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Decreased chloride levels of cerebrospinal fluid in patients with amyotrophic lateral sclerosis
Journal of the Neurological Sciences 285 (2009) 146–148
Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Decreased chloride levels of cerebrospinal fluid in patients with amyotrophic lateral sclerosis Shohei Watanabe ⁎, Takashi Kimura, Koichi Suenaga, Sayoko Wada, Kenkichi Tsuda, Shuhei Kasama, Toshio Takaoka, Koji Kajiyama, Masanaka Takeda, Hiroo Yoshikawa Department of Internal Medicine, Division of Neurology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan
a r t i c l e
i n f o
Article history: Received 11 March 2009 Received in revised form 11 June 2009 Accepted 12 June 2009 Available online 12 July 2009 Keywords: Amyotrophic lateral sclerosis Chloride Cerebrospinal fluid Serum Pathogenesis Diagnosis
a b s t r a c t Recent studies have suggested that the elevation of intracellular chloride contributes to excitotoxic cell death in motor neuron and can be related to the pathogenesis of amyotrophic lateral sclerosis (ALS). We investigated whether chloride levels in cerebrospinal fluid (CSF) and serum were lower in ALS patients than in control patients with other neurological diseases (OND). We also examined the relationship between chloride levels and clinical ALS phenotypes. We measured chloride levels (CSF and serum) in 27 ALS patients and 33 age- and gender-matched OND controls admitted to our hospital for diagnosis. The CSF chloride levels were lower in ALS patients (117 [range 102–130] mmol/L) than in OND controls (126 [range 114–134] mmol/L) (P b 0.0001). However, no significant difference was found in their serum chloride levels (P N 0.05). There was no significant difference in CSF chloride levels among the sub-groups of ALS patients classified according to their age, gender, duration of illness, clinical state and type of onset (P N 0.05). CSF chloride levels already significantly decreased in ALS patients at the time of diagnosis. We conclude that the elevation of intracellular chloride would cause the reduction of chloride in CSF and be related to the pathogenesis of ALS. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons in the spinal cord, brain stem, and motor area of the cerebrum [1]. Although the precise pathogenic mechanism of ALS has not been elucidated, it is well known that glutamatemediated excitotoxicity could play an important role in the neurodegenerative process of ALS [2]. Recently, several reports have suggested that excitotoxicity during glutamate receptor stimulation in cultured neurons is accompanied by chloride (Cl−) influx through multiple pathways [3–5]. A high concentration of intracellular Cl− would be detrimental to neuronal cells [6]. Cl− influx has also been reported in relation to γ-aminobutyric acid (GABA)A receptor, which is known to function as ligand-gated Cl− channel [7]. Cl− influx through GABAA receptors or through other Cl− channels during sustained glutamate receptor stimulation would be harmful to spinal motor neurons [5]. In fact, treatment of GABAA agonist muscimol was observed to shorten the survival of ALS model mice [5]. We hypothesized that intracellular Cl− would be accumulated in the motor neurons of ALS patients in advance of the appearance of their symptoms, and investigated whether the Cl− levels in cerebrospinal fluid (CSF) and serum decreased when the patients were hospi⁎ Corresponding author. Tel.: +81 798 45 6598; fax: +81 798 45 6597. E-mail address: [email protected] (S. Watanabe). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.06.026
talized for diagnosis as ALS. We also studied a possible association between Cl− levels of CSF and clinical ALS phenotypes. 2. Materials and methods We investigated 27 ALS patients (14 male, 13 female) whose Cl− levels of CSF and serum were examined for differential diagnosis during their hospitalization in The Hospital of Hyogo College of Medicine between 2000 and 2007. Cl− levels of both CSF and serum were determined by ion-selective electrode method. The clinical diagnosis of ALS was based on El Escorial Revised Criteria [8]. None of the ALS patients were treated with riluzole or any respiratory assistance when their Cl− levels were examined. We also checked their age, gender, type of ALS onset, duration of illness, and clinical state based on the medical records of their hospitalization. The duration of illness was defined as the interval between the first signs of the disease onset and the hospitalization when their CSF and serum were examined. The clinical state was defined according to the ALS Health State Scale (ALS/ HSS) [9,10]. The ALS patients were classified into the sub-groups according to their gender, age (young–up to 65 years, old–over 65 years), type of ALS onset (Bulbar or Spinal Onset), duration of illness (short– up to 6 months, long–over 6 months), and clinical state (mild, moderate, and severe). The control group consisted of 33 age- and gender-matched patients with OND (6 Guillain–Barré syndrome, 5 chronic inflammatory demyelinating polyradiculoneuropathy, 5 multiple sclerosis, 5
S. Watanabe et al. / Journal of the Neurological Sciences 285 (2009) 146–148 Table 1 Demographic and clinical characteristics, and chloride levels of cerebrospinal fluid (CSF) and serum in amyotrophic lateral sclerosis (ALS) patients and control patients with other neurological diseases (OND). Variable
ALS patients, n = 27 OND controls, n = 33
Median age (range), years Male/Female Bulbar onset Spinal onset Median duration of illness, months Definite ALS Probable ALS⁎ Possible ALS Clinical state Mild Moderate Severe Serum chloride median (range), mmol/L CSF chloride median (range), mmol/L#
66 (46–75) 14/13 8 (30%) 19 (70%) 6 (2–35) 1 (4%) 23 (85%) 3 (11%) 4 (15%) 19 (70%) 4 (15%) 105 (85–108) 117 (102–130)
64 (46–76) 18/15
104 (100–110) 126 (114–134)
⁎Including probable laboratory-supported ALS, according to El Escorial Revised Criteria. # P b 0.0001; Mann–Whitney U-test.
