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The pathology of diabetic neuropathy and the effects of aldose reductase inhibitors
8 The Pathology of Diabetic Neuropathy and the Effects of Aldose Reductase Inhibitors K. R. W. GILLON R. H. M. KING P. K. THOMAS
A wide range of manifestations related to disturbances of peripheral nerve function is encountered in patients with diabetes mellitus. The underlying pathological changes can be expected to be correspondingly varied. In order to discuss these it is necessary first to summarize the different clinical syndromes that can be identified. The causation of the diabetic neuropathies is still uncertain and it is thus not possible to group them in terms of .aetiology. Classifications based on their clinical features have therefore to be adopted. The most satisfactory categorization is a separation into two broad groups comprising generalized polyneuropathies on the one hand and focal and multifocal neuropathies on the other (Thomas and Eliasson, 1984). Both groups are capable of further subdivision.
CLINICAL SYNDROMES Generalized polyneuropathies An important problem that arises in relation to diabetic neuropathy is one of definition. Abnormalities may be found on neurological examination in the absence of symptoms, and nerve conduction is frequently abnormal in patients with diabetes without neurological symptoms or abnormal signs (subclinical neuropathy). In a recent large scale study (Dyck et ai, 1985a), an assessment of scaled symptoms and signs, and the results of nerve conduction studies, were correlated with quantitative morphometric observations on the pathological changes observed on nerve biopsy. The clinical and electrophysiological abnormalities were found to be correlated with the pathological changes, although not in a precise manner. The most common form of diabetic neuropathy is a distal sensory polyneuropathy, sometimes with minor accompanying motor involvement. It is of a length-related pattern, longer nerve fibres being more vulnerable Clinics in Endocrinology and Metabolism-Vol. 15, No.4, November 1986
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than shorter. The symptoms thus begin in the feet and spread proximally. The hands are affected later and again, with advancing disease, the se nsory changes spread up the arms. A typical 'stocking and glove' pattern thus arises. In more severe instances the changes also affect the anterior abdominal wall and extend laterally around the trunk. In the most severe cases the vertex of the head is affected, related to involvement of the longest fibres travelling in the first division of the trigeminal nerves and the second cervical nerve roots. Numbness, tingling and pain are the most frequent symptoms. Loss of pain and temperature sensibility is the most important contributory factor in the development of foot ulceration and neuropathic joint degeneration, although autonomic neuropathy is also implicated (see Chapter 10). Loss of postural afferents may result in sen sory ataxia with unsteadiness of gait that is worse in the dark. Sensory polyneuropathy is usually of insidious onset and tends to be persistent, although the occurrence of pain is often episodic. Reversible syndromes may also occur. Thus newly diagnosed diabetics not infrequently experience distal paraesthesiae in the limbs that clear relatively rapidly with the establishment of diabetic control. Newly diagnosed diabetics, whether or not they have sensory symptoms, frequently also show some reduction of nerve conduction velocity that is rapidly corrected by treatment of the hyperglycaernia (Gregerson, 1968; Ward et al , 1971). In established sensory polyneuropathy, reduction in amplitude or loss of sensory nerve action potentials is the most important change. Motor nerve conduction velocity is usually detected on electromyography of distal lower limb muscles. Acute painful diabetic neuropathy represents a distinct and fortunately uncommon syndrome (Archer et al , 1983). Following precipitate weight loss, patients develop severe pain and contact hypersensitivity of the skin, mainly affecting the lower limbs. With adequate diabetic control and weight gain, slow recovery occurs over a period of some months. Autonomic neuropathy is also classifiable as a length-related polyneuropathy. The initial manifestations are probably distally in the lower limbs, although symptoms related to defective innervation of the viscera arc often the most obtrusive manifestation . Autonomic neuropathy , which is' con sidered in Chapter 9, often coexists with the distal sen sory polyneuropathy. Motor syndromes are usually asymmetric. Lower limb proximal motor neuropathy of gradual onset may be bilaterally symmetrical . The occurrence of an acute distal motor polyneuropathy resembling th e GuillainBarre syndrome has been suggested but is a questionable entity. Focal and multi focal neuropathies Although not formally established by epidemiological studies, it is accepted that isolated peripheral nerve lesions are more common in patients with diabetes than in the general population. They affect the cranial nerves , and the limb and trunk nerves. The third and sixth cranial nerves are those most frequently affected. In the limbs, the lesions often
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occur at common sites of external compression or entrapment, accompanied by focal abnormalities of nerve conduction. In thoracoabdorninal neuropathies, lesions of the intercostal or spinal nerves give rise to focal symptoms including pain and other sensory disturbances, and also localized weakness of the anterior abdominal wall. Lesions of the third, sixth and seventh cranial nerves, as is sometimes true of those of limb nerves, are often of abrupt onset. Asymmetric proximal weakness of acute onset, frequently with pain, is another variant of the diabetic amyotrophy syndrome. Patchy distal lower limb weakness may also occur, especially affecting the anterior tibial and peroneal muscles.
HISTOPATHOLOGY Sensory polyneuropathy
The nature of any histopathological correlates of the distal sensory symptoms, or the reduced nerve conduction velocity, that rapidly improv.e with establishment of diabetic control is not known. Nerve biopsies have not been undertaken in such cases. In established sensory polyneuropathy there is a loss of myelinated and unmyelinated axons that is maximal distally in the lower limbs, corresponding to the distribution of the sensory loss clinically. It has been suggested (Brown et al, 1976) that there is a spectrum of cases varying from those with predominant loss of small myelinated and unmyelinated axons at one end to those with predominant loss of large myelinated fibres at the other. Cases of 'small fibre neuropathy' have heen found to show selective loss of pain and temperature sensibility and to be associated with autonomic neuropathy. The tendon reflexes tend to be preserved. Pain is often prominent as a symptom. This pattern of nerve fibre loss has heen convincingly established (Figure 1). Patients with large fibre neuropathy have been considered to have selective loss of joint position and vibration sense and to lose their tendon reflexes. Pain and temperature sensibility has been said to be relatively preserved and the occurrence of pain not to be a feature. This pattern of fibre loss has not so far been demonstrated from morphometric studies on nerve biopsies, although in cases with more severe neuropathy, fibres of all diameters are lost. The nature of the fibre loss is not fully established. Although there may be some loss of dorsal root ganglion, autonomic ganglion and anterior horn cells, this is not usually prominent. Axonal degeneration occurs in the peripheral nerves. There is some evidence that it is of 'dying-back' type, i.e. a distal degeneration that progresses centripetally towards the cell hody, but it could also represent focal destruction related to local factors within the nerve trunks. Loss of axons may also be evident in the posterior columns in the spinal cord but it is not established whether this is of dying-back pattern and therefore more severe rostrally, or whether it is related to loss of dorsal root ganglion cells.
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Figure I. Sizc-frequeney distribution for myelinated nerve fibres in the sural nerve for two cases of painful diabetic neuropathy (histograms on right). In comparison with the bimodal size distribution from a control nerve on the left, there is a relatively greater loss of the small fibre peak in the diabetic nerves. Reproduced from Brown et al (1976), with permission.
