Effect of aflibercept combined with triamcinolone acetonide on aqueous humor growth factor and inflammatory mediators in diabetic macular edema

AIM:To investigate the efficacy of aflibercept combined with sub-tenon injection of triamcinolone acetonide(TA)in treating diabetic macular edema(DME)and to examine changes in growth factors and inflammatory mediator levels in aqueous humor after injection.METHODS:Totally 67 DME patients(67 eyes)and 30 cataract patients(32 eyes)were enrolled as the DME group and the control group,respectively.The DME group was divided into the aflibercept group(34 cases)and the aflibercept combined with TA group(combined group,33 cases).The aqueous humor of both groups was collected during the study period.The aqueous levels of vascular endothelial growth factor(VEGF),monocyte chemoattractant protein-1(MCP-1),interleukin-6(IL-6),interleukin-8(IL-8),and interleukin-1β(IL-1β)were detected using a microsphere suspension array technology(Luminex 200TM).Aqueous cytokines,best-corrected visual acuity(BCVA),central macular thickness(CMT),and complications before and after treatment were compared between the aflibercept group and combined group.RESULTS:The concentrations of VEGF,MCP-1,IL-6,and IL-8 in the aqueous humor were significantly higher in the DME group than those of the control group(all P<0.01).After 1mo of surgery,the concentrations of VEGF,MCP-1,IL-6,and IL-8 in the aqueous humor were significantly lower in the combined group than those of the aflibercept group(all P<0.01).The BCVA and CMT values of the two groups were statistically different after 1 and 2mo of treatment(P<0.01).However,the difference was not statistically significant after 3mo of treatment(P>0.05).CONCLUSION:The cytokines VEGF,MCP-1,IL-6,and IL-8 in the aqueous humor of DME patients are significantly increased.Aflibercept and aflibercept combined with TA have good efficacy in DME patients,can effectively reduce CMT,improve the patient’s vision,and have high safety.Aflibercept combined with TA can quickly downregulate the aqueous humor cytokines and help to relieve macular edema rapidly.However,the long-term efficacy is comparable to that of aflibercept alone.

Research progress on the correlation between aqueous humor components and pathogenesis and postoperative complications in patients with different types of cataracts

Aqueous humor provides the necessary nutrition for the lens and transports the metabolites in the eye.It was a liquid that can directly reflect the microenvironment in the eye'and it can be easily obtained during the operation.This review intended to analyze the components of aqueous humor in patients with different types of cataracts'so as to reflect the pathogenesis and development of the disease'evaluate the incidence of postoperative complications and provide reference value for the surgical design of sequential cataract surgery.The aqueous humor components of different types of cataracts showed different degrees of inflammation'oxidative stress and extracellular matrix remodeling.The biomarker of early neuropathy in diabetic cataract was neural cell adhesion molecule-1(NCAM1).Transforming growth factor-β(TGF-β)was the evaluation factor of disease development in patients with pseudoexfoliation syndrome.The relationships between postoperative complications of different types of cataracts and aqueous humor components were as follows:Macular edema after diabetic cataract surgery was associated with tumor necrosis factor-alpha;capsular contraction after high myopic cataract surgery was related to monocyte chemoattractant protein-1(MCP-1)and TGF-β2;Klotho and glutathione S-transferase P 1(GSTP1)were associated with high intraocular pressure after primary open-angle glaucoma complicated by cataract surgery;capsular contraction after retinitis pigmentosa complicated by cataract surgery was associated with matrix metalloproteinases;pro-inflammatory cytokines and fibroblast growth factor 4 in the aqueous humor of congenital cataracts were associated with posterior capsular opacification after surgery.Granulocyte colony stimulating factor 3 and MCP-1 were the main cytokines mediating the pain of the second eye in the binocular sequential cataract surgery short interval(1 wk)'while MCP-1 mediated pain in the long interval(6 wk).The second eye after binocular sequential cataract surgery had a higher level of proinflammatory factors.The components of aqueous humor in patients with different types of cataracts were related to the pathogenesis and postoperative complications of the disease.Monitoring the components of the aqueous humor could help better understand the intraocular microenvironment of different types of cataracts and provide a reference for predicting the development of the disease and implementing relevant targeted therapy.