147
Table 2 Chloride levels in cerebrospinal fluid (CSF), and a comparative analysis among the groups of ALS patients. Variable Sex Male (n = 14) Female (n = 13) Age Young (n = 11) Old (n = 16) Type of onset Spinal (n = 19) Bulbar (n = 8) Duration of illness Short (n = 14) Long (n = 13) Clinical state Mild (n = 4) Moderate (n = 19) Severe (n = 4)
CSF chloride median (range), mmol/L
Comparisons
116 (102–126) 118 (111–130)
Male vs. female P = 0.43
121 (102–126) 116 (111–130)
Young vs. old P = 0.23
118.5 (111–130) 117.5 (115–126)
Spinal vs. bulbar P = 0.89
118.5 (111–130) 117 (102–126)
Short vs. long P = 0.77
118.5 (115–126) 118 (102–130) 116 (111–126)
Clinical state P = 0.82
Mann–Whitney U-test, and Kruskal–Wallis test.
transverse myelitis, 3 cervical spondylosis, 2 epilepsy, 1 Alzheimer disease, 1 Creutzfeldt–Jakob disease, 1 Charcot–Marie–Tooth disease, 1 normal-pressure hydrocephalus, 1 transient global amnesia, 1 Vitamin B12 deficiency, 1 neurosis). The Cl− levels of CSF and serum of OND patients were also examined for clinical diagnosis during their hospitalization. There was no significant difference in CSF and serum Cl− levels among the patients with each of OND in this study (data not shown). The patients with other diseases known to affect Cl− concentrations, such as tuberculous meningitis [11], were excluded from this study. This retrospective study was approved by Ethics Committee of Hyogo College of Medicine, and all of the CSF and serum samples were collected after obtaining informed consent from both the ALS patients and the OND controls. For statistical analysis, we used non-parametric tests since the data were not normally distributed; Mann–Whitney Utest and Kruskal–Wallis test. The values were expressed in mmol/L. P values b 0.05 were considered significant.
and gender distribution of these characteristics were detected. The median duration of ALS was 6 months, and the ratio of bulbar-onset one was 30% of the patients. According to El Escorial Revised Criteria, approximately 90% of the patients were classified into probable (including probable laboratory supported) and definite ALS. We found that CSF Cl− levels were significantly lower in ALS patients than in control patients with OND (P b 0.0001) (Table 1 & Fig. 1). On the other hand, there was no significant difference in serum Cl− levels between the ALS patients and the OND controls (P N 0.05). In the next step, we examined the relationship between CSF Cl− levels in the ALS patients and their clinical ALS phenotype. However, no significant correlation was observed between CSF Cl− levels and their age, gender, duration of illness, clinical state, or type of ALS onset (Table 2). There was also no significant association between serum Cl− levels in the ALS patients and their clinical phenotype (age, gender, duration of illness, clinical state, or type of ALS onset) (P N 0.05, data not shown).
