WO2005014105A1

WO2005014105A1 - Nerve function and tissue healing

Info

Publication number: WO2005014105A1
Application number: PCT/AU2004/001079
Authority: WO
Inventors: Zeinab Khalil
Original assignee: National Ageing Research Institute Incorporated
Priority date: 2003-08-12
Filing date: 2004-08-12
Publication date: 2005-02-17
Also published as: AU2003904303A0

Abstract

The present invention relates to method of improving the function of an impaired sensory nerve to release growth factors in response to injury, or promoting tissue healing in human patients with impaired sensory nerve function, the methods comprising applying a stimulus to the nerve to stimulate the nerve to release endogenous substance P followed by endogenous calcitonin gene related peptide.

Description

NERVE FUNCTION AND TISSUE HEALING

The present invention relates to nerve function and tissue healing, and more particularly although not exclusively, to means of improving nerve function and management of injured tissues and the prophylaxis and treatment of wounds and chronic pain in patients with peripheral neuropathy, including the elderly. BACKGROUND OF THE INVENTION

Wound repair similar to the repair of other injured tissues including injured nerves is a complex and multifaceted process that is initiated when tissue is injured. The primary goal in the treatment of wounds is to achieve wound closure . Open cutaneous wounds represent one major category of wounds and include burn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers, and diabetic ulcers. Inflammation is essentially a protective response that initiates the process of tissue repair. A major part of the normal inflammatory response is dependent upon intact unmyelinated primary afferent sensory innervation. These particular afferents are termed Capsaicin-sensitive primary afferent nerves (C fibres) because they are selectively destroyed by the neurotoxin Capsaicin, the pungent extract of red peppers of the genus Capsicum. Numerous factors can affect wound healing and repair of any injured tissue, including malnutrition, infection, pharmacological agents (e.g., actinomycin and steroids), diabetes, and neurodegenerative conditions, including advanced age . Neural innervation is an essential prerequisite for normal tissue repair. Upon tissue injury the body responds by increasing local blood supply to the injured area which bring in vital nutrients (including growth factors) and helps in the removal of waste products from the injured site. The increase in local blood supply to the injured tissue is an essential prerequisite for healing. Local blood supply to tissues is under the control of two sets of nerves which regulate the function of blood vessels. One set of nerves, the sensory nerves, increase blood flow by the action of growth factors or sensory neuropeotides such as substance P and calcitonin gene related protein (CGRP) , while the other set, the sympathetic nerves, decrease blood flow. Adequate balance between the function of these two sets of nerves is essential for proper control of local blood flow. The sensory nerves also play a major role in providing the injured tissue with growth factors. With ageing and other conditions like diabetes, there is a decrease in the capacity of individuals to maintain appropriate inflammatory and repair processes. It is known that neurogenic function is impaired with age and neuropathic conditions such as diabetes, HIV or AIDS. This in turn causes an associated impairment in tissue repair. Changes of the nervous system in ageing and neuropathy involve several mechanisms, including apoptosis in some parts of the nervous system and loss of dendrites and dendritic spines in the cortical neurones. The causes of peripheral nerve impairment are numerous . They originate in diminished blood flow through the vessels supplying the peripheral nerves and spinal cord, progressive reduction of myelinated and unmyelinated fibres, irregularities in internodal length, and changes in skin properties regarding the total number of cutaneous receptors, which are irregularly distributed and vary in size and shape. Decreased velocity of nerve conduction and slow central information processing can cause functional differences . Additionally, ageing and neuropathy may cause a fall in cell numbers of the central autonomic nuclei and first- and second-order autonomic neurones outside the central nervous system. The consequences are alterations in heart rate, postural hypotension and inability to recognise temperature changes. Further, the vasomotor and sudomotor responses may be impaired. The inventor has previously reported that exogenous administration of substance P and CGRP accelerated the healing of thermal burn wounds in aged rats to nearly equal the rate of wound healing in young rats (Khalil & Helme (1996) Journal of Gerontology: BIOLOGICAL SCIENCES Vol. 51A, No. 5, B354-B361) supporting the notion that sensory nerves are important for the initiation of wound healing and tissue repair. The inventor has also previously reported that non- invasive transcutaneous electrical nerve stimulation (TENS) could accelerate tissue repair in aged rats with thermal burns to a similar rate as that in young rats but only with low frequencies of activation (20V, 2 ms, 5 Hz, 1 minute) . Application of TENS at 20V, 2 ms, 15 Hz for 1 minute did not result in nerve activation (Khalil & Merhi (2000), Journal of Gerontology: BIOLOGICAL SCIENCES Vol. 55A, No. 6, B257-B263) . The authors also noted that low firing frequencies in nociceptors were sufficient to evoke flare (efferent response) but do not evoke a conscious perception of pain in humans (afferent response) , allowing the TENS method to be used without a concomitant afferent response . Regardless of the inventor's previous work, impaired wound healing in the aged and neuropathically impaired remains a primary concern. Along with the increased incidence and diagnosis of neuropathy, more and more people in the developed world are living into old age. In 1998 there were 2.3 million people aged 65 and over living in Australia and this number is expected to reach at least 6 million by 2051. Old age is associated with many illnesses, including chronic leg and foot wounds. Furthermore, delayed tissue repair in general is a major pathology underlying many neurodegenerative and vascular diseases associated with age. The prevalence of chronic leg wounds is strongly age dependent. There is a clear need to improve wound management in the elderly and other groups having impaired sensory nerve function. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY OF THE INVENTION

