WO2015135933A1 - Methods for treating respiratory failure in a subject afflicted with cervical spinal cord injury - Google Patents

Abstract

The present invention relates to methods for treating respiratory failure in a subject afflicted with cervical spinal cord injury. In particular, the present invention relates to a method for treating respiratory failure in a subject afflicted with cervical spinal cord injury comprising subjecting the subject to repetitive transcranial magnetic stimulation (rTMS) for a time sufficient to achieve an improvement of the subject's respiratory function.

Description

METHODS FOR TREATING RESPIRATORY FAILURE IN A SUBJECT AFFLICTED WITH CERVICAL SPINAL CORD INJURY

FIELD OF THE INVENTION:

The present invention relates to methods for treating respiratory failure in a subject afflicted with cervical spinal cord injury.

BACKGROUND OF THE INVENTION:

It is well known that pulmonary pathophysiology is one of the most significant factors associated with the morbidity and mortality of individuals afflicted with cervical spinal cord injury.

Diaphragm plays a fundamental role in ventilation, and bulbar motor control involving chemo reflexes seems to be sufficient to adapt diaphragm contractions to tidal ventilation and peripheral oxygenation demands. However, as any other skeletal muscle, diaphragm is involved in miscellaneous complex physiological activities such as speaking or singing, requiring voluntary respiratory muscle control. Cortical motor control has been demonstrated in several animal preparations (Planche 1972) as well as in humans (Gandevia and Rothwell 1987, Macefield and Gandevia 1991, Zifko et al 1996) by stimulating primary motor cortex electrically, and recording diaphragmatic motor responses. Although some questions concerning the relationship between cortical and bulbar motor controls are not totally elucidated (Davey et al. 1996), a descending corticospinal pathway seems to be dedicated to respiratory phrenic motoneuron pool (Lane 2011).

Studying diaphragmatic motor evoked potential (MEP) by using transcranial magnetic stimulation (TMS) in humans has been proved to be relevant (Lissens 1994, Maskill et al. 1991, Khedr and Trakhan 2001) allowing an easy non- invasive and painless clinical tool in humans for studying diaphragmatic corticospinal pathway excitability. Moreover, different pathological circumstances in humans have reported that an impaired cortical drive could explain, at least partly, how deep ventilatory parameters were altered regardless of bronchopulmonary abnormalities (Hopkinson et al. 2012). More recent studies (Raux et al. 2010, Azabou et al. 2013) have also demonstrated that neuromodulation techniques (repetitive transcranial magnetic stimulation: rTMS and transcranial direct current stimulation: tDCS, respectively) could increase or decrease diaphragmatic motor cortex excitability as proved by change of diaphragmatic testing MEP amplitude in response to single pulse TMS. Nevertheless, few preclinical studies in rats using TMS and questioning neuronal plasticity as well excitability changes were published. Technical considerations have been put forward regarding the size of rat cortex by comparison with the human one. However, some authors (Luft et al. 2001, Salvador and Miranda 2009) have shown that using conventional coil in humans for clinical purpose could be satisfactory in rat studies, allowing MEP recordings at least from forelimbs (Rotenberg et al, 2013) as well as hind limb muscles. Moreover, unilateral stimulation seems possible since Rotenberg et al. (2013) have shown that lateralization of the coil on one side allowed to record contralateral MEPs without any detectable signal on the ipsilateral side. It seems that this was not possible from hind limb muscle as a result of a smaller cortical area of these muscles than those corresponding to forelimb ones (Kolb & Tees, 1990), and/or because of different intrinsic excitability properties of their motor interneurons related to different motor activities specialization.

Despite numerous publications reporting the effect of rTMS on different cortical and brainstem structures (Gersner et al. 201 1, Wang et al. 2011), to date no report of exploring rat diaphragmatic corticospinal pathways by using TMS is published.

SUMMARY OF THE INVENTION:

The present invention relates to methods for treating respiratory failure in a subject afflicted with cervical spinal cord injury. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Cervical spinal cord injury (CSCI) is responsible of locomotor impairment but also to loss of respiratory muscles control leading to respiratory failure. Spontaneous partial restoration of respiratory muscles function may occur in close relation with activation of normally silent crossing pathways at each cervical metameric pool of phrenic motoneurons. New therapeutic strategies enhancing activation of such facilitating connections would be an interesting way of respiratory function recovery. Repetititve transcranial magnetic stimulation (rTMS) is known to change corticospinal excitability and would be an interesting tool to improve phrenic motoneurons reinnervation in CSCI. The inventors have shown that a diaphragmatic motor evoked potential could be evoked by single pulse TMS providing a good anatomic and functional rationale for applying rTMS for the treatment of respiratory failure in a subject afflicted with cervical spinal cord injury. Thus a first object of the present invention relates to a method for treating respiratory failure in a subject afflicted with cervical spinal cord injury comprising subjecting the subject to repetitive transcranial magnetic stimulation (rTMS) for a time sufficient to achieve an improvement of the subject's respiratory function.

Typically said subject is a mammal including a non-primate (e.g., pig, horse, goat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, a subject is a human. In some embodiments, a subject is a human infant. In some embodiments, a subject is a human child. In some embodiments, a subject is a human adult. In some embodiments, a subject is an elderly human.