3. Results
4. Discussion
The clinical characteristics of the ALS and the control subjects in this study are shown in Table 1. No significant differences in the age
Our study revealed that Cl− levels of CSF were significantly lower in the ALS patients than in the OND controls. And we also found that the reduction of CSF Cl− levels was not related to the clinical ALS phenotype, including duration of illness and clinical state. This result suggests that CSF Cl− levels have already decreased when the symptoms of ALS appear. Cl− is the primary permeant anion in mammals, and the homeostasis of its flux across cell membranes is important for a range of neurophysiological processes [7]. The concentration of Cl− in neurons is very low, and thus small changes in the concentration have a large impact on the transmembrane gradient [6]. Although the role of Cl− in the pathogenesis of ALS is not fully elucidated, several reports have suggested Cl− influx into motor neurons would be related to excitotoxicity. In cultured motor neurons, sustained stimulation of glutamate receptor causes accumulation of intracellular Cl−, and the Cl− influx aggravates excitotoxic motor neuron death [5]. This report also shows that administration of GABA enhances the Cl− influx. Another report has indicated that GABAA receptors (ligand-gated Cl− channels) demonstrate higher affinity with their agonist and the mRNA and protein levels of GABAA α1 subunit increase in cultured motor neurons derived from a genetic mouse model of ALS, which can induce a higher Cl− influx [12]. These previous reports about Cl− suggest that accumulation of intracellular Cl− can participate in the pathogenesis of ALS. The ion concentration of CSF truly reflects that of the extracellular space in the central nervous
Fig. 1. Chloride levels of cerebrospinal fluid (CSF Cl−) in patients with amyotrophic lateral sclerosis (ALS) and control patients with other neurological diseases (OND).
148
S. Watanabe et al. / Journal of the Neurological Sciences 285 (2009) 146–148
system (CNS) [13]. Therefore, the decrease in CSF Cl− levels observed in our study is consistent with a higher Cl− influx indicated by these in vitro findings. The reason why CSF Cl− levels decrease in ALS patients can be related to inflammatory mechanisms. Inflammatory mediators were reported to raise intracellular Cl− levels in dorsal ganglion neurons [14]. In fact, the levels of prostaglandin E2, one of the representative inflammatory mediators, were elevated in CSF in ALS patients [15]. The reduced CSF Cl− levels in ALS patients may reflect the response of prostaglandin E2. A previous report showed that there was no significant difference in CSF Cl− levels between ALS patients and controls, although CSF Cl− levels tended to decrease in ALS patients [16]. Probably, this discrepancy may be caused by too small number (n = 7) of ALS patients in the study. Serum Cl− levels are known to be prognostic indicators of survival in ALS [17]. Lower serum Cl− is associated with a faster rate of decline in percent predicted Vital Capacity (%VC) [18]. In the later stages of ALS, progressive respiratory failure causes respiratory acidosis, which is the most common cause of death [19,20]. Chronic respiratory acidosis causes a compensatory increase in serum bicarbonate levels that leads to Cl− excretion by the kidneys and decreased serum chloride levels [18]. In our study, the serum Cl−levels in the ALS patients did not significantly decrease. The reason for this may be because we investigated the ALS patients hospitalized for first diagnosis. Many of the ALS patients in our study were in the early stage of the disease and their respiratory function could comparatively be maintained, although the precise assessment (%VC or blood gas analysis) was not done in all of the subjects. Serum Cl− levels certainly influence CSF Cl− levels, but we found that there was significant difference in only CSF Cl− levels between the ALS patients and the OND controls. This fact suggests that the change of Cl− movement in CNS is important in ALS patients. There remain several questions about CSF Cl− levels in ALS patients. What happens to CSF Cl− levels upon riluzole treatment? Can Cl− channel agonists or blockers affect CSF Cl− levels? Investigating these questions may lead to elucidating the change of Cl−movement in CNS of ALS patients. In CNS, intracellular Cl− levels are regulated, in part, by cationchloride co-transporters (CCCs). CCCs consist of the inwardly directed Na–K–2Cl co-transporter NKCC1 and various outwardly directed K–Cl co-transporters, such as KCC2 and KCC3 [7]. Because the accumulation of intracellular Cl− and the Cl− influx were reported to aggravate excitotoxic motor neuron death [5], the control of CCCs for lowering the intracellular Cl− may lead to a new therapeutic approach for ALS. There are several limitations in our study. First, the size of our study is relatively small and our results may be accidental findings. Larger prospective studies are indispensable to ascertain our observations. However, our findings should not be overlooked, because they are consistent with several in vitro findings about the role of Cl− in excitotoxicity. Second, we did not compare ALS patients with normal controls, but with OND controls. Because lumber puncture is an invasive maneuver, it was difficult to examine CSF Cl− levels in normal controls. Although it is essential to investigate normal controls, CSF Cl− levels may be helpful for differential diagnosis in the future. Third, our
