Brain Research, 566 (1991) 54-60 1~ 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91l$03.50
54 BRES 17202
Amyotrophic lateral sclerosis: changes of noradrenergic and serotonergic transmitter systems in the spinal cord Oswald Bertel x, Susanne Malessa*, Elfriede Sluga2 and Oleh Hornykiewicz 1 tlnstitute of Biochemical Pharmacology and 2Neurological Institute, University of Vienna, Vienna (Austria) (Accepted 16 July 1991) Key words: Noradrenaline; Dopamine; Serotonin; 5.Hydroxyindoleacetic acid; Human spinal cord; Amyotrophic lateral sclerosis
Noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5.hydroxyindoleacetic acid (5-HIAA) were measured in discrete subdivisions of cervical, thoracic and lumbar spinal cord segments obtained at autopsy of 4 subjects with amyotrophic lateral sclerosis (ALS) and 7 control patients. NA concentrations in thoracic and lumbar spinal cord of ALS patients were 2- to 4-fold higher compared with values obtained in control patients. 5-HT levels were unchanged at the cervical and thoracic level and slightly above normal in lumbar spinal cord, while the concentration of 5-HIAA was lowered in cervical and thoracic, but within the control range, in lumbar spinal cord. As a result, the molar ratios of 5-HT/5-HIAA were increased at all spinal levels in ALS. No difference in spinal DA concentration was found between ALS and control patients. "I~e changes in the noradrenergic and serotonergic transmitter systems reported here most probably reflect a decreased release of these transmitter substances in ALS spinal cord. Since lack of the facilitatory monoaminergic influence would necessitate an increase in the excitatory, potentially neurotoxic glutamatergic input onto the motoneurones, we hypothesize that this could contribute to the progressive loss of spinal motoneurones in amyotrophic lateral sclerosis.
INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disorder of the human motor system. It always involves both upper and lower motoneurones while the pattern of cell loss and motor dysfunction varies from one ease to another. Although some authors point to an additional affection of non-motor systems, such us dorsal root ganglia =t, dorsal horns t s ' " and Clarke's column 2, the most prominent clinical signs of ALS are neurogenic wasting of muscles, spasticity and fasciculations without clear evidence of sensory dysfunction. The characteristic histopathologic features in the spinal cord are a substantial loss of large, and also small, motoneurones with astrocytic gliosis, and a loss of fibres in the pyramidal tract (for review, see refs. 6, 38). In accord with these changes, neurochemical analyses of ALS spinal cord tissue revealed a reduction of cholinergic markers, such as choline aeetyltransferase activity t6'26, acetylcholinesterase activity25, and muscarinic cholinergic receptors is'44 (Berger et al., submitted). Determination of the monoamines in the cerebrospinal fluid (CSF) has disclosed elevated concentrations of
noradrenaline (NA) in ALS patients 7'4s. In contrast, reduced homovanillic acid (HVA) levels in CSF as well as a reduced probe~lecid-induced accumulation of HVA in the CSF of ALS patients was measured suggesting an impaired central D A synthesis, A therapeutic trial with levodopa, however, remained unsuccessful u . The anecdotal nature of the available information prompted us to study the detailed distribution of the 3 major monoamine neurotransmitters, NA, dopamine (DA) and serotonin (5-HT) as well as the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in ALS-affected spinal cord tissue. MATERIALS AND METHODS Spinal cords from 4 ALS patients and 7 control subjects without signs of neurological impairment were obtained at autopsy. Age, sex, duration of ALS, cause of death and time interval between death and freezing of tissue (pro time) are given in Table I. ALS had been diagnosed on the basis of clinical symptoms and confirmed by histopathological examination. All 4 patients suffered from sporadic ALS with upper and lower motoneuron involvement. From ALS patient no. 4 and control patient no, 1 only thoracic and lumbar parts of spinal cord were available for neurochemical analyses. After removal, whole spinal cords were quickly frozen on dry ice, and stored at -80 *(2 until dissection. The dissection technique described by Otsuka2~ and Fleetwood-Walkert4 was used with small
* Present address: Institute of Neuropathology, University of Munich, Munich, ER.G. Correspondence: O. But'tel, Institute of Biochemical Pharmacology, University of Vienna, Borschkegasse 8a, A-1000 Vienna, Austria. Fax: (43) (222) 430790.
55 2
1
3
1
/
1
2
3
2
A
'
/
i,..
