Development of biodegradable controlled release scleral systems of triamcinolone acetonide.

PURPOSE: To develop scleral controlled-release-systems of triamcinolone acetonide (TA) based on biodegradable poly(lactide) (PLA). MATERIALS AND METHODS: (1) PLA microspheres containing TA were prepared by a single or double emulsification-solvent evaporation method. Morphology, size, effect of drug input and method of microsphere preparation on drug loading, and in vitro TA release of the microspheres were investigated. (2) Mini-tablets consisting of blank PLA-microspheres and TA (weight ratios of 1:1, 2:1, and 4:1, respectively) were developed and their release profile in vitro was evaluated. (3) The in vitro transscleral diffusion profile was evaluated by placing a PLA-TA (1:1) tablet in a donor chamber and measuring the TA concentration in a receptor chamber. Donor and receptor chambers were separated by rabbit sclera. (4) Two cadaver rabbit eyes received a 1:1 PLA-TA tablet episclerally, which was covered by a scleral patch. TA aqueous humor and vitreous concentrations were measured 5, 10, and 20 days post implantation. RESULTS: (1) Microsphere average size was 2 µm. The double emulsification method and increasing drug input led to an increase in drug loading and encapsulation. Sustained release of TA over several days from the microspheres in vitro was observed, with the rate of release being affected by their TA content. (2) TA exhibited sustained release profile from the PLA-TA tablets, with the rate of release being affected by the PLA:TA ratio. (3) TA could slowly cross the sclera tissue in vitro, with approximately 21% of the drug loaded in the donor compartment being diffused through the sclera in 45 days. (4) Following scleral administration of the PLA-TA mini-tablets, TA accumulated in the vitreous and aqueous humor of cadaver eyes. CONCLUSIONS: The PLA-TA microspheres and mini-tablets appear promising for the controlled transscleral delivery of TA and justify further investigation.

Ocular surface bacterial colonisation in sedated intensive care unit patients.

We investigated the time-dependent ocular surface bacterial colonisation of sedated patients hospitalised in an intensive care unit and aimed to evaluate whether proper topical antibiotic prophylaxis could prohibit corneal infection. The study lasted 12 months and included 134 patients undergoing sedation and mechanical respiratory support for various medical reasons. Patients hospitalised for less than seven days and those with pre-existing ocular surface pathology were excluded. All patients were examined on admission by inspecting the cornea for erosions. Followup examinations were performed each subsequent day. Cultures were also obtained from the conjunctival sac of both eyes on admission and every seventh day until the end of sedation. Standard laboratory techniques were used for isolation, identification and antibiotic susceptibility testing of bacteria. Antibiotic treatment for prophylaxis was administered accordingly. Analysis was carried out for 70 patients. Duration of sedation ranged from seven to 122 days. Fifty-four (77%) patients were colonised by at least one bacterial species other than normal flora within seven to 42 days. Multiple bacteria were isolated from 28 patients undergoing prolonged sedation. Prevalent isolates were Pseudomonas aeruginosa, Acinetobacter spp. and Staphylococcus epidermidis. Infectious keratitis was prohibited in all cases. Ocular surface of long-term sedated patients was found to be colonised by various bacterial species and their isolation was closely associated with the time period of hospitalisation. The results of this study suggest that the early identification of ocular surface bacteria colonisation and the administration of topical antibiotics for prophylaxis can prohibit corneal infection in these patients.

Pseudoexfoliation syndrome prevalence in Greek patients with cataract and its association to glaucoma and coronary artery disease.

