5-Lipoxygenase Inhibition Protects Retinal Pigment Epithelium from Sodium Iodate-Induced Ferroptosis and Prevents Retinal Degeneration.

Excessive reactive oxygen species (ROS) contribute to damage of retinal cells and the development of retinal diseases including age-related macular degeneration (AMD). ROS result in increased metabolites of lipoxygenases (LOXs), which react with ROS to induce lipid peroxidation and may lead to ferroptosis. In this study, the effect of 5-LOX inhibition on alleviating ROS-induced cell death was evaluated using sodium iodate (NaIO3) in the retinal pigment epithelium (RPE) cell line ARPE-19 and a mouse model investigating oxidative stress in AMD. We demonstrated that NaIO3 induced cell death in the RPE cells through mechanisms including ferroptosis. Inhibition of 5-LOX with specific inhibitor, Zileuton, or siRNA knockdown of ALXO5 mitigated NaIO3-induced lipid peroxidation, mitochondrial damage, DNA impairment, and cell death in ARPE-19 cells. Additionally, in the mouse model, pretreatment with Zileuton reduced the NaIO3-induced lipid peroxidation of RPE cells, cell death in the photoreceptor layer of the retina, inflammatory responses, and degeneration of both the neuroretina and RPE monolayer cells. Our results suggest that 5-LOX plays a crucial role in ROS-induced cell death in the RPE and that regulating 5-LOX activity could be a useful approach to control ROS and ferroptosis-induced damage, which promote degeneration in retinal diseases.

Nanoprotection Against Retinal Pigment Epithelium Degeneration via Ferroptosis Inhibition.

Lethal oxidative stress and ferrous ion accumulation-mediated degeneration/death in retinal pigment epithelium (RPE) exert an indispensable impact on retinal degenerative diseases with irreversible visual impairment, especially in age-related macular degeneration (AMD), but corresponding pathogenesis-oriented medical intervention remains controversial. In this study, the potent iron-binding nanoscale Prussian blue analogue KCa[FeIII (CN)6 ] (CaPB) with high biocompatibility is designed to inhibit RPE death and subsequently photoreceptor cell degeneration. In mice, CaPB effectively prevents RPE degeneration and ultimately fulfills superior therapeutic outcomes upon a single intravitreal injection: significant rescue of retinal structures and visual function. Through high-throughput RNA sequencing and sophisticated biochemistry evaluations, the findings initially unveil that CaPB nanoparticles protect against RPE degradation by inhibiting ferroptotic cell fate. Together with the facile, large-scale preparations and in vivo biosafety, it is believed that the synthesized CaPB therapeutic nanoparticles are promising for future clinical treatment of diverse retinal diseases involving pathological iron-dependent ferroptosis, including AMD.

Protection of Aronia melanocarpa Fruit Extract from Sodium-Iodate-Induced Damages in Rat Retina.

Age-related macular degeneration (AMD) is one of the major causes of blindness in elderly populations. However, the dry form of AMD has lack of effective treatments. The fruits of Aronia melanocarpa are rich in anthocyanins. In this study, the protective effects of aronia fruit extract on rat retina were investigated using a NaIO3-induced dry AMD model. Full-field electroretinograms (ERGs) showed that b-wave amplitudes were significantly decreased and the retina structures were disordered in the model. The extract treatment alleviated the injuries. The b-wave amplitudes increased 61.5% in Scotopic 0.01ERG, 122.0% in Photopic 3.0ERG, and 106.8% in Photopic 3.0 flicker; the retina structure disorder was improved with the thickness of outer nuclear layer increasing by 44.1%; and the malonaldehyde level was significantly reduced in extract-treated rat retinas compared to the model. The proteomics analysis showed the expressions of five crystallin proteins, α-crystallin A chain, ß-crystallin B2, ß-crystallin A3, α-crystallin B chain, and γ-crystallin S, which protect retina ganglion cells, were increased by 7.38-, 7.74-, 15.30-, 4.86-, and 9.14-fold, respectively, in the extract treatment compared to the control, which was also confirmed by immunoblotting. The results suggest that aronia fruit extract, probably due to its anthocyanins, could protect the rat retina by alleviating oxidative damages and by upregulating the crystallin proteins to protect its nerve system.

Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina.

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1ß, il-33 and tgf-ß were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.

