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Serotonergic receptors in the midbrain correlated with physiological data on sleep apnea in SIDS victims
Pathophysiology 10 (2004) 189–194
Serotonergic receptors in the midbrain correlated with physiological data on sleep apnea in SIDS victims Toshiko Sawaguchi a,∗ , Yuri Ozawa b , Patricia Franco c , Hazim Kadhim c , Jose Groswasser c , Martine Sottiaux c , Sachio Takashima d , Hiroshi Nishida e , Andre Kahn c a
Department of Legal Medicine, School of Medicine, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan b Department of Neonatology, Toho University School of Medicine, Tokyo, Japan c Children’s Hospital Reine Fabiola, Free University of Brussels, Brussels, Belgium d International University of Health and Welfare, Fukuoka, Japan e Maternal & Perinatal Center, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan
Abstract Background: Recently it has been reported that serotonin and related matters are associated with the sudden infant death syndrome (SIDS), which is still the main cause of postneonatal infant death. To further explore this claim, the correlation between serotonin receptors in the brainstem and sleep apnea in SIDS victims was investigated. Materials and methods: Among 27,000 infants studied prospectively to characterize their sleep–wake behavior, 38 infants died under 6 months of age including 26 cases of SIDS. All the infants had been recorded during one night in a pediatric sleep laboratory some 3–12 weeks before death. The frequency and duration of sleep apnea were analyzed. Brainstem material was collected and immunohistochemistry on 5-hydroxy tryptamine 1A (5HT1A) receptor was carried out. The density of 5HT1A receptor-positive neurons was measured quantitatively. Nonparametric analysis of the density of 5HT1A receptor-positive neurons was carried out between SIDS and non-SIDS cases. Correlation analyses were performed between the density of 5HT1A receptor-positive neurons and the data on sleep apnea. Results: There was no correlation between the pathological data on 5HT1A receptors and the physiological data on sleep apnea in SIDS victims. Conclusions: No correlation between pathological findings of serotonin and physiological findings of sleep apnea were not in agreement with the association of sleep apnea in pathophysiology of SIDS. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Serotonin receptor; Pons; Obstructive apnea; Central apnea; Sudden infant death syndrome
1. Introduction The sudden infant death syndrome (SIDS) is the main cause of postneonatal infant death, being defined as the sudden death of an infant under 1 year of age that remains unexplained after a complete clinical review, autopsy, and death scene investigation [1]. The role of sleep apnea [2–4] and of arousal [5–11] have been prominently advanced in the investigation of the pathophysiology of SIDS. There seems the possibility that abnormal development of the neuronal circuitry in the brainstem, the neurons of which regulate rhythmic breathing and ∗
Corresponding author. Tel./fax: +81-3-5269-7300. E-mail address: [email protected] (T. Sawaguchi).
arousal, underlies the cardio-respiratory instability in SIDS [12]. Abnormal early postnatal development of neurotransmitters in the central nervous system has been reported in SIDS [13–24]. The kinetics of serotonin, serotonergic neurons, serotonergic receptors, and the serotonin transporter have been claimed that this plays a major role in the pathophysiology of SIDS [22,25–29]. In this present report, the correlation between prospective physiological data on sleep apnea and retrospective pathological data on serotonergic receptors was investigated. As serotonergic receptors are involved in the arousal pathways, the results of this investigation might explain the association of serotonergic receptors with apnea and arousal in SIDS.
