Medir la funcionalidad de la placa neuromuscular

Resumen

Una evaluación funcional de la placa neuromuscular (NMJ) puede proporcionar información esencial sobre la comunicación entre el músculo y del nervio. Aquí se describe un protocolo para evaluar exhaustivamente el NMJ y músculo funcionalidad utilizando dos preparaciones diferentes músculo-nerviosa, es decir, sóleo-ciático y frénico-diafragma.

Resumen

Funcionalidad de la placa neuromuscular (NMJ) desempeña un papel fundamental al estudiar enfermedades en que la comunicación entre neurona motora y músculo se deteriora, como el envejecimiento y esclerosis lateral amiotrófica (ELA). Aquí describimos un protocolo experimental que puede utilizarse para medir funcionalidad NMJ combinando dos tipos de estimulación eléctrica: dirigir la estimulación de la membrana muscular y la estimulación a través del nervio. La comparación de la respuesta del músculo a estos dos estímulos diferentes puede ayudar a definir, a nivel funcional, posibles alteraciones en el NMJ que conducen a deterioro funcional en el músculo.

Preparaciones dex vivo E son adecuadas para estudios bien controlados. Aquí describimos un protocolo intensivo para medir varios parámetros de funcionalidad NMJ para la preparación del nervio ciático en sóleo y para la preparación del nervio frénico-diafragma y músculo. El protocolo dura aproximadamente 60 minutos y se lleva a cabo ininterrumpidamente por medio de una medida software que mide las propiedades de la cinética de contracción, la relación de la frecuencia de la fuerza para estimulación de músculo y nervio y dos parámetros específicos a la funcionalidad de la NMJ, es decir falta de neurotransmisión y fatiga intratetanic. Esta metodología fue utilizada para detectar daños en preparaciones de nervio Músculo sóleo y el diafragma mediante el uso de ratón transgénico de SOD1^G93A , un modelo experimental de esclerosis lateral amiotrófica que ubicuo overexpresses del mutante antioxidante enzima superóxido dismutasa 1 (SOD1).

Introducción

La Unión neuromuscular (NMJ) es una sinapsis química formada por la conexión entre la placa motora de la fibra muscular y el axón de la neurona motora terminal. El NMJ ha demostrado jugar un papel crucial cuando la comunicación entre el músculo y el nervio se deteriora, como ocurre en el envejecimiento o la esclerosis lateral amiotrófica (ELA). Como músculo y del nervio se comunican en forma bidireccional^1,2, pudiendo medir NMJ defectos por separado de defectos músculo puede proporcionar nuevas perspectivas sobre su interrelación fisiopatológica. De hecho, esta evaluación funcional puede ayudar a evaluar si alteraciones morfológicas o bioquímicas reducen la funcionalidad de señalización de la neurotransmisión.

La comparación de la respuesta contráctil muscular provocada por estimulación del nervio y la respuesta del músculo mismo evocado por estimulación directa de su membrana se ha propuesto como una medida indirecta de la funcionalidad de la NMJ. De hecho, desde membrana estímulo por pases neurotransmisión señalización, cualquier diferencia en las dos respuestas contráctiles puede atribuirse a los cambios en el NMJ. Este enfoque ha sido ampliamente propuesto para las ratas^3,4,5,6,7y también se utiliza para recopilar información sobre los modelos de ratón^8,9,10,11,12.

Aquí, describimos en detalle un procedimiento para suprimir dos preparaciones de músculo-nerviosa, e. i. los preparativos sóleo-ciático y frénico-diafragma. Mediante una medida software, diseñamos un protocolo de pruebas continuado que combina la medición de varios parámetros que caracterizan la funcionalidad de NMJ y músculo, lo que rinde una evaluación comprensiva del daño NMJ por separado del músculo. En particular, el protocolo mide la fuerza de contracción, la cinética muscular, la curva de frecuencia de la fuerza directa y estímulos del nervio, la neurotransmisión falta¹³ para un disparo y las frecuencias tetánicas y la fatiga intratetanic⁷.

