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May 2019

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Induction of Temporal Lobe Epilepsy in Mice with Pilocarpine
毛果芸香碱诱导小鼠颞叶癫痫   

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Abstract

In the pilocarpine model of temporal lobe epilepsy (TLE) in rodents, systemic injections of pilocarpine induce continuous, prolonged limbic seizures, a condition termed “Status Epilepticus” (SE). With appropriate doses, many inbred strains of mice show behavioral seizures within an hour after pilocarpine is injected. With the behavioral scoring system based on a modification of the original Racine scale, one can monitor the seizures behaviorally, as they develop into more prolonged seizures and SE. SE is typically associated with damage to subsets of hippocampal neurons and other structural changes in the hippocampus and generally subsides on its own. However, more precise control of the duration of SE is commonly achieved by injecting a benzodiazepine into the mouse 1 to 3 h after the onset of SE to suppress the seizures. Several days following pilocarpine-induced SE, electrographic and behavioral seizures begin to occur spontaneously. The goal of this protocol is to reliably generate mice that develop spontaneous recurrent seizures (SRS) and show the typical neuropathological changes in the brain characteristic of severe human mesial temporal lobe epilepsy (mTLE), without high mortality. To reduce mortality, multiple subthreshold injections of pilocarpine are administered, which increases the percentage of mice developing SE without concomitant mortality. Precise control of the duration of SE (1 or 3 h) is achieved by suppressing SE with the benzodiazepine Midazolam (Versed). We have found that this protocol is an efficient means for generating mice that subsequently develop characteristics of human mTLE including high-frequency interictal spike and wave activity and SRS. In addition, we and others have shown that this protocol produces mice that show excitotoxic cell death of subsets of hippocampal GABAergic interneurons, particularly in the dentate gyrus and compensatory sprouting of excitatory projections from dentate granule cells (mossy fiber sprouting). Aspects of this protocol have been described in several of our previous publications.

Keywords: Hippocampus (海马体), Seizures (癫痫), Neurodegeneration (神经退行性病变), Epileptogenesis (癫痫发生), Pilocarpine (毛果芸香碱)

Background

The mouse pilocarpine model of TLE is an experimental paradigm that has been widely used as an animal model of human mTLE. This model has been used for testing long-term efficacy of novel therapies for suppressing SRS and improving cognitive and neuropathological changes associated with severe mTLE. This rodent model of TLE recapitulates many of the neuropathological hallmarks in mTLE, including severe seizures, loss of GABAergic interneurons in the dentate gyrus, Ammon’s Horn sclerosis, and cognitive impairments (Swartz et al., 2006). Although some patients with mTLE can be treated successfully anti-convulsant medications, about one-third experience intractable pharmaco-resistant seizures and surgical removal of the seizure foci are required. If the foci are located in the mesial temporal lobes, surgical removal may not be feasible due to the role of the hippocampus in memory formation and consolidation. For these patient populations, novel treatments are needed. The cost-effective pilocarpine model of TLE in rodents provides an experimental system for testing novel therapies because a high percentage of rodents develop SRS that can be monitored by video electroencephalography (v-EEG) for weeks or months. Novel treatments that suppress seizures can also be studied for their effects on the neuropathological changes observed in this model, including mossy fiber sprouting (Henderson et al., 2014), dispersion of the granule cell layer (Scharfman et al., 2000), and aberrant neurogenesis (Parent et al., 1997).

Pilocarpine hydrochloride (referred to as pilocarpine) is a drug that directly stimulates muscarinic cholinergic receptors in the central nervous system and in the parasympathetic branch of the peripheral nervous system. As a parasympathomimetic drug, pilocarpine induces exocrine gland secretion and stimulates smooth muscle contractions in the gut and secretory glands. It is also a chemoconvulsant that is used to elicit limbic seizures in rodents. Pilocarpine is typically administered by subcutaneous (Walter et al., 2007) or intraperitoneal (i.p.) injections in rodents to induce a condition of continuous generalized seizures, called Status Epilepticus (SE). After the mice develop SE in this model, the experimenter terminates the seizures by injecting a sedative dose of benzodiazepine, a class of drugs that acts on gamma-aminobutyric acid-A (GABA-A) receptors in the brain. Typically, rodents that have experienced one or more hours of SE, will develop TLE with high-frequency interictal spike, wave activity, and SRS (Mello et al., 1993; Wozny et al., 2003; Knopp et al., 2005; Henderson et al., 2014).

In a comprehensive study of the multiple factors contributing to the development of SRS in the rodent pilocarpine model, the strongest correlations were found between dentate gyrus GABAergic neuron loss and seizure frequency (i.e., disease severity) (Buckmaster et al., 2017). Many human TLE patients and rodents subjected to pilocarpine-induce SE show loss of somatostatin-expressing GABAergic interneurons in the hippocampus (Robbins et al., 1991; Borges et al., 2003; Hoffman et al., 2016). In the mouse pilocarpine model, GABAergic interneurons in the hilus of the dentate gyrus and CA1 that co-express somatostatin and Striatal Enriched Tyrosine Phosphatase (STEP) were found to be highly vulnerable to SE (Buckmaster and Jongen-Relo, 1999; Choi et al., 2007; Zhang et al., 2009). Associated with the loss of GABAergic interneurons, studies reported reduced synaptic inhibition (Kobayashi and Buckmaster, 2003), despite the fact that many of the GABAergic interneurons in the dentate gyrus and CA1 that survive after SE sprout additional inhibitory projections (Wittner et al., 2002; Bausch, 2005; Zhang et al., 2009; Thind et al., 2010). Loss of both somatic and distal dendritic GABAergic inhibition to granule cells occurs shortly after SE, but precedes the development of SRS, suggesting that loss of GABAergic inhibition onto granule cells in the dentate gyrus is an important predictor of whether TLE will develop (Kobayashi and Buckmaster, 2003).

