Experimental Models Poster Presentation

P0981 - Monitoring of encephalitogenic cells treated with nanoparticles in adoptively transferred experimental autoimmune encephalomyelitis. (ID 1637)

Speakers
  • R. García-Hernández
Authors
  • R. García-Hernández
  • A. Nakal-Chidiac
  • A. Sánchez-Sanz
  • M. Aguilar
  • A. Garcia-Grande
  • M. Coronado-Albi
  • L. García-Fernández
  • M. Criado-González
  • C. Zamora Cañadas
  • J. García-Merino
  • A. Sánchez-López
Presentation Number
P0981
Presentation Topic
Experimental Models

Abstract

Background

The circulatory pattern of encephalitogenic cells (ECs) in the adoptively transferred experimental autoimmune encephalomyelitis (at-EAE) is not well known. Multimodal imaging nanoparticles (NPs), that combine more than one imaging moieties to be monitored by different imaging tecniques have gained special attention for cell monitoring.

Objectives

To evaluate the in vitro toxicity and internalization of new chitosan-based multimodal imaging NPs that incorporated IR820 NIR fluorescent probe, and superparamagnetic iron nanoparticles in ECs. To study the distribution of ECs labelled with internalized NPs after the induction of at-EAE.

Methods

Splenocytes and lymph nodes from C57BL/6 mice with actively induced EAE were cultured for 24, 48, 72 and 96 hours with MOG35-55 (25µg/ml), IL-12 (25ng/ml), IL-18 (25ng/ml) and different dilutions of NPs (1/50 and 1/100) which had different concentrations of Fe (1/10 and 1/100). ECs viability (up to 90%) was assessed by flow cytometry and Alamar blue. NPs internalization was analyzed by confocal microscopy and flow cytometry using 633nm red laser.

After selecting the best cell viability and IR emission culture condition with NPs, 2 x 106 ECs were injected intraperitoneally to induce at-EAE. Mice were sacrificed at different time points (1, 2, 3, 4, 7 and 24 days) and biodistribution of ECs was analyzed by confocal microscopy in heart, lymph nodes, lung, liver, kidney, spleen, brain and spinal cord..

To check whether the internalization of NPs affected the encephalitogenic capacity, clinical assessment was carried out for 24 days in two groups of animals. One of them was treated with ECs cultured with NPs, and the other with ECs without NPs.

Results

Confocal images obtained by fluorescence and reflection,corroborate the internalization of NPs and their persistence inside the cells over time. NPs did not affect the viability nor the encephalitogenic capacity of ECs. The highest fluorescence signal in ECs was obtained with the less diluted NPs (1/50) and the maximum concentration of Fe (1/10). These conditions were chosen for the induction of at-EAE. We have found the presence of ECs at days 1, 2 and 3 post injection in brain, spinal cord, lymph nodes and spleen.

Conclusions

Our findings suggest that activated ECs cross the blood-brain barrier rapidly after inoculation, locating themselves mainly in the central nervous system, where they trigger the inflammatory response that will produce the clinical characteristics of at-EAE. New multimodal imaging NPs allowed successful ECs monitoring due to their good tolerability and low toxicity.

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