In vivo gene therapy in young and adult RPE65-/- dogs produces long-term visual improvement.

Defects in the RPE65 gene, which is selectively expressed in the retinal pigment epithelium (RPE), result in blindness and gradual photoreceptor cell degeneration. Experiments were conducted to assess the efficacy of gene replacement therapy in restoring retinal function in RPE65-/- dogs. Long-term effects of RPE65 gene therapy were assessed using visual behavioral testing and electroretinographic (ERG) recordings at 10-12 weeks and 6-9 months after surgery in five affected dogs. Subretinal injections of similar dosages of two constructs were performed in affected dogs at the ages of 4-30 months: rAAV.RPE65 into one eye and, in four of five dogs, rAAV.GFP contralaterally. Before surgery all RPE65-/- dogs were behaviorally blind with either no or very low-amplitude ERG responses to light stimuli. Marked improvements in visual behavior and ERG responses were observed as early as 4 weeks after surgery in affected animals. Except for light-adapted 50 Hz ERG flicker responses, all ERG parameters tested increased significantly in the eyes treated with the rAAV.RPE65 construct at the early follow-up. Gradual progressive improvements in ERG responses were observed in the RPE65-treated eyes over time. An unexpected finding was that on long-term follow-up, marked improvement of photopic ERG responses were also observed in the contralateral control eye in both young and older dogs. These results are promising for future clinical trials of human patients with retinal degenerative diseases, such as Leber congenital amaurosis, that result from RPE65 gene defects.

Effects of pituitary adenylate-cyclase-activating polypeptide on the direct-current electroretinogram of the rabbit eye.

Pituitary adenylate-cyclase-activating polypeptide (PACAP) is a recently discovered neuropeptide present in two different forms, PACAP-27 and PACAP-38. Both peptides stimulate the catalytic enzyme adenylate cyclase in pituitary cells. This enzyme is important also regarding the function of the retinal pigment epithelium (RPE). The purpose of the study was to investigate possible influences of PACAP on the rabbit retina and the RPE as reflected in the direct-current (d.c.) electroretinogram (ERG) and the standing potential of the eye (SP). After unilateral sector vitrectomy, a continuous intraocular perfusion with a reference solution alternated with a test solution was established. The corneal d.c. ERG and the SP were recorded from both eyes with the contralateral eye as a control. Both PACAP-27 (0.1 and 1 microM) and PACAP-38 (1 microM) increased the c-wave amplitude significantly (p = 0.028, p = 0.013 and p = 0.024, respectively, n = 4) while neither PACAP-27 (0.1 and 1 microM, p > 0.05, n = 4) nor PACAP-38 (1 microM, p > 0.05, n = 4) produced any significant effects on the a- and b-wave amplitudes of the d.c. ERG. The SP response to the two substances differed with a significant elevation of the SP level with PACAP-27 (1 microM, p = 0.017, n = 4), while PACAP-38 induced a small, nonsignificant SP elevation (1 microM, p > 0.05, n = 4). Retinal penetrations during PACAP-27 (10 microM) perfusion showed an increase in transepithelial potential (TEP) c-wave (p = 0.003) as well as in slow PIII (p = 0.011, n = 3) amplitude level. The results support the presence of PACAP receptors both on the RPE and in the neural retina.

Serotonin elevates the c-wave of the electroretinogram of the rabbit eye by increasing the transepithelial potential.