The present invention addresses the problems cited above, and provides a method of improving the function of an impaired sensory nerve to release growth factors in response to injury, the method broadly comprising applying a stimulus to the nerve to stimulate the nerve to release endogenous substance P followed by endogenous calcitonin gene related peptide. In a second aspect the present invention provides a method for promoting tissue healing in human patients with impaired sensory nerve function, the method broadly comprising stimulating the impaired sensory nerve to release endogenous substance P followed by endogenous calcitonin gene related peptide. The sensory nerve plays a major role in supplying growth factors such as substance P and CGRP to injured tissue to initiate tissue healing, as well as increasing blood supply to the wound site. The inventor proposes that if the function of the sensory nerves is impaired a compromise of the balance needed to provide adequate blood supply to the wound site might occur causing a reduction in the supply of growth factors to the wound site.

Consequently the overall ability of the tissue to heal is reduced. The inventor has investigated the changes in sensory nerve function on ageing and neuropathy, for example as a result of diabetic neuropathy and has determined that impaired nerves have a reduced ability to produce substance P and CGRP and other growth promoting sensory neuropeptides in response to wound healing. The inventor has found that the ability can be restored at least partially by stimulating the sensory nerve to produce endogenous substance P and CGRP, which has the effect of healing the nerve function. Optionally the stimulus can also improve the ability of the sensory nerve to release other growth promoting sensory neuropeptides after the release of CGRP. The inventor has also investigated the local effect of a wound on normal sensory nerve function and has determined that upon wounding the sensory nerve servicing the wound area is stimulated to release growth factors and other agents to act on the blood vessels surrounding the nerve to increase blood flow and immune response and thus bring about tissue repair. The inventor has determined the magnitude and order of release of growth factors associated with wound healing and has investigated methods of mimicking this in impaired nerves. For purposes of illustration the invention will be described below largely in the context of ageing, which is a condition to which the invention has particular applicability; however, it will be understood that the invention has utility for all patients with impaired sensory nerve function, including but not limited to persons with peripheral neuropathy caused by diabetes, trauma, accidents, chemotherapy, HIV or AIDS.

