Role of Thermo-responsiveness and Poly(ethylene glycol) Diacrylate Cross-link Density on Protein Release from Poly(N-isopropylacrylamide) Hydrogels.

Thermo-responsive hydrogels have shown promise as injectable materials for local drug delivery. However, the phase-induced changes in polymer properties of N-isopropylacrylamide (NIPAAm) can pose additional challenges for achieving controlled protein release. In this work, thermo-responsive hydrogels derived from NIPAAm and cross-linked with poly(ethylene glycol) diacrylate (PEG-DA) were synthesized via free radical polymerization. The volume phase transition temperature (VPTT) of the hydrogels ranged from 32.9°C to 35.9°C. Below the VPTT, swelling ratios of the hydrogels decreased with cross-linker concentration, and showed a sharp drop (at least 4-fold) upon phase change. Protein encapsulation efficiency was high (84-90%) and decreased with cross-linker concentration. Release of bovine serum albumin, a model protein, at body temperature was significantly higher than at room temperature (67% at 37°C compared to 44% at 23°C after 48 h). The release kinetics of proteins from the hydrogels were initially expected to be a function of cross-link density. However, at the hydrogel compositions explored in this work, protein release did not change significantly with cross-linker mol fraction. The thermo-responsive hydrogels offer a promising platform for the localized delivery of proteins.

Effects of inhibition of neuronal nitric oxide synthase on basal retinal blood flow regulation.

Nitric oxide (NO) has been observed to regulate blood flow under basal and stimulated conditions in the retina. Recent evidence suggests that NO produced by neuronal nitric oxide synthase (nNOS) may regulate blood flow in addition to that produced by endothelial nitric oxide synthase (eNOS). The objective of the current study was to investigate the contribution of NO produced by nNOS in the regulation of basal retinal blood flow. A non-specific NOS inhibitor N (G)-nitro-l-arginine methyl ester (l-NAME) and the specific nNOS inhibitors 1-(2-trifluoromethylphenyl) imidazole (TRIM) and (4S)-N-(4-amino-5 [aminoethyl] aminopentyl)-N-nitroguanidine (AAAN) were injected into the vitreous (intravitreal) of Long-Evans rats. Vessel diameters, velocities and volumetric blood flow rates (VBF) in the retinal circulation were determined prior to and in 30-min intervals for 4-4.5h after injection. In addition, the basal amount of nNOS in the rat retina was quantified using a specific enzyme linked immunoassay (ELISA). Treatment with l-NAME and TRIM significantly decreased diameters and VBF. Compared with saline, treatment with l-NAME and TRIM produced a significant (p<0.001) decrease of approximately 12-17% in vessel diameters. Treatment with AAAN significantly decreased vessel diameters and venous VBF. Compared with saline AAAN produced a significant decrease of approximately 7% in arterial (p<0.001) and 5% in venous (p=0.011) diameters, respectively. The amount of nNOS in the rat retina was 0.17+/- 0.0147 pmol mg(-1) of dry retina. The results suggest that though inhibition of nNOS decreases basal diameters, constant VBF is maintained in the retinal circulation.

Preservation of intact adult rat photoreceptors in vitro: study of dissociation techniques and the effect of light.

PURPOSE: Intact adult photoreceptors in culture can be a valuable tool in the search of therapies for retinal degenerations. The major challenge in this technique is that photoreceptors undergo an alteration in cytoarchitecture and loss of outer segment during the cell culture process. This study compared techniques for the isolation of photoreceptor cells from adult rat retinas to determine which technique yields the highest percent of structurally well preserved cells in vitro. In addition, the role of light exposure during the dissociation and culture process was investigated to minimize photoreceptor cell deformation over time in culture. METHODS: Photoreceptor cells from adult rat retinas were isolated and quantified using three dissociation techniques: enzymatic dissociation with gentle pipeting; enzymatic dissociation with gentle pipeting and centrifugation; and non-enzymatic dissociation with gentle pipeting. To evaluate the effect of light exposure on cell deformation, we performed dissociations and cell seeding both in dark- and light-adapted conditions and measured the deformation of photoreceptors over a 12 h period right after dissociation. Cell viability in both conditions was evaluated after 4 and 7 days in culture. Preservation of cell structure in culture was assessed by immunofluorescence labeling of cells with anti-rhodopsin and 4',6-diamidino-2-phenylindole (DAPI) nuclear staining. RESULTS: An enzymatic technique followed by gentle pipeting or mechanical trituration yielded the highest number of intact elongated photoreceptors right after dissociation. Data suggested that centrifugation after the dissociation contributed to cell deformation immediately after isolation. Immunohistochemistry results showed that cells had deformed into a circular shape by 2 days after seeding. However, photoreceptors isolated in dark conditions maintained their elongated shape, even 7 days after seeding. Performing experiments in dark also promoted a higher number of cells to remain viable with time. CONCLUSIONS: The current study demonstrated the importance of proper isolation techniques to obtain the maximum amount of intact photoreceptor cells. The data suggested that a gentle dissociation technique, consisting of enzymatic treatment followed by moderate pipeting of the retinal tissue, may be the key to obtain a high number of intact or structurally preserved photoreceptors. Furthermore, isolation and cell culture procedures performed under dark conditions may facilitate to maintain high number of elongated photoreceptor cells in vitro.

