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Autonomic nervous function assessment using thermal reactivity of microcirculation
Clinical Neurophysiology 111 (2000) 1880±1888
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Autonomic nervous function assessment using thermal reactivity of microcirculation q Raphael M. Bonelli, Peter KoÈltringer* Department of Neurology and Psychiatry, Hospital BHB Eggenberg, Bergstraûe 27, 8021 Graz, Austria Accepted 18 July 2000
Abstract There are only a few reliable objective methods of diagnosing peripheral neuronal damage suitable for routine use; the most important is based on measurement of nerve conduction velocity, which only shows changes when severe disturbances are already present. However, it is precisely at this stage that the possibilities of therapy are no longer satisfactory. As small ®bres are affected earlier in the course of most forms of PNP than the large ones, assessment of afferent as well as efferent C-®bre function gains importance in the management of this widespread disease. In assessment of autonomic dysfunction, variability of the heartbeat with deep breathing or the Valsalva manoeuvre is a good and generally accepted test, although not strongly associated with other PNP test abnormalities. However, axonal degeneration starts in the most distal parts of the axon due to impaired axonal transport. Therefore, the longest C-®bres, i.e. in the lower extremities, are affected ®rst, and incipient changes are most prominent there. For this reason HLDF, a re¯ex response of the skin blood ¯ow stimulated by heat, has advantages in assessment of early C-®bre dysfunction. Considering the fact that the afferent and efferent sympathetic C-®bres are involved in regulation of microcirculation, the skin blood ¯ow regulation is investigated by means of laser Doppler ¯owmetry. The microcirculation is stimulated by heat and the reaction of microcirculation is assessed as a value for the function of afferent and efferent (sympathetic) C-®bres. The results of this method are in close correlation with electrophysiologic tests, which is not achieved with sudomotor function. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C-®bre function; Skin blood ¯ow; Stimulation by heat; Reactivity of microcirculation; Autonomic dysfunction; Autonomic PNP
1. Introduction During the last 3 decades, many attempts have been made to develop objective methods to diagnose different forms and symptoms of polyneuropathy (PNP) and other peripheral neuronal damage (Ward et al., 1971; Kennedy et al., 1984; Tobin et al., 1999). According to the American Diabetes Association (1988) and the Rochester Diabetic Neuropathy Study (Dyck et al., 1992), evaluations for diagnosis and staging of PNP should include assessment of (a) neuropathic symptoms, (b) neuropathic de®cits, (c) nerve conduction, (d) quantitative sensory examination, and (e) quantitative autonomic examination. The attributes of nerve conduction (Brown and Asbury, 1984) are the compound muscle action potential and distal latencies of the ulnar, median, peroneal, and tibial nerves, the motor nerve conduction velocity (Hodes et al., 1948) of q The two authors contributed equally to this work. * Corresponding author. Tel.: 143-316-59892000; fax: 143-31659892380. E-mail address: [email protected] (P. KoÈltringer).
the same nerves and the sensory nerve action potentials of the ulnar, median, and sural nerves (Dawson, 1956). Measurement of nerve conduction velocity, however, only shows changes when severe disturbances are already present. But it is precisely at this stage that the possibilities of therapy are no longer satisfactory. It is important to recognize neuropathic or polyneuropathic changes at an earlier stage, which can be carried out and used in routine operation and which thus makes a timely therapeutic treatment of the neuropathy possible. Indeed, 28.2% of diabetic PNP patients present only signs or symptoms of PNP without nerve conduction velocity abnormalities (Sangiorgio et al., 1997). We think that changes in small ®bres precede changes in the large ones, and this can be measured by studying C-®bre function. Quantitative autonomic examination of efferent ®bres is usually revealed nowadays by heartbeat variability on the one hand (parasympathetic C-®bres) (Bonnet and Arand, 1997; Vaughn et al., 1995), and the sudomotor axon re¯ex test or the sweat gland excretion tests on the other hand (sympathetic C-®bres) (Matsunaga et al., 1998; Kanzato et al., 1997; Rossini et al., 1993a,b; Elie and Guiheneuc,
1388-2457/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S13 88-2457(00)0042 4-7
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R.M. Bonelli, P. KoÈltringer / Clinical Neurophysiology 111 (2000) 1880±1888
1990). Variability of the heartbeat with deep breathing or the Valsalva manoeuvre is a good and generally accepted test, although not strongly associated with other PNP test abnormalities (Dyck et al., 1992). However, axonal degeneration starts in the most distal parts of the axon due to impaired axonal transport. Therefore, the longest C-®bres, i.e. in the lower extremities, are affected ®rst, and incipient changes are most prominent there. However, the results gained with sudomotor function are not in close correlation with electrophysiologic tests (Dyck et al., 1992), and sweat gland excretion tests (Kennedy et al., 1984; Raethjen et al., 1997), on the other hand, are very complicated in clinical practice, and most of them are not quantitative. For studying the function of afferent C-®bres, a single test based on pain stimulation has been developed that examines C-®bre together with Ad-®bre function (Bromm and Lorenz, 1998; Svensson et al., 1997; Siedenberg and Treede, 1996; Kanda et al., 1996; Kakigi et al., 1989; Treede et al., 1988), called laser evoked potentials. The big problem with this method ± where an ultra-short burning stimulus is achieved by CO2 laser ± is the local effects on the skin. In patients who are prone to skin ulceration, like diabetic PNP (but in fact in nearly every C-®bre pathology), this method is contraindicated. As a second disadvantage, the high costs of this method have to be mentioned, which additionally limit its use in clinical practice to a signi®cant degree. A method for assessment of efferent (sympathetic) and afferent C-®bre function has been developed based on the physiology of the peripheral re¯ex arc which induces vasodilatation in hyperthermia. To explain this method properly, the principles of this re¯ex arc have to be described ®rst. 2. Principles of the re¯ex arc Regulation of microcirculation in human tissue is in¯uenced by in¯ammatory, metabolic and toxic factors and mediated through sympathetic tonus and vasoactive agents (Kodama et al., 1995; Yamaguchi et al., 1994; Meyer et al., 1993; Iadecola, 1993; Hyslop and De-Nucci, 1993). Hyperthermia itself plays a major role in upregulation of microcirculation in the skin. During a hyperthermic challenge, the skin blood ¯ow increases primarily through activation of the cutaneous active vasodilator system (Magerl and Treede, 1996). However, mechanisms through which activation of this system elevates skin blood ¯ow remain unknown (Crandall et al., 1997). In the elderly the cutaneous circulation has shown a reduced vasoreactive capacity when stimulated by heat, and autonomic dysfunction developing in older age is suggested (Van-den-Brande et al., 1997b). This ageing phenomenon affecting the microvascular tone and reactivity might be one of the elements responsible for the attenuated cutaneous vasoreactivity in response to heat (Kenney et al., 1997; Inoue and Shibasaki, 1996).
