眼内麻醉剂对兔角膜内皮细胞影响的实验研究

目的:研究不同眼内麻醉剂对离体兔角膜内皮细胞的作用。方法:将实验用兔角膜取下后,分别用10g/L利多卡因、1g/L塞罗卡因及BSS处理。通过台盼蓝-茜素红联合染色观察兔角膜内皮细胞的形态学改变,并用计算机自动图像分析系统对兔角膜内皮细胞的损伤情况进行定量分析;对处理后的兔角膜内皮细胞进行扫描电镜观察,了解其超微形态学改变。结果:①各实验组中,10g/L利多卡因作用后兔角膜内皮细胞的损伤率分别为(0.91±0.12)%,(1.23±0.27)%,(2.42±0.31)%,(3.61±0.14)%;10g/L塞罗卡因作用后的兔角膜内皮细胞的损伤率分别为(0.68±0.16)%,(0.89±0.17)%,(1.84±0.34)%,(2.58±0.34)%。二者差异均具有显著性。(P<0.05,P<0.01)。②眼内麻醉剂的作用时间与兔角膜内皮细胞的损伤率呈正相关,相关系数分别为0.974、0.976。③眼内麻醉剂作用后,10g/L利多卡因造成的兔角膜内皮细胞的损伤情况较10g/L塞罗卡因更为严重。结论:10g/L利多卡因及10g/L塞罗卡因都会对兔角膜内皮细胞造成损伤。比较而言,不含防腐剂的塞罗卡因更安全。     

Short-term effect of intracameral triamcinolone acetonide on corneal endothelium using the rabbit model

PURPOSE: To investigate the effect of intracameral injection of triamcinolone acetonide on the corneal endothelium in rabbit eyes. METHODS: Triamcinolone acetonide (40 mg/ml, 0.2 cm3) after filtering and resuspension in balanced salt solution (BSS) was injected intracamerally for 3 min into 10 rabbit eyes and irrigated with 5 cm(3) of BSS. Triamcinolone without resuspension and BSS were injected, respectively, into five rabbit eyes. Endothelial toxicity was evaluated and compared by measurements of endothelial cell counts and central corneal thickness. The endothelial viability was determined using vital staining with alizarin red and trypan blue at 2 h after injection. The scanning electron microscopy (SEM) was performed in one cornea from each group. RESULTS: Endothelial cell counts and central corneal thickness following intracameral injection of triamcinolone acetonide did not significantly change when compared to controls. The mean percentage of viable endothelial cells was 99.50, 99.52, and 99.49% in the resuspended triamcinolone group, triamcinolone without resuspension group, and BSS group, respectively (P=0.46, Kruskall-Wallis test). But SEM showed reduced microvilli of endothelial surface in an eye of the triamcinolone without resuspension group. CONCLUSIONS: The intracameral injection of triamcinolone acetonide did not induce a significant visible change of endothelium in rabbit eyes. However, ultrastructural villi changes observed suggest a possibility of microstructural damages in endothelium with triamcinolone acetonide injection when used without filtering and resuspension.     

Diffusion if lidocaine after intracameral injection

PURPOSE: To determine the lidocaine diffusion space, we compared lidocaine aquous humor concentration in topical anesthesia with 1% lidocaine intracameral injection and in peribulbar anesthesia with 2% lidocaine prior phacoemulsification. MATERIAL AND METHOD: A gas chromatography technique of analyzing 100 microliters aqueous humor was used to detect the presence of lidocaine prior to phakoemulsification cataract surgery in two groups of patients: group A: after peribulbar anesthesia with 10 ml 2% lidocaine, group B: after 1% tetracaine topical anesthesia and 0.5 ml intracameral injection of 1% preservative-free lidocaine. The intracameral volume was estimated mathematically in group B. Endothelial cells loss was analyzed in two groups with non contact specular microscopy. RESULTS: Lidocaine was detected in aqueous humor with a good reliability. The mean concentration after intracameral injection was 6,300 micrograms/ml and was higher than after peribulbar injection. This concentration was near than theorical intracameral rate, suggesting that there was no diffusion in the posterior segment. There was no significant difference in the 2 groups in endothelial cells loss. CONCLUSION: Intracameral injection of lidocaine is an effective technique to anesthetize intracameral structures without diffusion in posterior segment prior to phakoemulsification.     

