J. D. Ward

Microangiopathy in human diabetic neuropathy: relationship between capillary abnormalities and the severity of neuropathy

SummaryClinical, electrophysiological and ultrastractural morphometric observations were made in 5 diabetic non-neuropathic patients, 5 diabetic patients with mild neuropathy and 11 diabetic patients with severe neuropathy. Capillary abnormalities were assessed in simultaneous nerve, muscle and skin biopsies and compared with results from 6 age-matched, non-diabetic control subjects.Nerve capillaries demonstrated markedly greater pathology than skin and muscle capillaries. Endoneurial capillary density was significantly reduced in severely neuropathic diabetic patients (p<0.01) when compared with control subjects. Capillary basement membrane (p<0.002), endothelial cell (p<0.003) and total diffusion barrier (endothelial cell, pericyte, basement membrane) (p<0.001) thickness were significantly increased, and oxygen diffusing capacity was significantly reduced (p<0.001) in the nerves of patients with severe diabetic neuropathy when compared to control subjects. Endothelial cell profile number and luminal perimeter were significantly increased in asymptomatic (p<0.01), (p<0.05) and severely neuropathic (p<0.001), (p<0.05) diabetic patients respectively. However, endothelial cell outer perimeter, a measure of capillary size, showed no significant increase in diabetic patients when compared with control subjects. An association was observed between neurophysiological and neuropathological measures of neuropathic severity. There was no significant correlation between the duration of diabetes and HbA1 levels with capillary pathology or with neuropathic severity. Very few abnormalities of muscle and skin correlated with neuropathic severity. However, all measures of nerve capillary pathology correlated significantly with neurophysiological and neuropathological measures of neuropathic severity.

Sural nerve oxygen tension in diabetes.

Peripheral nerve oxygen tensions were assessed in vivo by using microelectrodes to measure endoneurial oxygen tension in exposed sural nerve. In 11 diabetic patients with chronic sensorimotor neuropathy the mean endoneurial oxygen tension was 39.7 (SD 10.2) mm Hg. In all but one patient compared with none of four non-neuropathic subjects the mean nerve oxygen tensions were below dorsal foot vein values. This unphysiological state may have a role in the aetiology of diabetic neuropathy.

Vascular factors in diabetic neuropathy

SummaryDespite considerable research we still do not have a comprehensive explanation for the pathogenesis of diabetic neuropathy. Although chronic hyperglycaemia is almost certainly involved, it is not known whether the primary pathology is metabolic, microvascular, or an interaction between the two. Hyperglycaemia-induced polyol pathway hyperactivity associated with nerve sorbitol accumulation and myo-inositol depletion may play a part in the genesis of diabetic neuropathy. The case for microvascular disease in diabetic neuropathy is now strong. Fibre loss in human sural nerve is multifocal, suggesting ischaemia. The degree of vessel disease has been related to the severity of neuropathy. People with chronic obstructive pulmonary disease develop the so called “hypoxic neuropathy” in which similar microvascular changes occur as in diabetic neuropathy. In rats with experimental diabetic neuropathy nerve blood flow is reduced and oxygen supplementation or vasodilator treatment improved the deterioration in conduction velocity and nerve blood flow. Similarly, in human diabetic neuropathy, there is impaired nerve blood flow, epineurial arterio-venous shunting and a reduction in sural nerve oxygen tension. At what stage during the development of nerve damage these changes occur is yet to be determined.

Impaired blood flow and arterio-venous shunting in human diabetic neuropathy: a novel technique of nerve photography and fluorescein angiography.

