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.

Aldose reductase inhibition increases CNTF-like bioactivity and protein in sciatic nerves from galactose-fed and normal rats.

The impact of exaggerated polyol pathway flux on ciliary neurotrophic factor (CNTF)-like bioactivity and expression of CNTF in rat sciatic nerve was examined after 2 months of galactose intoxication. Polyol content was elevated (P < 0.001) and motor nerve conduction velocity reduced (P < 0.05) in galactose-fed rats compared with control animals or control and galactose-fed rats treated with the aldose reductase inhibitor (ARI) Ponalrestat. CNTF-like bioactivity in the galactose-fed group was reduced to 30% of that assayed in the control group (P < 0.001). ARI treatment significantly increased CNTF-like bioactivity by 60% compared with the untreated galactose group (P < 0.05) but did not restore it to control levels. Unexpectedly, bioactivity in ARI-treated control animals was increased by nearly 250% compared with untreated controls (P < 0.005). In addition to the deficit in CNTF bioactivity in untreated galactose rats, the expression of protein, but not of mRNA, was reduced (P < 0.05). In ARI-treated control and galactose-fed rats, the expression of CNTF peptide was significantly enhanced above control levels (both P < 0.05). Concomitant with the reduction in CNTF levels, there was a shift in the axonal size-frequency distribution of myelinated fibers toward smaller axons in galactose-fed rats that was prevented by ARI treatment. Since galactose feeding has little impact on levels of CNTF mRNA, these observations suggest that deficits in CNTF-like bioactivity may result from a posttranscriptional modification of neurotrophic protein expression or turnover. Unlike other functional and structural disorders in galactose neuropathy, factors other than polyol accumulation may contribute to the deficit in CNTF-like bioactivity.

Schwann cell injury is attenuated by aldose reductase inhibition in galactose intoxication.

Four months of galactose intoxication induces a dose-dependent osmotic imbalance of the nerve microenvironment characterized by polyol, water, and electrolyte accumulation. Recently, dose-dependent cellular lesions have been described in the sciatic nerves of galactose-intoxicated rats. The present study was designed to demonstrate that the cell injury and endoneurial osmotic imbalance in galactose intoxication are dependent on the subsequent metabolism of galactose by the polyol pathway. Three groups of age-matched, female Sprague-Dawley rats were fed a control diet or diets containing complete micronutrient supplements with 40% galactose or 40% galactose and 0.04% Ponalrestat, an aldose reductase inhibitor (ARI). After 4 to 5 months, sciatic nerves were analyzed for polyol, water and endoneurial electrolyte content and processed for light and electron microscopic examination. Ponalrestat prevented myo-inositol depletion and accumulations of dulcitol, water and endoneurial fluid electrolytes. Axonal size-frequency histograms revealed that Ponalrestat attenuated the shift toward smaller fibers and the decrease in mean axonal diameter seen in untreated galactose-fed rats. Electron microscopic examination showed widespread reactive and degenerative changes in Schwann cells of galactose-intoxicated rats that culminated in cytoplasmic disintegration. Quantitative electron microscopy revealed that ARI treatment significantly reduced the incidence of abnormal Schwann cells. These observations indicate that the osmotic imbalance and cell injury seen in galactose intoxication are dependent on the metabolism of galactose by the polyol pathway.

Comparative histology of experimental allergic neuritis induced with minimum length neuritogenic peptides by adoptive transfer with sensitized cells or direct sensitization.

Using synthetic peptides representing specific regions of the bovine myelin protein P2, the minimum peptide length requirement for the T-cell epitope necessary for successful production of experimental allergic neuritis (EAN) has been shown to involve residues 61-70 of the P2 protein. In this study, morphometric analysis was used to compare the histologic changes in sciatic nerves of Lewis rats after disease was induced by P2 specific neuritogenic T-cell lines (P(2)3) or, alternatively, by direct inoculation of synthetic peptides representing residues 60-70 or 61-70 of the P2 protein sequence. Inoculation with cell line P(2)3 stimulated with residue 61-70 failed to elicit demyelination in sciatic nerves. However, cells stimulated with residue 60-70 produced inflammation, endoneurial edema, mild demyelination and axonal degeneration within seven days. In contrast, disease induced with either peptide by direct sensitization was more severe. Morphometric analysis revealed that inflammation was most severe in animals sensitized to the decapeptide. In the sciatic nerve, axonal degeneration was proportional in frequency to the extent of inflammation. Inflammation was especially intense in spinal roots with extensive destruction of axons including unmyelinated fibers. Spinal root changes were associated with Wallerian degeneration in the posterior white matter tracts of the spinal cord and were apparently secondary to endoneurial inflammation. Disruption of the blood-nerve-barrier (BNB), evident as physical separation of endothelial cells in association with severe perivascular inflammation, was observed.

