Phenotyping animal models of diabetic neuropathy: a consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab).

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

Proteolipid protein is necessary in peripheral as well as central myelin.

Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are major components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.

Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat.

The effect of diabetes mellitus on opiate-mediated inhibition of calcium current density (I(D Ca) [pA pF-1]) and cytosolic calcium response ([Ca2+]i nM) to depolarization with elevated KCl and capsaicin was assessed. Experiments were performed on isolated, acutely dissociated dorsal root ganglion (DRG) neurons from diabetic, BioBreeding/Worcester (BB/W) rats and age-matched control animals. Sciatic nerve conduction velocity was significantly decreased in diabetic animals compared to controls. Mean I(DCa) and [Ca2+]i responses to capsaicin and elevated KCl recorded in DRGs from diabetic animals were significantly larger than those recorded in DRG neurons from controls. In neurons from diabetic animals, the opiate agonist dynorphin A (Dyn A; 1, 3, and 5 microM) had significantly less inhibitory effect on I(D Ca) and KCl-induced [Ca2+]i responses compared to controls. Omega-conotoxin GVIA (omega-CgTX; 10 microM) and pertussis toxin (PTX; 250 ng ml-1) abolished Dyn A-mediated inhibition of I(DCa) and [Ca2+]i in control and diabetic neurons, suggesting that Dyn A modulated predominantly N-type calcium channels coupled to opiate receptors via PTX-sensitive (Gi/o) inhibitory G proteins. These results suggest that opiate-mediated regulation of PTX-sensitive, G protein-coupled calcium channels is diminished in diabetes and that this correlates with impaired regulation of cytosolic calcium.

Histopathological heterogeneity of neuropathy in insulin-dependent and non-insulin-dependent diabetes, and demonstration of axo-glial dysjunction in human diabetic neuropathy.

Altered sorbitol and myo-inositol metabolism, (Na,K)-ATPase function, electrochemical sodium gradients, axonal swelling, and distortion and disruption of the node of Ranvier ("axo-glial dysjunction") directly implicate hyperglycemia in the pathogenesis of neuropathy in diabetic rats, but the relevance of this sequence to clinical neuropathy in heterogeneous groups of diabetic patients remains to be established. Fascicular sural nerve morphometry in 11 patients with neuropathy complicating insulin-dependent diabetes revealed a pattern of interrelated structural changes strikingly similar to that of the diabetic rat when compared to age-matched controls. 17 older non-insulin-dependent diabetic patients with comparable duration and severity of hyperglycemia and severity of neuropathy, displayed similar nerve fiber loss, paranodal demyelination, paranodal remyelination and segmental demyelination compared to age-matched controls, but axo-glial dysjunction was replaced by Wallerian degeneration as the primary manifestation of fiber damage, and fiber loss occurred in a spatial pattern consistent with an ischemic component. The mechanistic model developed from the diabetic rat does indeed appear to apply to human diabetic neuropathy, but superimposed hormonal, metabolic, vascular, and/or age-related effects alter the morphologic expression of the neuropathy in non-insulin dependent diabetes.

Axo-glial dysjunction. A novel structural lesion that accounts for poorly reversible slowing of nerve conduction in the spontaneously diabetic bio-breeding rat.

Biochemical abnormalities in peripheral nerve are thought to precede and condition the development of diabetic neuropathy, but metabolic intervention in chronic diabetic neuropathy produces only limited acute clinical response. The residual, metabolically unresponsive neurological deficits have never been rigorously defined in terms of either persistent metabolic derangements or irreversible structural defects because human nerve tissue is rarely accessible for anatomical and biochemical study and experimentally diabetic animals do not develop the structural hallmarks of human diabetic neuropathy. Detailed neuroanatomical-functional-biochemical correlation was therefore undertaken in long-term spontaneously diabetic BB-Wistar rats that functionally and structurally model human diabetic neuropathy. Vigorous insulin replacement in chronically diabetic BB rats essentially normalized both the sural nerve fiber caliber spectrum and the decreased sciatic nerve myo-inositol and (Na,K)-ATPase levels generally associated with conduction slowing in diabetic animals; yet, nerve conduction was only partially restored toward normal. Morphometric analysis revealed a striking disappearance of paranodal axo-glial junctional complexes that was not corrected by insulin replacement. Loss of these strategic junctional complexes, which are thought to limit lateral migration of axolemmal Na channels away from nodes of Ranvier, correlates with and can account for the diminished nodal Na permeability and resultant nodal conduction delay characteristic of chronic diabetic neuropathy in this animal model.

