Tear analysis of ascorbic acid, uric acid and malondialdehyde with capillary electrophoresis.

Tears have a significant role in antioxidant defense in ocular tissues and since their collection is quick and noninvasive, their analysis would facilitate monitoring of pathophysiological changes. However, their low volume and low content of antioxidants makes analysis difficult; methods of high sensitivity are needed. In this paper, we present a method for tear analysis of two antioxidant molecules (ascorbic and uric acid) and of a lipid peroxidation indicator (malondialdehyde) with capillary electrophoresis. Tears were collected with Schirmer strips, extracted with a low-pH phosphate buffer, centrifuged through membrane filters and an antioxidant was added. They were stable at -70 degrees C for 15 days. After pilot experiments, optimum electrophoretic separation was achieved in a 25 mM borate buffer, pH 10.0, containing 100 mM sodium dodecyl sulfate at 25 degrees C and 20 kV. The developed method has good repeatability (<5% RSD), precision (<15% relative error values) and high sensitivity (LLOQ values of 20, 2.3 and 2.5 microM for ascorbate, urate and malondialdehyde, respectively). It was applied to the analysis of tears from healthy individuals and the antioxidant levels are in agreement with those obtained with other techniques. This method might serve as a tool to clarify the role of endogenous antioxidants in the pathophysiology of ocular diseases.

Apoptotic mechanisms within the retina in Staphylococcus epidermidis experimental endophthalmitis.

AIM: To investigate the potential involvement of apoptosis and its regulators Bcl-2, Bax, and Fas within the retina in Staphylococcus epidermidis experimental endophthalmitis. METHODS: Endophthalmitis was induced in 48 male Lewis rats by unilateral 25-mircrol intravitreal injection of 7,000 viable organisms of slime-producing S. epidermidis strain ATCC 35983 (experimental group). Forty-eight other Lewis rats received a similar sterile normal saline injection (control group). The injected eyes were graded for clinical inflammation and were removed in groups at 6, 12, 24, 48, 72, and 168 hours post-injection. After surgical separation, retinal tissue specimens were fixed, and paraffin sections underwent hematoxylin-eosin staining, immunohistochemistry against Bcl-2, Bax, and Fas, and TUNEL assay for detection of apoptotic cells. Following morphometric analysis, the apoptotic body index (ABI) was calculated. RESULTS: While Bcl-2 expression was absent, Bax and Fas expression and apoptosis in ganglion cells, bipolar cells, and photoreceptors, were significantly higher in the experimental group compared to the control group (P < 0.05). In the experimental group, inflammation peaked at 24 hours, Bax and Fas expression at 48 hours and the ABI at 72 hours post-injection. CONCLUSION: Apoptosis is increased within the retina in S. epidermidis experimental endophthalmitis through upregulation of Bax and Fas, peaking soon after peak inflammation.

Effect of ketorolac 0.5% drops on patients' pain perception during intravitreal injection procedure.

PURPOSE: To evaluate the analgesic effect of ketorolac 0.5% drops during the intravitreal injection procedure. METHODS: Thirty patients (n=30) received topical ketorolac 0.5% or vehicle on subsequent intravitreal drug administrations. The procedure followed for the intravitreal injections was the same for all subsequent administrations with the use of tetracaine 0.5% drops as anesthetic. Ketorolac or vehicle was instilled before the injection, and pain perception was recorded on a 0 to 100 Visual Analog Scale (VAS) immediately after the intravitreal administration. RESULTS: Mean VAS pain score was 8.16±1.3 when patients received ketorolac and 12.33±1.41 when they received placebo, a difference that was statistically significant (P=0.0003) (paired t-test). CONCLUSIONS: Topical ketorolac 0.5% reduces patients' pain perception during intravitreal drug administration.

Pars plana vitrectomy in the treatment of severe complicated toxoplasmic retinochoroiditis.

PURPOSE: To present the anatomic and functional results of pars plana vitrectomy performed in severe complicated toxoplasmic retinochoroiditis. METHODS: Three patients, 2 women and 1 man aged 57, 22, and 57 years, are presented. The first patient was under immunosuppressive therapy for dermatomyositis and underwent diagnostic/therapeutic vitrectomy for severe toxoplasmic panuveitis with dense vitritis. The other 2 patients underwent vitrectomy for macula-off rhegmatogenous retinal detachment that developed after severe toxoplasmic panuveitis. RESULT: Preoperative visual acuity was hand movement for the first 2 patients and 20/400 for the third. All patients received pars plana vitrectomy with epiretinal membrane peeling, laser photocoagulation, and SF6 gas tamponade. The second and third patients needed 5 and 3 additional operations, respectively, including extensive retinotomies and silicone-oil tamponade, for recurrent retinal detachment due to proliferative vitreoretinopathy. At the end of the follow-up period (11, 5, and 1 year, respectively), the retina was attached and visual acuity was 20/30 for the first patient but counting fingers for the other 2 patients. CONCLUSIONS: Severe panuveitis and/or recurrent retinal detachment may develop in some cases of ocular toxoplasmosis, compromising the visual prognosis. Retinal detachment due to toxoplasmosis is generally complex, and long-acting tamponade with silicone oil should be contemplated for anatomic retinal reattachment.

