Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Marie Soukupov&#225;; Annunziata Guarino; Laila Asth; Pietro Marino; Mario Barbieri; Michele Simonato; Silvia Zucchini

doi:10.3791/65636

Amostragem de líquido cefalorraquidiano e sangue da veia lateral da cauda em ratos durante regist...

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JoVE Journal Neuroscience

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Amostragem de líquido cefalorraquidiano e sangue da veia lateral da cauda em ratos durante registros de EEG

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10:46 min

September 1, 2023

DOI: 10.3791/65636-v

Marie Soukupová¹, Annunziata Guarino¹, Laila Asth¹, Pietro Marino¹, Mario Barbieri¹, Michele Simonato¹, Silvia Zucchini¹

¹Department of Neuroscience and Rehabilitation, Section of Pharmacology,Universita degli Studi di Ferrara

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Overview

This study investigates potential biomarkers for epilepsy by comparing cerebrospinal fluid (CSF) and plasma samples taken from epileptic rats during spontaneous seizure monitoring using video-electroencephalogram (EEG). The approach aims to correlate the levels of different molecules with seizure activity while addressing concerns of sample quality and stress management in the animals.

Key Study Components

Area of Science

Neuroscience
Epilepsy research
Electrophysiology

Background

Identification of reliable biomarkers for epilepsy is crucial for diagnosis and prognosis.
Current research involves small non-coding ribonucleic acids as potential biomarkers.
Challenges exist in collecting high-quality samples without inducing stress-induced seizures.
Simultaneous sampling of CSF and plasma provides significant insights into seizure-related changes.

Purpose of Study

To identify diagnostic and prognostic biomarkers for epilepsy.
To correlate molecular changes in CSF and plasma with seizure occurrences.
To improve sampling techniques to ensure high data quality without inducing stress in rats.

Methods Used

Electrophysiological monitoring using video-EEG in epileptic rats.
Cerebrospinal fluid and plasma sampling at multiple time points for biomarker analysis.
Innovative blood collection methods to prevent hemolysis and enhance sample quality.
Behavioral acclimatization of rats and use of a Faraday cage to minimize external interference during EEG recording.

Main Results

Successful parallel collection of blood and CSF samples demonstrated improvements in sample quality with modified techniques.
Establishment of a method that produces hemolysis-free plasma samples for better analysis.
Correlations between marker levels and seizure occurrence are anticipated from repeated sampling.
Validation of sample collection techniques and their impact on data quality were highlighted.

Conclusions

This study provides foundational methodologies for exploring biomarkers in epilepsy that may lead to improved diagnostic tools.
Enhanced sampling techniques and analysis may enable better understanding of seizure mechanisms and lead to novel therapeutic strategies.
The findings underscore the importance of managing stress in animal models to yield reliable biological insights.

Frequently Asked Questions

What are the advantages of using the rat model in this study?

Rats offer a well-characterized model for epilepsy that allows for detailed monitoring of spontaneous seizures and the biological response to interventions.

How is the plasma and CSF sampling conducted?

Samples are collected simultaneously during EEG monitoring, using specific collection techniques to minimize stress and ensure high-quality samples.

What types of molecular analyses are performed on the samples?

The study focuses on identifying levels of biomarkers such as non-coding RNAs in plasma and CSF, which may correlate with seizure activity.

How do the sampling methods affect the quality of the plasma?

Modified sampling techniques were developed to produce hemolysis-free plasma, improving the reliability of subsequent analyses.

What are the implications of finding reliable biomarkers for epilepsy?

Identifying reliable biomarkers could greatly enhance diagnosis, prognosis, and the overall understanding of epilepsy, leading to better patient management.

O protocolo mostra repetidas coletas de líquido cefalorraquidiano e sangue de ratos epilépticos realizadas em paralelo com a monitorização contínua por vídeo-eletroencefalograma (EEG). Estes são instrumentais para explorar possíveis ligações entre alterações em várias moléculas de fluido corporal e atividade convulsiva.

Em nossa pesquisa, pretendemos encontrar biomarcadores diagnósticos e prognósticos para epilepsia. A fim de validá-los em líquidos biológicos, comparamos os níveis de substâncias específicas no líquido cefalorraquidiano e plasma com a ocorrência de convulsões espontâneas em ratos. Atualmente, alguns pequenos ácidos ribonucleicos não codificantes, como miRNAs ou fragmentos de RNAt, têm sido identificados como um biomarcador circulante para epilepsia, mas sua utilidade como uma ferramenta diagnóstica ou prognóstica eficaz e confiável na epilepsia deve ser confirmada.

diferentes marcadores circulantes para epilepsia, rotineiramente retiramos plasma e LCR de ratos epilépticos. Ao realizar o registro do EEG, o aluno encontra convulsões espontâneas. À medida que repetimos a amostragem ao longo de vários dias, podemos correlacionar os níveis de marcadores com a ocorrência de uma única crise ou seu efeito cumulativo.

O desafio atual é obter volume suficiente de amostras de alta qualidade para os seguintes procedimentos analíticos sem induzir convulsões provocadas por estresse em animais causadas por sua manipulação, que podem interferir nos níveis de moléculas sob investigação. Há uma necessidade médica urgente não atendida de descobrir biomarcadores para pessoas com epilepsia, especialmente os prognósticos e de risco. A amostragem de plasma e LCR em paralelo ao monitoramento de crises no EEG permite o avanço significativo para encontrar o marcador adequado.

Nossa técnica de coleta de sangue da veia caudal produz amostras de plasma sem hemólise de alta qualidade, pois não requer ordenha a vácuo ou cauda. A punção de Cisterna magna que usamos para coletar amostras de LCR tem melhor esterilidade e menor risco de perda do implante cefálico em comparação a um sistema canulado.

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