Effect of ALS IgG on motor neurons in organotypic spinal cord cultures

OBJECTIVE: Reports about the role of autoimmunity in amyotrophic lateral sclerosis (ALS) are inconsistent. The aim of this work was to investigate the effect of IgG from patients with ALS on motor neurons in a physiological-like surrounding. METHODS: Using affinity chromatography, IgG from six ALS patients, four disease controls and five healthy subjects was purified. Organotypic spinal cord cultures, which conserve the structure of the spinal cord in a horizontal plane and are suitable for studies with long-term treatment, were used and IgG with different concentrations ranging from 0.05 mg/mL to 0.5 mg/mL was added to the culture medium. Ventral motor neuron survival was evaluated by morphology and SMI-32 immunohistochemistry staining. Lactate dehydrogenase (LDH) level in the culture medium was measured by colorimetry. RESULTS: After cultures were treated with ALS IgG for three weeks, the number and morphology of motor neurons showed little change. In addition, there was no significant difference in lactate dehydrogenase release between cultures treated with medium alone, normal control IgG, disease control IgG or ALS IgG. CONCLUSIONS: The results indicate that IgG from these ALS patients was insufficient per se to induce motor neuron death in organotypic slice cultures. However, this does not preclude the possibility that other changes may have occurred in the motor neurons. This work offered a new model to evaluate the role of IgG in the pathogenesis of ALS. Organotypic cultures contribute to study of the impact of IgG on motor neurons by mimicking physiological conditions.     

Cholinergic function in cultures of mouse spinal cord neurons

Cholinergic synapses formed in cultures of fetal mouse spinal cord (SC) and superior cervical ganglion (SCG) were studied using intracellular and extracellular stimulation and recording as well as immunohistochemical staining for choline acetyltransferase (ChAT). Dissociated SC neurons and SC explants exhibited cholinergic terminals on SCG and SC neurons as demonstrated by ChAT immunoreactivity. Intracellular recordings showed that cholinergic inputs to SCG neurons were relatively common and that these synaptic inputs were blocked by the nicotinic acetylcholine (ACh) receptor blocker, tubocurarine. A comparison of three preparations indicated that the incidence of cholinergic activity recorded in SCG neurons was significantly higher in co-cultures of SCG with spinal cord ventral horn (VH) neurons grown on a substrate of non-neuronal cells from cerebral cortex, than in co-cultures with VH alone or with SC and dorsal root ganglion cells. Consistency between cholinergic physiology and staining for ChAT-positive terminals on SCG neuronal somata was obtained in cultures of SC explants grown with dissociated SCG. Application of acetylcholine, muscarine, and/or vasoactive intestinal polypeptide (VIP) produced slow excitation of SC neurons. Fast excitatory cholinergic interactions between SC neurons were not observed. Excitatory synaptic interactions between SC neurons were augmented by ACh or muscarine, while inhibitory synaptic interactions were diminished. Both types of synaptic modulation probably were produced by a presynaptic mechanism. Acetylcholine or muscarine affected synaptic interactions between SC neurons in only one-third of the synaptic connections tested, suggesting that the incidence of presynaptically cholinoceptive SC neurons is low in dissociated cell cultures. The experimental results show that a culture system incorporating dissociated fetal mouse SC neurons or explants of SC with sympathetic ganglion neurons expresses both nicotinic and muscarinic cholinergic function.     

Changes in corticospinal facilitation of lower limb spinal motor neurons after spinal cord lesions.

