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 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.
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-1β) levels in human CSF and serum, expressed as ratio of IL-1β 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'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' 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.
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 ribonucleic acid (sncRNAs) in the rat CSF and plasma obtained under EEG control in epileptic animals. At its option, please see the related JoVE video for a better understanding and improvements of the surgery19,20,21.
1. Preparation of animals for surgical implantation of electrodes or telemeters
NOTE: The stereotaxic surgery technique varies according to the EEG system used. The following method section provides a description of steps that are in common for the two types of surgeries.
2. Surgical implantation of tethered electrodes
NOTE: Before establishing the puncture CSF withdrawal procedure of this protocol (see step 9 for details), repeated CSF withdrawals via guide cannula in a few freely moving non-anesthetized rats were performed. Cannulated animals implanted with tethered electrodes were used in order to evaluate the impact of double-head implants on long-term EEG recording coupled with multiple CSF sampling. In these specific experiments, rats were implanted with a dummy guide cannula placed in the cisterna magna, the tip of which was inserted 7 mm into it stereotactically, according to previously published protocols22. Double implant surgery approaches were similar to those adopted by some workers in the past for microdialysis guide cannulas and tethered electrode implantation23,24.
3. Surgical implantation of the telemeters
NOTE: Use only sterile telemeters. If telemeters are reused, clean and sterilize them before the surgery according to their manufacturer instructions. In this protocol, a data science International (DSI) telemeters for EEG recording was used.
4. Post-operative care
5. Status epilepticus induction in rats
NOTE: For a detailed protocol of status epilepticus (SE) induction needed to reproduce the mesial temporal lobe epilepsy (mTLE) in rats, refer to Guarino et al.25.
6. Tethered video-EEG in epileptic rats and analyses of seizure activity
NOTE: This section describes the experimental procedure to record EEG signals in single-housed, freely moving rats under standard conditions. The cage should not contain objects where the animal or the recording cable can get stuck. Depending on the scientific question to be addressed, several parameters can be analyzed. In the case of epilepsy research, the EEG traces are screened to recognize electrical and motor seizures. The most common parameters used to identify a seizure are the amplitude, frequency, and duration of paroxysmal electrical activity.
7. Telemetry video-EEG in epileptic rats and analysis of seizure activity
NOTE: This section describes the experimental procedure to record radiotelemetry EEG signals in single-housed, freely moving rats under standard conditions. The protocol is based on a commercially available telemetry system. However, several telemetry systems differ slightly in their functional and technical specifications. The system should be chosen depending on lab requirements and research goals.
8. Procedure of blood collection from the tail vein
NOTE. The vacuum blood collection system consists of a butterfly needle (23 G x ¾ x 12 (0.8 mm x 19 mm x 305 mm). The blood collection technique can be easily performed by one operator and the procedure takes about 5 min.
9. CSF collection procedure
NOTE. The technique can be easily performed by a single operator, and the procedure requires around 2-4 min. The materials used for the collection of CSF are low-cost, single-use vacuum butterfly needles and extraction tubes. In this protocol, a butterfly-winged infusion set connected to a sterile syringe is used in order to create the vacuum (Figure 2A).
10. Spectrophotometry analysis of the sample's quality
NOTE: After proper collection of CSF and plasma samples, the samples are ready for spectrophotometer analyses and do not require any specific handling. Measure the hemoglobin absorbance by UV spectrophotometry at 414 nm to evaluate the hemolysis risk in samples. Use a cut-off absorbance value of 0.25 in rat samples. The choice of this limit may depend on subsequent qPCR analysis and its specific requirements for sncRNAs quantification.
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 collection in the double-head implant-endowed rats (cannulated for CSF withdrawal) with the success rate of CSF collection (carried out by cisterna magna puncture) in only tethered or telemetry electrodes implanted animals (Table 1). In different animals, the impact of vacuum blood collection or tail milking on the quality of plasma samples was evaluated (Table 2). For this purpose, UV spectrophotometry analysis at 414 nm was used for the detection of free hemoglobin. For statistical analyses, commercial software was used, and Kruskal-Wallis or one-way ANOVA with post-hoc Tukey's multiple comparison tests were used (p<0.05 considered statistically significant). The data are expressed as a mean ± SEM.
