Substance P and secretoneurin in vitreous aspirates of patients with various vitreoretinal diseases

By means of highly sensitive radioimmunoassays, the levels of substance P (SP) and secretoneurin (SN) were detected in vitreous aspirates of patients with macular holes which served as controls, in patients with nonproliferative diabetic retinopathy (DR), active proliferative diabetic retinopathy (active PDR), inactive PDR, rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR). Furthermore, SN-like immunoreactivities were characterized by reversed phase-HPLC. The concentration of SN was more than 20-fold higher in macular holes when compared with SP and reversed phase HPLC revealed evidence that the vitreous levels of SN represent authentic SN. SN was significantly decreased in patients with nonproliferative DR, active PDR and inactive PDR by more than 70% which seems to result from a reduced expression and/or secretion from the cilary epithelium and a reduced release from the retina both due to diabetes mellitus. By contrast SP was increased in rhegmatogenous retinal detachment most obviously due to an enhanced outflow of the peptide through retinal breaks. Despite their proangiogenic activities, SP and SN are unlikely to be involved in the pathogenesis of neovascularizations in DR because of their unchanged and reduced levels, respectively, but the low levels of both peptides may facilitate the regression of vasoproliferations following laser photocoagulation.     

Septin expression in proliferative retinal membranes.

We undertook this study to evaluate the expression of septin family members SEPT5, SEPT8, and SEPT11 in proliferative retinal membranes. Epiretinal membranes (ERM) were obtained from seven patients with proliferative vitreoretinopathy (PVR) and from four patients and four postmortem eyes with proliferative diabetic retinopathy (PDR). Subretinal membranes (SRM) were obtained from one patient and six postmortem eyes. Membranes were examined by immunohistochemical staining of paraffin sections using polyclonal antibodies against SEPT5, SEPT8, and SEPT11 and an ABC detection system. SEPT8 expression was detected in all ERM and SRM, with an exceptionally strong expression of 100% for ERM of PVR, 63% for PDR membranes, and 57% for SRM. SEPT11 was identified in 91% of all cases, with strong expression of 14%, 25%, and 14% in ERM of PVR, PDR, and SRM, respectively. SEPT5 was seen in 54% of all cases; strong immunostaining was found in only one case of PVR membranes. Our finding suggests a role for members of the septin family in the development of proliferative retinal membranes.     

康柏西普预防严重后巩膜裂伤玻璃体切除术后增生性玻璃体视网膜病变的临床疗效

摘要 目的：探讨玻璃体腔注射康柏西普对于严重后巩膜裂伤患者玻璃体切除术后增生性玻璃体视网膜病变发生的预防效果。方法：选取从2018年9月至2020年9月我院收治的40例（40眼）严重后巩膜裂伤患者进行研究,随机分为对照组20眼（行常规巩膜裂伤缝合术及经睫状体平坦部玻璃体切除术）和观察组20眼（行巩膜裂伤缝合术及经睫状体平坦部玻璃体切除术的同时联合玻璃体腔注射康柏西普治疗）。比较两组患者术前及术后的视力、眼压,以及术后增生性玻璃体视网膜病变的发生率、视网膜再脱离的发生率。结果：对照组及观察组术后的最佳矫正视力较术前均提高、术后眼压均正常,观察组术后的增生性玻璃体视网膜病变发生率（15.0 %）明显低于对照组（45.0 %, P<0.05）,观察组术后视网膜脱离复发率（5.0 %）低于对照组（30.0 %, P>0.05）。结论：严重后巩膜裂伤患者玻璃体切除术联合玻璃体注射康柏西普治疗能够有效降低增生性玻璃体视网膜病变的发生率和术后视网膜脱离的复发率,还可以改善患者的视力预后。     

Intraocular injection of an aptamer that binds PDGF-B: a potential treatment for proliferative retinopathies