B
,,, f
/Lw
C
O
\'k, f
E
',,.._
a: Cervical
b: T h o r a c i c
c: L u m b a r
Fig. 1. a-c show schematically the transverse section of the human spinal cord at the cervical (a), thoracic (b) and the lumbar level (c). The superimposed grid lines indicate the regional subdivisions as performed in a representative dissection. Each tissue sample is defined by two coordinates (IA-3E). LW, lateral white matter; VW, ventral white matter.
modifications. On a cold plate (~lVroximately -tO °C) a series of 4-6 transverse slices, 1-2 mm thick, was cut from cervical enlargements, mid-thoracic cord and lumbar enlargements. Cervical and lumbar slices were cut into 30, thoracic slices into 24 rectangular pieces of tissue to form a grid-like pattern as schematically indicated in the Fig, la-c. Corresponding tissue pieces from consecutive slices were pooled,
TABLE 1
ALS and control patients: clinical characteristics Comrols Sex
Age pm thne (years) (h)
Causeof death
1 2 3 4
f m f m
75 56 50 78
24 15 13.5 7
5 6
f m
83 48
11 10.5
7
f
59
7.5
malignant lymphoma melanoma myelocyticleukemia Non-Hodgkin lymphoma heart failure carcinomaof the tonsills breast carcinoma
mean age: 64 ± 5.8 years
mean pm time 12.6 ± 2.4 h
ALS
Sex
Age pm time Cause (years) (h) of death
Duration of ALS (years)
1
f
62
17.5
4
2
f
55
4.5
3
m
57
7
4
f
64
30
mean age 59.5 +-. 2.1 years
Respiratory failure Respiratory failure Respiratory failure Respiratory failure
1,5 4 2
mean pm time 14.5 -+ 5.8 h
combining also corresponding left and right parts. After weighing, samples were homogenized by sonication in 5 to 30 parts of ice cold deionized and degassed water, saturated with N2. These homogenization conditions were chosen to permit the additional analysis of enzyme activities in the homogenates (Berger et al., submitted). Aliquots of the homogenates were diluted with an equal volume of 0.2 M perchloric acid containing 0.8 mM NaHSO3 and centrifuged at 17,000 8 for 15 min. From the supernatants, aliquots were taken for determination of 5-HT and 5-HIAA. Catecholamines were extracted from the remaining supernatants by adsorption to A120~ and analysed on a high performance liquid chromatographic system with electrochemical detection according to Felice n with minor modifications as described previously'~':.7, One.tailed Student's t-test was used for statistical analyses of data. RESULTS
5 . H T and 5.HIAA In control subjects, the concentration of 5-HT was highest in the spinal cord samples comprising the ventral and the intermediate grey matter free of any admixture with white matter (range 300-1500 ng/g fresh tissue). The 5-HT levels (70-320 ng/g) in the dorsal horn samples, which as a rule contained some adjacent white matter did not substantially differ from the neighboring samples of pure white matter (range 30-340 ng/g fresh tissue). The lowest levels of 5-HT were found in the white matter of the dorsal funiculi (range 20-300 ng/g fresh tissue). This 5-HT pattern was seen at all spinal levels investigated, although in this respect there was a marked rostrocaudal concentration gradient, with the 5-HT levels in the lumbar enlargement 3-4 fold higher (900-1200 ng/g) than in the cervical cord (Fig. 2). In ALS patients, in most subdivisions of the spinal
56
Cervical 1201-
5-HT
NA 1000 F
100 -
!
800 r 600 r
'°°E ABCDE I
A B C DE 2
3
W
1
2
A B C DE 3
L D W
Thoracic
NA
5-HT
100 80 60 40
. .i:
:::t
20 ABCD 1
ABCD 2
ABCD 3
L D W
ABCD !
ABCD 2
ABCD 3
L D W
Lumbar
2~00
S-HT NA 2000
,°°I
1600
120
1200
80
800
40
400
160 r
ABCDE 1
ABCDE 2
ABCDE 3
LD W
ABCDE I
ABCDE 2
ABCDE 3
L O W
Fig. 2. Concentrations of NA and S-HT given as ng/g wet weight (means ± S,E.M.) in ALS patients (solid bars) versus control patients (open bars), For the exact anatomical positions of the coordinates IA-3E in the cross-sections of the spinal cord and abbreviations, see Fig. I. *P < 0,05: **P < 0.01,
57 TABLE II Molar ratios of 5-HTIS-HIAA (means ± S.E.M.) in control patients versus AYeS Fr~'tients
For the exact anatomical positions of the coordinates IA-3E in the cross-sections of the spinal cord, see Fig. 1. LW, lateral white matter, DW, dorsal white matter. Cervical
