Endotoxin-induced Uveitis Model Research Articles

OCTN2- and ATB0,+-targeted nanoemulsions for improving ocular drug delivery

Traditional eye drops are administered via topical instillation. However, frequent dosing is needed due to their relatively rapid precorneal removal and low ocular bioavailability. To address these issues, stearoyl L-carnitine-modified nanoemulsions (SC-NEs) were fabricated. The physicochemical properties of SC-NEs in terms of size, morphology, zeta potential, encapsulation efficiency, and in vitro drug release behavior were characterized. The cellular uptake and mechanisms of SC-NEs were comprehensively studied in human corneal epithelial cells and the stearoyl L-carnitine ratio in SC-NEs was optimized. The optimized SC-NEs could target the novel organic cation/carnitine transporter 2 (OCTN2) and amino acid transporter B (0 +) (ATB0,+) on the corneal epithelium, which led to superior corneal permeation, ocular surface retention ability, ocular bioavailability. Furthermore, SC-NEs showed excellent in vivo anti-inflammatory efficacy in a rabbit model of endotoxin-induced uveitis. The ocular safety test indicated that the SC-NEs were biocompatible. In general, the current study demonstrated that OCTN2 and ATB0,+-targeted nanoemulsions were promising ophthalmologic drug delivery systems that can improve ocular drug bioavailability and boost the therapeutic effects of drugs for eye diseases.Graphical

Roles of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor 2 in Endotoxin-Induced Acute Retinal Inflammation

ABSTRACT Purpose To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. Methods EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. Results Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. Conclusion SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases.

Supramolecular nanofiber of indomethacin derivative confers highly cyclooxygenase-2 (COX-2) selectivity and boosts anti-inflammatory efficacy

Herein, we report a facile method for converting carboxylate-containing indomethacin (Idm) into a cyclooxygenase-2 (COX-2) selective inhibitor via the amidation of an unnatural peptide sequence (Nal-Nal-Asp). The resulting indomethacin amides (i.e., Idm-Nal-Nal-Asp) have high selectivity for COX-2, and can self-assemble into a one-component supramolecular hydrogel that acts as a ‘self-delivery’ system for boosting anti-inflammatory efficacy. Self-assembled Idm-Nal-Nal-Asp hydrogel robustly inhibits COX-2 expression in lipopolysaccharide (LPS)-activated Raw 264.7 macrophages while also exhibits superior anti-inflammatory and antioxidant activities via reactive oxygen species (ROS)-related NF-κB and Nrf2/HO-1 pathways. Moreover, a rabbit model of endotoxin-induced uveitis (EIU) reveals that the Idm-Nal-Nal-Asp hydrogel outperforms clinically used 0.1 wt% diclofenac sodium eye drops in terms of in vivo anti-inflammatory efficacy via topical instillation route. As a rational approach to designing and applying COX-2 selective inhibitors, this work presents a simple method for converting non-selective nonsteriodal anti-inflammatory drugs (NSAIDs) into highly selective COX-2 inhibitors that can self-assemble into supramolecular hydrogel for anti-inflammation applications.

Topical Sustained-Release Dexamethasone-Loaded Chitosan Nanoparticles: Assessment of Drug Delivery Efficiency in a Rabbit Model of Endotoxin-Induced Uveitis.

