RESUMEN
Despite differences in the processes leading to tissue damage, the ocular irritation response to various surfactants, two concentrations of an acid and an alkali, and an acetone, alcohol, aromatic amine, and aldehyde has been shown to depend on the extent of initial injury. The purpose of this study was to assess the extent to which this fundamental relationship exists for bleaching agents in the rabbit low-volume eye test. Ten microl of sodium perborate monohydrate (NaBO3), sodium hypochlorite (NaOCl), 10% hydrogen peroxide (H2O2), and 15% H2O2 was applied directly to the cornea of the right eye of each rabbit. Macroscopic assessments for irritation were made 3 hours after dosing and periodically until 35 days. Light microscopic examinations were conducted on tissues obtained at 3 hr and on 1, 3, and 35 days. In vivo confocal microscopy (CM) and measurements of dead corneal epithelial cells and keratocytes at 3 hours and 1 day were used to characterize quantitatively initial corneal injury, while in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days was used to characterize quantitatively the corneal changes over time. The changes with NaBO3 and NaOCl were consistent with mild irritancy. For both, corneal injury was limited to the epithelium and superficial stroma. The changes with 10% H202 and 15% H2O2 were consistent with severe irritation. Both concentrations affected the epithelium and deep stroma, with 15% H2O2 also at times affecting the endothelium. However, unlike other irritants previously studied, with 10% H2O2 and 15% H2O2 there was an incongruity between the extent of epithelial and stromal injury, with stromal injury being more extensive than epithelial injury. A similar, although less dramatic, effect was observed with NaBO3. Additionally, there was still significant keratocyte loss at 35 days with 10% H2O2 and 15% H2O2 even though the eyes at times were considered to be macroscopically normal. These observations highlight the need to include both epithelial and stromal components in an ex vivo or in vitro alternative assay. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. Importantly, we have identified unique differences in the ocular injury and responses occurring with bleaching agents that are important to consider in the development and validation of alternative ocular irritation tests to characterize a broad range of materials differing in type and irritancy.
Asunto(s)
Boratos/toxicidad , Córnea/efectos de los fármacos , Enfermedades de la Córnea/patología , Peróxido de Hidrógeno/toxicidad , Irritantes/toxicidad , Hipoclorito de Sodio/toxicidad , Animales , Conjuntiva/efectos de los fármacos , Conjuntiva/patología , Córnea/patología , Enfermedades de la Córnea/inducido químicamente , Relación Dosis-Respuesta a Droga , Iris/efectos de los fármacos , Iris/patología , Masculino , Microscopía Confocal , Conejos , Factores de TiempoRESUMEN
The ocular irritation responses to 11 different surfactants and two concentrations of acetic acid and sodium hydroxide have been shown to depend on the extent of initial injury, despite marked differences in the processes leading to tissue damage. The purpose of these studies was to determine the extent to which this fundamental relationship applies to other nonsurfactants. Ten microl of acetone (ACT). cyclohexanol (CY), parafluoroaniline (PF), or 37% formaldehyde (FA) was directly applied to the cornea of the right eye of each rabbit. Eyes and eyelids were macroscopically scored for signs of irritation beginning 3 hours after dosing and periodically until recovery or 35 days. Tissues were obtained for light microscopic examination after 3 hours and on days 1, 3, and 35. Initial corneal injury was characterized quantitatively at 3 hours and I day using in vivo confocal microscopy (CM) and by postmortem quantitation of dead corneal epithelial cells and keratocytes using a Live Dead Assay (L/D, Molecular Probes) and scanning laser CM. Corneal changes over time were characterized quantitatively using in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days. The changes with ACT were consistent with mild irritation. Corneal injury was limited to the epithelium and superficial stroma, with the mean normalized depth of injury (NDI) being less than 10% with the majority of regions showing no stromal injury. Changes with CY and PF were consistent with moderate to severe irritation, and FA caused severe irritation. Specifically, corneal injury by CY and PF tended to involve the epithelium and anterior stroma, with the mean NDI being 10.4% to 23.8%, while injury with FA involved the epithelium, deep stroma, and at times the endothelium. Interestingly, with FA significantly less injury was observed at 3 hours with a dramatic increase in injury observed at 1 day and thereafter. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. We believe this approach can be applied to developing alternative assays based on injury to ex vivo eyes or injury to an in vitro corneal equivalent system.
