We have examined brainstem lesions in patients with refractory epilepsy disorders, including West syndrome (WS), Lennox-Gastaut syndrome (LGS), and dentatorubral-pallidoluysian atrophy (DRPLA). Acetylcholinergic neurons (AchNs) in the pedunculopontine tegmental nucleus (PPN) are involved in mental development, and disruption of neuronal nicotinic acetylcholine receptors can lead to epilepsy. In order to investigate the involvement of lesions of AchNs in refractory epilepsy, we performed immunohistochemical analyses of AchNs in the PPN in autopsy cases who had a past history of WS and/or LGS and in DRPLA cases who showed progressive myoclonic epilepsy. In addition, we performed a preliminary quantification of the levels of acetylcholine, neuropeptides, and monoamine metabolites in the cerebrospinal fluid (CSF) of patients with WS and benign convulsions associated with mild gastroenteritis (CwG). In the PPN analysis, the total number of neurons and the number of AchNs were reduced in WS/LGS and WS cases, while DRPLA cases showed a decrease in the number and percentage of AchNs. In the CSF analysis, WS patients demonstrated a reduction in the levels of inhibitory neuropeptides, while CwG patients showed increased levels of acetylcholine and decreased levels of serotonin metabolites. These data suggest the possible involvement of lesions of AchNs in WS and DRPLA.
West syndrome (WS), which consists of tonic spasms, psychomotor developmental delay, and characteristic electroencephalography changes involving hypsarrhythmia, can have various causes, including congenital brain anomalies and sequelae after perinatal hypoxic and ischemic encephalopathy (HIE) [
Acetylcholinergic neurons (AchNs) in the nucleus basalis of Meynert (NbM) and the pedunculopontine tegmental nucleus (PPN) are involved in mental development and learning abilities [
In order to investigate the involvement of the acetylcholinergic neuronal system in epileptogenesis, we performed an immunohistochemical analysis of AchNs, CANs, and GABAis in the PPN in autopsy cases with a history of WS and/or LGS and DRPLA. In addition, we preliminarily evaluated the CSF levels of acetylcholine, neuropeptides, and monoamine metabolites in patients with WS and benign convulsions associated with mild gastroenteritis (CwG) (disease controls).
The clinical subjects included 6 cases with a history of both WS and LGS (WS/LGS cases), 6 cases with a history of only WS (WS cases), and 7 cases with clinically and genetically confirmed juvenile and early adult types of DRPLA showing PME in addition to 6 controls with no pathological changes in the central nervous system; subjects were aged from 4 months to 40 years (Table
Summary of the subjects in the pathological analysis.
Age/sex | Cause of death |
---|---|
Control | |
4 m/male | Peritonitis |
1 y/male | Malignant lymphoma |
6 y/male | Acute leukemia |
22 y/male | DIC |
29 y/male | Migraine |
38 y/female | Heart failure |
| |
Etiology | |
| |
West syndrome/Lennox-Gastaut syndrome | |
5 y/female | Lissencephaly |
8 y/male | Lissencephaly |
10 y/female | Lissencephaly |
13 y/male | Perinatal HIE |
18 y/male | Perinatal HIE |
21 y/male | Lissencephaly |
West syndrome only | |
5 y/female |
Microdysgenesis |
13 y/female |
Microdysgenesis |
16 y/male |
Heterotopia |
20 y/male |
Perinatal HIE |
24 y/male |
Porencephaly |
26 y/Male | Microdysgenesis |
| |
Disease type | |
| |
Dentatorubral-pallidoluysian atrophy | |
16 y/female | Juvenile |
17 y/male | Juvenile |
21 y/male | Juvenile |
24 y/male | Juvenile |
27 y/male | Juvenile |
39 y/female | Early adult |
40 y/male | Early adult |
m: months; y: years; DIC: disseminated intravascular coagulation; HIE: hypoxic ischemic encephalopathy.
Brains were fixed in a buffered formalin solution. Each formalin-fixed brain was cut coronally and then embedded in paraffin. Six
The PPN was identified dorsolateral to the rostral superior cerebellar peduncle and the medial lemniscus in the lower midbrain, as previously reported [
Clinical subjects included 3 infants with clinically and electrophysiologically confirmed WS, 5 patients with CwG, and 5 normal controls with acute viral infections who lacked neurological abnormalities at the follow-up examination; subjects were aged from 1 month to 2 years (Table
Summary of the subjects in the analysis of cerebrospinal fluid.
Age/sex | Etiology |
---|---|
Control |
|
1 m/female |
Upper respiratory infection |
1 m/female |
Upper respiratory infection |
2 m/male |
Viral gastroenteritis |
2 m/male |
Viral gastroenteritis |
6 m/female | Upper respiratory infection |
Benign convulsion with mild gastroenteritis | |
8 m/male |
Viral gastroenteritis |
1 y/female | Viral gastroenteritis |
2 y/male |
Viral gastroenteritis |
2 y/male |
Viral gastroenteritis |
2 y/male |
Viral gastroenteritis |
| |
Disease type prognosis | |
| |
West syndrome |
|
5 m/female |
Cryptogenic |
Normal development |
|
6 m/female |
Symptomatic |
Normal development |
|
8 m/female |
Cryptogenic |
Delayed development |
m: months; y: years.
