In our research, we aim to find putative 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. To date, 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.
We study different circulating markers for epilepsy. We routinely withdraw plasma and CSF of epileptic rats while performing EEG recording to identify 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 product 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 access to find the suitable marker.
Our drop tail vein blocked 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. 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 it 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. 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 drop-wise. 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 animal's 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 micro tube. 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.
To begin, prepare a 203G butterfly needle by cutting its plastic sleeve protection such that the end of the bare needle is exposed by seven millimeters. Then connect the butterfly needle with polymer tubing to a one 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 micro tube 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 two microliters of the sample for quality control and store the rest at minus 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.
