We have developed an animal model of diabetic autonomic neuropathy that is characterized by neuroaxonal dystrophy (NAD) involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozotocin (STZ)-diabetic rats. Studies with the sorbitol dehydrogenase inhibitor SDI-158, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the sorbitol pathway), have shown a dramatically increased frequency of NAD in ileal mesenteric nerves and SMG of SDI-treated versus untreated diabetics. Although lesions developed prematurely and in greater numbers in SDI-treated diabetics, their distinctive ultrastructural appearance was identical to that previously reported in long-term untreated diabetics. An SDI effect was first demonstrated in the SMG of rats that were diabetic for as little as 5 wk and was maintained for at least 7.5 months. As in untreated diabetic rats, rats treated with SDI i) showed involvement of lengthy ileal, but not shorter, jejunal mesenteric nerves; ii) demonstrated NAD in paravascular mesenteric nerves distributed to myenteric ganglia while sparing adjacent perivascular axons ramifying within the vascular adventitia; and, iii) failed to develop NAD in the superior cervical ganglia (SCG). After only 2 months of SDI-treatment, tyrosine hydroxylase immunolocalization demonstrated marked dilatation of postganglionic noradrenergic axons in paravascular ileal mesenteric nerves and within the gut wall versus those innervating extramural mesenteric vasculature. The effect of SDI on diabetic NAD in SMG was completely prevented by concomitant administration of the aldose reductase inhibitor Sorbinil. Treatment of diabetic rats with Sorbinil also prevented NAD in diabetic rats not treated with SDI. These findings indicate that sorbitol pathway-linked metabolic imbalances play a critical role in the development of NAD in this model of diabetic sympathetic autonomic neuropathy.

Dysfunction of the autonomic nervous system is a recognized complication of diabetes, ranging in severity from relatively minor sweating and pupillomotor abnormality to debilitating interference with cardiovascular, genitourinary, and alimentary dysfunction. Neuroaxonal dystrophy (NAD), a distinctive distal axonopathy involving terminal axons and synapses, represents the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy in man and several insulinopenic experimental rodent models. Although the pathogenesis of diabetic sympathetic NAD is unknown, recent studies have suggested that loss of the neurotrophic effects of insulin and/or insulin-like growth factor-I (IGF-I) on sympathetic neurons rather than hyperglycemia per se, may be critical to its development. Therefore, in our current investigation we have compared the sympathetic neuropathology developing after 8 months of diabetes in the streptozotocin (STZ)-induced diabetic rat and BB/ Wor rat, both models of hypoinsulinemic type 1 diabetes, with the BBZDR/Wor rat, a hyperglycemic and hyperinsulinemic type 2 diabetes model. Both STZ- and BB/Wor-diabetic rats reproducibly developed NAD in nerve terminals in the prevertebral superior mesenteric sympathetic ganglia (SMG) and ileal mesenteric nerves. The BBZDR/Wor-diabetic rat, in comparison, failed to develop superior mesenteric ganglionic NAD in excess of that of age-matched controls. Similarly, NAD which developed in axons of ileal mesenteric nerves of BBZDR/Wor rats was substantially less frequent than in BB/Wor- and STZ-rats. These data, considered in the light of the results of previous experiments, argue that hyperglycemia alone is not sufficient to produce sympathetic ganglionic NAD, but rather that it may be the diabetes-induced superimposed loss of trophic support, likely of IGF-I, insulin, or C-peptide, that ultimately causes NAD.

Mitochondrial release of cytochrome c in apoptotic cells activates caspases, which execute apoptotic cell death. However, the events themselves that culminate in caspase activation can have deleterious effects because caspase inhibitor-saved cells ultimately die in a caspase-independent manner. To determine what events may underlie this form of cell death, we examined bioenergetic changes in sympathetic neurons deprived of NGF in the presence of a broad-spectrum caspase inhibitor, boc-aspartyl-(OMe)-fluoromethyl ketone. Here, we report that NGF-deprived, boc-aspartyl-(OMe)-fluoromethyl ketone-saved neurons rely heavily on glycolysis for ATP generation and for survival. Second, the activity of F0F1 contributes to caspase-independent death, but has only a minor role in the maintenance of mitochondrial membrane potential, which is maintained primarily by electron transport. Third, permeability transition pore inhibition by cyclosporin A attenuates NGF deprivation-induced loss of mitochondrial proteins, suggesting that permeability transition pore opening may have a function in regulating the degradation of mitochondria after cytochrome c release. Identification of changes in caspase inhibitor-saved cells may provide the basis for rational strategies to augment the effectiveness of the therapeutic use of postmitochondrial interventions.

