Toxicity and pharmacokinetics of intravitreally injected ciprofloxacin in rabbit eyes.

To assess the toxicity of intraocular injection of ciprofloxacin, 22 New Zealand white rabbits received midvitreal injections of 100, 200, 400, 800 or 3200 micrograms of ciprofloxacin in 0.1 mL of distilled water (39 eyes) or 0.1 mL of distilled water only (5 eyes). The ocular pharmacokinetics of intravitreally injected ciprofloxacin was determined by aqueous humour and vitreous sampling 1, 2, 4, 8, 12, 18 or 24 hours after midvitreal injection of 100 micrograms of the drug in one eye each of 25 New Zealand white rabbits. The samples were analysed by means of a disc diffusion bioassay. No ocular damage was noted on ophthalmoscopy at any of the concentrations tested. Histologic study showed mild, transient vacuolation of the nerve-fibre layer in all eyes, including the control eyes, 2 hours after injection; at 24 hours no vacuolation was evident except at concentrations of 800 and 3200 micrograms, at which plexiform layer damage was evident. Peak aqueous and vitreous levels of ciprofloxacin were obtained at 1 hour (0.59 and 27.26 micrograms/mL respectively); the vitreous level fell to below 1.0 micrograms/mL 12 hours after injection. We conclude that intravitreally injected ciprofloxacin may be a safe and useful antibiotic in the treatment of aminoglycoside-resistant bacterial endophthalmitis.     

The effect of intravitreally injected povidone-iodine on Staphylococcus epidermidis in rabbit eyes.

PURPOSE: Firstly, the aim of this study was to determine the maximally tolerated dose of intravitreally injected povidone-iodine (PVP-I). A second aim was to test the efficacy of PVP-I on rabbit eyes infected intravitreally with Staphylococcus epidermidis. METHODS: Phase I of the study used 16 New Zealand albino rabbits, divided into 4 groups (n=4 each). Animals were anesthetized and intravitreally injected with 0.1 mL of 50, 100, 200, or 400 micrograms (microg) of PVP-I in 1 eye, and with saline in the other. The animals were examined at days 1, 7, and 14, using indirect ophthalmoscopy and slit-lamp biomicroscopy; electroretinography (ERG) was performed before treatment and prior to euthanization. Histological preparations were examined to determine retinal damage. Phase II of the study divided 20 New Zealand albino rabbits into 4 groups (n=5 each). Animals were anesthetized and injected with 0.1 mL of S. epidermidis containing 3030 colony forming units (CFU) in 1 eye and saline in the other. Seven (7) h later, animals were treated with 0.1 mL of 20, 50, and 100 microg of PVP-I, or no treatment. Bacterial concentrations from extracted vitreous were determined 2 days following infection. Results were analyzed for statistical significance, using the Student t test and analysis of variance, and histologic preparations assessed the presence of endophthalmitis. RESULTS: Phase I of the study observed no retinal damage at any of the concentrations studied, as noted by indirect ophthalmoscopy, slit-lamp biomicroscopy, ERG, and histologic exam. Phase II of the study showed no statistical difference in bacterial counts between treatment and control groups. All infected eyes went on to develop endophthalmitis, as observed by indirect ophthalmoscopy and histologic preparations. CONCLUSIONS: These results suggest that 400_g of PVP-I can be tolerated intravitreally in rabbit eyes with no noticeable damage over a 14-day period. Results further showed that 100 microg of intravitreally injected PVP-I has no statistically significant effect on rabbit eyes injected intravitreally with 3030 CFU of S. epidermidis.     

Duration of therapeutic levels of intravitreally injected liposome-encapsulated clindamycin in the rabbit

We investigated an intravitreal preparation of liposome-encapsulated clindamycin phosphate to determine the duration of therapeutic levels of the drug in the vitreous cavity. Twenty New Zealand albino rabbits were given an intravitreal injection of 750 micrograms/0.1 mL of encapsulated clindamycin (10 animals) or 800 micrograms/0.1 mL of nonencapsulated clindamycin (10 animals) and then were killed immediately or 6, 12, 24 or 48 hours later. The mean concentration of encapsulated clindamycin in the vitreous at 48 hours was 28.4 micrograms/mL, while that of nonencapsulated clindamycin at 24 hours was 2.3 micrograms/mL. The estimated elimination rate of nonencapsulated clindamycin was 3 hours, compared with approximately 10 hours for the encapsulated preparation. This drug delivery system warrants further investigation for possible use in humans.     