Decreased contrast sensitivity in children and adolescents with type 1 diabetes mellitus.

PURPOSE: To evaluate contrast sensitivity in children and adolescents with diabetes mellitus without evidence of diabetic retinopathy. METHODS: Sixty patients with insulin-dependent diabetes mellitus (age range: 8 to 18 years) were studied. Their contrast sensitivity scores were obtained using the CSV-1000 device (Vector Vision, Dayton, OH) for four spatial frequencies and were compared with v scores of 45 age-matched and gender-matched "healthy" patients. Contrast sensitivity values were also correlated to patient's age, duration of disease, and metabolic control of diabetes mellitus. RESULTS: The patients with insulin-dependent diabetes mellitus had a significant contrast sensitivity score reduction at all spatial frequencies tested. Glycosylated hemoglobin levels were inversely related to the contrast sensitivity thresholds. No significant correlation was found between the contrast sensitivity scores and the patient's age or duration of disease. CONCLUSION: Contrast sensitivity defects are detected in patients with insulin-dependent diabetes mellitus. These defects may represent an early dysfunction of the retina, visual pathway, or both in patients with insulin-dependent diabetes mellitus who do not show any signs of diabetic retinopathy.

Non-invasive detection of antibiotics and physiological substances in the aqueous humor by Raman spectroscopy.

BACKGROUND AND OBJECTIVES: Laser Raman spectroscopy is an inelastic light scattering technique able to characterize molecules in aqueous environments. The purpose of this work is to develop a non-contact and non-invasive spectroscopic method to identify and eventually quantify the presence of medicines (e.g., antibiotics) and physiological substances (e.g., glucose) in the aqueous humor of the eye. STUDY DESIGN/MATERIALS AND METHODS: A new laser light delivery probe has been developed and adapted to a Raman spectroscopic system with the ability of favorable collection of the Raman light at 90 degrees scattering geometry while scanning the anterior chamber of the eye. Different amounts of ceftazidime, amphotericin B, and glucose had been injected in the aqueous humor of porcine eyes, maximum 24 hours after death and extraction, in-vitro. Raman measurements were excited with a visible (514.5 nm) laser beam at a power of 25 mW and an exposure/acquisition time of 1 second. RESULTS: The specific collection optics and Raman analysis components used in the present work have resolved the Raman signatures of probed molecules and low concentrations of ceftazidime (0.9 mg/mL), amphotericin B (9 microg/mL), and glucose (2 mg/ml) separately injected in the anterior chamber of porcine eyes were detected in vitro. CONCLUSION: This special illumination design gives the opportunity of avoiding the direct exposure to the laser light of basic cordial tissues of the eye, like lens and retina, although an optimum collection of scattered light is accomplished. Concentrations close to the minimum inhibitory concentration (MIC) have been detected for ceftazidime and amphotericin b; the detection of glucose has been realized at concentrations close to the early pathological levels of patients with diabetes.

Expression of TNF-alpha, IL-1beta, and IFN-gamma in Staphylococcus epidermidis slime-positive experimental endophthalmitis is closely related to clinical inflammatory scores.

PURPOSE: The purpose of this study was to investigate the expression of inflammatory cytokines TNF-alpha, IL-1beta, and IFN-gamma in the vitreous after experimentally induced endophthalmitis by a Staphylococcus epidermidis slime-producing strain. METHODS: Seventy-two experimental Lewis rats received an intravitreal injection of 7000 viable organisms of Staphylococcus epidermidis slime-producing ATCC strain 35983, while 72 control rats received an intravitreal injection of sterile normal saline. Eyes were graded daily for signs of clinical inflammation and were removed 6, 12, 24, 48, 72 h, and 7 days after injection. Vitreous was obtained and titers of TNF-alpha, IL-1beta, and IFN-gamma were measured with established enzyme-linked immunosorbent assays. RESULTS: In the experimental group, the clinical inflammatory score reached maximum (4+) within 24 h, while inflammation was almost abolished by day 7 (score 0-0.5+). Statistically increased levels of TNF-alpha and IL-1beta were detected in the experimental vitreous with maximum levels observed at 12 h. IFN-gamma was also detected in the experimental vitreous and reached maximum levels at 48 h. None of the cytokines examined was detected in sera at any time point in experimental or control rats. CONCLUSIONS: The results of this study suggest that Staphylococcus epidermidis experimental endophthalmitis induces the expression of cytokines TNF-alpha, IL-1beta, and IFN-gamma in the vitreous. The time course of those cytokine expression levels is closely associated to the clinical presentation of this endophthalmitis model.