Axonal changes short of complete breakdown may be present. It is not certain whether axonal atrophy occurs, although this is observed in experimental diabetes in animals. Abnormal intra-axonal inclusions may be encountered, including myelin figures, secondary lysosomal structures and increased quantities of glycogen granules, either membrane-bound or lying free in the axoplasm (Figure 2). Evidence of axonal regeneration is often conspicuous, seen as groups of myelinated and unmyelinated axons ('regenerative clusters') (Figure 3). Groups of small calibre regenerating unmyelinated axons arc also observed. Regenerating axon sprouts derived from injured small myelinated and unmyelinated axons subserving pain provide a possible explanation for the occurrence of pain in diabetic neuropathy (Asbury and Fields, 1984). There is some evidence that ectopic impulses arising in such sprouts may be responsible. Although regenerative activity is evident in the peripheral nerves, if degeneration occurs within the spinal cord in the centrally directed axons of the primary sensory neurons, effective regeneration will not be possible. This could provide one reason for the disappointing recovery in patients with sensory polyneuropathy despite good diabetic control. Segmental demyelination is also a feature of diabetic neuropathy. In untreated diabetic subjects without symptoms of neuropathy, it has been found from sural nerve biopsy (Dyck et al, 1980) that segmental demyelination and remyelination is the predominant abnormality. In
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Figure 2. Electron micrograph of an unmyelinated axon from a sural nerve biopsy in a patient with diabetic sensory polyneuropathy containing a collection of glycogen particles (g) and membrane profiles. x 27200.
untreated diabetics with symptomatic neuropathy, segmental demyelination was combined with axonal degeneration. Finally, in patients with long-standing treated diabetes, axonal degeneration was the salient abnormality. It is not yet clear how far the demyelination is a direct effect of the diabetic state on Schwann cell function or how far it may be secondary to axonal abnormalities. Autonomic neuropathy Observations on the histopathological changes in the autonomic nervous system have not so far kept pace with the extensive clinical studies on disturbances of autonomic function in diabetes that have been undertaken in recent years. Degenerative changes are seen in autonomic ganglion cells, together with demyelination and axonal loss in the white communicating rami, and in the vagus and splanchnic nerves. Fibre degeneration and loss have also been demonstrated in the bladder wall, the corpora cavernosa and lower limb blood vessels. An interesting observation has been the presence of inflammatory infiltrates in autonomic ganglia and in nerve bundles in the walls of the viscera in some cases (Duchen et al, 1980).
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Figure 3. Electron micrograph from a sural nerve biopsy from a patient with diabetic sensory polyneuropathy showing a group of regenerating axons ('regenerative cluster') within the persisting basal lamina (bl) that had surro unded the original fibre . x 17400.
Focal and multifocal neuropathy In patients with multifocallimb neuropathy, nerve biopsy may show patchy fibre loss , its severity varying between the component fascicles of the nerve . It is also evident that multifocal proximal lesions can summate to produce a clinically symmetric distal sensorimotor neuropathy. The histopathological changes have been studied in patients with acute third cranial nerve lesions who have come to autopsy (Asbury et ai, 1970) . Evidence of focal demyelination and remyelination was observed (Figure 4). It is of interest that this tended to affect the centre of the nerve, correlating with the preservation of pupillary function that characterizes diabetic third nerve lesions. The pupillomotor fibres are known to travel at the periphery of the nerve. The supposition is that such lesions have a vascular basis, although this has not been substantiated. The predominantly demyelinating nature of the pathology, with axonal preservation, equates with the usual natural history of diabetic third nerve lesions in which recovery tends to be rapid and complete. Vascular and connective tissue changes Abnormalities of the endoneurial and perineurial blood vessels are prominent in patients with established diabetic neuropathy. These consist
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Diagram of serial reconstruction from sections of thc intracavernous portion of the
left third cranial nerve of a diabetic patient who died three weeks after developing a third
nerve palsy. The stippled area indicates the site of a recent focal region of demyelination. A. Band C indicate the blood supply to the nerve. Reproduced from Asbury et al (1970), with permission .
of endothelial proliferation and consequent luminal narrowing. Thickening of the walls of these vessels also occurs as a consequence of reduplication of the surrounding basal lamina (Figures 5 and 6). In nerve biopsies, the number of capillaries in which the lumen is 'closed' is increased in diabetic subjects in comparison with controls, and the proportion of closed capillaries correlates with the severity of the neuropathy (Dyck et ai, 1985b) . Intrafascicular area may be increased and there is often extensive collagen deposition. The lamelIated perineurial sheath that surrounds the fascicles also displays abnormalities. The basal lamina of the perineurial celIs is thickened and calcification may be present in the connective tissue space between the celIular lamellae (Figure 7). A general review of the histopathological changes in diabetic neuropathy has been given by Thomas and Eliasson (1984). BIOCHEMICAL CHANGES The notion that the complications of diabetes, including neuropathy, are a consequence of the hyperglycaemic state remains controversial, but one which has gained significance from recent evidence from both experimental and clinical studies . Persisting hyperglycaemia can be induced in various animal species by the injection of either streptozotocin (streptozocin) or alloxan and these animals are useful models to study the effect of hyperglycaemia on nerve metabolism. The relevance of such
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.. Figure 5. Portion of sural nerve from a patient with diabetic sensory polyneuropathy showing severe loss of myelinated nerve fibres and thickening of the walls of four endoneurial capillaries (arrowed). Semithin section stained with acridine orange and thionin. X516.
Figure 6. Electron micrograph of a transverse section through an cndoneurial capillary from a patient with diabetic sensory polyneuropathy. There is reduplication of the surrounding basal lamina (bl). x5015.
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Figure 7. Electron micrograph of transverse section through perineurium from the sural nerve of a patient with diabetic sensory polyneuropathy showing thickening of the basal lamina (hi) on either side of the perineurial cells (pc) and multiple calcified deposits (c) in the interlamellar spaces. x 14440.
models to clinical diabetes has been questioned and has resulted in the increased use of animals genetically predisposed to development of hyperglycaemia which are considered more appropriate clinically. Studies on the peripheral nerves of both chemically and genetically diabetic animals have resulted in the emergence of differing, although not mutually exclusive, concepts on the aetiology of neuropathy (for detailed reviews, see Gabbay, 1973; Clements, 1979; Tomlinson and Mayer, 1984; Greene et ai, 1985). Tissues which do not depend on plasma insulin for glucose entry to their cells (notably, for the purpose of this review, peripheral nerve) have cytoplasmic glucose concentrations which reflect the degree of glycaemia. The enzyme aldose reductase has a low affinity for its substrates, hexose sugars (including glucose), and so, at the low nerve glucose concentrations in peripheral nerve during normoglycaemia, the concentrations of the product of the reaction, sorbitol, are low. The sorbitol formed is further metabolized to fructose by the enzyme sorbitol dehydrogenase. Induction of hyperglycaemia in animals results in markedly elevated concentrations of glucose in peripheral nerve (Stewart et al, 1966), the consequence of which is increased activity of aldose reductase and elevated concentrations of sorbitol and fructose (Gabbay, 1973). Motor nerve conduction velocity
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is reduced in such animals. It was postulated that since sorbitol and fructose are osmotically active their accumulation would cause cellular oedema and eventually structural damage. Because aldose reductase is confined to the Schwarm cell (Gabbay and O'Sullivan, 1968), this was thought to be the site of cell damage, resulting in slowed nerve conduction velocity. However, there is an approximately 30% shrinkage of Schwarm cell volume in diabetic rat nerve (Jakobsen, 1978), which refutes the Schwarm cell osmotic theory. It is possible, however, that the micromolar concentrations of polyol which do accumulate may be highly, and critically, localized in another nerve compartment. Greene and co-workers (1975) demonstrated that not only arc glucose, sorbitol and fructose concentrations elevated in the nerves of diabetic animals, but that this is accompanied by a decrease in the concentration of the sugar alcohol, lIlya-inositol. Moreover, they also showed that the functional deficit in these animals correlated with plasma insulin level and nerve lIlya-inositol depletion since treatment of the animals with insulin or a Illya-inositol supplemented diet resulted in correction of slowed motor nerve conduction velocity. Illya-Inositol is maintained in peripheral nerve at concentrations approximately 3D-100 times that of plasma by sodium dependent, energy consuming active transport which is inhibited by increasing glucose concentrations in vitro (Greene and Lattimer, 1982; Gillon and Hawthorne, 1983). Indeed, peripheral nerves from diabetic rats accumulate approximately 40% less lIlya-inositol than nerves from control animals (Gillon and Hawthorne, 1983), which may partly account for the reduced concentration of the compound in diabetic rat nerves. With the advent of aldose reductase inhibitors came the opportunity to test directly the hypothesis that polyol accumulation is involved in the pathophysiology of diabetic neuropathy. These compounds were clearly able to normalize or prevent elevated sorbitol concentrations in the nerves of diabetic animals (Cahill et ai, 1978). Administration of structurally unrelated inhibitors had similar effects on nerve sorbitol concentration, with no reduction of glycaemia (Tomlinson et al, 1982; Finegold et al, 1983; Gillon et aI, 1983; Poulsom and Heath, 1983). In two of these studies (Tomlinson et ai, 1982; Gillon et aI, 1983) the motor nerve defect, which develops during hyperglycaemia, was prevented or restored. In addition, aldose reductase inhibitor treatment normalized or prevented the decrease in Illya-inositol concentration. Since lIlya-inositol feeding or treatment also restores the functional deficit, presumably through correction of peripheral nerve lIlya-inositol concentration, it can be hypothesized that lIlyo-inositol loss, rather than polyol accumulation, is implicated in the abnormality of nerve conduction velocity in diabetic animals (Greene et aI, 1975). Thus polyol accumulation and Illyo-inositol loss are linked processes and appear to be causally related to the development of early neuropathy in diabetic animals. Das et al (1976) demonstrated that the activity of the membrane-bound enzyme, sodium- and potassium-dependent ATPase (Na+ ,K+ -ATPase), is reduced in the nerves of streptozotocin diabetic rats. This observation was not only confirmed some years later in streptozotocin diabetic rats but it