Retinal toxicity of intravitreally injected plain and liposome formulation of fluconazole in rabbit eye

PURPOSE: Candidal endophthalmitis is a sight-threatening ocular infection that most frequently occurs as a complication of candidemia. Fluconazole has been effective against Candida albicans in various animal models. Our objective was to evaluate retinal toxicity of plain and liposome formulation of fluconazole at various dose levels after intravitreal injection. MATERIALS AND METHODS: Twelve New Zealand albino rabbits weighing 2-2.5 kg were used. Two rabbits were used for every dose level. Liposome formulation containing 100 and 200 microg of fluconazole in sterile phosphate buffer solution and plain fluconazole at concentrations of 100, 200, 400 and 800 microg in 0.1 ml of sterile normal saline were injected intravitreally into the right eyes. The left eyes received 0.1 ml normal saline or 0.1 ml of liposome formulation without fluconazole. One week later, the animals were sacrificed, their eyes enucleated and processed for light microscopy and scanning electron microscopy. RESULTS: It showed that plain fluconazole at a concentration of 100 microg and above caused retinal changes, with disorganization of the photoreceptor outer segments. However, liposome formulation of fluconazole (200 microg/0.1 ml) did not show any significant microscopic changes of the retina. CONCLUSION: The liposome formulation decreased the retinal toxicity of fluconazole up to the studied concentration of 200 microg/0.1 ml.     

Biocompatibility and retinal support of a foldable capsular vitreous body injected with saline or silicone oil implanted in rabbit eyes

Introduction: The aim of this study was to evaluate over a 180‐day period the biocompatibility and retinal support of a foldable capsular vitreous body injected with either saline or silicone oil implanted in rabbit eyes. Methods: A standard three‐port pars plana vitrectomy was performed, and foldable capsular vitreous bodies were implanted into the vitreous cavity of rabbit eyes (n = 18). Silicone oil tamponade was used as the control group (n = 5). Of the foldable capsular vitreous body‐implanted eyes, either saline (n = 9) or silicone oil (n = 9) was injected into the foldable capsular vitreous body to support the retina. The treated eyes were examined using a slit lamp with a non‐contact slit‐lamp lens, a tonopen, a non‐contact specular microscope and a B‐scan ultrasound during the 180‐day implantation period. A histological examination was performed at 90 and 180 days. Results: During the 180‐day implantation period, no significant corneal keratopathy or intraocular inflammation was noted, and the intraocular pressure (IOP) and corneal endothelial numbers remained steady among the three groups. B‐scan ultrasonography showed a smoothly increased echogenicity in front of the retina in group of foldable capsular vitreous bodies injected with saline. Gross examination showed that the foldable capsular vitreous bodies injected with saline or silicone oil smoothly supported the retina. The saline or silicone oil inside the foldable capsular vitreous body was homogeneous, transparent and filled the foldable capsular vitreous body. Histological examination showed no obvious abnormality of the cornea, ciliary body or retina in the foldable capsular vitreous body‐implanted eyes. Conclusions: These results suggest that foldable capsular vitreous bodies injected with either saline or silicone oil showed good biocompatibility and retinal support in rabbit eyes over a 180‐day implantation time.     

硅油与重硅油对兔眼视网膜电生理影响的对比研究

目的:探讨硅油和重硅油两种眼内填充物对兔眼视网膜电生理的中长期影响.方法:选用标准家兔28只,右眼为术眼,随机分成3组, A组12只,B组12只,C组4只.A组:玻璃体切割联合玻璃体腔填充硅油组;B组:玻璃体切割联合玻璃体腔填充重硅油组;C组:玻璃体切割联合玻璃体腔填充平衡盐溶液组.对实验动物术眼术前与术后不同时间段测得的视网膜后极部厚度、眼压及视网膜电图b波振幅值进行统计分析.结果:实验A、B组及对照C组术前及术后不同时间点测得的术眼眼压经过两两比较,无统计学差异(P>0.05);A、B、C组经两两比较,在术后第24wk时经OCT测得的术眼视网膜后极部厚度值均有统计学差异(P<0.05);在术后第24wk时术眼ERG的b波振幅均值有显著统计学差异(P<0.01).结论:硅油或者重硅油在作为玻璃体腔内填充物后,对兔眼术后眼压中长期内的影响无明显差别;重硅油在填充后对兔眼视网膜视觉信息传导功能的负面影响,导致视网膜变薄的程度要明显大于普通硅油.     

