Mouse Phrenic Nerve Hemidiaphragm Assay (MPN)
小鼠中膈神经偏侧膈分析(MPN)   

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PLOS Pathogens
Aug 2017

 

Abstract

The neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their biochemical nature, the site they target (either the nerve or the muscle) and their mechanism of action, substances affecting NMJ produce very specific alterations of neuromuscular functionality.

Here we provide a detailed protocol to isolate the diaphragmatic muscle from mice and to set up two autonomously innervated hemidiaphragms. This preparation can be used to study bioactive substances like toxins, venoms and neuroactive molecules of various origin, or to measure the force of skeletal muscle contraction.

The ‘mouse phrenic nerve hemidiaphragm assay’ (MPN) is an established model of ex vivo NMJ and recapitulates the complexity of neuromuscular transmission in a system easy to control and to manipulate, thus representing a valuable tool to study both NMJ physiology and the mechanism of action of toxins and other molecules acting at this synapse.

Keywords: Electrophysiology (电生理), Neuromuscular junction (神经肌肉接头), Hemidiaphragm assay (偏侧膈分析), Phrenic nerve (膈神经), Toxins (毒素), Botulinum (肉毒杆菌), Inhibitors (抑制剂)

Background

The neuromuscular junction (NMJ) is the chemical synapse enabling communication between motor neurons and skeletal muscle fibers. This is the best characterized synapse and most of the knowledge on maturation, structure and function of synapses derives from its study (Li et al., 2017). At the NMJ, the action potential running along the motor axon invades the nerve terminal (presynaptic bouton) and induces the fusion of synaptic vesicles with the presynaptic membrane. This triggers the release of acetylcholine (ACh), the neurotransmitter that binds the nicotinic ionotropic ACh receptors (nAChRs) on the postsynaptic muscle fiber. Upon ACh binding to nAChRs, a postsynaptic action potential spreads out along the muscle fiber causing Ca2+ release from the sarcoplasmic reticulum into the cytosol, thereby inducing muscle contraction. At variance from central synapses, NMJs are not protected by anatomical barriers, like the blood brain barrier or the blood nerve barrier, and are exposed to the action of various pathogenic molecules, including chemicals, toxins from plant, animal and bacteria as well as autoantibodies raised against NMJ antigens. Depending on the way they act, these agents produce distinct kinds of injury to the NMJ, eventually leading to impairment of muscle contraction.

The ‘mouse phrenic nerve hemidiaphragm assay’ (MPN) is an established model to study ex vivo NMJ function and offers a valuable tool to investigate the mechanism of action of toxins and molecules that produce NMJ alteration. In addition, MPN can be used to evaluate skeletal muscle contractility and measure diaphragmatic muscle force elicited by electrical stimulation of its phrenic nerve, so providing a model which recapitulates the complexity of the neuromuscular system in a more accessible and isolated environment.

The MPN provided fundamental insights to tackle the mechanism of action of Botulinum Neurotoxins (BoNTs), and it is currently used in many laboratories worldwide to perform qualitative/quantitative analysis of BoNTs. Its employment allows a significant refinement and reduction in the use of animals and results are in good agreement with the classical mouse lethality bioassay (Rasetti-Escargueil et al., 2011; Bigalke and Rummel, 2015).

For this, and because the hemidiaphragm-phrenic nerve intoxicated by BoNTs closely mimics the failure of respiratory muscles occurring in vivo, the MPN is presently listed in the European Pharmacopeia as an alternative method to the mouse bioassay for assaying BoNT/A lots for human use (https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/eur/eph_botat-508.pdf). This system is also a valuable tool to test new BoNTs (Zanetti et al., 2017) and the neutralizing potency of antibodies and inhibitors (Rasetti-Escargueil et al., 2011; Azarnia Tehran et al., 2015; Beske et al., 2017).

Besides BoNTs, the MPN can be used to study many other bioactive substances, comprising toxins like phospholipases, myotoxins and complete venoms, neuroactive molecules like peptides, lipids and drugs (Rigoni et al., 2005; Caccin et al., 2006; Bercsenyi et al., 2014; Yan et al., 2014; Caccin et al., 2015), or to measure muscle force in diaphragms from mice pre-treated with autoantibodies involved in myasthenic syndromes (Klooster et al., 2012) or from animal models of neuromuscular diseases (Nascimento et al., 2014).

Here, we describe a very detailed protocol to successfully dissect the mouse diaphragmatic muscle with both the phrenic nerves completely functional. This is a remarkable advantage as it allows obtaining two autonomously innervated hemidiaphragms to be independently used, either as an internal control or to increment the number of experimental data. The small volume of muscle bath-chambers, the possibility of finely control bath concentration of substances used, the easy manipulability of experimental conditions (temperature, washes, etc.) and the possibility to use the muscles for further analysis (immunofluorescence, Western blot, etc.), represent significant advantages as well.