Differential analysis of aqueous humor cytokine levels in patients with macular edema secondary to diabetic retinopathy or retinal vein occlusion

AIM:To evaluate the difference and the correlation between the concentrations of cytokines in the aqueous humor of eyes with macular edema secondary to diabetic retinopathy(DR)or retinal vein occlusion(RVO).METHODS:This is a retrospective case control study.The aqueous humor samples were collected during intravitreal injection of anti-vascular endothelial growth factor(VEGF)for patients diagnosed with macular edema secondary to DR(DME)or RVO(RVO-ME)at Xijing Hospital from August 2021 to July 2022.Meanwhile,aqueous humor samples during vitrectomy from patients with idiopathic macular hole(IMH)were also collected and served as controls.The aqueous humor concentrations of VEGF,platelet-derived factor(PDGF),interleukin(IL)-6,IL-8,IL-18,tumor necrosis factor-α(TNF-α)and monocyte chemoattractant protein 1(MCP-1)were measured with Human Premixed Multi-Analyte Kit(Luminex).The difference of the aqueous cytokines and the correlation between the two diseases were analyzed.RESULTS:A total of 40 eyes of 38 patients were enrolled in the study,including 13 eyes of 11 DME patients(DME group),16 eyes of 16 RVO-ME patients(RVO-ME group)and 11 eyes of 11 IMH patients(control group).The VEGF,PDGF,IL-6,IL-8,and MCP-1 levels of the aqueous humor were higher in both DME and RVO-ME groups compared with the control group(all P<0.05),the levels of TNF-αwas higher in the DME group than in the control group(P<0.05).The VEGF,IL-6,MCP-1,and TNF-αlevels in the aqueous humor were significantly higher in the DR group than those in the RVO group(all P<0.05).Correlation analyses revealed that there were complex positive correlations between IL-6,IL-8,IL-18,MCP-1,and TNF-αlevels in the aqueous humor of eyes with two diseases.CONCLUSION:Although ischemic and inflammatory factors are similarly involved in the pathogenesis of DME and RVO-ME,the roles of these factors are more significant or more likely to be activated in DR patients,suggesting different treatment strategies should be considered for the two diseases.

Increased melatonin levels in aqueous humor of patients with proliferative retinopathy in type 2 diabetes mellitus

AIM： To report the association between melatonin levels in aqueous humor and serum, and diabetic retinopathy（DR） grade in type 2 diabetic patients.·METHODS： Aqueous humor and plasma samples from26 patients with DR（in nonproliferative and proliferative stages） and 14 control subjects were collected during cataract surgery after 6 p.m. Melatonin concentrations were determined using an enzyme-linked immunosorbent assay（ELISA）.·RESULTS： Melatonin levels were significantly higher in the aqueous humor of patients with proliferative diabetic retinopathy（PDR） [18.57 ±2.67 pg/m L（range 15.20-23.06）vs 13.63±2.71 pg/m L（range 10.20-20.20）, P =0.0001], but not in those with nonproliferative retinopathy（NPDR）[13.79±2.56 pg/m L（range 9.80-20.10） vs 13.63±2.71 pg/m L（range 10.20-20.20）, P =0.961] compared to controls. There was decrement in the plasma melatonin level of patients with PDR, but no significant differences between the plasma melatonin levels of the study groups [ 5. 37 ±1.74 pg/m L（range 2.85-8.65） vs 6.11±1.90 pg/m L（range3.13-9.41）, P =0.293], or between control and DR groups[NPDR 6.11 ±1.90 pg/m L（range 3.13-9.41） vs control6.15±1.91 pg/m L（range 2.18-9.86）; PDR（5.37±1.74 pg/m L（range 2.85-8.65） vs control 6.15 ±1.91 pg/m L（range2.18-9.86）, P =0.808, P =0.264].·CONCLUSION： Elevated melatonin levels in aqueous humor in PDR may indicate the level to be associated with DR severity.

