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Expression of nitric oxide synthases in the anterior horn cells of amyotrophic lateral sclerosis
Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 163 – 167
Expression of nitric oxide synthases in the anterior horn cells of amyotrophic lateral sclerosis Koichi Kashiwado*, Yasumasa Yoshiyama, Kimihito Arai, Takamichi Hattori Department of Neurology, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, Japan
Abstract The authors investigated for a correlation between the expression of nitric oxide synthases (NOSs) with the severity of motor neuronal loss in the anterior horns of patients with amyotrophic lateral sclerosis (ALS). Spinal cords from six patients with ALS and from three normal controls were examined. The sections of cervical, lumbar, and sacral cord including Onuf’s nucleus, which are seldom degenerated until the late stage, were stained with three antibodies against NOSs (anti-n-NOS, anti-e-NOS, and anti-i-NOS) using ABC methods. Perikarya of motor neurons in ALS, but not in controls, were immunoreactive against anti-n-NOS and e-NOS. Anti-i-NOS did not recognize the motor neurons of ALS or of controls. The immunoreactivity for n- and e-NOSs was approximately the same in the sections of cervical, lumbar, and sacral cord in ALS. No significant differences in immunoreactivity were observed among the patients with ALS. These results suggest that the expression of NOSs does not immediately affect neuronal loss in ALS. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Anterior horn cells; Nitric oxide synthase
1. Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive muscular weakness and atrophy. Although the cause of ALS is still unknown, oxidative stress is thought to be a participant of its pathogenesis (Beckman et al., 1993; Urushitani et al., 1997; Bredt, 1999). The discovery of mutations in the copper/zinc superoxide dismutase (SOD-1) in 15% of familial ALS led to the hypothesis that a loss of function of this enzyme might cause oxidative damage to the motor neurons. However, SOD mutations in sporadic ALS have not been observed. Glutamate activates calcium influx by stimulating N-methyl- D -aspartate (NMDA) receptors, and the intracellular calcium ion produces free radicals such as superoxide and nitric oxide (NO). Superoxide reacts with NO, which is produced by nitric oxide synthases (NOSs), and forms the powerful oxidant perox-
Abbreviations: ALS, amyotrophic lateral sclerosis; NMDA, N-methylD-aspartate; NOS, nitric oxide synthase
* Corresponding author. Tel.: +81-43-226-2129; fax: +81-43-2262160. E-mail address: [email protected] (K. Kashiwado).
ynitrite (ONOO –). SOD-1 catalyzes a reaction of peroxynitrite with protein –tyrosine, resulting in nitration of a residue that may be involved in the pathomechanism of selective motor neuron death (Beckman et al., 1993); notably, enhanced nitration is observed in the spinal cord of ALS (Abe et al., 1995, 1997; Beal et al., 1997; Bruijn et al., 1997; Strong et al., 1998). Generally, NO itself is a highly reactive and unstable gas, characterized by dual actions. At higher doses NO is cytotoxic, and at lower doses plays a protective role in neurotoxicity (Kashii et al., 1996). Its toxic effects consist of the depletion of cellular energy stores by inhibition of enzymes involved in mitochondrial respiration (Brorson et al., 1999). NO directly inhibits mitochondrial respiration by competing with molecular oxygen for binding to cytochrome c oxidase (CcO) (Giulivi, 1998). Endogenous NO is produced by NOSs, of which three types of isoforms, n-NOS, e-NOS, and i-NOS, are known. NOS from neurons (n-NOS) and endothelial cells (e-NOS) are constitutively expressed, and inducible NOS (i-NOS) mediates immune functions (Bredt, 1999). The expressions of n-NOS and e-NOS in motor neurons are enhanced in ALS compared to controls (Abe et al., 1995, 1997). If NO is synthesized in excess in ALS, the relationship between neuronal loss and NOS expres-
0278-5846/02/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 8 - 5 8 4 6 ( 0 1 ) 0 0 2 4 2 - 1
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Table 1 Background of cases Case
Age at death (years)
Gender
Disease duration (months)
ALS a1 a2 a3 a4 a5 a6
42 73 69 64 69 64
F F M M M M
26 22 9 30 22 21
Control c1 c2 c3
79 54 62
M F M
sion should be observed. Although patients with ALS suffer from widespread muscular atrophy, different clinical types are known; the conventional type (c-ALS), which begins with small hand muscle atrophy; the bulbar type, which begins with bulbar palsy; and the lumbar type (l-ALS), which begins with muscular atrophy in the lower limb (Brooks, 1996; Bonduelle, 1975). Clinically, the extraocular muscles and sphincter muscles of the bladder and rectum are characteristically spared until the latest stage of the illness. Pathologically, motor neurons in the oculomotor, trochlear, abducens, and Onuf’s nuclei are usually spared in ALS. These characteristics indicate that the loss of motor neurons depends on the vulnerability of the regions, and determines the clinical type of ALS. Therefore the authors investigated the correlation between NOSs and neuronal loss in the cervical and lumbar cord and in
Fig. 1. The anterior horns of the control and ALS cases. The upper, middle, and lower strands indicate C8, L2, and S2, respectively. (a – c) Control case (c1). The cell population is normal. (d – f) c-ALS (a2). Severe neuronal loss was seen in the cervical segment, but only moderate loss was seen in the lumbar segment. (g – i) l-ALS (a6). Severe neuronal loss was seen in the lumbar segment. The Onuf’s nucleus of the ALS cases appeared normal, and was similar to that in the control cases. Black bar indicates 200 mm. Klu¨ver – Barrera stain.
