RIL: Research

Anatomical Basis of Behavioral Impairments | Neural Stem Cells and Brain Repair | Immunological Properties of Neural Stem Cells | In Situ Tissue Regeneration After Stroke | Cellular MRI | Regenerative Imaging | Post-mortem MR Histology

Anatomical Basis of Behavioral Impairments

The anatomical and functional organization of the brain underpins behavior. Damage or dysfunction of the brain therefore leads to behavioral impairment. However, behavior is the product of a complex network of distributed systems. Importantly, damage or dysfunction in one system leads to anatomical and functional changes in other systems. To really understand how the changes in the brain lead to behavioral problems, therefore requires us to study the effect of damage over time and over the whole brain.

To achieve this, we need to use non-invasive imaging techniques that do not perturb the brain while we study it over time. It is also important to capture the whole brain as remote changes from the site of damage/dysfunction might contribute to the behavioral impairment. We therefore use non-invasive imaging techniques, such as MRI and PET, to visualize the presence and consequences of brain damage. To establish the significance of these changes to behavioral impairments, we use batteries of behavioral tests that probe sensory, motor, and cognitive functions. By combining these two separate sets of data using sophisticated image analysis, we are able to link changes in behavior with specific regional changes in the brain after acute brain injury (e.g. stroke), but also neurodegenerative disease (e.g. Huntington’s & Parkinson’s disease). This approach will potential provide us with early diagnostic tools to predict the progression of brain damage and the emergence of behavioral symptoms. Early non-invasive diagnostic markers are essential to potential treat a disease before the patient detects any symptom. These studies are essential to reveal the anatomical networks that underpin behavioral dysfunctions.

Key References:

Crum, W.R., Giampietro, V.P., Smith, E.J., Gorenkova, N., Stroemer, R.P., Modo, M. A comparison of automated anatomical-behavioural mapping methods in a rodent model of stroke. Journal of Neuroscience Methods. 2013: 218 (2); 170-183. PubMed PMID: 23727124

Rattray, I., Smith, E., Crum, W., Walker, T., Gale, R., Bates, G., Modo, M. Correlations of behavioral deficits with brain pathology assessed through longitudinal MRI and histopathology in the R6/1 mouse model of Huntington’s disease. PLOS One. 2013: 8(12): e84726. PubMed PMID: 24367693

Vernon AC, Crum WR, Johansson SM, Modo M. Evolution of extra-nigral damage predicts behavioural deficits in a rat proteasome inhibitor model of Parkinson's disease. PLoS One. 2011 Feb 25;6(2):e17269. PubMed PMID: 21364887

Neural Stem Cells and Brain Repair

During brain development, neural stem cells (NSCs) produce brain cells that are responsible for our behavior. However, do not produce vascular cells or microglia. These two cell populations are derived from peripheral organ systems and colonize the brain. Nevertheless, they are a key constituent to ensure proper development and functioning of the brain. In the case of neurological disease, clinical signs are commonly evident due to the dysfunction or loss of neurons. If blood supply to the brain is interrupted, as is the case in stroke, the energy demand of brain tissue can no longer be met and cells will die, eventually leaving behind a tissue cavity. In the peri-infarct are dramatic changes do also occur, some neurons will be lost, gliosis is creating a scar along the cavity, some blood vessels die and some new small blood vessels are also being formed. To improve the functioning of this peri-infarct tissue, we aim to implant neural stem cells that can develop into adult brain cells. These cells can improve functioning of this tissue in many ways, for instance by creating new neurons that integrate with others to support signaling across the brain, differentiate into astrocytes and provide a support function to other cells, but also by producing oligodendrocytes that myelinate axons and improve transmission speed of information carried between neurons. However, the NSCs also secrete chemical factors that rescue dysfunctional cells and they enhance the formation of small blood vessels. Our aims are to define the therapeutic parameters of the cell injection and investigate mechanisms that can explain how these cells are improving behavioral impairments.