Because of the strong link between loss of GABAergic inhibition and development of TLE, we and other groups have tested whether transplantation of GABAergic progenitors, from either the embryonic mouse forebrain or generated in vitro from human pluripotent stem cells, suppresses pilocarpine-induced TLE (Maisano et al., 2012; Henderson et al., 2014; Anderson et al., 2018). Additionally, recent work shows that this model can be used in combination with molecular and electrophysiological approaches, such as retroviral labeling and optogenetics, to evaluate cellular mechanisms responsible for seizure suppression following transplantation (Gupta et al., 2019). The methods detailed in this protocol for induction of SE in both male and female rodents can be adapted to different inbred strains of mice with low mortality; a high incidence of the mice that experience SE will subsequently develop SRS and the typical histopathological hallmarks of TLE (Walter et al., 2007; Henderson et al., 2014; Anderson et al., 2018; Gupta et al., 2019) .

In the following protocol, we provide the detailed methods for the successful induction of SE with multiple subthreshold doses of pilocarpine in inbred strains of mice. This method builds upon number of prior studies. In a comparison of 8 inbred strains of mice, Schauwecker and colleagues found significant differences in the susceptibility to pilocarpine, mortality and neuropathological changes after SE (Schauwecker, 2012). Doses ranging between 300 and 400 mg/kg were reported to induce SE successfully. Higher single doses of pilocarpine were correlated with more successful induction of SE but also increased mortality (Turski et al., 1984; Cavalheiro et al., 1991; Curia et al., 2008; Buckmaster and Haney, 2012; Kelly and Coulter, 2017). In contrast, multiple subthreshold doses of pilocarpine were effective for inducing SE and SRS, with much lower mortality rates (Glien et al., 2001; Groticke et al., 2007).