Uveitis is an ocular illness that if not treated properly can lead to a total loss of vision. In this study, we evaluated the utility of HA-coated Dexamethasone-sodium-phosphate (DEX)-chitosan nanoparticles (CSNPs) coated with hyaluronic acid (HA) as a sustained ocular delivery vehicle for the treatment of endotoxin-induced-uveitis (EIU) in rabbits. The CSNPs were characterized for particle size, zeta potential, polydispersity, surface morphology, and physicochemical properties. Drug encapsulation, in vitro drug release, and transcorneal permeation were also evaluated. Finally, eye irritation, ocular pharmacokinetics, and pharmacodynamics were in vivo. The CSNPs ranged from 310.4 nm and 379.3 nm pre-(uncoated) and post-lyophilization (with HA-coated), respectively. The zeta potentials were +32 mV (uncoated) and -5 mV (HA-uncoated), while polydispersity was 0.178-0.427. Drug encapsulation and loading in the CSNPs were 73.56% and 6.94% (uncoated) and 71.07% and 5.54% (HA-coated), respectively. The in vitro DEX release over 12 h was 77.1% from the HA-coated and 74.2% from the uncoated NPs. The physicochemical properties of the CSNPs were stable over a 3-month period when stored at 25 °C. Around a 10-fold increased transcorneal-flux and permeability of DEX was found with HA-CSNPs compared to the DEX-aqueous solution (DEX-AqS), and the eye-irritation experiment indicated its ocular safety. After the ocular application of the CSNPs, DEX was detected in the aqueous humor (AH) till 24 h. The area under the concentrations curve (AUC0-24h) for DEX from the CSNPs was 1.87-fold (uncoated) and 2.36-fold (HA-coated) higher than DEX-AqS. The half-life (t1/2) of DEX from the uncoated and HA-coated NPs was 2.49-and 3.36-fold higher, and the ocular MRT0-inf was 2.47- and 3.15-fold greater, than that of DEX-AqS, respectively. The EIU rabbit model showed increased levels of MPO, TNF-α, and IL-6 in AH. Topical DEX-loaded CSNPs reduced MPO, TNF-α, and IL-6 levels as well as inhibited NF-κB expression. Our findings demonstrate that the DEX-CSNPs platform has improved the delivery properties and, hence, the promising anti-inflammatory effects on EIU in rabbits.

The possible effect of topically applied azithromycin and moxifloxacin on the alleviation of uveitis.

To investigate the inhibitory effect of topically administered azithromycin (AZM), and moxifloxacin (MXF) against tumor necrosis factor-α (TNF-α) production in a rat model of endotoxin-induced uveitis (EIU). Thirty-six Wistar albino rats were divided into 6 equal groups. Groups 1, 2 and 3 were determined as sham, control group for topical AZM application and control group for topical MXF application, respectively. Sterile saline, topical AZM 1.5%, and topical MXF 0.5% were instilled 5 times daily for totally 6days on both eyes of the rats in Group 4, Group 5, and Group 6, before and after inducing EIU by intravitreal injections of lipopolysaccharide, respectively. At 24h after intravitreal injections, aqueous humor was collected from both eyes of each rat for the assessment of TNF-α concentration. Also, density of nuclear factor kappa B (NF-κB) in ciliary body, and the number of cells infiltrating the posterior segment of EIU rat eyes was assessed in one eye of each rat. There was a significant reduction in mean aqueous humor concentration of TNF-α in EIU rats pretreated with topical AZM in comparison with those pretreated with sterile saline (139 ± 38.6 in Group 4 vs. 72 ± 12.6 in Group 5, p = 0.006). There was also a marked decrease in mean aqueous humor concentration of TNF-α in EIU rats pretreated with topical MXF (139 ± 38.6 in Group 4 vs.86.1 ± 35.5 in Group 6, p = 0.025). Also, evident suppressions were determined in mean density of NF-κB, and in mean number of cells in EIU rats pretreated either with topical AZM, or topical MXF. Topically applied AZM or MXF may be beneficial in the suppression of TNF-α production in aqueous humor.

Development of Triamcinolone Acetonide Nanocrystals for Ocular Administration

Triamcinolone acetonide (TA) is a powerful anti-inflammatory drug used in the treatment of inflammatory ocular disorders; however, its poor aqueous solubility and ocular anatomical barriers hinder optimal treatment. The aim of this work was to obtain triamcinolone acetonide nanocrystals (TA-NC) to improve ocular corticosteroid therapy. Self-dispersible TA-NC were prepared by the bead milling technique followed by spray-drying, exhaustively characterized and then evaluated in vivo in an ocular model of endotoxin-induced uveitis (EIU). Self-dispersible TA-NC presented an average particle size of 257 ± 30 nm, a narrow size distribution and a zeta potential of -25 ± 3 mV, which remained unchanged for 120 days under storage conditions at 25 °C. In addition, SEM studies of the TA-NC showed uniform and spherical morphology, and FTIR and XRDP analyses indicated no apparent chemical and crystallinity changes. The subconjunctival administration of TA-NC in albino male white rabbits showed no clinical signs of ocular damage. In vivo studies proved that treatment with self-dispersible TA-NC alleviated the inflammatory response in the anterior chamber and iris in EUI rabbit eyes. Dispersible TA-NC are a promising approach to obtaining a novel nanometric TA formulation for ocular disorders.