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was also shown that administration of Illyo-inositol or an aldose reductase inhibitor could reverse the decrease in activity (Greene and Lattimer, 1984). Since this enzyme controls the sodium/potassium ion balance across the nodal axoplasmic membrane its activity is critical to the generation of the nerve action potential, and it is tempting to speculate that this defect is at the heart of the functional impairment in diabetic rat nerve. Protein synthesis in peripheral nerve trunks is limited and the nerve trunks are dependent on the process ofaxoplasmic transport for the supply of structural and functional proteins (and Na+,K+-ATPase may be no exception) from the cell body (Sidenius, 1982). This mechanism is deficient in the diabetic rat (Jakobsen and Sidenius, 1980) and the slowing in delivery of protein may be an important factor in diabetic neuropathy which involves primarily axonal degeneration. myo-Inositol treatment or aldose reductase inhibitor treatment can also prevent or reverse the deficit in slow axoplasmic transport in diabetic nerve and this effect is temporally related with the correction or prevention of the functional deficit (Tomlinson and Mayer, 1984). The defect in axonal transport may be present in nerve from human diabetics, since accumulation of the enzymes dopamine B-hydroxylase and acetylcholinesterase was reduced in nerve biopsies (Brimijoin and Dyck, 1979). A possible mechanism for the development of experimental diabetic neuropathy has been proposed by Greene et al (1985) in which nerve Illyo-inositol concentration is reduced by inhibition of active transport by hyperglycaemia. The reduced nerve Illyo-inositol concentration results in abnormal metabolism of membrane polyphosphoinositides, lipids long known to be important in the function of excitable membranes, and this may be a factor in the reduction in activity of membrane Na+ ,K+ -A'TPase. Since myo-inositol transport is also dependent on this enzyme, a self-reinforcing cycle of inositol loss and Na +,K+ -A'I'Pasc reduction is formed leading to axoplasmic sodium accumulation at the nodes of Ranvier and a deficit in action potential generation. The role of polyol accumulation in this theory is unclear but it is involved in reducing intracellular Illyo-inositol by an unexplained mechanism, perhaps by causing leakage of Illyo-inositol from the axon by localized osmotic effects. Localized swelling in the region of the nodes of Ranvier has been demonstrated recently in the nerve of the spontaneously diabetic BB Wistar rat (Greene, 1985 personal communication). Recent evidence has also shown that reduced blood flow and endoneurial oxygen tension may also contribute to the biochemical abnormalities, including the deficit in Na +,K +-A'TPase activity (Low et al, 1985). Indeed, maintaining diabetic animals in an oxygen-supplemented environment resulted in partial correction of some biochemical and functional abnormalities. Evidence for the existence of these biochemical abnormalities in nerves of diabetic individuals is scarce because of the obvious difficulty in obtaining suitable human material. Three notable studies have attempted to answer this question. Ward et al (1972) found increased concentrations of glucose, fructose and sorbitol in nerves post mortem. Evidence of increased concentrations of fructose and sorbitol in nerve biopsies in some
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patients (mainly non-insulin dependent diabetics) has been reported (Dyck et al, 1980). The concentrations of sugars and polyols were found to be much more variable in nerves from patients compared with controls presumably due to varying degrees of glycaemia. However, in this study, there was no indication of lowered nerve free 11Iyo-inositol levels. Increased glucose, fructose and sorbitol concentrations and reduced 11Iyo-inositol concentration have been demonstrated in human sciatic nerve obtained post mortem mainly from non-insulin dependent diabetic patients (Mayhew et aI, 1983). Therefore increasing evidence suggests the existence of abnormal sugar metabolism in the nerves from diabetic individuals, but the data from post-mortem material should be interpreted with caution and final confirmation awaits further studies on biopsied nerves. Data from work on animal models indicating a metabolic basis for some aspects of diabetic neuropathy have provided the impetus for clinical intervention trials on three broad fronts: improved control of glycaemia, treatment with lIlyo-inositol and treatment with aldose reductase inhibitors.
Control of glycaemia Evidence for beneficial effects on nerve function resulting from improved diabetic control is increasing. Slowed nerve conduction velocity is an early indication of diabetic neuropathy. Starting insulin in previously untreated patients resulted in rapid improvements in peroneal (Gregersen, 1968; Ward et aI, 1971) and median (Ward et al, 1971) nerves. Small improvements in median motor conduction velocity were sustained for 12 months following the initiation of insulin in non-insulin dependent diabetics, and the degree of improvement correlated with the reduction in glycaemia (Graf et aI, 1981). No improvement was found in sensory nerves in these patients. In patients already treated with insulin, two methods have been used to improve control aiming to achieve normoglycaemia. Intensive insulin therapy (i.e. multiple insulin injections) was compared with conventional insulin therapy in both insulin dependent and noninsulin dependent diabetic patients in a three-year randomized trial (Service et aI, 1983). Unfortunately, there was no clear discrimination of diabetic control between the groups owing to improvement of some patients in the conventionally treated group, and thus no difference in neurological function was discovered. This study was important in advocating methodology to study a wide range of nerve functions including sensory perception, and suggested that normoglycaemia may be required to benefit neuropathy. The best method of achieving this may be by continuous subcutaneous insulin infusion (CSII) using a portable insulin delivery pump and this method has been used extensively in recent studies. Boulton et al (1982) achieved near normal glycaemia in patients with painful neuropathy over a four-month period. Significant improvements in motor conduction velocity and vibration perception threshold occurred after six weeks of CSII and were maintained for at least four months. Pain scores in these patients were also significantly reduced, a finding also observed during an open trial of acute blood glucose lowering (Samanta
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and Burden, 1985). In another randomized study of CSII and conventional insulin treatment, near normoglycaemia was achieved in the CSII group throughout the study without improvement in the conventionally treated group (Service et aI, 1985): improvements in motor conduction velocity and vibration perception threshold were also demonstrated in this study but with a different time course, the effect not being evident after four months but only present after eight months of CSIl. Myo-inositol treatment As previously demonstrated in diabetic animals (Greene et ai, 1975), administration of a myo-inositol load to diabetic patients increases its plasma concentration despite increased urinary excretion (Clements and Reynertson, 1977; Gregersen et al, 1983) making possible an increase of peripheral nerve concentrations through increased active transport from plasma. Four studies have examined the effect of lIIyo-inositol treatment on diabetic neuropathy, all four of them using electrophysiological parameters (Gregersen et ai, 1978; Salway et ai, 1978; Clements et al, 1979; Gregersen et al, 1983), and one studying both electrophysiologicar changes together with signs and symptoms (Greene et al, 1981). In three of these studies (Gregersen et al, 1978, 1983; Greene et al, 1981) no improvement in the electrophysiological measurements was found. Two other studies, of two weeks and 16 weeks respectively, demonstrated improvement in the amplitude of the evoked action potential in the median, sural and tibial nerves (Salway et al, 1978) and in median and sural sensory nerve conduction velocity in neuropathic diabetic patients (Clements et al, 1979). Clinical improvement of symptoms (pain) and signs (pin prick, temperature perception) was also demonstrated in some patients after six months of myo-inositol therapy (Greene et ai, 1981). Aldose reductase inhibitors Rat peripheral nerve and red blood cell sorbitol concentrations are strongly correlated and red cell level is related to ambient plasma glucose levels (Malone et ai, 1984). Therefore investigators have used red cell sorbitol concentrations as an index of nerve levels (and hence drug inhibitory efficacy) during clinical studies with aldose reductase inhibitors. Several studies have now demonstrated that structurally distinct aldose reductase inhibitors reduce elevated sorbitol levels in the red cells of diabetic patients (Kilpenainen et al, 1983; Malone et al, 1984; Norris et aI, 1985; Raskin et al, 1985) and hence, by inference from the rat model, also in peripheral nerve. Clinical studies in neuropathy have been reported for three compounds, Alrestatin, Sorbinil and Tolrestat. Double-blind trials with Alrestatin indicated that three months of treatment in one study resulted in significant improvement in sensory and electrophysiological parameters (Fagius and Jameson, 1981), while a four-month study in symptomatic patients revealed no evidence of subjective or objective improvement consistent
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with a drug effect (Handelsman and Turtle, 1981). In controlled trials with Sorbinil, small but significant improvements in peroneal and median motor conduction velocity and median sensory conduction velocity have been reported after nine weeks treatment (Judzewitsch et aI, 1983) and in ulnar and tibial F wave latency and ulnar distal sensory latency after 24 weeks treatment (Fagius et al, 1985). In the latter study autonomic nervous function assessed by heart rate (R-R interval) variation also improved, and this has recently been confirmed (Jaspan et aI, 1985). In patients with severely painful neuropathy, substantial and rapid reduction in pain has been reported in an essentially uncontrolled study (Jaspan et aI, 1983), the effect being evident within a few days of starting treatment. In controlled trials on patients with painful neuropathy, confirmation of pain reduction has been obtained in a four-week cross-over study of Sorbinil and placebo although the effect was much more subtle (Young et aI, 1983). Recent controlled trials of Tolrestat suggest that this compound too has a beneficial effect on pain (Koglin et al, 1985).
