracking stem cells as they divide and differentiate while being able to track morpho-functional changes in the heart.16 Additional information on cell viability specific to the transplanted cells while maintaining MRI sensitivity would certainly be useful for cell transplantation in SCI models such as the ones here presented. This would help track changes in the cord and lesion while simultaneously track cell viability.

Better understanding macrophage response is important for planning anti-inflammatory treatments and cell therapy.17 Imaging macrophage infiltration in vivo will contribute to better understand their role in SCI and consider it when optimizing treatments. In the case of SCI where there can be different sources of hyper- and hypo- intensity due to pathological features of the injury, like edema and hemorrhage combined MRI protocols may also help distinguish between iron labeled cells and other sources of hypo-intensity. Susceptibility weighted imaging,18, 19 _{for instance, has shown the ability to detect}

loss from different sources. The development of new pulse sequences, which generate positive contrast from iron-labeled cells, could also be incorporated to the techniques here developed to better define the signal from iron-labeled cells.21, 22

The FSL measurement provided useful information for monitoring increased iron in the SCI lesion; it was significantly greater in the cord images after USPIO administration. However, FSL does not provide rigorous quantification of iron present. New algorithms are being developed for iron quantification,23 such as phase gradient mapping to estimate iron concentration. Implementing algorithms for cord registration and segmentation would certainly improve the ability to quantify the changes observed in signal due to USPIO. We have previously used lesion volume measurements in a rat model ex vivo to assess an anti-inflammatory treatment.24 A significant difference in the lesion volume was measured for treated versus untreated SCI rats. We have now shown that it is possible to image the rat in vivo and label cells in situ. Combining this technology of in vivo imaging with long-term follow-up of SCI pathological features could be used to compare treatments; for example, how stem cell therapy might impact cavity formation in the chronic phase.

SCI is certainly a complex condition and in the end, it is most likely that a combination of treatments and a comprehensive rehabilitation will maximize the chances of functional recovery and thus improve quality of life of individuals who deal daily with the obstacles it poses. There are still questions to be addressed in a wide variety of treatments25, 26 and in a field where clinical translation and longitudinal monitoring is so important to decide which of these treatments to take forward to clinical trials, cellular MRI could certainly be an important part of this process. As these tools are refined, they can be used to test different treatments (MSC, mobilization agents, anti-inflammatory or combinations) and use information obtained from MRI to establish best treatment time-points to optimize treatment effects.