Effects of intravitreally and intraperitoneally injected atropine on two types of experimental myopia in chicken

Atropine, a non-selective muscarinic receptor antagonist, is currently the most potent agent used to prevent myopia in animal models and children. However, the ocular target tissues are not well defined. To learn more about the effect of atropine on experimental myopia, atropine was applied both intravitreally and systemically (intraperitoneally) to chickens wearing either negative lenses or light diffusers. Furthermore, the effect of ipsilateral intravitreal atropine on myopia development in the saline-treated fellow eye was studied. Monocular intravitreal injections of atropine were performed daily for a period of 4 successive days, starting at day 8 post-hatching. Fellow eyes received saline injections. Chicks were fitted with -7D lenses, either over the atropine-injected eyes only (unilateral "lens-induced myopia (LIM)"), or over both eyes (bilateral LIM). Other groups of chicks were fitted with translucent diffusers over the atropine-injected eyes (unilateral "form deprivation myopia (FDM)"). Finally, atropine was intraperitoneally injected for 4 days in chicks that wore monocularly -7D lenses. Refractive errors (RE) were measured with infrared photoretinoscopy and axial length (AL) with A-scan ultrasonography. Atropine prevented development of myopia in both unilateral LIM and FDM in a dose-dependent fashion. Fifty percent inhibition of myopia was observed at a dose of 25 microg (unilateral LIM) or 90 microg atropine (bilateral LIM) and complete inhibition at 750 microg; in unilateral FDM, 50% inhibition occurred at 2.5 microg and almost 100% inhibition at 250 microg. Interestingly, at the highest dose of atropine (2500 microg), the treated eyes became even more hyperopic compared to the saline-injected contralateral eyes with normal visual experience. In the bilateral LIM model, atropine suppressed development of myopia in both the treated and the saline-injected control eye. However, about 8.3 times higher doses were necessary to achieve comparable contralateral suppression. Since this ratio is lower than the vitreous volume to blood volume ratio (about 1:23 in young chicks), it seems unlikely that systemic dilution of the intravitreally injected drug can fully account for the contralateral suppression. Intraperitoneal injection inhibited myopia development only at the highest dose (2500 microg) but, strikingly, this inhibition was still less when the same dose was provided through the vitreous of the fellow eye. Both eyes seem to be coupled by a yet unknown, perhaps neuronal pathway. Estimations of the scleral concentrations of atropine after intravitreal injection are compatible with the assumption that the suppression of myopia by atropine occurs by direct inhibition of scleral chondrocytes.     

Immunopathology of intraocular silicone oil: retina and epiretinal membranes

AIMS: To determine the inflammatory response in retina and epiretinal membranes after intraocular silicone oil tamponade. METHODS: 14 proliferative vitreoretinopathy (PVR) epiretinal membranes, 33 retro-oil epiretinal membranes, 19 retinectomies, 14 retro-oil retinectomies and 37 idiopathic epiretinal membranes (controls) underwent immunohistochemical analysis using the avidin-biotin complex technique and a panel of monoclonal and polyclonal antibodies. The number of positive cells counted in five 0.5 mm diameter fields of immunohistochemical sections was graded on a score of 1-4. RESULTS: Macrophage cell counts were significantly greater in membranes with a history of exposure to silicone oil (p<0.001). An inflammatory response could be observed within 1 month of silicone oil exchange, and the intensity seemed to be unrelated to the duration of exposure. Macrophages were confined to epiretinal membranes on the surface of retinectomy specimens in 10 of 14 cases and intraretinal macrophages were observed only in specimens with gliotic retina. T and B lymphocytes were rarely seen in the specimens examined. Marked glial cell up regulation was observed in 11 of 16 retinectomy specimens and in 8 of 11 retro-oil retinectomies. Glial cell content was variable in the membranes, but there was a trend of increased presence after exposure to silicone oil. CONCLUSION: This study has shown that the use of silicone oil is accompanied by an inflammatory reaction, primarily mediated by bloodborne macrophages. This response can be observed within 1 month of silicone oil injection and continues after silicone oil removal. Retinal surgeons should be aware of the potential secondary effects of intraocular silicone oil when they are considering its use (and removal) in vitreoretinal surgery.     