Typically, the method of the invention includes treating a subject with a treatment course which includes a plurality of (i.e., more than one) treatment regimens which consist of applying repetitive transcranial magnetic stimulation (rTMS) over a position on the subject's scalp corresponding to respiratory primary motor cortex area.

A treatment course according to the disclosed methods typically comprises a number of treatment regimens over a several week period. In some embodiments, a treatment course comprises at least two treatment regimens over the course of one week (7 day period), comprises at least three treatment regimens over the course of a week, comprises at least four treatment regimens over the course of a week, or comprises at least five treatment regimens over the course of a week. More treatments per week, including at least six treatments, at least seven treatments, at least eight treatments, at least nine treatments, at least ten treatments, at least twelve treatments, at least fourteen treatments, and at least sixteen treatments, are also included in the present invention. The higher number of treatments per week may result from more than one treatment per day. Accordingly, treatment regimens of the present invention include more than one treatment regimen per day. In some embodiments, two, three, four or even more treatments may be administered per day. The treatment course is, in some embodiments, organized into treatment regimens for a plurality of 7 day periods (weeks). In some embodiments, a treatment course include at least about two weeks, at least about three weeks, at least about four weeks, at least about six weeks, at least about eight weeks, at least about ten weeks, at least about twelve weeks, at least about fourteen weeks, at least about eighteen weeks, at least about twenty weeks, at least about twenty two weeks, at least about twenty four weeks, at least about twenty six weeks, at least about twenty eight weeks, at least about thirty weeks, at least about thirty two weeks, at least about thirty four weeks, and at least about thirty six weeks. In some embodiments the treatment course is about twelve weeks or more and about twenty four weeks or more. Treatment courses may be repeated either identical to the earlier treatment course or a new treatment course may be designed, depending on clinician assessment.

In some embodiments, a treatment course includes a first portion which will have a higher frequency of treatment regimens followed by a second portion which will have a lesser frequency of treatment regimens. Any variations of treatment courses which include a first portion and a second portion which result in the desired and/or maximum improvement in a cost effective manner can be desirable. In particular a treatment course which includes a first portion including at least about three treatment regimens per week for a period of weeks ranging from between about four weeks and about twelve weeks, followed by a second portion including two treatment regimens per week for a period of weeks ranging from between about four weeks and about twelve weeks. Another example consists of a treatment course which includes a treatment regimen which includes a first portion including at least about five treatments per week for a period of weeks ranging between about four weeks and about twelve weeks, followed by a second portion including two treatment regimens per week for a period of weeks ranging from between about four weeks and about twelve weeks. Generally speaking, the methods of the present invention can be re-initiated upon any decline in improvement noted or worsening of symptoms.

An rTMS system suitable for the present invention includes TMS units made by a number of manufacturers and include the MAGSTIM rapid stimulator connected to four booster modules (Magstim Company Ltd, Whitland, U.K.); the MAGSTIM 200 and Magstim QuadroPulse Model 500 made by the same manufacturer, and the MAGPRO stimulator such as MAGPRO XI 00 (Magventure, Farum, Denmark). The principles underlying TMS are well known. Briefly, a time varying current in a primary circuit (the coil) will induce an electric field and thereby a current flow in the brain. The interaction is mediated by the magnetic field generated by the changing current in the coil. At a microscopic level, the electric field affects the transmembrane potential, which may lead to local membrane depolarization and subsequent neural activation and/or inhibition. rTMS is known to either activate or suppress motor or sensory function, depending on the brain location for that motor or sensory function and parameters of rTMS delivery.

Macroscopic responses to rTMS can be detected with functional imaging tools such as electroencephalography, positron emission tomography, functional magnetic resonance imaging, motor electron potentials, or clinical changes. A more detailed discussion of an exemplary rTMS unit is contained within, for example, U.S. Patent Application Publication No. 20050154426; and U.S. Patent Application Publication No. 20050256539; all of which are incorporated by reference herein in their entireties. Generally, the circuit used consists of a discharge capacitor connected with the coil in series by a thyristor. The capacitor is initially charged to 2-3 kV then discharged through the coil as the gating of the thyristor converts to the conducting state. The current that is generated lasts about 300 microseconds (also known as the pulse width) with a peak value of 10 kA, for example, which creates a magnetic field strength of about 1 tesla. The shape of the electric field that is generated is dependent on factors such as the shape of the induction coil, the location and orientation of the coil with respect to the scalp, and the electrical conductivity of the tissue. The simplest shaped coil is a circular one, with round coils being relatively powerful, but they have a larger focal point than the figure of eight shaped coils or butterfly shaped coils which elicit a maximal current at the intersection of the two round or oval components. Typically the coil is a figure of eight shaped coil.

It has been found that different frequencies of magnetic stimulation could have different effects. In some embodiments, the magnetic field's properties are adjusted such that upon treatment, the effects are seen in the subject's respiratory primary motor cortex area. Typically, such effects are seen with rTMS frequency between 0.2 and 1 Hz (cycles per second), called low frequency rTMS, or between 5 and 25 Hz, called high frequency rTMS. Typically, the frequency is between about 0.2 and 0.5 Hz. The electrical current used for generating the magnetic field may be any that is known in the art, and typically is either monophasic, biphasic, or polyphasic. The intensity of the electrical field to use to generate the magnetic force can vary. As a rough guide, intensities of about 1 tesla (T) with an upper limit of about 2 T are typically generated.