PURPOSE: To investigate the prevalence of glaucoma and coronary artery disease (CAD) in patients with cataract and pseudoexfoliation (PEX) syndrome. METHODS: Cross-sectional study of 2140 consecutive patients with cataract admitted at the University Hospital of Patras, Greece, for cataract surgery. Only patients with senile cataract were included in this study. All patients underwent a complete ophthalmological examination that included slit-lamp evaluation with dilated pupil for PEX material in the anterior segment, intraocular pressure (IOP) measurements, and optic disc cup examination. They also underwent an evaluation for CAD by a cardiologist. CAD was considered present if a patient had a history of myocardial infarction, or ischaemia, or abnormal coronary angiography. The patients were classified into two groups: the PEX and the non-PEX group. RESULTS: One thousand and eighty-eight (50.8%) patients were men and 1052 (49.2%) were women. The overall prevalence of PEX syndrome was found to be 27.9% and it was found to increase with progressing age. Bilateral PEX was more frequent than unilateral PEX, with the percentage of bilateral PEX raising with progressing age. A total of 132 patients (22.1%) in the PEX group exhibited glaucoma, while in the non-PEX group only 2.5% suffered glaucoma. PEX was also found to be positively associated with the risk for CAD among subjects 50 years or older. No association between CAD and glaucoma was found. CONCLUSIONS: PEX syndrome constitutes a major glaucoma risk factor and a CAD risk factor. Patients with PEX should be informed and examined frequently as the risk is present throughout.

Evidence for oxidative stress in lens epithelial cells in pseudoexfoliation syndrome.

PURPOSE: To investigate the oxidative status in lens epithelial cells of patients with pseudoexfoliation (PEX) syndrome. METHODS: Lens capsule samples obtained during cataract surgery of patients with PEX syndrome and normal age-matched control subjects were examined for changes in the levels of glutathione (GSH), glutathione disulphide (GSSG), protein concentration, and lipid peroxidation. Concentrations of GSH, GSSG, lipid peroxidation, and protein concentration were determined by specific fluorescent assays. RESULTS: This study shows a 2.2- and 2.0-fold decrease in GSH and GSSG levels, respectively, in PEX lens epithelial lens compared with non-PEX lens epithelial cells, as well as a 2.5-fold increase in lipid peroxidation product malondialdehyde (MDA) levels. CONCLUSION: The increased MDA and decreased GSH levels indicate high oxidative stress. On the other hand, GSSG usually increases in cases of high-oxidative stress, but this is not always the case, as it may not always accumulate in cells. Our findings suggest a role for oxidative stress in the pathogenesis and the progression of PEX syndrome.

Immunization with specific polysaccharide antigen reduces alterations in corneal proteoglycans during experimental slime-producing Staphylococcus epidermidis keratitis.

PURPOSE: Staphylococcus epidermidis is a leading cause of bacterial keratitis associated with corneal damage. Corneal integrity is closely associated with matrix macromolecules, such as proteoglycans (PGs) and collagen. The aim of this study was to examine whether active immunization (AI) using a major immunogenic polysaccharide determinant of slime (20-kDa PS) as antigen, and passive immunization (PI) after administration of specific antibodies toward 20-kDa PS affect the distribution of PGs as well as corneal lesions in an experimental model of slime-producing S. epidermidis keratitis. METHODS: For AI, seven rabbits were immunized with 20-kDa PS, whereas for PI, seven rabbits received specific antibodies against 20-kDa PS. Lesions were graded clinically for a 21-day period. Levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by ELISA. The distribution of certain extracellular matrix PGs during corneal healing was analyzed immunohistochemically. RESULTS: Levels of specific anti-20-kDa PS antibodies in serum and aqueous humor obtained after either AI or PI were significantly higher as compared with those in the respective nonimmunized control groups (p<0.001). Clinical grading showed that both AI and PI rabbits had a significantly less corneal damage as compared with infected nontreated rabbits. Immunohistochemical analyses for PGs exhibited significant differences to the wounded regions as compared with noninfected corneal tissue. Accumulation of keratan sulfate PGs and decorin was observed in the corneal stroma of infected rabbits and of heparan sulfate PGs around the new-formed vessels. This phenomenon was significantly reduced in immunized animals in accordance with macroscopically decreased corneal damage observed in these animals. CONCLUSIONS: Results of this study suggest a key role of 20-kDa PS and its antibodies as prophylactic and therapeutic agents in keratitis caused by slime-producing S. epidermidis.

Collagen type IX and HNK-1 epitope in tears of patients with pseudoexfoliation syndrome.