Materials and Reagents

  1. 2 µl micropipette
  2. 200 µl micropipette
  3. 1,000 µl micropipette
  4. Tips for 2, 200 and 1,000 µl micropipettes
  5. Petri dish, 100 x 20 mm, coated with Sylgard (Dow Corning, Sylgard® 184 Silicone Elastomer kit)
  6. Surgical needles (Rudolf, catalog number: RU 5899-01 )
  7. Cotton thread
  8. Mice of desired genotype and age
  9. Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: H1758 )
  10. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  11. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  12. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  14. Magnesium chloride, standard solution 1 M (MgCl2) (Honeywell International, Fluka, catalog number: 63020 )
  15. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  16. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7528 )
  17. Ringer’s solution (see Recipes)

Equipment

  1. Volumetric flask
  2. Clamp (Bulldog Clamp, Diethrich) (Rudolf, catalog number: RU 3934-16 ; or similar)
  3. 2 x scissors (Delicate Surgical Scissors) (Rudolf, catalog number: RU 1503-12 )
  4. 2 x forceps (Micro Jewelers Forceps, curved) (Rudolf, catalog number: RU 4240-07 )
  5. Stimulator: 6002 Stimulator (Harvard Apparatus, model: 6002 Basic Stimulator )
  6. 2 x micromanipulator (three-dimensional coarse manual manipulator) (NARISHIGE, catalog number: M-152 )
  7. 2 x Tension transducer: isometric transducer (Harvard Apparatus, catalog number: 72-4494 )
  8. 2 x Lead Screw Positioner (Harvard Apparatus, catalog number: 53-2082W )
  9. 2 x Support system (to hold both stimulation chamber and transducer sensors)
  10. 2 x Static stimulation chamber (jacketed, total internal volume 5 ml)
  11. Thermostatic bath (Isco, catalog number: GTR190 )
  12. Recording unit: i-WORX 118 system (iWORX, model: iWORX 118 )
  13. Computer compatible with the software
  14. Gas tank (95% O2, 5% CO2) equipped with pressure control (Air Liquide, model: HBS 240-1-2 )

Software

  1. Recording and Analysis: i-WORX 118 system (Dover, NH, USA) interfaced via Labscribe software (iWorx Systems Inc., Dover, NH, USA)

Procedure

  1. Solutions and setup preparation
    1. Prepare Ringer’s solution (Recipe 1) and saturate it by aeration with 95% O2, 5% CO2, for at least 15 min to buffer pH at 7.4. During aeration, add 1 mg/ml of glucose (complete Ringer’s solution).
      Note: About 50 ml of solution is needed for one standard experiment.
    2. Switch on all the components of the setup and run Labscribe software (see Figure 1).


      Figure 1. Electrophysiological setup components. A. Setup overview. 1) Muscle chamber; 2) Stimulator; 3) Tension transducer; 4) Data acquisition system; 5) Computer system with Labscribe software. B. Muscle chamber details. 6) Support system; 7) Static stimulation chamber; 8) Muscle support with oxygen cannula; 9) Micromanipulator; 10) Platinum electrodes; 11) Screw Positioner; 12) Transducer sensor.

  2. Dissection
    1. Euthanize the mice, preferably by cervical dislocation.
      Notes:
      1. All procedures were performed in accordance with the Italian laws and policies (D.L. no 26 14th March 2014), with the guidelines established by the European Community Council Directive no 2010/63/UE and approved by the veterinary services of the University of Padova (O.P.B.A.-Organismo Preposto al Benessere degli Animali) (protocol 359/2015). All the procedures should be utilized according to the ethical standards of the Institution where experiments are carried out.
      2. Exsanguination with decapitation can be carried out to avoid the presence of bleeding and clot formations within the rib cage.
    2. Pin the mouse on a support in a supine position, remove the skin over the chest and the abdomen, and open the peritoneum under the rib cage.
    3. Remove pectoral muscles to expose the rib cage.
    4. Hold firmly the lower extremity of the sternum with a clamp, and make a central cut on it, approximately at the level of the second rib; keep on cutting the right and left side of the rib cage to get the diaphragm exposed (see Figure 2A).
      Note: While cutting, do not follow the course of the ribs, but chop them.
    5. Completely remove the superior part of the rib cage (see Figure 2B).


      Figure 2. Rib cage opening for diaphragm explantation

    6. Gently, move away the heart and the pulmonary lobes to spot the left phrenic nerve (it forms a white arch, see Figure 3A). Using forceps, insert the cotton thread under the nerve, in the upper part and make two knots. Carefully, cut the nerve behind the knots; holding the thread (avoid excessive stretching which may cause nerve damage), isolate the nerve removing adherent tissue as much as possibile; it is advisable to start from the knots and gently proceed toward the diaphragm muscle. A cleared nerve is easier to mount and stimolate, yet this requires carefull handling to avoid nerve stretching or damage, particularly close to the nerve insertion point into the muscle. Gently depose the isolated nerve on the muscle (see Figures 3B and 3C). Repeat these operations for the right phrenic nerve. Be careful: the right nerve is very close to blood vessels whose damage may lead to annoying hemorrhages (see Figures 3D and 3E). The result of these steps is shown in Figure 3F.


