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The mode of cell death in the developing brain
Placenta
680
The Mode
of Cell Death
in the Developing
(1998),
Vol. 19
London,
UK
Brain
A. D. Edwards Department
of Paediatrics
and Neonatal
Medicine,
Royal Postgraduate
The pathogenesis of brain damage following hypoxicischaemic injury in the developing brain is complex and incompletely understood (Vannucci, 1990). However, noninvasive studies using 31P magnetic resonance spectroscopy (MRS) have shown that hypoxia-ischaemia followed by resuscitation leads not only to primary cellular injury during the period of the insult, but also to a delayed secondary injury 24 to 48 h later (Wyatt et al., 1989). The degree of later neurodevelopmental impairment is directly related to the severity of the secondary injury (Roth et al., 1992). Measurements using the ‘13Xe clearance technique or nearinfrared spectroscopy have shown that cerebral blood flow is increased, although the normal response to changes in carbon dioxide is lost (Pryds, 1991; Reynolds et al., 1991). Despite the high cerebral blood flow during this period, there is an accumulation of lactate within the brain, and there is a direct correlation between the severity of delayed cerebral injury and the concentration of lactate in the brain soon after birth (Hanrahan et al., 1996). In infants who go on to develop neurodevelopmental impairment cerebral lactate concentrations remain high for many weeks. Magnetic resonance imaging has also helped to demonstrate the process of brain injury. Soon after an insult, diffusion weighted images are abnormal, while conventional Tl and T2 images become abnormal some days later (Cowan et al., 1994). It is also clear that brain injury and repair is a prolonged process: the concentration of lactate remains high for many weeks in the brains of infants who develop neurodevelopmental impairment (Yue et al., 1996), and brain structure shows significant alterations, including both the development of atrophy distant from the original site of injury and in some cases accelerated growth in areas of focal infarction (Rutherford et al., 1996). The mechanisms of secondary injury are poorly understood, but probably involve cellular injury mediated by excess concentrations of excitatory neurotransmitters (Rotman and Olney, 1987), free radical formation and lipid peroxidation (Watson and Ginsberg, 1989), activation of immune mechanisms (Giulian et al., 1991), and removal of trophic factors which normally support cell survival (Raff, 1992). Recent studies have demonstrated that the mechanisms of programmed cell death are activated after hypoxia-ischaemia, and a considerable proportion of cells die not by necrosis but by apoptosis (Mehmet et al., 1994). Apoptosis was discovered in 1972 by Kerr, Wyllie and Currie who noted that many tumour cells underwent a different and unusual mode of death without membrane rupture and inflammation. It is now under-
Medical
School,
Hammersmith
Hospitals,
stood that these apoptotic cells were demonstrating a physiological cell death that is ubiquitous and essential to the development and survival of multicellular organisms (Raff, 1992). This mode of death involves the activation of an intrinsic, constitutive ‘death programme’ that can be triggered by multiple stimuli, including loss of trophic factors, activation of specific membrane receptors, viral infection and cellular injury resulting from insults such as hypoxia-ischaemia or ionizing radiation. Apoptotic death is distinct from necrosis, requiring time, energy, and in some cases, gene transcription and translation. In a pathological study of infants who either died after birth asphyxia or underwent a sudden unexpected infant death, between one and three-quarters of all dead cells had the morphology of apoptosis (Edwards et al., 1997). Experimental data are accumulating which demonstrate that interruption of the apoptotic cascade will ameliorate brain injury after hypoxia-ischaemia. Studies in rodents have shown that: overexpression of the anti-apoptotic gene bcl-2 (Martinou et al., 1994), attenuation of pro-apoptotic genes such as P53 (Crumrine, Thomas and Morgan, 1994), or the administration of survival factors such as insulin-like growth factor-l (Gluckman et al., 1992) ameliorates neural damage after hypoxia-ischaemia. As apoptosis is an important part of the human response to intrauterine cerebral injury, clinically applicable anti-apoptotic strategies, such as moderate brain cooling (Edwards et al., 1995), may be worth evaluating for their therapeutic potential.
REFERENCES Cowan F, Pennock JM, Hanrahan D, Manji K & Edwards AD (1994) Early detection of infarction and hypoxic-ischaemic encephalopathy in neonates using diffusion weighted magnetic resonance imaging Neuropediatria,
25, 172-115.
Crumrine RC, Thomas AL & Morgan PF (1994) Attenuation of ~53 expression protects against focal ischemic damage in transgenic mice. 3 Cereb Blood Flow Metab, 14, 887-891. Edwards AD, Yue X, Squier MV, Thoresen M, Cady EB, Penrice J, Cooper C, Wyatt JS, Reynolds EOR & Mehmet H (1995) Specific inhibition of apoptosis after cerebral hypoxia-ischaemia by moderate postinsult hypothermia. Biochem Biophys Res Commun, 217, 1193-l 199. Edwards AD, Yue X, Cox P, Hope PL, Azzopardi D, Squier MV & Mehmet H. (1997) Apoptosis in the brains of infants suffering intrauterine cerebral injury. Pediatric Research, 42, 684689. Giulian D, Johnson B, Krebs JF, George JK & Tapscott M (1991) Microglial mitogens are produced in the developing and injured mammalian brain. J Cell Biol, 112, 323-333. Gluckman PD, Klempt N, Guan J, Mallard C, Sirimanne E, Dragunow M. Klempt M. Singh K. Williams CE & Nikolics K (1992) A role for 16F-l in ihe rescue-of 6NS neurons following hypoxiclischemic injury. B&hem Biophys Res Commun, 182, 593-599.
