FK506眼液防治同种异体角膜缘移植免疫排斥反应的实验研究

目的　探索同种异体角膜缘移植排斥反应规律及FK5 0 6眼液的免疫抑制作用。方法4 8只新西兰白兔随机均分成FK5 0 6组、环孢素A组及非治疗组 ,建立右眼角膜缘缺陷症动物模型 ,1个月后行同种异体角膜缘移植术 ,术后分别用 0 5 %FK5 0 6眼液、1%环孢素A眼液及生理盐水滴眼4周。角膜缘移植术前 1d ,术后 2、4周分别进行角膜中央印迹细胞学检查 ;术前 1d及术后 1～ 8周动态监测外周血T淋巴细胞CD2 5的表达 ;移植术后每日观察植片存活、角膜新生血管和上皮再生情况 ,共 10周 ;分别取术后第 4、8及 10周的角膜缘植片观测淋巴细胞浸润及T淋巴细胞上CD2 5的表达。结果　角膜缘创伤后 1个月 ,所有兔眼角膜中央印迹细胞学检查均为结膜表型 ;同种异体角膜缘移植术后 4周 ,FK5 0 6组和环孢素A组角膜中央保持角膜细胞表型 ,非治疗组呈结膜细胞表型。非治疗组最早出现上皮排斥反应 ,FK5 0 6与环孢素A均可抑制排斥反应的发生 ,停药后环孢素A组先于FK5 0 6组出现上皮排斥反应 (P <0 0 5 )。FK5 0 6组外周血、角膜缘植片T淋巴细胞CD2 5表达及角膜缘植片中淋巴细胞浸润程度低于环孢素A组 ,两组均低于非治疗组 (P <0 0 5 )。结论　同种异体角膜缘移植术后早期应用 0 5 %FK5 0 6眼液滴眼可抑制外周血及植片T淋巴细胞CD2 5     

Effect of immunosuppression on outcome measures in a model of rat limbal transplantation

PURPOSE: To examine the effect of immunosuppression with intramuscularly injected cyclosporine and topical corticosteroid on limbal allograft survival in a new model in the inbred rat. METHODS: Orthotopic limbal tissue harvested from male Fischer 344 (isografts) or male Wistar-Firth donors (allografts) was sutured into superior and inferior lamellar excision sites in female recipient Fischer 344 rats. Grafts were examined clinically for at least 55 days. Superficial epithelial cells were sampled weekly, and the DNA extracted and probed for the male-specific gene Sry by polymerase chain reaction. Recipients were killed at established intervals for immunohistochemistry. Graft-recipient animals were randomly assigned to receive either intramuscular cyclosporine plus topical prednisolone phosphate or vehicle for 4 weeks from the time of transplantation. RESULTS: Isografts survived for a median of more than 55 days. Allografts underwent clinical rejection at a median of 6 to 7 days after grafting. Acutely rejecting allografts showed a dense mononuclear infiltrate consisting of activated CD4(+) and CD8(+) T cells with some macrophages. Genomic Sry was usually detectable in cells sampled from the ocular surface for more than 55 days in isografts, but not beyond 7 days in allografts. Immunosuppression prolonged allograft survival significantly, as assessed clinically, but did not prolong donor cell survival on the ocular surface, as assessed by detection of genomic Sry. CONCLUSIONS: A robust model of limbal transplantation was developed in the rat. Isografts survived for the long term, whereas allografts underwent rapid rejection. Although clinical allograft survival was prolonged to a modest extent by immunosuppression, donor cell survival on the ocular surface was not improved.     

