Correction of altered metabolic activities in sciatic nerves of streptozocin-induced diabetic rats. Effect of ganglioside treatment.

The effect of ganglioside administration to nondiabetic and streptozocin-induced diabetic rats on sciatic nerve Na(+)-K(+)-ATPase, polyphosphoinositide (PPI) turnover, and protein phosphorylation was investigated. Gangliosides were injected (10 mg/kg body wt i.p.) for 10 or 30 days beginning 20 days after induction of diabetes. Na(+)-K(+)-ATPase activity was reduced nearly 50% in diabetic nerve and was restored to normal by both ganglioside treatments. The elevated levels of fructose and sorbitol and depressed content of myoinositol in diabetic nerve were unaffected by 30 days of ganglioside treatment, indicating that the restoration of Na(+)-K(+)-ATPase activity is not dependent on normal concentrations of these compounds. In the same nerves, 32P incorporation into phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate increased 73-76 and 39-53%, respectively, in diabetic compared with nondiabetic tissue. Ganglioside administration abolished the elevated labeling of PPIs after 30 days but was ineffective after only 10 days. Neither ganglioside regimen was able to reverse enhanced phosphorylation of the major peripheral nerve myelin protein P0. The finding that gangliosides can more quickly correct the effects of diabetes on Na(+)-K(+)-ATPase activity than on PPI turnover suggests that the mechanisms underlying these two phenomena are not closely related and are distinct from the sequence of events responsible for altered myelin protein phosphorylation.

ATPase activity defects in alloxan-induced diabetic sciatic nerve recovered by ganglioside treatment.

ATPase activities were measured in sciatic nerves from rats with alloxan-induced diabetes (ALX-D) of various duration (2 wk, 5 wk, 9 wk, and 6 mo). Our data confirm that sciatic nerve Na+-K+-ATPase abnormalities are present very early in ALX-D rats, similar to results previously described in streptozocin-induced diabetic rats, spontaneously diabetic BB Wistar rats, and ALX-D rabbits. Na+-K+-ATPase activity decreased by 26-47% in ALX-D rats compared with age-matched controls. Ganglioside treatment (10 mg/kg i.p. for 10 or 30 days starting 1 wk after ALX injection) completely impeded the enzyme reduction. The effect observed at the end of either 10 or 30 days of treatment lasted greater than or equal to 1 mo. Chronic diabetic groups treated for 30 days before killing also presented normal ATPase activity at the end of treatment. Therefore, gangliosides are effective on Na+-K+-ATPase even in animals with a longer duration of diabetes. The maintenance of fairly normal ATPase activity by ganglioside treatment could mirror a more general recovery from early metabolic dysfunction and/or late structural abnormalities in diabetic nerve fibers.

Experimental diabetic neuropathy. Effect of ganglioside treatment on axonal transport of cytoskeletal proteins.

Abnormalities in axonal transport of proteins are thought to play an important role in the pathogenesis of diabetic neuropathy. Gangliosides exert a positive action on numerous alterations in biochemistry and physiology of diabetic nerves. This study was undertaken to assess the effects of exogenous gangliosides on the axonal transport of structural proteins such as actin and tubulin in the sensory fibers of short-term (9-wk) and long-term (6-mo) diabetic rats. Adult Sprague-Dawley rats were made diabetic with a single injection of 70 mg/kg streptozocin i.p. Subgroups were injected daily with either highly purified ganglioside mixture (10 mg/kg i.p.) or saline for 1 mo, beginning either 2 or 17 wk after streptozocin injection. Age-matched rats were used as controls. Axonal transport was studied by the pulse-labeling technique. Three weeks after labeling, sciatic nerves were dissected out and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In diabetic rats of both experimental designs, the transport rate of tubulin and actin was decreased by approximately 30% compared with control rats. Ganglioside treatment counteracted such alterations in both 9-wk and 6-mo diabetic rats. These data suggest a pharmacological effect that could be correlated with molecular interactions between integral membrane glycolipids and cytoskeletal elements.

Ganglioside treatment and improved axonal regeneration capacity in experimental diabetic neuropathy.

