Pharmacologic vitreolysis with microplasmin increases vitreous diffusion coefficients

Background Pharmacologic vitreolysis is a new approach to improve vitreo-retinal surgery and ultimately to liquefy and detach the vitreous from the retina to eliminate the contribution of the vitreous to retinopathy. The mechanism of action of the agents being developed for pharmacologic vitreolysis remains unclear. The effect of microplasmin on vitreous diffusion coefficients was investigated using the non-invasive technique of dynamic light scattering (DLS). Methods Vitreous diffusion coefficients in 18 intact porcine eyes were measured in vitro with dynamic light scattering (DLS). DLS was performed on all specimens at 37 °C 30 min after injections of human recombinant microplasmin at doses ranging from 0.125 to 0.8 mg, with 20-nm tracer nanospheres. Results DLS findings in untreated porcine vitreous were similar to the previously described findings in bovine and human vitreous. Microplasmin increased porcine vitreous diffusion coefficients in a dose-dependent manner (correlation coefficient, r=0.93), with an 85% increase after a 30-min exposure to the maximum dose. Conclusions Pharmacologic vitreolysis with human recombinant microplasmin increases vitreous diffusion coefficients in vitro. The results of these studies have implications for the dosing, route of administration, duration of action and methods of determining efficacy in future studies of pharmacologic vitreolysis to enhance vitreo-retinal surgery, as well as the design of clinical trials to induce prophylactic posterior vitreous detachment. This work is an abridgement of a thesis submitted in partial fulfillment of requirements for membership in the American Ophthalmological Society, May 2005.     

Safety and efficacy of dispase and plasmin in pharmacologic vitreolysis

PURPOSE: To evaluate the safety and efficacy of dispase and plasmin when inducing posterior vitreous detachment (PVD) by intravitreous injection in rabbit eyes. METHODS: Forty-eight young pigmented rabbits were randomized into six groups. Groups 1 and 5 received 0.025 U dispase in test eyes; group 2, 0.1 U dispase; groups 3 and 6, 1 U plasmin; and group 4, 4 U plasmin. All groups received PBS in control eyes. Groups 5 and 6 were euthanatized 15 minutes after surgery for ocular histologic examination. The remaining groups (groups 1-4) received indirect ophthalmoscope and biomicroscopy 15 and 30 minutes; 1, 2, and 8 hours; and 1, 3, and 7 days after surgery. Ultrasonography and electroretinogram were performed 1 hour and 1 and 7 days after surgery. The eyes then were examined by scanning and transmission electron microscopy. RESULTS: Partial or complete PVDs were observed in the eyes that received dispase and plasmin, confirmed by the results of scanning electron microscopy. Light microscopy showed inflammation in both dispase- and plasmin-treated eyes of groups 5 and 6. However, whereas in plasmin-treated eyes the ERG and cell ultrastructure showed no significant changes, in dispase-treated eyes, the amplitudes of ERG showed a significant reduction from baseline and ultrastructural damage to the retina was detected by transmission electron microscopy. Cell damage, preretinal hemorrhage, and cataract were also observed in these eyes. No changes were observed in the control eyes. CONCLUSIONS: Intravitreal injection of dispase at 0.025 U or more can induce PVD, but it is not safe. Plasmin (1-4 U) is safer, except for the potential risk of inducing intraocular inflammation.     

Long-term effect of plasmin on the vitreolysis in rabbit eyes

The aim was to investigate the proteolytic activity of plasmin and its long-term complications. Plasmin was injected into the vitreous cavity of rabbits' eyes. Slit lamp biomicroscopy and electroretinography were performed. Rabbits were serially sacrificed at four months, and globes fixated and prepared for light and transmission electron microscopy. In both the plasmin-injected and control eyes, electroretinography showed a transient decrease in the amplitude, but this recovered to the baseline level in a week. Under the light microscope, the plasmin-treated eyes had a smooth retinal surface, implying separation of the vitreous cortex from the retina. In the control eyes, the collagen fibers remained on the retinal surface. By transmission electron microscopy, the plasmin-treated eyes showed a vitreous-free retinal surface, but no vitreoretinal separation was observed in the control eyes. Plasmin induces a cleavage between the vitreous and the internal limiting membrane, with no long-term complications, so may be a useful pharmacologic adjunct to vitrectomy.     

