Name: Collection of Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings
Uploaded: 2023-09-01T13:40:00
Description: Watch this Scientific Journal Video about Collection of Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings at JoVE.com

collection of cerebrospinal fluid from rat cisterna magna 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 Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings at JoVE.com

Collection of Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings  

Insamling av cerebrospinalvätska från råtta Cisterna Magna under EEG-inspelningar

Börja med att förbereda en 23G fjärilsnål genom att klippa av plasthylsan, så att änden av den nakna nålen exponeras med 7 millimeter. Anslut sedan fjärilsnålen med polymerslang till en 1 milliliters spruta. Placera en sövd råtta på en stereotaktisk ram och upprätthåll bedövning genom en ansiktsmask.Ta sedan bort pälsen på råttans bakre huvud och hals. Fäst råttans huvud med öronstänger. Flytta nosstången på den stereotaktiska ramen för att sänka djurets huvud med cirka 45 grader vertikalt.Hitta en något nedtryckt yta på det bakre huvudet med aspekten av en romb mellan nackutbuktningen och atlasryggraden. Gnid in ytan med 70% etanol. För CSF-samling, stick in fjärilsnålen vertikalt i mitten av den rombformade nedtryckta ytan i cisterna magna tills rörelsen blockeras av nålens plasthylsa.Dra försiktigt upp sprutkolven så att den cerebrospinalvätska långsamt rinner genom nålen. Samla upp cirka 100 mikroliter av CSF i polymerrör. Nyp ihop polymerslangen mycket nära fjärilsnålen och klipp av slangen vid denna tidpunkt.Dra upp det klara provet i sprutan. Skjut ut provet i det sterila 0,2 ml mikroröret och förvara på is i upp till en timme. Desinficera platsen för CSF-uttaget på djurets huvud.Ta bort råttan från stereotaktisk ram och sätt tillbaka den i buren. Lägg åt sidan 2 mikroliter av provet för kvalitetskontroll och förvara resten vid 80 grader Celsius för vidare analys. Upprepad utsättning av cerebrospinalvätskan utfördes under fem dagar i tre experimentella grupper av råttor och uttrycktes som en procentandel av lyckade utsättningar som resulterade i tydlig cerebrospinalvätska.I råttor med dubbla huvudimplantat som kanylerades för att dras ut ur ryggmärgsvätskan var framgångsfrekvensen 71,1 %, vilket tyder på att kanylen på djurets huvud kan störa upprepad provtagning av cerebrospinalvätska. Däremot, när CSF-insamlingen utfördes genom cisterna magna-punktion i endast bundna eller telemetrielektroder implanterade djur, var framgångsfrekvensen 86,7 % och 88,9 %Dessa resultat tyder på att punktionstekniken är mer lämplig för multipla CSF-uttag hos elektrodimplanterade djur.

Research

JoVE Journal

Neuroscience

Collection of Cerebrospinal Fluid from Rat Cisterna Magna 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 ...