Neurotrophic action of autopsied ventral spinal cord extracts in patients with amyotrophic lateral sclerosis on the ventral spinal cord of rat embryo

The effects of ventral spinal cord extracts from normal controls and patients with amyotrophic lateral sclerosis (ALS) on neurite appearance in ventral spinal cord explant of 13-day-old Sprague-Dawley rats were investigated. Ventral spinal cord extracts from normal controls and ALS were significantly more effective in stimulating growth than control medium only. There was no statistically significant difference between normal and ALS ventral spinal cord extracts in growth of neurites. Our results may be an important for the consideration of the hypothesis that ALS may be a disorder of motor neuron growth factors.     

Extracts of muscle from patients with amyotrophic lateral sclerosis enhance neurite outgrowth from ventral spinal cord explants of rat embryo

The effects of muscle extracts from normal controls and patients with amyotrophic lateral sclerosis (ALS) on neurite appearance in ventral spinal cord explants of 13-15-day-old Sprague-Dawley rats were investigated. Muscle extracts from normal controls and ALS muscle were significantly more effective in stimulating growth than control medium alone. There was no statistically significant difference between normal muscle extracts and ALS muscle extracts in growth of neurties. Our results may contribute to the hypothesis that ALS may be a disorder of motor neuron growth factors.     

Serum markers of type I collagen synthesis and degradation in amyotrophic lateral sclerosis

Collagen abnormalities in the skin and spinal cord have been reported in amyotrophic lateral sclerosis (ALS) patients. Serum carboxyterminal propeptide of type I procollagen (PICP) and the carboxyterminal cross-linked telopeptide of type I collagen (ICTP) reflect type I collagen synthesis and degradation, respectively. However, there has been no study concerning PICP or ICTP in ALS. We studied collagen contents of the skin and measured serum levels of PICP and ICTP in patients with ALS and control subjects. Serum PICP levels were significantly lower in ALS patients than in controls. Serum ICTP levels were significantly higher in ALS patients than in controls, and there was an appreciable positive correlation between serum ICTP levels and the duration of illness in ALS patients. In ALS patients, the collagen content of the skin was significantly smaller than in controls and indicated a progressive decrease in relation to illness. In addition, there was a significant negative correlation between serum ICTP concentrations and the collagen content of the skin in ALS patients. These data suggest that increased ICTP levels and decreased serum PICP levels may reflect unique changes in the skin, with a predominance of degradation compared to the synthesis of type I collagen in ALS.     

Amyotrophic lateral sclerosis: glutamate dehydrogenase and transmitter amino acids in the spinal cord.

Measurements were taken of the activity of glutamate dehydrogenase (GDH) and the levels of transmitter amino acids in anatomically dissected regions of cervical and lumbar spinal cord in eight patients dying with amyotrophic lateral sclerosis (ALS) and in 11 neurologically normal controls. GDH activity was considerably increased in lateral and ventral white matter and in the dorsal horn of the ALS cervical spinal cord, but normal in the ventral horn and the dorsal columns. Similar, although less pronounced, GDH changes were found in the lumbar enlargement. The mean concentrations of aspartate and glutamate were reduced in all regions of ALS spinal cord investigated. Taurine concentrations were significantly increased in several subdivisions of cervical spinal cord, but normal in lumbar regions. Glycine levels were significantly reduced in lumbar ventral and dorsal horns. There was no striking change in spinal cord GABA levels in our ALS patients. It is suggested that the reduced levels of glutamate and aspartate as well as the elevated GDH activity in the spinal cord of ALS patients may reflect an overactivity of the neurons releasing these potentially excitotoxic amino acids and thus may be causally related to the spinal neuro-degenerative changes characteristic of ALS.     

Elevation of MCP-1 and MCP-1/VEGF ratio in cerebrospinal fluid of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with progressive cell death of upper and lower motor neurons. In this study, we measured monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) levels in cerebrospinal fluid (CSF) and serum by enzyme-linked immunosorbent assay (ELISA) in 42 ALS patients, and compared these levels with those of control subjects with other neurodegenerative disorders or with those of normal controls. MCP-1 levels in CSF were significantly higher in ALS patients than in the control group. VEGF levels in CSF tended to be lower in ALS patients than in the control group, but not significantly. A positive correlation was found between MCP-1 levels in CSF of ALS patients and the total Norris scale. The elevation of MCP-1/VEGF ratio in CSF was more specific to ALS patients compared to other neurological diseases such as Parkinson's disease (PD) and spinocerebellar ataxia (SCA) and to controls. Our data suggested that both MCP-1 levels and MCP-1/VEGF ratio in CSF may be useful markers for the clinical diagnosis of ALS.     

