Effects of amniotic membrane extract on primary human corneal epithelial and limbal cells.

BACKGROUND: To assess the effects of amniotic membrane extract (AMX) on cellular activity of primary human corneal epithelial (HCE) cells under mechanical and oxidative stress, and on human limbal cells under oxidative stress. METHODS: Corneal mechanical stress was simulated with a linear scratch in confluent HCE cell plates, then incubated with 0.1% AMX for 48 and 72 h. Subjecting HCE cultures to 0.5 mmol/L tertiary-butylhydroperoxide for 1 h simulated an oxidative stress. 0.1% AMX-treated cultures were compared with controls at 24 and 48 h using cellular viability assay, along with 12-h AMX pretreatment and human limbal cell comparisons. RESULTS: Mechanical stress on HCE cultures revealed a statistically significant distance ratio at 48 and 72 h in favour of 0.1% AMX-treated cultures (P = 0.021 and 0.035, respectively). Oxidative stress did not reveal any significant difference in cellular viability of AMX-treated versus control cultures. Twelve hour AMX pre-treatment prior to oxidative stress revealed a significant difference after 24 h from oxidative injury (73.3% AMX vs. 66.0% control, P = 0.035), but not after 48 h. Human limbal cells demonstrated significantly improved oxidative viability compared with HCE cells, with (91.0% vs. 82.0% control, P = 0.017) and without 0.1% AMX pre-treatment (91.2% vs. 83.7% control, P = 0.019). CONCLUSIONS: HCE cells treated with AMX healed faster after mechanical insult, suggesting a potential benefit in acute corneal injuries. Under oxidative stress, human limbal cells, a more proliferative cell type, showed superior viability compared with HCE cells.

Topical treatment with nerve growth factor in an animal model of herpetic keratitis.

BACKGROUND: In vitro and in vivo studies demonstrated the antiviral efficacy of nerve growth factor (NGF) and its cyto-protective effect in herpes simplex virus (HSV)-infected cells. The aims of this study were to evaluate the role of endogenous NGF in HSV corneal infection, and the effects of topical NGF treatment on herpetic keratitis. METHODS: Herpetic keratitis was induced in 40 rabbits with the HSV-1 McKrae strain. Animals were divided into four groups, and treated with topical neutralizing anti-NGF antibodies, NGF, acyclovir or balanced salt solution (BSS) respectively. The clinical course of HSV keratitis was evaluated and scored by slit-lamp examination. In addition, biochemical (immunohistochemistry for glycoprotein D) and molecular (nested PCR for glycoprotein D) analyses were carried out to estimate viral replication. RESULTS: Treatment with anti-NGF antibodies induced a more severe keratitis associated with increased biochemical and molecular markers of active viral replication. Two animals in this group developed lethal HSV encephalitis. Conversely, topical treatment with NGF induced a significant amelioration of clinical and laboratory parameters when compared to the BSS treated group (control). No significant differences were observed between NGF- and acyclovir-treated groups. CONCLUSIONS: This study demonstrated the crucial role of endogenous NGF in herpetic keratitis. The comparable effects of NGF and acyclovir confirm the antiviral activity of NGF, and indicate a potential use of topical NGF in herpetic keratitis.

Role of neurotrophins and neurotrophin receptors in rat conjunctival goblet cell secretion and proliferation.