The efficacy of gangliosides in enhancing axonal regeneration and maturation in the early stages of diabetic neuropathy was assessed by quantitative analysis of immunostained serial sections of the sciatic nerve. Sprague-Dawley rats were made diabetic with a single injection of alloxan (100 mg/kg). One week later they were injected daily intraperitoneally with either a highly purified ganglioside mixture (10 mg/kg) or sterile saline for 4 wk. At the end of the treatment, sciatic nerves were crushed and allowed to regenerate for 1 wk without ganglioside treatment. The animals were then killed, and the nerves were frozen and processed for immunohistochemistry and electron microscopy. The number of regrowing axons was counted with a computerized image-analysis system on cross sections taken at predefined distances along the regenerating stump and stained with monoclonal antibody iC8 specific for the 145,000-Mr subunit of the neurofilaments. In untreated diabetic animals the number of axons able to regenerate and sustain elongation for greater than or equal to 13 mm from the crush point was reduced by 40% with respect to control rats. Ganglioside treatment was effective in compensating almost completely for this dramatic reduction. Electron microscopy confirmed that the immunofluorescence counts corresponded to regenerating axons containing neurofilaments. These results suggest that gangliosides are able to compensate for the derangements of axonal transport of cytoskeletal proteins reported in experimental diabetic neuropathy.

Effect of parenteral administration of GM1 on cytokines and anti-ganglioside antibody patterns. Preliminary report in normal human individuals.

To assess the effects of monosialoganglioside GM1 on some immunological parameters, 12 healthy men were treated with 100 mg GM1 i.m. daily for 15 days. Before and after treatment, the following were studied: (1) serum levels of antibodies against GM1, asialo-GM1 (aGM1), GM2 and GD1b; (2) serum levels of interleukin (IL)-1 beta, IL-2, soluble IL-2 receptor (sIL-2R), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma); (3) IL-1 beta and TNF-alpha production by peripheral blood monocytes (PBMO). Anti-ganglioside antibody and cytokine serum levels were not affected by exogenous GM1 administration with the exception of a transient increase in anti-GM1 antibody titer observed in one subject. In addition, no inhibition of IL-1 beta and TNF-alpha production by PBMO was observed. These preliminary data do not support a potential immunogenic or immunomodulatory function for in vivo administered GM1.

Neurotoxicity of nonionic low-osmolar contrast media. A receptor binding study.

RATIONALE AND OBJECTIVES: The use of the newest nonionic, water-soluble, low-osmolar radiographic contrast media (CM) is still associated with occasional adverse reactions affecting the neural tissues. Because these CM display lipophilic potential in their interactions with biological membranes when diffusing within the brain parenchyma, they could affect neurotransmitter binding to the receptors. Two representative nonionic CM, iopamidol and iohexol, were studied to assess whether CM-related neurotoxicity derived from their interactions with specific receptors on neural membranes. METHODS: Binding assays were carried out in vitro on crude total membrane or crude synaptic membrane preparations from selected brain areas (cortex, striatum, hippocampus, cerebellum). The concentrations of CM and reference drugs that reduce specific binding of each ligand by 50% of its maximum value (IC50) were determined using radioligands to the receptors of the most common neurotransmitters in the central nervous system, including excitatory amino acids. RESULTS: Neither iopamidol nor iohexol inhibited the (3H) ligand binding to any kind of receptor up to very high concentrations (100 microM). CONCLUSIONS: The nonionic, low-osmolar CM did not influence the normal functions of neural membranes in our model. This suggests that occasional neurotoxic effects do not occur as a consequence of specific action on brain receptors. These CM may have an indirect, postmembrane site of action.

Occurrence of lipofuscin pigment granules and "dense microspheres" in the spinal cord of young cats treated with beta,beta'-iminodipropionitrile (IDPN).

Spinal cords of cats treated with the neurotoxic compound beta,beta'-iminodipropionitrile (IDPN) were observed to contain rounded homogeneous bodies, 1-12 microns in diameter, termed "dense microspheres" (DMS). These bodies, absent in control animals, were consistently found only in the ventral horns. No relationship with blood vessels was evident. When stained with PAS and a modified von Kossa's silver nitrate technique, DMS remained negative, showing only very slight metachromasia in some toluidine blue-stained sections. They were consistently acidophilic as evidenced by destaining and differentiation investigations. DMS were observed more frequently in the proximity of nerve cell bodies or closely adjacent to dendrites and their location was mainly extracytoplasmic; with the electron microscope, however, some DMS were also found in glial processes. Rounded osmiophilic bodies, 0.1-0.8 microns in diameter, were noticed in mitochondria of both neurons and glial cells; however, whether they were special forms of DMS or different inclusions was not assessed. Both intra- and extracytoplasmic DMS were similar in ultrastructure, appearing as single membrane-bound spherical or pear-shaped bodies containing a cottony or finely granular matrix. Additionally, both perikaryon and processes of large motoneurons were found to contain pigment granules identified as lipofuscin, which seemed to increase in number and to spread centrifugally in the processes in correlation with duration of the intoxication and size of axonal swellings induced by IDPN.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of ganglioside treatment in reducing functional alterations induced by vincristine in rabbit peripheral nerves.