Key References:

Rossetti, T., Nicholls, F., Modo, M. Intracerebral cell transplantation: Preparation and characterization of cell suspensions. Cell Transplantation. 2016; 25: 645-664. PubMed PMID: 26720923

Chou, C-H., Modo, M. Human neural stem cell-induced endothelial morphogenesis requires paracrine and juxtacrine signaling. Scientific Reports. 2016; 6: 29029. PubMed PMID: 27374240

El-Akabawy, G, Rattray, I., Johansson, S.M., Gale, R., Bates, G., Modo, M. Implantation of undifferentiated and pre-differentiated human neural stem cells in the R6/2 transgenic mouse model of Huntington’s disease. BMC Neuroscience 2012: 13(1); 97. PubMed PMID: 22876937

Smith, E.J., Stroemer, R.P., Gorenkova, N., Nakajima, M., Crum, W.R., Tang, E., Stevanato, L., Sinden, J.D., Modo, M. Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of stroke. Stem Cells, 30(4), 785-796. PubMed PMID: 22213183

Immunological Properties of Neural Stem Cells

The immune system recognizes foreign antigens presented on implanted cells and can lead to their destruction. To ensure a long-lasting benefit of implanted cells, it is hence essential to ensure that these are not rejected by the host’s immune system. Neural stem cells, like all other cells, have a particular immunological profile, meaning that molecules responsible for recognition by the immune system, so called Major Histocompatibility Complexes (MHC), are expressed at different levels depending on the environment. In the normal brain, the expression of these MHC molecules is very low, but in the case of injury or disease, these typically get upregulated at the site of injury. Indeed, neural stem cells under normal conditions also express low levels of MHC, but when stimulated with cytokines, the expression of the molecules becomes more prominent. Implantation of these cells into sites of injury where inflammatory cytokines are readily available could hence lead to an upregulation of these molecules and make them vulnerable to rejection.

Our laboratory therefor investigates how inflammatory cytokines influence the expression of MHC molecules on neural stem cells and how this regulation affects in vivo immune responses to the cells. We further investigate if there is a need for immunosuppressant and/or anti-inflammatory medication to overcome the potential expression of foreign MHC antigens in a host.

Key References:

Johansson S, Price J, Modo M. Effect of inflammatory cytokines on major histocompatibility complex expression and differentiation of human neural stem/progenitor cells. Stem Cells. 2008 Sep;26(9):2444-54. Epub 2008 Jul 17. PubMed PMID: 18635871

Modo M, Mellodew K, Rezaie P. In vitro expression of major histocompatibility class I and class II antigens by conditionally immortalized murine neural stem cells. Neurosci Lett. 2003 Feb 6;337(2):85-8. PubMed PMID: 12527394

Modo M, Rezaie P, Heuschling P, Patel S, Male DK, Hodges H. Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response. Brain Res. 2002 Dec 20;958(1):70-82. PubMed PMID: 12468031

In Situ Tissue Regeneration After Stroke

Stroke is a devastating acute brain injury. However, no efficacious treatment to reverse the damage caused by stroke is available. Typically, stroke produces a dramatic loss of cells in the brain that eventually will lead to a cavitation where even the support structure (the so called extra-cellular matrix) is degraded. Our laboratories aim is to eventually replace this lost tissue and recover lost functions. However, major technical and biological challenges need to be overcome to achieve this long-term goal.

Replacement of lost cells can be achieved using neural stem cells. These are the cells producing the brain during normal development. When implanted into the brain next to the cavity caused by a stroke, these cells can differentiate into appropriate phenotypes and promote some degree of recovery. The precise mechanisms that lead to recovery remain, nevertheless, largely unknown. It is important to note as well that repair and recovery are far from complete. We therefore need to investigate how these cells promote recovery and how this can be optimized.

One key aspect is that when neural stem cells are transplanted into the stroke-caused cavity, they migrate into the remaining host brain rather than regenerating new tissue, hence leaving a large area of damage without repair. One reason for this is that the cells need to be embedded within an appropriate microenvironment, the so called neuro-vascular niche. Apart of neural cells, this niche also contains vascular cells and extra-cellular matrix (ECM) that keep the unit together. Re-creating such a neuro-vascular niche could hence potentially provide a means to regenerate lost brain tissue. However, it is a major challenge to recreating this niche and to implant it into the brain. Importantly, just recreating this niche might be insufficient to get cells to adopt the appropriate phenotypes as there are no local cues that guide the cells to induce a site-appropriate differentiation. Additional cues, such as chemical factors directing positional gene specification, might therefore be required.

