The Repeated Flurothyl Seizure Model in Mice

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Jul 2016



Development of spontaneous seizures is the hallmark of human epilepsy. There is a critical need for new epilepsy models in order to elucidate mechanisms responsible for leading to the development of spontaneous seizures and for testing new anti-epileptic compounds. Moreover, rodent models of epilepsy have clearly demonstrated that there are two independent seizure systems in the brain: 1) the forebrain seizure network required for the expression of clonic seizures mediated by forebrain neurocircuitry, and 2) the brainstem seizure network necessary for the expression of brainstem or tonic seizures mediated by brainstem neurocircuitry. In seizure naïve animals, these two systems are separate, but developing models that can explore the intersection of the forebrain and brainstem seizure systems or for elucidating mechanisms responsible for bringing these two seizure systems together may aid in our understanding of: 1) how seizures can become more complex over time, and 2) sudden unexpected death in epilepsy (SUDEP) since propagation of seizure discharge from the forebrain seizure system to the brainstem seizure system may have an important role in SUDEP because many cardiorespiratory systems are localized in the brainstem. The repeated flurothyl seizure model of epileptogenesis, as described here, may aid in providing insight into these important epilepsy issues in addition to understanding how spontaneous seizures develop.

Keywords: Epilepsy (癫痫), Epileptogenesis (癫痫发生), Flurothyl (三氟乙醚), Mouse (小鼠), Spontaneous seizures (自发性癫痫发作), Seizure semiology changes (癫痫发作症状变化)


Epilepsy is a complex and multifactorial disease defined by unprovoked spontaneous seizures. Approximately two-thirds of the epilepsy population are successfully treated with anticonvulsant drug regimens, but the remaining one third continue to experience seizures (Kwan and Brodie, 2000; Lindsten et al., 2001; Kwan et al., 2010; Loscher et al., 2013; Brodie, 2017). Given the complexities of genetic heterogeneity and inherent difficulties in studying the pathophysiology of epileptogenesis in humans, animal models of epilepsy have served important roles for understanding how spontaneous seizures develop. The mainstays of animal models of spontaneous seizures are either 1) chemically-induced status epilepticus models (SE: a condition characterized by continuous seizure activity) or electrically-induced SE models, or 2) traumatic brain injury (TBI) models. However, there are caveats with these models (Pitkänen et al., 2006). For instance, at least 30 min of SE (but more typically 1-2 h of SE) are required for the appearance of spontaneous seizures with the presence of spontaneous seizures being highly dependent on the duration of SE (Lemos and Cavalheiro, 1995; Gorter et al., 2003; Curia et al., 2008; Loscher, 2013; Kandratavicius et al., 2014; Polli et al., 2014; Gorter et al., 2016). Following these prolonged bouts of SE, there can be a significant increase in mortality (Goodman, 1998; Curia et al., 2008; Scorza et al., 2009; Loscher, 2013; Reddy and Kuruba, 2013; Kandratavicius et al., 2014). Given that SE is a significant seizure event, substantial neuronal death occurs (Goodman, 1998; Curia et al., 2008; Scorza et al., 2009; Loscher, 2013; Reddy and Kuruba, 2013; Kandratavicius et al., 2014). Importantly, both SE and substantial neuronal death are not common findings in most human epilepsies. Lastly, TBI models in rodents also have caveats in that very large regions of the brain require damage to produce spontaneous seizures, and substantial brain damage is not a common observation found in human epilepsy (Pitkänen et al., 2006). Therefore, new rodent models are needed limiting these caveats to continue to advance our understanding of epileptogenesis.

Experimental evidence suggests that there are two largely independent seizure systems that are responsible for the expression of generalized seizures (Kreindler et al., 1958; Browning et al., 1981; Browning and Nelson, 1986; Magistris et al., 1988; Applegate et al., 1991). These two seizure systems are referred to as the forebrain seizure network and the brainstem seizure network. Whereas the forebrain seizure network is responsible for the expression of clonic seizures, the brainstem seizure network is responsible for the expression of brainstem (tonic) seizures (Kreindler et al., 1958; Browning et al., 1981; Browning and Nelson, 1986; Magistris et al., 1988; Applegate et al., 1991). As such, forebrain neurocircuitry modulates the expression of clonic seizures, while brainstem neurocircuitry is both necessary and sufficient for the expression of a variety of tonic-brainstem seizure types. Notably, these seizure systems are mostly independent and the seizures elicited in one network do not readily spread to the other in seizure naïve rodents (Kreindler et al., 1958; Browning et al., 1981; Browning and Nelson, 1986; Magistris et al., 1988; Applegate et al., 1991). Interestingly, BOLD fMRI and SPECT imaging has revealed the critical nature of brainstem structures in the expression of tonic seizures in humans and in animal models (Blumenfeld et al., 2009; Varghese et al., 2009; DeSalvo et al., 2010). However, little is known regarding reorganizations that occur in the brainstem seizure network, or at the intersection of the forebrain seizure network and brainstem seizure network, which can both give rise to brainstem seizure expression.

