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Efecto de la aplicación intravítrea de células estromales mesenquimales en la respuesta inflamatoria intraocular en un modelo animal experimental de hipertensión ocular

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dc.contributor.advisorDe La Torre Cifuentes, Ligia Alejandra
dc.contributor.advisorRamírez Santana, Heily Carolina
dc.creatorDos Santos Evangelho, Karine
dc.creator.degreeDoctor en Ciencias Biomédicas y Biológicas
dc.creator.degreeLevelDoctorado
dc.creator.degreetypeFull time
dc.date.accessioned2025-02-11T20:08:24Z
dc.date.available2025-02-11T20:08:24Z
dc.date.created2024-06-24
dc.descriptionSe proyecta que más de 80 millones de personas padecerán glaucoma al final de esta década. Esta condición, que aún no cuenta con una solución definitiva, requiere el desarrollo de alternativas terapéuticas más eficaces. Recientemente, la investigación sobre la fisiopatología del glaucoma se ha centrado en comprender los efectos de la respuesta inmune en el daño a las células ganglionares de la retina (CGRs) y en su protección. Las CGRs desempeñan un papel crucial, ya que son las neuronas aferentes responsables de transmitir la información visual al cerebro a través de sus axones, los cuales forman el nervio óptico. La muerte de estas células conlleva ceguera irreversible. El estudio de los mecanismos inmunológicos involucrados en el glaucoma podría proporcionar nuevas vías para el tratamiento y la prevención de esta enfermedad, que puede llevar a la discapacidad visual. Se ha encontrado que un desequilibrio en el perfil de citoquinas producidas por las células T puede modificar el microambiente inmunológico en ojos glaucomatosos, influyendo así en la neuropatía óptica. Por lo tanto, las estrategias para bloquear o neutralizar las citoquinas relacionadas con el proceso neurodegenerativo en el glaucoma podrían representar un mecanismo terapéutico prometedor para proteger las neuronas y prevenir una mayor pérdida de la capacidad visual. Este hallazgo sugiere avances hacia tratamientos más efectivos que podrían mejorar la calidad de vida de los pacientes con glaucoma y reducir la gravedad de esta enfermedad. En este contexto neuroprotector, las células estromales mesenquimales (MSCs) están emergiendo como fuertes candidatas para ser utilizadas como terapia celular en el tratamiento de enfermedades neurodegenerativas, dado su considerable potencial regenerativo y su capacidad inmunosupresora. Esta investigación tiene como objetivo principal evaluar el perfil de determinadas citoquinas en el humor acuoso y vítreo en un modelo prospectivo, in vivo, experimental de hipertensión ocular (HO), así como su relación con la respuesta inmune intraocular en ojos tratados con inyección intravítrea de células estromales mesenquimales de gelatina de Wharton humana (WJ-MSCs). Esta investigación se llevó a cabo en el Bioterio y la Sala de Cirugía Experimental de la Escuela Superior de Oftalmología del Instituto Barraquer de América, tras recibir la aprobación del Comité de Ética en Investigación con Animales (CEIA-ESO-IBA). El estudio se dividió en dos etapas experimentales: un estudio piloto y una fase experimental. En el estudio piloto, se realizó la estandarización del modelo de HO para ajustar las dosis del medicamento y la medición de la presión intraocular (PIO). Se utilizaron cuatro conejos machos de la raza Nueva Zelanda, divididos en dos grupos experimentales asignados aleatoriamente durante un período de 4 semanas. Además de estandarizar el modelo de HO mediante la instilación de acetato de prednisolona tópica dos veces al día y la aplicación semanal de acetato de betametasona subconjuntival, se adaptaron técnicas para evaluar la respuesta pupilar fotocromática, realizar estudios histológicos y definir los marcadores inflamatorios utilizados en la investigación. Basándose en los resultados obtenidos en el estudio piloto, se desarrolló la fase experimental. Todos los