Gliomas with hybrid oligodendroglial/astrocytic features are diagnostically problematic, and our ability to predict tumor behavior is limited. Some likely represent intermingled mixed oligoastrocytomas (MOAs), though precise diagnostic criteria and specific markers for this lesion are lacking. From the files at Washington University (1987–2000), 155 "ambiguous" glioma/intermingled MOA candidates were independently classified and graded by 5 neuropathologists, with consensus-derived pure oligodendrogliomas and astrocytomas excluded from further study. The 90 remaining cases (grades II = 29, III = 44, IV = 17) were analyzed by FISH on formalin-fixed, paraffin-embedded sections. Detectable deletions included combined 1p/19q (9%), solitary 19q (22%), PTEN/DMBT1 (26%), and p16 (32%). EGFR amplification was found in 11%. Patients were followed until death (47%) or a median of 3.3 years. Similar to prior glioma series, patient age (p < 0.0001) and tumor grade (p < 0.0001) were strongly associated with survival times. EGFR amplification (p = 0.0007) and deletions of PTEN/DMBT1 (p = 0.016) or p16 (p = 0.014), either individually or as a group (p = 0.04), portended a shorter median survival compared with tumors lacking these alterations. We conclude that 1) distinct genetic subsets are identifiable by FISH in morphologically ambiguous gliomas, and 2) both histological grading and molecular analysis yield prognostically useful information.

To address the pathogenesis of diabetic autonomic neuropathy, we have examined the sympathetic nervous system in non-obese diabetic (NOD) and streptozotocin (STZ)-induced diabetic mice, two models of type 1 diabetes, and the db/db mouse, a model of type 2 diabetes. After only 3 to 5 weeks of diabetes, NOD mice developed markedly swollen axons and dendrites ("neuritic dystrophy") in the prevertebral superior mesenteric and celiac ganglia (SMG-CG), similar to the pathology described in diabetic STZ- and BBW-rat and man. Comparable changes failed to develop in the superior cervical ganglia of the NOD mouse or in the SMG-CG of non-diabetic NOD siblings. STZ-induced diabetic mice develop identical changes, although at a much slower pace and to a lesser degree than NOD mice. NOD-SCID mice, which are genetically identical to NOD mice except for the absence of T and B cells, do not develop diabetes or neuropathology comparable to diabetic NOD mice. However, STZ-treated NOD-SCID mice develop severe neuritic dystrophy, evidence against an exclusively autoimmune pathogenesis for autonomic neuropathy in this model. Chronically diabetic type 2 db/db mice fail to develop neuritic dystrophy, suggesting that hyperglycemia alone may not be the critical and sufficient element. The NOD mouse appears to be a valuable model of diabetic sympathetic autonomic neuropathy with unambiguous, rapidly developing neuropathology which corresponds closely to the characteristic pathology of other rodent models and man.

