Induction of Temporal Lobe Epilepsy in Mice with Pilocarpine

Materials and Reagents

1. Sterile disposable bottle top filter (0.2 µm) (Thermo Scientific, catalog number: 5680020)
2. 27 G needles (BD Biosciences, catalog number: 305109)
3. 1cc syringes (BD Biosciences, Luer-Lok tip, catalog number: 309628)
4. Pipette tips (Axygen, catalog number: 14-222-721)
5. Puralube Vet Ophthalmic Ointment (Dechra, catalog number: 17033-211-38)
6. Eppendorf tubes (VWR, catalog number: 89000-030)
7. Cotton tipped applicators (Thomas Scientific, catalog number: 1213Q08)
8. Nesting square (Ancare, catalog number: NES3600)
9. Paper house (Shepherd specialty papers, catalog number: Shepherd shack reg)
10. Lab markers (Sharpie, Amazon, Permanent ultrafine black 12 count)
11. C57Bl/6NHsd mice (Envigo, catalog number: 47)
12. Isothesia (Isoflurane) (Henry Schein, catalog number: SKU 029405)
13. 2% topical lidocaine (Hi Tech, catalog number: 00603-1393-64)
14. Povidone iodine (CVS, catalog number: 955338)
15. Sodium chloride (NaCl) (Sigma, catalog number: S9888-5KG)
16. Scopolamine methyl bromide (Sigma, catalog number: S85002), 4 °C, dispose 9 months after opening, 4 °C
17. Pilocarpine hydrochloride (Sigma, catalog number: P6503-SG), dispose 2 months after opening, 4 °C
18. Midazolam (Henry Schein, catalog number: 17478052410), dispose 2 months after opening, store at RT
19. Sterile lactated ringers (Henry Schein, catalog number: 065888), store at RT
20. Square container (Rubbermaid, catalog number: 1776401 1-1/4-Cup square container)
21. 0.9% Saline solution (see Recipes)
22. Scopolamine stock solution (see Recipes)
23. Scopolamine working solution (see Recipes)
24. Pilocarpine stock solution (see Recipes)
    

Equipment

1. Calculator (Casio, catalog number: FX-991EX)
2. P1000 pipette (Eppendorf, catalog number: 2231000601)
3. P200 pipette (Eppendorf, catalog number: 2231000601)
4. Weighing balance (Metler, catalog number: AE 240)
5. Vortex (Fisher Scientific, Genie 2, catalog number: 12-812)
6. Heating pad (Sunbeam, catalog number: 731-500)
7. Barnstead Nanopure (Thermo Fisher Scientific, catalog number: D11921)
8. Silver wire (World Precision Instruments, catalog number: AGW1030)
9. Six-pin connectors (Allied Electronics, catalog number: R1023090
10. Stainless steel screws (Plastics One, catalog number: 00-96x3-32)
11. Silver print (GC Electronics, catalog number: 22-023)
12. Dental acrylic cement (Lang Dental Manufacturing, catalog number: 0830 Fibred Pink)
13. Timer (Fisher Scientific, catalog number: 14-648-17)
14. Preamplifier (Pinnacle Technology, catalog number: 8406-SE4)
15. Electrical commutator (Pinnacle Technology, catalog number: 8408)
16. Data acquisition device (Pinnacle Technology, catalog number: 8401)
17. Automated stereotaxic injection system (Stoelting Quintessential, catalog number: 53311)
18. Charcoal filter (VetEquip, catalog number: 931401)
19. Dissecting microscope (Zeiss OPMI 6, catalog number: 93088)
    

Software

1. Sirenia Seizure Pro Software, including Sirenia Acquisition and Sirenia Seizure Detection modules (Pinnacle Technology, software version 2.0.5; https://www.pinnaclet.com/seizurePRO.html)

Procedure

1. Mouse ordering, housing and handling
   1. 3-4-week-old C57Bl/6 mice from Envigo or Jackson Labs.
   2. House the mice in individual cages with a single nesting square and a paper house for 1-2 weeks before experimentation on a 12-h light/dark cycle.
   3. Give free access to food and water. Weigh mice at 5-6 weeks of age. Handle the mice daily for ~5 min for a few days prior to SE induction, as handling appears to reduce the stress response.
      Note: This protocol works well in mice weighing between 18-22 g and is suitable for both male and female C57Bl/6N mice from (Envigo) as well as some transgenic inbred strains from Jackson Labs (e.g., NSG). Research personnel should be well-trained in procedures for safe and appropriate handling and restraint of mice when administering substances. The use of animal models of epilepsy should be guided by the goals of the research, while also giving priority to animal welfare and effective animal treatment and care (Lidster et al., 2016).
      
