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.

Severe myositis associated with Sarcocystis spp. infection in 2 dogs.

BACKGROUND: Dogs are definitive hosts for numerous species of the intracellular protozoan parasite Sarcocystis. Reports of sarcocysts in muscles of dogs most often represent incidental findings. HYPOTHESIS/OBJECTIVES: To report the clinicopathologic, ultrastructural, and molecular findings in 2 dogs with myositis associated with Sarcocystis spp. infection, as well as the response to treatment with antiprotozoal drugs. ANIMALS: Two dogs with severe myositis in association with massive sarcocystosis. METHODS: Retrospective case review. Affected dogs were identified by a diagnostic laboratory. Attending clinicians were contacted, and the medical records reviewed. Immunostaining and electron microscopy were performed on muscle biopsies. Biopsies also were subjected to 18S rRNA gene PCR. RESULTS: Both dogs had fever, lymphopenia, thrombocytopenia, and increased serum alanine aminotransferase (ALT) activity when first evaluated. One dog developed hyperbilirubinemia. Subsequently, both dogs had increased serum creatine kinase activity and clinical signs of myositis, with reluctance to move, generalized pain, and muscle wasting. Histopathology of muscle biopsies showed severe inflammatory and necrotizing myopathy with numerous sarcocysts. Ultrastructural studies and 18S rRNA gene sequence results were consistent with infection with a Sarcocystis spp. other than Sarcocystis neurona. Both dogs initially were treated unsuccessfully with clindamycin and anti-inflammatory drugs. One dog died. The other dog subsequently responded to treatment with decoquinate. CONCLUSIONS AND CLINICAL IMPORTANCE: Sarcocystis spp. infection should be included in the differential diagnosis for dogs that develop fever, thrombocytopenia, increased liver enzyme activities, and clinical and biochemical evidence of myositis. Although additional studies are required, decoquinate holds promise as an effective treatment for the disease.

Recurrent demyelination and remyelination in 37 young Bengal cats with polyneuropathy.

BACKGROUND: With the exception of diabetic neuropathy, polyneuropathy associated with hyperchylomicronemia, and a few inherited polyneuropathies, peripheral neuropathies are poorly characterized in cats. A chronic polyneuropathy is described in a cohort of young Bengal cats. OBJECTIVE: To characterize the clinical and histopathological features of a chronic-relapsing peripheral neuropathy in young Bengal cats. ANIMALS: Thirty-seven young Bengal cats with clinical weakness consistent with peripheral neuropathy. METHODS: Bengal cats were included in this study after a diagnosis of polyneuropathy was confirmed by muscle and peripheral nerve biopsy specimens. Pathological changes were characterized at the light and electron microscopic level and by morphometry. Clinical information and long-term outcome from case records of Bengal cats with histologically confirmed peripheral neuropathy were then assessed. RESULTS: Nerve fiber loss within distal intramuscular nerve branches was a consistent finding in young Bengal cats with polyneuropathy. The most common abnormalities in peripheral nerve biopsies included inappropriately thin myelin sheaths and thinly myelinated fibers surrounded by supernumerary Schwann cell processes, indicative of repeated cycles of demyelination and remyelination. Recovery was common. Response to treatment could not be determined. CONCLUSIONS AND CLINICAL IMPORTANCE: A chronic-relapsing form of polyneuropathy associated primarily with episodes of demyelination and remyelination was identified in young Bengal cats. The prognosis for recovery is good, although relapses are possible and there can be residual motor deficits.

Diabetes induces changes in ILK, PINCH and components of related pathways in the spinal cord of rats.