Typically, the intensity of electrical field used to generate the magnetic field (and generate the eddy current in the brain) is about 60-90% of the maximal output of the magnetic stimulator for the diaphragm. Motor threshold is a measure that varies considerably between subjects due to factors such as skull thickness, head shape, cortical excitability, medication and acute brain state. Gauging the motor threshold of the subject enables comparable strengths of stimuli to be employed between subjects leading to more predictable therapeutic outcomes. Methods by which to determine motor threshold are known in the art, and include determining the minimum stimulation intensity over a motor hot spot that can elicit an motor evoked potential (MEP) of no less than 50 microV in 5 of 10 trials. MEP may be recorded via electromyogram (EMG) measurement, in particular diaphragm EMG. EMG measurements may be taken by applying conductive elements or electrodes to the skin surface, or invasively within the muscle. Surface EMG is the more common method of measurement, since it is non-invasive. The EMG signals are amplified and filtered to graphically record and quantify the degree of muscle activity. The micro voltage of EMG measurement is directly proportional to the mechanical muscle contraction. EMG measurements are useful to both help confirm coil positioning and assess cerebral cortical excitability.

Current direction and coil direction are other variables that one of skill in the art can vary depending on the subject. The number of pulses to deliver may also be determined by one of skill in the art. Typically, the number of pulses to deliver comprises the number of pulses capable of being delivered in a time period of between about ten minutes and about 120 minutes, between about twenty minutes and about ninety minutes, or most typically between about thirty minutes and about sixty minutes. The number of pulses delivered in that time period can be determined with a simple calculation. For example, the number of pulses delivered at a frequency of 0.5 Hz will typically comprise between about five pulses and about sixty pulses, between about ten pulses and about forty five pulses, or most typically between about fifteen pulses and about twenty pulses. Frequency may change during a particular treatment regimen and/or during a treatment course.

Optimal positioning of the coil over the scalp may readily be determined by one of skill in the art, and will generally comprise positioning to correspond with the respiratory primary motor cortex area. Standard charts and descriptions of the locations are freely available. In order to determine the proper scalp positioning to maximal stimulation of a particular brain coordinate, a number of algorithms known in the art may be used, for example, the T2T-Converter (Talairach-to-T en-Twenty-Converter) which calculates the optimal stimulation positions for TMS studies by projecting brain coordinates to scalp coordinates.

In some embodiments, the treatment course leads to clinically relevant improvement in a symptom of the respiratory function as measured by one of skill in the art. In some embodiments, clinically relevant improvement is a functional improvement. Functional improvement may be evaluated by any method known in the art, and can be evaluated either subjectively (e.g., subject generated feedback or clinician observation) or objectively (e.g. rating/evaluation methods or symptoms that are known in the art). Typically, improvement is evaluated both subjectively and objectively. Additionally, various markers of improvement are known to those of skill in the art, and include surrogate markers of improvement as well as the clinically relevant improvement(s) as discussed above. Examples of markers of improvement typically include diaphragmatic motor response. Respiratory function can also be measured by, for example, spirometry, plethysmography, peak flows, symptom scores, physical signs (i.e., respiratory rate), and blood gases.

Typically, after a course of treatment according to the method disclosed herein, the improvement in a symptom or marker of improvement of the respiratory function is a non- transient improvement (for example, a non-transient effect). In some embodiments, a non- transient improvement is an improvement in a symptom or marker lasts for longer than one hour, longer than four hours, longer than eight hours, longer than twelve hours, longer than eighteen hours, longer than twenty four hours, longer than thirty six hours, longer than forty eight hours, longer than sixty hours, longer than seventy two hours, longer than four days, longer than five days, longer than six days, longer than a week, longer than two weeks, longer than three weeks, longer than a month, longer than two months, longer than three months, longer than four months, longer than five months, longer than six months, longer than nine months, and longer than year.

In some embodiments, wherein the respiratory function is progressive or is potentially progressive, the treatment course leads to a slowing in the time course of the progression of the respiratory function. Such slowing of the time course of progression may be evaluated by a clinician using methods known in the art, such as evaluating or monitoring the subject using methods such as clinical assessment and/or electrophysiological testing. In some embodiments, the progression of the respiratory function is slowed by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%, as compared with no treatment or sham treatment. In some embodiments, rTMS may be used in combination with other forms of stimulation. In some embodiments, a technique called transcranial direct current stimulation (tDCS) is used in addition to rTMS. Thus in some embodiments, the method of the present invention comprises a treatment course which includes a plurality of (i.e., more than one) treatment regimens which consist of applying tDCS.