Autonomic neuropathy is a significant complication of diabetes resulting in increased patient morbidity and mortality. A number of studies, which have shown correspondence between neuropathologic findings in experimental animals and human subjects, have demonstrated that axonal and dendritic pathology in sympathetic ganglia in the absence of significant neuron loss represents a neuropathologic hallmark of diabetic autonomic neuropathy. A recurring theme in sympathetic ganglia, as well as in the pot-ganglionic autonomic innervation of various end organs, is the involvement of distal portions of axons and nerve terminals by degenerative or dystrophic changes. In both animals and humans, there is a surprising selectivity of the diabetic process for subpopulations of autonomic ganglia, nerve terminals within sympathetic ganglia and end organs, from end organ to end organ, and between vascular and other targets within individual end organs. Although the involvement or autonomic axons in somatic nerves may reflect an ischemic pathogenesis, the selectivity of the diabetic process confounds simple global explanations of diabetic autonomic neuropathy as the result of diminished blood flow with resultant tissue hypoxia. A single unifying pathogenetic hypothesis has not yet emerged from clinical and experimental animal studies, and it is likely that diabetic autonomic neuropathy will be shown to have multiple causative mechanisms, which will interact to result in the variety of presentations of autonomic injury in diabetes. Some of these mechanisms will be shared with aging changes in the autonomic nervous system. The role of various neurotrophic substances and the polyol pathway in the pathogenesis and treatment of diabetic neuropathy likely represents a two-edged sword with both salutary and exacerbating effects. The basic neurobiologic process underlying the diabetes-induced development of neuroaxonal dystrophy, synaptic dysplasia, defective axonal regeneration, and alterations in neurotrophic substance may be mechanistically related.

Systematic studies of the autonomic nervous system of human subjects and development of well-defined animal models have begun to substantially improve our understanding of the pathogenesis of autonomic dysfunction in aging and may eventually provide strategies for intervention. Neuropathological studies of the sympathetic ganglia of aged human subjects and rodent models have demonstrated that neuroaxonal dystrophy involving intraganglionic terminal axons and synapses is a robust, unequivocal and consistent neuropathological finding in the aged sympathetic nervous system of man and animals. Quantitative studies have demonstrated that markedly swollen argyrophilic dystrophic axon terminals develop in the prevertebral superior mesenteric (SMG) and coeliac, but to a much lesser degree in the superior cervical ganglia (SCG) as a function of age, sex (males more than females) and diabetes. Dystrophic axons were immunoreactive for neuropeptide Y, tyrosine hydroxylase, dopamine-beta-hydroxylase, trkA and p75NTR, an immunophenotype consistent with their origin from postganglionic sympathetic neurons, and contained large numbers of highly phosphorylated neurofilaments or tubulovesicular elements. The sympathetic ganglia of aged rodents also showed the hallmark changes of neuroaxonal dystrophy as a function of age and location (many more in the SMG than in the SCG). Plasticity-related synaptic remodeling could represent a highly vulnerable target of the aging process. The fidelity of animal models to the neuropathology of aged humans suggests that similar pathogenetic mechanisms may be involved in both and that therapeutic advances in animal studies may have human application.

A nine-octapeptide insertional mutation in the prion protein (PrP) gene is associated with an inherited variant of Creutzfeldt–Jakob disease in humans. Transgenic mice that express the mouse PrP homologue of this mutation (designated PG14) under control of a PrP promoter display a progressive neurological disorder characterized by ataxia, apoptosis of cerebellar granule cells, and accumulation in the brain of mutant PrP molecules that display the biochemical hallmarks of PrPSc, the pathogenic isoform of PrP. In this report, we have investigated the expression of PG14 PrP in the peripheral tissues of these mice. We found highest levels of mutant PrP in the brain and spinal cord, intermediate levels in skeletal muscle, heart, and testis and low levels in kidney, lung, spleen, intestine, and stomach. Up to 70% of the PG14 PrP expressed in peripheral tissues was detergent-insoluble, and digestion with low concentrations of proteinase K yielded a PrP 27–30 fragment. These results suggest that the mutant protein was converted to a physical state reminiscent of PrPSc, although its infectivity remains to be determined. Histological analysis of skeletal muscle, one of the peripheral tissues with the highest level of PG14 PrP, revealed features indicative of a progressive, primary myopathy, including central nuclei, necrotic and regenerating fibers, and variable fiber size. These results indicate that the PG14 mutation structurally alters the protein in a way that promotes conversion to a PrPSc-like state, regardless of the tissue context, and suggest that accumulation of PrPSc can have deleterious effects on skeletal muscle cells as well as on neurons.