Pseudoexfoliation syndrome (PEX) is an age-related condition, which may cause open-angle glaucoma and has increasing interest since it seems to affect additional human tissues, i.e., cardiovascular tissue, skin, and still lacks elucidated pathogenesis. Collagen type IX and HNK-1 epitope have been considered characteristic constituents of the aqueous humour of PEX patients, since their amounts were increased in PEX aqueous humour compared to normal eyes. Since it has been proposed that the initial manifestations of PEX syndrome occur in conjunctiva, the present study was undertaken to investigate the presence of the same antigens in tears of PEX patients and their possible use as the biochemical markers for early diagnosis. Tears of PEX patients and healthy individuals were subjected to western blotting analysis for various basement membrane components identified in aqueous humour. It was found that collagen type IX and HNK-1 epitope were present in tears, the amount of the former being increased 2.7 times compared to normal (P<0.05), surprisingly high as compared with total protein or lysozyme activity in tears, which were found to be increased in PEX patients about 25% with no statistical differences (P approximately 0.4). The results suggest the possible use of tears' collagen type IX for the diagnosis of PEX syndrome.

Glutathione and lipid peroxide changes in pseudoexfoliation syndrome.

PURPOSE: To investigate the oxidative status of the aqueous humor of patients with pseudoexfoliation (PEX) syndrome. METHODS: Aqueous humor samples obtained during cataract surgery of patients with PEX syndrome and normal age-matched control subjects were examined for changes in the levels of glutathione (GSH), glutathione disulfide (GSSG), and TBA reactive species (TBARS), products of lipid peroxidation. GSH, GSSG, and TBARS were determined by specific fluorescent assays. RESULTS: Compared to normal controls, PEX syndrome aqueous humor samples showed a decrease of up to 28% of GSH concentration, and GSSG was increased up to 23%. The ratio of GSH/GSSG was 1.7-fold decreased in PEX syndrome samples. TBARS levels were increased by 100% in the PEX aqueous humor samples as compared to the controls. CONCLUSIONS: High levels of GSSG and TBARS indicate high oxidative stress, as well as the decrease in the ratio of GSH/GSSG. Our findings suggest a role for oxidation stress in the pathogenesis and the progression of PEX syndrome.

Changes in HNK-1 epitope and collagen type IX in the aqueous humour of patients with pseudoexfoliation syndrome.

PURPOSE: To investigate alterations in the proteoglycan (PG) and glycosaminoglycan (GAG) content of the aqueous humour in patients with pseudoexfoliation syndrome (PEX). MATERIALS AND METHODS: Aqueous humor samples were obtained during cataract surgery from nineteen patients bearing PEX features and twenty-three age-matched normal controls. Protein and IgG were quantified densitometrically after their electrophoretic separation. Collagen type IX, 3-sulphoglucuronic acid (HNK-1 epitope), biglycan and heparan sulphate proteoglycans were detected in Western and dot blots by using specific monoclonal antibodies (MAbs). The immunochemical analysis was performed in native aqueous humour or after degradation of the glycosaminoglycans with chondroitinases. RESULTS: Degradation of the samples with chondroitinases ABC, AC and B revealed that, in the aqueous humour from PEX eyes, collagen type IX and biglycan had a more dermatan sulphate than did normal eyes. In addition, more HNK-1 epitope was observed in PEX eyes, which after similar enzymatic treatment was found to be located mainly in dermatan sulphate sequences. 3-sulphoglucuronic acid was a constituent of the GAG chains of the collagen type IX. We found that the electrophoretic mobility of the bands of collagen type IX and HNK-1 epitope was exactly the same in the aqueous humour of normal and PEX samples; both migrated as four bands at 120, 113, 92.6 and 56 kDa. The PGs bearing heparan sulphate were found only in normal samples. Other PGs were not detected. CONCLUSIONS: Because no significant difference was observed in the concentration of albumin and IgG in PEX and normal samples, the blood-aqueous barrier was probably not significantly compromised in PEX patients with cataract but without open-angle glaucoma. The results support the hypothesis that the pathogenesis of PEX can be linked to disturbed metabolism of GAGs and PGs.

Alterations in conjunctival cytology and tear film dysfunction in patients with beta-thalassemia.