硅油填充术后的角膜内皮改变

目的评价硅油对有晶状体及无晶状体眼角膜内皮的影响。方法对45例(45眼)视网膜脱离患者行玻璃体切割联合硅油填充术,其中有晶状体组(22眼)及无晶状体组(23眼),比较其术前,术后1、3、6、9个月的改变。了解前房硅油对角膜内皮的影响。结果两组患者手术前后角膜内皮细胞的密度及角膜厚度的改变均无统计学差异,但两组同期角膜内皮细胞密度比较在术后1个月、6个月呈现显著差异(P=0.002,P=0.034)。硅油接触角膜内皮时,呈现“亮度颠倒”现象。末次随访时(术后9个月)的内皮细胞密度与无前房硅油组相比有极显著差异(P=0.000)。结论无论晶状体是否存在,玻璃体腔内的硅油对角膜内皮无明显的损害。而当硅油进入前房则将对角膜内皮造成严重的损害。     

Comparison of the influence of intracameral gentamicin, gatifloxacin, and moxifloxacin on the corneal endothelium in a rabbit model

Purpose

To compare the effect of three intracameral antibiotics, gentamicin (GM), gatifloxacin (GFLX), and moxifloxacin (MFLX), on the rabbit corneal endothelium.

Methods

Twenty-four eyes from 18 rabbits were used. In the GM treatment group of 12 eyes, a dose of 20 mg/ml, 2 mg/ml, 200 μg/ml, or 20 μg/ml of GM was injected into the anterior chamber. In the GFLX and MFLX treatment groups were injected into the anterior chamber of three eyes. The central corneal thickness was measured. The eyes were then enucleated for observation under scanning electron microscopy.

Results

Three days after the intracameral injection, a significant difference in central corneal thickness was found between the GM 20 mg/ml group and the control group (P < 0.05), but not between any other groups. The damage rate at the endothelial cell level was 67% in the GM 20 mg/ml group, 56% in the GM 2 mg/ml group, 33% in the GM 200 μg/ml group, 22% in the GM 20 μg/ml group, 22% in the GFLX group, and 0% in the MFLX group.

Conclusions

Intracameral GFLX or MFLX was almost nontoxic to the rabbit corneal endothelium, in contrast to the toxic results of intracameral GM 20 and 2 mg/ml.     

Morphogenesis of rabbit corneal endothelium

We studied ultrastructurally the development of rabbit corneal endothelium from the 13th day of gestation to 3 days after birth. Precursor corneal endothelial cells, stromal cells, and a vascular network migrate in close association with each other between the developing corneal and lens epithelia. During development, newly deposited extracellular fibrous matrices separate the prospective endothelium from the capillaries and corneal stroma. The extracellular matrix between the apical endothelial surface and the vascular network loses its fibrous appearance early in development. Simultaneously, randomly organized fibrils are deposited on the basal endothelial surface facing the stroma. These fibrils, gradually obscured by the deposition of a nonfibrous component, eventually become part of Descemet's membrane. Early in development, prospective endothelial cells cannot be distinguished morphologically from the overlying corneal stromal cells. Morphologic differentiation of the endothelial cell is characterized by the formation of sinuous lateral borders that interdigitate with those of adjacent cells to form a continuous single-cell layer of tissue. The basal endothelial membrane forms a pitted surface, distinguishing it from the apical cell membrane. Intercellular junctions between lateral membranes, a cilium projecting into the anterior chamber, and deposition of Descemet's membrane on the basal endothelial surface contribute to the polarization of the endothelium. Throughout most of corneal development the vascular pupillary membrane maintains a close association with the apical surface of the differentiating endothelium. We conclude that fetal corneal endothelium develops within a complex extracellular matrix environment and in proximity to the underlying vascular network. These structures play an important role in the morphogenesis of corneal endothelium.     

