Rabbit Aqueous Humor Research Articles

Secretoneurin in the human aqueous humor and the absence of an effect of frequently used eye drops on the levels

Secretoneurin (SN) was detected in the aqueous humor (AH) of patients treated topically with tobramycine eye drops alone or tobramycine and cyclosporine A, tobramycine and diclofenac or tobramycine and rimexolone. The levels of the peptide were found to be higher in the uninflamed human than in the rabbit aqueous humor which may be the result of species differences and/or age-related circumstances. Furthermore, they are approximately one hundred times higher than those of classical neuropeptides indicating release from nerve fibers and/or secretion from non-pigmented ciliary epithelium cells. Despite a slight tendency by rimexolone to decrease the levels, there was no significant effect seen for either of the drops. It must be considered that aminoglycosides are known to have toxic side effects and that they can influence the levels of SN which may be not diminished by low topical doses of corticosteroids or nonsteroidal antiinflammatory drugs. The high levels of the peptide are of relevance and may indicate a significant role of secretoneurin in the anterior segment of the eye. This should encourage performing functional studies.

Effect of dexamethasone on NO and Ca2 + levels in rabbit aqueous humor

目的 观测兔眼球结膜下注射地塞米松后其眼压(P)、房水流畅系数(C)及房水中一氧化氮(NO)浓度和Ca2 +含量的变化,探讨NO 、Ca2 +在皮质类固醇性青光眼(GIG)发病中的可能作用.方法隔日定时给新西兰幼龄白兔双眼球结膜下注射地塞米松0.5 mg,共15次,30d,隔日定时测定双眼眼压及C值,实验结束时抽取房水测定房水中NO及Ca2+浓度.结果地塞米松使兔眼眼压升高(P＜0.05)、C值及房水中NO浓度下降(P＜0.05);但对兔眼房水中Ca2+的含量无明显影响.结论眼局部长期应用地塞米松可诱导高眼压,引起房水中NO含量显著降低,提示NO参与了GIG的发病。

Dynamic changes of prostaglandin E2 and protein level in rabbit aqueous humor following penetrating keratoplasty

目的 通过对穿透角膜移植术后房水前列腺素E2(PGE2)和蛋白质量浓度的测定,探讨PGE2对炎症反应和免疫反应的作用.方法42只健康新西兰白兔,按穿透角膜移植术的类型和处理方法不同,随机分为6组,分别于术后第10、20、30、40d抽取各组动物的房水,用放射免疫法检测PGE2质量浓度,用紫外分光光度计法测定蛋白质量浓度.结果自体移植组术后10d房水PGE2质量浓度高于对照组;同种异体移植组术后10、20d房水PGE2质量浓度高于对照组,1只兔眼于术后20d出现排斥反应;异种移植组术后10、20、30d房水PGE2质量浓度均高于对照组,有6只兔眼于观察期内出现明显排斥反应;异种移植双氯芬酸钠和地塞米松(DFNa+Dex)治疗组术后10d房水PGE2质量浓度低于异种移植Dex治疗组(P＜0.05).房水蛋白质量浓度的变化情况与PGE2相一致.结论PGE2与穿透角膜移植术后的炎症反应和免疫排斥反应密切相关.Dex可抑制异种穿透角膜移植术后的排斥反应.DFNa对Dex有协同作用,可减少Dex的用量。

Aqueous penetration of gatifloxacin and levofloxacin into the rabbit aqueous humor following topical dosing.

The aqueous penetration of the commercial preparations of the fluoroquinolone antibiotics ofloxacin, ciprofloxacin, levofloxacin, and gatifloxacin were compared following topical dosing in a rabbit model. Levofloxacin achieved the highest aqueous concentrations, with a mean aqueous level of 4.8014 microcg/mL (p = 0.002, p = 0.00002, p = 0.015.) Ofloxacin (2.5136 microcg/mL) and gatifloxacin (2.4817 microcg/mL) achieved statistically equal aqueous concentrations (p = 0.479). Ciprofloxacin reached the lowest levels in the aqueous humor (0.9616 microcg/mL, p = 0.00002, 0.00004, 0.008). Gatifloxacin alone achieved concentrations in excess of the MIC90s of gram-positive pathogens of concern.