Flurothyl is a volatile chemoconvulsant acting as a GABAA antagonist that was extensively used historically to induce seizures in severely depressed patients as an alternative to electroconvulsive shock therapy (Krasowski, 2000; Fink, 2014). There are three primary advantages of flurothyl as a chemoconvulsant. First, there are minimal stressors imparted on the rodents since flurothyl is highly volatile. It is infused into a chamber wherein the animal inhales the flurothyl thereby eliminating the need for injections. Second, flurothyl is rapidly eliminated unmetabolized through the lungs, thus eliminating potential confounds of residual convulsant remaining in the body (Krantz et al., 1957; Dolenz, 1967). Finally, flurothyl-induced seizure durations are short (e.g., typically 15-60 sec depending on the seizure type expressed) due to the ease of controlling seizures by simply exposing the animals to room air.

The repeated flurothyl seizure model can be used to understand how seizures develop and become more complex over time, and to explore the mechanistic intersections of the forebrain seizure network and brainstem seizure network that may lead to more complex seizure types (Applegate et al., 1997; Samoriski and Applegate, 1997; Samoriski et al., 1997; Ferland and Applegate, 1998a; 1998b and 1999). With the repeated flurothyl seizure model, C57BL/6J mice express clonic-forebrain seizures during eight flurothyl induction trials (Samoriski and Applegate, 1997; Papandrea et al., 2009). Following a one month incubation period and a rechallenge with flurothyl, C57BL/6J mice express a clonic-forebrain seizure that rapidly and uninterruptedly transitions into a tonic-brainstem seizure (Samoriski and Applegate, 1997; Ferland and Applegate, 1998b). We refer to these seizures as forebrainbrainstem seizures denoting the ictal progression from the forebrain seizure network to the brainstem seizure network (Papandrea et al., 2009; Kadiyala et al., 2015). Lastly, C57BL6/J mice exposed to the repeated flurothyl seizure model rapidly develop spontaneous seizures that appear to remit without treatment following 1 month (Kadiyala et al., 2016), in contrast to DBA2/J mice which also rapidly develop spontaneous seizures that do not remit (Kadiyala and Ferland, 2017). Here, we describe the methods for assaying mice in the repeated flurothyl seizure model, which was originally described 20 years ago (Applegate et al., 1997; Samoriski and Applegate, 1997) and continues to be characterized.

Materials and Reagents

  1. 18 G needle
  2. 3 x 3 in. medium gauze pads (CVS, catalog number: 893120 )
  3. C57BL/6J male mice (6-7 weeks on arrival) (THE JACKSON LABORATORY, catalog number: 000664 )
  4. Aquarium sealant
  5. Flurothyl (Bis(2,2,2-trifluoroethyl) ether or 2,2,2-trifluoroethyl ether) (Sigma-Aldrich, catalog number: 287571 )
    IMPORTANT: Perform all flurothyl exposures in a certified chemical fume hood with exhaust out of the laboratory, since the inhalation of flurothyl will result in seizures in humans.
  6. 95% ethanol (ethyl alcohol 190 proof) (PHARMCO-AAPER, catalog number: 111000190 )
  7. Petroleum jelly
  8. 10% flurothyl solution (see Recipes)


  1. All-clear vacuum Plexiglas desiccator chamber (Ted Pella, model: 2240-1 )
  2. 20 ml glass syringe (Sigma-Aldrich, catalog number: Z101079 )
  3. Syringe pump (Kent Scientific, model: GENIE Plus )
  4. Forceps (for removing the flurothyl saturated gauze pad from the chamber)
  5. Wire mesh colander with at least ¼” square mesh
  6. Chemical fume hood