DETAILED DESCRIPTION OF THE INVENTION

The peripheral nerve trunk is capable of sustaining a flare response as observed in injured skin. Manipulation of the local injury response of the nerve trunk by pharmacological or electrical means may provide one strategy in the treatment of neuropathy. The proposal that activating C fibres innervating the actual nerve affected by the neuropathy is based on a sound understanding of the physiology of the microcirculation of the peripheral nerve (vasa nervorum) . The inventor proposes that the function of impaired sensory nerve function in tissue healing may be improved by stimulating the nerve to produce endogenous growth factors . It is essential that the nerve stimulus provided is selective to provide an afferent response without an efferent response. This is achieved by providing a stimulus which selectively activates the local effector function of C fibres only. The inventor has found that selective activation of C fibres may be achieved by applying an electrical or chemical stimulus to the nerve. Surprisingly the inventor has found that electrical or chemical stimulation is capable of regenerating the activity of an impaired sensory nerve to produce substance P and CGRP in a manner similar to that in a normal sensory nerve. The inventor provides evidence below to show that electrical stimulation with particular parameters when applied to a sensory nerve stimulates the production of endogenous substance P and CGRP in C fibres and improves the function of said nerve to produce substance P and CGRP and other sensory neuropeptides that exhibit growth promoting activities in response to a wound. The inventor also provides evidence below to show that electrical stimulation with particular parameters when applied at the site of a wound accelerates wound healing in aged patients who have chronic leg or foot ulcers, when applied to major sensory nerves that innervate wounds. The stimulation accelerates wound repair in aged people who have chronic leg or foot ulcers, when applied to aged patients with chronic leg or foot ulcers and improves vascular responsiveness in the area innervated by the nerve. Also when the stimulus is applied to major nerves in aged patients with chronic leg or foot ulcers it improves sensory nerve function in the area innervated by the nerves. It is known that C-afferent fibres are involved in wound healing and trophic responses to varying degrees. Further evidence to support the importance of sensory nerves in wound healing come from experimental studies using the sensory neurotoxin Capsaicin, which has been shown to accelerate wound contraction and re- epithelialization of full-thickness wounds following topical application, suggesting that this action was achieved via the release of substance P and/or CGRP from sensory nerves . Peripheral nerves have two vascular components: an extrinsic epineurial vascular plexus and intrinsic endoneurial microvessels . Endonurial blood flow does not autoregulate but is determined by the epinurial blood flow. Epinural vasa nervorum are innervated by vasodilatory peptidergic fibres. The peptidergic perivascular epineurial innervation includes terminals that contain substance P, CGRP and Vasocative Intestinal Polypeptide (VIP) . Like other tissue, peripheral nerves undergo both non-neurogenic and neurogenic forms of inflammation both contributing towards vasodilation, plasma extravasation and the generation of local ectopic discharges from the axons . Neurogenic inflammation of the nerve trunk is associated with activation of perivascular peptidergic afferents to release CGRP and substance P and induce hyperemic response of the nerve trunk. Unlike other tissues the nerve trunk may actually be able to participate in its own injury response in an additional way; when the axons within the nerve trunk are affected they add further peptides, beyond those released by perivascular afferents to the injury milieu and continue to deposit them by means of axoplasmic transport. CGRP may aid in the regeneration of injured nerves via a vasodilatory response as epineurial peptidergic terminals increase blood flow in the endoneurial compartment. An intact epineurial plexus is required which feeds endoneurial icrovessels . Diabetic neuropathy is associated with ischaemia of endoneurial microvessels and reduction in CGRP vasodilation may account for part of this ischaemia. The other major sensory neuropepide, substance P can also exert a powerful mitogenic action on different cell types involved in tissue repair such as fibroblasts, endothelial cells and macrophages . In addition, substance P can stimulate cytokine production by mononuclear cells, as well as stimulating the production of interleukin-1, transforming growth factor-α and epidermal growth factor by eosinophils and macrophages. A further property of substance P is its capacity to induce neovascularization in the cornea, and stimulate DNA synthesis in cultured arterial smooth muscle cells and human skin fibroblasts, the latter effect inhibited by a tachykinin antagonist. The nerve may be stimulated either electrically or chemically. The stimulation must be such as to effect release of endogenous substance P and then CGRP from the sensory nerve . The importance of the order of release of the neuropeptides is that when released as substance P and then CGRP, the effect of substance P is allowed to potentiate. If CGRP were released first substance P would block the continued release of CGRP and the duration of flare would be reduced. By stimulating release of substance P followed by CGRP synergy is provided. As outlined above substance P increases blood flow and is involved in an immune response in response to tissue damage. CGRP is involved in the release of growth factors . Other growth factors and cytokines are produced due to the action of substance P and CGRP. Preferably the electrical stimulation comprises a pulsed signal. The critical parameter for such electrical stimulation is the application to the sensory nerve of a current capable of exciting the nerve fibre. The effective voltage (and hence current) of the pulsed signal needed to excite