Vincristine (VCR) administration to rabbits resulted in severe electrophysiologic alterations of peripheral nerves. Sciatic nerve conduction velocity, compound action potential (CAP) amplitude, and area under the CAP waveform were all reduced in a dose-dependent fashion. In addition, the pattern of conduction velocity of both motor and sensory fibers was altered and shifted toward slow conduction classes. Simultaneous treatment with gangliosides limited significantly the changes in electrophysiologic parameters induced by VCR. It is suggested that gangliosides be given in the clinical setting as protection for the peripheral nerves against the side effects of antiblastic therapy.

Functional correlates of a three-component spatial model of the alpha rhythm.

We applied a three-component (temporal, occipital and parietal) spatial model to EEG data obtained from 46 young and 11 elderly subjects. With closed eyes the mean alpha frequency was 9.51 Hz for the temporal, 9.88 Hz for the occipital and 10.14 Hz for the parietal component. The power of the occipital component was larger than that of the others. With open eyes it was reduced to the same level as the temporal and parietal components. The power of the occipital component decreased significantly with age.

Regional changes in spinal cord glucose metabolism in a rat model of painful neuropathy.

Spinal cord patterns of metabolic activity in a model of neuropathic pain were assessed in unanesthetized rats by the [14C]-2-deoxyglucose (2-DG) technique. Rats used in this procedure had demonstrable thermal hyperalgesia ipsilateral to sciatic nerve ligation and ipsilateral hindpaws that were lifted in a guarded position. The latter indicated possible spontaneous pain. Sciatic nerve ligation produced significant increases in glucose utilization in the dorsal and ventral horns of both sides, with greater activity present on the ipsilateral as compared to the contralateral side. Peak activity was in laminae V-VI, a region involved in nociceptive processing. Thus, a chronic increase in neuronal activity in these regions may reflect spontaneous neuropathic pain.

Effect of gangliosides on diacylglycerol content and molecular species in nerve from diabetic rats.

The effects of ganglioside treatment on 1,2-diacylglycerol content and on molecular species in 1,2-diacylglycerol, phosphatidic acid and total diacylglycerolipids, as well as Na+,K(+)-ATPase activity, were examined in sciatic nerves from streptozotocin-induced diabetic rats. Beginning 2 weeks after induction of diabetes, animals were administered mixed bovine brain gangliosides, AGF1, an inner ester derivative of this mixture, or saline for 5 weeks. The levels of 1,2-diacylglycerol and arachidonoyl-containing molecular species in age-matched non-diabetic animals were not affected by ganglioside treatment. In nerves from saline-treated diabetic animals, 1,2-diacylglycerol levels were not reduced, but both Na+,K(+)-ATPase activity and all arachidonyl-containing species except for 18:0/20:4 1,2-diacylglycerol were significantly decreased. The content of 1,2-diacylglycerol was lowered by 23 and 16% in bovine brain ganglioside and AGF1-treated diabetic animals, respectively, and the quantity of 18:0/20:4 1,2-diacylglycerol was also selectively reduced. Ganglioside administration did not affect the diminished levels of arachidonoyl-containing molecular species in 1,2-diacylglycerol, phosphatidic acid or diacylglycerolipids in nerve from diabetic rats. In the same nerves, bovine brain gangliosides partially and AGF1 completely restored Na+,K(+)-ATPase activity. The results suggest that gangliosides depress the content of total 1,2-diacylglycerol and the quantity of 18:0/20:4 1,2-diacylglycerol, specifically, in diabetic nerve. The possible relationship between the corrective action of gangliosides on Na+,K(+)-ATPase activity and the effect of these substances on 1,2-diacylglycerol molecular species composition and metabolism is discussed.

Gangliosides prevent the inhibition by K-252a of NGF responses in PC12 cells.