Maintained regulation of polyamines in spinal cord from patients with amyotrophic lateral sclerosis

Levels of the polyamines putrescine, spermidine, and spermine were investigated in postmortem spinal cord from seven patients with amyotrophic lateral sclerosis (ALS) and seven control subjects. The method consisted of precolumn derivatization of the polyamines, followed by high-performance liquid chromatography (HPLC) analysis and fluorescence detection. The stability of the polyamines was examined in rat spinal cord during the interval of 0-36 h postmortem. The levels of putrescine, spermidine, and spermine increased by 32%, 15%, and 2%, respectively. Polyamine levels did not differ significantly between the ALS group and the control group, suggesting a maintained regulation of polyamines in the end stage of the disease. However, an effect of gender on the levels of spermidine and spermine was observed. Levels of spermidine and spermine in the ventral horn region of female ALS patients were significantly higher in comparison with the same region of the male ALS group (p<0.05). The female ALS group also presented significantly higher levels of spermidine in comparison with female controls (p<0.05).     

Altered metabolism of excitatory amino acids, N-acetyl-aspartate and N-acetyl-aspartylglutamate in amyotrophic lateral sclerosis

Since recent studies provided evidence for abnormal glutamate metabolism in amyotrophic lateral sclerosis, we measured amino acid levels in the fasting plasma of 52 ALS patients and an equal number of controls of a similar age. In addition, the content of amino acids, N-acetyl-aspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG) were measured in spinal cord and brain tissue obtained at autopsy from patients dying of ALS. Results showed significant elevations (by about 70%) in the plasma levels of glutamate in the ALS patients as compared to controls. In contrast, glutamate levels were significantly decreased in all CNS regions studied of ALS patients (by 21–40%), with the greatest changes occurring in the spinal cord. The ratio of glutamine to glutamate was altered significantly in the spinal cord ALS tissue. In addition, reductions in the levels of aspartate (by 32–35%), NAA, and NAAG (by 40–48%) were found in the spinal cord of ALS patients. These results are consistent with a generalized defect in the metabolism of neuroexcitotoxic amino acids. An altered distribution of these compounds may occur between their intracellular and extracellular pools with resultant abnormal potentiation of excitatory transmission mediated by glutamate receptors and selective degeneration of motor neurons.     

Immunohistochemical localization of vascular endothelial growth factor receptors-1, -2 and -3 in human spinal cord: altered expression in amyotrophic lateral sclerosis

Vascular endothelial growth factor (VEGF) has recently been implicated in several neurological disorders. Apart from its prominent role in angiogenesis, VEGF has been shown to have direct effects on neuronal and glial cells through activation of different VEGF receptor (VEGFR) types. In the present study the expression patterns of VEGFR-1, -2 and -3 were investigated in the spinal cord of control and both sporadic and familial amyotrophic lateral sclerosis (ALS) patients. Immunocytochemical analysis of control human spinal cord demonstrated that VEGFR-1, but not VEGFR-2 or -3 was found to be present in blood vessels of both white and grey matter. All three VGEFRs were not detectable in resting glial cells of control tissue. Diffuse neuropil staining was observed in the control spinal cord grey matter for VEGFR-3. Regional differences in VEGFRs immunoreactivity (IR) were apparent in ALS compared to controls. In particular, VEGFR-1 expression was increased in reactive astroglial cells in both grey (ventral horn) and white matter of ALS spinal cord. In addition to the astroglial labelling, increased expression of VEGFR-1 and, to a less extent also of VEGFR-2, was observed in blood vessels of the ALS spinal cord. No changes in VEGFR-3 IR were detected in blood vessels or reactive astroglial cells, whereas VEGFR-3 neuropil expression was reduced and paralleled the distribution of neuronal loss in the ventral horn of ALS spinal cord. These findings indicate that VEGFRs have specific distribution patterns, suggesting different physiological functions in human spinal cord. Moreover, the altered expression observed in ALS supports a role for these receptors in the complex reactive processes that are associated with the progression of spinal cord damage.     

The serum level of free testosterone is reduced in amyotrophic lateral sclerosis

Sporadic amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motoneurons. There is an approximately 2:1 higher incidence of ALS in men compared to women, and this has raised the hypothesis of an involvement of sex hormones in the etiopathogenesis of the disorder. In this work, the serum levels of dehydroepiandrosterone sulphate (DHEAS), 17-betaestradiol, free and total testosterone were measured in 35 patients with defined or probable ALS, according to the El-Escorial/WFN revisited criteria, and compared to those obtained from 57 disease controls, matched for age and gender to the ALS group.We found no differences between ALS cases and disease controls in the serum levels of DHEAS, 17-betaestradiol and total testosterone. Conversely, free testosterone was significantly decreased in the ALS group. Given that testosterone crosses the blood-brain barrier only as unbound form, we suggest a possible involvement of this sex hormone in the pathophysiology of this severe motor neuron disease.     