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2.1. Vasomotion and microcirculation Vasomotion is the rhythmic contraction exhibited by the resistance vessels (i.e. small arteries and arterioles) (Tenland et al., 1983; Bernardi et al., 1996). These ¯uctuations seem to be under central autonomic in¯uence (Bernardi et al., 1997a) and can be synchronized by hypersystolic leg compression and by thermal stimulation (Jahnukainen et al., 1997). Cholinergic nerve activation seems to mediate cutaneous active vasodilatation through release of an unknown co-transmitter, not through acetylcholine (Kellogg et al., 1995). Recent ®ndings emphasize a role of neuropeptide Y in the regulation of the cutaneous microcirculation (Pinter et al., 1996, 1997). On the other hand, the effect of sympathetic stimulation seems to be mediated by the direct vasoconstrictor action of catecholamines on the vascular smooth muscle cells (Schachinger and Zeiher, 1996). Some authors suggest that this microvascular motion is impaired in diabetic subjects compared with healthy control subjects due to sympathetic dysfunction (Bernardi et al., 1997b). In a recent study, these oscillations in laser Doppler microvascular measurements taken from the pulpar surface were impaired in diabetic subjects in comparison with healthy controls (Stansberry et al., 1996). Cutaneous arteriovenous anastomoses modulated by sympathetic stimuli (Du-Buf-Vereijken et al., 1997) regulate the regional blood ¯ow in the skin and play an important part in regulating the body temperature both generally and locally. If the local temperature is raised above 408C, the connecting vessel relaxes and an increased ¯ow results, with a consequent cooling effect (Williams and Warwick, 1980). In the feet of patients with diabetic neuropathy in the resting state, total skin blood ¯ow was shown to be increased due to an increased anastomotic shunt ¯ow (i.e. lower sympathetic tonus). Some studies suggest that this increased anastomotic shunt ¯ow leads to an overperfused nutritive capillary circulation in the feet of patients with diabetic neuropathy (Netten et al., 1996). This is why in feet of some patients with an autonomic polyneuropathy syndrome a higher baseline skin temperature is found (Netten et al., 1996), which is called rubeosis diabetica when clinically apparent. 2.2. The role of C-®bres in the regulation of microcirculation Also sensory C-®bres have been implicated in the control of microvascular tone, in¯uencing both arteriolar and venous microvessels (Ahluwalia and Vallance, 1997). Electrical antidromic stimulation of cutaneous C-®bres evokes vasodilatation preceded by a short vasoconstriction in the skin (Pinter et al., 1996, 1997; Yonehara et al., 1992), especially C-®bres of the polymodel nociceptive class (Gee et al., 1997). This effect was attenuated by pre-medication with capsaicin (Yonehara et al., 1992), a strong inactivator of C-®bre function (Gronroos and Pertovaara, 1993). The possible release of vasoactive substances from C-®bres may
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play a role in local microcirculation (Vass et al., 1994; Whittle and Lopez-Belmonte, 1993), i.e. calcitonin generelated peptide (Whittle and Lopez-Belmonte, 1993; Brain et al., 1993; Guth, 1992; Hong et al., 1997), substance P (Yonehara et al., 1992; Piedimonte et al., 1992; Milner et al., 1995; Vass et al., 1995) and nitric oxide (NO) (Vass et al., 1995; Konturek, 1997; Merchant et al., 1995). Other theories suggest that sympathetic ®bres activate endothelium cells, which themselves release NO to counteract the sympathetic vasoconstriction (Schachinger and Zeiher, 1996; Habler et al., 1997; Nase and Boegehold, 1997). However, the exact mechanism of interaction between afferent C-®bres and microcirculation is far from being clari®ed. Very confusing data about the role of C-®bers and their potent inactivator capsaicin (the main pungent ingredient in hot chilli peppers) (Caterina et al., 1997) on skin perfusion are available today. C-®bers are very sensitive to the neurotoxic capsaicin, which can deplete neuropeptide stores in C-®bers and in high doses cause degeneration of C-®bre afferent pathways (Vizzard et al., 1995). On the one hand, several studies on rats (Ahluwalia and Vallance, 1997; Hong et al., 1997; Piedimonte et al., 1992; Schaafsma et al., 1997; Abdel Salam et al., 1996; Herbert et al., 1995) and rabbits (Brain et al., 1993) revealed a local vasodilatation caused by capsaicin, maybe due to neurogenic in¯ammation (Schaafsma et al., 1997; Zochodne and Ho, 1994; Petersen et al., 1997) (proportions of nociceptors (naked endings of C-®bers) seem to contain neuropeptides such as substance P and calcitonin gene-related peptide, which are released from the activated nociceptive terminals and cause neurogenic in¯ammation, including pre-capillary vasodilatation and post-capillary plasma extravasation (Messlinger, 1997)). In a recent study in intact human skin in vivo capsaicin 2% ointment applied on the skin for 2.5 h increased skin blood ¯ow by 300±400% as measured by laser Doppler ¯owmetry (Petersen et al., 1997). According to some authors, intradermal injections of capsaicin in humans induces an axon re¯ex vasodilatation mediated by C-®bers (Jolliffe et al., 1995). This effect of vasodilatation is changed to vasoconstriction in higher doses of capsaicin (Vass et al., 1994; Dembinski et al., 1995), which could be explained as a depletion of vasodilatative neuropeptides in C-®bre endings (Whittle and Lopez-Belmonte, 1993; Tepperman and Whittle, 1992). On the other hand, in the pig vasodilatation (a result of a release of vasoactive substances from capsaicin-sensitive nerve endings) was blocked by capsaicin (Bartho et al., 1992). The same results were observed in humans, where capsaicin delayed (KoÈltringer, 1996) or even abolished (Magnusson and Koskinen, 1996) the vasodilatative response to local heat and other vasodilatative stimuli in some studies (Del-Bianco et al., 1996). 2.3. Sense organs and afferent root Because the sense organs are located subepithelially, it is
the temperature of the subcutaneous tissues that determines the responses. Cool metal objects feel colder that wooden objects of the same temperature because the metal conducts heat away from the skin more rapidly, cooling the subcutaneous tissues to a greater degree. Below a skin temperature of 208C and above 408C, there is no adaptation, but between 20 and 408C there is adaptation, so that the sensation produced by a temperature change gradually fades to one of thermal neutrality (Ganong, 1987). In the skin there are two types of temperature sense organs: those responding maximally to temperature slightly above body temperature (so-called mechanoheat nocireceptors) (Schmidt et al., 1997; Kirschstein et al., 1997), and those responding maximally to temperatures slightly below body temperature (so-called mechanocold nocireceptors) (Simone and Kajander, 1997). Mapping experiments show that there are discrete cold-sensitive and warmthsensitive spots in the skin (Ganong, 1987). There are 4±10 times as many cold spots as warm. Some authors distinguish slowly adapting warm-sensitive units from rapidly adapting warm-sensitive units (Adelson et al., 1997). Nociceptors can be de®ned as sensory receptors that are activated by noxious stimuli that damage or threaten the body's integrity. Nociceptors belong to the slowly conducting afferent Ad- and C®bres. They are classi®ed according to their responses to mechanical, thermal, and chemical stimuli. In the skin high-threshold mechano-nociceptors and mechano-heat