K-252a, a general kinase inhibitor, selectively blocks the actions of nerve growth factor (NGF) in PC12 cells. Since gangliosides have been reported to modulate neuronal cell responsiveness to NGF and to regulate several protein kinases, the ability of these compounds to reverse the inhibition by K-252a was tested. Parameters at both short- and long-term times following treatment of PC12 cells with NGF were analyzed which are known to be either transcription-dependent or -independent events. Gangliosides were found to completely prevent the inhibition by K-252a of NGF-induced neurite regeneration and c-fos induction, and partially also that of protein kinase N activation. The ganglioside protective effects were concentration-dependent and required the intact molecule. These findings raise the possibility that gangliosides might affect a specific pathway of NGF responses sensitive to inhibition by K-252a.

Defective activity of Na+,K(+)-ATPase in peripheral nerve of diabetic rats is independent of the axonal transport of the enzyme.

This study addressed the question as to whether the reduced activity of Na+,K(+)-ATPase reported to occur in diabetic nerves and to play a crucial role in the pathogenesis of diabetic neuropathy could be due to derangements in the axonal transport of the enzyme. A micromethod was developed to evaluate the ATPase accumulation in individual segments of ligated sciatic nerves from streptozotocin-induced diabetic rats. The results confirmed a approximately 40% decrease in the background activity, but showed that the enzyme was transported at similar rates in both anterograde and retrograde directions, suggesting that the decrease in its activity does not depend on an altered delivery along the axons.

Vasotocin fibers in the mesencephalon and pons of the domestic fowl. An immunohistochemical study.

Immunohistochemical analysis of the extrahypothalamic distribution of vasotocin-like immunoreactive elements within the brainstem of the domestic fowl revealed several, topographically identifiable, mesencephalic and pontine target areas. In the considered regions numerous nerve endings were surrounding perikarya or large dendritic trunks. No extrahypothalamic immunopositive perikarya have been observed in normal birds.

A sexually dimorphic nucleus in the quail preoptic area.

The cytoarchitectural analysis of the preoptic-anterior hypothalamic region of the Japanese quail reveals a sexual dimorphism in the total volume of the medial preoptic nucleus (significantly larger in males than in females). Different nuclei of the region (dorsal preopticus, suprachiasmaticus) do not show any statistically significant difference. The sex-related difference is more consistent comparing the distribution of dark volume. This last is due to a larger number of cells containing high amount of Nissl's substance in male than in female. Present findings represent the first example of sexual dimorphism in the avian hypothalamus.

Effect of intracarotid injection of iopamidol on local cerebral glucose utilization in rat brain.

We assessed, by means of the [14C]-2-deoxy-D-glucose autoradiography method, the effect of intracarotid injection of a nonionic, low-osmolar contrast medium (iopamidol) on local cerebral glucose utilization in the rat brain. Contrast medium was injected at 20 degrees C and at 37 degrees C, and the relative changes in local cerebral glucose utilization were measured. At 20 degrees C the viscosity of the contrast agent was about twice that of the same solution at 37 degrees C, and resulted in a statistically significant increase in local cerebral glucose utilization in the hemisphere ipsilateral to the side of intracarotid infusion. Saline control studies showed that the metabolic change was not related to either the solution temperature or the osmolality. These findings suggest that increased viscosity of a contrast medium may contribute to its neurotoxic effects during cerebral angiography, hence emphasizing the importance of preheating contrast material to avoid adverse reactions.

Motor unit function during evolution of proximal axonal swellings.

beta,beta'-Iminodipropionitrile (IDPN) impairs axonal transport of neurofilaments; their accumulation leads to the formation of proximal swellings in motor axons. Similar proximal swellings are a feature of some cases of motor neuron disease such as amyotrophic lateral sclerosis (ALS). Motor units in IDPN-treated animals were assessed to determine their relative susceptibilities to impaired function and whether the functional changes resulting from proximal axonal swellings share certain electromyographic features with ALS. Intrinsic properties of medial gastrocnemius motoneurones (MN) and contractile responses of their motor units were examined during the evolution of proximal axonal swellings in cats administered IDPN (50 mg/kg once weekly) for 7, 14 or 35 days. While conduction velocities were significantly decreased in all motor unit types by 35 days, the conduction slowing was greater in fast fatigable (types FF and FI) motor units than in fatigue resistant (types FR and S) motor units. Normal correlations between axonal conduction velocity and MN input resistance (Rin) and the inverse relationship between Rin and rheobase were lost with progression of the neuropathy. Twitch and maximum tetanic tension developed by fast-fatigable motor units declined early in the neuropathy, whereas fatigue-resistant units did not show similar changes until later stages of the intoxication. In some motor units, irregular and abnormal tetanic tensions were elicited by repetitive MN discharge. At 14 and 35 days, a novel, intermediate motor unit response classified as slow and fatigable (SF) was observed. Conduction block, characterized by repetitive MN firing without a corresponding contractile response, was observed in some type FF and S units by 35 days. Morphometric analysis of muscle fiber types showed significant atrophy, particularly in the type I fibers at 14-35 days; the atrophy reversed following cessation of IDPN administration. The influence of proximal axonal swellings on motor unit function in IDPN neuropathy is discussed in terms of reported electrophysiological alterations in motoneurone disease.