  1. Construction of flurothyl chamber
    1. The Plexiglas chamber needs to have a small hole drilled in the top of the chamber to allow for the fixing of an 18 G needle in this hole (the 18 G needle is cut at the luer lock end of the plastic edge of the needle and at the beveled edge of the needle resulting in a short 18 G blunted steel tube). The 18 G needle tube, upon fixing it to the top of the chamber, is connected to small diameter tubing on the outside of the chamber. At this point, seal the connection with the tubing and the top of the chamber with aquarium sealant to ensure that the chamber is air-tight. The no-tubing end of the 18 G tube should now be extended into the top of the chamber. Also, be sure to seal any other openings in the top half of the chamber with aquarium sealant (this is usually where the vacuum valve would be attached).
    2. Next, tape a screen support to hold a gauze pad below where the 18 G needle tubing is hanging. The top and bottom of the chamber should be regularly greased with petroleum jelly to ensure an adequate seal between the top, bottom and O-ring of the chamber. The other end of the tubing is connected to a 20 ml glass syringe that is attached to a syringe pump (Figure 1).

      Figure 1. The repeated flurothyl seizure model set-up. Black arrows are pointing to the tubing with slack running from the syringe to the 18 G steel tube in the top of the chamber.

  2. Repeated flurothyl seizure model
    1. Mice are allowed ad libitum access to food and water, and are maintained on a standard 12 h light-dark cycle with lights on at 7:00 AM. Mice are allowed to acclimate to the animal facility for ~1 week before seizure testing. Individual mice (7-8 weeks old) are exposed to 10% flurothyl (see Recipes section). Flurothyl is delivered to mice in a closed Plexiglas chamber. Flurothyl binds at the GABAA receptor where it acts as a noncompetitive antagonist (Krasowski, 2000). Since a receptor bound by a non-competitive antagonist will not be activated by binding of an agonist, and since ethanol is a positive allosteric modulator of the GABAA receptor, ethanol is unlikely to exert a major effect at the GABAA receptor in the presence of flurothyl.
    2. Be sure to perform all flurothyl exposures in a certified chemical fume hood with exhaust out of the laboratory, since the inhalation of flurothyl will result in seizures in humans.
    3. Ten percent flurothyl is infused via a syringe pump and glass syringe at a flow rate of 6 ml/h onto a gauze pad (folded in half) suspended from the top of the chamber by a screen (Figure 1). Since flurothyl is highly volatile, it rapidly vaporizes, leading to inhalation and subsequent seizure expression. The generalized seizure threshold (GST) is defined as the latency from the commencement of the infusion of flurothyl to the occurrence of an animal’s loss of postural control.
    4. Once the animal losses its posture (i.e., expresses a generalized clonic seizure; grades 1-2 [see Table 1]), the chamber is opened to fresh air, resulting in the rapid elimination of flurothyl (and the flurothyl infusion pump is turned off). Mice with different genetic backgrounds will respond with different latencies to loss of postural control (i.e., GSTs). In different mouse strains, trial 1 GSTs can range from 200-500 sec (Papandrea et al., 2009). However, the variability within strains is quite small given that this is a behavioral analysis. It is good practice to place a wire mesh colander over the bottom of the chamber, when the top is removed, to prevent the mouse from jumping out of the chamber. This is particularly important following the 28-day flurothyl incubation period and flurothyl rechallenge, since mice will often have brainstem seizures which can result in the mouse having wild running and bouncing seizures in which they can escape from the bottom half of the chamber. The wire mesh colander helps to contain the mouse in the bottom half of the chamber, while also exposing the animal to room air.
    5. The latency to the first myoclonic jerk (behaviorally, myoclonic jerks are brief, but significant, contractions of the neck and body musculature, while maintaining postural control [Applegate et al., 1997; Samoriski and Applegate, 1997]), the number of myoclonic jerks expressed before the onset of a generalized seizure, the latency to the loss of postural control (GST), the time to regain posture, the duration of the seizure (calculated as the time from the start of the generalized seizure to the time when the animal regains its posture and stops showing clonus of the limbs), and the type of seizure is recorded (Table 1) for each trial.