Cellular pathology of the nerve microenvironment in galactose intoxication.

The effect of chronic hyperglycemia and polyol pathway activation on the Schwann cell has not been resolved although injury to this cell has long been suspected in diabetic neuropathy. Hyperglycemia, resulting from galactose intoxication of four months duration, induces dose-dependent accumulations of endoneurial fluid sodium and chloride that are linked to polyol pathway activity and associated with dose-dependent increases in sciatic nerve water content, endoneurial fluid pressure and (Na+, K+)-ATPase activity. In order to understand the impact of these changes on the nerve microenvironment, cellular elements of the endoneurium were quantitatively and qualitatively assessed in rats receiving 0%, 10%, 20% or 40% galactose diets. After four months of galactose intoxication, dose-dependent changes in the size distribution of myelinated nerve fibers were apparent. A shift in size-frequency histograms of galactose-intoxicated animals towards smaller fibers was accompanied by a decrease in axon diameter and the volume fraction ratio of axon to myelinated nerve fibers. In the sciatic nerve of all 40% galactose-fed rats examined by electron microscopy, Schwann cells of myelinated fibers showed both reactive and degenerative changes. Demyelination was preceded by splitting at the intraperiod line. Remyelination was identified by axons with disproportionately thin myelin sheaths. Axonal dystrophy and degeneration were infrequently seen, but there was axonal regeneration. Dose-dependent increases in mast cell number were observed with degranulation apparent in rats receiving 20% and 40% galactose. Endothelial cell number and basal lamina thickness were increased in the endoneurial vessels of galactose-intoxicated rats. Increased cytoplasmic area and degenerative changes in pericytes were also noted. These observations indicate that significant morphologic changes accompany the hyperosmotic imbalance resulting from galactose intoxication of four months duration. Schwann cell injury and demyelination are present in a disorder linked to polyol metabolism since aldose reductase, the anabolic enzyme of the polyol pathway, is localized to this myelin-forming cell.

TX14(A), a prosaposin-derived peptide, reverses established nerve disorders in streptozotocin-diabetic rats and prevents them in galactose-fed rats.

Recently, TX14(A), a prosaposin-derived neurotrophic peptide, was shown to prevent both large and small fiber deficits in streptozotocin diabetes. Here, the efficacy of TX14(A) in reversing established nerve conduction disorders in streptozotocin diabetes, a model of insulin deficiency, and preventing them in galactose feeding, an insulin-replete model of polyol pathway flux, was investigated. Following streptozotocin injection (50 mg/kg ip), TX14(A) treatment (1 mg/kg ip thrice weekly) was initiated in half of the animals. After 8 wk, treatment was begun in half of the untreated animals and discontinued in half of the treated animals, and the experiment continued for 6 wk. TX14(A) reversed established motor and sensory nerve conduction deficits in streptozotocin-diabetic rats and the impact of previous treatment was still evident 3 wk after withdrawal. With the onset of 40% galactose feeding, the same dose of TX14(A) was given to half of the control and half of the galactose-fed animals for 16 wk. TX14(A) was without effect in control animals but it attenuated motor and sensory nerve conduction deficits in galactose-fed rats, an effect associated with amelioration of axonal dwindling in the sciatic nerve. These observations extend the therapeutic utility of TX14(A) and highlight its potential in treating established diabetic neuropathy.

Evolution of neuropathologic abnormalities associated with blood-brain barrier breakdown in transgenic mice expressing interleukin-6 in astrocytes.