a given nerve fibre decreases with increasing pulse duration and increasing pulse frequency. The impedance of the effective circuit formed by the skin and tissue layers is also prone to variation owing to, among other variables, the size, type and depth of the fibres and thickness and age of skin. A preferred range of current to evoke a local effector function of the C fibres without sending a pain signal is 0.5-5.0Ma, the exact current varying from patient to patient due to the difference in resistance between patients. Treatment preferably involves generating peak pulse currents up to 5mA flowing through the nerve fibre to be treated. When a peak pulse current of 2.5mA is required, for example, it is apparent using Ohm's law that a 48V pulse must be applied to the electrodes if the impedance of the effective circuit formed is about 20kΩ. Traditionally in electrical stimulation work, voltage was more often cited than current in terms of the stimulus applied. This was done because most researchers considered that voltage was the important parameter and perhaps because it is an easier parameter to measure while being delivered. However this approach presents problems when delivering electrical pulses to the body, particularly through the skin. The skin presents an impedance (Z) to the passing of current (I) and this impedance (Z) can be markedly different between individuals. This becomes an issue when the same voltage (V) is set to be delivered by a stimulator to 2 patients but one of the patients has a much higher skin impedance (for a range of reasons including dryness, oils etc.). What happens is the patient with the lower impedance (Z) , although receiving pulses of the same voltage, will receive more current (I) (milliamps) than the other patient. [This is due to Ohms Law, V = I x Z meaning simply and intuitively that for a set voltage (V) , a higher resistance (Z) of the skin will permit less Current (I) to pass] . More recent thinking suggests that it is not voltage but current (amount of charged particles) which is the 'physiological catalyst' i.e. the more the charge, the greater the effect (or sometimes an inverse relationship) but the size of effect is dependent on amount of charge. So two people being stimulated with identical voltage value pulses may be receiving different amounts of charge over the treatment time and so the outcomes can not be compared. Accordingly in the present invention we talk of the application of current, rather than voltage. Treatment preferably involves the application of a pulsed electrical signal applied twice daily, although not exclusively, each application having a duration of between 1 to 10 minutes with 5 minutes being preferred. The duration between each application may be up to 6 hours, preferably between 3 to 4 hours. The pulsed electrical signal is most preferably a unipolar square wave having about a 1% duty cycle (i.e. 2msec ON, 200msec OFF), a fixed frequency between 1Hz to 10Hz, and a voltage sufficient to cause up to 5mA peak current to flow through said nerve fibre. Successful trials have involved using a fixed 0.5mA, 5Hz unipolar square wave having about a 1% duty cycle. The foregoing parameters remain fixed for the treatment duration once the desired current level is initially set because significantly varying the current during treatment may have detrimental effects. The electrical stimulation may be provided to the nerve peripherally and proximally, via electrodes attached to the skin. Electrodes are conducting pads which are connected to the electrical pulse generator via electrical wires . When used to regenerate nerve function each of the electrode pads is positioned on the skin at a site below which a sensory nerve is located, e.g. in the leg. Preferably the electrodes are positioned axially over the nerve fibre to be treated. When used to invoke wound healing each of the electrode pads may positioned non-invasively on the skin axial to the wound or may be positioned on either side of the wound to be treated. The electrodes may be fastened to the skin surface using non-conductive adhesive tape or Velcro™ which may be incorporated within a dressing or pressure stocking and applied at particular points to stimulate specific peripheral nerves. For example electrodes may be attached to a Velcro™ strap so that they straddle either side of the common peronal nerve or saphenous nerve. The skin may be prepared for attachment of the electrodes using an alcohol swap. Electric conducting paste may be applied to the electrode prior to the application of the electrode to the skin. Once the electrodes have been attached to a patient's skin as prescribed, an operator sets the required pulse frequency of the electrical pulse generator and ensures that the application voltage is initially set to zero. The pulse generator is subsequently switched on and the voltage is gradually increased, using a voltage dial of the electrical pulse generator, until the required level of peak current flows in the effective circuit including the nerve fibre being treated. The applied current can be read by the operator from an ammeter provided, which measures the peak current flowing through either electrode. The patient may feel a slight tingling sensation in the region being treated during treatment . After the treatment period has concluded, the operator switches off the pulse generator. The electrodes may remain in their position within the dressing or pressure stocking although it is preferred that the generated is disconnected from the electrodes. The foregoing procedure may be performed twice a day, although not exclusively, with between three to four hours break in between. The operator may be the patient or may be a skilled or non skilled operator, a nurse or a doctor. It is the intention that once the initial parameters for a patient have been determined that the patient may be able to administer the treatment regime themselves, preferably in a home environment, thus reducing the burden on hospitals and doctor's surgeries. A suitable means for applying the electrical parameters according to an embodiment of the invention is by use of an adapted