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Protocolo

All the animal experiments were approved by the ethics committee of Sapienza University of Rome-Unit of Histology and Medical Embryology and were performed in accordance with the current version of the Italian Law on the Protection of Animals.

1. Experimental set-up

1. Set-up the experimental system composed of 1 actuator/transducer, 2 stimulators, 1 in-vitro muscle apparatus, 1 preparatory tissue bath, 1 suction electrode, 1 digital oscilloscope, 1 stereomicroscope, 1 cold light illuminator, 1 acquisition board, 1 connector block and a personal computer.
   NOTE: Here the command software was programmed in LabView and was designed to guarantee high flexibility, accuracy, and repeatability of the stimulations. For each stimulation, the user can select the pulse width, frequency, duration, and decide whether it is delivered to the membrane or through the nerve. The user can also select the length of the rest period before each stimulation.

2. Evaluations of the NMJ contractile properties of soleus and diaphragm muscles

1. System warm-up
   1. Prepare the Krebs Ringer buffer¹⁴ and place it in a bath of the mounting apparatus and the preparatory tissue bath. Place a 60 mm dish prepared with 4 ml of silicon in the preparatory tissue bath.
   2. Turn on the circulating water bath and set the temperature to 30 °C for the soleus muscle and to 27 °C for the diaphragm muscle.
   3. Allow 95% O₂ - 5% CO₂ mixture to flow into both the baths.
   4. Turn on the actuator/transducer and the two pulse stimulators and set the current values to 300 mA for membrane stimulation and to 5 mA for nerve stimulation.
      NOTE: The 300 mA current value has previously been shown not to induce any significant contraction on the nerve branches when D-tubocurarine is added to the solution¹⁵. The 5 mA current value corresponds to the amount of current required to stimulate the muscle through the nerve without creating an overlap with the membrane stimulation induced by means of the solution. Since the distance between the suction electrode and the muscle is dependent on the nerve length, this current value must be carefully adjusted before each experiment, as described below.
2. Soleus and diaphragm dissection
   1. Sacrifice the mouse by cervical dislocation to minimize suffering, and immediately excise the muscles for testing as follows.
   2. Soleus-sciatic preparation, dissection, and set-up
      1. Spray 70% ethanol on the mouse leg and remove the skin covering the leg. Make an incision in the upper part of the leg with a pair of scissors and then pull the skin firmly. Isolate the Achilles' tendon from the tibia and then cut the Achilles' tendon with a pair of scissors.
      2. Tightly clamp the tendon with a pair of forceps and gently pull the gastrocnemius muscle together with the soleus. Once the proximal tendon of the soleus is exposed, cut the entire calf with a pair of scissors while still holding the Achilles' tendon. Immediately place the tissue sample in the preparatory tissue bath.
      3. Place the preparatory bath under the stereomicroscope and fix the calf to the silicon dish using 0.20 mm diameter stainless steel pins. Using a pair of forceps clamp the proximal tendon of the soleus and gently pull it to expose the sciatic innervation.
      4. Once the innervation is exposed (Figure 1), carefully remove the surrounding tissue to expose about 5 mm of the nerve. Use a fine pair of scissors to cut the nerve. While tightly clamping the soleus on the proximal tendon with a pair of forceps, pull it again and excise it by cutting the Achilles' tendon with a pair of scissors.
      5. Pass a nylon wire through the hole of the lever arm of the actuator/transducer. Create a noose at the end of the wire and grasp the Achilles' tendon before pulling the wire back until the muscle reaches the bath of the mounting apparatus. Clamp the proximal tendon within the fixed clamp and tie the nylon wire around the lever arm. Allow the muscle to equilibrate in the solution.
      6. Determine the initial optimal length (L₀).
         1. First, stretch the muscle to preload it at 10 mN (this value guarantees the maximum twitch force (based on experience)). Then, gently stretch the muscle to change the preload and stimulate it with a series of single pulses. The optimal length is achieved when the maximum twitch force is measured.
   3. Diaphragm-phrenic preparation, dissection, and set-up
      1. Spray 70% ethanol on the mouse skin. Remove the skin covering the diaphragm muscle by making an incision over the sternum with a pair of scissors and by pulling the skin firmly.
      2. With a pair of scissors, cut the abdomen horizontally below the sternum and gently remove the intestine and the organs with a pair of forceps. Use a pair of thick scissors to cut the spine.
      3. Cut the ribs about 1 cm over the sternum and proceed horizontally. Gently remove the outer organs with a pair of forceps and cut the spine to obtain a circular ring of ribs containing the diaphragm.
      4. Wash the preparation in a dish containing the Krebs Ringer buffer and then place it in the silicon dish inside the preparatory tissue bath, with the upper side facing up and the sternum facing forward.
      5. Using the stereomicroscope, carefully remove the lungs and the remaining organs; the phrenic nerve will be visible on the right side (Figure 2A).
      6. Trim the ribs to leave a ring of about 2 mm all along the diaphragm.
      7. Using forceps, fix one 0.20 mm diameter stainless steel pin on the diaphragm central tendon at the point corresponding to the phrenic nerve innervation, and another pin on the ribs on the same axis to identify the diaphragm strip of interest.
      8. Fix two additional pins on the central tendon and another two pins on the ribs on either side of the selected strip to lay out the diaphragm. If necessary, clean the phrenic nerve from the surrounding connective tissue using forceps.
      9. Using a curved blade, cut the diaphragm on either sides of the innervation to identify a tendon-muscle-rib strip as shown in Figure 2B.
      10. Pass a nylon wire through the hole of the lever arm of the actuator/transducer. Create a noose at the end of the wire and grasp the tendon before pulling the wire back until the muscle reaches the bath of the mounting apparatus.
      11. Clamp the ribs within the fixed clamp and tie the nylon wire around the lever arm.
      12. Allow the muscle to equilibrate in the solution and determine the initial optimal length (L0).
          1. Stretch the muscle to preload it at 5 mN. Gently stretch the muscle to change the preload and stimulate it with a series of single pulses. The optimal length is achieved when the maximum twitch force is measured.

