Richard L. Benton

Stem cell repair of central nervous system injury

Neural stem cells (NSCs) have great potential as a therapeutic tool for the repair of a number of CNS disorders. NSCs can either be isolated from embryonic and adult brain tissue or be induced from both mouse and human ES cells. These cells proliferate in vitro through many passages without losing their multipotentiality. Following engraftment into the adult CNS, NSCs differentiate mainly into glia, except in neurogenic areas. After engraftment into the injured and diseased CNS, their differentiation is further retarded. In vitro manipulation of NSC fate prior to transplantation and/or modification of the host environment may be necessary to control the terminal lineage of the transplanted cells to obtain functionally significant numbers of neurons. NSCs and a few types of glial precursors have shown the capability to differentiate into oligodendrocytes and to remyeliate the demyelinated axons in the CNS, but the functional extent of remyelination achieved by these transplants is limited. Manipulation of endogenous neural precursors may be an alternative therapy or a complimentary therapy to stem cell transplantation for neurodegenerative disease and CNS injury. However, this at present is challenging and so far has been unsuccessful. Understanding mechanisms of NSC differentiation in the context of the injured CNS will be critical to achieving these therapeutic strategies.

VEGF165 therapy exacerbates secondary damage following spinal cord injury.

Vascular endothelial growth factor (VEGF) demonstrates potent and well-characterized effects on endothelial cytoprotection and angiogenesis. In an attempt to preserve spinal microvasculature and prolong the endogenous neovascular response observed transiently following experimental spinal cord injury (SCI), exogenous recombinant human VEGF (rhVEGF165) was injected into the injured rat spinal cord. Adult female Fischer 344 rats were subjected to moderate SCI (12.5 g-cm) using the NYU impactor. At 72 h after injury, animals were randomly assigned to three experimental groups receiving no microinjection or injection of saline or saline containing 2 μg of rhVEGF165. Acutely, VEGF injection resulted in significant microvascular permeability and infiltration of leukocytes into spinal cord parenchyma. 6 weeks postinjection, no significant differences were observed in most measures of microvascular architecture following VEGF treatment, but analysis of histopathology in spinal cord tissue revealed profound exacerbation of lesion volume. These results support the idea that intraparenchymal application of the proangiogenic factor VEGF may exacerbate SCI, likely through its effect on vessel permeability.

Griffonia simplicifolia Isolectin B4 Identifies a Specific Subpopulation of Angiogenic Blood Vessels Following Contusive Spinal Cord Injury in the Adult Mouse

After traumatic spinal cord injury (SCI), disruption and plasticity of the microvasculature within injured spinal tissue contribute to the pathological cascades associated with the evolution of both primary and secondary injury. Conversely, preserved vascular function most likely results in tissue sparing and subsequent functional recovery. It has been difficult to identify subclasses of damaged or regenerating blood vessels at the cellular level. Here, adult mice received a single intravenous injection of the Griffonia simplicifolia isolectin B4 (IB4) at 1–28 days following a moderate thoracic (T9) contusion. Vascular binding of IB4 was maximally observed 7 days following injury, a time associated with multiple pathologic aspects of the intrinsic adaptive angiogenesis, with numbers of IB4 vascular profiles decreasing by 21 days postinjury. Quantitative assessment of IB4 binding shows that it occurs within the evolving lesion epicenter, with affected vessels expressing a temporally specific dysfunctional tight junctional phenotype as assessed by occludin, claudin‐5, and ZO‐1 immunoreactivities. Taken together, these results demonstrate that intravascular lectin delivery following SCI is a useful approach not only for observing the functional status of neovascular formation but also for definitively identifying specific subpopulations of reactive spinal microvascular elements. J. Comp. Neurol. 507:1031–1052, 2008.

Consequences of noggin expression by neural stem, glial, and neuronal precursor cells engrafted into the injured spinal cord

