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The relationship between neuronal plasticity and serotonergic neurons in the brainstem of SIDS victims
Early Human Development 75 Suppl. (2003) S139 – S146 www.elsevier.com/locate/earlhumdev
The relationship between neuronal plasticity and serotonergic neurons in the brainstem of SIDS victims Toshiko Sawaguchi a,*, Franco Patricia 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-ku, 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 Background: The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death and its cause is still unknown. Recently, the medullary serotonergic network deficiency theory has been proposed and an association between SIDS and neuronal plasticity has also been suggested. The growth-associated phosphoprotein 43 (GAP43) is a marker of synaptic plasticity and is critical for normal development of the serotonergic innervation. Therefore, the characteristics of GAP43-positive elements and their association with serotonergic neurons were here investigated in the brainstem of SIDS victims. Materials and methods: The materials of this study included 26 cases of SIDS and 12 control cases. The brainstem material was collected and the immunohistochemistry of GAP43 and tryptophan hydroxylase (TrypH) carried out. The density of GAP43-positive neurons and dendrites and of TrypH-positive neurons were measured quntitatively. Nonparametric analyses of GAP43 between SIDS and non-SIDS and correlation analyses between GAP43 and TrypH were performed. Results: No significant difference in GAP43-associated findings was found between SIDS and non-SIDS nor any significant correlation between GAP43-associated findings and TrypHpositive neurons. Conclusions:. The results of this study were not in agreement with the association of GAP43 with SIDS and with serotonergic innervation in SIDS. D 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Growth-associated phosphoprotein 43 (GAP43); Tryptophan hydroxylase (TrypH); Serotonin; Brainstem; Sudden infant death syndrome (SIDS)
* Corresponding author. Tel./fax: +81-3-5269-7300. E-mail address: [email protected] (T. Sawaguchi). 0378-3782/$ - see front matter D 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2003.08.017
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1. Introduction Several etiological hypotheses have been proposed to explain the mechanisms responsible for the sudden infant death syndrome (SIDS), which is the main cause of postneonatal infant deaths and 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 been investigated for many years and a number of causative hypotheses advanced, including the roles of apnea [2– 4] and of arousal [5– 11]. Neuropathological studies have shown 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 insults. It has been reported that TrypH activity decreased markedly in all brain areas, at all ages following hypoxia [27 –29], and an hypoxic environment may provoke both early and long-term alterations in serotonin metabolism [30]. In addition, the medullary serotonergic network deficiency theory in SIDS has been proposed [31] and the possibility has been discussed that serotonin maybe involved in the pathophysiology of SIDS [31 –37]. The growth-associated phosphoprotein 43 (GAP43) is a marker of synaptic plasticity and a key regulator in normal pathfinding and arborization of serotonergic axons during early brain development [38]. GAP43 is critical for normal development of the serotonergic innervation in the forebrain [38]. Therefore, the immunohistochemical appearance of GAP43 and TrypH in the brainstem in SIDS victims was investigated and their significance and association in SIDS were analyzed in this report. The hypothesis for this study was that ‘‘serotonergic deficiency in SIDS occurs from the effect of GAP43’’.
2. Materials and methods 2.1. 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 [26], of the remaining infants, 3 died from bronchopneumopathy, 2 from myocarditis, 1 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.
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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
M
39
24
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
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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A total of 48 paraffin blocks of brainstems were collected from each of the autopsied brains of the 38 infants who died unexpectedly: 7 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. Neurohistological examination Hematoxylin – eosin stain was the standard staining method for neurohistological examination. 2.3. Immunohistochemical examination The blocks were subjected to immunohistochemical examination, using an anti-GAP43 monoclonal antibody (YLEM, Italy) and an anti-TrypH monoclonal antibody (Oncogene, USA). For the antigen retrieval, the 4-Am-thick sections from each block were preincubated with 1 mM of an EDTA solution (pH, 7.0), using a microwave oven (Panasonic) at 800 W for 10 min to stain GAP43. After blocking intrinsic peroxidase by 3% hydrogen peroxide for 5 min and washing, the sections were incubated with the antibody for 3 days for TrypH and overnight for GAP43, both at 4 jC. Finally, GAP43 and TrypH were immunohistochemically examined with the aid of a LSAB2 kit (Dako) followed by the DAB reaction. 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 TrypH-positive neurons and GAP43-positive neurons and dendrites were counted manually. Each counting procedure in an area 625 102 Am2 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 dendrites divided by the number of total neurons. The pathological measurements were made twice by the same pathologist and data with large standard deviations were recounted or rejected. 2.5. Data analysis 2.5.1. 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. 2.5.2. 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 GAP43-positive neurons and dendrites in each site of the brainstem between SIDS and non-SIDS groups using SPSS ver. 8.0.