Recent work suggests that diabetes affects processing of peripheral, spinal and supraspinal signals in the spinal cord. However, there is little evidence for spinal cord lesions that would account for alterations in behavioral responses induced by experimental diabetes. Therefore, we assessed the expression of proteins that might affect neuronal cytoskeletal stability and thus promote dendritic and synaptic reorganization in diabetic rats. Expression of ILK, PINCH, PI3K, GSK-3beta, tau, MAP2, synaptophysin and drebrin in the lumbar spinal cord of non-diabetic and streptozotocin-diabetic rats was assessed by Western-blot analysis and immunocytochemistry after 8 and 20weeks of diabetes. The impact of diabetes on the proteins studied was duration-dependent with changes observed after 20 but not 8weeks of diabetes. ILK and PINCH proteins levels were significantly decreased and both colocalized to neurons and oligodendrocytes. PI3K protein levels were also significantly decreased, while GSK-3beta activity tended to be increased. Phosphorylation of tau and MAP2A/B protein expression were significantly increased, and expression of synaptophysin and drebrin were reduced in diabetic rats. Decreased ILK and PINCH as well as alterations of components of related signaling pathways are associated with tau hyperphosphorylation, MAP2 overexpression and reduction of synaptic proteins in the spinal cord of diabetic rats, suggesting that ILK and PINCH contribute to stabilization of axonal and dendritic structures. However, these changes are not likely the cause of altered behavioral responses in diabetic rats that occur after short-term diabetes, but may contribute to structural changes occurring in long-term diabetes.

Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor.

AIMS/HYPOTHESIS: Sensory neuropathy in diabetic patients frequently presents itself as progressive loss of thermal perception, while some patients describe concurrent spontaneous pain, allodynia or hyperalgesia. Diabetic rats develop thermal hypoalgesia and tactile allodynia by unknown mechanisms. We investigated whether sensory disorders in rats were related to glucose metabolism by aldose reductase. We also explored the therapeutic potential of exogenous neurotrophic factors. METHODS: Behavioural assessments of thermal and tactile sensitivity were performed in normal rats and in rats with streptozotocin-induced diabetes. Some of the rats were treated with insulin, aldose reductase inhibitors, ciliary neurotrophic factor or brain-derived neurotrophic factor. RESULTS: Thermal hypoalgesia was present after 8 weeks of diabetes and was prevented by insulin treatment, which maintained normoglycaemia, by the aldose reductase inhibitor Statil or by ciliary neurotrophic factor. Brain-derived neurotrophic factor did not have an effect. When diabetic rats were tested after shorter durations of diabetes, they showed transient thermal hyperalgesia after 4 weeks which progressed to thermal hypoalgesia after 8 weeks. The aldose reductase inhibitor IDD 676 (Lidorestat), given from the onset of diabetes, prevented the development of thermal hyperalgesia and also stopped progression to thermal hypoalgesia when delivered in the last 4 weeks of an 8-week period of diabetes. Tactile allodynia was not prevented by neurotrophic factor or aldose reductase inhibitor treatment. CONCLUSIONS/INTERPRETATION: Transient thermal hyperalgesia and subsequent progressive thermal hypoalgesia occur in diabetic rats secondary to exaggerated flux through the polyol pathway. A depletion of ciliary neurotrophic factor mediated by the polyol pathway may be involved in the aetiology of thermal hypoalgesia.

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.

Nerve growth factor and neurotrophin-3 promote chemotaxis of mouse macrophages in vitro.

Microchemotaxis chambers were used to explore macrophage chemotaxis in response to the neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). Both NGF and NT-3, but not BDNF, promoted macrophage chemotaxis that was receptor mediated and dependent on protein phosphorylation. These in vitro observations point to novel roles for neurotrophins that are present in nerve prior to and immediately after injury.

Prosaposin is immunolocalized to muscle and prosaptides promote myoblast fusion and attenuate loss of muscle mass after nerve injury.

Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick-embryo myoblasts showed similar prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose-dependent fashion by addition of a neutralizing anti-prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, prosaposin has myotrophic properties.

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.

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.

Prosaposin: a myelinotrophic protein that promotes expression of myelin constituents and is secreted after nerve injury.