Safety of intravitreal clindamycin in albino rabbit eyes

Purpose

To study the potential toxic effects of intravitreal clindamycin on the retina of albino rabbits, by assessing functional and morphological retinal changes.

Methods

Eight albino rabbits were included in the study. In each rabbit, 1 mg/0.1 ml clindamycin was injected into the vitreous of the right (experimental) eye, and 0.1 ml saline was injected into the vitreous of the left (control) eye. The electroretinogram (ERG) was recorded before injection, 3 days, 1, 2, and 4 weeks post-injection. The visual evoked potential (VEP) was recorded 4 weeks post-injection. Clinical examination was conducted at all time points. The eyes were enucleated at the termination of the follow-up period in order to prepare the retinas for histology in order to assess retinal structure.

Results

ERG and VEP responses that were recorded from the experimental eye at different times following intravitreal clindamycin injection were very similar to the corresponding responses that were recorded from the control eyes. Clinical examination was normal in all eyes, and no histological damage was observed.

Conclusions

Intravitreal injection of 1 mg clindamycin does not cause functional or morphological signs of retinal toxicity in albino rabbits, during a period of 4 weeks post-injection. These findings support the clinical use of 1 mg intravitreal clindamycin.

     

Safety of intravitreal injection of bevacizumab in rabbit eyes

PURPOSE: To evaluate the safety of intravitreal injection of bevacizumab in rabbits using electrophysiological testing and histopathologic analysis. METHODS: New Zealand albino rabbits were injected in one eye with control antibody (n = 2), 0.05 mL of bevacizumab (n = 3), or 0.2 mL of bevacizumab (n = 3). Electroretinograms were obtained 1 week and 4 weeks after injection. Histologic analysis was performed after completion of the electroretinographic studies. RESULTS: No statistical differences were seen in scotopic and photopic a- and b-wave amplitudes between untreated control and bevacizumab-injected eyes. No histopathologic differences were identified between untreated control and bevacizumab-injected eyes. CONCLUSION: Our study did not find evidence of retinal toxicity from a single intravitreal injection of bevacizumab in rabbits.     

Enzymatic vitreolysis with recombinant microplasminogen and tissue plasminogen activator

PURPOSE: To generate microplasmin (microPlm) using recombinant microplasminogen (microPlg) and recombinant tissue plasminogen activator (rt-PA) before intravitreous injection and to investigate the efficacy of microPlm in inducing posterior vitreous detachment (PVD). METHODS: Forty-eight female or male New Zealand white rabbits were randomized into three groups. Recombinant human microPlg was incubated with rt-PA with a 200:1 molar ratio at 37 degrees C for 40 min. The right eyes of groups 1, 2, and 3, were injected with 0.5, 1.0, and 1.5 U microPlm in 0.1 ml respectively, and 0.1 ml balanced salt solution (BSS) was injected into the left eye as controls. Scanning electron microscopy (SEM), gross specimen examination, B-ultrasonography and optical coherence tomography (OCT) were performed to detect vitreoretinal interface. RESULTS: Over eighty percent of recombinant human microPlg could be activated to active microPlm by rt-PA after 40 min incubation. Complete PVD was found at vitreous posterior pole of microPlm-treated eyes without morphological change of retina. Complete PVD of 25, 75, and 87.5% rabbit eyes was induced by 0.5, 1.0 and 1.5 U recombinant microPlm respectively on day 1. The remnants of vitreous cortex at the posterior pole were dependent on the concentration of microPlm. Among the four approaches for detecting PVD, SEM, gross specimen examination, and B-ultrasonography were more effective methods than OCT. CONCLUSION: Intravitreous injection of 1.5 U microPlm can effectively induce complete PVD in rabbit eyes on day 1 without morphological change of retina.     

Safety and pharmokinetics of triamcinolone hexacetonide in rabbit eyes.

