Physiological and physiopathological signs of respiration
Respiration, the alternation of effective inspiration and expiration, requires the functioning of various nerve and muscle structures: cerebral cortex, brain stem, descending nerve pathways, phrenic nerves and intercostal respiratory muscles.
The electrical impulse, which is affected by both voluntary and involuntary input, originates at the level of respiratory neurones in the brain stem and is then transmitted via motor nerves (especially the phrenic nerves). Once past the neuromuscular junction, it is propagated through the neuromuscular membrane causing the contraction. Disturbances at any one of these sites can lead to anomalies in co-ordination of the system or to muscle weakness [1,2].
The phrenic nerves (motor component) originate at the C3-C5 level, and innervate the two hemidiaphragms ipsilaterally.
The diaphragm is the most important respiratory muscle, and alone is responsible for the movement of 50% of air volume during normal breathing. The contraction of the diaphragm creates negative intrapleural pressure (measured as oesophageal pressure, esP), and positive abdominal pressure (measured as gastric pressure, gaP). The resulting trans-diaphragmatic pressure (Pdi, Pdi = gaP − esP) has been used to indicate the tension, or rather the force, exerted by the diaphragm [3,4].
Weakness of the respiratory muscles, which frequently occurs with many neuromuscular illnesses, means that it is impossible to generate adequate levels of pressure or inspiratory and expiratory air flow . The contribution of the thoraco-pulmonary system is also reduced in neuromuscular illnesses, and this causes an increase in the mechanical load placed on respiratory muscles. When there is a mismatch between muscle load and capacity, it leads to muscle fatigue and then to respiratory insufficiency.
If the respiratory muscle weakness is only slight or moderate, there is an increase in the central respiratory drive, which causes alveolar hyperventilation with a reduction in CO2 partial pressure, while the alveolar-arterial oxygen gradient may be normal. In cases of severe weakness, the system is no longer capable of adequate ventilation and thus retention of CO2 results, and, as a final consequence, clear respiratory insufficiency.
Weakness of the diaphragm and other respiratory muscles presents clinically with a reduction in vital capacity (VC) and total lung volume (TLV), with a reduction in maximum inspiratory pressure (max IP) and the presence of a paradoxical abdominal respiration where the abdomen moves inwards during inspiration. A reduction of 25% or more in VC from erect to supine is a good indicator of diaphragm weakness .
Weakness of the expiratory muscles (abdominal muscles and internal intercostals) causes inadequate clearance of the airways with secretion retention, which leads to an increase in airways resistance, thus altering the respiratory mechanics, making it easier for infections or atelectasia to set in. A reduction in the maximum expiratory pressure (max EP) and the ability to cough are also observed, but these are not considered sufficient by themselves to account for dependence on ventilation .
Phrenic nerve stimulation techniques
The diaphragm is only innervated by the phrenic nerve, which is why phrenic nerve stimulation enables us to analyse the diaphragm independently of the other respiratory muscles. The other important feature of phrenic nerve stimulation is that it enables us to eliminate the effect of the central nervous system. The effects of phrenic nerve stimulation can be studied either electrophysiologically or mechanically.
Electrophysiologic tests evaluate the integrity of the neuromuscular respiratory apparatus. There are two types of tests that assess respiratory muscle function: electromyography and respiratory nerve stimulation tests. Stimulation of the respiratory nerves, especially the phrenic nerve, can be carried out using electric or magnetic stimulators.