nociceptors of A- and C-®bres are frequently found. The latter are usually called polymodal C-®bres if they also show chemosensitive properties (Messlinger, 1997; Toda et al., 1997). Recent ®ndings in the animal model indicate that heat stimuli may directly activate capsaicin-sensitive primary nociceptive afferents (i.e. C®bers). Using temperatures between 41 and 538C rat dorsal root ganglion capsaicin-sensitive small neurons exhibited a heat-evoked inward current in vitro, whereas large neurons did not respond to heat and were not sensitive to capsaicin either (Kirschstein et al., 1997). However, the temperature sense organs are naked nerve endings (synonym: free nerve endings) that respond to absolute temperature, not the temperature gradient across the skin (Ganong, 1987). Free nerve endings, which are regarded as the morphological correlatives of nociceptors, usually consist of bundles of unmyelinated ®bres. With electron microscopy varicose segments of the sensory axon are visible that are characterized by free areas of axolemma, accumulations of mitochondria and vesicles, and a modi®ed axoplasm. These presumptive receptive sites are periodically arranged along the whole course of the sensory endings at a length of up to several hundred microns. Additionally, the ®ne sensory endings are branched, forming tree-like structures, and frequently innervate different types of tissues (Messlinger, 1997). Sensations of sharp pain (®rst pain) are evoked by intraneural microstimulation of nociceptive Ad-®bres, whereas stimulation of C-®bres causes dull pain sensations (second
R.M. Bonelli, P. KoÈltringer / Clinical Neurophysiology 111 (2000) 1880±1888
pain). Also thermal stimuli delivered to the skin of the arms or legs can produce these two distinct pains (Yeomans and Proud®t, 1996; Yeomans et al., 1996; Adelson et al., 1997). Under appropriate conditions ®rst pain decreases in intensity (adaptation) while second pain increases in intensity (slow temporal summation) (Harkins et al., 1996). In animal models, behavioural nociceptive responses evoked by relatively low rates (0.98C/s) of skin heating appear to be mediated by the activation of C-®bre nociceptors, but evoke only a few action potentials in Ad nociceptors. C-®bers begin ®ring at a rate of less than 1 Hz between 8 and 10 s after the onset of heating and ®re at a mean rate of 1.5 Hz between 10 and 12 s, which corresponds to the latency of the foot withdrawal response (Yeomans and Proud®t, 1996; Yeomans et al., 1996). Noxious heat stimuli (15 s ramp from 32 to 478C measured at the corium side of the skin of 2-week-old mice) excited 26% of Ad-®bres with a threshold of 42.58C and 41% of C-®bers with a mean threshold of 37.68C (Koltzenburg et al., 1997). It could be concluded that painful thermal stimuli are mediated by Ad-®bres and temperature is mediated through C-®bers. Scienti®c research will provide new understanding in the following years. However, the recently identi®ed capsaicin receptor in sensory C-®bers is thought to function as a transducer of thermal stimuli in vivo in humans (Caterina et al., 1997). The latter afferents, conducting information on second pain and temperature, are small, unmyelinated ®bres of 0.2±1 mm in diameter in Erlanger and Gasser's C group (Erlanger and Gasser, 1937) (Lloyd's group IV ®bres; Lloyd, 1943) with a conduction velocity of 0.6 m/s (Zenker, 1985). This nerve ®bre quality plays an important role in the theoretical fundamentation of our new method of hyperthermal laser Doppler ¯owmetry. 2.4. Central nervous system and efferent root From C-®bers, impulses (e.g. information on hyperthermia) are conducted in the peripheral and the central axon of the pseudounipolar spinal ganglion cell in the dorsal root ganglion (Zhang et al., 1997) through the posterior radix to the substancia gelatinosa of the spinal cord where the neurite ends, one or two segments above the entrance of the posterior radix (Duus, 1987). A recent study in an animal model revealed that some kinds of nociception appear to be partially mediated by the release of substance P in the spinal cord dorsal horn from terminals of primary afferent nociceptors (Zachariou et al., 1997). Speci®cally, substance P appears to be released by unmyelinated (C) nociceptive afferents when activated by noxious heat stimulation to the skin, but does not appear to be contained in cutaneous myelinated (Ad) nociceptive afferents. From the substancia gelatinosa impulses pass on the one hand via the lateral spinothalamic tract (temperature and pain) and the lemniscus spinalis to the thalamus. On the
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other hand, another path could lead as part of a vegetative re¯ex arc (Duus, 1987) through the various synapses in the substancia gelatinosa to the visceromotor cells in the lateral horn of the spinal cord, from where the efferent sympathetic nerves make their way to the vessels of microcirculation. In the thalamus, the second neuron ends. From here, information reaches consciousness via the thalamic radiation to the post-central gyrus of the parietal lobe. Another path goes from the thalamus to the hypothalamic paraventricular and posterior nucleus. Also afferent ®bres from the limbic system end here. The central temperature regulation is located in this area. From here, efferent paths go through the lateral horn of the spinal cord to the visceromotor cells in the ganglia of the sympathetic trunk chain, from where the cutaneous blood vessel diameter and the arteriovenous anastomoses (and thus perfusion) are mediated through sympathetic tonus (Brandt et al., 1993). 2.5. Conclusion To summarize, parts of the re¯ex arc are still not clear today. What we do not know is whether impulses (e.g. information about local hyperthermia) pass through the spinal cord to the hypothalamic temperature centre to initiate local vasodilatation, or if these impulses take the short way via the synapses in the substancia gelatinosa of the spinal cord with the same result. Maybe both paths are involved. However, the afferent arm to the spinal cord, represented by C-®bers, and the efferent part from the spinal cord, represented by sympathetic nerve ®bres (also C-®bers), is clear in all cases. These two paths are the theoretical fundamentation of our new method of hyperthermal laser Doppler ¯owmetry. 3. New methods using laser Doppler ¯owmetry (LDF) 3.1. Sympathetic nerve function assessment The measurement of vasoconstrictor responses to deep inspiration by using LDF is quite a novel method for detecting peripheral sympathetic failure (Baron and Engler, 1996; Schurmann et al., 1996; Valensi et al., 1997; Abbot et al., 1993, 1996; Baron et al., 1993; Ando et al., 1992). After deep inspiration, the vasoconstrictor response in the ®ngers or tips of the toes is measured (Wilder-Smith and WilderSmith, 1996; Wilder-Smith et al., 1996, 1997). A skin temperature of approximately 348C is the optimal temperature for the evaluation of skin vasomotor re¯exes. The vasoconstrictor response to deep inspiration in the big toe was signi®cantly decreased in NIDDM patients compared with healthy subjects in a recent study (Asob et al., 1997). However, during hyperventilation no changes in microcirculation can be measured (Steurer et al., 1995). The vasomotor response was achieved only on the ®ngers, but not on
