Avaliação histomorfométrica e da proliferação celular em úlceras de córnea superficiais, induzidas em coelhos, após o uso de colírio de Citrus lemon / Histomorphometry evaluation and cellular proliferation in superficial corneal ulcer, induced in rabbits, after using eyedrops of Citrus lemon

O objetivo do presente estudo foi avaliar e comparar, por meio de histomorfometria e imuno-histoquímica para PCNA (Antígeno Nuclear de Proliferação Celular), o processo de reparação corneal de úlceras superficiais induzidas em coelhos e tratadas com colírios de óleo essencial de Citrus lemon (CL). Foram utilizadas 40 fêmeas da espécie leporina, constituindo-se quatro grupos experimentais de 10 animais cada. Todos os animais foram submetidos à indução da úlcera superficial experimental por meio da aplicação tópica de n-heptanol. Em dois grupos foram instilados colírios à base de óleo essencial de Citrus lemon, em diferentes concentrações, sendo 3% (GL3) e 5% (GL5). Outro grupo foi tratado com Tween 80 8% (GT), que é o diluente utilizado na produção dos colírios de CL; o grupo controle (GC) recebeu apenas substituto da lágrima. Todos os colírios foram aplicados quatro vezes ao dia. Os grupos experimentais foram distribuídos em dois subgrupos, com cinco animais cada, de acordo com os períodos finais de avaliação. O primeiro subgrupo (M1) foi avaliado após 24 horas e o segundo (M2), após cinco dias. Nas comparações entre os momentos iniciais e finais, os grupos tratados com substituto da lágrima, Tween 80 8% e colírio à base de óleo essencial de Citrus lemon 5% promoveram aumento na espessura epitelial na periferia da córnea e maior percentual de proliferação celular. Não houve diferença de celularidade entre os tratamentos. Os colírios à base de óleo essencial de Citrus lemon, nas diferentes concentrações, promoveram a reepitelização corneal, sem causar lesões adicionais ao epitélio ou estroma corneal, podendo ser utilizado na superfície ocular.

The aim of this study was to evaluate and compare through histomorphometry and immunohistochemistry for PCNA (Proliferating Cell Nuclear Antigen), the repair process in superficial corneal ulcers induced in rabbits and treated with eyedrops of Citrus lemon (CL) essential oil. Fifty female rabbits were used and divided into 4 experimental groups of 10 animals each one. Every animal underwent induction of experimental superficial ulcer by topical application of n-heptanol. Three groups were treated with eyedrops of Citrus lemon essential oil in two different concentrations: 3% (GL3) and 5% (GL5). Another group was treated with Tween 80 8% (GT), which is the solvent used in the production of eyedrops of CL; the control group (CG) received only tear substitute. All eyedrops were applied four times daily. The experimental groups were divided into two subgroups with five animals in each one, according to the final evaluation periods. The first subgroup (M1) was evaluated after 24 hours and the second (M2) after 5 days. In the comparison between the initial and final moments, the groups treated with tear substitute, Tween 80 8% and eyedrops of Citrus lemon essential oil 5% had an increase in epithelial thickness at the periphery of the cornea and a higher percentage of cell proliferation. There was no difference in cellularity between treatments. The eyedrops of Citrus lemon essential oil, at different concentrations, promoted corneal reepithelialization without causing further injury to the epithelium and corneal stroma, so they can be used on the ocular surface.

Effect of gap junction on the cardioprotection of ischemic postconditioning in rat heart / 中国应用生理学杂志