Asunto(s)
Acetona/toxicidad , Compuestos de Anilina/toxicidad , Enfermedades de la Córnea/inducido químicamente , Ciclohexanoles/toxicidad , Formaldehído/toxicidad , Irritantes/toxicidad , Acetona/administración & dosificación , Administración Tópica , Compuestos de Anilina/administración & dosificación , Alternativas a las Pruebas en Animales , Animales , Muerte Celular , Conjuntiva/efectos de los fármacos , Conjuntiva/patología , Enfermedades de la Córnea/patología , Sustancia Propia/efectos de los fármacos , Sustancia Propia/patología , Ciclohexanoles/administración & dosificación , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/patología , Párpados/efectos de los fármacos , Párpados/patología , Femenino , Fluoruros/toxicidad , Formaldehído/administración & dosificación , Irritantes/administración & dosificación , Masculino , Microscopía Confocal , Conejos , Factores de Tiempo , Pruebas de Toxicidad , Cicatrización de HeridasRESUMEN
Based on studies that have characterized the extent of injury occurring with irritants of differing type and severity, we have proposed that extent of initial injury is the principal mechanism underlying ocular irritation. We report here our efforts to apply this hypothesis, as a mechanistic basis, to the development of an alternative eye irritation assay using an ex vivo rabbit corneal model. Rabbit eyes were obtained immediately after sacrifice or from an abattoir and 8.5-mm diameter corneal buttons were removed and cultured overnight at an air-liquid interface under serum-free conditions. Buttons were exposed to materials of differing type (surfactant, acid, base, alcohol and aldehyde) and irritancy (slight to severe) that had been previously characterized microscopically in the rabbit low-volume eye test. Exposure was accomplished by applying 1.5 microl of an irritant to a sterile, 3 mm diameter, filter paper disk and then placing the disk on the center of the corneal button for 10 s. After removal of the disk, buttons were washed and cultured for 3, 24 or 48 h. Buttons were then evaluated for extent of injury using a Live/Dead staining kit and fluorescent microscopy to measure cell size of live surface epithelial cells, area of epithelial denudation and depth of stromal injury. Ex vivo exposure to slight irritants generally reduced surface epithelial cell size (i.e. erosion) while exposure to mild irritants produced epithelial denudation with variable injury to the corneal stroma. Severe irritants generally produced extensive epithelial denudation and damaged the corneal stroma and endothelium. Overall, ex vivo extent of injury significantly correlated with in vivo extent of injury as measured in previous animal tests (r=0.81, P<0.001). These findings indicate that extent of corneal injury, as shown to be associated with ocular irritation occurring in vivo, can be applied to the development of a mechanistically-based alternative eye irritation model. We believe that this approach may ultimately lead to an alternative assay to replace the use of animals in ocular irritation testing.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Córnea/efectos de los fármacos , Ojo/efectos de los fármacos , Irritantes/toxicidad , Pruebas de Toxicidad/métodos , Ácido Acético/toxicidad , Acetona/toxicidad , Animales , Colorantes , Ciclohexanoles/toxicidad , Fluoresceínas , Formaldehído/toxicidad , Microscopía Confocal/métodos , Valor Predictivo de las Pruebas , Conejos , Hidróxido de Sodio/toxicidad , Tensoactivos/toxicidadRESUMEN
Defining the extent of initial injury has proven to be a useful basis for differentiating the ocular irritation potential of surfactants; however, the applicability of this method to other types of irritants has not been demonstrated. In the following studies we characterized the extent of corneal injury following exposure to different concentrations of acetic acid and sodium hydroxide (NaOH) in the rabbit low-volume eye test. Groups of rabbits received 3% acetic acid, 10% acetic acid, 2% NaOH, or 8% NaOH and were evaluated in vivo by macroscopic and in vivo confocal microscopic examination and postmortem using a live/dead staining kit and scanning laser confocal microscopic examination. Quantitative assessment of macroscopic scores, corneal surface epithelial cell size, corneal epithelial thickness, corneal thickness, depth of stromal injury, corneal light scattering (confocal microscopy through focusing, CMTF), and number of dead cells was conducted at various times, including the following: at 3 hours and at 1, 3, 7, 14, and 35 days. Based on macroscopic scores, the order of ocular irritancy potential was 3% acetic acid < 2% NaOH < 10% acetic acid < 8% NaOH. Evaluation of the quantitative in vivo and postmortem microscopic live/dead data revealed a slight decrease in epithelial thickness and an increase in dead epithelial cell numbers with 3% acetic acid. With 2% NaOH, significant focal changes in epithelial cell size, epithelial thickness, corneal thickness, and number of dead surface epithelial cells occurred at 3 hours and at 1 day, with injury to only a very small number of corneal stromal keratocytes, despite the presence of epithelial denudation. Changes with 10% acetic acid were similar to those noted with 2% NaOH at 3 hours and 1 day, but these changes were more diffuse and included stromal injury to a depth of 7.2 +/- 9.3% of the corneal thickness, with significant numbers of dead keratocytes. Eight percent NaOH, on the other hand, caused focally extensive injury that averaged 26.3 +/- 18.4% of the corneal thickness at 1 day, with significant light scattering from the cornea, which did not return to normal by 35 days postinjury. Overall, these data indicate that ocular irritation as a result of acetic acid and NaOH was associated with changes similar to those observed with surfactants (ie, slight irritants damage the corneal epithelium, mild and moderate irritants damage the corneal epithelium and anterior stromal cells, and severe irritants damage the corneal epithelium and deep stroma). To our knowledge, this is the first time that the ocular irritation potential for different types of materials (acid/alkali, surfactants) has been shown to be primarily dependent on the initial area and depth of injury.
