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

Prelievo di liquido cerebrospinale e sangue dai ratti con registrazione EEG concomitante

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.

Autore in primo piano: Ottenere campioni di sangue e CSF di alta qualità per la scoperta di biomarcatori dell'epilessia 

Campionamento del liquido cerebrospinale e del sangue dalla vena caudale laterale nei ratti durante le registrazioni 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  

Raccolta di sangue dalla vena caudale dei ratti durante le registrazioni EEG

Per iniziare, rivestire un ago a farfalla e il suo tubo con una soluzione di EDTA all'1% di potassio. Tagliare il tubo appena dietro l'ago per raccogliere il sangue goccia a goccia. Posizionare un ratto anestetizzato sul telaio stereotassico mantenendo l'anestesia attraverso una maschera facciale.Metti un termoforo sotto l'animale, mantenendo una parte della sua coda a diretto contatto con il tappetino. Sposta gli animali indietro in modo che siano rivolti lateralmente e la vena caudale laterale sia visibile in alto. Immergere la coda in acqua tiepida per dilatare la vena laterale.Quindi pulire la coda con etanolo al 70%. Metti una luce calda sulla coda usando una normale lampadina a incandescenza. Inserire l'ago a farfalla 21G nella vena caudale laterale con un angolo di 20 gradi a una profondità di cinque millimetri.Successivamente, raccogliere il sangue in una provetta di raccolta sottovuoto da 500 microlitri contenente cinque milligrammi di EDTA di potassio come anticoagulante. Rimuovere l'ago e interrompere il flusso sanguigno esercitando pressione sul sito della puntura. Riporta il ratto nella sua gabbia di casa.Quindi, capovolgere delicatamente la provetta 10 volte per mescolare l'anticoagulante nel sangue. Fai dei buchi profondi 1,5 centimetri nel ghiaccio e posiziona i tubi verticalmente. Entro un'ora dalla raccolta, centrifugare il campione di sangue in una centrifuga refrigerata per separare il plasma.Quindi aspirare circa 200 microlitri di plasma, evitando lo strato di globuli rossi e bianchi. Mettere il plasma prelevato in una microprovetta sterile da 0,2 millilitri. Mettere da parte cinque microlitri del campione per il controllo di qualità e conservare il campione rimanente a meno 80 gradi Celsius fino all'analisi.Il sangue è stato prelevato dai ratti in cinque momenti utilizzando metodi diversi, come la tecnica del vuoto il giorno 52, la mungitura della coda il giorno 55 e le tecniche di goccia nei giorni da 58 a 64. La qualità dei campioni di plasma valutati utilizzando la spettrofotometria UV ha mostrato che il plasma era di colore rosa con un valore medio di assorbanza di 0,647 quando si utilizzava la tecnica del vuoto. Allo stesso modo, la tecnica di mungitura della coda ha prodotto plasma di colore rosa con un'assorbanza media di 0,620.Al contrario, con il sistema di prelievo a goccia abilitato dalla gravità, sono stati ottenuti campioni di plasma trasparenti con valori medi di assorbanza plasmatica significativamente ridotti, suggerendo che questa procedura è ottimale per ottenere campioni di alta qualità per le analisi.

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

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 experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

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

Ricerca

Neuroscienze

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

To begin, place the animal in a clean cage in the recording room to allow habituation to the new environment. Place the cage into a Faraday cage to avoid interference of the environmental electromagnetic field in the EEG signal. Connect one end of the recording cable to the recording device.Use a volt meter to measure the electric potential and to differentiate between ground and reference electrodes. Then hold the cement cover on the animal's head, and insert the other end of the recording cable into the electrode connector fixed on the head of the rat. Use a commutator to counterbalance the weight of the recording cable and to allow free movement of the animal without twisting the cable.Before starting the registration, check all the settings used to acquire and process the data from the EEG. Ensure adequate filtering and sampling rates are applied to avoid artifacts and noise in EEG signals. Start the video recording and the EEG recordings on the monitor.Check the power in specific frequency bands across time to ensure that the traces match an expected EEG signal. Check the EEG traces periodically. Also, check the animals.If a recording cable detaches from the electrode connector, reconnect and check for a clear EEG signal. Analyze the EEG signal by screening through the EEG recording and identify seizure-like activities. Confirm the potential convulsive seizures by checking the synchronized video recording collected simultaneously with EEG.