animales del estudio fueron sometidos a un examen clínico detallado, que incluyó la inspección de mucosas, dentición, piel y anexos, además de un examen oftalmológico completo. Aquellos que presentaron enfermedades de la superficie ocular, opacidad del cristalino, enfermedades inflamatorias sistémicas, lesión retiniana o pérdida de peso severa (≥10% del peso inicial) fueron excluidos del estudio. En la fase experimental, se utilizaron 15 animales, divididos aleatoriamente en tres grupos: grupo 1 (HO), grupo 2 (trasplante intravítreo de WJ-MSCs) y grupo 3 (HO y trasplante intravítreo de WJ-MSCs). Se inyectaron 10⁵/100μL de WJ-MSCs intravítreas en la semana 7 del estudio, asignando el ojo izquierdo como control. En este estudio, se evaluaron los efectos de las WJ-MSCs en la retina mediante Tomografía de Coherencia Óptica (OCT), PEV flash y Respuesta Pupilar Fotocromática (RPF). La retinografía reveló cambios vasculares en la retina, excavación de la copa óptica y alteraciones en la coloración del disco óptico en el grupo 1, mientras que en el grupo 3 se observó una solución de WJ-MSCs localizada sobre el nervio óptico. La OCT mostró una reducción en la capa de células ganglionares de la retina (CCGRs) en el grupo 1, pero un ligero aumento en el grupo 3. Además, los animales tratados con WJ-MSCs presentaron menos alteraciones en la retina y el nervio óptico en comparación con el grupo de HO, incluida una mayor preservación de las CGRs observada mediante histología, lo que indica un efecto beneficioso de la terapia celular. Se observó un posible efecto terapéutico del trasplante celular en la RPF, con un aumento significativo en la respuesta de contracción pupilar a la luz azul solo en las primeras semanas del estudio en el grupo de HO que recibió WJ-MSCs, lo que podría indicar un efecto neuroprotector y axogénico de la terapia celular en la retina. Sin embargo, las WJ-MSCs no lograron restablecer la función de las células ganglionares ni del nervio óptico según el PEV flash. Además, se midieron los niveles de citoquinas en humor acuoso (IL-6, IL-8, factor de necrosis tumoral alfa (TNF-α)) y humor vítreo (interferón (IFN-γ), IL-10 y factor de crecimiento transformante beta (TGF-β)) mediante la técnica de inmunoensayo ELISA. Se evaluó también la expresión de células TCD3+, TCD3+/TCD4+ y TCD3+/TCD8+, así como de la proteína ácida fibrilar glial (GFAP) e inmunohistoquímica en retina y en el nervio óptico después de la aplicación intravítrea de WJ-MSCs, con el fin de analizar el efecto neuroprotector de la terapia celular y su relación con las respuestas inmune e inflamatoria. Se evidenció una disminución en la secreción de citoquinas específicas, como TNF-α, IL-6, IFN-γ, IL-10 e IL-8, en el grupo 3 en comparación con el grupo 1. Además, se observó un aumento en la concentración de TGF-β en humor vítreo después de la aplicación de WJ-MSCs. Al evaluar la infiltración de células TCD3+/TCD4+ en la CCGRs, se observó que el grupo 3 presentaba una infiltración leve, mientras que el grupo 1 no mostró infiltración celular, lo que sugiere un posible efecto inmunomodulador sobre las células TCD4+ mediante la neuroprotección en esta capa de la retina. Por otro lado, la infiltración retiniana de células TCD3+ en todos los grupos tendía a pertenecer más a la subpoblación de células TCD8+ que a las células TCD4+ en varias capas de la retina, lo que indica que la inmunidad celular puede desempeñar un papel en la neuropatía óptica glaucomatosa. Asimismo, se observó una fuerte expresión de GFAP en la retina y en el nervio óptico en todos los grupos del estudio. Además de los resultados descritos, el trasplante de WJ-MSCs de cordón umbilical mostró buenos perfiles de seguridad y pocos eventos adversos en el modelo experimental propuesto. En conjunto, los hallazgos de este estudio sugieren que las células estromales derivadas del cordón umbilical pueden ser una fuente celular prometedora para la neuroprotección en la enfermedad glaucomatosa.