      Figure 3. Procedure for isolation and ligation of the two phrenic nerves. D: diaphragm; P: phrenic nerve. White arrows: knots position. White dotted lines: position of the phrenic nerves. Red dotted line: blood vessel running along the right phrenic nerve. Yellow dotted line in A: whole diaphragmatic muscle area.

    7. Hold the diaphragm using the lower part of the sternum, to avoid damage of muscle fibers; remove the connective tissue adhering the abdominal side of the diaphragm (arrow in Figure 4A). Explant the diaphragm by cutting the dorsal part of the muscle (arrow in Figure 4B).
      Note: Pay close attention to spot nerve insertion point and avoid their cleavage.


      Figure 4. Diaphragm explantation

    8. Transfer the preparation into a Petri dish (10 cm diameter), bottomed with sylgard and filled with 5-10 ml of complete Ringer’s solution; pin the muscle as shown in Figure 5A.
    9. Split the whole muscle into two hemidiaphragms, each innervated by its own phrenic nerve, by cutting along the dotted lines shown in Figure 5A and discarding the central wedge with the sternum attachment. The result of this processing is two triangles, one of which is shown in Figure 5B.
      Note: With the present dissection procedure, two hemidiaphragms are obtained allowing two parallel but independent analyses, which is a remarkable advantage.
    10. Tie a cotton thread at the apex of the triangle to connect the preparation to the transducer, making double knots without drilling the tissue. Use the ribs at the base of the triangle to secure the preparation to the support: drill the tissue between the ribs, pass a cotton thread and make a double knot around the lower rib (Figure 5B). Position the support and tie the base of the triangle via a double knot around the holder (Figure 5C).


      Figure 5. Hemidiaphragms separation and mounting on the tissue holder

  3. Nerve stimulation and acquisition
    1. Very carefully, transfer the preparation into the stimulation chamber (filled with 4 ml of complete Ringer’s solution, at 37 °C). Set O2 pressure to have a continuous but gentle bubbling of gas. Fix the thread knotted at the triangle apex to the transducer sensor with a double knot (Figure 6A).
      Notes:
      1. For a complete force transduction, the muscle must be perpendicular to the axis of the transducer sensor (thread vertical as much as possible); thread contact with other parts of the setup may alter the measurement and must be avoided (Figure 6B). Muscle twitching is recorded by an isometric transducer that translates the mechanical force in electrical signal and commutated into a digital signal displayed by the Labscribe software.
      2. A calibration curve can be built by applying weights of known value to the transducer and recording the corresponding Volt values; this procedure allows to express the force of muscle contraction in Newton (or in grams). The unit of measurement is irrelevant when results are provided as normalized values (see below).
    2. Stretch the muscles, using the lead screw positioner (Figure 6A), until its tension reaches about 0.2 V (resting tension).
    3. Pass the cotton wire connected to the phrenic nerve through the two platinum rings of the stimulating electrode (arrow in Figure 6C). Using the micromanipulator, carefully move the electrode inside the stimulation chamber. Pull the cotton thread until the phrenic nerve gets in contact with the rings (Figure 6C).
      Note: The nerve must be completely immersed into the solution and ideally perpendicular to the muscle, avoiding excessive tension. The electrode must not be in contact with the muscle, to prevent direct stimulation of muscle fibers.


      Figure 6. Details of the experimental setup. A. Transducer sensor with the cotton thread knotted. The asterisk indicates the screw positioner. B. Connection between the cotton thread (parallel to the white dotted line) and the hemidiaphragm. C. Final positioning of the phrenic nerve in the chamber. Black arrow: electrode rings with the nerve passing through and contacting it.

    4. Set the standard parameters for phrenic nerve stimulation. Amplitude: 5 V (supramaximal stimulus); pulse width: 0.1 msec; frequency: 0.1 Hz. Start the stimulation and test for muscle twitching.
      Note: In these conditions, a hemidiaphragm muscle preparation can twitch at least 4 h.
    5. If the muscle contracts properly, adjust the optimal tension for twitch response using the screw positioner. Slowly increase the muscle tension repeated times, alternating with 10-15 min periods of muscle stabilization, until, incrementing the resting tension, twitch amplitude does not increase any more. An optimal resting tension is around 0.3 V.
    6. Let muscle twitch stabilize for further 20 min at 37 °C before starting the experiment or before adding a treatment.
      Note: The mean twitch amplitude measured at the end of this period is used to normalize twitch values upon treatment.
    7. Add the treatment directly into the stimulation chamber.
    8. Using i-WORX 118 system with Labscribe software, record muscle twitch for the desired time or until muscle force completely drops.

Data analysis

  1. Twitch amplitude is calculated as the difference between the tension peak value after stimulation (an in Figure 7) and the average basal tension before stimulus (mean value of resting tension in the time interval bn in Figure 7). This value, in standard conditions, is maintained for at least 4 h.