      
      
2. Day of seizure induction
   Induce SE between 08:00 A.M. and 12:00 P.M. during the light portion of the dark/light cycle (07:00 A.M. to 07:00 P.M.) to control for diurnal variations (Walter et al., 2007; Henderson et al., 2014; Anderson et al., 2018; Gupta et al., 2019). The optimal dose of pilocarpine is critical for successful SE induction in mice and should be worked out based on the strain used in the study.
     To induce SE in mice, first inject scopolamine (0.5 mg/ml, i.p.), followed by an injection of pilocarpine (280 mg/kg; i.p.). If the mouse fails to develop seizure in 30 min after the first injection, 1 or 2 supplemental doses of pilocarpine 30-60 mg/kg, i.p. can be administered to induce SE. Mice injected with pilocarpine at these doses will generally develop generalized motor seizures that may include jumping and running. During induction of SE, the experimenter monitors behavioral seizures visually, based on a modification of a behavioral scoring system originally developed by Racine (Racine, 1972). In the modified version, the severity of behavioral seizures are scored (Shibley and Smith, 2002) (see Table 1). Mice that develop SE exhibit repetitive head bobbing and a lack of motor control that continues after the seizures are attenuated with Midazolam. The rhythmic head movements typically subside after ~6 h but body tics or brief limb/body twitches may continue.
   
   Table 1. Modified Racine scale for scoring seizures
   
   
   Monitoring and scoring pilocarpine-induced seizures and SE: The modified Racine Scale shown in Table 1 is used during pilocarpine induction of SE to observe and classify different levels of seizures (Shibley and Smith, 2002). The original scale included 5 behavior categories of seizures. In the pilocarpine model in mice, several stages may occur together in rapid succession, making it difficult to distinguish stage 4. This rating system is convenient, but less sensitive than EEG detection of seizures. The following video recordings illustrate three stages of seizures, stage 3 (Video 1), stage 5 (Video 2), and SE (Video 3).
   
   
   
   Video 1. Video-EEG of a mouse exhibiting Modified Racine Stage 3 seizures. In this stage, pilocarpine-treated mice develop generalized tonic-clonic seizures associated with a stiff tail, involuntary movements of the forelimbs, and orofacial movements of mouth and whiskers (Video 1). (The behavioral seizure begins at 14 s in the video. Wesleyan University Institutional Animal Care and Use Committee confirms that this protocol (Protocol # 2017_0531_Naegele(C), Project; valid through 05/30/20) conforms to all Federal and State laws and guidelines and all institutional policies and procedures concerning the care and use of animal in research, teaching, and testing.)
   
   
   
   Video 2. Video-EEG of a mouse exhibiting Modified Racine Scale Stage 5 seizures. The seizure begins at 22 s in this video and shows the characteristic behaviors of mice in Stage 5 seizures. These include rearing up onto hind legs and falling, as well as involuntary, bilateral, tonic-clonic movements of the limbs. In this video, the mouse briefly loses postural control and then regains it, which is also typical. (The behavioral seizure begins at 14 s in the video. Wesleyan University Institutional Animal Care and Use Committee confirms that this protocol (Protocol # 2017_0531_Naegele(C), Project; valid through 05/30/20) conforms to all Federal and State laws and guidelines and all institutional policies and procedures concerning the care and use of animal in research, teaching, and testing.)
   
   
   
   Video 3. Video-EEG of a mouse exhibiting SE. In this stage called SE, mice have experienced 3 or more stage 3-5 seizures and develop continuous seizures, or SE. In this stage, the mice become immobile and exhibit prominent head-tics. (The behavioral seizure begins at 14 s in the video. Wesleyan University Institutional Animal Care and Use Committee confirms that this protocol (Protocol # 2017_0531_Naegele(C), Project; valid through 05/30/20) conforms to all Federal and State laws and guidelines and all institutional policies and procedures concerning the care and use of animal in research, teaching, and testing.)
   