Asunto(s)
Ácido Acético/toxicidad , Enfermedades de la Córnea/inducido químicamente , Topografía de la Córnea/métodos , Microscopía Confocal/métodos , Hidróxido de Sodio/toxicidad , Ácido Acético/administración & dosificación , Enfermedad Aguda , Animales , Muerte Celular , Tamaño de la Célula , Córnea/efectos de los fármacos , Córnea/patología , Enfermedades de la Córnea/patología , Sustancia Propia/efectos de los fármacos , Sustancia Propia/patología , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/patología , Ojo/efectos de los fármacos , Ojo/patología , Conejos , Hidróxido de Sodio/administración & dosificación , Factores de TiempoRESUMEN
Differences in ocular irritancy have been hypothesized to reflect differences in the extent of initial injury. Although differences in the processes leading to tissue damage may exist, extent of injury is believed to be the principal factor determining final outcome of ocular irritation. Previous studies characterizing the pathology of surfactant-induced ocular irritation support this premise. The purpose of this study was to begin to determine the applicability of this premise in terms of nonsurfactants; we planned to accomplish this by assessing the ocular irritancy of different concentrations of an acid and an alkali. Ten microliters of 3 or 10% acetic acid (C2H4O2) or 2 or 8% sodium hydroxide (NaOH) were directly applied to the cornea of the right eye of each test rabbit. Untreated left eyes served as the controls. Eyes and eyelids were macroscopically examined for signs of irritation beginning 3 hours after dosing and periodically until recovery or day 35. Eyes and eyelids from animals in each group were collected for microscopic examination after 3 hours and on days 1, 3, and 35. The macroscopic and microscopic changes were consistent with slight (3% C2H4O2), mild (2% NaOH, 10% C2H4O2), and severe (8% NaOH) irritancy. The spectra of changes were similar to those previously reported for surfactants of differing types and irritancies. As with surfactants, as the extent of initial injury increased, the intensity and duration of the subsequent responses increased. These results indicate that our hypothesis also applies to nonsurfactants. The results also support our belief that the initial extent of injury associated with ocular irritation may be used to predict the subsequent responses and final outcome. Finally, our results further indicate that such an approach may be applicable to the development of alternative assays that are based on either injury to ex vivo eyes or injury to an in vitro corneal equivalent system.
Asunto(s)
Ácido Acético/toxicidad , Oftalmopatías/inducido químicamente , Oftalmopatías/patología , Hidróxido de Sodio/toxicidad , Ácido Acético/administración & dosificación , Animales , Conjuntiva/efectos de los fármacos , Conjuntiva/patología , Córnea/efectos de los fármacos , Córnea/patología , Ojo/efectos de los fármacos , Ojo/patología , Párpados/efectos de los fármacos , Párpados/patología , Masculino , Conejos , Hidróxido de Sodio/administración & dosificaciónRESUMEN
We have hypothesized that differences in ocular irritancy are related to differences in extent of initial injury and that, regardless of the processes leading to tissue damage, extent of injury is the primary factor that determines the final outcome of ocular irritation. In previous in vivo confocal microscopic (CM) studies we identified quantifiable differences in the extent of corneal injury occurring with four surfactants (three anionic, one cationic) known to cause different levels of ocular irritation and demonstrated that extent of initial corneal injury was related to the magnitude of cell death. The purpose of this study was to assess the applicability of this hypothesis to a broad sampling of surfactants. Specifically, initial corneal changes induced by seven different surfactants (one anionic, three cationic, three nonionic) were measured by in vivo CM and cell death was measured by an ex vivo live/dead assay. The right eye of each rabbit was treated by placing 10 microl of a surfactant directly on the cornea. Eyes were examined macroscopically and scored for irritation at 3 h and 1 day. At 3 h and 1 day, in vivo CM was used to examine the corneas and quantitate epithelial cell size, epithelial thickness, corneal thickness, and depth of stromal injury. At 3 h and/or at 1 day, corneas were removed and excised regions were placed in culture media containing 2 microM calcein AM and 4 microM ethidium homodimer. Using laser scanning CM, the number of dead epithelial and/or stromal cells in a 300 x 300 x 170-microm3 (xyz) volume of the cornea was determined. In vivo CM and live/dead assay findings revealed three surfactants to affect only the epithelium, three surfactants to affect the epithelium and superficial stroma, and one surfactant to affect the epithelium and deep stroma. Extent of initial corneal injury reflected level of ocular irritation, and magnitude of cell death was related to the extent of initial corneal injury. These findings are consistent with those for known slight, mild, and moderate to severe irritants, respectively. They suggest that our hypothesis is broadly applicable to surfactants. Additionally, we believe these surfactants should be included as part of a new "gold standard" for use in developing and validating in vitro tests to replace the use of animals in ocular irritancy testing.
Asunto(s)
Lesiones de la Cornea , Irritantes/toxicidad , Tensoactivos/toxicidad , Animales , Córnea/efectos de los fármacos , Lesiones Oculares/inducido químicamente , Femenino , Irritantes/clasificación , Masculino , Microscopía Confocal , Necrosis , Conejos , Tensoactivos/clasificaciónRESUMEN
In vivo confocal microscopy (CM) provides a unique ability to section optically through living, intact tissues and organs to characterize qualitatively and quantitatively pathological changes in 4 dimensions (x, y, and z, and time). It involves the capture of real-time images without the need for excision, fixation and processing. In vivo CM principally has been used for evaluation of eyes in patients and laboratory animals but has potential application to studies of other tissues/organs. In vivo CM is being used in human ophthalmology clinics. It has been used as a research tool for quantitative, in situ measurement of corneal wound contraction, fibroblast migration, corneal endothelial cell migration, corneal epithelial cell size and desquamation following contact lens wear and surgery, and the assessment of corneal surface toxicity following application of commonly used ophthalmic preservatives. In vivo CM allows us to (a) characterize changes to a light microscopic (i.e., cellular) level; (b) quantify changes objectively: (c) conduct studies of injury and repair in the same animal and directly correlate microscopic changes to clinical observations over time as this technique is used in the living animal; and (d) conduct comparative studies in humans. Here we present a brief overview of in vivo CM and how we are using it to provide noninvasive, in situ qualitative and quantitative histopathologic characterization of accidental ocular irritation. Our intent is to provide an awareness of this relatively new methodology and one practical application of its use in research. The goal of our work is to provide objective, quantitative data for use in developing and validating mechanistically based in vitro replacement tests.
