Intracellular potassium activity of isolated human and rabbit corneal epithelium

Single-barrelled open tip microelectrodes containing a potassium-selective liquid ion exchanger (Corning 477 317) were used to determine intracellular K-activity a_Kⁱ in squamous and basal cells of isolated rabbit and human corneal epithelium. The intracellular potassium activity a_Kⁱ during the five hours of incubation in Ringer solution was 114·3±23·6 mmol/l and 107·6±26·2 mmol/l for the basal cell layer of rabbit and human epithelia respectively. The corresponding values for the squamous cells of the superficial cell layer were 41·2±14·2 mmol/l and 50·2±11·1 mmol/l. Based on intracellular chemical concentrations of potassium c_Kⁱ an apparent activity coefficient could be calculated that is close to that of a dilute solution of the same ionic strength. This indicates that intracellular potassium is essentially free in both epithelia. Ouabain (5 × 10^?5 mol/l) added to the bathing solution of the rabbit cornea lowered the transmembranal voltage Ψ_M as well as the intracellular K-activities of squamous and basal cells. Three hours after application of ouabain a_Kⁱ of the basal cells had decreased to about one third of its initial value. Under all experimental conditions the potassium equilibrium potential E_K considerably exceeded Ψ_M.Our data indicate: (1) The high absolute values for a_Kⁱ imply that almost no potassium is bound or sequestered within the epithelial cells. (2) An active potassium uptake is necessary to explain the high K-activity in relation to the transmembranal voltage Ψ_M. (3) There is an intraepithelial gradient of a_Kⁱ within the corneal epithelia.     

The effect of timolol,betaxolol and levobunolol on the surface of rabbit cornea

Pigmented rabbits were treated with timolol maleate, betaxolol hydrochloride or levobunolol hydrochloride eye drops twice a day for six months. Animals of the same age group and breed were used as controls. There were no differences observed in corneal epithelium with light and transmission electron microscopy. With scanning electron microscopy, in the timolol and betaxolol treated animals the picture of the corneal surface was similar to that of normal rabbit corneas after exposure to air. In scanning electron micrographs of the levobunolol treated animals, the corneal surface resembled the corneas of normal rabbits treated with artificial tears.     

The deleterious effects of exogenous bicarbonate on the rabbit cornea undergoing prolonged refrigerated storage

The functional integrity of stored corneas is investigated, using the criterion of final minimum thickness achieved by the corneas when incubated under standardized conditions. This criterion indicates that rabbit corneas stored in low bicarbonate media function better in vitro than those stored in high bicarbonate media. Reasons are advanced which might explain the phenomenon. It is proposed that a relatively low concentration of bicarbonate in McCarey-Kaufman medium (1974) is a major factor in the success of their procedure.     

Energetic requirements of active transepithelial Na and Cl transport in the isolated bullfrog cornea.

In the isolated bullfrog cornea, the energetic requirements were estimated of anaerobic active transepithelial Na and Cl transport (i.e. $\text{Δ}\text{Na}\text{Δ}\text{ATP}$ and $\text{Δ}\text{Cl}\text{Δ}\text{ATP}$). These estimates were done by measuring the corresponding effects of various stimulators and inhibitors of active transepithelial Na and Cl transport on anaerobic lactate efflux and the short-circuit current (s.c.c.). In NaCl Ringer's solution, 10^?5m-amphotericin B stimulated the calculated rate of active transepithelial Na transport by between 0·86 and 1·12 μEq/hr.cm². The corresponding increase in lactate efflux was 0·29 μmol/hr.cm². Assuming that glucose is the sole substrate for anaerobic glycolysis, the energetic requirement as estimated from these stimulatory effects on active transepithelial Na transport is between 3·0 and 3·9. The energetic requirement of active transepithelial Cl transport was studied by considering the corresponding effects on lactate efflux and the Cl-originated s.c.c. of various known selective inhibitors and stimulators of the Cl-originated s.c.c. The Cl energetic requirement could not be estimated from either the inhibitory effects of 10^?5m-bumetanide or 3 × 10^?4m-furosemide since both of these drugs only inhibited the Cl-originated s.c.c. without affecting lactate efflux. Only 10^?3m-ouabain had a slight inhibitory effect on lactate efflux. The selective stimulants of active transepithelial Cl transport, 10^?4m-epinephrine and 2 × 10^?3m-theophylline had large stimulatory effects on the s.c.c. but did not affect lactate efflux. Only the stimulant 10^?5m-A23187 (calcium ionophore), which had the largest stimulatory effect on the Cl-originated s.c.c, significantly stimulated the lactate efflux. From the inhibitory effects of 10^?3m-ouabain on the A23187 stimulated s.c.c. and lactate efflux, the energetic requirement of active transepithelial Cl transport is equivalent to a movement of 4·0 mol of Cl per mol of ATP.     