Effect of intracameral carbachol on intraocular pressure after cataract extraction

Thirty-two patients were randomly assigned to a treatment or a control group to determine the dose-response and duration of action of intracameral carbachol on immediate postoperative intraocular pressure after extracapsular cataract extraction using a viscoelastic substance. Patients in the treatment group received 0.5, 0.25, or 0.1 ml of 0.01% intracameral carbachol. Patients in the control group received 0.1 or 0.5 ml of balanced salt solution. Intraocular pressures of all patients were measured preoperatively and at three, six, 12, 24, and 48 hours postoperatively. The control group as a whole showed a 9.5-mm Hg intraocular pressure rise at three hours, a 10.0-mm Hg rise at six hours, a 9.0-mm Hg rise at 12 hours, and a 7.2-mm Hg rise at 24 hours postoperatively. The group treated with 0.5 ml of carbachol maintained stable intraocular pressures through the 48-hour measurement period. The groups treated with 0.25 and 0.1 ml of carbachol maintained stable intraocular pressures through 24 hours postoperatively. The differences in intraocular pressure were statistically significant for all treated groups through the 24-hour measurement.     

The histopathological effect of intracameral ropivacaine in different concentrations on corneal endothelium

We evaluated the histopathological changes occurring in corneal endothelium after intracameral injection ropivacaine into rats. Intracamerally administered ropivacaine in 1, 0.5, and 0.1% concentrations resulted in impairment of hexagonal structure of corneal endothelial cells and inter-cellular junctions, destruction of microvilli on the cell surface, roughness of cell borders, picnotic nucleus, diffuse vacuolization, and crystalysis in mitochondria. The authors have stated that they do not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article. The authors also do not discuss the use of off-label products, which include unlabeled, unapproved, or investigative products or devices.     

Riboflavin concentration in corneal stroma after intracameral injection

AIM:To evaluate the enrichment of riboflavin in the corneal stroma after intracameral injection to research the barrier ability of the corneal endothelium to riboflavin in vivo.METHODS:The right eyes of 30 New Zealand white rabbits were divided into three groups. Different concentrations riboflavin-balanced salt solutions (BSS) were injected into the anterior chamber (10 with 0.5%, 10 with 1%, and 10 with 2%). Eight corneal buttons of 8.5 mm in diameter from each group were dissected at 30min after injection and the riboflavin concentrations in the corneal stroma were determined using high-performance liquid chromatography (HPLC) after removing the epithelium and endothelium. The other two rabbits in every group were observed for 24h and sacrificed. As a comparison, the riboflavin concentrations from 16 corneal stromal samples were determined using HPLC after instillation of 0.1% riboflavin-BSS solution for 30min on the corneal surface (8 without epithelium and 8 with intact epithelium).RESULTS: The mean riboflavin concentrations were 11.19, 18.97, 25.08, 20.18, and 1.13 μg/g for 0.5%, 1%, 2%, de-epithelialzed samples, and the transepithelial groups, respectively. The color change of the corneal stroma and the HPLC results showed that enrichment with riboflavin similar to classical de-epithelialized corneal collagen crosslinking (CXL) could be achieved by intracameral1% riboflavin-BSS solution after 30min; the effect appeared to be continuous for at least 30min.CONCLUSION:Riboflavin can effectively penetrate the corneal stroma through the endothelium after an intracameral injection in vivo, so it could be an enhancing method that could improve the corneal riboflavin concentration in transepithelial CXL.     

A method for registration of toxic drug effects on corneal endothelium. Effect of gentamicin on rabbit corneal endothelium.

