Name: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery (Video) | JoVE
Uploaded: 2023-09-01T13:40:00
Description: Watch this Scientific Journal Video about Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery at JoVE.com

This study was supported by a grant from the European Union&#39;s Horizon 2020 Work Programme (call H2020-FETOPEN-2018-2020) under grant agreement 964712 (PRIME; to M. Simonato).

All experimental procedures have been approved by the University of Ferrara Institutional Animal Care and Use Committee and by the Italian Ministry of Health (authorization: D.M. 603/2022-PR) in accordance with guidelines outlined in the European Communities Council Directive of 24 November 1986 (86/609/EEC) on the protection of animals used for experimental and other scientific purposes. This protocol is specifically adjusted for further quantitative polymerase chain reaction (qPCR) analyses of small non-coding ribonucl...

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+physiological+parameters+of+rat+cerebrospinal+fluid+during+chronic+sampling:+evaluation+of+two+sampling+methods.">Changes in physiological parameters of rat cerebrospinal fluid during chronic sampling: evaluation of two sampling methods.</a> Brain Research Bulletin. 27 (2), 283-286 (1991).</li><li>Soukupov&#225;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+GABA+release+in+the+hippocampus+at+the+time+of+the+first+spontaneous+seizure+in+the+pilocarpine+model+of+temporal+lobe+epilepsy.">Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy.</a> Experimental Neurology. 257, 39-49 (2014).</li><li>Soukupov&#225;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+Excitatory+Amino+Acids+During+EEG+Recordings+in+Freely+Moving+Rats.">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> Journal of Visualized Experiments. 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The present work illustrates an easy-to-master technique of CSF and blood collection in rats, which may be useful not only for studies in models of epilepsy but also of other neurological conditions or diseases such as Alzheimer, Parkinson, or multiple sclerosis. In epilepsy research, both sampling procedures coupled with video-EEG are ideal when a correlation between the levels of different soluble molecules and seizure activity is pursued. For this specific reason, a continuous video-EEG recording was employed: i) in o...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blood collection set BD Vacutainer Safety-Lok</td>
			<td>BD Italy SpA, Milan, Italy</td>
			<td>367246</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Blood Collection tubes (Microtainer K2E)</td>
			<td>BD Italy SpA, Milan, Italy</td>
			<td>365975</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Butterfly Winged Infusion Set 23G x 3/4&#39;&#39; 0.6 x 19 mm</td>
			<td>Nipro, Osaka, Japan&#160;</td>
			<td>PSY-23-ET-ICU</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Centrifuge refrigerated ALC PK 130R</td>
			<td>DJB Labcare Ltd, Buckinghamshire, England</td>
			<td>112000033</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Cotton suture 3-0</td>
			<td>Ethicon, Johnson &#38; Johnson surgical technologies, Raritan, New Jersey, USA</td>
			<td>7343H</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Diazepam 5 mg/2ml, Solupam</td>
			<td>Dechra Veterinary Products, Torino, Italy</td>
			<td>105183014 (AIC)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Digital video 8-channel media recorder system of telemetry EEG set up</td>
			<td>Data Sciences International (DSI), St Paul, MN, USA</td>
			<td>PNM-VIDEO-008</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Digital video surveillance system of tethered EEG set up</td>
			<td>EZVIZ Network, Hangzhou, Cina</td>
			<td>EZVIZ (V5.3.2)</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Disinfectant based on stabilized peroxides and quaternary ammonium activity</td>
			<td>Laboratoire Garcin-Bactinyl, France</td>
			<td>LB 920111</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Dummy guide cannula 8 mm</td>
			<td>Agn Tho&#39;s, Lindig&#246;, Sweden</td>
			<td>CXD-8</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Electrode 3-channel two-twisted</td>
			<td>Invivo1, Plastic One, Roanoke, Virginia, USA</td>
			<td>MS333/3-B/SPC</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Electrode holder for stereotxic surgery</td>
			<td>Agn Tho&#39;s, Lindig&#246;, Sweden</td>
			<td>1776-P1</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Eppendorf BioSpectrometer basic</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>6137</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td> 