Suppressors of cytokine-signaling proteins induce insulin resistance in the retina and promote survival of retinal cells.

OBJECTIVE: Suppressors of cytokine signaling (SOCS) are implicated in the etiology of diabetes, obesity, and metabolic syndrome. Here, we show that some SOCS members are induced, while others are constitutively expressed, in retina and examine whether persistent elevation of SOCS levels in retina by chronic inflammation or cellular stress predisposes to developing insulin resistance in retina, a condition implicated in diabetic retinopathy. RESEARCH DESIGN AND METHODS: SOCS-mediated insulin resistance and neuroprotection in retina were investigated in 1) an experimental uveitis model, 2) SOCS1 transgenic rats, 3) insulin-deficient diabetic rats, 4) retinal cells depleted of SOCS6 or overexpressing SOCS1/SOCS3, and 5) oxidative stress and light-induced retinal degeneration models. RESULTS: We show that constitutive expression of SOCS6 protein in retinal neurons may improve glucose metabolism, while elevated SOCS1/SOCS3 expression during uveitis induces insulin resistance in neuroretina. SOCS-mediated insulin resistance, as indicated by its inhibition of basally active phosphoinositide 3-kinase/AKT signaling in retina, is validated in retina-specific SOCS1 transgenic rats and retinal cells overexpressing SOCS1/SOCS3. We further show that the SOCS3 level is elevated in retina by oxidative stress, metabolic stress of insulin-deficient diabetes, or light-induced retinal damage and protects ganglion cells from apoptosis, suggesting that upregulation of SOCS3 may be a common physiologic response of neuroretinal cells to cellular stress. CONCLUSIONS: Our data suggest two-sided roles of SOCS proteins in retina. Whereas SOCS proteins may improve glucose metabolism, mitigate deleterious effects of inflammation, and promote neuroprotection, persistent SOCS3 expression caused by chronic inflammation or cellular stress can induce insulin resistance and inhibit neurotrophic factors, such as ciliary neurotrophic factor, leukemia inhibitory factor, and insulin, that are essential for retinal cell survival.

Scanning laser ophthalmoscope-particle tracking method to assess blood velocity during hypoxia and hyperoxia.

The main objective was to evaluate a Scanning Laser Ophthalmoscope (SLO) based particle tracking method as a means of quantitative assessment of retinal blood velocity and vessel diameter changes in response to hypoxia and hyperoxia. Retinal blood velocities were measured by tracking fluorescent microspheres (1.0 microm diameter) in anesthetized adult pigmented rats. Velocities were calculated based on microsphere position changes and the recording frame rate. Hypoxia was induced by inspiring a mixture of nitrogen and air and hyperoxia was induced by inspiring 100% oxygen. Average blood velocities during hypoxia obtained for arteries, veins, and small vessels (diameter < 40 microm) were 39.9 +/- 9.9, 34.9 +/- 2.7, and 8.8 +/- 1.8 mm/sec, respectively, whereas during hyperoxia, the average blood velocities obtained were 23.7 +/- 6.2, 28.2 +/- 2.7, and 7.6 +/- 0.7 mm/sec. Hypoxia was found to increase the diameters of arteries by 25% but did not change the diameters of veins; whereas, hyperoxia was found to decrease their diameters by 25% and 18%. Changes detected in vessel diameter and blood velocity suggest that the level of oxygen tension alters retinal hemodynamics. Dynamics of retinal hemodynamics in response to hypoxia and hyperoxia can be assessed using the SLO imaging method.

Thermoresponsive hydrogels as a new ocular drug delivery platform to the posterior segment of the eye.