The present invention also relates to a rTMS system (as above described) for use in a method for treating respiratory failure in a subject afflicted with cervical spinal cord injury wherein the method comprises subjecting the subject to repetitive transcranial magnetic stimulation (rTMS) with said rTMS system for a time sufficient to achieve an improvement of the subj ect ' s respiratory function.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Effect of magnetic shield on the induced magnetic field intensity from the TMS coil. A) 3D representation and its 2D projection (bottom of the graph) of the whole magnetic field intensity (represented as intensity colors) recorded in volts with the solenoid from the naked TMS coil. B) 3D representation and its 2D projection (bottom of the graph) of the whole magnetic field intensity (represented as intensity colors) recorded with the shield on the TMS coil. Note that black square in the 2D projection representing the 3x3 cm window. The intensity is statistically reduced by 27% (p<0.001) compared to the one obtained without shielding, and better concentrated where the rat head will be placed on (Middle of the window). Horizontal and Lateral scale represent the side of the coil, which each measured point represents half centimeter. Figure 2: Antero-posterior stimulation with a single TMS pulse on the amplitude and latence of the observed MEPDia. A) Representation of the different stimulated sites on a rat brain skull with a representation of the rat brain and upper spinal cord. B) Example of a representative MEPDia induced with a single stimulation using the naked coil from different antero-posterior stimulation sites. C) Example of a representative MEPDia induced with a single stimulation using the shield on the coil from different antero-posterior stimulation sites. D) Histogram of the MEPDia amplitude (in μν) from the different antero-posterior stimulated position with shield (white) and no shield (black) in all animals. E) Histogram of the MEPDia latence (in ms) from the different antero-posterior stimulated position with shield (white) and no shield (black) in all animals. *: p<0.05.

Figure 3: Effect of the stimulus intensity on the MEPdia following a single TMS in the occipital cortex. A) Representative example of MEPdia obtained with different stimulus intensities (60 to 100% MO) using the naked coil. B) Representative example of MEPdia obtained with different stimulus intensities (60 to 100% MO) with the magnetic shield on the coil. C) Correlation between MEPdia amplitude (in μν) and stimulus intensity (%MO) for the animals stimulated with no shield (black circles) or with the magnetic shield (cross) on the coil. D) Correlation between MEPdia latence (in ms) and stimulus intensity (%MO) for the animals stimulated with no shield (black circles) or with the magnetic shield (cross) on the coil. MO: Maximal output; *: p<0.05.

Figure 4: Effect of the coil rotation on the MEPdia. A) Representative MEPdia recorded after a single TMS pulse with the naked coil at different orientation degrees (from 0° to 360°, 45° steps) of the coil (represented on the top of the occipital cortex in black). B) Representative MEPdia recorded after a single TMS pulse with the magnetic shield on the coil at different orientation degrees (from 0° to 360°, 45° steps).

Figure 5: Effect of vagotomy and muscular paralysis on the MEPdia induced by a single TMS pulse. A) Representative MEPdia before vagotomy, after vagotomy and injection of a paralytic agent (gallamine trietiodide) obtained with the naked coil. B) Representative MEPdia before vagotomy, after vagotomy and injection of a paralytic agent (gallamine trietiodide) obtained with the shield placed on the coil. Note the absence of the vagotomy on the MEPdia, and the loss of MEPdia following paralysis in both conditions in A and B. C) Histogram of the MEPdia amplitude before vagotomy, after vagotomy and following the injection of paralytic agent in the no shield and with shield on the TMS coil. *: p<0.001.

Figure 6: Effect in a total transection of the spinal cord at the C2 level on the MEPdia when stimulated at the occipital cortex and the spinal cord level. A)

Representatives MEPdia (black arrows) obtained after a single TMS pulse at the occipital cortex (up trace) and spinal cord level (bottom trace) with a magnetic shield on the coil before the complete C2 spinal cord transection. B) Representatives MEPdia (black arrows) obtained after a single TMS pulse at the occipital cortex (up trace) and spinal cord level (bottom trace) with a magnetic shield on the coil after the complete C2 spinal cord transection. Note the absence of MEPdia when the occipital cortex is stimulated, and the enhanced MEPdia when the spinal cord is stimulated.

EXAMPLE: Methods:

Animals

Experiments were performed on 2 month-old male Sprague-Dawley rats (Janvier, France). All experiments were approved by the Ethics committee of the RBUCE-UP chair of Excellence (University of Paris-Sud, grant agreement No. 246556), the University of Versailles Saint-Quentin en Yvelines and conformed to policies laid out by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals. Transcranial magnetic stimulation (TMS)

TMS was performed by using a magnetic stimulator MAGPRO XI 00 (Magventure, Farum, Denmark) connected to a figure-of-eight coil (CB60; dimensions: 165x85x20 mm) delivering biphasic single pulse (380 in duration) with an intensity of the stimulus expressed as a percentage of maximum output of the stimulator (%MO), from 60 to 100%MO in this study. Magnetic shielding has been used by inserting between the coil and the animal scalp, a 2mm thick plate of mu-metal having a 3x3 cm centered open window (labeled "with shield" in the figure). The "no shield" labeled throughout the manuscript refers to the coil without the magnetic shielding. Using such magnetic shield was chosen in line with previous work (Nakatoh et al. 1998) suggesting that focality and efficiency of magnetic field was significantly improved while using it, with lower motor threshold values and higher amplitude MEPs.