Rapid determination of nimesulide in rabbit aqueous humor by liquid chromatography

A rapid method was developed for quantification of nimesulide (methanesulfonamide, N-[4-nitro-2-phenoxyphenil]) in rabbit aqueous humor. The analyses were performed by high-performance liquid chromatography using a C 18 reversed-phase column (Ultracarb ODS) with UV detection at 300 nm. The mobile phase consisted of acetonitrile-water containing 1% triethylamine (TEA) adjusted to pH 3.2 with orthophosphoric acid. The retention time was 4.5 min. A simple pre-treatment with acetonitrile was used to deproteinize aqueous humor samples. The limit of quantitation was 50 ng/ml. The recovery was over 90%. The relationship between peak areas and concentration was linear over the range between 0.05 and 2.5 μg/ml, with r 2 values over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of nimesulide.

PLGA microspheres for the ocular delivery of a peptide drug, vancomycin using emulsification/spray-drying as the preparation method: in vitro/in vivo studies

The aim of this study was an in vitro/in vivo investigation on poly(lactide-co-glycolide) (PLGA) microspheres as carriers for the topical ocular delivery of a peptide drug vancomycin (VA). The microspheres were prepared by an emulsification/spray-drying technique that can be proposed as an alternative to the double emulsion method for preparation of peptide-loaded microparticles. The drug encapsulation efficiencies were close to the theoretical values (84.2–99.5%); the average particle size, expressed as d vs, was about 11 μm. The microspheres were able to modulate the in vitro drug release of VA with a behavior dependent on their composition: the highest drug content corresponded to the highest release rate. In vivo studies were carried out by assessing the pharmacokinetic profile of VA in the aqueous humor of rabbits after topical administration of aqueous suspensions of microspheres. High and prolonged VA concentrations and increased AUC values (2-fold) with respect to an aqueous solution of the drug were observed. Increasing the viscosity of the microsphere suspension by addition of a suspending-viscosizing agent (hydroxypropylcellulose) did not produce an increase of the ocular bioavailability. PLGA microspheres can be proposed as a system for ocular delivery of peptide drugs.

Improvement of the ocular bioavailability of timolol by sorbic acid

The ocular bioavailability of timolol increased in sorbic acid solution due to ion pair formation. Its octanol/water partition coefficient also increased, suggesting the formation of a more lipophilic complex. The concentration of timolol in rabbit aqueous humor was determined after instillation of timolol ophthalmic solution containing sorbic acid. When the molar ratio of sorbic acid to timolol was two or higher, the concentration of timolol in the aqueous humor was higher than with timolol alone. In the presence of sorbic acid the maximal aqueous humor concentration and the area under the curve were more than two-fold higher than those of Timoptol ®, a timolol maleate ophthalmic solution, and similar in value to TIMOPTIC-XE ®, a gel-forming ophthalmic solution. To investigate the transcorneal absorption mechanism, in vitro permeation profiles across the intact and de-epithelialyzed cornea were analyzed on the basis of the bilayer diffusion model. The partition coefficient in the epithelium was about twice as high in the presence of sorbic acid than with timolol alone, although the diffusion coefficient in the epithelium did not change. We conclude that the improved ocular bioavailability in the presence of sorbic acid is due to increased partitioning of timolol in the corneal epithelium.

Bremazocine increases C-type natriuretic peptide levels in aqueous humor and enhances outflow facility.

A relatively selective agonist of kappa opioid receptors (KOR), bremazocine (BRE), lowers intraocular pressure in rabbits, in part, by increasing natriuretic peptide levels in aqueous humor and by enhancing total outflow facility (TOF). Natriuretic peptide (NP) levels [atrial NP (ANP), brain NP (BNP), and C-type NP (CNP)] were measured in aqueous humor of rabbits either by radioimmunoassay or enzyme immunoassay. TOF was determined in rabbits by two-level constant pressure perfusion of the anterior chamber. Experimental regimens included topical treatment with BRE in the presence or absence of KOR antagonist (norbinaltorphimine), protein kinase C inhibitor (chelerythrine), and natriuretic peptide receptor antagonist (isatin). The rank order of basal NP levels in aqueous humor of rabbits was BNP CNP > ANP. Topical administration of BRE (1-100 microg) caused dose-related elevations of CNP levels in aqueous humor that were inhibited by topical pretreatment with either norbinaltorphimine (100 microg, bilaterally) or chelerythrine (10 microg, bilaterally). Topically administered BRE (100 microg) also elevated levels of ANP and BNP in aqueous humor and evoked an 80% increase in TOF. The increase in TOF was antagonized by topical pretreatment with either norbinaltorphimine (100 microg, bilaterally) or isatin (100 microg, bilaterally). Bremazocine induced an increase in NP (ANP, BNP, and CNP) levels and TOF in rabbits by activating KOR. The increase in CNP levels elicited by BRE was inhibited by norbinaltorphimine and chelerythrine; therefore, this event is most likely mediated by a KOR-linked activation of protein kinase C. These data provide evidence that the increase in TOF elicited by BRE was mediated by a KOR-activated paracrine effect of NPs on tissues within ocular outflow tract(s).