      Table 1. Behavioral seizure classification grading scale
      Seizure grade
      Description of corresponding seizure behavior*

      Grade 1
      Loss of posture associated with facial clonus, including chewing, and clonus of forelimbs and/or hindlimbs
      Grade 2
      Grade 1 seizure followed by recovery of the righting reflex and low intensity bouncing
      Grade 3
      Grade 1 and/or 2 features with recovery of the righting reflex followed by wild running and popcorning
      Grade 4
      Grade 3 followed by forelimb and/or hindlimb treading
      Grade 5
      Grades 3 and/or 4 followed by bilateral tonic extension of the forelimbs
      Grade 6
      Grade 5 followed by bilateral tonic extension of the hindlimbs
      Grade 7
      Grade 6 followed by immediate death
      *In general, grades 1 and 2 are clonic-forebrain seizures comparable to a grade 5 seizure on the Racine scale for electrical kindling (Racine, 1972). Grades 3-7 are brainstem seizures. Importantly, mice recover their righting reflex before transitioning into grades 3-7 (Kreindler et al., 1958; Browning and Nelson, 1986). We categorize mice having seizure grades 3-7 as forebrainbrainstem seizures to denote the progression of this type of seizure.

    6. Upon seizure resolution, the animal is returned to its home cage until the next day. One mouse is tested at a time in the flurothyl chamber using a new gauze pad each time. Mice are given one 10% flurothyl-induced seizure per day for 8 days (the flurothyl induction phase) followed by a 4 week rest period (the flurothyl incubation period). We routinely try to keep the time intervals between induction trials as close to 24 h as possible (24 ± 4 h). Previous work has demonstrated the necessity of 8 induction trials and a 28-day incubation period for the effects observed (Samoriski and Applegate, 1997). The gauze pad, saturated in flurothyl, is removed from the top half of the chamber from the screening, using forceps, so as to not contaminate one’s gloves with flurothyl. The gauze pads are laid out under the fume hood to allow for flurothyl evaporation. Gauze pads can be reused the next day, since flurothyl will have evaporated completely from the gauze pad by this time.
    7. Following the 28-day incubation period, mice are again given a flurothyl exposure (retest/rechallenge) and the criteria described above are recorded. Representative examples from C57BL6/J mice of the observed changes in the latency to the first myoclonic jerk, the number of myoclonic jerks, and generalized seizure thresholds are shown (Figure 2). C57BL6/J mice typically do not express forebrainbrainstem seizures during the 8 flurothyl induction trials, but upon a 28-day incubation period and flurothyl retest, a significant percentage of mice now express this new seizure behavior (Figure 3).

      Figure 2. Myoclonic jerk threshold, the number of myoclonic jerks, and the generalized seizure threshold (GST) in C57BL6/J mice. The latency to the first myoclonic jerk (myoclonic jerk threshold in seconds), the number of myoclonic jerks expressed before the onset of a generalized seizure, and the latency to a generalized seizure (generalized seizure threshold) on each seizure trial in C57BL6/J mice. The retest trial is a rechallenge to flurothyl following a 28-day incubation period.

      Figure 3. Flurothyl-induced seizure behaviors in C57BL6/J mice during 8 induction phase seizures, and a final flurothyl rechallenge (retest) after a 28-day incubation phase. While none of the C57BL6/J mice expressed a complex forebrain→brainstem seizure during the flurothyl induction trials (these mice only expressed clonic-forebrain seizures), 75-100% of C57BL6/J mice will express forebrain→brainstem seizures following a rechallenge to flurothyl following a 28-day incubation period.

    8. Additionally, local field potential recordings from the cerebral cortex and hippocampus of C57BL6/J mice, during the 28-day incubation period, reveals that flurothyl-exposed mice develop spontaneous seizures following the 8th flurothyl induction trial with relatively high seizure occurrences (Kadiyala et al., 2016). However, these seizures begin to remit over the 28-day incubation phase (Kadiyala et al., 2016). Interestingly, DBA/2J mice similarly treated also develop spontaneous seizures with fewer seizure occurrences than C57BL6/J mice, but the spontaneous seizures observed in DBA/2J mice do not appear to remit (Kadiyala and Ferland, 2017).

Data analysis

Repeated measures analyses of variance (ANOVA) are employed for comparisons between flurothyl-exposed and control groups across trials followed by post-hoc tests. Student’s t-tests are used to determine differences in seizure characteristics between trial 8 of the induction phase and the retest/rechallenge that followed the 28-day incubation period. Evaluation of differences (percentage) in the numbers of mice exhibiting clonic–forebrain seizures and the numbers of mice exhibiting forebrainbrainstem seizures, after flurothyl exposure, utilize Chi-square or Fisher Exact tests.


