Aqueous Humor Of Rabbit Eyes Research Articles

The generation of fluorometholone nanocrystal eye drops, their metabolization to dihydrofluorometholone and penetration into rabbit eyes

Fluorometholone is a widely used anti-inflammatory ophthalmic formulation, which elicits a lower ocular hypertensive response than other glucocorticoid medications. This serves to mitigate against the risk of steroid-induced glaucoma. Based on the hypothesis that an improved corneal permeability can increase the bioavailability of a drug, we sought to obtain fluorometholone in suspension with a small particle size. Accordingly, we describe the formulation of fluorometholone nanocrystal eye drops, which have a mean particle size of 201.2 ± 14.1 nm (standard deviation (s.d.)) when measured by dynamic light scattering. Scanning electron microscopy further indicates that fluorometholone nanocrystals are predominantly rectangular in shape. Fluorometholone microcrystals, on the other hand, with a mean particle size of 9.24 ± 4.51 µm (s.d.), tend to have a rod-like morphology. Powder x-ray diffraction revealed that fluorometholone microcrystal and nanocrystal formulations have the same crystal structure, with the main diffraction peaks at 2θ = 10.4 and 15.3°. The nanocrystal formulation was found to be stable, long-term, when stored at 10 °C for up to 6-months. High pressure liquid chromatography (HPLC) of the aqueous humor of rabbit eyes 15–240 mins after the in vivo application of fluorometholone eye drops to the ocular surface revealed that the molecule had been converted to 20α-dihydrofluorometholone (with no evidence of a 20β-dihydrofluorometholone fraction), and that penetration was 2–6 fold higher and longer lasting with the nanocrystal, rather than the microcrystal, formulation. In current study we show how newly generated fluorometholone nanocrystals when administered as eye drops enter the anterior chamber of the eye and become metabolized to dihydrofluorometholone.

Evaluation of D‐dimer levels in aqueous humor of rabbit eyes with and without induced intraocular fibrin and fibrinolytic treatment

To analyze D-dimer concentrations in aqueous humor (AH) of rabbit eyes under physiological conditions, after induction of fibrin clots, and following fibrinolytic therapy. Prospective study measuring D-dimers in aqueous humor of rabbit eyes with induced fibrin clots (n=44). Rabbits were purchased in two groups, which led to two temporally separated experimentation groups. Different treatment protocols were compared for their efficacy in fibrin reduction (slit-lamp examination, high-resolution ultrasound). AH was taken from left eyes before clot induction (baseline, day 1), 24hours later after clot establishment/prior to drug administration (post-induction, day 2) and 48hours after clot induction (post-treatment, day 3). An enzyme-linked immunosorbent assay (ELISA) was performed to measure intraocular D-dimer concentrations RESULTS: D-dimer concentrations were measurable in all samples. There were no differences in D-dimer levels across time points or treatments within the arrival groups. However, a significant difference in mean D-dimer levels was observed between the two arrival groups (group 1:3.1µg/mL; group 2:6.1µg/mL; P<.0001), which made a direct comparison of treatment groups impossible. Clinically, all eyes displayed fibrin clots in the anterior chamber and different treatment types led to significant differences in clot resolution (clot size reduction after intracameral treatment: 98%, topical treatment: 60%, no treatment: 40%). D-dimers were identified in all AH samples of rabbits with large variability between samples. D-dimer levels were neither predictive for differences in induced fibrin formation nor for drug efficacy.

Protective Role of Oral Bupropion in Prevention of Cataract Induced Experimentally in Rabbits

Cataract is one of the chief causes of blindness and visual impairment in the elderly people throughout the developing world. The aim of the study was to investigate the possible protective role of oral bupropion against selenite induced cataract in rabbits. Adult rabbits were used in the present study. Groups of study were: Apparently normal group, Cataract group and oral bupropion group. Cataract induction was done by a single intravitreal injection of 0.1 ml (0.01% w/v) of sodium selenite solution in the right eyes. Bupropion (50 mg/kg two times daily) was given for five days before intravitreal injection sodium selenite solution and 21 days after. The parameters were: Lens opacity, pupil diameter, light reflex, corneal sensation, conjunctival redness, the level of malondialdehyde (MDA) and reduced glutathione (GSH) in aqueous humor of rabbit eyes. Oral bupropion resulted in high significant protection from cataract development and there was no change in pupil diameter, positive light reflex, positive corneal sensation, no conjunctival redness, decreased level of MDA and increased level of GSH.