0928-4680/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2004.01.009
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2. Materials and methods 2.1. Physiological analyses 2.1.1. Subjects The sleep characteristics of 38 apparently healthy infants were prospectively recorded some 3–12 weeks before their death. They were taken from over 27,000 infants who had undergone polysomnographies prospectively during a period of over 20 years in various pediatric sleep laboratories to determine infant sleep–wake characteristics. Informed consent was obtained from their families before leaving the maternity ward. The infants selected for this study met the following criteria: they were born at term after a normal gestation and had no past and family history of apnea, apparent life-threatening event (ALTE) or SIDS. At the time of recording, the infants were 2–27-weeks-old, healthy and not under medication. Two to twelve weeks after the sleep recording, 38 infants died suddenly and unexpectedly. Autopsies suggested that 26 had died of SIDS [11,29]; of the remaining infants, three died from bronchopneumopathy, two from myocarditis, one each from pneumonia, varicella, cardiopathy with pulmonary hypertension, Opitz syndrome, hepatitis, and general infection, infanticide and meningitis complicated by brain infarction. These 12 infants form the control group. The general profiles of the subjects are shown in Table 1. 2.1.2. Polysomnography Eight-hour overnight sleep studies were conducted in a sleep laboratory, following standard techniques [30–33]. The recordings were made in a quiet and darkened room at an ambient temperature between 20 and 23 ◦ C. All infants slept in a supine position without restraints. Recording started around 9.00 p.m. The infants were observed continuously during recording and were fed on demand. Their behavior and any nursing intervention were recorded manually. Pacifiers were withheld during the recording. The following variables were recorded simultaneously: two-channel scalp electroencephalograms from unilateral central and occipital areas, horizontal and vertical electroculograms and an electrocardiogram. As for variables of respiration, thoracic respiratory movements were measured by impedance and airflow with thermistors taped under both nostrils and on the side of the mouth. Oxygen saturation was recorded continuously by a transcutaneous sensor (Nellcor, USA). Gross body movements were measured with an actigram placed on one arm. The data were collected on a computerized infant sleep recorder (Alice recording system III, Healthdyne, USA). Electromyographic recording of the mentalis muscle was not done. 2.1.3. Method and standard of analysis Based on the polygraphic recordings, sleep stages, and sleep apneas (when lasting 3 s or longer) were rated according to standard definitions [30–35]. Arousals from sleep
Table 1 General profiles of each case Case number Sex Gestational Postneonatal age (weeks) age (weeks) SIDS cases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Cause of death
F F M F F M M F M M F M M M M F F M M M M M M F F M
40 38 40 41 37 40 39 40 39 38 40 39 40 38 40 36 40 38 3 37 35 40 40 40 37 31
16 13 12 18 19 16 12 14 21 10 11 22 40 36 10 19 3 4 6 18 6 22 31 31 20 20
SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS
Control cases 1 M
39
24
Meningitis and brain infarction Pneumonia Myocarditis Varicella Cardiopathy with pulmonary hypertension Syndrome of Opitz Hepatitis and general infection Bronchopneumopathy Myocarditis Infanticide Bronchopneumopathy Bronchopneumopathy
2 3 4 5
M F M M
40 40 33 40
11 6 21 14
6 7
M F
37 37
4 5
8 9 10 11 12
M M F M F
39 40 37 39 40
6 7 7 9 9
were determined by direct observation by the technicians and by the recordings of breathing and heart rate changes as well as eye movements. Arousals were also accompanied by activation of the actigrams and movement artifacts were identified on the cardiac and saturation recordings. Apneas were designated as central apnea when flat tracings were obtained simultaneously from the strain gauges and the thermistors. Periodic breathing was defined as a succession of more than two central apneas separated by a period of less than 20 s. Obstructive apneas were defined as a flat tracing recorded from the thermistors with continuous deflections from the strain gauges. A mixed apnea was scored when a central apnea was followed immediately by an obstructive
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episode. Mixed apneas were scored together with the obstructive episodes. The frequency of central and obstructive apneas was measured by dividing the total number of each apnea by the total sleep time in minutes and multiplied by 60. The type, frequency (number per hour of sleep) and duration (in s) of sleep apneas were computed. All recordings were analyzed visually by two independent scorers without knowledge of the subject’s sex and attribution to either the SIDS or the control group. Discrepancies between scorers were discussed before the data were computed.
3. Pathological analyses 3.1. Subjects A total of 48 paraffin blocks of brainstems were collected from the brain of each of the 38 autopsied infants who died unexpectedly: seven blocks from the midbrain, 22 from the pons, and 19 from the medulla oblongata. The maximum time that elapsed between the estimated time of death and the postmortem examination was 24 h. 3.2. Neurohistological examination Hematoxylin–eosin was the standard staining method for neurohistological examination.
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nuclei of the pons and hypoglossal nucleus, dorsal nucleus of the vagus, solitary nucleus, and the ventolateral medulla (VLM) in the medulla oblongata. The density of 5HT1A receptor-immunoreactive neurons were graded as mild, (±: a few neurons were weakly stained), as moderate (+: several neurons were stained weakly to strong) and marked (+: most neurons were well-stained). For the statistical calculation, it was semi-quantitized that (−) as 0, (−)–(±) as 1, (±) as 2, (±)–(+) as 3,(+) as 4, (+)–(+) as 5, and (+) as 6. Each measurement procedure in an area 625 m × 102 m was repeated five times at different overlapping sites, and an average was recorded. The pathological measurements were made twice by the same pathologist, and data with large standard deviations were recounted or rejected.