Concentrations of acetylcholine, neuropeptides, including aspartate, glutamate, glycine, and GABA, and monoamine metabolites, such as 3-methoxy-4-hydroxyphenylethylglycol (MHPG), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), in the CSF were determined with high performance liquid chromatography (ECD-300, Eicom Corporation, Kyoto, Japan). All results are presented as the mean (SD), and Kruskal-Wallis tests were used to analyze the differences in the data among controls and patients with WS and CwG.
The numbers of MAP2-immunoreactive neurons and AchNs immunoreactive for AchE were reduced significantly in WS/LGS and WS cases (Table
Results of the quantitative analysis in the pedunculopontine tegmental nucleus in cases with a history of West syndrome.
Age | MAP2 | AchE | %AchE | TH | %TH | CD | %CD | |
---|---|---|---|---|---|---|---|---|
Controls ( |
4 m–38 y |
243 (41) |
68 (16) |
29 (9) |
25 (13) |
10 (5) |
27 (17) |
11 (6) |
WS/LGS ( |
5–18 y |
181 (39) |
12 (15) |
8 (12) |
35 (10) |
19 (5) |
18 (20) |
9 (10) |
WS ( |
5–26 y |
184 (38) |
29 (39) |
13 (18) |
27 (18) |
16 (10) |
31 (14) |
17 (8) |
Kruskal-Wallis |
|
|
“WS/LGS” and “WS” denote cases with a history of West syndrome (WS) and Lennox-Gastaut syndrome (LGS) and cases with a history of only WS, respectively. All results are presented as the mean (SD), and both the total number of neurons that were immunoreactive for each marker and the percentage of neurons immunoreactive for each marker relative to the total neurons are shown. m: months; y: years; MAP2: microtubule-associated protein 2; AchE: acetylcholinesterase; TH: tyrosine hydroxylase; CD: calbindin-D28K.
Representative illustrations of immunohistochemistry in the pedunculopontine tegmental nucleus. Photographs (a) and (c) are from a 22-year-old control who died of disseminated intravascular coagulation syndrome. Photographs (b) and (d) are from a 21-year-old case of lissencephaly with a history of West syndrome and Lennox-Gastaut syndrome. In the immunohistochemistry for acetylcholinesterase, the control showed many immunoreactive neurons (a), which were reduced in case (b). In the immunohistochemistry for tyrosine hydroxylase, immunoreactive neurons with neuronal processes were identified commonly in the control (c) and the case (d). Bars = 100
Data of the quantitative analysis in the pedunculopontine tegmental nucleus of cases with dentatorubral-pallidoluysian atrophy (DRPLA). Open and closed columns denote the numbers and percentages of each marker in controls and DRPLA cases, respectively. M: months; y: years; MAP2: microtubule-associated protein 2; AchE: acetylcholinesterase; TH: tyrosine hydroxylase; CD: calbindin-D28K.
WS patients demonstrated a significant reduction in the levels of inhibitory neuropeptides (glycine and GABA) but not of those of acetylcholine, excitatory amino acids, or monoamine metabolites (Table
Summary of the data in the analysis of the cerebrospinal fluid.
Acetylcholine |
Aspartate |
Glutamate |
Glycine |
GABA |
MHPG |
HVA |
5-HIAA |
||
---|---|---|---|---|---|---|---|---|---|
Age | (fmol) | (pmol) | (pmol) | (pmol) | (pg/ |
(pg/ |
(pg/ |
(pg/ | |
Controls ( |
0–6 m |
1.8 (1.2) |
305 (179) |
94 (39) |
477 (214) |
44 (13) |
46 (33) |
204 (133) |
912 (520) |
West syndrome ( |
5–8 m |
5.8 (4.3) |
81 (25) |
37 (7) |
137 (49) |
22 (7) |
77 (24) |
203 (64) |
698 (225) |
CwG ( |
8 m–2 y |
231 (199) |
223 (108) |
116 (50) |
408 (105) |
70 (27) |
43 (12) |
86 (21) |
36 (13) |
Kruskal-Wallis |
|
|
|
|
All results are presented as the mean (SD). m: months; y: years; MHPG: 3-methoxy-4-hydroxyphenylethylglycol; HVA: homovanillic acid; 5-HIAA: 5-hydroxyindoleacetic acid; CwG: benign convulsions associated with mild gastroenteritis.
Mutations in the neuronal nAChR subunit genes
The cases of WS/LGS and WS had a reduction of AchNs in addition to decreases in total neurons in the PPN (Table
The Prader-Willi syndrome case showed a reduction of AchNs in the PPN but not in the NbM [
The CSF levels of neurotransmitters, neuropeptides, and their metabolites have been examined in patients with refractory epilepsy, for example, WS patients tend to show changes that are related to an impairment of serotonergic neurons and GABAis [
In our preliminary CSF analysis, we examined 3 WS infants just before treatment and CwG children as disease controls, but not patients with DRPLA, in which the CSF samples were not acquired. It is interesting that 3 WS patients demonstrated changes in the CSF levels of glycine and GABA, whereas there were no significant differences in those of excitatory neuropeptides, acetylcholine, and monoamine metabolites (Table
In conclusion, the pathological study and preliminary CSF analysis suggested the possible involvement of lesions of AchNs in the pathogenesis of WS/LGS, DRPLA, and CwG. The selective reduction of AchNs was verified in the PPN in WS and DRPLA. In order to reach a definite conclusion, we should perform similar immunohistochemical examinations in cases with PME other than DRPLA and should expand the number of patients in the CSF analysis.
None of the authors has any conflicts of interests to disclose.