study is a retrospective study. Prospective studies are necessary to confirm whether CSF Cl− levels in ALS patients are prognostic factors. In summary, we have found that CSF Cl− levels significantly decreased in ALS patients. The measurement of CSF Cl− levels might be a useful method for diagnosis of ALS in the future, and the regulation of Cl− levels in CNS may be a therapeutic target for ALS. Acknowledgement We thank Dr. Seiichi Nagano (Department of Neurology, Osaka University Graduate School of Medicine) for his valuable comments in the preparation of the manuscript. References [1] Rowland LP, Shneider NA. Medical progress: amyotrophic lateral sclerosis. N Engl J Med 2001;344(22):1688–700. [2] Heath PR, Shaw PJ. Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis. Muscle Nerve 2002;26(4): 438–58. [3] Chen Q, Moulder K, Tenkova T, Hardy K, Olney JW, Romano C. Excitotoxic cell death dependent an inhibitory receptor activation. Exp Neurol 1999;160(1):215–25. [4] Beck J, Lenart B, Kintner DB, Sun D. Na–K–Cl cotransporter contributes to glutamatemediated excitotoxicity. J Neurosci 2003;23(12):5061–8. [5] Van Damme P, Callewaert G, Eggermont J, Robberecht W, Van Den Bosch L. Chloride influx aggravates Ca2+-dependent AMPA receptor-mediated motoneuron death. J Neurosci 2003;23(12):4942–50. [6] De Koninck Y. Altered chloride homeostasis in neurological disorders: a new target. Curr Opin Pharmacol 2007;7(1):93–9. [7] Kahle KT, Staley KJ, Nahed BV, Gamba G, Hebert SC, Lifton RP, et al. Roles of the cationchloride cotransporters in neurological disease. Nat Clin Pract Neurol 2008;4(9): 490–503. [8] Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2000;1(5):293–9. [9] Riviere M, Meininger V, Zeisser P, Munsat T. An analysis of extended survival in patients with amyotrophic lateral sclerosis treated with riluzole. Arch Neurol 1998;55(4): 526–8. [10] Ilzecka J, Stelmasiak Z. Serum bilirubin concentration in patients with amyotrophic lateral sclerosis. Clin Neurol Neurosurg 2003;105(4):237–40. [11] Jaffe IP. Tuberculous meningitis in childhood. Lancet 1982;1(8274):738. [12] Carunchio I, Mollinari C, Pieri M, Merlo D, Zona C. GABA(A) receptors present higher affinity and modified subunit composition in spinal motor neurons from a genetic model of amyotrophic lateral sclerosis. Eur J Neurosci 2008;28(7):1275–85. [13] Plum F, Siesjo BK. Recent advances in CSF physiology. Anesthesiology 1975;42(6): 708–30. [14] Funk K, Woitecki A, Franjic-Wurtz C, Gensch T, Mohrlen F, Frings S. Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons. Mol Pain 2008;32(4). [15] Almer G, Teismann P, Stevic Z, Halaschek-Wiener J, Deecke L, Kostic V, et al. Increased levels of the pro-inflammatory prostaglandin PGE2 in CSF from ALS patients. Neurology 2002;58(8):1277–9. [16] Csenker E, Dioszeghy P, Fekete I, Mechler F. Ion concentrations in serum and cerebrospinal fluid of patients with neuromuscular diseases. Arch Psychiatr Nervenkr 1982;231(3):251–8. [17] Stambler N, Charatan M, Cedarbaum JM, Grp ACTS. Prognostic indicators of survival in ALS. Neurology 1998;50(1):66–72. [18] Qureshi M, Shui A, Dibernardo AB, Brown Jr RH, Schoenfeld DA, Cudkowicz ME. Medications and laboratory parameters as prognostic factors in amyotrophic lateral sclerosis. Amyotroph Lateral Scler 2008;9(6):369–74. [19] Traynor BJ, Zhang H, Shefner JM, Schoenfeld D, Cudkowicz ME, Consortium N. Functional outcome measures as clinical trial endpoints in ALS. Neurology 2004;63 (10):1933–5. [20] Caroscio JT, Mulvihill MN, Sterling R, Abrams B. Amyotrophic lateral sclerosis. Its natural history. Neurol Clin 1987;5(1):1–8.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Decreased chloride levels of cerebrospinal fluid in patients with amyotrophic lateral sclerosis Shohei Watanabe ⁎, Takashi Kimura, Koichi Suenaga, Sayoko Wada, Kenkichi Tsuda, Shuhei Kasama, Toshio Takaoka, Koji Kajiyama, Masanaka Takeda, Hiroo Yoshikawa Department of Internal Medicine, Division of Neurology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan
a r t i c l e
i n f o
Article history: Received 11 March 2009 Received in revised form 11 June 2009 Accepted 12 June 2009 Available online 12 July 2009 Keywords: Amyotrophic lateral sclerosis Chloride Cerebrospinal fluid Serum Pathogenesis Diagnosis