The projections from the cortex to the motor neurons of lower limb muscles were examined in 33 normal subjects and 16 patients with incomplete spinal cord lesions. Corticospinal neurons were excited by transcranial magnetic stimulation and the effects on single spinal motor neurons determined from peristimulus time histograms (PSTHs) of single tibialis anterior (TA) and soleus (SOL) motor units. In normal subjects magnetic stimulation produced a short latency facilitation of TA motor units but had little or no effect on SOL motor units. In the patients with spinal cord lesions magnetic stimulation also produced facilitation of TA but not SOL motor units; however, the mean latency of the TA facilitation was significantly longer (by about 14 ms) in the patient group. The F wave latencies were normal in all patients tested, suggesting that central rather than peripheral conduction was slowed. The duration of the period of increased firing probability (in TA motor units) was also significantly longer in the patients with spinal cord lesions. These changes may reflect the slowing of conduction and dispersal of conduction velocities in the corticospinal pathways as a consequence of the spinal cord lesion. No significant correlations were found between the delay of the TA facilitation and the clinical deficits in this group of patients.     

Prenatal development of the human spinal cord. I. Ventral motor neurons

Differentiation of the ventral motor neurons were followed in the developing human spinal cord from week 8 to week 26 of intrauterine life by thionin staining and the rapid Golgi method. Ventral roots were seen as translucent rootlets by week 8 and the ventral motor neurons were clearly identifiable by week 10, indicating that the ventral grey was earlier to differentiate than the dorsal grey, which showed a small, darkly stained, undifferentiated cell population. Lateral groupings of the motor neurons in the cervical and lumbar enlargements were obvious by week 10. Nissl substance and dendrites had reached an adult pattern by about week 18 of intrauterine life.     

Long term follow up of spinal cord injury caused by penetrating missiles.

Eighty-four spinal cord injured patients (SCIP) injured as a result of penetrating missiles were categorised according to: neurological level of injury, age at time of injury, circumstances of injury, missile type, initial treatment, duration of injury, and ethnic background. Evaluations and comparisons were made concerning: life habits, family status, education, employment, and mental well being. A discussion of complicating factors, both physical and psychological, and their relation to the final rehabilitation result is presented.     

Autophagy in spinal cord motor neurons in sporadic amyotrophic lateral sclerosis

To assess the potential role of autophagy in amyotrophic lateral sclerosis (ALS), lumbar spinal cords in a total of 19 sporadic ALS cases and 27 age-matched controls were investigated. Immunohistochemical analysis using antibodies to the markers of autophagy microtubule-associated protein light chain 3 (LC3) and p62 was performed on samples from 12 ALS and 15 controls. Electron microscopy was performed on samples from 16 ALS and 15 controls, including overlapping cases. In the ALS cases, the somata of normal-appearing and degenerated motor neurons and round bodies were occasionally immunostained for LC3; round bodies and skein-like inclusions were immunostained for p62. By electron microscopy, all 16 ALS patients showed features of autophagy in the cytoplasm of normal-appearing motor neurons and, more frequently, in degenerated motor neurons. Autophagosomes surrounded by a double-membrane and autolysosomes isolated by a single membrane contained sequestered cytoplasmic organelles, such as mitochondria and ribosome-like structures. These autophagy features were also found in close association with the characteristic inclusions of ALS(i.e. round bodies, skein-like inclusions, and Bunina bodies); honeycomb-like structures also occasionally showed autophagy-associated features. Normal-appearing anterior horn neurons in control patients showed no autophagy features. Thus, autophagy seems to be activated and upregulated in the cytoplasm of motor neurons and may be involved in the mechanisms of neurodegeneration of motor neurons in sporadic ALS.     

Glycogen accumulation in spinal cord motor neurons due to partial ischemia

Summary Partial ischemia of the spinal cord in adult cats was induced by abdominal aortic ligation. The most striking abnormality was an accumulation of glycogen in large motor neurons and astroglia in the peripheral anterior horns. Little or no histological and ultrastructural abnormalities were present in these regions. The first glycogen deposits appeared after 1/2 h in glial cells, whereas glycogen accumulation in neurons was first noticeable 1 h after ligation reaching a maximum in 24 h. A gradual decrease occurred with disappearance of glycogen at 10 days. Increase in UDPG-transferase was found preceeding glycogen appearance, and increase in glycogen-phosphorylase activity occurred later concurrent with glycogen accumulation. This unique neuronal glycogen deposition may be due to the UDPG-transferase normally present in -motor neurons of the anterior horns. Other possible mechanisms are also considered.     