Efficacy of systemic cyclosporine A and amniotic membrane on rabbit conjunctival limbal allograft rejection

PURPOSE: To evaluate the effect of intramuscular cyclosporine A (CsA) and amniotic membrane (AM) on conjunctival limbal allograft survival in a rabbit model. METHODS: Eighty-two female rabbits (59 New Zealand white rabbits, 23 Dutch pigmented rabbits) were used. The New Zealand white rabbits were divided into 4 treatment groups: group 1 (n=13), conjunctival limbal autograft transplantation; group 2 (n=12), conjunctival limbal allograft transplantation without additional treatment; group 3 (n=18), conjunctival limbal allograft transplantation and human AM; and group 4 (n=16), conjunctival limbal allograft transplantation and systemic CsA (10 mg/kg/day intramuscularly). The 23 Dutch pigmented rabbits were used as limbal stem cell allograft donors. The rejection index, the mean survival time, and the rejection rates were calculated for each group. RESULTS: After 28 days of follow-up, there were no episodes of limbal rejection in groups 1 and 4, whereas the rejection rate was 100% in groups 2 and 3. There was no significant difference in mean survival time of the rejected grafts between groups 2 and 3. CONCLUSIONS: A model of rejection of conjunctival limbal transplantation was developed in the rabbit. Intramuscularly injected CsA effectively prevents limbal allograft rejection. Human AM is not useful for this purpose.     

CsA滴眼液防治角膜缘移植排斥反应的实验研究

目的 探讨环孢霉素A(CsA)滴眼液对同种异体角膜缘移植术后的免疫抑制作用。方法 32只新西兰白兔随机分成CsA组和生理盐水对照组,每组16只。建立右眼角膜缘缺陷症动物模型,1月后实施同种异体角膜缘移植术,术后分别给予1％CsA滴眼液及生理盐水治疗,共4周。移植术后每日观察植片存活、角膜新生血管和上皮再生情况,共8周;术前1日及术后1～8周动态监测外周血T淋巴细胞CD25的表达。结果 对照组先于CsA组出现上皮排斥反应(P<0.05)。CsA组外周血T淋巴细胞CD25表达低于对照组(P<0.05)。结论 同种异体角膜缘移植术后早期应用CsA滴眼液可抑制外周血T淋巴细胞CD25的表达从而抑制排斥反应发生。     

Topical and systemic absorption in delivery of dexamethasone to the anterior and posterior segments of the eye

PURPOSE: This study aimed to: (1) determine the relative efficiencies of topical and systemic absorption of drugs delivered by eyedrops to the anterior and posterior segments of the eye; (2) establish whether dexamethasone-cyclodextrin eyedrops deliver significant levels of drug to the retina and vitreous in the rabbit eye, and (3) compare systemic absorption following topical application to the eye versus intranasal or intravenous delivery. METHODS: In order to distinguish between topical and systemic absorption in the eye, we applied 0.5% dexamethasone-cyclodextrin eyedrops to one (study) eye of rabbits and not to the contralateral (control) eye. Drug levels were measured in each eye. The study eye showed the result of the combination of topical and systemic absorption, whereas the control eye showed the result of systemic absorption only. Systemic absorption was also examined after intranasal and intravenous administration of the same dose of dexamethasone. RESULTS: In the aqueous humour dexamethasone levels were 170 +/- 76 ng/g (mean +/- standard deviation) in the study eye and 6 +/- 2 ng/g in the control eye. Similar ratios were seen in the iris and ciliary body. In the retina the dexamethasone level was 33 +/- 7 ng/g in the study eye and 14 +/- 3 ng/g in the control eye. Similar ratios were seen in the vitreous humour. Systemic absorption was similar from ocular, intranasal and intravenous administration. CONCLUSIONS: Absorption after topical application dominates in the anterior segment. Topical absorption also plays a significant role in delivering dexamethasone to the posterior segment of the rabbit eye. In medication administered to the retina, 40% of the drug reaches the retina via the systemic route and 60% via topical penetration. Dexamethasone-cyclodextrin eyedrops deliver a significant amount of drug to the rabbit retina.     