PURPOSE: To characterize thermoresponsive hydrogels (liquids at room temperature, gels at body temperature) as a novel drug delivery platform to the posterior segment. METHODS: Thermoresponsive hydrogels were synthesized using poly(N-isopropylacrylamide) (PNIPAAm), cross-linked with poly(ethylene glycol) diacrylate (PEG-DA). Proteins were then encapsulated into the hydrogels, including bovine serum albumin (BSA), immunoglobulin G (IgG), and, finally, bevacizumab and ranibiumab. By varying the degree of cross-linker density, the rate of protein release could be adjusted. The rate of release was assessed at various time points with Bradford assay, and the bioactivity of the released anti-vascular endothelial growth factor agents was studied in an in vitro cell culture assay. RESULTS: Cross-linked PNIPAAm hydrogel exhibited a fast and reversible phase change with alteration in temperature. The rate of protein release was examined as a function of cross-link density. Release profiles of the proteins showed that there was an initial burst of release within 48 hours, and then a steady state was reached, which was sustained for approximately 3 weeks. Hydrogels with less cross-linking showed faster release and yielded a more pliable gel for intravitreal injection via small-gauge needles. Examination of the gels after the release experiment revealed significant residual entrapped protein. CONCLUSION: Thermoresponsive hydrogels were successfully synthesized and exhibited fast and reversible phase changes. The gel was able to encapsulate and release various proteins. Current formulation of the gel will be modified to extend the release time and to be made fully biodegradable. Thermoresponsive hydrogels appear to be a promising, minimally invasive platform for extended drug delivery to the posterior segment.

Test of the paired-flash electroretinographic method in mice lacking b-waves.

Previous studies of rod photoreceptors in vivo have employed a paired-flash electroretinographic (ERG) technique to determine rod response properties. To test whether absence versus presence of the ERG b-wave affects the photoreceptor response derived by the paired-flash method, we examined paired-flash-derived responses obtained from nob mice, a mutant strain with a defect in signal transduction between photoreceptors and ON bipolar cells that causes a lack of the b-wave. Normal littermates of the nob mice served as controls. The normalized amplitude-intensity relation of the derived response determined in nob mice at the near-peak time of 86 ms was similar to that determined for the controls. The full time course of the derived rod response was obtained for test flash strengths ranging from 0.11 to 17.38 scotopic cd s m(-2) (sc cd s m(-2)). Time-course data obtained from nob and control mice exhibited significant but generally modest differences. With saturating test flash strengths, half-recovery times for the derived response of nob versus control mice differed by approximately 60 ms or less about the combined (nob and control) average respective values. Time course data also were obtained before versus after intravitreal injection of L-2-amino-4-phosphonobutyrate (APB) (which blocks transmission from photoreceptors to depolarizing bipolar cells) and of cis 2,3-piperidine dicarboxylic acid (PDA) (which blocks transmission to OFF bipolar cells, and to horizontal, amacrine and ganglion cells). Neither APB nor PDA substantially affected derived responses obtained from nob or control mice. The results provide quantitative information on the effect of b-wave removal on the paired-flash-derived response in mouse. They argue against a substantial skewing effect of the b-wave on the paired-flash-derived response obtained in normal mice and are consistent with the notion that, to good approximation, this derived response represents the isolated flash response of the photoreceptors in both nob and normal mice.

The electroretinogram components in Abyssinian cats with hereditary retinal degeneration.

PURPOSE: To examine phototransduction using the a-wave and other aspects of retinal function with the intraretinal b- and c-waves at different stages of an inherited photoreceptor degeneration in Abyssinian cats. METHODS: Vitreal and intraretinal ERGs were recorded from eight dark-adapted, anesthetized Abyssinian cats. Brief bright flashes were used to elicit vitreal a- and b-waves. Longer, weaker flashes were used to elicit intraretinal b- and c-waves. Stages 1 through 4 of the disease were characterized ophthalmoscopically. Parameters of the Lamb and Pugh a-wave model (a(max), A, and t(eff)) for the Abyssinian cats were compared with those for normal cats. Light microscopy was used to count photoreceptor nuclei. RESULTS: The maximum a-wave amplitude, a(max), was significantly smaller in stage 1, and continued to decrease (stage 1: 50% of normal, stage 2: 28%, stage 3: 27%; and stage 4: unrecordable). There was a small, but not significant, decrease in the amplification constant A from 0.24 +/- 0.11 s(-2) in normal cats to 0.16 +/- 0.08 s(-2) in Abyssinian cats. The intraretinal b- and c-wave amplitudes decreased most dramatically during the early stage of the disease. Affected animals had fewer photoreceptors than unaffected Abyssinians or control animals. The number of photoreceptors declined most rapidly in the inferior periphery. CONCLUSIONS: The amplitudes of all ERG components were already reduced significantly by stage 1 and progressively declined. The lack of major changes in a-wave model parameters indicates that the degeneration is probably not due to a mutation in transduction proteins. Losses of photoreceptor function were larger than losses of photoreceptor nuclei.

Retinal oxygenation and oxygen metabolism in Abyssinian cats with a hereditary retinal degeneration.