Bioavailability and anticataract effects of a topical ocular drug delivery system containing disulfiram and hydroxypropyl-beta-cyclodextrin on selenite-treated rats

Purpose. To test the effect of aqueous eye drops containing a high concentration of disulfiram (DSF) in a cyclodextrin-based drug delivery system. This system increases both the drug solubility in aqueous eye drops and the permeability of drug into the rabbit eye, by the formation of a drug-cyclodextrin inclusion complex, and so enhances the ocular bioavailability and anti-cataract effect of DSF.Methods. The DSF and hydroxypropyl-β-cyclodextrin (HPβCD) inclusion (DSF/HPβCD) was studied using solubility methods, IR spectra and X-ray diffraction patterns. Suitable formulations for DSF eye drops were first identified by a trans-corneal penetration experiment in vitro. Finding a new p-bromophenacyl bromide (p-BPB) derivative reagent for diethyldithiocarbamic acid (DDC), which was a metabolite of DSF, allowed precise determination of the contents of DSF in aqueous humor. The ocular bioavailability was calculated by a transcorneal experiment of DSF in vivo. The lens opacity of a selenite-induced cataract in rat pups was monitored using a slit lamp with an anterior eye segment analysis system.Results. The formation of DSF/HPβCD inclusion and the addition of hydroxypropylmethylcellulose (HPMC), as a penetration enhancer, played very important roles in increasing the ocular bioavailability of DSF. DSF eye drops, with a formulation of 1.26% (w/v) DSF/HPβCD inclusion, 0.01% (w/v) HPMC, 0.005% (w/v) benzalkonium chloride and 0.9% (w/v) sodium chloride, inhibited the onset of selenite-induced cataracts effectively.Conclusions. The cyclodextrin-based drug delivery system enhances both the solubility of DSF in aqueous eye drops and permeability of the drug into the rabbit eye. DSF ocular bioavailability in rabbit aqueous humor exceeded those reported for the DSF ophthalmic preparation. DSF eye drops effectively prevent the development of selenite-induced cataracts.

Calcium and magnesium enhance the production of Pseudomonas aeruginosa protease IV, a corneal virulence factor.

The effect of calcium and magnesium on protease IV production during the growth of Pseudomonas aeruginosa was investigated. Strain PA103 was grown to stationary phase in medium containing various concentrations of either calcium or magnesium. Culture supernatants were concentrated, standardized relative to cell density, and the pyoverdine concentrations were measured. Overall extracellular protease activity and specific protease IV (lysine endoproteinase) activity were measured with or without TLCK, a serine protease inhibitor effective against protease IV activity. Protease IV activity was also observed by casein zymography. Calcium and magnesium were quantified in the corneas and aqueous humor of rabbits that were inoculated intrastromally with strain PA103. Pyoverdine production was not significantly different in cultures grown in medium with added calcium or magnesium, but extracellular caseinase activity increased in these cultures. Susceptibility of caseinase activity to TLCK inhibition and a specific assay for protease IV indicated that protease IV activity increased in cultures grown in calcium or magnesium. Casein zymography supported the observation that protease IV activity increased in the cultures with added calcium and magnesium. Addition of calcium or magnesium to the protease IV-specific assay had no effect on the catalytic activity of pure protease IV. Infection of rabbit corneas with PA103 did not change the magnesium concentration in either corneas or aqueous humor, but significantly increased the concentration of calcium in corneas. These results indicate that calcium and magnesium enhance the production of protease IV, but not pyoverdine production. Calcium increases in the cornea following infection with P. aeruginosa could favor production of protease IV.

Elevated levels of secretoneurin in the rabbit aqueous humor in response to formaldehyde irritation.