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
    Reagent Amount:
    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
    950 µl
    0.9% sterile saline
    50 µl
    This amount is sufficient for 8 mice
  4. Pilocarpine stock solution
    Reagent Amount:
    1 ml
    Pilocarpine HCl
    140 mg
    0.9% sterile saline to 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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  2. Bausch, S. B. (2005). Axonal sprouting of GABAergic interneurons in temporal lobe epilepsy. Epilepsy Behav 7: 390-400.
  3. Borges, K., Gearing, M., McDermott, D. L., Smith, A. B., Almonte, A. G., Wainer, B. H. and Dingledine, R. (2003). Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model. Exp Neurol 182(1): 21-34. 
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  5. Buckmaster, P. S., Abrams, E. and Wen, X. (2017). Seizure frequency correlates with loss of dentate gyrus GABAergic neurons in a mouse model of temporal lobe epilepsy. J Comp Neurol 525(11): 2592-2610. 
  6. Buckmaster, P. S. and Haney, M. M. (2012). Factors affecting outcomes of pilocarpine treatment in a mouse model of temporal lobe epilepsy. Epilepsy Res 102(3): 153-159.
  7. Cavalheiro, E. A., Leite, J. P., Bortolotto, Z. A., Turski, W. A., Ikonomidou, C. and Turski, L. (1991). Long-term effects of pilocarpine in rats: structural damage of the brain triggers kindling and spontaneous recurrent seizures. Epilepsia 32(6): 778-782.
  8. Choi, Y. S., Lin, S. L., Lee, B., Kurup, P., Cho, H. Y., Naegele, J. R., Lombroso, P. J. and Obrietan, K. (2007). Status epilepticus-induced somatostatinergic hilar interneuron degeneration is regulated by striatal enriched protein tyrosine phosphatase. J Neurosci 27(11): 2999-3009. 
  9. Curia, G., Longo, D., Biagini, G., Jones, R. S. and Avoli, M. (2008). The pilocarpine model of temporal lobe epilepsy. J Neurosci Methods 172(2): 143-157. 
  10. Glien, M., Brandt, C., Potschka, H., Voigt, H., Ebert, U. and Loscher, W. (2001). Repeated low-dose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy. Epilepsy Res 46(2): 111-119.
  11. Groticke, I., Hoffmann, K. and Loscher, W. (2007). Behavioral alterations in the pilocarpine model of temporal lobe epilepsy in mice. Exp Neurol 207(2): 329-349.
  12. Gupta, J., Bromwich, M., Radell, J., Arshad, M. N., Gonzalez, S., Luikart, B. W., Aaron, G. B. and Naegele, J. R. (2019). Restrained dendritic growth of adult-born granule cells innervated by transplanted fetal GABAergic interneurons in mice with temporal lobe epilepsy. eNeuro 6(2): pii: ENEURO.0110-18.2019.
  13. Henderson, K. W., Gupta, J., Tagliatela, S., Litvina, E., Zheng, X., Van Zandt, M. A., Woods, N., Grund, E., Lin, D., Royston, S., Yanagawa, Y., Aaron, G. B. and Naegele, J. R. (2014). Long-term seizure suppression and optogenetic analyses of synaptic connectivity in epileptic mice with hippocampal grafts of GABAergic interneurons. J Neurosci 34(40): 13492-13504.
  14. Kelly, M. E., and Coulter, D. A. (2017). Chapter 42: The pilocarpine model of acquired epilepsy. In: Models of Seizures and Epilepsy. 2nd Edition. Pitkänen, A., Buckmaster, P. S., Galanopoulou, A. S., and Moshé, L. S. (Eds.). London, UK: Academic Press, 625-636.
  15. Knopp, A., Kivi, A., Wozny, C., Heinemann, U. and Behr, J. (2005). Cellular and network properties of the subiculum in the pilocarpine model of temporal lobe epilepsy. J Comp Neurol 483(4): 476-488. 
  16. Kobayashi, M. and Buckmaster, P. S. (2003). Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy. J Neurosci 23(6): 2440-2452.
  17. Lidster, K., Jefferys, J. G., Blumcke, I., Crunelli, V., Flecknell, P., Frenguelli, B. G., Gray, W. P., Kaminski, R., Pitkanen, A., Ragan, I., Shah, M., Simonato, M., Trevelyan, A., Volk, H., Walker, M., Yates, N. and Prescott, M. J. (2016). Opportunities for improving animal welfare in rodent models of epilepsy and seizures. J Neurosci Methods 260: 2-25. 
  18. Maisano, X., Litvina, E., Tagliatela, S., Aaron, G. B., Grabel, L. B. and Naegele, J. R. (2012). Differentiation and functional incorporation of embryonic stem cell-derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy. J Neurosci 32(1): 46-61.
  19. Mello, L. E., Cavalheiro, E. A., Tan, A. M., Kupfer, W. R., Pretorius, J. K., Babb, T. L. and Finch, D. M. (1993). Circuit mechanisms of seizures in the pilocarpine model of chronic epilepsy: cell loss and mossy fiber sprouting. Epilepsia 34(6): 985-995.
  20. Parent, J. M., Yu, T. W., Leibowitz, R. T., Geschwind, D. H., Sloviter, R. S. and Lowenstein, D. H. (1997). Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci 17(10): 3727-3738.
  21. Robbins, R. J., Brines, M. L., Kim, J. H., Adrian, T., de Lanerolle, N., Welsh, S. and Spencer, D. D. (1991). A selective loss of somatostatin in the hippocampus of patients with temporal lobe epilepsy. Ann Neurol 29(3): 325-332. 
  22. Scharfman, H. E., Goodman, J. H. and Sollas, A. L. (2000). Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis. J Neurosci 20(16): 6144-6158.
  23. Schauwecker, P. E. (2012). Strain differences in seizure-induced cell death following pilocarpine-induced status epilepticus. Neurobiol Dis 45(1): 297-304.
  24. Shibley, H. and Smith, B. N. (2002). Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice. Epilepsy Res 49(2): 109-120.
  25. Swartz, B. E., Houser, C. R., Tomiyasu, U., Walsh, G. O., DeSalles, A., Rich, J. R. and Delgado-Escueta, A. (2006). Hippocampal cell loss in posttraumatic human epilepsy. Epilepsia 47(8): 1373-1382. 
  26. Thind, K. K., Yamawaki, R., Phanwar, I., Zhang, G., Wen, X. and Buckmaster, P. S. (2010). Initial loss but later excess of GABAergic synapses with dentate granule cells in a rat model of temporal lobe epilepsy. J Comp Neurol 518(5): 647-667. 
  27. Turski, W. A., Cavalheiro, E. A., Bortolotto, Z. A., Mello, L. M., Schwarz, M. and Turski, L. (1984). Seizures produced by pilocarpine in mice: a behavioral, electroencephalographic and morphological analysis. Brain Res 321(2): 237-253.
  28. Walter, C., Murphy, B. L., Pun, R. Y., Spieles-Engemann, A. L. and Danzer, S. C. (2007). Pilocarpine-induced seizures cause selective time-dependent changes to adult-generated hippocampal dentate granule cells. J Neurosci 27(28): 7541-7552.
  29. Wittner, L., Eross, L., Szabo, Z., Toth, S., Czirjak, S., Halasz, P., Freund, T. F. and Magloczky, Z. S. (2002). Synaptic reorganization of calbindin-positive neurons in the human hippocampal CA1 region in temporal lobe epilepsy. Neuroscience 115(3): 961-978.
  30. Wozny, C., Kivi, A., Lehmann, T. N., Dehnicke, C., Heinemann, U. and Behr, J. (2003). Comment on "On the origin of interictal activity in human temporal lobe epilepsy in vitro". Science 301(5632): 463; author reply 463. 
  31. Zhang, W., Yamawaki, R., Wen, X., Uhl, J., Diaz, J., Prince, D. A. and Buckmaster, P. S. (2009). Surviving hilar somatostatin interneurons enlarge, sprout axons, and form new synapses with granule cells in a mouse model of temporal lobe epilepsy. J Neurosci 29(45): 14247-14256.

简介

[摘要 ] 在啮齿类动物颞叶癫痫(TLE)的毛果芸香碱模型中,系统性注射毛果芸香碱会诱发连续的,延长的边缘性癫痫发作,这种情况称为“癫痫持续状态”(SE)。适当剂量的许多近交系小鼠在注射毛果芸香碱后一小时内表现出行为性癫痫发作。借助基于原始Racine量表的改进的行为评分系统,随着发作发展为更长的发作和SE,人们可以从行为上监测癫痫发作。SE通常与损害海马神经元和 海马的其他结构变化通常会自行消退。但是,SE持续时间的更精确控制通常是通过在SE发作后1至3小时向小鼠注射苯二氮卓以抑制癫痫发作来实现的。毛果芸香碱诱发的SE后几天,电图和行为性癫痫发作开始自发发生。该协议的目标是可靠地生成可自发复发性癫痫发作(SRS)并显示严重人中颞叶癫痫(mTLE )的脑部特征的典型神经病理变化而无高死亡率的小鼠。为了降低死亡率,进行了多次阈下的毛果芸香碱注射,这增加了患有SE而没有伴随死亡率的小鼠的百分比。通过用苯二氮卓咪达唑仑(Versed)抑制SE,可以精确控制SE的持续时间(1或3小时)。我们已经发现,该协议是一种生成小鼠的有效手段,该小鼠随后会发展出人类mTLE的特征,包括高频发作频率和波活动以及SRS。此外,我们和其他人已经证明,该协议产生的小鼠表现出海马GABA能性中间神经元子集的兴奋性细胞死亡,特别是在齿状回和齿状颗粒细胞的兴奋性投射的补偿性发芽(苔藓纤维发芽)。此协议的各个方面已在我们之前的几篇出版物中进行了介绍。