Transplantation of cultured rabbit retinal epithelium to rabbit retina using a closed-eye method

We have developed a closed-eye technique for transplanting cultured rabbit retinal epithelial cells to Bruch's membrane of the rabbit. A glass micropipette containing a suspension of 3H-thymidine-labeled, cultured retinal pigment epithelial (RPE) cells is inserted through a pars plana incision and positioned adjacent to the neural retina. A jet stream from the pipette is used to make a small retinal hole and bleb detachment. Patches of host retinal epithelium lift off with the neural retina, creating areas of bare Bruch's membrane. The cell suspension is injected into the subretinal space, and labeled cells can be seen attached to Bruch's membrane as early as 1 hr later. The neural retina spontaneously reattaches within 24 to 48 hr, bringing photoreceptor outer segments in direct contact with the transplanted cells. Phagocytosis of outer segment material by transplanted cells can be seen as early as 24 hr after surgery. This closed-eye technique offers an advantage over the open-sky method used previously in that it allows for reattachment of the neural retina and at least a partial return of function in the transplanted retinal epithelium.     

Effects of fluorouracil and fluorouridine on protein synthesis in rabbit retina.

5-Fluorouracil (5-FU) and its active metabolite 5-fluorouridine (FUR) are currently being evaluated for the treatment of proliferative vitreoretinopathy and the control of scarring after glaucoma filtering procedures. To test for retinal toxicity, the authors examined the effect of intravitreal injections of 5-FU and FUR on protein synthesis in rabbit retinal photoreceptors and ganglion cells. In addition, the toxic effect of subconjunctival 5-FU injections, after a trephine filtering procedure, on ganglion cell protein synthesis was examined. Albino rabbit eyes were given either unilateral intravitreal injections of 1 mg of 5-FU, 2.5 mg of 5-FU, or 0.1 mg of FUR, or subconjunctival injections of 3 mg of 5-FU twice daily after a trephine procedure. Quantitative autoradiography was used to study ganglion cells and photoreceptor outer segment renewal, and scintillation counting was used to quantify newly synthesized protein transported axonally from ganglion cell bodies to the superior colliculus (SC). Marked reduction of labeled protein reaching the SC was noted after either intravitreal 0.1 mg of FUR (41% inhibition after a single injection and 53% after two injections) or 2.5 mg of 5-FU (41% after one injection and 26% after two injections). This reduction was still present after 8 days in eyes receiving 0.1 mg of FUR (32%) and 2.5 mg of 5-FU (22%). Quantitative autoradiography of retinal photoreceptors and ganglion cells corroborated these data, demonstrating inhibition of outer segment renewal after one or two injections of either 0.1 mg of FUR or 2.5 mg of 5-FU. This inhibitory effect was statistically significant using the paired t-test for both drugs. No mean inhibition was observed after intravitreal 1 mg of 5-FU injections or after subconjunctival injections of 5-FU.     

Cyclic nucleotide distribution in identified layers of suprafused rabbit retinas

To assess the effects of pharmacologic agents on metabolite levels in identified retinal layers we have devised an apparatus that allows suprafusion of everted eye cups (vitreal surface exposed) or isolated retinas (photoreceptor surface exposed), recording of electroretinograms (ERG), and rapid freezing in a configuration which permits obtaining frozen tangential sections of the retina. Samples from identified layers of the freeze-dried sections can be subsequently weighed and processed for biochemistry by Lowry methods (Lowry and Passonneau , 1972), or cyclic nucleotides can be extracted from weighed samples and assayed. To test the usefulness of the apparatus, we have compared levels of guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP), measured in defined layers of suprafused rabbit retinas, to concentrations reported for retinal layers from eyes removed from light- and dark-adapted rabbits and quickly frozen. For most layers the concentrations of cyclic nucleotides were similar in both the suprafused and whole eye preparations. In experimental applications we suprafused dark-adapted eye cups and retinas with a physiological saline whose free calcium level was sharply reduced by chelation, and observed that cGMP levels were elevated only in those retinal layers containing photoreceptor portions. Ethyleneglycol bis-(beta-amino-ethyl ether)N,N'-tetraacetic acid (EGTA) (3 mM) yielded a threefold increase in cGMP in 10 min when applied to the photoreceptor surface of isolated retinas. However, when the vitreal surfaces of everted eye cups were suprafused with calcium-chelated medium for 10 min, a threefold increase in cGMP required 20 mM EGTA, 3 mM EGTA being ineffective. In another study, vitreal surfaces of eye cups or photoreceptor surfaces of retinas were suprafused with physiological saline containing 3 mM 3-isobutyl-1-methylxanthine (IBMX) in order to alter cAMP retinal levels. In both cases, eightfold increases in cAMP were measured in the inner nuclear and inner plexiform layers, while three- to fourfold increases occurred in the outer nuclear and outer plexiform layers. This agent also caused a large increase in cAMP in the pigment epithelium.     