Role of sorbitol accumulation and myo-inositol depletion in paranodal swelling of large myelinated nerve fibers in the insulin-deficient spontaneously diabetic bio-breeding rat. Reversal by insulin replacement, an aldose reductase inhibitor, and myo-inositol.

Axo-glial dysjunction refers to the disruption of important junctional complexes that anchor terminal loops of myelin to the paranodal axolemma in diabetic human and animal peripheral nerve. Neither axo-glial dysjunction nor the preceeding acute localized paranodal swelling has been specifically attributed to discrete metabolic consequences of insulin deficiency or hyperglycemia. Two metabolic sequelae of hyperglycemia in diabetic nerve, sorbitol accumulation via aldose reductase, and (Na,K)-ATPase deficiency related to myo-inositol depletion, were explored as possible underlying causes of acute paranodal swelling in the spontaneously diabetic bio-breeding rat. 3 wk of insulin replacement, or therapy with an aldose reductase inhibitor or myo-inositol completely reversed paranodal swelling in sural nerve fibers after 3 wk of untreated insulin deficiency. These observations suggest that insulin deficiency and hyperglycemia cause reversible paranodal swelling, and ultimately poorly reversible axo-glial dysjunction, via the myo-inositol-related (Na,K)-ATPase defect rather than by the osmotic effects of sorbitol accumulation within nerve fibers.

Preventive effect of long-term aldose reductase inhibition (ponalrestat) on nerve conduction and sural nerve structure in the spontaneously diabetic Bio-Breeding rat.

To test the hypothesis that aldose reductase inhibition may prevent or delay the development of functional and structural neuropathy in the insulin-deficient diabetic Bio-Breeding rat (BB-rat), hyperglycemic rats were begun on the aldose reductase inhibitor (ARI) ponalrestat 25 mg/kg body wt soon after the onset of diabetes and followed for 4 or 6 mo. Ponalrestat treatment completely prevented the characteristic nerve conduction slowing and structural abnormalities of the node of Ranvier for 4 mo despite only partial preservation of axonal integrity. Ponalrestat treatment for 6 mo achieved a partial but significant prevention of nerve conduction slowing, axoglial dysjunction, and axonal degenerative changes. This incomplete but significant prevention of neuropathy by ponalrestat suggests that additional mechanisms besides polyol-pathway activation may be of importance in the pathogenesis of diabetic neuropathy. Alternatively, the dosage used in the present study may not have been sufficient to achieve a complete prevention. Despite the only partial protective effect of ARI treatment on degenerative peripheral nerve changes in hyperglycemic BB-rats, 6 mo of treatment resulted in a more than threefold increase in regenerating nerve fibers. These data suggest that prophylactic ARI treatment may be efficacious in delaying the development of diabetic neuropathy.

Primary preventive and secondary interventionary effects of acetyl-L-carnitine on diabetic neuropathy in the bio-breeding Worcester rat.

The abnormalities underlying diabetic neuropathy appear to be multiple and involve metabolic neuronal and vasomediated defects. The accumulation of long-chain fatty acids and impaired beta-oxidation due to deficiencies in carnitine and/or its esterified derivatives, such as acetyl-L-carnitine, may have deleterious effects. In the present study, we examined, in the diabetic bio-breeding Worcester rat, the short- and long-term effects of acetyl-L-carnitine administration on peripheral nerve polyols, myoinositol, Na+/K+ -ATPase, vasoactive prostaglandins, nerve conduction velocity, and pathologic changes. Short-term prevention (4 mo) with acetyl-L-carnitine had no effects on nerve polyols, but corrected the Na+/K+ -ATPase defect and was associated with 63% prevention of the nerve conduction defect and complete prevention of structural changes. Long-term prevention (8 mo) and intervention (from 4 to 8 mo) with acetyl-L-carnitine treatment normalized nerve PGE(1) whereas 6-keto PGF(1-alpha) and PGE(2) were unaffected. In the prevention study, the conduction defect was 73% prevented and structural abnormalities attenuated. Intervention with acetyl-L-carnitine resulted in 76% recovery of the conduction defect and corrected neuropathologic changes characteristic of 4-mo diabetic rats. Acetyl-L-carnitine treatment promoted nerve fiber regeneration, which was increased two-fold compared to nontreated diabetic rats. These results demonstrate that acetyl-L-carnitine has a preventive effect on the acute Na+/- K+_ATPase defect and a preventive and corrective effect on PGE1 in chronically diabetic nerve associated with improvements of nerve conduction velocity and pathologic changes.