  1. Be sure to perform ALL flurothyl exposures in a functional and annually certified chemical fume hood to ensure safety of laboratory personnel, given that flurothyl is an inhalant chemoconvulsant. Moreover, do not remove any materials that were exposed to flurothyl (i.e., gauze pads, etc.) in a liquid state, for at least 24 h, to ensure evaporation of flurothyl in the chemical fume hood.
  2. As mice get older than 7-8 weeks of age, some mice do not have an obvious loss of posture with flurothyl exposure. They can present with very severe myoclonic jerks and even some bilateral forelimb clonus in the absence of loss of posture. Mice experiencing these behaviors often result in brainstem seizure expression, since the mice are continually exposed to flurothyl because the top of the chamber was not removed. Missing the clonic seizure (as defined by loss of posture) results in the chamber top not being removed and exposing the mice to room air. Continual flurothyl exposure will always result in a brainstem seizure, therefore, it is important to detect the generalized clonic seizure to terminate the trial, which may involve having to record EEGs to detect generalized seizures.


  1. Preparing the 10% flurothyl solution
    10% flurothyl is made by diluting a 5 g ampule (~5 ml) of liquid flurothyl into 45 ml of 95% ethanol in a glass container
    The flurothyl/ethanol mixture is then shaken to mix the flurothyl
    Note: Flurothyl is always prepared and stored in a chemical fume hood (with the bottle and cap sealed with Parafilm).Be sure that the glass container is clearly labeled as a potential hazard as an inhalant chemoconvulsant.


This work was supported by an NIH/NINDS R01NS064283 grant to RJF. This work was adapted from previous publications (Samoriski and Applegate, 1997; Kadiyala et al., 2016).


