Neurochem. Int. Vol. 20, No. 1, pp. 97 102, 1992
0197-0186/9255.00+0.00 Pergamon Press plc
Printed in Great Britain
BETA-ENDORPHIN A N D N E U R O T E N S I N IN BRAINSTEM A N D CEREBROSPINAL FLUID IN THE S U D D E N I N F A N T DEATH SYNDROME* A. COQUEREL, M. BUSER,J. TAYOT, F. PFAFF, F. MATRAYand B. PROUST
Centre Hospitalier Universitaire, 76031 Rouen, France (Received 13 January 1991 ; accepted 3 April 1991)
Abstract--Beta-endorphin (BE) and neurotensin (NT) are two neuropeptides which induce apneas. In infants who died of Sudden Infant Death Syndrome (SIDS) we measured, in brainstem and CSF, BE and NT by IRMA and RIA respectively. BE and NT levels are compared to same aged infant and adult controls. CSF BE level was significantlyhigher in SIDS than in the two control groups (86 ± 14 vs 33 ± 13 and 16±5 pmol/l). In 6 SIDS victims NT and BE were assayed in 5 brainstem sections, each of them divided in median, intermediate and lateral parts. We found high levels of BE in every fragment (3-11 pmol/mg protein) while NT elevated values were restricted to the mesencephalicregions (I .4-12 pmol/mg), the medial pons (6 pmol/mg) and the intermediate parts of the medulla (including the olive: 1.3-1.6 pmol/mg). These'results support the hypothesis that NT and/or BE could induce or participate to the fatal issue of SIDS.
we studied BE and, comparatively, a non-opioid peptide : neurotensin (NT). This tridecapeptide produces hypotensive and kinine-like properties after i.v. administration. NT has also numerous powerful effects after central administration like sedation and hypothermia (Nemeroff et al., 1977), and antinocisponsive properties (Clineschmidt et al., 1979). Opioid peptides and NT are involved in respiratory control. The central respiratory rhythm is produced by neurons located :
The Sudden Infant Death Syndrome (SIDS) is the primary cause of death of infants between one month and one year of age in developed countries. To demonstrate evidence of SIDS, the unexpected death has to occur in apparently healthy infants and must be characterized by a negative necropsic examination. Until today the etiology(ies) remain(s) unknown even if for two decades apnea has been supposed to be the common feature. In 1972 Steinschneider observed and documented several prolonged apneas which could lead to SIDS unless rescuscitation was attempted. Kuich and Zimmerman (1981) noticed that there are many analogies between SIDS and the morphine overdose syndrome and they proposed the hypothesis that potent opioid peptide(s), like beta-endorphin (BE), could be involved in SIDS apneas. Orlowski (1986) found high BE levels in the cerebrospinal fluid (CSF) of infants who presented apnea. In 1983, Pasi et al. reported 2 cases of SIDS with high BE contents in several discrete brainstem areas. To test whether the endorphin hypothesis could be confirmed
(i) in the bulbar dorsal and ventral respiratory nuclei [i.e. nucleus of the tractus solitarius (NTS) and the nuclei ambiguus plus retroambigualis, respectively] ; (ii) at the pontine level (pneumotaxic centre) by the parabrachialis medialis and KollikerFuse nuclei. These respiratory neurons are depressed by microiontophoretic applications of both mu and delta opiate agonists such as morphine or levorphanol and methionine-enkephaline, respectively (DenavitSaubi~ et al., 1978). With autoradiographic studies Sales et al. (1985) have shown that delta agonist ligands bound preferentially to the pneumotaxic centre while mu agonists were preponderant in the NTS. In unanesthetized rats intraperitoneal opiate administrations induce different types of respiratory depression: mu agonists reduce the tidal volume while delta opiates reduce preferentially the respira-
* This paper presents parts of a talk given at a workshop on Nucleus tractus solitarius : Anatomy, Physiology and Implications in Sudden Infant Death Syndrome held at the lOth European Winter Conference on Brain Research, Les Arcs, Val d'Is6re, France, 3-10 March, 1990. The workshop was organized by N. Kopp, Laboratories of Pathology, Alexis Carrel Medical Faculty, Lyon, France and by M. Denavit-Saubi6, Neurophysiology Laboratories of "C.N.R.S.", 91190 Gif sur Yvette, France. 97