Fig. 2. Immunostaining for n-NOS in the control and ALS cases. Each figure represents the same segments indicated in Fig. 1. n-NOS expression in ALS cases was very strong and diffuse even in the neurons of the Onuf’s nucleus (f,i).On the other hand, control cases showed no immunoreactivity for n-NOS (a – c). The magnification of inset pictures is the same as (c), (f), and (i). Black bar indicates 100 mm.
Onuf’s nucleus from patients with c-ALS and from those with l-ALS.
2. Methods 2.1. Human autopsy cases The authors examined the cervical cord (C7, C8), the lumbar cord (L1, L2, L3), and the sacral cord (S2, S3) from three autopsied cases with c-ALS, three autopsied cases with l-ALS, and three normal controls (Table 1). Autopsy was performed within 5 h of death in each case. Spinal cord was fixed in 10% formaldehyde, embedded in paraffin, cut into sections 6 mm thick, and stained by HE and Klu¨ver –
Fig. 3. Immunostaining for n-NOS in the intermediolateral cell column of the control (a) and ALS (b) cases. n-NOS expression was seen strongly and diffusely in the lateral horn cells. Black bar indicates 50 mm.
K. Kashiwado et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 163–167 Table 2 Expression of n-NOS
165
Table 4 Expression of i-NOS
Case
Cervical cord
Lumbar cord
Onuf’s nucleus
Case
Cervical cord
Lumbar cord
Onuf’s nucleus
a1 a2 a3 a4 a5 a6 c1 c2 c3
2+ 2+ 1+ 1+ 2+ 2+ 0 0 0
2+ 2+ 1+ 1+ 2+ 2+ 0 0 1+
2+ 2+ 1+ 1+ 1+ 2+ 0 0 0
a1 a2 a3 a4 a5 a6 c1 c2 c3
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0: none, 1+: sparsely present, 2+: markedly present.
0: none, 1+: sparsely present, 2+: markedly present.
Barrera. For the immunohistochemistry, sections were autoclaved (120 C, 6 min), incubated with 1% H2O2 in methyl alcohol for 30 min at room temperature, incubated with 10% normal swine serum for 30 min at room temperature, then incubated with anti-NOS antibodies (n-, e-, i-NOS all polyclonal, Biogenesis, 1:200) for 48 h at 4 C. They were then treated for 2 h at room temperature with biotinylated secondary antibody (Vector). Washing between steps was performed with PBS. Peroxidase labeling was visualized by incubating with a solution containing 0.001% 3.30-diaminobenzidine, 0.6% nickel ammonium sulfate, 0.05% imidazole, and 0.0003% H2O2. When a dark purple product formed, the reaction was terminated. Sections were washed, dehydrated with graded alcohol, then coverslipped. We also stained sections with the Klu¨ver –Barrera methods. For the immunohistochemistry, 20 serial sections were stained every 4 sections.
In the c-ALS, the neuronal loss in the cervical anterior horn cells was more severe than that in the lumbar cord (Fig. 1d,e). On the other hand, the lumber type showed more severe loss in the lumbar cord (Fig. 1g,h). The Onuf’s nucleus in the ALS cases, without regard to the clinical type, was well preserved, and similar to that in the controls (Fig. 1c,f,i).