Comparison of Aqueous Humor Nitric Oxide Levels After Different Corneal Collagen Cross-Linking Methods

ABSTRACTPurpose: Nitric oxide production can cause either apoptotic or necrotic cell death through oxidative stress. We aimed to investigate the nitrite oxide metabolites (NOx) and nitrite levels in the aqueous humor of rabbit eyes after different methods of corneal collagen cross-linking (CXL).Materials and methods: Twenty-four eyes of 12 adult New Zealand rabbits were used. They were assigned into four groups, each including six eyes. Group 1 (control) consisted of eyes with no treatment. Group 2 received UV-A power setting at 3 mW/cm2 for 30 minutes of continuous exposure and named as standard CXL group. Group 3 received UV-A power setting at 30 mW/cm2 for 3 minutes of continuous exposure and named as accelerated CXL (A-CXL) group. Group 4 received UV-A power setting at 30 mW/cm2 for 6 minutes of pulsed exposure (1 sec on, 1 sec off) and named as pulse-light accelerated CXL (PLA-CXL). Aqueous humors were aspirated from anterior chamber with a 27G needle after 1 hour UV-A exposure. NOx and nitrite levels were measuredResults: The nitrite levels in aqueous humor were significantly increased in Group 2 and Group 3 when compared with Group 1 (p = 0.000, p = 0.036, respectively). When treatment modalities were compared with each other, high nitrite level in Group 2 was statistically significant when compared with Group 4 (p = 0.019). NOx levels were higher in Group 2 when compared with Group 1 (p = 0.006).Conclusions: Numerous studies investigated the physiological and pathophysiological roles of NO. NO is considered one of the most important molecule for ocular health. According to NOx level in aqueous humor, it seems that PLA-CXL is the safest method due to the similar results with control group.

Effects of honey to acyclovir in the rabbit eye transport kinetics

Using pharmacokinetics to explore the mechanism of honey to enhance the efficacy of acyclovir (ACV) treatment of herpes simplex keratitis (HSK), providing the basis for combination of the prescription of two drugs and dosage regimen designed. Single dosages of 5% honey and 0% honey Meyasu eye ointment are injected into rabbit eyes. The aqueous humor of rabbit eye is measured at different times, specifically the content of ACV in aqueous humor by HPLC. Mathematical models are established, from which pharmacokinetic parameters are extracted and compared by mathematics and statistics methods. Both the 5% and 0% honey Meyasu eye ointment in rabbit eyes are belong to a two-compartment model. The absorption half-life of the 5% Meyasu eye ointment in aqueous humor is as 2.30 times longer, the distribution half-life is 2.12 times longer, the peak concentration is 1.17 times longer, the peak time is 1.36 times longer, AUC is 1.41 times longer when compared to the 0% Meyasu eye ointment. Honey can significantly increase the ACV concentration and bioavailability in the eye, extend the action time of ACV in target cells and increase the retention capacity of ACV in the target tissue; thereby improving treatment success.

Chitosan Nanoparticles of 5-Fluorouracil for Ophthalmic Delivery: Characterization, in-Vitro and in-Vivo Study

The aim of this investigation was to develop 5-fluorouracil (5-FU) loaded chitosan nanoparticles (CH-DNPs) for ophthalmic delivery. CH-DNPs were fabricated by ionotropic gelation mechanism using chitosan (CH) and a polyanion (TPP). The nanoparticles were smooth and spherical, confirmed by scanning electron microscopy (SEM) and atomic force microscope (AFM). CH/TPP mass ratio and TPP significantly changed the particles size morphology and encapsulation efficiency. The nanoparticles size ranged from approximately 114 to 192 nm and had a positive zeta potential (30±4 mV). The encapsulation efficiency, loading capacity and recovery of DNPs were 8.12-34.32%, 3.14-15.24% and 24.22 to 67% respectively. Physical characterization was done by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD). No interaction was observed in between drug and polymer and crystallinity of drug was not changed in drug loaded nanoparticles. In-vitro release study of DNPs showed diffusion controlled release. Bioavailability study of batch CS9 was studied in rabbit eye and compare to 5-FU solution. 5-FU level was significantly higher in aqueous humor of rabbit eye. Ocular tolerance was studied in the eye of New Zealand rabbits and tested formulation was non-irritant with no sign of inflammation.

Non-invasive monitoring of commonly used intraocular drugs against endophthalmitis by Raman spectroscopy.