<p><b>AIM</b>To determine whether the cardioprotection of ischemic postconditioning and heptanol in ischemic heart against ischemia/reperfusion (I/R) is mediated by gap junction.</p><p><b>METHODS</b>The effect of ischemic postconditioning, heptanol at different doses (0.03, 0.06, 0.30, and 0.60 mg/kg) and AAP10 (10 mg/kg) on the intact rat heart during 30 min ischemia and 2 h of reperfusion was observed. Ischemic postconditioning was achieved by 3 cycles of 10 s reperfusion/10 s regional ischemia starting at the beginning of the reperfusion. The infarct size and the arrhythmia scores were measured. The effect of ischemic postconditioning, heptanol at different doses (0.05, 0.10, 0.50 and 1.00 mmol/L) and AAP10 (1 x 10(-7)mol/L) on the isolated heart during 30 min ischemia and 2 h of reperfusion was observed. Ischemic postconditioning was achieved by 6 cycles of 10 s reperfusion/10 s global ischemia starting at the beginning of the reperfusion. The arrhythmia scores and conduction velocity of ventricle muscle were measured.</p><p><b>RESULTS</b>In the intact rat heart model, ischemic postconditioning and heptanol reduced infarct size and arrhythmia scores. In the Langendorff perfused rat heart model, ischemic postconditioning and heptanol reduced arrhythmia scores and conduction velocity of ventricle muscle. Administration of AAP10, an opener of gap junction attenuated the cardioprotection of ischemic postconditioning and heptanol.</p><p><b>CONCLUSION</b>The cardioprotection of ischemic postconditioning and heptanol may be related to the attenuation of gap junction communication on myocardiac ischemia/reperfusion injury.</p>

Functional Connectivity Map of Retinal Ganglion Cells for Retinal Prosthesis

Retinal prostheses are being developed to restore vision for the blind with retinal diseases such as retinitis pigmentosa (RP) or age-related macular degeneration (AMD). Among the many issues for prosthesis development, stimulation encoding strategy is one of the most essential electrophysiological issues. The more we understand the retinal circuitry how it encodes and processes visual information, the greater it could help decide stimulation encoding strategy for retinal prosthesis. Therefore, we examined how retinal ganglion cells (RGCs) in in-vitro retinal preparation act together to encode a visual scene with multielectrode array (MEA). Simultaneous recording of many RGCs with MEA showed that nearby neurons often fired synchronously, with spike delays mostly within 1 ms range. This synchronized firing - narrow correlation - was blocked by gap junction blocker, heptanol, but not by glutamatergic synapse blocker, kynurenic acid. By tracking down all the RGC pairs which showed narrow correlation, we could harvest 40 functional connectivity maps of RGCs which showed the cell cluster firing together. We suggest that finding functional connectivity map would be useful in stimulation encoding strategy for the retinal prosthesis since stimulating the cluster of RGCs would be more efficient than separately stimulating each individual RGC.

Role of Gap Junction in the Regulation of Renin Release and Intracellular Calcium in As 4.1 Cell Line

Gap junction protein, connexin, is expressed in endothelial cells of vessels, glomerulus, and renin secreting cells of the kidney. The purpose of this study was to investigate the role of gap junction in renin secretion and its underlying mechanisms using As 4.1 cell line, a renin-expressing clonal cell line. Renin release was increased proportionately to incubation time. The specific gap junction inhibitor, 18-beta glycyrrhetinic acid (GA) increased renin release in dose-dependent and time- dependent manners. Heptanol and octanol, gap junction blockers, also increased renin release, which were less potent than GA. GA-stimulated renin release was attenuated by pretreatment of the cells with amiloride, nifedipine, ryanodine, and thapsigargin. GA dose-dependently increased intracellular Ca2+ concentration, which was attenuated by nifedipine, nimodipine, ryanodine, and thapsigargin. However, RP-cAMP, chelerythrine, tyrphostin A23, or phenylarsine oxide did not induced any significant change in GA-stimulated increase of intracellular Ca2+ concentration. These results suggest that gap junction plays an important role on the regulation of renin release and intracellular Ca2+ concentration in As 4.1 cells.

Inhibition of Corneal Neovascularization by Subconjunctival Injection of alphaVbeta5 Integrin Antibody in Rabbit