Treatment of the sequelae of ocular burns using limbal transplantation

PURPOSE: To report the results of limbal transplantation in patients with severe ocular burns and limbal stem cell deficiency. PATIENTS AND METHODS: This series includes six autografts (unilateral ocular burns) and five allografts (bilateral ocular burns) performed in ten eyes of ten males with an average age of 43 years. The origin of the ocular burn was chemical in eight cases and thermal in the remaining two cases. The average time between the initial trauma and limbal transplantation was 79 months. The average size of limbal grafts was 190 degrees (range: 80-20 degrees for autografts and 120-360 degrees for allografts). Patients with allografts received oral cyclosporine in three cases, topical cyclosporine in one case, and intravenous methylprednisolone in one case. Eight patients underwent penetrating keratoplasty an average of 11 months after limbal transplantation (range: 5-24 months). RESULTS: The average follow-up time was 36 months (range: 7-77 months). The overall success rate of limbal transplantation (ocular surface improvement) was 73% (8/11). The success rate of penetrating keratoplasty was 63% (5/8). The average initial visual acuity was 0.4/10 and the average final visual acuity was 1.6/10. Visual acuity improved by two lines or more in seven cases. DISCUSSION: Limbal transplantation is a useful surgical technique in patients with severe ocular burns. However, results remain insufficient and new techniques such as limbal stem cell culture and transplantation are needed to improve the visual prognosis of these patients.     

Reconstruction of ocular surface with heterologous limbal epithelium and amniotic membrane in a rabbit model

PURPOSE: To report in vivo reconstruction of the ocular surface using amniotic membrane and heterologous transplants of epithelial limbal cells in rabbits with chemical burns. METHODS: After severe damage to the ocular surface with n-heptanol and keratectomy, 15 rabbits developed total limbal deficiency with conjunctival epithelialization, vascularization, and chronic inflammation. One month later, a complete keratectomy was performed in all eyes: 12 received additional transplantation of human amniotic membrane and heterologous limbal epithelial cells in a double amniotic membrane layer, 2 received amniotic membrane only, and 1 control eye received no procedure. RESULTS: After 1 month of follow-up, corneas in eight of the operated eyes presented minimal vascularization, without signs of rejection. Corneal surface reconstruction was demonstrated with the growth of new corneal-like epithelial phenotype and integration of amniotic membrane to the basal corneal surface. A superficial amniotic membrane (with the amnion side up as a dressing) peeled off after 7 to 10 days. The epithelialization with heterologous limbal epithelial cells was evident underneath. The other four operated eyes were followed for 6 months; the ocular surface was also stable with a corneal-like epithelial phenotype. CONCLUSION: Simultaneous transplantation of amniotic membrane and heterologous limbal epithelial cells in severe ocular surface disorders could restore ocular surface and may be useful in patients with severe bilateral limbal epithelial loss, giving new perspectives for the treatment of severe ocular surface disorders.     

Ciliary tissue transplantation in the rabbit

Irreversible damage of the ciliary body can be responsible for prolonged ocular hypotony and phthisis bulbi, which, currently, cannot be treated. The aim of this study was to achieve survival of morphologically normal ciliary tissue (CT) transplants in the anterior chamber of a rabbit's eye. Outbred female New Zealand albino rabbits received CT allografts, which were placed on to the surface of the host iris. We evaluated the influence of ciclosporin (CsA), VEGF and donor perfusion on graft survival. Operated eyes were assessed clinically and histologically, and revascularization of the grafts was determined by fluorescein angiography. All grafts became dark and ischemic during the first five to seven days after transplantation. Reperfusion of the grafted tissue was complete at approximately ten days after transplantation. In untreated animals, transplants became infiltrated by inflammatory cells, which led to destruction of the tissue. This was prevented by systemic use of CsA. Transplants treated with VEGF prior to transplantation had fewer ischemic areas but epithelial cell survival was not improved. Whole body donor perfusion prior to preparation of the grafts resulted in less inflammation and, histologically, in a better quantity and quality of the epithelial cells in the CT transplants. Ciliary tissue can be successfully transplanted but the ciliary epithelium suffers from ischemia and in untreated animals the whole transplant is rejected in the classical fashion. If the donor is perfused and the host immunosuppressed, histologically normal ciliary epithelium can be preserved together with rapid revascularization, minimal inflammation and good survival of the transplant, although fibrosis continued to occur during the two months after transplantation.     

