Effect of intranasal administration of NeuroEPO in the histological structure of the olfactory mucosa of Wistar rats / Efecto de la administración intranasal de NeuroEPO en la estructura histológica de la mucosa olfatoria de ratas Wistar

Introduction: Strokes and neurodegenerative diseases are major global health problems, not only because they cause high mortality and disability, but due to the lack of effective therapies. NeuroEPO, a variant of recombinant human erythropoietin (rHu-EPO) with a low sialic acid content, has shown encouraging results as a potential neuroprotective agent when administered intranasally. Objective: To determine the effect of intranasal administration of NeuroEPO on the histological structure of the olfactory mucosa of Wistar rats. Materials and Methods: An experimental, prospective, and longitudinal study was conducted on Wistar rats. Ten healthy animals were randomly distributed into two groups of five each. The control group received a vehicle (0.3 μl/g/day) and the treated group received NeuroEPO (300 μg/kg/day). Both treatments were administered intranasally for 28 days. The histological characteristics of the olfactory mucosa were evaluated. The medians between the study groups were compared using the Mann-Whitney U test. Results: There were no alterations in the histological characteristics of the olfactory epithelium. However, slight hypertrophy and hyperplasia of the Bowman's glands were observed at the level of the lamina propria in the group treated with NeuroEPO. Conclusions: The administration of the nasal formulation of NeuroEPO did not induce histological alterations of the olfactory mucosa of Wistar rats under the experimental conditions of this research.

Introducción: Los accidentes cerebrovasculares y las enfermedades neurodegenerativas constituyen un importante problema de salud mundial. No solo porque causan una alta mortalidad y discapacidad, sino por la falta de terapias eficaces para tratarlos. La NeuroEPO, una variante de la eritropoyetina humana recombinante (rHu-EPO) con bajo contenido en ácido siálico, ha mostrado resultados alentadores como potencial agente neuroprotector al ser administrada por vía intranasal. Objetivo: Determinar el efecto de la administración intranasal de NeuroEPO en la estructura histológica de la mucosa olfatoria de ratas Wistar. Materiales y Métodos: Se realizó un estudio experimental, prospectivo y de corte longitudinal en ratas Wistar. Se utilizaron diez animales sanos distribuidos aleatoriamente en dos grupos de cinco cada uno. El grupo control recibió vehículo (0,3 μl/g/día) y el grupo tratado recibió NeuroEPO (300 μg/kg/día). Ambos tratamientos fueron administrados por vía intranasal durante 28 días. Fueron evaluadas las características histológicas de la mucosa olfatoria. Las medianas de los grupos del estudio fueron comparadas mediante la prueba U de Mann-Whitney. Resultados: No se evidenciaron alteraciones en las características histológicas del epitelio olfatorio. Sin embargo, a nivel de la lámina propia en el grupo tratado con NeuroEPO, se observó una ligera hipertrofia e hiperplasia de las glándulas de Bowman. Conclusiones: La administración de la formulación nasal de NeuroEPO no indujo alteraciones histopatológicas de la mucosa olfatoria de ratas Wistar en las condiciones experimentales de esta investigación.

Biopsy of the olfactory epithelium from the superior nasal septum: is it possible to obtain neurons without damaging olfaction? / Biópsia do epitélio olfatório do septo nasal superior: é possível obter neurônios sem prejudicar o olfato?

Abstract Introduction Olfactory epithelium biopsy has been useful for studying diverse otorhinolaryngological and neurological diseases, including the potential to better understand the pathophysiology behind COVID-19 olfactory manifestations. However, the safety and efficacy of the technique for obtaining human olfactory epithelium are still not fully established. Objective This study aimed to determine the safety and efficacy of harvesting olfactory epithelium cells, nerve bundles, and olfactory epithelium proper for morphological analysis from the superior nasal septum. Methods During nasal surgery, 22 individuals without olfactory complaints underwent olfactory epithelium biopsies from the superior nasal septum. The efficacy of obtaining olfactory epithelium, verification of intact olfactory epithelium and the presence of nerve bundles in biopsies were assessed using immunofluorescence. Safety for the olfactory function was tested psychophysically using both unilateral and bilateral tests before and 1 month after the operative procedure. Results Olfactory epithelium was found in 59.1% of the subjects. Of the samples, 50% were of the quality necessary for morphological characterization and 90.9% had nerve bundles. There was no difference in the psychophysical scores obtained in the bilateral olfactory test (University of Pennsylvania Smell Identification Test [UPSIT®]) between means before biopsy: 32.3 vs. postoperative: 32.5, p= 0.81. Also, no significant decrease occurred in unilateral testing (mean unilateral test scores 6 vs. 6.2, p= 0.46). None out of the 56 different odorant identification significantly diminished (p> 0.05). Conclusion The technique depicted for olfactory epithelium biopsy is highly effective in obtaining neuronal olfactory tissue, but it has moderate efficacy in achieving samples useful for morphological analysis. Olfactory sensitivity remained intact.