As both astrocytes and cytokines modulate the permeability of cerebral endothelial cells, transgenic animal models which overexpress cytokines, such as interleukin-6 (IL-6), may provide insight into the neuropathological consequences of increased BBB permeability. In this study, a GFAP-IL6 transgenic mouse model and horseradish peroxidase (HRP) were used to investigate BBB permeability and associated neuropathologic changes. In the cerebellum of control mice, the BBB developed between postnatal days 7 and 14. In transgenic mice, the BBB never developed and extensive breakdown was evident in both high- and low-expressor animals by 1 month after birth. Vascular proliferation was apparent from birth in association with development and retention of normal cerebellar architecture until 3 and 6 months in high- and low-expressor animals, respectively. At these times, a leptomeningeal inflammatory infiltrate, vacuolated astrocytic foot processes and endothelial abnormalities were apparent in the cerebellum. At 6 months in high-expressor and 12 months in low-expressor animals, parenchymal inflammation, gliosis, spongiform change, axonal degeneration and macrophage accumulation were evident. The findings suggest that increased production of IL-6 can influence the development and physiologic function of the BBB as well as contribute to parenchymal central nervous system injury.

BDNF attenuates functional and structural disorders in nerves of galactose-fed rats.

Galactose intoxication of rats was used to disrupt metabolism of Schwann cells and skeletal muscle, two sites that contain the polyol-forming enzyme aldose reductase (AR). Galactose-fed rats develop a neuropathy characterized by nerve conduction deficits and axonal atrophy. To investigate the possibility that galactose metabolism by AR influences axonal function and structure by altering production of neurotrophic factors, the impact of galactose intoxication on nerve and muscle BDNF levels and the effects of exogenous BDNF treatment on galactose neuropathy were examined using biochemical, electrophysiologic and morphometric techniques. Galactose feeding increased BDNF protein in peripheral nerve and muscle. Exogenous BDNF treatment attenuated motor nerve conduction velocity deficits in the sciatic nerve of galactose-fed animals and myelin splitting of motor axons in the ventral root. In contrast, sensory nerve conduction velocity (SNCV) deficits in the sciatic nerve and myelin splitting in the central projections of sensory neurons were not prevented by BDNF treatment. BDNF treatment did not attenuate reduced axonal caliber in the sciatic nerve, but did ameliorate the diminution of the caliber of central sensory projections in the dorsal root. These findings point to the potential use of BDNF in the treatment of peripheral neuropathies.

NT-3 attenuates functional and structural disorders in sensory nerves of galactose-fed rats.

The present study investigated the effect of NT-3, a neurotrophin expressed in nerve and skeletal muscle, on myelinated fiber disorders of galactose-fed rats. Adult, female Sprague-Dawley rats were fed diets containing complete micronutrient supplements and either 0% D-galactose (control) or 40% D-galactose. Treated controls received 20 mg/kg NT-3 and treated galactose-fed rats received 1, 5, or 20 mg/kg NT-3 three times per week by subcutaneous injections. After 2 months, sciatic and saphenous sensory nerve conduction velocity (SNCV) and sciatic motor nerve conduction velocity (MNCV) were measured and the sciatic, sural, peroneal and saphenous nerves and dorsal and ventral roots processed for light microscopy. Treatment of control animals with NT-3 had no effect on any functional or structural parameter. Compared to control values, galactose feeding induced a sensory and motor nerve conduction deficit and a reduction in axonal caliber. Treatment with 5 and 20 mg/kg NT-3 ameliorated deficits in sciatic and saphenous SNCV in galactose-fed rats but had no effect on the MNCV deficit. NT-3 treatment also attenuated the decrease in mean axonal caliber in the dorsal root and sural nerve but not in the saphenous nerve, ventral root and peroneal nerve. These observations show that NT-3 can selectively attenuate the sensory conduction deficit of galactose neuropathy in a dose-dependent manner that depends only in part on restoration of axonal caliber of large-fiber sensory neurons.

Reduced nerve blood flow in hexachlorophene neuropathy: relationship to elevated endoneurial fluid pressure.

To test the hypothesis that increased endoneurial fluid pressure (EFP) causes a reduction in nerve blood flow (NBF) in the vasa nervorum, we adapted a noninvasive method for measurement of nerve blood flow which was originally developed for measurement of local cerebral blood flow. This technique measures tissue distribution to the radioisotope, 14C-iodoantipyrine, and was used to compare NBF in sciatic nerves of rats with increased EFP induced by feeding them hexachlorophene (HCP), a neurotoxin which causes edema exclusively to the nervous system and confined to the myelin sheath. Elevation of interstitial fluid pressure in peripheral nerves from control values of 2.0 +/- 1.0 cm H2O to over approximately 6 cm H2O was associated with a statistically significant reduction in nerve blood flow from 14.8 +/- 5.9 to 7.8 +/- 2.5 ml/100 g of tissue/minute (min). These results support the hypothesis that increased endoneurial fluid pressure exacerbates the neuropathy by diminishing local blood flow.