PURPOSE: Patients with beta-thalassemia (beta-tha) represent a group with lifelong transfusion-dependent anemias. This study aimed to describe the conjunctival changes and tear film parameters in these patients. METHODS: A total of 52 patients (104 eyes) with beta-tha major and 22 normal control subjects (44 eyes) were studied during 1999 through 2000. Tear film break-up time (BUT), Schirmer test, rose Bengal staining, and cytologic evaluation of the conjunctival epithelium were performed in all subjects. The Papanicolaou and May-Grümwald-Giemsa staining procedures were performed on all smears. Patients and control subjects were compared for tear function parameters and conjunctival changes. RESULTS: The BUT, Schirmer test, and rose Bengal staining values were significantly lower (P < 0.001) in beta-tha patients than in control subjects. Keratinized cells were observed in conjunctival samples in 41% of patients, with a decrease in the number of goblet cells per slide in 64% of patients. In 9% of beta-tha patients, there were a slightly greater number of inflammatory cells than in control eyes. CONCLUSION: Ocular surface disorder of these patients was characterized by goblet cell loss and conjunctival squamous metaplasia. Our findings were correlated positively with the variable age. Epithelial damage by toxic reaction and disorder of tear quality and quantity are implicated as important factors in the pathogenesis of the ocular surface disease in beta-tha patients.

Matrix metalloproteinases and their inhibitors in exfoliation syndrome.

The purpose of this study was to determine the expression of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the aqueous humour of patients with exfoliation syndrome (XFS). XFS and control samples were analysed for their MMP content by zymography and for their tissue inhibitors by ELISA. In XFS eyes, an increase for up to 60% in almost all MMPs was observed, as compared to the controls. MMP-2 and MMP-9 were found to predominate. TIMP-1 levels in XFS samples were slightly decreased, while TIMP-2 levels were similar to those of the controls. Our findings suggest that MMPs may be crucial in the progression of XFS, by degrading the abnormal fibrillar matrix components in the anterior segment tissues of XFS eyes. However, the increased levels of MMPs seem not to be able to overcome the overproduction and accumulation of the exfoliative material.

Levels of specific antibodies towards the major antigenic determinant of slime-producing Staphylococcus epidermidis determined by an enzyme immunoassay and their protective effect in experimental keratitis.

Staphylococcus epidermidis is an important cause of bacterial keratitis. Certain S. epidermidis strains produce an extracellular slime layer rich in an acidic polysaccharide with a molecular size of 20 kDa (20-kDa PS). We have demonstrated that the level of 20-kDa PS-specific antibodies significantly rises after establishment of slime-producing S. epidermidis bacteraemia and, furthermore, that rabbit polyclonal antibodies to 20-kDa PS opsonize cells of slime-producing S. epidermidis to a great degree and promote their clearance by polymorphonuclear cells (Arch. Biochem. Biophys. 342 (1997) 389; J. Pharm. Biomed. Anal. 22 (2000) 1029). The purpose of this study was to examine the protective and therapeutic effects both of active immunization, using 20-kDa PS as antigen, and of passive administration of specific antibodies towards the 20-kDa PS in a rabbit keratitis model. For active immunization, 20 rabbits were subcutaneously immunized with 20-kDa PS, whereas for passive immunization specific polyclonal IgG antibodies against 20-kDa PS were administered to 20 rabbits 1 day before induction of infection. Clinical observations were made weekly for 1 month and levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by an enzyme immunoassay. The levels of specific anti-20-kDa PS IgG in serum and aqueous humor following either active or passive immunization were significantly higher as compared with control groups (P<0.001). Although, actively immunized rabbits showed significantly less corneal damage than control animals, passively immunized ones were significantly better protected as compared with both control and those actively immunized. Obtained results suggest that 20-kDa PS plays crucial role in the pathogenesis of S. epidermidis keratitis and that both types of immunization significantly protect against corneal S. epidermidis pathology and damage.

Effects of oral acitretin on contrast sensitivity and tear film function: a prospective study.