2.2. Data analysis Histopathological evaluation included the degree of NOS expression (frequency and extent) semiquantitatively. 0: none, 1+: sparsely present, 2+: markedly present. The evaluation was performed by three experienced researchers.
3. Results In normal controls, the anterior horn cells were well preserved in cervical, lumbar, and sacral segments (Fig. 1a – c).
3.1. Immunohistochemistry for NOS (Figs. 2 and 3) In normal controls, the expression of NOSs (n-NOS, e-NOS, and i-NOS) was not detected in the anterior horn cells (Fig. 2a – c), though n-NOS expression was seen strongly and diffusely in the lateral horn cells (Fig. 3a). The lateral horn cells in the ALS cases showed the same tendency (Fig. 3b). In ALS cases, n-NOS and e-NOS expression in the residual anterior horn cell cytoplasms was strong and diffuse, and this type of expression was also seen in some neurites. NOS immunoreactivity was seen more than half of the residual cells. Immunoreaction of the nuclei and the surface of the anterior horn cells could not be seen. In relation to the n-NOS and e-NOS expression, no difference was observed in the staining ability between the conventional type and the lumbar type in any segment of the animal cord examined (Fig. 2d– i, Tables 2 and 3). Namely, the expression of NOSs (n-NOS and e-NOS) in the preserved Onuf’s nucleus was equivalent to that in the severely degenerated areas. i-NOS did not express in any type of ALS (Table 4).
4. Discussion
Table 3 Expression of e-NOS Case
Cervical cord
Lumbar cord
Onuf’s nucleus
a1 a2 a3 a4 a5 a6 c1 c2 c3
2+ 1+ 1+ 2+ 1+ 2+ 1+ 0 0
2+ 2+ 1+ 2+ 1+ 1+ 0 0 0
1+ 1+ 1+ 2+ 1+ 1+ 0 0 0
0: none, 1+: sparsely present, 2+: markedly present.
Some researchers have found that NO is involved in the process of various neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease (Beal, 1998; Bredt, 1999). Because NO is produced by NOS and NOS expresses in the anterior horn cells of the spinal cord in ALS, there is a possibility that NOS plays an important role in the pathogenesis of ALS (Beckman et al., 1993). If the hypothesis that NOS is involved in the process of ALS is correct, the expression of NOS has to be strong in the severely afflicted lesions. Therefore, it is important to
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investigate the severity of lesions and whether it is associated with the strength of NOS expression. To our knowledge, no previous report has focused on the differences of NOS expression between clinical types of ALS and the spinal cord segments. First, we compared NOS expressions in the anterior horn cells of ALS with those of controls. Our findings showed that n-NOS and e-NOS expressions in motor neurons were almost always detected in ALS. Abe et al. (1995, 1997) showed that the expressions of NOS (not i-NOS but n-NOS and e-NOS) in motor neurons were enhanced in ALS compared to those in controls. Up to this point, our result is in accord with theirs. It supports the hypothesis that NOS expression is involved with the disease process at first glance. Second, to clarify this hypothesis, we compared clinical types of ALS in terms of NOS expressions in the spinal cord segments. Before discussing this point, some discussion is warranted on the significance of the Onuf’s nucleus in this study. 4.1. Onuf’s nucleus in ALS It is well known that external ocular muscles and external sphincter muscles of the bladder and rectum are spared in ALS until the terminal stage. Onuf’s nucleus, located in the ventral margin of the anterior horn at the second sacral cord level, may control strained muscles in the pelvic floor (Mannen et al., 1977; Schroder and Reske-Nielsen, 1984). Onuf’s nucleus in ALS are usually preserved in comparison to anterior horn cells of other parts (Schroder and ReskeNielsen, 1984). We confirmed this in this study by the Klu¨ver – Barrera method. Therefore, Onuf’s nucleus is a suitable region to use for a comparison of the expression of NOS in anterior horn cells of ALS. The authors examined not only the cervical and lumbar segments but also the sacral cord where the Onuf’s nucleus is situated. 4.2. Clinical types of ALS and NOSs expression The apparent difference of neuronal loss was observed according to the clinical types of ALS. In c-ALS, the number of motor neurons left in the cervical cord was lower than that left in the lumbar cord. On the other hand, in l-ALS, the number of motor neurons left in the lumbar cord was lower than that left in the cervical cord. However, the neurons in the Onuf’s nuclei were well preserved in both types. The expression of n-NOS and e-NOS in anterior horns of the cervical cord, lumbar cord, and the Onuf’s nucleus was up-regulated, but there was no difference among segments (Tables 2 and 3). There are three possibilities for the discrepancy between NOS expression and neuronal loss. One possibility is that the selective loss of motor neurons in ALS is due to vulnerability to NO. If this possibility is correct, there must be only one clinical type of ALS, and the types extant reflect an ordered vulnerability to NO in each spinal cord segment.