Bone morphogenetic proteins (BMPs) are a large class of secreted factors, which serve as modulators of development in multiple organ systems, including the CNS. Studies investigating the potential of stem cell transplantation for restoration of function and cellular replacement following traumatic spinal cord injury (SCI) have demonstrated that the injured adult spinal cord is not conducive to neurogenesis or oligodendrogenesis of engrafted CNS precursors. In light of recent findings that BMP expression is modulated by SCI, we hypothesized that they may play a role in lineage restriction of multipotent grafts. To test this hypothesis, neural stem or precursor cells were engineered to express noggin, an endogenous antagonist of BMP action, prior to transplantation or in vitro challenge with recombinant BMPs. Adult rats were subjected to both contusion and focal ischemic SCI. One week following injury, the animals were transplanted with either EGFP- or noggin-expressing neural stem or precursor cells. Results demonstrate that noggin expression does not antagonize terminal astroglial differentiation in the engrafted stem cells. Furthermore, neutralizing endogenous BMP in the injured spinal cord significantly increased both the lesion volume and the number of infiltrating macrophages in injured spinal cords receiving noggin-expressing stem cell grafts compared with EGFP controls. These data strongly suggest that endogenous factors in the injured spinal microenvironment other than the BMPs restrict the differentiation of engrafted pluripotent neural stem cells as well as suggest other roles for BMPs in tissue protection in the injured CNS.

Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems.

Viral gene delivery for spinal cord injury (SCI) is a promising approach for enhancing axonal regeneration and neuroprotection. An understanding of spatio‐temporal transgene expression in the spinal cord is essential for future studies of SCI therapies. Commonly, intracellular marker proteins (e.g., EGFP) were used as indicators of transgene levels after viral delivery, which may not accurately reflect levels of secreted transgene. This study examined transgene expression using ELISA after viral delivery of D15A, a neurotrophin with BDNF and NT‐3 activities, at 1, 2, and 4weeks after in vivo and ex vivo delivery using lentiviral, adenoviral, and retroviral vectors. Further, the inflammatory responses and viral infection patterns after in vivo delivery were examined. Lentiviral vectors had the most stable pattern of gene expression, with D15A levels of 536 ± 38 and 363 ± 47 pg/mg protein seen at 4 weeks after the in vivo and ex vivo delivery, respectively. Our results show that protein levels downregulate disproportionately to levels of EGFP after adenoviral vectors both in vivo and ex vivo. D15A dropped from initial levels of 422 ± 87 to 153 ± 18 pg/mg protein at 4 weeks after in vivo administration. Similarly, ex vivo retrovirus‐mediated transgene expression exhibited rapid downregulation by 2 weeks post‐grafting. Compared to adenoviral infection, macrophage activation was attenuated after lentiviral infection. These results suggest that lentiviral vectors are most suitable in situations where stable long‐term transgene expression is needed. Retroviral ex vivo delivery is optional when transient expression within targeted spinal tissue is desired, with adenoviral vectors in between.

Schwann cell-like differentiation by adult oligodendrocyte precursor cells following engraftment into the demyelinated spinal cord is BMP-dependent

The development of remyelinating strategies designed to enhance recruitment and differentiation of endogenous precursor cells available to a site of demyelination in the adult spinal cord will require a fundamental understanding of the potential for adult spinal cord precursor cells to remyelinate as well as an insight into epigenetic cues that regulate their mobilization and differentiation. The ability of embryonic and postnatal neural precursor cell transplants to remyelinate the adult central nervous system is well documented, while no transplantation studies to date have examined the remyelinating potential of adult spinal‐cord‐derived oligodendrocyte precursor cells (adult OPCs). In the present study, we demonstrate that, when transplanted subacutely into spinal ethidium bromide/X‐irradiated (EB‐X) lesions, adult OPCs display a limited capacity for oligodendrocyte remyelination. Interestingly, the glia‐free environment of EB lesions promotes engrafted adult OPCs to differentiate primarily into cells with immunophenotypic and ultrastructural characteristics of myelinating Schwann cells (SCs). Astrocytes modulate this potential, as evidenced by the demonstration that SC‐like differentiation is blocked when adult OPCs are co‐transplanted with astrocytes. We further show that inhibition of bone morphogenetic protein (BMP) signaling through noggin overexpression by engrafted adult OPCs is sufficient to block SC‐like differentiation within EB‐X lesions. Present data suggest that the macroglial‐free environment of acute EB lesions in the ventrolateral funiculus is inhibitory to adult spinal cord‐derived OPC differentiation into remyelinating oligodendrocytes, while the presence of BMPs and absence of noggin promotes SC‐like differentiation, thereby unmasking a surprising lineage fate for these cells.

CNTF promotes the survival and differentiation of adult spinal cord-derived oligodendrocyte precursor cells in vitro but fails to promote remyelination in vivo

Delivery of factors capable of promoting oligodendrocyte precursor cell (OPC) survival and differentiation in vivo is an important therapeutic strategy for a variety of pathologies in which demyelination is a component, including multiple sclerosis and spinal cord injury. Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that promotes both survival and maturation of a variety of neuronal and glial cell populations, including oligodendrocytes. Present results suggest that, although CNTF has a potent survival and differentiation promoting effect in vitro on OPCs isolated from the adult spinal cord, CNTF administration in vivo is not sufficient to promote oligodendrocyte remyelination in the glial-depleted environment of unilateral ethidium bromide (EB) lesions.