PURPOSE: To investigate the efficacy of a subconjunctival injection of alphaVbeta5 integrin antibody on corneal angiogenesis induced by chemical epithelial denudation in a rabbit eye model. METHODS: One week after debridement by heptanol, rabbits were treated with a subconjunctival injection of alphaVbeta5 integrin antibody or control immunoglobulin G weekly for 2 weeks. Rabbits that did not receive injection after debridement served as the untreated group. The percentage of neovascularized corneal area was calculated by biomicroscopy, and the sectioned area and number of new vessels were calculated by histological examinations. RESULTS: At 7 days after the first injection, alphaVbeta5 integrin antibody-treated eyes had 9.5% (P=0.02) and 6.8% (P=0.03) less neovascularized corneal area than vector-treated eyes and untreated eyes, respectively. At 7 days after the second injection, alphaVbeta5 integrin antibody-treated eyes had 21.1% (P=0.02) and 18.3% (P=0.02) less neovascularized corneal area than vector-treated eyes and untreated eyes, respectively. Light microscopic examination showed a smaller neovascularized corneal area and a reduced number of new vessels in the alphaVbeta5 integrin antibody-treated eyes compared to the control eyes. CONCLUSIONS: Subconjunctival injection of alphaVbeta5 integrin antibody effectively reduces experimental corneal neovascularization induced by chemical injury, and could be used as a corneal angiogenesis inhibitor in the future.

Modelos experimentais de deficiência limbar em coelhos: análise clínica / Experimental models of limbal deficiency in rabbits: clinical analysis

Objetivo: Compararem coelhos três modelos experimentais de destruiçäo das células germinativas (CG) do limbo corneano quanto a aspectos clínicos. Métodos: Foram utilizados 54 coelhos, 108 olhos, subdivididos em 3 grupos experimentais: (G 1), (G2) e (G3), cada um formado pelos OE de 18 coelhos, submetidos às técnicas experimentais (T1), (T2) e (T3), respectivamente, e um grupo controle, formado pelos 54 olhos contralaterais (OD). Nas três técnicas foi utilizado o n-heptanol. Na T1, o n-heptanol foi aplicado por 5 minutos, para remoçäo do epitélio límbico. Na T2, além da aplicaçäo do n-heptanol, realizou-se peritomia e remoçäo da conjuntiva perilímbica até 4 mm do limbo, juntamente com a escarificaçäo do tecido episcleral. Na T3, além dos procedimentos da T2, foi realizada dissecçäo lamelar do limbo abrangendo aproximadamente 1,5 mm na periferia da córnea e 2 mm na superfície escleral. Em todas as córneas dos animais foram estudados seis parâmetros clínicos: neovascularizaçäo, perda da transparência, irregularidade da superfície, reparaçäo epitelial, erosäo ou defeito epitelial, granuloma e outras lesöes corneanas. Resultados: A neovascularizaçäo corneana iniciou-se mais precocemente com a T1 e T2; ocorreu em 100 por cento dos casos com as três técnicas, de forma näo homogênea, variando de leve a intensa; permaneceu estável a partir do 28 dia até o final do experimento (56 dia), foi maior nos quadrantes superior e inferior e menor nos quadrantes nasal e temporal. A perda da transparência e a irregularidade da superfície corneana foram menores com a T 1 que com a T2 e T3, que foram similares entre si. Houve, nas três técnicas experimentais, latência de três dias para o início da reepitelizaçäo, que se completou com a T1 no 7 dia e com a T2 e T3 no 14 dia. Erosäo epitelial corneana e granuloma corneano foram encontradas de forma similar nas três técnicas experimentais. Conclusöes: A T2 e T3 mostraram-se adequadas como possíveis modelos de ampla remoçäo das CG límbicas, levando a resultados similares nos diversos parâmetros estudados. A Tl se mostrou adequada como modelo de remoçäo parcial do epitélio límbico. Ocorreu conjuntivalizaçäo e neovascularizaçäo nas três técnicas experimentais.

Early Effect of Autologous Limbal Transplantation Immediately following Total Limbal Injury in Rabbits