    Figure 7. Typical trace displayed by Labscribe software during data recording

  2. By adding a treatment impinging on neuromuscular transmission (both presynaptic or postsynaptic compartment), twitch amplitude drops over the time (Figure 8A). In case of complete paralysis, the paralytic half-time (T50%), defined as the time required to decrease twitch amplitude to 50% of the initial value (Figure 8B), is commonly used to evaluate the potency of the treatment.


    Figure 8. Typical experiment using MPN with Botulinum neurotoxin serotype A. Red arrow: toxin addition (200 pM), assumed as time = 0. A. Raw data recorded with Labscribe Software; the mean value of twitch amplitude in the time interval from -10 to 0’ (Tm), calculated as in Figure 7, is used to normalize subsequent amplitude values. B. Plot of normalized twitch amplitude against function of time, and calculation of paralytic half-time (T50%, black arrow).

  3. For comparative studies (for example mutant toxin vs. wild-type toxin), it is advisable to perform parallel experiments using the two hemidiaphragms harvested from the same animal to reduce variability, and to report data as paired observations. Alternatively, a potency calibration curve can be carried out with a toxin considered as reference and then used to extrapolate the relative potency of other toxin batches or variants (Bigalke and Rummel, 2015)
  4. Different toxins (or other substances) could require various experimental conditions (Caccin et al., 2006).
    Note: Muscle fibers functionality could be detected using additional electrodes (not shown) to directly stimulate the muscle.

Recipes

  1. Ringer’s solution

    Notes:
    1. Use ddH2O. Glassware must be washed with hydrogen chloride 0.1 N to remove any trace of carbonate and rinsed with ddH2O very well. Do not use solutions in presence of any deposit or opalescence.
    2. The buffer used is a customized Ringer’s solution; throughout the protocol it is indicated as ‘Ringer’s’ for simplicity.
    3. Before the analysis, Ringer’s solution is saturated with 95% O2, 5% CO2, by aeration for at least 15 min to obtain pH of 7.4 and supplemented with glucose (final concentration 1 mg/ml, 11 mM) (the buffer after oxygen saturation and glucose supplementation is indicated as ‘complete Ringer’s solution’ in this protocol). Glucose is necessary for recordings longer than 1 h.

Acknowledgments

This work was supported by the University of Padova, with ‘Senior Research Grant for young people not employed in the University of Padova’ granted to M. Pirazzini and with a ‘junior fellowship’ to S. Negro and by Fondazione Caritro with ‘Bando 2017 per giovani ricercatori coinvolti in progetti di eccellenza’ granted to G. Zanetti. All the procedures were performed in the laboratory of ‘Neurotoxins, Neuroparalysis and Regeneration’ headed by Prof. Cesare Montecucco at the Department of Biomedical Sciences (University of Padova). The authors declare no competing interests. PC performed the procedure for hemidiaphragm preparation. SN and GZ took the pictures to describe all the procedures and prepared the figures. GZ and PC wrote the paper, with the help of SN and MP. All authors reviewed the manuscript and approved the final version.