   Before starting seizure induction
   1. Prepare scopolamine stock solution, scopolamine working solution, and the pilocarpine working solution, as detailed in Recipes. Prepare these solutions on the day of SE induction and discard after using.
   2. Put all the liquid reagents (saline, scopolamine, pilocarpine) on ice.
   3. Bring reagents, 0.5 ml Eppendorf tubes, calculator, lab markers, pipette, and pipette tips up to the procedure room to induce SE.
   4. Approximately 15 mice are induced in the same session. Typically, 4 trained observers are needed to monitor seizures for 15 mice. Each observer then monitors 3 or 4 mice.
   5. Weigh the mice individually in a square container with a lid (Rubbermaid container).
   6. Remove extra bedding, food, water, and environmental enrichment from the cages prior to seizure induction.
   7. Label each cage with the experimental number of the mouse, label the Eppendorf tubes that will contain the injectables for that mouse, and the disposable syringe for that mouse.
   8. Prepare the pilocarpine working solutions for each mouse (calculations are provided below).
   9. Calculations for pilocarpine working solution:
      1. Prepare the Eppendorf tube for each mouse by diluting pilocarpine stock solution with 0.9% sterile saline, in a final volume of 300 µl.
      2. The volume of pilocarpine stock solution should be calculated as follows:
         6 x (mouse weight) = X pilocarpine (µl)
      3. The volume of 0.9% sterile saline is calculated as follows:
         300 µl minus volume of pilocarpine stock (µl)
         Example: 21.2-gram mouse
         Volume of Pilocarpine (µl): 6 x 21.2 (g) = 127.2
         0.9% sterile saline: 300 µl – 127.2 µl = 172.8 µl
         
         
         
   SE induction procedure
   1. Inject each mouse with 0.07 ml scopolamine (~0.5 mg/ml; i.p.) and note the time of injection.
   2. 30 min after the scopolamine injection, inject the working pilocarpine solution (0.1 ml; i.p.). Note that the dilution is specific for each mouse (see “Before starting seizure induction” of Section B, Step 9).
   3. Note the time of each seizure event and the duration (count motor seizures lasting 15 s or longer only).
   4. If mouse does not exhibit any stage 4 or stage 5 seizures within 20-30 min of the first injection, inject a supplemental dose of pilocarpine (0.01-0.02 ml, i.p.) using the solution prepared for that mouse. If the mouse has one or more stage 3 seizures, but no stage 4 or 5 seizures, the supplemental dose is 0.01 ml. However, if a mouse exhibits stage 0, 1 or 2 but no stage 3, 4, or 5 seizures, the mouse is given a supplemental dose of 0.02 ml (i.p.).
   5. After 3-5 times stage 3, 4, or 5 Racine scale seizures, the mouse will typically develop SE, defined as a continuous state of seizures characterized by a prominent head tic or “hiccup”, rigidity (body and tail), and immobility. Note the time of SE.
   6. SE will typically continue unabated. One to 3 h after the time of the initial onset of SE, inject 0.04 ml Midazolam (i.p.) to attenuate the seizures (Figure 1). Gently scruff mouse and apply ophthalmic ointment with cotton swab to each eye to prevent the corneas from drying out, as mice that develop SE blink infrequently.
   7. Monitor each mouse in its home cage for 6 h following attenuation of SE with midazolam. Provide additional warmth for several hours using a heating pad under the cage, until the mouse is walking and drinking.
   8. Provide a nesting square, a paper house, food and water ad libitum, and maintain the mice in the 12-h light/12-h dark cycle. We routinely provide all mice with moistened/softened food for several days, as mice may become constipated following induction of SE.
   9. Monitor the mice and administer Ringer’s solution daily for several days (1 ml, i.p.). Inject 2x/day only when a mouse becomes immobile or fails to eat or groom.
   Notes:
   1. One hour of SE is sufficient for generating mice that reliably develop TLE with recurrent spontaneous seizures. In our laboratory, the typical success rate for inducing SE with this method is ~70% or higher. Three hours of SE results in a more severe disease phenotype, but longer periods of SE are associated with increased mortality rates.
   2. Animal welfare assessments must be conducted multiple times per day for several days after the induction of SE. The mice should be provided ad libitum with softened food and hydrated with sterile Ringer’s lactate injections (i.p.) for several days after induction of SE. Mice may lose weight and show increased aggression after SE, necessitating individual housing. If a mouse becomes immobile or fails to eat or groom for more than 24 h, the mouse should be euthanized. Mortality and suffering of animals can be minimized by ensuring that the same experimenter handles the mice and by avoiding unnecessary loud noises and startling of the animals. Additional considerations regarding use of rodents in epilepsy research models may be found on the web at the National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs).
   