Recently, we demonstrated that prosaposin and prosaptides (peptides encompassing the neurotrophic sequence in prosaposin) prevent cell death and increase extracellular regulated kinase (ERK) phosphorylation and sulfatide content in primary Schwann cells or oligodendrocytes (Hiraiwa et al., 1997a). Here, we examine the effect of prosaptide on other myelin constituents, on Schwann cell morphology and proliferation, and characterize the time course of expression of prosaposin protein after sciatic nerve injury. After 24 h of treatment with 10 nM TX14(A), a 14-mer prosaptide, the specific activity of UDP-galactose:ceramide galactosyltransferase (GalT) in primary Schwann cells was increased by 150% over controls. Under the same conditions, the maximum content of sulfatide increased 3-fold over controls after 48 h of treatment. Northern blot analysis, probed with oligonucleotide sequences from the GalT and P0 cDNAs, revealed that the mRNA levels of GalT and P0 protein were elevated about 30 and 200%, respectively, over controls after 24 h of treatment with TX14(A). Treatment of primary Schwann cells with TX14(A) also induced a morphological change at 10 nM; the peptide-treated cells had a bipolar (spindle-shaped) appearance after 48 h of treatment, compared to control cells which were irregular and multipolar. TX14(A) did not induce cell proliferation, indicating that TX14(A), unlike IGF-I, is not mitogenic. After sciatic nerve transection, Western blot analysis demonstrated the presence of intact prosaposin in tubular fluid in a silicon chamber into which the proximal and distal nerve stumps were sutured. The concentration of prosaposin in the fluid was maximum after 9 days post-surgery and returned to normal after 28 days post-surgery. In uninjured and injured nerve, prosaposin immunolocalized to the smooth muscle of epineurial and endoneurial vessels. These findings indicated that sciatic nerve secreted prosaposin after injury and that prosaposin is a naturally occurring injury-repair protein which acts to prevent degeneration and to promote regeneration of peripheral nerves.

Effects of hindlimb temperature on sciatic nerve laser Doppler vascular conductance in control and streptozotocin-diabetic rats.

The effects of hindlimb temperature on sciatic nerve and skeletal muscle laser Doppler vascular conductance (LDVC) were assessed in anesthetized control and streptozotocin (STZ)-diabetic rats. With core temperature at 37 degrees C and exposed hindlimb temperature at 32 degrees C, nerve LDVC was significantly lower in rats after 8 weeks of STZ diabetes than in age-matched control rats. Subsequent warming of the exposed hindlimb of control rats from 32 degrees C to 37 degrees C significantly decreased nerve LDVC by 41% and increased muscle LDVC by 48%. Because nerve LDVC was unchanged by hindlimb warming in STZ-diabetic rats, there was no significant difference between control and diabetic nerve LDVC at 37 degrees C. In a second study, after 6 weeks of STZ diabetes, changes from control nerve LDVC were shown to depend on temperature rather than the duration of surgical exposure. These findings emphasize that information about hindlimb temperature is a prerequisite for interpreting the effects of experimental diabetes on hindlimb nerve blood flow.

Decreased accumulation of endogenous brain-derived neurotrophic factor against constricting sciatic nerve ligatures in streptozotocin-diabetic and galactose-fed rats.

Anterograde and retrograde trafficking of brain-derived neurotrophic factor (BDNF) was examined in streptozotocin-diabetic and galactose-fed rats by measuring accumulation of endogenous neurotrophin proximal and distal to two constricting sciatic nerve ligatures and by direct injection of radiolabeled neurotrophin into the sciatic nerve. Compared to controls, accumulation of endogenous BDNF proximal and distal to the ligatures as well as basal levels in non-ligated nerve segments were decreased in streptozotocin-diabetic and galactose-fed rats. Neither streptozotocin diabetes nor galactose intoxication affected the amount of 125I-labeled BDNF retrogradely transported to the DRG after injection into the sciatic nerve. These results suggest that reduced anterograde and retrograde accumulations of BDNF in experimental diabetes are not a result of impaired capacity for receptor-mediated transport.

Histomorphometric analysis of optic nerve changes in experimental glaucoma.