To develop a non-contact and non-invasive method for quantification of the local concentration of certain antibiotic and antifungal drugs in the eye. An integrated CCD-based Raman spectroscopic system designed specifically for ophthalmic applications was used to non-invasively detect the presence of ceftazidime and amphotericin B in ocular media. Specific Raman signatures of the above named drugs were determined for various concentrations that were injected through a needle in the aqueous humor of rabbit eyes in vivo. Raman spectra were subsequently acquired by focusing an argon laser beam within the anterior chamber of the eye. Compared to ocular tissue, unique spectral features of ceftazidime appeared near 1,028, 1,506, 1,586, and 1,641 cm(-1). Amphotericin B exhibited its characteristic peaks at 1,156.5 and 1,556 cm(-1). The amplitude of the spectral peak corresponding to these drugs (acquired by 1 second exposure time and 25 mW of laser power) were determined to be linearly dependent on their local concentration in the anterior chamber of the eye. Raman spectroscopy may offer an effective tool to non-invasively assess the local concentration of the delivered drugs within the ocular media. This technique potentially could be used to investigate the pharmacokinetics of intraocular drugs in vivo either from a releasing implant or a direct injection.

Spectroscopic detection of a therapeutic drug in the mammalian eye

We have developed a novel and fast ocular spectrometer and demonstrated its ability to perform real time spectroscopic measurements in the mammalian eye. The instrument comprises a hand-held sensor head which, when placed on the cornea, effectively turns the anterior chamber into a cuvette. This sensor head is connected to the rest of the instrument via two optical fibers. Ex vivo and in vivo evaluation demonstrated the ability of the instrument to detect compounds via both absorption and fluorescence spectroscopy. In vivo evaluation demonstrated the ability of the instrument to detect brimonidine in the cornea and aqueous humor of rabbit eyes.

Nitric oxide levels in aqueous humor after lens extraction and poly(methyl methacrylate) and foldable acrylic intraocular lens implantation in rabbit eyes

Purpose To measure the nitric oxide (NO) level in the aqueous humor of rabbit eyes after common cataract surgery techniques. Setting Department of Ophthalmology, Military Medical University, and Department of Immunology and Immunotoxicology of Respiratory System, Clinic of Pneumonology and Allergology, Medical University of Łódź, Łódź, Poland. Methods Thirty rabbits were randomly divided into 4 groups, and surgery was performed as follows: Group 1, extracapsular cataract extraction (ECCE) and poly(methyl methacrylate) (PMMA) intraocular lens (IOL) implantation; Group 2, ECCE; Group 3, phacoemulsification and foldable acrylic IOL implantation; Group 4, phacoemulsification. The aqueous humor specimens were collected on days 1, 3, and 5. Nitric oxide measurements were performed indirectly with a spectrophotometer. The NO level in the aqueous humor collected before surgery served as a control group. Results In the physiological condition, the concentration of NO was 26.52 nm/dL ± 4.86 (SD). Postoperatively, the highest NO level in each groups was as follows: Group 1, 46.26 ± 5.23 nm/dL on day 3; Group 2, 72.46 ± 6.94 nm/dL on day 1; Group 3, 41.04 ± 7.31 nm/dL on day 1; and Group 4, 32.67 ± 2.84 nm/dL on day 3. Significantly higher levels of NO (P < .05) were observed in Groups 1 and 2 at each follow-up day; in Group 3, on days 1 and 3; and in Group 4, on day 3. Conclusions Techniques of cataract extraction caused the NO level to increase; however, phacoemulsification with a foldable IOL caused the smallest release of NO. Rapid postoperative NO changes may theoretically contribute to blood−aqueous barrier breakdown secondary to cataract surgery.

Noninvasive Detection of Ganciclovir in Ocular Tissue by Raman Spectroscopy: Implication for Monitoring of Drug Release

Treatment of various pathological conditions in ophthalmology, such as cytomegalovirus (CMV) retinopathy and endophthalmitis, requires a local drug intervention rather than a systemic approach. Accurate knowledge of intraocular drug concentration can permit the ophthalmologist to maintain drug levels within the therapeutic levels necessary for an optimal prognosis, while preventing or minimizing toxicity associated with drug overdose. To develop a noninvasive/noncontact method for quantification of the local concentration of ganciclovir in the ocular media. An integrated CCD-based Raman spectroscopic system designed specifically for ophthalmic applications was used to noninvasively detect the presence of ganciclovir in the ocular media. Various known concentrations of ganciclovir were injected into the aqueous humor of rabbit eyes in a pilot study, in vivo. Raman spectra were then acquired by focusing an argon laser beam within the anterior chamber of the eye. The specific Raman signature of ganciclovir was assessed at several concentrations. Spectral features unique to ganciclovir were identified and distinguished from those of ocular tissue. The amplitudes of the spectral peaks corresponding to ganciclovir exhibited a linear dependence on the local concentration of the drug in the anterior chamber of the eye. Raman spectroscopy may offer an effective tool for the noninvasive assessment of the local concentration of ganciclovir in the ocular media. This technique offers the potential to determine both the amount and the rate of the drug release from implants designed to deliver antiviral drugs locally within the eye. The availability of such data could enable the ophthalmologist to improve treatment efficacy by avoiding premature or late surgical replacement of the implants.