4. Data analysis 4.1. Double-blind analyses The scorers of the sleep recordings and the pathologist were not aware of the causes of the infants’ deaths. The scorers of the sleep recordings had no contact with the pathologist. 4.2. Matching the physiological and pathological data
3.3. Immunohistochemical examination The sections for immunohistochemical examination with 5-hydroxy tryptamine 1A (5HT1A) receptor antibodies were deparaffinized and pretreated with 0.3% hydrogen peroxide in methanol for 15 min and then subjected to microwave irradiation for 9 min at 90 ◦ C and rinsing with PBS (pH 7.4). They were then preincubated for 3 min in the presence of 10% normal rabbit serum and incubated with goat polyclonal antibodies against a peptide corresponding to amino acids 394–416 mapped to the carboxy terminus of the 5-HT receptor 1A precursor of human origin (Santa Cruz Biotechnology), diluted 1:100 overnight twice at 4 ◦ C, followed by biotinylated rabbit anti-goat IgG (Vector Laboratories), diluted 1:500 for 1 h at room temperature and then stained with peroxidase-conjugated streptavidin (Nichirei) for 30 min at room temperature. Each step was followed by washing with PBS. The immunoproducts were visualized using 0.02 M diaminobenzidine tetrahydrochloride as the chromogen in 0.05 M Tris buffer, pH 7.4, containing 0.006% hydrogen peroxide.
For each infant, the type and scores of apneas (frequency or duration of obstructive or central apnea) were correlated with the neuropathological findings of 5HT1A receptor obtained from the postmortem studies. Correlation was analyzed and Pearson’s correlation coefficient was computed by SPSS ver. 8.0. In addition, the correlation between the physiological and pathological data were separately analyzed for the SIDS group.
5. Ethical issues This study was approved by the Ethical Committee of the University Children’s Hospital Reine Fabiola and conducted in accordance with the ethical standards prescribed by the 1964 Declaration of Helsinki.
6. Results
3.4. Cell density analysis
6.1. Standard neurohistological examination
Measurements of 5HT1A receptor immunoreactivity were made in the periaqueductal gray (PAG) matter, oculomotor nucleus, raphe nucleus, and substantia nigra of the midbrain, and the locus coeruleus, reticular formation, and pontine
Hematoxylin–eosin staining exhibited definite neuropathological findings in three cases. One infant clinically diagnosed as having SIDS was shown to have meningitis and brain infarction. In the control group, one infant with
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Table 2 Correlation coefficients between pathological data on 5-hydroxyttryptamine 1A receptor and physiological data on sleep apnea in SIDS victims Obstructive apnea
Central apnea
Frequency (number/s)
Duration (s)
Frequency (number/s)
Medulla oblongata Nucleus hypoglossus Correlation coefficient Significant value
−0.132 0.778
−0.321 0.482
−0.138 0.769
0.073 0.876
Dorsal nucleus vagus Correlation coefficient Significant value
0.515 0.296
−0.739 0.093
−0.489 0.325
−0.342 0.507
Solitary nucleus Correlation coefficient Significant value
0.012 0.979
−0.191 0.682
−0.250 0.589
0.098 0.835
Ambiguus nucleus Correlation coefficient Significant value
0.179 0.645
−0.383 0.309
0.127 0.744
−0.220 0.569
Ventrolateral medulla Correlation coefficient Significant value
−0.163 0.614
−0.462 0.131
0.194 0.546
−0.120 0.710
Pons Locus coeruleus Correlation coefficient Significant value
−0.026 0.948
0.284 0.068
0.012 0.655
−0.528 0.130
Reticular formation Correlation coefficient Significant value
0.432 0.094
−0.031 0.908
−0.026 0.925
−0.330 0.212
Pontine nucleus Correlation coefficient Significant value
0.048 0.855
0.055 0.835
0.065 0.804
−0.414 0.098
Midbrain Periaqueductal gray matter Correlation coefficient Significant value
−0.208 0.621
0.033 0.938
0.015 0.972
−0.619 0.102
Raphe nucleus Correlation coefficient Significant value
−0.452 0.445
0.664 0.222
0.112 0.858
−0.677 0.209
Substantia nigra Correlation coefficient Significant value
0.041 0.923
−0.022 0.959
−0.461 0.251
−0.666 0.072
a tumor with hemorrhagic infarct and polymicrogyria were found. The former two infants were reclassified as control cases. 6.2. Matching of physiological and pathological data No significant SIDS-specific correlation between the density of 5HT1A receptor positive neurons in each site of the brainstem (medulla oblongata, pons, and midbrain) and the characteristic of sleep apnea was found (Table 2). 6.3. Pathological findings of 5HT1A receptors in SIDS and non-SIDS Pathological findings of 5HT1AR receptors in SIDS and non-SIDS are shown in Table 3.
