Real Time <em>In Vivo</em> Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

Elisa Oltra; Alejandro Caicedo

doi:10.3791/58236

No Vivo controle de timócitos na Câmara Anterior do olho a Laser, microscopia eletrônica...

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JoVE Journal Biology

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

No Vivo controle de timócitos na Câmara Anterior do olho a Laser, microscopia eletrônica de varredura em tempo real

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08:21 min

October 2, 2018

DOI: 10.3791/58236-v

Elisa Oltra^1,2, Alejandro Caicedo³

¹School of Medicine and Dentistry,Universidad Católica de Valencia San Vicente Mártir, ²Unidad Mixta CIPF-UCV,Centro de Investigación Príncipe Felipe, ³Department of Medicine,University of Miami

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Overview

This study demonstrates a protocol for longitudinal intravital real-time tracking of thymocytes using laser scanning microscopy in thymic implants within the anterior chamber of the mouse eye. This innovative approach allows for continuous monitoring of progenitor cell recruitment and the egress of mature T-cells, providing insights into the mechanisms of autoimmunity and immunodeficiency.

Key Study Components

Research Area

Immunology
Thymocyte behavior
In vivo microscopy techniques

Background

Importance of studying thymus function
Role of thymocytes in immune response
Mechanisms of autoimmunity and immunodeficiency

Methods Used

Longitudinal intravital imaging
Mouse models with thymic implants
Laser scanning microscopy

Main Results

Real-time visualization of thymocyte dynamics
Tracking of cell recruitment and egress
Insights into thymus physiology and its dependence on vascularization

Conclusions

This study highlights a novel method for observing thymus activity in a living organism.
It opens avenues for further research into immunological disorders and cell behavior in grafted tissues.

Frequently Asked Questions

What is the purpose of the thymic implant in this study?

The thymic implant allows researchers to observe the behavior of thymocytes in a living system, providing insights into their roles in immune responses.

How does laser scanning microscopy contribute to this research?

Laser scanning microscopy enables real-time visualization of cellular dynamics and interactions within the thymic graft, allowing for detailed analysis of immunological processes.

What are the implications of tracking T-cell egress?

Tracking T-cell egress is crucial for understanding the development of the immune system and the potential dysregulation in autoimmune diseases.

Can this technique be applied to other tissues?

Yes, the method can also be adapted for studying other tissues such as pancreatic islets and kidney glomeruli.

What is the significance of vascularization in this context?

Vascularization is important as it influences thymus physiology and is critical for the delivery of nutrients and immune factors necessary for thymocyte development.

What challenges are associated with thymus transplantation?

Challenges include ensuring proper engraftment, avoiding immune rejection, and maintaining tissue integrity during surgical procedures.

What advancements does this protocol represent in immunology research?

This protocol represents a significant advancement by enabling longitudinal studies of thymocyte behavior in a living organism, thereby providing unprecedented insights into immune system functioning.

O objetivo do protocolo é mostrar longitudinal intravital acompanhamento em tempo real de timócitos por laser, microscopia em implantes do timo na câmara anterior do olho do rato. A transparência da córnea e vascularização do enxerto permitem gravar continuamente recrutamento de células progenitoras e egresso de células T maduro.

Este método pode ajudar a responder perguntas-chave no campo da imunologia sobre os mecanismos de autoimunidade, imunodeficiência, tolerância central e involução de timo. A principal vantagem dessa técnica é que permite o registro longitudinal in vivo do recrutamento progenitor e a egressão madura de células T nos segmentos de timo de camundongos vascularizados e enxertados. Embora este método possa fornecer uma visão sobre o desenvolvimento e a função do timo, também pode ser aplicado a outros tecidos, como ilhotas pancreáticas ou glomeruli renal.

Para isolar o timo, coloque o rato em toalhas de papel absorvente estéreis na posição dorsal vertical em um capô de fluxo laminar e pulverize e limpe o abdômen do rato com 70% de etanol. Faça uma incisão superficial em forma de V no abdômen inferior para expor a cavidade torácica e use um par de tesouras dissecando 10 centímetros em linha reta para fazer uma incisão de 0,5 a um centímetro no peito ao longo da linha média ventral. Dobre a pele sobre cada lado do peito para expor a cavidade torácica e faça duas incisões laterais adicionais de 0,5 a um centímetro através do diafragma e da caixa torácica para acessar o mediastino superior na cavidade torácica anterior.

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