The influence of serotonin (5-hydroxytryptamine, 5-HT) and serotonin analogues on the direct current electroretinogram (d.c. ERG) and the standing potential of the albino rabbit eye (SP) was studied. After unilateral vitrectomy, corneal recordings were obtained during simultaneous intravitreal perfusion with a control solution alternating with 5-HT at concentrations of 25, 120 and 200 microM. The c-wave increased at 25 and 120 microM when changing from control solution to test solution (P < 0.05) but did not decrease significantly when changing back to control solution (P > 0.05). The c-wave was reversibly elevated at 200 microM (PHS-5-HT, P < 0.01; 5-HT-PHS, P < 0.05). To analyse further the influence on the c-wave, in vivo intraretinal microelectrode recordings were obtained during intravitreal perfusion with 5-HT. The transepithelial potential (TEP) increased (P < 0.01), while the slow PIII was not significantly affected (P > 0.05). The serotonin receptor agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, 5-methoxytryptamine, alpha-methyl-5-hydroxytryptamine and 2-methyl-5-hydroxytryptamine, caused a significant reversible elevation of the c-wave, whereas 5-carboxyamidotryptamine did not. Tropisetron did not block the serotonin effect and LY53857 had an effect of its own on the c-wave. The results seem to indicate that the influence of serotonin on the c-wave is mainly due to an effect on the retinal pigment epithelium (RPE) and that more than one type of serotonin receptor may be involved.

Effects of PhXA41, a prostaglandin analogue, and PGF2 alpha on the corneal and intraretinal d.c. electroretinogram (ERG) of the albino rabbit eye.

PhXA41, a prostaglandin F2 alpha analogue, is a new drug alternative for medical treatment of glaucoma. From earlier studies, it is known that certain prostaglandins (pgs) exert an influence on the d.c. electroretinogram (ERG). In the present study, possible effects of intraocular perfusion with PhXA41 (free acid) or PGF2 alpha on the rabbit retina and retinal pigment epithelium (RPE) were evaluated. Unilateral vitrectomy was performed and a continuous intraocular perfusion was established, in which a reference solution was alternated with the test solution. In part one of the experiments, the corneal d.c. ERG and the standing potential of the eye (SP) were recorded from both eyes. In part two, vitreal and intraretinal d.c. ERG were recorded in the vitrectomized eye. PhXA41 and PGF2 alpha produced similar effects, i.e. a significant and reversible c-wave increase at concentrations of 1-10 micrograms/ml. No influence was found in the concentration interval 10-100 ng/ml. There were no changes in a- and b-wave amplitudes at any of the concentrations tested. At a concentration of 10 micrograms/ml, the SP increased significantly. The intraretinal d.c. ERG showed a large reduction of slow PIII accompanied by a small change in transepithelial potential (TEP). Theoretically, a vitreous concentration of less than 1 ng/ml may be reached after topical application. This concentration level does not influence the d.c. ERG in this animal model. At higher concentrations, a significant and reversible c-wave increase seemed to be generated mainly in the inner retina by a reduction of slow PIII generated in the Müller cells. The effect of PhXA41 appears to be mediated by an FP receptor.

Adrenergic effects on the corneal and intraretinal direct-current electroretinogram and on the standing potential of albino rabbit eyes.

This study was undertaken to investigate further the responsiveness of the albino rabbit retinal pigment epithelium and the inner retina to adrenergic agents as reflected in changes of the direct-current electroretinogram and of the standing potential of the eye. After unilateral vitrectomy on albino rabbits, a continuous intraocular perfusion with a reference solution was established. The reference solution was then alternated with the test solution. The direct-current electroretinogram and the standing potential were recorded from both eyes with a scleral contact lens and a reference electrode connected to matched calomel half-cells. An in vivo experimental technique that allows intraocular perfusion of a test substance and simultaneous intraretinal microelectrode measurements was also used. The alpha-adrenergic agonist phenylephrine (0.04 microM, n = 8) produced a reversible increase in c-wave amplitude (48%, p < 0.001) and also a small increase in b-wave amplitude (12%, p < 0.002). There was no significant influence on the a-wave amplitude. The standing potential was elevated at 1694 +/- 362 microV (mean +/- SEM) (p < 0.002). The alpha 2-adrenergic agonist clonidine caused similar effects on the electroretinogram, although at a higher concentration (40 microM, n = 5), with an elevation of the c-wave (25%, p < 0.001) and a small b-wave increase (12% p < 0.002). No significant influence on the a-wave or on the standing potential was found. Intraretinal direct-current electroretinogram-recordings during intraocular perfusion with phenylephrine showed an increase in transepithelial potential (p < 0.004; n = 6), accompanied by a reduction of the slow PIII (p < 0.0035; n = 6). The c-wave increase resulting from alpha-adrenergic stimulation seems to be generated partly across the retinal pigment epithelium, with an increase in transepithelial potential, combined with a reduction of the slow PIII. The elevation of the b-wave amplitude, together with the influence on the slow PIII, suggests alpha-adrenergic effects also on the inner retina. The experimental technique used in this study with intraocular perfusion after vitrectomy and simultaneous intraretinal direct-current recordings seems to be a practicable method for studies of the influence of pharmacologic agents on the retina and the retinal pigment epithelium.