CONCLUSIONS Therapeutic studies have demonstrated that nerve function and clinical symptoms can be improved, lending weight to the argument for a metabolic contribution to diabetic polyneuropathy, but the effects on objective parameters are small and on subjective parameters, subtle. Problems remain in terms of patient selection, methodology for assessment and duration of treatment. Vascular factors and an increased liability to compression injury are likely to be important in the origin of focal nerve lesions, and there is increasing evidence that ischaemia may be involved in the causation of generalized polyneuropathies.
REFERENCES Archer A, Watkins Pl, Thomas PK et al (1983) The natural history of acute painful diabetic neuropathy. Journal of Neurology, Neurosurgery and Psychiatry -l6: .191-504. Asbury AK & Fields HL (1984) Pain due to peripheral nerve damage: a hypothesis. Neurology [Cleveland} 34: 1587-1590. Asbury AK, Aldredge H, Hershberg R & Fisher CM (1970) Oculomotor palsy in diabetes mellitus: a clinicopathological study. Brain 93: 555-566. Boulton A1M, Drury 1, Clarke B & Ward lD (1982) Continuous subcutaneous insulin infusion in the management of painful diabetic neuropathy. Diabetes Care 5: 386--390. Brimijoin S & Dyck Pl (1979) Axonal transport of dopamine-beta-hydroxylase and acetylcholinesterase in human peripheral neuropathy. Experimental Neurology 66: 467-478. Brown Ml, Martin lR & Asbury AK (1976) Painful diabetic neuropathy: a morphometric study. Archives of Neurology 33: 16-t-171. Cahill GF, Field 113 & Wiseman EH (1978) Progress towards understanding and treating diabetic complications. Metabolism 28(supplement I). Clements RS lr (1979) Diabetic neuropathy-new concepts of its etiology. Diabetes 28: 60+-611.
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Clements RS Jr & Rcyncrtson R (1977) mJo-lnositol mctabol ism in diabetes mellitus. E ffect of insulin treatment. Diabetes 26: 215-221. . Clem ents RS , Jr , Vourganti B, Kuba T et al (1979) Dietary mJo-inositol intake and peripheral nerve function in diabet ic neuropathy. Metabolism 28(supplemcnt 1): 477-483. Das PK,Dray GM, A guayo AJ & Ra smin sky M (1976) Diminished ouabain-sensitive, sod ium pot assium ATPase activity in sciatic nerves of rats with streptozotocin-induccd diabetes. Experimental Neurology 53: 285-288. Duchen LW , Anjorin A, Watk ins pJ & Mackay JD (1980) Pathology of a uto no mic neuropathy in diabetes. Annals of Internal Medicine 92: 301-303. Dyck PJ , Sherman WR, Hallcher LM et al (1980) Human diabetic endoneuria! sorbitol, fructose , and mJo·inositol related to sural nerve morphometry. Annals of Neurology 8: 590-596. Dyck PJ, Karnes JL , Daube Jet al (1985a) Clinical and neuropathological criteria for thc diagnosis and staging of diabetic polyneuropathy. Brain 108: 861- 880. Dyck PJ, Hansen S, Karnes Jet al (1985b) Capillary number and percentage closed in human diabetic sural nerve. Proceedings of the National Academy of S cien ces (USA) 82: 2513-2517. Fagiu s J & Jameson S (1981) Effect of aldose reductase inhibitor treatment in d iabetic polyn europathy-a clinical neurophysiological study. Journal of Neurology, Neurosurgery and Psychiatry M: 991-1001. Fagius J, Brattbcrg A, Berne C & Jameson S (1985) Limited benefit of treatment of diabetic polyneuropathy with an aldose reductase inhibitor-a 24 week controlled trial. Diabetologia 28: 323-329. Fineg old D, Lattimer S, Nolle S et al (1983) Polyol pathway activity and rnyo-inositol met aboli sm. Diabetes 32: 988-992. Gabbay KH (1973) The sorbitol pathway ami the complications of diabetes. Ne w En gland Journal vf Medicine 288: 831-83 6. Gabbay KH & O'Sullivan Jil (1968) The sorbitol pathway. Enz yme localisat ion and content in normal ami d iabetic nerve and cord . Diabetes 17: 239-243. G illon KRW & Hawthorne IN (1983) Transport of IIIJo-ino sitol into endoneurial preparations of sciatic nerve from normal and streptozotocin diabetic rats . Biochemical Journal 210: 775-781. Gillon KRW, Hawthorne IN & Tomlinson DR (1983) mJo-lnositol and sorbitol metabolism in rel ation to peripheral nerve function in experimental diabetes in the rat-the effect of aldose reductase inhibition. Diabetologia 25: 365-371. Graf RJ, Halter JB, Pfeifer MA et al (1981) Glycemic control and nerve conduction abnormalities in non-insulin dependent diabetic subjects. Annals of Internal Medicine 94: 307-311. Greene DA & Lattimer SA (1982) Sod ium and energy dependent upt ake of IIIJo-inos it ol by rabbit peripheral nerve: competitive inhibition by glucose and lack o f an insulin effect. Journal of Clinical Investigation 70: 1009-1018 . Greene DA & Lattimer SA (1984) Action of sorbinil in diabetic peripheral nerve. Rel ationship of 1'01)'01 (sorb itol) pathway inhibitio n to a lIlyo-inosito1·mediated defect in sodium-potassium ATPase activity. Diabetes 33: 712-716. Greene DA, De Jesus PV & Winograd AI (1975) Effects of insulin and dietary myoinositol on impaired peripheral motor nerve conduction velocity in acute streptozotocin diabetes. Journal of Clinical Investigation 55: 1326-1336. Greene DA, Drown MJ, Braunstein SN et al (1981) Comparison of clinical course and sequenti al elcctrophysiological tests in diabetics with symptomatic polyneuropathy and its implications for clinical trials. Diabetes 30: 139-147. Greene DA, Lattimer S, Ulbrecht J & Carroll P (1985) Glucose-induced alterations in nerve met abol ism : current perspective on the path ogenesis of diabetic neuropathy and future directions for research and therapy. Diab etes Care 8: 29(}....299. Gregersen G (1968) Variations in motor conduction velocity produced by acute changes of the metabolic state in diabet ic patients. Diabetologia 4: 273-277. Gregersen G, Bursting H, Theil I' & Servo C (1978) lII)'o-lnositol and function of peripheral nerves in human diabetics. A controlled clinical tr ial. Acta Ncurologica Scandinavica 58: 241-248.