Elevation of MCP-1 and MCP-1/VEGF ratio in cerebrospinal fluid of amyotrophic lateral sclerosis patients

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with progressive cell death of upper and lower motor neurons. In this study, we measured monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) levels in cerebrospinal fluid (CSF) and serum by enzyme-linked immunosorbent assay (ELISA) in 42 ALS patients, and compared these levels with those of control subjects with other neurodegenerative disorders or with those of normal controls. MCP-1 levels in CSF were significantly higher in ALS patients than in the control group. VEGF levels in CSF tended to be lower in ALS patients than in the control group, but not significantly. A positive correlation was found between MCP-1 levels in CSF of ALS patients and the total Norris scale. The elevation of MCP-1/VEGF ratio in CSF was more specific to ALS patients compared to other neurological diseases such as Parkinson's disease (PD) and spinocerebellar ataxia (SCA) and to controls. Our data suggested that both MCP-1 levels and MCP-1/VEGF ratio in CSF may be useful markers for the clinical diagnosis of ALS.     

Paradoxical response of VEGF expression to hypoxia in CSF of patients with ALS

Vascular endothelial growth factor (VEGF) is implicated in motor neurone degeneration. In normal individuals, hypoxia is known to induce an overexpression of VEGF, as measured in CSF. We show that patients with ALS do not manifest this VEGF overexpression in the presence of hypoxia. Although VEGF gene expression is mainly stimulated by hypoxia, we have measured lower VEGF levels in cerebrospinal fluid (CSF) from hypoxaemic patients with amyotrophic lateral sclerosis (ALS) than in CSF from normoxaemic patients with ALS. In contrast, hypoxaemic neurological controls displayed higher levels than normoxaemic neurological controls. There was a negative correlation between VEGF levels and the severity of hypoxaemia in patients with ALS, suggesting deregulation of VEGF in ALS.     

Cerebrospinal fluid vascular endothelial growth factor in patients with amyotrophic lateral sclerosis

Oxidative stress and glutamate-mediated toxicity may play an important role in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). The vascular endothelial growth factor (VEGF) is a neuroprotective cytokine activated by hypoxia. The aim of this study was to measure VEGF levels in the cerebrospinal fluid (CSF) of ALS patients. The study concerned 30 ALS patients and 30 control subjects. The VEGF was measured by the enzyme-linked immunosorbent assay. The results have shown that CSF VEGF levels are significantly increased in patients with long duration of ALS and in patients with limb-onset of the disease compared with controls (P<0.05). Moreover, the type of ALS patients’ subgroup significantly influences CSF VEGF levels (P=0.05). The CSF VEGF levels were significantly increased in patients with limb-onset compared to patients with bulbar-onset of ALS, and in patients with long duration of ALS compared to patients with its short duration (P<0.05). There was a significant correlation between CSF VEGF levels and duration of ALS (P<0.05). It seems that a significant increase in CSF VEGF levels in patients with limb-onset of ALS and in patients with long duration of the disease may have a protective role against glutamate-mediated toxicity and oxidative damage of motor neurons. However, the conclusions are limited due to relatively small subgroups of ALS patients and by lack of a control group consisting of healthy persons. Further investigations could help to confirm the results from this preliminary report.     

Alterations in receptors for thyrotropin-releasing hormone, serotonin, and acetylcholine in amyotrophic lateral sclerosis

We utilized quantitative autoradiography to examine thyrotropin-releasing hormone (TRH) receptors, serotonin type 1A (5-HT1A) receptors, muscarinic cholinergic receptors, choline uptake sites, beta-adrenergic receptors, and norepinephrine uptake sites in discrete laminae of spinal cord from patients with amyotrophic lateral sclerosis (ALS) and non-neurologic controls. We found decreases of over 50% in the concentration of TRH receptors in lamina IX of cervical, thoracic, and lumbar spinal cord from ALS patients. Similar reductions were noted in concentrations of muscarinic cholinergic receptors in lamina IX of spinal cords from ALS patients. Significant increases of up to 140% in 5-HT1A receptor densities were noted in lamina IX of spinal cords from ALS patients. No differences were noted between the concentrations of beta-adrenergic receptors or norepinephrine uptake sites in patients with ALS and controls. These findings suggest that TRH and 5-HT may be involved in the pathophysiology of ALS, and act in a comodulatory role in the normal spinal cord.     