Platelet-derived growth factor-B (PDGF-B) has been implicated in the pathogenesis of proliferative retinopathies and other scarring disorders in the eye. In this study, we sought to test the therapeutic potential of an aptamer that selectively binds PDGF-B, ARC126, and its PEGylated derivative, ARC127. Both ARC126 and ARC127 blocked PDGF-B-induced proliferation of cultured fibroblasts with an IC50 of 4 nM. Pharmacokinetic studies in rabbits showed similar peak vitreous concentrations of approximately 110 microM after intravitreous injection of 1 mg of either ARC126 or ARC127, but the terminal half-life was longer for ARC127 (98 versus 43 h). Efficacy was tested in rho/PDGF-B transgenic mice that express PDGF-B in photoreceptors and develop severe proliferative retinopathy resulting in retinal detachment. Compared to eyes injected with 20 microg of scrambled aptamer in which five of six developed detachments (three total and two partial), eyes injected with ARC126 (no detachment in five of six and one partial detachment), or ARC127 (no detachment in six of six) had significantly fewer retinal detachments. They also showed a significant reduction in epiretinal membrane formation. These data demonstrate that a single intravitreous injection of an aptamer that specifically binds PDGF-B is able to significantly reduce epiretinal membrane formation and retinal detachment in rho/PDGF-B mice. These striking effects in an aggressive model of proliferative retinopathy suggest that ARC126 and ARC127 should be considered for treatment of diseases in which PDGF-B has been implicated, including ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), and choroidal neovascularization.     

兔眼增殖性玻璃体视网膜病变模型的建立

目的探讨兔眼增殖性玻璃体视网膜病变模型的建立方法。方法①体外培养兔眼视网膜色素上皮细胞;②兔眼3组,每组6只,分别在玻璃体内注射0.1 mL的生理盐水、1×106细胞及2×106细胞,在不同时间段进行裂隙灯显微镜、间接检眼镜、眼底照像和B超检查,观察成模情况。结果注射后28 d,生理盐水组成模0眼;1×106细胞组成模5眼,其中Ⅰ级眼1只,Ⅱ级眼3只,Ⅲ级眼1只;2×106细胞组成模6眼,其中Ⅱ级眼2只,Ⅲ级眼4只。结论兔眼玻璃体内注射2×106同种视网膜色素上皮细胞建立增殖性玻璃体视网膜病变模型,符合病变发展规律,而且稳定可靠,成模较快,简单易行。     

Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy

PURPOSE: To determine levels of the chemokines CCL1/I-309, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL8/MCP-2, CXCL5/ENA-78, CXCL6/GCP-2, CXCL10/IP-10, and CXCL11/I-TAC in the vitreous humor and serum, from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD), and to investigate the expression of MCP-1, CXCL12/SDF-1, and the chemokine receptor CXCR3 in epiretinal membranes. METHODS: Paired vitreous humor and serum samples were obtained from patients undergoing vitrectomy for the treatment of RD (57 specimens), PVR (32 specimens), and PDR (88 specimens). The levels of chemokines were measured by enzyme-linked immunosorbent assays. Eighteen PDR and 5 PVR membranes were studied by immunohistochemical techniques. RESULTS: Of all the chemokines studied, only MCP-1 and IP-10 were detected in vitreous humor samples. MCP-1 levels in vitreous humor samples were significantly higher than in serum samples (p < 0.001). MCP-1 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0002). MCP-1 levels in vitreous humor samples from patients with active PDR were significantly higher than in inactive PDR cases (p = 0.0224). IP-10 levels in vitreous humor samples were significantly higher than in serum samples (p = 0.0035). IP-10 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0083). The incidence of IP-10 detection in vitreous humor samples was significantly higher in active PDR cases compared with inactive cases (p = 0.0214). There was a significant association between the incidence of IP-10 detection and increased levels of MCP-1 in vitreous humor samples from all patients, and patients with RD and PDR (p < 0.001 for all comparisons). MCP-1, and SDF-1 were localized in myofibroblasts in PVR and PDR membranes and in vascular endothelial cells in PDR membranes. CXCR3 was expressed by vascular endothelial cells in PDR membranes. CONCLUSION: MCP-1, IP-10 and SDF-1 may participate in pathogenesis of PVR and PDR. Myofibroblasts and vascular endothelial cells are the major cell types expressing MCP-1, SDF-1, and CXCR3 in epiretinal membranes.     