[背景] TLE的小鼠毛果芸香碱模型是一种实验范式,已被广泛用作人类mTLE 的动物模型。该模型已用于测试新型疗法的长期疗效,该疗法可抑制SRS并改善与严重mTLE 相关的认知和神经病理变化。TLE的这种啮齿动物模型概括了mTLE中的许多神经病理学特征,包括严重的癫痫发作,齿状回中GABA能神经元的丢失,Ammon的Horn硬化和认知障碍(Swartz 等,2006)。尽管一些mTLE 患者可以成功治疗抗惊厥药物,但约有1/3的顽固性耐药性癫痫发作需要手术治疗,并且需要通过手术切除癫痫发作灶。如果病灶位于中颞叶,由于海马在记忆形成和巩固中的作用,可能无法进行手术切除。对于这些患者人群,需要新颖的治疗方法。具有成本效益的TLE在啮齿动物中的毛果芸香碱模型提供了一种测试新疗法的实验系统,因为高比例的啮齿动物会发展出可以通过视频脑电图(v-EEG)监测数周或数月的SRS 。还可以研究抑制癫痫发作的新型疗法对这种模型中观察到的神经病理变化的影响,包括苔藓纤维萌芽(Henderson 等,2014),颗粒细胞层的分散(Scharfman 等,2000),和异常神经发生(Parent 等,1997)。

盐酸毛果芸香碱(称为毛果芸香碱)是一种直接刺激中枢神经系统和周围神经系统副交感神经分支的毒蕈碱胆碱能受体的药物。作为拟副交感神经药,毛果芸香碱可诱导外分泌腺分泌,并刺激肠道和分泌腺的平滑肌收缩。它也是一种化学惊厥剂,用于引起啮齿动物的边缘性癫痫发作。毛果芸香碱通常通过在啮齿动物中皮下注射(Walter 等,2007)或腹膜内注射(ip 。)来引起持续性全身性癫痫发作,称为癫痫持续状态(SE)。小鼠在此模型中发展为SE后,实验者通过注射镇静剂量的苯二氮卓来终止癫痫发作,苯二氮卓是一种作用于大脑中γ-氨基丁酸-A(GABA-A)受体的药物。通常,经历了一个或多个SE的啮齿动物会发展为TLE,并伴有高频高频尖峰信号,波活动和SRS (Mello 等,1993;Wozny 等,2003;Knopp 等,2005;Met al 。,1993;Wolsny 等,2003;Konpp 等,2005)。亨德森(Henderson)等人,2014年)。

在促进SRS的啮齿动物模型毛果芸香碱发展的多种因素的综合研究中,齿状回神经元的GABA能损失和癫痫发作频率之间发现的最强相关性(即,疾病的严重程度)(巴克马斯特等人。,2017)。许多接受皮毛果树素诱导SE的人类TLE患者和啮齿类动物在海马体中都表达了表达他汀类的他汀类药物GABA能神经元(Robbins 等,1991 ; Borges 等,2003 ; Hoffman 等,2016 )。在小鼠毛果芸香碱模型中,齿状回和希拉里的GABA能神经元与生长抑素和纹状体富集酪氨酸磷酸酶(STEP)共表达的CA1 极易被SE (Buckmaster和Jongen-Relo,1999;Choi 等)等人,2007;Zhang 等,2009)。与GABA能中间的损失有关,研究报告减少突触抑制(小林和巴克马斯特,2003),尽管事实上,许多在齿状回和CA1幸存SE萌芽额外的抑制作用预测后的GABA能中间的(Wittner 等人, 2002;Bausch,2005;Zhang 等,2009;Thind 等,2010)。大号既躯体和远端树突GABA能抑制到颗粒细胞的OSS SE后不久发生,但先于SRS的发展,表明GABA能抑制的齿状回中的损失到颗粒细胞是TLE是否会发展的重要预测指标(Kobayashi和Buckmaster,2003年)。

由于GABA能抑制的丧失与TLE的发展之间有着密切的联系,我们和其他研究小组已经测试了从胚胎小鼠前脑移植或从人多能干细胞体外产生的GABA能祖细胞的移植是否抑制毛果芸香碱诱导的TLE (Maisano 等人,2012;亨德森等人,2014;安德森等人,2018)。此外,最近的工作表明该模型可以与分子和电生理学方法(例如逆转录病毒标记和光遗传学)结合使用,以评估负责移植后癫痫发作抑制的细胞机制(Gupta 等人,2019)。该协议中详述的在雄性和雌性啮齿动物中诱导SE的方法可适用于低死亡率的不同自交系小鼠。经历SE的小鼠的高发病率随后会发展成SRS和TLE的典型组织病理学特征(Walter 等,2007;Henderson 等,2014;Anderson 等,2018;Gupta 等,2019)。