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Gregersen G. Bertelsen B & lIarbo II (1983) Oral supplementation of myo-inositol-effeets on peripheral nerve function in human diabetics and on the concentrations in plasma. erythrocytes. urine and muscle tissue in human diabetics and normals. Acta Neurologica Scandinavica 67: 16+-172. Handelsman DJ & Turtle JR (1981) Clinical trial of an aldose reductase inhibitor in diabetic neuropathy. Diabetes 30: 459-tM. Jakobsen J (1978) Peripheral nerves in early experimental diabetes. Expansion of the endoneuria! space as a cause of increased water content. Diabetologia 14: 113-119. Jakobsen J & Sidcnius P (1980) Decreased axonal transport of structural proteins in strcptozotocin diabetic rats. Journal of Clinical lnvcstigation 66: 292-297. Jaspan J. Herold K. Maselli R & Bartkus C (1983) Treatment of severely painful diabetic neuropathy with an aldose reductase inhibitor-relief of pain and improved somatic and autonomic nerve function. Lancet ii: 75f1.-762. Jaspan JB. Herold K & Bartkus C (1985) Effects of sorbiniltherapy in diabetic patients with painful peripheral neuropathy and autonomic neuropathy. American Journal of Medicine 79: 25-37. Judzcwitsch RII. Jaspan J. Polonsky KS ct al (1983) Aldose reductase inhibition improves nerve conduction velocity in diabetic patients. New England Journal of Medicine 308: 119-125. Kilpclaincn II. Puhakaincn E. Saamanen AM ct al (1983) The effect of aldose reductase inhibitor (sorbinil) on erythrocyte sorbitol concentration in diabetic neuropathy. Journal of Neurochemistry 41: S27. Koglin L. Clarke C. Ryder S & Mullane JF (1985) The results of thc long-term open-label administration of Alredase' I M in the treatment of diabetic neuropathy. Diabetes 34(supplement I): 202A. Low PA. Tuck RR. Dyck PJ et al (1985) Prevention of some c1ectrophysiologic and biochemical abnormalities with oxygen supplementation in experimental diabetic neuropathy. Proceedings of the National Academy of Sciences (USA) 81: 6894-6898. Malone JI. Lcavengood II & Peterson MJ et al (1984) Red blood cell sorbitol as an indicator of polyol pathway activity. Inhibition of sorbinil in insulin-dependent subjects. Diabetes 33: 45-49. Mayhew JA. Gillon KRW & Hawthorne IN (1983) Free and lipid inositol, sorbitol and sugars in sciatic nerve obtained post-mortem from diabetic patients and control subjects. Diabetologia 24: 13-15. Norris SC. Perkins CM. Bastain W et al (1985) Studies on the pharmacokinetics and pharmacodynamics of Statil (ICI 128,436). an aldose reductase inhibitor. Clinical Science 69(supplement 12): 22P. Poulsom R & Heath II (1983) Inhibition of aldose reductase in five tissues of the strcptozotocin diabetic rat. Biochemistry and Pharmacology 32: 1495-1499. Raskin P, Rosenstock J. Challis Pet al (1985) Effect of tolrestat on red blood cell sorbitol levels in patients with diabetes. Clinical Pharmacology antl Therapy 38: 625-630. Salway JG. Whitehead L. Finnegan JA et al (1978) Effect of myo-inositol on peripheral nerve function in diabetes. Lancet ii: 1282-1284. Sarnanta A & Burden C (1985) Rapid improvement in pain of diabetic neuropathy using an open-loop intravenous infusion system. Practical Diabetes 2: 22-23. Service FJ. Daube JR. O'Brien PC et al (1983) Effect of blood glucose control on peripheral nerve function in diabetic patients. Mayo Clinic Proceedings 58: 283-289. Service FJ. Rizza RA. Daube JR et al (1985) Near norrnoglycacmia improves nerve conduction and vibration sensation in diabetic neuropathy. Diabetologia 28: 722-727. Sidcnius P (1982) The axonopathy of diabetic neuropathy. Diabetes 31: 356-363. Stewart MA. Sherman WR & Anthony S (1966) Free sugars in alloxan diabetic rat nerve. Biochemistry and Biophysics Research Communications 22: 488-l93. Thomas PK & Eliasson SG (1984) Diabetic neuropathy. In Dyck PJ. Thomas PK. Lambert EH & Bunge R (cds) Peripheral Neuropathy, 2nd cdn, pp 1773-1810. Philadelphia: WB Saunders. Tomlinson DR & Mayer JH (1984) Defects of axonal transport in diabetes mellitus-a possible contribution to the aetiology of diabetic neuropathy. Journal of All/anomie Pharmacology 4: 59-72.
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Tomlinson DR, Holmes PR & Mayer HI (1982) Reversal. by treatment with an aldose reductase inhibitor. of impaired axonal transport and motor nerve conduction velocity in experimental diabetes. Neuroscience Letters 31: 189-193. Ward OJ, Baker R WP & Davis BH (1972) Effect of blood sugar control on the accumulation of sorbitol and fructose in nerve tissue. Diabetes 21: 1173-1178. Ward JD, Barnes eG, Fisher OJ et al (1971) Improvement in nerve conduction following treatment in newly diagnosed diabetics. Lancet i: 428-t31. Young RJ. Ewing DJ & Clarke BF (1983) A controlled trial of sorbinil, an aldose reductase inhibitor. in chronic painful neuropathy. Diabetes 32: 938-9-t2.
A wide range of manifestations related to disturbances of peripheral nerve function is encountered in patients with diabetes mellitus. The underlying pathological changes can be expected to be correspondingly varied. In order to discuss these it is necessary first to summarize the different clinical syndromes that can be identified. The causation of the diabetic neuropathies is still uncertain and it is thus not possible to group them in terms of .aetiology. Classifications based on their clinical features have therefore to be adopted. The most satisfactory categorization is a separation into two broad groups comprising generalized polyneuropathies on the one hand and focal and multifocal neuropathies on the other (Thomas and Eliasson, 1984). Both groups are capable of further subdivision.
