Corneal permeability in a redesigned corneal holder for the bovine cornea opacity and permeability assay

The bovine cornea opacity and permeability assay (BCOP) is a proposed alternative to the Draize rabbit test for potential eye irritants. In the standard BCOP, bovine corneas are mounted in a holder on a flat surface between two identical chambers. The flat configuration of the standard holder does not conform to the normal curved shape of the bovine cornea and it comes into direct contact with the cornea tissue. Mounting corneas in this holder causes extensive damage to both epithelial and endothelial corneal cell layers. Our laboratory has designed a new holder that allows the cornea to maintain its natural curvature and does not damage the cornea. Previous tests, using both the new and standard holders, and comparing corneal opacity, hydration and endothelial morphology, have shown that the new holder is a significant improvement over the standard holder. The present study extends the comparisons of the new and standard holders to measurement of corneal fluorescein permeability. The permeability (ng/cm²/min) of intact corneas, corneas with no epithelium, and corneas treated with 1% NaOH, isopropanol, acetone, 30% trichloroacetic acid or 30% sodium dodecysulfate for either 1 or 10 min was determined by measuring fluorescence of samples taken from the endothelial chamber after 90 min epithelial exposure to 0.04% sodium fluorescein. In all trials, the redesigned holders yielded not only lower permeability measurements but also decreased measurement variability. The data provide further evidence that the new holder is an improvement over the standard holder and should be incorporated into a new protocol for the BCOP.     

Corneal opacity, hydration and endothelial morphology in the bovine cornea opacity and permeability assay using reduced treatment times.

The purpose of this study was to determine whether the standard bovine cornea opacity and permeability (BCOP) assay exposure time of 10 minutes overestimates the ocular irritancy of chemical substances. Corneas were subjected to BCOP protocol following 30-second and 1-minute exposures to irritants. Corneal opacity and hydration (mg H(2)O/mg cornea) were then measured and compared to data obtained after 10 minute irritant treatments. For most test substances corneal opacity and hydration were lower following reduced exposure times. It is suggested that using shorter exposure times in BCOP protocol may be more predictive of human response to ocular irritants, since irritants are usually in brief contact with the ocular surface during accidental exposure. A second purpose of this study was to examine effects of irritants on the corneal endothelium. Corneas were treated according to BCOP protocol following exposure to irritants for 1 or 10 minutes. The endothelium was stained with Alizarin Red and trypan blue, and examined using light microscopy. Severe irritants, such as NaOH and trichloroacetic acid, cause endothelial cell death. It was also determined that simply mounting the cornea in the BCOP assay holders caused damage to 20% of the endothelial cells. Because the endothelium is essential for normal corneal transparency and hydration, it is suggested that examination of the endothelium be added to the BCOP assay and that optimization of the assay will require modification of the cornea holders.     

A redesigned corneal holder for the bovine cornea opacity and permeability assay that maintains normal corneal morphology.

The bovine cornea opacity and permeability assay (BCOP) has been in use for nearly 10 years but has not been submitted for regulatory approval. In previous reports we have presented corneal hydration and endothelial damage as additional endpoints in this assay and have suggested that the design of the BCOP's corneal holder should be modified. The standard holder used in the BCOP assay induces physical damage to the cornea because it contacts clear cornea causing edge damage to the epithelial, stromal and endothelial layers. Second, by forcing a curved, oval-shaped bovine cornea into a flat, circular opening, corneal wrinkling occurs which can alter the cornea's optical characteristics and, most importantly, induces endothelial damage. We now report on a redesigned BCOP corneal holder that clamps onto the sclera, maintains normal corneal shape and does not cause damage to the endothelium. This ensures that irritancy tests are conducted using healthy, anatomically normal tissue. Tests of this holder using acetone, trichloroacetic acid, isopropanol and benzalkonium chloride show that it is now possible to evaluate effects of chemical substances on the endothelium. The effects of these compounds on corneal opacity and hydration in the new holder are similar to their effects on the cornea in the standard holder.     

A simple method to assess surfactant-induced bovine corneal opacity in vitro: Preliminary findings

The in vitro development of bovine corneal opacity induced by two anionic surfactants, sodium deeyl sulphate (NaDS) and sodium lauryl sulphate (NaLS), was monitored using a simple, specially constructed instrument, the opacitometer. Results show clearly that NaDS was more potent, and acted more rapidly, than NaLS, an order of potency which has been shown by in vivo ocular irritancy tests with these agents but not in cytotoxicity tests. The measurement of irritant-induced opacity of bovine isolated cornea may therefore offer another approach to the search for viable in vitro alternatives to in vivo ocular irritancy testing.     

Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy

Most of the published in vitro tests of ocular irritancy investigate a single parameter, generally cytotoxicity, using different cell types in culture. Although good correlations with in vivo data have been reported by some investigators, many of these studies examined only limited classes of products, mainly surfactants and cosmetic ingredients. To predict the irritant potential of compounds in development and process intermediates (which include a wide variety of chemical classes with variable physical characteristics), an assay which would allow great flexibility was needed. A recently published model of corneal opacity was appropriate for this purpose and therefore investigated. The method was substantially modified and extended to study, in the same assay, two important components of irritation, i.e., opacity and permeability. For opacity alone, values obtained for 44 common chemicals showed a correlation of r = 0.73 with published in vivo data. However, compounds like sodium lauryl sulfate and some medium-chain alcohols gave false-negative results, apparently because they produced destruction of corneal epithelium. Such an effect was quantified by the measure of corneal permeability to fluorescein, and changes observed were found to be consistent with the known irritant potential of the compounds. In combination, the measurement of these two endpoints thus appeared to be sufficient to accurately predict ocular irritancy. This was further verified with 15 process intermediates. In short, the bovine corneal opacity and permeability assay allows investigation of two important components of eye irritation, in a one-day experiment, using an ocular tissue. It represents a useful approach to assess ocular irritation at least for our needs.     

Development of a new opacitometer for the bovine corneal opacity and permeability (BCOP) assay

The bovine corneal opacity and permeability (BCOP) assay is an in vitro eye irritation test that relies on the measurement of induced opacity and permeability evaluated in isolated bovine corneas. Routinely, opacity is measured by an OP-KIT opacitometer which provides a centre-weighted reading of light transmission through the corneal tissue. However, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of isolated cornea. Consequently, these findings question the reliability of the obtained data when opacity is manifested in a non-homogeneous manner. In addition, the BCOP test is only recommended to identify corrosive and severe eye irritants, and has not proven as sensitive in distinguishing among mild and moderate eye irritants. Within this context, a new opacitometer was developed which uses an adjustable laser beam in combination with a calibrated photometer. This work focused on a technical optimisation and describes the optical characteristics of the new device. The laser-based opacitometer allowed the analysis of the complete corneal surface, showed improved sensitivity to detect subtle changes in corneal transparency and was able to detect more efficiently opaque spots located along the sides of the excised corneas.     

Performance of porcine corneal opacity and permeability assay to predict eye irritation for water-soluble cosmetic ingredients.

The purpose of this paper is to report on the ability of an in-house porcine corneal opacity and permeability assay (PCOP) to predict eye irritation for cosmetic ingredients. Preliminary studies showed that the PCOP assay could accurately predict eye irritation class for liquid and water soluble materials. To broaden our experience a larger study on 50 cosmetic ingredients of this group was conducted. A prediction model (PM) was obtained based on only one endpoint-permeability measured after 30-min exposure O.D.30. This PM allows to distinguish nonirritating compounds (if O.D.30 < 0.35) from irritating (if O.D.30 > or = 0.35). Forty-nine of the 50 ingredients tested in the PCOP assay were accurately classified. The agreement was high (concordance 98%-kappa = 0.96). For 43 of the test substances an equation PM was obtained to predict the MAS. Despite satisfactory statistical coefficients this algorithm is not recommended due to wide 95% confidence intervals. These results confirm the usefulness of the PCOP for water-soluble cosmetic ingredients to discriminate nonirritants (MAS < or = 15) and irritants (MAS >15). For this type of ingredients the PCOP seems to be better than the BCOP to predict irritation class. Future work will be done to compare the BCOP and PCOP performances and to develop an appropriate protocol for water insoluble compounds.     