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背景 癫痫是由无端自发性癫痫发作定义的复杂多因素疾病。大约三分之二的癫痫患者成功地用抗惊厥药物治疗方案治疗,其余三分之一的人仍然发现癫痫发作(Kwan and Brodie,2000; Lindsten等人,2001; Kwan et al。 al。,2010; Loscher等人,2013; Brodie,2017)。鉴于遗传异质性的复杂性和研究人类癫痫发生的病理生理学的固有困难,癫痫动物模型对于了解自发性发作发展起着重要作用。自发性癫痫发作的动物模型的主要内容是1)化学诱导的癫痫持续状态模型(SE:特征为持续性发作活动的病症)或电诱发的SE模型,或2)创伤性脑损伤(TBI)模型。然而,这些模型有注意事项(Pitkänenet al。,2006)。例如,出现自发性发作需要至少30分钟的SE(但更典型地为1-2小时的SE),存在自发性发作高度依赖于SE的持续时间(Lemos和Cavalheiro,1995; Gorter ,2003; Curia等人,2008; Loscher,2013; Kandratavicius等人,2014; Polli et al。,2003; ,2014; Gorter等人,2016)。在这些长期的SE之后,死亡率可能会大大增加(Goodman,1998; Curia et al。,2008; Scorza等人,2009; Loscher, 2013; Reddy和Kuruba,2013; Kandratavicius等人,2014)。鉴于SE是一个重大的癫痫事件,发生重大的神经元死亡(Goodman,1998; Curia等人,2008; Scorza等人,2009; Loscher,2013; Reddy和Kuruba,2013; Kandratavicius等人,2014)。重要的是,在大多数人类癫痫中,SE和大量神经元死亡都不是常见的发现。最后,啮齿类动物的TBI模型也有注意事项,因为大面积的大面积区域需要损伤才能产生自发性癫痫发作,并且大量的脑损伤不是人类癫痫发现中常见的一种观察(Pitkänen等人, 2006)。因此,新的啮齿动物模型需要限制这些注意事项,以继续提高我们对癫痫发生的理解。
 实验证据表明,有两个主要独立的发作系统负责广泛性癫痫发作(Kreindler等人,1958年; Browning等人,1981年) ; Browning和Nelson,1986; Magistris等人,1988; Applegate等人,1991)。这两种癫痫发作系统被称为前脑癫痫发作网络和脑干癫痫发作网络。前脑癫痫发作网络负责表达阵挛性发作,脑干癫痫发作网络负责脑干(强直)癫痫发作的表达(Kreindler等,1958; Browning等1981年; Browning和Nelson,1986; Magistris等人,1988; Applegate等人,1991)。因此,前脑神经电路调节阵挛性发作的表达,而脑干神经电路对于表达各种强直性脑干癫痫发作类型是必需的和足够的。值得注意的是,这些癫痫发作系统大多是独立的,在一个网络中引发的癫痫发作在癫痫幼稚的啮齿动物(Kreindler等人,1958年)中不容易传播到另一个网络中; Browning等人1981年; Browning和Nelson,1986; Magistris等人,1988; Applegate等人,1991)。有趣的是,BOLD fMRI和SPECT成像已经揭示了脑干结构在人类和动物模型中强直性癫痫发作表现的关键性质(Blumenfeld et al。,2009; Varghese等人, ,2009; DeSalvo等人,2010)。然而,关于在脑干癫痫发作网络中发生的重组,或前脑癫痫发作网络和脑干癫痫发作网络的交叉点,这两者都可以引起脑干癫痫发作表达,知之甚少。
 氟乙基是挥发性化学惊厥剂,其作为GABA A A拮抗剂,其历史上被广泛用于在严重抑郁症患者中诱发癫痫发作,作为电惊厥性休克治疗的替代方案(Krasowski,2000; Fink,2014)。氟乙基作为化学消毒剂有三个主要优点。首先,由于氟乙烯是挥发性高的,因此对啮齿动物施加最小的压力。将其注入到室内,其中动物吸入氟乙基,从而消除了对注射的需要。其次,通过肺部快速消除未代谢的氟乙基,从而消除残留在身体中的残留惊厥药的潜在混乱(Krantz等人,1957; Dolenz,1967)。最后,由于通过简单地将动物暴露于室内空气来容易地控制癫痫发作,氟乙基引起的发作持续时间短(例如,通常为15-60秒,取决于表达的发作类型)。
 重复的氟乙基缉获模型可用于了解癫痫发作随着时间的推移发展和变得更加复杂,并探索前脑癫痫发作网络和脑干癫痫发作网络的机械交叉点,这可能导致更复杂的癫痫发作类型(Applegate 1997; Samoriski和Applegate,1997; Samoriski等人,1997; Ferland和Applegate,1998a; 1998b和1999)。随着氟尿嘧啶重复模型的重复,C57BL / 6J小鼠在八次氟乙基诱导试验(Samoriski和Applegate,1997; Papandrea等人,2009)中表达克隆前脑癫痫发作。经过一个月的潜伏期和用氟乙基重新激发,C57BL / 6J小鼠表达快速而不间断地转变为强直性脑干癫痫发作的阵挛前脑癫痫发作(Samoriski和Applegate,1997; Ferland and Applegate,1998b)。我们将这些癫痫发作称为前脑癫痫发作,表示从前脑癫痫发作网络到脑干癫痫发作网络的发作情况(Papandrea et al。,2009; Kadiyala等人)。 ,2015)。最后,与DBA2 / J小鼠相反,暴露于重复氟乙基癫痫发作模型的C57BL6 / J小鼠在1个月后快速发展出自发性癫痫发作(Kadiyala等,2016)也迅速发展自发性癫痫发作(Kadiyala和Ferland,2017)。在这里,我们描述了在20年前(Applegate等人,1997; Samoriski和Applegate,1997)最初描述的重复氟乙基捕获模型中测定小鼠的方法,并继续表征。

关键字:癫痫, 癫痫发生, 三氟乙醚, 小鼠, 自发性癫痫发作, 癫痫发作症状变化


  1. 18 G针
  2. 3 x 3 in。中等纱布垫(CVS,目录号:893120)
  3. C57BL / 6J雄性小鼠(抵达6-7周)(JACKSON LABORATORY,目录号:000664)
  4. 水族馆密封胶
  5. (二(2,2,2-三氟乙基)醚或2,2,2-三氟乙基醚)(Sigma-Aldrich,目录号:287571)
  6. 95%乙醇(乙醇190证明)(PHARMCO-AAPER,目录号:111000190)
  7. 石油果冻
  8. 10%氟乙基溶液(见配方)


  1. 全透明真空有机玻璃干燥器室(Ted Pella,型号:2240-1)
  2. 20毫升玻璃注射器(Sigma-Aldrich,目录号:Z101079)
  3. 注射泵(Kent Scientific,型号:GENIE Plus)
  4. 镊子(用于从室中除去氟乙烯饱和纱布垫)
  5. 具有至少1/4“方形网格的网状漏勺
  6. 化学通风柜