Secretoneurin, a 33-amino-acid neuropeptide, is generated by proteolytic processing of secretogranin II, which belongs to the chromogranin family. This study aimed to investigate whether secretoneurin is present in the uninflamed rabbit aqueous humor and whether it is released in response to treatment with topical formaldehyde, an agent known to release sensory peptides originating from the trigeminal ganglion. Blood samples and aqueous humor of eyes pretreated with neutral formaldehyde and untreated controls were analyzed for secretoneurin immunoreactivity by a highly sensitive radioimmunoassay. Furthermore, the molecular form of the secretoneurin immunoreactivity was characterized by gel filtration high-performance liquid chromatography (HPLC). In the blood, secretoneurin levels were found to be below the detection limit of 2 fmol/100 microl. In the aqueous humor, secretoneurin-immunoreactivity was detected in moderate but significant amounts. The mean concentration of secretoneurin was 8.1 (+/-0.34) fmol/100 microl in controls and 7.8 (+/-0.1) fmol/100 microl 15 min after formaldehyde application. Thirty minutes after treatment, the secretoneurin levels were significantly elevated by 63%. Gel filtration HPLC revealed that the main molecular form corresponded to the free peptide secretoneurin. The neuropeptide secretoneurin has been detected in the anterior segment of the eye for the first time. The elevation of secretoneurin in formaldehyde-treated eyes may be induced by an enhanced release from the iris/ciliary body complex, as formaldehyde is known to provoke neurogenic inflammation in the anterior segment via release of sensory peptides originating from the trigeminal ganglion. This is why our results indicate a sensory origin of secretoneurin in the eye.

Somatostatin is an immunosuppressive factor in aqueous humor.

To detect the presence of somatostatin (SOM) in normal aqueous humor and to characterize its immunosuppressive activity. Fresh rabbit aqueous humor was assayed for SOM by competitive ELISA. Primed T cells stimulated through their T-cell receptor (TCR) were treated with SOM at concentrations that ranged the level of SOM found in normal aqueous humor. The T cells were assayed for proliferation, lymphokine production, and immunosuppressive activity. Normal rabbit aqueous humor contained 196 +/- 45 pg/mL (10(-10) M) of SOM. At concentrations between 10 and 300 pg/mL, SOM suppressed IFN-gamma production by TCR-stimulated primed T cells in culture. Frozen and thawed aqueous humor depleted of SOM no longer suppressed IFN-gamma production by the TCR-stimulated primed T cells. SOM induced TGF-beta but not IL-4 production, nor did it suppress proliferation by TCR-stimulated primed T cells. The SOM-treated T cells functioned as regulatory T cells, and this regulatory activity was neutralized by anti-alpha-MSH antibodies. Furthermore, SOM induced alpha-MSH production by the TCR-stimulated primed T cells. SOM is present in aqueous humor and contributes to the immunosuppressive activity of aqueous humor. Moreover, SOM induces the production of the potent immunomodulating factor alpha-MSH by TCR-stimulated primed T cells through which the SOM-treated T cells suppress other T cells. Thus, SOM can contribute to the ocular immunosuppressive microenvironment by promoting the production of immunosuppressive cytokines and inducing the activation of regulatory T cells.

Vitamin C Metabolomic Mapping in the Lens with 6-Deoxy-6-fluoro-ascorbic Acid and High-Resolution19F-NMR Spectroscopy

Metabolomics, or metabolic profiling, is an emerging discipline geared to providing information on a large number of metabolites, as a complement to genomics and proteomics. In the current study, a fluorine-labeled derivative of ascorbic acid (F-ASA), a major antioxidant- and UV-trapping molecule in the aqueous humor and the lens, was used to investigate the extent to which the lens accumulates potentially toxic degradation products of vitamin C. Human lens epithelial cells (HLE-B3) and rat lenses were exposed to hyperglycemic or oxidative stress in vitro or in vivo and probed for accumulation of F-ASA, fluoro-dehydroascorbate (F-DHA), fluoro-2,3-diketogulonate (F-DKG), and their degradation products in protein-free extracts, by proton-decoupled 750-MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy. F-ASA and F-DHA were taken up into HLE B-3 cells by an Na(+)-dependent transporter. Their uptake was unexpectedly only slightly affected by hyperglycemia in vitro, unless glutathione was severely depleted. Glycemic stress catalyzed oxidation of F-ASA into a single novel F-compound at -212.4 ppm, whereas F-DHA and F-DKG were the major degradation products observed after GSH depletion. In contrast, F-ASA uptake was markedly suppressed in diabetic cataractous rat lenses, which accumulated both the F-DHA and the -212.4-ppm compound. In an unexpected finding, the latter formed only from F-ASA and not F-DHA or F-DKG, suggesting a novel pathway of in vivo F-ASA degradation. Both the cells and the intact rat and human lenses were permeable to several advanced F-ASA and F-DHA degradation products, except F-DKG. The unknown compound at -212.4 ppm was the only F-ASA degradation product that spontaneously formed in rabbit aqueous humor upon incubation with F-ASA. These studies suggest the existence of a novel ascorbic-acid-degradation pathway in the lens and aqueous humor that is influenced by the nature of the oxidant stress. Under similar culture conditions, intact lenses are more prone to hyperglycemia-mediated oxidant stress than are lens epithelial cells, but both are permeable to various F-ASA degradation products, the structure and biological roles of which remain to be established.