TENS machine. Electrotherapies such as transcutaneous electrical nerve stimulation (TENS) are predominantly used for the symptomatic management of acute and non-malignant chronic pain. TENS is a non-invasive electrotherapy treatment involving the electrical stimulation of cutaneous electrode pads positioned in a dermatomal pattern on the surface of the skin. During TENS, pulsed currents are generated by a portable pulse generator and delivered across the intact surface of the skin via the electrodes. The electrical characteristics are chosen with a view to selectively activate different populations of nerve fibres thereby producing different analgesic outcomes . TENS machines generally available are not adapted for low frequency. Application of electrical stimulation via a TENS machine set at high frequency (as is used for pain relief) provides a reduction of function of sensory nerve fibres. TENS machines currently available are not suitable for use according to the present invention as the parameters needed can not be produced. As an alternative to TENS other electrical therapies are available to deliver the required current to the nerve fibre being treated. For example, percutaneous electrical nerve stimulation (PENS) involves the invasive positioning of acupuncture-like needle probes in soft tissue to stimulate peripheral sensory nerves and has the benefit of bypassing the resistance of the cutaneous barrier thereby delivering the electrical stimulus in closer proximity to the nerve endings . Although testing to date has involved the application of a unipolar pulse signal during treatment, other alternating current (AC) signals could be used. Using rectified sine pulses may benefit the effectiveness of the treatment by removing the high frequency harmonics associated with square pulses. In addition, using a bipolar signal may have the benefit of reducing the build up of ion concentrations forming beneath the electrodes thereby preventing adverse skin reactions which result due to polar concentrations. The inventor also contemplates that method of the present invention may be administered by means of electrodes provided on a strap or in a surgical stocking for example, which electrodes may be connected to means for generating the electrical stimulation. The method of the present invention may also be carried out by chemical or pharmacological means. For example the application of Capsaicin peripheral to a nerve may have the effect of stimulating the production of endogenous substance P and then CGRP effective to regenerate nerve function. Capsaicin may also be administered to promote wound healing in patients with damaged sensory nerves. Due to the potency of Capsaicin it preferably is not applied directly to the wound as this would prove too painful . It may however be applied to nerves away from the wound site. Capsiacin may be administered topically as a cream. Preferably the cream contains only a low dose of Capsaicin, so as to avoid stinging and irritation. Alternatively the cream may contain a less potent analogue of Capsaicin. The invention here is based on the idea of sensitising C fibres rather than desensitizing them. The method of the present invention may be carried out on any person with impaired sensory nerve function. Preferably the patient has impaired function in unmyelinated afferent C fibres. Impaired sensory function is associated with neuropathy, including diabetic neuropathy, and peripheral neuropathy caused by ageing, chemotherapy, autoimmune diseases, HIV or AIDS. When applied for wound healing the method of the present invention may be used to treat wounds, particularly those of the limbs and back, such as ulcers, including venous ulcers, pressure sores such as bed sores, burns, cuts and abrasions. In order to carry out the method of the present invention the inventor had had to create a new electrical device. Accordingly in a second aspect the present invention provides an electrical apparatus comprising an electrical generator for generating a stimulus capable of inducing flare when applied to a nerve C fibre, the electrical pulse generator having two or more application electrodes, an anode (+) and a cathode (-) , wherein the electrical generator is adapted to produce an electrical signal comprising a unipolar square wave having about a 1% duty cycle, a fixed frequency between 1Hz to 10Hz, and a voltage sufficient to cause up to 5mA peak current to flow though a nerve C fibre . Preferably, the electrical signal has a frequency of 5Hz and is capable of causing at least 0.5mA peak current to flow through a nerve fibre . Preferably the application electrodes are releasably attachable to the electrical generator. The application electrodes may take the form of a series of electrodes provided on a strap or bandage . Preferably the application electrodes are provided in the form of a stocking, sock, garter or cuff. Most preferably the application electrodes are provided in compression stocking. Preferably, the electrical pulse generator is adapted to sense the skin impedance and adjust the voltage of the pulses generated accordingly. For example when the stimulator is set to 2 millamps, it will deliver 2 milliamps regardless of skin impedance as it will increase the voltage if the impedance is high OR reduce the voltage if the skin impedance is lower as indicated by V = I x Z. In a further aspect the present invention provides a method for the prevention or treatment of pressure sores comprising administering a low level dose of topical Capsaicin to a patient to stimulate sensory nerves. This method is particularly relevant for bedridden patients and for patients left for long periods on trolleys awaiting treatment. Such patients may be prone to pressure sores (including bed sores) . Capsaicin administration is capable of providing a flare in nerve function and this acts to prevent and/or treat pressure sores . For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning. It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification. The invention will now be described in detail by way of reference only to the following non-limiting examples and drawings .