Melatonin receptor agonist ramelteon alleviates experimental acute ocular inflammation via HIF-1Α/VEGF/E-NOS signaling.

Ramelteon (RML) is a potent, selective agonist of the high-affinity melatonin receptor 1 and 2 receptors. In addition, RML is known to have antioxidant and anti-inflammatory effects. In this study, we aimed to investigate the effects of RML on HIF-1α, VEGF and e-NOS signaling pathway in a rat model of endotoxin-induced uveitis (EIU). Twenty-eight Wistar albino rats were divided into 4 groups as controls, lypopolysaccharide (LPS) group (5 mg/kg i.p.), LPS + RML group (5 mg/kg i.p and 8 mg/kg orally, respectively) and RML group (8 mg/kg orally). EIU was induced by a single intraperitoneal LPS injection. Histopathological and genetical analyses were performed. In histopathological analysis, LPS caused mild anterior uveitis characterized by increased surface area of iris leaflets and ciliary body due to edema, mild to moderate congestion, and inflammatory infiltrate 6 h following the injection. The pathological findings were reduced by RML. Higher inflammation levels seen in LPS group were significantly reduced in LPS + RML group. Also, HIF-1 α, eNOS and VEGF expressions increased in LPS and decreased in LPS + RML group. RML treatment reversed the changes in the HIF-1α /eNOS/ VEGF signaling pathway in LPS-induced uveitis in rats, preventing the progression of the damage and showed positive effects.

“Spanlastic nanovesicles for enhanced ocular delivery of vanillic acid: design, in vitro characterization, and in vivo anti-inflammatory evaluation”

Spanlastics are novel surfactant-based, elastic vesicular nanocarriers composed of spans and edge activators. The present work aims to exploit their special penetration enhancing properties to enhance the ophthalmic delivery of the versatile nutraceutical vanillic acid (VA), for treatment of ocular inflammation. VA-loaded spanlastics were formulated by ethanol injection method using Tween 80, sodium deoxy cholate or Tween 60 as edge activators (EA) at various Span 60: EA mass ratios. Vesicles were characterized for their particle size (PS), polydispersity index (PDI), zeta potential, entrapment efficiency (EE%), surface morphology, in vitro release profile, thermal properties and long-term stability, in addition to in vivo anti-inflammatory efficacy of the selected formula in an endotoxin-induced uveitis model. Selected formulation composed of Span 60: Tween 80 at a mass ratio of 70:30 displayed smallest PS of 299.8 ± 9.97 nm, PDI of 0.386 ± 0.047, an acceptable EE%, as well as good physical stability for 3 months. According to clinical scoring, inflammatory mediators levels and histopathological examination, VA-loaded spanlastic formulation resulted in significant alleviation of inflammation compared to drug suspension (p < 0.05). Formulation of VA into spanlastic nanoformulation is a promising approach to improve its ocular permeability, absorption and anti-inflammatory activity providing a safer alternative to current regimens.

Retinal Proteomic Analysis in a Mouse Model of Endotoxin-Induced Uveitis Using Data-Independent Acquisition-Based Mass Spectrometry