Electric stimulation of phrenic nerves
Phrenic nerve conduction tests were, unfortunately, ignored for many years. One of the reasons may have been fear of a technique that seemed less precise than standard limb nerve conduction tests, coupled with the difficulty in actually carrying them out on non-co-operative patients. In 1993, Bolton  changed the technique used by Markand and colleagues , showing the repeatability of the DCAP measure (diaphragm compound action potential) after electric stimulation of the phrenic nerve with the same accuracy as the TCAP (thenar compound action potential) measure after electric stimulation of the median nerve. The technique consisted of direct stimulation of the nerve at the postero-lateral level of the sternal head of the sternocleidomastoid muscle, with the compound motor action potential recorded at the level of the diaphragm using surface electrodes positioned at the thoracic level (the first on the sternum 5 cm away from the xyphoid process and the second at a distance of 16 cm from the first on the costal margin on the same side). The point of stimulation is approximately 3 cm above the clavicula, and the cathode is placed below the anode. The electric stimulus lasts between 0.1 and 0.2 ms and varies in intensity between 30 and 50 mA. A simple technique for locating the nerve is by doing repeated stimulation at a frequency of, say, 1 Hz at a relatively low intensity and trying various sites. To make sure the site is the correct one, observation of the abdomen may prove useful because the contraction of the diaphragm results in small movements of the abdominal wall. Small changes to the position of the stimulator and its voltage guarantee a supramaximal response. Stimuli lasting 0.5 ms may be necessary in patients with a large neck. The neck needs to be in the neutral position or in slight extension. Given how close the stimulation point of the phrenic nerve is to the neck and brachial plexus, it is important that co-stimulation of the plexus is avoided. If this happens, the compound action potential that is recorded may turn out to have an initial positive phase and a very short latency period. If the stimulating electrode is repositioned so that only the phrenic nerve is stimulated, the problem disappears. The amplitude of the combined action potential also varies according to the phase of respiration and the positioning of the electrodes. With the Bolton method, the amplitude is greater during inspiration. With the Swensen method, on the other hand, where the recording electrode is placed on the costal margin and the reference electrode placed below the navel, the amplitude is greater during expiration . The average is not necessary. The distance between the point of registration of the phrenic nerve and the recording electrode (at the xyphoid level) is measured, even if has been shown that there is little variation in this figure in adults. Control values are: latency, 6.3 ± 0.8 ms; amplitude, 597 ± 139 μV; average ± SD. Given that the right phrenic nerve is shorter than the left, latency is less on the right.
Electric stimulation of the phrenic nerve can reveal a longer latency period if there is reduced conduction speed, which, in turn, is indicative of demyelinating myopathy (usually Guillain-Barré syndrome), or a reduction in amplitude of the combined motor action potential (CMAP) if there is a problem with the axon, as is typical in axonal neuropathies like critical illness polyneuropathy (CIP) or traumatic neuropathy. The diagnostic limitations of electromyography (EMG) for axonal neuropathies (resolved after needle EMG of the diaphragm) are revealed when a reduction in amplitude of the compound motor action potential is recorded even when diaphragm myopathy is present and axons are intact.
Finally, repeated stimulation of the nerve and recording of the diaphragm action potential evaluates the neuromuscular junction, which is useful if neuromusclar blocks are being used or if the neuromuscular plate is affected by the disease as in myasthenia gravis. In these cases, there is a progressive, reversible reduction in the compound motor action potential from the diaphragm.
The advantage of electric stimulation of the phrenic nerve is that it produces ‘pure' contraction of the diaphragm.
Assessment of the pressure generated (trans-diaphragmatic, if mechanical properties are being assessed) or the compound motor action potential from the diaphragm (if electrophysiologic properties are being assessed) relates only to the diaphragm muscle or phrenic nerve. What is more, the test is reproducible in the right hands.
Limitations of the technique are the discomfort for the patient caused by supramaximal electric stimuli, the difficulty in distinguishing phrenic stimulation from that of the brachial plexus and the impossibility of actually locating the nerve in some cases.
The problem of co-stimulation of the brachial plexus is particularly significant if the phrenic nerve is stimulated using cervical magnetic stimulation.
Axonal neuropathies and neuromuscular transmission defects are associated with a reduction in amplitude of the CMAP and a reduction in trans-diaphragmatic pressure, whereas contractile problems are associated with a reduction in twitch trans-diaphragmatic pressure.