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the volar forearm (Kurvers et al., 1996) and was suppressed by amitriptyline (Muck-Weymann and Rechlin, 1996). Another method uses severe and prolonged local cold to assess the changes on the cutaneous microcirculation. In a single study of 10 healthy probands, perfusion was measured by LDF at the calf during 20 min of local ice cooling and 15 min of subsequent recovery. In the sixth minute of cooling, the mean skin ¯ux decreased to 58% of the resting value and then increased and reached 129% of the resting value at the end of the cooling period, followed by a phase of reactive hyperaemia with a maximum of 225% (Van-den-Brande et al., 1997a). No clinical study has been done (nor will be done) with this method, but similar methods may be useful in patients with Raynaud's phenomenon (Klyscz et al., 1997). Some other forms of sympathetic activation have been found, including the contralateral cooling described in some studies (Schurmann et al., 1996; Abbot et al., 1993), the visuogenic re¯ex via ¯ashes of light (Kolev et al., 1997), the postural vasoconstriction arteriolar re¯ex (Cacciatori et al., 1997; Valensi et al., 1997), the Valsalva manoeuvre (Valensi et al., 1997; Abraham et al., 1986), or the microcirculation change of an extremity in dependency (Gordon, 1996). 3.2. Assessment of C-®ber function Some studies describe a determination of C-®ber function by measurement of axon re¯ex vasodilatation and ¯are size induced by histamine iontophoresis (Baron et al., 1993; Baron and Engler, 1996). The problem of this method is the responsiveness to iontophoresis of histamine: 41.1% of the units are unresponsive to histamine, 44.6% respond only weakly with a few spike discharges after iontophoresis, and only 14.3% of the units respond with sustained discharges to histamine, and their discharge patterns match the time course of the itch sensations (Schmelz et al., 1997). So the function of only a small part of C-®bers is measured with this method. Another possibility would be the painful axon re¯ex vasodilatation in response to intradermal capsaicin, as measured by laser Doppler ¯owmetry (Raethjen et al., 1997; Abbot et al., 1996). Some electromyographic experiments in arti®cially ventilated and anaesthetised rats elicited a two-component re¯ex response in the ipsilateral biceps femoris muscle when stimulated electrically to the sural nerve territory. The second, late component had a longer latency, longer duration, and higher threshold and is thought to be due to afferent signals transmitted via C-®bers (Falinower et al., 1994). As is evident, this method is far from clinical use.
ting such exact results, because in HLDF the perfusion oscillations due to the phenomenon of vasomotion are corrected mathematically (and by that objecti®ed and so made comparable) by the special curve-®tting program. In the vasoconstrictor response to deep inspiration, per de®nitionem only the time of one inspiration can be measured, which leads to quite variable data even when standardization of the inspiratory gasp test by spirometric control has taken place (Du-Buf-Vereijken et al., 1997). Therefore, severe disadvantages are obvious in the alternative methods using LDF.
4. Hyperthermal laser Doppler ¯owmetry (HLDF) 4.1. Methodology of HLDF Considering the fact that the autonomic nerve ®bres and C-®bres are involved in microcirculation, our method investigates the skin blood ¯ow regulation. Peter KoÈltringer developed a standardized method which he called hyperthermal laser Doppler ¯owmetry (HLDF). In this method the microcirculation is stimulated by heat and the time between the onset of heat induction and the de®nitive increase of microcirculation is measured (KoÈltringer et al., 1989). This de®nitive increase represents a parameter for autonomic nerve and C-®ber function in skin (see Fig. 1). To stabilize microcirculation we use an adaptation time of 20 min in a standard room climate (238C and 60±65% relative humidity) prior to the onset of measurement, where the patient is lying in the prone position. The measurement itself is then carried out in 5 min. The skin microcirculation is tested by means of LDF (Holloway and Watkins, 1977; Stern, 1975; Tooke et al., 1983; Stern et al., 1977) with 650 nm (He/Ne-Laser, Typ Per¯ux PF3, Fa. Peri¯ux, Sweden). For inducing hyperthermia, an infrared lamp (100 W, Fa. Phillips, Germany) is used at a distance of 35 cm from the skin of dorsum pedis. Blood ¯ow measurement begins 30 s before the onset of hyperthermia and it ends 5 min later. If the skin temperature exceeds 378C, the infrared bulb is switched off automatically. It is necessary for the reliability of data to achieve a 48C difference (as a minimum) in skin temperature between the start and the end of the test.
3.3. Valuation of the alternative methods using LDF All these methods have been developed several years after the establishment of HLDF in 1988. In contrast to the latter, no method is able to study both ®bre functions simultaneously. Moreover, no method is capable of permit-
Fig. 1. Methodology of HLDF.
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Fig. 2. Parameters of HLDF.
4.2. Parameters of HLDF With specially developed software, including a curve®tting program and an artefact ®lter, it is possible to distinguish between two parameters: Hyperthermal Perfusion Latency and Fluctuation Index (see Fig. 2). The Hyperthermal Perfusion Latency is de®ned as the delay between the onset of hyperthermia and the ®rst reaching of perfusion of the line of 10% of the maximum value of perfusion reached in the whole experiment. It is a value for the function of afferent C-®bers, as we saw in clinical practice. This suggestion was veri®ed by a pilot study with selective blocking of the C-®bers by capsaicin (Gronroos and Pertovaara, 1993), which abolishes the vasodilatative response to local heat (Magnusson and Koskinen, 1996; KoÈltringer and Reisecker, 1997). In this project (KoÈltringer, 1996), the application of capsaicin increased the HTPL from 50 to 200 s within 20 min. As perfusion increases steadily during hyperthermia, maximum perfusion will be reached at the end of the measurement in healthy probands. The Fluctuation Index (FI) is de®ned as the maximal decline of perfusion from an already reached value. It is a value for the function of efferent sympathetic C-®bers. Again this was veri®ed by the same pilot study (KoÈltringer, 1996), where we selectively blocked the sympathetic ®bres by Levobunolol (Egorov and Shmeleve, 1992; Bosen et al., 1992). Here, the application of the substance caused a marked increase in the FI from 10% of the maximum value of perfusion at the beginning to 60% ¯uctuation after 20 min. 4.3. Clinical relevance of HLDF HLDF seems to be a reliable method in clinical practice in the following conditions: 1. HLDF could be an important diagnostic method to prevent diabetic foot ulceration, as autonomic PNP is a principal cause of foot ulceration and no substance has proven to be effective in autonomic PNP when symptoms are established. 2. Furthermore, HLDF seems to be a useful method in preventing other late symptoms of autonomic PNP. As
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regulation of cutaneous perfusion is often part of the symptoms of autonomic PNP, marked defects in cardiovascular, thermoregulatory, gastrointestinal, urogenital, or respiratory regulation (depressed CO2 response), erectile impotence, vesical atonia, and the lacking recognition of hypoglycaemia could possibly be predicted. 3. HLDF is possibly the best method for the early diagnosis of re¯ex sympathetic dystrophy, since early treatment signi®cantly improves the chances for a successful outcome. 4. HLDF could be an important help in patients suffering from suspect carpal tunnel syndrome with normal median nerve conduction velocity.