Duration (s)
7. Discussion Some authors have described changes in serotonergic receptors in the brainstems of SIDS victims. Kinney et al. has investigated 19 regions of the brainstem of SIDS and claimed that six medullary areas (arcuate nucleus, principal inferior olive, solitary nucleus, intermediate reticular zone, nucleus paragigantocellularis lateralis, nucleus gigantocellularis, and the nucelus raphe obscurus) showed reduced serotonin receptor binding in a subset of SIDS victims [25,36]. Based on this research, they have proposed a medullary serotonergic network-deficiency theory, focused upon a specific neurotransmitter (serotonin) and specific territories, namely, the ventral medulla and regions of the medullary reticular formation that contain serotonergic neurons [22]. In this theory, SIDS is due to a developmental abnormality in a medullary
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Table 3 Comparison of 5HT1AR immunoreactivity in the brainstem between SIDS cases and controls Medulla oblongata Nucleus hypoglossus
SIDS Controls
Dorsal vagal nucleus
Solitary Nucleus
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
0 0
3 0
0 0
5 0
0 0
1 1
3 0
2 0
0 0
6 2
1 0
1 0
Medulla oblongata
Pons
Ambiguous nucleus
SIDS Controls
Ventrolateral medulla
Locus coeruleus
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
0 0
3 2
2 1
4 0
0 0
4 0
3 3
4 0
0 0
2 5
3 2
6 0
Pons
Midbrain
Reticular formation
SIDS Controls
Pontine nucleus
Periaqueductal gray matter
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
2 0
5 5
5 0
7 0
2 0
6 5
6 0
3 0
0 0
3 0
3 0
4 0
Midbrain Oculomotor nucleus
SIDS Controls
Raphe nucleus
Substantia nigra
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
0 0
4 0
0 0
0 0
0 0
2 0
1 0
2 0
0 0
5 0
2 0
1 0
±: Mild: a few neurons were weakly stained; +: moderate, several neurons were well stained. Abbreviations: 5HT1AR: 5-hydroxytryptamine 1A receptor; SIDS: sudden infant death syndrome.
network composed of rhombic lip-derived, serotonergic neurons including in the caudal raphe and arcuate nucleus [22]. To substantiate this theory, only a decrease of serotonergic neurons and serotonin receptors have been reported, mainly in the medulla oblongata. On the other hand, Ozawa and Okado [26] have reported not only a decrease, but also an increase in the serotonin receptors in the brainstem of SIDS victims. In their study, they found a significant decrease of 5HT1A and 5HT2A receptor immunoreactivity in the dorsal nucleus of the vagus, solitary nucleus and VLM in the medulla oblongata and an increase in 5HT1A and 5HT2A receptor immunoreactivity in PAG matter [26]. Regarding developmental changes in serotonin receptors, 5HT1A receptor is highest in the fetal period and then decreases [37]. The higher level of 5HT1A receptor in SIDS victims may suggest delayed neuronal maturation [26]. Descending projections from the PAG to the medulla are necessary links for PAG evoked analgesic and autonomic responses [38] and the serotonergic input to PAG through the projections, arise from pontine and medullary reticular and raphe nuclei in the rat [39]. From this morphological background, it might be inferred that the increase of 5HT1A receptor immunoreactivity in the PAG [26] of the midbrain and the pontine nuclei of the pons, may reflect compensatory
changes in response to a malfunction of serotonergic neurons in the medulla oblongata [26]. There was no correlation between the pathological findings of 5HT1A receptor and physiological findings of sleep apnea in SIDS victims in our study here. This fact is not agreement with association of sleep apnea in SIDS pathophysiology. The negative results in this report might have been brought about because many of the controls were in a hypoxic state.
Acknowledgements The authors sincerely thank Professor C. DePrez for the kind help extended by Health Sciences Research Grants for Research on Children and Families from the Japanese Ministry of Health and Welfare.