Given that loss was comparable among the segments in all three types, this possibility is unlikely. The second possibility is that the expression of NOSs occurs upstream of the degenerative processes, and NOSs-bearing anterior horn cells eventually die. According to this hypothesis, neurons of the Onuf’s nucleus inevitably degenerate in cases with long-standing life support. Because the expression of NOSs was not related to the severity of the neuronal loss, upregulation of NOSs may not immediately cause neuronal loss. The equal expression of n-NOS and e-NOS among cervical, lumbar, and sacral segments suggests a third possibility; given that no remarkable neuronal loss was found in the Onuf’s nucleus, it is possible that NOS plays a protective role in neurons. A low concentration of NO plays a protective role in glutamate neurotoxicity by inhibiting NMDA-channel activity (Kashii et al., 1996). This protective mechanism constitutes one aspect of the dual actions of nitric oxide. Lehrer-Graiwer et al. (2000) found that n-NOS-expressing neurons were selectively spared from NO-induced cytotoxicity in chronic neurodegenerative conditions. Although NO directly inhibits the mitochondrial enzyme CcO, it simultaneously induces mRNA and protein for CcO. Neurons chronically exposed to low concentrations of NO through endogenous n-NOS activity may up-regulate CcO levels, and thereby contribute to selective sparing of n-NOS neurons (LehrerGraiwer et al., 2000). Our data support these previous reports that NOS works neuro-protectively in the process of neuronal death.
5. Conclusion The expression of NOSs does not immediately affect neuronal loss in sporadic ALS.
Acknowledgments We would like to thank Mr. H. Nagase for his technical assistance. This study was supported in part by a grant from the Ministry of Education, Japan.
References Abe, K., Pan, L.-H., Watanabe, M., Kato, T., Itoyama, Y., 1995. Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis. Neurosci. Lett. 199, 152 – 154. Abe, K., Pan, L.-H., Watanabe, M., Konno, H., Kato, T., Itoyama, Y., 1997. Upregulation of protein – tyrosine nitration in the anterior horn cells of amyotrophic lateral sclerosis. Neurol. Res. 19, 124 – 128. Beal, M.F., 1998. Exitotoxicity and nitric oxide in Parkinson’s disease pathogenesis. Ann. Neurol. 44, S110 – S114. Beal, M.F., Ferrante, R.J., Browne, S.E., Matthews, R.T., Kowall, N.W., Brown Jr., R.H. 1997. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis. Ann. Neurol. 42, 644 – 654.