Resumo Introdução A biópsia do epitélio olfatório tem sido útil para estudar diversas doenças otorrinolaringológicas e neurológicas, incluindo seu potencial para melhor compreender a fisiopatologia por trás das manifestações olfatórias na COVID‐19. No entanto, a segurança e eficácia da técnica de obtenção de epitélio olfatório humano ainda não estão totalmente estabelecidas. Objetivos Este estudo teve como objetivo determinar a segurança e eficácia da coleta de células do epitélio olfatório, feixes nervosos e epitélio olfatório adequados para análise morfológica, no septo nasal superior. Método Durante a cirurgia nasal, 22 indivíduos sem queixas olfatórias foram submetidos a biópsias de epitélio olfatório do septo nasal superior. A eficácia da obtenção de epitélio olfatório, a verificação de epitélio olfatório íntegro e a presença de feixes nervosos nas biópsias foram avaliadas por imunofluorescência. A segurança da função olfatória foi testada psicofisicamente usando testes unilaterais e bilaterais antes e um mês após o procedimento cirúrgico. Resultados Epitélio olfatório foi encontrado em 59,1% dos sujeitos. Das amostras, 50% apresentaram a qualidade necessária para a caracterização morfológica e 90,9% continham feixes nervosos. Não houve diferença nos escores psicofísicos obtidos no teste olfatório bilateral (University of Pennsylvania Smell Identification Test [UPSIT®]) entre as médias antes da biópsia: 32,3 vs. pós‐operatório: 32,5, p = 0,81. Além disso, nenhuma diminuição significante ocorreu no teste unilateral (escore médio do teste unilateral 6 vs. 6,2, p = 0,46). Não houve redução significante na identificação de nenhum dos 56 odorantes diferentes (p > 0,05). Conclusão A técnica descrita para biópsia de epitélio olfatório é altamente eficaz na obtenção de tecido olfatório neuronal, mas tem eficácia moderada na obtenção de amostras adequadas para análise morfológica. A capacidade olfativa permaneceu intacta.

Human olfactory mesenchymal stromal cells co-expressing horizontal basal and ensheathing cell proteins in culture / Las células mesenquimales del estroma olfatorio humano coexpresan proteínas de las células basales horizontales y de recubrimiento neural en cultivo

Introduction: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells. Objective: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation. Materials and methods: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors. Results: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample. Conclusions: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells.

Introducción. El recambio celular del neuroepitelio olfatorio ocurre durante la vida del individuo gracias a precursores olfatorios. Además, las células mesenquimales del estroma también contribuyen a la homeostasis de la mucosa. Cuando un explante de una biopsia de mucosa se cultiva en un medio esencial mínimo, se genera una población predominante de células adherentes que expresan proteínas típicas de las células mesenquimales del estroma. La coexpresión de marcadores fenotípicos de precursores olfatorios y de células del recubrimiento del nervio olfatorio constituiría una propiedad única de las células mesenquimales del estroma. Objetivo. Determinar si la diferenciación celular de las células mesenquimales hacia fenotipos neurales modula la expresión de los marcadores mesenquimales característicos. Materiales y métodos. Se compararon las células aisladas de la mucosa olfatoria en un medio de cultivo con suplemento de 10 % de suero fetal bovino con esferas generadas en un medio sin suero más factores de crecimiento. Resultados. Se determinó la expresión de proteínas de las células mesenquimales del estroma (CD29+, CD73+, CD90+, CD45-), de las basales horizontales (ICAM-1/CD54+, p63+, p75NGFr+), y de las del recubrimiento del nervio olfatorio (nestin+, GFAP+) en la misma población cultivada. La determinación de Oct 3/4, Sox-2 y Mash-1, así como de las neurotrofinas BDNF, NT3 y NT4, sugirió que las células del estroma son funcionales. La expresión de las proteínas de las células mesenquimales y los precursores olfatorios, disminuyó en las células de las mesenesferas inducidas por ausencia de suero en el medio de cultivo. Conclusión. Las células mesenquimales del estroma de la mucosa olfatoria presentan una tendencia dominante hacia la diferenciación neural.

Olfactory epithelium as an infinitive source of neural stem cells for derivation of inner ear hair cells / El epitelio olfatorio como fuente infinita de células madre neurales para la derivación de los vestibulocitos del oído interno

Surgical techniques for treatment of sensory neural hearing loss (SNHL) have unpredictable outcomes and in recent years cell therapy investigated for treatment of SNHL. Olfactory epithelium proceed neurogenesis during life time and provide an easily accessible source of neural stem cells. So the aim of this study was isolating neural stem cells from olfactory epithelium of rat and differentiation of these cells into hair cells of inner ear in vitro. The epithelium tissue of olfactory mucosa of rats were removed and digested by collagenase H. The digested tissue was cultured in flasks in suspension forms to create spheres. Spheres were passaged and from passage 2 spheres selected for differentiation. At this stage cells of spheres isolated from each other and placed in flask containing defined differentiation medium. Cells at this stage cultured in adhesive form. Immunohistochemistry and RT-PCR were used for neural stem cells and hair cells identification. Spheres formed from olfactory epithelium culture and immunohistochemistry revealed that cells of spheres from passage one and two expressed the neural stem cells markers. After culture of isolated cells in differentiation medium, the morphology of cells begun to change. The cells presented neural cells projections and after 10 days the projections elongated more and interact to each other in multi layers. RT-PCR and immunohistochemistry revealed that differentiated cells expressed hair cells specific genes. In this study we showed that neural stem cells of olfactory epithelium can differentiate into hair cells of inner ear and therefore can be used for treatment of SNHL.