Early experiments from our group using poly(D,L-lactic co-glycolic acid) (PLGA) microparticles to which cells were attached for transplantation into the stroke cavity are encouraging, at least from a technical point of view. However, there is no ingrowth of blood vessels and long-term survival might therefore be a further challenge. It is important to develop roadmap that would allow us to identify what the key aspects of in situ brain regeneration might be and to then devise appropriate bio-engineering strategies to overcome these obstacles. However, without a doubt each one of these will be a major undertaking.

Key References:

Ghuman, H., Massensini, A, Kim, T., Badylak, S.F., Modo, M. ECM hydrogel injection for the treatment of stroke: Characterization of the acute host cell invasion. Biomaterials. 2016; 91: 166-181. PubMed PMID: 27031811

Massensini, A., Ghuman, H., Medberry, C.J., Ling, W., Nicholls, F.J., Badylak, S.F., Modo, M. Concentration-dependent intracerebral delivery of ECM hydrogel to a stroke cavity. Acta Biomaterialia. 2015; 27:116-130. PubMed PMID: 26318805

Bible, E., Qutachi, O., Chau, D.Y., Alexander, M.R., Shakesheff, K.M., Modo, M. Neo-vascularization of the stroke cavity by implantation of human neural stem cells on VEGF-releasing PLGA microparticles. Biomaterials 2012: 33; 7435-7446. PubMed PMID: 22818980

Bible E, Chau DY, Alexander MR, Price J, Shakesheff KM, Modo M. Attachment of stem cells to scaffold particles for intra-cerebral transplantation. Nat Protoc. 2009;4(10):1440-53. Epub 2009 Sep 17. PubMed PMID: 19798079

Cellular MRI

The improvement of behavioral functions after an intracerebral injection of neural stem cells is time-dependent process. It takes several weeks before changes in behavior are apparent and this is a gradual process that last several weeks at least. However, during this time of recovery, it is currently not possible to investigate over time in the same host what these cells are doing. Non-invasive imaging however can provide an insight into these brains, but conventional techniques do not afford a distinction of implanted cells from the host tissue. We are therefore using MRI contrast agents that are incorporated into the cells prior to implantation. These MRI contrast agents (e.g. Gadolinium, Iorn Oxides) are detectable against the background of the normal brain and hence allow us to map the distribution of cells over time. To ensure that this labeling does not affect the properties and functions of cells, we conduct extensive in vitro studies, but also in vivo investigations determining if labeled cells perform in the same fashion than unlabeled cells. The ultimate aim of these experiments is to develop tools that would allow us to study how the evolution of the distribution, survival and differentiation of implanted cells relates to observed changes in behavioral impairments.

Key References:

Nicholls, F., Rotz, M.W., Ling, W., Ghuman, H., MacRenaris, K.W., Meade, T.J., Modo, M. DNA-Gadolinium-Gold nanoparticles for in vivo T1 MR imaging of transplanted human neural stem cells. Biomaterials. 2016; 77: 291-306. PubMed PMID: 26615367

Nicholls, F., Ling, W., Ferrauto, G., Aime, S., Modo, M. Simultaneous MR imaging for tissue engineering in a rat model of stroke. Scientific Reports. 2015; 5: 14597. PubMed PMID: 26419200

Chung, Y.-L., El Akabawy, G., So, P-W., Solanky, B., Leach, M.O., Modo, M. Profiling metabolite change in the neuronal differentiation of human striatal neural stem cells using 1H-magnetic resonance spectroscopy. NeuroReport. 2013: 24(18);1035-1040. PubMed PMID: 24145773

Naumova, A.V., Modo, M., Moore, A., Murry, C.E., Frank, J.A. Clinical imaging in regenerative medicine. Nature Biotechnology. 2014; 32(8): 804-818. PubMed PMID: 25093889

Modo, M., Kolosnjaj, J., Nicholls, F., Ling, W., Wilhelm, C., Debarge, O., Gazeau, F., Clement, O. Considerations for the clinical use of MRI contrast agents for cell tracking in regenerative medicine. Contrast Media and Molecular Imaging. 2013: 8(6); 439-455. PubMed PMID: 24375900

Regenerative Imaging

Transplantation of neural stem cells and tissue engineering are exciting new avenues to promote recovery from brain damage. However, very little is known about these cells promote this recovery. One key challenge is to monitor what these cells are doing in vivo. Survival, location, migration, differentiation and growth factor are key aspects of their function, but at present the only reliable way to measure these is using post-mortem histology.