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Serotonergic receptors in the midbrain correlated with physiological data on sleep apnea in SIDS victims Toshiko Sawaguchi a,∗ , Yuri Ozawa b , Patricia Franco c , Hazim Kadhim c , Jose Groswasser c , Martine Sottiaux c , Sachio Takashima d , Hiroshi Nishida e , Andre Kahn c a
Department of Legal Medicine, School of Medicine, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan b Department of Neonatology, Toho University School of Medicine, Tokyo, Japan c Children’s Hospital Reine Fabiola, Free University of Brussels, Brussels, Belgium d International University of Health and Welfare, Fukuoka, Japan e Maternal & Perinatal Center, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan
Abstract Background: Recently it has been reported that serotonin and related matters are associated with the sudden infant death syndrome (SIDS), which is still the main cause of postneonatal infant death. To further explore this claim, the correlation between serotonin receptors in the brainstem and sleep apnea in SIDS victims was investigated. Materials and methods: Among 27,000 infants studied prospectively to characterize their sleep–wake behavior, 38 infants died under 6 months of age including 26 cases of SIDS. All the infants had been recorded during one night in a pediatric sleep laboratory some 3–12 weeks before death. The frequency and duration of sleep apnea were analyzed. Brainstem material was collected and immunohistochemistry on 5-hydroxy tryptamine 1A (5HT1A) receptor was carried out. The density of 5HT1A receptor-positive neurons was measured quantitatively. Nonparametric analysis of the density of 5HT1A receptor-positive neurons was carried out between SIDS and non-SIDS cases. Correlation analyses were performed between the density of 5HT1A receptor-positive neurons and the data on sleep apnea. Results: There was no correlation between the pathological data on 5HT1A receptors and the physiological data on sleep apnea in SIDS victims. Conclusions: No correlation between pathological findings of serotonin and physiological findings of sleep apnea were not in agreement with the association of sleep apnea in pathophysiology of SIDS. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Serotonin receptor; Pons; Obstructive apnea; Central apnea; Sudden infant death syndrome
1. Introduction The sudden infant death syndrome (SIDS) is the main cause of postneonatal infant death, being defined as the sudden death of an infant under 1 year of age that remains unexplained after a complete clinical review, autopsy, and death scene investigation [1]. The role of sleep apnea [2–4] and of arousal [5–11] have been prominently advanced in the investigation of the pathophysiology of SIDS. There seems the possibility that abnormal development of the neuronal circuitry in the brainstem, the neurons of which regulate rhythmic breathing and ∗
Corresponding author. Tel./fax: +81-3-5269-7300. E-mail address: [email protected] (T. Sawaguchi).
arousal, underlies the cardio-respiratory instability in SIDS [12]. Abnormal early postnatal development of neurotransmitters in the central nervous system has been reported in SIDS [13–24]. The kinetics of serotonin, serotonergic neurons, serotonergic receptors, and the serotonin transporter have been claimed that this plays a major role in the pathophysiology of SIDS [22,25–29]. In this present report, the correlation between prospective physiological data on sleep apnea and retrospective pathological data on serotonergic receptors was investigated. As serotonergic receptors are involved in the arousal pathways, the results of this investigation might explain the association of serotonergic receptors with apnea and arousal in SIDS.