Peripheral neuropathy is a significant complication of diabetes resulting in increased patient morbidity and mortality. Deficiencies of neurotrophic substances (e.g. NGF, NT-3, and IGF-I) have been proposed as pathogenetic mechanisms in the development of distal symmetrical sensory diabetic polyneuropathy, and salutary effects of exogenous NGF administration have been reported in animal models. In comparison, relatively little is known concerning the effect of NGF on experimental diabetic sympathetic autonomic neuropathy. We have developed an experimental animal model of diabetic autonomic neuropathy characterized by the regular occurrence of pathologically distinctive dystrophic axons in prevertebral sympathetic ganglia and ileal mesenteric nerves of rats with chronic streptozotocin (STZ)-induced diabetes. Treatment of STZ-diabetic rats for 2–3 months with pharmacologic doses of NGF or NT-3, neurotrophic substances with known effects on the adult sympathetic nervous system, did not normalize established neuroaxonal dystrophy (NAD) in diabetic rats in the prevertebral superior mesenteric ganglia (SMG) and ileal mesenteric nerves as had pancreatic islet transplantation and IGF-I in earlier experiments. NGF treatment of control animals actually increased the frequency of NAD in the SMG. New data suggests that, in adult sympathetic ganglia, NGF may contribute to the pathogenesis of NAD rather than its amelioration, perhaps as the result of inducing intraganglionic axonal sprouts in which dystrophic changes are superimposed. NT-3 administration did not alter the frequency of NAD in diabetic animals, although it resulted in a significant decrease in NAD in control SMG. Although deficiencies of neurotrophic substances may represent the underlying pathogenesis of a variety of experimental neuropathies, delivery of excessive levels of selected substances may produce untoward effects.

Two adult brothers, one documented to have methylmalonic acidemia with homocystinuria, or cobalamin C deficiency, after autopsy, displayed severe but divergent neurological presentations. One exhibited a myelopathy and the other chronic endocrine problems (Schmidt's syndrome) followed by a neuropsychiatric and dementing disorder owing to cerebral perivascular demyelination. The recognition of cobalamin C deficiency has practical implications because it is one of the few inherited diseases of central white matter that is treatable. Ann Neurol 2001;49:396–400

The authors report on two patients with classic medulloblastoma, each of whom underwent extensive therapy-associated neuronal maturation. The first patient presented at 3 months of age with hydrocephalus caused by a 5-cm tumor in the cerebellar vermis. He underwent a gross-total resection of a desmoplastic medulloblastoma. No mature elements were identified. Despite adjuvant chemotherapy, a 1.5-cm recurrent tumor developed 6 months later. Sections from the subtotally resected tumor demonstrated exclusively mature neuronal elements, ranging from neurocytes to ganglion cells. Four months later, a second recurrent tumor was resected. The specimen collected this time demonstrated classic medulloblastoma morphological characteristics. The patient was subsequently treated with radiation therapy, which seemed to have an effect; however, the tumor eventually progressed and the patient died. The second patient presented at 3 years of age with a midline medulloblastoma and was treated with subtotal resection, radiation therapy, and chemotherapy. Although the tumor remained stable on radiographic imaging, a second resection was performed 8 years later to alleviate hydrocephalus. Histological examination revealed predominantly small mature neurons with scattered ganglion cells and extensive calcification. No adjuvant therapy was given and the patient is alive and well as of his last follow-up examination. The mature neuronal neoplasms resected in both patients demonstrated negligible proliferative indices and stained appropriately with neuronal immunohistochemical markers. The smaller neuronal population resembled those of a central neurocytoma and medullocytoma/cerebellar neurocytoma. Analogous to neuroblastoma, our cases suggest that adjuvant therapy can induce extensive or complete neuronal maturation in medulloblastoma. Additional cases must be studied to determine the prognostic significance of this rare phenomenon.

