Sistema de Prueba

Bayliss (1900-1) recognised the importance of Strieker’s observation (in 1876) that electrical or mechanical stimulation of the distal end of the divided posterior lumbar root in the dog caused ipsilateral hind-limb vasodilatation as measured by temperature rise in the paw. He concluded from his own work that chemical and thermal excitation were equally effective stimuli, and proposed the term “antidromic” for such impulses that were conveyed from the proximal end of afferent fibres to produce vascular dilatation peripherally within the tissues of the body and so travelled contrary to the Bell-Majendie law. He also noted that the fibres of the posterior root system degenerated when the root ganglion was removed but not when the root was cut between the spinal cord and the ganglion.

Bruce (1913) showed conjunctival reddening and swelling in response to mustard oil which was independent of the central nervous system. He showed the local vessel vasodilatation in skin following irritants was unaffected by spinal cord sectioning, nerve section of the posterior root proximal to the dorsal root ganglion (DRG) or section of peripheral nerve fibres distal to the DRG if those fibres had not yet degenerated. However it was stopped if they had degenerated or were treated with local anaesthetic. He proposed that these responses were due to impulses that travelled up one branch of a sensory nerve and down a second to a blood vessel, rather than via a spinal reflex arc. As such he was probably the first to describe and provide a pictorial representation of the axon reflex as is it is understood today. He borrowed the term “axon-reflex” from earlier work by Langley (1900) who had

used “axon reflex” and “pseudo reflex” to describe reflexes, including piloerection, in preganglionic sympathetic nerves.

Langley (1923, 1923-24), who believed the flush to be due to capillaries, subsequently confirmed the absence of the vasodilatory response when peripheral nerves had degenerated following section and that the response was still present even if the aorta was clamped. (Langley credited the first observation of the flushing to Morat in 1892).

In his book “Blood Vessels of the Human Skin and Their Responses” Lewis (1927) summarised his work with Grant, Marvin, Cotton and others from the preceding 12 years and brought a consensus of observations of vasodilatation in the skin. In it he described what he termed the “Triple Response” which consisted of local vasodilatation, flush and local oedema resulting in a wheal. He showed the local “red reaction” or “flush” that followed firm mechanical stroking of the skin (or surface of the liver, spleen or brain) was independent of the central and peripheral nervous systems, i.e. non- neurogenic, and due to primary dilation of arterioles, capillaries and venules. Distinct from this simple flush was a local, spreading flush or “flare” which was due to arteriolar dilation only and was dependent upon the integrity of cutaneous nerves; it was lost only if cutaneous nerves had had time to degenerate but not if sympathetic nerves had degenerated; it was also lost if the dorsal root ganglion was destroyed or following application of local anaesthetic. The flare occurred only in the skin or conjunctiva and not on viscera. It could be induced by stronger or repeated strokes, as well as intradermal histamine, faradic or galvanic electrical stimulation, poisonous and irritant substances and injury. Most of these were painful. The flare was mediated by an axon reflex rather than a spinal cord reflex. It was probably transmitted by collateral branches of single sensory fibres which by division supplied both the arteriolar wall and sensory nerve endings, as had been reported to exist anatomically by Woollard (1926) as the possible morphological basis for the axon reflex. Lewis proposed a unifying mechanism for the flare irrespective of the stimulus whereby an antidromic

impulse released a common chemical, the H-substance (H meaning histamine-like), into the skin.

Later, Lewis (1937) described a “Nocifensor System”, reactions of which included the flare produced around a local injury or distal stimulation of posterior nerve roots or cutaneous nerves, and the hyperalgesia that spreads around a local injury and around distally stimulated cutaneous nerves. He considered the vasodilator response that occurs in extremities when they are exposed to cold to be a similar local axon reflex of this system which like the flare was independent of sympathetic nerves and reliant upon the integrity of somatic sensory nerves. Bonney (1954) used this “cold vasodilatation response” in tandem with Lewis’ histamine skin prick test to investigate patients with brachial plexus traction lesions. Heat-flow across a metallic disc from finger-tips to the cold water in which they were immersed was measured as a means of showing the vasodilatation; galvanometer readings from the disc were proportional to the heat flow across it.