Asunto(s)
Enfermedades de la Córnea/inducido químicamente , Enfermedades de la Córnea/patología , Irritantes/toxicidad , Microscopía Confocal/métodos , Animales , HumanosRESUMEN
PURPOSE: To correlate area and depth of initial corneal injury induced by surfactants of differing type and irritant properties with corneal responses and outcome in the same animals over time by using in vivo confocal microscopy (CM). METHODS: Six groups of six adult rabbits were treated with anionic, cationic, and nonionic surfactants that caused different levels of ocular irritation. Test materials included slight irritants: 5% sodium lauryl sulfate (SLS), polyoxyethylene glycol monoalkyl ether (POE), and 5% 3-isotridecyloxypropyl-bis(polyoxyethylene) ammonium chloride (ITDOP); mild irritants: 5% 3-decyloxypropyl-bis(polyoxyethylene) amine (DOP) and sodium linear alkylbenzene sulfonate (LAS); and a moderate irritant: a proprietary detergent (DTRGT). Ten microliters surfactant were directly applied to the cornea of one eye of each rabbit. Ten untreated rabbits served as control subjects. Area and depth of initial injury was determined by using in vivo CM to measure epithelial thickness, epithelial cell size, corneal thickness, and depth of stromal injury in four corneal regions at 3 hours and at day 1. Area and depth of corneal responses to injury were evaluated at various times from days 3 through 35 by macroscopic grading and quantitative confocal microscopy through-focusing (CMTF). RESULTS: In vivo CM revealed corneal injury with slight irritants to be restricted to the epithelium, whereas the mild and moderate irritants caused complete epithelial cell loss with increasing anterior stromal damage: DOP < LAS < DTRGT. With the slight ocular irritants there was little or no change in corneal thickness or the CMTF intensity profiles. Three hours after treatment, mild and moderate ocular irritants caused a significant increase in corneal thickness, which peaked at day 1 with DOP (483.3+/-80.1 microm) and LAS (572.3+/-60.0 microm) and day 3 with DTRGT (601.4+/-68.7 microm); returning to normal (similar to control values) by day 7 with DOP and day 35 with LAS and DTRGT. The CMTF intensity profiles also showed significant elevation over that in the anterior stroma, which peaked at day 1 with DOP (14,608+/-4,306 U [U is defined as micrometers X pixel intensity]) and day 3 with LAS and DTRGT (18,471+/-6,581 U and 22,424+/-3,704 U, respectively) and returned toward normal by day 7 with DOP and day 14 with LAS and DTRGT. Elevated CMTF profiles principally reflected the presence of hyperreflective, punctate keratocytes and inflammatory cells at days 1 and 3 and the presence of activated keratocytes at day 7. There was a significant correlation between the elevated CMTF intensity profile and the corresponding macroscopic total score in each eye (r = 0.839; P < 0.001). More important, there was a significant correlation between area and depth of initial stromal injury measured at day 1, regardless of ocular irritant and the stromal response measured by the area under the CMTF intensity profile curve in each cornea (r = 0.87; P < 0.0005). A significant correlation between the area and depth of injury and the area under the corneal thickness curve was also observed in each cornea (r = 0.75; P < 0.0005). CONCLUSIONS: In individual animals, the extent of initial stromal injury correlated with the magnitude of the corneal responses, measured by the change in corneal thickness and the CMTF depth intensity profile. These findings further support the hypothesis that area and depth of injury are the principal factors determining the early responses and eventual repair processes after accidental eye irritation. They also support the proposed use of area and depth of acute injury as a mechanistic correlate to ocular irritation in the development and validation of potential in vitro ocular irritation tests.
Asunto(s)
Córnea/efectos de los fármacos , Córnea/patología , Enfermedades de la Córnea/inducido químicamente , Enfermedades de la Córnea/patología , Tensoactivos/toxicidad , Animales , Recuento de Células , Tamaño de la Célula , Sustancia Propia/efectos de los fármacos , Sustancia Propia/patología , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/patología , Microscopía Confocal , ConejosRESUMEN
PURPOSE: In previous studies in which in vivo confocal microscopy (CM) was used, quantifiable differences were identified in the corneal epithelium and stroma for surfactants producing different degrees of ocular irritation. In the present study, in vivo confocal microscopy was used to determine area and depth of the initial corneal changes, and the correlation of the data to cell death was characterized by ex vivo live-dead assay. METHODS: In four groups of rabbits (12 animals each), 10 microl surfactants known to produce slight, mild, moderate, or severe irritation was applied to the central cornea of one eye; 4 untreated rabbits served as controls. Measurements of group total mean epithelial thickness, epithelial cell area, and depth of keratocyte loss in four corneal regions were made by in vivo CM in 6 rabbits of each group and in 4 control animals at 3 hours and in the remaining rabbits at 3 hours and 1 day. Corneas were then removed and fixed for conventional histologic examination (two eyes/treatment/group), or regions were excised and placed in culture media containing 2 microM calcein-acetoxymethyl ester (calcein-AM) and 4 microM ethidium homodimer. Using laser scanning CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 microm (in the x, y, and z axes, respectively) volume of the cornea was determined. RESULTS: Confocal microscopy showed that application of the slight irritant resulted in decreased epithelial thickness at 3 hours (41.2+/-2.6 microm in treated eyes versus 43.6+/-3 microm in control eyes; n=6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630+/-203 microm2 versus 1427.2+/-90.7 microm2). On day 1, mild, moderate, and severe irritants caused complete loss of epithelium and disappearance of keratocytes to a depth of 30.8+/-10.7 microm, 47.2+/-10.4 microm, and 764.6+/-159.6 microm (n=6, 5, and 6), respectively. At 3 hours, live-dead assay detected more dead epithelial cells as a percentage of total surface cells (49.2+/-4.5% in slightly irritated eyes versus 20.9+/-3.2% in control eyes), significantly correlating with the measurement by in vivo CM of average epithelial cell size in each eye (r=-0.96; P < 0.005). On day 1, mild and moderate irritants showed increasing stromal cell death from 9.8+/-16.2 cells to 36.4+/-17.7 cells, which significantly correlated with the depth of stromal injury determined by in vivo CM (r=0.79; P < 0.00001). No surviving keratocytes were detected in severely irritated eyes. CONCLUSIONS: The data support the hypothesis that differences in surfactant-induced ocular irritation are directly related to area and depth of acute corneal injury.