We have developed an animal model of diabetic autonomic neuropathy characterized by neuroaxonal dystrophy (NAD) involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozotocin‐diabetic rats. We have examined the effect of SDI‐158, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the polyol pathway), on NAD in control and diabetic rats. SDI‐treatment (SDI‐Rx) did not produce NAD in control animals despite the fact that sciatic nerve sorbitol levels reached the same levels as untreated diabetic animals. SDI‐Rx resulted in a dramatically increased frequency of NAD in ileal mesenteric nerves and SMG. SDI‐Rx diabetic rats developed lesions prematurely, after only one month of diabetes, and in greater numbers than untreated diabetics. SDI‐Rx for 1,2,3 and 6 months showed a statistically significant but progressively smaller effect on NAD. The ultrastructural appearance of SDI‐Rx ganglia and mesenteric nerves were identical to that previously reported in long‐term untreated diabetics; however, short term SDI‐Rx resulted in less compacted tubulovesicular elements in the SMG. Three month SDI‐Rx superior cervical ganglia failed to develop NAD in the same animals in which large numbers of lesions were found in the SMG. Dystrophic axons involved ileal mesenteric nerves while sparing those to the jejunum. Dystrophic tyrosine hydroxylase immunoreactive sympathetic axons were distributed to myenteric and submucosal ganglia within the gut wall but not to the mesenteric vasculature. Therefore, although SDI‐Rx diabetic rats show an exaggerated severity and accelerated time course of NAD compared to untreated diabetics, lesion appearance, immunoreactivity and distribution are comparable to those found in untreated STZ‐diabetic rats of much longer durations.

Diabetic autonomic neuropathy results in significant morbidity and mortality. Both diabetic humans and experimental animals show neuroaxonal dystrophy of autonomic nerve terminals, particularly in the prevertebral superior mesenteric ganglia (SMG) and celiac ganglia (CG) which innervate the hyperplastic/hypertrophic diabetic small intestine. Previously, investigators suggested that disturbances in ganglionic nerve growth factor (NGF) content or transport might play a pathogenetic role in diabetic autonomic pathology. To test this hypothesis, we measured NGF content and NGF receptor expression, p75(NTR) (low affinity neurotrophin receptor) and trkA (high affinity NGF receptor), in control and diabetic rat SMG, CG and superior cervical ganglia (SCG). Surprisingly, rather than a decrease, we observed an approximate doubling of NGF content in the diabetic SMG and CG, a result which reflects increased NGF content in the hyperplastic diabetic alimentary tract. No change in NGF content was detected in the diabetic SCG which is relatively spared in experimental diabetic autonomic neuropathy. NGF receptor expression was not consistently altered in any of the autonomic ganglia. These observations suggest that increased NGF content in sympathetic ganglia innervating the diabetic alimentary tract coupled with intact receptor expression may produce aberrant axonal sprouting and neuroaxonal dystrophy.

Recent studies have suggested a role for neurotrophic substances in the pathogenesis and treatment of diabetic neuropathy. In this study, the effect of insulin-like growth factor I (IGF-I) on diabetic sympathetic autonomic neuropathy was examined in an experimental streptozotocin-induced diabetic rat model. Two months of IGF-I treatment of chronically diabetic rats with established neuroaxonal dystrophy (the neuropathological hallmark of the disease) involving the superior mesenteric ganglion and ileal mesenteric nerves resulted in nearly complete normalization of the frequency of neuroaxonal dystrophy in both sites without altering the severity of diabetes. Treatment with low-dose insulin (to control for the transient glucose-lowering effects of IGF-I) failed to affect the frequency of ganglionic or mesenteric nerve neuroaxonal dystrophy or the severity of diabetes. The striking improvement in the severity of diabetic autonomic neuropathy shown with IGF-I treatment in these studies and the fidelity of the rat model to findings in diabetic human sympathetic ganglia provide promise for the development of new clinical therapeutic strategies.

We have developed a model of autonomic nervous system aging using the mouse superior cervical sympathetic ganglion (SCG) which is characterized by the reproducible development of distinctive, markedly-enlarged neuritic swellings (vacuolar neuritic dystrophy, VND). These structures contained an admixture of lucent vacuoles and subcellular organelles, and involved both presynaptic and postsynaptic ganglionic elements. Quantitation of the frequency of VND was accomplished at the light microscopic level and validated by ultrastructural examination. VND lesions were 30–100-fold more frequent in the aged mouse paravertebral SCG than in the prevertebral celiac/superior mesenteric (C/SMG) sympathetic ganglia. Although VND was identified in all ages of mice examined, the number of lesions increased significantly with age. The frequency of VND was a function of the strain of mouse examined with a 40-fold difference in VND frequency between C57BL6 mice, the least involved strain, and the DBA/2J strain, which was most affected and began to develop significant numbers of lesions at an early age. As in our human studies of aging in the sympathetic nervous system, there was a prominent gender effect with males developing twofold greater numbers of VND lesions than females. Mice maintained on a significant calorie restricted diet for 30 months developed 70% fewer lesions than ad libitum-fed, age and sex matched controls. The aging mouse SCG, therefore, represents a robust animal model with reproducible, quantifiable and unambiguous neuropathology. Insights into pathogenetic mechanisms gained in the subsequent analysis of this relatively simple peripheral sympathetic nervous system model may contribute to the understanding of some of the most complex and significant problems involving higher brain function.