  1. 氟乙烯室的构造
    1. 有机玻璃室需要在室顶部钻出一个小孔,以便将18 G针固定在该孔中(18 G针在针的塑料边缘的鲁尔锁定端处切割,针的斜边缘产生短的18G钝钢管)。 18G针管在将其固定到腔室的顶部时连接到腔室外部的小直径管道。此时,用水族密封剂密封与管道和腔室的顶部的连接,以确保腔室是气密的。 18 G管的无管端应该延伸到腔室的顶部。另外,请务必用水族箱密封剂(这通常是真空阀附着的地方)密封上半部分的其他开口。
    2. 接下来,将屏幕支撑带固定在18 G针管悬挂下方的纱布垫上。室内的顶部和底部应经常用油膏润滑,以确保腔室的顶部,底部和O形环之间的充分密封。管的另一端连接到连接到注射泵的20ml玻璃注射器(图1)。

      图1.重复的氟乙烯吸收模型设置。黑色箭头指向从注射器的松弛管道到腔室顶部的18 G钢管。

  2. 重复荧光素检测模型
    1. 允许小鼠随意获取食物和水,并保持在标准的12小时黑暗周期,上午7:00点亮。在癫痫发作测试前,允许小鼠适应动物设施约1周。将单个小鼠(7-8周龄)暴露于10%氟乙基(参见食谱部分)。氟化乙基在封闭的有机玻璃室中输送给小鼠。氟乙基在GABA A A受体上结合,其中它作为非竞争性拮抗剂(Krasowski,2000)。由于受非竞争性拮抗剂结合的受体不会通过激动剂的结合而被活化,并且由于乙醇是GABA A A受体的正变构调节剂,所以乙醇不太可能在在氟乙基存在下的GABA A A受体。
    2. 确保在认证的化学通风橱中进行所有氟烷基曝光,并排出实验室,因为吸入氟乙基会导致人体癫痫发作。
    3. 通过注射泵和玻璃注射器,以6ml / h的流速将10%的氟乙基通过筛网(图1)输入到从室顶部悬浮的纱布垫(折叠成两半)。由于氟乙基具有高挥发性,因此其迅速蒸发,导致吸入和随后的发作表达。广义发作阈值(GST)定义为从氟氯乙烯注入开始到发生动物失去姿势控制的潜伏期。
    4. 一旦动物失去其姿势(即,,表示广泛的阵挛性发作;等级1-2 [参见表1]),将该腔室打开至新鲜空气,导致快速消除氟乙基(氟氯乙酸输液泵关闭)。具有不同遗传背景的小鼠将以不同的延迟对姿势控制的丧失作出反应(即,,GST)。在不同的小鼠品系中,试验1 GST可以在200-500秒的范围内(Papandrea等人,2009)。然而,鉴于这是行为分析,菌株内的变异性相当小。当顶部被移除时,将丝网滤网放置在腔室的底部是很好的做法,以防止鼠标从腔室中跳出。这在28天的氟乙基孵育期和氟乙基重新触发之后尤为重要,因为小鼠经常会发生脑干癫痫发作,这可导致小鼠具有野蛮的运动和弹跳癫痫发作,其中它们可以从腔室的下半部分逸出。丝网滤网有助于将鼠标放在腔室的下半部分,同时还将动物暴露于室内空气。
    5. 第一个肌阵挛跳动的延迟(行为上,肌阵挛的冲动是短暂的,但重要的是颈部和身体肌肉组织的收缩,同时保持姿势控制[Applegate等人,1997年; Samoriski和Applegate,1997年]),在广泛性癫痫发作发作之前表达的肌阵挛爆发的数量,姿势控制丧失的延迟(GST),恢复姿势的时间,癫痫发作的持续时间(以从全身癫痫发作到动物恢复其姿势并停止显示四肢的克隆),并且每次试验记录癫痫发作的类型(表1)。

      < *一般来说,1级和2级是克隆前脑癫痫发作,相当于Racine电烙术的5级癫痫发作(Racine,1972年)。 3-7年级脑干癫痫发作。重要的是,小鼠恢复正常反射,然后转为3-7年级(Kreindler等,1958; Browning和Nelson,1986)。我们将具有3-7级癫痫发作的小鼠归类为前脑脑干癫痫发作,以表示这种癫痫发作的进展。