Limbal epithelium plays a great role in reconstruction of damaged corneal epithelium and early limbal transplantation showed better results in limbal injury.However, there is no consensus about the appropriate time for limbal transplantation.We, therefore, investigated the results of autologous limbal transplantation (ALT)immediately following limbal epithelial injury in rabbits.We classified the rabbits in three groups whether the application of ALT and therapeutic contact lens were done or not.Injury of limbal and corneal epithelium was made by application of n-heptanol and tarsorrhaphy was done in all groups.ALT from the healthy fellow eye was done in Group 1 and Group 2 but not in Group 3, control group.Therapeutic soft contact lens was applied to Group 2 after ALT.We evaluated epithelial defect, haze, and neovascularization of corneas at 3 days, 1 week, and 2 weeks after operation.We also examined tissue specimen of corneas after two weeks of operation.Epithelial defect was almost healed within 2 weeks after operation in Group 1 and Group 2, but Group 3 showed persistent epithelial defect. Corneal neovascularization and haze were the most severe in Group 3, but was not so severe in Group 1 and Group 2, and there was no significant difference between the two groups.On histopathologic examination, Group 1 and Group 2 showed almost normal corneal epithelium though a few inflammatory cells and goblet cells were observed in some cases but control group showed severe inflammaton and many new vessels and goblet cells.In conclusion, ALT immediately following severe limbal injury is effective in reconstructing corneal epithelium.

p53 Suppressor Gene Ove rex p ression in the Cornea after Mechanical-and Chemical-induced Epithelial Injury

p53 suppressor gene expression and protein production increases in response to DNA damage and the subsequent cell cycle prolongation permits DNA repair or, in the severe cases, leads to apoptosis. These concepts led us to investigate the expression of p53 as a potential key regulator of DNA repair in the cornea after mechanical-and chemical-induced injury. Mechanical-induced injury was performed by denuding a4mmdiameter area of the central epithelium from the corneas of Sprague Dawley rats. Chemical-induced injury was performed by applying a 3.5 mmdiameter filter paper disc of saturated n-heptanol to the cornea. Samples were collected on the 1st 3rd and 7th day of treatment. We performed immunohistochemistry and Western blot analysis on corneal buttons. Control group did not receive any treatment. The immunohistochemical analysis demonstrated that p53 is localized in the anterior stromal keratocytes. Western blot analysis indicated p53 bandsin the lanes of the mechanically and the chemically injured corneas. Our results suggest that injury to the corneal epithelium triggers the activation of p53.

The Anti-angiogenic Effect of Recombinant Tissue Inhibitor of Metalloproteinase Type 2 (TIMP-2) in N-heptanol Induced Neovascularization of Rat Cornea

Corneal neovascularization is a challenging problem in ophthalmologic practice. We evaluated the anti-angiogenic effect of recombinant tissue inhibitor of metalloproteinase type 2 (rTIMP-2) which is an endogenous inhibitor of matrix metalloproteinases. Sprague-Dawley rats of 6 weeks of age were used in this study. N-heptanol was applied to the eyes of the rats to induce chemical injury and eventual neovascularization. The rats were divided into 4 groups, 5 rats for each. We instilled only the phosphate buffered solution once a day for 10 days to the eyes of rats in the control group. Rats in group 1 received subconjunctival injection of 0.05 mgof rTIMP-2, those in group 2 received intraperitoneal injection of 0.05 mg of rTIMP-2, and those in group 3 received intraperitoneal injection of 0.1 mg of rTIMP-2 once a day for 10 days respectively. After 4 weeks, photographs of corneas were taken with a built-in camera of the slit lamp, and the eyes were enucleated. We made histologic specimens of the corneas and examined them with a light microscope. The severity of the neovascularization was quantified with angiogenesis scoring system. The group 1, 2 and 3 showed significant suppression of angiogenesis compared with the control respectively, but there was no statistically significant difference among their angiogenesis scores. Under the light microscope, the corneas of control group showed much more severe infiltration of inflammatory cells and higher density of new vessels compared with group 1, 2 and 3. We hypothesize that TIMP-2 suppressed the angiogenesis in chemical injury of cornea and TIMP-2 might benefit those patients with corneal neo-vascularization, although careful further studies are required in humans.