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Autonomic nervous function assessment using thermal reactivity of microcirculation q Raphael M. Bonelli, Peter KoÈltringer* Department of Neurology and Psychiatry, Hospital BHB Eggenberg, Bergstraûe 27, 8021 Graz, Austria Accepted 18 July 2000
Abstract There are only a few reliable objective methods of diagnosing peripheral neuronal damage suitable for routine use; the most important is based on measurement of nerve conduction velocity, which only shows changes when severe disturbances are already present. However, it is precisely at this stage that the possibilities of therapy are no longer satisfactory. As small ®bres are affected earlier in the course of most forms of PNP than the large ones, assessment of afferent as well as efferent C-®bre function gains importance in the management of this widespread disease. In assessment of autonomic dysfunction, variability of the heartbeat with deep breathing or the Valsalva manoeuvre is a good and generally accepted test, although not strongly associated with other PNP test abnormalities. However, axonal degeneration starts in the most distal parts of the axon due to impaired axonal transport. Therefore, the longest C-®bres, i.e. in the lower extremities, are affected ®rst, and incipient changes are most prominent there. For this reason HLDF, a re¯ex response of the skin blood ¯ow stimulated by heat, has advantages in assessment of early C-®bre dysfunction. Considering the fact that the afferent and efferent sympathetic C-®bres are involved in regulation of microcirculation, the skin blood ¯ow regulation is investigated by means of laser Doppler ¯owmetry. The microcirculation is stimulated by heat and the reaction of microcirculation is assessed as a value for the function of afferent and efferent (sympathetic) C-®bres. The results of this method are in close correlation with electrophysiologic tests, which is not achieved with sudomotor function. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C-®bre function; Skin blood ¯ow; Stimulation by heat; Reactivity of microcirculation; Autonomic dysfunction; Autonomic PNP
1. Introduction During the last 3 decades, many attempts have been made to develop objective methods to diagnose different forms and symptoms of polyneuropathy (PNP) and other peripheral neuronal damage (Ward et al., 1971; Kennedy et al., 1984; Tobin et al., 1999). According to the American Diabetes Association (1988) and the Rochester Diabetic Neuropathy Study (Dyck et al., 1992), evaluations for diagnosis and staging of PNP should include assessment of (a) neuropathic symptoms, (b) neuropathic de®cits, (c) nerve conduction, (d) quantitative sensory examination, and (e) quantitative autonomic examination. The attributes of nerve conduction (Brown and Asbury, 1984) are the compound muscle action potential and distal latencies of the ulnar, median, peroneal, and tibial nerves, the motor nerve conduction velocity (Hodes et al., 1948) of q The two authors contributed equally to this work. * Corresponding author. Tel.: 143-316-59892000; fax: 143-31659892380. E-mail address: [email protected] (P. KoÈltringer).
the same nerves and the sensory nerve action potentials of the ulnar, median, and sural nerves (Dawson, 1956). Measurement of nerve conduction velocity, however, only shows changes when severe disturbances are already present. But it is precisely at this stage that the possibilities of therapy are no longer satisfactory. It is important to recognize neuropathic or polyneuropathic changes at an earlier stage, which can be carried out and used in routine operation and which thus makes a timely therapeutic treatment of the neuropathy possible. Indeed, 28.2% of diabetic PNP patients present only signs or symptoms of PNP without nerve conduction velocity abnormalities (Sangiorgio et al., 1997). We think that changes in small ®bres precede changes in the large ones, and this can be measured by studying C-®bre function. Quantitative autonomic examination of efferent ®bres is usually revealed nowadays by heartbeat variability on the one hand (parasympathetic C-®bres) (Bonnet and Arand, 1997; Vaughn et al., 1995), and the sudomotor axon re¯ex test or the sweat gland excretion tests on the other hand (sympathetic C-®bres) (Matsunaga et al., 1998; Kanzato et al., 1997; Rossini et al., 1993a,b; Elie and Guiheneuc,
1388-2457/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S13 88-2457(00)0042 4-7
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1990). Variability of the heartbeat with deep breathing or the Valsalva manoeuvre is a good and generally accepted test, although not strongly associated with other PNP test abnormalities (Dyck et al., 1992). However, axonal degeneration starts in the most distal parts of the axon due to impaired axonal transport. Therefore, the longest C-®bres, i.e. in the lower extremities, are affected ®rst, and incipient changes are most prominent there. However, the results gained with sudomotor function are not in close correlation with electrophysiologic tests (Dyck et al., 1992), and sweat gland excretion tests (Kennedy et al., 1984; Raethjen et al., 1997), on the other hand, are very complicated in clinical practice, and most of them are not quantitative. For studying the function of afferent C-®bres, a single test based on pain stimulation has been developed that examines C-®bre together with Ad-®bre function (Bromm and Lorenz, 1998; Svensson et al., 1997; Siedenberg and Treede, 1996; Kanda et al., 1996; Kakigi et al., 1989; Treede et al., 1988), called laser evoked potentials. The big problem with this method ± where an ultra-short burning stimulus is achieved by CO2 laser ± is the local effects on the skin. In patients who are prone to skin ulceration, like diabetic PNP (but in fact in nearly every C-®bre pathology), this method is contraindicated. As a second disadvantage, the high costs of this method have to be mentioned, which additionally limit its use in clinical practice to a signi®cant degree. A method for assessment of efferent (sympathetic) and afferent C-®bre function has been developed based on the physiology of the peripheral re¯ex arc which induces vasodilatation in hyperthermia. To explain this method properly, the principles of this re¯ex arc have to be described ®rst. 2. Principles of the re¯ex arc Regulation of microcirculation in human tissue is in¯uenced by in¯ammatory, metabolic and toxic factors and mediated through sympathetic tonus and vasoactive agents (Kodama et al., 1995; Yamaguchi et al., 1994; Meyer et al., 1993; Iadecola, 1993; Hyslop and De-Nucci, 1993). Hyperthermia itself plays a major role in upregulation of microcirculation in the skin. During a hyperthermic challenge, the skin blood ¯ow increases primarily through activation of the cutaneous active vasodilator system (Magerl and Treede, 1996). However, mechanisms through which activation of this system elevates skin blood ¯ow remain unknown (Crandall et al., 1997). In the elderly the cutaneous circulation has shown a reduced vasoreactive capacity when stimulated by heat, and autonomic dysfunction developing in older age is suggested (Van-den-Brande et al., 1997b). This ageing phenomenon affecting the microvascular tone and reactivity might be one of the elements responsible for the attenuated cutaneous vasoreactivity in response to heat (Kenney et al., 1997; Inoue and Shibasaki, 1996).