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows bar graphs demonstrating changes in sensory nerve activity in response to LF-TENS in two groups of diabetic patients with peripheral neuropathy that received sham and active treatments respectively. Figure la demonstrates that the active group showed a reduction in reaction time required to respond, indicating improvement in nerve function. Figure lb demonstrates that the active group showed an increase in vascular response in response to a sensory stimulus indicating improvement in nerve function. Figure 2 shows bar graphs indicating the rate of healing of leg ulcers in patients who received active and sham treatments respectively. Figure 3 shows bar graphs indicating the change of transcutaneous oxygen tension in patients with diabetic patients who received active and sham treatments respectively. Figure 4 shows bar graphs indicating any change in microvascular blood flow in diabetic patients with peripheral neuropathy who received active and sham treatments respectively.

EXAMPLES EXAMPLE 1

THE STUDY Study design - efficacy study - randomised placebo controlled double blind trial of parallel group design. Study design - descriptive study - descriptive study of the alteration in vascular responsiveness and in sensation for the modalities of vibration (A beta fibres) and temperature sensation (Adelta fibres for cold and C fibres for warmth) following Large Fibre Trans Electrical Stimulation (LF-TENS) . Use of a between groups comparison and within subject comparisons, comparing LF-TENS treated legs with non treated legs. Study participants - inclusion criteria - persons 55 or older having a venous leg ulcer for at least 6 weeks. Study participants - exclusion criteria - inability to consent to the research; an above or below knee amputation stump wound; an ABI of <0.8; living more than 20 km from the study centre; judged too sick to take part in the study (calculated creatine clearance less than 30 ml/min; albumin concentration less than 25 g/1; severe immobility or other serious medical condition judged by experienced clinicians as at a significant risk of death during the study) have vasculitis or who smoke. Setting - The National Ageing Research Institute (NARI) , in a climate controlled room and at the Melbourne Extended Care and Rehabilitation Service Wound Clinic (MECRS) . Recruitment - participants were recruited from the Royal District Nursing Service and MECRS. Randomisation - after consent was obtained the patients were randomly allocated to one of two groups: A - active LF-TENS applied proximally to nerves that innervate the wound, B - sham LF-TENS applied proximally to nerves that innervate the wound. Block randomisation was used with classification by presence or absence of neuropathy to ensure study participants with neuropathy were equally distributed between groups. Allocation was in sealed, sequentially numbered opaque envelopes. Allocation was generated from a random nuber table. Allocation remained masked throughout the study. Baseline measures - sensory nerve function - nerve fibres were measured using the sensory nerve stimulator at NARI. Large fibres tested are the Aδ and Aβ fibres at a testing frequency of 250 and 2000 Hz respectively. These nerves mediate pressure and vibration. Small fibres tested were the C fibres, these having a testing frequency of 5 Hz. Each nerve threshold was measured separately using the double random staircase procedure (Gracely et al . , 1988 Pain 32:55-63) minimising the trails required. Testing was 5cm from the rostral border from the wound and was performed on a mirror image location on the other limb. Transcutaneous oxygen tension was tested using Radiometer™ machine. At the same time Laser Digital Flowmetry was used on a site 5cm from the rostral edge of the wound. The flare response 5cm from the rostral edge of he wound was established using capsaicin. This produced an area of eythema that was traced onto clear acetate at 15 minute intervals until the flare started to reduce in size. The area of maximum erythema was measured using a digital planimeter (Tamaya, Japan) . Vascular responsiveness at a mirror image site on the other limb was also established. Baseline measurements - wound variables - the wounds and medical conditions of the study participants was as required for clinical management. Arterial ankle-brachial doppler pressure ratios were measured. ENS Units - the study used purpose built active and sham ENS units. Active units were capable of stimulation at the following parameters: lOv (equivalent to between 0.5 and 5 mA depending on the impedence of the skin of the patient) , 5Hz, 2ms square waves for 5- minutes, twice daily. Active and sham ENS had two light emitting diodes (LEDs) that flash when the units are "activated" . The green light flashes when the unit is working and a red light flashed when the unit is no longer providing an appropriate charge. The sham ENS was electrically inactive. Group 1 - active ENS applied proximally to nerves that innervate the wound: Electrodes stimulated the common peroneal and saphenous nerves. Electrodes were affixed to a strap, appropriately locating them for each individual when the strap is affixed such that marks straddle the tibial tuberosity. Study participants affixed the strap twice daily. Group 2 - sham ENS applied proximal to the nerve that innervate the wound: Electrodes for sham LF-TENS units, indistinguishable from the active units were applied as in Group 1. All participants received standard wound care of ProFore™ and Allevyn™. Sample size estimation - we extrapolated that LF-TENS would result in healing in close to 100% of wounds by 12 weeks, with a conservative sample size estimation of 80%. A sample size of 60 per limb was seen as adequate.