在以下协议中,我们提供了在自交系小鼠中成功诱导多亚阈剂量毛果芸香碱SE的详细方法。该方法建立在先前研究的数量上。在比较8个自交系小鼠后,Schauwecker 及其同事发现SE后对毛果芸香碱的敏感性,死亡率和神经病理学变化存在显着差异(Schauwecker ,2012)。据报道,剂量范围在300至400 mg / kg之间可成功诱导SE。较高剂量的毛果芸香碱与更成功的SE诱导相关,但也增加了死亡率(Turski 等,1984; Cavalheiro 等,1991; Curia 等,2008; Buckmaster和Haney,2012; Kelly和Coulter,2017 )。相比而言,多个亚阈值剂量毛果芸香碱是有效的诱导SE和SRS,以低得多的死亡率(Glien 等,2001; Groticke 等,2007)。

关键字:海马体, 癫痫, 神经退行性病变, 癫痫发生, 毛果芸香碱

材料和试剂


 


无菌d isposable b ottle 吨运算˚F ILTER(0.2微米)(Thermo Scientific的,目录号:5680020)
27 G针(BD Biosciences,目录号:305109)
1cc注射器(BD Biosciences,Luer-Lok 吸头,目录号:309628)
移液器吸头(Axygen ,目录号14-222-721)
Puralube Vet O 眼药膏(Dechra ,目录号:17033-211-38)
Eppendorf管(V WR,货号:89000-030)
棉签涂抹器(              Thomas Scientific,目录号:1213Q08)
嵌套广场(Ancare ,CATA 登录号:NES3600)
纸房(Shepherd专业论文,目录号:Shepherd shack reg)
实验室标记(Sharpie,Amazon,永久超细黑12计数)
C57Bl / 6NHsd小鼠(Envigo ,目录号:47)
Isothesia (异氟烷)(Henry Schein,目录号:SKU 029405)                           
2%的局部利多卡因(高科技,目录号:00603-1393-64)                           
聚维酮我odine(CVS,目录号:955338)
钠Ç hloride(氯化钠)(Sigma,目录号:S9888-5KG)
东pol碱甲基溴(Sigma,目录号:S85002),4 °C,开封后9个月处置,4 °C
盐酸匹罗卡品(Sigma,目录号:P6503-SG),开封后2个月处置,4 °C
咪达唑仑(Henry Schein,货号:17478052410),开业后两个月处置,在RT存放
无菌乳酸林格氏菌(Henry Schein,目录号:065888),在RT存放
方形容器(Rubbermaid,货号:1776401 1-1 / 4杯子方形容器)
0.9%盐溶液(请参阅食谱)
东co碱储备溶液(请参阅配方)
东co碱工作溶液(请参阅食谱)
毛果芸香碱储备液(请参阅食谱)
 


设备


 


计算器(卡西欧(Casio),货号:FX-991EX)
P1000移液器(Eppendorf,货号:2231000601)
P200移液器(Eppendorf,目录号:2231000601)
称重天平(米特勒,货号:AE 240)
Vortex(Fisher Scientific,Genie 2,目录号:12-812)
加热垫(光束,目录号:731-500)
Barnstead Nanopure (Thermo Fisher Scientific,目录号:D11921)
银线(世界精密仪器,目录号:AGW1030)
六针连接器(联合电子,目录号:R1023090
不锈钢螺钉(塑料一号,目录号:00-96x3-32)
银版印刷(GC电子产品,目录号:22-023)
牙科丙烯酸水泥(Lang Dental Manufacturing,目录号:0830 Fieded Pink)
计时器(Fisher Scientific,目录号:14-648-17)
前置放大器(Pinnacle Technology,目录号:8406-SE4)
电气换向器(Pinnacle Technology,目录号:8408)
数据采集设备(Pinnacle Technology,目录号:8401)
自动立体定位注射SYSTE 米(Stoelting 典型,目录号:53311)                           
木炭过滤器(VetEquip ,目录号:931401 )             
解剖显微镜(Zeiss OPMI 6,目录号:93088)
 


软件


 


1. Sirenia Seizure Pro软件,包括Sirenia Acquisition和Sirenia Seizure Detection模块(Pinnacle Technology,软件版本2.0.5        ; https://www.pinnaclet.com/seizurePRO.html)






程序


 


鼠标订购,安装和处理
来自Envigo 或Jackson Labs的3-4周龄的C57Bl / 6小鼠。
在12小时的明/暗周期进行实验之前,将小鼠放在一个带有单个嵌套正方形和纸房子的笼子中放置1-2周。
免费获得食物和水。称量5-6周龄的小鼠。SE诱导前几天每天处理小鼠约5分钟,因为处理应用程序的耳朵可减少应激反应。
注意:此协议可很好小鼠18-22克之间的称重并适于从(雄性和雌性C57BL / 6N小鼠Envigo )以及一些TRA 从杰克逊实验室(例如nsgenic近交系,NSG)。研究人员应接受过良好的程序训练,以确保在给药时安全,适当地处理和约束小鼠。癫痫动物模型的使用应以研究目标为指导,同时也应优先考虑动物福利以及有效的动物治疗和护理(Lidster等,2016)。


 


诱发癫痫发作的日子
在黑暗/光照周期的光部分期间08:00 AM和12:00 PM之间诱导SE(07:00上午。至下午7时。以控制昼夜变化)(沃尔特等人,2007。 ; 亨德森等等人,2014;Anderson 等人,2018;Gupta 等人,2019)。毛果芸香碱的最佳剂量对于成功诱导小鼠SE是至关重要的,应根据研究中使用的菌株制定出最佳剂量。