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2.1. Vasomotion and microcirculation Vasomotion is the rhythmic contraction exhibited by the resistance vessels (i.e. small arteries and arterioles) (Tenland et al., 1983; Bernardi et al., 1996). These ¯uctuations seem to be under central autonomic in¯uence (Bernardi et al., 1997a) and can be synchronized by hypersystolic leg compression and by thermal stimulation (Jahnukainen et al., 1997). Cholinergic nerve activation seems to mediate cutaneous active vasodilatation through release of an unknown co-transmitter, not through acetylcholine (Kellogg et al., 1995). Recent ®ndings emphasize a role of neuropeptide Y in the regulation of the cutaneous microcirculation (Pinter et al., 1996, 1997). On the other hand, the effect of sympathetic stimulation seems to be mediated by the direct vasoconstrictor action of catecholamines on the vascular smooth muscle cells (Schachinger and Zeiher, 1996). Some authors suggest that this microvascular motion is impaired in diabetic subjects compared with healthy control subjects due to sympathetic dysfunction (Bernardi et al., 1997b). In a recent study, these oscillations in laser Doppler microvascular measurements taken from the pulpar surface were impaired in diabetic subjects in comparison with healthy controls (Stansberry et al., 1996). Cutaneous arteriovenous anastomoses modulated by sympathetic stimuli (Du-Buf-Vereijken et al., 1997) regulate the regional blood ¯ow in the skin and play an important part in regulating the body temperature both generally and locally. If the local temperature is raised above 408C, the connecting vessel relaxes and an increased ¯ow results, with a consequent cooling effect (Williams and Warwick, 1980). In the feet of patients with diabetic neuropathy in the resting state, total skin blood ¯ow was shown to be increased due to an increased anastomotic shunt ¯ow (i.e. lower sympathetic tonus). Some studies suggest that this increased anastomotic shunt ¯ow leads to an overperfused nutritive capillary circulation in the feet of patients with diabetic neuropathy (Netten et al., 1996). This is why in feet of some patients with an autonomic polyneuropathy syndrome a higher baseline skin temperature is found (Netten et al., 1996), which is called rubeosis diabetica when clinically apparent. 2.2. The role of C-®bres in the regulation of microcirculation Also sensory C-®bres have been implicated in the control of microvascular tone, in¯uencing both arteriolar and venous microvessels (Ahluwalia and Vallance, 1997). Electrical antidromic stimulation of cutaneous C-®bres evokes vasodilatation preceded by a short vasoconstriction in the skin (Pinter et al., 1996, 1997; Yonehara et al., 1992), especially C-®bres of the polymodel nociceptive class (Gee et al., 1997). This effect was attenuated by pre-medication with capsaicin (Yonehara et al., 1992), a strong inactivator of C-®bre function (Gronroos and Pertovaara, 1993). The possible release of vasoactive substances from C-®bres may
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play a role in local microcirculation (Vass et al., 1994; Whittle and Lopez-Belmonte, 1993), i.e. calcitonin generelated peptide (Whittle and Lopez-Belmonte, 1993; Brain et al., 1993; Guth, 1992; Hong et al., 1997), substance P (Yonehara et al., 1992; Piedimonte et al., 1992; Milner et al., 1995; Vass et al., 1995) and nitric oxide (NO) (Vass et al., 1995; Konturek, 1997; Merchant et al., 1995). Other theories suggest that sympathetic ®bres activate endothelium cells, which themselves release NO to counteract the sympathetic vasoconstriction (Schachinger and Zeiher, 1996; Habler et al., 1997; Nase and Boegehold, 1997). However, the exact mechanism of interaction between afferent C-®bres and microcirculation is far from being clari®ed. Very confusing data about the role of C-®bers and their potent inactivator capsaicin (the main pungent ingredient in hot chilli peppers) (Caterina et al., 1997) on skin perfusion are available today. C-®bers are very sensitive to the neurotoxic capsaicin, which can deplete neuropeptide stores in C-®bers and in high doses cause degeneration of C-®bre afferent pathways (Vizzard et al., 1995). On the one hand, several studies on rats (Ahluwalia and Vallance, 1997; Hong et al., 1997; Piedimonte et al., 1992; Schaafsma et al., 1997; Abdel Salam et al., 1996; Herbert et al., 1995) and rabbits (Brain et al., 1993) revealed a local vasodilatation caused by capsaicin, maybe due to neurogenic in¯ammation (Schaafsma et al., 1997; Zochodne and Ho, 1994; Petersen et al., 1997) (proportions of nociceptors (naked endings of C-®bers) seem to contain neuropeptides such as substance P and calcitonin gene-related peptide, which are released from the activated nociceptive terminals and cause neurogenic in¯ammation, including pre-capillary vasodilatation and post-capillary plasma extravasation (Messlinger, 1997)). In a recent study in intact human skin in vivo capsaicin 2% ointment applied on the skin for 2.5 h increased skin blood ¯ow by 300±400% as measured by laser Doppler ¯owmetry (Petersen et al., 1997). According to some authors, intradermal injections of capsaicin in humans induces an axon re¯ex vasodilatation mediated by C-®bers (Jolliffe et al., 1995). This effect of vasodilatation is changed to vasoconstriction in higher doses of capsaicin (Vass et al., 1994; Dembinski et al., 1995), which could be explained as a depletion of vasodilatative neuropeptides in C-®bre endings (Whittle and Lopez-Belmonte, 1993; Tepperman and Whittle, 1992). On the other hand, in the pig vasodilatation (a result of a release of vasoactive substances from capsaicin-sensitive nerve endings) was blocked by capsaicin (Bartho et al., 1992). The same results were observed in humans, where capsaicin delayed (KoÈltringer, 1996) or even abolished (Magnusson and Koskinen, 1996) the vasodilatative response to local heat and other vasodilatative stimuli in some studies (Del-Bianco et al., 1996). 2.3. Sense organs and afferent root Because the sense organs are located subepithelially, it is
the temperature of the subcutaneous tissues that determines the responses. Cool metal objects feel colder that wooden objects of the same temperature because the metal conducts heat away from the skin more rapidly, cooling the subcutaneous tissues to a greater degree. Below a skin temperature of 208C and above 408C, there is no adaptation, but between 20 and 408C there is adaptation, so that the sensation produced by a temperature change gradually fades to one of thermal neutrality (Ganong, 1987). In the skin there are two types of temperature sense organs: those responding maximally to temperature slightly above body temperature (so-called mechanoheat nocireceptors) (Schmidt et al., 1997; Kirschstein et al., 1997), and those responding maximally to temperatures slightly below body temperature (so-called mechanocold nocireceptors) (Simone and Kajander, 1997). Mapping experiments show that there are discrete cold-sensitive and warmthsensitive spots in the skin (Ganong, 1987). There are 4±10 times as many cold spots as warm. Some authors distinguish slowly adapting warm-sensitive units from rapidly adapting warm-sensitive units (Adelson et al., 1997). Nociceptors can be de®ned as sensory receptors that are activated by noxious stimuli that damage or threaten the body's integrity. Nociceptors belong to the slowly conducting afferent Ad- and C®bres. They are classi®ed according to their responses to mechanical, thermal, and chemical stimuli. In the skin high-threshold mechano-nociceptors and mechano-heat nociceptors