References

  1. Azarnia Tehran, D., Zanetti, G., Leka, O., Lista, F., Fillo, S., Binz, T., Shone, C. C., Rossetto, O., Montecucco, C., Paradisi, C., Mattarei, A. and Pirazzini, M. (2015). A novel inhibitor prevents the peripheral neuroparalysis of botulinum neurotoxins. Sci Rep 5: 17513.
  2. Bercsenyi, K., Schmieg, N., Bryson, J. B., Wallace, M., Caccin, P., Golding, M., Zanotti, G., Greensmith, L., Nischt, R. and Schiavo, G. (2014). Nidogens are therapeutic targets for the prevention of tetanus. Science 346(6213): 1118-1123.
  3. Beske, P. H., Bradford, A. B., Hoffman, K. M., Mason, S. J. and McNutt, P. M. (2017). In vitro and ex vivo screening of candidate therapeutics to restore neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A1. Toxicon. doi: 10.1016/j.toxicon.2017.10.017.
  4. Bigalke, H. and Rummel, A. (2015). Botulinum neurotoxins: qualitative and quantitative analysis using the mouse phrenic nerve hemidiaphragm assay (MPN). Toxins (Basel) 7(12): 4895-4905.
  5. Caccin, P., Rigoni, M., Bisceglie, A., Rossetto, O. and Montecucco, C. (2006). Reversible skeletal neuromuscular paralysis induced by different lysophospholipids. FEBS Lett 580(27): 6317-6321.
  6. Caccin, P., Scorzeto, M., Damiano, N., Marin, O., Megighian, A. and Montecucco, C. (2015). The synaptotagmin juxtamembrane domain is involved in neuroexocytosis. FEBS Open Bio 5: 388-396.
  7. Klooster, R., Plomp, J. J., Huijbers, M. G., Niks, E. H., Straasheijm, K. R., Detmers, F. J., Hermans, P. W., Sleijpen, K., Verrips, A., Losen, M., Martinez-Martinez, P., De Baets, M. H., van der Maarel, S. M. and Verschuuren, J. J. (2012). Muscle-specific kinase myasthenia gravis IgG4 autoantibodies cause severe neuromuscular junction dysfunction in mice. Brain 135(Pt 4): 1081-1101.
  8. Li, L., Xiong, W. C. and Mei, L. (2017). Neuromuscular junction formation, aging, and disorders. Annu Rev Physiol.
  9. Nascimento, F., Pousinha, P. A., Correia, A. M., Gomes, R., Sebastiao, A. M. and Ribeiro, J. A. (2014). Adenosine A2A receptors activation facilitates neuromuscular transmission in the pre-symptomatic phase of the SOD1(G93A) ALS mice, but not in the symptomatic phase. PLoS One 9(8): e104081.
  10. Rasetti-Escargueil, C., Liu, Y., Rigsby, P., Jones, R. G. and Sesardic, D. (2011). Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies. Toxicon 57(7-8): 1008-1016.
  11. Rigoni, M., Caccin, P., Gschmeissner, S., Koster, G., Postle, A. D., Rossetto, O., Schiavo, G. and Montecucco, C. (2005). Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid mixtures. Science 310(5754): 1678-1680.
  12. Yan, Y., Li, J., Zhang, Y., Peng, X., Guo, T., Wang, J., Hu, W., Duan, Z. and Wang, X. (2014). Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity. Biol Res 47: 17.
  13. Zanetti, G., Sikorra, S., Rummel, A., Krez, N., Duregotti, E., Negro, S., Henke, T., Rossetto, O., Binz, T. and Pirazzini, M. (2017). Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission. PLoS Pathog 13(8): e1006567.

简介

神经肌肉接头(NMJ)是外周运动神经元支配肌纤维和控制骨骼肌收缩的特化突触。 NMJ是几种异生素的靶标,包括化学品,植物,动物和细菌毒素,以及针对NMJ抗原产生的自身抗体。根据它们的生物化学性质,它们靶向的部位(神经或肌肉)及其作用机制,影响NMJ的物质产生神经肌肉功能的非常特定的改变。

在这里,我们提供了一个详细的协议来隔离小鼠的膈肌,并建立两个自主神经支配的半膈。这种制剂可用于研究生物活性物质,如毒素,毒液和各种起源的神经活性分子,或测量骨骼肌收缩力。

“小鼠膈神经半膈膜测定法”(MPN)是一种建立的离体NMJ模型,并概括了容易控制和操纵的系统中神经肌肉传导的复杂性,因此代表了一种有价值的研究工具NMJ生理学和毒素和其他分子作用于该突触的作用机制。

【背景】神经肌肉接头(NMJ)是使运动神经元和骨骼肌纤维之间能够交流的化学突触。这是最好的特征性突触,关于突触的成熟,结构和功能的大部分知识来源于其研究(Li等人,2017)。在NMJ,沿着运动轴突运动的动作电位侵入神经末梢(突触前布顿)并诱导突触小泡与突触前膜融合。这触发乙酰胆碱(ACh)的释放,该乙酰胆碱是结合突触后肌肉纤维上的烟碱离子型ACh受体(nAChR)的神经递质。 ACh与nAChRs结合后,突触后动作电位沿着肌纤维向外扩散,从肌浆网释放Ca2 +释放入胞质溶胶,从而诱导肌肉收缩。与中枢突触不同,NMJ不受像血脑屏障或血液神经屏障之类的解剖障碍的保护,并暴露于各种致病分子的作用,包括化学物质,来自植物,动物和细菌的毒素以及自身抗体针对NMJ抗原产生。根据它们的作用方式,这些制剂对NMJ产生不同的损伤,最终导致肌肉收缩受损。

关键字:电生理, 神经肌肉接头, 偏侧膈分析, 膈神经, 毒素, 肉毒杆菌, 抑制剂

材料和试剂

  1. 2μl微量移液器
  2. 200μl微量移液器
  3. 1,000μl微量移液器
  4. 2,200和1,000微升微量移液器的技巧
  5. 用Sylgard(Dow Corning,Sylgard 184 Silicone Elastomer试剂盒)包被的培养皿,100×20mm
  6. 外科手术针(Rudolf,目录号:RU 5899-01)
  7. 棉线
  8. 所需基因型和年龄的小鼠
  9. 氯化氢(HCl)(Sigma-Aldrich,目录号:H1758)
  10. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  11. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  12. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  14. 氯化镁,标准溶液1M(MgCl 2)(Honeywell International,Fluka,目录号:63020)
  15. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C5080)
  16. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G7528)
  17. 林格的解决方案(见食谱)

