Outcomes of different concentrations of human amniotic fluid in a keratoconjunctivitis sicca-induced mouse model.

To compare the effects of different concentrations of topical human amniotic fluid (HAF) in a mouse model of dry eye, forty C57BL/6 mice were divided into 4 treatment groups: 20 % HAF, 50 % HAF, 100 % HAF, and isotonic salt solution (control). Dry eye was induced by an injection of botulinum toxin B into the lacrimal gland. Tear production, ocular surface fluorescein staining, and blink rate were evaluated in each mouse at 5 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Regarding tear production, 20, 50, and 100 % HAF groups were all different from the control group (P < 0.001) at week 1. However, there were no statistically significant differences between the 20, 50, and 100 % HAF groups. At week 2, 20, 50, and 100 % HAF groups had significant improvement in staining score and were significantly different from the control group (P = 0.047, P = 0.005, and P = 0.001, respectively). No difference in spontaneous blink rate was observed between groups, at any time point. Goblet cell density was significantly decreased in the control group compared to the HAF treatment groups. All tested concentrations of topical HAF were effective and superior than the control in this keratoconjunctivitis sicca-induced mouse model. Further studies are needed to evaluate the effects of HAF on the human ocular surface.

Impaired viscosity of gastric secretion and its mucin content as potential contributing factors to the development of chronic constipation.

BACKGROUND: The alimentary tract mucosa continuously releases mucus-rich secretion. Mucin, the major component of mucus, determines its viscosity and provides lubrication for the luminal content of indigestible food particles. AIMS: To measure mucin secretion rate and its viscosity in patients with chronic constipation (CC) and in asymptomatic volunteers. METHODS: Nineteen patients with symptoms of CC and 19 controls were included in the study. Mucin secretion and viscosity were assessed in aspirated gastric juice in basal conditions and after stimulation with pentagastrin (1 h each). Mucin content was tested by PAS methodology. Viscosity was measured using cone/plate digital viscometer. RESULTS: Mucin secretion rates in basal and stimulated conditions in controls were 65 and 42 % higher than in patients with CC (P < 0.05 and P < 0.001, respectively). Basal viscosity in controls was 48 % higher than in CC (P < 0.05) at the lowest and 55 % higher (P < 0.05) at the middle velocities. Viscosity in pentagastrin-stimulated conditions in controls was 71 % higher than in CC (P < 0.01) at the lowest and 35 % higher (P < 0.05) at the middle velocities. CONCLUSIONS: (1) The significantly lower rate of soluble mucin secretion in patients with CC than in normal volunteers may reflect impairment in mucin-related lubrication. (2) Significantly lower viscosity of gastric secretion in patients with CC may result from the lower rate of mucin secretion and may also diminish lubrication within the alimentary tract. (3) This may potentially set the stage for the development of symptoms related to chronic constipation and open a new therapeutic avenue for this patient population.

Riboflavin and ultraviolet light a therapy as an adjuvant treatment for medically refractive Acanthamoeba keratitis: report of 3 cases.

PURPOSE: To present the first 3 cases of Acanthamoeba keratitis (AK), unresponsive to medical treatment, that were successfully treated with a novel adjunctive therapy using ultraviolet light A (UVA) and riboflavin (B2). DESIGN: Interventional case series. PARTICIPANTS: Two patients with confirmed AK and 1 patient with presumptive AK, which were all refractive to multidrug conventional therapy. INTERVENTION: Two treatment sessions involving topical application of 0.1% B2 solution to the ocular surface combined with 30 minutes of UVA irradiation focused on the corneal ulcer. MAIN OUTCOME MEASURES: Clinical examination by slit lamp, confocal microscopy, and histopathology, when available. RESULTS: All patients in these series showed a rapid reduction in their symptoms and decreased ulcer size after the first treatment session. The progress of the clinical improvement began to slow after 1 to 3 weeks of the first application and was then renewed after the second application. All ancillary signs of inflammation mostly resolved after the second treatment session. The ulcers in all patients continued to decrease and were closed within 3 to 7 weeks of the first application. Two patients developed dense central corneal scars, and penetrating keratoplasty was performed for visual rehabilitation. Histopathologic examination of the excised tissue revealed no Acanthamoeba organisms. The remaining patient had no symptoms or signs of infection, both clinically and by confocal microscopy, and was left with a semitransparent eccentric scar that did not affect visual acuity. CONCLUSIONS: The adjunctive use of UVA and B2 therapy seems to be a possible alternative for selected cases of medication-resistant AK.