Morphologic and electrophysiologic findings of retinal degeneration after intravitreal sodium iodate injection following vitrectomy in canines.

We developed and characterized a canine model of outer retinal degeneration induced by sodium iodate (SI) intravitreal injection after vitrectomy. In the preliminary study, we repeatedly injected SI intravitreally into the eyes of three canines to develop outer retinal degeneration two weeks after vitrectomy. Based on the preliminary study, a single dose of either 1.2 mg or 1.0 mg SI/0.05 mL was also injected (1.2 mg in n = 5 canines, 1.0 mg in n = 2 canines). Spectral domain-optical coherence tomography (OCT), electroretinography (ERG), and histological examinations were performed at baseline and following intravitreal injection. In the preliminary study, after a 0.5-mg SI injection and a 1.0-mg SI injection and after two 0.8-mg SI injections, retinal degeneration with retinal thinning was observed on OCT imaging. In the second study, after a single 1.0- or 1.2-mg SI injection, outer retinal degeneration was induced. All eyes showed diffuse outer retinal degeneration on OCT and a loss of both cone and rod responses in ERG. Histological examination also showed the loss of outer retinal layer. Intravitreally injected SI (1.0-1.2 mg) in a vitrectomized canine model induced outer retinal degeneration effectively, and could be evaluated through in vivo ophthalmic examination.

Establishment of Retinal Degeneration Model in Rat and Monkey by Intravitreal Injection of Sodium Iodate.

BACKGROUND: Animal models play critical roles in studies of the etiology and therapy of retinal degeneration (RD). OBJECTIVE: To establish an RD model without severe systemic side effects in monkeys. METHODS: Cynomolgus monkeys and Sprague-Dawley rats were treated with intravenous and intravitreal sodium iodate (SI). Electroretinographic (ERG) recording, fluorescein fundus angiography (FFA), optical coherence tomography (OCT) and a retinal morphology examination were conducted to evaluate retinal function and structure. ARPE-19 cells were treated with SI to assess cell viability and morphology. Glutathione (GSH) was administered to SI-treated cultured cells and rats for mechanistic studies. RESULTS: Intravenous SI failed to induce RD in monkeys due to its lethal toxicity and the spontaneous recovery of visual function. However, intravitreal SI injection induced very rapid and severe retinal damage in both monkeys and rats. Different doses of SI were tested in both rats and monkeys, and the SI dose appropriate for the model was calculated. GSH partially rescued oxidative damage to SI-treated retinas. A combination of the appropriate dose of intravitreal SI and intravenous GSH generated moderate subacute RD. CONCLUSIONS: An RD model was established in cynomolgus monkeys by intravitreal SI injection. The key advantages of this model are that lethal SI side effects can be avoided and that the structural and functional changes are similar to those in patients with RD, although the development of RD in the model is too rapid and more severe. An appropriate dose of SI plus systemic GSH generates delayed and moderate RD; this prolonged therapeutic window allows the development of new therapies, such as gene or stem cell-based therapy, for RD.

Nitric oxide is involved in the hypothyroidism with significant morphology changes in female Wistar rats induced by chronic exposure to high water iodine from potassium iodate.

Epidemiological studies indicated that chronic exposure to high water iodine is associated with primary hypothyroidism (PH) and subclinical hypothyroidism (SCH). However, the mechanism is not well understood. In this study, we explored whether chronic exposure to high water iodine from potassium iodate (KIO3) can induce hypothyroidism in addition to determining if nitric oxide (NO) is involved in the pathogenesis. 96 female Wistar rats were divided into six groups: control, I1000µg/L, I3000µg/L, I6000µg/L, N-nitro-L-arginine methylester (L-NAME) and L-NAME+I6000µg/L. After 3 months, urine iodine concentration, thyroid hormone, NO and nitric oxide synthase (NOS) serum levels were determined. Additionally, thyroid expression of inducible nitric oxide synthase (iNOS) was also investigated. Thyroid morphology was observed under light microscopy and transmission electron microscope. SCH as indicated by elevated serum thyrotropin (TSH) was induced among rats exposed to 3000 µg/L I-, while rats treated with 6000 µg/L I- presented PH characterized by elevated TSH and lowered total thyroxine in serum. Moreover, serum NO, NOS and iNOS expression in the thyroid were significantly increased in I3000µg/L and I6000µg/L groups. Changes in thyroid function and morphology in the L-NAME+I6000µg/L group were extenuated compared to I6000µg/L group. These findings suggested that chronic exposure to high water iodine from KIO3 likely induces hypothyroidism with significant morphology changes in female Wistar rats and NO appears to be involved in the pathogenesis.