PURPOSE: The aim of this study was to evaluate whether intravitreal triamcinolone hexacetonide (TH) is a safe, longer lasting alternative to intravitreal triamcinolone acetonide (TA) in the rabbit eye. METHODS: Three groups, each comprising of 15 Dutch-belted rabbits, received a unilateral injection of 0.1 mL of drug and 0.1 mL of physiologic salt solution in the fellow eye. Group I received TA, group II received commercially available TH, and group III received reformulated iso-osmolar triamcinolone hexacetonide (rTH). Simultaneous bilateral dark-adapted electroretinography was performed following the injection. Retinal morphology was assessed by using histopathology in each group enucleated 12 weeks after injection. High-performance liquid chromatography of vitreous isolated from the enucleated eyes was used to determine drug concentrations. RESULTS: A significant reduction in saturated a-wave and maximal scotopic b-wave was observed in the group II eyes relative to the fellow control eyes at both 2 and 12 weeks postinjection (P < 0.001 for each comparison) but not in the other groups. Histopathology showed no differences between drug-injected eyes and fellow control eyes in groups I and III, but in group II there was severe degeneration of all retina layers. In group I, the drug half-life was 17.7 +/- 1.7 days, group II 44 +/- 13 days, and group III 12.8 +/- 2.3 days. CONCLUSIONS: The half-life of commercially available TH in the vitreous is double that of TA, but the former is toxic to the retina in this rabbit model. Reformulated iso-osmolar TH showed no evidence of deleterious effects to retina function or structure but had a similar half-life to TA.     

In vivo transscleral iontophoresis of amikacin to rabbit eyes.

The objectives of these studies were to determine the amount and distribution of the aminoglycoside antibiotic amikacin delivered to rabbit eyes following transscleral iontophoresis and to determine the inter-study reproducibility of delivery over three identical studies. New Zealand White rabbits (N = 6 per dose group) were treated with a 200-mg/mL amikacin solution at 0, 2, 3 or 4 mA of (+) DC current for 20 minutes. Amikacin concentrations in eye tissues were highest with the 4-mA treatment. Concentrations for all three studies at this current were approximately 5.4, 40, 41, 343, and 92 mcg/g in the vitreous humor, anterior segment, non-treated hemisphere of the sclera, treated hemisphere of the sclera, and retina/choroid, respectively. These values were approximately 27, 50, 40, 10, and 13 fold greater than in the 0-mA control group and are well above the in vitro minimum inhibitory concentrations (MICs) for this drug. Inter-study reproducibility (measured as %CV) depended on the tissue type and treatment group and ranged from 8% for the retina/choroid to 51% for the anterior segment in the 4-mA group. Pretreatment with topical proparacaine hydrochloride local anesthetic did not affect amikacin delivery and total drug delivered was not affected by delivery time for the same total charge administered. Therapeutically relevant amounts of amikacin were delivered into eye tissues in a reproducible and controllable manner.     

Safety and pharmacokinetics of an intravitreal biodegradable implant of dexamethasone acetate in rabbit eyes

The treatment of vitreoretinal diseases is limited and, nowadays, new drug delivery approaches have been reported in order to increase drug bioavailability. The objective of the current study was to determine the pharmacokinetic profile of a biodegradable dexamethasone acetate implant inserted into the vitreous of rabbits and to evaluate its potential signs of toxicity to the rabbits' eyes. The results showed that the intravitreous drug concentration remained within the therapeutic range along the 8-week period of evaluation. The system under study was not toxic to the normal rabbit retina, and no significant increase in intraocular pressure was observed.     