The effect of duration and timing of systemic cyclosporine therapy on corneal allograft survival in a rat model

BACKGROUND: Systemic cyclosporine A (CsA) remains a valuable treatment option in the prevention of corneal graft rejection, but the question of timing and duration of this systemic therapy remains unresolved. The effect of a pre- and postoperative dosing schedule, related to the expected moment of rejection, was examined in a rat model. METHODS: All AO (strain) recipients of PVG grafts were assigned to the following treatment groups: Group 1 (controls), groups 2-5 (a postoperative treatment regimen of CsA for 5, 10, 15 and 30 days respectively) and groups 6 and 7 (CsA preoperatively for 5 days and postoperatively for another 5 or 10 days respectively). Corneal allografts were clinically evaluated and blood CsA levels were measured at various time points. RESULTS: Untreated controls rejected their allografts after 13 days. Regression analysis showed a strongly significant positive correlation between graft survival time and duration of cyclosporine therapy. There was no difference in graft survival between groups 3 (CsA 10 days) and 4 (CsA 15 days). A pre-operative dosing schedule of CsA followed by postoperative treatment had no advantage over a solely postoperative treatment regimen. The moment of rejection was characterized by a low to undetectable CsA concentration. CONCLUSION: The present study demonstrates a significant influence of the duration of systemic CsA administration on allograft survival time. However, preoperative administration of CsA does not seem to have an additional influence on graft survival, which is in line with the biological evidence of the mechanism of action of CsA on the efferent arm of graft rejection.     

深低温保存角膜缘干细胞自体移植实验研究

目的:评价深低温冷冻保存兔眼角膜缘干细胞活性及自体移植治疗角膜干细胞缺乏眼表疾病的疗效。方法:12只新西兰白兔用刀片制备带2mm周边角膜和2mm球结膜的环行浅层角膜缘植片,去除余下的角膜上皮,制成角膜干细胞缺乏眼表疾病模型。角膜缘植片通过程序降温仪程序降温后-196℃深低温保存。30天和60天后作自体移植行兔眼眼表重建术。结果:角膜干细胞缺乏导致角膜上皮愈合延迟,7眼在术后22～26天愈合,平均24±1天,4眼复发性上皮糜烂;基质混浊水肿;角膜血管化,平均5±1天出现新生血管。深低温保存角膜缘干细胞自体移植能促进角膜上皮愈合,平均10±2天愈合,基质混浊逐渐吸收,新生血管消退、变细,2眼消失。结论:深低温保存法能保存角膜缘干细胞活性;能随时按需为临床提供活性角膜缘材料;为临床深低温保存角膜缘干细胞移植治疗眼表疾病提供了实验依据。眼科学报1998;14:224～226。     

FK-506抑制同种异体角膜缘移植术后免疫排斥反应的研究

目的：探讨ＦＫ－５０６抑制角膜缘干细胞移植术后免疫排斥反应的临床可行性与有效性。方法：应用前瞻性评估研究方法,将角膜缘移植术后病例６４只眼按随机原则分投药组及对照组各半,投药组应用０．５％ＦＫ－５０６滴眼液,对照组应用１％ＣｓＡ滴眼液。平均随访期半年,以术后角膜缘植片新生血管、水肿、混浊及溶解程度为临床主要评估指标。结果：随访期内在新生血管指数、上皮排斥发生率、移植排斥指数及假性胬肉指数四项指标投     

Cyclosporin A: tissue levels following topical and systemic administration to rabbits.

Plasma and aqueous levels of cyclosporin A (CsA) were determined following topical administration of CsA 1% to healthy rabbit eyes and compared with levels obtained when administered to rabbit eyes which had received corneal grafts 7-10 days previously. In addition plasma levels were determined following intramuscular administration of 50 mg CsA and the results compared with those obtained following topical administration. Topical administration to healthy rabbit eyes five times a day for three days resulted in plasma levels of CsA which were similar to those obtained over three days following an intramuscular administration of 50 mg CsA. The plasma levels in both were significantly higher than those obtained following topical administration to rabbit eyes which had received corneal grafts 7-10 days previously. Aqueous levels of CsA were lower than plasma levels, and there was no significant difference between levels obtained when CsA was administered topically to healthy eyes, to eyes which had received corneal grafts, or to the fellow eye.     