Magnetic field quantification

Mapping of the magnetic field intensity has been tested in vitro to evaluate the influence of magnetic field shape over the coil relative to rat central nervous system structures, and the effect of magnetic shielding with the centered open window. Single magnetic pulse was recorded by using a solenoid (Magprobe, Magventure, Farum, Denmark). The solenoid is a 2.5cm diameter copper coil converting a magnetic field into a measurable electrical current (in Volts). The solenoid was placed over the CB60 coil as close as possible (570 different points of recording, 30x19 matrix) in all three dimensional directions of the magnetic field (x, y and z axes). The induced electrical current for each direction (x, y and z) was recorded by using the Powerlab and LabChart 7 Pro (AD Instrument). The whole magnetic field recorded with the solenoid was calculated using the following formula:jx² + y² + z². Each dimension of the electrical field induced in the solenoid was reconstructed in 3D with a 2D projection (Sigmaplot 12.5 software).

Electrophysiological recordings

A total of 20 animals were used in this study, separated in 2 groups depending on the use of magnetic shielding of the coil while performing a single pulse of TMS ("with shield" n=9; "no shield" n=l l). As described previously (Vinit et al, 201 1), anesthesia was induced using isoflurane (100% 0₂ balanced). A 25 G catheter was placed in the tail vein, then the rats were tracheotomized and pump ventilated (Rodent Ventilator, model 683; Harvard Apparatus, South Natick, MA, USA). EtC02 was monitored by using an infrared capnograph (Viamed, VM-2500-M). Rats were maintained on isoflurane (3.5% in 21% 02, continuously monitored by an oxygen sensor (Viamed, AX300), placed on a heating pad to maintain body temperature constant throughout the experiment. Rectal temperature was monitored throughout the procedure. A catheter was inserted in the right femoral artery. Arterial pressure and tracheal pressure was monitored continuously with pressure transducers connected to a bridge amplifier (AD Instruments). The isoflurane anesthesia was then slowly converted to urethane anesthesia (1.8 g/kg, i.v.; Sigma-Aldrich). A laparotomy was performed, and the liver was gently pulled down to have a better access to the diaphragm. A gauze soaked with warm saline was placed on the liver to prevent dehydration. A custom-made silver bipolar electrode was placed on the right mid-costal part of the diaphragm. Diaphragm EMG and motor evoked potential induced by a single pulse of TMS (MEPdia) were amplified (gain, lk; A-M Systems, Everett, WA, USA) and band pass-filtered (100 Hz to 10 kHz). The signal was digitized with an 8 channels Powerlab data acquisition system (Acquisition rate: lOOk/s; AD Instruments) connected to a PC, recorded and analyzed using LabChart 7 Pro (AD Instruments). The head of the animal was placed on a nonmagnetic custom made stereotaxic apparatus which allowed moving the head of the animal from the center of the figure-of-eight coil in several antero-posterior positions (see Figure 2A for the positions) and rotational positions (Figure 3A and 3B) in line with what had been published earlier (Maskill et al. 1991 , Brasil-Neto et al. 1992, Mills et al. 1992, Niehaus et al. 2000, Dubach et al. 2004, Ni et al. 2011). At the end of the study, a bilateral vagotomy then paralytic agent (Gallamine trietiodide, lmg, Sigma, n=8, 4 from each group) or a complete transection of the spinal cord at the C2 level (n=8, 4 from each group) was performed in all animals.

Data processing

A minimum average of 5 MEPdia for each condition was calculated with LabChart Pro (AD Instruments). The peak-to-peak amplitude, the latency of each averaged MEPdia were measured. Normality of data distribution was assessed by using a Kolmogorov-Smirnoff test Two way ANOVA followed by post hoc Student paired t-test, with Bonferonni correction for multiple comparisons were performed. All the data are presented as mean ± one SEM. A test was considered significant if p<0.05.

Results:

Effect of magnetic shield on the magnetic field of the coil

The whole magnetic field recorded is located in the middle of the coil without the magnetic shield, with 2 hot spots located in the middle of the 2 holes of the figure-of-eight (represented in red, Figure 1A). The whole recorded magnetic field from the coil is statistically reduced by 32% with the magnetic shield (average "no shield" = 5.76 ± 0.1 V, average "with shield" = 3.89 ± 0.06 V, p<0.001, paired t-test; Figure 1A and IB) and better concentrated in the middle of the 3x3 cm window (Figure IB). All the 3 dimensional x, y and z of the magnetic field are statistically reduced with the magnetic shield (average "no shield" in x = 3 ± 0.09 V, average "with shield" in x = 2.03 ± 0.06 V, reduction of 32%, pO.001, paired t-test; average "no shield" in y = 2.74 ± 0.07 V, average "with shield" in y = 1.78 ± 0.04 V, reduction of 35%, pO.001, paired t-test; average "no shield" in z = 2.75 ± 0.11 V, average "with shield" in z = 2.07 ± 0.07 V, reduction of 24%, p<0.001, paired t-test; data not shown).

Physiological effect of a single magnetic pulse on the diaphragmatic motor evoked response (MEPDia)

No statistically difference were observed in different recorded physiological parameters (body weight, body temperature, EtC02, blood pressure) between the treatment group ("no shield" versus "with shield") used in this study (Table 1).