Analysis of clear corneal incision integrity in an ex vivo model.

PURPOSE: To determine the most favorable sutureless incision configuration to minimize extraocular fluid inflow after cataract surgery. SETTING: The Wilmer Eye Institute, Baltimore, Maryland, USA. METHODS: Five fresh human eyes were used in the study. Two 27-gauge needles connected to a saline solution bag and a digital manometer were inserted through the limbus 180 degrees from each other. Intraocular pressure (IOP) was maintained at 15 to 20 mm Hg. Three incisions were performed in different quadrants of each cornea: uniplanar 1.0 mm and 3.0 mm tunnel lengths and 2-step 3.0 mm tunnel length. India ink was applied to the incision site, and IOP fluctuation was induced by applying pressure to the limbal area of the opposite quadrant using an ophthalmodynamometer. Imaging was performed before and after pressure application. RESULTS: The linear distance of India ink inflow after pressure application was higher than the prepressure measurements in the 1.0 mm and 3.0 mm incision groups (P = .039 and P = .023, respectively). The maximum mean of inflow after pressure application was not higher than the prepressure measurement in the 2-step incision group (P = .105). The total ink area measured before and after pressure applications in the incisions of the 3 groups was not significantly different (P = .285). CONCLUSIONS: Intraocular pressure fluctuations may promote entry of bacteria-size particles into the eye when 1.0 mm and 3.0 mm single-plane incisions are performed. Stepped incisions seem to be more resistant to inflow in the presence of IOP fluctuation.

Novel laser-activated solder for sealing corneal wounds.

PURPOSE: To compare the effectiveness of a laser-activated biological tissue solder with that of standard sutures for sealing corneal incisions. METHODS: Two keratome knives measuring either 3.0 mm or 2.85 mm were used to create a non-self-sealing peripheral oblique corneal wound (POCW) or a central perpendicular corneal wound (CPCW) in fresh rabbit cadaver eyes. Wounds were sealed with a solder strip (POCW; n = 5), a solder patch (CPCW; n = 5), or three interrupted nylon 10-0 sutures (n = 5). After the solder was placed on the wound, a diode laser was used to activate the solder, resulting in cross-linking to tissue. Wound stability was tested by a stepwise infusion of saline, and pressure changes were monitored with a digital manometer. Leaking pressure was recorded. RESULTS: The pressurized mean baseline IOP in the intact globe was 131.13 mm Hg (SD, 4.66). Mean IOP after CPCW was 1.7 mm Hg (SD, 0.13); for POCW it was 3.62 mm Hg (SD, 3.09). For the CPCW group, the mean leaking pressure in the sutured eyes was 82.76 mm Hg (SD, 6.55), whereas in the solder patch it was 101.42 mm Hg (SD, 29.92; P = 0.2222). For the POCW group, the mean leaking pressure in the sutured eyes was 33.44 mm Hg (SD, 9.38), and the mean IOP achieved in the solder repaired eyes was 125.16 mm Hg (SD, 9.85; P = 0.0079). CONCLUSIONS: The tested laser-activated solder was as effective as sutures when used as a patch and superior to sutures for clear corneal incisions in this animal ex vivo model.

Sutured clear corneal incision: wound apposition and permeability to bacterial-sized particles.

PURPOSE: To determine the effects of single radial or horizontal suture placement in 2-step clear corneal incision (CCI) wound apposition and permeability to particles of India ink. METHODS: Five fresh human globes were included. Two 25-gauge needles connected to a saline solution bag and to a digital manometer were inserted through the limbus, 120 degrees apart from each other. Four 2-step CCIs (2.75 mm wide and 3 mm length) were constructed in each cornea. Incisions were divided into 3 groups: single radial suture (SRS), single horizontal suture (SHS), and unsutured group. Optical coherence tomography (OCT) was performed before and after suture placement. With a preset 10 mm Hg intraocular pressure (IOP), India ink was applied to the incision site and a standardized sudden IOP fluctuation was induced. OCT and superficial images were recorded before and after suture placement. India ink inflow and internal and external CCI gapping were outlined and measured by planimeter. RESULTS: The area and linear distance of India ink inflow after pressure challenge in all study groups were higher when compared with pre-pressure measurements; however, this increase was significant in the SRS and SHS groups (P < 0.05). Additionally, SRS placement significantly increased inner wound gapping (P = 0.018), and SHS significantly widened outer wound gape (P = 0.02). CONCLUSIONS: Well-constructed unsutured 2-step CCI seems to be more efficient at preventing bacterial-sized particles inflow during sudden changes in IOP, and it seems to offer better wound apposition as assessed by OCT.

