Glucagon secretion from the pancreatic alpha cells is crucial to prevent hypoglycemia. People with type 1 diabetes, however, lose this glucoregulatory mechanism and are susceptible to dangerous insulin treatment-induced hypoglycemia. We established that activating glutamate receptors of the AMPA/kainate type in alpha cells is needed for decreases in glucose levels to elicit full glucagon responses from mouse and human islets. We performed functional studies using living pancreas slices from donors with type 1 diabetes and found that alpha cells had normal glucagon content and responded typically to KCl depolarization, but failed to respond to decreases in glucose concentration and had severely impaired AMPA/kainate receptor signaling. Reactivating residual AMPA/kainate receptor function with the positive allosteric modulators cyclothiazide and aniracetam partially rescued glucagon secretion in response to hypoglycemia. Positive allosteric modulators of AMPA/kainate receptors already approved to treat other conditions could thus be repurposed to prevent hypoglycemia and improve management of diabetes.

It is now well established that beta cell replacement through pancreatic islet transplantation results in significant improvement in the quality-of-life of type 1 diabetes (T1D) patients. This is achieved through improved control and prevention of severe drops in blood sugar levels. Islet transplant therapy is on the verge of becoming standard-of-care in the USA. Yet, as with other established transplantation therapies, there remain hurdles to overcome to bring islet transplantation to full fruition as a long-lasting therapy of T1D. One of these hurdles is establishing reliable new sites, other than the liver, where durable efficacy and survival of transplanted islets can be achieved. In this article, we discuss the anterior chamber of the eye as a new site for clinical islet transplantation in the treatment of T1D. We specifically focus on the common conceptions, and preconceptions, on the requirements of islet mass, and whether or not the anterior chamber can accommodate sufficient islets to achieve meaningful efficacy and significant impact on hyperglycemia in clinical application.

The World Health Organization projects diabetes prevalence worldwide to be at 4.4% in 2030 compared to 2.8% in the year 2000. These alarming predictions come amid vigorous efforts in diabetes research which have failed so far to deliver effective therapies. Our incomplete understanding of the pathogenesis of diabetes is likely to contribute to the "disconnect" between our research efforts and their translation into successful therapies. Technically, studying the pathophysiology of the pancreatic islets is hindered by the anatomical location of the pancreas, which is deeply embedded in the body, and by lack of experimental tools that enable comprehensive interrogation of the pancreatic islets with sufficient resolution in the context of the natural in vivo environment non-invasively and longitudinally. Emerging evidence also indicates that challenges in successful translation of findings in animal models to the human setting are complicated by some inherent structural and functional differences between the mouse and human islets. In this review, we briefly describe the advantages and shortcomings of existing intravital imaging approaches used to study the pancreatic islet biology in vivo, and we contrast such techniques with a recently established intravital approach using pancreatic islet transplantation into the anterior chamber of the eye. We also provide a summary of recent structure-function studies in the human pancreas to reveal distinctive features of human islets compared with mouse islets. We finally touch on a recently renewed discussion of the validity of animal models in studying human health and disease, and we highlight the potential utility of "humanized" animal models in studying different aspects of human islet biology and improving our understanding of diabetes.

Aims/hypothesis The aim of this study was to provide evidence that the anterior chamber of the eye serves as a novel clinical islet implantation site. Methods In a preclinical model, allogeneic pancreatic islets were transplanted into the anterior chamber of the eye of a baboon model for diabetes, and metabolic and ophthalmological outcomes were assessed. Results Islets readily engrafted on the iris and there was a decrease in exogenous insulin requirements due to insulin secretion from the intraocular grafts. No major adverse effects on eye structure and function could be observed during the transplantation period. Conclusions/interpretation Our study demonstrates the long-term survival and function of allogeneic islets after transplantation into the anterior chamber of the eye. The safety and simplicity of this procedure provides support for further studies aimed at translating this technology into the clinic.

A major problem in medical research is to translate in vitro observations into the living organism. In this perspective, we discuss ongoing efforts to non-invasively image pancreatic islets/β-cells by techniques, such as magnetic resonance imaging and positron emission tomography, and present an experimental platform, which allows in vivo imaging of pancreatic β-cell mass and function longitudinally and at the single-cell level. Following transplantation of pancreatic islets into the anterior chamber of the eye of mice and rats, these islets are studied by functional microscopic imaging. This imaging platform can be utilized to address fundamental aspects of pancreatic islet cell biology in vivo in health and disease. These include the dynamics of pancreatic islet vascularization, islet cell innervation, signal-transduction, change in functional β-cell mass and immune responses. Moreover, we discuss the feasibility of studying human islet cell physiology and pathology in vivo as well as the potential of using the anterior chamber of the eye as a site for therapeutic transplantation in type 1 diabetes mellitus.