The linked roles of nitric oxide, aldose reductase and, (Na+,K+)-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat.

Metabolic and vascular factors have been invoked in the pathogenesis of diabetic neuropathy but their interrelationships are poorly understood. Both aldose reductase inhibitors and vasodilators improve nerve conduction velocity, blood flow, and (Na+,K+)-ATPase activity in the streptozotocin diabetic rat, implying a metabolic-vascular interaction. NADPH is an obligate cofactor for both aldose reductase and nitric oxide synthase such that activation of aldose reductase by hyperglycemia could limit nitric oxide synthesis by cofactor competition, producing vasoconstriction, ischemia, and slowing of nerve conduction. In accordance with this construct, N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase reversed the increased nerve conduction velocity afforded by aldose reductase inhibitor treatment in the acutely diabetic rat without affecting the attendant correction of nerve sorbitol and myo-inositol. With prolonged administration, N-nitro-L-arginine methyl ester fully reproduced the nerve conduction slowing and (Na+,K+)-ATPase impairment characteristic of diabetes. Thus the aldose reductase-inhibitor-sensitive component of conduction slowing and the reduced (Na+,K+)-ATPase activity in the diabetic rat may reflect in part impaired nitric oxide activity, thus comprising a dual metabolic-ischemic pathogenesis.

The preventive and therapeutic effects of GCPII (NAALADase) inhibition on painful and sensory diabetic neuropathy.

Excitotoxic glutamate release occurs in several neurological disorders. One source is derived from the hydrolysis of the neuropeptide N-acetyl aspartyl glutamate (NAAG) by glutamate carboxypeptidase II (GCPII, also known as NAALADase). Drugs that attenuate glutamate transmission have been shown to relieve neuropathic pain, however side effects have limited their clinical use. It appears that GCPII is exclusively recruited to provide a glutamate source in hyperglutamatergic, excitotoxic conditions and therefore would be devoid of such side effects. Here we report on the therapeutic effects of an orally bio-available GCP II inhibitor on established painful and sensory neuropathy in the spontaneously diabetic BB/Wor rat. It significantly improved hyperalgesia, nerve conduction velocity and underlying myelinated fiber atrophy. The data suggest that GCP II inhibition may provide a meaningful and effective approach to the treatment of painful diabetic neuropathy.

Intramedullary spinal cord metastases from small cell carcinoma of the lung.

Four patients with intramedullary spinal cord metastases from small cell carcinoma of the lung (SCCL) are described, with emphasis on clinical presentation and treatment. All patients developed the Brown-Séquard syndrome due to intramedullary tumor in the cervical spinal cord, three within 2 mo after combined modality treatment using chemotherapy and radiotherapy. One patient presented with a Brown-Séquard syndrome and an extradural spinal cord compression from tumor. The radiological and cerebrospinal fluid findings are presented and discussed. Radiation treatment was administered to the involved segments of spinal cord in each patient. All patients responded; two for 3 and 7 mo, two for 3 and 4 wk, respectively. There was significant improvement in the quality of life for three of the four patients.

Autonomic neuropathy in BB rat. Assessment by improved method for measuring heart-rate variability.

Sinus arrhythmia is a normal respiration-related variation in heart rate that is diminished or absent in autonomic nerve dysfunction. The conventionally used measurements, R-R intervals and their standard deviations (RRSD), were made and compared with a computed statistic (R) in spontaneously diabetic BB rats and in age-matched nondiabetes-prone control rats. In addition, the effects of pentobarbital sodium anesthesia on RRSD and R values in normal control rats were compared with those of nonanesthetized animals. BB rats demonstrated a decrease in R values after 8 wk of diabetes. This decrease became more marked with increased duration of diabetes. No differences in mean R-R intervals or RRSD were obtained between diabetic and nondiabetic rats until 24 wk of diabetes. Normal control rats showed a decrease in sinus arrythmia with age. Both RRSD and R values were greatly decreased in anesthetized animals.