Changes of total antioxidant capacity and total oxidant status of aqueous humor in diabetes patients and correlations with diabetic retinopathy

AIM:To measure changes of total oxidant status(TOS)and total antioxidant capacity(TAC) of aqueous humor(AH) in diabetic retinopathy(DR) patients,and to determine if there were any differences in TOS and TAC of AH in diabetic patients without retinopathy compared with non-diabetic patients.·METHODS:One hundred and three eyes of 103patients who were enrolled for cataract surgery were included in this study.Patients were grouped according to presence of diabetes and stage of DR.Prior to cataract surgery,0.1mL to 0.2mL of AH was aspirated and analyzed for TAC and TOS level using a colorimetric method.·RESULTS:TOS levels were highest among proliferative diabetic retinopathy(PDR) patients and lowest in patients with only cataracts.Results were statistically significant between all groups(P 【0.05).Whereas result between diabetic without retinopathy patients and non-proliferative diabetic retinopathy(NPDR) patients was not statistically significant(P =0.757).TAC levels were highest in patients with only cataract and lowest among PDR patients and results were statistically significant between all groups(P 【0.05).·CONCLUSION:Aqueous humor TAC levels are low in diabetic patients and reduced further in DR patients,TOS levels are increased in diabetic patients and this is exacerbated in DR patients.

Aqueous proinflammatory cytokines in acute primary angle-closure eyes

AIM：To evaluate changes of proinflammatory cytokines in aqueous humor of patients with acute primary angleclosure（APAC） and age-related cataracts. METHODS：Twenty eyes of 20 APAC patients and 15 eyes of 15 age-related cataract patients were included in this cross-sectional study. Aqueous humor samples were collected prospectively. The levels of 20 proinflammatory cytokines were evaluated in the aqueous humor of the APAC and cataract patients using the multiplex bead immunoassay technique. Clinical data were collected for correlation analysis.RESULTS：Seven of the 20 proinflammatory cytokines included in the magnetic bead panel were detectable in both APAC eyes and cataract eyes：interleukin（IL）-10, IL-12, IL-15, IL-21, IL-6, chemokine（C-C motif） ligand 20, and tumor necrosis factor alpha（TNF-α）. IL-27 was only detectable in APAC eyes. Compared with the cataract eyes, the APAC eyes had significantly elevated concentrations of IL-12（P=0.036）, IL-15（P=0.001）, IL-6（P=0.012）, and IL-27（only detectable in APAC eyes）. Age was positively correlated with IL-12（P=0.022） and IL-6（P=0.037）, and time elapsed between APAC onset and aqueous humor samples collection was positively correlated with IL-15（P=0.037）, IL-27（P=0.040）, and TNF-α（P=0.042）.CONCLUSION：Several proinflammatory cytokines including IL-12,IL-15, IL-6 and IL-27, were elevated in the APAC eyes and may be implicated in its pathologic mechanism.

Proteome alterations in aqueous humour of primary open angle glaucoma patients

AIM: To unravel the primary open angle glaucoma(POAG) related proteomic changes in aqueous humour(AH).METHODS: Totally 35 patients listed for cataract surgery(controls: n=12, age: 67.4±13.6 y) or trabeculectomy for POAG(n=23, age: 72.5±8.3 y) were included. AH samples of those patients were obtained during cataract surgery or trabeculectomy. AH samples were subsequently pooled into the experimental groups under equal contribution in terms of protein amount of each individual patient. Protein samples were analyzed by a linear trap quadrupol Orbitrap Mass Spectrometry device with an upstream liquid chromatography system. The obtained raw data were analyzed using the Maxquant proteome software and compared. Proteins with a fold-change ratio higher than a cut-off of 2 were considered as noticeably altered.RESULTS: A total number of 175 proteins could be identified out of the AH from POAG and cataract by means of quantitative mass spectrometric analysis. Apolipoprotein D(fold change, 3.16 times), complement C3(2.96), pigment epithelium-derived factor(2.86), dickkopf-related protein 3(2.18) and wingless-related integration(Wnt) inhibitory factor 1(2.35) were significantly upregulated within the AH of glaucoma compared to cataract serving as controls.CONCLUSION: AH provides a tool to analyze changes in glaucoma and shows striking changes in Wnt signaling inhibitory molecules and other proteins.