Ocular trace metal kinetics and toxicology. I. The distribution of intravitreally injected 67Cu++ within intraocular compartments and its loss from the globe

Radioactive copper (67Cu++) was injected into the center of the vitreous body of rabbits. The relatively rapid initial loss of 67Cu from the vitreous was associated with its accumulation in intraocular tissues. At 24 hr, 20% of the injected 67Cu was found in the retina, representing the highest 67Cu concentration among all ocular tissues, and this high 67Cu concentration was maintained in this tissue throughout the 10-day observation period. Significant amounts of 67Cu were not detected in the aqueous humor at any time. About one-half of the injected 67Cu was lost from the whole globe in 5 days, but the remaining Cu was retained in the globe during the next 5 days. Eyes that received a large dose of CuSO4 in addition to the tracer showed decreased 67Cu activity in the retina and a slight increase in the aqueous humor. Endotoxin-induced ocular inflammation decreased the rate of 67Cu loss from the vitreous, reduced its accumulation by the retina, and increased 67Cu entry into the aqueous humor. It is concluded that 67Cu is retained in the vitreous and the globe due to its binding by, and/or uptake into, intraocular tissues, especially the retina. Cu does not effectively enter the anterior chamber from the vitreous, apparently due to its effective removal by the ciliary processes, thus ruling out the possibility of identifying the existence of Cu-containing intraocular foreign bodies in the posterior segment of the eye by analysis of Cu in the aqueous humor.     

高原缺氧对兔视网膜Nogo表达的影响

目的:观察在高原环境下Nogo在北京青紫蓝兔视网膜中的表达和分布。方法:北京青紫蓝兔24只随机分为实验组20只,对照组4只,分别喂养于高原环境和亚高原环境中1,3,7,10,14d取出兔眼球,制成蜡片,应用免疫组化的方法检测视网膜中Nogo的表达及分布。结果:在高原环境下Nogo在兔视网膜中的表达增多,7d达到高峰,持续到10,14d下降,与对照组比较,差异有统计学意义。结论:在高原环境下,Nogo在视网膜急性缺血缺氧损伤可能发挥了重要作用。     

Intraocular distribution of intravitreally administered amphotericin B in normal and vitrectomized eyes

A study was performed to determine how amphotericin B is distributed in the eye after direct intravitreal injection. Radiolabeled amphotericin B was administered by direct injection into the vitreous space of unmodified phakic control or vitrectomized aphakic eyes in the rabbit model. The eyes were removed at different post-injection times, frozen and dissected into anatomical subparts of cornea, aqueous, iris, lens, vitreous and sclera-choroid-retina. The parts were assayed for total radioactivity (expressed as remaining amphotericin B). No accumulation of drug was observed in the cornea, lens, iris or aqueous region. The majority of drug was found in the vitreous cavity. The rate of disappearance of radiolabeled drug or radiolabeled drug degradation products from the vitreous space was similar to the rate of disappearance from the eye. However, progressive accumulation of radioactivity was observed in the sclera-choroid-retina tissue in the unmodified phakic eyes. This was not observed in vitrectomized aphakic eyes. The accumulated radioactivity could have represented drug degradation products or active drug. These results shed light on the distribution of amphotericin B in the eye after direct intravitreal injection, a procedure often employed clinically for fungal endophthalmitis.     

