Name: Collection of Blood from the Tail Vein of Rats During EEG Recordings
Uploaded: 2023-09-01T13:40:00
Description: Watch this Scientific Journal Video about Collection of Blood from the Tail Vein of Rats During EEG Recordings at JoVE.com

collection of blood from the tail vein of rats during eeg recordings

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Cerebrospinal fluid (CSF) and blood sampling are important to identify and validate biomarkers of epilepsy, in both preclinical and clinical research1,2. Nowadays, the diagnosis of epilepsy and most of the research on epilepsy biomarkers focus on EEG and neuroimaging3,4,5. These approaches, however, present several limitations. Apart from routine scalp measurements, in many cases, EEG requires invasive techniques like depth electrodes6. Brain imaging methods have poor temporal and spatial resolution and are relatively expensive and time-consuming7,8. For this reason, the identification of non-invasive, low-cost, and biofluid-based biomarkers would provide a very attractive alternative. In addition, these biofluid biomarkers could be combined with available diagnostic approaches to sharpen their predictiveness.
Patients diagnosed with epilepsy are routinely submitted to EEG9,10 and blood sampling11,12,13,14, and many also to CSF withdrawal to exclude life-threatening causes (i.e., acute infections, autoimmune encephalitis)15. These blood and CSF samples can be used in clinical research aiming to identify biomarkers for epilepsy. For example, Hogg and co-workers have found that an increase in three plasma tRNA fragments precedes seizure occurrence in human epilepsy14. Similarly, interleukin-1beta (IL-1&#946;) levels in human CSF and serum, expressed as ratio of IL-1&#946; levels in CSF over serum, can predict post-traumatic epilepsy development after traumatic brain injury16. These studies highlight the importance of biofluids sampling for epilepsy biomarkers research, but they face multiple limitations intrinsic to clinical trials, e.g., the cofounding factor of anti-epileptic drugs (AEDs) in blood, the frequent lack of etiology information, inadequate controls, modest numbers of patients, and others17,18.
Pre-clinical research offers other opportunities for investigating molecules in biofluids as potential biomarkers for epilepsy. In fact, it is possible to withdraw plasma and/or CSF from animals while performing EEG recordings. Moreover, sampling can be performed repeatedly across multiple days of the experiment, and a number of age, sex, and epileptic insult-matched controls can be used to improve the study&#39;s robustness. Here, a flexible technique to obtain CSF from cisterna magna with parallel withdrawal of plasma from the tail vein in EEG-monitored rats is described in detail. The presented technique has several advantages over alternative methods. By using a butterfly needle approach, it is possible to collect CSF several times without compromising the function of EEG electrodes or similar head implants. This represents a refinement of intrathecal catheter withdrawal procedures, which are associated with a relatively high risk of infection. In addition, the reported free fall dropping approach used for blood collection is superior to other approaches of tail vein blood withdrawal because of the highly reduced risk of hemolysis, due to the fact that blood does not pass through tubing and no vacuum pressure is applied. If performed under strict germ-free conditions, there is a particularly low risk of infection for animals. In addition, by starting the blood withdrawals at the very end of the animals&#39; tails, sampling can be repeated several times. Such techniques are easy to master and can be applied in many preclinical studies of central nervous system disorders.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

Toma de muestras de líquido cefalorraquídeo y sangre de la vena lateral de la cola en ratas durante los registros de EEG