This nocifensor system as such is today known to be mediated by small nerve fibres. Indeed, as long ago as 1930, the vasodilatation of posterior root stimulation was associated with slow-conducting fibres and the C-wave of the action potential (Hinsey and Gasser 1930) and Celandar and Folkow (1953a & b) showed the spread of vasodilatation was dependent upon peripheral nociceptive C-fibres. They proposed, that since the flare could occur even with negligible pain stimuli that caused very low rates of neuronal discharge, and that only pain fibres were involved, the function of the response was to increase blood flow to the area of injury. They did not believe the vasodilator substance to be acetylcholine or histamine.

The flare was measured by Lewis as area or rise in skin temperature. Le Quesne and Parkhouse (1987) used laser Doppler to measure the flares produced by iontophoresed acetylcholine. This method has the advantage of detecting more subtle changes in cutaneous blood flow especially in pigmented skin.

Scanning laser Doppler has shown marked variation in flux rise within an axon reflex skin flare in response to electrical stimulation, with rises from base-line of

between 32-48% and 80-100% of maximum flux/perfusion between individual neighbouring measurement sites (approximately 0.4 mm^), i.e. differences of up to 100%. Thus a “microstructure” of the response to the stimulus can be discerned probably reflecting the microanatomy of skin vasculature (Wardel et al, 1993). Careful review of these scans also shows a rise in flux at the site of needle (electrode) entry.

Innumerable substances have been shown to produce an axon reflex flare including substance-P (Hagermark, Hokfelt, Pernow 1978), CGRP, VIP and somatostatin. Capsaicin can by pre-treatment block this action of these substances (Anand, Bloom and McGregor 1983), probably by depletion of substance-P from nerve terminals (Carpenter and Lynn 1981). Capsaicin, by its selective action on small unmyelinated fibres to produce pain and axon reflex vasodilatation (Holtzer 1991), probably by this same substance-P release, is therefore a useful and selective tool to induce flares. Use of capsaicin in this context was usually by topical application, but intradermal administration has been found to be equally if not more useful (Simone et al 1987). lontophoretic application of capsaicin (or other substances) is less painful than intradermal injection or topical application but flare onset is slower than injection and the technique is subject to electrical artefact and a consequent flare induced by electrical stimulation.

3.3 Sympathetic Reflexes

3.3.1 —Sweating

Pilomotor responses were described by Lewis and Marvin (1927) as “goose skin” following strong faradic stimulation which depended on an intact sympathetic nerve supply in the skin.

Faradic stimuli can also produce local sweating via post ganglionic cholinergic sympathetic nerves involving many overlapping axon systems as described by Bickford (1938) and Wilkins, Newman and Doupe (1938).

Other stimuli were found to induce piloerection and sweating including intradermal acetylcholine, both directly by a muscarinic action and indirectly via axon reflex (Coon and Rothman 1940), and nicotine by axon reflex (Coon

and Rothman 1941). The pilomotor and sweat responses were independent of each other since ergotamine abolished the piloerection only, and hence was adrenergic at some point along the reflex, and atropine abolished the sweating only, and hence was cholinergic at some independent point (Coon and Rothman 1940). Both reactions were blocked by local anaesthetics or peripheral nerve degeneration, suggesting the role of neurons, and both, by

virtue of their presence in in vitro preparations or peripheral nerve which had

been sectioned or undergone anaesthetic block, were independent of the central nervous system. Coon and Rothman coined the term "nicotine test”, akin to Lewis’ histamine test but to confirm the integrity of the sympathetic nervous system in the skin rather than nociceptor fibres.