Figure 1 - Soleus-sciatic nerve preparation. Soleus-sciatic nerve during surgical operation for the functional tests. The sciatic is exposed using a pair of forceps. Please click here to view a larger version of this figure.

Figure 2 - Diaphragm-phrenic nerve preparation. Picture shows a phase of the diaphram-phrenic nerve preparation excision (A) and the strip to be mounted for functional tests (B). Please click here to view a larger version of this figure.

3. Measuring NMJ properties separately from muscle contractility
   1. Set the best current intensity for the stimulation trough the nerve and verify that the current pulses delivered through the suction electrode do not elicit muscle contraction by the spread of current in the bath.
      1. Place the suction electrode near the muscle and pull the nerve in. While gently increasing the current intensity (starting from 5 mA), stimulate the muscle with a series of single pulses. The optimal current value is determined when the twitch force is maximum.
      2. Push the nerve out of the electrode and deliver a few single pulses. Ensure that the muscle does not contract due to overlap with the membrane stimulation by means of the solution. Pull the nerve in again.
   2. Launch the program and select the input file to run the desired testing protocol.
      NOTE: The input file includes all the stimulation parameters needed to run the entire protocol. Although the same protocol structure is used for the two muscles the stimulation parameters differ depending on the protocol refers to the soleus or diaphragm muscles.
      1. Soleus-sciatic nerve stimulation (see Table 1)
         1. Use the following pulse widths for all the electrical stimulations: 1.4 ms for sciatic nerve stimulation and 0.1 ms for direct soleus stimulation. The full protocol lasts approximately 65 min.
      2. Diaphragm-phrenic nerve stimulation (see Table 2)
         1. Use the following pulse widths for all the electrical stimulations: 1.8 ms for phrenic nerve stimulation and 0.2 ms for direct diaphragm stimulation. The full protocol lasts approximately 55 min.
      3. At the end of the experimental protocol, measure the length and the wet weight of the muscle using an analog caliper with an 0.05 mm accuracy and a precision scale with an 0.1 mg accuracy, respectively.
         1. Compute the cross sectional area (CSA) by dividing the muscle mass by the product of optimal fiber length (L_f) and the density of mammalian skeletal muscle (1.06 mg/mm³)¹⁶.
            NOTE: L_f is obtained by multiplying L₀ by the fiber length/muscle length ratio (0.71 for soleus muscle¹⁶ and 1 for the diaphragm muscle¹⁷).

