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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 resol....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
Blood collection set BD Vacutainer Safety-LokBD Italy SpA, Milan, Italy367246Material
Blood Collection tubes (Microtainer K2E)BD Italy SpA, Milan, Italy365975Material
Butterfly Winged Infusion Set 23G x 3/4'' 0.6 x 19 mmNipro, Osaka, Japan PSY-23-ET-ICUMaterial
Centrifuge refrigerated ALC PK 130RDJB Labcare Ltd, Buckinghamshire, England112000033Material
Cotton suture 3-0Ethicon, Johnson & Johnson surgical technologies, Raritan, New Jersey, USA7343HMaterial
Diazepam 5 mg/2ml, SolupamDechra Veterinary Products, Torino, Italy105183014 (AIC)Solution
Digital video 8-channel media recorder system of telemetry EEG set upData Sciences International (DSI), St Paul, MN, USAPNM-VIDEO-008Equipment
Digital video surveillance system of tethered EEG set upEZVIZ Network, Hangzhou, CinaEZVIZ (V5.3.2)Equipment
Disinfectant based on stabilized peroxides and quaternary ammonium activityLaboratoire Garcin-Bactinyl, FranceLB 920111Solution
Dummy guide cannula 8 mmAgn Tho's, Lindigö, SwedenCXD-8Material
Electrode 3-channel two-twistedInvivo1, Plastic One, Roanoke, Virginia, USAMS333/3-B/SPCMaterial
Electrode holder for stereotxic surgeryAgn Tho's, Lindigö, Sweden1776-P1Equipment
Eppendorf BioSpectrometer basicEppendorf AG, Hamburg, Germany6137Equipment

Eppendorf PCR Tubes 0.2 mL
Eppendorf Srl, Milan, Italy30124332Material
Eppendorf μCuvette G1.0Eppendorf AG, Hamburg, Germany6138Equipment
Feeding needle flexible 17G for ratAgn Tho's, Lindigö Sweden7206Material
Grass Technology apparatusGrass Technologies, Natus Neurology Incorporated, Pleasanton, California, USAM665G08Equipment (AS40 amplifier, head box, interconnecting cables, telefactor model RPSA S40)
Isoflurane 100%, IsoFloZoetis, Rome, Italy103287025 (AIC)Solution
Ketamine (Imalgene)Merial, Toulouse, France221300288 (AIC)Solution
Lithium chloride Sigma-Aldrich, Milan, ItalyL9650Material
Microinjection cannula 31G 9 mmAgn Tho's, Lindigö SwedenCXMI-9Material
MP150 modular data acquisition and analysis system Biopac, Goleta, California, USAMP150WSWEquipment
Ophthalmic vet ointment, Hylo nightUrsapharm, Milan, Italy941791927 (AIC)Material
Pilocarpine hydrochlorideSigma-Aldrich, Milan, ItalyP6503Material
PTFE Tube with jointAgn Tho's, Lindigö, SwedenJT-10Material
Saline0.9% NaCl, pH adjusted to 7.0Solution
Scopolamine hydrobromide trihydrateSigma-Aldrich, Milan, ItalyS2250Material
Scopolamine methyl nitrateSigma-Aldrich, Milan, ItalyS1876Material
Silver sulfadiazine 1% cream Sofar, Trezzano Rosa, Milan, Italy025561010 (AIC)Material
Simplex rapid dental methacrylic cement  Kemdent, Associated Dental Products Ltd, Swindon, United KingdomACR811Material
Stereotaxic apparatusDavid Kopf Instruments, Los Angeles, CA, USAModel 963Equipment
Sucrose solution10% sucrose in distilled waterHome-madeSolution
Syringe 1 mL Biosigma, Cona, Venezia, Italy20,71,26,03,00,350Material
TelemetersData Sciences International (DSI), St Paul, MN, USACTA-F40Material
Telemetry EEG traces analyzerData Sciences International (DSI), St Paul, MN, USANeuroScore v3-0Equipment
Telemetry systemData Sciences International (DSI), St Paul, MN, USAHardware plus software Ponemah core 6.51Equipment
Xylazine hydrochlorideSigma-Aldrich, Milan, ItalyX1251Material

References

  1. Hanin, A., et al. Cerebrospinal fluid and blood biomarkers of status epilepticus. Epilepsia. 61 (1), 6-18 (2020).
  2. Pitkänen, A., et al. Advances in the development of biomarkers for epilepsy. The ....

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