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2.5.3. Correlation analysis between GAP43-positive findings and TrypH-positive neurons Correlation analysis was carried out between the density of GAP43-positive neurons or dendrites and the density of TrypH-positive neurons using SPSS ver. 8.0. 2.6. 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.
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. Confirmation of the pathological characteristics of the SIDS and non-SIDS groups As the result of the nonparametric test (Kruskal –Wallis test), no significant correlation between the density of GAP43-positive elements was found between SIDS and non-SIDS groups.
Table 2 Correaltion coefficients between GAP43-positive neuron or dendrite and tryptophan hydroxylase (TrypH)positive neuron in SIDS victims Density of GAP43-positive neurons and density of TrypH-positive neurons Dorsal raphe nucleus Correlation coefficient Significant value Periaqueductal gray matter Correlation coefficient Significant value Pedunculopontine tegmentum Correlation coefficient Significant value Pedunculopontine tegmentum Correlation coefficient Significant value
0.4188 0.350 0.4426 0.320 nucleus (compact part) 0.4435 0.149 nucleus (dissipated part) 0.0270 0.960
Density of GAP43-positive dendrites and density of TrypH-positive neurons 0.4501 0.311 – – 0.1426 0.659 0.2214 0.673
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3.3. Correlation analysis between GAP43-positive findings and TrypH-positive neurons No SIDS-specific or non-SIDS-specific significant correlation was found between the density of GAP43-positive neurons or dendrites and the density of TrypH-positive neurons (Table 2).
4. Discussion GAP43 is a developmentally regulated phosphoprotein and also a presynaptic protein. Its distribution is largely restricted to the nervous system and it is frequently used as a marker for sprouting, because it is located in growth cones, maximally present during nervous system development and re-induced in injured and regenerating neural tissues [39]. The various interactions are thought to underlie the role of GAP43 in synaptic plasticity, participating in membrane extension during neuritogenesis, in neurotransmitter release and long-term potentiation [39]. GAP43 depletion affects neuronal pathfindings and reduces postnatal survival [39]. It is present at high levels during neurite extension, axonal elongation and synaptogenesis and peaks in postnatal days [40]. The high levels of GAP43 mRNA correlate strongly with the later stages of axon outgrowth and with the early stages of synapse formation [40]. During late synaptogenesis, growth-regulated proteins decrease in abundance, and growth-cone functions of motility and organelle assembly are being replaced by junctional contact and transmitter release [41]. There is a stage during which growth cone and synaptic properties overlap to some extent [41]. This overlap and its continuation allows for synaptic plasticity in both developing and adult nervous systems [41]. Recently, it has been proposed an association exists between neuronal plasticity and SIDS [42,43]. In these reports, immunohistochemistry of GAP43 and synaptophysin and Bielshowsky staining was used for the detection of neuronal plasticity. SIDS-specific correlations were made between the pathological data of neuronal plasticity in the arousal pathway and the physiological data of sleep apnea [40]. However, the induction or inhibition of GAP43-positivity by the hypoxia resulted by sleep apnea has not yet been proven. All the results of this present study were negative. In addition, there was no statistically significant difference between the density of TrypH-neurons in SIDS and non-SIDS [44]. These results did not support the serotonergic deficiency theory or the neuronal plasticity-deficiency theory of SIDS. However, further investigation of other sites in the brainstem and cerebrum should be carried out before a final conclusion is reached.