Success rate of multiple CSF sampling in cannulated and punctured rats
CSF has been sampled 5x within 2 weeks in 3 groups of rats: (i) cannulated and tethered electrode implanted rats (CT group of animals); in these, the CSF withdrawal was performed via dummy guide cannula and PTFE tubing joint to 1 mL syringe when they were non-anesthetized and freely moving under video-EEG; (ii) punctured (step 9) and tethered electrode implanted rats (PT group); (iii) punctured and telemetry electrode implanted rats (PTe group). A total of 9 animals per group (6 epileptic and 3 control rats) were used. The number of successful collections over 5 times was evaluated. The success rate was similar in punctured rats: 86.7% ± 5.8% in tethered and 88.9% ± 4.8% in telemetry electrode implanted animals. Instead, in the cannulated rats, the rate was reduced even if not significantly different (71.1% ± 8.9%, Table 1). Such results indicate that the cannula on animals' heads may interfere with repeated CSF sampling and compromise longitudinal studies. The puncture technique is more suitable for multiple CSF withdrawals in electrodes implanted animals.
Impact of vacuum and tail milking on the plasma collection method
Blood was collected 5x from 9 rats (6 epileptic and 3 control rats) at days 52, 55, 58, 61, and 64 post-SE and the plasma quality was evaluated for hemolysis visually and by UV spectrophotometry at 414 nm. To obtain the first sample in each rat, the vacuum withdrawal via a 21G butterfly needle attached to a 1 mL syringe was employed. With the second sample, the drop withdrawal and 21G butterfly needle system were employed when milking the tail simultaneously. To get the 3rd-5th sample, the drop withdrawal procedure without milking the tail (described in step 9) was used.
When employing a vacuum, the plasma was pink colored under the visual inspection, and the mean absorbance value of 9 rats' samples was 0.647 ± 0.067 (Table 2, Figure 3). Similar results were obtained if employing the tail milking during the procedure: pink-colored plasma with 0.620 ± 0.043 mean absorbance (Table 2, Figure 3). In contrast, with the gravity-enabled drop withdrawal and 21G butterfly needle system, the mean plasma absorbance values were significantly reduced (0.226 ± 0.017 at 58 dpSE; 0.223 ± -0.09 at 61 dpSE; 0.226 ± 0.018 at 64 dpSE; Table 2, Figure 3) with respect to vacuum or tail milking method. Moreover, the drop plasma samples were mainly transparent. Higher values of absorbance (52 and 55 dpSE) correlated with the pink color of samples (data not shown). These results may suggest that the last method is the best to get samples of very high quality for analyses.
Figure 1: Key steps of plasma sampling workflow. (A) Materials necessary for blood withdrawal and rat in the stereotaxic frame, ready for collection; (B, C) Magnifications of the tail with 21G butterfly needle inserted into the lateral tail vein and the blood drop falling down the walls of the collection tube with an anticoagulant. Please click here to view a larger version of this figure.
Figure 2: Key steps of cerebrospinal fluid (CSF) sampling workflow. (A) Materials necessary for the CSF withdrawal and rat in the stereotaxic frame, shortly before collection; (B) The 23G butterfly needle preparation by cutting its plastic sleeve protection so that the end of the bare needle is exposed for 7 mm to ensure correct penetration into the cisterna magna; (C) The rat head is inclined downwards by 45° during withdrawal. (D) Magnification on the rhomboid site with a butterfly needle inserted in the cisterna magna. Note the CSF that rises in the tubing, indicated by the tip of the marker. Please click here to view a larger version of this figure.
Figure 3: Quality evaluation of plasma samples. Degree of hemolysis measured at 414 nm for free hemoglobin by UV spectroscopy in plasma samples of 9 animals at 5 time-points (52, 55, 58, 61, and 64 days' post status epilepticus, dpSE) using different methods: day 52 – the vacuum technique; day 55 – the tail milking; days 58-64 the drop techniques were employed. The decrease in free hemoglobin in plasma obtained by drop technique compared to vacuum and tail milking methods was significant (*p <0.05 according to one-way ANOVA and post-hoc Tukey's multiple comparison test). Please click here to view a larger version of this figure.
Table 1: Success rates of CSF withdrawals. Comparison of the success rates of repeated CSF withdrawal in three experimental groups of animals expressed as a percentage of successful withdrawals across 5 days. The value 1 was assigned to successful withdrawal of > 100 µL of clear CSF; the zero value was assigned to withdrawals < 100 µL and/or of unclear CSF. Abbreviations: N/A – the absence of collection due to the loss of cannula during the sampling procedure (CT animals only); CT – cannulated tethered; PT – punctured tethered; PTe – punctured telemetry electrodes implanted. Please click here to download this Table.
Table 2: Evaluation of hemolysis in plasma samples. Results of the hemolysis measurements at 5 time-points using three different methods of blood sampling: day 52 – the vacuum technique; day 55 – the tail milking; days 58-64 the drop techniques. Values >0.3 of absorbance correlated with pink color of samples. Please click here to download this Table.