The Expression of NADPH-diaphorase in Corneal Neovascularization of streptozotocin Induced Diabetic Rat

We evaluated the effect of nitric oxide on the corneal neovascularization in diabetic rats. The NADPH-diaphorase histochemistry was used to investigate the relationship between the nitric oxide synthase activity and the corneal neovascularzation of disbetic rats. Sprague-Dawley rats of 6 weeks of age were use in this study. Streptozotocin(65mm/kg, Sigma, USA) was injected intraperitoneally to induce diabetes. N-heptanol was applied to the right eyes of the diabetic rats to induce neovascularization and the left eyes were remained uninjured. After four weeks, 5 eyes of the non-diabetic rats, 5 eyes of the diabetic rats, and 5 eyes of the neovascularized diavetic rats were enucleated. The enucleated eyes were stained with NADPH-diaphorase histochemistry method and examined under the light microscope. Neovascularized corneas showed intense expression of NADPH-diaphorase at the whole layer of epithelium, the superficial stroma, and the walls of new vessels. Corneas of the nondiabetic rats and uninjured those of diabetic rats showed only mild expression of NADPH-diaphorase, and there were no significant difference of expression between them. According to these results, we think that the increased expression of nitric oxide synthase or nitric oxide may be related to the corneal neovascularization in diabetic rats.

Effect of Topical Na-Hyaluronan on Epithelial Cell Morphogenesis and Superficial Stromal Healing in Corneal Wound

The effect of 1% Na-Hyaluronan(Na-HA) on the morphogenesis of microvilli in superficial epithelial cells and of hemidesmosomes in basal epithelial cells together with the organization of superficial stromal collagen were evaluated in n-heptanol induced corneal epithelial wounds. Epithelial wounds were produced by applying a 5.5mm round filter paper, soaked in n-heptanol, on the central cornea for 60 seconds. 1% Na-HA in phosphate buffered saline(PBS) or PBS alone were instilled 4 times a day for 3 days. Epithelial healing rates determined during the first two days were not altered by Na-HA. The number of microvilli in superficial epithelial cells and of collagens fibers in superficial stroma were approximately the same between two groups. However, the number of hemidesmosome in the central cornea, which was counted in 2micrometer length of the basement membrane, significantly increased by the treatment with 1% Na-HA, being 10.0+/-1.1 in the 1% Na-HA treated group and 6.5+/-2.5 in the control group. The results suggest that topically applied 1% Na-HA may enhance the formation of hemidesmosome in n-heptanol wounded cornea.

Influencia del tejido limbal en la regeneración epitelial corneal / Influence of the limbal tissue in the corneal ephitelium regeneration

Las células madres del epitelio corneal se piensa que están localizadas en el limbo. El tejido limbal fue removido quirúrgicamente en uno de los ojos de 56 conejos New Zealand y se practicó la desepitelización corneal en ambos ojos utilizando n-heptanol. Comparamos el crecimiento epitelial entre ambos ojos a las 24 horas, 48 horas, 72 horas, 7 días y ocho semanas. En un grupo constituído por 5 conejos practicamos sólo la desepitelización corneal en 7 mm. centrales. Evidenciamos un crecimiento mayor, estadísticamente significativo, en los ojos controles en los cuales se conservó el tejido limbal que en aquellos en los cuales se removió y mayor en los defectos epiteliales menores que en aquellos extensos. Observamos además mayor conjuntivalización y vascularización en los ojos queratolimbectomizados que en los controles, fenómeno éste que fue inversamente proporcional a la reepitelización

Comparison of Central Corneal Epithelial Healing Rates Between Different Wound Conditions

The role of limbal epithelial cells on the initial re-surfacing of the central epithelial defect was evaluated. Central or peripheral corneal wounds were produced by using n-heptanol in New Zealand white rabbit, and the epithelial healing rates were evaluated. Central corneal wounds made by applying a filter paper soaked in 0.2 N NaOH with vs without limbal damage were produced for the paralleled set of experiment. A 5.5 mm round filter paper for central wound and a 2 mm filter paper strip for limbal damage were used for the initial wounding. Epithelial defect areas were photodocumented after fluorescein stainmg at 0.6, 18, 30, 42, 48 hours following initial damage. Epithelial healing rates were measured by linear regression analysis, The epithelial healing rates of central and peripheral heptatlol wounds were 0.71 +/- 0.14 mm2/h, and 0.79 +/- 0.10 mm2/h(p>0.05), respectively, NaOH central wound without limbal damage and with limbal damage were 0.77 +/- 0.11 mm2/h, and 0.76 +/- 0.11 mm2/h, respectively(p>0.05). These data suggest that the limbal epithelium has no influence on the initial re-surfacing of the central epithelial defect.