Thoracic
Lumbar
Controls
ALS
Controls
ALS
IA IB IC ID IE
0.251 0.229 0.338 0.610 0.490
.4. 0.063 - 0.042 ± 0.087 -- 0.097 ± 0.079
0.465 ..4-0.093 0.500 ± 0.058 0.699 - 0.093 1.136 ± 0.142 1.176 - 0.415"
0.331 - 0.068 0.341 ± 0.087 0.450 -+ 0.118 0.532 ± 0.122
0.629 0.617 0.870 0.935
0.115 0.118 0.227 0.122
0.469 0.552 0.710 0.893 0.962
± ± ±
0.051 0.043 0.463 0.096 0.120
0.645 0.649 0.909 1.235 1.515
2A 2B 2C 2D 2E
0.189 0.286 0.500 0.163 0.510
± ± ± ± ±
0.043 0.043 0.068 0.098 0.073
0.284 ± 0.446 ± 0.926 ± 1.000 1.266 ±
0.347 ± 0.490 ± 0.763 0.794 ±
0.314 ± 0.019 0.794 -+ 0.107 1.333 ± 0.249 1.316 ± 0.294
0.418 0.526 0.699 0.719 0.800
± ± -
0.028 0.050 0.059 0.067 0.090
0.476 ± 0.048 0.621 --. 0.073 0.826 - 0.116 0.990 - 0.255 1.075 ± 0.197
3A 3B 3C 3D 3E
0.534 0.114 0.184 0.361 0.314
± ± ± ±
0.567 0.036 0.056 0.026 0.031
0.337 ± 0.056 0.136 4- 0.062 0.300 ± 0.045 0.546 ± 0.021" 0.813 ± 0.264**
0.218 ± 0.030 0.266 - 0.054 0.592 ± 0.109 0.280 +-. 0.153
0.225 0.249 ± 0.934 1.099 -
0.302 ± 0.047 0.281 - 0.050 0.446 - 0.058 0.546.4-0.069 0.568 - 0.090
0.320 0.279 0.592 0.769 0.671
LW DW
0.242 • 0.071 0.395 ± 0.091
0.478 ± 0.149 0.980 :t: 0.404
0.321 "" 0.116 0.433 ± 0.139
0.787 ± 0.118 1.099 - 0.18
0.763 - 0.116 0.592 - 0.119
1.667 -+ 0.500* 1.282 - 1.052
0.036 0.038 0.028 0.19 0.240*
0.077 0.070 0.099 0.095
Controls
± ±
0.059 0.050 0.096 0.109
ALS
± 0.162 _+ 0.059 ± 0.107 -+ 0.320 -+ 0.390
- 0.036 - 0.071 +-- 0.070 - 0.024 - 0.059
*P < 0.05; **P < 0.01.
cord the mean 5-HT concentrations were above normal, but the differences reached statistical significance (P < 0.05) only in the two lumbar subdivisions containing the lateral white matter and the lateral part of the ventral horn (Fig. 2). The distribution of 5.HIAA in white and grey matter as well as at the 3 rostrocaudal levels of spinal cord examined, closely paralleled that of 5-HT, ranging in controis from 200 to 800 ng/g fresh tissue in white matter, and from 400 to 2400 ng/g fresh tissue in grey matter (Table il). In contrast to 5.HT, in the ALS patients the mean concentrations of 5-HIAA were decreased. This decrease was most pronounced in cervical cord, where the levels of 5 - H I A A were reduced to about 50% of the control levels. Due to great interindividual variations, the differences did not reach statistical significance. However, the molar ratio of 5-HT/5-HIAA was above normal throughout all spinal levels (Table II) the increase being significant in 3 ventral horn parts of the cervical spinal cord (P < 0.05 and P < 0.01, respectively). N A and D A
The N A levels found in the spinal cord of control patients were considerably lower than the spinal 5-HT levels (5-25% w/w of 5-HT). The highest NA concentrations were measured in the ventral and the intermediate
grey matter (range 15-200 ng/g fresh tissue). Samples containing the dorsal horn showed considerably lower NA concentrations which in some instances did not differ from NA levels in adjacent white matter (range 3-30 ng/g fresh tissue). In respect to the rostrocaudal gradient, NA concentrations were highest in the lumbar enlargement, somewhat lower in the cervical enlargement and lowest in the thoracic segments (Fig. 2). Compared with controls, in ALS spinal cord the NA concentrations were consistently above normal. The changes were most pronounced in lumbar and thoracic spinal cord, where a 2- to 4-fold increase, significant at the P < 0.01 confidence level, was observed in ventral and lateral areas (Fig. 2). Spinal D A concentrations were the lowest of the 3 monoamine neurotransmitter substances measured. Highest levels of D A were found in the dorsal horn region (mean 12 ng/g fresh tissue in lumbar spinal cord) and in the intermediate grey (mean 8 ng/g fresh tissue in cervical and thoracic spinal cord). In many samples of both control and ALS cases, the concentration of DA was below the detection limits of our assay of about 15 pg/sample (typical sample weight 15 mg); in other samples up to 1 ng were found (data not shown in detail). No differences in mean D A concentrations were found between the different spinal cord levels or between ALS and control cases.