K. Kashiwado et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 163–167 Beckman, J.S., Carson, M., Smith, C.D., Koppenol, W.H., 1993. ALS, SOD and peroxynitrite. Nature 364, 584. Bonduelle, M., 1975. Amyotrophic lateral sclerosis. In: Vinken, P.J., Bruyn, G.W. (Eds.), Handbook of Clinical Neurology, vol. 22. North-Holland Publishing, Amsterdam/Elsevier, New York, pp. 281 – 338. Bredt, D.S., 1999. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radical Res. 31, 577 – 596. Brooks, B.R., 1996. Natural history of ALS: symptoms, strength, pulmonary function, and disability. Neurology 47, S71 – S82. Brorson, J.R., Schumacker, P.T., Zhang, H., 1999. Nitric oxide acutely inhibits neuronal energy production. The committees on neurobiology and cell physiology. J. Neurosci. 19, 147 – 158. Bruijn, L.I., Beal, M.F., Becher, M.W., Schulz, J.B., Wong, P.C., Price, D.L., Cleveland, D.W., 1997. Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals through out amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant. Proc. Natl. Acad. Sci. USA 94, 7606 – 7611. Giulivi, C., 1998. Functional implications of nitric oxide produced by mitochondria in mitochondrial metabolism. Biochem. J. 332, 673 – 679. Kashii, S., Mandai, M., Kikuchi, M., Honda, Y., Tamura, Y., Kaneda, K.,
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Akaike, A., 1996. Dual actions of nitric oxide in N-methyl-D-aspartate receptor-mediated neurotoxicity in cultured retinal neurons. Brain Res. 711, 93 – 101. Lehrer-Graiwer, J.E., Firestein, B.L., Bredt, D.S., 2000. Nitric oxide-mediated induction of cytochrome c oxidase mRNA and protein in a mouse macrophage cell line. Neurosci. Lett. 288, 107 – 110. Mannen, T., Iwata, M., Toyokura, Y., Nagashima, K., 1977. Preservation of a certain motorneuron group of the sacral chord in amyotrophic lateral sclerosis: its clinical significance. J. Neurol. Neurosurg. Psychiatry 40, 464 – 469. Schroder, H.D., Reske-Nielsen, E., 1984. Preservation of the nucleus X-pelvic floor motorsystem in amyotrophic lateral sclerosi. Clin. Neuropathol. 3, 210 – 216. Strong, M.J., Sopper, M.M., Crow, J.P., Strong, W.L., Beckman, J.S., 1998. Nitration of the low molecular weight neurofilament is equivalent in sporadic amyotrophic lateral sclerosis and control cervical spinal cord. Biochem. Biophys. Res. Commun. 248, 157 – 164. Urushitani, M., Shimohama, S., Kihara, T., Sawada, H., Akaike, A., Ibi, M., Inoue, R., Kitamura, Y., Taniguchi, T., Kimura, J., 1997. Mechanism of selective motor neuronal death after exposure of spinal cord to glutamate: involvement of glutamate-induced nitric oxide in motor neuron toxicity and nonmotor neuron protection. Ann. Neurol. 44, 796 – 807.
Expression of nitric oxide synthases in the anterior horn cells of amyotrophic lateral sclerosis Koichi Kashiwado*, Yasumasa Yoshiyama, Kimihito Arai, Takamichi Hattori Department of Neurology, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, Japan
Abstract The authors investigated for a correlation between the expression of nitric oxide synthases (NOSs) with the severity of motor neuronal loss in the anterior horns of patients with amyotrophic lateral sclerosis (ALS). Spinal cords from six patients with ALS and from three normal controls were examined. The sections of cervical, lumbar, and sacral cord including Onuf’s nucleus, which are seldom degenerated until the late stage, were stained with three antibodies against NOSs (anti-n-NOS, anti-e-NOS, and anti-i-NOS) using ABC methods. Perikarya of motor neurons in ALS, but not in controls, were immunoreactive against anti-n-NOS and e-NOS. Anti-i-NOS did not recognize the motor neurons of ALS or of controls. The immunoreactivity for n- and e-NOSs was approximately the same in the sections of cervical, lumbar, and sacral cord in ALS. No significant differences in immunoreactivity were observed among the patients with ALS. These results suggest that the expression of NOSs does not immediately affect neuronal loss in ALS. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Anterior horn cells; Nitric oxide synthase
1. Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive muscular weakness and atrophy. Although the cause of ALS is still unknown, oxidative stress is thought to be a participant of its pathogenesis (Beckman et al., 1993; Urushitani et al., 1997; Bredt, 1999). The discovery of mutations in the copper/zinc superoxide dismutase (SOD-1) in 15% of familial ALS led to the hypothesis that a loss of function of this enzyme might cause oxidative damage to the motor neurons. However, SOD mutations in sporadic ALS have not been observed. Glutamate activates calcium influx by stimulating N-methyl- D -aspartate (NMDA) receptors, and the intracellular calcium ion produces free radicals such as superoxide and nitric oxide (NO). Superoxide reacts with NO, which is produced by nitric oxide synthases (NOSs), and forms the powerful oxidant perox-
Abbreviations: ALS, amyotrophic lateral sclerosis; NMDA, N-methylD-aspartate; NOS, nitric oxide synthase
* Corresponding author. Tel.: +81-43-226-2129; fax: +81-43-2262160. E-mail address: [email protected] (K. Kashiwado).