兔眼玻璃体腔注射替考拉宁眼内药代动力学研究

背景 眼内炎是严重的感染性眼病,早期有效的药物治疗至关重要.替考拉宁眼内用药治疗眼内炎及眼内药代动力学方面的研究文献报道较少.目的 探讨兔眼玻璃体腔注射替考拉宁眼内药代动力学过程及特点.方法 取日本大耳白兔33只,右眼玻璃体腔注射替考拉宁0.5 mg后,分别于15 min、30 min和1、2、4、6、12、24、48、96、192 h抽取玻璃体及房水各0.1ml,采用微生物检定法测定替考拉宁的质量浓度.结果 替考拉宁标准品质量浓度的对数值随着抑菌圈直径的增加而增加,其标准品回归曲线方程:Y=0.174X-0.813(R2 =0.999),在替考拉宁质量浓度为1.0-80.0 mg/L范围内线性关系良好.替考拉宁玻璃体腔单次注射符合开放性二室模型,玻璃体腔分布相Tα1/2与消除相Tβ1/2分别为1.68 h、152.15 h,房水中分布相T1/2与消除相Tβ1/2分别为2.83 h、70.56h.替考拉宁在玻璃体、房水中的峰质量浓度分别为(358.47±21.53)mg/L、(102.17±9.54)mg/L,达峰时间分别为1h;在192 h时,玻璃体、房水中替考拉宁质量浓度分别为(4.38±0.68)mg/L、(2.38±0.38)mg/L.结论 替考拉宁0.50 mg玻璃体腔注射后能在玻璃体、房水中维持较长时间的药物治疗质量浓度.     

毛果芸香碱透明质酸钠药膜药代动力学研究

目的 探讨2%毛果芸香碱透明质酸钠药膜在兔眼的药代动力学特征,评价其相对生物利用度.设计实验性研究.研究对象 48只健康白兔.方法 48只兔随机分成实验组和对照组,每组24只兔.分别给兔眼结膜囊内嵌入2%毛果芸香碱透明质酸钠药膜和滴入4%毛果芸香碱凝胶滴眼液.评价用药后兔眼刺激症状得分、瞳孔大小,并于给药后0.5、1、3、6、12、24、48及72h时抽取房水,每组各时间点随机选取3只兔(6眼),用反相高效液相色谱法(HPLC)测定房水中的毛果芸香碱浓度.主要指标兔眼刺激症状,瞳孔大小,房水药物浓度.结果 实验组与对照组比较兔结膜充血症状无显著性差异(P＞0.05);瞳孔缩小持续时间分别为(54.78±4.52)h、(21.33±2.28)h(P＜0.01);药物房水达峰时间分别为30min和3h,达峰浓度分别为(18.44 9.56)μg/ml、(16.61±4.92)μg/ml.结论 2%毛果芸香碱透明质酸钠药膜可明显提高毛果芸香碱的生物利用度,减少用药频率,并具有临床开发应用的前景.(眼科,2008,17:283-285)     

Endophotocoagulation through perfluorodecalin in rabbit eyes

Retinal laser endophotocoagulation through perfluorodecalin was studied in six eyes of three Dutch-belted rabbits after vitrectomy. Both the energy density threshold (EDT/50) and the energy threshold necessary to obtain a therapeutic lesion were evaluated. Both argon and semiconductor diode laser endophotocoagulators were used. The amount of laser power energy and the histology of chorioretinal lesions were similar when photocoagulating through perfluorodecalin, compared to photocoagulation through balanced salt citrate-buffered solution. This experimental study indicates that no extra care is necessary when retinal endophotocoagulation is performed through perfluorodecalin, as long as circular spots are obtained and energy is delivered symmetrically to the target site.     

汉防己甲素的兔眼内药代动力学研究

目的 研究汉防己甲素(Tet)滴眼液单次滴眼后在兔眼内的药代动力学行为.方法 采用0.3%Tet滴眼液单次滴眼后,利用高效液相色谱法(HPLC)检测其在兔眼内各组织中的药物浓度变化过程,并计算其在眼内各组织中的药代动力学参数.结果 Tet在眼内各组织中的浓度变化均符合血管外给药一室模型一级动力学过程.其在角膜组织中的浓度最高,消除半衰期为115.140min,消除速率常数为0.060/min.Tel在角膜中的AUC为2336.294μg/(g·min),约为房水中AUC的8倍,虹膜睫状体的5倍,晶状体的15倍,玻璃体的25倍.结论 Tet滴眼液单次滴眼后,在角膜中能够达到较高的浓度,Tet在眼内的药代动力学参数为其临床应用以及用药方案选择提供了实验依据.     