Diabetic autonomic neuropathy in BB rat. Ultrastructural and morphometric changes in parasympathetic nerves.

Longitudinal electron-microscopic and morphometric studies of autonomic nerves containing predominantly parasympathetic fibers were undertaken in the spontaneously diabetic BB rat. Unmyelinated fibers of the diabetic vagus nerve and myelinated fibers of the penile nerve showed increased numbers of axonal glycogenosomes and axonal sequestration. Morphometric examination of myelinated and unmyelinated fibers of the vagus nerve revealed diminished fiber size compared with age-matched control animals. The distal myenteric nerve showed marked degenerative changes, whereas no structural changes could be demonstrated in intra-myenteric ganglion cells. These changes are similar to those described previously in somatic nerves of this model but different from those seen in sympathetic nerves of the diabetic BB rat.

Effect of aldose reductase inhibition and insulin treatment on retinal capillary basement membrane thickening in BB rats.

This study compared the effect of glycemic control with insulin to that of the aldose reductase inhibitor ponalrestat on capillary basement membrane thickening in the retinas of diabetic BB rats. Diabetic animals with or without ponalrestat treatment were compared with diabetic rats subjected to vigorous insulin therapy or nondiabetic control rats with or without ponalrestat treatment after 6 mo of follow-up. Untreated diabetic animals showed the characteristic capillary basement membrane thickening in both the superficial and deep capillary beds of the retina. Vigorous blood glucose control with insulin therapy was accompanied by a complete prevention of capillary basement membrane thickening in both capillary beds, whereas aldose reductase inhibitor treatment achieved a complete prevention of basement membrane thickening in the deep capillary bed but not in the superficial capillary bed of the diabetic retina. These findings suggest that the mechanisms responsible for capillary basement membrane thickening in diabetes may be varied and modulated by topographical peculiarities in various capillary beds.

Diabetic neuropathy in the mutant mouse [C57BL/ks(db/db)]: a morphometric study.

Detailed studies of peripheral nerves were undertaken in the mutant diabetic mouse of the [C57BL/ks(db/db)] strain using electrophysiologic and morphometric techniques. Electrophysiologic studies showed severely impaired motor nerve conduction velocity (MNCV), which developed promptly during the early phase of the diabetic syndrome. Morphometric changes occurred first after 20 wk of diabetes in both myelinated and unmyelinated fibers. There were both loss and shrinkage of myelinated fibers, most pronounced in the sural nerve and the ventral root. Changes appeared late in the dorsal root and in the peroneal and vagus nerves. Unmyelinated fibers showed both shrinkage and loss of axons, presumably involving sympathetic and afferent somatic fibers. Teased fiber studies and calculations of axon-myelin ratios confirmed our earlier suggestion that the neuropathy is primarily axonal in nature. The temporal discrepancy between functional and structural impairments in the present model strongly suggests a metabolic cause of the early neuropathy. This was further supported by the positive effect of insulin treatment on MNCV during the early phase of diabetes, whereas, during the late phase, treatment failed to show any effect.

Proximal motor neuropathy in the BB-Wistar rat.

Proximal motor neuropathy is a well-recognized neuropathic complication in human diabetes mellitus, due to microvasculopathic changes. Spontaneously diabetic BB-Wistar rats maintained at a moderate severity of diabetes developed structural proximal motor neuropathy after long-standing diabetes. This was caused by multiple infarcts in the spinal ventral roots. Endoneurial vessels showed occlusions by platelet aggregates and unstriated fibrin, indicative of a hypercoagulability.

Diabetic autonomic neuropathy in the BB rat. Ultrastructural and morphometric changes in sympathetic nerves.

Autonomic neuropathy occurring in sympathetic nerves of the spontaneously diabetic BB rat has been characterized using ultrastructural and morphometric techniques. Paravertebral thoracic ganglion cells, preganglionic myelinated fibers of the white ramus, and postganglionic unmyelinated fibers of the gray ramus communicans were examined in a longitudinal fashion. The main structural abnormality consisted of expanded axons containing a variety of normal and abnormal subcellular structures, so-called dystrophic axonal changes. These increased with duration of diabetes and were found in close proximity to the somata of ganglion cells, suggesting a preganglionic origin. The ganglion cells showed no alteration in perikaryal or nuclear volume but exhibited decreased number of synapses, which correlated with the progressive increase of dystrophic changes. While no qualitative or quantitative abnormalities could be demonstrated in preganglionic myelinated fibers of the white ramus, postganglionic fibers in the gray ramus showed an increased number of glycogenosomes, axonal sequestration, and reduction in axonal size. The present findings suggest an axonopathy in sympathetic nerves of the diabetic BB rat.