CLINICAL SYNDROMES Generalized polyneuropathies An important problem that arises in relation to diabetic neuropathy is one of definition. Abnormalities may be found on neurological examination in the absence of symptoms, and nerve conduction is frequently abnormal in patients with diabetes without neurological symptoms or abnormal signs (subclinical neuropathy). In a recent large scale study (Dyck et ai, 1985a), an assessment of scaled symptoms and signs, and the results of nerve conduction studies, were correlated with quantitative morphometric observations on the pathological changes observed on nerve biopsy. The clinical and electrophysiological abnormalities were found to be correlated with the pathological changes, although not in a precise manner. The most common form of diabetic neuropathy is a distal sensory polyneuropathy, sometimes with minor accompanying motor involvement. It is of a length-related pattern, longer nerve fibres being more vulnerable Clinics in Endocrinology and Metabolism-Vol. 15, No.4, November 1986
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than shorter. The symptoms thus begin in the feet and spread proximally. The hands are affected later and again, with advancing disease, the se nsory changes spread up the arms. A typical 'stocking and glove' pattern thus arises. In more severe instances the changes also affect the anterior abdominal wall and extend laterally around the trunk. In the most severe cases the vertex of the head is affected, related to involvement of the longest fibres travelling in the first division of the trigeminal nerves and the second cervical nerve roots. Numbness, tingling and pain are the most frequent symptoms. Loss of pain and temperature sensibility is the most important contributory factor in the development of foot ulceration and neuropathic joint degeneration, although autonomic neuropathy is also implicated (see Chapter 10). Loss of postural afferents may result in sen sory ataxia with unsteadiness of gait that is worse in the dark. Sensory polyneuropathy is usually of insidious onset and tends to be persistent, although the occurrence of pain is often episodic. Reversible syndromes may also occur. Thus newly diagnosed diabetics not infrequently experience distal paraesthesiae in the limbs that clear relatively rapidly with the establishment of diabetic control. Newly diagnosed diabetics, whether or not they have sensory symptoms, frequently also show some reduction of nerve conduction velocity that is rapidly corrected by treatment of the hyperglycaernia (Gregerson, 1968; Ward et al , 1971). In established sensory polyneuropathy, reduction in amplitude or loss of sensory nerve action potentials is the most important change. Motor nerve conduction velocity is usually detected on electromyography of distal lower limb muscles. Acute painful diabetic neuropathy represents a distinct and fortunately uncommon syndrome (Archer et al , 1983). Following precipitate weight loss, patients develop severe pain and contact hypersensitivity of the skin, mainly affecting the lower limbs. With adequate diabetic control and weight gain, slow recovery occurs over a period of some months. Autonomic neuropathy is also classifiable as a length-related polyneuropathy. The initial manifestations are probably distally in the lower limbs, although symptoms related to defective innervation of the viscera arc often the most obtrusive manifestation . Autonomic neuropathy , which is' con sidered in Chapter 9, often coexists with the distal sen sory polyneuropathy. Motor syndromes are usually asymmetric. Lower limb proximal motor neuropathy of gradual onset may be bilaterally symmetrical . The occurrence of an acute distal motor polyneuropathy resembling th e GuillainBarre syndrome has been suggested but is a questionable entity. Focal and multi focal neuropathies Although not formally established by epidemiological studies, it is accepted that isolated peripheral nerve lesions are more common in patients with diabetes than in the general population. They affect the cranial nerves , and the limb and trunk nerves. The third and sixth cranial nerves are those most frequently affected. In the limbs, the lesions often
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occur at common sites of external compression or entrapment, accompanied by focal abnormalities of nerve conduction. In thoracoabdorninal neuropathies, lesions of the intercostal or spinal nerves give rise to focal symptoms including pain and other sensory disturbances, and also localized weakness of the anterior abdominal wall. Lesions of the third, sixth and seventh cranial nerves, as is sometimes true of those of limb nerves, are often of abrupt onset. Asymmetric proximal weakness of acute onset, frequently with pain, is another variant of the diabetic amyotrophy syndrome. Patchy distal lower limb weakness may also occur, especially affecting the anterior tibial and peroneal muscles.
HISTOPATHOLOGY Sensory polyneuropathy
The nature of any histopathological correlates of the distal sensory symptoms, or the reduced nerve conduction velocity, that rapidly improv.e with establishment of diabetic control is not known. Nerve biopsies have not been undertaken in such cases. In established sensory polyneuropathy there is a loss of myelinated and unmyelinated axons that is maximal distally in the lower limbs, corresponding to the distribution of the sensory loss clinically. It has been suggested (Brown et al, 1976) that there is a spectrum of cases varying from those with predominant loss of small myelinated and unmyelinated axons at one end to those with predominant loss of large myelinated fibres at the other. Cases of 'small fibre neuropathy' have heen found to show selective loss of pain and temperature sensibility and to be associated with autonomic neuropathy. The tendon reflexes tend to be preserved. Pain is often prominent as a symptom. This pattern of nerve fibre loss has heen convincingly established (Figure 1). Patients with large fibre neuropathy have been considered to have selective loss of joint position and vibration sense and to lose their tendon reflexes. Pain and temperature sensibility has been said to be relatively preserved and the occurrence of pain not to be a feature. This pattern of fibre loss has not so far been demonstrated from morphometric studies on nerve biopsies, although in cases with more severe neuropathy, fibres of all diameters are lost. The nature of the fibre loss is not fully established. Although there may be some loss of dorsal root ganglion, autonomic ganglion and anterior horn cells, this is not usually prominent. Axonal degeneration occurs in the peripheral nerves. There is some evidence that it is of 'dying-back' type, i.e. a distal degeneration that progresses centripetally towards the cell hody, but it could also represent focal destruction related to local factors within the nerve trunks. Loss of axons may also be evident in the posterior columns in the spinal cord but it is not established whether this is of dying-back pattern and therefore more severe rostrally, or whether it is related to loss of dorsal root ganglion cells.
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Figure I. Sizc-frequeney distribution for myelinated nerve fibres in the sural nerve for two cases of painful diabetic neuropathy (histograms on right). In comparison with the bimodal size distribution from a control nerve on the left, there is a relatively greater loss of the small fibre peak in the diabetic nerves. Reproduced from Brown et al (1976), with permission.
Axonal changes short of complete breakdown may be present. It is not certain whether axonal atrophy occurs, although this is observed in experimental diabetes in animals. Abnormal intra-axonal inclusions may be encountered, including myelin figures, secondary lysosomal structures and increased quantities of glycogen granules, either membrane-bound or lying free in the axoplasm (Figure 2). Evidence of axonal regeneration is often conspicuous, seen as groups of myelinated and unmyelinated axons ('regenerative clusters') (Figure 3). Groups of small calibre regenerating unmyelinated axons arc also observed. Regenerating axon sprouts derived from injured small myelinated and unmyelinated axons subserving pain provide a possible explanation for the occurrence of pain in diabetic neuropathy (Asbury and Fields, 1984). There is some evidence that ectopic impulses arising in such sprouts may be responsible. Although regenerative activity is evident in the peripheral nerves, if degeneration occurs within the spinal cord in the centrally directed axons of the primary sensory neurons, effective regeneration will not be possible. This could provide one reason for the disappointing recovery in patients with sensory polyneuropathy despite good diabetic control. Segmental demyelination is also a feature of diabetic neuropathy. In untreated diabetic subjects without symptoms of neuropathy, it has been found from sural nerve biopsy (Dyck et al, 1980) that segmental demyelination and remyelination is the predominant abnormality. In
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I
Figure 2. Electron micrograph of an unmyelinated axon from a sural nerve biopsy in a patient with diabetic sensory polyneuropathy containing a collection of glycogen particles (g) and membrane profiles. x 27200.
untreated diabetics with symptomatic neuropathy, segmental demyelination was combined with axonal degeneration. Finally, in patients with long-standing treated diabetes, axonal degeneration was the salient abnormality. It is not yet clear how far the demyelination is a direct effect of the diabetic state on Schwann cell function or how far it may be secondary to axonal abnormalities. Autonomic neuropathy Observations on the histopathological changes in the autonomic nervous system have not so far kept pace with the extensive clinical studies on disturbances of autonomic function in diabetes that have been undertaken in recent years. Degenerative changes are seen in autonomic ganglion cells, together with demyelination and axonal loss in the white communicating rami, and in the vagus and splanchnic nerves. Fibre degeneration and loss have also been demonstrated in the bladder wall, the corpora cavernosa and lower limb blood vessels. An interesting observation has been the presence of inflammatory infiltrates in autonomic ganglia and in nerve bundles in the walls of the viscera in some cases (Duchen et al, 1980).
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Figure 3. Electron micrograph from a sural nerve biopsy from a patient with diabetic sensory polyneuropathy showing a group of regenerating axons ('regenerative cluster') within the persisting basal lamina (bl) that had surro unded the original fibre . x 17400.