Spinal Cord Contusion Based on Precise Vertebral Stabilization and Tissue Displacement Measured by Combined Assessment to Discriminate Small Functional Differences

Contusive spinal cord injury (SCI) is the most common type of spinal injury seen clinically. Several rat contusion SCI models have been described, and all have strengths and weaknesses with respect to sensitivity, reproducibility, and clinical relevance. We developed the Louisville Injury System Apparatus (LISA), which contains a novel spine-stabilizing device that enables precise and stable spine fixation, and is based on tissue displacement to determine the severity of injury. Injuries graded from mild to moderately severe were produced using 0.2-, 0.4-, 0.6-, 0.8-, 1.0-, and 1.2-mm spinal cord displacement in rats. Basso, Beattie, and Bresnahan (BBB) and Louisville Swim Score (LSS) could not significantly distinguish between 0.2-mm lesion severities, except those of 0.6- and 0.8-mm BBB scores, but could between 0.4-mm injury differences or if the data were grouped (0.2-0.4, 0.6-0.8, and 1.0-1.2). Transcranial magnetic motor evoked potential (tcMMEP) response amplitudes were decreased 10-fold at 0.2-mm displacement, barely detected at 0.4-mm displacement, and absent with greater displacement injuries. In contrast, somatosensory evoked potentials (SSEPs) were recorded at 0.2- and 0.4-mm displacements with normal amplitudes and latencies but were detected at lower amplitudes at 0.6-mm displacement and absent with more severe injuries. Analyzing combined BBB, tcMMEP, and SSEP results enabled statistically significant discrimination between 0.2-, 0.4-, 0.6-, and 0.8-mm displacement injuries but not the more severe injuries. Present data document that the LISA produces reliable and reproducible SCI whose parameters of injury can be adjusted to more accurately reflect clinical SCI. Moreover, multiple outcome measures are necessary to accurately detect small differences in functional deficits and/or recovery. This is of crucial importance when trying to detect functional improvement after therapeutic intervention to treat SCI.

ADAM8 is selectively up-regulated in endothelial cells and is associated with angiogenesis after spinal cord injury in adult mice.

Endothelial cell (EC) loss and subsequent angiogenesis occur over the first week after spinal cord injury (SCI). To identify molecular mechanisms that could be targeted with intravenous (i.v.) treatments, we determined whether transmembrane “a disintegrin and metalloprotease” (ADAM) proteins are expressed in ECs of the injured spinal cord. ADAMs bind to integrins, which are important for EC survival and angiogenesis. Female adult C57Bl/6 mice with a spinal cord contusion had progressively more ADAM8 (CD156) immunostaining in blood vessels and individual ECs between 1 and 28 days following injury. Uninjured spinal cords had little ADAM8 staining. The increase in ADAM8 mRNA and protein was confirmed in spinal cord lysates, and ADAM8 mRNA was present in FACS‐enriched ECs. ADAM8 colocalized extensively and exclusively with the EC marker PECAM and also with i.v.‐injected lectins. Intravenous isolectin B4 (IB4) labels a subpopulation of blood vessels at and within the injury epicenter 3–7 days after injury, coincident with angiogenesis. Both ADAM8 and the proliferation marker Ki‐67 were present in IB4‐positive microvessels. ADAM8‐positive proliferating cells were seen at the leading end of IB4‐positive blood vessels. Angiogenesis was confirmed by BrdU incorporation, binding of i.v.‐injected nucleolin antibodies, and MT1‐MMP immunostaining in a subset of blood vessels. These data suggest that ADAM8 is vascular selective and plays a role in proliferation and/or migration of ECs during angiogenesis following SCI. J. Comp. Neurol. 512:243–255, 2009.

Angiogenic Potential of Microvessel Fragments is Independent of the Tissue of Origin and can be Influenced by the Cellular Composition of the Implants

Please cite this paper as: Nunes, Krishnan, Gerard, Dale, Maddie, Benton and Hoying (2010). Angiogenic Potential of Microvessel Fragments is Independent of the Tissue of Origin and can be Influenced by the Cellular Composition of the Implants. Microcirculation17(7), 557–567.