Asunto(s)
Quemaduras Químicas/patología , Córnea/patología , Quemaduras Oculares/inducido químicamente , Tensoactivos/toxicidad , Animales , Bencenosulfonatos/toxicidad , Recuento de Células , Muerte Celular , Supervivencia Celular , Cetrimonio , Compuestos de Cetrimonio/toxicidad , Córnea/efectos de los fármacos , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/patología , Quemaduras Oculares/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Conejos , Dodecil Sulfato de Sodio/toxicidadRESUMEN
The pathology of surfactant-induced ocular irritation, especially in the context of accidental human exposures and animal tests used to assess a surfactant's potential ocular irritation, is not well understood. The purpose of this study was to characterize the microscopic changes in rats at 3 hr and on days 1, 2, 3, 4, 7, 14, and 35 following treatment with anionic, cationic, and nonionic surfactants of differing irritancy. The right eye of each rat was treated by placing 10 microliters of a surfactant directly on the cornea. Untreated left eyes served as the controls. At each time point, eyes and eyelids were macroscopically examined and collected for microscopic examination. Macroscopically, the differing levels of irritation were characterized by differences in incidence and magnitude of scores, reflecting involvement of the cornea, conjunctiva, and iris, as well as by the incidence of neovascularization and time to recovery. Microscopically, differences in the area and depth of injury paralleled the differences seen grossly and the relative irritancy of the various surfactants. All surfactants affected the corneal and conjunctival epithelium. All surfactants, except the slightly irritating anionic surfactant, caused corneal stromal changes, with this involvement being proportional to their overall level of irritation. Corneal endothelial cell effects principally occurred with only the severely irritating cationic surfactant. Over time, responses to surfactants of differing irritancy were qualitatively and quantitatively different, and these differences correlated with the extent of initial injury. Qualitative differences in response included presence of keratocyte regeneration, corneal neovascularization, and conjunctivalization of the corneal epithelium with all of the surfactants except the slight irritant. Quantitative differences in response occurred in the extent of epithelial regeneration, edema, and inflammation for surfactants of slight to severe irritancy, and with neovascularization, keratocyte regeneration, and conjunctivalization for surfactants of mild to severe irritancy. These results suggest that by defining initial area and depth of injury associated with an ocular irritant, it may be possible to predict the subsequent response and final outcome. Such an approach would be applicable to the development of mechanistically based in vitro assays.
Asunto(s)
Oftalmopatías/inducido químicamente , Ojo/patología , Irritantes/toxicidad , Tensoactivos/toxicidad , Administración Tópica , Animales , Oftalmopatías/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
We believe the development and validation of in vitro alternatives to eliminate the need to use animals in ocular irritation testing must be based on a thorough understanding of the mechanisms of ocular irritation. We have recently undertaken the task of developing such an understanding for a panel of surfactants. The purpose of this study was to expand our current panel of surfactants for which the microscopic changes occurring over time have been characterized. Macroscopic and microscopic findings regarding the ocular irritation of 6 surfactants of relatively unknown irritancy were compared to those of 6 surfactants of known irritancy. The right eye of each rat was treated by placing 10 microliters of a surfactant directly on the cornea. Untreated left eyes served as the controls. At 3 hr and on days 1, 3, and 35, eyes and eyelids were collected for microscopic examination. Collectively, the macroscopic and microscopic findings revealed 3 surfactants to be similar to the mildly irritating surfactants previously studied, and 3 surfactants to be similar to the moderately irritating surfactant previously studied. Information such as this will be important to develop mechanistically based in vitro alternatives to replace the use of animals for ocular irritation testing.
Asunto(s)
Oftalmopatías/inducido químicamente , Ojo/patología , Irritantes/toxicidad , Tensoactivos/toxicidad , Administración Tópica , Animales , Oftalmopatías/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
We have previously demonstrated with slightly and severely irritating surfactants that the new technology of noninvasive, in vivo confocal microscopy (CM) can be a useful approach to a better understanding of the pathobiology of ocular irritation in situ. In this study, in vivo CM was used to qualitatively and quantitatively characterize the initial microscopic corneal changes occurring with surfactants of slight, mild, moderate, and severe irritation. Surfactants were directly applied to the corneas of rabbits (6/group) at a dose of 10 microl. Eyes and eyelids were examined macroscopically and scored for irritation beginning at 3 hr after dosing and periodically through Day 35. Concurrently, the corneas were evaluated by in vivo CM; 3D data sets extending from the surface epithelium to the endothelium were assessed for surface epithelial cell size, epithelial layer thickness, total corneal thickness, and depth of keratocyte necrosis. The average macroscopic scores at 3 hr for the slight, mild, moderate, and severe irritants were 6.0, 39.3, 48.5, and 68.7, respectively, of a possible 110. At 3 hr, in vivo CM revealed corneal injury with the slight irritant limited to the epithelium, resulting in reductions in epithelial cell size and thickness to 59.0 and 82.4% of controls (p < 0.001 and p < 0.01, respectively). These parameters returned to normal by Day 3. For the mild irritant, at 3 hr the epithelium was absent, corneal thickness was increased to 157.6% of controls (p < 0.001), and necrosis of keratocytes extended to an average depth of 4.3 microm (0.8% of the corneal thickness); these parameters were essentially normal by Day 14. For the moderate irritant, at 3 hr the epithelium was markedly attenuated, corneal thickness was increased to 155.8% of controls (p < 0.001), and keratocyte necrosis extended to an average depth of 19.0 microm (3.6% of corneal thickness; statistically greater than with the mild irritant, p < 0.001); these parameters were essentially normal by Day 14. For the severe irritant, at 3 hr the epithelium was significantly thinned, corneal thickness was increased to 165.9% of controls (p < 0.001), and keratocyte necrosis occurred to an average depth of 391.1 microm (70.1% of corneal thickness). These findings demonstrate that significant differences in area and depth of injury occur with surfactants of differing irritancy. The data suggest that differences at 3 hr can be used to distinguish different levels of ocular irritation. Data such as these will be important in the development and evaluation of future mechanistically based in vitro alternatives for ocular irritancy testing.