TESTING Patients were given a small nerve stimulator unit (ENS unit as described above) and asked to apply this to the appropriate nerve twice daily for 12 weeks. Electrodes were attached to a Velcro strap. The strap was applied in such a way that the electrodes straddle either side of the common peroneal nerve or saphenous nerve. The skin was prepared using an alcohol swab. Electric conducting paste was applied to the electrode prior to the electrode being applied to the skin. The stimulation was applied for five minutes, twice daily for 12 weeks. The patients were reviewed at week 0 and week 12 to undertake tests for blood flow and nerve fuction. Sensory nerve function of the three different sensory nerves was ascertained using an electrical nerve threshold stimulator Neurometer. Skin blood flow was measured using laser doppler flowmetry. Neurogenic flare response was assessed using Capsaicin. Figure 1 shows bar graphs demonstrating changes in sensory nerve activity in response to stimulation with the ENS unit in the two groups of diabetic patients with peripheral neuropathy that received sham and active treatments respectively. Figure la demonstrates that the active group showed a reduction in reaction time required to respond indicating improvement in nerve function. Figure lb demonstrates that the active group showed an increase in vascular response in response to a sensory stimulus indicating improvement in nerve function.

EXAMPLE 2 Further studies were carried out, testing for improved wound healing in patients with leg ulcers and separately for improved sensory nerve function in patients with diabetic peripheral neuropathy. The studies both used purpose built sensory nerve stimulation (SNS) units that could be internally set to active or placebo (inactive) settings. Active units were set to provide 4 mA stimulation at 5 Hz via a 2 ms square wave. The on-off switch is the only external control. Both active and placebo SNS units have green light emitting diodes that flash when the units are turned on. Patients were told to apply the stimulus for 5 minutes, twice daily. All participants were asked to place electrodes at the knee to "stimulate" the common peroneal nerve. Electrodes were affixed to a strap, approximately locating them for each individual when the strap is affixed such that marks straddle the tibial tuberosity or other obvious landmark. Study participants activated the unit for five minutes, twice daily. In a normal volunteer, such SNS increased capillary blood flow in the foot (by laser Doppler flowmetry - LDF) and caused almost imperceptible sensation at the knee and trivial discomfort distally. The placebo SNS machines produce no electrical stimulation. All participants were told that they may or may not feel any sensation when the machine is working. Self-reported adherence to the SNS protocol was assessed by questionnaire.

VENOUS ULCER NERVE STIMULATION RESULTS This study was a randomized, double blind, placebo controlled trial. A total of 29 subjects were recruited for the study. Fourteen subjects received active treatment and 15 subjects received sham treatment. Univariate ANOVAs indicated no significant effects of nerve stimulation on Tcp02 (transcutaneous oxygen tension), ECPT or LDF. ECPT and LDF didn't appear to change from baseline to week 12, and although Tcp02 increased it did so to a similar extent in both groups. However, inspection of data concerning severity of ulcers at initial appointment (duration of ulcer and size at initial appointment) indicated that participants in the active group had more severe ulcers than those in the sham group. The average duration of ulcers for the active group (44 months, SD 39) was at least twice that of those in the sham group (20 months, SD 19) . Similarly the average initial size of ulcers for the active group was 7.5 cm2 (SD 8.5 cm2) more than twice that for the sham group (3.2 cm2 , SD 2.8 cm2) . Despite these differences in ulcer severity, the number of weeks taken for the ulcers to heal was virtually identical for both groups (active group, 5.75 weeks, SD 0.67 weeks; sham group 5.2 weeks, SD 0.6) . Although none of these group differences were statistically significant, it is clear from the mean data that ulcers healed at a faster rate in the active group (1.3 cm2 per week) compared to the sham group (0.6 cm2 per week) - see Figure 2. These data suggest a promising therapeutic effect of nerve stimulation on venous ulcers . Further studies using greater numbers of subjects and attempting to match groups for wound size will be carried out to confirm this effect.

RESULTS OF DIABETIC PERIPHERAL NEUROPATHY POST NERVE STIMULATION TREATMENT The participants in the study suffered from diabetic peripheral neuropathy. This study was a randomized, double blind, placebo controlled trial to test if any improvement in sensory nerve function resulted from electrical stimulation. A total of 66 subjects were recruited for the study. Thirty five subjects received active treatment and 31 subjects received sham treatment. Results of univariate ANOVAs indicated that post nerve stimulation had significant effects on Tcp02 and there was a tendency for an effect in the predicted direction on LDF. The magnitude of the change in Tcp02 was significantly greater in the active group compared to the sham group (F(l,57 ) = 4.481, p = 0.04), indicating a greater effect of post nerve stimulation in the active group (see Figure 3) . Although there was no significant group effect for LDF [F(l,65) = 2.794, p = 0.1], the magnitude of the change in LDF was greatest in the active group, and was more than three times greater than that for the sham group. The mean change in LDF for the active group was 89% (SD 197) compared to 25.3 % (SD 84) for the sham group (see Figure 4) . Inspection of raw data indicated that some of the participants in the active group failed to show a response to post nerve stimulation. For this reason a second univariate ANOVA was performed, comparing LDF values for responders (participants who responded to stimulation in the active stimulation group) to non responders (participants who failed to respond in the active stimulation group) . Results indicated a highly significant effect of electrical stimulation on LDF [F ( 1,34 ) = 11.697 , p < 0.002] . Mean change in LDF for responders was 180% (SD 230) compared to -19% (SD 30.8 in the non-responders group) . These results suggest that post nerve stimulation increases Tcp02 and possibly also LDF. As power was low for both these variables (ranging from 0.38 to 0.55) it is likely that a larger sample would result in significant effects of nerve stimulation on LDF as well as Tcp02.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of improving the function of an impaired sensory nerve to release growth factors in response to injury, the method comprising applying a stimulus to the nerve to stimulate the nerve to release endogenous substance P followed by endogenous calcitonin gene related peptide .