PURPOSE: To investigate the effects of a hereditary retinal degeneration on retinal oxygenation and determine whether it is responsible for the severe attenuation of retinal circulation in hereditary photoreceptor degenerations. METHODS: Seven adult Abyssinian cats affected by hereditary retinal degeneration were studied. Oxygen microelectrodes were used to collect spatial profiles of retinal oxygenation in anesthetized animals. A one-dimensional model of oxygen diffusion was fitted to the data to quantify photoreceptor oxygen utilization (Qo(2)). RESULTS: Photoreceptor Qo(2) progressively decreased until it reached zero in the end stage of the disease. Average inner retinal oxygen tension remained within normal limits at all disease stages, despite the observed progressive retinal vessel attenuation. Light affected photoreceptors normally, decreasing Qo(2) by approximately 50% at all stages of the disease. CONCLUSIONS: Loss of photoreceptor metabolism allows choroidal oxygen to reach the inner retina, attenuating the retinal circulation in this animal model of retinitis pigmentosa (RP) and probably also in human RP. As the degeneration progresses, there is a strong relationship between changes in the a-wave of the ERG and changes in rod oxidative metabolism, indicating that these two functional measures change together.

Dark adaptation of rod photoreceptors in normal subjects, and in patients with Stargardt disease and an ABCA4 mutation.

PURPOSE: Psychophysical and electroretinographic (ERG) studies indicate that patients with Stargardt disease exhibit abnormally slow rod dark adaptation after illumination that bleaches a substantial fraction of rhodopsin. However, relatively little information is available concerning rod recovery in this disease after weaker adapting (i.e., conditioning) light. With the use of a paired-flash ERG method, properties of the derived rod response to a low-bleach (<1%) but rod-saturating conditioning flash were investigated in seven normal subjects and in five Stargardt patients with identified sequence variations in the ABCA4 gene. METHODS: In the first of two experiments, the interval between a fixed conditioning flash (67 or 670 scotopic cd s m(-2)) and a bright probe flash of fixed strength was varied to determine the falling-phase kinetics of the derived rod response to the conditioning flash. In the second, the instantaneous amplitude-intensity function for the rod response at an intermediate stage of recovery from the conditioning flash was determined by presenting a test flash of various strengths at a fixed time after the conditioning flash, and a probe flash at 200 ms after the test flash. RESULTS: The maximum peak amplitude of the dark-adapted, rod-mediated a-wave determined in Stargardt patients (211 +/- 87 microV) was on average lower than that determined in normal subjects (325 +/- 91 microV; P = 0.06). The derived rod response to the 670 scotopic cd s m(-2) conditioning flash determined in normal subjects and Stargardt patients exhibited a biphasic recovery, and the kinetics of the early stage of this recovery were similar in the two subject groups. For both normal subjects and patients, normalized amplitude-intensity functions describing the dark-adapted derived rod response exhibited half-saturation at approximately 1.5 log scotopic troland second. In both groups, the normalized amplitude-intensity function determined at approximately 2 seconds after the 67 scotopic cd s m(-2) conditioning flash and at approximately 9 seconds after the 670 scotopic cd s m(-2) conditioning flash exhibited an average desensitization (i.e., an increase of test flash strength at half-saturation) of approximately 0.5 to 0.6 log unit relative to that determined under dark-adapted conditions. CONCLUSIONS: The results indicate that, despite a reduction in the average dark-adapted maximum a-wave amplitude in the Stargardt/ABCA4 patients, the early-stage recovery kinetics of the derived rod response to a low-bleaching conditioning flash as well as the lingering rod desensitization produced by such a flash are similar to those determined in normal subjects.

Excitation and desensitization of mouse rod photoreceptors in vivo following bright adapting light.

Electroretinographic (ERG) methods were used to determine response properties of mouse rod photoreceptors in vivo following adapting illumination that produced a significant extent of rhodopsin bleaching. Bleaching levels prevailing at approximately 10 min and approximately 20 min after the adapting exposure were on average 14 % and 9 %, respectively, based on the analysis of visual cycle retinoids in the eye tissues. Recovery of the rod response to the adapting light was monitored by analysing the ERG a-wave response to a bright probe flash presented at varying times during dark adaptation. A paired-flash procedure, in which the probe flash was presented at defined times after a weak test flash of fixed strength, was used to determine sensitivity of the rod response to the test flash. Recovery of the response to the adapting light was 80 % complete at 13.5 +/- 3.0 min (mean +/- S.D.; n = 7) after adapting light offset. The adapting light caused prolonged desensitization of the weak-flash response derived from paired-flash data. By comparison with results obtained in the absence of the adapting exposure, desensitization determined with a test-probe interval of 80 ms was ~fourfold after 5 min of dark adaptation and approximately twofold after 20 min. The results indicate, for mouse rods in vivo, that the time scale for recovery of weak-flash sensitivity substantially exceeds that for the recovery of circulating current following significant rhodopsin bleaching. The lingering desensitization may reflect a reduced efficiency of signal transmission in the phototransduction cascade distinct from that due to residual excitation.