Peroxynitrite, generated by the reaction of nitric oxide (NO) with superoxide at sites of inflammation, is a strong oxidant capable of damaging tissues and cells. Detection of nitrotyrosine (NT) at inflammatory sites serves as a biochemical marker for peroxynitrite-mediated damage. In this study, NT was detected immunohistochemically within autopsied CNS tissues from six of nine multiple sclerosis (MS) patients, and in most of the MS sections displaying inflammation. Nitrite and nitrate, the stable oxidation products of NO and peroxynitrite, respectively, were measured in cerebrospinal fluid samples obtained from MS patients and controls. Levels of nitrate were elevated significantly during clinical relapses of MS. These data suggest that peroxynitrite formation is a major consequence of NO produced in MS-affected CNS and implicate a role for this powerful oxidant in the pathogenesis of MS.

The polyol pathway and its dependent biochemical pathways are thought to play a role in the pathogenesis of diabetic neuropathy. We have developed an animal model of diabetic autonomic neuropathy characterized by neuroaxonal dystrophy involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozocin-diabetic rats. Our previous studies have shown a salutary effect of aldose reductase inhibitors on experimental autonomic neuropathy, suggesting a role for the polyol pathway in its pathogenesis. In the current studies we have examined the effect of the sorbitol dehydrogenase inhibitor (SDI) CP-166,572, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the polyol pathway) resulting in markedly increased levels of sorbitol in peripheral nerve. Fourteen weeks of treatment with CP-166,572 resulted in a dramatically increased frequency of neuroaxonal dystrophy in ileal mesenteric nerves and SMG. Although lesions developed prematurely and in greater numbers in SDI-treated diabetics than untreated diabetics did, their anatomic distribution and ultrastructural appearance were identical to that previously reported in long-term untreated diabetics. CP-166,572 treatment did not produce neuroaxonal dystrophy in control animals despite the fact that sciatic nerve sorbitol levels were markedly increased, reaching the same levels as untreated diabetic animals. Treatment of diabetic rats for 14 weeks with the aldose reductase inhibitor zopolrestat resulted in a significant decrease in the frequency of neuroaxonal dystrophy compared with untreated diabetics.

Prevertebral and paravertebral sympathetic autonomic ganglia respond differently to a large number of experimental and clinical insults. The selective involvement of subpopulations of sympathetic neurons may reflect differences in their response to neurotrophic substances. To test this hypothesis, we investigated the response of prevertebral and paravertebral rat sympathetic ganglia to selected neurotrophic substances in vivo and in vitro and identified the ganglionic distribution of neurons expressing high affinity neurotrophin receptor mRNAs. Dissociated cultures of embryonic prevertebral and paravertebral ganglionic neurons showed comparable responses to NGF deprivation and only small differences in their response to rescue with other trophic substances. In situ hybridization studies of adult rat sympathetic ganglia using probes specific for the high-affinity neurotrophin receptor transcripts trks A, B, and C demonstrated that neurons in both prevertebral and paravertebral sympathetic ganglia express predominantly trkA receptors in vivo. In addition, increased tyrosine hydroxylase (TOH) activity was induced only by doses of neurotrophic substances that activate trkA and showed only small differences between neonatal prevertebral and paravertebral ganglia. Although small differences in the sensitivity of pre- and paravertebral sympathetic neurons to various neurotrophins have been identified in our studies, they are unlikely, in isolation, to explain major differences in the sensitivity of these ganglia to neuropathologic processes.