Thomas V. Johnson

Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in experimental glaucoma.

Purpose. Retrograde neurotrophic factor transport blockade has been implicated in the pathophysiology of glaucoma. Stem cell transplantation appears to ameliorate some neurodegenerative conditions in the brain and spinal cord, in part by neurotrophic factor secretion. The present study was conducted to determine whether local or systemic bone marrow-derived mesenchymal stem cell (MSC) transplantation can confer neuroprotection in a rat model of laser-induced ocular hypertensive glaucoma. Methods. MSCs were isolated from the bone marrow of adult wild-type and transgenic rats that ubiquitously express green fluorescent protein. MSCs were transplanted intravitreally 1 week before, or intravenously on the day of, ocular hypertension induction by laser photocoagulation of the trabecular meshwork. Ocular MSC localization and integration were determined by immunohistochemistry. Optic nerve damage was quantified by counting axons within optic nerve cross-sections 4 weeks after laser treatment. Results. After intravitreal transplantation, MSCs survived for at least 5 weeks. Cells were found mainly in the vitreous cavity, though a small proportion of discrete cells migrated into the host retina. Intravitreal MSC transplantation resulted in a statistically significant increase in overall RGC axon survival and a significant decrease in the rate of RGC axon loss normalized to cumulative intraocular pressure exposure. After intravenous transplantation, MSCs did not migrate to the injured eye. Intravenous transplantation had no effect on optic nerve damage. Conclusions. Local, but not systemic, transplantation of MSCs was neuroprotective in a rat glaucoma model. Autologous intravitreal transplantation of MSCs should be investigated further as a potential neuroprotective therapy for glaucoma.

Rodent models of glaucoma

Glaucoma is a progressive, age-related optic neuropathy and a leading cause of irreversible blindness in the world. Animal models of glaucoma are essential to our continued efforts of elucidating the natural course of the disease and to developing therapeutic interventions to halt or reverse the progression of the condition. Over the past 10-15 years, rodents have become a popular model organism to study glaucoma, because of their high degree of availability, relatively low cost, short life-span, and amenability to experimental and genetic manipulation. In this review, we examine the numerous in vivo and in vitro rodent models of glaucoma, discuss the methods used to generate them, summarize some of the major findings obtained in these models, and identify individual strengths and weaknesses for the various systems.

Development and Characterization of an Adult Retinal Explant Organotypic Tissue Culture System as an In Vitro Intraocular Stem Cell Transplantation Model

PURPOSE To develop and characterize a retinal explant culture system to facilitate investigation of novel methods of improving retinal stem cell therapy. METHODS Retinas explanted from adult rats were cultured in serum-free medium (B27/N2) or medium containing normal horse serum (NHS). Tissue viability was assessed by gross morphology, propidium iodide (PI) uptake, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. Müller progenitor cells (hMIO-M1), or mesenchymal stem cells (MSC) were placed on explants, to model intravitreal cell transplantation. Explants were compared with whole eyes, with or without experimental glaucoma and/or intravitreal cell transplantation. RESULTS Explants cultured in B27/N2 medium were viable for at least 17 days, as assessed by the aforementioned parameters. NHS medium was associated with obvious tissue degradation, greater/more diffuse PI uptake, significant cell loss over time, and temporal increase in activated caspase-3(+) cells. Explants in B27/N2 medium strongly expressed beta-III-tubulin, neurofilament, NeuN, Brn3a, Thy-1, GFAP, vimentin, nestin, and glutamine synthetase, whereas immunoreactivity was weak in NHS medium and decreased further with time. Seven and 14 days after coculture or transplantation, glial reactivity (GFAP/vimentin expression) was highly upregulated in explants and eyes, respectively. Some grafted cells migrated into the retina, but most remained outside the inner limiting membrane. CONCLUSIONS Retinal explants prepared using the described techniques and cultured in B27/N2 medium are viable for at least 2 weeks and mimic in vivo glial reactivity to transplantation while allowing few grafted cells to integrate. This system may be a useful in vitro model for investigating methods of enhancing retinal stem cell therapy.

Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome

The development of neuroprotective strategies to attenuate retinal ganglion cell death could lead to novel therapies for chronic optic neuropathies such as glaucoma. Intravitreal transplantation of mesenchymal stem cells slows retinal ganglion cell death in models of optic nerve injury, but the mechanism of action remains unclear. Here we characterized the neuroprotective effects of mesenchymal stem cells and mesenchymal stem cell-derived factors in organotypic retinal explant culture and an in vivo model of ocular hypertensive glaucoma. Co-culture of rat and human bone marrow-derived mesenchymal stem cells with retinal explants increased retinal ganglion cell survival, after 7 days ex vivo, by ∼2-fold and was associated with reduced apoptosis and increased nerve fibre layer and inner plexiform layer thicknesses. These effects were not demonstrated by co-culture with human or mouse fibroblasts. Conditioned media from mesenchymal stem cells conferred neuroprotection, suggesting that the neuroprotection is mediated, at least partly, by secreted factors. We compared the concentrations of 29 factors in human mesenchymal stem cell and fibroblast conditioned media, and identified 11 enriched in the mesenchymal stem cell secretome. Treatment of retinal explants with a cocktail of these factors conferred retinal ganglion cell neuroprotection, with factors from the platelet-derived growth factor family being the most potent. Blockade of platelet-derived growth factor signalling with neutralizing antibody or with small molecule inhibitors of platelet-derived growth factor receptor kinase or downstream phosphatidylinositol 3 kinase eliminated retinal ganglion cell neuroprotection conferred by mesenchymal stem cell co-culture. Intravitreal injection of platelet-derived growth factor -AA or -AB led to profound optic nerve neuroprotection in vivo following experimental induction of elevated intraocular pressure. These data demonstrate that mesenchymal stem cells secrete a number of neuroprotective proteins and suggest that platelet-derived growth factor secretion in particular may play an important role in mesenchymal stem cell-mediated retinal ganglion cell neuroprotection. Furthermore, platelet-derived growth factor may represent an independent target for achieving retinal ganglion cell neuroprotection.

Identification of Barriers to Retinal Engraftment of Transplanted Stem Cells

PURPOSE Intraocular stem cell transplantation may be therapeutic for retinal neurodegenerative diseases such as glaucoma via neuronal replacement and/or neuroprotection. However, efficacy is hindered by extremely poor retinal graft integration. The purpose was to identify the major barrier to retinal integration of intravitreally transplanted stem cells, which was hypothesized to include the cellular and/or extracellular matrix (ECM) components of the inner limiting membrane (ILM). METHODS Mesenchymal stem cells (MSCs) were cocultured on the vitreal surface of retinal explants. Retinal MSC migration was compared between control explants and explants in which portions of the ILM were removed by mechanical peeling; the inner basal lamina was digested with collagenase; and glial cell reactivity was selectively modulated with alpha-aminoadipic acid (AAA). In vivo, the MSCs were transplanted after intravitreal AAA or saline injection into glaucomatous rat eyes. RESULTS Retinal MSC migration correlated positively with the amount of peeled ILM, whereas enzymatic digestion of the basal lamina was robust but did not enhance MSC entry. In contrast, AAA treatment suppressed glial cell reactivity and facilitated a >50-fold increase in MSC migration into retinal explants. In vivo analysis showed that AAA treatment led to a more than fourfold increase in retinal engraftment. CONCLUSIONS The results demonstrated that the ECM of the inner basal lamina is neither necessary nor sufficient to prevent migration of transplanted cells into the neural retina. In contrast, glial reactivity was associated with poor graft migration. Targeted disruption of glial reactivity dramatically improved the structural integration of intravitreally transplanted cells.

Neurotrophic factor delivery as a protective treatment for glaucoma

Glaucoma is a progressive optic neuropathy and a major cause of visual impairment worldwide. Neuroprotective therapies for glaucoma aim to ameliorate retinal ganglion cell degeneration through direct or indirect action on these neurons. Neurotrophic factor (NTF) delivery is a key target for the development of potential neuroprotective glaucoma treatments. This article will critically summarize the evidence that NTF deprivation and/or dysfunction plays a role in the pathogenesis of glaucoma. Experimental support for the neuroprotective potential of NTF supplementation in animal models of glaucoma will be reviewed, in particular for brain-derived neurotrophic factor, ciliary neurotrophic factor, and glial cell line-derived neurotrophic factor. Finally, the challenges of clinical translation will be considered with an emphasis on the most promising NTF delivery strategies including slow-release drug delivery, gene therapy, and cell transplantation.

Toll-like receptors: roles in neuroprotection?