			Eppendorf PCR Tubes 0.2 mL</td>
			<td>Eppendorf Srl, Milan, Italy</td>
			<td>30124332</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Eppendorf &#956;Cuvette G1.0</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>6138</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Feeding needle flexible 17G for rat</td>
			<td>Agn Tho&#39;s, Lindig&#246; Sweden</td>
			<td>7206</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Grass Technology apparatus</td>
			<td>Grass Technologies, Natus Neurology Incorporated, Pleasanton, California, USA</td>
			<td>M665G08</td>
			<td>Equipment (AS40 amplifier, head box, interconnecting cables, telefactor model RPSA S40)</td>
		</tr>
		<tr>
			<td>Isoflurane 100%, IsoFlo</td>
			<td>Zoetis, Rome, Italy</td>
			<td>103287025 (AIC)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Ketamine (Imalgene)</td>
			<td>Merial, Toulouse, France</td>
			<td>221300288 (AIC)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Lithium chloride&#160;</td>
			<td>Sigma-Aldrich, Milan, Italy</td>
			<td>L9650</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Microinjection cannula 31G 9 mm</td>
			<td>Agn Tho&#39;s, Lindig&#246; Sweden</td>
			<td>CXMI-9</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>MP150 modular data acquisition and&#160;analysis system&#160;</td>
			<td>Biopac, Goleta, California, USA</td>
			<td>MP150WSW</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Ophthalmic vet ointment, Hylo night</td>
			<td>Ursapharm, Milan, Italy</td>
			<td>941791927 (AIC)</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Pilocarpine hydrochloride</td>
			<td>Sigma-Aldrich, Milan, Italy</td>
			<td>P6503</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>PTFE Tube with joint</td>
			<td>Agn Tho&#39;s, Lindig&#246;, Sweden</td>
			<td>JT-10</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>0.9% NaCl, pH adjusted to 7.0</td>
			<td></td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Scopolamine hydrobromide trihydrate</td>
			<td>Sigma-Aldrich, Milan, Italy</td>
			<td>S2250</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Scopolamine methyl nitrate</td>
			<td>Sigma-Aldrich, Milan, Italy</td>
			<td>S1876</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Silver&#160;sulfadiazine 1% cream&#160;</td>
			<td>Sofar, Trezzano Rosa, Milan, Italy</td>
			<td>025561010 (AIC)</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Simplex rapid dental methacrylic cement&#160;&#160;</td>
			<td>Kemdent, Associated Dental Products Ltd, Swindon, United Kingdom</td>
			<td>ACR811</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>David Kopf Instruments, Los Angeles, CA, USA</td>
			<td>Model 963</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Sucrose solution</td>
			<td>10% sucrose in distilled water</td>
			<td>Home-made</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Syringe 1 mL&#160;</td>
			<td>Biosigma, Cona, Venezia, Italy</td>
			<td>20,71,26,03,00,350</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Telemeters</td>
			<td>Data Sciences International (DSI), St Paul, MN, USA</td>
			<td>CTA-F40</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Telemetry EEG traces analyzer</td>
			<td>Data Sciences International (DSI), St Paul, MN, USA</td>
			<td>NeuroScore v3-0</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Telemetry system</td>
			<td>Data Sciences International (DSI), St Paul, MN, USA</td>
			<td>Hardware plus software Ponemah core 6.51</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride</td>
			<td>Sigma-Aldrich, Milan, Italy</td>
			<td>X1251</td>
			<td>Material</td>
		</tr>
	</tbody>
</table>

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.

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

The outcome of different CSF and blood withdrawal procedures performed in 9 control and 18 chronic epileptic rats, all implanted with electrodes at 1-month post-SE, is reported in terms of success rate. After implantation, all rats were video-EEG monitored for 1 month, during which the CSF plus blood was withdrawn 5x every 3 days during the two last weeks of the experiment (i.e., at days 52, 55, 58, 61, and 64 post-SE; dpSE). Data from multiple withdrawals in different animals were used to compare the success rate of CSF...

Watch this Scientific Journal Video about Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery at JoVE.com

Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery (Video) | JoVE

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 ...