Prosaposin gene expression and the efficacy of a prosaposin-derived peptide in preventing structural and functional disorders of peripheral nerve in diabetic rats.

We have recently demonstrated that prosaposin is a neurotrophic and myelinotrophic factor with the active trophic sequence located at the N-terminal region of the saposin C domain. There are also reports that prosaposin mRNA is increased distal to a physical nerve injury and that exogenous prosaposin treatment induces subsequent neuronal sprouting, suggesting involvement in repair processes. In the present study, we show that prosaposin mRNA is significantly (p < 0.05) elevated in the peripheral nerve of streptozotocin-diabetic rats, a model of insulin-deficient diabetes in which nerve injury arises from the metabolic trauma of hyperglycemia and its consequences. A 14 amino acid peptide derived from the neurotrophic region of prosaposin prevented the development of deficits in both large and small fiber function caused by diabetes in rats. The dose-dependent prevention of nerve conduction slowing by TX 14(A) was accompanied by preservation of axonal caliber and sodium-potassium ATPase activity, while prevention of thermal hypoalgesia was associated with attenuation of the decline in nerve substance P levels. It is concluded that nerve subject to the metabolic injury of uncontrolled diabetes responds by increasing prosaposin gene expression, and that prosaposin-derived neurotrophic peptides may provide a novel therapeutic approach to treatment of diabetic and other peripheral neuropathies.

Peripheral nerve demyelination in rabbits after inoculation with Freund's complete adjuvant alone or in combination with lipid haptens.

The pathology of demyelination in rabbits with experimental allergic neuritis (EAN) or galactocerebroside-induced neuritis was compared to that in rabbits inoculated with either an emulsion of lipid haptens (gangliosides, lecithin and cholesterol) and Freund's complete adjuvant or Freund's complete adjuvant (FCA) alone. In rabbits inoculated with bovine peripheral myelin in FCA, perivenular demyelination associated with infiltrates of lymphocytes and macrophages occurred after 30 days, while those animals inoculated with galactocerebroside (GC) in Freund's adjuvant did not develop lesions until 60-90 days. GC rabbits had demyelination and severe nerve edema without cellular infiltrates. In rabbits inoculated with FCA alone, demyelination was restricted to ganglia and proximal nerve roots. Myelin basic protein (MBP) and GC antibodies from EAN, GC and lipid hapten-inoculated rabbits were detected by ELISA in sera at all post-inoculation time points. Appreciable P0 and P2 antibody titers were detected only in EAN animals. The results indicate that Freund's complete adjuvant alone or in combination with lipid haptens is capable of producing neuropathic effects in the rabbit independent of those produced by EAN or galactocerebroside neuritis.

Relationship of optic disc topography to optic nerve fiber number in glaucoma.

OBJECTIVE: To assess the relationship between in vivo measurements of optic disc topography and histomorphometric measurements of optic nerve fiber number in glaucoma. METHODS: Both eyes of 10 monkeys (Macaca fascicularis) with laser-induced glaucoma in the right eye were studied. Optic disc topography was measured in vivo with a confocal scanning laser ophthalmoscope. Histomorphometry was performed on optic nerve cross sections using bright-field microscopy with camera lucida. Nerve fiber density was estimated by unbiased random sampling. Nerve fiber number was estimated for each sector by multiplying nerve fiber density with neuroglial area. Nerve fiber count was compared with each of 13 global optic disc topographic parameters. RESULTS: For neuroretinal measurements in the glaucomatous eyes, rim area, retinal nerve fiber layer (RNFL) cross-sectional area, rim volume, and RNFL thickness correlated significantly with optic nerve fiber number. Differences in nerve fiber count between control and glaucomatous optic nerves showed the strongest correlation with differences in mean height contour; this was followed by RNFL cross-sectional area, RNFL thickness, rim volume, and differences in rim area. For cup measurements in the glaucomatous eyes, cup volume below reference, cup area, mean cup depth, the ratio of cup area to disc area, and cup shape correlated significantly with nerve fiber number. Differences in nerve fiber number between control and glaucomatous optic nerves showed the strongest correlation with differences in cup shape; this was followed by mean cup depth, cup volume below reference, the ratio of cup area to disc area, cup area, and differences in cup volume below surface. No association was found between optic nerve fiber number and optic disc area in glaucomatous eyes. CONCLUSIONS: In experimental glaucoma, most optic disc topography measures correlated significantly with optic nerve fiber number. The results of this histomorphometric study support the use of confocal scanning laser ophthalmoscopy to evaluate optic nerve damage in glaucoma.