Uveitis is a group of sight-threatening ocular inflammatory diseases, potentially leading to permanent vision loss in patients. However, it remains largely unknown how uveitis causes retinal malfunction and vision loss. Endotoxin-induced uveitis (EIU) in rodents is a good animal model to study uveitis and associated acute retinal inflammation. To understand the pathogenic mechanism of uveitis and screen potential targets for treatment, we analyzed the retinal proteomic profile of the EIU mouse model using a data-independent acquisition-based mass spectrometry (SWATH-MS). After systemic LPS administration, we observed activation of microglial cells accompanied with the elevation of pro-inflammatory mediators and visual function declines. In total, we observed 79 upregulated and 90 downregulated differentially expressed proteins (DEPs). Among the DEPs, we found that histone family members (histone H1, H2A, H2B) and blood proteins including haptoglobin (HP), hemopexin (HPX), and fibrinogen gamma chain (FGG) were dramatically increased in EIU groups relative to those in control groups. We identified phototransduction and synaptic vesicle cycle as the top two significant KEGG pathways. Moreover, canonical pathway analysis on DEPs using Ingenuity Pathway Analysis revealed top three most significant enriched pathways related to acute phase response signaling, synaptogenesis signaling, and eif2 signaling. We further confirmed upregulation of several DEPs associated with the acute phase response signaling including HP, HPX, and FGG in LPS-treated retinas by qPCR and Western blot. In summary, this study serves as the first report to detect retinal proteome changes in the EIU model. The study provides several potential candidates for exploring the mechanism and novel therapeutic targets for uveitis and other retinal inflammatory diseases.

Enzyme-instructed self-assembly of peptide-drug conjugates in tear fluids for ocular drug delivery

In vivo self-assembly of small molecules offers an excellent opportunity for targeted and long-term accumulation of a therapeutic agent at the lesion site. Here we demonstrate the strategy of enzyme-instructed self-assembly (EISA) by designing a phosphorylated peptide-drug (IBF-HYD-GFFpY) precursor through the ester bond to release active drugs at the target site. Meanwhile, the in vivo assembly can be achieved by the catalysis of alkaline phosphatase (ALP) in the tear fluid for ocular drug delivery efficiently. The in vitro enzymatic experiments indicate that the dephosphorylation of IBF-HYD-GFFpY occurs firstly with the yield of IBF-HYD-GFFY which subsequently self-assembles into the supramolecular hydrogel to afford sustained drug release over 96 h. In the treatment of lipopolysaccharide (LPS)-activated Raw 264.7 macrophages, IBF-HYD-GFFpY exerts the more potent anti-inflammatory efficacy than that of free ibuprofen (IBF) at the concentration of 200 μM. Moreover, the aqueous solution of IBF-HYD-GFFpY via topical instillation hardly causes ocular irritation, and displays longer precorneal retention compared to the conventional eye drop formulation. In addition, in the in vivo study, a rabbit model of endotoxin-induced uveitis (EIU) evidences the comparable therapeutic efficacy of IBF-HYD-GFFpY eye drops with that of clinically used 0.1 wt% diclofenac (DIC) sodium eye drops by the reduction of macrophage and leukocyte influx. This work, in situ EISA in the tear microenvironment directing in vivo self-assembly of small molecules, may guide a powerful approach for developing enzymatic self-assembled molecules as an efficient delivery system of ocular drugs.

γ-Cyclodextrin-based polypseudorotaxane hydrogels for ophthalmic delivery of flurbiprofen to treat anterior uveitis

Anterior uveitis is a sight-threatening inflammation inside the eyes. Conventional eye drops for anti-inflammatory therapy need to be administered frequently owing to the rapid elimination and corneal barrier. To address these issues, polypseudorotaxane hydrogels were developed by mixing Soluplus micelles (99.4 nm) and cyclodextrins solution. The optimized hydrogels exhibited shear-thinning and sustained release properties. The hydrogels exhibited higher transcorneal permeability coefficient (Papp, 1.84 folds) than that of drug solutions. Moreover, animal study indicated that the hydrogels significantly increased the precorneal retention (AUC, 21.2 folds) and intraocular bioavailability of flurbiprofen (AUCAqueous humor, 17.8 folds) in comparison with drug solutions. Importantly, the hydrogels obviously boosted anti-inflammatory efficacy in rabbit model of endotoxin-induced uveitis at a reduced administration frequency. Additionally, the safety of hydrogels was confirmed by cytotoxicity and ocular irritation studies. In all, the present study demonstrates a friendly non-invasive strategy based on γ-CD-based polypseudorotaxane hydrogels for ocular drug delivery.

Anti-Inflammatory Effect of Tacrolimus/Hydroxypropyl-β-Cyclodextrin Eye Drops in an Endotoxin-Induced Uveitis Model.