Focal and multifocal neuropathy In patients with multifocallimb neuropathy, nerve biopsy may show patchy fibre loss , its severity varying between the component fascicles of the nerve . It is also evident that multifocal proximal lesions can summate to produce a clinically symmetric distal sensorimotor neuropathy. The histopathological changes have been studied in patients with acute third cranial nerve lesions who have come to autopsy (Asbury et ai, 1970) . Evidence of focal demyelination and remyelination was observed (Figure 4). It is of interest that this tended to affect the centre of the nerve, correlating with the preservation of pupillary function that characterizes diabetic third nerve lesions. The pupillomotor fibres are known to travel at the periphery of the nerve. The supposition is that such lesions have a vascular basis, although this has not been substantiated. The predominantly demyelinating nature of the pathology, with axonal preservation, equates with the usual natural history of diabetic third nerve lesions in which recovery tends to be rapid and complete. Vascular and connective tissue changes Abnormalities of the endoneurial and perineurial blood vessels are prominent in patients with established diabetic neuropathy. These consist
843
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L
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Diagram of serial reconstruction from sections of thc intracavernous portion of the
left third cranial nerve of a diabetic patient who died three weeks after developing a third
nerve palsy. The stippled area indicates the site of a recent focal region of demyelination. A. Band C indicate the blood supply to the nerve. Reproduced from Asbury et al (1970), with permission .
of endothelial proliferation and consequent luminal narrowing. Thickening of the walls of these vessels also occurs as a consequence of reduplication of the surrounding basal lamina (Figures 5 and 6). In nerve biopsies, the number of capillaries in which the lumen is 'closed' is increased in diabetic subjects in comparison with controls, and the proportion of closed capillaries correlates with the severity of the neuropathy (Dyck et ai, 1985b) . Intrafascicular area may be increased and there is often extensive collagen deposition. The lamelIated perineurial sheath that surrounds the fascicles also displays abnormalities. The basal lamina of the perineurial celIs is thickened and calcification may be present in the connective tissue space between the celIular lamellae (Figure 7). A general review of the histopathological changes in diabetic neuropathy has been given by Thomas and Eliasson (1984). BIOCHEMICAL CHANGES The notion that the complications of diabetes, including neuropathy, are a consequence of the hyperglycaemic state remains controversial, but one which has gained significance from recent evidence from both experimental and clinical studies . Persisting hyperglycaemia can be induced in various animal species by the injection of either streptozotocin (streptozocin) or alloxan and these animals are useful models to study the effect of hyperglycaemia on nerve metabolism. The relevance of such
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.. Figure 5. Portion of sural nerve from a patient with diabetic sensory polyneuropathy showing severe loss of myelinated nerve fibres and thickening of the walls of four endoneurial capillaries (arrowed). Semithin section stained with acridine orange and thionin. X516.
Figure 6. Electron micrograph of a transverse section through an cndoneurial capillary from a patient with diabetic sensory polyneuropathy. There is reduplication of the surrounding basal lamina (bl). x5015.
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Figure 7. Electron micrograph of transverse section through perineurium from the sural nerve of a patient with diabetic sensory polyneuropathy showing thickening of the basal lamina (hi) on either side of the perineurial cells (pc) and multiple calcified deposits (c) in the interlamellar spaces. x 14440.
models to clinical diabetes has been questioned and has resulted in the increased use of animals genetically predisposed to development of hyperglycaemia which are considered more appropriate clinically. Studies on the peripheral nerves of both chemically and genetically diabetic animals have resulted in the emergence of differing, although not mutually exclusive, concepts on the aetiology of neuropathy (for detailed reviews, see Gabbay, 1973; Clements, 1979; Tomlinson and Mayer, 1984; Greene et ai, 1985). Tissues which do not depend on plasma insulin for glucose entry to their cells (notably, for the purpose of this review, peripheral nerve) have cytoplasmic glucose concentrations which reflect the degree of glycaemia. The enzyme aldose reductase has a low affinity for its substrates, hexose sugars (including glucose), and so, at the low nerve glucose concentrations in peripheral nerve during normoglycaemia, the concentrations of the product of the reaction, sorbitol, are low. The sorbitol formed is further metabolized to fructose by the enzyme sorbitol dehydrogenase. Induction of hyperglycaemia in animals results in markedly elevated concentrations of glucose in peripheral nerve (Stewart et al, 1966), the consequence of which is increased activity of aldose reductase and elevated concentrations of sorbitol and fructose (Gabbay, 1973). Motor nerve conduction velocity
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is reduced in such animals. It was postulated that since sorbitol and fructose are osmotically active their accumulation would cause cellular oedema and eventually structural damage. Because aldose reductase is confined to the Schwarm cell (Gabbay and O'Sullivan, 1968), this was thought to be the site of cell damage, resulting in slowed nerve conduction velocity. However, there is an approximately 30% shrinkage of Schwarm cell volume in diabetic rat nerve (Jakobsen, 1978), which refutes the Schwarm cell osmotic theory. It is possible, however, that the micromolar concentrations of polyol which do accumulate may be highly, and critically, localized in another nerve compartment. Greene and co-workers (1975) demonstrated that not only arc glucose, sorbitol and fructose concentrations elevated in the nerves of diabetic animals, but that this is accompanied by a decrease in the concentration of the sugar alcohol, lIlya-inositol. Moreover, they also showed that the functional deficit in these animals correlated with plasma insulin level and nerve lIlya-inositol depletion since treatment of the animals with insulin or a Illya-inositol supplemented diet resulted in correction of slowed motor nerve conduction velocity. Illya-Inositol is maintained in peripheral nerve at concentrations approximately 3D-100 times that of plasma by sodium dependent, energy consuming active transport which is inhibited by increasing glucose concentrations in vitro (Greene and Lattimer, 1982; Gillon and Hawthorne, 1983). Indeed, peripheral nerves from diabetic rats accumulate approximately 40% less lIlya-inositol than nerves from control animals (Gillon and Hawthorne, 1983), which may partly account for the reduced concentration of the compound in diabetic rat nerves. With the advent of aldose reductase inhibitors came the opportunity to test directly the hypothesis that polyol accumulation is involved in the pathophysiology of diabetic neuropathy. These compounds were clearly able to normalize or prevent elevated sorbitol concentrations in the nerves of diabetic animals (Cahill et ai, 1978). Administration of structurally unrelated inhibitors had similar effects on nerve sorbitol concentration, with no reduction of glycaemia (Tomlinson et al, 1982; Finegold et al, 1983; Gillon et aI, 1983; Poulsom and Heath, 1983). In two of these studies (Tomlinson et ai, 1982; Gillon et aI, 1983) the motor nerve defect, which develops during hyperglycaemia, was prevented or restored. In addition, aldose reductase inhibitor treatment normalized or prevented the decrease in Illya-inositol concentration. Since lIlya-inositol feeding or treatment also restores the functional deficit, presumably through correction of peripheral nerve lIlya-inositol concentration, it can be hypothesized that lIlyo-inositol loss, rather than polyol accumulation, is implicated in the abnormality of nerve conduction velocity in diabetic animals (Greene et aI, 1975). Thus polyol accumulation and Illyo-inositol loss are linked processes and appear to be causally related to the development of early neuropathy in diabetic animals. Das et al (1976) demonstrated that the activity of the membrane-bound enzyme, sodium- and potassium-dependent ATPase (Na+ ,K+ -ATPase), is reduced in the nerves of streptozotocin diabetic rats. This observation was not only confirmed some years later in streptozotocin diabetic rats but it
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was also shown that administration of Illyo-inositol or an aldose reductase inhibitor could reverse the decrease in activity (Greene and Lattimer, 1984). Since this enzyme controls the sodium/potassium ion balance across the nodal axoplasmic membrane its activity is critical to the generation of the nerve action potential, and it is tempting to speculate that this defect is at the heart of the functional impairment in diabetic rat nerve. Protein synthesis in peripheral nerve trunks is limited and the nerve trunks are dependent on the process ofaxoplasmic transport for the supply of structural and functional proteins (and Na+,K+-ATPase may be no exception) from the cell body (Sidenius, 1982). This mechanism is deficient in the diabetic rat (Jakobsen and Sidenius, 1980) and the slowing in delivery of protein may be an important factor in diabetic neuropathy which involves primarily axonal degeneration. myo-Inositol treatment or aldose reductase inhibitor treatment can also prevent or reverse the deficit in slow axoplasmic transport in diabetic nerve and this effect is temporally related with the correction or prevention of the functional deficit (Tomlinson and Mayer, 1984). The defect in axonal transport may be present in nerve from human diabetics, since accumulation of the enzymes dopamine B-hydroxylase and acetylcholinesterase was reduced in nerve biopsies (Brimijoin and Dyck, 1979). A possible mechanism for the development of experimental diabetic neuropathy has been proposed by Greene et al (1985) in which nerve Illyo-inositol concentration is reduced by inhibition of active transport by hyperglycaemia. The reduced nerve Illyo-inositol concentration results in abnormal metabolism of membrane polyphosphoinositides, lipids long known to be important in the function of excitable membranes, and this may be a factor in the reduction in activity of membrane Na+ ,K+ -A'TPase. Since myo-inositol transport is also dependent on this enzyme, a self-reinforcing cycle of inositol loss and Na +,K+ -A'I'Pasc reduction is formed leading to axoplasmic sodium accumulation at the nodes of Ranvier and a deficit in action potential generation. The role of polyol accumulation in this theory is unclear but it is involved in reducing intracellular Illyo-inositol by an unexplained mechanism, perhaps by causing leakage of Illyo-inositol from the axon by localized osmotic effects. Localized swelling in the region of the nodes of Ranvier has been demonstrated recently in the nerve of the spontaneously diabetic BB Wistar rat (Greene, 1985 personal communication). Recent evidence has also shown that reduced blood flow and endoneurial oxygen tension may also contribute to the biochemical abnormalities, including the deficit in Na +,K +-A'TPase activity (Low et al, 1985). Indeed, maintaining diabetic animals in an oxygen-supplemented environment resulted in partial correction of some biochemical and functional abnormalities. Evidence for the existence of these biochemical abnormalities in nerves of diabetic individuals is scarce because of the obvious difficulty in obtaining suitable human material. Three notable studies have attempted to answer this question. Ward et al (1972) found increased concentrations of glucose, fructose and sorbitol in nerves post mortem. Evidence of increased concentrations of fructose and sorbitol in nerve biopsies in some
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patients (mainly non-insulin dependent diabetics) has been reported (Dyck et al, 1980). The concentrations of sugars and polyols were found to be much more variable in nerves from patients compared with controls presumably due to varying degrees of glycaemia. However, in this study, there was no indication of lowered nerve free 11Iyo-inositol levels. Increased glucose, fructose and sorbitol concentrations and reduced 11Iyo-inositol concentration have been demonstrated in human sciatic nerve obtained post mortem mainly from non-insulin dependent diabetic patients (Mayhew et aI, 1983). Therefore increasing evidence suggests the existence of abnormal sugar metabolism in the nerves from diabetic individuals, but the data from post-mortem material should be interpreted with caution and final confirmation awaits further studies on biopsied nerves. Data from work on animal models indicating a metabolic basis for some aspects of diabetic neuropathy have provided the impetus for clinical intervention trials on three broad fronts: improved control of glycaemia, treatment with lIlyo-inositol and treatment with aldose reductase inhibitors.
