Akademik Veri Yönetim Sistemi
36th National Congress of Neurology, İstanbul, Türkiye, 01 Kasım 2000, ss.73, (Özet Bildiri)
INTRODUCTION:
Morvan’s syndrome (Ms) is a rare disease
characterised by neuromyotonia (spontaneous muscular activity resulting from
repetitive motor unit action potentials of peripheral origin), dysfunction of
autonomic nervous system and central nervous system (CNS) involvement (1). The
cardinal symptoms of autonomic and central nervous system dysfunction are hyperhidrosis
and insomnia, hallucinosis, respectively (2). Some cases with Ms have been
suggested to have an autoimmune origin, depending on associated findings such
as concurrent myasthenia gravis and other autoimmune disorders, thymoma, serum
antibodies to acetylcholine receptors, titin, N-type calcium channels and
voltage gated potassium channels, resolution of symptoms by plasmapheresis,
thymectomy or immunosuppression (1,3).
Ms cases with insomnia have rarely been
investigated with polysomnographic studies (4,5) and anatomical structures
involved in pathophysiology of sleep disorder have not been analysed in detail.
Other reported major clinical syndromes that can manifest with organic insomnia
are vascular, traumatic or degenerative lesions of the brainstem, bilateral
stereotatic thalamic injury, trypanosomiasis, Von Economo encephalitis and
fatal familial insomnia (5). Lesions of raphe, thalamic or hypothalamic nuclei
seem to play an important role in these clinical situations (5).
There are prominent differences between
sleep-wake cycles of adults and newborns. The newborn infant spends three
quarters of his time asleep, while at adulthood sleep occupies only one third
of all the day. The development of sleep and wakefulness is continuous and
follows a progressive pattern: first rapid in the perinatal period and until
one year of age, then much slower until puberty. Main steps in the ontogenetic
development of sleep-wake cycle are the building up of a circadian sleep-wake
rhythm, the lengthening of the daytime awakening period, the reduction of REM
sleep time and the relative increase of non-REM sleep duration. This
ontogenetic process may be observed in almost all mammals (6).
The aim of this study was to identify the anatomical
features underlying these differences from an immunological point of view. For
this purpose, serum of a 76-year old male patient presenting with
neuromyotonia, excessive sweating, salivation, insomnia (Morvan’s syndrome) and
pulmonary adenocarcinoma was used. The serum was known to be positive for
anti-voltage-gated potassium channel (VGKC) antibodies. Immunohistochemical
studies were compared with the results of autoradiographic studies performed
with spesific K channel blockers.
CASE
REPORT:
A 76 year old man
Muscle weakness, fatigue, excessive
sweating, salivation, small joint pain, itching erythema and progressive weight
loss at age 75.
Three months later à diffuse muscular twitching, episodes of acute confusional syndrome,
visual and auditory hallucinations and complex nocturnal behaviour during
sleep.
Later à progressive nocturnal insomnia, impairment of memory, constipation,
urinary incontinence and excessive lacrymation.
The patient died after a number of
plasmapheresis sessions and progressive worsening of all clinical findings.
Neurologic examination à myokymia in the face, tongue, trunk and all limbs, fasciculations,
spontaneous and reflex myoclonus, slight muscular atrophy and absence of tendon
reflexes. Pin-prick, touch and vibration senses and muscle strength were
normal. Plantar reflexes in flexion.
Bedside neuropsychological examination à Psychomotor agitation, poor cooperation, hallucinations, impairment
of memory (consistent with organic brain syndrome).
Detailed neuropsychological examination
after plasmapheresis à
normal.
Routine laboratory examination:
Haemogram, serum electrolytes, thyroid
function, liver function, lactate dehydrogenase, creatine kinase, pyruvic and
lactic acid, anti-nuclear, anti-smooth muscle, anti-DNA, anti-thyroglobulin and
anti-microsomal antibodies and tumoral markers (CEA, PAP, alpha1 fetoprotein,
PSA, CA19-9, TPA, CT, SCG, TG) were all normal. Sedimentation rate à (49 mm/h).