The purpose of this prospective study was to investigate the ocular side effects of short-term therapy with oral acitretin (1 mg/kg/day) in 24 patients with severe and recalcitrant dermatoses. Apart from the routine ophthalmological examination, the following tests were performed: break-up time of tear film for the determination of its stability, Schirmer test for the assessment of lacrimal gland function, rose bengal staining for the detection of possible ocular surface damage and contrast sensitivity test for the evaluation of visual function. No statistically significant differences could be found between the pretreatment values of the assessed parameters and those obtained after 1 and 2 months of therapy. It seems reasonable, therefore, to suggest that ocular surface integrity and tear film and visual function are not affected by short-term oral acitretin administration.

Bilateral panophthalmitis as the initial presentation of meningococcal meningitis in an infant.

Endophthalmitis is a well-recognized, frequently devastating ophthalmic disease. The colonization of the eye and the subsequent development of endophthalmitis may be exogenous (including postsurgical and post-traumatic infections) or it may be of endogenous origin, representing a metastasis from a focus of infection elsewhere in the body associated with bacteremia (such as meningitis or cellulitis).

Determination of malondialdehyde by capillary electrophoresis, application to human plasma and relation of its levels with prematurity.

Malondialdehyde (MDA) is considered as the most important marker for monitoring lipid peroxidation, which is strongly associated with the development of serious diseases in adults and premature neonates. In this paper we report a method for determination of free MDA in human plasma using capillary zone electrophoresis. MDA was separated and determined as conjugate with tetrabutylammonium hydrogen sulphate (TBAS). Analysis was performed using 20 mM borare, pH = 9.3, as operating buffer and detection of the MDA-TBAS adduct at 267 nm. The method has a linear range up to 80 microM with a detection limit of 0.2 microM. The method was applied to the analysis of MDA in plasma of healthy adults, normal-gestation infants and of preterm neonates. Plasma proteins were successfully removed following centrifugation through a centricon-3 membrane. Results showed that the method can be easily and accurate applied for the determination of MDA in human plasma and that the level of MDA in pretern neonates is significantly higher (p

Contrast sensitivity function in patients with impaired oral glucose tolerance.

PURPOSE: To evaluate contrast sensitivity function in patients with impaired oral glucose tolerance test (OGTT) compared with normal subjects. METHODS: Sixteen patients with impaired OGTT and 11 normal control subjects were tested for contrast sensitivity function at four spatial frequencies. Glucose intolerance was established by the 1985 World Health Organization criteria. RESULTS: The two groups were similar in terms of age, visual acuity, refractive correction, and lens opacities. A statistically significant loss of contrast sensitivity was associated with impaired OGTT (p < 0.001) in every spatial frequency tested. CONCLUSIONS: Functional visual loss in patients with impaired glucose tolerance using the 1985 World Health Organization criteria indicates that at least part of those patients should be classified as diabetic according to the 1997 American Diabetic Association criteria. It seems that the 1997 American Diabetic Association criteria are more efficient at detecting patients with abnormal visual function.

Increased aqueous humor basic fibroblast growth factor and hyaluronan levels in relation to the exfoliation syndrome and exfoliative glaucoma.

PURPOSE: To quantify the concentrations of basic fibroblast growth factor (bFGF) and hyaluronan (HA) in the aqueous humor of patients with the exfoliation syndrome (XFS) or exfoliative glaucoma (XFG). METHODS: Aqueous humor bFGF and HA levels were measured in 13 patients with XFS and in 7 patients with XFG. The results were compared with those obtained from 17 healthy controls. RESULTS: Mean bFGF levels were significantly higher in the XFG patients than those in the XFS patients, which in turn were higher than the bFGF levels in the healthy individuals. Aqueous humor HA levels in both patients with the XFS and the XFG were significantly higher compared to the controls. CONCLUSION: We suggest that bFGF plays an important role in the pathogenesis of XFS and XFG, as well as in the synthesis of secreted HA, which may result in connective tissue degradation that affects the ocular anterior segment.

Contrast sensitivity function in patients with beta-thalassemia major.

