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

Cerebrospinalvätska och bloduttag från råttor med samtidig EEG-registrering

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.

Författare i fokus: Erhållande av högkvalitativa CSF- och blodprover för upptäckt av biomarkörer för epilepsi 

Provtagning av cerebrospinalvätska och blod från lateral svansven hos råttor under EEG-inspelningar

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  

Insamling av blod från svansvenen hos råttor under EEG-inspelningar

Börja med att täcka en fjärilsnål och dess slang med 1 % kalium-EDTA-lösning. Klipp av slangen precis bakom nålen för att samla upp blod droppvis. Placera en sövd råtta på den stereotaktiska ramen samtidigt som du bibehåller bedövning genom en ansiktsmask.Lägg en värmedyna under djuret och håll en del av svansen i direkt kontakt med dynan. Flytta djuren bakåt så att de är vända åt sidan, och den laterala svansvenen är synlig upptill. Doppa svansen i varmt vatten för att vidga sidovenen.Torka sedan av svansen med 70% etanol. Sätt varmt ljus på svansen med en vanlig glödlampa. Sätt in 21G fjärilsnålen i den laterala svansvenen i en vinkel på 20 grader till ett djup av fem millimeter.Samla sedan blod i ett 500 mikroliters vakuumuppsamlingsrör som innehåller fem milligram kalium EDTA som antikoagulantia. Ta bort nålen och stoppa blodflödet genom att trycka på stickstället. Sätt tillbaka råttan i sin hembur.Vänd sedan försiktigt upp och ner på slangen 10 gånger för att blanda antikoagulantia i blodet. Gör 1,5 centimeter djupa hål i isen och placera rören vertikalt i dem. Inom en timme efter insamlingen, centrifugera blodprovet i en kyld centrifug för att separera plasman.Aspirera sedan cirka 200 mikroliter plasma och undvik det röda och vita blodkroppsskiktet. Placera den utdragna plasman i ett 0,2 ml sterilt mikrorör. Lägg undan fem mikroliter av provet för kvalitetskontroll och förvara det återstående provet vid minus 80 grader Celsius fram till analys.Blod tappades från råttor vid fem tidpunkter med hjälp av olika metoder, såsom vakuumteknik på dag 52, svansmjölkning på dag 55 och droppteknik på dag 58 till 64. Kvaliteten på plasmaprover som utvärderades med UV-spektrofotometri visade att plasman var rosafärgad med ett medelabsorbansvärde på 0,647 vid användning av vakuumtekniken. På samma sätt resulterade svansmjölkningstekniken i rosafärgad plasma med en genomsnittlig absorbans på 0,620.Däremot, med det gravitationsaktiverade dropputtagssystemet, erhölls klara plasmaprover med signifikant reducerade genomsnittliga plasmaabsorbansvärden, vilket tyder på att denna procedur är optimal för att få högkvalitativa prover för analyser.

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 ...