Dose-dependent alterations in nerve polyols and (Na+,K+)-ATPase activity in galactose intoxication.

Nerve polyol content and (Na+,K+)-adenosine triphosphatase (ATPase) activity of nerve homogenates were studied in a colony of rats fed diets containing either 0%, 10%, 20%, or 40% galactose for 4 months. Nerve water and dulcitol content exhibited dose-dependent increases, whereas nerve myo-inositol content declined with increasing dietary galactose. Homogenate (Na+,K+)-ATPase activity increased with increasing galactose consumption of up to 20% dietary intake and thereafter remained consistently elevated at twice the activity of 0% galactose-fed values. Nerves of rats fed 40% galactose were also examined at the light microscope level and showed evidence of both edema and myelin splitting. These data demonstrate that increased nerve water content, dulcitol accumulation, and myo-inositol depletion parallel the previously reported dose-related increase of endoneurial fluid sodium and chloride in nerves of galactose-fed rats and suggest that elevated nerve homogenate (Na+,K+)-ATPase activity may be related to one or more of these consequences of exaggerated polyol pathway flux.

CNTFR alpha alone or in combination with CNTF promotes macrophage chemotaxis in vitro.

Microchemotaxis chambers were used to investigate whether one aspect of ciliary neurotrophic factor CNTF's role as a lesion factor might be to promote the initial early recruitment of macrophages, which express the signal transducing receptor components, gp130 and LIFRbeta. CNTFRalpha alone, or in combination with CNTF, elicited concentration-dependent macrophage chemotaxis that was inhibited by a neutralizing gp 130 antibody. IL-6, but not LIF, similarly promoted gp 130-dependent macrophage chemotaxis. Stimulation of macrophages with either CNTFRalpha in combination with CNTF or IL-6 alone resulted in tyrosine phosphorylation of an approximately 130 kD protein, presumed to be gp130. Macrophage chemotaxis induced by the combination of CNTFRalpha and CNTF was inhibited in a dose-dependent fashion by wortmannin, LY294002 or PD98059, suggesting the involvement of the phosphoinositide-3 kinase and mitogen-activated protein kinase signaling proteins. As CNTFRalpha and CNTF are present, or have immediate access to nerves after injury, these data point to the possibility that this soluble receptor alone or in combination with its ligand may promote the initial early recruitment of macrophages in vivo.

Permeability and surface area of the blood-nerve barrier in galactose intoxication.

The blood-nerve movement of a small molecular weight non-electrolyte was studied in control and galactose-fed rats by measuring the permeability-surface area (PSA) product of the blood-nerve interface to [14C]mannitol in sciatic nerve using an in vivo injection method. PSA products were measured after 9 to 11 months of feeding control rats a diet containing 0% galactose and galactose-intoxicated rats a diet containing 40% galactose. Nerves of the galactose-fed group were hydrated as reflected by a significant increase in nerve water content and wet weight to dry weight ratio (both P < 0.05). Compared to controls, PSA products were increased by 51% (P < 0.01) in galactose-fed animals when referenced to nerve dry weight (13.59 +/- 2.90 x 10(-5) ml/s/g dry wt. versus 8.99 +/- 1.59 x 10(-5) ml/s/g dry wt.; mean +/- S.D.; galactose vs. control, respectively) or by 30% (P < 0.001) when referenced to nerve length (2.43 +/- 0.43 x 10(-5) ml/s/mm vs. 1.87 +/- 0.48 x 10(-5) ml/s/mm) but not when referenced to nerve wet weight. It is suggested that in galactose intoxication, where endoneurial volume changes reflect increases in nerve water content, PSA products are best normalized to dry weight or length, which are not affected by volume changes. Normalized to dry weight, the blood-nerve barrier surface area (i.e. vessels and perineurium) was determined by morphometric methods to be increased by 34% in the galactose-intoxicated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced ciliary neuronotrophic factor-like activity in nerves from diabetic or galactose-fed rats.