Effects of lipid composition on the transcorneal penetration of liposomes containing disulfiram, a potential anti-cataract agent, in the rabbit.

We previously demonstrated that topical treatment with disulfiram (DSF) prevented the development of cataracts in sodium selenite-injected rat pups. In biological systems, DSF is rapidly reduced to diethyldithiocarbamate (DDC), a potent antioxidant. In this study, we investigated the effect of altering the lipid composition of liposomes containing DSF on the transcorneal transit of DDC. Liposomes containing DSF were prepared with various molar ratios of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and cetylpyridinum chloride (CPC) by reverse-phase evaporation. Liposomes with a DMPC to DPPC molar ratio of 5:5, examined by differential scanning calorimetry, had the highest enthalpy of transition and the presence of one molar ratio of CPC further enhanced the enthalpy value. The addition of bovine serum albumin or a homogenate of rabbit cornea to the incubation buffer resulted in the release of DDC, but not DSF from the liposomes. The amount of DDC present in the aqueous humor of rabbit eyes following topical administration increased with increase in DMPC to DPPC ratios and was also enhanced by the addition of CPC to the liposomes. The results of this study suggest that liposome formulations are effective for transcorneal drug delivery of anticataract agents such as DSF. DSF in liposomes consisting of DMPC, DPPC, and CPC with a molar ratio of 8:2:1 may be a potential drug formulation for the prevention and/or treatment of cataracts.

The pharmacokinetics and effects of diltiazem in rabbits.

To investigate the effect of diltiazem on wound healing after the creation of conjunctival flaps in rabbit eyes. Also, to investigate the pharmacokinetics of diltiazem in rabbits after subconjunctival and topical administration. For the histopathological study, a limbal-based flap was prepared and diltiazem was injected subconjunctivally for five days after the surgery. The rabbits were euthanised 20 days after surgery. The effectiveness of diltiazem on wound healing was evaluated by histopathological examination and measurement of the thickness of subconjunctival fibrous tissue. For the pharmacokinetic study, diltiazem was applied topically or injected subconjunctivally. Aqueous paracenteses were performed 0.5, 1, 2, 4 hours thereafter. The histopathological study found no difference in thickness of the subconjunctival fibrous tissue in control and diltiazem-treated eyes. No significant toxicity was observed in eyes treated with diltiazem. The peak aqueous concentration was 3.8 +/- 0.4 microg/ml after topical application and 15.3 +/- 1.1 microg/ml after subconjunctival injection. The peak aqueous concentration was achieved 1/2 hours after administration in both cases. Diltiazem did not appear to affect wound healing at the dose tested. Topical and subconjunctival diltiazem successfully penetrated the aqueous humor of rabbit eyes.

The effect of antiglaucoma drugs on rabbit aqueous humor proteins determined by gel electrophoresis.

Previous studies indicate that there may be differences in the protein composition of the aqueous humor in normal and glaucomatous human eyes. These differences in protein composition and concentration may be due to the topical antiglaucoma medications used to treat glaucoma. These differences should be distinguished from any possible protein composition changes due to glaucoma. In order to study the effects of topical antiglaucoma medications on aqueous humor protein composition, we analyzed the aqueous humor of rabbit eyes topically treated with various antiglaucoma medications (timolol, pilocarpine, and dipivefrin). One eye of each rabbit was treated with the experimental drug, and the fellow eye was treated with saline solution (control). Thirty-six aqueous humor samples from 18 rabbits were obtained after 24 hours and 36 samples were obtained from 18 additional rabbits after 7 days of topical drug treatment. The protein composition of the aqueous humor samples was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Significant protein differences in aqueous humor samples were found between the eyes treated with timolol and the control eyes, whereas no significant differences were found between the eyes treated with pilocarpine or dipivefrin and the controls. These protein differences ranged from 14,000 to 18,000 daltons in molecular weight.