Impairment of salivary mucin production resulting in declined salivary viscosity during naproxen administration as a potential link to upper alimentary tract mucosal injury.

OBJECTIVES: Nonsteroidal anti-inflammatory drugs (NSAIDs) contribute to the esophageal mucosal injury through its direct topical impact on the luminal aspect of the surface epithelium. Its indirect, systemic impact, however, on salivary component of the esophageal pre-epithelial barrier remains to be explored. Therefore, salivary mucin secretion and viscosity at baseline and during naproxen-placebo, as well as naproxen-rabeprazole, administration were investigated. METHODS: Twenty-one asymptomatic volunteers were included in this double-blind, placebo-controlled, crossover designed study. Salivary samples were obtained in basal and pentagastrin-stimulated conditions (6 mg/kg s.c.) mimicking the food-stimulated conditions. Patients received 7 days of naproxen-placebo or naproxen-rabeprazole with a 2-week washout period in between. Salivary mucin content and viscosity were measured before and after treatment using periodic acid/Schiff's methodology and Cone/Plate Digital Viscometer, respectively. RESULTS: The rate of salivary mucin secretion in basal condition declined by 32% during administration of naproxen-placebo (11.3±1.7 vs. 16.8±3.3 mg/h). Salivary mucin secretion in pentagastrin-stimulated condition declined significantly (by 34%) during the administration of naproxen-placebo (13.6±1.5 vs. 20.7±3.0 mg/h; P<0.05). Viscosity significantly decreased after naproxen-placebo administration in basal (by 60%) and stimulated conditions (by 56%) (P<0.001). Coadministration of rabeprazole at least partly restored the naproxen-induced decline of salivary mucin in basal condition (by 8%), and pentagastrin-stimulated conditions (by 30%). CONCLUSIONS: A significant decline of salivary mucin and viscosity during administration of naproxen may at least partly explain a propensity of patients on chronic therapy with NSAIDs to the development of esophageal mucosal injury and complications. In addition the trend to restorative capacity of rabeprazole on the quantitative impairment of salivary mucin during administration of naproxen may potentially translate into its tangible clinical benefit but it requires further investigation.

Effects of topical human amniotic fluid and human serum in a mouse model of keratoconjunctivitis sicca.

PURPOSE: To compare the effects of topical human amniotic fluid (HAF), topical human serum (HS), and topical artificial tears in a mouse model of dry eye. METHODS: Thirty C57BL/6 mice were divided into 3 treatment groups: HAF, HS, and preservative-free artificial tears. Dry eye was induced by an injection of botulinum toxin B (BTX-B) into the lacrimal gland. Tear production and ocular surface fluorescein staining were evaluated in each mouse at 6 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Apoptotic keratocytes were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling test assay. RESULTS: A significant decrease in tear production was observed 3 days after BTX-B injection in all groups. At week 1, the HAF and HS groups had improved tear production compared with the control group (P < 0.001 and P = 0.003, respectively). HAF had a significantly improved fluorescein staining score compared with the HS (P = 0.043) and control (P = 0.007) groups at week 2. Goblet cell density was significantly decreased in the control group compared with the HAF and HS groups (P < 0.001). No difference in the amount of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive keratocytes was observed among the groups. CONCLUSION: HAF was superior to HS and artificial tears for improving corneal staining within 2 weeks of therapy in this induced mouse model of keratoconjunctivitis sicca. Clinical studies are needed to ascertain the benefits of these therapies in patients with ocular surface disorders associated with dry eye.

Bacterial-sized particle inflow through sutured clear corneal incisions in a laboratory human model.