A cyclic adenosine monophosphate agonist elevates the b- and c-waves of the rabbit direct-current electroretinogram.

The effects of the stable cyclic adenosine monophosphate analogue adenosine 3',5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMPS) on the direct-current electroretinogram and the standing potential of the eye were studied. Corneal recordings were obtained from unilaterally vitrectomized albino rabbit eyes during alternating intravitreal perfusions with Sp-cAMPS and a control solution (Pharmacia eye irrigating solution). The contralateral eye was used as a control. To evaluate further the effects on the c-wave, in vivo intraretinal microelectrode measurements were made during simultaneous intravitreal perfusion of Sp-cAMPS and irrigating solution, respectively. Sp-cAMPS in concentrations of 1, 10 and 100 microM was tested by corneal direct-current electroretinography. There was no significant effect on the a-wave amplitude. The b-wave amplitude was reversibly elevated at an Sp-cAMPS concentration of 100 microM was tested by corneal direct-current electroretinography. There was no significant effect on the a-wave amplitude. The b-wave amplitude was reversibly elevated at an Sp-cAMPS concentration of 100 microM (p < 0.01, n = 7). The c-wave amplitude was reversibly elevated at an Sp-cAMPS concentration of 100 microM (p < 0.01, n = 7). The c-wave amplitude was reversibly elevated at a concentration of 10 microM (p < 0.001, n = 8), and this effect was more pronounced at 100 microM (p < 0.001, n = 7). The SP increased reversibly at a concentration of 100 microM (p < 0.001, n = 7). Microelectrode recordings were performed with Sp-cAMPS at a concentration of 100 microM. The recordings showed significant increases in both the transepithelial potential (p < 0.01, n = 3) and the slow PIII (p < 0.01, n = 3). The effects of Sp-cAMPS on the b-wave as well as the two components of the c-wave suggest influences on both the inner retina and the retinal pigment epithelium of the rabbit eye.

Effects of quisqualic acid on the corneal and intraretinal direct-current electroretinogram and on the standing potential of the rabbit eye.

Quisqualic acid, an excitatory amino acid agonist, has been shown to stimulate inositol phosphate production in the rabbit retina. Inositol trisphosphate serves as a second messenger and increases intracellular calcium. We investigated the influence of quisqualic acid on the direct-current electroretinogram and on the standing potential of the rabbit eye. After unilateral vitrectomy, the corneal direct-current electroretinogram and the standing potential were recorded from both eyes of albino rabbits during simultaneous unilateral intravitreal perfusion with quisqualic acid alternating with control solution. The contralateral eye was used as a control. Intravitreal perfusion with 100-microM and 200-microM quisqualic acid elevated the standing potential significantly. This elevation was accompanied by a significant increase in c-wave amplitude and a significant decrease in b-wave amplitude. Quisqualic acid at 200-microM concentration decreased the a-wave amplitude also. In vivo intraretinal recordings showed that intravitreal perfusion with quisqualic acid at 200-microM concentration significantly increased the retinal pigment epithelial component of the c-wave. We conclude that quisqualic acid influences the direct-current electroretinogram and the standing potential apparently through its action on the retinal pigment epithelium. A possible mode of action is increased production of inositol trisphosphate, followed by an increase in intracellular release of calcium ions and an increase in basal chloride conductance. The decrease in a- and b-wave amplitudes indicates direct effects of quisqualic acid also on the neural retina.