激光性角膜手术与眼内屈光手术矫治超高度近视临床效果观察

目的探讨激光性角膜手术与眼内屈光手术矫治超高度近视的临床效果和安全性。方法选择笔者所在医院近年来收治的超高度近视患者行各类屈光手术患者74例共108眼,按照随机分组的原则将之分为A、B、C 3组各36眼,其中A组患者行LASIK术进行治疗;B组患者行透明晶状体超声乳化摘除加后房型人工晶状体植入术;C组患者行有晶状体眼虹膜固定型人工晶状体植入术,术后进行各项指标的统计对比。结果 3组患者在治疗之后视力均比术前有明显提高,A、B、C 3组患者的术后屈光度分别为(-2.10±2.38)D、(-1.75±1.33)D和(0.83±1.16)D。结论对于超高度近视患者而言,晶状体性屈光手术的效果要明显优于LASIK术,而且对于超高度近视患者而言应当根据患者的年龄、眼部情况等进行具体的手术计划制定。     

Orbscan-Ⅱ在LASIK手术前后角膜检测中的应用研究

目的探讨Orbscan-Ⅱ在准分子激光原位角膜磨镶(LASIK)手术前后检查中的应用价值。方法对84例患者(160只眼)于LASIK术前和术后1个月分别用Orbscan-Ⅱ和超声角膜测厚仪测量角膜厚度并进行统计学比较。结果超声测量瞳孔中央角膜厚度,术前为(539.1±32.2)μm。术后1月为(464.4±31.1)μm;术前Orbscan-Ⅱ测量的瞳孔中央角膜厚度、角膜最薄点厚度与超声结果无统计学差异(P>0.05),术后1个月Orbscan-Ⅱ的测量结果与超声结果有非常显著性差异(P<0.01),Orbscan-Ⅱ同时显示了Diff值等其它厚度相关参数。结论Orbscan-Ⅱ角膜检测对LASIK手术具有重要指导意义。     

Corneal cell culture models: a tool to study corneal drug absorption

In recent times, there has been an ever increasing demand for ocular drugs to treat sight threatening diseases such as glaucoma, age-related macular degeneration and diabetic retinopathy. As more drugs are developed, there is a great need to test in vitro permeability of these drugs to predict their efficacy and bioavailability in vivo. Corneal cell culture models are the only tool that can predict drug absorption across ocular layers accurately and rapidly. Cell culture studies are also valuable in reducing the number of animals needed for in vivo studies which can increase the cost of the drug developmental process. Currently, rabbit corneal cell culture models are used to predict human corneal absorption due to the difficulty in human corneal studies. More recently, a three dimensional human corneal equivalent has been developed using three different cell types to mimic the human cornea. In the future, human corneal cell culture systems need to be developed to be used as a standardized model for drug permeation.     

玻璃体浑浊临床治疗分析

目的探讨玻璃体混浊的临床治疗方法。方法本次研究选择本院2008～2011年收治的玻璃体混浊患者86例。随机分为观察组45例,对照组41例,所有患者首先实行常规的基础治疗,在此基础上,观察组加用氨碘肽治疗,对照组则采用平地木颗粒治疗,跟踪观察两组治疗情况,对所得数据进行记录。结果观察组45例患者,显效23例,有效18例,无效4例,有效率为91.1%;对照组41例患者,显效17例,有效15例,无效9例,有效率为78.0%,差异具有统计学意义（P〈0.05）。结论在治疗玻璃体混浊疾病的过程中,在常规治疗的基础上加入氨碘肽治疗的效果较为显著,可以有效促进患者病情的改善,并提高患者的生活质量,值得在临床推广应用。     

青光眼术后白内障不同手术方式角膜内皮细胞的变化

目的探讨青光眼术后白内障不同手术方式角膜内皮细胞密度的变化。方法将58例(58眼)青光眼术后白内障患者分为两组。A组:采用透明角膜切口超声乳化术;B组:采用颞侧巩膜隧道切口非超声乳化术。分别观察两组术前及术后的角膜内皮细胞密度。结果术前角膜内皮细胞密度:A组(2231±192)个/mm2、B组(2042±141)个/mm2,二者差异无统计学意义;术后1月角膜内皮细胞密度:A组(2004±152)个/mm2、B组(1825±191)个/mm2。二者差异无统计学意义。结论两种手术方式对角膜内皮损害基本相同。     

胸骨下段小切口在心脏直视手术中的应用

目的探讨胸骨下段小切口在心脏直视手术中的应用。方法对296例心脏病患者采用胸骨下段小切口，保持胸骨柄完整性，在停跳或不停跳下完成心脏直视手术，其中先天性心脏病250例，风湿性心脏病40例，左房粘液瘤5例，冠心病1例。结果全组患者成功施行胸骨下段小切口心脏直视手术，没有死亡及后遗症。结论胸骨下段小切口适用于大多数心脏直视手术，有良好的手术和美容效果，但不适合于部分心脏复杂畸形及巨大心脏的手术矫治。     