Collins and Weiner (1961) were able to show that there was a bell-shaped dose-response relationship between intradermal nicotine concentration and sweat reaction. They noted that whilst nicotine produced 3 rapid reactions, piloerection, flare and sweating, all maximal within 1 minute, methacholine gave a direct muscarinic-like sweat response only, which was slow in onset as the drug diffused from the injection site. Local anaesthetic could abolish the effect of nicotine but not that of methacholine. Acetylcholine gave a reaction composed of both muscarinic and nicotine-like effects. Since stimulation of the dorsal root in the cat did not give a sweat reaction, they suggested that nicotine’s action was at “free” nerve endings in the skin acting like sensory/mechanoreceptors, rather than at any other point along the axon reflex pathway and also in preference to a theoretical peripheral nerve ganglion. This would spread along the system of interdigitating axon branches innervating sweat glands visualised by Wilkins, Newman and Doupe (1938). Sweating had been measured by obtaining sweat prints from applying starch- iodine paper to the skin for a couple of seconds, silastic imprint, colour changes in an indicator such as quinizarin powder in response to moistness and alterations in skin electrical resistance. However, an alternative method was described by Low and colleagues (1983), the Quantitative Sudomotor Axon Reflex Test or Q-SART where a cell consisting of 3 concentric compartments was placed on the skin. In the outer-most was placed

acetylcholine which could be iontophoresed into the skin. Both the drug and the electrical stimulation induced axon reflex sweating, with a latency of 1 to 2 minutes, and sweat output was measured as the rise in humidity in a constant flow of nitrogen through the inner-most compartment. They compared Q- SART responses with Thermoregulatory sweat tests (TST), sweating in response to ambient temperature. In combination the tests differentiated the site of sweating disorders: when both pre- and post-ganlionic fibres were involved TST was less than Q-SART (compared to normal subjects when TST exceeded Q-SART); post-ganglionic denervation was suggested by a reduced or absent Q-SART. Complementary information on sweating is also available using the other methods mentioned earlier (Low 1994).

More immediate assessment of changes in humidity relating to sweat responses can be obtained with an evaporimeter (Abdel-Rahman et al 1992).

3.3.2 —The venoarteriolar axon reflex

The venoarteriolar axon reflex occurs when the venous pressure in small veins rises by 25 mmHg in response to limb dependency. Impulses along local sympathetic 0 fibres result in arteriolar vasoconstriction which reduces blood flow by about 50% (Henriksen 1977). Its clinical usefulness has been questioned by Moy et al (1989) who confirmed that reflex vasoconstriction was significantly less in diabetic subjects when compared to controls but remarked on the considerable overlap among groups.

4. METHODS

4.1 SUBJECTS

4.1.1 Diabetic Subjects:

Fifty-five diabetic patients were enrolled from a diabetic outpatients clinic or following neurological referral for neuropathic symptoms. Subjects were initially selected by review of their notes to include the age range 20 to 70 years and to confirm suitability for investigation. In order to minimise possible confounding clinical features subjects were excluded if they were undergoing neurological investigation or review for disorders other than diabetic neuropathy, if they had a past history of strokes or TIAs, or if they were known to suffer peripheral vascular disease or malignancy. Other exclusion criteria were previous psychiatric disease and severe English language difficulties so that a full and complete clinical assessment could be made, sensory tests performed and informed consent obtained. Patients were also excluded if they had major neuropathic complications including neuropathic ulceration so that the risks of poor healing following skin biopsy could be minimised. From this selection, patients were presented randomly to the clinician.

Of the 55 patients recruited to the study over a 1 year period, 28 suffered IDDM (defined as having required insulin treatment from diagnosis) and 27 NIDDM , of which 20 were controlled with insulin (having been managed adequately on diet/hypoglycaemic agents for at least 1 year in the past). Table 4 below further defines these populations.

Table 4: Age and Duration of Diabetes in Insulin and Non-Insulin Dependent Diabetic Groups. A G E D U R A T 1 O N Number Group Sub­ group Mean (years) Range (years) Standard Deviation (years) Mean (years) Range (years) Standard Deviation (years) IDDM All 39.82 23-68 11.99 18.77 1-52 11.38 28 Males 40.94 23-68 13.13 19.15 1.5-52 12.44 17 Females 38.09 27-55 10.33 18.18 1-33 10.05 11 NIDDM All 56.04 30-68 9.81 11.41 1-23 6.74 27 Males 56.83 30-68 10.74 12.14 1-23 7.19 18 Females 54.44 43-65 7.95 9.94 1.5-17 5.85 9

The IDDM group were significantly younger (p<0.0001) and had diabetes for significantly longer (p=0.005) than the NIDDM group.