Body weight (g) Temperature (°C) EtCO_? (mmHg) MAP Start (mmHg) MAP End (mmHg)

Treatment Group Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM

No shield 339 16 37,3 0,2 38,8 0,8 108 4 111 5

With shield 327 21 36,9 0,1 38 1,8 90 5 93 5

Table 1: Physiological parameters.

A single pulse of magnetic stimulation at different location of the brain and spinal cord (Figure 2A) induces different MEPdia depending of the origin of the stimulus. A stimulation (100%MO for the with shield group; 85%MO for the no shield group, representing 140%) of resting MT value in each condition) applied at the spinal cord, cerebellum, occipital, mid and from cortex induce an observable MEPdia whereas a snout stimulation does not in both groups (Figure 2B and 2C). The magnetic shield does not affect the presence/absence of the MEPdia. However, the magnetic shield statistically reduces the amplitude of the recorded MEPdia for the cerebellum stimulation location ("no shield": 2.49 ± 0.48 μν compared to "with shield": 1.11 ± 0.26 μν, p=0.04; Figure 2D) and spinal cord stimulation location ("no shield": 5.95 ± 1.34 _μν compared to "with shield": 2.15 ± 0.51 μν, p=0.045; Figure 2D). No statistical differences are observed on the MEPdia amplitude for the occipital cortex stimulation ("no shield": 1.23 ± 0.36 μν compared to "with shield": 0.62 ± 0.06 μν, p=0.751; Figure 2D), for the mid cortex stimulation ("no shield": 0.67 ± 0.19 μν compared to "with shield": 0.74 ± 0.24 μν, p=0.597; Figure 2D) and the front cortex stimulation ("no shield": 0.27 ± 0.05 μν compared to "with shield": 0.47 ± 0.09 μν, p=0.102; Figure 2D) locations. The magnetic shield statistically increases the latency of the recorded MEPdia for the spinal cord ("no shield": 7.84 ± 0.98 ms compared to "with shield": 11.07 ± 0.86 ms, p=0.009; Figure 2E), cerebellum ("no shield": 7.71 ± 0.81 ms compared to "with shield": 10.58 ± 0.77 ms, p=0.031; Figure 2E) and front cortex stimulation ("no shield": 6.36 ± 0.39 ms compared to "with shield": 9.59 ± 0.82 ms, p=0.003; Figure 2E). No differences are observed for the latency of the MEPdia for the mid cortex ("no shield": 7.71 ± 0.79 ms compared to "with shield": 9.42 ± 1.14 ms, p=0.161 ; Figure 2E) and occipital cortex stimulation locations ("no shield": 9 ± 0.77 ms compared to "with shield": 10.41 ± 0.83 ms, p=0.252; Figure 2E). However, no difference in MEPdia latency is also observed across the groups "no shield" and "with shield" for the different locations of the stimuli (p<0.05; Figure 2E).

The amplitude of the MEPdia is also dependant of the stimulus intensity from the TMS machine (express as %MO). The MEPdia starts to be recorded at a stimulus intensity of 70%MO in both groups ("no shield" Figure 4A; "with shield" Figure 4B) at the occipital cortex stimulated location. The amplitude of the observed MEPdia increases with the stimulus intensity in both "no shield" and "with shield" groups (Figure 4A and 4B respectively). Quantitatively, the amplitude of the recorded MEPdia for the "no shield" group (Figure 4C, black circles) follow a S-shape curve, with a plateau at a stimulus intensity of 85%MO, whereas, for the "with shield" group (black x), the plateau is not yet reached at a stimulus of 100%MO (MEPdia amplitude at 95%MO= 0.49 ± 0.02 μν compared to 100%MO= 0.81 ± 0.1 μν, p<0.05). A statistical difference between the 2 groups for the MEPdia amplitude is observed, started from 80%MO to 100%MO (p<0.05 for each stimulus intensity from 80 to 100 %MO). The stimulus intensity does not have any statistical effects on the MEPdia latency in each group or between the two groups (Figure 4D) at the occipital cortex stimulus location.

The orientation of the coil also play a role in the amplitude, shape and latency of the MEPdia recorded after occipital cortex stimulation (Figure 5). The amplitude of the MEPdia in the "no shield" group varies with the coil rotation, with a maximum amplitude at +90° and -90° (Figure 5 A), which is correlated with the shape of the magnetic field (Figure 1 A) where the maximal field intensity (in red) is represented in both holes of the eight-shape coil. Same trend is also observed in the "with shield" group with an evident reduced MEPdia amplitude (Figure 5B). Specificity of the diaphragmatic motor evoked potential

A single magnetic stimulation at the occipital cortex location induces an observable MEPdia before vagotomy for both groups (Figure 6A "no shield" and 6B "with shield"). A bilateral vagotomy do not affect the amplitude of the recorded MEPdia in the "no shield" (before vagotomy: 0.74 ± 0.17 μΥ, after vagotomy: 0.84 ± 0.24 μΥ, p=0.25; Figure 6C) and "with shield" groups (before vagotomy: 0.59 ± 0.12 μν, after vagotomy: 0.45 ± 0.04 μν, p=0.36; Figure 6C). No difference in latency is also observed (data not shown). However, an injection of a paralytic agent (Gallamine trietiodide) abolish the MEPdia in the "no shield" (after vagotomy: 0.84 ± 0.24 μν, after muscular paralysis: 0.03 ± 0.008 μν, p=0.018) and "with shield" (after vagotomy: 0.45 ± 0.04 μν, after muscular paralysis: 0.03 ± 0.01 μν, p=0.001) groups (Figure 6C). An occipital cortex single magnetic stimulation (100%MO) as well as spinal cord magnetic stimulation induces a MEPdia in the "no shield" group (Figure 7A). However, a complete transection of the cervical spinal cord at the C2 level abolishes the observed MEPdia after an occipital cortex stimulation location whereas the observed MEPdia is enhanced following a spinal cord magnetic stimulation (Figure 7B). Similar results for all the animals are observed in the "with shield" group (data not shown).