PURPOSE: To determine which neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4)-and their receptors (NTrs) TrkA, TrkB, TrkC, and p75 are present in rat conjunctiva and cultured rat goblet cells (CGCs) and whether NTs stimulate glycoconjugate secretion or cell proliferation. METHODS: Western blot analysis and immunofluorescence microscopy determined presence and location of NTs and NTrs. CGCs were incubated with NTs (10(-12)-10(-8) M) for 2 or 24 hours to measure secretion or proliferation, respectively. An enzyme-linked lectin assay analyzed glycoconjugate secretion. WST-8 determined cell proliferation. RESULTS: Western blot analysis showed all NTs and NTrs in both conjunctiva and CGCs. The cytoplasm of conjunctival stratified squamous cells and goblet cell lateral membranes contained NGF, BDNF, and NT4. Stratified squamous cell membranes contained NT3. In CGCs, NGF and BDNF had punctuate perinuclear staining. The nucleus contained NT3 and cytosol contained NT4. TrkA, TrkB, and p75 immunoreactivity was on conjunctival goblet cell lateral membranes. Plasma membranes of the basal layer of stratified squamous cells contained TrkA. Stratified squamous cell and goblet cell nuclei contained TrkC. In CGCs, all NTrs were present in the nucleus. NGF and BDNF, but not NT3 and NT4, induced a concentration-dependent stimulation of secretion from CGCs with a maximum increase of 10(-9) M each. No effect on cell proliferation was detected with any NTs. CONCLUSIONS: Rat conjunctival goblet cells and CGCs contain all NTs and NTrs. Only NGF and BDNF stimulated goblet cell glycoconjugate secretion, and none induced CGC proliferation.

Theoretical model and design of a device to reduce the influence of environmental factors on refractive surgery outcomes.

Recently many different groups show that environmental temperature and relative humidity affects the outcomes of refractive surgery performed by excimer lasers in an unpredictable fashion. A theoretical model of the water vapor absorption at 193 nm wavelength is presented and discussed in order to quantitatively assess the influence of environmental parameters on the laser energy that actually reaches the corneal surface. Model simulations show that laser energy absorption (up to 7% of the available energy) occurs along the path of laser beam, into the existent space between the laser beam source and the patient's eye, and is caused by environmental temperature and relative humidity (respectively 35 degrees C and 95%). Our findings suggest that this energy loss reduces the ablation rate, producing a significant under-correction of the treated corneas. A cost effective device which keeps thermo hygrometric parameters constant along the laser beam path, is also provided.

Nerve growth factor (NGF) and lenses: effects of NGF in an in vitro rat model of cataract.

BACKGROUND: The aims of this study are to investigate the presence and production of nerve growth factor (NGF) in the rat lens in basal conditions and to evaluate, in vitro, the role of NGF in a model of xylose-induced cataract. METHODS: Rat lenses were dissected and the expression of NGF, NGF mRNA and high-affinity NGF-receptor (TrkA) was evaluated by immunohistochemistry, immunoenzymatic assay (ELISA) and in-situ hybridization (ISH) techniques. To investigate the role of NGF in cataract formation we used an in vitro model of sugar-induced cataract by culturing rat lenses for 48 h in Eagle's minimum essential medium (MEM) supplemented with xylose. To evaluate the potential protective effect of NGF on xylose-induced cataract formation, exogenous NGF at different concentrations or antibodies neutralizing endogenous NGF (NGF-Ab) or aspecific antibodies were added to xylose-cultured lenses, and the following cataract-related parameters were evaluated and compared to xylose-treated lenses. Cataract formation was evaluated using three different parameters: staging of the cataract by lens photography, quantification of lens transparency in terms of gray level medium (GLM) and evaluation of the hydration percentage (H%) of the lens. To investigate the role of endogenous NGF in cataract onset, NGF levels were evaluated and compared in lenses cultured in xylose supplemented medium versus lenses cultured in control culture medium. RESULTS: The epithelium from fresh rat lenses expresses NGF-receptor, NGF protein and NGF-mRNA. NGF levels in fresh lens were 54.0 +/- 24.5 pg/g as quantified by ELISA. Xylose-cultured lenses develop cataract changes, including a decrease of GLM and an increase in hydration percentage, associated with a decrease in NGF levels when compared to lenses cultured in the control culture medium. The addition of NGF to xylose-cultured lenses reduces cataract formation, increasing GLM and decreasing the hydration percentage as compared to xylose-treated lenses. On the other hand, the addition of NGF-Ab induces an increase in cataract formation and lens hydration. CONCLUSIONS: This study demonstrates that rat lens epithelium expresses and synthesizes NGF. Moreover, immunohistochemistry shows that lens epithelial cells also express the NGF receptor. Although the functional significance of TrkA on lens epithelium is at present not clear, the expression of NGF and its high-affinity receptor on the same cells together with our experimental results suggest that NGF is involved in supporting trophism and/or the function of the lens epithelium.