0928-4680/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2004.01.009
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2. Materials and methods 2.1. Physiological analyses 2.1.1. Subjects The sleep characteristics of 38 apparently healthy infants were prospectively recorded some 3–12 weeks before their death. They were taken from over 27,000 infants who had undergone polysomnographies prospectively during a period of over 20 years in various pediatric sleep laboratories to determine infant sleep–wake characteristics. Informed consent was obtained from their families before leaving the maternity ward. The infants selected for this study met the following criteria: they were born at term after a normal gestation and had no past and family history of apnea, apparent life-threatening event (ALTE) or SIDS. At the time of recording, the infants were 2–27-weeks-old, healthy and not under medication. Two to twelve weeks after the sleep recording, 38 infants died suddenly and unexpectedly. Autopsies suggested that 26 had died of SIDS [11,29]; of the remaining infants, three died from bronchopneumopathy, two from myocarditis, one each from pneumonia, varicella, cardiopathy with pulmonary hypertension, Opitz syndrome, hepatitis, and general infection, infanticide and meningitis complicated by brain infarction. These 12 infants form the control group. The general profiles of the subjects are shown in Table 1. 2.1.2. Polysomnography Eight-hour overnight sleep studies were conducted in a sleep laboratory, following standard techniques [30–33]. The recordings were made in a quiet and darkened room at an ambient temperature between 20 and 23 ◦ C. All infants slept in a supine position without restraints. Recording started around 9.00 p.m. The infants were observed continuously during recording and were fed on demand. Their behavior and any nursing intervention were recorded manually. Pacifiers were withheld during the recording. The following variables were recorded simultaneously: two-channel scalp electroencephalograms from unilateral central and occipital areas, horizontal and vertical electroculograms and an electrocardiogram. As for variables of respiration, thoracic respiratory movements were measured by impedance and airflow with thermistors taped under both nostrils and on the side of the mouth. Oxygen saturation was recorded continuously by a transcutaneous sensor (Nellcor, USA). Gross body movements were measured with an actigram placed on one arm. The data were collected on a computerized infant sleep recorder (Alice recording system III, Healthdyne, USA). Electromyographic recording of the mentalis muscle was not done. 2.1.3. Method and standard of analysis Based on the polygraphic recordings, sleep stages, and sleep apneas (when lasting 3 s or longer) were rated according to standard definitions [30–35]. Arousals from sleep
Table 1 General profiles of each case Case number Sex Gestational Postneonatal age (weeks) age (weeks) SIDS cases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Cause of death
F F M F F M M F M M F M M M M F F M M M M M M F F M
40 38 40 41 37 40 39 40 39 38 40 39 40 38 40 36 40 38 3 37 35 40 40 40 37 31
16 13 12 18 19 16 12 14 21 10 11 22 40 36 10 19 3 4 6 18 6 22 31 31 20 20
SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS SIDS
Control cases 1 M
39
24
Meningitis and brain infarction Pneumonia Myocarditis Varicella Cardiopathy with pulmonary hypertension Syndrome of Opitz Hepatitis and general infection Bronchopneumopathy Myocarditis Infanticide Bronchopneumopathy Bronchopneumopathy
2 3 4 5
M F M M
40 40 33 40
11 6 21 14
6 7
M F
37 37
4 5
8 9 10 11 12
M M F M F
39 40 37 39 40
6 7 7 9 9
were determined by direct observation by the technicians and by the recordings of breathing and heart rate changes as well as eye movements. Arousals were also accompanied by activation of the actigrams and movement artifacts were identified on the cardiac and saturation recordings. Apneas were designated as central apnea when flat tracings were obtained simultaneously from the strain gauges and the thermistors. Periodic breathing was defined as a succession of more than two central apneas separated by a period of less than 20 s. Obstructive apneas were defined as a flat tracing recorded from the thermistors with continuous deflections from the strain gauges. A mixed apnea was scored when a central apnea was followed immediately by an obstructive
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episode. Mixed apneas were scored together with the obstructive episodes. The frequency of central and obstructive apneas was measured by dividing the total number of each apnea by the total sleep time in minutes and multiplied by 60. The type, frequency (number per hour of sleep) and duration (in s) of sleep apneas were computed. All recordings were analyzed visually by two independent scorers without knowledge of the subject’s sex and attribution to either the SIDS or the control group. Discrepancies between scorers were discussed before the data were computed.
3. Pathological analyses 3.1. Subjects A total of 48 paraffin blocks of brainstems were collected from the brain of each of the 38 autopsied infants who died unexpectedly: seven blocks from the midbrain, 22 from the pons, and 19 from the medulla oblongata. The maximum time that elapsed between the estimated time of death and the postmortem examination was 24 h. 3.2. Neurohistological examination Hematoxylin–eosin was the standard staining method for neurohistological examination.
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nuclei of the pons and hypoglossal nucleus, dorsal nucleus of the vagus, solitary nucleus, and the ventolateral medulla (VLM) in the medulla oblongata. The density of 5HT1A receptor-immunoreactive neurons were graded as mild, (±: a few neurons were weakly stained), as moderate (+: several neurons were stained weakly to strong) and marked (+: most neurons were well-stained). For the statistical calculation, it was semi-quantitized that (−) as 0, (−)–(±) as 1, (±) as 2, (±)–(+) as 3,(+) as 4, (+)–(+) as 5, and (+) as 6. Each measurement procedure in an area 625 m × 102 m was repeated five times at different overlapping sites, and an average was recorded. The pathological measurements were made twice by the same pathologist, and data with large standard deviations were recounted or rejected.