设备

  1. 容量瓶
  2. Clamp(Bulldog Clamp,Diethrich)(Rudolf,目录号:RU 3934-16;或类似)
  3. 2把剪刀(精密手术剪刀)(Rudolf,目录号:RU 1503-12)
  4. 2把镊子(微型珠宝镊子,弯曲)(Rudolf,目录号:RU 4240-07)
  5. 刺激器:6002刺激器(哈佛仪器,型号:6002基本刺激器)
  6. 2×显微操作器(三维粗手动操作器)(NARISHIGE,目录号:M-152)
  7. 2 x张力传感器:等轴传感器(Harvard Apparatus,目录号:72-4494)
  8. 2 x前置螺丝定位器(Harvard Apparatus,目录号:53-2082W)
  9. 2个支持系统(用于容纳刺激室和换能器传感器)
  10. 2×静电刺激室(夹套,总内容积5毫升)
  11. 恒温浴(Isco,目录号:GTR190)
  12. 录音单元:i-WORX 118系统(iWORX,型号:iWORX 118)
  13. 计算机与软件兼容
  14. 装有压力控制器的空气罐(95%O 2,5%CO 2)(液化空气,型号:HBS 240-1-2)

软件

  1. 记录和分析:i-WORX 118系统(Dover,NH,USA)通过Labscribe软件(iWorx Systems Inc.,多佛尔,新罕布什尔州,美国)

程序

  1. 解决方案和设置准备
    1. 准备林格溶液(配方1),并通过95%O 2,5%CO 2通气至少15分钟使其饱和,以将pH缓冲至7.4。通气过程中,加入1 mg / ml葡萄糖(完全林格溶液)。
      注意:一次标准实验需要约50 ml的溶液。
    2. 打开设置的所有组件并运行Labscribe软件(参见图1)。


      图1.电生理设置组件。 A.设置概述。 1)肌肉室; 2)刺激器; 3)张力传感器; 4)数据采集系统; 5)带有Labscribe软件的计算机系统。 B.肌肉腔室细节。 6)支持系统; 7)静电刺激室; 8)用氧气插管肌肉支持; 9)微操纵器; 10)铂电极; 11)螺丝定位器; 12)传感器传感器。

  2. 解剖
    1. 安乐死小鼠,最好是颈椎脱臼。
      备注:
      1. 所有程序均按照意大利法律和政策(2014年3月14日第26号法令)执行,其中欧洲共同体委员会指令no 2010/63 / UE制定的指南以及帕多瓦(OPBA-Organismo Preposto al Benessere degli Animali)(议定书359/2015)。所有的程序应该根据进行实验的机构的道德标准来使用。
      2. 可以进行断头放血,以避免肋骨内出血和凝块形成。
    2. 将鼠标以仰卧位固定在支架上,取出胸部和腹部的皮肤,打开肋骨下的腹膜。

    3. 删除胸肌以暴露肋骨。
    4. 用夹子牢牢固定胸骨的下肢,并在其上做一个中心切口,大约在第二根肋骨的水平面上;继续切割胸廓的右侧和左侧,使隔膜暴露(见图2A)。
      注意:在切割时,不要按照肋骨的走向,而要切碎它们。

    5. 完全移除肋骨的上部(见图2B)。


      图2.用于隔膜移植的肋骨开口

    6. 轻轻地移开心脏和肺叶以发现左侧膈神经(它形成白色的弓,见图3A)。使用镊子,将棉线插入神经下方,上部并做两个结。小心地切开结的背后的神经;抓住线(避免可能导致神经损伤的过度拉伸),尽可能隔离除去附着组织的神经;建议从结节开始,轻轻向膈肌进行。已清除的神经更容易安装和刺激,但这需要谨慎处理以避免神经伸展或损伤,特别是靠近神经插入点进入肌肉。轻轻地将孤立的神经放在肌肉上(见图3B和3C)。对右侧膈神经重复这些操作。注意:右侧神经与血管非常接近,血管的损伤可能导致恼人的出血(见图3D和3E)。这些步骤的结果如图3F所示。


      图3.两个膈神经的分离和结扎程序。 D:隔膜; P:膈神经。白色箭头:结位置。白色虚线:膈神经的位置。红色虚线:沿右侧膈神经的血管。 A中的黄色虚线:整个膈肌区域。

    7. 使用胸骨下部保持膈肌,以避免损伤肌肉纤维;去除粘附在隔膜腹侧的结缔组织(图4A中的箭头)。通过切割肌肉的背部部分(图4B中的箭头)来移除隔膜。
      注意:密切注意斑点神经插入点并避免它们分裂。


      图4.膈肌移植

    8. 将制剂转移到培养皿(直径10厘米)中,用sylgard制成底部并装满5-10ml完全林格溶液;钉住肌肉,如图5A所示。
    9. 通过沿着图5A中所示的虚线切割并且用胸骨附件丢弃中心楔,将整个肌肉分成两个半膈,每个半膈都由其自己的膈神经支配。这个处理的结果是两个三角形,其中之一显示在图5B中。
      注:通过目前的解剖程序,获得两个半中膜,允许两个平行但独立的分析,这是一个显着的优势。
    10. 在三角形的顶端系上一根棉线,将制剂连接到换能器上,在不钻组织的情况下制作双结。使用三角形底部的肋骨将准备工作固定到支撑上:在肋骨之间钻取组织,穿过棉线并在下肋条周围形成双结(图5B)。
      定位支架并通过支架上的双结系住三角形的底部(图5C)。