2. A method for promoting tissue healing in human patients with impaired sensory nerve function, the method comprising stimulating the impaired sensory nerve to release endogenous substance P followed by endogenous calcitonin gene related peptide.

3. A method according to claim 1 or claim 2, in which impairment to the sensory nerve function is due to aging, peripheral neuropathy caused by diabetes, trauma, accidents, chemotherapy, HIV or AIDS.

4. A method according to any preceding claim, in which the impaired sensory nerve function is due to impaired function in unmyelinated afferent C fibres.

5. A method according to claim 1 or claim 2, in which stimulus provided is selective to provide an afferent response without an efferent response.

6. A method according to claim 5, in which the stimulus selectively activates the local effector function of C fibres only.

7. A method according to any preceding claim, in which the stimulus is an electrical signal.

8. A method according to claim 7, in which the stimulus signal comprises a pulsed signal.

9. A method according to claim 7, in which the stimulus provides a range of current to evoke a local effector function of the C fibres without sending a pain signal is 0.5 - 5.0 mA.

10. A method according to claim 7, in which the stimulus equates to a peak pulse current up to 5mA flowing through the nerve fibre to be treated.

11. A method according to claim 8, in which the pulsed signal is applied twice daily.

12. A method according to claim 7, in which the electrical signal has a frequency between 1Hz to 10Hz.

13. A method according to claim 12, in which the electrical signal includes a fixed 0.5mA, 5Hz unipolar square wave having about a 1% duty cycle.

14. A method according to claim 7, in which the electrical stimulus is applied to the nerve peripherally and proximally, via electrodes attached to the skin.

15. A method according to any one of claim 1 to 6, in which the stimulus is chemical.

16. A method according to claim 15, in which the chemical stimulus comprises administering Capsaicin peripheral to a nerve .

17. A method according to claim 16, in which Capsiacin is administered topically as a cream.

18. A method according to claim 2 for use in treating wounds .

19. A method according to claim 18, in which the wounds are those of the limbs and back.

20. A method according to claim 19, in which the wounds are selected from the group including ulcers, venous ulcers, diabetic ulcers, pressure sores, bed sores, burns, cuts and abrasions.

21. An electrical apparatus comprising an electrical generator for generating a stimulus capable of inducing flare when applied to a nerve C fibre, the electrical pulse generator having two or more application electrodes, an anode (+) and a cathode (-) , wherein the electrical generator is adapted to produce an electrical signal comprising a unipolar square wave having about a 1% duty cycle, a fixed frequency between 1Hz to 10Hz, and a voltage sufficient to cause up to 5mA peak current to flow though a nerve C fibre.

22. An electrical apparatus according to claim 21, in which the electrical signal has a frequency of 5Hz and is capable of causing at least 0.5mA peak current to flow through a nerve fibre .

23. An electrical apparatus according to claim 21 or claim 22, in which the application electrodes are releasably attachable to the electrical generator.

24. An electrical apparatus according to claim 23, in which the application electrodes take the form of a series of electrodes provided on a strap or bandage .

25. An electrical apparatus according to claim 24, in which the application electrodes are provided in the form of a stocking, sock, garter or cuff.

26. An electrical apparatus according to claim 25, in which the application electrodes are provided in compression stocking.

27. A method according to any one of claims 1 to 14, wherein the stimulus is provided by the apparatus according to any one of claims 21 to 26.

28. An electrical apparatus according to any one of claims 21 to 26, when used in the method according to any one of claims 1 to 14.

29. A method for the prevention or treatment of pressure sores comprising administering a low level dose of topical Capsaicin to a patient to stimulate sensory nerves.

30. A method according to any preceding method claim carried out on a human.