What Can Pharmacological Models of Retinal Degeneration Tell Us?

Animal models with pharmacologically induced retinal degeneration including sodium iodate (NaIO3) and N-methyl-N-nitrosourea (MNU) have been extensively used in ophthalmic research to investigate retinal degeneration. NaIO3 induces degeneration of the retinal pigment epithelium (RPE) followed by photoreceptor (PRC) cell death, mimicking features of age-related macular degeneration. In contrast, MNU leads to rapid destruction of the PRCs only, enabling the use of the MNU model to investigate degeneration induced in retinitis pigmentosa. It has been shown that multiple cell death pathways are involved in the cell-specific effects of the toxins. Necrosis has been identified as the cause of the NaIO3-induced RPE loss. PRC degeneration in the described models is mainly induced by programmed cell death, indicated by the upregulation of conventional apoptosis initiator and effector caspases. However, recent research points to the additional involvement of caspase-independent processes as endoplasmic reticulum stress and calpain activation. Since there is still a substantial amount of contradictory hypotheses concerning triggers of cell death, the use of pharmacological models is controversial. Thereby, the advantages of such models like the application reaching across species and strains as well as modulation of onset and severity of damage are not exploited to a full extent. Thus, the present review aims to give more insight into the involved cell death pathways and discusses recent findings in the most widely used retinal degeneration models. It might facilitate further studies aiming to develop putative therapeutic approaches for retinal degenerative diseases including combinatory treatment with cell death inhibitors and cell transplantation therapy.

Course of Sodium Iodate-Induced Retinal Degeneration in Albino and Pigmented Mice.

Purpose: To characterize the course of sodium iodate (SI)-induced retinal degeneration in young adult albino and pigmented mice. Methods: Single intraperitoneal (IP) injections of SI (25, 50, and 100 mg/kg) were performed in 7- to 8-week-old BALB/c and C57Bl/6J mice. Retinal function and structure was assessed at baseline, 24 hours, 3 days, 1, 2, 3, and 4 weeks postinjection by optokinetic tracking response, ERG, optical coherence tomography (OCT), and histologic and immunohistochemical techniques. Results: The 50 mg/kg SI dosage was selected after dose ranging due to consistent retinal effects and lack of systemic toxicity. Time-dependent deterioration in retinal function and morphology was consistently observed between 1 and 4 weeks in all measured parameters. These include reduction of ERG responses, thinning of retinal layers as observed by OCT and histology, and loss of RPE nuclei. Immunohistochemistry revealed rapid RPE disorganization with loss of tight junctions and markedly reduced expression of RPE65 and rod opsin, accompanied by mislocalization of cone opsins. Earlier time points displayed variable results, including partial recovery of visual acuity at 1 week and supranormal ERG cone responses at 24 hours, suggesting possible limitations of early intervention and assessment in the SI model. Conclusions: A single IP injection of 50 mg/kg SI leads to severe RPE injury followed by vision impairment, dysfunction, and loss of photoreceptors in both BALB/c and C57Bl/6J mice. This easily induced and reproducible noninherited model may serve as a useful tool for seeking and evaluating novel therapeutic modalities for the treatment of retinal degenerations caused by primary failure of the RPE.

Protective effect of KI in mtDNA in porcine thyroid: comparison with KIO3 and nDNA.