Discussion:

The first aim of this study was to determine whether diaphragmatic motor response to TMS in rat was recordable and may be used in animal studies involving neuroplasticity and excitability change of the corticospinal pathways. Previous studies had demonstrated in rat that limb muscles MEP evoked by figure-of-eight coil were recordable and suggest that pyramidal tract cells were recruited (Fishback et al, 1995, Kamida et al., 1998, Linden et al, 1999, Luft et al, 2001, Rotenberg et al, 2010). For the first time we report here the recordings of diaphragmatic MEP in response to magnetic stimulation. Although forepaw or hind paw muscle MEP has been described in awaked animals (Linden et al, 1999), most of the studies were carried out in anesthetized rats. Most common anesthetics are known to reduce or abolish MEP, however propofol (Fishback et al, 1995, Luft et al, 2001), ketamine (Kamida et al, 1998), pentobarbital (Rotenberg et al, 2010) or urethane (Kamida et al, 1998) have been used successfully with different rat lineages (Wistar, Long-Evans). In our study, blood injection of urethane in Sprague-Dawley rats did not prevent to record MEPdia evoked by TMS. Moreover, vital parameters were constant all along the experiments with no blood flow limitation which could have influence ours results. However, several technical and physiological features have to be discussed regarding either the unusual involvement of a magnetic stimulator connected to a large figure-of-eight coil usually used in humans or the specificity of respiratory corticospinal pathways.

Despite the fact that our main goal was to determine the feasibility or evoking a diaphragmatic MEP in rat with a conventional magnetic stimulator, we also tested the influence of magnetic shielding designed to focalize or improve efficiency of magnetic field over respiratory primary motor cortex area to depolarize diaphragmatic cortical cells. In a previous single study Nakatoh et al. (1998) had reported the beneficial use of such device in cats, with lower resting motor threshold values et increased MEP amplitude. However, our results in rat with a much smaller brain size relative to coil dimensions, both in vitro and in vivo, are less univocal and need to be commented. In vitro measurements yield that focality of magnetic field density is slightly improved by the shield in the middle of the coil surface whereas the intensity is then globally reduced. These results may explain at least partly the reason why with shield recruitment curve in figure 4 looks very different in with shield condition compared with the no shield one. Firstly, it may be noticed that the diaphragmatic resting motor threshold values obtained in rat are almost in the same range as for rats for limb muscles (Kamida et al, 1998, Luft et al, 2001)as well as in humans in more recent published studies (Sharshar et al. 2003, Azabou et al. 2013). Moreover, there is an increase of about 10% of resting motor threshold value by adding the magnetic shield. By increasing stimulation intensities, there is an obvious recruitment effect with a curvilinear relationship which implies that the magnetic field at the centre of the coil is high enough to recruit the most excitable motor cortex cells able do depolarize phrenic motoneurons. However, shielding seems to limit the effect of ectopic stimulation of extra-cortical cell populations as the with shield curve displays a continuous increase up to maximal with relatively small SEM values, whereas no shield recruitment curve exhibits an upward shift as stimulation intensity reaches a threshold value of 80%MO. Moreover, beyond 80%MO, an increase in SEM amplitude values was also observed suggesting that a first recruitment mode changes. In that prospect, regarding the very small rat primary motor cortex area devoted to respiratory muscles by comparison with leg muscles (Kolb and Tees, 1990) shielding seems interesting because it limits the extra cortical excitable cells recruitment by lowering the magnetic field intensity of the peripheral regions of the coil. Interaction between shielding and position of the coil was also present. In no shield conditions, no MEP was recordable forwarding the coil over the snout even for the highest stimulation intensities, whereas by moving backwards there was an optimal site to get recordable MEP over the brain. These results fit well the previous report of a most excitable area at the bregma to electrically or magnetically evoked MEP (Kamida et al, 1998). However, a huge electrical response was recorded since the coil was centred over cervico medullary regions suggesting that neuro foramina stimulation of cervical root fibres occurs, in addition or in combination with cortical cell stimulation. Cervical neuroforamina root stimulation at high stimulation intensities (almost around 80- 90%MO) is possible in animal model (Bader et al, 2011), and is known to spread out stimulation sites of peripheral motor fibres (Cros et al. 1990). Our results concerning the effect of acute cervical cord section (figure 7) enhance this view since MEP was not recordable any longer with TMS performed over motor cortex. Nevertheless, high amplitude response was still present, and may be was magnified, following spinal cord section for magnetic stimulation performed over cervicomedullary region. These results as well as those from antero-posterior stimulation site shifting indicated that MEPdia has an optimal site to be recorded without any contamination from spinal cord excitable cells. All these results concerning MEP amplitude were confirmed by focussing upon latency values. Data from figure 2, examining the effect of antero-posterior position of the coil, do not retrieve significant change in MEP Nl latency mean values for the most posterior sites - over cerebellum and cervicomedullary sites - suggesting that closer to the diaphragm, different cell populations could be depolarized with action potential volleys having lower propagation velocity, and the likelihood of an antidromic volley cancelling those coming from stimulated motor cortex. Then, it seems important to care about the position over the rat brain to prevent this phenomenon. This interpretation may be extended to latency data related to magnetic shielding. Despite a lack of statistical significance, MEPdia latency mean values were always shorter in no shield condition than those measured with shield, suggesting that corticospinal fibres depolarization site would be closer to diaphragm, and that shielding would definitively induce a small delay due to the fact that magnetic stimulation would be more effective at the motor cortex level than elsewhere. In addition, latency mean values are in the same range as those reported for limb muscles taken either the onset latency (Fishback et al., 1995; Kamida et al, 1998; Linden et al, 1999) or the MEPdia first negative wave latency (Nl) (Luft et al, 2001) suggesting that evoked potentials originates in the same central nervous system structure rather than at spinal or phrenic nerve locations. Definitively, our data suggest that position of the coil is then crucial to record diaphragmatic MEP in rat, and as in humans MEP amplitude seems to be the highest while centring the coil over motor cortex area defining a hot spot.