Type of experiment	Frequency	Duration	Repetitions	Muscle or Nerve stimulation	Purpose
	(Hz)	(s)
Twitch	Single pulse		1	Muscle	Twitch force and kinetics
Rest		30
Twitch	Single pulse		1	Nerve
Rest		30
Twitch	Single pulse		1	Muscle
Rest		30
Twitch	Single pulse		1	Nerve
Rest		120
Unfused tetanus	40	0.8	1	Nerve	Force/frequency curves for nerve and muscle stimulations
Rest		180
Unfused tetanus	60	0.8	1	Muscle
Rest		180
Fused tetanus	80	0.8	1	Nerve
Rest		180
Unfused tetanus	20	0.8	1	Muscle
Rest		180
Unfused tetanus	60	0.8	1	Nerve
Rest		180
Fused tetanus	80	0.8	1	Muscle
Rest		180
Unfused tetanus	20	0.8	1	Nerve
Rest		180
Unfused tetanus	40	0.8	1	Muscle
Rest		300
Fatigue paradigm	35	0.8	1 muscle stimulation followed by 14 nerve stimulations with a rest time of 1.2 s each, repeated 20 times		Neurotransmission failure (NF)
Rest		900
Fatigue paradigm	80	0.8	1 muscle stimulation followed by 14 nerve stimulations with a rest time of 1.2 s each, repeated 20 times		Neurotransmission failure (NF) and Intratetanic fatigue (IF)

Table 1 - Soleus-sciatic nerve stimulation protocol. The table lists the sequence of tests that form the complete protocol for testing soleus-sciatic nerve preparations.

Type of experiment	Frequency	Duration	Repetitions	Muscle or Nerve stimulation	Purpose
	(Hz)	(s)
Twitch	Single pulse		1	Muscle	Twitch force and kinetics
Rest		30
Twitch	Single pulse		1	Nerve
Rest		30
Twitch	Single pulse		1	Muscle
Rest		30
Twitch	Single pulse		1	Nerve
Rest		120
Unfused tetanus	60	0.5	1	Nerve	Force/frequency curves for nerve and muscle stimulations
Rest		120
Fused tetanus	100	0.5	1	Muscle
Rest		180
Unfused tetanus	40	0.5	1	Nerve
Rest		120
Unfused tetanus	20	0.5	1	Muscle
Rest		120
Unfused tetanus	80	0.5	1	Nerve
Rest		150
Unfused tetanus	80	0.5	1	Muscle
Rest		150
Unfused tetanus	20	0.5	1	Nerve
Rest		120
Fused tetanus	100	0.5	1	Muscle
Rest		150
Fused tetanus	100	0.5	1	Nerve
Rest		180
Unfused tetanus	60	0.5	1	Muscle
Rest		300
Fatigue paradigm	35	0.33	1 muscle stimulation followed by 14 nerve stimulations with a rest time of 0.67 s each, repeated 20 times		Neurotransmission failure (NF)
Rest		900
Fatigue paradigm	80	0.33	1 muscle stimulation followed by 14 nerve stimulations with a rest time of 0.67 s each, repeated 20 times		Neurotransmission failure (NF) and Intratetanic fatigue (IF)

Table 2 - Diaphragm-phrenic nerve stimulation protocol. The table lists the sequence of tests that form the complete protocol for testing diaphragm-phrenic nerve preparations.