Toll-like receptors (TLRs) enable mammalian cells to sense pathogenic challenges. They are essential for appropriate initiation, execution and regulation of innate and adaptive immune responses. Whereas TLR-mediated processes in the central nervous system (CNS) might contribute to detrimental (auto)immune reactions, they are unlikely to have exclusively neurodestructive consequences. Indeed, appropriately controlled TLR signaling might be crucial for preserving CNS structure and function in certain contexts. Recent findings illustrate neuroprotective capacities for TLRs, mediated by containment of trauma-associated infection or by recruitment of neuroprotective T lymphocytes. By the latter mechanism, endogenous or therapeutically administered TLR ligands could conceivably generate neuroprotective benefits in noninfectious CNS disorders. This article focuses on the yet less-addressed protective potential of TLR engagement within the CNS.

Use of an Adult Rat Retinal Explant Model for Screening of Potential Retinal Ganglion Cell Neuroprotective Therapies

PURPOSE. To validate an established adult organotypic retinal explant culture system for use as an efficient medium-throughput screening tool to investigate novel retinal ganglion cell (RGC) neuroprotective therapies. METHODS. Optimal culture conditions for detecting RGC neuroprotection in rat retinal explants were identified. Retinal explants were treated with various recognized, or purported, neuroprotective agents and cultured for either 4 or 7 days ex vivo. The number of cells surviving in the RGC layer (RGCL) was quantified using histologic and immunohistochemical techniques, and statistical analyses were applied to detect neuroprotective effects. RESULTS. The ability to replicate previously reported in vivo RGC neuroprotection in retinal explants was verified by demonstrating that caspase inhibition, brain-derived neurotrophic factor treatment, and stem cell transplantation all reduced RGCL cell loss in this model. Further screening of potential neuroprotective pharmacologic agents demonstrated that betaxolol, losartan, tafluprost, and simvastatin all alleviated RGCL cell loss in retinal explants, supporting previous reports. However, treatment with brimonidine did not protect RGCL neurons from death in retinal explant cultures. Explants cultured for 4 days ex vivo proved most sensitive for detecting neuroprotection. CONCLUSIONS. The current adult rat retinal explant culture model offers advantages over other models for screening potential neuroprotective drugs, including maintenance of neurons in situ, control of environmental conditions, and dissociation from other factors such as intraocular pressure. Verification that neuroprotection by previously identified RGC-protective therapies could be replicated in adult retinal explant cultures suggests that this model could be used for efficient medium-throughput screening of novel neuroprotective therapies for retinal neurodegenerative disease.

Cell transplantation approaches to retinal ganglion cell neuroprotection in glaucoma.

Glaucoma is a complex neurodegenerative disease that involves interactions among multiple signaling pathways, ultimately leading to progressive retinal ganglion cell (RGC) death. The development of neuroprotective approaches to glaucoma therapy could preserve vision by modulating these pathologic pathways or by acting directly on RGCs to attenuate cell death and maintain function. Intraocular cell transplantation is being evaluated as one approach to achieve sustained RGC neuroprotection. Unlike traditional pharmacological approaches, transplanted cells might be capable of simultaneously targeting multiple pro-survival pathways via local delivery of secreted factors and/or via modulation of the intraocular microenvironment. Elucidating the mechanisms by which different cell types attenuate RGC death in models of glaucoma may uncover additional novel mechanisms of neuroprotection. In this review, we will discuss the rationale for transplantation-based approaches to neuroprotection for glaucoma and explore the various mechanisms of action proposed to account for RGC neuroprotection achieved by two distinct cell classes that have been studied most extensively for this purpose: glial cells and mesenchymal stem cells.

Stem cells for neuroprotection in glaucoma

Stem cell transplantation is currently being explored as a therapy for many neurodegenerative diseases including glaucoma. Cellular therapies have the potential to provide chronic neuroprotection after a single treatment, and early results have been encouraging in models of spinal cord injury and Parkinsons disease. Stem cells may prove ideal for use in such treatments as they can accumulate at sites of injury in the central nervous system (CNS) and may also offer the possibility of targeted treatment delivery. Numerous stem cell sources exist, with embryonic and fetal stem cells liable to be superseded by adult-derived cells as techniques to modify the potency and differentiation of somatic cells improve. Possible neuroprotective mechanisms offered by stem cell transplantation include the supply of neurotrophic factors and the modulation of matrix metalloproteinases and other components of the CNS environment to facilitate endogenous repair. Though formidable challenges remain, stem cell transplantation remains a promising therapeutic approach in glaucoma. In addition, such studies may also provide important insights relevant to other neurodegenerative diseases.