The appearance and possible role of plasminogen activator of urokinase type (u-PA) activity in the cornea related to soft contact lens wear in rabbits

This is the first study in which u-PA activity is detected in situ during SCL wear. The histochemical localization of u-PA activity is done by the methods of Lojda using unfixed cryostat sections on semipermeable membranes and a gel incubation medium containing sensitive substrates with the 7-amino-4-trifluoromethylcoumarin (AFC) (Enzyme Systems Products, Sierra Lane, Dublin, CA, USA) leaving groups. Z-Gly-GIy-Arg-AFC and Glut-Gly-Arg-AFC were employed as the substrates. The results show that in the normal cornea u-PA activity is absent. Also the wearing of SCL does not evoke the appearance of u-PA activity in the cornea within the first three days. On day 4 the first u-PA activity appears; it is located in the superficial layers of the corneal epithelium. On day 7 of SCL wear, u-PA activity is present in all layers of the corneal epithelium and (to a lesser extent) also in the comeal endothelium; keratocytes of the corneal stroma are only slightly active for u-PA. Extended SCL wear (for two weeks) leads to an increase of u-PA activity in keratocytes beneath the epithelium. Also, some inflammatory cells (mainly polymorphonuclear leukocytes, PMNs) present in the corneal stroma are enzymatically active. After three weeks of SCL wear the number of PMNs in the corneal stroma increases; some PMNs are highly active for u-PA. In the corneal endothelium the u-PA activity is also highly pronounced. It can be concluded that extended SCL wear leads to the gradual increase of u-PA activity in the rabbit cornea. It is suggested that active u-PA is involved in the corneal damage related to SCL wear. This revised version was published online in July 2006 with corrections to the Cover Date.     

几种抗炎滴眼液对兔角膜上皮损伤和愈合影响的实验研究

目的 探讨双氯芬酸钠、妥布霉素地塞米松、普拉洛芬、溴芬酸钠滴眼液频繁点眼对兔角膜上皮的副作用,并观察其常规使用对兔角膜上皮创伤愈合的影响。设计 实验性研究。研究对象80只新西兰大白兔。方法 将兔分为2组,每组40只,一组为频点组（点滴眼液每小时1次）;另一组行直径6 mm的角膜上皮刮除模型并予点滴眼液每日4次。每组兔再随机分为5组,每组8只,分别给予生理盐水、双氯芬酸钠、妥布霉素地塞米松、普拉洛芬、溴芬酸钠滴眼液。选择右眼为观察眼。在角膜上皮损伤前、损伤后12、24、48、72、96小时及第5、6、7天进行观察,并在观察结束后摘除眼球行组织病理学检查。主要指标 眼表刺激症状、角膜上皮损伤、愈合情况和角膜上皮厚度。结果 与生理盐水组比较,四种滴眼液频繁点滴兔眼均未出现明显刺激症状且未见上皮缺损形成,但荧光素染色均可见角膜上皮点染,以第3天明显,且双氯芬酸钠组角膜上皮损伤的积分（9分）明显高于对照组（0分）和溴芬酸钠组（1分）（P<0.05）;病理学检查显示点药5天后,妥布霉素地塞米松组角膜上皮细胞层数（3.67±0.52层）少于对照组（4.17±0.41层）（P<0.05）,其余各用药组与对照组的角膜上皮细胞层数未见明显差异。创伤后兔角膜上皮的平均修复时间在双氯芬酸钠组和妥布霉素地塞米松组分别为（75.0±27.0）小时和（75.0±8.5）小时,而生理盐水、普拉诺芬和溴酚酸钠组分别为（66.0±11.1）小时、（69.0±15.4）小时和 （66.0±11.1）小时,各组比较差异无统计学意义（P>0.05）,但角膜上皮愈合后妥布霉素地塞米松组（2.00±0.00层）和双氯芬酸钠组（2.50±0.55层）上皮细胞的层数少于对照组（5.00±0.00层）（P<0.05）。结论  频繁滴用抗炎滴眼液可导致兔角膜上皮细胞潜在的损伤;双氯芬酸钠和妥布霉素地塞米松滴眼液抑制兔角膜上皮创伤的愈合作用略强于普拉洛芬和溴芬酸钠。     

Partial purification and characterization of keratokinase, the fibrinolytic activator of the cornea.