Video-EEG legato nei ratti epilettici per l'analisi dell'attività convulsiva

To begin, coat a butterfly needle and its tubing with 1%potassium EDTA solution. Cut the tubing just behind the needle in order to collect blood dropwise. Place an anesthetized rat on the stereotaxic frame while maintaining anesthesia through a face mask.Put a heating pad under the animal, keeping a part of its tail in direct contact with the pad. Move the animals back such that it faces sideways, and the lateral tail vein is visible at the top. Dip the tail in warm water to dilate the lateral vein.Then wipe the tail with 70%ethanol. Put warm light onto the tail using a regular incandescent bulb. Insert the 21G butterfly needle into the lateral tail vein at an angle of 20 degrees to a depth of five millimeters.Next, collect blood in a 500 microliter vacuum collection tube containing five milligrams of potassium EDTA as an anticoagulant. Remove the needle and stop the flow of the blood by putting pressure on the puncture site. Return the rat to its home cage.Next, gently invert the tube 10 times to mix anticoagulant in the blood. Make 1.5 centimeter deep holes in the ice and place the tubes in them vertically. Within one hour of collection, centrifuge the blood sample in a refrigerated centrifuge to separate the plasma.Then aspirate about 200 microliters of plasma, avoiding the red and white blood cell layer. Place the withdrawn plasma into a 0.2 milliliter sterile microtube. Put aside five microliters of the sample for quality control and store the remaining sample at minus 80 degrees Celsius until analysis.Blood was withdrawn from rats at five time points using different methods, such as the vacuum technique on day 52, tail milking on day 55, and drop techniques on days 58 to 64. The quality of plasma samples evaluated using UV spectrophotometry showed that the plasma was pink-colored with a mean absorbance value of 0.647 when using the vacuum technique. Similarly, the tail milking technique resulted in pink-colored plasma with mean absorbance of 0.620.In contrast, with the gravity-enabled drop withdrawal system, clear plasma samples were obtained with significantly reduced mean plasma absorbance values, suggesting that this procedure is optimal for getting high quality samples for analyses.

Collection of Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings

To begin, prepare a 23G butterfly needle by cutting its plastic sleeve protection, such that the end of the bare needle is exposed by 7 millimeters. Then, connect the butterfly needle with polymer tubing to a 1 milliliter syringe. Place an anesthetized rat on a stereotaxic frame and maintain anesthesia through a face mask.Next, remove the fur on the rear head and neck of the rat. Fix the head of the rat with ear bars. Move the nose bar of the stereotaxic frame to lower the animal's head by approximately 45 degrees vertically.Find a slightly depressed surface on the rear head with the aspect of a rhombus between the occipital protuberance and the atlas spine. Rub the surface with 70%ethanol. For CSF collection, insert the butterfly needle vertically into the center of the rhombus-shaped depressed surface into the cisterna magna til the movement is blocked by the plastic sleeve protection of the needle.Gently draw the syringe piston in order to let the CSF slowly flow through the needle. Collect about 100 microliters of the CSF into polymer tubing. Pinch the polymer tubing very close to the butterfly needle and cut the tubing at this point.Draw the clear sample into the syringe. Expel the sample into the sterile 0.2 milliliter microtube and store on ice for up to one hour. Disinfect the site of CSF withdrawal on the animal's head.Remove the rat from stereotaxic frame and put it back in its cage. Put aside 2 microliters of the sample for quality control and store the rest at 80 degrees Celsius for further analysis. Repeated CSF withdrawal was performed across five days in three experimental groups of rats and expressed as a percentage of successful withdrawals that resulted in clear CSF.In the double head implant endowed rats cannulated for CSF withdrawal, the success rate was 71.1%indicating that the cannula on the animal's head may interfere with repeated CSF sampling. In contrast, when CSF collection was carried out by cisterna magna puncture in only tethered or telemetry electrodes implanted animals, the success rate was 86.7%and 88.9%These results suggest that the puncture technique is more suitable for multiple CSF withdrawals in electrode implanted animals.