Age-related pro-inflammatory and pro-angiogenic changes in human aqueous humor

AIM： To reveal age-related aqueous cytokine changes in human aqueous humor.METHODS： Aqueous humor was collected from 12 young children（3-6.5 years old） and 71 healthy adults（22-106 years old） with cataract but without other systemic or ocular disorders. Levels of 22 cytokines, chemokines and vascular endothelial growth factor（VEGF） were measured and analyzed.RESULTS： The following proteins showed significant increase from childhood to adult： interferon-gamma（IFN-γ）, interleukin（IL）-13, IL-6, IL-12（p70）, IL-10, CCL2, CCL3, CCL4, CXCL8, CXCL9, CXCL10, IFN-α2 and VEGF（all P〈0.05）. IFN-γ, IL-13, IL-12（p70）, IL-10, CCL3, CXCL9 and VEGF also showed moderate strength age-related increase in the adult group（r〉0.5）. The strength of correlation between aging and CCL4 were fair（r=0.398）. The concentrations of IL-2, IL-4, IL-5, IL-1β and TNF-α were low in both groups.CONCLUSION： From childhood to adult, the immunological milieu of the anterior chamber become more pro-inflammatory and pro-angiogenic. Such changes may represent the parainflammation state of the human eye.

Relationship between raised intraocular pressure and ischemia-modified albumin in serum and humor aqueous: a pilot study in rabbits

AIM:To evaluate the relationship between increased intraocular pressure(IOP),is chemia-modified albumin levels in serum(IMA-s)and in humor aqueous(IMA-HA)in rabbits.METHODS:Twenty-five albino New Zealand rabbits weighing between 2.0 and 2.8 kg were used in this pilot study.With permission from Canakkale Onsekiz Mart University Animal Ethics Committee,the IOP of both eyes of each rabbit were recorded with a Tonopen(Tono-Pen XL,Reichart Inc.,Depew,NY,USA)after the application of topical proparacaine 0.5%HCl anesthesia.Blood(4 mL)was collected from the marginal ear vein and an intracameral injection of 2.3 mg/mL sodium hyaluronate and subconjunctival dexamethasone was given in the right eye.Anterior chamber aqueous fluid was obtained using a limbal approach with a 27 gauge needle from both eyes.The left eyes were used as controls.IOP was measured on the 1st,3rdand 10thday after the initial injection,with Tonopen,IMA-s levels and IMA-HA examined simultaneously.RESULTS:Beforetheinjections,IOPwas11.4±3.0mmHg in the right eye and 11.3±3.1 mm Hg in the left eye(P】0.05).There was a statistically significant difference between IMA-s levels before the IOP increase(IMA-s0)and IMA-s levels on the 1stand 3rddays after the increase in IOP(P=0.012 and P=0.01,respectively).No difference was observed between IMA-s0and serum IMA levels on the 10thday(IMA-s10)after IOP increase(P=0.989).IMA-HA in the right eye in the first day after the injection was positively correlated with IOP(r=0.748;P=0.02).No othercorrelation is found between any other parameter with IMA-HAlevels at any test time.A statistically significant positive correlation was observed between IMA-s values and IOP on the 1stand 3rddays(r=0.398,P=0.04 and r=0.382,P=0.04,respectively).There was no correlation between IMA-s levels and increased IOP on the 10thday after IOP increase(r=0.026,P=0.902).CONCLUSION:IMA may be an important indicator of acute damage caused by diseases involving ischemic damage to the eye,especially in case of increased intraocular pressure.

LMWH inhibits anterior chamber inflammation after extra capsular lens extraction through down regulation of bFGF content in aqueous humor

AIM: To observe the changes of basic fibroblast growth factor (bFGF) content in anterior chamber before and after extra capsular lens extraction for investigating the mechanism of low molecular weight heparin (LMWH) inhibiting anterior chamber inflammation. METHODS: Eighty-four rabbits were randomly divided into control and experimental group, 42 rabbits in each group. Extra capsular lens extraction was done on unilateral eye in each rabbit. LMWH was perfused into anterior chamber by the concentration of 50U/mL at the end of operation in experimental group. The degrees of corneal edema, aqueous flare and fibrin were evaluated with slit lamp microscope in postoperative day 1, 3, 6, 15, 30, 45 and 60, respectively. Six eyes of each group were at each time point. Contents of bFGF in aqueous humor were determined by ELISA after animals were killed. Another six eyes were used for determining the base line level of bFGF in aqueous humor. RESULTS: The degrees of corneal edema, aqueous flare and fibrin in experimental group were significantly lighter than those in control group (p<0.01) on postoperative day 1, 3 and 6, respectively. No difference was showed between the two groups at other point time. Contents of bFGF in aqueous humor increased at the same time. bFGF content was reached peal on postoperative day 1 in experimental group, while on postoperative day 6 in control group. Contents of bFGF in the two groups declined slowly after reaching peak. The bFGF content in control group were significantly higher than that in experimental group 1-30 days after surgery (p<0.05). No significant differences were shown between the two groups on postoperative day 45 and 60, respectively. CONCLUSION: Perfusion with LMWH by the concentration of 50U/mL can significantly reduce anterior chamber inflammation after extra capsular lens extraction in rabbits, which may be related to down regulation of bFGF content in aqueous humor.