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 order to correctly diagnose epilepsy or ii) to monitor the different phases of the disease progression, and/or iii) to correlate sampling with the occurrence of spontaneous seizures. Such sampling techniques can be performed in anesthetized rats, thus causing minimal stress.
Critical steps, troubleshooting, method limitations
The protocol has some critical technical steps. Firstly, it can be difficult to find the correct place for CSF collection at the first attempt. If the operator misses the cisterna magna at the first attempt, any subsequent trial would be blood contaminated, as the animal will bleed from the needle wound. From this point of view, the success in the collection is heavily dependent on the operator's skill. Secondly, some steps of blood withdrawal need special attention. In particular, there is a high risk of hemolysis if the operator rubs the tail too vigorously with ethanol, if the temperature of the water used for the tail vein vasodilation is higher than 42 °C, or if the blood in the collection tube is mixed with anticoagulant too energetically. Another peculiarity of blood collection in chronic epileptic animals is the influence of their bradycardia on the rate at which blood drops out of the tail vein33. If this is too slow, the blood may coagulate on the walls of the collection tube. To avoid this problem, one option is to split the sampling into two collection tubes, reducing the volume of blood/tube. Finally, there is one pitfall that is intrinsic to epilepsy studies. The stress provoked by animal manipulation before sampling may induce seizures, which in turn may interfere with the levels of molecules under the investigation34. Whenever it is possible, place the anesthesia induction chamber into the home cage and allow the animal to enter it spontaneously. As a modification of the proposed protocol, a restrainer can be used to perform blood withdrawal without isoflurane anesthesia. However, this can be done in telemetry, but not in tethered animals, because tethered rats may lose their head implants during this procedure.
Having a well-trained operator and posing maximum effort to avoid stress, the only limitation of the present protocol is the maximal volume that can be withdrawn without compromising the animal's health. According to current standards, it is recommended to collect a maximum of 100 µL of CSF for 4x over 15 days in a single animal32. Similarly, it is suggested to collect less than 10% of total blood body volume on a single sampling and less than 15% of total blood body volume in 28 days30,31.
Comparison of the method with other techniques
Proposed time-resolved CSF and plasma sampling approaches have several advantages with respect to existing alternative methods. First, a cisterna magna puncture used to sample CSF in epileptic rats has a lower risk of head implant loss in comparison to a cannulated system if coupled to tethered EEG. In contrast to puncture procedures, the cannula attached to the electrode by dental cement (bulky and heavy for animals' heads), while solicited by repeated attachments/detachments to the CSF withdrawals, is much more prone to being lost over multiple days of sampling. Indeed, the success rate results show how some cannulated animals (N/A) do not arrive at the advanced sampling time points, thus their respective samples are lost (Table 1). Additionally, the puncture method seems to be superior to cannulated approach in terms of better sterility and reduced meningeal reaction with limited increase of cell and albumin content in CSF, as has been previously documented by other22,35,36. The degree of CSF leukocytes and albumin contamination may be important for the validity of methods used for epilepsy biomarkers quantification35. Second, the free fall dropping method of blood plasma sampling used for repeated measures is superior to any other withdrawal method as it is not terminal (non-recovery), unlike the decapitation, cardiac puncture, abdominal/thoracic blood vessel or retro-orbital withdrawal and allows multiple blood sampling. It is simpler than many tail vein blood vacuum withdrawal techniques, as it does not require tubing28,37 and produces hemolysis-free plasma samples of high quality for further sncRNA analyses focused on identifying putative biomarkers of epileptogenesis38. The absence of free hemoglobin in the samples, when employing the drop sampling technique or avoiding the tail milking, was confirmed by low absorbance results of the plasma samples (Table 2) in line with previously published procedures suitable for the evaluation of sncRNA plasma content39,40.
Applications and future directions
The above-described methods may be applied to measure soluble molecules of interest in any model of neurological diseases. A specific example is the sampling of biological liquids for the identification of potential/putative epilepsy biomarkers. There is an urgent unmet medical need to discover these biomarkers for people with epilepsy, especially prognostic and susceptibility/risk biomarkers, as they do not yet exist.
In conclusion, the present protocol is feasible in rats, including epileptic rats, and is easy to actuate for trained individuals. Moreover, it permits multiple high-quality sampling in longitudinal studies in compliance with the principle of 3Rs (i.e., replacement, reduction, and refinement)41.
The authors have nothing to disclose.
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).