Decrease of nerve Na+,K(+)-ATPase activity in the pathogenesis of human diabetic neuropathy.

A decrease in Na+,K(+)-ATPase activity is claimed to play a central role in the pathogenesis of electrophysiological and morphological abnormalities that characterize the neuropathic complications in different animal models of diabetes mellitus. The peripheral nerves from 17 patients with either type I or type II diabetes mellitus were studied to assess the importance of changes in Na+,K(+)-ATPase activity in chronic human diabetic neuropathy. Sixteen nerves from age- and sex-matched normal individuals, and 12 nerves from non-diabetic neuropathic subjects undergoing vascular or orthopedic surgery served as negative and positive controls, respectively. All specimens were processed blind. Ouabain-sensitive ATPase activity was measured by a modified spectrophotometric coupled-enzyme assay. Standard histology, fiber teasing and electron microscopy were used to establish the normal or neuropathological patterns of surgical material. Morphometric analysis permitted calculation of fiber density in each nerve specimen and correlation of this figure with the relevant enzymatic activity. Na+,K(+)-ATPase activity was approximately 59% lower in nerves from diabetic patients than in normal controls (P < 0.01) and approximately 38% lower in nerves from non-diabetic patients with neuropathy (P < 0.01). Although nerves from both neuropathic conditions had significantly fewer fibers than those from normal individuals (diabetic -33%, and non-diabetic -22%), the decreases in Na+,K(+)-ATPase activity and fiber density were not correlated only in specimens from diabetic patients (r2 = 0.096; P = 0.22). Taken together with data from experimental animal models, these results suggest that the reduction in Na+,K(+)-ATPase activity in diabetic nerves is not an epiphenomenon secondary to fiber loss; rather, it may be an important factor in the pathogenesis and self-maintenance of human diabetic neuropathy.

Brainstem auditory evoked potentials in rats with streptozotocin-induced diabetes.

Brainstem acoustic evoked potentials (BAEPs) were studied in streptozotocin (STZ)-diabetic rats and age-matched controls at 3 and 5 months from induction of the pathology. The diabetic status of the animals was kept uncontrolled throughout the study. Body weight and glycosylated hemoglobin were markedly altered in the diabetic animals (-42%, and +120% of control values, respectively). Neurophysiological results showed an increase in the latency of the major components of BAEPs; this increase was clearly time-dependent for the peripheral component (peak I). The central component (peak IV) was also significantly delayed. However, no significant impairment of the central conduction time was demonstrated by examining the interpeak I-IV latency. In conclusion, BAEPs prove to be a useful non-invasive neurophysiological technique that may help unravel both the relative involvement of the peripheral and central nervous systems in the course of diabetes mellitus, and the evolution of diabetic neuropathy.

Inner ester derivatives of gangliosides protect autonomic nerves of alloxan-diabetic rats against Na+, K(+)-ATPase activity defects.

Bovine brain gangliosides have been shown to prevent decay in Na+,K(+)-ATPase activity in sciatic and optic nerves of alloxan- and streptozotocin-diabetic rats. In the search for a drug with greater bioavailability and increased incorporation into neural tissue, ganglioside inner ester derivatives (AGF1) were recently developed. We evaluated the effect of AGF1 treatment on Na+,K(+)-ATPase activity in homogenates of vagus nerve from alloxan-diabetic rats (100 mg/kg s.c.). Animals were treated with AGF1: 10 mg/kg 6 days/week i.p., or 30 mg/kg biweekly i.p. Treatment began 10 d post-alloxan and continued for 8 consecutive weeks. Normal age- and sex-matched rats were used as controls. Alloxan intoxication produced a 39% decrease in Na+,K(+)-ATPase activity of the vagus nerve, which was completely restored (96-97% recovery) by both AGF1 regimes. Results suggest that ganglioside inner ester derivatives may be used in the clinical setting for the management of diabetic autonomic neuropathy.