a b s t r a c t Recent studies have suggested that the elevation of intracellular chloride contributes to excitotoxic cell death in motor neuron and can be related to the pathogenesis of amyotrophic lateral sclerosis (ALS). We investigated whether chloride levels in cerebrospinal fluid (CSF) and serum were lower in ALS patients than in control patients with other neurological diseases (OND). We also examined the relationship between chloride levels and clinical ALS phenotypes. We measured chloride levels (CSF and serum) in 27 ALS patients and 33 age- and gender-matched OND controls admitted to our hospital for diagnosis. The CSF chloride levels were lower in ALS patients (117 [range 102–130] mmol/L) than in OND controls (126 [range 114–134] mmol/L) (P b 0.0001). However, no significant difference was found in their serum chloride levels (P N 0.05). There was no significant difference in CSF chloride levels among the sub-groups of ALS patients classified according to their age, gender, duration of illness, clinical state and type of onset (P N 0.05). CSF chloride levels already significantly decreased in ALS patients at the time of diagnosis. We conclude that the elevation of intracellular chloride would cause the reduction of chloride in CSF and be related to the pathogenesis of ALS. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons in the spinal cord, brain stem, and motor area of the cerebrum [1]. Although the precise pathogenic mechanism of ALS has not been elucidated, it is well known that glutamatemediated excitotoxicity could play an important role in the neurodegenerative process of ALS [2]. Recently, several reports have suggested that excitotoxicity during glutamate receptor stimulation in cultured neurons is accompanied by chloride (Cl−) influx through multiple pathways [3–5]. A high concentration of intracellular Cl− would be detrimental to neuronal cells [6]. Cl− influx has also been reported in relation to γ-aminobutyric acid (GABA)A receptor, which is known to function as ligand-gated Cl− channel [7]. Cl− influx through GABAA receptors or through other Cl− channels during sustained glutamate receptor stimulation would be harmful to spinal motor neurons [5]. In fact, treatment of GABAA agonist muscimol was observed to shorten the survival of ALS model mice [5]. We hypothesized that intracellular Cl− would be accumulated in the motor neurons of ALS patients in advance of the appearance of their symptoms, and investigated whether the Cl− levels in cerebrospinal fluid (CSF) and serum decreased when the patients were hospi⁎ Corresponding author. Tel.: +81 798 45 6598; fax: +81 798 45 6597. E-mail address: [email protected] (S. Watanabe). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.06.026
talized for diagnosis as ALS. We also studied a possible association between Cl− levels of CSF and clinical ALS phenotypes. 2. Materials and methods We investigated 27 ALS patients (14 male, 13 female) whose Cl− levels of CSF and serum were examined for differential diagnosis during their hospitalization in The Hospital of Hyogo College of Medicine between 2000 and 2007. Cl− levels of both CSF and serum were determined by ion-selective electrode method. The clinical diagnosis of ALS was based on El Escorial Revised Criteria [8]. None of the ALS patients were treated with riluzole or any respiratory assistance when their Cl− levels were examined. We also checked their age, gender, type of ALS onset, duration of illness, and clinical state based on the medical records of their hospitalization. The duration of illness was defined as the interval between the first signs of the disease onset and the hospitalization when their CSF and serum were examined. The clinical state was defined according to the ALS Health State Scale (ALS/ HSS) [9,10]. The ALS patients were classified into the sub-groups according to their gender, age (young–up to 65 years, old–over 65 years), type of ALS onset (Bulbar or Spinal Onset), duration of illness (short– up to 6 months, long–over 6 months), and clinical state (mild, moderate, and severe). The control group consisted of 33 age- and gender-matched patients with OND (6 Guillain–Barré syndrome, 5 chronic inflammatory demyelinating polyradiculoneuropathy, 5 multiple sclerosis, 5
S. Watanabe et al. / Journal of the Neurological Sciences 285 (2009) 146–148 Table 1 Demographic and clinical characteristics, and chloride levels of cerebrospinal fluid (CSF) and serum in amyotrophic lateral sclerosis (ALS) patients and control patients with other neurological diseases (OND). Variable
ALS patients, n = 27 OND controls, n = 33
Median age (range), years Male/Female Bulbar onset Spinal onset Median duration of illness, months Definite ALS Probable ALS⁎ Possible ALS Clinical state Mild Moderate Severe Serum chloride median (range), mmol/L CSF chloride median (range), mmol/L#
66 (46–75) 14/13 8 (30%) 19 (70%) 6 (2–35) 1 (4%) 23 (85%) 3 (11%) 4 (15%) 19 (70%) 4 (15%) 105 (85–108) 117 (102–130)
64 (46–76) 18/15
104 (100–110) 126 (114–134)
⁎Including probable laboratory-supported ALS, according to El Escorial Revised Criteria. # P b 0.0001; Mann–Whitney U-test.