MicroRNAs as biomarkers in glaucoma and potential therapeutic targets

Glaucoma is a neurodegenerative disease in which optic nerve damage and visual field defects occur.It is a leading cause of irreversible blindness.Its pathogenesis is largely unknown although several risk factors have been identified,with an increase in intraocular pressure being the main one.Lowering of intraocular pressure is the only treatment available.Open-angle glaucoma is the most common form of the condition,accounting for~90%of all cases of glaucoma,with primary open-angle glaucoma and exfoliation glaucoma being the most frequent types.There are strong indications that microRNAs play important roles in the pathogenesis of primary open-angle glaucoma.Most of the recent studies reviewed had performed microRNA profiling in aqueous humor from glaucoma patients compared to controls who were chiefly cataract patients.A very large number of microRNAs were dysregulated but with limited overlap between individual studies.MiRNAs in aqueous humor that could be possible targets for therapeutic intervention are miR-143-3p,miR-125b-5p,and miR-1260b.No ove rlap of findings occurred within the dysregulated miRNAs for blood plasma,blood serum,peripheral blood mononuclear cells,and tears of primary open-angle glaucoma patients.Seve ral impo rtant limitations were identified in these studies.Further studies are warranted of mic roRNA expression in aqueous humor and blood samples of primary open-angle glaucoma patients in the early stages of the disease so that validated biomarkers can be identified and treatment initiated.In addition,whether modifying the levels of specific microRNAs in aqueous humor or tears has a beneficial effect on intraocular pressure and ophthalmic examination of the eyes should be investigated using suitable animal models of glaucoma.

Intraocular pressure control of a novel glaucoma drainage device-in vitro and in vivo studies

AIM：To evaluate the intraocular pressure（IOP）control of an artificial trabeculum drainage system（ATDS）,a newly designed glaucoma drainage device,and postoperative complications in normal rabbit eyes.METHODS：Pressure drops in air and fluid of 30 ATDS were measured after being connected to a closed manometric system.Twenty of them were then chosen and implanted randomly into the eyes of 20 rabbits.Postoperative slitlamp,gonioscopic examination and IOP measurements were recorded periodically.Ultrasound biomicroscopy and B-scan ultrasonography were also used to observe the complications.Eyes were enucleated on day 60.RESULTS：Pressure drops of 4.6-9.4 mm Hg were obtained at physiological aqueous flow rates in the tests in vitro.The average postoperative IOP of the experimental eyes（11.6-12.8 mm Hg）was lower than the controls significantly（P〈0.05）at each time point.Complications of hemorrhage（n=1）,cellulosic exudation（two cases）and local iris congestion（two cases）were observed.The lumina of the devices were devoid of obstructions in all specimens examined and a thin fibrous capsule was found around the endplate.CONCLUSION：ATDS reduce IOP effectively.However,further studies on the structure are needed to reduce complications.

Analogs of microgravity: the function of Schlemm's canal, intraocular pressure and autonomic nervous during the head-down tilt test in healthy subjects

AIM:To evaluate the ocular outcomes and to elucidate possible mechanisms underlying intraocular pressure(IOP)change following the head-down tilt(HDT)test.METHODS:The study included 21 participants at the Department of Ophthalmology of Tongji Hospital.Subjects received the test of I-care tonometry,enhanced depth imaging optical coherence tomography and heart rate variability(HRV)analysis before and after 15 min HDT test.The lumen area of Schlemm’s canal(SCAR),IOP,HRV were calculated.RESULTS:IOP increased significantly after 20°head down position from 14.0±3.0 to 17.0±3.3 mm Hg(P<0.001).SCAR decreased from 13449.0±5454.9μm^(2) at sitting condition to 9576.6±4130.9μm^(2) post 15 min HDT test.High frequency(HF)indices increased significantly from 1462±865 Hz at baseline to 2128±824 Hz.Heart rate(HR)decreased significantly from 76±11.48 to 70±11.52 bpm after the HDT.The linear regression analysis showed that the difference of HF and SCAR significantly correlated with each other during the HDT(R^(2)=20%,P=0.04).CONCLUSION:These outcomes perform the first evidence of the activation of autonomic nervous system of HDT may cause the collapse of Schlemm’s canal lumen,which in turn leading to the increased IOP.