3. Data analysis

NOTE: At the end of the protocol compute all the desired parameters as follow.

1. From single pulse stimulation
   1. Compute twitch force (mN; maximum active force generated during twitch) and specific twitch force (mN/mm²). Compute it by subtracting the initial preload value to the maximum force generated by the muscle. Compute the specific twitch force as twitch force divided by the muscle CSA.
   2. Compute time to peak tension (TTP) (ms) as the time from pulse release to twitch force.
   3. Compute half relaxation time (1/2RT) (ms) as the time from twitch force to 50% of twitch force.
   4. Compute dF/dt (mN/ms) as the maximum value of force derivative during the contractile phase.
   5. Compute -dF/dt (mN/ms) as the maximum value of force derivative during the relaxing phase.
2. From pulse train stimulations
   1. Compute unfused force (mN) and specific unfused force (mN/mm²). Compute it by subtracting the initial preload value to the maximum force generated by the muscle. Compute the specific unfused force as the unfused force divided by the muscle CSA.
      NOTE: The unfused force is the maximum active force generated during a pulse train with a frequency lower than the tetanic frequency.
   2. Compute tetanic force (mN) and specific tetanic force (mN/mm2); the maximum force is the maximum active force generated during a pulse train delivered at tetanic frequency. Compute it by subtracting the initial preload value to the maximum force generated by the muscle. Compute the specific tetanic force as the tetanic force divided by the muscle CSA.
   3. Compute force-frequency curve. Plot the value of force (or specific force) obtained for each stimulation versus the increasing frequency of stimulation. Typically, it begins with the single pulse stimulation and ends up with the tetanic frequency.
3. From fatigue paradigms
   1. Calculate neurotransmission failure (NF) as:
      
      where MF is the force decrease following muscle stimulation and F is the force decrease following nerve stimulation, measured on the first pulse train after muscle stimulation.
   2. Calculate intratetanic fatigue (IF) as:
      
      where F_lp is the force generated by the muscle at the last pulse of stimulation, and F_m is the maximum force generated by the muscle during the same pulse train. Regarding to the nerve stimulation, compute the intratetanic fatigue on the first pulse train immediately after the direct stimulation.

4. Statistical analysis

NOTE: The statistical analysis models must be chosen according to whether muscle response to both nerve and membrane stimulations is compared within the same animal model or between 2 different animal models^18,19.

1. Use the Student's t test for TTP, 1/2RT, dF/dt, -dF/dt if comparing only muscle responses to direct and indirect stimulations. If comparing muscle preparations obtained from 2 different mouse strains, use 2-way ANOVA with stimulation type and mouse strain as fixed factors. When the 2-way ANOVA returns a significant outcome, use multiple comparison tests to search for statistical differences.
2. For force-frequency curve (if comparing only muscle responses to direct and indirect stimulations), use 2-way ANOVA with stimulation type and frequency of stimulation as fixed factors. Use multiple comparison tests if required.
   1. If comparing muscle preparations obtained from 2 different mouse strains, use 3-way ANOVA with stimulation type, frequency of stimulation and mouse strain as fixed factors.
      1. When the 3-way ANOVA yields a significant effect of the factors animal strain and stimulation type, use 2-way ANOVA to identify significant differences between 2 groups at a time. For example, differences between the muscle response of the 2 groups to membrane stimulation, or between muscle response to direct and indirect stimulations within one group can be assessed.
3. Intratetanic fatigue
   NOTE: Since the protocol was designed to induce fatigue in response to nerve stimulation even in control mice, this parameter can only be used to investigate differences between 2 mouse strains, and a statistical analysis within a single group would always yield significant differences.
   1. To compare two groups, use 3-way ANOVA with stimulation type, time, and mouse strain as fixed factors. When the 3-way ANOVA yields a significant effect of the factors animal strain and stimulation type, use 2-way ANOVA to identify significant differences between 2 groups at a time, as for the force-frequency curve.
4. For neurotransmission failure, use 2-way ANOVA with animal strain and time as fixed factors. If it returns significant outcomes, use multiple comparison tests to search for statistical differences.
   NOTE: Since this parameter only returns one value for each mouse strain, it allows different mouse models to be compared.
5. For muscle weight, length, and CSA, use Student's t test to investigate differences when comparing muscle-nerve preparations from different animal models.