PURPOSE: To investigate contrast sensitivity function in patients with beta-thalassemia major, after regular transfusion and chelation therapy. METHODS: We measured contrast sensitivity at four spatial frequencies in 30 patients with beta-thalassemia major and in 30 matched normal control subjects. All subjects underwent an ophthalmic examination that included fluorescein angiography. The contrast sensitivity results from the two groups were compared between them. Patients' contrast sensitivity values were correlated to the variables age, duration of transfusion, duration of chelation therapy and serum ferritin levels, to select the important predictors. RESULTS: Contrast sensitivity function in all beta-thalassemic patients was significantly lower (p<0.0001) compared to the normal control subjects, for all spatial frequencies tested. The most important predictor of contrast sensitivity loss was patients' age. CONCLUSION: Contrast sensitivity testing can detect early changes in the visual function of beta-thalassemic patients and should be considered as a monitor for patients under chronic transfusion-chelation therapy.

Capillary electrophoretic analysis of brimonidine in aqueous humor of the eye and blood sera and relation of its levels with intraocular pressure.

The aim of this study was the development of a capillary electrophoretic method for the determination of the levels of the selective alpha(2)-adrenergic receptor agonist brimonidine in aqueous humor of the eye and blood sera and their relation to its efficacy in reducing the intraocular pressure (IOP). Analysis of brimonidine was performed by capillary zone electrophoresis using 20 mM borate, pH 9.3, as operating buffer and detection at 255 nm. Brimonidine levels were determined in aqueous humor and blood sera from seven patients admitted for cataract extraction following ocular administration of the ophthalmic Alphagantrade mark solution. Levels of brimonidine and IOP values were recorded for a 24 h period. Alphagantrade mark administration resulted in a significant reduction of IOP, from within 30 min up to 4-5 h, whereafter a stepwise increase was recorded until 24 h, where mean IOP value returned to that before administration. The IOP reduction was related to the levels of brimonidine in aqueous humor, where maximal levels (80-100%) were obtained within 1-3 h. A 50% amount of the solution was determined after 4-5 h, whereas it reached the minimum level after 12 h. Serum levels reached maximum within 3-4 h, a 50% reduction was recorded in 12 h and minimum level in 24 h. It is concluded that brimonidine administration may significantly reduce IOP in patients when its level is maintained >/=50% of the maximum present in aqueous humor, i.e within a 4-6 h period. Since at this time the level of brimonidine in blood serum has reached maximum value, administration of brimonidine every 6 h may be used to obtain adequate brimonidine levels to maintain a constantly lowered IOP.

Vascular retinal abnormalities in neonates of mothers who smoked during pregnancy.

OBJECTIVE: To investigate whether maternal smoking during pregnancy causes retinal abnormalities in the newborn. STUDY DESIGN: One hundred sixty-two neonates of smoking mothers and 162 matched neonates of nonsmoking mothers (112 appropriate for gestational age [AGA], 30 small for gestational age [SGA], 20 large for gestational age [LGA] in each group) were studied. RESULTS: Retinal arterial narrowing and straightening (RANS) was observed in 52 and 10 eyes of the newborns of smoking and nonsmoking mothers, respectively (P <. 000001) in association with elevated blood pressure in the neonates. The frequency of RANS was more than 3-fold greater in the SGA neonates than in the AGA and LGA neonates of the smoking mothers. Retinal venous dilatation and tortuosity (RVDT) was found in 100 and 36 eyes of neonates of smoking and nonsmoking mothers, respectively (P <.000001). The frequency of RVDT in the SGA neonates of the smoking mothers was 2.5-fold and 4.2-fold greater than in the AGA infants and the LGA infants, respectively. Also, intraretinal hemorrhages were found in 61 and 31 eyes of neonates of smoking and nonsmoking mothers, respectively (P =.0007) in association with elevated hematocrit and RVDT, whereas no intraretinal hemorrhages were found when RANS was present. All retinal abnormalities resolved by 6 months in infants of smoking mothers and by 2 months in infants of nonsmoking mothers. CONCLUSIONS: Maternal smoking during pregnancy causes increased frequency of RANS, RVDT, and intraretinal hemorrhages; but these retinal abnormalities resolve by 6 months of age.