  为了在小鼠中诱发SE,首先注射东碱(0.5 mg / ml,ip 。),然后注射毛果芸香碱(280 mg / kg; ip 。)。如果鼠标未能发展癫痫发作在30分钟内第一次注射后,1个或2个剂量补充毛果芸香碱30 - 60毫克/千克,腹膜。可以被诱导SE。用这些剂量的毛果芸香碱注射的小鼠通常会发展为全身性运动性癫痫发作,可能包括跳跃和奔跑。在诱发SE的过程中,实验人员根据最初由Racine (Racine,1972)开发的行为评分系统的修改,以视觉方式监视行为发作。在修改后的版本中,对行为性癫痫发作的严重程度进行了评分(Shibley和Smith,2002年)(请参见表1)。咪达唑仑减轻癫痫发作后,形成SE的小鼠表现出反复的头部抖动,并且缺乏运动控制。节律性头部运动通常在约6小时后消退,但身体抽动或短时的四肢/身体抽搐可能会继续。






表1.用于评分癫痫发作的改良拉辛量表


展示行为


量表分数


正常活动


0


固定(冻结姿势)


1个


面部自动机(晶须抽搐,张开和闭合嘴巴)


2


加硬/延伸的尾巴(Straub)


2.5


部分身体阵挛- 前肢或后肢


3


饲养


4


阵挛性强直/姿势不稳(一侧小鼠)


5


 


  监测和评分毛果芸香碱诱发的癫痫发作和SE :在毛果芸香碱诱发SE期间使用表1所示的改良Racine量表,以观察和分类不同程度的癫痫发作(Shibley和Smith,2002)。原始量表包括5种癫痫发作的行为类别。在小鼠毛果芸香碱模型中,可能会快速连续地出现多个阶段,从而难以区分第4阶段。此评级系统很方便,但不如癫痫发作的EEG检测灵敏。以下视频记录说明了癫痫发作的三个阶段:阶段3 (V ideo 1),阶段5 (V ideo 2)和SE (V ideo 3)。


 






视频1. 表现出改良的Racine 3期癫痫发作的小鼠的Video-EEG。在此阶段,经毛果芸香碱治疗的小鼠出现全身性强直性阵挛性癫痫发作,伴有僵硬的尾巴,前肢的不自主运动以及口和胡须的口面部运动(视频1)。行为性癫痫发作在视频中的14秒开始。


 






视频2. 表现出改良的Racine Scale阶段5发作的小鼠的Video-EEG。该视频中的癫痫发作开始于22 s,显示了处于第5阶段癫痫发作的小鼠的典型行为。这些措施包括饲养起来到后腿和下降,以及非自愿,双边,强直阵挛性四肢的运动。在此视频中,鼠标短暂失去姿势控制,然后重新获得姿势控制,这也是典型的情况。


 






录像3 。展示SE的鼠标的Video-EEG。在这个称为SE的阶段中,小鼠经历了3次或更多的3-5阶段癫痫发作,并持续发作。在这一阶段,小鼠变得不动并表现出明显的头晕。


 


开始诱发癫痫发作之前


准备东莨菪碱股票如此lution,东莨菪碱工作液,和毛果芸香碱的工作方案,在详细介绍[R ecipe 小号。在SE诱导的当天准备这些溶液,并在使用后丢弃。
将所有液体试剂(盐水,东pol碱,毛果芸香碱)放在冰上。
将试剂,0.5 ml Eppendorf管,计算器,实验室标记物,移液器和移液器吸头带到手术室以诱发SE。
在同一阶段中诱导出约15只小鼠。通常,需要4位训练有素的观察员来监视15只小鼠的癫痫发作。然后每个观察者监视3或4只小鼠。
在带有盖的方形容器(Rubbermaid容器)中分别称重小鼠。
诱发癫痫发作之前,请从笼子中取出多余的被褥,食物,水和丰富的环境。
用鼠标的实验编号标记每个笼子,标记将包含该小鼠注射剂的Eppendorf管和该小鼠的一次性注射器。
为每只小鼠准备毛果芸香碱工作溶液(计算在下面提供)。
毛果芸香碱工作溶液的计算:
通过用0.9%的无菌盐水稀释毛果芸香碱储备溶液,最终体积为300 µl,为每只小鼠准备Eppendorf管。
毛果芸香碱储备溶液的体积应按以下方式计算:
6 x (小鼠体重)= X毛果芸香碱(µl)


0.9%无菌盐水的体积计算如下:
300 µl减去毛果芸香碱储备液的体积(µl)


示例:21.2克鼠标


                            毛果芸香碱的体积(μl):6 x 21.2 (g )= 127.2


                            0.9%的无菌盐水:300 µl – 127.2 µl = 172.8 µl


 


SE诱导程序


给每只小鼠注射0.07 ml东pol碱(〜0.5 mg / ml; ip 。),并注意注射时间。
东碱注射后30分钟,注射毛果芸香碱溶液(0.1 ml; ip 。)。注意,稀释是特定于每个小鼠(见“ 开始发作诱导前” S的挠度B,小号TEP 9)。
注意每次癫痫发作的时间和持续时间(仅算出持续15 s或更长时间的运动性癫痫发作)。
如果小鼠在第一次注射后20-30分钟内未出现任何第4阶段或第5阶段癫痫发作,请使用为该小鼠准备的溶液注射补充剂量的毛果芸香碱(0.01-0.02 ml,腹膜内)。如果小鼠患有一次或多次3期癫痫发作,但没有4期或5期癫痫发作,则补充剂量为0.01 ml。但是,如果小鼠表现出0、1或2期癫痫发作,而没有3、4或5期癫痫发作,则给小鼠补充剂量0.02 ml(ip 。)。
在第3、4或5次Racine鳞状癫痫发作3-5 次后,小鼠通常会发展为SE,定义为癫痫发作的连续状态,其特征是头部抽动或“打””,僵硬(身体和尾巴)和固定不动。注意SE的时间。
SE通常将继续保持不变。SE初次发作后1至3小时,注射0.04 ml 咪达唑仑(ip 。)以减轻癫痫发作(图1)。轻轻擦伤小鼠,并用棉签将眼药膏涂在每只眼睛上,以防止角膜干燥,因为形成SE的小鼠很少眨眼。
用咪达唑仑减弱SE后,在其家笼中监视每只小鼠6小时。使用笼子下面的加热垫,为几个小时提供更多的热量,直到鼠标走路和喝水为止。
提供一个嵌套的正方形,一个纸房子,食物和水,并保持小鼠处于12小时光照/ 12小时黑暗周期。我们通常为所有小鼠提供几天的弄湿/软化食物,因为诱导SE后小鼠可能会变得便秘。
监测小鼠并每天服用林格氏液几天(1毫升,腹腔注射)。仅当鼠标变得不动或无法进食或修饰时,才每天注入2x。
笔记:


