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

Liquor- und Blutentnahme bei Ratten mit gleichzeitiger EEG-Aufzeichnung

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.

Autor im Rampenlicht: Gewinnung hochwertiger Liquor- und Blutproben für die Entdeckung von Epilepsie-Biomarkern 

Entnahme von Liquor und Blut aus der seitlichen Schwanzvene bei Ratten während EEG-Aufzeichnungen

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  

Entnahme von Liquor aus der Cisterna Magna der Ratte während der EEG-Aufzeichnungen

Bereiten Sie zunächst eine 23G-Schmetterlingsnadel vor, indem Sie den Plastikhüllenschutz so abschneiden, dass das Ende der blanken Nadel 7 Millimeter freiliegt. Verbinden Sie dann die Schmetterlingsnadel mit einem Polymerschlauch mit einer 1-Milliliter-Spritze. Legen Sie eine betäubte Ratte auf einen stereotaktischen Rahmen und halten Sie die Anästhesie durch eine Gesichtsmaske aufrecht.Als nächstes entfernst du das Fell am Hinterkopf und Hals der Ratte. Befestigen Sie den Kopf der Ratte mit Ohrstangen. Bewegen Sie die Nasenleiste des stereotaktischen Rahmens, um den Kopf des Tieres um ca. 45 Grad vertikal abzusenken.Finde eine leicht eingedrückte Oberfläche am Hinterkopf mit dem Aspekt einer Raute zwischen dem Hinterhauptsvorsprung und der Atlaswirbelsäule. Reiben Sie die Oberfläche mit 70%igem Ethanol ein. Für die Liquorentnahme wird die Schmetterlingsnadel senkrecht in die Mitte der rautenförmigen Eingedrücktenfläche in die Cisterna magna eingeführt, bis die Bewegung durch den Kunststoffhülsenschutz der Nadel blockiert wird.Ziehen Sie den Spritzenkolben vorsichtig ab, damit der Liquor langsam durch die Nadel fließen kann. Sammeln Sie etwa 100 Mikroliter des Liquors in Polymerschläuchen. Drücken Sie den Polymerschlauch ganz nah an die Butterfly-Nadel und schneiden Sie den Schlauch an dieser Stelle ab.Ziehen Sie die durchsichtige Probe in die Spritze. Die Probe wird in das sterile 0,2-Milliliter-Mikroröhrchen ausgestoßen und bis zu einer Stunde auf Eis gelagert. Desinfizieren Sie die Liquorentnahmestelle am Kopf des Tieres.Entferne die Ratte aus dem stereotaktischen Rahmen und setze sie zurück in ihren Käfig. Legen Sie 2 Mikroliter der Probe zur Qualitätskontrolle beiseite und lagern Sie den Rest zur weiteren Analyse bei 80 Grad Celsius. Ein wiederholter Liquorentzug wurde über fünf Tage in drei experimentellen Gruppen von Ratten durchgeführt und als Prozentsatz der erfolgreichen Entzüge ausgedrückt, die zu einem deutlichen Liquor führten.Bei den mit Doppelkopfimplantaten ausgestatteten Ratten, die für die Liquorentnahme kanüliert wurden, lag die Erfolgsrate bei 71,1 %, was darauf hindeutet, dass die Kanüle am Kopf des Tieres die wiederholte Liquorentnahme beeinträchtigen kann. Im Gegensatz dazu betrug die Erfolgsrate bei Tieren mit Elektrodenimplantaten 86,7 % und 88,9 %, wenn die Liquorentnahme durch Cisterna-Magna-Punktion durchgeführt wurde.

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 ...