dc.description.abstractIt is estimated that more than 80 million people will suffer from glaucoma by the end of this decade. This condition, which still lacks a definitive cure, demands the development of more effective therapeutic alternatives. Recent research on the pathophysiology of glaucoma has focused on understanding the impact of immune responses on retinal ganglion cell (RGC) damage and how these cells can be protected. RGCs play a crucial role as afferent neurons responsible for transmitting visual information to the brain through their axons, which form the optic nerve. The loss of these cells results in irreversible blindness. Studying the immunological mechanisms involved in glaucoma may provide new avenues for treatment and prevention, helping mitigate visual deterioration. An imbalance in the cytokine profile produced by T cells has been shown to alter the immune microenvironment in glaucomatous eyes, thereby influencing optic neuropathy. Strategies to block or neutralize cytokines involved in the neurodegenerative processes of glaucoma could represent a promising therapeutic approach to protect neurons and prevent further vision loss. This insight suggests progress toward more effective treatments that could improve the quality of life for glaucoma patients and help reduce the severity of the disease. In this neuroprotective context, mesenchymal stromal cells (MSCs) have emerged as strong candidates for use in cell therapy for neurodegenerative diseases due to their significant regenerative potential and immunosuppressive properties. This study aims to evaluate the profile of certain cytokines in the aqueous and vitreous humor of a prospective in vivo experimental model of ocular hypertension (OH) and explore their relationship with the intraocular immune response in eyes treated with intravitreal injections of human Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs). The research was conducted at the Animal Facility and Experimental Surgery Room of the Superior School of Ophthalmology at the Barraquer Institute of America, following approval from the Animal Research Ethics Committee (CEIA-ESO-IBA). The study was divided into two experimental stages: a pilot study and the experimental phase. During the pilot phase, the OH model was standardized to adjust drug doses and intraocular pressure (IOP) measurements. Four male New Zealand rabbits were used, randomly assigned to two experimental groups over a four-week period. In addition to standardizing the OH model through the topical application of prednisolone acetate twice daily and weekly subconjunctival injections of betamethasone acetate, techniques were adapted to assess the photopic pupillary response, conduct histological studies, and define the inflammatory markers used in the research. Based on the pilot study’s results, the experimental phase was developed. All animals underwent a detailed clinical examination, including inspection of mucous membranes, teeth, skin, and appendages, along with a comprehensive ophthalmological evaluation. Animals with ocular surface disease, lens opacity, systemic inflammatory conditions, retinal damage, or significant weight loss (≥10% of their initial weight) were excluded from the study. During the experimental phase, 15 animals were randomly divided into three groups: Group 1 (OH), Group 2 (intravitreal transplantation of WJ-MSCs), and Group 3 (OH and intravitreal transplantation of WJ-MSCs). Intravitreal injections of 10⁵/100μL of WJ-MSCs were administered in the seventh week, with the left eye used as a control. The effects of WJ-MSCs on the retina were evaluated using Optical Coherence Tomography (OCT), flash Visual Evoked Potential (VEP), and Photopic Pupillary Response (PPR). Retinography revealed vascular changes, optic nerve cupping, and alterations in optic disc coloration in Group 1, while a localized WJ-MSC solution was observed over the optic nerve in Group 3. OCT showed a reduction in the RGC layer in Group 1 but a slight increase in Group 3. Furthermore, animals treated with WJ-MSCs exhibited fewer retinal and optic nerve alterations compared to the OH group, including better preservation of RGCs, as confirmed through histology. This finding indicates a beneficial effect of cell therapy. A potential therapeutic impact of the cell transplant was also observed in the PPR, with a significant improvement in the contraction response of the pupils.
dc.format.extent284 pp
dc.format.mimetypeapplication/pdf
dc.identifier.doihttps://doi.org/10.48713/10336_44970
dc.identifier.urihttps://repository.urosario.edu.co/handle/10336/44970
dc.language.isospa
dc.publisherUniversidad del Rosario
dc.publisher.departmentEscuela de Medicina y Ciencias de la Salud
dc.publisher.programDoctorado en Ciencias Biomédicas y Biológicas
dc.rightsAttribution-NoDerivatives 4.0 International*
dc.rights.accesRightsinfo:eu-repo/semantics/openAccess
dc.rights.accesoAbierto (Texto Completo)
dc.rights.licenciaPARGRAFO: En caso de presentarse cualquier reclamación o acción por parte de un tercero en cuanto a los derechos de autor sobre la obra en cuestión, EL AUTOR, asumirá toda la responsabilidad, y saldrá en defensa de los derechos aquí autorizados; para todos los efectos la universidad actúa como un tercero de buena fe.
dc.rights.urihttp://creativecommons.org/licenses/by-nd/4.0/*
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dc.source.instnameinstname:Universidad del Rosario
dc.source.reponamereponame:Repositorio Institucional EdocUR
dc.subjectCelulas madre
dc.subjectGlaucoma
dc.subjectCitocinas
dc.subjectRespuesta inmune
dc.subjectCélulas estromales mesenquimales
dc.subject.keywordStem cells
dc.subject.keywordGlaucoma
dc.subject.keywordCytokines
dc.subject.keywordImmune response
dc.subject.keywordMesenchymal stromal cells
dc.titleEfecto de la aplicación intravítrea de células estromales mesenquimales en la respuesta inflamatoria intraocular en un modelo animal experimental de hipertensión ocular
dc.title.TranslatedTitleEffect of intravitreal application of mesenchymal stroma cells on the intraocular inflammatory response in an experimental animal model of ocular hypertension
dc.typedoctoralThesis
dc.type.hasVersioninfo:eu-repo/semantics/acceptedVersion
dc.type.spaTesis de doctorado
local.department.reportEscuela de Medicina y Ciencias de la Salud
local.regionesBogotá
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Doctorado en Ciencias Biomédicas y Biológicas