Background: Uveitis is an infrequent disease which constitutes a major cause of ocular morbidity. Correct management is essential, being corticosteroids its cornerstone. In case of contraindication to corticosteroids or treatment failure, the use of topical tacrolimus (TAC) could be an alternative which has already demonstrated safety and effectiveness in other ocular pathologies. However, TAC eye drops are not marketed, thus their elaboration must be carried out in Hospital Pharmacy Departments (HPDs). Methods: 32 Sprague-Dawley rats were divided into 4 groups of 8 rats each: (a) untreated healthy rats (Healthy); (b) untreated Endotoxin-Induced Uveitis model-rats (EIU); (c) EIU-rats treated with standard treatment of dexamethasone ophthalmic drops (DXM) and (d) EIU-rats treated with TAC-hydroxypropyl-β-cyclodextrin eye drops previously developed by our group (TAC-HPβCD). The mRNA expression levels of IL-6, IL-8, MIP-1α and TNF-α, quantitative analysis of leucocytes in aqueous humor and histological evaluation were performed. Results: TAC-HPβCD eye drops demonstrated to reduce ocular inflammation, expression of IL-6, TNF-α, MIP-1α and leukocyte infiltration in aqueous humor. Conclusions: TAC-HPβCD eye drops showed beneficial effect in EIU model in rats, positioning as an alternative for uveitis treatment in case of corticosteroids resistance or intolerance.

Protective effect of filgotinib in rat endotoxin-induced uveitis model

To investigate the protective effect of filgotinib in endotoxin-induced uveitis model in rats. This study used 24 Wistar Albino rats. Group I (control group) included the healthy controls; in Group II (sham group), only 300µg/kg intraperitoneal (ip) lipopolysaccharide (LPS) was administered; and in Group III (treatment group), 3mg/kg/day filgotinib was administered orally for 10days followed by 300µg/kg ip LPS. In all groups, clinical activity scores were evaluated after 24h. Moreover, histopathological and immunological examinations were performed. In Groups I, II, and III, the mean clinical activity and histopathological examination scores were 0.00, 3.25 ± 0.70, and 1.89 ± 0.60 and 0.00, 2.88 ± 1.12, and 1.44 ± 0.52, respectively. The clinical activity and histopathological examination scores were significantly increased in the sham group compared to the control group (p < 0.05); these findings were significantly reduced in the treatment group (p < 0.05). The mean TNF-α and IL-6 ELISA levels in all groups were 50.20 ± 3.24, 59.87 ± 2.98, and 54.34 ± 4.62 and 30.88 ± 1.79, 36.77 ± 1.21, and 33.66 ± 1.86, respectively. The TNF-α and IL-6 ELISA levels were significantly decreased in the treatment group compared to the sham group (p < 0.05); there was no significant difference between the treatment group and the control group (p = 0.105, p = 0.067, respectively) CONCLUSION: Filgotinib may be an alternative treatment option in preventing the development of noninfectious uveitis.

The Protective Effects of VVN001 on LPS-Induced Inflammatory Responses in Human RPE Cells and in a Mouse Model of EIU.

To investigate protective effects of VVN001 on lipopolysaccharide (LPS)-induced inflammatory response in human retinal pigment epithelial (RPE) cells and in a mouse model of endotoxin-induced uveitis (EIU), and to explore the underlying mechanisms. Human primary RPE (hRPE) and ARPE-19 cells were pretreated with or without VVN001 for 1h followed by 10μg/mL LPS stimulation for 24h. mRNA, and protein levels of inflammatory cytokines were analyzed with real-time PCR, western blotting, and ELISA. EIU was induced by intravitreal injection of 125ng LPS in female BALB/c mice. VVN001 eye drops (1%) were locally administrated every 4h for 24h after LPS injection. Clinical scores were assessed with a slit lamp. mRNA and protein levels of inflammatory cytokines were investigated simultaneously. Compared with the LPS group, VVN001 pretreatment significantly reduced mRNA expressions of intercellular adhesion molecule-1 (ICAM-1), IL-6, IL-8, TNF-α, IL-1β, IL-18, caspase-1 in hRPE, and ARPE-19 cells. Protein overproduction of ICAM-1, TNF-α, IL-1β, NLRP3, caspase-1 P20, and p-IκBα/IκBα stimulated by LPS was suppressed by VVN001 pretreatment. In vivo, VVN001 significantly reduced the average clinical score from 5.0 to 1.3 in EIU mice. Furthermore, overproduction of ICAM-1, IL-1β, NLRP3, caspase-1 P20, and p-IκBα/IκBα at mRNA and protein levels were remarkably suppressed by VVN001. VVN001 alleviated the inflammatory response induced by LPS both in vitro and in vivo. The effect of anti-inflammation is associated with inhibiting the overproduction of ICAM-1 and blocking the activation of NLRP3 inflammasome and the NF-κB signaling pathway.