Control of glycaemia Evidence for beneficial effects on nerve function resulting from improved diabetic control is increasing. Slowed nerve conduction velocity is an early indication of diabetic neuropathy. Starting insulin in previously untreated patients resulted in rapid improvements in peroneal (Gregersen, 1968; Ward et aI, 1971) and median (Ward et al, 1971) nerves. Small improvements in median motor conduction velocity were sustained for 12 months following the initiation of insulin in non-insulin dependent diabetics, and the degree of improvement correlated with the reduction in glycaemia (Graf et aI, 1981). No improvement was found in sensory nerves in these patients. In patients already treated with insulin, two methods have been used to improve control aiming to achieve normoglycaemia. Intensive insulin therapy (i.e. multiple insulin injections) was compared with conventional insulin therapy in both insulin dependent and noninsulin dependent diabetic patients in a three-year randomized trial (Service et aI, 1983). Unfortunately, there was no clear discrimination of diabetic control between the groups owing to improvement of some patients in the conventionally treated group, and thus no difference in neurological function was discovered. This study was important in advocating methodology to study a wide range of nerve functions including sensory perception, and suggested that normoglycaemia may be required to benefit neuropathy. The best method of achieving this may be by continuous subcutaneous insulin infusion (CSII) using a portable insulin delivery pump and this method has been used extensively in recent studies. Boulton et al (1982) achieved near normal glycaemia in patients with painful neuropathy over a four-month period. Significant improvements in motor conduction velocity and vibration perception threshold occurred after six weeks of CSII and were maintained for at least four months. Pain scores in these patients were also significantly reduced, a finding also observed during an open trial of acute blood glucose lowering (Samanta
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and Burden, 1985). In another randomized study of CSII and conventional insulin treatment, near normoglycaemia was achieved in the CSII group throughout the study without improvement in the conventionally treated group (Service et aI, 1985): improvements in motor conduction velocity and vibration perception threshold were also demonstrated in this study but with a different time course, the effect not being evident after four months but only present after eight months of CSIl. Myo-inositol treatment As previously demonstrated in diabetic animals (Greene et ai, 1975), administration of a myo-inositol load to diabetic patients increases its plasma concentration despite increased urinary excretion (Clements and Reynertson, 1977; Gregersen et al, 1983) making possible an increase of peripheral nerve concentrations through increased active transport from plasma. Four studies have examined the effect of lIIyo-inositol treatment on diabetic neuropathy, all four of them using electrophysiological parameters (Gregersen et ai, 1978; Salway et ai, 1978; Clements et al, 1979; Gregersen et al, 1983), and one studying both electrophysiologicar changes together with signs and symptoms (Greene et al, 1981). In three of these studies (Gregersen et al, 1978, 1983; Greene et al, 1981) no improvement in the electrophysiological measurements was found. Two other studies, of two weeks and 16 weeks respectively, demonstrated improvement in the amplitude of the evoked action potential in the median, sural and tibial nerves (Salway et al, 1978) and in median and sural sensory nerve conduction velocity in neuropathic diabetic patients (Clements et al, 1979). Clinical improvement of symptoms (pain) and signs (pin prick, temperature perception) was also demonstrated in some patients after six months of myo-inositol therapy (Greene et ai, 1981). Aldose reductase inhibitors Rat peripheral nerve and red blood cell sorbitol concentrations are strongly correlated and red cell level is related to ambient plasma glucose levels (Malone et ai, 1984). Therefore investigators have used red cell sorbitol concentrations as an index of nerve levels (and hence drug inhibitory efficacy) during clinical studies with aldose reductase inhibitors. Several studies have now demonstrated that structurally distinct aldose reductase inhibitors reduce elevated sorbitol levels in the red cells of diabetic patients (Kilpenainen et al, 1983; Malone et al, 1984; Norris et aI, 1985; Raskin et al, 1985) and hence, by inference from the rat model, also in peripheral nerve. Clinical studies in neuropathy have been reported for three compounds, Alrestatin, Sorbinil and Tolrestat. Double-blind trials with Alrestatin indicated that three months of treatment in one study resulted in significant improvement in sensory and electrophysiological parameters (Fagius and Jameson, 1981), while a four-month study in symptomatic patients revealed no evidence of subjective or objective improvement consistent
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with a drug effect (Handelsman and Turtle, 1981). In controlled trials with Sorbinil, small but significant improvements in peroneal and median motor conduction velocity and median sensory conduction velocity have been reported after nine weeks treatment (Judzewitsch et aI, 1983) and in ulnar and tibial F wave latency and ulnar distal sensory latency after 24 weeks treatment (Fagius et al, 1985). In the latter study autonomic nervous function assessed by heart rate (R-R interval) variation also improved, and this has recently been confirmed (Jaspan et aI, 1985). In patients with severely painful neuropathy, substantial and rapid reduction in pain has been reported in an essentially uncontrolled study (Jaspan et aI, 1983), the effect being evident within a few days of starting treatment. In controlled trials on patients with painful neuropathy, confirmation of pain reduction has been obtained in a four-week cross-over study of Sorbinil and placebo although the effect was much more subtle (Young et aI, 1983). Recent controlled trials of Tolrestat suggest that this compound too has a beneficial effect on pain (Koglin et al, 1985).
CONCLUSIONS Therapeutic studies have demonstrated that nerve function and clinical symptoms can be improved, lending weight to the argument for a metabolic contribution to diabetic polyneuropathy, but the effects on objective parameters are small and on subjective parameters, subtle. Problems remain in terms of patient selection, methodology for assessment and duration of treatment. Vascular factors and an increased liability to compression injury are likely to be important in the origin of focal nerve lesions, and there is increasing evidence that ischaemia may be involved in the causation of generalized polyneuropathies.
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