Limbus transplantation for reconstruction of the ocular surface

Proliferation of the corneal epithelium originates in undifferentiated, long-lived stem cells that are located in the basal limbal epithelium. Stem cells are important for corneal epithelial regeneration and wound healing. Depletion of stem cells due to accidents as well as malfunctions of stem cells due to inborn or inflammatory diseases result in limbal stem cell deficiency. Limbal deficiency is characterized by conjunctivalization of the cornea with vascularization and opacification. Partial limbal deficiency can be treated by removing ingrown conjunctival epithelium thus allowing normal limbal epithelium to repopulate the cornea. Unilateral limbus-derived stem cell disease requires either limbal autograft transplantation from the healthy partner eye or kerato-limbal allograft transplantation. Several modifications of the latter technique have been performed including large kerato-limbal lamellar grafts and central penetrating kerato-limbal allografts. All homologous procedures render a very high risk of immunological reactions that require long term systemic immunosuppression. The use of amniotic membrane, better pharmacological drugs for immunosuppression and improvements in the HLA-matching of limbal allografts as well as ex vivo expansion of corneal stem cells should allow for better reconstruction of the ocular surface in limbal deficiency.     

Prolonged survival of allogeneic corneal grafts in rabbits treated with topically applied cyclosporin A: systemic absorption and local immunosuppressive effect.

The modes of action of topical cyclosporin A were studied in rabbits. Immunorejection of corneal allografts was provoked by placing the grafts eccentrically, in contact with the limbus. Topical application of cyclosporin A five times daily for 28 days prevented the rejection of corneal allografts. All grafts were rejected in the control animals. The seven rabbits of the cyclosporin A group were subsequently treated for six months with a lower dosage of cyclosporin A 1%. In six rabbits the graft remained clear. One rabbit treated with two drops a day showed an allograft reaction that could be suppressed by increasing the dosage. After six months, discontinuation of the therapy resulted in rejection of all grafts within four weeks. Cyclosporin A could be detected in the plasma and aqueous humour of both eyes at the end of the treatment, raising the question whether the immunosuppressive effect of topically applied cyclosporin A was due to local or systemic action. Cyclosporin A 1% was therefore applied to the fellow eye five times daily following transplantation, and this treatment, producing similar plasma levels of cyclosporin A, failed to delay the rejection of eccentric corneal allografts. Consequently the suppression of the allograft rejection by topical cyclosporin A is primarily a local immunosuppressive effect, though systemic influence is not ruled out.     

Cyclosporine treatment of RPE allografts in the rabbit subretinal space

PURPOSE: To determine the effects of systemic cyclosporine A (CsA) on the survival of retinal pigment epithelial (RPE) allografts in the subretinal space in an animal model using atraumatic transplantation surgery. METHODS: Following pars plana vitrectomy, an RPE cell suspension from brown rabbits was injected with a glass micropipette into the subretinal space of 39 albino rabbits. For immunosuppression, 22 rabbits were given an injection of CsA, 20 mg daily intramuscularly, 17 rabbits with RPE grafts were controls. The grafts were monitored by biomicroscopy, color fundus photography, and fluorescein angiography. Rabbits were sacrificed at 1, 3 and 6 months, respectively, and the eyes processed for light and electron microscopy including immunohistochemistry. RESULTS: After three months, the transplanted RPE cells, in both the CsA group and the controls, formed a monolayer in the subretinal space. Although a few macrophages were encountered, there was no massive cellular infiltration and the photoreceptor layer was well preserved. After six months, however, there was a disruption of grafted RPE cells in both groups, characterized by dispersion of melanin pigment in the subretinal space, and invasion of macrophages with focal photoreceptor damage but no infiltration of lymphocytes in the retina or choroid. No significant differences between the CsA treated and the control eyes were discernible. CONCLUSION: Although the subretinal space has been considered an immunologically privileged site, we found that the survival of RPE allografts was limited. CsA did not prevent RPE allograft destruction in the subretinal space. The transplant seems to be disrupted either by immunological mechanisms that are not inhibited by CsA, or by nonimmunologic events.     