58 DISCUSSION
The present study gives detailed and extensive information on the concentrations and distribution pattern of the monoamines NA, 5-HT and the 5-HT metabolite 5-HIAA in the normal regionally subdivided human spinal cord. Our data are in good agreement with the respective data of the few earlier investigations27'2s'34. Compared with other species, such as the rabbit, rat 47 or cat t4. human spinal cord contains lower concentrations of NA and DA but similar levels of 5-HT. The serotonergic innervation of the mammalian spinal cord has its origin in the caudal raphe nuclei and the adjoining medial ventral reticular formation 5'ts. A large subset of these 5-HT projections also contains substance P and/or thyrotropin releasing hormone (TRH). Its terminals are seen around motoneurones, in the intermediolateral cell column t, and also in the intermediate spinal greys'ts'tg. In various electrophysiological experiments, substance P, TRH and 5-HT have been shown to enhance the glutamate-evoked excitation of motoneurones 3'!s'3~-43'45. The main source of the spinal NA is thought to be the nucleus coeruleus and subcoeruleus in the lower brainstem and the A5 and A7 cell groups t3. Noradrenergic influences on sensory as well as motor functions at the spinal cord level have been reported 9'ts' 42. NA increase~, independently of 5-HT, the excitability of ~.motoneurones to glutamate 42'43, The increased 5-HT/5-HIAA molar ratios observed in our study of ALS spinal cords were due to lowered 5.HIAA concentrations in the cervical and the thoracic cord. This is in good agreement with the work of Oshugi et al. 2s who measured a decrease of 5-HIAA, but unchanged levels of 5.HT, in ALS cervical segments. In the lumbar enlargement, however, we found only small changes of the 5-HIAA concentrations, so that here the significantly increased ratios are largely a consequence of the increased levels of 5-HT. Our observation of abovenormal NA concentrations at all spinal cord levels represents, to our knowledge, the first such report in patients with ALS. In the CSF of patients with ALS. elevated NA has previously been reported 46. Since in many spinal subdivisions the increased NA levels were not accompanied by analogous changes in 5-HT and/or 5-HIAA, it is unlikely that the observed NA changes were due to the effect of tissue shrinkage. In all probability, the changes of both the 5-HT and the NA were the result of their decreased release, being possibly consequent to the loss of a large number of their target cells, i.e. the motoneurones. Regarding the increased tissue concentrations of NA measured in our study of the ALS spinal cord, an additional possibility is denser noradrenergic innervation. In
the transected spinal cord of rats and dogs neosympathetic innervation has been shown to occur caudal to the site of transection s'23, and sympathetic sprouting into affected areas of the CNS, suffering experimental cholinergic deafferentation has also been observed t°'ll'l~. Since in ALS spinal cholinergic cells are lost, similar sprouting of noradrenergic (sympathetic) fibres may also take place in the course of this disease and thus in part contribute to the above normal spinal (and CSF) NA levels. Decreased TRH content of cervical ventral horn 25, loss of substance P-, but not of 5-HT-containing fibers35, have been reported in ALS. Our present observations, together with those in the literature, document widespread alterations in the monoaminergic/peptidergic systems of the spinal cord in ALS. In addition, we have recently described significantly reduced levels of glutamate as well as glycine in regionally subdivided ALS spinal cord 22 (see also refs. 31, 32). Since in the CSF of patients with ALS above-normal glutamate levels have been found 33 an increased release of this excitatory amino acid in spinal cord tissue of ALS patients can be assumed. Putting together the above findings, the following pattern may be envisaged regarding the possible neurotransmitter influences on the remaining spinal motoneurones in advanced stages of ALS: compared with normal spinal cord, the spinal motoneurones in ALS may be exposed to fewer monoaminergic and peptidergic signals, thus being forced to integrate more glutamatergic input in order to reach effective depolarization and release of acetylcholine at the neuromuscular junction. This would result in overexposure of the spinal motoneurones to potentially neurotoxic amounts of glutamate. This effect may be aggravated by concomitantly increased release of glycine, which we found to be selectively reduced in the dorsal and ventral horn in our ALS patients 2z. Giycine has been shown to be essential for the excitotoxic effects of glutamate (NMDA) receptor activation 2°'3°. Although our present observations leave the question of the pathogenesis of ALS unresolved, they do suggest the possibility that re-institution of the 5.1-11" and/or NA influence on the spinal motoneurones by administration of appropriate drugs may reduce the necessity for excessive glutamatergic activity and thus slow down the progress of this neurodegenerative condition.
Acknowledgements,We wish to thank Drs. E.M, Maskin, M. Schmidbauer and Prof, K, Jellinger for providing material and for histopathological assessment of cases. The technical assistance of G. Sobal, P, Rebernik and H, Reither is greatly appreciated. We thank Mrs. W. Krivanek for secreterial help. S.M. was supported by Dr. CarI-Duisberg-Fund (Bayer Pharmaceuticals, F,R.G.) and the German Academic Exchange Program (DAAD).