SE的一小时足以生成可靠地发展为TLE并反复发作的小鼠。在我们的实验室中,用这种方法诱导SE的典型成功率为〜70%或更高。SE的三个小时导致更严重的疾病表型,但是SE的时间越长,死亡率越高。
诱发SE后的几天内,每天必须进行多次动物福利评估。诱发SE后,应为小鼠随意提供软化食物,并用无菌林格氏乳酸盐注射液(ip 。)补水几天。SE后小鼠可能会体重减轻并表现出更大的攻击性,因此需要单独饲养。如果鼠标静止不动或无法进食或修饰超过24小时,则应对小鼠实施安乐死。通过确保同一位实验人员处理老鼠并避免不必要的大声喧and和惊吓动物,可以使动物的死亡率和痛苦降到最低。有关在啮齿动物研究模型中使用啮齿类动物的其他注意事项,可以在国家替代,精制和减少动物研究中心(NC3R)的网站上找到。
 


D:\ Reformatting \ 2020-1-6 \ 1902749--1286 Janice Naegele 793138 \ Figs jpg \图1.jpg


图1. 诱发癫痫持续状态的实验方案流程图


 


电极植入
通过在感应室中用异氟烷气体麻醉来准备要进行手术的鼠标。通过捏脚确认动物已正确麻醉。
将鼠标放在立体定位设备中。将前门牙对准鼻夹的开口,并将鼻锥固定在适当的位置,以在手术期间进行异氟烷气体麻醉。
调整耳棒,确保它们固定在鼠标的头骨上,且不会过分用力。
用消毒的棉签将眼药膏涂在小鼠的眼睛上。
通过使用无菌棉签涂抹聚维酮碘和利多卡因来消毒皮肤,以减轻疼痛。
用手术刀刀沿颅骨的中线轴向切开一条切口,露出前reg和lambda,然后轻轻推开附着在颅骨顶部的肌肉。
用无菌棉签将H 2 O 2 涂在颅骨上,以通过解剖显微镜帮助观察前reg和λ。
对于外科手术植入硬膜下电极,在颅骨上钻两个中线孔。前reg前一个,lam bda后一。参照b子钻四个附加孔(每侧两个),坐标AP:-1.5 mm;L:± 2.5毫米和AP:-3.0毫米; L ± 2.5毫米
电极头安装座由0.25毫米银线焊接到六针连接器上组成。加热四根硬脑膜下电极银线的末端,以在末端形成光滑的球形,以避免导线损坏皮层。
将不锈钢螺钉插入前reg前部和λ后部的两个颅骨孔中。将两根电极锚定银线缠绕在这些螺钉上,以用作参考和接地连接。
在这些连接上涂上银色以确保良好的电气接触。
将四根硬脑膜下电极线插入位于坐标AP 处的颅骨孔中:-1.5 mm;L:± 2.5毫米,AP:-3.0毫米;L ± 2.5 mm ,以前reg为基准。确保电极线末端的球与大脑表面轻轻接触。
涂上牙科丙烯酸水泥以覆盖并固定电极头支架。
从立体定位设备上取下鼠标,并将其放在加热垫上的干净笼子中。监视鼠标,直到它恢复意识并开始四处移动。将鼠标返回到家中的笼子里。
 


SE诱导后通过Video-EEG对SRS进行数据采集和评分
毛果芸香碱模型通常用作TLE的慢性模型,因为小鼠会开始自发性发作,通常发生在持续4-5天的发作丛中,每天发作多次,并且无发作间隔可能持续一段时间几天甚至几周。尽管SRS的发作有所不同,但我们发现在SE诱导后3周开始进行监测很有用,因为小鼠在3-4周开始表现出更频繁的癫痫发作。可以使用视频(V)-EEG记录准确监测特定实验治疗对癫痫发作的影响(Curia 等,2008)。当评估特定治疗方法的疗效时,我们通常会连续进行3周或更长时间的v-EEG记录。


  进行多次立体定向手术时,我们通常会在SE后至少等待1周。在我们先前的工作中,我们在SE后约2 周进行了干细胞的立体定向注射,在干细胞移植之前或之后的1周进行了逆转录病毒的立体定向注射。我们通常有一个实验治疗组(例如,接受干细胞移植的小鼠),并将它们与接受赋形剂(或立体定向注射培养基减去干细胞)的对照组进行比较。然后,我们在SE后约3周植入硬膜下电极以进行慢性v-EEG记录。为了检测和量化SRS,连续进行3周或更长时间的v-EEG记录非常有用。对于v-EEG记录,EEG信号通过连接到数据采集设备的前置放大器进行放大,并使用Sirenia Seizure Pro软件(Pinnacle Technology)进行记录,该软件是一种分析套件,可根据功率和线路长度快速识别和分析用户定义的发作事件。使用Sirenia Acquisition软件以1,000 Hz,10倍的增益捕获数据,分别使用分别设置为0.5 Hz和100 Hz的高通和低通滤波器。Sirenia癫痫发作监测软件将视频记录与EEG信号同步。我们使用基线脑电图脑电活动的功率和线长偏差的变化来检测该软件中的癫痫发作(Henderson 等,2014 ; Anderson 等,2018)。使用软件检测潜在的癫痫发作后,应由实验人员手动验证每个癫痫发作事件,该实验者查看EEG和相关的视频记录,并根据改良的Racine量表对癫痫发作的严重程度进行评分(表1;图2)。必须进行验证,以区分真正的癫痫发作与头戴式电极修饰或其他问题引起的偶尔的EEG伪影。