Asunto(s)
Córnea/patología , Irritantes/toxicidad , Microscopía Confocal/métodos , Tensoactivos/toxicidad , Animales , Tamaño de la Célula , Conjuntiva/efectos de los fármacos , Conjuntiva/patología , Córnea/efectos de los fármacos , Iris/efectos de los fármacos , Iris/patología , Masculino , Necrosis , Conejos , Tensoactivos/administración & dosificaciónRESUMEN
PURPOSE: To study the feasibility of measuring total corneal thickness, as well as the thickness of the epithelium and Bowman's layer, using a novel in vivo confocal microscopy through-focusing (CMTF) methodology. METHODS: The central cornea was scanned from the epithelium to endothelium at an average focal plane speed of 32 microns/sec for rabbits, and 64 microns/sec for humans. Scans were initially video-recorded and later digitized. From digital images, CMTF intensity curves were generated by calculating the average pixel intensity in the central 180 x 180 pixel region (285 microns x 285 microns) of each image in the scan, and plotting as a function of z-depth. Peaks in this intensity profile were then empirically correlated to unique corneal layers using a program which interactively displayed images corresponding to the mouse cursor position along the intensity profile curve. Sublayer thickness values were then calculated from the z-axis positions of the relevant peaks in the intensity curve. Ten normal rabbits and seven human volunteers were evaluated in the study. Both CMTF and ultrasonic pachymetry (UP) measurements were performed on rabbit eyes to determine the agreement between CMTF and UP. RESULTS: Distinct epithelial, basal lamina, and endothelial peaks were identified for all 10 rabbit eyes. The mean central corneal thickness in the rabbit was 381.6 +/- 27.3 microns by CMTF and 384.4 +/- 28.7 microns by UP. The mean difference in central corneal thickness between CMTF and UP was -2.8 +/- 7.1 microns which was not statistically significant (p > 0.2 by paired t-test). Central epithelial thickness in the rabbit measured by CMTF was 47.7 +/- 2.2 microns. The average coefficients of variation for repeated scans were 2.5% and 0.7% for epithelial and corneal thickness, respectively. The standard errors for both epithelial and corneal thickness were less than 1.5 microns for all rabbits. The reproducibilities for epithelial and corneal thickness measurements were 2.2 microns and 2.6 microns, respectively, calculated as the square root of the within group variances of One-Way ANOVA. Intensity profiles for human corneas showed strong epithelial and endothelial peaks, as well as smaller peaks corresponding to the basal-epithelial nerve plexus and the denser anterior layer of stromal keratocyte nuclei. The mean central corneal thickness in the human was 532.1 +/- 18.8 microns; central epithelial thickness was 50.6 +/- 3.9 microns; central Bowman's layer thickness was 16.6 +/- 1.1 microns. The average coefficients of variation for repeated scans were 5.9%, 13.2%, and 1.6% for epithelial, Bowman's layer, and corneal thickness, respectively. The standard errors for all measurements were less than 2.4 microns. The reproducibilities for epithelial, Bowman's layer, and corneal thickness measurements were 3.2 microns, 2.3 microns, and 10.0 microns, respectively. CONCLUSIONS: CMTF is a novel, reproducible technique for obtaining epithelial and corneal thickness measurements during clinical in vivo confocal microscopy of the cornea. More importantly, this methodology provides the first objective, quantitative approach for measurement and analysis of depth and thickness of corneal sub-layers which may prove uniquely valuable in temporally assessing corneal function.