98
A. COQUEREL fl ~://.
tory frequency (Morin-Surun et al., 1984). In cats NT injections in the NTS induce apneustic breathing by increasing the firing o f respiratory related neurons (Morin-Surun et al., 1986). We thus assayed BE and N T levels in C S F and brainstem o f SIDS victims. EXPERIMENTAL PROCEDURES
Ii~[~lrlt.s' Studies were conducted with 1 5-months-old infants. The consent of the parents was obtained prior to post-mortem examinations. The SIDS diagnosis was established on the basis of both necropsic examination and clinical investigation negativities: these consisted in bacterial and viral samples and the interview of parents. We pointed out the lack of antecedent of convulsion, dyspnea, vomiting and the absence of fever or acute disease during the days preceding the death. CSF studies ( i/ In S1DS inIbnts : lumbar punctures were done to verify CSF sterility and biochemical normality (protein, glucose and chloride contents). (ii) Control values: CSF samples were obtained from infants hospitalized for acute benign diseases (such as diarrhea and/or vomiting with or without fever, upper airways or bronchial infections). (iii) We also measured CSF from adults who were examined for a post-meningitis control or during neuroradiological examinations. All these CSF samples were normal for cytology and classical biochemical parameters. SIDS CSF were obtained within a 3 24 h post-mortem delay, by lumbar route. They were centrifuged (5 min at 500 g) and the supernatant was immediately frozen and then kept at - 25°C until assay. Control CSF of infants and adults were frozen within a delay of 3 ~ h after puncture. Brainstem studies SIDS brainstems were removed within a 6 24 h postmortem delay and kept at -80°C until BE and NT assays. After thawing, each brainstem was cut into 5 sections (upper and lower mesencephalon, middle pons, upper and lower medulla) and each of them divided in 3 parts : median, intermediate and lateral (see Fig. 1). During brainstem studies we did not test infant or adult controls because we did not obtain any brain in good conditions (i.e. without previous brain suffering or damage, lack of prolonged agony or post mortem delay shorter than 24 h). For each piece of brainstem, peptide extraction was obtained in a 5 10 vol. HCI 0.1 M solution, at 95'C during 5 min. After sonication (1 min 20,000 Hz at 100% of a 60 W sonotrode) samples were centrifuged twice. The second supernatant was buffered with NaOH and Tris in order to adjust pH (7.60+0.05). The regional distribution of immunoreactive BE and NT were expressed in pmol/mg of protein. Protein concentrations were determined by the method of Lowry. Radio-immunoassay procedures BE (61 91 beta-lipotropin hormone, b-LPH) "~as measured by an Immuno-Radio-Metric Assay (IRMA) adapted from the BE kit of Nichols Institute (San Juan Capistrano, CA, U.S.A.). Briefly, the test principle consists in a binding
of the N and C paris of BE in a sandwich of two specific rabbit antibodies: the first antibody is immobilized on a plastic bead and the second is labelled with 125 Iodine. Incubation conditions were done with 300 /xl sample volume. The standard curve was obtained with synthetic human BE (Sigma, Paris, France) diluted in artificial CSF (NaCI: I(10 mmol/1, Na2HPO4/NaH2PO4 50 mmol/l, bovine serum albumin 1 g/l ; pH = 7.4). The standard curve range was 4400 pmol/I. The cross reactivities were 16% (w/w) with b-LPH and
0197-0186/9255.00+0.00 Pergamon Press plc
Printed in Great Britain
BETA-ENDORPHIN A N D N E U R O T E N S I N IN BRAINSTEM A N D CEREBROSPINAL FLUID IN THE S U D D E N I N F A N T DEATH SYNDROME* A. COQUEREL, M. BUSER,J. TAYOT, F. PFAFF, F. MATRAYand B. PROUST
Centre Hospitalier Universitaire, 76031 Rouen, France (Received 13 January 1991 ; accepted 3 April 1991)
Abstract--Beta-endorphin (BE) and neurotensin (NT) are two neuropeptides which induce apneas. In infants who died of Sudden Infant Death Syndrome (SIDS) we measured, in brainstem and CSF, BE and NT by IRMA and RIA respectively. BE and NT levels are compared to same aged infant and adult controls. CSF BE level was significantlyhigher in SIDS than in the two control groups (86 ± 14 vs 33 ± 13 and 16±5 pmol/l). In 6 SIDS victims NT and BE were assayed in 5 brainstem sections, each of them divided in median, intermediate and lateral parts. We found high levels of BE in every fragment (3-11 pmol/mg protein) while NT elevated values were restricted to the mesencephalicregions (I .4-12 pmol/mg), the medial pons (6 pmol/mg) and the intermediate parts of the medulla (including the olive: 1.3-1.6 pmol/mg). These'results support the hypothesis that NT and/or BE could induce or participate to the fatal issue of SIDS.