Keratokinase (KK) is a fibrinolytic enzyme released by in vitro cultures of cornea. KK converts plasminogen into plasmin producing on the molecule of plasminogen the same pattern of limited proteolysis as urinary activator, urokinase (UK) does. The mol. wt. of KK is around 55 000 daltons, i.e. in the same range of that of UK. Like UK, but unlike tissue activator (TA, derived from vascular endothelium) KK shows a biphasic activity response to increasing concentrations of epsilon aminocaproic acid (EACA) and is scarcely bound to fibrin during clotting.KK may play a role in a number of pathophysiological states of the cornea such as corneal swelling, inflammation and collagen metabolism.     

微型角膜刀法准分子激光角膜上皮瓣下磨镶术后兔角膜上皮瓣结构及活性的研究

目的 探讨角膜微型刀上皮瓣下准分子激光原位角膜磨镶术（Epi-LASIK）术后上皮瓣形态结构、活性的变化、周边上皮（上皮瓣切口缘与角膜缘之间的区域）增生情况及其对细胞凋亡的影响。方法对29只新西兰白兔的双眼进行手术,28只眼行Epi—LASIK,24只眼行PRK,随机分为4组,在术后1、3、5、7d取标本,6只未手术眼作为空白对照组。采用透射电镜、光镜观察形态结构的变化;冰冻切片行酶组织化学检测上皮瓣细胞活性的变化;石蜡切片行凋亡及增殖细胞核抗原免疫组织化学检测。结果透射电镜发现KM5000D型上皮刀分离的上皮瓣基底膜完整,细胞之间结合紧密,术后上皮瓣与基质粘合牢固。1、3、5、7d上皮瓣细胞三磷酸腺苷酶和葡萄糖．6．磷酸酶活性（上皮瓣与周边上皮细胞酶活性比值）分别为79％、58％、69％、86％和79％、63％、77％、97％;各组Epi-LASIK眼周边上皮细胞活性与空白对照组差异无统计学意义（F=1．09,P〉0．05）。各组Epi-LASIK眼周边上皮增生与空白对照差异无统计学意义（F=1．10,P〉0．05）。Epi-LASIK和PRK组在术后1d基质细胞凋亡数为（3．429±1．693）和（3．796±1．998）个／10000μm^2,差异无统计学意义（t=-0．33,P〉0．05）;而Epi-LASIK眼术后3、5、7d基质细胞凋亡少于PRK,差异均有统计学意义（P〈0．01）。结论KM5000D型上皮刀分离的上皮瓣结构完整,细胞结合紧密,能保持较高活性,无明显刺激周边角膜上皮增生,有助于抑制凋亡的发生。（中华腠科杂志,2007,43：651-657）     

The influence of topical prostaglandins on HSA-induced uveitis in the rabbit

In this study the effect of topical administration of prostaglandins (PGs) on a human serum albumin (HSA)-induced uveitis is evaluated. Topical prostaglandin E₁ (PGE,) and prostaglandin F₂ (PGF₂) partly inhibited hyperaemia and flare in the anterior chamber after the induction of immune complex uveitis. A marked increase in the cellular response was observed in the aqueous humour after topical PGE₁ and PGF₂. Topical prostaglandins may decrease endogenous prostaglandin formation and reduce the prostaglandin-mediated inflammatory symptoms; on the other hand, they also stimulate the aqueous cellular response, possibly by facilitation of leukotriene formation.These results indicate that topical prostaglandins should not be used to treat immunogenic uveitis.     

Dopamine stimulation of passive permeability and secretion in the isolated rabbit ciliary epithelium.

Dopamine, at concentrations between 1·25 × 10^?7m and 1·25 × 10^?3m, increases both fluid secretion and passive fluid permeability across the isolated rabbit ciliary epithelium. These effects can be blocked by α- and β-adrenergic antagonists but not by specific dopaminergic antagonists. The present data suggest that dopaminergic receptors are not present in the ciliary epithelium.     

Tear plasminogen activators — indicators of epithelial cell destruction. The effect of scraping,n-heptanol debridement,and alkali burn of the cornea on the plasminogen activator activity of rabbit tears

Plasminogen activator (uPA) activities were determined in the tears of rabbits following mechanical (scraping) or chemical (n-heptanol) debridement and alkali burn of the central part of the corneal epithelium. All three types of injury enhanced the plasminogen activator activities in the tears. The increase in uPA activity was highest in alkali burn, lowest for n-heptanol debridement. Scraping yielded an intermediate increase in uPA activity. The maximum value in activator activity was reached at 5 hours for mechanical injury and at 24 hours for chemical injuries. uPA activity values returned to the normal range by the time of re-epithelialization for mechanical scraping and 1–3 days following re-epithelialization for heptanol debridement and alkali burn. A trend was observed between uPA activity level and the size of the wound but the correlation was not pronounced (R = 0.538).     