Presence of diadenosine polyphosphates in the aqueous humor: their effect on intraocular pressure.

Adenine dinucleotides are present in many biological systems and may serve as physiological regulators of processes such as neurotransmitter release, vascular tone or corneal hydration. The presence of diadenosine polyphosphates was investigated in New Zealand White rabbit aqueous humor. Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) were identified and quantified in the aqueous humor with concentrations of 0.34 +/- 0.1 and 0.08 +/- 0.01 microM, respectively. The effects of topical corneal application of diadenosine pyrophosphate (Ap2A), diadenosine triphosphate (Ap3A), Ap4A, and Ap5A on intraocular pressure in rabbits were also studied. Ap2A, Ap3A, and Ap5A increased intraocular pressure with threshold doses of approximately 0.1 to 1.0 micro g. 10 microl(-1). Ap4A decreased intraocular pressure with an IC50 value of 0.12 micro g. 10 microl(-1) (or 0.13 nmol). Cross-desensitization studies suggested the activation of a P2X receptor for the hypotensive effect of Ap4A and a P2Y receptor in the case of Ap5A. The ATP receptor antagonists (all 100 micro g. 10 microl(-1)), pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suramin, and reactive blue 2 (RB-2) alone had no effect on intraocular pressure but attenuated responses to diadenosine polyphosphates by approximately 80%. It is concluded that Ap2A, Ap3A, and Ap5A increase intraocular pressure, and Ap4A decreases intraocular pressure via mechanisms that involve P2 receptors, and that Ap4A present in aqueous humor may serve to regulate intraocular pressure. Furthermore, we suggest that topical application of Ap4A to the cornea has therapeutic potential for lowering intraocular pressure, a major risk factor for glaucoma.

Analysis of an anti-inflammatory steroidal drug, difluprednate, in aqueous humor by combination of semi-micro HPLC and column switching method

A specific and sensitive method for the determination for difluprednate (DFBA) and its metabolite (deacetylated DFBA, DFB) in aqueous humor was developed. DFBA and DFB were initially absorbed on a Pinkerton-type column, then analyzed by high-performance liquid chromatography using a semi-micro column after column switching. Under the optimized conditions, calibration curves for DFBA and DFB showed good linearity over the range of 1.0–50 and 0.5–50 ng/ml, respectively. We applied the method to the analysis of DFBA and DFB in rabbit aqueous humor, and found that DFBA in rabbit aqueous humor 1 h after instillation of 0.002% DFBA ophthalmic emulsion was not detected, but DFB was present at the concentration of 4.3±3.1 ng/ml.

Safety Studies on Collagen Viscoelastic Substance as an Auxiliary Agent in Anterior Segment Surgery: Assays of Anti-collagen Antibodies in Blood and Residual Concentration of Collagen in the Anterior Chamber of Rabbits

Alkali-soluble collagen solution was assessed as a possible viscoelastic substance in anterior segment surgery, in terms of its safe applicability by assaying antigenicity, disappearance rate of collagen from the anterior chamber, and histopathological effect on the corneal tissue. The aqueous humor of rabbits was replaced with collagen solution three times. Then, follow-up clinical examinations with hand-slitlamp-microscopy, tonometry, pachymetry, and specular microscopy as well as ophthalmic histopathological examination were performed. Remaining collagen concentration in the aqueous humor at 1, 3, 5, 8, 24 and 72 hours after injection was determined to evaluate the disappearance rate of collagen from the aqueous humor with time. In vivo effects of chemical modification of collagen and buffer concentration on the corneal tissue were further studied by using transmission electron microscope (TEM) to find an optimum condition for collagen application. Neither anti-collagen antibody formation, nor inflammatory responses in the anterior segment and systemic symptoms were observed even after 3 injections of collagen solution, except for 1 case which showed corneal opacity. As much as 97.4% of the collagen injected into the anterior chamber disappeared from the eyeball. On the basis of TEM findings, succinylated collagen in diluted phosphate buffer seems to be superior to alkali soluble collagen in terms of corneal tissue protection. Collagen specifically prepared for this study showed no antigenecity and disappeared promptly from the anterior chamber. The optimal form of collagen in terms of corneal protection was discussed.

