Name: Collection of Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings
Uploaded: 2023-09-01T13:40:00
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collection of cerebrospinal fluid from rat cisterna magna during eeg recordings

נוזל מוחי ונסיגת דם מחולדות עם רישום EEG במקביל

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.

מחבר זרקור: השגת CSF באיכות גבוהה ודגימות דם לגילוי סמנים ביולוגיים לאפילפסיה 

דגימת נוזל מוחי ודם מווריד זנב לטרלי בחולדות במהלך רישומי 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 Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings at JoVE.com

Collection of Cerebrospinal Fluid from Rat Cisterna Magna During EEG Recordings  

איסוף נוזל מוחי שדרתי מחולדה Cisterna Magna במהלך רישומי EEG

כדי להתחיל, להכין מחט פרפר 23G על ידי חיתוך ההגנה שרוול פלסטיק שלה, כך קצה המחט החשופה נחשף על ידי 7 מ"מ. לאחר מכן, חבר את מחט הפרפר עם צינורות פולימר מזרק 1 מיליליטר. הניחו חולדה מורדמת על מסגרת סטריאוטקסית ושמרו על הרדמה באמצעות מסכת פנים.לאחר מכן, להסיר את הפרווה על הראש האחורי והצוואר של החולדה. תקן את ראש החולדה עם מוטות אוזניים. הזיזו את מוט האף של המסגרת הסטריאוטקסית כדי להוריד את ראש החיה בכ-45 מעלות אנכית.מצא משטח מעט מדוכא על הראש האחורי עם היבט של מעוין בין הבליטה העורפית לבין עמוד השדרה האטלס. שפשפו את פני השטח עם 70% אתנול. לאיסוף CSF, הכנס את מחט הפרפר אנכית למרכז המשטח המדוכא בצורת מעוין לתוך מגנה cisterna עד שהתנועה תיחסם על ידי הגנת שרוול הפלסטיק של המחט.צייר בעדינות את בוכנה מזרק על מנת לאפשר CSF לזרום לאט דרך המחט. לאסוף כ 100 מיקרוליטר של CSF לתוך צינורות פולימר. צבטו את צינור הפולימר קרוב מאוד למחט הפרפר וחתכו את הצינור בשלב זה.ציירו את הדגימה השקופה לתוך המזרק. יש להוציא את הדגימה לתוך המיקרו-צינורית הסטרילית של 0.2 מיליליטר ולאחסן על קרח למשך עד שעה. לחטא את האתר של נסיגה CSF על הראש של החיה.הוציאו את החולדה מהמסגרת הסטריאוטקסית והחזירו אותה לכלוב שלה. שים בצד 2 מיקרוליטר של המדגם לבקרת איכות ולאחסן את השאר ב 80 מעלות צלזיוס לניתוח נוסף. נסיגה חוזרת של CSF בוצעה במשך חמישה ימים בשלוש קבוצות ניסוי של חולדות והתבטאה באחוז משיכות מוצלחות שהביאו ל-CSF ברור.בחולדות בעלות שתל ראש כפול שהוענקו לנסיגת CSF, שיעור ההצלחה היה 71.1%, מה שמצביע על כך שהצינורית שעל ראשו של בעל החיים עלולה להפריע לדגימת CSF חוזרת. לעומת זאת, כאשר איסוף CSF בוצע על ידי ניקוב cisterna magna רק באלקטרודות קשורות או טלמטריה שהושתלו בבעלי חיים, שיעור ההצלחה היה 86.7% ו 88.9% תוצאות אלה מצביעות על כך שטכניקת הניקוב מתאימה יותר למשיכות CSF מרובות בבעלי חיים מושתלי אלקטרודות.

collection-cerebrospinal-fluid-from-rat-cisterna-magna-during-eeg

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 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 ...

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.

וידאו EEG קשור בחולדות אפילפטיות, לניתוח פעילות התקפים

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

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.

איסוף דם מווריד הזנב של חולדות במהלך רישומי EEG

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.