147
Table 2 Chloride levels in cerebrospinal fluid (CSF), and a comparative analysis among the groups of ALS patients. Variable Sex Male (n = 14) Female (n = 13) Age Young (n = 11) Old (n = 16) Type of onset Spinal (n = 19) Bulbar (n = 8) Duration of illness Short (n = 14) Long (n = 13) Clinical state Mild (n = 4) Moderate (n = 19) Severe (n = 4)
CSF chloride median (range), mmol/L
Comparisons
116 (102–126) 118 (111–130)
Male vs. female P = 0.43
121 (102–126) 116 (111–130)
Young vs. old P = 0.23
118.5 (111–130) 117.5 (115–126)
Spinal vs. bulbar P = 0.89
118.5 (111–130) 117 (102–126)
Short vs. long P = 0.77
118.5 (115–126) 118 (102–130) 116 (111–126)
Clinical state P = 0.82
Mann–Whitney U-test, and Kruskal–Wallis test.
transverse myelitis, 3 cervical spondylosis, 2 epilepsy, 1 Alzheimer disease, 1 Creutzfeldt–Jakob disease, 1 Charcot–Marie–Tooth disease, 1 normal-pressure hydrocephalus, 1 transient global amnesia, 1 Vitamin B12 deficiency, 1 neurosis). The Cl− levels of CSF and serum of OND patients were also examined for clinical diagnosis during their hospitalization. There was no significant difference in CSF and serum Cl− levels among the patients with each of OND in this study (data not shown). The patients with other diseases known to affect Cl− concentrations, such as tuberculous meningitis [11], were excluded from this study. This retrospective study was approved by Ethics Committee of Hyogo College of Medicine, and all of the CSF and serum samples were collected after obtaining informed consent from both the ALS patients and the OND controls. For statistical analysis, we used non-parametric tests since the data were not normally distributed; Mann–Whitney Utest and Kruskal–Wallis test. The values were expressed in mmol/L. P values b 0.05 were considered significant.
and gender distribution of these characteristics were detected. The median duration of ALS was 6 months, and the ratio of bulbar-onset one was 30% of the patients. According to El Escorial Revised Criteria, approximately 90% of the patients were classified into probable (including probable laboratory supported) and definite ALS. We found that CSF Cl− levels were significantly lower in ALS patients than in control patients with OND (P b 0.0001) (Table 1 & Fig. 1). On the other hand, there was no significant difference in serum Cl− levels between the ALS patients and the OND controls (P N 0.05). In the next step, we examined the relationship between CSF Cl− levels in the ALS patients and their clinical ALS phenotype. However, no significant correlation was observed between CSF Cl− levels and their age, gender, duration of illness, clinical state, or type of ALS onset (Table 2). There was also no significant association between serum Cl− levels in the ALS patients and their clinical phenotype (age, gender, duration of illness, clinical state, or type of ALS onset) (P N 0.05, data not shown).