Effect of sensory and sympathetic denervation on substance P immunoreactivity in nerve fibres of the rabbit eye

Substance P (SP) immunoreactivity was demonstrated in ocular tissues of rabbit. SP was found in nerve fibres of cornea iris, ciliary body, choroid and in the inner plexiform layer of the retina.In order to verify the origin of these nerves the animals were subjected to two different denervation procedures: intracranial combined maxillary and ophthalmic neurotomy or superior cervical ganglionectomy. The former operation destroyed all SP immunoreactive nerves of the ipsilateral eye except for the retina, whereas the latter had no effect. It is concluded that the ocular SP immunoreactive nerves are sensory trigeminal fibres. SP immunoreactivity in the retina is not due to sensory nerves but probably to amacrine cells.     

姜黄素对兔视网膜色素上皮细胞DNA含量、线粒体膜电位及胞质内钙离子的影响

目的 探讨姜黄素对兔视网膜色素上皮细胞(RPE)DNA含量(凋亡率)、线粒体膜电位(Aψm)和胞质内钙离子(Ca2+)的影响.方法 实验研究.选取体外培养的第4代RPE细胞进行试验,分为姜黄素组和空白对照组(10% FBS-DMEM含二甲基业砜0.5‰),姜黄素组设10、15、20 mg/L 3个质量浓度.噻唑蓝(MTT)法检测姜黄素10、15、20 mg/L 3个质量浓度分别作用24、48、72及96 h后对体外培养的RPE细胞增殖72及96 h后对体外培养的RPE细胞增殖的抑制率.相关回归分析计算24、48、72及96 h的半数抑制率(IC50)剂量.流式细胞仪检测姜黄素15 mg/L作用8、24、48及72 h,后RPE细胞DNA含量(凋亡率)、△Ψm和胞质内Ca2+的变化.对抑制率分别在同浓度不同时间组间、同时间不同浓度组间进行方差分析(多个样本问两两比较);采用相关回归分析计算姜黄素各时间段半数抑制率剂最;对凋亡率、线粒体△Ψm、细胞质内Ca2+等对照组与姜黄素组间进行两独市样本t检验;对凋亡率、线粒体△Ψm、细胞质内Ca2+等姜黄素不同时间组间进行方差分析(多个样本间两两比较).结果姜黄素对RPE细胞有明显抑制作用,呈时间和剂量依赖件.姜黄素在24、48、72及96 h对RPE细胞的IC50分别是29.31、17.50、13.24及10.99 mg/L.姜黄素作用8、24、48、72 h后,RPE细胞质内Ca2+浓度明显升高与空白对照组相比,差异有统计学意义(t=7.50,10.61,20.74,21.14;P＜0.01)△Ψm明显下降,与对照组相比均有统计学意义(t=7.50,11.74,14.91,15.29;P＜0.01).姜黄素作用8 h DNA含量未见明显下降,24、48及72 h DNA含量明显下降(即凋亡率升高),与空白对照组相比均有统计学意义(t=10.00,14.68,13.68;P＜0.01).结论姜黄素可以使RPE细胞质内Ca2+浓度升高,△Ψm下降,进而使DNA含量明显下降,诱导RPE细胞凋亡.姜黄素可以有效抑制RPE细胞增殖,有望成为防治增生性玻璃体视网膜病变的理想天然药物.(中华眼科杂志.2009,45:210-215)     

模拟远程投送对甘油长期冷冻保存兔角膜内皮细胞的影响

目的:探讨在实验室模拟远程运输状态下机械振动对改良后甘油长期保存兔角膜内皮细胞(CEC)活性的影响。方法:通过改良后的甘油保存角膜的方法,实验室中保存兔角膜片3mo,实验室用摇床模拟远程运输环境,结合实际按不同时间、不同振动强度进行分组,分别进行CEC活性染色并行实验性PKP术,对比各组间模拟运输对角膜内皮的影响。结果:实验条件下观察指标并不随振动强度和时间变化,组间各项指标差异无显著性。结论:实验室模拟远程投送对甘油长期保存兔角膜的CEC无显著性影响。     

Selective actions of barium on the c-wave and slow negative potential of the rabbit eye

The c-wave and slow negative potential (incorporating the slow P111) are both thought to be generated by light-evoked changes in the concentration of potassium surrounding the photoreceptors. We find the intravitreal barium, a blocker of potassium conductance, eliminates the c-wave from the intact rabbit eye but not the slow negative response. However, if the pigment epithelium is damaged by systemic administration of sodium iodate, barium then eliminates the slow negative response.