玻璃体视网膜手术治疗先天性视网膜劈裂及其并发症的临床疗效

目的:评价玻璃体视网膜手术治疗先天性视网膜劈裂及其并发症的临床疗效。方法:选择2009年1月-2012年1月于我院进行玻璃体视网膜手术的先天性视网膜劈裂患者30例(42只眼),患者均接受了闭合式睫状体经扁平部三切口入路保留晶状体的玻璃体切割手术,并分析其术前及术后情况。结果:先天性视网膜劈裂患者中发生孔源性视网膜脱离19眼,牵拉性视网膜脱离8眼,玻璃体积血10眼,同时伴有视网膜脱离和玻璃体积血有5眼;在末次随访时视力提高者有36只眼,占85.71%,无提高者有6只眼,占14.29%;术前平均视力为(0.15±0.09),末次随访时平均视力提高至(0.31±0.16),两者平均视力差异具有统计学意义(t=5.649,P0.001);42只眼视网膜解剖结构复位良好,视网膜平伏;OCT检查结果显示,末次随访时黄斑劈裂平均面积(0.22±0.18)mm2,与术前黄斑劈裂平均面积(1.07±0.52)mm2比较,差异有统计学意义(t=10.011,P0.001),黄斑微囊样改变有改善;随访期间5只眼出现并发症,占11.90%,其中2眼术后发生PVR且伴牵拉性视网膜脱离,2只眼发生白内障,1只眼出现玻璃体积血,术后视网膜解剖均复位良好。结论:玻璃体视网膜手术可以帮助患者进行视网膜解剖复位及提高其先天性视网膜劈裂患者视功能,具有良好的临床疗效。     

复杂性眼外伤患者玻璃体切割围手术期护理体会

目的:玻璃体切割术是眼科手术中比较复杂精细显手术,主要应用于治疗眼外伤导致的玻璃体出血,玻璃体浑浊,复杂性视网膜脱离以及增殖性玻璃体视网膜病变。本文主要探讨玻璃体切割术治疗复杂性眼外伤患者的围手术期护理的临床效应。方法:总结作者所在科室多年来护理眼外伤患者的临床经验,对2013年3月至2013年7月入院的75例(75只眼)复杂性眼外伤患者进行悉心的术前心理护理,完善的用药指导,充分的术前准备,术后特殊卧位的指导,预防并发症的护理及出院指导。结果:术后视力提高57例(76.0%),视力不变13(17.3%)例,视力下降5例(6.7%),28例眼内异物患者均经手术顺利取出异物。相关手术并发症经医护人员的对症治疗后均得到有效控制。患者术后满意度达98%以上,医生满意度达100%。结论:复杂性眼外伤患者玻璃体切割手术围手术期进行全方位的护理服务,对促进病人康复起到至关重要的作用。     

In vivo models of proliferative vitreoretinopathy

We outline current in vitro and in vivo models for experimental proliferative vitreoretinopathy (PVR) and provide a detailed protocol of our standardized in vivo PVR model. PVR is the leading cause of failed surgical procedures for the correction of rhegmatogenous retinal detachment. The pathogenesis of this multifactorial condition is still not completely understood. Experimental models for PVR help us understand the factors that play a role in the pathogenesis of the disease process in a controlled manner and allow for reproducible preclinical assessment of novel therapeutic interventions. We describe a cell injection model in detail that uses homologous retinal pigment epithelial (RPE) cell cultures to induce PVR over a 2-8 week period.     

Retinal photocoagulation does not influence intraocular levels of IGF-I, IGF-II and IGF-BP3 in proliferative diabetic retinopathy-evidence for combined treatment of PDR with somatostatin analogues and retinal photocoagulation?

Retinal photocoagulation reduces the incidence of severe visual loss in proliferative diabetic retinopathy (PDR). Reduced levels of VEGF/VPF might result in an improved function of the blood-retina barrier and cause a decrease of blood derived intraocular growth factors such as IGF-I. This study investigates whether retinal photocoagulation is able to normalize the concentrations of IGF-I, IGF-II and IGF-BP3 in the vitreous humor of patients undergoing vitrectomy. Levels of IGFs and the permeability marker, albumin, were measured in serum and vitreous of 52 patients. Three groups were compared: controls without proliferating eye disease (n = 19) and patients with PDR with (PDR+; n = 25) and without (PDR-; n = 8) previous retinal photocoagulation. IGF-I, IGF-II, IGF-BP3 and albumin were determined by immunological methods and were confirmed to be increased in patients with PDR compared to controls. Retinal photocoagulation influenced neither the intraocular concentration of the permeability marker albumin (PDR+: 253.2 +/- 46 mg/dl; PDR-: 256.4 +/- 66.5 mg/dl) nor the levels of IGFs (PDR+: IGF-I: 1.2 +/- 0.1 ng/ml; p = 0.38; IGF-II: 34.8 +/- 2.2 ng/ml; p = 0.1; IGF-BP3: 75.7 +/- 9.7 ng/ml; p = 0.27; PDR-: IGF-I: 1.1 +/- 0.2ng/ml; IGF-II: 29.3 +/- 5.2 ng/ml; IGF-BP3: 61.5 +/- 18.3 ng/ml). Systemic levels of albumin and IGFs were not changed significantly by retinal photocoagulation. These results demonstrate that previous retinal photocoagulation in patients undergoing vitrectomy does not functionally reestablish the blood-retina barrier despite decreases in VEGF/VPF. The lack of influence on intraocular concentrations of the serum-derived growth factors, IGF-I, IGF-II and IGF-BP3, might in part explain the failure of previous photocoagulation in the investigated patients. These results suggest that a combined treatment with retinal photocoagulation and growth hormone-lowering drugs, such as somatostatin analogues, could be a useful treatment, which may prevent further loss of visual acuity in patients with PDR.     