   
   Figure 1. Flow chart diagram of experimental protocol for inducing status epilepticus
   
   
3. Electrode implantation
   1. Prepare the mouse for surgery by anesthetizing with isoflurane gas in an induction chamber. Confirm that the animal is properly anesthetized by pinching foot.
   2. Place the mouse in the stereotaxic apparatus. Line up the front incisors in the opening in nose clamp and fix the nose cone in place to deliver isoflurane gas anesthesia during surgery.
   3. Adjust the ear bars, making sure that they immobilize the mouse’s skull, without excessive pressure.
   4. Apply ophthalmic ointment to the mouse’s eyes with a sterile cotton swab.
   5. Sterilize the skin by applying povidone-iodine and lidocaine with sterile cotton swab, to reduce pain.
   6. Make a longitudinal incision along the midline axis of the skull with the scalpel blade, exposing bregma and lambda and gently push aside muscles that are attached to the top of the skull.
   7. Apply H₂O₂ to the skull with a sterile cotton swab to aid in bregma and lambda visualization through dissecting microscope.
   8. For surgical implantation of subdural electrodes, drill two midline holes in the skull; one anterior to bregma and one posterior to lambda. Drill four additional holes (two per side) with reference to bregma, at coordinates AP: -1.5 mm; L: ±2.5 mm and AP: -3.0 mm; L ±2.5 mm.
   9. The electrode head mount consists of 0.25 mm silver wires soldered to six-pin connectors. The ends of the four subdural electrode silver wires are heated to form smooth balls at the ends, to avoid damage to the cortex by the wires.
   10. Insert stainless steel screws into the two holes in the skull that are anterior to bregma and posterior to lambda. Wrap the two electrode anchor silver wires serving as reference and ground connections around these screws.
   11. Apply silver print to these connections to ensure good electrical contacts.
   12. Insert the four subdural electrode wires into the holes in the skull positioned at coordinates AP: -1.5 mm; L: ±2.5 mm and AP: -3.0 mm; L ±2.5 mm, with reference to the bregma. Make certain that the balls at the ends of the electrode wires are in light contact with the surface of the brain.
   13. Apply dental acrylic cement to cover and hold the electrode head mount in place.
   14. Remove the mouse from the stereotaxic apparatus and place it in a clean cage on a heating pad. Monitor the mouse until it regains consciousness and begins to move around. Return the mouse to the home cage.
       
       
       
4. Data acquisition and scoring of SRS by Video-EEG following SE induction
   The pilocarpine model is often used as a chronic model of TLE because the mice will begin to spontaneous seizures, typically occurring in clusters of seizures that last 4-5 days with multiple brief seizures/day, and seizure-free intervals that can last for periods of days or even weeks. Although the onset of SRS varies, we find it useful to begin to monitor 3 weeks after SE induction because the mice begin to exhibit more frequent seizures by 3-4 weeks. The effects of a particular experimental treatment on seizures can be accurately monitored using video (V)-EEG recordings (Curia et al., 2008). We typically conduct continuous v-EEG recordings for 3 weeks or longer, when assessing the efficacy of a particular therapeutic treatment.
   
   When performing multiple stereotaxic surgeries, we typically wait at least 1 week after SE. In our prior work, we made stereotaxic injections of stem cells at ~2 weeks after SE and stereotaxic injections of retrovirus at 1 week before, or after, stem cell transplantation. We typically have an experimental treatment group (e.g., mice that receive stem cell transplants) and compare them to a control group that receives vehicle (or stereotaxic injections of the media minus stem cells). We then implant subdural electrodes for chronic v-EEG recordings at ~3 weeks after SE. To detect and quantify SRS, it is useful to carry out continuous v-EEG recordings for 3 weeks or longer. For v-EEG recordings, EEG signals are amplified with a preamplifier connected to the data acquisition device and recorded with Sirenia Seizure Pro software (Pinnacle Technology), an analysis package for quickly identifying and analyzing user-defined seizure events based on power and line length. Data are captured using Sirenia Acquisition software at 1,000 Hz with a gain of 10x, using high and low pass-filters set at 0.5 Hz and 100 Hz respectively. Video recordings are synchronized with EEG signals by the Sirenia seizure monitoring software. We use changes in power and line length deviations from baseline EEG brain activity to detect seizures in this software (Henderson et al., 2014; Anderson et al., 2018). After detecting potential seizures with the software, each of the seizure events should be verified manually by an experimenter who views the EEG and associated video recording and scores the severity of the seizures based on the modified Racine scale (Table 1; Figure 2). Verification is necessary to distinguish true seizures from occasional EEG artifacts caused by grooming or other issues with the head-mounted electrodes.
   