3. Results
4. Discussion
The clinical characteristics of the ALS and the control subjects in this study are shown in Table 1. No significant differences in the age
Our study revealed that Cl− levels of CSF were significantly lower in the ALS patients than in the OND controls. And we also found that the reduction of CSF Cl− levels was not related to the clinical ALS phenotype, including duration of illness and clinical state. This result suggests that CSF Cl− levels have already decreased when the symptoms of ALS appear. Cl− is the primary permeant anion in mammals, and the homeostasis of its flux across cell membranes is important for a range of neurophysiological processes [7]. The concentration of Cl− in neurons is very low, and thus small changes in the concentration have a large impact on the transmembrane gradient [6]. Although the role of Cl− in the pathogenesis of ALS is not fully elucidated, several reports have suggested Cl− influx into motor neurons would be related to excitotoxicity. In cultured motor neurons, sustained stimulation of glutamate receptor causes accumulation of intracellular Cl−, and the Cl− influx aggravates excitotoxic motor neuron death [5]. This report also shows that administration of GABA enhances the Cl− influx. Another report has indicated that GABAA receptors (ligand-gated Cl− channels) demonstrate higher affinity with their agonist and the mRNA and protein levels of GABAA α1 subunit increase in cultured motor neurons derived from a genetic mouse model of ALS, which can induce a higher Cl− influx [12]. These previous reports about Cl− suggest that accumulation of intracellular Cl− can participate in the pathogenesis of ALS. The ion concentration of CSF truly reflects that of the extracellular space in the central nervous
Fig. 1. Chloride levels of cerebrospinal fluid (CSF Cl−) in patients with amyotrophic lateral sclerosis (ALS) and control patients with other neurological diseases (OND).
148
S. Watanabe et al. / Journal of the Neurological Sciences 285 (2009) 146–148
system (CNS) [13]. Therefore, the decrease in CSF Cl− levels observed in our study is consistent with a higher Cl− influx indicated by these in vitro findings. The reason why CSF Cl− levels decrease in ALS patients can be related to inflammatory mechanisms. Inflammatory mediators were reported to raise intracellular Cl− levels in dorsal ganglion neurons [14]. In fact, the levels of prostaglandin E2, one of the representative inflammatory mediators, were elevated in CSF in ALS patients [15]. The reduced CSF Cl− levels in ALS patients may reflect the response of prostaglandin E2. A previous report showed that there was no significant difference in CSF Cl− levels between ALS patients and controls, although CSF Cl− levels tended to decrease in ALS patients [16]. Probably, this discrepancy may be caused by too small number (n = 7) of ALS patients in the study. Serum Cl− levels are known to be prognostic indicators of survival in ALS [17]. Lower serum Cl− is associated with a faster rate of decline in percent predicted Vital Capacity (%VC) [18]. In the later stages of ALS, progressive respiratory failure causes respiratory acidosis, which is the most common cause of death [19,20]. Chronic respiratory acidosis causes a compensatory increase in serum bicarbonate levels that leads to Cl− excretion by the kidneys and decreased serum chloride levels [18]. In our study, the serum Cl−levels in the ALS patients did not significantly decrease. The reason for this may be because we investigated the ALS patients hospitalized for first diagnosis. Many of the ALS patients in our study were in the early stage of the disease and their respiratory function could comparatively be maintained, although the precise assessment (%VC or blood gas analysis) was not done in all of the subjects. Serum Cl− levels certainly influence CSF Cl− levels, but we found that there was significant difference in only CSF Cl− levels between the ALS patients and the OND controls. This fact suggests that the change of Cl− movement in CNS is important in ALS patients. There remain several questions about CSF Cl− levels in ALS patients. What happens to CSF Cl− levels upon riluzole treatment? Can Cl− channel agonists or blockers affect CSF Cl− levels? Investigating these questions may lead to elucidating the change of Cl−movement in CNS of ALS patients. In CNS, intracellular Cl− levels are regulated, in part, by cationchloride co-transporters (CCCs). CCCs consist of the inwardly directed Na–K–2Cl co-transporter NKCC1 and various outwardly directed K–Cl