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| July 2002 | Inside IOVS | Volume 43/7 |
| Novel Basic Approaches to Treating Ocular Diseases |
Propagation of Corneal Stem Cells
Cultivated corneal epithelial cell transplantation is a recently developed treatment for severe ocular surface diseases. Most groups using this technique culture their corneal epithelium from limbal explants. In this study, Koizumi et al. (p. 2114), describe a new culture system involving the in vitro propagation of epithelial cells, on amniotic membrane, from enzymatically dissociated limbal epithelial cells. This new cell-suspension culture system produced a healthy epithelial cell layer which should contain stem cells and which was morphologically superior to explant-cultured epithelium. This new technique has considerable potential for use in cultivated corneal limbal epithelial transplantation.
Corneal Lymphangiogenesis
The normal human cornea is devoid of blood and lymphatic vessels, but (hem)angiogenesis accompanies certain corneal diseases. Using novel lymphendothelial markers (LYVE-1, Podoplanin), Cursiefen et al. (p. 2127) present ultrastructural and immunohistochemical evidence for lymphangiogenesis occurring alongside corneal hemangiogenesis. Human corneal lymphangiogenesis, being correlated with the degree of hemangiogenesis, may be regulated by lymphangiogenic growth factor VEGF-C via VEGF receptor 3. In relation to subsequent corneal transplantation, lymphatics in vascularized human host beds adjacent to grafted tissue could enhance antigen presentation in regional lymph nodes and promote rejection. Inhibition of corneal lymphangiogenesis or recruitment of antigen presenting cells into these vessels might enhance transplant survival in the high-risk setting.
Corneal Cryopreservation
Ice-free cryopreservation of cornea below -100ºC has been achieved by vitrification for the first time. As reported by Armitage et al. (p. 2160), this glassy state avoids the inevitable damage associated with ice formation, but requires exposure of the tissue to high concentrations of solutes in order to vitrify at practicable cooling rates. The results, based on morphology and function of the endothelium, are encouraging and could lead the way to the development of an alternative method of cryopreservation for corneas and other tissues.
Slipping and Sliding Causes Squinting?
Connective tissue pulleys inflect the paths of the rectus extraocular muscles, serving as their functional mechanical origins and determining pulling directions. Oh et al. (p. 2169) used quantitative, high resolution magnetic resonance imaging to study subjects with strabismus associated with gaze-dependent sideslip of rectus muscle paths due to instability of one or more pulleys. The strabismic people had horizontal or vertical ocular misalignments that would previously have been blamed on other causes, such as pathology of the trochlea or superior oblique tendon. The imaging findings suggest that instability of rectus pulleys should be added to the list of causes of strabismus.
A Mouse Model of Outflow Function
Lowered intraocular pressure following topical application of latanoprost to the mouse eye raises the possibility of a uveoscleral outflow pathway in this species. To investigate this possibility, Lindsey and Weinreb (p. 2201) injected fluorescent dextran into the anterior chamber of mouse eyes and then examined the distribution of this tracer within paraffin sections of the eyes. Over the course of an hour, the tracer appeared sequentially in the trabecular meshwork and ciliary process stroma, in the anterior choroid, and in the equatorial choroid and adjacent sclera. These tracer movements indicate that at least a portion of aqueous outflow in the mouse eye is through the uveoscleral outflow pathway. This supports use of the mouse as a model in which to study uveoscleral outflow function.
Glutamate Transporter Changes in Glaucoma
Glutamate transporters help protect against the toxic effects of excess extracellular glutamate throughout the central nervous system. Glutamate-induced excitotoxicity has been proposed as a possible cause of retinal ganglion cell death in glaucoma. Martin et al. (p. 2236) report significant reductions in the retinal levels of two glutamate transporter proteins, GLT-1 and GLAST, in an experimental rat glaucoma model. The fall in glutamate transporter levels preceded significant retinal ganglion cell loss and was not observed following optic nerve transection. It is therefore suggested that reductions in glutamate transporters may play a causative role in glaucomatous retinal ganglion cell death.
HSV-1 Latency in the Ciliary Ganglion
The trigeminal ganglion is a site of HSV-1 latency. To determine whether HSV-1 may be latent in other ganglia, PCR was used by Bustos and Atherton (p. 2244) to amplify HSV-1 sequences from formalin fixed human ciliary and trigeminal ganglia. The prevalence of virus-positive ciliary ganglia (66.6%) was approximately the same as the prevalence of virus-positive trigeminal ganglia (68.0%) suggesting that the ciliary ganglion may be an additional site of herpesvirus latency. Spread of reactivated HSV-1 from the ciliary ganglion to neuronal pathways synaptically connected to the optic nerve and retina resulting in retinal infection may be one explanation for the pathogenesis of acute retinal necrosis (ARN) caused by HSV-1.