we studied BE and, comparatively, a non-opioid peptide : neurotensin (NT). This tridecapeptide produces hypotensive and kinine-like properties after i.v. administration. NT has also numerous powerful effects after central administration like sedation and hypothermia (Nemeroff et al., 1977), and antinocisponsive properties (Clineschmidt et al., 1979). Opioid peptides and NT are involved in respiratory control. The central respiratory rhythm is produced by neurons located :
The Sudden Infant Death Syndrome (SIDS) is the primary cause of death of infants between one month and one year of age in developed countries. To demonstrate evidence of SIDS, the unexpected death has to occur in apparently healthy infants and must be characterized by a negative necropsic examination. Until today the etiology(ies) remain(s) unknown even if for two decades apnea has been supposed to be the common feature. In 1972 Steinschneider observed and documented several prolonged apneas which could lead to SIDS unless rescuscitation was attempted. Kuich and Zimmerman (1981) noticed that there are many analogies between SIDS and the morphine overdose syndrome and they proposed the hypothesis that potent opioid peptide(s), like beta-endorphin (BE), could be involved in SIDS apneas. Orlowski (1986) found high BE levels in the cerebrospinal fluid (CSF) of infants who presented apnea. In 1983, Pasi et al. reported 2 cases of SIDS with high BE contents in several discrete brainstem areas. To test whether the endorphin hypothesis could be confirmed
(i) in the bulbar dorsal and ventral respiratory nuclei [i.e. nucleus of the tractus solitarius (NTS) and the nuclei ambiguus plus retroambigualis, respectively] ; (ii) at the pontine level (pneumotaxic centre) by the parabrachialis medialis and KollikerFuse nuclei. These respiratory neurons are depressed by microiontophoretic applications of both mu and delta opiate agonists such as morphine or levorphanol and methionine-enkephaline, respectively (DenavitSaubi~ et al., 1978). With autoradiographic studies Sales et al. (1985) have shown that delta agonist ligands bound preferentially to the pneumotaxic centre while mu agonists were preponderant in the NTS. In unanesthetized rats intraperitoneal opiate administrations induce different types of respiratory depression: mu agonists reduce the tidal volume while delta opiates reduce preferentially the respira-
* This paper presents parts of a talk given at a workshop on Nucleus tractus solitarius : Anatomy, Physiology and Implications in Sudden Infant Death Syndrome held at the lOth European Winter Conference on Brain Research, Les Arcs, Val d'Is6re, France, 3-10 March, 1990. The workshop was organized by N. Kopp, Laboratories of Pathology, Alexis Carrel Medical Faculty, Lyon, France and by M. Denavit-Saubi6, Neurophysiology Laboratories of "C.N.R.S.", 91190 Gif sur Yvette, France. 97
98
A. COQUEREL fl ~://.
tory frequency (Morin-Surun et al., 1984). In cats NT injections in the NTS induce apneustic breathing by increasing the firing o f respiratory related neurons (Morin-Surun et al., 1986). We thus assayed BE and N T levels in C S F and brainstem o f SIDS victims. EXPERIMENTAL PROCEDURES
Ii~[~lrlt.s' Studies were conducted with 1 5-months-old infants. The consent of the parents was obtained prior to post-mortem examinations. The SIDS diagnosis was established on the basis of both necropsic examination and clinical investigation negativities: these consisted in bacterial and viral samples and the interview of parents. We pointed out the lack of antecedent of convulsion, dyspnea, vomiting and the absence of fever or acute disease during the days preceding the death. CSF studies ( i/ In S1DS inIbnts : lumbar punctures were done to verify CSF sterility and biochemical normality (protein, glucose and chloride contents). (ii) Control values: CSF samples were obtained from infants hospitalized for acute benign diseases (such as diarrhea and/or vomiting with or without fever, upper airways or bronchial infections). (iii) We also measured CSF from adults who were examined for a post-meningitis control or during neuroradiological examinations. All these CSF samples were normal for cytology and classical biochemical parameters. SIDS CSF were obtained within a 3 24 h post-mortem delay, by lumbar route. They were centrifuged (5 min at 500 g) and the supernatant was immediately frozen and then kept at - 25°C until assay. Control CSF of infants and adults were frozen within a delay of 3 ~ h after puncture. Brainstem studies SIDS brainstems were removed within a 6 24 h postmortem delay and kept at -80°C until BE and NT assays. After thawing, each brainstem was cut into 5 sections (upper and lower mesencephalon, middle pons, upper and lower medulla) and each of them divided in 3 parts : median, intermediate and lateral (see Fig. 1). During brainstem studies we did not test infant or adult controls because we did not obtain any brain in good conditions (i.e. without previous brain suffering or damage, lack of prolonged agony or post mortem delay shorter than 24 h). For each piece of brainstem, peptide extraction was obtained in a 5 10 vol. HCI 0.1 M solution, at 95'C during 5 min. After sonication (1 min 20,000 Hz at 100% of a 60 W sonotrode) samples were centrifuged twice. The second supernatant was buffered with NaOH and Tris in order to adjust pH (7.60+0.05). The regional distribution of immunoreactive BE and NT were expressed in pmol/mg of protein. Protein concentrations were determined by the method of Lowry. Radio-immunoassay procedures BE (61 91 beta-lipotropin hormone, b-LPH) "~as measured by an Immuno-Radio-Metric Assay (IRMA) adapted from the BE kit of Nichols Institute (San Juan Capistrano, CA, U.S.A.). Briefly, the test principle consists in a binding
of the N and C paris of BE in a sandwich of two specific rabbit antibodies: the first antibody is immobilized on a plastic bead and the second is labelled with 125 Iodine. Incubation conditions were done with 300 /xl sample volume. The standard curve was obtained with synthetic human BE (Sigma, Paris, France) diluted in artificial CSF (NaCI: I(10 mmol/1, Na2HPO4/NaH2PO4 50 mmol/l, bovine serum albumin 1 g/l ; pH = 7.4). The standard curve range was 4400 pmol/I. The cross reactivities were 16% (w/w) with b-LPH and