Phacoemulsification and silicone oil removal through the planned posterior capsulorhexis

The purpose of the study was to present operative technique and results of a passive hydrodynamic expression of silicone oil through planned posterior capsulorhexis during cataract surgery in patients after pars plana vitrectomy. The retrospective analysis was done on 57 eyes with cataract after a previous pars plana vitrectomy, operated on between 2001 and 2004 at the Clinical hospital "Sestre milosrdnice" Zagreb. Preoperative and postoperative best corrected visual acuity (BCVA), preoperative and postoperative intraocular pressure (IOP), and postoperative complications were reviewed. Visual acuity improved or stabilized in all patients with an attached retina. Retinal detachment occurred in 11 eyes. Transient vitreous hemorrhage, that resolved within 1 week of surgery without treatment, was observed in 4 eyes. Asymptomatic intraocular lens (IOL) decentration occurred in 2 eyes. Our findings suggest that silicone oil removal and cataract surgery can be performed as a single procedure in selected patients in the absence of macular pucker and retinal reproliferation, and in a presence of a stable retina.     

Comparison of Combined and Sequential Surgery for Proliferative Diabetic Retinopathy: A Single Surgeon Study

Purpose

To compare the results of combined and consecutive surgeries to treat proliferative diabetic retinopathy and cataract.

Methods

Retrospective comparative study. Forty-one patients with proliferative diabetic retinopathy (PDR) were enrolled. Twenty-nine eyes for the combined surgery group and twelve eyes for the sequential group were included. All surgeries were performed by one surgeon. Phacoemulsification was performed using a clear cornea incision. The vitrectomy was performed using a 20-gauge vitreous cutter.

Results

The best corrected visual acuity (BCVA) and intra- and post-operative complications were the main outcome measures. In the combined surgery group, the BCVA increased in 18 (62.1%) eyes, while eight (27.6%) eyes remained stable and three (10.3%) eyes decreased. Postoperative complications included fibrinous exudation in nine eyes, macular edema in three eyes and vitreous hemorrhage in three eyes. In the sequential surgery group, the BCVA increased in seven (58.3%) eyes, remained the same in four (33.3%) eyes and was reduced in one (8.3%) eye. Postoperative complications included macular edema in two eyes, neovascular glaucoma in two eyes and vitreous hemorrhage in one eye.

Conclusions

Both combined and sequential surgeries are safe and effective for treating PDR and cataracts. The combined surgery had a higher incidence of fibrinous exudation.     

Thrombin activation of PI3K/PDK1/Akt signaling promotes cyclin D1 upregulation and RPE cell proliferation

The retinal pigment epithelium (RPE) plays an essential role in the survival and function of the neural retina. RPE uncontrolled proliferation leads to the development of proliferative ocular pathologies, among which proliferative vitreoretinopathy (PVR) is the main cause of retinal surgery failure. Upon the breakdown of the BRB due to trauma or metabolic imbalance the contact of RPE with serum-contained thrombin has been shown to stimulate the proliferation of otherwise quiescent RPE cells. Although the molecular mechanisms involved in this effect are still undetermined, thrombin proteolytic activation of protease-activated G protein coupled receptor-1 (PAR-1) activates PI3K and Akt, known to play an essential role in proliferation. The present study demonstrates that: 1) thrombin stimulates Ser 473 Akt phosphorylation without affecting Thr 308 basal phosphorylation in RPE cells; 2) thrombin-induced Akt stimulation promotes cyclin D1 accumulation through the phosphorylation/ inhibition of GSK-3β, thus preventing Thr 286 cyclin D1 phosphorylation, nuclear export and degradation; 3) Akt signaling requires the upstream activation of PI3K and PLC. Since the pharmacological inhibition of these pathways or the silencing of cyclin expression prevent thrombin-induced RPE cell proliferation, these results contribute relevant evidence for establishing the mechanism involved in the development of proliferative eye diseases.     