   
   1. Open “Sirenia Seizure”. Click “File”, then select the file to analyze.
   2. Click “Time Span” and choose “1 Minute”. Click “Time Step” and choose “10 s”.
   3. Right-click on the y-axis tab to the left of each EEG channel and click “Manual”.
   4. Click in the text boxes that appear on the top and bottom of each y-axis tab. Change the top box to “500” and the bottom box to “-500” to scale down the EEG trace for easier viewing of seizure activity. Seizure activity is usually about ± 500 μV or more than twice baseline amplitude, for each mouse.
   5. Click the “Seizure Settings” icon. Change the “Window Size” and “Step Size” to 5.00.
   6. Select the “Line Length” search option and adjust the threshold to 6,000.
   7. Make sure all EEG channels are selected under “Use Channels”.
   8. Click the “Next Possible Seizure” button to begin searching for seizures.
   9. The “Search Progress” window will open. When a seizure is found, the search will pause, and the region of activity detected as greater than the specified line length in the window size and step will be highlighted in pink.
   10. Use the “Pan” tool to identify the beginning and the end of the seizure event.
   11. Begin ~10 s prior to detected seizures and click the “Play” icon to start the playback for scoring behavioral seizures.
   12. Watch the camera recording to score the mouse’s behavior.
   13. EEG 1 and 2 in the Sirenia seizure software measure the EEG activity in the right and left hemisphere respectively of the mouse in the camera’s view. However, EEG 3 is a reference electrode. See Figure 3.
   14. Click the “Next Possible Seizure” icon to begin searching for the next seizure.
       
       
       Figure 2. Examples of spontaneous epileptiform activity in mice following pilocarpine-induced SE. The EEGs were acquired using Sirenia Acquisition and Sirenia Seizure Detection software.
       
       
       Figure 3. The graphical user interface in the Sirenia seizure detection software. The setting for detection of seizures is based on the EEG line length, with a threshold setting of 6000.000 len/sec. The mouse is shown in a video camera view that has been superimposed onto the screenshot of the graphical user interface.
       

Recipes

1. 0.9% sterile saline solution
   

   ReagentAmount:	500 ml
   NaCl	4.5 g

   Add 500 ml MilliQddH₂O
   Sterilize the solution by passing through a 0.2 µm bottle top filter
2. Scopolamine stock solution
   

   Reagent Amount:	1 ml
   Scopolamine Methyl Bromide	10 mg

   Dissolve scopolamine in 998 µl of 0.9% sterile saline, for a final volume of 1 ml
3. Scopolamine working solution
   

   Reagent Amount:	1 ml
   Scopolamine stock solution	50 µl
   0.9% sterile saline	950 µl

   This amount is sufficient for 8 mice
4. Pilocarpine stock solution
   

   Reagent Amount:	1 ml
   Pilocarpine HCl	140 mg

   0.9% sterile salineto 1 ml
   Dissolve pilocarpine in 888 µl of 0.9% sterile saline, for a final volume of 1 ml
   

Acknowledgments

This work was funded by grants from National Institutes of Health (NINDS grant R15NS072879-01A1), McKnight Foundation Brain Disorders Award (JRN), A Challenge Award from Citizens United for Research in Epilepsy (JRN), The Regenerative Medicine Research Fund of Connecticut Established Investigator Grant (JRN), and the Alan M. Dachs Endowed Professorship at Wesleyan University (JRN). We also thank our colleagues and collaborators who contributed to published work that was based on variations of this protocol: Bryan Luikart, Karl Obrietan, Yun-Sik Choi, Paul Lombroso, Laura Grabel, and Gloster Aaron. We thank lab members Jaye Jeong and Nicolas Cimino for contributing to discussions about this manuscript. We thank former lab technicians who helped us develop this detailed protocol: Sara Royston, Stephanie Tagliatela, Katharine Henderson, Dan Lawrence, and Kevin Cobbol.

Competing interests

The authors have no competing interests.

Ethics

Wesleyan University Institutional Animal Care and Use Committee confirms that this protocol (Protocol # 2017_0531_Naegele(C), Project; valid through 05/30/20) conforms to all Federal and State laws and guidelines and all institutional policies and procedures concerning the care and use of animal in research, teaching, and testing.

References

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