Innovative therapeutic strategies for restoring lymphocyte functions in septic patients

Septic syndromes still remain a major but largely under-recognized healthcare problem worldwide accounting for thousands of deaths every year. Despite numerous clinical trials, therapies have failed to mitigate the devastating effects of these conditions. It is now agreed that the initial hypotheses for sepsis pathophysiology have been misconstrued. Sepsis deeply perturbs immune homeostasis by concomitantly inducing a strong inflammatory response and a major anti-inflammatory process, acting as a negative feedback. Several lines of evidences indicate that this inhibitory response secondly may be deleterious in patients who survived initial resuscitation, as it may be directly responsible for worsening outcome by decreasing resistance to secondary nosocomial infections. In this context, while the majority of clinical and basic science conducted so far has focused on innate immune cell depressed functions (especially monocytes), the contribution of T lymphocyte anergy has been somewhat ignored. This review focuses on lymphocyte dysfunctions described so far in patients and on potential new therapeutic strategies aimed at restoring a functional lymphocytic response after sepsis.     

Opacification test by using the pig isolated cornea and its application to a test of corneal opacity induced by befunolol hydrochloride

Corneas isolated from the pig eyeball have been treated in vitro with varying concentrations of befunolol hydrochloride, in order to test the topical effect of befunolol on the cornea in terms of opacity. Experiments were carried out with the corneas treated as follows: intact, epithelium-removed, endothelium-removed, and both epithelium- and endothelium-removed. Solutions of the drug were applied to both epithelial and endothelial surfaces, to the epithelial surface only, or to the endothelial surface only. When the drug was applied either to both surfaces or to the endothelial surface only, there was a significant increase in opacity. However, when applied to the epithelial surface only, befunolol caused an insignificant increase in opacity as compared with that of control. It is suggested, therefore, that corneal opacity due to befunolol hydrochloride topically administered to the eye, is concerned mainly with the epithelium in cornea, since the drug is uncapable of passing barrier probably existing in surface layer of epithelium but it easily penetrates in corneal layers from endothelial side to attain reversely to epithelial cells. Thus, it is probable that the risk to induce opacity is minor in case that befunolol is topically given in drops to the eye.     

依诺沙星滴眼液治疗兔眼细菌性角膜溃疡的疗效观察

选用家兔角膜实层内注射菌液,制备膜溃疡病理模型。考察依诺沙星滴眼液对兔眼角膜溃疡的研究,以市售氟沙星滴眼液作阳性对照,生理盐水作阴性对照。结果表明,０．３％以上浓度依诺沙星滴眼液对Ｇ＾＋和Ｇ＾－菌所致角膜溃疡有较好的治疗作用。     

Prediction of eye irritation potential of surfactant-based rinse-off personal care formulations by the bovine corneal opacity and permeability (BCOP) assay

Evaluation of eye irritation potential is a routine part of product safety testing. Many companies have elected to use in vitro methods for this evaluation. We used the bovine corneal opacity and permeability (BCOP) assay for the prediction of the eye irritation potential of surfactant-based rinse-off personal care formulations in this study because of its positive performance in previous studies (1), and its potential to measure depth of injury through histological evaluation. The BCOP uses isolated corneas maintained in short-term organ culture and measures changes in opacity and epithelial barrier integrity (as determined by fluorescein passage through the cornea). Surfactants (anionic and nonionic) used in personal care formulations induce only small increases in direct opacity but the degree of epithelial damage is reflected in permeability measurement (OD490). The permeability measurement is the primary endpoint for this class of products. Marketed liquid hand soap was selected as the benchmark due to extensive in vivo and market history data. Using the liquid hand soap, a 25% v/v aqueous dilution with a 30-minute exposure produced the optimal resolution. A series of products with known in vivo eye irritation scores were evaluated to establish a prediction model. Histological evaluation was used to compare the degree of tissue damage to the permeability scores. Based on the permeability scores and histological review, the testing protocol for a surfactant-based rinse-off personal care formulation was developed using a 25% v/v aqueous dilution, a 30-minute exposure, concurrent testing of the benchmark control, and use of permeability measurements as the endpoint for the evaluation of eye irritation potential.