PURPOSE: To assess relative changes in different tissue components of optic nerve and their relationship to nerve fiber loss in the experimental monkey model of glaucoma. METHODS: Chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the right eye of eight monkeys (Macaca fascicularis). Both experimental right optic nerves and control left optic nerves were studied. Histomorphometric analysis was performed on optic nerve cross-sections using bright field microscopy with camera lucida. Cross-sectional areas of optic nerve tissue components were estimated by point counting. Nerve fiber density was estimated by unbiased random sampling. Nerve fiber number was calculated by multiplying nerve fiber density with neuroglial area. RESULTS: Varying degrees of nerve fiber loss were seen in eight optic nerves with chronic IOP elevation. More than 50% nerve fiber loss was noted in four of eight experimental optic nerves. In these severely affected optic nerves, total optic nerve area was significantly decreased compared with control optic nerves. Among the optic nerve tissue components, only the ratio of myelinated fiber area to total optic nerve area was significantly decreased. The ratio of extraaxonal area to total optic nerve area was significantly increased, whereas the ratio of interfascicular septal area to total optic nerve area did not change significantly. For all optic nerves, differences in nerve fiber count between control and experimental optic nerves showed the strongest correlation with differences in myelinated fiber area, followed by differences in extraaxonal area and total optic nerve area. CONCLUSION: This histomorphometric study suggests the validity of the experimental monkey model of glaucoma in studying changes occurring in the nonaxonal optic nerve tissue components in human glaucomatous optic neuropathy. Glial scar tissue area was significantly increased in optic nerves with severe glaucomatous damage. Although a decrease in total optic nerve area was observed, among the optic nerve tissue components only myelinated nerve fiber area decreased significantly. Myelinated nerve fiber area also showed the strongest association with nerve fiber loss in experimental glaucoma.

Neurotrophin-3 reverses nerve conduction velocity deficits in streptozotocin-diabetic rats.

The ability of neurotrophin-3 (NT-3) to reverse established nerve disorders was investigated in the peripheral neuraxis of streptozotocin-diabetic rats. Sciatic sensory and motor nerve conduction velocity deficits established after 2 months of diabetes were completely normalized by one further month of treatment with either NT-3 or insulin. None of these conduction velocity changes were associated with altered mean axonal caliber in the sciatic nerve. In the dorsal and ventral roots, mean axonal caliber was significantly decreased after 8 weeks of diabetes (both P < 0.05). Subsequently, one month of insulin, but not NT-3, treatment increased mean axonal caliber to age-matched control values. NT-3 treatment was also without effect on the significant (both P < 0.05) decrease in phosphorylated heavy neurofilament (NFH) subunits seen in dorsal and ventral roots of 12 week diabetic rats. In the sural nerve, diabetes attenuated a maturation-associated increase in mean axonal caliber over the first 8 weeks of diabetes, and induced atrophy between weeks 8 and 12 that was ameliorated by both NT-3 and insulin treatment. Reductions in sural nerve axonal caliber were associated with a tendency for elevation of both phosphorylated NFH levels in large fibers and the ratio of phosphorylated to nonphosphorylated NFH that was attenuated by NT-3. These data demonstrate that NT-3 corrects established sciatic nerve conduction deficits in diabetic rats in a manner independent of changes in axonal caliber in this nerve. Further, although NT-3 was without effect on decreases in axonal caliber and NFH subunit phosphorylation in the spinal roots, reversal of axonal caliber deficits in peripheral nerves of sensory fibers may involve NT-3-mediated normalization of aberrant neurofilament phosphorylation.

Classification of teased nerve fibers for multicenter clinical trials.

Teased nerve fibers are used widely in both clinical and experimental neuropathology, but anecdotal evidence indicates that even experienced readers find little agreement on categories for teased fiber classification. To develop a classification scheme that could be used and understood by both experienced and naive readers, specific criteria were developed for normal fibers and those exhibiting Wallerian degeneration, demyelination, hypomyelination, remyelination, and abnormal paranodal myelination. Twenty fibers teased from human sural nerve biopsies were selected as examples of one or more of these categories. Ten readers, including seven having no previous experience with teased fibers, were given a set of instructions and asked to score each fiber for all matching categories. These readers averaged high rates of true positive (56-85%) classifications, while average false positive (3-18%) rates were much lower. Among the three experienced readers, true positive agreements averaged between 75 and 100% across the fiber classifications. False positives were correspondingly low, ranging between 0 and 8%. These results suggest that it is possible to design an easily learned, meaningful scheme for classifying teased nerve fibers.

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.

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.

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.

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.