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54 BRES 17202
Amyotrophic lateral sclerosis: changes of noradrenergic and serotonergic transmitter systems in the spinal cord Oswald Bertel x, Susanne Malessa*, Elfriede Sluga2 and Oleh Hornykiewicz 1 tlnstitute of Biochemical Pharmacology and 2Neurological Institute, University of Vienna, Vienna (Austria) (Accepted 16 July 1991) Key words: Noradrenaline; Dopamine; Serotonin; 5.Hydroxyindoleacetic acid; Human spinal cord; Amyotrophic lateral sclerosis
Noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5.hydroxyindoleacetic acid (5-HIAA) were measured in discrete subdivisions of cervical, thoracic and lumbar spinal cord segments obtained at autopsy of 4 subjects with amyotrophic lateral sclerosis (ALS) and 7 control patients. NA concentrations in thoracic and lumbar spinal cord of ALS patients were 2- to 4-fold higher compared with values obtained in control patients. 5-HT levels were unchanged at the cervical and thoracic level and slightly above normal in lumbar spinal cord, while the concentration of 5-HIAA was lowered in cervical and thoracic, but within the control range, in lumbar spinal cord. As a result, the molar ratios of 5-HT/5-HIAA were increased at all spinal levels in ALS. No difference in spinal DA concentration was found between ALS and control patients. "I~e changes in the noradrenergic and serotonergic transmitter systems reported here most probably reflect a decreased release of these transmitter substances in ALS spinal cord. Since lack of the facilitatory monoaminergic influence would necessitate an increase in the excitatory, potentially neurotoxic glutamatergic input onto the motoneurones, we hypothesize that this could contribute to the progressive loss of spinal motoneurones in amyotrophic lateral sclerosis.
INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disorder of the human motor system. It always involves both upper and lower motoneurones while the pattern of cell loss and motor dysfunction varies from one ease to another. Although some authors point to an additional affection of non-motor systems, such us dorsal root ganglia =t, dorsal horns t s ' " and Clarke's column 2, the most prominent clinical signs of ALS are neurogenic wasting of muscles, spasticity and fasciculations without clear evidence of sensory dysfunction. The characteristic histopathologic features in the spinal cord are a substantial loss of large, and also small, motoneurones with astrocytic gliosis, and a loss of fibres in the pyramidal tract (for review, see refs. 6, 38). In accord with these changes, neurochemical analyses of ALS spinal cord tissue revealed a reduction of cholinergic markers, such as choline aeetyltransferase activity t6'26, acetylcholinesterase activity25, and muscarinic cholinergic receptors is'44 (Berger et al., submitted). Determination of the monoamines in the cerebrospinal fluid (CSF) has disclosed elevated concentrations of
noradrenaline (NA) in ALS patients 7'4s. In contrast, reduced homovanillic acid (HVA) levels in CSF as well as a reduced probe~lecid-induced accumulation of HVA in the CSF of ALS patients was measured suggesting an impaired central D A synthesis, A therapeutic trial with levodopa, however, remained unsuccessful u . The anecdotal nature of the available information prompted us to study the detailed distribution of the 3 major monoamine neurotransmitters, NA, dopamine (DA) and serotonin (5-HT) as well as the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in ALS-affected spinal cord tissue. MATERIALS AND METHODS Spinal cords from 4 ALS patients and 7 control subjects without signs of neurological impairment were obtained at autopsy. Age, sex, duration of ALS, cause of death and time interval between death and freezing of tissue (pro time) are given in Table I. ALS had been diagnosed on the basis of clinical symptoms and confirmed by histopathological examination. All 4 patients suffered from sporadic ALS with upper and lower motoneuron involvement. From ALS patient no. 4 and control patient no, 1 only thoracic and lumbar parts of spinal cord were available for neurochemical analyses. After removal, whole spinal cords were quickly frozen on dry ice, and stored at -80 *(2 until dissection. The dissection technique described by Otsuka2~ and Fleetwood-Walkert4 was used with small
* Present address: Institute of Neuropathology, University of Munich, Munich, ER.G. Correspondence: O. But'tel, Institute of Biochemical Pharmacology, University of Vienna, Borschkegasse 8a, A-1000 Vienna, Austria. Fax: (43) (222) 430790.
55 2
1
3
1
/
1
2
3
2
A
'
/
i,..