Extracellular matrix gene expression in human trabecular meshwork cells following mechanical fluid flow stimulation

AIM: To investigate changes in extracellular matrix(ECM) gene expression in human trabecular meshwork(HTM) cells in response to mechanical fluid flow stimulation.METHODS: HTM cells were grown on a glass plate coated with 0.02% type Ⅰ collagen(COL) and exposed to shear stress(0, 0.2, 1.0 dyne/cm;) for 12 h.Changes in genes related to the ECM were evaluated by real-time reverse transcriptase-polymerase chain reaction.Phosphorylation of Smad2 protein was investigated by Western blotting.RESULTS: After mechanical stimulation, COL type 4 alpha 2, COL type 6 alpha 1, and fibronectin-1 mRNA were significantly higher than the static control(P<0.05, <0.05, and <0.01, respectively).The metalloproteinase-2 and plasminogen activator inhibitor-1 mRNA were significantly higher than the static control(P<0.05 and <0.01, respectively), while the differences in the tissue inhibitors of metalloproteinases-2 mRNA were not significant.The phosphorylation of Smad2 levels was significantly higher compared to the static control cells.CONCLUSION: Changes in the expressions of genes associated ECM metabolism result in HTM cells after mechanical stimulation.The mechanical stimulation of the aqueous humor to the trabecular meshwork may promote ECM turnover and contribute to intraocular pressure homeostasis.

Risk factors for the long-term prognosis and recurrence of HIV-negative cytomegalovirus retinitis in North China

AIM: To demonstrate the clinical features, the risk factors, the visual prognosis and the recurrence of cytomegalovirus(CMV) retinitis(CMVR) in HIV-negative patients.METHODS: HIV-negative patients with CMVR were involved in this study. Best corrected visual acuity(BCVA), intraocular pressure(IOP), CMV-DNA load in aqueous and/or serum samples, treatment, follow-up time, recurrence and complications were recorded. Ocular characteristics were evaluated by fundus photographs. Association between ocular factors and visual prognosis were analyzed by regression analysis.RESULTS: Twenty-five eyes of 16 patients were included. All 25 eyes underwent intravitreal injections of anti-viral agents. The mean log MAR BCVA improved from 0.94±0.98(0.98-0.78) initially to 0.77±0.73(0.82-0.68) at last visit, but not significantly. After antiviral treatment, the aqueous CMV DNA load significantly reduced to(3.42±1.47)×10~2 copies/m L(P=0.001), compared with(2.51±3.11)×10~5 copies/m L at baseline. Macular involvement(R~2=0.475, P=0.049) and initial visual acuity(R~2=0.475, P=0.017) were significantly associated with the poor visual prognosis(BCVA<20/400). The extent of retinal lesions(R~2=0.064, P=0.04) was significant associated with the risk of recurrence of CMVR.CONCLUSION: Intravitreal injection of anti-viral agents offers a safe and effective treatment for CMVR. Macular involvement and initial visual acuity significantly associate with visual prognosis. The extent of retinal lesions is significantly associated with the recurrence of CMVR. These ocular factors can be used as predictive risk factors for long term visual prognosis in HIV-negative CMVR patients.

AN EXPERIMENTAL STUDY ON UVEOSCLERAL OUTFLOW WITH FITC-BSA AS A TRACER IN RABBIT EYE

It is to discuss the method for studying uveoscleral pathway and the influence of uveoscleral outflow on aqueous humor drainage. Seventeen rabbits were randomly divided into the flow group that included 5 rabbits and the morphological group that included 12. Fluoresceinisothiocyanate bovine serum albumin (FITCBSA) was perfused into anterior chambers of flow group rabbit eyes as the tracer for measuring uveoscleral outflow. This tracer was infused into anterior chambers of morphological group rabbit eyes for observing fluorescent intensity of all tissue of the uveoscleral pathway with fluorescence microscopy. Fluorescence was seen in all sites of uveoscleral pathway. The fluorescent intensity of ciliary body and suprachoroidal space was significantly stronger than that of anterior sclera (P<005). The intensity of choroid and posterior sclera was significantly weaker than that of other tissues (P<005). The average value of uveoscleral outflow was (0180031) l/min. This study demonstrates that uveoscleral pathway plays an important role in aqueous humor drainage. Anterior sclera is the main site where aqueous humor flows out. FITCBSA is an effective tracer for studying uveoscleral pathway from both morphological and functional aspects.