of A- and C-®bres are frequently found. The latter are usually called polymodal C-®bres if they also show chemosensitive properties (Messlinger, 1997; Toda et al., 1997). Recent ®ndings in the animal model indicate that heat stimuli may directly activate capsaicin-sensitive primary nociceptive afferents (i.e. C®bers). Using temperatures between 41 and 538C rat dorsal root ganglion capsaicin-sensitive small neurons exhibited a heat-evoked inward current in vitro, whereas large neurons did not respond to heat and were not sensitive to capsaicin either (Kirschstein et al., 1997). However, the temperature sense organs are naked nerve endings (synonym: free nerve endings) that respond to absolute temperature, not the temperature gradient across the skin (Ganong, 1987). Free nerve endings, which are regarded as the morphological correlatives of nociceptors, usually consist of bundles of unmyelinated ®bres. With electron microscopy varicose segments of the sensory axon are visible that are characterized by free areas of axolemma, accumulations of mitochondria and vesicles, and a modi®ed axoplasm. These presumptive receptive sites are periodically arranged along the whole course of the sensory endings at a length of up to several hundred microns. Additionally, the ®ne sensory endings are branched, forming tree-like structures, and frequently innervate different types of tissues (Messlinger, 1997). Sensations of sharp pain (®rst pain) are evoked by intraneural microstimulation of nociceptive Ad-®bres, whereas stimulation of C-®bres causes dull pain sensations (second
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pain). Also thermal stimuli delivered to the skin of the arms or legs can produce these two distinct pains (Yeomans and Proud®t, 1996; Yeomans et al., 1996; Adelson et al., 1997). Under appropriate conditions ®rst pain decreases in intensity (adaptation) while second pain increases in intensity (slow temporal summation) (Harkins et al., 1996). In animal models, behavioural nociceptive responses evoked by relatively low rates (0.98C/s) of skin heating appear to be mediated by the activation of C-®bre nociceptors, but evoke only a few action potentials in Ad nociceptors. C-®bers begin ®ring at a rate of less than 1 Hz between 8 and 10 s after the onset of heating and ®re at a mean rate of 1.5 Hz between 10 and 12 s, which corresponds to the latency of the foot withdrawal response (Yeomans and Proud®t, 1996; Yeomans et al., 1996). Noxious heat stimuli (15 s ramp from 32 to 478C measured at the corium side of the skin of 2-week-old mice) excited 26% of Ad-®bres with a threshold of 42.58C and 41% of C-®bers with a mean threshold of 37.68C (Koltzenburg et al., 1997). It could be concluded that painful thermal stimuli are mediated by Ad-®bres and temperature is mediated through C-®bers. Scienti®c research will provide new understanding in the following years. However, the recently identi®ed capsaicin receptor in sensory C-®bers is thought to function as a transducer of thermal stimuli in vivo in humans (Caterina et al., 1997). The latter afferents, conducting information on second pain and temperature, are small, unmyelinated ®bres of 0.2±1 mm in diameter in Erlanger and Gasser's C group (Erlanger and Gasser, 1937) (Lloyd's group IV ®bres; Lloyd, 1943) with a conduction velocity of 0.6 m/s (Zenker, 1985). This nerve ®bre quality plays an important role in the theoretical fundamentation of our new method of hyperthermal laser Doppler ¯owmetry. 2.4. Central nervous system and efferent root From C-®bers, impulses (e.g. information on hyperthermia) are conducted in the peripheral and the central axon of the pseudounipolar spinal ganglion cell in the dorsal root ganglion (Zhang et al., 1997) through the posterior radix to the substancia gelatinosa of the spinal cord where the neurite ends, one or two segments above the entrance of the posterior radix (Duus, 1987). A recent study in an animal model revealed that some kinds of nociception appear to be partially mediated by the release of substance P in the spinal cord dorsal horn from terminals of primary afferent nociceptors (Zachariou et al., 1997). Speci®cally, substance P appears to be released by unmyelinated (C) nociceptive afferents when activated by noxious heat stimulation to the skin, but does not appear to be contained in cutaneous myelinated (Ad) nociceptive afferents. From the substancia gelatinosa impulses pass on the one hand via the lateral spinothalamic tract (temperature and pain) and the lemniscus spinalis to the thalamus. On the
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other hand, another path could lead as part of a vegetative re¯ex arc (Duus, 1987) through the various synapses in the substancia gelatinosa to the visceromotor cells in the lateral horn of the spinal cord, from where the efferent sympathetic nerves make their way to the vessels of microcirculation. In the thalamus, the second neuron ends. From here, information reaches consciousness via the thalamic radiation to the post-central gyrus of the parietal lobe. Another path goes from the thalamus to the hypothalamic paraventricular and posterior nucleus. Also afferent ®bres from the limbic system end here. The central temperature regulation is located in this area. From here, efferent paths go through the lateral horn of the spinal cord to the visceromotor cells in the ganglia of the sympathetic trunk chain, from where the cutaneous blood vessel diameter and the arteriovenous anastomoses (and thus perfusion) are mediated through sympathetic tonus (Brandt et al., 1993). 2.5. Conclusion To summarize, parts of the re¯ex arc are still not clear today. What we do not know is whether impulses (e.g. information about local hyperthermia) pass through the spinal cord to the hypothalamic temperature centre to initiate local vasodilatation, or if these impulses take the short way via the synapses in the substancia gelatinosa of the spinal cord with the same result. Maybe both paths are involved. However, the afferent arm to the spinal cord, represented by C-®bers, and the efferent part from the spinal cord, represented by sympathetic nerve ®bres (also C-®bers), is clear in all cases. These two paths are the theoretical fundamentation of our new method of hyperthermal laser Doppler ¯owmetry. 3. New methods using laser Doppler ¯owmetry (LDF) 3.1. Sympathetic nerve function assessment The measurement of vasoconstrictor responses to deep inspiration by using LDF is quite a novel method for detecting peripheral sympathetic failure (Baron and Engler, 1996; Schurmann et al., 1996; Valensi et al., 1997; Abbot et al., 1993, 1996; Baron et al., 1993; Ando et al., 1992). After deep inspiration, the vasoconstrictor response in the ®ngers or tips of the toes is measured (Wilder-Smith and WilderSmith, 1996; Wilder-Smith et al., 1996, 1997). A skin temperature of approximately 348C is the optimal temperature for the evaluation of skin vasomotor re¯exes. The vasoconstrictor response to deep inspiration in the big toe was signi®cantly decreased in NIDDM patients compared with healthy subjects in a recent study (Asob et al., 1997). However, during hyperventilation no changes in microcirculation can be measured (Steurer et al., 1995). The vasomotor response was achieved only on the ®ngers, but not on