 


打开“ Sirenia Seizure”。单击“文件”,然后选择要分析的文件。
单击“时间跨度”,然后选择“ 1分钟”。单击“时间步长”,然后选择“ 10 s”。
右键单击每个EEG通道左侧的y轴选项卡,然后单击“手动”。
单击出现在每个y轴选项卡顶部和底部的文本框。将顶部框更改为“ 500”,将底部框更改为“ -500”以按比例缩小EEG迹线,以便于查看癫痫发作活动。每只小鼠的癫痫发作活动通常约为± 500μV 或基线幅度的两倍以上。
单击“癫痫发作设置”图标。将“窗口大小”和“步长”更改为5.00。
选择“线长”搜索选项,并将阈值调整为6,000。
确保在“使用通道”下选择了所有EEG通道。
单击“下一次可能的癫痫发作”按钮开始搜索癫痫发作。
“搜索进度”窗口将打开。当发现癫痫发作时,搜索将暂停,并且在窗口大小和步长中检测到的活动区域大于指定的行长时,将以粉色突出显示。
使用“平移”工具来识别发作事件的开始和结束。
在检测到癫痫发作之前约10 s开始,然后单击“播放”图标开始播放,以对行为性癫痫发作进行评分。
观看摄像机记录,以对鼠标的行为进行评分。
Sirenia癫痫发作软件中的EEG 1和EEG 2分别在相机视图中测量鼠标左右半球的EEG活动。但是,EEG 3是参比电极。参见图3。
单击“下一次可能的癫痫发作”图标,开始搜索下一次癫痫发作。
 


D:\ Reformatting \ 2020-1-6 \ 1902749--1286 Janice Naegele 793138 \ Figs jpg \图2.jpg


图2. 毛果芸香碱诱导的SE后小鼠自发性癫痫样活性的例子。使用Sirenia采集和Sirenia癫痫发作检测软件采集EEG。


D:\ Reformatting \ 2020-1-6 \ 1902749--1286 Janice Naegele 793138 \ Figs jpg \图3.jpg


图3. Sirenia癫痫发作检测软件中的图形用户界面。癫痫发作检测的设置基于EEG线长,阈值设置为6000.000 len / sec。鼠标显示在摄像机视图中,该视图已叠加到图形用户界面的屏幕截图上。


 


菜谱


 


0.9%无菌盐溶液
试剂量:500毫升             


氯化钠4.5克             


加入500 ml M illiQddH 2 O


通过0.2 µm瓶顶过滤器对溶液进行灭菌


东co碱储备溶液
试剂量:1毫升             


东pol碱甲基溴化物10毫克             


将东pol碱溶解于998 µl 0.9%的无菌盐中,最终体积为1 ml


东pol碱工作溶液
试剂量:1毫升             


东co碱储备溶液950 µl             


0.9%无菌盐水50 µl             


这个数量足以容纳8只老鼠


毛果芸香碱储备液
试剂量:1毫升             


盐酸匹罗卡品140 mg             


0.9%的无菌盐水至1毫升             


将毛果芸香碱溶于888 µl 0.9%的无菌盐水中,最终体积为1 ml


 


致谢


 


这项工作由美国国立卫生研究院(NINDS赠款R15NS072879-01A1),麦克奈特基金会脑部疾病奖(JRN),公民癫痫研究联合会(JRN)挑战奖,康涅狄格州再生医学研究基金资助格兰特(JRN)研究员和卫斯理大学(JRN)的Alan M. Dachs 授予教授职位。我们还要感谢我们的同事和合作者,他们基于该协议的不同版本为已发表的工作做出了贡献:Bryan Luikart ,Karl Obrietan ,Yun- Sik Choi,Paul Lombroso,Laura Grabel 和Gloster Aaron。我们感谢实验室成员Jeye Jeong 和Nicolas Cimino 为有关此手稿的讨论做出了贡献。我们感谢帮助我们开发此详细协议的前实验室技术人员:Sara Royston,Stephanie Tagliatela ,Katharine Henderson,Dan Lawrence和Kevin Cobbol 。


 


利益争夺


 


作者没有竞争利益。


 


伦理


 


卫斯理大学机构动物护理和使用委员会确认该协议(协议编号2017_0531_Naegele(C),项目;有效期至05/30/20)符合所有联邦和州法律和准则以及有关护理和使用的所有机构政策和程序动物的研究,教学和测试。


 


参考文献


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Arshad, M. N. and Naegele, J. R. (2020). Induction of Temporal Lobe Epilepsy in Mice with Pilocarpine. Bio-protocol 10(4): e3533. DOI: 10.21769/BioProtoc.3533.
  2. Gupta, J., Bromwich, M., Radell, J., Arshad, M. N., Gonzalez, S., Luikart, B. W., Aaron, G. B. and Naegele, J. R. (2019). Restrained dendritic growth of adult-born granule cells innervated by transplanted fetal GABAergic interneurons in mice with temporal lobe epilepsy. eNeuro 6(2): pii: ENEURO.0110-18.2019.
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