Enalaprilat and enalapril maleate eyedrops lower intraocular pressure in rabbits

Purpose: This study aimed to develop low‐viscosity aqueous eyedrops containing enalaprilat and its prodrug enalapril maleate in solution, and to evaluate the eyedrops in rabbits. Methods: Aqueous eyedrops with hydroxypropyl‐β‐cyclodextrin containing 0.01–2.9% (w/v) enalaprilat, 1.0% (w/v) enalapril maleate with cyclodextrin or 0.5% (w/v) timolol were prepared. The eyedrops were administered to rabbits and intraocular pressure (IOP) was measured at various time intervals after the administration and the results (mean of 10 experiments ± standard error of the mean) are expressed as the change from baseline (24.7 ± 3.3 mmHg). Results: Enalaprilat possessed sufficient stability to be formulated as an aqueous eyedrop solution with a shelf‐life of several years at room temperature. The maximum decline in IOP after topical administration of one drop of 2.9% enalaprilat solution was 6.2 ± 0.7 mmHg at 4 hours after administration. Duration of activity exceeded 10 hours. A 1% enalaprilat solution lowered IOP by 4.4 ± 0.8 mmHg at 4 hours after administration and had similar duration, and was more potent than 0.5% timolol. The enalapril maleate eyedrops resulted in delayed action, showing maximum potency at 10–22 hours after administration and duration of up to 32 hours. Conclusions: Enalaprilat eyedrops lower IOP in rabbits. The decline in IOP is proportional to the concentration of dissolved enalaprilat in low‐viscosity aqueous eyedrop formulations.     

Long-term survival of allogeneic donor cell-derived corneal epithelium in limbal deficient rabbits

PURPOSE: To investigate the capability of cultivated allogeneic epithelial stem cells to restore a functional ocular surface in a limbal deficient cornea; to verify the long term survival of epithelial allograft; and to examine the host immune response to heterologous cell transplant in a rabbit model. METHODS: Limbal deficiency was established by performing limbectomy on rabbits (n = 100). Corneal epithelial stem cells were obtained from the limbus and replicated in vitro without a supporting layer. The cell (3 x 10(5)) suspension was then transplanted via topical application as eye drops. Animals were divided into allograft, autograft, and control groups. Females were used as recipients and males as donors for the allograft. Corneas were collected at 7, 14, 21, 40 days as well as 2, 3, 7 and 8 months after cell transplantation. Experimental corneas were evaluated by histology, immunofluorescence, immunohistochemistry and Y chromosome analysis. RESULTS: A well-differentiated corneal epithelium was recognized at 14 to 40 days after cell transfer overlying an infiltrated corneal stroma. Corneal re-epitheliazation was confirmed in 31 of 36 allograft corneas. No significant immune rejection was noted. Stromal abnormality caused by previous limbal deficiency was mostly resolved three months after the regeneration of corneal epithelium. CONCLUSIONS: Transplanted corneal epithelial stem cells were able to differentiate into normal corneal epithelium in vivo without the use of membrane scaffolding. This non-autologous donor cell-derived corneal epithelium survived up to 8 months without immunosuppression and was able to reverse the stromal scarring. Thus, cultivated epithelial stem cells have great potential as an alternative to multiple-surgical procedures in the treatment of limbal deficiency states.     

Experimental transplantation of cultured human limbal and amniotic epithelial cells onto the corneal surface.