Effect of insulin and statil on aldose reductase expression in diabetic rats.

Tissue accumulation of sorbitol secondary to enhanced polyol-pathway activity is believed to play an important role in the development of diabetic complications. We previously demonstrated sorbitol accumulation, due in part to enhanced expression of aldose reductase (AR) in the diabetic kidney. In this study, we quantitated AR enzyme activity, immunoreactivity, and mRNA in various tissues from nondiabetic and diabetic BB/Wor rats 3 mo after onset of diabetes. In addition, the effects of intensive insulin treatment (3-6 U/day) and the effects of the AR inhibitor Statil (25 mg.kg-1.day-1) on AR expression were determined. Of 13 tissues examined, AR activity was significantly increased in the lens, kidney, sciatic nerve, skeletal muscle, retina, and spinal cord from diabetic rats compared with age-matched nondiabetic control rats. In most tissues, AR immunoreactivity and AR mRNA were proportionately elevated. Intensive insulin treatment, which normalized blood glucose and glycosylated hemoglobin, significantly reduced AR activity and immunoreactivity. AR mRNA abundance was also reduced in tissues from insulin-treated diabetic rats. Statil treatment had no significant effect on AR immunoreactivity or AR mRNA abundance, although AR activity in tissues from Statil-treated diabetic rats was significantly reduced compared with untreated diabetic rats. These studies demonstrate that the expression of the AR gene is upregulated in most tissues of the diabetic rat, that insulin treatment reverses this phenomenon, and that AR inhibition has no effect on AR gene expression.

Endoneurial microvessels in human diabetic neuropathy. Endothelial cell dysjunction and lack of treatment effect by aldose reductase inhibitor.

Endoneurial microvascular abnormalities have been invoked in the pathogenesis of diabetic distal symmetric polyneuropathy. Detailed morphometric analysis of the endoneurial microvasculature was correlated with previously published data on nerve fiber morphometry and teased fiber analysis obtained from the same sural nerve biopsies. Biopsy specimens from neuropathic diabetic patients were obtained before and after 12 mo of aldose reductase inhibitor (ARI) treatment and compared to 15 carefully age-matched control subjects. Diabetic microvessels showed basement membrane thickening and loss of endothelial cell tight junctions. Microvascular density and the frequency of microvessels closed by endothelial cells increased with age in diabetic and control nerves and were unaffected by diabetes. The density of microvessels showing patent lumina did not differ between control and diabetic subjects and was not related to age or diabetes. Closed microvessels were composed of postcapillary venules that were otherwise devoid of ultrastructural abnormalities. We suggest that microvascular closure by endothelial cells may be a physiological condition and is unlikely to have any pathogenetic significance in diabetic neuropathy. Based on the current limited biopsy material, we conclude that 12 mo of ARI treatment that induced significant fiber repair and regeneration had no detectable effect on endoneurial microvascular abnormalities. These data suggest that endoneurial vascular pathology is not a rate-limiting factor in fiber damage or repair at this stage of diabetic neuropathy.

Are disturbances of sorbitol, phosphoinositide, and Na+-K+-ATPase regulation involved in pathogenesis of diabetic neuropathy?

Alterations in myo-inositol and phosphoinositide metabolism, induced by hyperglycemia and prevented by aldose reductase inhibitors, are implicated in impaired Na+-K+-ATPase regulation in peripheral nerve and other tissues prone to diabetic complications by an increasing range of scientific observations. However, the precise role of these related metabolic derangements in various stages of clinical complications is complex. For instance, it appears that these biochemical defects may play a role not only in the initiation of diabetic neuropathy but also in its later progression. Therefore, full appreciation of the potential pathogenetic role of altered phosphoinositide metabolism in diabetic complications requires detailed studies of both the earliest and the more mature stages of these disease processes.