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Pathological data on apoptosis in the brainstem and physiological data on sleep apnea in SIDS victims
Pathophysiology 10 (2004) 155–159
Pathological data on apoptosis in the brainstem and physiological data on sleep apnea in SIDS victims Toshiko Sawaguchi a,∗ , Patricia Franco b , Hazim Kadhim b , Jose Groswasser b , Martine Sottiaux b , Hiroshi Nishida c , Andre Kahn b a
Department of Legal Medicine, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan b Children’s Hospital Reine Fabiola, Free University of Brussels, Brussels, Belgium c Maternal and Perinatal Center, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan
Abstract The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death and its cause is still unknown. A chronic hypoxic situation has been shown to exist in the brains of SIDS victims and apoptosis has been demonstrated in hypoxic situations. In this study, the correlation between apoptotic neurons or glias and sleep apnea in SIDS was investigated in the brainstem of SIDS victims. Materials and methods: In a cohort of 27,000 infants studied prospectively to characterize their sleep–wake behavior, 38 infants died under 6 months of age. They included 26 cases of SIDS. The frequency and duration of sleep apnea were analyzed. The brainstem material was collected and terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method was carried out. The density of TUNEL-positive neurons or glias was measured quantitatively. Correlation analyses were carried out between the apoptosis-associated pathological data and the physiological data of sleep apnea. Results: No significant negative or positive correlation between the density of TUNEL-positive neurons or glias and the characteristics of sleep apnea was observed in SIDS victims. No statistically significant differences associated with apoptotic neurons and glias were observed between SIDS and non-SIDS. Conclusions: The pathological findings of apoptosis were not in agreement with the hypothesis refer to apnea and arousal phenomenon in pathophysiology of SIDS. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Apoptosis; Terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL); Brainstem; Obstructive apnea; Central apnea; Arousal; Sudden infant death syndrome (SIDS)
1. Introduction The sudden infant death syndrome (SIDS) is the main cause of postneonatal infant deaths. SIDS is 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 causes and pathophysiology of SIDS have long been investigated without any definite conclusions being reached. Several etiological hypotheses have been proposed to explain the mechanisms responsible for SIDS, including the role of apnea [2–4] and of arousal [5–11]. Neuropathological studies showed that the brainstems of SIDS victims were characterized by minute changes [12–23], such as gliosis [12–14,17,18,20,22] or apoptosis [24–26], attributed to hypoxic insult. ∗ Corresponding author. Tel.: +81-3-5269-7300; fax: +81-3-5269-7300. E-mail address: [email protected] (T. Sawaguchi).
Therefore, to investigate the apnea and the arousal deficiency theories of SIDS, the presence of apoptosis in the arousal pathway of the brainstem in SIDS victims was investigated and their association with sleep apnea was analyzed.
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. These were obtained 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
0928-4680/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2004.01.003
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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 or family history of apnea, apparent life-threatening events (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. Postmortem examination suggested that 26 had died of SIDS [26]; 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 [27–30]. The recordings were made in a quiet and darkened room at an ambient temperature between 20 and 23 ◦ C. All infants slept in supine position without restraint. 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 electrooculograms 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
Table 1 General profiles of each case Case no.
Sex
Gestational age (weeks)
Postneonatal age (weeks)
Cause of death
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
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 2 3 4 5 6 7 8 9 10 11 12
M M F M M M F M M F M F
39 40 40 33 40 37 37 39 40 37 39 40
24 11 6 21 14 4 5 6 7 7 9 9
Meningitis and brain infarction Pneumonia Myocarditis Varicella Cardiopathy with pulmonary hypertension Syndrome of Opitz Hepatitis and general infection Bronchopneumopathy Myocarditis Infanticide Bronchopneumopathy Bronchopneumopathy
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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.
Mannheim, Germany) [33–36]. Prior to the TUNEL method, the sections were pretreated by microwave irradiation in a 0.01 M citric acid buffer (pH 6.0) at 800 W for 5 min in a microwave device (Panasonic), then the TUNEL reaction was allowed to take place in the same manner described in previously published papers [34,35].
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 [27–32]. Arousals from sleep 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 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 seconds) 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 was computed.
2.2.4. Quantification of immunohistochemical presentations Measurements were made in the periaqueductal gray (PAG) matter and dorsal raphe nucleus of the midbrain and the PPTN (compact part: PPTNc and dissipated parts: PPTNd). The numbers of TUNEL-positive neurons and glias were counted manually. Each counting procedure in an area 625 m × 102 m was repeated five times at different overlapping sites and an average was recorded. The density was expressed as a percentage of the number of reaction-positive neurons and glias divided by the number of total neurons and glias. The pathological measurements were made twice by the same pathologist and data with large standard deviations were recounted or rejected.