ynitrite (ONOO –). SOD-1 catalyzes a reaction of peroxynitrite with protein –tyrosine, resulting in nitration of a residue that may be involved in the pathomechanism of selective motor neuron death (Beckman et al., 1993); notably, enhanced nitration is observed in the spinal cord of ALS (Abe et al., 1995, 1997; Beal et al., 1997; Bruijn et al., 1997; Strong et al., 1998). Generally, NO itself is a highly reactive and unstable gas, characterized by dual actions. At higher doses NO is cytotoxic, and at lower doses plays a protective role in neurotoxicity (Kashii et al., 1996). Its toxic effects consist of the depletion of cellular energy stores by inhibition of enzymes involved in mitochondrial respiration (Brorson et al., 1999). NO directly inhibits mitochondrial respiration by competing with molecular oxygen for binding to cytochrome c oxidase (CcO) (Giulivi, 1998). Endogenous NO is produced by NOSs, of which three types of isoforms, n-NOS, e-NOS, and i-NOS, are known. NOS from neurons (n-NOS) and endothelial cells (e-NOS) are constitutively expressed, and inducible NOS (i-NOS) mediates immune functions (Bredt, 1999). The expressions of n-NOS and e-NOS in motor neurons are enhanced in ALS compared to controls (Abe et al., 1995, 1997). If NO is synthesized in excess in ALS, the relationship between neuronal loss and NOS expres-
0278-5846/02/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 8 - 5 8 4 6 ( 0 1 ) 0 0 2 4 2 - 1
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Table 1 Background of cases Case
Age at death (years)
Gender
Disease duration (months)
ALS a1 a2 a3 a4 a5 a6
42 73 69 64 69 64
F F M M M M
26 22 9 30 22 21
Control c1 c2 c3
79 54 62
M F M
sion should be observed. Although patients with ALS suffer from widespread muscular atrophy, different clinical types are known; the conventional type (c-ALS), which begins with small hand muscle atrophy; the bulbar type, which begins with bulbar palsy; and the lumbar type (l-ALS), which begins with muscular atrophy in the lower limb (Brooks, 1996; Bonduelle, 1975). Clinically, the extraocular muscles and sphincter muscles of the bladder and rectum are characteristically spared until the latest stage of the illness. Pathologically, motor neurons in the oculomotor, trochlear, abducens, and Onuf’s nuclei are usually spared in ALS. These characteristics indicate that the loss of motor neurons depends on the vulnerability of the regions, and determines the clinical type of ALS. Therefore the authors investigated the correlation between NOSs and neuronal loss in the cervical and lumbar cord and in
Fig. 1. The anterior horns of the control and ALS cases. The upper, middle, and lower strands indicate C8, L2, and S2, respectively. (a – c) Control case (c1). The cell population is normal. (d – f) c-ALS (a2). Severe neuronal loss was seen in the cervical segment, but only moderate loss was seen in the lumbar segment. (g – i) l-ALS (a6). Severe neuronal loss was seen in the lumbar segment. The Onuf’s nucleus of the ALS cases appeared normal, and was similar to that in the control cases. Black bar indicates 200 mm. Klu¨ver – Barrera stain.
Fig. 2. Immunostaining for n-NOS in the control and ALS cases. Each figure represents the same segments indicated in Fig. 1. n-NOS expression in ALS cases was very strong and diffuse even in the neurons of the Onuf’s nucleus (f,i).On the other hand, control cases showed no immunoreactivity for n-NOS (a – c). The magnification of inset pictures is the same as (c), (f), and (i). Black bar indicates 100 mm.
Onuf’s nucleus from patients with c-ALS and from those with l-ALS.
2. Methods 2.1. Human autopsy cases The authors examined the cervical cord (C7, C8), the lumbar cord (L1, L2, L3), and the sacral cord (S2, S3) from three autopsied cases with c-ALS, three autopsied cases with l-ALS, and three normal controls (Table 1). Autopsy was performed within 5 h of death in each case. Spinal cord was fixed in 10% formaldehyde, embedded in paraffin, cut into sections 6 mm thick, and stained by HE and Klu¨ver –
Fig. 3. Immunostaining for n-NOS in the intermediolateral cell column of the control (a) and ALS (b) cases. n-NOS expression was seen strongly and diffusely in the lateral horn cells. Black bar indicates 50 mm.