Acknowledgements The authors sincerely thank Prof. C. De Prez 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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The relationship between neuronal plasticity and serotonergic neurons in the brainstem of SIDS victims Toshiko Sawaguchi a,*, Franco Patricia 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-ku, 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 Background: The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death and its cause is still unknown. Recently, the medullary serotonergic network deficiency theory has been proposed and an association between SIDS and neuronal plasticity has also been suggested. The growth-associated phosphoprotein 43 (GAP43) is a marker of synaptic plasticity and is critical for normal development of the serotonergic innervation. Therefore, the characteristics of GAP43-positive elements and their association with serotonergic neurons were here investigated in the brainstem of SIDS victims. Materials and methods: The materials of this study included 26 cases of SIDS and 12 control cases. The brainstem material was collected and the immunohistochemistry of GAP43 and tryptophan hydroxylase (TrypH) carried out. The density of GAP43-positive neurons and dendrites and of TrypH-positive neurons were measured quntitatively. Nonparametric analyses of GAP43 between SIDS and non-SIDS and correlation analyses between GAP43 and TrypH were performed. Results: No significant difference in GAP43-associated findings was found between SIDS and non-SIDS nor any significant correlation between GAP43-associated findings and TrypHpositive neurons. Conclusions:. The results of this study were not in agreement with the association of GAP43 with SIDS and with serotonergic innervation in SIDS. D 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Growth-associated phosphoprotein 43 (GAP43); Tryptophan hydroxylase (TrypH); Serotonin; Brainstem; Sudden infant death syndrome (SIDS)
* Corresponding author. Tel./fax: +81-3-5269-7300. E-mail address: [email protected] (T. Sawaguchi). 0378-3782/$ - see front matter D 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2003.08.017
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1. Introduction Several etiological hypotheses have been proposed to explain the mechanisms responsible for the sudden infant death syndrome (SIDS), which is the main cause of postneonatal infant deaths and 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 been investigated for many years and a number of causative hypotheses advanced, including the roles of apnea [2– 4] and of arousal [5– 11]. Neuropathological studies have shown 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 insults. It has been reported that TrypH activity decreased markedly in all brain areas, at all ages following hypoxia [27 –29], and an hypoxic environment may provoke both early and long-term alterations in serotonin metabolism [30]. In addition, the medullary serotonergic network deficiency theory in SIDS has been proposed [31] and the possibility has been discussed that serotonin maybe involved in the pathophysiology of SIDS [31 –37]. The growth-associated phosphoprotein 43 (GAP43) is a marker of synaptic plasticity and a key regulator in normal pathfinding and arborization of serotonergic axons during early brain development [38]. GAP43 is critical for normal development of the serotonergic innervation in the forebrain [38]. Therefore, the immunohistochemical appearance of GAP43 and TrypH in the brainstem in SIDS victims was investigated and their significance and association in SIDS were analyzed in this report. The hypothesis for this study was that ‘‘serotonergic deficiency in SIDS occurs from the effect of GAP43’’.
2. Materials and methods 2.1. 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 [26], of the remaining infants, 3 died from bronchopneumopathy, 2 from myocarditis, 1 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.
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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
M
39
24
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
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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A total of 48 paraffin blocks of brainstems were collected from each of the autopsied brains of the 38 infants who died unexpectedly: 7 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. Neurohistological examination Hematoxylin – eosin stain was the standard staining method for neurohistological examination. 2.3. Immunohistochemical examination The blocks were subjected to immunohistochemical examination, using an anti-GAP43 monoclonal antibody (YLEM, Italy) and an anti-TrypH monoclonal antibody (Oncogene, USA). For the antigen retrieval, the 4-Am-thick sections from each block were preincubated with 1 mM of an EDTA solution (pH, 7.0), using a microwave oven (Panasonic) at 800 W for 10 min to stain GAP43. After blocking intrinsic peroxidase by 3% hydrogen peroxide for 5 min and washing, the sections were incubated with the antibody for 3 days for TrypH and overnight for GAP43, both at 4 jC. Finally, GAP43 and TrypH were immunohistochemically examined with the aid of a LSAB2 kit (Dako) followed by the DAB reaction. 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 TrypH-positive neurons and GAP43-positive neurons and dendrites were counted manually. Each counting procedure in an area 625 102 Am2 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 dendrites divided by the number of total neurons. The pathological measurements were made twice by the same pathologist and data with large standard deviations were recounted or rejected. 2.5. Data analysis 2.5.1. 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. 2.5.2. 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 GAP43-positive neurons and dendrites in each site of the brainstem between SIDS and non-SIDS groups using SPSS ver. 8.0.