2.2. Pathological analyses 2.2.1. Subjects A total of 48 paraffin blocks of brainstems were collected from the autopsied brains of the 38 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. 2.2.2. Neurohistological examination Hematoxylin–eosin (HE) stain was the standard staining method for neurohistological examination. 2.2.3. Immunohistochemical examination The blocks were subjected to terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method using in situ cell death detection kit (Boehringer
2.3. Data analysis 2.3.1. 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 not contact with the pathologist. 2.3.2. Matching the physiological and pathological data For each infant, the type and scores of apneas were correlated with the neuropatho-logical findings obtained from the postmortem studies. Correlation was analyzed and Pearson’s correlation coefficient was computed by SPSS Version 8.0. In addition, the intercorrelations between the physiological and pathological data were separately analyzed for the SIDS group and non-SIDS group. 2.4. Confirmation of the pathological characteristics of the SIDS and non-SIDS groups Nonparametric test (Kruskal–Wallis test) was carried out to evaluate the significant difference of density of TUNEL-positive neurons and glias in each site of the brainstem between SIDS and non-SIDS groups. 2.5. 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.
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Table 2 Correlation coefficients between pathological data on TUNEL-positive glia and physiological data on sleep apnea in SIDS victims Obstructive apnea Frequency (no./s)
Central apnea Duration (s)
Frequency (no./s)
0.1660 0.722
−0.0818 0.862
−0.0048 0.992
0.4280 0.338
−0.1138 0.808
−0.1454 0.756
−0.2319 0.617
−0.2417 0.602
Pedunculopontine tegmentum nucleus (compact part) Correlation coefficient 0.1316 Significant value 0.668
−0.1530 0.618
−0.1848 0.546
−0.2989 0.321
Pedunculopontine tegmentum nucleus (dissipated part) Correlation coefficient −0.3079 Significant value 0.553
−0.6501 0.162
0.0331 0.950
0.3311 0.521
Dorsal raphe nucleus Correlation coefficient Significant value Periaqueductal gray matter Correlation coefficient Significant value
3. Results 3.1. Standard neurohistological examination 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 a tumor with hemorrhagic infarct and polymicrogyria were found. The former two infants were reclassified as control cases. 3.2. Matching of physiological and pathological data 3.2.1. SIDS cases No significant positive or negative correlations were found between the density of TUNEL-positive neurons or glias in the arousal pathway of the midbrain and the each characteristic of sleep apnea were found in SIDS victims (Table 2). 3.2.2. Non-SIDS cases In non-SIDS cases, there is no significant correlation between pathological data on apoptosis in each site and physiological data of each sleep apnea. 3.3. Confirmation of the pathological characteristics of the SIDS and non-SIDS groups As the result of the nonparametric test (Kruskal–Wallis test), no significant difference of pathological data associated with apoptosis was found between in SIDS group and in non-SIDS groups.
Duration (s)
the brainstem of SIDS victims, mainly within the gracile and cuneate nuclei, spinal trigeminal tract neurons, tractus solitatius nucleus, lateral reticular formation and lateral cuneate nucleus [24]. It was hypothesized that hypoxia was responsible for the increase in TUNEL-positive cells in the brains of SIDS victims [24]. In contrast to this present study, no apoptosis was reported, however, in the dorsal raphe nucleus and PAG matter of the midbrain and the PPTN. These arousal pathways were not investigated in the previous study. In the present study, no significant correlation was found between apoptosis and the characteristics of sleep apnea in the arousal pathway of SIDS. In the previously reported paper, the increase of apoptotic neurons was observed in the watershed area in the brain and, consequently, the appearance of apoptosis in SIDS has been assumed to be hypoxic [25]. In the present study, the contribution of hypoxia to the development of apoptosis in the arousal pathway was not confirmed. In conclusion, no SIDS-specific correlation between the pathological findings of apoptosis and the physiological data of sleep apnea and no SIDS-specific significance of the pathological data of apoptosis was found and we therefore could not support either the traditional apnea-related hypothesis of SIDS and the recent arousal deficiency theory of SIDS. Further investigation of the distribution of apoptosis in other sites within the brainstem seems desirable.
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.