co-transporters, such as KCC2 and KCC3 [7]. Because the accumulation of intracellular Cl− and the Cl− influx were reported to aggravate excitotoxic motor neuron death [5], the control of CCCs for lowering the intracellular Cl− may lead to a new therapeutic approach for ALS. There are several limitations in our study. First, the size of our study is relatively small and our results may be accidental findings. Larger prospective studies are indispensable to ascertain our observations. However, our findings should not be overlooked, because they are consistent with several in vitro findings about the role of Cl− in excitotoxicity. Second, we did not compare ALS patients with normal controls, but with OND controls. Because lumber puncture is an invasive maneuver, it was difficult to examine CSF Cl− levels in normal controls. Although it is essential to investigate normal controls, CSF Cl− levels may be helpful for differential diagnosis in the future. Third, our
study is a retrospective study. Prospective studies are necessary to confirm whether CSF Cl− levels in ALS patients are prognostic factors. In summary, we have found that CSF Cl− levels significantly decreased in ALS patients. The measurement of CSF Cl− levels might be a useful method for diagnosis of ALS in the future, and the regulation of Cl− levels in CNS may be a therapeutic target for ALS. Acknowledgement We thank Dr. Seiichi Nagano (Department of Neurology, Osaka University Graduate School of Medicine) for his valuable comments in the preparation of the manuscript. References [1] Rowland LP, Shneider NA. Medical progress: amyotrophic lateral sclerosis. N Engl J Med 2001;344(22):1688–700. [2] Heath PR, Shaw PJ. Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis. Muscle Nerve 2002;26(4): 438–58. [3] Chen Q, Moulder K, Tenkova T, Hardy K, Olney JW, Romano C. Excitotoxic cell death dependent an inhibitory receptor activation. Exp Neurol 1999;160(1):215–25. [4] Beck J, Lenart B, Kintner DB, Sun D. Na–K–Cl cotransporter contributes to glutamatemediated excitotoxicity. J Neurosci 2003;23(12):5061–8. [5] Van Damme P, Callewaert G, Eggermont J, Robberecht W, Van Den Bosch L. Chloride influx aggravates Ca2+-dependent AMPA receptor-mediated motoneuron death. J Neurosci 2003;23(12):4942–50. [6] De Koninck Y. Altered chloride homeostasis in neurological disorders: a new target. Curr Opin Pharmacol 2007;7(1):93–9. [7] Kahle KT, Staley KJ, Nahed BV, Gamba G, Hebert SC, Lifton RP, et al. Roles of the cationchloride cotransporters in neurological disease. Nat Clin Pract Neurol 2008;4(9): 490–503. [8] Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2000;1(5):293–9. [9] Riviere M, Meininger V, Zeisser P, Munsat T. An analysis of extended survival in patients with amyotrophic lateral sclerosis treated with riluzole. Arch Neurol 1998;55(4): 526–8. [10] Ilzecka J, Stelmasiak Z. Serum bilirubin concentration in patients with amyotrophic lateral sclerosis. Clin Neurol Neurosurg 2003;105(4):237–40. [11] Jaffe IP. Tuberculous meningitis in childhood. Lancet 1982;1(8274):738. [12] Carunchio I, Mollinari C, Pieri M, Merlo D, Zona C. GABA(A) receptors present higher affinity and modified subunit composition in spinal motor neurons from a genetic model of amyotrophic lateral sclerosis. Eur J Neurosci 2008;28(7):1275–85. [13] Plum F, Siesjo BK. Recent advances in CSF physiology. Anesthesiology 1975;42(6): 708–30. [14] Funk K, Woitecki A, Franjic-Wurtz C, Gensch T, Mohrlen F, Frings S. Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons. Mol Pain 2008;32(4). [15] Almer G, Teismann P, Stevic Z, Halaschek-Wiener J, Deecke L, Kostic V, et al. Increased levels of the pro-inflammatory prostaglandin PGE2 in CSF from ALS patients. Neurology 2002;58(8):1277–9. [16] Csenker E, Dioszeghy P, Fekete I, Mechler F. Ion concentrations in serum and cerebrospinal fluid of patients with neuromuscular diseases. Arch Psychiatr Nervenkr 1982;231(3):251–8. [17] Stambler N, Charatan M, Cedarbaum JM, Grp ACTS. Prognostic indicators of survival in ALS. Neurology 1998;50(1):66–72. [18] Qureshi M, Shui A, Dibernardo AB, Brown Jr RH, Schoenfeld DA, Cudkowicz ME. Medications and laboratory parameters as prognostic factors in amyotrophic lateral sclerosis. Amyotroph Lateral Scler 2008;9(6):369–74. [19] Traynor BJ, Zhang H, Shefner JM, Schoenfeld D, Cudkowicz ME, Consortium N. Functional outcome measures as clinical trial endpoints in ALS. Neurology 2004;63 (10):1933–5. [20] Caroscio JT, Mulvihill MN, Sterling R, Abrams B. Amyotrophic lateral sclerosis. Its natural history. Neurol Clin 1987;5(1):1–8.