Magnetic stimulation of the phrenic nerve
The technique of magnetic cervical stimulation was introduced by Similowski and colleagues  in 1989 and consists of stimulating the nerve at the cervical level using a bobbin, which creates a magnetic field. Magnetic stimulation creates intense, brief magnetic fields, which, unlike electric currents, are only slightly affected by skin and bone. They can thus reach deep-lying nerve structures, which is difficult with surface electric stimulation. Nerve response mechanisms to magnetic stimulation are different from those to electric stimulation [12,13]. This is why results obtained using the two different techniques can be interpreted differently. For bilateral stimulation of the phrenic nerve, the bobbin is placed at the level of the transverse process of the seventh cervical vertebra and the magnetic stimulus depolarizes the roots of the third, fourth and fifth pairs of cervical nerves causing a bilateral contraction of the diaphragm [14,15]. Magnetic cervical stimulation also stimulates other elements of the cervical roots and neighbouring nerves, so that not only the diaphragm but also the neck and muscles in the upper part of the thoracic cage contract [11,16,17]. The technique is easy to carry out and is less painful for the patient, but it is not selective, cannot be carried out repeatedly and is expensive .
The disadvantages are reduced with focal magnetic stimulation of the phrenic nerve [19,20]. Bobbins, which are 8-shaped, are positioned at the neck level, either unilaterally or bilaterally, at the same point as for electric stimulation.
Neuromuscular respiratory insufficiency and the use of electric stimulation of the phrenic nerve in Intensive Care
Neuromuscular respiratory insufficiency is caused by a process involving motor neurones, peripheral nerves, neuromuscular transmission or respiratory muscles. One of the most frequent causes of neuromuscular insufficiency presenting in Intensive Care is CIP [21-24] (Tables 1 and 2). Involvement of the neuromuscular respiratory system in CIP manifests through difficulty in weaning the patient off mechanical ventilation and through muscle weakness . The patient complains of feeling unwell and tired, but unlike patients with pulmonary parenchymal pathologies, or airways obstruction, they are not hypoxic nor do they necessarily present with dyspnoea. Breathing is rapid and superficial and heart rate is sometimes increased because of autonomic nervous system involvement. Other clinical features that suggest that neuromuscular mechanisms are responsible for the respiratory insufficiency are paradoxical breathing and the absence of abdominal distension during inspiration, increased breathing difficulty when supine compared to when sitting and a weak or absent cough. In critically ill patients on mechanical ventilation, these signs are hard to identify and interpret. Other lung-function tests, like VC and max IP and max EP, are difficult to carry out in Intensive Care as they rely on patient collaboration, which is not possible when patients are critically ill . As a result, the extent to which these tests are able to determine neuromuscular respiratory system involvement when it is difficult to wean the patient off mechanical ventilation has often been questioned [27,28]. Electrophysiologic tests are the only way of recognizing the nature and level of the anatomical lesion responsible for neuromuscular respiratory insufficiency.
Respiratory insufficiency intended as difficulty weaning the patient off the ventilator is of historic importance because it was the clinical problem that led to the discovery and description of CIP in the early 1980s . Despite this, it is only recently that CIP has been shown to be responsible for a longer weaning process. Only a few studies [30-35] have shown a direct link between CIP and the duration of mechanical ventilation, probably because there are many factors that influence the length of time a patient needs ventilation, not least of which is the overall seriousness of the case and the reason for hospitalisation in the first place . Two recent studies showed the role of possible confounders, showing that CIP is an independent risk factor for weaning failure [35,37]. Curiously, neither of the two studies assessed disturbances in the phrenic nerve or diaphragm, only the lower limbs.
The first study, co-ordinated by De Jonghe and colleagues , showed that in 95 patients on mechanical ventilation for more than 7 days, there were two variables that were independently associated with length of time on ventilation: diagnosis of existing chronic obstructive pulmonary disease and ICU-acquired paresis defined by an MRC (Medical Research Council) score of <48 for 7 or more days after return to consciousness.