B
,,, f
/Lw
C
O
\'k, f
E
',,.._
a: Cervical
b: T h o r a c i c
c: L u m b a r
Fig. 1. a-c show schematically the transverse section of the human spinal cord at the cervical (a), thoracic (b) and the lumbar level (c). The superimposed grid lines indicate the regional subdivisions as performed in a representative dissection. Each tissue sample is defined by two coordinates (IA-3E). LW, lateral white matter; VW, ventral white matter.
modifications. On a cold plate (~lVroximately -tO °C) a series of 4-6 transverse slices, 1-2 mm thick, was cut from cervical enlargements, mid-thoracic cord and lumbar enlargements. Cervical and lumbar slices were cut into 30, thoracic slices into 24 rectangular pieces of tissue to form a grid-like pattern as schematically indicated in the Fig, la-c. Corresponding tissue pieces from consecutive slices were pooled,
TABLE 1
ALS and control patients: clinical characteristics Comrols Sex
Age pm thne (years) (h)
Causeof death
1 2 3 4
f m f m
75 56 50 78
24 15 13.5 7
5 6
f m
83 48
11 10.5
7
f
59
7.5
malignant lymphoma melanoma myelocyticleukemia Non-Hodgkin lymphoma heart failure carcinomaof the tonsills breast carcinoma
mean age: 64 ± 5.8 years
mean pm time 12.6 ± 2.4 h
ALS
Sex
Age pm time Cause (years) (h) of death
Duration of ALS (years)
1
f
62
17.5
4
2
f
55
4.5
3
m
57
7
4
f
64
30
mean age 59.5 +-. 2.1 years
Respiratory failure Respiratory failure Respiratory failure Respiratory failure
1,5 4 2
mean pm time 14.5 -+ 5.8 h
combining also corresponding left and right parts. After weighing, samples were homogenized by sonication in 5 to 30 parts of ice cold deionized and degassed water, saturated with N2. These homogenization conditions were chosen to permit the additional analysis of enzyme activities in the homogenates (Berger et al., submitted). Aliquots of the homogenates were diluted with an equal volume of 0.2 M perchloric acid containing 0.8 mM NaHSO3 and centrifuged at 17,000 8 for 15 min. From the supernatants, aliquots were taken for determination of 5-HT and 5-HIAA. Catecholamines were extracted from the remaining supernatants by adsorption to A120~ and analysed on a high performance liquid chromatographic system with electrochemical detection according to Felice n with minor modifications as described previously'~':.7, One.tailed Student's t-test was used for statistical analyses of data. RESULTS
5 . H T and 5.HIAA In control subjects, the concentration of 5-HT was highest in the spinal cord samples comprising the ventral and the intermediate grey matter free of any admixture with white matter (range 300-1500 ng/g fresh tissue). The 5-HT levels (70-320 ng/g) in the dorsal horn samples, which as a rule contained some adjacent white matter did not substantially differ from the neighboring samples of pure white matter (range 30-340 ng/g fresh tissue). The lowest levels of 5-HT were found in the white matter of the dorsal funiculi (range 20-300 ng/g fresh tissue). This 5-HT pattern was seen at all spinal levels investigated, although in this respect there was a marked rostrocaudal concentration gradient, with the 5-HT levels in the lumbar enlargement 3-4 fold higher (900-1200 ng/g) than in the cervical cord (Fig. 2). In ALS patients, in most subdivisions of the spinal
56
Cervical 1201-
5-HT
NA 1000 F
100 -
!
800 r 600 r
'°°E ABCDE I
A B C DE 2
3
W
1
2
A B C DE 3
L D W
Thoracic
NA
5-HT
100 80 60 40
. .i:
:::t
20 ABCD 1
ABCD 2
ABCD 3
L D W
ABCD !
ABCD 2
ABCD 3
L D W
Lumbar
2~00
S-HT NA 2000
,°°I
1600
120
1200
80
800
40
400
160 r
ABCDE 1
ABCDE 2
ABCDE 3
LD W
ABCDE I
ABCDE 2
ABCDE 3
L O W
Fig. 2. Concentrations of NA and S-HT given as ng/g wet weight (means ± S,E.M.) in ALS patients (solid bars) versus control patients (open bars), For the exact anatomical positions of the coordinates IA-3E in the cross-sections of the spinal cord and abbreviations, see Fig. I. *P < 0,05: **P < 0.01,
57 TABLE II Molar ratios of 5-HTIS-HIAA (means ± S.E.M.) in control patients versus AYeS Fr~'tients
For the exact anatomical positions of the coordinates IA-3E in the cross-sections of the spinal cord, see Fig. 1. LW, lateral white matter, DW, dorsal white matter. Cervical