    6. 缉拿后,将动物送回家中笼,直到第二天。每次使用新的纱布垫,在氟乙烯室中一次测试一只鼠标。每天给予10%氟乙基引起的癫痫发作8天(荧光素诱导期),随后4周休息期(氟乙烯潜伏期)。我们经常尝试将诱导试验之间的时间间隔尽可能接近24小时(24±4 h)。以前的工作已经证明了8次诱导试验的必要性和观察到的效果的28天潜伏期(Samoriski和Applegate,1997)。氟化乙烯饱和的纱布垫,使用镊子从筛子上取下室的上半部分,以免用氟乙烯污染手套。纱布垫布置在通风柜下面,以使氟乙烯蒸发。第二天纱布垫可以再次使用,因为这个时候氟乙烯将完全从纱布上蒸发出来。
    7. 在28天的潜伏期后,再次给予小鼠氟尿素暴露(重新测试/重新触发),并记录上述标准。来自C57BL6 / J小鼠的代表性例子显示了对第一次肌阵挛反应的延迟观察到的变化,肌阵挛性跳动的数量和广泛发作的癫痫发作阈值(图2)。 C57BL6 / J小鼠通常在8次氟化诱导试验期间不表达前脑脑干癫痫发作,但是在28天的潜伏期和氟乙基复苏中,相当一部分的小鼠现在表现出这种新的癫痫发作行为(图3)。 >

      图2.肌力振幅阈值,肌阵挛跳数,以及C57BL6 / J小鼠中的广泛发作阈值(GST)。第一次肌阵挛反应的潜伏期(肌阵挛刺激阈值(以秒为单位)),在全身癫痫发作发生之前表达的肌阵挛反应的数量,以及C57BL6 / J小鼠每次癫痫发作的广泛性癫痫发作(广泛性发作阈值)的潜伏期。再次测试是在28天潜伏期后对氟乙的重新测试。

      图3.在8次诱导期癫痫发作期间C57BL6 / J小鼠中的氟乙基引起的发作行为,以及28天孵育阶段后的最终氟乙基重新振荡(重新测试)。虽然没有一个C57BL6 / J小鼠在氟烷基诱导试验(这些小鼠只表达阵挛前脑癫痫发作)期间,表达复杂的前脑→脑干癫痫发作,75-100%的C57BL6 / J小鼠将在28天的潜伏期内对氟乙烯进行再刺激后表达前脑→脑干癫痫发作。

    8. 另外,在28天的培养期间,来自C57BL6 / J小鼠的大脑皮层和海马的局部场电位记录显示,氟乙基暴露的小鼠在第8次氟乙基诱导试验后发生自发性发作,相对较高的发作发生(Kadiyala等人,2016)。然而,这些癫痫发作开始于28天的孵化阶段(Kadiyala等人,2016)。有趣的是,类似治疗的DBA / 2J小鼠也发生了比C57BL6 / J小鼠更少的发作发作的自发性发作,但DBA / 2J小鼠中观察到的自发性发作似乎不显示(Kadiyala和Ferland,2017)。
    9. 数据分析



      1. 确保在功能性和年度认证的化学通风橱中进行所有氟烷基曝光,以确保实验室人员的安全,因为氟乙基是吸入化学消毒剂。此外,不要将任何暴露于氟乙烯( ie ,纱布垫,等等)的材料以液态移除至少24小时,以确保蒸发在化学通风橱中的氟乙烯。
      2. 当老鼠年龄大于7-8周龄时,一些小鼠在氟乙烯暴露时并没有明显的姿势丧失。在没有失去姿势的情况下,他们可以呈现非常严重的肌阵挛,甚至一些双侧前肢克隆。经历这些行为的小鼠经常导致脑干发作表达,因为小鼠不连续地暴露于氟乙基,因为室的顶部没有被去除。缺少阵挛性发作(由失去姿势所定义)导致腔室顶部不被移除,并将小鼠暴露于室内空气。持续的氟乙烯暴露将总是导致脑干发作,因此,重要的是检测广泛的阵挛性发作以终止试验,其可能涉及记录脑电图以检测广泛性癫痫发作。


      1. 准备10%氟乙基溶液
        10%氟乙基通过在玻璃容器中稀释5g安瓿(约5ml)液体氟乙基酯至45ml 95%乙醇中而制成,
        注意:氟乙基总是制备并储存在化学通风橱中(瓶子和盖子用Parafilm密封)。确保玻璃容器被清楚地标记为吸入化学惊厥剂的潜在危险。 br />


      这项工作得到了NIH / NINDS R01NS064283授予RJF的支持。这项工作改编自以前的出版物(Samoriski和Applegate,1997; Kadiyala等人,2016)。


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引用:Ferland, R. J. (2017). The Repeated Flurothyl Seizure Model in Mice. Bio-protocol 7(11): e2309. DOI: 10.21769/BioProtoc.2309.