Cobalt prevents nitric oxide-induced apoptotic motoneuron death in vitro.

We studied the mechanism of nitric oxide (NO) toxicity in cultured rat spinal motoneurons. Treatment with the NO donor NOC-18 (NOC) resulted in slow motoneuron death, ending in apoptosis. The observed motoneuron death was completely prevented by hemoglobin. Treatment with inhibitors of the known intracellular targets of NO, soluble guanylate cyclase, polyADP-ribose polymerase (PARP) and superoxide, did not result in any significant protection against NOC-induced motoneuron death. ATP levels were reduced as soon as 3 h after the start of NOC treatment, suggesting a direct inhibition of cellular energy production. NOC toxicity could be blocked by the general voltage-gated calcium channel blocker cobalt, but not by specific blockers of various subtypes of calcium channels.

Trophic influences of alpha-MSH and ACTH4-10 on neuronal outgrowth in vitro.

Slices of foetal spinal cords in culture were used to establish possible trophic effects of alpha-melanocyte stimulating hormone (alpha-MSH) and a fragment of the adrenocorticotropic hormone (ACTH4-10) on the outgrowth of neurites from spinal neurons. The spinal cord slices were treated with peptides over a wide concentration range. Using monoclonal antibodies against (subunits of) neurofilament followed by immunofluorescence, we could show that the extension consisted mainly of axons. After 5 and 7 days, outgrowth was quantified with 2 different techniques, namely by visual scoring under phase contrast and by means of an ELISA for neurofilament protein. Both methods yielded the same dose-response profile. Both alpha-MSH and ACTH4-10 stimulated the formation of neurites in a dose-dependent manner, with a maximal stimulatory effect at 0.001-0.01 nM (ACTH4-10) or 0.1-1.0 nM (alpha-MSH). The maximal effect of the peptides was 30-40% compared to controls. We conclude that alpha-MSH and ACTH4-10 stimulate axonal outgrowth from foetal spinal cord slices in vitro in a dose-dependent way.

Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment.

This paper presents a new and gentle method to separate Schwann cells from fibroblasts obtained from foetal rat dorsal root ganglia (DRG). The method exploits the different growth and adhesion characteristics of fibroblasts and Schwann cells under different experimental conditions such that antiproliferative (cytotoxic) drugs or time-consuming centrifugation is not needed. Standard procedures were used to obtain mixed cultures of Schwann cells, fibroblasts and neurons. After about 5 days further purification of the cells was achieved by exploiting the different responses of Schwann cells and fibroblasts to a temperature shock. Cooling the cells with cold phosphate-buffered saline (PBS), followed by pipetting cold medium directly on top of the cells ('cold jet'), resulted in specific detachment of Schwann cells and neurons, whereas fibroblasts remained securely attached. Schwann cells attached to the surface of new, uncoated culture dishes whereas neurons did not. Two cycles of the cold jet procedure resulted in nearly pure (98-100%) cultures of Schwann cells. Besides being gentle, this method is easy and fast, and because cytotoxic drugs are not used, it does not affect cell survival negatively.

Protection by an ACTH4-9 analogue against the toxic effects of cisplatin and taxol on sensory neurons and glial cells in vitro.

Sensory neuropathy is a serious side effect of anti-tumour drugs such as cisplatin and taxol. There are indications that an analogue of the adrenocorticotrophic hormone 4-9 fragment (ACTH4-9: Met(O2)-Glu-His-Phe-D-Lys-Phe) can prevent these neurotoxic effects. We studied the potential protective effects of this analogue in cultures of chick dorsal root ganglia and rat Schwann cells treated with cisplatin or taxol to gain insight into the mode of action and characteristics of this neuroprotection. Neurite outgrowth of sensory neurons in vitro was dose-dependently inhibited by cisplatin and taxol; after 48 hr, 10 micrograms/ml cisplatin reduced outgrowth from 431 +/- 17 microns to 220 +/- 6 microns and 0.01 micrograms/ml taxol from 344 +/- 3 microns to 200 +/- 43 microns. Co-treatment of 10 micrograms/ml cisplatin with the ACTH4-9 analogue (0.1 nM-1 nM) resulted in about 35% more outgrowth than cisplatin alone. In contrast, the analogue could not prevent taxol neurotoxicity. Migration of neurons and satellite cells from the DRG-body is completely inhibited by 10 micrograms/ml cisplatin. Taxol had no effect on the migration of these cells. In addition, cisplatin was more toxic to Schwann cells than taxol; 3-10 micrograms/ml cisplatin significantly reduced their laminin content, total protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase activity, and cell division. The ACTH4-9 analogue (0.01 nM-100 nM) had no effect on the migration of cells out of the DRGs and could not prevent the toxic effect on the Schwann cells. These data support our hypothesis that the neuroprotective effect of ACTH4-9 analogue is brought about by a direct action on neurons, possibly by replacing a Schwann-/satellite-cell derived trophic factor.