The cerebrospinal fluid (CSF) à normal,
VDRL in CSF à negative
IgG index à normal
Weak
oligoclonal bands in CSF. Absent in the serum.
Chest x-rays and CT à normal.
ECG
à frequent supraventricular extrasystoles.
Urodynamic
test à hyperreflexic bladder
Pathologic examination:
Muscle
biopsy (left vastus lateralis) à angular and atrophic fibres (mild neurogenic involvement)
Autopsy
à pulmonary adenocarcinoma. Adrenal
glands are normal.
Pathologic examination of brain à no significant pathology
Neuroradiologic examination:
Brain
MRI à high intensity lesions in white matter of both cerebral hemispheres
PET (after plasmapheresis) à normal
Electrophysiologic examination:
EMGà
spontaneous occurrence of irregularly repetitive MUPs, complex repetitive
discharges. No fibrillation potentials, normal
insertional activity. No decrement
or increment.
ENG à normal.
Video-PSG
à NREM sleep findings (spindles, K complexes, delta waves) are
absent, abnormal halucinatory movements (dream-like behavioural episodes).
EEG
à wakefulness (abundant theta activity), short REM sleep phases,
short muscle atonia duration
After plasmapheresis à almost normal
METHODS:
Radioimmunoassay
(RIA): The presence of anti-voltage gated potassium
channels anti-calcium activated potassium channels was assessed in all serum
samples by RIA. Five ml of serum samples (diluted 1/10 in
PTX) were incubated with 2.5 ml of 125I-labelled
charybdotoxin (ChTx) (125000 cpm/assay tube) and 8 ml of 125I-labelled DTx (40000 cpm/assay tube) for 2 hours
at room temperature. Then 500 ml of sheep anti-human IgG was added and
the mixture was incubated overnight at 4°C. After centrifugation at 13000 rpm for 5 minutes, the precipitates
were counted for 125I with a gamma scintillation counter.
Serum antibodies to AChR, VGCC, GAD,
gangliosides, paraneoplastic antigens were also detected.
Immunohistochemistry: Serum of the patient was used for immunohistochemistry studies.
Adult rat brain and foetal mouse head tissues were immediately frozen at -70°C until used for immunohistochemical studies. Consecutive 10 mm frozen sections were air dried. After fixation with 4%
paraformaldehyde for 3 minutes and inhibition of endogenous peroxidase with 3%
hydrogen peroxide in methanol for 10 minutes, the sections were incubated with
5% fetal coated serum for 10 minutes, serum samples (dilution 1/100) for 1 hour
and horseradish peroxidase labelled rat anti-human IgG for 30 minutes at room
temperature. The reaction was developed with AEC stock with hydrogen peroxide
in acetate buffer in 20 minutes. Dilution of sera was done in PBS.
Identification of anatomical localizations were performed by comparisons with
an adult rat brain and foetal mouse brain atlas.
Autoradiography: Adult rat brain was used for 125I-labelled dendrotoxin
(DTx) autoradiography and foetal mouse head tissues were used for both DTx and 125I-labelled
charybdotoxin (ChTx) autoradiography. Ten mm frozen sections were mounted on slides and air dried. After fixation
with aceton for 10 minutes, sections were incubated overnight with ChTx (14000
cpm/assay tube) and DTx (14000 to 70000 cpm/assay tube) at 4°C. After fixation with 4% paraformaldehyde for 5 minutes, the
sections were rinsed with PBS and rehydrated with 70% and 95% ethanol,
respectively. The sections were placed in the emulsion. The autoradiograms were
obtained by using a developer and fixer. In order to identify anatomical
localizations, sections were stained with hematoxylen eosin and compared with
the adult rat brain and foetal mouse brain atlas.
RESULTS:
RIA:
Serum antibodies to VGKC were strongly positive (633 pM). Anti-Ca activated K channel antibodies could
not be detected. Serum antibodies to AChR, VGCC, GAD, gangliosides,
paraneoplastic antigens were all negative.
IMMUNOHISTOCHEMISTRY:
Adult
rat brain sections: Antibodies (1/800) bound
strongly to neuronal cells in the cortex, hippocampus, medial thalamic
structures and midbrain sparing the white matter.