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Resultados

El protocolo que se describe proporciona información sobre la denervación funcional en varias enfermedades neuromusculares o envejecimiento-sarcopenia. Este protocolo puede utilizarse para determinar si (y, en caso afirmativo, en qué nivel) alteraciones de la musculatura se deben a cambios selectivos que ocurren en el músculo sí mismo o en la transmisión neuromuscular. Los datos que se muestran a continuación son los resultados de un trabajo previo por nuestro grupo

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Discusión

El protocolo experimental descrito proporciona una manera ideal de medir y discriminar alteraciones funcionales que se han producido directamente en el músculo o indirectamente en el nivel de placa neuromuscular. Puesto que esta técnica se basa en una medición indirecta de la funcionalidad de la NMJ, no puede utilizarse para establecer si un defecto está relacionado con cambios morfológicos o a cambios bioquímicos. Por el contrario, proporciona una manera eficaz de determinar si las alteraciones morfológicas o bio...

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Materiales

Name	Company	Catalog Number	Comments
Dual-Mode Lever System	Aurora Scientific Inc.	300B	actuator/transducer
High-Power Bi-Phase Stimulator	Aurora Scientific Inc.	701B	pulse stimulator (nerve)
High-Power Bi-Phase Stimulator	Aurora Scientific Inc.	701C	pulse stimulator (muscle)
In vitro Muscle Apparatus	Aurora Scientific Inc.	800A
Preparatory tissue bath	Radnoti	158400
Monopolar Suction Electrode	A-M Systems	573000	with a home-made reference
Oscilloscope	Tektronix	TDS2014
Stereomicroscope	Nikon	SMZ 800
Cold light illuminator	Photonic Optics	PL 3000
Acquisition board	National Instruments	NI PCIe-6353
Connector block	National Instruments	NI 2110
Personal computer	AMD Phenom II x4 970		Processor 3.50 Ghz with Windows 7
LabView 2012 software	National Instruments
Krebs-Ringer Bicarbonate Buffer	Sigma-Aldrich	K4002	physiological buffer
Sodium bicarbonate	Sigma-Aldrich	S5761
Calcium chloride CaCl2	Sigma-Aldrich	C4901	anhydrous, powder, ≥97%
Buffer HEPES	Sigma-Aldrich	H3375	≥99.5% (titration)
Dishes 60mm x 15mm	Falcon	353004	Polystyrene
Silicone	Sylgard	184 Silicone	Elastomer Kit  0.5Kg.
Thermostat	Dennerle	DigitalDuomat 1200
Pump	Newa Mini	MN 606	for aquarium
Heat resistance Thermocable	Lucky Reptile	61403-1	50/60Hz 50W
Bucket	any 10 liters		Polypropylene
O2 + 5%CO2	siad		Mix gas
#5 Forceps	Fine Science Tools	11252-20	2 items
Spring Scissors - 8 mm Blades	Fine Science Tools	15024-10	nerve excision
Sharp Scissors	Fine Science Tools	14059-11	muscle removal
Delicate Scissors	Wagner	02.06.32	external of the animal
Student Scalpel Handle #3	Fine Science Tools	91003-12
Scalpel Blades #10	Fine Science Tools	10010-00
Scalpel Blades #11	Fine Science Tools	10011-00
nylon wire Ø0.16 mm	any