Effect of cAMP and cGMP on Connexin43 Expression in Isolated Human and Bovine Ciliary Epithelium

The aim of the study was to assess the distribution of connexin43 (Cx43) and connexin40 (Cx40) in human and bovine ciliary bodies. The effect of the second messengers cAMP and cyclic cGMP on Cx43 protein expression was also investigated. Enucleated human eyes (remnant after corneal transplantation) and bovine eyes were used. Tissue preparations of the anterior segments of the eyes have proceeded for immunohistochemical staining with polyclonal antibodies of Cx43 and Cx40. Isolated ciliary bodies of human and bovine eyes were incubated with cAMP analog 8-Bromo-cAMP or the cGMP analog 8-Bromo-cGMP, the expression of Cx43 protein in the tissues was then assessed by Western blot assay. Both in human and bovine ciliary bodies, strong immunoreactivity of Cx43, but not Cx40, was observed predominantly in the apical cytoplasmic portions of the pigment ciliary epithelial cells and non-pigmented ciliary epithelial cells. In human ciliary body both cAMP and cGMP up-regulated Cx43 expression, while in the bovine ciliary body, cGMP increased Cx43 expression but cAMP decreased it. Cx43 is the major component of human and bovine gap junctions in the ciliary epithelium. The regulation on the Cx43 expression by cAMP and cGMP in human and bovine ciliary bodies suggests the possibly different roles of these signal messengers in the intracellular communication.

Reversible changes in aqueous outflow facility, hydrodynamics, and morphology following acute intraocular pressure variation in bovine eyes

Background Elevated intraocular pressure （lOP） is primarily due to increased aqueous outflow resistance, but how aqueous outflow resistance is generated and regulated are still not fully understood. The aim of this study is to determine whether changes in outflow facility, outflow pattern, and morphology following acute lOP elevation were reversible when the lOP was returned to a normal level in bovine eyes using a two-color tracer technique to label outflow patterns within the same eye. Methods Twelve fresh enucleated bovine eyes were perfused with Dulbecco＇s phosphate buffer saline （PBS） containing 5.5 mmol/L glucose （DBG） at 30 mmHg first to establish the baseline outflow facility followed by a fixed volume of red fluorescent microspheres （0.5 μm, 0.002% v/v）. After the red tracer being replaced with DBG in the anterior chamber, perfusion was continued at 7 mmHg with the same volume of green tracer, followed by a fixative. In two control groups, the eyes were constantly perfused at either 30 mmHg （n=6） or 7 mmHg （n=6） using the same methods. The outflow facility （C, pJ.min.-lmmHg-1）, was continuously recorded. Confocal images were taken along the inner wall （IW） of the aqueous plexus （AP） in frontal sections. The percent of the effective filtration length （PEFL, PEFL=IW length exhibiting tracer labeling/total length of IW） was measured. Sections with AP were processed and examined by light microscopy. The total length of IW and the length exhibiting separation （SL） in the juxtacanalicular connective tissue （JCT） were measured. A minimum of eight collector channel （CC） ostia per eye were analyzed for herniations. Results In the experimental （30-7 mmHg） group, the outflow facility was significantly higher at 7 mmHg （（4.81±1.33） #lmin-1 mmHg-1） than that at 30 mmHg （（0.99±0.15） μl.min-1 mmHg-1, P=-0.002）, corresponding to a significant increase in the PEFL （P=-0.0003）. The percent of CC ostia exhibiting herniations in the experimental group （（67.40±8.90） μl.min·-1mmHg-1） decreased significantly compared to that in the control at 30 mmHg （（94.44±3.33） μl.min-lmmHg-1, P=-0.03）, but higher than that in the control at 7 mmHg （（29.43±4.60） μl.min-1mmHg-1, P=0.01）. Washout-associated separation between the IW and JCT was found by light microscopy and percent separation length （PSL, PSL=SL/total length of IW） was decreased in the control at 30 mmHg compared to that in the experimental group and control at 7 mmHg. Conclusions The pressure-induced morphological and hydrodynamic changes were reversible. Changes （collapse of AP, separation between the JCT and IW, and herniation into CC ostia） influence the effective filtration area that regulates outflow facility.