      图5.半隔膜分离和安装在纸巾架上

  3. 神经刺激和采集
    1. 非常小心地将制剂转移到刺激室中(在37℃下用4ml完全林格溶液填充)。设置O 2 压力使气体连续但温和地起泡。用三角结把三角形顶端的线固定到传感器传感器上(图6A)。
      注意:
      1. 对于完整的力传导,肌肉必须垂直于传感器传感器的轴线(尽可能多的垂直线);螺纹与设置的其他部分接触可能会改变测量结果并且必须避免(图6B)。肌肉抽动由等距传感器记录,该传感器将电信号中的机械力转换成由Labscribe软件显示的数字信号。
      2. 通过向换能器施加已知值的权重并记录相应的伏特值,可以建立校准曲线;这个程序允许表示牛顿肌肉收缩力(或克)。当结果作为标准值提供时,测量单位无关紧要(见下文)。

    2. 使用丝杠定位器(图6A)拉伸肌肉,直到其张力达到约0.2 V(静止张力)。
    3. 将连接到膈神经的棉线穿过刺激电极的两个铂环(图6C中的箭头)。使用显微操作器,小心地移动刺激室内的电极。拉动棉线,直到膈神经与环接触(图6C)。
      注意:神经必须完全浸入溶液中,理想的是垂直于肌肉,避免过度紧张。电极不能与肌肉接触,以防止直接刺激肌肉 s。


      图6.实验装置的细节 A.打结棉线的传感器传感器。星号表示螺丝定位器。 B.棉线(平行于白色虚线)和半隔膜之间的连接。 C.室内膈神经的最终定位。黑色箭头:电极环神经通过并接触它。

    4. 设置膈神经刺激的标准参数。幅度:5 V(超最大刺激);脉冲宽度:0.1毫秒;频率:0.1Hz。开始肌肉抽搐的刺激和测试。
      注意:在这些情况下,膈肌肌肉准备可以抽搐至少4小时。
    5. 如果肌肉正常收缩,请使用螺丝定位器调整最适合抽搐反应的张力。缓慢增加肌肉张力反复次数,交替10-15分钟的肌肉稳定期,直到增加静息张力,抽搐幅度不再增加。
      最佳静息张力约为0.3 V.
    6. 让肌肉抽搐在开始实验之前或在加入治疗前在37°C稳定20分钟。
      注意:在这段时间结束时测量的平均抽搐幅度用于标准化治疗后的抽搐值。
    7. 将治疗直接加入刺激室。
    8. 使用带有Labscribe软件的i-WORX 118系统,记录所需时间的肌肉抽搐,或者直到肌肉力量完全消失。

数据分析

  1. 抽搐幅度计算为刺激后的张力峰值(图7中的αn)与刺激前的平均基础张力(时间间隔bn中的静息张力的平均值图7中的)。在标准条件下,该值至少保持4小时。


    图7. Labscribe软件在数据记录过程中显示的典型痕迹

  2. 通过添加冲击神经肌肉传输(突触前或突触后间隔)的治疗,颤搐振幅随时间下降(图8A)。在完全瘫痪的情况下,麻痹半时间(T 50%)被定义为将颤搐幅度减小到初始值的50%所需的时间(图8B),通常用于评估治疗的效力。


    图8.使用MPN与肉毒杆菌神经毒素血清型A进行的典型实验。红色箭头:加入毒素(200pM),假定时间= 0. A.用Labscribe软件记录的原始数据;在如图7中计算的从-10到0'的时间间隔(T min)中颤搐幅度的平均值被用于归一化随后的幅度值。 B.相对于时间函数的标准化颤搐幅度图和麻痹性半衰期(T 50%,黑色箭头)的计算。

  3. 对于比较研究(例如突变体毒素与野生型毒素),建议使用从同一动物收获的两个半膈进行平行实验以减少变异性,并将数据报告为配对观察值。或者,可以将毒素作为参照进行效价校准曲线,然后用于推断其他毒素批次或变体的相对效力(Bigalke and Rummel,2015)
  4. 不同的毒素(或其他物质)可能需要各种实验条件(Caccin等人,2006年)。
    注:使用附加电极(未显示)可以检测肌肉纤维功能,以直接刺激肌肉。