Presence and localization of neurotrophins and neurotrophin receptors in rat lacrimal gland.

PURPOSE: To determine which of the neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4)-and their receptors (NTrs), TrkA, TrkB, TrkC, and p75, are present in the adult rat lacrimal gland. METHODS: RT-PCR was performed on RNA isolated from male rat lacrimal gland, using oligonucleotides specific to each NT and NTr. The presence of NT and NTr protein, was determined by Western blot analysis of lacrimal gland homogenate or membranes. The location of NTs and NTrs was determined by immunofluorescence histochemistry. Western blot analyses and immunofluorescence microscopy were performed using primary rabbit polyclonal antibodies raised against NTs and NTrs. RESULTS: RT-PCR showed positive bands at the appropriate sizes for NGF, BDNF, NT3, and NT4, and for the receptors TrkA, TrkB, TrkC, and p75. Western blot analysis confirmed these results, showing that the lacrimal gland expresses NGF, BDNF, NT3, and NT4 as well as the NTrs TrkA, TrkB, and TrkC and the p75 protein. NGF, BDNF, NT3, and NT4 were localized in the lacrimal gland acini with differing cellular distributions, whereas TrkA, TrkB, and TrkC, were localized in myoepithelial cell and ductal cell membranes. The protein p75 was expressed only on myoepithelial cell membranes. CONCLUSIONS: Members of the neurotrophin family of growth factors and their receptors are present in rat lacrimal gland, which suggests a role for NTs and their receptors in the lacrimal gland.

Intraocular production and release of nerve growth factor after iridectomy.

PURPOSE: To determine the presence of nerve growth factor (NGF), NGF mRNA, and NGF receptor (TrkA) in rabbit ocular tissues, and whether changes occur in NGF and NGF mRNA levels after experimental iridectomy. METHODS: Immunohistochemistry for NGF and TrkA and in situ hybridization for NGF mRNA were performed on rabbit cornea, iris, ciliary body, and lens in the basal state. Quantification of NGF mRNA and NGF protein levels in these tissues was performed by RT-PCR and immunoenzymatic assay, respectively. A time course of NGF concentration in the aqueous humor and the expression of NGF mRNA in iris and ciliary body were performed after the iridectomy and were compared with levels in a sham-treated group (paracentesis). RESULTS: Cornea, iris, ciliary body, and lens expressed NGF mRNA, NGF protein, and TrkA in the basal state. The highest levels of NGF were detected in the iris (8938.0 +/- 3968.1 pg/g), and the lowest were in the aqueous humor (22.8 +/- 9.7 pg/mL). Experimental iridectomy induced a transient increase of NGF concentration in the aqueous humor that reached its peak 4 hours after the experimental injury (464.4 +/- 29.9 pg/mL versus the control group 101.6 +/- 18.8 pg/mL; P < 0.001) and returned to baseline value after 7 days. A significant increase of NGF mRNA was also observed 1 hour and 4 hours after the iridectomy in the iris (1 hour, 788 +/- 85 OD; 4 hours, 760 +/- 81 OD versus baseline, 246 +/- 32 OD; P < 0.0001) and ciliary body (1 hour, 330 +/- 19 OD; 4 hours, 453 +/- 52 OD versus baseline, 219 +/- 37 OD; P < 0.05), but not in the cornea, lens, or any tissues from the control group. CONCLUSIONS: NGF is present and produced in the anterior segment of the eye and is released in the aqueous humor in the basal state. Experimental iridectomy induces increased production of NGF in the iris and in the ciliary body and an increased concentration of NGF in the aqueous humor.