Full clinical examinations were performed on all diabetic individuals and were assigned “Neurological Symptom and Disability Scores” for symptoms and signs respectively according to Dyck et al (1980). This provided a scoring system for the number and severity of symptoms and signs for each subject. (See Appendix 2). Abnormality was accepted as NSS >0 and NDS >6 (Dyck et al 1985).

To provide a simple assessment of peripheral vascular insufficiency, the presence of dorsalis pedis and posterior tibial pulses for right and left feet was noted and each subject scored 1 per pulse, to give a crude over all score with a maximum of four. A score of 2/4 and over was accepted as normal.

Diabetic control was assessed by measuring H b A lc. Only the results from the 26 IDDM subjects and 17 NIDDM subjects from the diabetes clinic have been used in this analysis since a different laboratory technique was employed for samples taken from patients referred to the neurology clinic. Mean HbAlc values (±SEM) for the IDDM group were 7.62(±0.32) and for NIDDM 8.18(±0.46). There was no significant difference between the diabetic groups (unpaired t-test). Reference laboratory range was 4.2-5.9%.

Blood tests excluded significant renal impairment in all diabetic patients. However, 3 subjects had serum creatinines just above the reference range of 62-106 pmol/l, 2 of which were shown to have proteinuria of 1.76g/l (female IDDM) and 0.88g/l (male NIDDM) respectively, although specific treatment had not been required. Of the other 40 whose urine was tested, 16 had microalbuminuria above 20 mg/l. (Urine samples could not obtained at clinic for the other subjects.)

All subjects undenA/ent nerve function testing as described below.

Thirty-eight proceeded to skin biopsy, 18 subjects with IDDM (11 male, 7 female) and 20 with NIDDM (13 male, 7 female). Mean ages and duration of diabetes were similar to diabetic populations as defined above (table 5).

Table 5: Age and Duration of Diabetes in Insulin and Non-Insulin Dependent Diabetic Individuals who underwent Skin Biopsy

A G E D U R A T 1 O N Number Group Sub­ group Mean (years) Range (years) Standard Deviation (years) Mean (years) Range (years) Standard Deviation (years) IDDM All 38.41 23-68 11.78 19.73 1-37 9.08 18 Males 37.60 23-68 12.55 20.25 1.5-37 8.63 11 Females 39.57 29-55 11.46 19.00 1-33 10.34 7 NIDDM All 55.55 30-68 10.68 10.30 5.91 1-23 20 Males 57.15 30-68 11.82 10.50 1-23 6.03 13 Females 52.57 43-65 8.10 9.93 1.5-17 6.11 7 4.1.2 Control Subjects:

Small and large fibre function tests were carried out on subjects from a pool of

75 controls 43 males (mean (±SD) age 40.37±13.13; range 22-69) and 32

females (40.06±14.54; 20-68) who were hospital or research staff or relatives of patient subjects. A sub-group of these subjects were used as age-matched controls for the NIDDM groups (table 6). The number of control values obtained for each test and site was comparable to the number of diabetics results available.

Table 6: Details of Control Groups

Group Sub­ group Mean (years) Range (years) Standard Deviation (years) Number All All 38.24 20-69 13.75 75 Controls Males 40.37 22-69 13.13 43 Females 40.06 20-68 14.54 32 Controls All 56.69 46-69 6.34 26 Aged Males 56.33 46-69 6.48 15 45-70 Females 57.18 48-68 6.43 11

Skin biopsies were obtained from normal non-diabetic subjects undergoing operative procedures to the lower limb under GA. Eight males (mean age ±

standard deviation 29.25±9.77 years, age range 20-46 years) had dorso-lateral

calf skin taken at sural nerve harvesting prior to brachial plexus nerve grafting

for brachial plexus injury and from four females (42.50±17.46; 27-63) 2

provided lateral calf skin during exploration of the common peroneal nerve, 1 medial calfskin during exploration of the Achilles tendon and one mid-thigh skin as a skin flap was fashioned for the lower leg skin grafting.

METHODS