Development of a novel two color tracer perfusion technique for the hydrodynamic study of aqueous outflow in bovine eyes

Background Elevation of intraocular pressure is usually associated with primary open angle glaucoma and caused by increased outflow resistance. A two-color fluorescent tracer technique was developed to investigate the hydrodynamics of aqueous humor outflow with changing intraocular pressure within the same eye, to better understand the relationship between outflow facility and effective filtration area. Methods Eighteen enucleated bovine eyes were first perfused at 30 mmHg with Dulbecco＇s phosphate-buffered saline containing 5.5 mmol/L D-glucose. After a stable baseline facility, red fluorescent microspheres （0.5 um, 0.002% v/v） were exchanged and perfused. Eyes in the one-color control group （n=6） were immediately perfused with fixative. In the experimental group （n=6）, eyes were perfused with green tracer after intraocular pressure reduced to 7 mmHg, while in the two-color control group （n=6）, eyes were perfused with green tracer with intraocular pressure remaining at 30 mmHg. All 12 eyes were then per＇fusion-fixed. Outflow facility was continuously recorded in all eyes. Confocal images were taken along the inner wall of the aqueous plexus and the percent of the effective filtration length （PEFL; length of inner wall exhibiting tracer labeling/total length of inner wall） was measured. The relationships between outflow facility and PEFL were analyzed statistically. Results No significant differences were found in baseline facilities （ul.min-1.mmHg-1） among the three groups （the experimental group： 0.93±0.12; the two-color control group： 0.90±0.19; the one-color control group： 0.98±0.13）. In the experimental group, the outflow facility was significantly higher at 7 mmHg （4.29±1.01） than that at 30 mmHg （1.90±0.67, P 〈0.001）, which corresponded to a significant increase in the PEFL at 7 mmHg （54.70±8.42） from that at 30 mmHg （（11.76±4.56）%, P 〈0.001）. The PEFL labeled by red fluorescent microspheres in the experimental group （（11.76±4.56）%） showed no significant difference from that of the one-color control group （（13.39±2.19）%, P=-0.473） or the two-color control group （（11.49±4.95）%, P=-0.930）. The PEFL labeled by green fluorescent microspheres in the experimental group （（54.70±8.42）%） was significantly higher than that of the two color control group （（37.34±8.17）%, P=0.010）. A positive correlation was found between outflow facility and PEFL（r=0.897, R2=0.804） in the experimental group. Conclusions Changes in aqueous humor outflow patterns before and after a change in intraocular pressure can be successfully distinguished within the same eye using our newly developed two-color tracer perfusion technique. The PEFL showed positive correlation with the outflow facility.

Real-time and label-free biosensing using moiré pattern generated by bioresponsive hydrogel

Bioresponsive hydrogels are smart materials that respond to various external stimuli and exhibit great potential as biosensors owing to their capability of real-time and label-free detection.Here,we propose a sensing platform based on bioresponsive hydrogels,employing the concept of moiré patterns.Two sets of line patterns with different pitch sizes are prepared;a hydrogel grating whose pitch size changes according to external stimuli and a reference grating with constant pitch size.The volume changes of the hydrogel caused by external stimuli changes the pitch size of the hydrogel grating,and subsequently,the pitch sizes of the moiré patterns(moiré signal),whose values can be obtained in a real-time and label-free manner through customized moiré microscopy and signal processing.After confirming that the pH-induced swelling of hydrogel could be monitored using moiré patterns,we performed moiré pattern-based detection of specific proteins using protein-responsive hydrogel that underwent shrinking via interaction with target proteins.Brain-derived neurotrophic factor and platelet-derived growth factor were selected as the model proteins,and our proposed system successfully detected both proteins at nanomolar levels.In both cases,the pitch size change of hydrogel grating was monitored much more sensitively using moiré patterns than through direct measurements.The changes in the moiré signals caused by target proteins were detected in ex-vivo environments using a custom-made intraocular lens incorporating the hydrogel grating,demonstrating the capability of the proposed system to detect various markers in intraocular aqueous humor,when implanted in the eye.