Tolerance of intravitreal povidone-iodine in rabbit eyes

Povidone-iodine is frequently instilled on to the conjunctival surface prior to intraocular surgery in order to prevent septic endophthalmitis. A small amount of povidone-iodine is inevitably introduced into the eye when it is used in this manner. The toxicity of intravitreal povidone-iodine was assessed in rabbit eyes by injecting 0.1 ml of povidone-iodine in concentrations of 0.05%, 0.5% and 5% into the vitreous cavity. The injected eyes were evaluated by clinical examination, anterior segment and fundus photography, endothelial cell counts, electroretinography and histopathology. Compared to control eyes, no changes were observed in all 6 eyes injected with 0.1 ml of 0.05% povidone-iodine solution. 9 of 10 eyes tolerated a concentration of 0.5% with no detectable adverse changes. One eye developed a temporary mild iritis and mild suppression of the ERG. Intra-retinal hemorrhages, edema, arteriolar narrowing and retinal edema were seen one week following injection. Mild retinal necrosis of the same area was seen on histology. All 4 eyes injected with 5% povidone-iodine developed temporary hypotony and iridocyclitis. A dense cataract developed in all eyes. Full thickness retinal necrosis and a profound lasting reduction in the ERG was produced in all of these eyes. No corneal epithelial or endothelial changes were observed in any eye in this series. Low concentrations of intravitreal povidone-iodine likely to be produced by instillation prior to surgery are tolerated by rabbit eyes. The concentrations tolerated by the studied eyes are near reported bactericidal levels.     

Cytotoxicity of mitomycin C in rabbit eyes

The cytotoxic effects of mitomycin C (MMC) on intact rabbit eyes were studied using light microscopy and transmission electron microscopy. A volume of 0.1 ml MMC solution (0.4 mg/ml) was given to rabbits subconjunctivally once a day. The eyes were enucleated at 1, 2, 3, and 4 weeks after subconjunctival injection. MMC showed considerable cytotoxicity in most tissues of the rabbit eyes, such as the cornea, the iris, especially to the choroid and the ciliary body. However, MMC cytotoxicity in the sclera was slight. Based on the above results it is concluded that MMC should not be used when the cornea or sclera is invaded, for example after pterygial resection, and scleral necrosis due to MMC could occur due to the damage of alimentary vessels of the sclera.     

In vivo toxicity of netilmicin and ofloxacin on intact and mechanically damaged eyes of rabbit

PURPOSE: To evaluate the in vivo toxicity of netilmicin and ofloxacin using both normal and mechanically damaged eyes of rabbit. METHODS: Male albino New Zealand rabbits were given either 0.3% netilmicin, 0.3% ofloxacin, or 0.9% sodium chloride solution by topical instillation (50 microL) into the conjunctival sac 6 times daily for 5 days. In some animals a 6-mm-diameter epithelial wound was mechanically made to the center of the cornea. Ocular toxicity on normal eyes was evaluated by impression cytology of the conjunctiva, histology of the entire globes, and scanning electron microscopy (SEM) of the cornea. Analysis of toxicity and reepithelialization on wounded corneas was evaluated by SEM with observations being made 48 and 72 hours after induction of the wound. RESULTS: Cytologic, histopathologic, and SEM analyses of normal healthy eyes following netilmicin treatment revealed no signs of toxicity, whereas those treated with ofloxacin revealed alterations in the cornea (stromal swelling) and conjunctiva (infiltration of polymorphonuclear cells) with reduced goblet cell numbers. Wounded corneas treated with netilmicin exhibited normal morphology and reepithelialization, whereas the administration of ofloxacin resulted in disordered cellular organisation and slower rates of epithelial recovery. CONCLUSIONS: Netilmicin, an antibacterial aminoglycoside, is well tolerated even in an experimental wound-healing model where the integrity of the ocular surface is compromised, whereas ofloxacin, a fluoroquinolone, appears to provoke an inflammatory response in the normal eye and a clear alteration of reepithelialization in the wounded eye. These findings suggest that netilmicin may offer a superior toxicological profile in both normal eyes and clinical situations where the integrity of the ocular epithelium is suspect.