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the volar forearm (Kurvers et al., 1996) and was suppressed by amitriptyline (Muck-Weymann and Rechlin, 1996). Another method uses severe and prolonged local cold to assess the changes on the cutaneous microcirculation. In a single study of 10 healthy probands, perfusion was measured by LDF at the calf during 20 min of local ice cooling and 15 min of subsequent recovery. In the sixth minute of cooling, the mean skin ¯ux decreased to 58% of the resting value and then increased and reached 129% of the resting value at the end of the cooling period, followed by a phase of reactive hyperaemia with a maximum of 225% (Van-den-Brande et al., 1997a). No clinical study has been done (nor will be done) with this method, but similar methods may be useful in patients with Raynaud's phenomenon (Klyscz et al., 1997). Some other forms of sympathetic activation have been found, including the contralateral cooling described in some studies (Schurmann et al., 1996; Abbot et al., 1993), the visuogenic re¯ex via ¯ashes of light (Kolev et al., 1997), the postural vasoconstriction arteriolar re¯ex (Cacciatori et al., 1997; Valensi et al., 1997), the Valsalva manoeuvre (Valensi et al., 1997; Abraham et al., 1986), or the microcirculation change of an extremity in dependency (Gordon, 1996). 3.2. Assessment of C-®ber function Some studies describe a determination of C-®ber function by measurement of axon re¯ex vasodilatation and ¯are size induced by histamine iontophoresis (Baron et al., 1993; Baron and Engler, 1996). The problem of this method is the responsiveness to iontophoresis of histamine: 41.1% of the units are unresponsive to histamine, 44.6% respond only weakly with a few spike discharges after iontophoresis, and only 14.3% of the units respond with sustained discharges to histamine, and their discharge patterns match the time course of the itch sensations (Schmelz et al., 1997). So the function of only a small part of C-®bers is measured with this method. Another possibility would be the painful axon re¯ex vasodilatation in response to intradermal capsaicin, as measured by laser Doppler ¯owmetry (Raethjen et al., 1997; Abbot et al., 1996). Some electromyographic experiments in arti®cially ventilated and anaesthetised rats elicited a two-component re¯ex response in the ipsilateral biceps femoris muscle when stimulated electrically to the sural nerve territory. The second, late component had a longer latency, longer duration, and higher threshold and is thought to be due to afferent signals transmitted via C-®bers (Falinower et al., 1994). As is evident, this method is far from clinical use.
ting such exact results, because in HLDF the perfusion oscillations due to the phenomenon of vasomotion are corrected mathematically (and by that objecti®ed and so made comparable) by the special curve-®tting program. In the vasoconstrictor response to deep inspiration, per de®nitionem only the time of one inspiration can be measured, which leads to quite variable data even when standardization of the inspiratory gasp test by spirometric control has taken place (Du-Buf-Vereijken et al., 1997). Therefore, severe disadvantages are obvious in the alternative methods using LDF.
4. Hyperthermal laser Doppler ¯owmetry (HLDF) 4.1. Methodology of HLDF Considering the fact that the autonomic nerve ®bres and C-®bres are involved in microcirculation, our method investigates the skin blood ¯ow regulation. Peter KoÈltringer developed a standardized method which he called hyperthermal laser Doppler ¯owmetry (HLDF). In this method the microcirculation is stimulated by heat and the time between the onset of heat induction and the de®nitive increase of microcirculation is measured (KoÈltringer et al., 1989). This de®nitive increase represents a parameter for autonomic nerve and C-®ber function in skin (see Fig. 1). To stabilize microcirculation we use an adaptation time of 20 min in a standard room climate (238C and 60±65% relative humidity) prior to the onset of measurement, where the patient is lying in the prone position. The measurement itself is then carried out in 5 min. The skin microcirculation is tested by means of LDF (Holloway and Watkins, 1977; Stern, 1975; Tooke et al., 1983; Stern et al., 1977) with 650 nm (He/Ne-Laser, Typ Per¯ux PF3, Fa. Peri¯ux, Sweden). For inducing hyperthermia, an infrared lamp (100 W, Fa. Phillips, Germany) is used at a distance of 35 cm from the skin of dorsum pedis. Blood ¯ow measurement begins 30 s before the onset of hyperthermia and it ends 5 min later. If the skin temperature exceeds 378C, the infrared bulb is switched off automatically. It is necessary for the reliability of data to achieve a 48C difference (as a minimum) in skin temperature between the start and the end of the test.
3.3. Valuation of the alternative methods using LDF All these methods have been developed several years after the establishment of HLDF in 1988. In contrast to the latter, no method is able to study both ®bre functions simultaneously. Moreover, no method is capable of permit-
Fig. 1. Methodology of HLDF.
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Fig. 2. Parameters of HLDF.
4.2. Parameters of HLDF With specially developed software, including a curve®tting program and an artefact ®lter, it is possible to distinguish between two parameters: Hyperthermal Perfusion Latency and Fluctuation Index (see Fig. 2). The Hyperthermal Perfusion Latency is de®ned as the delay between the onset of hyperthermia and the ®rst reaching of perfusion of the line of 10% of the maximum value of perfusion reached in the whole experiment. It is a value for the function of afferent C-®bers, as we saw in clinical practice. This suggestion was veri®ed by a pilot study with selective blocking of the C-®bers by capsaicin (Gronroos and Pertovaara, 1993), which abolishes the vasodilatative response to local heat (Magnusson and Koskinen, 1996; KoÈltringer and Reisecker, 1997). In this project (KoÈltringer, 1996), the application of capsaicin increased the HTPL from 50 to 200 s within 20 min. As perfusion increases steadily during hyperthermia, maximum perfusion will be reached at the end of the measurement in healthy probands. The Fluctuation Index (FI) is de®ned as the maximal decline of perfusion from an already reached value. It is a value for the function of efferent sympathetic C-®bers. Again this was veri®ed by the same pilot study (KoÈltringer, 1996), where we selectively blocked the sympathetic ®bres by Levobunolol (Egorov and Shmeleve, 1992; Bosen et al., 1992). Here, the application of the substance caused a marked increase in the FI from 10% of the maximum value of perfusion at the beginning to 60% ¯uctuation after 20 min. 4.3. Clinical relevance of HLDF HLDF seems to be a reliable method in clinical practice in the following conditions: 1. HLDF could be an important diagnostic method to prevent diabetic foot ulceration, as autonomic PNP is a principal cause of foot ulceration and no substance has proven to be effective in autonomic PNP when symptoms are established. 2. Furthermore, HLDF seems to be a useful method in preventing other late symptoms of autonomic PNP. As
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regulation of cutaneous perfusion is often part of the symptoms of autonomic PNP, marked defects in cardiovascular, thermoregulatory, gastrointestinal, urogenital, or respiratory regulation (depressed CO2 response), erectile impotence, vesical atonia, and the lacking recognition of hypoglycaemia could possibly be predicted. 3. HLDF is possibly the best method for the early diagnosis of re¯ex sympathetic dystrophy, since early treatment signi®cantly improves the chances for a successful outcome. 4. HLDF could be an important help in patients suffering from suspect carpal tunnel syndrome with normal median nerve conduction velocity.
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