High-performance liquid chromatographic determination of 2-hydroxypropyl-γ-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence

A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-γ-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5–15 μg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.

Rapid high-performance liquid chromatographic assay of dorzolamide in rabbit aqueous humor.

A rapid high-performance liquid chromatographic method using a C(18) reversed-phase column (Hypersil ODS) with UV detection at 254 nm and a simple pre-treatment of samples is presented for the analysis of dorzolamide, a carbonic anhydrase inhibitor, in rabbit aqueous humor. A water solution containing 2% ZnSO(4) small middle dot7H(2)O was used to deproteinize aqueous humor samples. The mobile phase consisted of 7% CH(3)CN and 93% of a solution containing 1% TEA adjusted to pH = 3.5 with H(3)PO(4). Paracetamol was found to be a suitable internal standard. The standard curves were linear in the detection range. The precision and the accuracy were <5% for both intra- and inter-day assays.

Simultaneous determination of cloricromene and its active metabolite in rabbit aqueous humor by high-performance liquid chromatography

A rapid and simple method was developed for the simultaneous separation and quantification of cloricromene, a coumarine derivative, and its active metabolite, cloricromene acid, in rabbit aqueous humor. The analyses were performed by high-performance liquid chromatography using a C 18 reversed-phase column (Hypersil ODS) with UV detection at 318 nm. The mobile phase consisted of acetonitrile–water containing 1% triethylamine pH 3.5, adjusted with orthophosphoric acid. An acetonitrile gradient was necessary to achieve good separation within 13 min. Timolol was found to be a suitable internal standard. The retention times ranged from 5.72 to 11.25 min. A simple pre-treatment with acetonitrile containing 0.6% HClO 4 was used to deproteinize aqueous humor samples. The limit of quantitation ranged between 10 and 20 ng/ml. The recovery was >90%. The relationship between peak areas and concentration was linear over the range between 0.01 and 3.8 μg/ml, with r 2>0.99. The assay provided good reproducibility and accuracy for both analytes and proved to be suitable for pharmacokinetic studies of cloricromene.

Circadian variations of transforming growth factor-ß2 and basic fibroblast growth factor in the rabbit aqueous humor

Purpose. To compare levels of transforming growth factor-ß2 (TGF-ß2) and basic fibroblast growth factor (bFGF) in the ocular fluids of rabbits at various times of the day. Methods. Seventy-two young adult New Zealand albino rabbits were entrained to a daily 12-hour light (6 AM to 6 PM) and 12-hour dark cycle. Eight rabbits were sacrificed at each of 6 time points: 4 AM, 8 AM, noon, 4 PM, 8 PM, and midnight. Under an acute 24-hour constant dark condition, 8 additional rabbits were sacrificed at each of 3 time points in the accustomed light period (8 AM, noon, and 4 PM). Samples of aqueous humor and vitreous humor were collected from both eyes. Using enzyme immunoassay, samples from one eye were assayed for total TGF-ß2 and active form of TGF-ß2 and samples from the other eye were assayed for bFGF. Concentrations at various time points were analyzed using one-way ANOVA and post-hoc Bonferroni t-test for multiple comparisons. Results. Aqueous humor levels of total TGF-ß2, active TGF-ß2, and bFGF changed during the 24-hour period. Total TGF-ß2 and bFGF levels increased between 4 AM and 8 AM. Active TGF-ß2 level increased between 8 PM and midnight. Similar patterns of changes were found when considering aqueous humor samples collected under the acute constant dark condition. In the vitreous humor, total TGF-ß2, active TGF-ß2, and bFGF remained unchanged throughout the 24 hours. Conclusions. Endogenous circadian rhythms of total TGF-ß2, active TGF-ß2, and bFGF occur in the rabbit aqueous humor. These two extracellular signaling molecules may be associated with specific physiological functions in the anterior segment according to the time of the day.