SummaryNew techniques of sural nerve photography and fluorescein angiography which are able to provide an index of nerve blood flow have been developed. Under local anaesthetic, 3 cm of sural nerve was exposed at the ankle using an operating microscope. Without disturbing the epineurium, vessels were identified and photographed at a standard magnification (× 30). These were independently graded by an ophthalmologist not otherwise involved with the study. Fluorescein angiography was then carried out on the exposed nerve. The fluorescein appearance time and intensity of fluorescence were quantified, using computer analysis of digitised images. Thirteen subjects with chronic sensory motor neuropathy, five non-neuropathic diabetic and nine normal control subjects were studied. The mean epineurial vessel pathology score of the neuropathic group was significantly higher than the combined normal control and non-neuropathic diabetic groups (p <0.01). Direct epineurial arteriovenous shunting was observed in six neuropathic and one non-neuropathic diabetic patients and not in any of the normal control subjects. The nerve fluorescein appearance time was significantly delayed in subjects with chronic sensory motor neuropathy (51.5 ± 12 s) compared to both normal (34.7 ± 9 s, p <0.01) and non-neuropathic diabetic subjects (33.4 ± 11 s, p <0.025). The mean intensity of fluorescence at 96, 252 and 576 s, was significantly lower in subjects with chronic sensory motor neuropathy compared with both of the other groups (p <0.05). The epineurial vessel pathology score was significantly related to reduced sural (p <0.01) and peroneal (p <0.001) nerve conduction velocities, elevated vibration (p <0.01) and thermal (p <0.001) perception and the severity of retinopathy (p <0.002). The fluorescein appearance time was significantly related to reduced sural sensory (p <0.02) conduction velocity, elevated vibration (p <0.01) perception and epineurial vessel (p <0.002) pathology score, but it failed to relate to peroneal motor (p = 0.06) conduction velocity, thermal (p = 0.1) perception and the severity of retinopathy (p = 0.3). Intensity of fluorescence was significantly related to fluorescein appearance time (at 96 s, p <0.001; at 576 s, p <0.05) but did not relate to measures of neuropathic severity. These techniques have enabled us to observe that epineurial vessel anatomy is abnormal and that nerve blood flow is impaired in subjects with chronic sensory motor neuropathy. In addition epineurial arterio-venous shunting may be a feature of diabetic neuropathy. These techniques may further be applied to study nerve blood flow in early diabetic neuropathy.

Endoneurial localisation of microvascular damage in human diabetic neuropathy.

SummaryTwenty diabetic patients with neuropathy underwent clinical and neurophysiological evaluation together with a detailed morphometric assessment of capillary pathology in endoneurial and epineurial microvascular beds of the sural nerve. Morphological data were compared with ten non-diabetic control subjects. There were no significant differences in control subjects between basement membrane area, endothelial cell area, endothelial cell profile number or luminal area of endoneurial when compared with epineurial capillaries. In contrast, when compared with epineurial capillaries, endoneurial capillaries from diabetic patients demonstrated a significant increase in basement membrane (p<0.001) and endothelial cell (p<0.001) area and a significant reduction in luminal area (p<0.001). There was no significant difference in endothelial cell profile number between endoneurial and epineurial capillaries amongst diabetic patients. Previous studies have demonstrated a good correlation between the degree of microangiopathy and measures of neuropathic severity. In the present study increased endoneurial capillary basement membrane area was significantly related to reduced peroneal nerve conduction velocity (p<0.001), myelinated fibre density (p<0.001) and elevated vibration (p<0.05) and thermal (p<0.001) perception. Increased endothelial cell area and reduced luminal size were related to a reduced peroneal nerve conduction (p<0.05, p<0.01, respectively), reduced myelinated fibre density (p<0.05, p<0.01) and elevated thermal perception (p<0.05, p<0.001). Epineurial capillary basement membrane, endothelial cell and luminal area failed to relate to measures of neuropathic severity. This study has demonstrated more advanced microangiopathy and a more significant relationship to neuropathic severity in endoneurial compared with epineurial capillaries, thus providing further support for the role of microangiopathy in the pathogenesis of human diabetic neuropathy.

Electron microscopical studies of vessels in diabetic peripheral neuropathy.

the results of an electron microscopical study of sural nerve biopsies from 11 patients with diabetic neuropathy are presented. Thrombi were seen in six cases in at least one intraneural vessel; nine cases showed hyperplasia of endothelial cells, and in seven out of these nine the hyperplasia was sufficient to occlude completely the lumen of small vessels; six cases showed degenerate pericytes and endothelial cells, and in some cases endothelial cells had been shed from the vessel wall, exposing the blood within the vessel to the underlying basement membrane; in five cases large lipid droplets were seen within endothelial cells. Abnormalities of the vessel wall would result in decreased fibrinolytic activity and a reduction of the antiplatelet aggregating proprties of the vessel. Desquamation of endothelial cells from the vessel wall, with exposure of platelets to underlying collagen, may act as a trigger for thrombus formation, particularly as the blood of diabetic patients is often in a hypercoagulable state. The significance of hyperplasia of endothelial cells is at present unknown but, once established, this too would result in profound alterations of loal blood flow and ischaemia of nerve. Damage to endothelial cells may also allow seepage of haematological constituents into the vessel wall, resulting in its progressive thickening.

Small vessel disease in progressive diabetic neuropathy associated with good metabolic control.