LPS - Induced Keratitis
Contact lens associated peripheral ulcers (CLPU) and contact lens associated red eye (CLARE), often occur in the absence of live bacteria and are associated with sterile infiltrates. Khatri et al. (p. 2278) have developed a murine model for lipopolysaccharide (LPS, endotoxin) – induced keratitis, which is measured quantitatively by in vivo scanning confocal microscopy. Using neutralizing antibodies, they demonstrated an essential role for neutrophils, and identified adhesion molecules and chemokines that mediate neutrophil infiltration, and an essential role for Toll like receptor 4. Taken together, this study may lead to new therapeutic approaches to these manifestations of keratitis.
Cataract Prevention by Src Kinase Inhibition
While many studies suggest that stress factors cause lens opacities, little is known about the pathways involved in this process. Zhou and Menko (p. 2293) examined whether the Src kinases, which are activated downstream of many stress factors, mediate the signaling pathways that lead to lens opacification. Their studies show that cataract formation could be prevented by exposure of cultured lenses to specific inhibitors of Src kinase activity. This treatment allowed the lenses to maintain their normal cytoarchitecture. By deciphering the components of this Src signaling pathway it should be possible to develop therapies that target these molecules and prevent the progression of cataract.
TGFb2 AND PCO
A critical aspect of posterior capsule opacification (PCO) development following cataract surgery is deformation of the lens capsule. Wormstone et al. (p. 2301) provide evidence using a human culture system, based on a cataract operation, that exposure to TGFb2 can stimulate lens cells to both increase expression of alpha smooth muscle actin (a transdifferentiation marker) and to form light scattering wrinkles. These changes were successfully inhibited by the fully human monoclonal anti-TGFb2 antibody, CAT-152, and thus application at the time of surgery could provide therapeutic benefit for cataract patients.
Nucleotide Regulation of Aqueous Humor
It has been recently reported that topical application of ATP or some of its derivatives increases the intraocular pressure in rabbit, while other ATP analogues lower it. Not much is known about the cellular location of the purinergic receptors, and signaling pathways mediating these effects. Farahbakhsh and Cilluffo (p. 2317) provide evidence for the presence of two subtypes of purinergic receptors in nonpigmented epithelial cells of the rabbit ciliary body, differentially coupled to the intracellular second messenger, Ca2+. These results suggest a possible mechanism for regulation of the aqueous humor formation by purine and pyrimidine nucleotides.
Glucose Uptake in Diabetic Retinopathy
Hyperglycemia is well known to be a major causative factor for the development of diabetic retinopathy; however, the regulation of retinal glucose uptake in diabetes remains an area of controversy. Results from this study by Busik et al. (p. 2356) demonstrate that short-term exposure cells isolated from human inner (retinal vascular endothelial) and outer (retinal pigmented epithelial) blood-retinal barrier (BRB) to elevated glucose induce increases in the Vmax of glucose. The observed increase in the velocity of glucose uptake might explain how high levels of glucose initially enter the retina, before BRB is compromised in diabetes.
Gene Therapy for Axotomized RGCs
Brain-derived neurotrophic factor (BDNF) gene was injected intravitreally followed by in vivo electroporation, and the over-expression of BDNF was confirmed by western immunoblotting and immunohistochemistry. The survival of axotomized RGCs was estimated by the TdT-dUTP terminal nick-end labeling (TUNEL) method and measured by counting the number of DiI labeled RGCs. These findings by Mo et al. (p. 2401) demonstrated that electroporation effectively delivered gene into RGCs, and the axotomized RGCs were rescued by transferred BDNF gene. The survival effect produced by these methods might be useful in treating the diseases of RGC.
Gene Therapy for PVR
Growth factors are widely believed to contribute to the development of proliferative vitreoretinopathy (PVR). As shown in Ikuno and Kazlauskas (p. 2406), blocking the function of platelet-derived growth factor was found to attenuate experimental PVR. This gene therapy-based strategy may turn out to be an effective new approach to treat PVR in the future.
Gene Therapy Causing NV Regression
Mori et al. (p. 2428) show that in mice with established ocular neovascularization, intraocular injection of an adenoviral vector encoding pigment epithelium-derived factor (PEDF) results in regression of the neovascularization by inducing apoptosis in vascular cells participating in the neovascularization. There was no apoptosis of vascular cells of normal retinal or choroidal vessels, indicating that PEDF selectively affects new vessels. This suggests that PEDF gene transfer may be useful for both treatment and prevention of ocular neovascularization.
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