Selective vulnerability of spinal cord motor neurons to non-NMDA toxicity

We previously reported that alpha-motor neurons in organotypic cultures of rat spinal cord (OTC-SC) are resistant to excitotoxicity induced through NMDA receptors. Here we describe the effects of non-NMDA glutamate receptor agonists kainic acid (KA) and quisqualic acid (QUIS) on motor neurons in OTC-SC. Large ventral horn acetylcholinesterase-positive neurons (VHANs), most of which are motor neurons, were quite sensitive to QUIS and KA toxicity and displayed losses of 95% and 94%, respectively. Small VHANs were reduced by 41% and 61% only. Identical results were obtained in cultures stained for non-phosphorylated neurofilaments. These observations demonstrate that alpha-motor neurons are considerably more sensitive to KA and QUIS than to NMDA toxicity. The proposed excitotoxic mechanism of ALS, therefore, is most likely mediated through non-NMDA glutamate receptors.     

GABA, not glycine, mediates inhibition of latent respiratory motor pathways after spinal cord injury

Previous work has shown that latent respiratory motor pathways known as crossed phrenic pathways are inhibited via a spinal inhibitory process; however, the underlying mechanisms remain unknown. The present study investigated whether spinal GABA-A and/or glycine receptors are involved in the inhibition of the crossed phrenic pathways after a C2 spinal cord hemisection injury. Under ketamine/xylazine anesthesia, adult, female, Sprague-Dawley rats were hemisected at the C2 spinal cord level. Following 1 week post injury, rats were anesthetized with urethane, vagotomized, paralyzed and ventilated. GABA-A receptor (bicuculline and Gabazine) and glycine receptor (strychnine) antagonists were applied directly to the cervical spinal cord (C3-C7), while bilateral phrenic nerve motor output was recorded. GABA-A receptor antagonists significantly increased peak phrenic amplitude bilaterally and induced crossed phrenic activity in spinal-injured rats. Muscimol, a specific GABA-A receptor agonist, blocked these effects. Glycine receptor antagonists applied directly to the spinal cord had no significant effect on phrenic motor output. These results indicate that phrenic motor neurons are inhibited via a GABA-A mediated receptor mechanism located within the spinal cord to inhibit the expression of crossed phrenic pathways.     

Chronic spinal cord transection does not affect peripheral nerve regeneration

Spinal cord transection is known to cause progressive changes in motor neurons and hind limb muscles. In the present study, regeneration of the peroneal nerve was examined in rats 25 weeks after a T9 spinal cord transection. Successful regeneration and innervation of the target muscle was observed after crush injury to the nerve in the spinal cord transected animals. It is concluded that the ability of peripheral nerve to regenerate remains preserved after spinal cord injury.     

Intrathecal oxymetazoline does not produce neurotoxicity in the spinal cord of the rat.