This study examined the levels of ciliary neuronotrophic factor (CNTF)-like activity in the sciatic nerve of rats with short-term streptozotocin-induced diabetes or dietary supplementation with 40% galactose. CNTF-like activity, found in nerve extracts by in vitro bioassay, was reduced to 20% of control values after 1 or 2 months of galactose feeding (both P less than 0.01) and to 70% of control values after two months of streptozotocin diabetes (P less than 0.01). These data demonstrate that short-term hyperglycemia or its consequences can reduce extractable levels of Schwann cell-derived neuronotrophic factor and raise the possibility that impaired Schwann cell production of CNTF may contribute to the development of experimental diabetic neuropathy.

Nerve conduction velocity, laser Doppler flow, and axonal caliber in galactose and streptozotocin diabetes.

In an initial study, the effects of galactose intoxication on nerve laser Doppler blood flow (NLDF) and nerve conduction velocity (NCV) were assessed after 1-16 weeks of galactose feeding in pentobarbital-anesthetized rats. NLDF was not significantly changed at any time point. NCV was significantly reduced after 16, but not 1 or 4, weeks of galactose feeding. In a second study, NLDF was not significantly changed by 4 weeks of galactose intoxication, but streptozotocin-diabetic NLDF was significantly reduced compared to both control (P<0.001) and galactose-intoxicated rats (P<0.05). Compared to control animals, sciatic motor NCV was significantly (P<0.001) reduced in the galactose group, while sciatic and saphenous sensory NCVs were not significantly changed. In the streptozotocin-diabetic rats, motor and sensory NCVs were all significantly reduced (P<0.001). In contrast to the NCV findings, mean caliber of myelinated axons in both the saphenous and sciatic nerves was reduced in galactose-intoxicated, but not streptozotocin-diabetic rats. The observed sequence of changes associated with these two models of diabetic neuropathy is not consistent with the proposed roles of ischemia and axonal dwindling in the reported nerve conduction deficits.

Tactile hyperesthesia, altered epidermal innervation and plantar nerve injury in the hindfeet of rats housed on wire grates.

The effects of wire grates on nerve injury and recovery were examined in rats housed in cages with sawdust-covered solid flooring. For the first 3 weeks of the study, 20 rats were housed on sawdust alone and 20 rats were housed in cages with wire grates placed over the sawdust. For the remaining 9 weeks, 10 animals housed on sawdust had wire grates added to their cages, while grates were removed from the cages of 10 animals. The effects of tactile stimulation on hindpaw plantar skin was measured weekly using the Von Frey filament test. Intraepidermal innervation using PGP 9.5 immunostaining and plantar nerve histology were assessed at the end of the 12-week study. After just 1 week on grates, hindpaw withdrawal thresholds were already markedly decreased and remained low until the grates were removed at 3 weeks. Thresholds returned to normal by 4 weeks after removal of the grates. Wire grates also induced increases in PGP 9.5 immunoreactive intraepidermal fine nerve endings that were normalized after grate removal. Demyelination, Wallerian degeneration and Renaut bodies were induced in the medial plantar nerve in rats housed in cages with wire-grate flooring. Nerve injury was largely resolved after 9 weeks on sawdust flooring. These data demonstrate that wire grates rapidly induce hindpaw tactile hyperesthesia and plantar neuropathy in rats and emphasize a risk of using wire-grate cage flooring in studies assessing hindlimb function and structure.

Aldose reductase inhibition, Doppler flux and conduction in diabetic rat nerve.

Two chemically distinct aldose reductase inhibitors, ponalrestat and tolrestat, were tested against laser Doppler blood flow and conduction deficits in the sciatic nerve of diabetic rats. The effects of two months of streptozotocin-induced diabetes and aldose reductase inhibition on body weight, plasma glucose, and nerve sugars and polyols were comparable to those reported previously. Nerve blood flow, reflected by laser Doppler flow measurements, and motor nerve conduction velocity were both significantly less in diabetic than in control animals. Both of these reductions were prevented by ponalrestat, but not tolrestat. Thus, either deficits in laser Doppler blood flow and conduction are not aldose reductase inhibitor-dependent or tolrestat has some other property which offsets the beneficial effects of aldose reductase inhibition. In either case, these data are consistent with the hypothesis that reduced nerve blood flow contributes to conduction deficits in diabetes.