A weight recording system (Medin & Davanger 1988, 1989), was used to demonstrate possible toxic damage of a medicament to the endothelium of rabbit corneas stored in organ culture. Seven corneas were stored in organ culture medium containing gentamicin (3.0 mg/ml). Seven other corneas stored in identical organ culture medium without gentamicin served as controls. The corneas were followed with weight recordings for up to 76 h. A toxic effect of gentamicin was demonstrated by a rapid weight increase in the corneas stored in the presence of gentamicin. After 5.3 h there was a significant difference (P = 0.0002) between the average weights of the two groups, and this difference increased during the following 2-3 days. Corneas from the two groups were also examined by light microscopy and scanning electron microscopy. There was good accordance between the weight recordings and the morphology. The weight recording system detects clearly the toxic effect of gentamicin (3.0 mg/ml).     

Effect of intracameral carbachol given during cataract surgery on macular thickness

To evaluate the effect of intracameral carbachol on foveal thickness in patients who underwent uneventful cataract surgery. This retrospective study included two groups: the study group patients (group 1, n?=?47 eyes) had uneventful cataract surgery and received only carbachol 0.01?% for miosis; the control group patients (group 2, n?=?49 eyes) had uneventful cataract surgery without carbachol or any intracameral medication(s). The groups were compared for foveal thickness after cataract surgery. All phacoemulsification plus intraocular lens implantation surgeries were performed under local anesthesia via temporal clear corneal tunnel incisions. Mean values and standard deviations were calculated for preoperative and postoperative visual acuity (VA) and foveal thickness (FT) at 1 and 4?weeks. Optical coherence tomography was used for the FT measurements, with the MM6 map program. The patients in the study and control groups had a mean age of 57.78?±?9.07 and 59.72?±?8.96, respectively (p?=?0.355). All eyes had a significant improvement in VA. In the study group, the mean FT at the visits before and 1 and 4?weeks after surgery was 216.87?±?21.06, 228.81?±?30.52, and 222.94?±?29.91?μm, respectively. For the control group, the mean FT, before and 1 and 4?weeks after surgery, was 222.53?±?17.66, 231.67?±?23.08, and 225.41?±?22.59?μm, respectively. Intracameral carbachol 0.01?% had no effect on foveal thickness in patients who underwent uneventful cataract surgery.     

The healing of rabbit corneal endothelium

A circular 4 mm endothelial defect was induced by transcorneal freezing. The experimental damage and the healing took place in the living rabbit in 15 eyes, and in the isolated cornea in organ culture in further 20 eyes. The reparative process was studied by SEM, and proved to be the same in vivo and in vitro. The defect was covered with endothelial cells after 3 days. The normal hexagonal pattern was regained after 3 weeks. Both cell migration and cell division were involved in the reparative process. Only cells recruited from a zone close to the defect were active; the cells situated more than a few cell diameters from the original edge maintained their form and size unchanged. The first phase of cell division was the formation of a spherical cell with numerous blebs on its surface.     

Effect of fluorescein on the electrical potential difference across isolated rabbit corneal endothelium

The authors investigated whether fluorescein sodium affects the in vitro endothelial function of rabbit corneas. As an index of this function, the transendothelial electrical potential difference (TEPD) was used. The TEPD in a balanced salts and glucose (BSG) control solution increased for the first 30 min and then decayed slowly, reaching about 60% of its original value after 5 hr. When a BSG solution containing 5 micrograms/ml of fluorescein sodium was used, the TEPD time course was similar to the control solution. Since this fluorescein sodium concentration is about sevenfold higher than that seen in the anterior chamber of ocular patients, these results reassure users that no toxic effect of fluorescein is discernible at concentrations relevant to ophthalmic practice. With a fluorescein sodium concentration of 500 micrograms/ml, the TEPD decreased below control values after 4 hr of exposure, but such a concentration is approximately 5000-fold higher than that seen in the anterior chamber of patients. The adverse effect of fluorescein on TEPD is probably irrelevant for standard systemic clinical use.     

Systemic levels of lidocaine after intracameral injection during cataract surgery.