Data from rotating the coil over the midbrain reveal interesting features too. Because MEP amplitude was tremendously increased for -90° as well as +90° positions, it is likely that this is due to the geometry of the magnetic field under the coil, with or without shielding. These two positions correspond to the alignment of the great axis of the coil and the central nervous system antero-posterior axis, namely extending the magnetic field over the spinal cord area, and increasing the ability to depolarize phrenic motoneuron axons at neuroforamina portions of the cervical roots. This is not shield dependant, and it is likely that the magnetic field is less reduced at the lateral winged part of the coil than in central position. Moreover, the fact that almost no signal was recorded for +180° gives some insight about the influence of current direction, independently. As for human motor cortex magnetic stimulation (Niehaus et al, 2000, Maskill et al, 1991), motor cortex is sensitive to current direction and preferentially recruited for 45° position. This had been interpreted as the orientation of the magnetic field, being perpendicular to primary motor cortex cell orientation in frontal sulcus, also demonstrated for human diaphragmatic motor cortex (Maskill et al, 1991; Dubach et al, 2004). Nevertheless, rat motor cortex is very different from human one with less complex cerebral convolutions and sulcus organisation. This result showing that rat motor cortex shares this property, suggests a strong link between magnetic field geometry and motor cell intrinsic properties rather than the consequence of differential brain surface architecture.

Indeed, the major concern of this paper was to demonstrate that recorded MEPdia does reflect diaphragmatic fibres depolarisation in response to corticospinal activation following motor cortex stimulation and nothing else. As previously discussed, MEPdia latency and amplitude values related to increasing magnetic stimulation output, suggests a cortical origin (Devanne et al, 1997). However, regarding technical and physiological considerations, it cannot be ruled out that MEPs might be contaminated by artefacts. First of all, stimulation of other miscellaneous nervous structures such as vagus nerve could be put forward as a confounding factor. The size of the magnetic field was large and powerful enough to depolarize vagus nerve fibres antidromically, performing an ectopic activation of some diaphragmatic motor units or muscle fibres. Recordings of unchanged diaphragmatic MEP following vagotomy, ruled out this hypothesis. Moreover, by inhibiting motor end plates with a cholinergic blocker, magnetic stimulation was then unable to record any MEP, suggesting that diaphragmatic motor unit recruitment was performed with TMS through corticospinal tract activation, and not directly or by local electrical currents induced by the magnetic field depolarizing motor axons (Maccabee et al, 1998). Finally, we may infer that MEPdia actually reflect corticospinal pathways activation due to an action potential volley from motor cortex to diaphragm, rather than an artefact mimicking any diaphragmatic motor response.

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Claims

CLAIMS:

1. A method for treating respiratory failure in a subject afflicted with cervical spinal cord injury comprising subjecting the subject to repetitive transcranial magnetic stimulation (rTMS) for a time sufficient to achieve an improvement of the subject's respiratory function.

2. The method of claim 1 wherein the subject is a human infant, a human child, a human adult or an elderly human.

3. The method of claim 1 which includes treating the subject with a treatment course which includes a plurality of treatment regimens which consist of applying repetitive transcranial magnetic stimulation (rTMS) over a position on the subject's scalp corresponding to respiratory primary motor cortex area.

4. The method of claim 1 wherein the rTMS is used in combination with transcranial direct current stimulation (tDCS).

5. A rTMS system (for use in a method for treating respiratory failure in a subject afflicted with cervical spinal cord injury wherein the method comprises subjecting the subject to repetitive transcranial magnetic stimulation (rTMS) with said rTMS system for a time sufficient to achieve an improvement of the subject's respiratory function.