Retinal toxicity of intravitreal ganciclovir in rabbit eyes following vitrectomy and insertion of silicone oil

BACKGROUND: Although intravitreal ganciclovir dosages up to 500 microg have been demonstrated to be safe in some studies, other studies have shown toxic retinal effects in rabbit eyes without silicone oil at lower dosages. In current clinical practice, the same dosage of intravitreal antiviral agent is given regardless of whether there has been retinal detachment repair with silicone oil. We performed a study to investigate, in rabbit eyes following vitrectomy and silicone oil insertion, the retinal toxicity of serial intravitreal injections of ganciclovir, using dosages previously found not to produce significant toxic effects in nonvitrectomized eyes. METHODS: Twenty-eight eyes of 14 New Zealand pigmented rabbits underwent pars plana vitrectomy and silicone oil insertion. One eye of each animal received an intravitreal ganciclovir injection twice weekly for 2 weeks.The other eye received 0.1 mL of normal saline as a control. Three dosages of ganciclovir (50, 100 or 200 microg/0.1 mL) were used in three groups of three to six animals. Scotopic electroretinography and histologic examination were performed 2 weeks postoperatively. RESULTS: No differences in scotopic b-wave threshold (p = 0.23, 0.78 and 0.50 for ganciclovir dosages of 50, 100 and 200 microg/0.1 mL respectively, Mann-Whitney U test) or in light microscopy findings were noted between the treatment and control eyes at any dosage of ganciclovir. Surgical complications were observed in eight eyes; the data for these eyes were not used for analysis. INTERPRETATION: Ganciclovir dosages of up to 200 microg/0.1 mL appear to be safe for serial intravitreal injection in rabbit eyes following vitrectomy and silicone oil insertion.     

Effects of continuous darkness on ERG correlates of disc shedding in rabbit retina.

The electroretinogram (ERG) was recorded in the dark from photo-entrained albino rabbits, using a constant-intensity, 500-nm, 50- or 100-msec stimulus at 1-min intervals. Under these conditions, the b-wave of the ERG was previously shown to decrease in amplitude at the time of the morning rod photoreceptor disc shedding event. We have extended our observations to rabbits housed in continuous darkness. The present data show that in constant darkness the change in retinal sensitivity continues to occur, but with a period slightly less than 24 hr. Unilateral dark adaptation, achieved by eyepatching, alters the timing of the event only in the retina of the occluded eye. Thus, the change in retinal sensitivity shares the characteristics of endogenous rhythmicity and intraocular control that have been demonstrated by histologic methods for mammalian rod disc shedding. We also report elongation of rod outer segments in some regions of the albino rabbit retina after continuous darkness.     

Verapamil substantially increases the chemomyectomy effect of doxorubicin injected into rabbit or monkey eyelid.

Local doxorubicin injections have been used clinically to treat blepharospasm, hemifacial spasm, and other related disorders permanently and nonsurgically. Doxorubicin is an effective myotoxic agent for the removal of the orbicularis oculi muscle in the eyelid after local injection. Injections of this drug alone resulted in removal of up to 70% of the muscle fibers from the treated eyelids in monkeys. The authors attempted to optimize the conditions for doxorubicin myotoxicity of the orbicularis oculi. Doxorubicin was injected shortly after local verapamil injection in rabbits and a monkey in an attempt to maximize the muscle injury in the eyelid. Verapamil (dose, 0.5 mg or 1.6 mg in the rabbits), injected with a range of doses of doxorubicin, caused substantially increased muscle loss in the eyelid compared with doxorubicin alone. In the monkey, verapamil (dose, 0.25 mg) injection was followed by an injection of 1 mg of doxorubicin. Verapamil cotreatment resulted in increased muscle loss over that caused by doxorubicin alone in both rabbits and the monkey. Injection of verapamil alone also caused muscle loss, and this was quantified. The muscle loss with doxorubicin and verapamil injections included muscle in the preseptal portion of the muscle and even in the pretarsal muscle (which previously was difficult to destroy). This technique clinically might be used to decrease the dose of doxorubicin injected and/or decrease the total number of injections necessary but still retain a clinically effective treatment for blepharospasm and hemifacial spasm. The reduction in the dose of doxorubicin also may decrease the risk of skin injury from doxorubicin chemomyectomy in these patients.