PURPOSE: To evaluate the systemic concentrations of lidocaine after intracameral injection of a 1% solution during cataract surgery and the safety of this application mode. SETTING: Department of Ophthalmology, Medical University of Lübeck, Lübeck, Germany. METHODS: This prospective study included 10 patients who had phacoemulsification and posterior chamber lens implantation with a self-sealing scleral tunnel incision. Topical anesthesia was achieved using cocaine 10% eyedrops combined with 0.5mL of unpreserved lidocaine 1% injected into the anterior chamber. Blood samples were taken from all patients at predetermined intervals before and during the procedure. Consecutive analysis for lidocaine was performed with gas chromatography. RESULTS: In all samples, serum lidocaine concentrations were below a minimum detectable level of 100 ng/mL. No local or systemic intraoperative or postoperative complications were noted. CONCLUSIONS: Intracameral injection of 0.5 mL lidocaine 1% revealed no systemic therapeutic concentrations. In patients in whom other forms of needle-delivered local anesthesia are contraindicated, intracameral injection of lidocaine should be considered to enhance topical anesthesia.     

Active and passive properties of the rabbit corneal endothelium

Some of the physiological properties of rabbit corneal endothelium were investigated by studying: (a) fluid transport; (b) osmotic water flow; (c) electrical potential difference; and (d) electrical impedance, all of them across the endothelial layer; (e) biochemical parameters (ATP level and Na⁺+K⁺-activated ATPase activity) of endothelial cells, and (f) morphological (light and electronmicroscopic) appearance of the endothelium. Several experimental manipulations which decreased or a bolished fluid transport (total replacement of 43 mm bicarbonate by Cl, of 5 mm K by Na and of 150 mm Na by Li; treatment with 5 × 10^?5m ouabain and with 20 μ/ml cytochalasin B) also decreased or abolished the electrical potential difference. The electrical resistance of fresh preparations was 41 ± 5 Ωcm² (s.e.m., n = 14; range: 16–81 Ωcm²); scraping off the endothelium and treating it with Ca-free media decreased the resistance to that of the supporting stromal layer (3–10 Ωcm²) Cytochalasin B (20 μg/ml) markedly decreased the resistance, while ouabain did not affect it. Omission of all metabolites normally present in the perfusion medium (reduced glutathione, adenosine, and glucose) decreased the endothelial ATP level markedly, while addition of reduced glutathione spared ATP from depletion. Since the transport ceased even in the presence of near normal ATP levels and (Na⁺+K⁺)-activated ATPase activity, other yet undefined factors are also probably involved in pump function. Osmotically induced water flows across the endothelium were not linear but followed a square root function of the driving force. Cytochalasin B induced profound changes in the shape of the endothelial cells, causing some of them to clump together. The values of electrical resistance, capacitance and water flows experimentally measured agree reasonably well with the numerical values calculated from a model. The results are consistent with the possibility that the fluid transport across the endothelium would be due to an electrogenic ionic pump, and that the intercellular spaces and their gap junctions would constitute the dominant “shunt” pathway for water and electrolytes.     

Na+ transport across the rabbit corneal endothelium

It has been reported so far that the Na+ transport across the corneal endothelium is symmetrical and therefore, that there is no net transport. We now report that there is a net Na+ transport across the rabbit corneal endothelium. Instead of measuring the conventional steady-state fluxes, pre-steady state fluxes were measured as a function of time (readings were taken at two minute intervals). This technique is based on a theory, proposed very recently, that the flux ratio (efflux/influx) is independent of time. Both the efflux and influx reached their steady state values about fifteen minutes after the addition of the isotope to one side of the chamber. The net sodium flux was obtained from the difference between the steady state efflux and influx values. The direction of the net Na+ flux was from the stromal to the aqueous side, with a magnitude of 2.3 +/- 0.4 microEq/h.cm2 (n = 11, S.E.M.). The net Na+ transport was inhibited by the presence of ouabain (10(-4)M).