4. Discussion The presence of apoptosis in the brainstem of the SIDS victims had also been reported previously [24–26]. A recent report [24] described a high incidence of apoptosis within
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Pathological data on apoptosis in the brainstem and physiological data on sleep apnea in SIDS victims Toshiko Sawaguchi a,∗ , Patricia Franco b , Hazim Kadhim b , Jose Groswasser b , Martine Sottiaux b , Hiroshi Nishida c , Andre Kahn b a
Department of Legal Medicine, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan b Children’s Hospital Reine Fabiola, Free University of Brussels, Brussels, Belgium c Maternal and Perinatal Center, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan
Abstract The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death and its cause is still unknown. A chronic hypoxic situation has been shown to exist in the brains of SIDS victims and apoptosis has been demonstrated in hypoxic situations. In this study, the correlation between apoptotic neurons or glias and sleep apnea in SIDS was investigated in the brainstem of SIDS victims. Materials and methods: In a cohort of 27,000 infants studied prospectively to characterize their sleep–wake behavior, 38 infants died under 6 months of age. They included 26 cases of SIDS. The frequency and duration of sleep apnea were analyzed. The brainstem material was collected and terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method was carried out. The density of TUNEL-positive neurons or glias was measured quantitatively. Correlation analyses were carried out between the apoptosis-associated pathological data and the physiological data of sleep apnea. Results: No significant negative or positive correlation between the density of TUNEL-positive neurons or glias and the characteristics of sleep apnea was observed in SIDS victims. No statistically significant differences associated with apoptotic neurons and glias were observed between SIDS and non-SIDS. Conclusions: The pathological findings of apoptosis were not in agreement with the hypothesis refer to apnea and arousal phenomenon in pathophysiology of SIDS. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Apoptosis; Terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL); Brainstem; Obstructive apnea; Central apnea; Arousal; Sudden infant death syndrome (SIDS)
1. Introduction The sudden infant death syndrome (SIDS) is the main cause of postneonatal infant deaths. SIDS is 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 causes and pathophysiology of SIDS have long been investigated without any definite conclusions being reached. Several etiological hypotheses have been proposed to explain the mechanisms responsible for SIDS, including the role of apnea [2–4] and of arousal [5–11]. Neuropathological studies showed that the brainstems of SIDS victims were characterized by minute changes [12–23], such as gliosis [12–14,17,18,20,22] or apoptosis [24–26], attributed to hypoxic insult. ∗ Corresponding author. Tel.: +81-3-5269-7300; fax: +81-3-5269-7300. E-mail address: [email protected] (T. Sawaguchi).
Therefore, to investigate the apnea and the arousal deficiency theories of SIDS, the presence of apoptosis in the arousal pathway of the brainstem in SIDS victims was investigated and their association with sleep apnea was analyzed.
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. These were obtained 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
0928-4680/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2004.01.003
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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 or family history of apnea, apparent life-threatening events (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. Postmortem examination suggested that 26 had died of SIDS [26]; 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 [27–30]. The recordings were made in a quiet and darkened room at an ambient temperature between 20 and 23 ◦ C. All infants slept in supine position without restraint. 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 electrooculograms 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
Table 1 General profiles of each case Case no.
Sex
Gestational age (weeks)
Postneonatal age (weeks)
Cause of death
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
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 2 3 4 5 6 7 8 9 10 11 12
M M F M M M F M M F M F
39 40 40 33 40 37 37 39 40 37 39 40
24 11 6 21 14 4 5 6 7 7 9 9
Meningitis and brain infarction Pneumonia Myocarditis Varicella Cardiopathy with pulmonary hypertension Syndrome of Opitz Hepatitis and general infection Bronchopneumopathy Myocarditis Infanticide Bronchopneumopathy Bronchopneumopathy
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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.
Mannheim, Germany) [33–36]. Prior to the TUNEL method, the sections were pretreated by microwave irradiation in a 0.01 M citric acid buffer (pH 6.0) at 800 W for 5 min in a microwave device (Panasonic), then the TUNEL reaction was allowed to take place in the same manner described in previously published papers [34,35].
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 [27–32]. Arousals from sleep 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 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 seconds) 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 was computed.
2.2.4. Quantification of immunohistochemical presentations Measurements were made in the periaqueductal gray (PAG) matter and dorsal raphe nucleus of the midbrain and the PPTN (compact part: PPTNc and dissipated parts: PPTNd). The numbers of TUNEL-positive neurons and glias were counted manually. Each counting procedure in an area 625 m × 102 m was repeated five times at different overlapping sites and an average was recorded. The density was expressed as a percentage of the number of reaction-positive neurons and glias divided by the number of total neurons and glias. The pathological measurements were made twice by the same pathologist and data with large standard deviations were recounted or rejected.