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.     

Elevated levels of IFNγ and LIGHT in the spinal cord of patients with sporadic amyotrophic lateral sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is a paralytic and fatal neurodegenerative disorder caused by the gradual loss of both upper and lower motoneurons. There is compelling evidence from ALS experimental models that neuroinflammation actively contributes to motoneuron damage. We recently proposed that interferon gamma (IFNγ), a potent proinflammatory cytokine, induces motoneuron death by eliciting the activation of the lymphotoxin beta receptor (LT‐βR) through its ligand LIGHT. Here, we explore the pertinence of this non‐cell‐autonomous mechanism in human ALS. Methods: The levels and expression pattern of IFNγ, LIGHT, and LT‐βR were investigated by Western blot and immunohistochemical analysis in spinal cord of patients with sporadic ALS. Results: We observed significant increased levels of IFNγ in human ALS spinal cords compared to control cases. We found that large ventral horn neurons as well as glial cells were immunoreactive for IFNγ in sporadic ALS spinal cord. We further observed that LIGHT and LT‐βR were expressed mainly by motoneurons in both ALS and control cases, and while LT‐βR levels remained constant between ALS and control cases, LIGHT levels were increased in human ALS spinal cords. Conclusion: These findings in sporadic ALS cases, which are consistent with the observation made in ALS experimental models, propose that the IFNγ‐triggered LIGHT/LT‐βR‐mediated death pathway may contribute to human ALS pathogenesis.     

ALS serum has no effect on three enzymatic activities in cultured human spinal cord neurons

Dissociated human spinal cord cells were grown in monolayer cultures in the presence of serum from normal controls or from patients with ALS or other neurologic diseases. After 20 to 24 days, the levels of choline acetyltransferase, glutamic acid decarboxylase, and lactate dehydrogenase activities were determined in the cultures. On the basis of these biochemical measurements, there was no detectable difference between the effects of the three types of serum on the cultures.     

Insulin-like growth factor-1 receptors in human spinal cord: changes in amyotrophic lateral sclerosis

Summary Neurotrophic factors are important for neuronal survival and maintenance in the adult nervous system. The regional distribution of insulin-like growth factor-1 (IGF-1) receptors in human spinal cords from controls and amyotrophic lateral sclerosis (ALS) patients was studied by immunohistochemistry and quantitative autoradiography. When comparing¹²⁵I-IGF-1 binding in the different spinal levels of normal spinal cord the same distribution pattern was found in which the binding was highest in the central canal > dorsal horn > ventral horn > white matter. In the ALS cases although a general upregulation of IGF-1 receptors was observed throughout the spinal cord, significant increases were observed in the cervical and sacral segments compared to controls. IGF-1 receptor immunoreactivity showed a similar pattern to that for¹²⁵I-IGF-1 binding, with immunoreactivity being found in the gray matter of the spinal cord and enhanced immunoreactivity occuring in ALS patients compared to controls. In agreement with the distribution of IGF-1 receptors, IGF-1 immunoreactivity was found within the gray matter of the spinal cord. The cartography of IGF-1 receptors in the normal spinal cord as well as the change of these receptors in diseased spinal cord may be of importance in future treatment strategies of ALS.     

Cerebrospinal fluid and serum vascular endothelial growth factor and nitric oxide levels in newborns with hypoxic ischemic encephalopathy

Excitatory amino acids, cytokines and nitric oxide (NO) have been studied in the etiology and pathogenesis of hypoxic ischemic encephalopathy (HIE) of the newborn. Vascular endothelial growth factor (VEGF) is a known mediator of angiogenesis and has been shown to induce vascular proliferation and permeability via NO-mediated mechanism during hypoxia. The objective of this study was to investigate the cerebrospinal fluid and serum VEGF and NO levels in different stages of HIE and the correlation between the two mediators. Cerebrospinal fluid (CSF) and serum samples of 19 newborns with HIE and 13 controls were obtained within the first 24 h of life and kept at -70 degrees C until the time of measurement. NO levels were determined by Sievers NOA by chemiluminescence method and VEGF levels were measured by the enzyme-linked immunosorbent assay double sandwich method. The NO levels in CSF were higher than the control and mild HIE group in newborns with moderate to severe HIE, and VEGF levels in CSF were higher in the mild HIE group compared to controls but similar in the moderate to severe HIE group compared to mild HIE and control patients. There was no difference between groups with regard to serum NO or VEGF levels, and no correlation was observed between NO and VEGF levels both in CSF and serum samples. Depending on the severity of the hypoxic insult the stimulus for NO production by VEGF may have variable effects on endothelial cells which may give rise to the current results.