PURPOSE: Iodine, bivalent iron (Fe²âº), and hydrogen peroxide (H2O2), all significantly affecting the red-ox balance, are required for thyroid hormone synthesis. Intracellular iodine excess (≥10⁻³ M) transiently blocks thyroid hormonogenesis (an adaptive mechanism called Wolff-Chaikoff effect). The aim of the study was to evaluate the effects of iodine, used as potassium iodide (KI) or potassium iodate (KIO3), in concentrations corresponding to those typical for Wolff-Chaikoff effect, on the level of oxidative damage to nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) isolated from porcine thyroid under basal conditions and in the presence of Fenton reaction (Fe²âº+H2O2 → Fe³âº+(·)OH + OH⁻) substrates. METHODS: Thyroid nDNA and mtDNA were incubated in the presence of either KI or KIO3 (2.5-50 mM), without/with FeSO4 (30 µM) + H2O2 (0.5 mM). Index of DNA damage, i.e., 8-oxo-7,8-dihydro-2'-deoxyguanosine, was measured by HPLC. RESULTS: Neither KI nor KIO3 increased the basal level of 8-oxodG in both nDNA and mtDNA. KI-in all used concentrations-completely prevented the damaging effect of Fenton reaction substrates in mtDNA, and it partially prevented this damage in nDNA. KIO3 partially prevented Fe²âº+H2O2-induced oxidative damage in both DNA only in its highest used concentrations (≥25 mM). CONCLUSIONS: Without additional prooxidative abuse, both iodine compounds, i.e., KI and KIO3, seem to be safe in terms of their potential oxidative damage to DNA in the thyroid. The superiority of KI over KIO3 relies on its stronger protective effects against oxidative damage to mtDNA, which constitutes an argument for its preferential utility in iodine prophylaxis.

Protection of retina by αB crystallin in sodium iodate induced retinal degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD and αB crystallin expression is increased in RPE and associated drusen in AMD. The purpose of this study was to investigate the role of αB crystallin in sodium iodate (NaIO3)-induced retinal degeneration, a model of AMD in which the primary site of pathology is the RPE. Dose dependent effects of intravenous NaIO3 (20-70 mg/kg) on development of retinal degeneration (fundus photography) and RPE and retinal neuronal loss (histology) were determined in wild type and αB crystallin knockout mice. Absence of αB crystallin augmented retinal degeneration in low dose (20 mg/kg) NaIO3-treated mice and increased retinal cell apoptosis which was mainly localized to the RPE layer. Generation of reactive oxygen species (ROS) was observed with NaIO3 in mouse and human RPE which increased further after αB crystallin knockout or siRNA knockdown, respectively. NaIO3 upregulated AKT phosphorylation and peroxisome proliferator-activator receptor-γ (PPARγ) which was suppressed after αB crystallin siRNA knockdown. Further, PPARγ ligand inhibited NaIO3-induced ROS generation. Our data suggest that αB crystallin plays a critical role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression.

Effect of iodine source and dose on growth and iodine content in tissue and plasma thyroid hormones in fattening pigs.

PURPOSE: The aim of the present feeding trial with iodine was to assess pigs' growth performance and carcass characteristics, the iodine accumulation in tissues, and their influences on the thyroid hormones in plasma. METHODS: Eighty pigs (33-115 kg body weight) were allotted to 5 dietary treatments: a control group (150 µg I/kg), two potassium iodide [KI] groups (4,000 and 10,000 µg I/kg), and two potassium iodate [KIO3] groups (4,000 and 10,000 µg I/kg). Iodine concentration was determined in thyroid gland, liver, kidney, muscle, fat, and skin by ICP-MS. Furthermore, thyroxine (T4) and triiodothyronine (T3) in plasma were evaluated. RESULTS: High dietary iodine tended to have a negative effect on younger animals' growth (average daily gain, ADG). However, during the entire growth period, the growth performance and carcass characteristics were not influenced by iodine dosages or sources. Irrespective of iodine source, higher iodine doses of diets affected higher iodine stores in all tested tissues except for abdominal fat. Thus, iodine supplementation with 10,000 µg I/kg feed significantly increased iodine content in thyroid gland (+122%), liver (+260%), kidney (+522%), muscle (+131%), and skin (+321%) compared to the control group. However, there was no significance of thyroid hormones in plasma. CONCLUSIONS: As a result, pork and fat of pigs showed only low iodine accumulation even in the high-iodine groups. Thus, there should be no risk of an iodine excess in human nutrition and animal health, and the EU-upper level for iodine in pig feed can be maintained.

Full-field ERGs obtained using a contact lens electrode with built-in high intensity white light-emitting diodes in beagle dogs can be applied to toxicological assessments.

We investigated full-field ERGs in beagle dogs using a contact lens electrode with built-in LED. Experiment 1 was performed to determine the appropriate conditions for stimulus intensity and background illumination. We found that full-field ERGs could be recorded under the following conditions: stimulus intensity: -2.5logcd*s/m(2) in rod responses (RRs), 1.2logcd*s/m(2) in maximal responses (MRs), oscillatory potentials (OPs), cone responses (CRs), 30-Hz flicker responses (FRs), and background illumination: more than 25cd/m(2) in CRs and FRs. Experiment 2 was performed to apply full-field ERGs in beagle dogs to the detection of retinal toxicities. A dog was given one 30mg/kg dose of sodium iodate (NaIO(3)) intravenously. ERGs were recorded before administration and 1, 3, 5, 8, 24h, 7 and 14 days after administration of NaIO(3). The RRs disappeared completely at 1h when MRs and OPs decreased. On the other hand, CRs and FRs were recorded even at 8h. All responses disappeared at 24h. These findings indicate that retinal toxicity by NaIO(3) is first expressed in rods, followed by cones. These results suggest that full-field ERGs in beagle dogs using an LED contact lens can be used to evaluate toxic effects on rods and cones separately, with the potential to prove more useful than conventional methods for toxicological assessments of developing pharmaceuticals, and can be applied to it.

[Scanning electron microscopic observation on altered fenestration of the choriocapillaris in sodium iodate-treated rats].

Fenestration of the choriocapillaris may be lost and regained in various pathologic conditions. The purpose of this study was to observe the gradual disappearance of fenestrae with the scanning electron microscope. Pigmented rats were treated with an intravenous injection of sodium iodate. Changes in the choriocapillaris were produced by damaging retinal pigment epithelium. The eyes were processed for styrene embedded cracking on the third, fifth, and seventh days. Dense clusters of fenestrae were seen at the luminal surface facing retina on the third day. These clusters were surrounded by protrusion of cellular surface. The clusters of fenestrae were smaller with some variation in size on the fifth and seventh days. The protrusion between the clusters became flatter and wider. Some endothelial cells had a flat surface with tiny clusters of fenestrae. Although there was loss of fenestrae with smaller clusters and some variation in size, the tendency of the fenestrae to cluster was sustained in the same way as during the reformation of fenestrae in the choriocapillaris after laser photocoagulation.

Visualization of subtle contrast-related intensity changes using temporal correlation.

Contrast-enhanced magnetic resonance imaging (MRI) is a promising method for investigating the breakdown of the blood-retinal barrier (BRB). However, subtle intensity changes due to low concentrations of contrast agent can be difficult to detect without observer bias. In this study, we developed a temporal correlation method for detecting these subtle signal intensity changes. The method was evaluated in eyes with chemically induced retinal lesions of known size. A time series of MRI data were collected following i.v. administration of different doses of gadolinium-diethylaminetriaminepentaacetic acid (0.05, 0.1, 0.5 mmol/kg). These time course images were analyzed by temporal correlation to a reference enhancement curve. The reference curve was generated based on a validated theoretical enhancement curve. The temporal correlation method detected signal intensity changes in cases where the changes were too subtle to be visible on a postinjection image or a subtraction image (obtained by subtracting the precontrast image from the final image in the time course set). In addition, assessment of leakage was performed by viewing each image in the set with an eight gray-level palette. Areas of leakage identified in this manner corresponded to those identified by temporal correlation, a finding which supports the validity of the temporal correlation method. These results suggest that temporal correlation may be a time-efficient way to screen large numbers of image data sets using an objective, user-independent criterion.

[Early effects of sodium iodate shown by detection of the destruction of anionic sites in outer blood-retinal barrier].

In order to demonstrate the early effect of sodium iodate (NaIO3) on retinal pigment epithelium (RPE), Bruch's membrane and choriocapillaries, we examined the alteration of their anionic sites using a cationic probe, polyethyleneimine (PEI). We injected 10 microliters of 3% NaIO3 solution into the vitreous of pigmented rabbits and after thirty minutes or one hour, administered 0.5% PEI intravenously. In the eyes injected with intravitreously saline solution or without injection, PEI-positive particles (diameter 15-20 nm) were located in a row on both sides of the basement membrane of the RPE and choriocapillaries and periodically at the collagen fiber of inner and outer collagenous zone of Bruch's membrane. However no particle was observed on the cell membrane of the basal infolding of the RPE. Thirty minutes after injection of NaIO3, PEI particle positive sites were reduced at the basement membrane of the RPE. One hour after injection they reduced in number but remained at the basement membrane of choriocapillaris and collagens in Bruch's membrane. We showed the early effect of NaIO3 to the RPE by examining the alterations of the anionic sites.