2.2. Pathological analyses 2.2.1. Subjects A total of 48 paraffin blocks of brainstems were collected from the autopsied brains of the 38 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. 2.2.2. Neurohistological examination Hematoxylin–eosin (HE) stain was the standard staining method for neurohistological examination. 2.2.3. Immunohistochemical examination The blocks were subjected to terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method using in situ cell death detection kit (Boehringer
2.3. Data analysis 2.3.1. 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 not contact with the pathologist. 2.3.2. Matching the physiological and pathological data For each infant, the type and scores of apneas were correlated with the neuropatho-logical findings obtained from the postmortem studies. Correlation was analyzed and Pearson’s correlation coefficient was computed by SPSS Version 8.0. In addition, the intercorrelations between the physiological and pathological data were separately analyzed for the SIDS group and non-SIDS group. 2.4. Confirmation of the pathological characteristics of the SIDS and non-SIDS groups Nonparametric test (Kruskal–Wallis test) was carried out to evaluate the significant difference of density of TUNEL-positive neurons and glias in each site of the brainstem between SIDS and non-SIDS groups. 2.5. 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.
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Table 2 Correlation coefficients between pathological data on TUNEL-positive glia and physiological data on sleep apnea in SIDS victims Obstructive apnea Frequency (no./s)
Central apnea Duration (s)
Frequency (no./s)
0.1660 0.722
−0.0818 0.862
−0.0048 0.992
0.4280 0.338
−0.1138 0.808
−0.1454 0.756
−0.2319 0.617
−0.2417 0.602
Pedunculopontine tegmentum nucleus (compact part) Correlation coefficient 0.1316 Significant value 0.668
−0.1530 0.618
−0.1848 0.546
−0.2989 0.321
Pedunculopontine tegmentum nucleus (dissipated part) Correlation coefficient −0.3079 Significant value 0.553
−0.6501 0.162
0.0331 0.950
0.3311 0.521
Dorsal raphe nucleus Correlation coefficient Significant value Periaqueductal gray matter Correlation coefficient Significant value
3. Results 3.1. Standard neurohistological examination 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 a tumor with hemorrhagic infarct and polymicrogyria were found. The former two infants were reclassified as control cases. 3.2. Matching of physiological and pathological data 3.2.1. SIDS cases No significant positive or negative correlations were found between the density of TUNEL-positive neurons or glias in the arousal pathway of the midbrain and the each characteristic of sleep apnea were found in SIDS victims (Table 2). 3.2.2. Non-SIDS cases In non-SIDS cases, there is no significant correlation between pathological data on apoptosis in each site and physiological data of each sleep apnea. 3.3. Confirmation of the pathological characteristics of the SIDS and non-SIDS groups As the result of the nonparametric test (Kruskal–Wallis test), no significant difference of pathological data associated with apoptosis was found between in SIDS group and in non-SIDS groups.
Duration (s)
the brainstem of SIDS victims, mainly within the gracile and cuneate nuclei, spinal trigeminal tract neurons, tractus solitatius nucleus, lateral reticular formation and lateral cuneate nucleus [24]. It was hypothesized that hypoxia was responsible for the increase in TUNEL-positive cells in the brains of SIDS victims [24]. In contrast to this present study, no apoptosis was reported, however, in the dorsal raphe nucleus and PAG matter of the midbrain and the PPTN. These arousal pathways were not investigated in the previous study. In the present study, no significant correlation was found between apoptosis and the characteristics of sleep apnea in the arousal pathway of SIDS. In the previously reported paper, the increase of apoptotic neurons was observed in the watershed area in the brain and, consequently, the appearance of apoptosis in SIDS has been assumed to be hypoxic [25]. In the present study, the contribution of hypoxia to the development of apoptosis in the arousal pathway was not confirmed. In conclusion, no SIDS-specific correlation between the pathological findings of apoptosis and the physiological data of sleep apnea and no SIDS-specific significance of the pathological data of apoptosis was found and we therefore could not support either the traditional apnea-related hypothesis of SIDS and the recent arousal deficiency theory of SIDS. Further investigation of the distribution of apoptosis in other sites within the brainstem seems desirable.
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.
4. Discussion The presence of apoptosis in the brainstem of the SIDS victims had also been reported previously [24–26]. A recent report [24] described a high incidence of apoptosis within
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