K. Kashiwado et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 163–167 Table 2 Expression of n-NOS
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Table 4 Expression of i-NOS
Case
Cervical cord
Lumbar cord
Onuf’s nucleus
Case
Cervical cord
Lumbar cord
Onuf’s nucleus
a1 a2 a3 a4 a5 a6 c1 c2 c3
2+ 2+ 1+ 1+ 2+ 2+ 0 0 0
2+ 2+ 1+ 1+ 2+ 2+ 0 0 1+
2+ 2+ 1+ 1+ 1+ 2+ 0 0 0
a1 a2 a3 a4 a5 a6 c1 c2 c3
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0: none, 1+: sparsely present, 2+: markedly present.
0: none, 1+: sparsely present, 2+: markedly present.
Barrera. For the immunohistochemistry, sections were autoclaved (120 C, 6 min), incubated with 1% H2O2 in methyl alcohol for 30 min at room temperature, incubated with 10% normal swine serum for 30 min at room temperature, then incubated with anti-NOS antibodies (n-, e-, i-NOS all polyclonal, Biogenesis, 1:200) for 48 h at 4 C. They were then treated for 2 h at room temperature with biotinylated secondary antibody (Vector). Washing between steps was performed with PBS. Peroxidase labeling was visualized by incubating with a solution containing 0.001% 3.30-diaminobenzidine, 0.6% nickel ammonium sulfate, 0.05% imidazole, and 0.0003% H2O2. When a dark purple product formed, the reaction was terminated. Sections were washed, dehydrated with graded alcohol, then coverslipped. We also stained sections with the Klu¨ver –Barrera methods. For the immunohistochemistry, 20 serial sections were stained every 4 sections.
In the c-ALS, the neuronal loss in the cervical anterior horn cells was more severe than that in the lumbar cord (Fig. 1d,e). On the other hand, the lumber type showed more severe loss in the lumbar cord (Fig. 1g,h). The Onuf’s nucleus in the ALS cases, without regard to the clinical type, was well preserved, and similar to that in the controls (Fig. 1c,f,i).
2.2. Data analysis Histopathological evaluation included the degree of NOS expression (frequency and extent) semiquantitatively. 0: none, 1+: sparsely present, 2+: markedly present. The evaluation was performed by three experienced researchers.
3. Results In normal controls, the anterior horn cells were well preserved in cervical, lumbar, and sacral segments (Fig. 1a – c).
3.1. Immunohistochemistry for NOS (Figs. 2 and 3) In normal controls, the expression of NOSs (n-NOS, e-NOS, and i-NOS) was not detected in the anterior horn cells (Fig. 2a – c), though n-NOS expression was seen strongly and diffusely in the lateral horn cells (Fig. 3a). The lateral horn cells in the ALS cases showed the same tendency (Fig. 3b). In ALS cases, n-NOS and e-NOS expression in the residual anterior horn cell cytoplasms was strong and diffuse, and this type of expression was also seen in some neurites. NOS immunoreactivity was seen more than half of the residual cells. Immunoreaction of the nuclei and the surface of the anterior horn cells could not be seen. In relation to the n-NOS and e-NOS expression, no difference was observed in the staining ability between the conventional type and the lumbar type in any segment of the animal cord examined (Fig. 2d– i, Tables 2 and 3). Namely, the expression of NOSs (n-NOS and e-NOS) in the preserved Onuf’s nucleus was equivalent to that in the severely degenerated areas. i-NOS did not express in any type of ALS (Table 4).
4. Discussion
Table 3 Expression of e-NOS Case
Cervical cord
Lumbar cord
Onuf’s nucleus
a1 a2 a3 a4 a5 a6 c1 c2 c3
2+ 1+ 1+ 2+ 1+ 2+ 1+ 0 0
2+ 2+ 1+ 2+ 1+ 1+ 0 0 0
1+ 1+ 1+ 2+ 1+ 1+ 0 0 0
0: none, 1+: sparsely present, 2+: markedly present.