4. Data analysis 4.1. Double-blind analyses The scorers of the sleep recordings and the pathologist were not aware of the causes of the infants’ deaths. The scorers of the sleep recordings had no contact with the pathologist. 4.2. Matching the physiological and pathological data
3.3. Immunohistochemical examination The sections for immunohistochemical examination with 5-hydroxy tryptamine 1A (5HT1A) receptor antibodies were deparaffinized and pretreated with 0.3% hydrogen peroxide in methanol for 15 min and then subjected to microwave irradiation for 9 min at 90 ◦ C and rinsing with PBS (pH 7.4). They were then preincubated for 3 min in the presence of 10% normal rabbit serum and incubated with goat polyclonal antibodies against a peptide corresponding to amino acids 394–416 mapped to the carboxy terminus of the 5-HT receptor 1A precursor of human origin (Santa Cruz Biotechnology), diluted 1:100 overnight twice at 4 ◦ C, followed by biotinylated rabbit anti-goat IgG (Vector Laboratories), diluted 1:500 for 1 h at room temperature and then stained with peroxidase-conjugated streptavidin (Nichirei) for 30 min at room temperature. Each step was followed by washing with PBS. The immunoproducts were visualized using 0.02 M diaminobenzidine tetrahydrochloride as the chromogen in 0.05 M Tris buffer, pH 7.4, containing 0.006% hydrogen peroxide.
For each infant, the type and scores of apneas (frequency or duration of obstructive or central apnea) were correlated with the neuropathological findings of 5HT1A receptor obtained from the postmortem studies. Correlation was analyzed and Pearson’s correlation coefficient was computed by SPSS ver. 8.0. In addition, the correlation between the physiological and pathological data were separately analyzed for the SIDS group.
5. Ethical issues This study was approved by the Ethical Committee of the University Children’s Hospital Reine Fabiola and conducted in accordance with the ethical standards prescribed by the 1964 Declaration of Helsinki.
6. Results
3.4. Cell density analysis
6.1. Standard neurohistological examination
Measurements of 5HT1A receptor immunoreactivity were made in the periaqueductal gray (PAG) matter, oculomotor nucleus, raphe nucleus, and substantia nigra of the midbrain, and the locus coeruleus, reticular formation, and pontine
Hematoxylin–eosin staining exhibited definite neuropathological findings in three cases. One infant clinically diagnosed as having SIDS was shown to have meningitis and brain infarction. In the control group, one infant with
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Table 2 Correlation coefficients between pathological data on 5-hydroxyttryptamine 1A receptor and physiological data on sleep apnea in SIDS victims Obstructive apnea
Central apnea
Frequency (number/s)
Duration (s)
Frequency (number/s)
Medulla oblongata Nucleus hypoglossus Correlation coefficient Significant value
−0.132 0.778
−0.321 0.482
−0.138 0.769
0.073 0.876
Dorsal nucleus vagus Correlation coefficient Significant value
0.515 0.296
−0.739 0.093
−0.489 0.325
−0.342 0.507
Solitary nucleus Correlation coefficient Significant value
0.012 0.979
−0.191 0.682
−0.250 0.589
0.098 0.835
Ambiguus nucleus Correlation coefficient Significant value
0.179 0.645
−0.383 0.309
0.127 0.744
−0.220 0.569
Ventrolateral medulla Correlation coefficient Significant value
−0.163 0.614
−0.462 0.131
0.194 0.546
−0.120 0.710
Pons Locus coeruleus Correlation coefficient Significant value
−0.026 0.948
0.284 0.068
0.012 0.655
−0.528 0.130
Reticular formation Correlation coefficient Significant value
0.432 0.094
−0.031 0.908
−0.026 0.925
−0.330 0.212
Pontine nucleus Correlation coefficient Significant value
0.048 0.855
0.055 0.835
0.065 0.804
−0.414 0.098
Midbrain Periaqueductal gray matter Correlation coefficient Significant value
−0.208 0.621
0.033 0.938
0.015 0.972
−0.619 0.102
Raphe nucleus Correlation coefficient Significant value
−0.452 0.445
0.664 0.222
0.112 0.858
−0.677 0.209
Substantia nigra Correlation coefficient Significant value
0.041 0.923
−0.022 0.959
−0.461 0.251
−0.666 0.072
a tumor with hemorrhagic infarct and polymicrogyria were found. The former two infants were reclassified as control cases. 6.2. Matching of physiological and pathological data No significant SIDS-specific correlation between the density of 5HT1A receptor positive neurons in each site of the brainstem (medulla oblongata, pons, and midbrain) and the characteristic of sleep apnea was found (Table 2). 6.3. Pathological findings of 5HT1A receptors in SIDS and non-SIDS Pathological findings of 5HT1AR receptors in SIDS and non-SIDS are shown in Table 3.