To identify and monitor these processes, we are currently developing cellular and molecular imaging, techniques that visualize cells or molecules by in vivo imaging, such as MRI. These techniques will be key to gain a better mechanistic understanding of what transplanted cells do in vivo, but is also quintessential to monitor this therapy upon clinical translation.

As imaging techniques typically do not distinguish transplanted from host cells, we need to tag these cells using material that distinguished transplanted from host cells. The contrast agents are then incorporated into the cells prior to transplantation. We use a variety of metal-based particles, such as iron oxide or gadolinium-based contrast agents, but also fluorinated compounds. Fluorinated compounds (19F) has the advantage that it does not interfere with more conventional scans (1H) that are used to identify pathology. However, the disadvantage is that the signal that can be gained from these agents is much lower, hence fewer cells can be detected in the same amount of time. We therefore also need to improve our acquisition strategies to provide a better detection of these cells in vivo.

Although cellular MRI will allow us to track transplanted cells, the advent of in situ tissue engineering puts additional demands on our imaging. Not only will we need to detect transplanted neural stem cells, but we will transplant multiple cell types at the same time and incorporate these within biomaterials that support the formation of new tissue. This Regenerative Imaging therefore required multiple types of scans to detect these different elements and to monitor their progression over time. However, we are just at the beginning of sketching out these new demands, but it will certainly be an interesting time for imaging to develop new tools to monitor a regenerating tissue.

Key References:

Jin, T., Nicholls, F.J., Ghuman, H., Badylak, S.F., Modo, M. Diamagnetic chemical exchange saturation transfer (dia-CEST) affords magnetic resonance imaging of extracellular matrix hydrogel implantation in a rat model of stroke. Biomaterials. 2017; 113:176-190. PubMed PMID: 27816001

Nicholls, F., Ling, W., Ferrauto, G., Aime, S., Modo, M. Simultaneous MR imaging for tissue engineering in a rat model of stroke. Scientific Reports. 2015; 5: 14597. PubMed PMID: 26419200

Massensini, A., Ghuman, H., Medberry, C.J., Ling, W., Nicholls, F.J., Badylak, S.F., Modo, M. Concentration-dependent intracerebral delivery of ECM hydrogel to a stroke cavity. Acta Biomaterialia. 2015; 27:116-130. PubMed PMID: 26318805

Bible, E., Dell’Acqua, F., Solanky, B., Bladucci, A., Crapo, P., Badylak, S.F., Ahrens, E.T., Modo, M. Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodelling in the stroke-damaged rat brain by 19F- and diffusion-MRI. Biomaterials. 2012; 33, 2858-2871. PubMed PMID: 22244696

Post-mortem MR Histology

The anatomical composition of the brain has mostly relied on immunohistochemical analyses. However, this approach cannot be achieved in a living individual. Non-invasive imaging, however, can provide a view inside the living brain to reveal its anatomy and molecular composition. Spatial resolution is typically very low in comparison to post-mortem histology and also information about the different types of cells is rather limited. In contrast, in vivo imaging can provide indications as to the white matter pathways that connect different regions, whereas histology is more limited in re-constructing these in 3 dimensions.

Post-mortem imaging can potential bridge this chiasm between histology and in vivo imaging. A high spatial resolution can be achieved and it is possible to perform immunohistochemical investigations on the brain samples to validate the “MR histology”. By further developing MR histology, we aim to develop methods that will eventually allow us to reliably described a detailed 3 dimensional volume of a tissue that has been regenerate in vivo by in situ tissue engineering.

Key References:

Modo, M., Hitchens, T.K., Liu, J., Richardson, R.M. Ex vivo mesoscale diffusion MRI reveals an aberrant collateral mossy fiber connection between dentate gyrus and the stratum moleculare layer in a patient with uncontrolled temporal lobe epilepsy. Human Brain Mapping. 2016; 37(2):780-795. PubMed PMID: 26611565

Stille, M., Smith, E., Crum, W.R., Modo, M. 3D reconstruction of 2D fluorescence histology images and registration with in vivo MR images: Application in a rodent model of stroke. Journal of Neuroscience Methods. 2013: 219 (1); 27-40. PubMed PMID: 23816399

Dell’Acqua, F, Bodi, I., Slater, D., Catani, M., Modo, M. MR diffusion-based histology and micro-tractography reveal mesoscale features of the human cerebellum. Cerebellum, 2013:12(6); 923-931. PubMed PMID: 23907655