食谱

  1. 林格的解决方案

    备注:
    1. 使用ddH 2 O。玻璃器皿必须用0.1N氯化氢洗涤以除去任何痕量碳酸盐,并用ddH 2 O em冲洗。存在任何沉积物或乳光时,请勿使用溶液。
    2. 所使用的缓冲区是一种定制的林格解决方案;在整个协议中,为简单起见,它被表示为“Ringer's”。
    3. 在分析之前,林格氏溶液被95%O饱和,5%CO 2 通过通气至少15分钟以获得7.4的pH并补充有葡萄糖(终浓度1mg / ml,11mM)(氧饱和度和葡萄糖补充后的缓冲液在这个协议中被表示为'完整的林格溶液')。葡萄糖对于记录时间超过1小时是必需的。

致谢

这项工作得到了帕多瓦大学的支持,其中'帕多瓦大学未就业青年高级研究资助'授予M. Pirazzini,并与S. Negro授予“初级研究员”,Fondazione Caritro授予'Bando 2017每Giovani ricercatori coinvolti in progetti di eccellenza'授予G. Zanetti。所有程序均在由生物医学科学系(帕多瓦大学)Cesare Montecucco教授领导的'神经毒素,神经麻痹和再生'实验室中进行。作者声明没有竞争利益。 PC进行半膈肌制备的程序。 SN和GZ拍照描述所有的程序并准备好数字。 GZ和PC在SN和MP的帮助下写了这篇文章。所有作者都回顾了手稿并批准了最终版本。

参考

  1. Azarnia德黑兰,D.,Zanetti,G.,Leka,O.,Lista,F.,Fillo,S.,Binz,T.,Shone,CC,Rossetto,O.,Montecucco,C.,Paradisi,C., Mattarei,A.和Pirazzini,M.(2015)。 一种新型抑制剂可预防肉毒杆菌神经毒素的外周神经麻痹。 5:17513.
  2. Bercsenyi,K.,Schmieg,N.,Bryson,JB,Wallace,M.,Caccin,P.,Golding,M.,Zanotti,G.,Greensmith,L.,Nischt,R.和Schiavo,G。(2014年)。 Nidogens是预防破伤风的治疗目标。 科学 > 346(6213):1118-1123。
  3. Beske,P.H.,Bradford,A.B.,Hoffman,K.M.,Mason,S.J。和McNutt,P.M。(2017)。 体外和 ex vivo 筛选候选治疗剂可恢复由肉毒杆菌神经毒素血清型A1导致的神经末梢神经传递。 Toxicon 。 doi:10.1016 / j.toxicon.2017.10.017。
  4. Bigalke,H.和Rummel,A。(2015)。 肉毒神经毒素:使用鼠标膈神经偏侧膈测定(MPN) 毒素(巴塞尔) 7(12):4895-4905。
  5. Caccin,P.,Rigoni,M.,Bisceglie,A.,Rossetto,O。和Montecucco,C。(2006)。 不同溶血磷脂诱导的可逆性骨骼肌肉麻痹 FEBS Lett 580(27):6317-6321。
  6. Caccin,P.,Scorzeto,M.,Damiano,N.,Marin,O.,Megighian,A.和Montecucco,C.(2015年)。 突触前膜近膜区涉及神经细胞增生 FEBS Open Bio 5:388-396。
  7. Klooster,R.,Plomp,JJ,Huijbers,MG,Niks,EH,Straasheijm,KR,Detmers,FJ,Hermans,PW,Sleijpen,K.,Verrips,A.,Losen,M.,Martinez-Martinez,P. ,De Baets,MH,van der Maarel,SM和Verschuuren,JJ(2012)。 肌肉特异性激酶重症肌无力IgG4自身抗体在小鼠中引起严重的神经肌肉接头功能障碍 Brain 135(Pt 4):1081-1101。
  8. Li,L.,Xiong,W.C.和Mei,L。(2017)。 神经肌肉接头的形成,衰老和障碍 Annu Rev Physiol 。
  9. Nascimento,F.,Pousinha,P. A.,Correia,A. M.,Gomes,R.,Sebastiao,A. M.和Ribeiro,J. A.(2014)。 腺苷A2A受体激活促进SOD1(G93A)ALS小鼠的症状前阶段的神经肌肉传递,但不是在症状阶段。 PLoS One 9(8):e104081。
  10. Rasetti-Escargueil,C.,Liu,Y.,Rigsby,P.,Jones,R.G和Sesardic,D。(2011)。 膈神经 - 膈肌作为测定功能性肉毒杆菌毒素抗体的高度敏感的替代测定。 Toxicon 57(7-8):1008-1016。
  11. Rigoni,M.,Caccin,P.,Gschmeissner,S.,Koster,G.,Postle,A. D.,Rossetto,O.,Schiavo,G。和Montecucco,C。(2005)。 蛇PLA2神经毒素和溶血磷脂脂肪酸混合物的等效效应 科学 310(5754):1678-1680。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zanetti, G., Negro, S., Pirazzini, M. and Caccin, P. (2018). Mouse Phrenic Nerve Hemidiaphragm Assay (MPN). Bio-protocol 8(5): e2759. DOI: 10.21769/BioProtoc.2759.
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