PDGF stimulation of Müller cell proliferation: Contributions of c-JNK and the PI3K/Akt pathway

Platelet-derived growth factor (PDGF) has a critical role in proliferative vitreoretinopathy (PVR) as a chemoattractant and mitogen for retinal pigment epithelial cells and retinal glial cells. Here, we investigated the potential effects of PDGF on the proliferation of Müller cells and the intracellular signaling pathway mediating these changes. PDGF induced Müller cell proliferation and increased phosphorylation of the PDGF receptor (PDGFR), as shown by an MTT assay and immunoprecipitation analyses. Both effects were blocked by JNJ, a PDGFR-selective tyrosine kinase inhibitor. PDGF also stimulated phosphorylation of c-JNK and Akt. PDGF-induced Müller cell proliferation was significantly reduced by pre-treatment with SP600125 and LY294002, inhibitors of c-JNK and Akt phosphorylation, respectively. Our findings collectively indicate that PDGF-stimulated Müller cell proliferation occurs via activation of the c-JNK and PI3K/Akt signaling pathways. These data provide useful information in establishing the role of Müller cells in the development of proliferative vitreoretinopathy.     

Vascular endothelial growth factor A competitively inhibits platelet-derived growth factor (PDGF)-dependent activation of PDGF receptor and subsequent signaling events and cellular responses

Certain platelet-derived growth factor (PDGF) isoforms are associated with proliferative vitreoretinopathy (PVR), a sight-threatening complication that develops in a subset of patients recovering from retinal reattachment surgery. Although these PDGF isoforms are abundant in the vitreous of patients and experimental animals with PVR, they make only a minor contribution to activating PDGF receptor α (PDGFRα) and driving experimental PVR. Rather, growth factors outside of the PDGF family are the primary (and indirect) agonists of PDGFRα. These observations beg the question of why vitreal PDGFs fail to activate PDGFRα. We report here that vitreous contains an inhibitor of PDGF-dependent activation of PDGFRα and that a major portion of this inhibitory activity is due to vascular endothelial cell growth factor A (VEGF-A). Furthermore, recombinant VEGF-A competitively blocks PDGF-dependent binding and activation of PDGFR, signaling events, and cellular responses. These findings unveil a previously unappreciated relationship between distant members of the PDGF/VEGF family that may contribute to pathogenesis of a blinding eye disease.     

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.^1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.^1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.⁵ This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.     

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. ^1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. ^1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.     

增生性玻璃体视网膜病变动物模型的改进和评价

增生性玻璃体视网膜病变是导致视网膜脱离手术失败的主要原因,建立动物模型能更好的研究其发病机理,从而进行防治。本文就PVR动物模型的所用动物、模型的建立方法以及评价和分级进行综述。     

Tumor necrosis factor-alpha (TNF-α)-mediated in vitro human retinal pigment epithelial (RPE) cell migration mainly requires Akt/mTOR complex 1 (mTORC1), but not mTOR complex 2 (mTORC2) signaling

When rhegmatogenous retinal detachment occurs, tumor necrosis factor-alpha (TNF-α) among other cytokines leaks into the subretinal space, induces resident retinal pigment epithelial (RPE) cells to migrate, which is the initial step of proliferative vitreoretinopathy (PVR). In the current study, we aim to understand how this is regulated by focusing the cellular mechanisms involved. Here we identified an Akt/Tuberous sclerosis protein 2 (TSC2)/mTOR complex1 (mTORC1) signaling pathway after TNF-α treatment to mediate RPE cell migration. Suppression of mTORC1 activation, either by its inhibitor rapamycin, or by activation of its suppressor AMP activated protein kinase (AMPK), inhibited TNF-α-mediated RPE cell migration, while RNA interference (RNAi)-mediated knocking-down of SIN1 or Rictor, two key components of mTOR complex 2 (mTORC2), had no significant effect on TNF-α-induced RPE cell migration. Our data provide initial evidence that TNF-α-mediated in vitro RPE cell migration mainly requires Akt/mTORC1, but not mTORC2 signaling. The results of this study may lead to indentify novel signaling targets against PVR.