Las técnicas quirúrgicas para el tratamiento de la pérdida auditiva neural sensorial (PANS) tienen resultados impredecibles y en los últimos años la terapia celular ha sido investigada para su tratamiento. El epitelio olfatorio se forma durante la neurogénesis y proporciona una fuente fácilmente accesible de células madre neurales. El objetivo de este estudio fue aislar las células madre neurales del epitelio olfativo de la rata y la diferenciación de estas células en vestibulocitos del oído interno in vitro. Se retiró el tejido del epitelio de la mucosa olfatoria de ratas y fue digerido con colagenasa H. El tejido se cultivó en forma de suspensión para crear esferas. Se seleccionaron dos esferas para la diferenciación. En esta fase, las células de esferas fueron aisladas unas de otras y colocadas en un medio de diferenciación definido. Células en esta etapa fueron cultivadas en forma adhesiva. Inmunohistoquímica y RT-PCR se utilizó para las células madre neurales y la identificación de células ciliadas. Las esferas formadas a partir del cultivo del epitelio olfatorio y la inmunohistoquímica revelaron que las células de esferas en etapas uno y dos expresaban los marcadores de células madre neurales. Se observaron cambios en la morfología de las células después del cultivo de células aisladas. RT-PCR e inmunohistoquímica revelaron que las células diferenciadas expresaron células específicas de gen de vestibulocitos. Se observó que las células madre neuronales de epitelio olfatorio puede diferenciarse en células en forma de cabello del oído interno y por lo tanto puede ser utilizado para el tratamiento de PANS.

Evaluación del olfato en pacientes con patología tumoral en la región selar, intervenidos mediante abordaje endoscópico transesfenoidal transnasal / Assessment of smell in patients with tumor pathology in the sellar region, operated by transnasal transsphenoidal endoscopic approach

Introducción: Para acceder a la región selar, podemos utilizar las técnicas transcraneal, transeptal, o transnasal endoscópica, pudiendo provocar diferentes grados de hiposmia. Se ha descrito menor morbilidad al utilizar la técnica endoscópica, pero faltan estudios dirigidos a los resultados olfatorios. Objetivo: Determinar la presencia de deterioro olfatorio en los pacientes sometidos a un abordaje transnasal endoscópico. Material y método: Se reclutaron 12 pacientes con tumores en la región selar durante 8 meses. Se les realizó un test de olfato preoperatorio, fueron intervenidos mediante abordaje transnasal endoscópico y controlados al mes posoperatorio. Resultados: Se logró seguimiento a 10 pacientes. Seis (60%) presentaron un test de olfato preoperatorio normal. Al mes posoperatorio, se constató mejoría olfatoria en 1 (10%) paciente, 8 (80%) se mantuvieron en la misma categoría y 1 (10%) presentó deterioro olfatorio. En suma, 9 de 10 pacientes (90%) mantienen o mejoran su olfato al mes posoperatorio. Conclusión: Nuestros resultados sugieren que el abordaje transnasal endoscópico utilizado en este estudio no produce deterioro olfatorio. Dado que además es una técnica de abordaje efectiva y relativamente segura, consideramos que constituye una alternativa factible para utilizar en pacientes con patología tumoral en la región selar.

Introduction: To access the sellar region we can use the transcranial, transeptal, or transnasal endoscopic approaches, which may cause different degrees of hyposmia. It has described less morbidity to use the endocopic technique, however, there are few studies directed at olfactory outcomes. Aim: To determine the presence of olfactory impairment secondary to endoscopic transnasal approach. Material and method: 12 patients with tumors in the sellar region were enrolled during 8 months. They were underwent a smell test preoperatively, operated by endoscopic transnasal approach, and controlled with postoperative retesting, after one month. Results: Follow-up was achieved to 10 patients. 6 (60%) presented a normal preoperative smell test. Within one postoperative month, olfactory improvement was found in 1 (10%) patient, 8 (80%) remained in the same category and 1 (10%) had olfactory impairment. In all, 9 out of 10 patients (90%) maintain or improve their sense of smell after surgery. Conclusions: Our results suggest that the transnasal endoscopic approach used in this study, doesn't produce olfactory impairment. Given that is also an effective and relatively safe approach, we believe that is a feasible alternative for use in patients with tumor pathology in the sellar region.