The second study by Garnacho-Montero and colleagues  showed that in 64 patients with severe sepsis or septic shock and on mechanical ventilation for more than 7 days; CIP increased the length of time on ventilation and was an independent factor in weaning failure. Out of 34 patients (53%) with diagnosed CIP as the start of the weaning period, the average length of weaning was significantly longer compared with that of patients who did not have CIP (15 vs. 2 days, P < 0,001). What is more, 14 of the 34 patients (41.2%) with CIP needed to be intubated again compared to only 4 of the 30 patients (13.3%) without CIP. Given that the neuromuscular respiratory system was not investigated in either of the two studies, we are left wondering as to whether the development of CIP led to the increased duration of mechanical ventilation or whether it was prolonged ventilation that led to the CIP. The De Jonghe and colleagues' study, in fact, shows that length of time on mechanical ventilation is an independent predictor of ICU-acquired paresis. The age-old question remains as to which came first, the chicken or the egg.
Even if it is difficult to assess the extent to which the neuromuscular respiratory system is involved in CIP patients, the extent to which CIP interferes with the weaning process and its role in prolonging mechanical ventilation are important questions. Very few studies have assessed the electrophysiology of respiratory muscles in patients in Intensive Care [38-42].
The first study to demonstrate the existence of axonal generation in the phrenic and intercostal nerves and respiratory muscle atrophy in CIP was an autopsical one by Zochodne and colleagues  in 1987.
Subsequently, Witt and colleagues , in 1991, documented significant correlation between reduction in the diaphragm compound action potential and the seriousness of CIP.
Spitzer and colleagues , in 1992, carried out a prospective study on 21 patients on prolonged ventilation at an advanced stage of their illness. Of their patients, 62% showed signs of neuropathy, particularly axonal neuropathy. The intercostal muscle EMG showed pathology in 6 out of the 10 patients studied (60%), with important implications for the risk of respiratory insufficiency and difficulty weaning patients off the ventilator. The quantitative analysis of motor unit potentials, which was carried out on 57% of the patients, showed that half of these had myopathies. This shows how much higher the incidence of myopathy is than was previously thought, and how phrenic nerve neuropathy is also present as an explanation for the difficulty in weaning patients off ventilation.
Maher and colleagues , in 1995, studied 40 Intensive Care patients over a course of 3 yr where a neurological cause was suspected for their difficulty with the weaning process. Limbs and phrenic nerve were tested with EMG and intercostal muscles and diaphragm with EMG. The prevalence of CIP was high (25 patients, 62.5%), and more than half of them (20 patients) presented with bilateral neuropathy of the phrenic nerve (only six of them at history-/ did not present with a clinical condition, which would explain the neuropathy); bilateral neuropathy of the phrenic nerve was only seen in patients who had peripheral neuropathy (except in one case with a subdiaphragmatic abscess). Of the 21 patients who had moderate electroneurographic disturbances to their lower limbs, only 6 (28.6%) had diaphragmatic involvement, while of the 10 patients with serious electroneurographic disturbances to their lower limbs, 9 (90%) had diaphragmatic involvement. These figures also showed correlation between the seriousness of CIP and the duration of mechanical ventilation, which was obviously much longer for the patients with serious forms of CIP (136 days) as compared to patients with slight or moderate forms (52 days). Zifko and colleagues , in 1998, found electroneuromyographic disturbances in 48 (77%) out of 62 patients with CIP. Patients with a reduction in amplitude of the compound muscle action potential from the diaphragm spend more time on mechanical ventilation than patients with normal amplitude (62 vs. 55 days on average generally) even if the difference is not statistically significant. These results suggest that electrophysiologic disturbances of the diaphragm are common in patients with EMG disturbances of the lower limbs, especially when the disturbances are severe.
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