PURPOSE: Tissue-cultured corneal epithelial transplantation is a novel procedure that uses tissue-cultured epithelial cells to restore severely damaged ocular surfaces. In this study, we used tissue-cultured human limbal and amniotic epithelial cells as donor cells to investigate the feasibility of this procedure for reestablishment of a damaged ocular surface in experimental conditions. METHODS: Primary human limbal epithelial cultures were established from banked limbal tissue. Amniotic epithelial cells were isolated from serologically screened human placenta and maintained in a specialized nutrient medium. Suspended cells (5 x 10(5)/ml) were seeded onto the concave surface of collagen corneal shields and incubated at 37 degrees C for 2-3 days. These cell-covered shields were then placed on a denuded stromal surface in organ culture and on New Zealand albino rabbit ocular surfaces that had the native epithelium previously removed. Specimens were collected 24, 48, 72, and 96 h later from organ-cultured corneal buttons and recipient animals, processed, and evaluated histologically. RESULTS: The cells grown on the collagen shield were spread uniformly and unpolarized after 48 h in culture. They were repolarized and tightly adhered to the recipient corneal stroma 24 h after transplantation, as demonstrated by formation of cell-substrate hemidesmosomes (HDs) and donor-specific antigen immunostaining. The donor cells were retained in six of 15 rabbits receiving limbal cells and four of 12 rabbits receiving amniotic cells for as long as 10 days after surgery. CONCLUSION: Cultured human limbal and amniotic epithelial cells can be successfully transplanted onto a denuded corneal surface where they adhere tightly to underlying stroma by hemidesmosomes.     

Improving effects of topical administration of iganidipine, a new calcium channel blocker, on the impaired visual evoked potential after endothelin-1 injection into the vitreous body of rabbits

PURPOSE: To investigate the effect of topical iganidipine, a new dihydropyridine derivative calcium channel blocker, on impairment of the ocular circulation caused by endothelin-1 (ET-1), we examined modification of the effect of ET-1 on visual-evoked potential (VEP) in albino rabbits. METHODS: To clarify whether VEP could be used to indicate the extent of ocular circulatory impairment, we evaluated the dose dependency of changes in VEP over 2 hours after intravitreal injection of 3 ET-1 doses (1.0, 3.3, or 10 pmol) and the vehicle. Then modification of the effect of ET-1 on the VEP by iganidipine was examined. One hour after the topical instillation of 0.1% iganidipine (20 microl) or its vehicle, 10 pmol of ET-1 was injected into the vitreous in rabbits. The VEP was measured for 2 hours after ET-1 application, and the response was compared between the eyes with iganidipine and vehicle pretreatment. RESULTS: Intravitreal injection of ET-1 dose-dependently reduced the VEP amplitude. Topical administration of 0.1% iganidipine significantly suppressed the reduction of VEP amplitude for the entire 2-hour monitoring period after intravitreal injection of 10 pmol ET-1. There was no significant change of the systemic blood pressure as well as intraocular pressure after topical administration of iganidipine. CONCLUSIONS: Iganidipine eyedrops may be useful for the treatment of ischemic retinal and optic nerve disorders related to abnormal ET-1 production for the maintenance of visual function.     

Prolonged, contemporaneous administration of pilocarpine and timolol increases the aqueous humor pilocarpine levels in rabbits.

The purpose of this study was to gather information on the mechanism by which timolol/pilocarpine (TI/PI) combination eyedrops provide additive ocular hypotensive effects. An hypothesis, according to which the combination eyedrops prolong the intraocular permanence of PI as a consequence of decreased aqueous humor secretion induced by TI, was not supported by clear-cut literature evidence. It was thus sought to verify if repeated instillations in albino rabbits of combination TI/PI eyedrops do effectively prolong the turnover of PI. Commercial eyedrops containing 0.68% w/v TI maleate and 2.0% w/v PI hydrochloride, buffered at pH 6.8, and two reference solutions containing PI hydrochloride alone (2% w/v), buffered at pH 5.5 and 6.8, were instilled b.i.d. in albino rabbits for five days. Aqueous humor samples, analyzed after the last treatment, showed that the aqueous humor PI levels observed after administration of the combination eyedrops were significantly higher than those resulting from administration of the reference formulations. When compared with the pH 6.8 reference solution, the pH 5.5 one produced slightly higher and more sustained drug levels in the aqueous humor. The present results appear to confirm the assumption that an increased retention of PI in the aqueous humor is responsible for the additive effects on intraocular pressure reported by several authors for the combination TI/PI eyedrops.