Foetal
mouse head sections: There was a high background in
all gray matter including neocortex, hippocampus, caudate, putamen, globus
pallidus, various nuclei of thalamus, hypothalamus, brain stem and cerebellum.
Antibodies binded most strongly to
parafascicular nuclei, fasciculus retroflexus and lateral
geniculate nuclei in thalamus, central
nucleus of the inferior colliculus in midbrain,
middle cerebellar peduncle and principal sensory trigeminal nucleus
in pons and olivary nucleus
in medulla.
The most prominent finding was the
staining of both parafascicular nuclei
and their tractus (fasciculus
retroflexus), which are important structures in sleep physiology. Lateral
geniculate nuclei were other sleep related structures in thalamus that are
also stained by patient’s serum.
AUTORADIOGRAPHY:
Adult
rat brain sections: DTx showed a distribution in
the rat brain, including many areas that serum antibodies bound. DTx showed
more binding to the axonal tracts (e.g. corpus callosum), not as much binding
to outer layers of cortex.
Foetal
mouse head sections:
ChTx: There was a strong binding pattern on all cortical structures including hippocampus. Weak to moderate binding was observed in caudate-putamen
and globus pallidus. There was weak staining on some hypothalamic
nuclei and cerebellum. There was always some binding on tongue,
muscles and salivary glands. There was no binding on choroid plexus, pineal gland and subcutaneus tissue.
DTx: There was a strong binding pattern on all cortical structures including hippocampus. Moderate to strong binding on hypothalamic nuclei,
caudate-putamen, globus pallidus, nucleus accumbens, pituitary
gland was also present. There was always some binding on tongue, muscles
and salivary glands and their ducts. Pineal
gland, choroid plexus and
subcutaneus tissue demonstrated strong binding, as well.
Parafascicular nucleus and fasciculus
retroflexus did not reveal any binding with both ChTx and DTx.
The results of our study revealed two
major evidences for differences between antigen expressions of adult and foetal
medial thalamic structures: 1.Although patient’s serum stained parafascicular
nuclei and their tractus strongly, no binding could be observed on these
structures by autoradiography. So, foetal parafascicular nuclei did not seem to
contain potassium channels. 2.The staining patterns observed on similar adult
rat sections were different. Fasciculus retroflexus could not be recognized
among various stained structures located in medial thalamus. Most probably,
antibodies in patient’s serum were binding to antigens (potassium channel or
other antigens) distributed in a more widespread area in medial thalamus and
this prevented the identification of these structures.
DISCUSSION:
1.There is a large number of reports
demonstrating the immaturity of thalamic nuclei and differences in antigen
expression of newborn and foetal rodents. Voltage responses and current
thresholds of VGKC of rat geniculate body reach to adult levels only after 13th
postnatal day (7). Neurons of zona incerta exhibit very low amounts of surface
proteoglycans in newborn mice, as compared to adults (8). Parvalbumin
reactivity of thalamic reticular nuclei of rat increase gradually during
maturation (9). All these observations and the results of our study may be
possible indicators of observed differences between sleep-wake cycles of adults
and newborns.
2.Some of the strongly stained structures
(lateral geniculate nuclei, parafascicular nucleus and fasciculus retroflexus)
contain high amounts of neuropeptide Y (NPY). NPY is usually an inhibitory
neurotransmitter, plays an important role in sleep physiology and exert its
effects via voltage-gated or calcium activated potassium channels in these
anatomical structures (10). The foetal tissue antigens that were observed in
fasciculus retroflexus and lateral geniculate nuclei but did not reveal any
binding by VGKC blockers may be NPY or calcium activated potassium channels.
REFERENCES:
2.
Haug BA, Schoenle PW, Karch
BJ, Bardosi A, Holzgraefe M. Morvan's fibrillary chorea. A case with possible
manganese poisoning. Clin Neurol Neurosurg 1989, 91(1): 53-9.
Hall AC, Earle-Cruikshanks G, Harrington ME. Role of membrane conductances and protein synthesis in subjective day phase advances of the hamster circadian clock by neuropeptide Y. Eur J Neurosci 1999, 11(10): 3424-32.