A cell-permeable peptide inhibitor of p55PIK signaling alleviates ocular inflammation in mouse models of uveitis

PurposePreviously we developed TAT-N24 as a synthetic cell-permeable peptide inhibitor of p55PIK signaling and demonstrated its anti-inflammatory effects. This study aimed to evaluate the potential of TAT-N24 as a new agent for the treatment of ocular inflammatory diseases. MethodsThe endotoxin-induced uveitis (EIU) model was established by intravitreal injection of lipopolysaccharide (LPS) in BALB/c mice and experimental autoimmune uveitis (EAU) model was established by subcutaneous injection of a peptide spanning amino acid residues 161–180 of interphotoreceptor retinoid binding protein (IRBP161-180) with complete Freund's adjuvant (CFA) in B10.RIII mice. TAT-N24 was topically administered in EIU model and intraperitoneally administered in EAU model. The severity levels of uveitis were assessed by clinical and histopathological scores. The mRNA levels of inflammatory cytokines in iris-ciliary body (ICB) and retina were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The protein levels of inflammatory factors were determined by ELISA or Western blotting. ResultsThe results showed that TAT-N24 alleviated clinical signs, decreased inflammatory cell infiltration and the expression of inflammatory cytokines in both EIU and EAU models. Furthermore, protein levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in aqueous humor and mRNA and protein levels of NF-κB p65 in the ICB significantly decreased in EIU model. In EAU model, TAT-N24 application induced a significant decrease of IFN-gamma (IFN-γ) and interleukin-17 (IL-17) in the retina, which were secreted by Th1 and Th17 cells, respectively. ConclusionIn conclusion, TAT-N24 suppressed intraocular inflammation in both EIU and EAU models, and the anti-inflammatory effects were mediated by suppressing the expression of inflammatory cytokines by PI3K/NF-κB signaling pathway. TAT-N24 could be potential candidate for the treatment of ocular inflammatory diseases.

Signaling mechanisms of growth hormone-releasing hormone receptor in LPS-induced acute ocular inflammation

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.

Anti-inflammatory effect of cortistatin in rat endotoxin-induced uveitis model.

Purpose:To evaluate the anti-inflammatory effect of cortistatin (CST) in endotoxin-induced uveitis (EIU) model and to compare the results with corticosteroid treatment.Methods:A total of 35 healthy Wistar albino rats were randomly divided into five groups. EIU was induced by a single subcutaneous injection of lipopolysaccharide (LPS). Group I received intraperitoneal (ip) normal saline (NS), Group II received ip 150 μg LPS plus NS, Group III received ip 150 μg LPS plus 250 μg/kg CST, Group IV received ip 150 μg LPS plus 1mg/kg dexamethasone, and Group V received ip 250 μg/kg CST only. The aqueous humor was collected 24 h after injection and the infiltrating cells were determined. Moreover, histopathological and immunohistochemical examinations were also performed.Results:The clinical score and infiltrated cell count were reduced in Groups III and IV compared with Group II (P < 0.001). The pathological findings of Groups III and IV were significantly reduced compared with Group II (P < 0.001). These findings were similar between Groups III and IV (P = 1.000). Tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL-1β) immunoreactivity in the ciliary body of Group III and Group IV were significantly reduced compared with Group II (P < 0.001). TNF-α and IL-1β immunoreactivity in the ciliary body of Group III and Group IV were similar compared with Group I and Group V (range of P values was 0.539–0.958).Conclusion:CST administration as a therapeutic agent might ameliorate the severity of intraocular inflammation in uveitis patients. In conclusion, effect of CST and dexamethasone in EIU model was comparable.