Thoracic
Lumbar
Controls
ALS
Controls
ALS
IA IB IC ID IE
0.251 0.229 0.338 0.610 0.490
.4. 0.063 - 0.042 ± 0.087 -- 0.097 ± 0.079
0.465 ..4-0.093 0.500 ± 0.058 0.699 - 0.093 1.136 ± 0.142 1.176 - 0.415"
0.331 - 0.068 0.341 ± 0.087 0.450 -+ 0.118 0.532 ± 0.122
0.629 0.617 0.870 0.935
0.115 0.118 0.227 0.122
0.469 0.552 0.710 0.893 0.962
± ± ±
0.051 0.043 0.463 0.096 0.120
0.645 0.649 0.909 1.235 1.515
2A 2B 2C 2D 2E
0.189 0.286 0.500 0.163 0.510
± ± ± ± ±
0.043 0.043 0.068 0.098 0.073
0.284 ± 0.446 ± 0.926 ± 1.000 1.266 ±
0.347 ± 0.490 ± 0.763 0.794 ±
0.314 ± 0.019 0.794 -+ 0.107 1.333 ± 0.249 1.316 ± 0.294
0.418 0.526 0.699 0.719 0.800
± ± -
0.028 0.050 0.059 0.067 0.090
0.476 ± 0.048 0.621 --. 0.073 0.826 - 0.116 0.990 - 0.255 1.075 ± 0.197
3A 3B 3C 3D 3E
0.534 0.114 0.184 0.361 0.314
± ± ± ±
0.567 0.036 0.056 0.026 0.031
0.337 ± 0.056 0.136 4- 0.062 0.300 ± 0.045 0.546 ± 0.021" 0.813 ± 0.264**
0.218 ± 0.030 0.266 - 0.054 0.592 ± 0.109 0.280 +-. 0.153
0.225 0.249 ± 0.934 1.099 -
0.302 ± 0.047 0.281 - 0.050 0.446 - 0.058 0.546.4-0.069 0.568 - 0.090
0.320 0.279 0.592 0.769 0.671
LW DW
0.242 • 0.071 0.395 ± 0.091
0.478 ± 0.149 0.980 :t: 0.404
0.321 "" 0.116 0.433 ± 0.139
0.787 ± 0.118 1.099 - 0.18
0.763 - 0.116 0.592 - 0.119
1.667 -+ 0.500* 1.282 - 1.052
0.036 0.038 0.028 0.19 0.240*
0.077 0.070 0.099 0.095
Controls
± ±
0.059 0.050 0.096 0.109
ALS
± 0.162 _+ 0.059 ± 0.107 -+ 0.320 -+ 0.390
- 0.036 - 0.071 +-- 0.070 - 0.024 - 0.059
*P < 0.05; **P < 0.01.
cord the mean 5-HT concentrations were above normal, but the differences reached statistical significance (P < 0.05) only in the two lumbar subdivisions containing the lateral white matter and the lateral part of the ventral horn (Fig. 2). The distribution of 5.HIAA in white and grey matter as well as at the 3 rostrocaudal levels of spinal cord examined, closely paralleled that of 5-HT, ranging in controis from 200 to 800 ng/g fresh tissue in white matter, and from 400 to 2400 ng/g fresh tissue in grey matter (Table il). In contrast to 5.HT, in the ALS patients the mean concentrations of 5-HIAA were decreased. This decrease was most pronounced in cervical cord, where the levels of 5 - H I A A were reduced to about 50% of the control levels. Due to great interindividual variations, the differences did not reach statistical significance. However, the molar ratio of 5-HT/5-HIAA was above normal throughout all spinal levels (Table II) the increase being significant in 3 ventral horn parts of the cervical spinal cord (P < 0.05 and P < 0.01, respectively). N A and D A
The N A levels found in the spinal cord of control patients were considerably lower than the spinal 5-HT levels (5-25% w/w of 5-HT). The highest NA concentrations were measured in the ventral and the intermediate
grey matter (range 15-200 ng/g fresh tissue). Samples containing the dorsal horn showed considerably lower NA concentrations which in some instances did not differ from NA levels in adjacent white matter (range 3-30 ng/g fresh tissue). In respect to the rostrocaudal gradient, NA concentrations were highest in the lumbar enlargement, somewhat lower in the cervical enlargement and lowest in the thoracic segments (Fig. 2). Compared with controls, in ALS spinal cord the NA concentrations were consistently above normal. The changes were most pronounced in lumbar and thoracic spinal cord, where a 2- to 4-fold increase, significant at the P < 0.01 confidence level, was observed in ventral and lateral areas (Fig. 2). Spinal D A concentrations were the lowest of the 3 monoamine neurotransmitter substances measured. Highest levels of D A were found in the dorsal horn region (mean 12 ng/g fresh tissue in lumbar spinal cord) and in the intermediate grey (mean 8 ng/g fresh tissue in cervical and thoracic spinal cord). In many samples of both control and ALS cases, the concentration of DA was below the detection limits of our assay of about 15 pg/sample (typical sample weight 15 mg); in other samples up to 1 ng were found (data not shown in detail). No differences in mean D A concentrations were found between the different spinal cord levels or between ALS and control cases.