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2.5.3. Correlation analysis between GAP43-positive findings and TrypH-positive neurons Correlation analysis was carried out between the density of GAP43-positive neurons or dendrites and the density of TrypH-positive neurons using SPSS ver. 8.0. 2.6. 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.
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. Confirmation of the pathological characteristics of the SIDS and non-SIDS groups As the result of the nonparametric test (Kruskal –Wallis test), no significant correlation between the density of GAP43-positive elements was found between SIDS and non-SIDS groups.
Table 2 Correaltion coefficients between GAP43-positive neuron or dendrite and tryptophan hydroxylase (TrypH)positive neuron in SIDS victims Density of GAP43-positive neurons and density of TrypH-positive neurons Dorsal raphe nucleus Correlation coefficient Significant value Periaqueductal gray matter Correlation coefficient Significant value Pedunculopontine tegmentum Correlation coefficient Significant value Pedunculopontine tegmentum Correlation coefficient Significant value
0.4188 0.350 0.4426 0.320 nucleus (compact part) 0.4435 0.149 nucleus (dissipated part) 0.0270 0.960
Density of GAP43-positive dendrites and density of TrypH-positive neurons 0.4501 0.311 – – 0.1426 0.659 0.2214 0.673
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3.3. Correlation analysis between GAP43-positive findings and TrypH-positive neurons No SIDS-specific or non-SIDS-specific significant correlation was found between the density of GAP43-positive neurons or dendrites and the density of TrypH-positive neurons (Table 2).
4. Discussion GAP43 is a developmentally regulated phosphoprotein and also a presynaptic protein. Its distribution is largely restricted to the nervous system and it is frequently used as a marker for sprouting, because it is located in growth cones, maximally present during nervous system development and re-induced in injured and regenerating neural tissues [39]. The various interactions are thought to underlie the role of GAP43 in synaptic plasticity, participating in membrane extension during neuritogenesis, in neurotransmitter release and long-term potentiation [39]. GAP43 depletion affects neuronal pathfindings and reduces postnatal survival [39]. It is present at high levels during neurite extension, axonal elongation and synaptogenesis and peaks in postnatal days [40]. The high levels of GAP43 mRNA correlate strongly with the later stages of axon outgrowth and with the early stages of synapse formation [40]. During late synaptogenesis, growth-regulated proteins decrease in abundance, and growth-cone functions of motility and organelle assembly are being replaced by junctional contact and transmitter release [41]. There is a stage during which growth cone and synaptic properties overlap to some extent [41]. This overlap and its continuation allows for synaptic plasticity in both developing and adult nervous systems [41]. Recently, it has been proposed an association exists between neuronal plasticity and SIDS [42,43]. In these reports, immunohistochemistry of GAP43 and synaptophysin and Bielshowsky staining was used for the detection of neuronal plasticity. SIDS-specific correlations were made between the pathological data of neuronal plasticity in the arousal pathway and the physiological data of sleep apnea [40]. However, the induction or inhibition of GAP43-positivity by the hypoxia resulted by sleep apnea has not yet been proven. All the results of this present study were negative. In addition, there was no statistically significant difference between the density of TrypH-neurons in SIDS and non-SIDS [44]. These results did not support the serotonergic deficiency theory or the neuronal plasticity-deficiency theory of SIDS. However, further investigation of other sites in the brainstem and cerebrum should be carried out before a final conclusion is reached.
Acknowledgements The authors sincerely thank Prof. C. De Prez 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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