Mast cells profiles and heterogeneity in lipopolysaccharide-induced uveitis model

Backgound: Uveitis is an important inflammation of the middle tunica of the eye which can lead to blindness. Mast cells (MCs) participate in the development of the inflammatory process by attracting other cell types. Methods: The MCs profile was evaluated in the endotoxin-induced uveitis model (EIU), in the acute (24 hours) and late (48 hours) phases of the inflammatory process. EIU was induced by inoculation of lipopolysaccharide, subcutaneously, into the paw of the rats. After 24 and 48 hours of the induction, the eyes were enucleated for paraffin inclusion and histopathological studies of MCs in the anterior chamber of the eye. MCs were morphologically evaluated using Toluidine blue dye, their histamine accumulation was analyzed by Alcian blue and Safranin-O method, and the presence of tryptase and chymase by immunohistochemistry. Results: The results showed larger amount of total and degranulated MCs, as well as Alcian blue and Safranin positive MCs in the acute phase of EIU, especially in the ciliary body. Few tryptase positive MCs were observed in acute and late phases of EIU, but numerous chymase positive MCs were found in the 24-hour group. Conclusions: The data highlight the MC modulation during EIU and indicate these cells closely linked to the inflammatory process in uveitis.

Synthetic Glucocorticoid-Induced Leucine Zipper Peptide Inhibits Lipopolysaccharide-Induced Ocular Inflammation in Rats

Purpose: To demonstrate the anti-inflammatory action of a synthetic glucocorticoid-induced leucine zipper (GILZ^98–134) peptide (GILZ-p) in a model of endotoxin-induced uveitis (EIU) in rats. Methods: The EIU model was induced in Sprague Dawley rats with an intravitreal injection of lipopolysaccharide (LPS). Synthetic GILZ-p was injected intravitreally 6 h after the LPS injection. To evaluate the anti-inflammatory effects of GILZ-p, the inflammatory response in the anterior chamber and iris of the rat eyes was evaluated with a slitlamp microscope on days 0, 1, 2, 3, and 4 after GILZ-p injection. The retinal expression of inflammatory cytokines was measured on days 0, 1, 2, 3, and 4 after GILZ-p injection. Müller cell gliosis was also detected at planned time points after GILZ-p injection. Results: Anterior segment inflammation peaked at 24 h after LPS injection in the EIU model. Compared with the controls, intravitreal GILZ-p significantly suppressed LPS-induced anterior segment inflammation in the EIU rats. The levels of retinal inflammatory factors IL-1β, TNF-α, MCP-1, and ICAM-1 were simultaneously reduced by the intravitreal GILZ-p injection. The expression of vimentin in the EIU retina was significantly reduced by GILZ-p, and the downregulated aquaporin 4 in the EIU retina was significantly restored by GILZ-p. Conclusion: The synthetic GILZ-p inhibited the inflammatory reaction in the EIU model and may have utility in the treatment of inflammatory ocular disease.

Comparative analysis of inflammatory response in the BALB/c and C57BL/6 mouse strains in an endotoxin-induced uveitis model

Uveitis is an inflammatory disease associated with diverse systemic and autoimmune diseases, defined as the inflammation of any given segment of the uveal tract, uveitis contributes with 5–20% cases of blindness in the USA/Europe and >25% of cases in third-world countries. To understand its pathogenic mechanisms, BALB/c and C57BL/6 mice were induced to develop the condition by a single intraperitoneal injection of E. coli lipopolysaccharide, the aim of the present work is to determine leukocyte infiltration in an endotoxin-induced uveitis (EIU) in two non-related mouse strains. Histopathological findings and clinical analysis were conducted 24 and 48 h postinjection. Both strains presented conventional clinical signs of uveitis 24 h post LPS injection and the highest inflammatory leukocyte infiltration in the uveal tract was found in the BALB/c mouse strain. This article will give an insight on the difference of the inflammatory response in the EIU model in two different mouse strains and the relationship between the pathologic response.