W. Graham McLean

Effects of graded experimental compression on slow and fast axonal transport in rabbit vagus nerve

Effects of compression at low pressures on slow and fast axonal transport was investigated in rabbit vagus nerve. Proteins in the sensory fibres were radiolabelled by injection of [3H]leucine or [35S]methionine into the nodose ganglion. A small compression chamber and/or ligatures were applied around the cervical part of the vagus nerve for 8 h, at an appropriate time for the subsequent analysis of the effects of compression on both slow and fast transport of radiolabelled proteins. In normal nerves there were two waves of slowly transported proteins with rates of about 12-15 and 25-30 mm/day, respectively. SDS-polyacrylamide gel electrophoresis was used and confirmed that the main proteins which accumulated proximal to the ligatures had a molecular weight of 54 000-56 000. Neither compression of the nerve at 20 mm Hg nor sham-compression induced any statistically significant accumulation of slowly transported proteins at the site of compression. A higher pressure, i.e. 30 mm Hg, induced a marked but incomplete accumulation of slowly transported proteins. Fast transport was partially inhibited in some, but not all, nerves, when 20 mm Hg was applied for 8 h, in contrast to the lack of effect found previously with the same pressure applied for only 2 h. Despite these slight differences, the results indicate that both slow and fast transport are impaired by low pressure levels of around 20-30 mm Hg, which are comparable with those found in human compression neuropathies. The impaired provision of cytoskeletal elements to the distal axon may be of significance in the pathophysiology of nerve entrapment syndromes.

Changes in fast axonal transport during experimental nerve compression at low pressures

The minimal pressure for impairment of fast anterograde axonal transport was determined in rabbit vagus nerve. Proteins, transported by fast anterograde axonal transport, were labeled by a microinjection of [3H]leucine into the nodose ganglion, and a small compression chamber was applied around the cervical vagus nerve. In this way the nerve was subjected to acute, graded compression. Compression at 20 mm Hg for 2 h as well as sham compression did not induce accumulation of axonally transported proteins at the level of compression. However, a pressure of 30 mm Hg for 2 h induced a block of axonal transport at the site of compression. The causes of the axonal transport block are discussed as well as the minimal pressure level in relation to pressures found in clinical nerve compression lesions.

The toxicity of artemisinin and related compounds on neuronal and glial cells in culture

The antimalarial drug artemisinin and a number of its derivatives were tested for their effects on proliferation of undifferentiated neuroblastoma Nb2a cells and glioma C6 cells in culture as well as their ability to inhibit neurite outgrowth from Nb2a cells differentiated by removal of serum and addition of dibutyryl cyclic AMP. In the Nb2a and C6 cell cultures, all drugs except desoxyartemisinin significantly inhibited cell proliferation in a dose-related manner with the lowest effective concentration being that of artemisinin at 0.1 microM. Artemether, arteether, artemisinin and dihydroartemisinin also produced a dose-related decrease in the number of neurites/extensions formed by differentiating Nb2a cells, with an effect of dihydroartemisinin at a concentration as low as 1 nM. Desoxyartemisinin had no effect on extension/neurite formation. We propose a potential mechanism for neurotoxicity of artemisinin and its derivatives that involves the endoperoxide bridge which is also known to be necessary for their antimalarial action.

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of32P into purified neurofilament proteins when they were incubated with32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.

The role of glutathione in the neurotoxicity of artemisinin derivatives in vitro

The role of antioxidants in the neurotoxicity of the antimalarial endoperoxides artemether and dihydroartemisinin was studied in vitro by quantitative image analysis of neurite outgrowth in the neuroblastoma cell line NB2a. Intracellular glutathione concentrations were measured by high performance liquid chromatography with fluorescence detection. Both dihydroartemisinin (1 microM) and a combination of artemether (0.3 microM) plus haemin (2 microM) significantly inhibited neurite outgrowth from differentiating NB2a cells to 11.5 +/- 11.0% (SD) and 19.6 +/- 15.2% of controls, respectively. The inhibition by artemether/haemin was prevented by the antioxidants superoxide dismutase (109.7 +/- 47.8% of control), catalase (107.0 +/- 29.3%) glutathione (123.8 +/- 12.4%), L-cysteine (88.0 +/- 6.3%), N-acetyl-L-cysteine (107.8 +/- 14.9%), and ascorbic acid (104.3 +/- 12.7%). Dihydroartemisinin-induced neurotoxicity was completely or partially prevented by L-cysteine (99.5 +/- 17.7% of control), glutathione (57.9 +/- 23.4% of control), and N-acetyl-L-cysteine (57.3 +/- 9.5%), but was not prevented by superoxide dismutase, catalase, or ascorbic acid. Buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, significantly increased the neurotoxic effect of non-toxic concentrations of artemether/haemin (0.1 microM/2 microM) and dihydroartemisinin (0.2 microM), suggesting that endogenous glutathione participates in the prevention of the neurotoxicity of artemether/haemin and dihydroartemisinin. Artemether/haemin completely depleted intracellular glutathione levels, whereas dihydroartemisinin had no effect. We conclude that although glutathione status is an important determinant in the neurotoxicity of endoperoxides, depletion of glutathione is not a prerequisite for their toxicity. This is consistent with their mechanisms of toxicity being free radical-mediated damage to redox-sensitive proteins essential for neurite outgrowth, or alteration of a redox-sensitive signalling system which regulates neurite outgrowth.