Oxidant treatment causes a dose-dependent phenotype of apoptosis in cultured motoneurons.

Evidence is growing that reactive oxygen species (ROS), by-products of (normal) cellular aerobic metabolism, are involved in the pathogenesis of neurodegenerative diseases. One of these diseases is amyotrophic lateral sclerosis (ALS), in which motoneurons die, leading to paralysis and death. It remains uncertain whether ROS are the cause of (apoptotic) motoneuron death in ALS. To further understand the role of ROS in motoneuron death, we investigated the effects of ROS on isolated spinal rat motoneurons in culture. ROS were generated with a combination of iron(III) and ascorbate, or with hydrogen peroxide. Both toxic treatments resulted in a dose-dependent motoneuron death. Iron(III)/ascorbate toxicity was completely prevented with the hydrogen peroxide detoxifying enzyme catalase and partially prevented with the antioxidant vitamin E. SOD1, the enzyme that removes superoxide, did not protect against iron(III)/ascorbate toxicity. ROS treatment caused apoptotic motoneuron death: low doses of iron(III)/ ascorbate or hydrogen peroxide resulted in complete apoptosis ending in nuclear fragmentation, while high doses of ROS resulted in incomplete apoptosis (nuclear condensation). Thus, depending on the dose of ROS, the motoneurons complete the apoptotic pathway (low dose) or are stopped somewhere during this route (high dose).

Selective expansion and long-term culture of human Schwann cells from sural nerve biopsies.

Fragments of sural nerve biopsy specimens were cultured in the presence of the supernatant of lymphokine-activated killer cells, resulting in the selective outgrowth of cells with bipolar or tripolar morphology, reminiscent of that of Schwann cells. Immunofluorescent staining with antibodies to the S-100 protein, the low-affinity nerve growth factor receptor, and the surface Thy-1 antigen confirmed that these cultures contained more than 99% Schwann cells and no detectable fibroblasts. The mitotic activity of Schwann cells was measured by bromodeoxyuridine labeling, and was increased when the cells were grown in medium with lymphokine-activated killer cell supernatant compared with medium without this supernatant. In the presence of lymphokine-activated killer cell supernatant, Schwann cells could be maintained in continuous culture for a minimum of 8 months.

Anti-sulphatide antibodies in peripheral neuropathy.

A study was carried out on 135 patients with chronic idiopathic neuropathy (63), neuropathy associated with monoclonal gammopathy (51, including eight with anti-MAG antibody activity) and the Guillain-Barré syndrome (GBS) (21). Serum IgM, IgG and IgA anti-sulphatide antibody titres were compared with titres in 304 patients with other neurological or immunological diseases and in 50 normal subjects. Titres were presented a) as the highest serum dilution at which reactivity could be detected, and b) in the linear region of the optical density curve. A substantial number of patients with neurological or immunological diseases had higher titres than normal subjects. Compared with normal and disease controls, five patients with neuropathy associated with IgMk monoclonal gammopathy had raised titres of IgM anti-sulphatide antibodies and one patient with GBS had raised IgM, IgG and IgA anti-sulphatide antibodies in the acute phase of the disease. Two patients had a predominantly axonal sensory neuropathy with presenting symptoms of painful paresthesiae and minimal neurological deficit. Three patients had a predominantly demyelinating sensorimotor neuropathy associated with anti-MAG antibody activity. The patient with GBS had extensive sensory loss and antibody titres returned to normal within three weeks. Raised titres of anti-sulphatide antibodies occurred in several types of neuropathy, but all had some degree of sensory impairment and associated immunological abnormality.