Asunto(s)
Córnea/anatomía & histología , Adulto , Animales , Endotelio Corneal/anatomía & histología , Análisis Factorial , Estudios de Factibilidad , Humanos , Masculino , Microscopía Confocal/métodos , Persona de Mediana Edad , ConejosRESUMEN
Limited information exists on the pathologic changes occurring with surfactant-induced ocular irritation in the context of accidental human exposures and animal tests used to assess for such irritation. The purpose of this study was to begin to characterize the pathologic changes that occur with surfactants in the context of standard animal tests and compare the response in rats to that in rabbits. Representative anionic, cationic, and nonionic surfactants causing slight to severe ocular irritation were directly applied to the corneas of rabbits and rats at a dose of 10 microliters. Eyes and eyelids of each animal were macroscopically examined for signs of irritation beginning 3 hr after dosing and periodically until recovery or day 35. Eyes and eyelids from animals in each group were collected for microscopic examination after 3 hr and at recovery or day 35. Microscopically, all of the surfactants caused erosion, denudation, and/or necrosis of the conjunctival and corneal epithelium in rabbits and rats. Necrosis of keratocytes was observed in rabbits and rats treated with the severely irritating cationic surfactant and in rats treated with anionic surfactants that were mildly irritating and moderately irritating. Corneal endothelial changes were observed in rabbits and rats with only the cationic surfactant. Changes in eyes of rabbits and rats that had not recovered by day 35 included decreased prominence of goblet cells, conjunctivalization of the corneal epithelium, neovascularization and fibrosis of the cornea, and presence of devitalized stroma. Overall, the changes in rabbits and rats were similar and suggest that the rat may be used as a surrogate for the rabbit in studies to understand better mechanisms of surfactant-induced eye irritation.
Asunto(s)
Oftalmopatías/inducido químicamente , Irritantes/toxicidad , Tensoactivos/toxicidad , Animales , Córnea/patología , Endotelio Corneal/patología , Ojo/patología , Oftalmopatías/patología , Enfermedades de los Párpados/inducido químicamente , Enfermedades de los Párpados/patología , Párpados/patología , Iris/patología , Masculino , Conejos , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Factores de TiempoRESUMEN
The purpose of this study was to assess the ability of in vivo confocal microscopy (CM) to provide noninvasively derived histopathologic correlates of surfactant-induced eye irritation from which specific pathologic mechanisms can be identified and later evaluated in alternative in vitro models. Rats and rabbits, divided into groups of 5, received 10 microliters of an anionic or cationic surfactant in one eye with the other eye used as a control. At specified times, eyes were examined and scored for ocular irritancy using a penlight and slit-lamp. Subsequently, corneas were evaluated by in vivo CM to evaluate epithelial layer thickness and surface epithelial cell area, corneal thickness, depth of necrosis, inflammation, fibrosis, and endothelial injury. At 3 hr, the anionic surfactant produced slight irritation with peak scores of 12.4 and 8.0 out of a possible 110 in the rats and rabbits, respectively. In vivo CM revealed changes limited to the corneal epithelium that decreased in thickness to 78% in rats and 81% in rabbits at 3 hr. This decrease in the thickness correlated with a significant decrease in surface epithelial cell area from 2,061 +/- 395 microns2 to 567 +/- 330 microns2 in the rats and 1,523 +/- 185 microns2 to 934 +/- 71 microns2 in the rabbits (p < 0.005 and 0.005, respectively). The cationic surfactant produced severe irritation in both the rats and rabbits with peak scores of 85.4 and 80.2 occurring at day 2, respectively. In vivo CM in the rats showed complete loss of corneal epithelium, lysis of keratocytes, and loss of corneal endothelium. In the rabbits, injury appeared limited to the anterior cornea with complete loss of epithelium and loss of keratocytes extending to 52% of the corneal thickness. These findings establish the application of noninvasive, in vivo CM to qualitatively and quantitatively characterize the pathobiology of ocular irritation in situ. This information will be important in the development and evaluation of mechanistically based in vitro alternatives for ocular irritancy testing.
Asunto(s)
Oftalmopatías/inducido químicamente , Oftalmopatías/patología , Irritantes/toxicidad , Tensoactivos/toxicidad , Animales , Córnea/patología , Endotelio Corneal/patología , Ojo/metabolismo , Ojo/patología , Oftalmopatías/metabolismo , Procesamiento de Imagen Asistido por Computador , Masculino , Microscopía Confocal , Conejos , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The present study was undertaken to further define the role of alveolar macrophages (AM) in the pulmonary response to crocidolite fibers. Briefly, groups of 4 male F344 rats were intratracheally instilled with saline or saline suspensions of crocidolite at 2 or 20 mg/kg body weight. Animals were sacrificed 3, 7, 14, and 28 d after exposure and the lung response was characterized by analysis of bronchoalveolar lavage fluid (BALF) for markers of lung injury and inflammation. AM obtained in BALF were cultured and their production of the pro-inflammatory cytokines, tumor necrosis factor alpha (TNF alpha), and interleukin-1 (IL-1) were characterized along with fibronectin, a protein known to stimulate fibroblast migration and proliferation. Lung hydroxyproline content was determined 28 d after exposure and lung histopathology was characterized on d 28 and 90 after exposure. Crocidolite instillation resulted in transient dose-related pulmonary inflammation as evidenced by increased numbers of BALF neutrophils at the low dose and neutrophils, macrophages, and lymphocytes at the high dose. Cytotoxicity and increased permeability were demonstrated by increased levels of BALF lactate dehydrogenase (LDH) and total protein, respectively. AM TNF alpha and IL-1 production were increased only at the high crocidolite dose. This cytokine response was greatest at d 3 and decreased thereafter. AM TNF alpha and IL-1 release were positively correlated with the increased BALF neutrophils. In contrast to TNF alpha and IL-1, AM fibronectin release was increased at both the low and high doses, with the magnitude of response increasing over time. Consistent with previous acute asbestos inhalation studies, histopathology revealed inflammation localized at the level of the terminal bronchioles and alveolar ducts. Fibrosis was demonstrated at both doses by increased trichrome staining of lung tissue sections. Only the high dose resulted in a detectable increase in lung hydroxyproline. Given the bioactivities of TNF alpha, IL-1, and fibronectin, their increased production after crocidolite exposure indicates they contribute to the pulmonary inflammation and fibrosis occurring with this mineral fiber. In addition, the correlation of increased AM TNF alpha and IL-1 production with increased BALF neutrophils supports a role for these cytokines in crocidolite-induced inflammatory cell recruitment. Lastly, association of a persistent increase in AM fibronectin production with an eventual increase in lung collagen deposition extends the growing database indicating this response is a predictive marker of pulmonary fibrosis.