58 DISCUSSION
The present study gives detailed and extensive information on the concentrations and distribution pattern of the monoamines NA, 5-HT and the 5-HT metabolite 5-HIAA in the normal regionally subdivided human spinal cord. Our data are in good agreement with the respective data of the few earlier investigations27'2s'34. Compared with other species, such as the rabbit, rat 47 or cat t4. human spinal cord contains lower concentrations of NA and DA but similar levels of 5-HT. The serotonergic innervation of the mammalian spinal cord has its origin in the caudal raphe nuclei and the adjoining medial ventral reticular formation 5'ts. A large subset of these 5-HT projections also contains substance P and/or thyrotropin releasing hormone (TRH). Its terminals are seen around motoneurones, in the intermediolateral cell column t, and also in the intermediate spinal greys'ts'tg. In various electrophysiological experiments, substance P, TRH and 5-HT have been shown to enhance the glutamate-evoked excitation of motoneurones 3'!s'3~-43'45. The main source of the spinal NA is thought to be the nucleus coeruleus and subcoeruleus in the lower brainstem and the A5 and A7 cell groups t3. Noradrenergic influences on sensory as well as motor functions at the spinal cord level have been reported 9'ts' 42. NA increase~, independently of 5-HT, the excitability of ~.motoneurones to glutamate 42'43, The increased 5-HT/5-HIAA molar ratios observed in our study of ALS spinal cords were due to lowered 5.HIAA concentrations in the cervical and the thoracic cord. This is in good agreement with the work of Oshugi et al. 2s who measured a decrease of 5-HIAA, but unchanged levels of 5.HT, in ALS cervical segments. In the lumbar enlargement, however, we found only small changes of the 5-HIAA concentrations, so that here the significantly increased ratios are largely a consequence of the increased levels of 5-HT. Our observation of abovenormal NA concentrations at all spinal cord levels represents, to our knowledge, the first such report in patients with ALS. In the CSF of patients with ALS. elevated NA has previously been reported 46. Since in many spinal subdivisions the increased NA levels were not accompanied by analogous changes in 5-HT and/or 5-HIAA, it is unlikely that the observed NA changes were due to the effect of tissue shrinkage. In all probability, the changes of both the 5-HT and the NA were the result of their decreased release, being possibly consequent to the loss of a large number of their target cells, i.e. the motoneurones. Regarding the increased tissue concentrations of NA measured in our study of the ALS spinal cord, an additional possibility is denser noradrenergic innervation. In
the transected spinal cord of rats and dogs neosympathetic innervation has been shown to occur caudal to the site of transection s'23, and sympathetic sprouting into affected areas of the CNS, suffering experimental cholinergic deafferentation has also been observed t°'ll'l~. Since in ALS spinal cholinergic cells are lost, similar sprouting of noradrenergic (sympathetic) fibres may also take place in the course of this disease and thus in part contribute to the above normal spinal (and CSF) NA levels. Decreased TRH content of cervical ventral horn 25, loss of substance P-, but not of 5-HT-containing fibers35, have been reported in ALS. Our present observations, together with those in the literature, document widespread alterations in the monoaminergic/peptidergic systems of the spinal cord in ALS. In addition, we have recently described significantly reduced levels of glutamate as well as glycine in regionally subdivided ALS spinal cord 22 (see also refs. 31, 32). Since in the CSF of patients with ALS above-normal glutamate levels have been found 33 an increased release of this excitatory amino acid in spinal cord tissue of ALS patients can be assumed. Putting together the above findings, the following pattern may be envisaged regarding the possible neurotransmitter influences on the remaining spinal motoneurones in advanced stages of ALS: compared with normal spinal cord, the spinal motoneurones in ALS may be exposed to fewer monoaminergic and peptidergic signals, thus being forced to integrate more glutamatergic input in order to reach effective depolarization and release of acetylcholine at the neuromuscular junction. This would result in overexposure of the spinal motoneurones to potentially neurotoxic amounts of glutamate. This effect may be aggravated by concomitantly increased release of glycine, which we found to be selectively reduced in the dorsal and ventral horn in our ALS patients 2z. Giycine has been shown to be essential for the excitotoxic effects of glutamate (NMDA) receptor activation 2°'3°. Although our present observations leave the question of the pathogenesis of ALS unresolved, they do suggest the possibility that re-institution of the 5.1-11" and/or NA influence on the spinal motoneurones by administration of appropriate drugs may reduce the necessity for excessive glutamatergic activity and thus slow down the progress of this neurodegenerative condition.
Acknowledgements,We wish to thank Drs. E.M, Maskin, M. Schmidbauer and Prof, K, Jellinger for providing material and for histopathological assessment of cases. The technical assistance of G. Sobal, P, Rebernik and H, Reither is greatly appreciated. We thank Mrs. W. Krivanek for secreterial help. S.M. was supported by Dr. CarI-Duisberg-Fund (Bayer Pharmaceuticals, F,R.G.) and the German Academic Exchange Program (DAAD).
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