In our research, we aim to find diagnostic and prognostic biomarkers for epilepsy. In order to validate them in biological liquids, we compare the levels of specific substances in the cerebrospinal fluid and plasma with the occurrence of spontaneous seizures in rats. Today some small non-coding ribonucleic acids like miRNAs or tRNA fragments have been identified as a circulating biomarker for epilepsy, but their utility as an effective and reliable diagnostic or prognostic tool in epilepsy must be confirmed.different circulating markers for epilepsy, we routinely withdraw plasma and CSF of epileptic rats. While performing EEG recording, student find spontaneous seizures. As we repeat the sampling across multiple days, we can correlate the levels of markers with single seizure occurrence or their cumulative effect.The current challenge is to obtain sufficient volume of high quality samples for the following analytical procedures without inducing stress-provoked seizures in animals caused by their manipulation, which may interfere with the levels of molecules under investigation. There is an urgent unmet medical need to discover biomarkers for people with epilepsy, especially the prognostic and risky ones. Plasma and CSF sampling in parallel to EEG seizure monitoring allow the significant advance to find the suitable marker.Our draw tail vein blood withdrawal technique produces high quality hemolysis-free plasma samples, as it does not require a vacuum or tail milking. Cisterna magna puncture that we use to sample CSF has better sterility and a lower risk of head implant loss in comparison to a cannulated system.

Watch this Scientific Journal Video about Collection of Blood from the Tail Vein of Rats During EEG Recordings at JoVE.com

Collection of Blood from the Tail Vein of Rats During EEG Recordings  

Recolección de sangre de la vena de la cola de ratas durante los registros de EEG

Para comenzar, cubra una aguja de mariposa y su tubo con una solución de EDTA al 1% de potasio. Corta el tubo justo detrás de la aguja para recoger la sangre gota a gota. Coloque una rata anestesiada en el marco estereotáxico mientras mantiene la anestesia a través de una máscara facial.Coloque una almohadilla térmica debajo del animal, manteniendo una parte de su cola en contacto directo con la almohadilla. Mueva a los animales hacia atrás de modo que miren hacia los lados y la vena lateral de la cola sea visible en la parte superior. Sumerge la cola en agua tibia para dilatar la vena lateral.A continuación, limpie la cola con etanol al 70%. Ponga una luz cálida en la cola con una bombilla incandescente normal. Inserte la aguja de mariposa 21G en la vena lateral de la cola en un ángulo de 20 grados a una profundidad de cinco milímetros.A continuación, recoja sangre en un tubo de recolección al vacío de 500 microlitros que contenga cinco miligramos de EDTA de potasio como anticoagulante. Retire la aguja y detenga el flujo de sangre ejerciendo presión en el lugar de la punción. Regresa a la rata a su jaula de origen.A continuación, invierta suavemente el tubo 10 veces para mezclar el anticoagulante en la sangre. Haz agujeros de 1,5 centímetros de profundidad en el hielo y coloca los tubos en ellos verticalmente. Dentro de una hora de la recolección, centrifugue la muestra de sangre en una centrífuga refrigerada para separar el plasma.Luego aspire unos 200 microlitros de plasma, evitando la capa de glóbulos rojos y blancos. Coloque el plasma extraído en un microtubo estéril de 0,2 mililitros. Reserve cinco microlitros de la muestra para el control de calidad y almacene la muestra restante a menos 80 grados centígrados hasta el análisis.La sangre se extrajo de las ratas en cinco puntos de tiempo utilizando diferentes métodos, como la técnica de vacío en el día 52, el ordeño de la cola en el día 55 y las técnicas de gota en los días 58 a 64. La calidad de las muestras de plasma evaluadas mediante espectrofotometría UV mostró que el plasma era de color rosa con un valor medio de absorbancia de 0,647 cuando se utilizó la técnica de vacío. Del mismo modo, la técnica de ordeño de cola dio como resultado un plasma de color rosado con una absorbancia media de 0,620.Por el contrario, con el sistema de extracción de gotas habilitado por gravedad, se obtuvieron muestras de plasma claro con valores medios de absorbancia plasmática significativamente reducidos, lo que sugiere que este procedimiento es óptimo para obtener muestras de alta calidad para análisis.

Research

JoVE Journal

Neuroscience

Collection of Blood from the Tail Vein of Rats During EEG Recordings

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

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Investigación

Neurociencias

Tethered Video-EEG in Epileptic Rats for Analyses of Seizure Activity

Video-EEG atado en ratas epilépticas para el análisis de la actividad convulsiva

Collection of Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings

Recolección de líquido cefalorraquídeo de rata Cisterna Magna durante registros de EEG