Enhanced in vitro neurotoxicity of artemisinin derivatives in the presence of haemin.

The role of haem in the neurotoxicity of artemisinin derivatives has been studied in vitro by examining neurite outgrowth measured by image analysis and cellular metabolism of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) measured spectrophotometrically in the neuroblastoma cell line NB2a, and by examining binding of radiolabelled dihydroartemisinin to NB2a cell and rat brain proteins. In the cases of artemether, dihydroartemisinin, and arteether, haemin (ferriprotoporphyrin IX) significantly increased the dose-related inhibition of neurite outgrowth from differentiating NB2a cells and significantly increased the dose-dependent inhibition of MTT metabolism. Inhibition of neurite outgrowth and metabolism of MTT in the presence or absence of haemin ranged from 72% to 93% and from 27% to 49% at a drug concentration of 300 nM. Haemin also significantly increased the dose-related binding of radiolabelled dihydroartemisinin to proteins from NB2a cells approximately twofold and to rat brain between three- and sixfold. Haemin did not enhance the neurotoxicity of desoxyarteether, a structural analogue of arteether with an ether linkage in the place of the endoperoxide bridge. It is suggested that haemin may catalyse the transformation of these derivatives via an interaction with the endoperoxide bridge of the artemisinin derivative to produce free radicals or electrophilic intermediates that are toxic to neuronal cells.

Graded inhibition of retrograde axonal transport by compression of rabbit vagus nerve

Effects of experimental compression at different pressures on retrograde axonal transport were studied in rabbit vagus nerve. Proteins in the sensory neurones were radiolabelled by injection of [3H]leucine into the nodose ganglion. Sixteen hours after labelling, a small compression chamber and/or ligatures were applied around the cervical part of the vagus nerve for 8 h. Compression of the vagus nerve at 20, 30 and 200 mm Hg pressure induced a graded inhibition of both retrograde and anterograde transport of the radiolabelled proteins. Neither retrograde nor anterograde transport was affected by the presence of the non-inflated chamber. The results indicate that compression at pressures similar to those found in human carpal tunnel syndrome can block retrograde axonal transport. The consequences of inhibition of retrograde and anterograde axonal transport for the metabolism in the nerve cell bodies are discussed.

Slow Orthograde Axonal Transport of Radiolabelled Protein in Sciatic Motoneurones of Rats with Short‐Term Experimental Diabetes: Effects of Treatment with an Aldose Reductase Inhibitor or myo‐Inositol

Abstract: This study examined the effect of streptozotocin diabetes of 5 weeks duration on the profile of slow orthogradely transported radiolabelled protein in rat sciatic motoneurones. The diabetic rats showed a retardation of the tail of the slow‐component profile. This selective retardation was unaffected by treatment with an aldose reductase inhibitor, although this treatment reduced the accumulation of sorbitol and prevented the depletion of myo‐inositol in the sciatic nerves of the treated diabetic rats. Other groups, treated with myo‐inositol, had normal or elevated sciatic nerve myo‐inositol levels in the presence of accumulated sorbitol. The axonal transport profiles from both control and diabetic myo‐inositol‐treated groups gave normal tail velocities but an altered shape such that retardation of the tail of the profile may have been present in both. The study concludes that rats with 5 weeks streptozotocin diabetes show retardation of the velocity of the most slowly transported proteins in sciatic motoneurones, and that this defect is not linked to the polyol pathway.

Effect of a Single Dose of β,β′-Iminodipropionitrile In Vivo on the Properties of Neurofilaments In Vitro: Comparison with the Effect of Iminodipropionitrile Added Directly to Neurofilaments In Vitro

Abstract: The biochemical properties of neurofilaments isolated from control and iminodipropionitrile‐treated rats were compared with regard to autophosphorylation capacity, hydrolysis of ATP, and the formation of a viscous gel between filaments. Both preparations exhibited a similar polypeptide composition, and no covalent cross‐Unking between neuro‐filament subunits was induced by iminodipropionitrile in vivo. An ATPase activity, systematically present in all preparations, was unaffected by the administration of iminodipropionitrile to the rats. Conversely, the autophosphorylation of neurofilament subunits in vitro was significantly higher in preparations from iminodipropionitrile‐treated rats than from control animals, with a marked increase of the phosphorylation of a high molecular weight neurofilament‐associated protein. Iminodipropionitrile provoked a higher gelation capacity of neurofilaments as measured in vitro, with a lower critical concentration for the preparation from treated animals. A similar increased interaction was obtained with millimolar concentrations of iminodipropionitrile added to bovine neurofilaments in vitro, involving likely neurofilamentassociated molecules, because the effect of the drug was lost after their extraction by 0.8 M KCl. These results support the hypothesis that iminodipropionitrile interferes with the neurofilament networks through a preferential interaction with the neurofilament‐associated proteins, resulting in a change in their properties and consequently in an increased capacity of interaction between the polymers.

Eosinophil and airway nerve interactions

In vivo, eosinophils localise to airway nerves in patients with asthma as well as in animal models of hyperreactivity. In both, in vivo and in vitro studies, we have shown that this localisation changes both cholinergic nerve and eosinophil function. In particular, it leads to an increase in acetylcholine release due to loss of function of a neuronal autoreceptor, the M(2) muscarinic receptor. This loss of M(2) receptor function occurs because eosinophils become activated and degranulate as a result of interactions that occur via specific adhesion molecules expressed on nerves that are recognised by counter ligands on eosinophils.