To determine if intrathecal (i.t.) oxymetazoline (OXY) induces histological evidence of spinal neurotoxicity, male, Sprague-Dawley rats (300-450 g; implanted with an i.t. catheter) were treated with i.t. saline or 100 nmol OXY twice daily for 3 days, or 200 or 300 nmol OXY once daily for 3 days. Spantide (D-Arg1, D-Try7,9, Leu11-substance P; 0.067 nmol = 0.1 microgram, 0.167 nmol = 0.25 microgram or 0.334 nmol = 0.5 microgram) or capsaicin (0.164 mumol = 50 micrograms), given as a single i.t. injection, were used as positive controls. Animals were killed 12 h after the last injection of saline or OXY, and 72 h after spantide or capsaicin. Spinal cord sections (L1 and adjacent segments) were examined by light microscopy for changes in gross morphology, substance P-like immunoreactivity (SP-IR) and calcitonin gene related peptide-like immunoreactivity (CGRP-IR). All doses of i.t. OXY produced antinociception (tail-flick ED50 = 53.7 nmol, paw pressure withdrawal ED50 = 93.3 nmol). Rectal temperature decreased by 1.5-2.4 degrees C up to 12 h after 100 nmol of i.t. OXY. There were no signs of inflammation or necrosis, and no detectable loss or damage to either spinal afferents or motor neurons as judged by SP-IR and CGRP-IR structures in spinal cords of OXY-treated animals (all doses) as compared to i.t. saline controls. Spantide (0.1 microgram) had no antinociceptive or neurotoxic effect; 0.25 microgram induced irreversible loss of the TF reflex and transient hind limb paralysis; 0.5 microgram induced irreversible loss of TF and PP responses, permanent hind limb paralysis, bladder and bowel dysfunction. The spinal cords from these animals showed signs of extensive necrosis, cavitation, and haemorrhage in the ventral horn accompanied by a loss of CGRP-IR motor neurons. Capsaicin-treated rats exhibited a permanent loss of the TF but not the PP response and a marked reduction of SP-IR spinal afferents in the dorsal horn. It is concluded that i.t. OXY produces antinociception in the rat with no detectable spinal neurotoxicity as assessed by parameters which are sensitive to the neurotoxins, spantide and capsaicin.     

Effect of geranylgeranylaceton on cellular damage induced by proteasome inhibition in cultured spinal neurons

We investigated the effect of two proteasome inhibitors, lactacystin and epoxomicin, on cultured spinal cord neurons. The incubation of spinal neurons with proteasome inhibitors for 24 hr induced neurotoxicity in a dose-dependent manner. We found motor neurons to be more vulnerable to proteasome-induced neurotoxicity than nonmotor neurons. The staining of cell bodies in treated motor neurons was markedly disrupted and showed characteristic granular patterns. Proteasome-induced neurotoxicity is accompanied by apoptotic nuclear changes, posttranslational modification of the cellular proteins, generation of intracellular free radicals, reduction in the amount of reduced glutathione, and mitochondrial dysfunction. Neurotoxicity was reduced by the administration of low concentrations (1-100 nM) of geranylgeranylacetone (GGA), which is widely used as an antiulcer drug, although higher concentrations of this drug produced neurotoxicity in spinal cord neurons. GGA was found to induce the expression of heat shock protein 70 as well as thioredoxin, which may partly contribute to the protective effect of GGA. These data suggest that the inhibition of proteasome may play a role in the mechanism of neurodegenerative diseases of the spinal cord, such as amyotrophic lateral sclerosis, and that the use of GGA may be effective in the treatment of these conditions.     

Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons

In decerebrate cats, electrical stimulation of nucleus raphe magnus (NRM) of the medulla produced marked inhibition of spinal neurons in lumbosacral dorsal horn. Only neurons with high threshold inputs were inhibited. These cells were located in lamina I and in or near laminae V and VI.The duration of inhibition produced was related to the stimulus train length. An ipsilateral lesion of the dorsolateral funiculus at L1 markedly reduced the inhibition of neurons caudal to the lesion.Although NRM stimulation was the most effective, inhibition from more lateral sites could be obtained at higher stimulus intensities.NRM induced inhibition is probably mediated by a direct projection via the dorsolateral funiculus to spinal dorsal horn laminae I, II, V and VI.The results are discussed in relation to proposed mechanisms underlying the analgesia produced by NRM stimulation.     

Calpain inhibitors delay injury-induced apoptosis in adult mouse spinal cord motor neurons

Here, we investigated the effect of calpain inhibitors on apoptosis in organotypic adult spinal cord slices from mice. An increase in calpain I immunoreactivity was found in the nuclei of motor neurons from slices cultured for 30 min. After 4 h, the immunopositive motor neurons exhibited apoptotic changes including nuclear and chromatin condensation. Eight hours after excision, most motor neurons showed nuclear apoptotic features. Two calpain inhibitors, leupeptin and calpain inhibitor XI, inhibited apoptosis in the motor neurons while the caspase inhibitor Z-VAD.fmk had no effect. Leupeptin, but not calpain inhibitor XI and Z-VAD.fmk, also inhibited nucleosomal DNA fragmentation. These results suggest the involvement of calpain I in the induction of apoptosis in motor neurons of adult spinal cord and that apoptosis can be triggered independent of caspase activation.     