The in vivo study of human islet cell biology relies mostly on cumbersome methods and generally islet cell function can only be inferred based on indirect measurements of plasma hormone concentrations. Unfortunately, functional imaging of islets in a living organism remains challenging, since non-invasive or minimally-invasive approaches to monitor islets at cellular resolution are not fully developed. We have previously described that the anterior chamber of the eye (ACE) represents a valuable implantation site for the study of mouse islet structure and function in vivo. In the present study, we have extended the use of the ACE to study human islet cell biology in vivo. Human islets were transplanted into the ACE of streptozotocin-induced diabetic athymic nude mice. After transplantation of 500 islet equivalents per eye, recipient mice achieved and maintained normoglycemia for over 150 days. Tight control of plasma glucose concentrations by transplanted islets was observed during intraperitoneal glucose tolerance tests performed two months after implant. Longitudinal imaging of human islet revascularization was performed by confocal laser scanning microscopy following intravenous injection of fluorescein dextran. Islets were visualized with reflected light. Human islet neovascularization was completed within one month from implantation into the ACE. The time to diabetes reversal paralleled the progression of neovascularization.

The cytoarchitecture of human islets has been examined, focusing on cellular associations that provide the anatomical framework for paracrine interactions. By using confocal microscopy and multiple immunofluorescence, we found that, contrary to descriptions of prototypical islets in textbooks and in the literature, human islets did not show anatomical subdivisions. Insulin-immunoreactive β cells, glucagon-immunoreactive α cells, and somatostatin-containing δ cells were found scattered throughout the human islet. Human β cells were not clustered, and most (71%) showed associations with other endocrine cells, suggesting unique paracrine interactions in human islets. Human islets contained proportionally fewer β cells and more α cells than did mouse islets. In human islets, most β, α, and δ cells were aligned along blood vessels with no particular order or arrangement, indicating that islet microcirculation likely does not determine the order of paracrine interactions. We further investigated whether the unique human islet cytoarchitecture had functional implications. Applying imaging of cytoplasmic free Ca2+ concentration, [Ca2+]i, we found that β cell oscillatory activity was not coordinated throughout the human islet as it was in mouse islets. Furthermore, human islets responded with an increase in [Ca2+]i when lowering the glucose concentration to 1 mM, which can be attributed to the large contribution of α cells to the islet composition. We conclude that the unique cellular arrangement of human islets has functional implications for islet cell function.

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA-binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining beta-cell viability. For these reasons we have developed novel methods for the specific assessment of beta-cell content and viability in human islets based on cellular composition analysis through laser scanning cytometry (LSC) coupled with identification of beta-cell-specific apoptosis at the mitochondrial level. Our novel analytical methods hold promise to prospectively analyze clinical islet transplantation preparations and predict functional performance, as suggested by the observed correlation with in vivo analysis of islet potency in immunodeficient rodents.

Severe visual loss in patients with age‐related macular degeneration is associated with the development of choroidal neovascularization (CNV). The pathogenic mechanisms for CNV formation have been extensively investigated, but remarkably little research has addressed the mechanisms for dysfunction of the retina in CNV. Using laser‐induced CNV in mice, we evaluated the mechanisms of retinal dysfunction. At 3 days, 1 week, 2 weeks, and 4 weeks after laser application, retinas under experimental CNV were characterized physiologically (ERG recordings, synaptic uptake of the exocytotic marker FM1‐43, and light‐induced translocation of transducin), histologically, and immunohistochemically. ERG amplitudes were reduced by 20% at 1 week after CNV. Depolarization‐induced FM1‐43 uptake in photoreceptor synapses was selectively reduced by 45% at 1 week after CNV. Although photoreceptor outer segments were shortened by 36%, light adaptation as measured by transducin translocation was mostly preserved. Early in CNV (3 days to 1 week), Muller cells demonstrated induction of c‐fos and pERK expression. Also, the density of macrophage‐like, F4/80 immunoreactive cells increased ∼3‐fold. Minimal photoreceptor death occurred during the first week, and was variable thereafter. At later times in CNV formation (≥2 weeks), expression of photoreceptor synaptic markers was reduced in the outer plexiform layer, indicating loss of photoreceptor synaptic terminals. ERG amplitudes, synaptic uptake of FM1‐43, and the induction of c‐fos and pERK in Muller cells were altered within 1 week of experimental CNV, suggesting that during CNV formation, deficits in retinal function, in particular photoreceptor synaptic function, precede degeneration of photoreceptor terminals and photoreceptor cell death. J. Comp. Neurol. 483:263–277, 2005. © 2005 Wiley‐Liss, Inc.