PURPOSE: To determine the effectiveness of a single radial suture placement in 2 clear corneal incision (CCI) configurations in preventing inflow of bacterial-sized particles. SETTING: Wilmer Eye Institute, Baltimore, Maryland, USA. DESIGN: Experimental study. METHODS: Ten human globes were used. Two 25-gauge needles connected to a saline solution bag and to a digital manometer were inserted through the limbus 120 degrees apart. Four incision-suture combinations were evaluated sequentially in each cornea. All incisions had the same dimensions (2.75 mm width, 3.00 mm length). Two incisions were single plane, and 2 were biplane. One incision from each configuration was left unsutured, and the other was sutured (10-0 nylon). With a preset 10 mm Hg intraocular pressure (IOP), India ink was applied to the incision site and a sudden IOP fluctuation was induced. Inflow was outlined and measured by planimetry. RESULTS: There was a significant increase in area and linear distance of India-ink inflow after pressure challenge in all groups (P < .05), but with important differences among them. When the sutured and unsutured groups from each CCI were compared, there was a significantly smaller area of inflow in the 2-step unsutured group (P < .05). The linear inflow was higher in both sutured groups; however, the difference was significant for the 2-step incision configuration only (P < .05). CONCLUSIONS: A single radial suture reduced the area of inflow of bacterial-sized particles but increased the linear distance of inflow in single-plane 3.0 mm incisions. Suture placement in a stepped incision increased inflow of bacterial-sized particles. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Standardized Seidel test to evaluate different sutureless cataract incision configurations.

PURPOSE: To compare aqueous outflow and India ink inflow to evaluate clear corneal incisions (CCIs). SETTING: Wilmer Eye Institute, Baltimore, Maryland, USA. METHODS: Two 25-gauge needles connected to a salt solution bag and to a digital manometer were inserted through the limbus of human donor corneas 120 degrees apart from each other. Three 2.75 mm wide incisions were created in a different third of each cornea as follows: single-plane 1.50 mm tunnel length, single-plane 3.00 mm tunnel length, and 2-step 3.00 mm tunnel length. The Seidel test was evaluated at 5 intraocular pressures (IOPs) in the physiologic range. A masked observer evaluated the recorded tests. With a preset 10 mm Hg IOP, India ink was applied to the incision site and a sudden IOP fluctuation induced. India ink influx was outlined and measured by planimetry. RESULTS: With the 1.5 mm single-plain incisions, all 6 globes showed inflow and outflow. With the 3.0 mm single-plain incisions, all 6 globes showed inflow and 2 showed outflow by the Seidel test. With the 3.0 mm 2-step incisions, 2 globes showed inflow and 3 had positive Seidel test results. Area and length of inflow were statistically significantly greater with the 2 single-planed incisions than with the 2-step incisions. CONCLUSION: This human ex vivo model showed that wound deformation produced during Seidel testing might not be an accurate way to predict the risk for bacterial invasion in the early postoperative period.

Corneal wound healing is modulated by topical application of amniotic fluid in an ex vivo organ culture model.

The purpose of this study was to evaluate the effects of topical human amniotic fluid (HAF) and equine amniotic fluid (EAF) on corneal reepithelialization and stromal wound healing. New Zealand white rabbit corneas (n=52) were placed in an ex vivo air-interface organ culture. An 8.5mm-diameter mark in the center of the cornea was produced with a hand trephine to select the area for epithelial scraping. A number 15 surgical blade was used to remove the epithelial layer within the demarcated area in a standardized fashion. The corneas were assigned to one of four treatment groups (n=8): fetal bovine serum (FBS), HAF, EAF, and a control group that was exposed to phosphate buffer solution (PBS). Corneal epithelial defects were imaged every 8 h for 72 h after the application of a 30 microl drop of 0.015% fluorescein. Five corneas of each treatment group were used for histology, proliferation, and apoptosis assay at 72 h after the epithelial defect was created. There was no significant difference in the mean rate of closure of the corneal epithelial defect between FBS treated corneas and controls (P>0.06). The mean epithelial defect area (MEDA) was significantly smaller in the EAF group as compared to control corneas at 24 h (P=0.016), 40 h (P=0.032), 64 h (P=0.008) and 72 h (P=0.007) following epithelial scrape. The MEDA in the HAF group was significantly smaller at 16 h (P=0.008), 64 h (P=0.0072), and 72 h (P=0.016) compared to the control group. The MEDA in the HAF and EAF groups was smaller at all time points as compared to the FBS group, but the difference was not significant. At histology, the mean keratocyte density was significantly higher in the anterior stroma in the HAF (P<0.001) and EAF groups (P=0.001) as compared to control group. The number of BrdU positive keratocytes was significantly higher in the superficial and deep stromal sub-areas in the HAF group as compared to control (P<0.001 and P=0.002, respectively). EAF and FBS treated corneas also showed a higher number of BrdU positive cells compared to control, but this difference was not significant. Finally, we did not observe any difference in the amount of TUNEL positive keratocytes among the different groups. Our data indicates that the topical application of HAF and EAF is associated with accelerated reepithelialization in this cornea organ culture model. Similarly, corneal keratocyte density appears to be less affected after epithelial injury using this treatment.