Asunto(s)
Asbesto Crocidolita , Asbestosis/inmunología , Citocinas/biosíntesis , Sustancias de Crecimiento/biosíntesis , Macrófagos Alveolares/inmunología , Fibrosis Pulmonar/inmunología , Análisis de Varianza , Animales , Asbestosis/etiología , Asbestosis/patología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Fibronectinas/análisis , Hidroxiprolina/análisis , Interleucina-1/análisis , Intubación Intratraqueal , Masculino , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Ratas , Ratas Endogámicas F344 , Organismos Libres de Patógenos Específicos , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Animal models historically used to assess the acute gastric irritation potential of accidental ingestion of consumer products include the dog, pig, rabbit, and cat. In looking at alternative methods that are of shorter duration and more cost-effective, the rat is being evaluated as a potential model. Acute gastric irritation is known to increase as the reserve alkalinity of the formulation increases. In initial experiments to assess the rat as a potential model, animals were dosed via oral gavage with 1 of 4 formulations ranging in reserve alkalinity from 4.0 to 10.8. Necropsies were performed at 15 and 60 min after dosing. Macroscopic and microscopic evaluations of the stomach revealed morphological differences in the various treatment groups that distinguish granular formulations having either a low (R.A. = 4.0), moderate (R.A. = 7.1), or higher (R.A. = 10.8) reserve alkalinity. Additionally, it was observed that the acute gastric changes in rats dosed with a liquid formulation having a low (R.A. = 4.2) reserve alkalinity were similar to those in rats dosed with a granular formulation having a moderate reserve alkalinity (R.A. = 7.1). This suggests that other factors such as types of ingredients, pH, and physical form influence the extent of acute gastric irritation and demonstrates that an evaluation of only reserve alkalinity is not sufficient to ensure the safety of these products. This preliminary work supports the rat as a potential model to assess the acute gastric irritation potential of alkaline formulations or substances.
Asunto(s)
Detergentes/toxicidad , Irritantes/toxicidad , Estómago/efectos de los fármacos , Animales , Masculino , Modelos Biológicos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Estómago/patología , Toxicología/métodosRESUMEN
To determine its carcinogenic potential, sodium fluoride (NaF) was fed to CD-1 mice for up to 97 weeks. Mice given NaF at a dose of 4, 10, or 25 mg/kg of body weight per day added to a low-fluoride diet were compared to controls given either an unsupplemented low-fluoride diet or laboratory chow. Nonneoplastic changes consistent with those previously recognized from fluoride toxicity were observed in teeth, bones, and joints. Unexpectedly, osteomas occurred in all groups. The incidence of osteomas was similar in groups given the low-fluoride control diet, laboratory chow, or NaF doses of 4 or 10 mg/kg per day. The incidence of osteomas in these groups was increased over that historically experienced at the laboratory and reported in the literature for CD-1 mice. The incidence of osteomas in the mice given 25 mg NaF/kg per day added to a low-fluoride diet was increased over that in the other groups. Osteomas were first observed at Week 55. No malignant bone tumors were observed during the course of the study. The locations, multiplicity, and morphologic features of the osteomas in all groups were similar to those associated with virus-induced bone tumors. Electron microscopic examination revealed abundant retrovirus particles in all osteomas examined from control and test mice. It was concluded that the study was confounded by a retrovirus which contributed to the induction of the osteomas. Because the study was confounded, it cannot be considered a valid bioassay to be used for risk assessment.
Asunto(s)
Carcinógenos/toxicidad , Fluoruro de Sodio/toxicidad , Animales , Neoplasias Óseas/inducido químicamente , Neoplasias Óseas/complicaciones , Neoplasias Óseas/microbiología , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Osteoma/inducido químicamente , Osteoma/complicaciones , Osteoma/microbiología , Virus de la Parainfluenza 1 Humana , Infecciones por Paramyxoviridae/complicaciones , Infecciones por Paramyxoviridae/patología , Factores de Riesgo , Fluoruro de Sodio/metabolismoRESUMEN
Rats were exposed to saline or cadmium chloride (CdCl2) at 25, 100, or 400 micrograms/kg body weight by intratracheal instillation. At 3, 7, 14, and 28 days after exposure five animals/treatment were euthanized, the lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, N-acetylglucosamindase (NAG), and cell number, type, and viability. Lung hydroxyproline concentration was characterized as a marker of lung collagen. Alveolar macrophages (AM) obtained in BALF were cultured and the release of fibronectin and TNF was determined. Lung tissue was examined microscopically at 28 and 90 days after exposure. Exposure to CdCl2 resulted in lung injury and inflammation demonstrated by increases in BALF LDH, total protein, NAG, and inflammatory cells. AM TNF release was not significantly changed by CdCl2 treatment. All doses of CdCl2 stimulated AM fibronectin secretion, a response which persisted throughout the 28-day postexposure period examined. Pulmonary fibrosis was demonstrated biochemically and/or histologically (trichrome staining tissue) at all CdCl2 dose levels. The association of CdCl2-induced AM fibronectin release with lung fibrosis confirms and extends previous observations relating AM-derived fibronectin to the development of interstitial lung disease and provides further evidence that the persistent increase in AM fibronectin release represents an early indicator of fibrosis.