Blood collection set BD Vacutainer Safety-Lok | BD Italy SpA, Milan, Italy | 367246 | Material |
Blood Collection tubes (Microtainer K2E) | BD Italy SpA, Milan, Italy | 365975 | Material |
Butterfly Winged Infusion Set 23G x 3/4'' 0.6 x 19 mm | Nipro, Osaka, Japan | PSY-23-ET-ICU | Material |
Centrifuge refrigerated ALC PK 130R | DJB Labcare Ltd, Buckinghamshire, England | 112000033 | Material |
Cotton suture 3-0 | Ethicon, Johnson & Johnson surgical technologies, Raritan, New Jersey, USA | 7343H | Material |
Diazepam 5 mg/2ml, Solupam | Dechra Veterinary Products, Torino, Italy | 105183014 (AIC) | Solution |
Digital video 8-channel media recorder system of telemetry EEG set up | Data Sciences International (DSI), St Paul, MN, USA | PNM-VIDEO-008 | Equipment |
Digital video surveillance system of tethered EEG set up | EZVIZ Network, Hangzhou, Cina | EZVIZ (V5.3.2) | Equipment |
Disinfectant based on stabilized peroxides and quaternary ammonium activity | Laboratoire Garcin-Bactinyl, France | LB 920111 | Solution |
Dummy guide cannula 8 mm | Agn Tho's, Lindigö, Sweden | CXD-8 | Material |
Electrode 3-channel two-twisted | Invivo1, Plastic One, Roanoke, Virginia, USA | MS333/3-B/SPC | Material |
Electrode holder for stereotxic surgery | Agn Tho's, Lindigö, Sweden | 1776-P1 | Equipment |
Eppendorf BioSpectrometer basic | Eppendorf AG, Hamburg, Germany | 6137 | Equipment |
Eppendorf PCR Tubes 0.2 mL |
Eppendorf Srl, Milan, Italy | 30124332 | Material |
Eppendorf μCuvette G1.0 | Eppendorf AG, Hamburg, Germany | 6138 | Equipment |
Feeding needle flexible 17G for rat | Agn Tho's, Lindigö Sweden | 7206 | Material |
Grass Technology apparatus | Grass Technologies, Natus Neurology Incorporated, Pleasanton, California, USA | M665G08 | Equipment (AS40 amplifier, head box, interconnecting cables, telefactor model RPSA S40) |
Isoflurane 100%, IsoFlo | Zoetis, Rome, Italy | 103287025 (AIC) | Solution |
Ketamine (Imalgene) | Merial, Toulouse, France | 221300288 (AIC) | Solution |
Lithium chloride | Sigma-Aldrich, Milan, Italy | L9650 | Material |
Microinjection cannula 31G 9 mm | Agn Tho's, Lindigö Sweden | CXMI-9 | Material |
MP150 modular data acquisition and analysis system | Biopac, Goleta, California, USA | MP150WSW | Equipment |
Ophthalmic vet ointment, Hylo night | Ursapharm, Milan, Italy | 941791927 (AIC) | Material |
Pilocarpine hydrochloride | Sigma-Aldrich, Milan, Italy | P6503 | Material |
PTFE Tube with joint | Agn Tho's, Lindigö, Sweden | JT-10 | Material |
Saline | 0.9% NaCl, pH adjusted to 7.0 | Solution | |
Scopolamine hydrobromide trihydrate | Sigma-Aldrich, Milan, Italy | S2250 | Material |
Scopolamine methyl nitrate | Sigma-Aldrich, Milan, Italy | S1876 | Material |
Silver sulfadiazine 1% cream | Sofar, Trezzano Rosa, Milan, Italy | 025561010 (AIC) | Material |
Simplex rapid dental methacrylic cement | Kemdent, Associated Dental Products Ltd, Swindon, United Kingdom | ACR811 | Material |
Stereotaxic apparatus | David Kopf Instruments, Los Angeles, CA, USA | Model 963 | Equipment |
Sucrose solution | 10% sucrose in distilled water | Home-made | Solution |
Syringe 1 mL | Biosigma, Cona, Venezia, Italy | 20,71,26,03,00,350 | Material |
Telemeters | Data Sciences International (DSI), St Paul, MN, USA | CTA-F40 | Material |
Telemetry EEG traces analyzer | Data Sciences International (DSI), St Paul, MN, USA | NeuroScore v3-0 | Equipment |
Telemetry system | Data Sciences International (DSI), St Paul, MN, USA | Hardware plus software Ponemah core 6.51 | Equipment |
Xylazine hydrochloride | Sigma-Aldrich, Milan, Italy | X1251 | Material |