Clinical, electrophysiological, and electron microscopical data are presented on 10 diabetic patients with severe progressive neuropathy, predominantly motor in type, in the presence of good blood glucose control, and for one patient with painful neuropathy and third cranial nerve palsy. Endothelial cell hyperplasia was seen in small vessels in all cases, and seven patients showed plugging of the vascular lumen by degenerate cellular material and electron dense protein. It is suggested that these cells desquamate and occlude smaller peripheral vessels at a point of narrowing. In one case the lumen of a vessel was occluded by thrombus. Electron microscopical examination showed a vessel occluded by degranulated platelets. Electrophysiological studies showed a pattern of denervation that was asymmetrical and distally predominant in some patients, suggesting that the neuropathy, at least in part, relates to multiple small infarcts.

Exercise-induced conduction velocity increment: a marker of impaired peripheral nerve blood flow in diabetic neuropathy

SummarySevere microvascular disease exists at the stage of clinical diabetic neuropathy. A non-invasive test that will identify those diabetic subjects who will eventually develop neuropathy is essential for early intervention. Sural sensory conduction velocity was recorded (x 3) in 12 non-neuropathic diabetic subjects, 15 diabetic subjects with established neuropathy and 16 age-matched normal control subjects, before and after exercise to 80% age/sex predicted maximum heart rate. Fixed sural electrodes were used. Subcutaneous temperature was recorded by a needle thermocouple placed near the sural nerve. Sural sensory conduction velocity increased significantly after exercise in normal subjects (p<0.01, mean increase 5.07 m/s) and non-neuropathic diabetic subjects (p<0.02, mean increase 3.99 m/s) but not in neuropathic subjects (mean increase 0.99 m/s). Subcutaneous temperature rose significantly in normal subjects (p<0.01, mean increase 2.07°C) and non-neuropathic diabetic subjects (p<0.001, mean increase 2.52 °C) but not in neuropathic subjects (mean increase 0.15 °C). However, sural sensory conduction velocity increased by 1.2 m · s−1. °C−1 following direct warming of the limb in six neuropathic subjects which was comparable to that of normal and non-neuropathic subjects (1.49 and 1.48 m · s−1. °C−1). The impairment of exercise conduction increment in diabetic neuropathy suggests impaired nerve blood flow in diabetic neuropathy.

Comparative analysis of the genetic associations of HLA-DR3 and tumour necrosis factor alpha with human IDDM

SummaryInsulin-dependent diabetes mellitus (IDDM) is associated with class II molecules of the MHC on chromosome 6, in particular HLA-DR and -DQ al-leles, but a pathogenic role for TNF-a in the class III region of the MHC has also been implied. We therefore tested whether there was any independent association between a biallelic TNF polymorphism and IDDM. The TNF2 allele was present in 61 of 114 (54 %) IDDM patients compared to 101 of 253 (40 %) control subjects (odds ratio 1.73; p > 0.02). Stratification analysis in individuals matched for HLA-DR3 revealed, however, that this association was not independent of HLA-DR3 and is most likely to be a result of linkage disequilibrium between these alleles.

Activation of Coagulation in Diabetes Mellitus in Relation to the Presence of Vascular Complications

To examine the relationship between diabetic vascular disease and haemostasis, a set of sensitive assays has been used to assess in vivo activation of coagulation in 62 diabetic patients (41 Type 1 and 21 Type 2), aged 19–68 years, who had been screened for the presence of complications. Fibrinopeptide A, an index of thrombin activity, was significantly increased in diabetic patients compared with control subjects (p<0.05), in both plasma (with complications mean 8.04 ± 11.87 (± SD); without complications 7.21 ± 10.13; control subjects 2.11 ± 1.40 μg l−1) and urine (with complications mean 1.48 ± 0.74; without complications 1.35 ± 0.62; control subjects 0.98 ± 0.39 μg l−1). Activated factor VII (VIIa ratio 1.21 ± 0.39; 1.13 ± 0.23; 1.01 ± 0.11) and fibrinogen (3.15 ± 0.59; 3.11 ± 0.69; 2.70 ± 0.57 g I−1) were also elevated in diabetic patients with and without complications (VIIa p<0.05, fibrinogen p<0.01). The only difference between Type 1 and Type 2 patients was in fibrin degradation products (Type 1 0.28 ± 0.18; Type 2 0.40 ± 0.18 mg I−1, p<0.01). Plasma levels of fibrin degradation products were elevated in diabetic patients (p<0.05 vs control subjects), and correlated with age (r=0.44, p<0.01) but were unrelated to the presence of complications. There were no significant differences in any coagulation variables between diabetic patients with and without complications. HbA1 was correlated with fibrin degradation products only (r=0.30, p<0.05), and there were no correlations with current blood glucose concentration. Our results show activation of the coagulation system in diabetes which precedes the appearance of clinically detectable complications and suggests early vascular damage.