Some researchers have found that NO is involved in the process of various neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease (Beal, 1998; Bredt, 1999). Because NO is produced by NOS and NOS expresses in the anterior horn cells of the spinal cord in ALS, there is a possibility that NOS plays an important role in the pathogenesis of ALS (Beckman et al., 1993). If the hypothesis that NOS is involved in the process of ALS is correct, the expression of NOS has to be strong in the severely afflicted lesions. Therefore, it is important to
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investigate the severity of lesions and whether it is associated with the strength of NOS expression. To our knowledge, no previous report has focused on the differences of NOS expression between clinical types of ALS and the spinal cord segments. First, we compared NOS expressions in the anterior horn cells of ALS with those of controls. Our findings showed that n-NOS and e-NOS expressions in motor neurons were almost always detected in ALS. Abe et al. (1995, 1997) showed that the expressions of NOS (not i-NOS but n-NOS and e-NOS) in motor neurons were enhanced in ALS compared to those in controls. Up to this point, our result is in accord with theirs. It supports the hypothesis that NOS expression is involved with the disease process at first glance. Second, to clarify this hypothesis, we compared clinical types of ALS in terms of NOS expressions in the spinal cord segments. Before discussing this point, some discussion is warranted on the significance of the Onuf’s nucleus in this study. 4.1. Onuf’s nucleus in ALS It is well known that external ocular muscles and external sphincter muscles of the bladder and rectum are spared in ALS until the terminal stage. Onuf’s nucleus, located in the ventral margin of the anterior horn at the second sacral cord level, may control strained muscles in the pelvic floor (Mannen et al., 1977; Schroder and Reske-Nielsen, 1984). Onuf’s nucleus in ALS are usually preserved in comparison to anterior horn cells of other parts (Schroder and ReskeNielsen, 1984). We confirmed this in this study by the Klu¨ver – Barrera method. Therefore, Onuf’s nucleus is a suitable region to use for a comparison of the expression of NOS in anterior horn cells of ALS. The authors examined not only the cervical and lumbar segments but also the sacral cord where the Onuf’s nucleus is situated. 4.2. Clinical types of ALS and NOSs expression The apparent difference of neuronal loss was observed according to the clinical types of ALS. In c-ALS, the number of motor neurons left in the cervical cord was lower than that left in the lumbar cord. On the other hand, in l-ALS, the number of motor neurons left in the lumbar cord was lower than that left in the cervical cord. However, the neurons in the Onuf’s nuclei were well preserved in both types. The expression of n-NOS and e-NOS in anterior horns of the cervical cord, lumbar cord, and the Onuf’s nucleus was up-regulated, but there was no difference among segments (Tables 2 and 3). There are three possibilities for the discrepancy between NOS expression and neuronal loss. One possibility is that the selective loss of motor neurons in ALS is due to vulnerability to NO. If this possibility is correct, there must be only one clinical type of ALS, and the types extant reflect an ordered vulnerability to NO in each spinal cord segment.
Given that loss was comparable among the segments in all three types, this possibility is unlikely. The second possibility is that the expression of NOSs occurs upstream of the degenerative processes, and NOSs-bearing anterior horn cells eventually die. According to this hypothesis, neurons of the Onuf’s nucleus inevitably degenerate in cases with long-standing life support. Because the expression of NOSs was not related to the severity of the neuronal loss, upregulation of NOSs may not immediately cause neuronal loss. The equal expression of n-NOS and e-NOS among cervical, lumbar, and sacral segments suggests a third possibility; given that no remarkable neuronal loss was found in the Onuf’s nucleus, it is possible that NOS plays a protective role in neurons. A low concentration of NO plays a protective role in glutamate neurotoxicity by inhibiting NMDA-channel activity (Kashii et al., 1996). This protective mechanism constitutes one aspect of the dual actions of nitric oxide. Lehrer-Graiwer et al. (2000) found that n-NOS-expressing neurons were selectively spared from NO-induced cytotoxicity in chronic neurodegenerative conditions. Although NO directly inhibits the mitochondrial enzyme CcO, it simultaneously induces mRNA and protein for CcO. Neurons chronically exposed to low concentrations of NO through endogenous n-NOS activity may up-regulate CcO levels, and thereby contribute to selective sparing of n-NOS neurons (LehrerGraiwer et al., 2000). Our data support these previous reports that NOS works neuro-protectively in the process of neuronal death.
5. Conclusion The expression of NOSs does not immediately affect neuronal loss in sporadic ALS.
Acknowledgments We would like to thank Mr. H. Nagase for his technical assistance. This study was supported in part by a grant from the Ministry of Education, Japan.
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