Choroidal neovascularization (CNV) is characterized by the subretinal invasion of a pathologic new vessel complex from the choriocapillaris. Although CNV is traditionally considered to consist of endothelial cells, the cellular population of CNV is likely more complex in nature, comprising several different cell types. In addition, recent studies suggest that the CNV cell population has a dual origin (circulating versus resident populations). In this study we sought to determine the contribution and origin of different cell types in experimental CNV. Laser-induced CNV was performed on chimeric mice generated by reconstituting C57BL/6 mice with bone marrow from green fluorescent protein (GFP)-transgenic mice. In these mice, bone marrow-derived cells are GFP-labeled. Immunofluorescence staining was used to examine both flatmount preparations of the choroid and cross sections of the posterior pole for macrophages, endothelial cells, vascular smooth muscle cells, retinal pigment epithelial (RPE) cells, lymphocytes, or neutrophils at day 3, 7, 14 and 28 post-laser (n=5 per group). Cell types present in CNV included macrophages (20% of the cells in CNV), endothelial cells (25%), vascular smooth muscle cells (11%), RPE cells (12%) and non-labeled cells (32%). The macrophage population was mostly derived from circulating monocytes at all timepoints studied (70% were GFP labeled), while endothelial and vascular smooth muscle cells were partly bone marrow derived (50–60% were GFP labeled), and RPE cells appeared to be entirely derived from preexisting tissue resident cells. These results demonstrate that bone marrow-derived progenitor cells contribute significantly to the vascular and inflammatory components of CNV. Knowledge of the cellular composition and origin might help understand the pathogenic mechanisms controlling CNV severity as well as indicate potential targets for therapeutic intervention.

Taste receptor cells are innervated by primary gustatory neurons that relay sensory information to the central nervous system. The transmitter(s) at synapses between taste receptor cells and primary afferent fibers is (are) not yet known. By analogy with other sensory organs, glutamate might a transmitter in taste buds. We examined the presence of AMPA and NMDA receptor subunits in rat gustatory primary neurons in the ganglion that innervates the anterior tongue (geniculate ganglion). AMPA and NMDA type subunits were immunohistochemically detected with antibodies against GluR1, GluR2, GluR2/3, GluR4 and NR1 subunits. Gustatory neurons were specifically identified by retrograde tracing with fluorogold from injections made into the anterior portion of the tongue. Most gustatory neurons in the geniculate ganglion were strongly immunoreactive for GluR2/3 (68%), GluR4 (78%) or NR1 (71%). GluR1 was seen in few cells (16%). We further examined if glutamate receptors were present in the peripheral terminals of primary gustatory neurons in taste buds. Many axonal varicosities in fungiform and vallate taste buds were immunoreactive for GluR2/3 but not for NR1. We conclude that gustatory neurons express glutamate receptors and that glutamate receptors of the AMPA type are likely targeted to synapses within taste buds.

Inflammation is a major mechanism in the pathogenesis of age-related macular degeneration, the most important cause of blindness in the elderly. Previous studies have focused on the role of macrophages in regulating the growth of pathological new vessels over the retina, called choroidal neovascularization (CNV). However, no research has been done to evaluate the role of inflammation as a mechanism of vision loss and retinal degeneration in the retina underlying CNV. In other neuropathological conditions, hematogenous macrophages and/or resident microglia contribute to neurodegeneration. We have combined laser-induced CNV in mice and bone marrow transplantation with GFP-labeled bone marrow to determine the relative role of recruited blood-derived macrophages versus resident microglia in the retina associated with CNV. Using these chimeric mice, we have found that many GFP-labeled cells infiltrated the retina underlying CNV but not the retina unaffected by CNV. Immunostaining for the cell adhesion molecules VCAM 1, ICAM 1, and PECAM was strongly upregulated in retinal blood vessels under CNV. All GFP-labeled cells were immunoreactive for the macrophage marker F4/80. Most (70%) of the F4/80 immunoreactive cells were GFP-labeled under CNV. The density of resident microglia did not increase. Most GFP-labeled cells were found in close proximity to activated Muller cells. Depleting circulating macrophages with clodronic acid diminished the density of F4/80 immunoreactive cells as well as the density of pERK immunoreactive Muller cells in the retina under CNV. Thus, recruitment of blood-derived macrophages more than resident microglia seems to be associated with CNV.

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 muM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 muM) and blocked by GYKI 52466 (80 muM) and Joro spider toxin (1 muM). It was unaffected by D-AP5 (50 muM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 muM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynatically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.