Aqueous and Vitreous Concentration of Mitomycin C by Topical Administration after Glaucoma Filtration Surgery in Rabbits

The authors investigated the aqueous and vitreous pharmacokinetics of mitomycin after postoperative topical administration in rabbits. Filtration surgery was performed in one eye of each rabbit. On the first postoperative day, mitomycin solution (0.4 mg/ml) was administered by either topical drop or cellulose sponge in both eyes of each rabbit. Aqueous and vitreous paracenteses were performed at 0.25, 0.5, 1, 2, 3, and 4 hours thereafter. Three rabbits were tested at each time interval for each type of administration. After topical drop administration, the peak aqueous concentration was 0.03 +/- 0.02 microgram/ml (mean +/- standard error) in surgical eyes and 0.06 +/- 0.03 microgram/ml in control eyes. After sponge administration, the peak aqueous concentration was 0.10 +/- 0.03 microgram/ml in surgical eyes and 0.08 +/- 0.04 microgram/ml in control eyes. Peak aqueous concentrations from drop and sponge applications were achieved at 1 to 2 hours after administration. Vitreous levels were well below the concentration known to cause retinal toxicity at all time intervals tested. Postoperative topical administration of mitomycin was successful in delivering mitomycin into the aqueous humor of rabbit eyes. Alternative methods of mitomycin application from the currently popular intraoperative administration may be beneficial in situations in which mitomycin delivery across intact conjunctiva may be desirable.

Evaluations on the Usefulness of Viscous Agents in Anterior Segment Surgery. II. Effect on Intraocular Pressure and Clearance from the Anterior Chamber

Various viscous agents in anterior segment surgery were replaced with the aqueous humor of rabbit eyes. The effects of these agents on intraocular pressure (IOP) and their clearance from the anterior chamber were studied. Furthermore, the degradation of these agents in ocular and hepatic tissues was investigated. The IOP after the injection of a 1% solution of sodium hyaluronate with a molecular weight of 790 x 10(3) (Na-HA (790 x 10(3), 1%], fell to the normal level after 12 hours; however, after a 1% solution of sodium hyaluronate with a molecular weight of 2160 x 10(3) (Na-HA (2160 x 10(3), 1%], a 2% solution of methylcellulose with a molecular weight of 86 x 10(3) (MC (86 x 10(3), 2%] or a 30% solution of sodium chondroitin sulfate with a molecular weight of 30 x 10(3) (Na-Chs (30 x 10(3), 30%], the return of IOP to normal level required 24 hours. Six hours after the injection of MC (86 x 10(3), 2%), the IOP was significantly higher than those of other agents. Na-Chs (30 x 10(3), 30%) and Na-HA (790 x 10(3), 1%) were eliminated from the anterior chamber within 12 and 24 hours, respectively. Na-HA (2160 x 10(3), 1%) and MC (86 x 10(3), 2%) required 72 hours to be eliminated from the anterior chamber. The degradation of Na-HA was detected in extracts of the iris, corneo-scleral junction, and liver. The degradation of Na-Chs was also detected in the liver extract, but the degradation of MC was not detected. On the basis of these findings, and the results in our previous report with respect to the efficacy of the viscous agents, it is concluded that Na-HA is the best viscous agent to be used in anterior segment surgery.

Leukotrienes levels in the aqueous humor following experimental ocular trauma.

Using radioimmunoassay technique, levels of leukotriene B4 and C4 (LTB4 and LTC4) in the aqueous humor of rabbit eyes were measured following experimental nonpenetrating ocular trauma. Slit lamp examination showed a time-dependent increase of flare, cells, and fibrin in the anterior chamber of the traumatized eyes. Polymorphonuclear (PMN) leukocyte influx into the aqueous humor was not seen at 6 hours but increased significantly in traumatized eyes after 12 hours. No inflammatory cells were observed in control eyes. Histopathologic studies demonstrated injuries of the ciliary body, ruptures of the retina and choroid, with intraocular hemorrhage. LTB4 values peaked at 6 hours, prior to PMN cell infiltration and remained significantly higher than controls, which remained undetectable at all intervals after injury. LTC4 values also peaked by 6 hours in the traumatized eyes. These data demonstrate that elevations in LTB4 and LTC4 are associated with blunt trauma, and this precedes PMN cell infiltration. Leukotrienes may play a role in the early inflammatory response following concussive ocular injuries.

Determination of metipranolol and desacetylmetipranolol in aqueous humor of rabbit eye by gas chromatography with electron-capture detection

Determination of metipranolol and desacetylmetipranolol in aqueous humor of rabbit eye by gas chromatography with electron-capture detection