Primary cortical motor neurons undergo apoptosis after axotomizing spinal cord injury

Spinal cord injury (SCI) results in loss of voluntary motor control followed by incomplete recovery, which is partly mediated by the descending corticospinal tract (CST). This system is an important target for therapeutic repair strategies after SCI; however, the question of whether apoptotic cell death occurs in these axotomized neurons remains unanswered. In this study, adult (150-175 g) male Sprague-Dawley rats underwent T9 transection of the dorsal funiculus, which axotomizes the dorsal CST, and introduction of the retrograde tracer Fluoro-Gold into the lesion site. Primary motor cortex (M1) was then examined for evidence of apoptosis weekly for 4 weeks after injury. Axotomized pyramidal cells, identified by retrograde transport of Fluoro-Gold, were found in M1 (57.5 +/- 9.6/median section, 6127 +/- 292 total), and a significant proportion were terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP)-rhodamine nick end labeling (TUNEL) -positive at 1 week after injury (39.3 +/- 5.6%), compared with animals undergoing sham surgery (1.2 +/- 1.4%). At 2-4 weeks, fewer cells were Fluoro-Gold-positive (24.6 +/- 65.06 to 25.3 +/- 6.4/median section, 2338 +/- 233 to 2393 +/- 124 total), of which very few were TUNEL-positive. In TUNEL-positive cells, Hoechst 33342 staining revealed nuclear morphology consistent with apoptosis, chromatin condensation, and formation of apoptotic bodies. Fluoro-Gold-positive cells showed increased caspase-3 and Bax immunoreactivity. Hematoxylin and eosin staining revealed similar nuclear changes and dystrophic cells. Internucleosomal DNA fragmentation was detected by gel electrophoresis at the 1-week time point. Lesioned animals not receiving Fluoro-Gold exhibited the same markers of apoptosis. These results document, for the first time, features of apoptotic cell death in a proportion of axotomized cortical motor neurons after SCI, suggesting that protection from apoptosis may be a prerequisite for regenerative approaches to SCI.     

Bcl-2 overexpression does not protect neurons from mutant neurofilament-mediated motor neuron degeneration.

Transgenic mice with a point mutation in the light neurofilament gene develop amyotrophic lateral sclerosis-like motor neuron disease characterized by selective spinal motor neuron loss, neurofilamentous accumulations, and severe muscle atrophy. To test whether the large motor neurons at risk in this disease could be protected from mutant neurofilament-mediated killing, these mice were bred to mice overexpressing the human Bcl-2 proto-oncogene. Elevated levels of Bcl-2 increased the numbers of motor and sensory axons surviving after the developmental period of naturally occurring cell death but did not greatly reduce the number of degenerating axons or protect the large motor neurons from mutant neurofilament-mediated death.     

Alteration in amino acids in motor neurons of the spinal cord in amyotrophic lateral sclerosis.

Little is known concerning the changes of amino acid composition in different regions of the spinal cord in patients with amyotrophic lateral sclerosis (ALS). We performed quantitative amino acid analyses in the posterior funiculus, the lateral corticospinal tract, and the anterior horn of cervical enlargement of the spinal cord from seven ALS patients, and the results were compared with those of seven patients with other neurologic diseases (control A) and seven patients without neurologic diseases (control B). The levels of collagen-associated amino acids, hydroxyproline, proline, glycine, and hydroxylysine, were markedly lower in the lateral corticospinal tract and the anterior horn of ALS patients than in controls A and B. The contents of the acidic amino acids glutamate and aspartate were also significantly decreased in the lateral corticospinal tract and the anterior horn of ALS patients as compared with those of controls A and B. These data suggest that decreased contents of collagen-associated amino acids and excitatory amino acids are related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS.