| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Introduction : It was reported that histamine neurotransmission is decreased in hypocretin deficient narcoleptic subjects. Thus, impaired histaminergic neurotransmission may be an important pathophysiological mechanism in narcolepsy and other sleep disorders. In the current study, we have measured CSF histamine and hypocretin (orexin) content in patients with narcolepsy with and without cataplexy, idiopathic hypersomnia (IHS), recurrent hypersomnia (RH), obstructive sleep apnea syndrome (OSAS) and in the controls to examine whether histaminergic neurotransmissions are altered in these sleep disorders.
Methods : Fifty narcolepsy, 45 IHS, 18 OSAS and 69 neurological control subjects were included. Narcoleptic subjects include both with and without cataplexy. Thirty five narcolepsy were shown to have very low CSF hypocretin levels (mean+/-SEM: 43+/-1.8pg/ml), while hypocretin levels in 15 narcolepsy (242+1-11pg/ml) were in the same range of normal controls (289+/-11pg/ml), and thus defined
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as a non-deficient narcolepsy. CSF samples, collected between 9:00 am to 5:00 p.m., were frozen (-80C) immediately and kept until the measurements were done. Hypocretin-1 was measured in the unextracted CSF samples using commercially available radioimmunoassay kits (Phoenix Pharmaceuticals, Belmont, CA), as previously reported. Histamine concentrations in the CSF were detected by a HPLC(ECD-300, Eicom, Kyoto)-fluorescence spectromonitor (L7485, Hitachi ; Ex:355, Em:450 nm) system ; The CSF samples were separated in a reversed-phase separation column (SC-5ODS, Eicom) using mobile phase of 5 % methanol in 0.16 M KH2PO4 containing 0.8 mM sodium octanesulfonate (pH 4.5) at a flow rate 0.3 ml/ min. Then eluate was subjected to the postcolumn derivatization with mixture of 2.5 M NaOH (0.06 ml/min) and 0.05 % OPA (0.06 ml/min) in a reaction coil (5 m x 0.25 mm I.D.) at 42 degree C, then added 2 M H3PO4(0.13 ml/min) to stop the reaction. The concentrations of histamine were quantified with a PowerChrom (AD Instruments, Australia), using pure histamine standards (Sigma). The detection limit was 10 pg (50 fmol). The hypocretin levels of other sleep disorders (IHS: 240+/-14 pg/ml, OSAS: 265+/-26 pg/ml) were as same as those with neurological controls (289+/-11 pg/ml). Histamine measurements were done with a fluorometric HPLC. All measurement was done blindly, and each CSF sample was measured twice, and the mean values of two measurements are reported. For statistical analysis, ANOVA with Donferroni/Dunn were used. The statistical significance was set to <0.003.
Results : We found significantly lower histamine levels in narcoleptic subjects with hypocretin-deficient (197+/-40pg/ml, p=0.0001), without hypocretin-deficient (193+/-57pg/ml, p=0.002) and IHS (144+/-35pg/ml, p=0.0002) compered to neurological controls (409+/-31pg/ml). Histamine levels in patients with OSAS (382+/-59pg/ml, p=0.66) were as same as those in neurological controls. CSF histamine values were not significantly correlated with hypocretin levels and gender, age and status of the medication.
Conclusions : Similar to findings obtained by Nishino et al., histamine neurotransmission is decreased in hypocretin deficient narcoleptic subjects. In addition, we found that the histamine levels in the patients with narcolepsy without hypocretin-deficient and IHS were also significantly decreased. In the previous report, histamine levels in narcolepsy without hypocretin-deficient were relatively low but did not reach statistical significance levels. The discrepancy of these results should be further studied. However our results indicated that the hypersomnia expressed by the patients with narcolepsy and IHS are explained by the low histamine levels. On the other hand, the hypersomnia expressed by the patients with OSAS may have another mechanism. Less
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