Duration (s)
7. Discussion Some authors have described changes in serotonergic receptors in the brainstems of SIDS victims. Kinney et al. has investigated 19 regions of the brainstem of SIDS and claimed that six medullary areas (arcuate nucleus, principal inferior olive, solitary nucleus, intermediate reticular zone, nucleus paragigantocellularis lateralis, nucleus gigantocellularis, and the nucelus raphe obscurus) showed reduced serotonin receptor binding in a subset of SIDS victims [25,36]. Based on this research, they have proposed a medullary serotonergic network-deficiency theory, focused upon a specific neurotransmitter (serotonin) and specific territories, namely, the ventral medulla and regions of the medullary reticular formation that contain serotonergic neurons [22]. In this theory, SIDS is due to a developmental abnormality in a medullary
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Table 3 Comparison of 5HT1AR immunoreactivity in the brainstem between SIDS cases and controls Medulla oblongata Nucleus hypoglossus
SIDS Controls
Dorsal vagal nucleus
Solitary Nucleus
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
0 0
3 0
0 0
5 0
0 0
1 1
3 0
2 0
0 0
6 2
1 0
1 0
Medulla oblongata
Pons
Ambiguous nucleus
SIDS Controls
Ventrolateral medulla
Locus coeruleus
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
0 0
3 2
2 1
4 0
0 0
4 0
3 3
4 0
0 0
2 5
3 2
6 0
Pons
Midbrain
Reticular formation
SIDS Controls
Pontine nucleus
Periaqueductal gray matter
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
2 0
5 5
5 0
7 0
2 0
6 5
6 0
3 0
0 0
3 0
3 0
4 0
Midbrain Oculomotor nucleus
SIDS Controls
Raphe nucleus
Substantia nigra
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
(−)–(±)
(±)
(±)–(+)
(+)
0 0
4 0
0 0
0 0
0 0
2 0
1 0
2 0
0 0
5 0
2 0
1 0
±: Mild: a few neurons were weakly stained; +: moderate, several neurons were well stained. Abbreviations: 5HT1AR: 5-hydroxytryptamine 1A receptor; SIDS: sudden infant death syndrome.
network composed of rhombic lip-derived, serotonergic neurons including in the caudal raphe and arcuate nucleus [22]. To substantiate this theory, only a decrease of serotonergic neurons and serotonin receptors have been reported, mainly in the medulla oblongata. On the other hand, Ozawa and Okado [26] have reported not only a decrease, but also an increase in the serotonin receptors in the brainstem of SIDS victims. In their study, they found a significant decrease of 5HT1A and 5HT2A receptor immunoreactivity in the dorsal nucleus of the vagus, solitary nucleus and VLM in the medulla oblongata and an increase in 5HT1A and 5HT2A receptor immunoreactivity in PAG matter [26]. Regarding developmental changes in serotonin receptors, 5HT1A receptor is highest in the fetal period and then decreases [37]. The higher level of 5HT1A receptor in SIDS victims may suggest delayed neuronal maturation [26]. Descending projections from the PAG to the medulla are necessary links for PAG evoked analgesic and autonomic responses [38] and the serotonergic input to PAG through the projections, arise from pontine and medullary reticular and raphe nuclei in the rat [39]. From this morphological background, it might be inferred that the increase of 5HT1A receptor immunoreactivity in the PAG [26] of the midbrain and the pontine nuclei of the pons, may reflect compensatory
changes in response to a malfunction of serotonergic neurons in the medulla oblongata [26]. There was no correlation between the pathological findings of 5HT1A receptor and physiological findings of sleep apnea in SIDS victims in our study here. This fact is not agreement with association of sleep apnea in SIDS pathophysiology. The negative results in this report might have been brought about because many of the controls were in a hypoxic state.
Acknowledgements The authors sincerely thank Professor C. DePrez for the kind help extended by Health Sciences Research Grants for Research on Children and Families from the Japanese Ministry of Health and Welfare.
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