Conference
681
Report
Hanrahan D, Sargentoni J, Azzopardi D, Manji K, Cowan F, Rutherford MA, Cox IJ, Bell JD, Bryant D & Edwards AD (1996) Cerebral metabolism within 18 hours of birth asphyxia: a proton magnetic resonance spectroscopy study. Pediarr Res, 39, W-590. Kerr JF, Wyllie AH & Currie AR (1972) Apoptosis, a basic biological phenomenon with wide-ranging implications in human tissue kinetics. Br3 Cancer, 36, 239-257. Martinou JC, Dubois Dauphin M, Staple JK, Rodriguez I, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S, Pietra C & Huarte J (1994) Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental &hernia. Neuron, 13, 1017-1030. Mehmet H, Yue X, Squier MV, Lorek A, Cady E, Penrice J, Sarraf C, Wylezinska M, Kirkbride V, Cooper C, Brown GC, Wyatt JS, Reynolds EOR & Edwards AD (1994) Increased apoptosis in the cingulate sulcus of newborn piglets following transient hypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult. Neurosci Lett, 181, 121-125. Pryds 0 (1991) Control of cerebral circulation in the high risk neonate. Ann Neural, 30, 321-329. Raff MC (1982) Social controls on cell survival and cell death. Narure, 356, 397-400. Reynolds EOR, McCormich DC, Roth SC, Edwards AD & Wyatt JS (1991) New non-invasive methods for the investigation of cerebral oxidative metabolism and haemodynamics in newborn infants. Ann Med, 23,681-686. Roth SC, Edwards AD, Cady EB, Delpy DT, Wyatt JS, Azzsopardi D, Baudin J, Townsend J, Stewart AL & Reynolds EOR (1992) Relation between cerebral oxidative metabolism following birth asphyxia and neurodevelopmental outcome and brain growth at one year. Dev Med Child Neural, 43, 285-295.
Apoptosis
P. N. Baker, Department
in the Human
S. C. Smith,
of Obstetrics
Rotman S & Olney JW (1987) Excitotoxicity and the NMDA receptor. TINS, 10, 299-302. Rutherford MA, Pennock JM, Schweiso J, Cowan F & Dubowitz L (1996) Hypoxic-ischaemic encephalopathy: early and late magnetic resonance imaging findings in relation to outcome. Arch Dis Child, 75, F145-F151. Vannucci RC (1990) Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage. Pediutr Rex, 27, 317-326. Watson BD & Ginsberg MD (1989) Ischemic injury in the brain. Role of oxygen radical-mediated processes. Ann NY Acad Sci, 559, 269-281. Wyatt JS, Edwards AD, Azzopardi D & Reynolds EOR (1989) Magnetic resonance and near infrared spectroscopy for investigation of perinatal hypoxic-ischaemic brain injury. Arch Dis Child, 64, 953-963. Yue X, Mehmet H, Penrice J, Cooper C, Cady E, Wyatt JS, Reynolds EOR, Edwards AD & Squier MV (1996) Apoptosis and necrosis in the newborn piglet brain following transient cerebral hypoxia-ischaemia. Neuropathol Appl Neurobiol, 22, 482-503.
DISCUSSION The following points were made. Magnesium is not protective against apoptosis. Steroids have not been fully evaluated, therefore there is conflicting interest. Antioxidants reduce apoptotic damage.
Placenta
E. Price and E. M. Symonds
and Gynaecology,
University
Hospital,
INTRODUCTION Apoptosis is associated with physiological or ‘programmed’ cell death, in contrast to necrosis which is associated with cell injury. It was first described in 1972 by Kerr et al. Apoptosis is involved in many physiological processes: normal tissue turnover, embryonic development, atrophy of tissues following removal of endocrine stimulus, etc. Defects in the process of apoptosis can lead to disease states (Savill, 1994). Apoptosis within the placenta has only recently been described (Smith, Baker and Symonds, 1997), and has not previously been quantified. Apoptotic cells are rapidly cleared (l-2 h), thus small changes in the absolute number of apoptotic cells are of great significance. There are no distinct features within the placentae of pregnancies complicated by intrauterine growth restriction (IUGR), but these placentae do tend to be smaller than those from normal pregnancies (Baker et al., 1995). Does an increase in the incidence of apoptosis account for this difference? The aims of this study were (1) to demonstrate apoptosis in the human placenta; (2) to quantify apoptosis throughout
Nottingham,
UK
pregnancy (see below); and (3) to investigate the role of apoptosis in the pathophysiology of IUGR.
METHODS We have quantified the incidence of apoptosis in 28 firsttrimester placental samples, and in the third-trimester placentae of 35 normal pregnancies. The percentage of apoptotic cells was determined by use of light microscopy. Paraffin-embedded sections were stained with haematoxylin and eosin. For each placenta, 50 high-power fields ( x 1000, oil immersion) were examined, and the number of apoptotic cells identified was expressed as a percentage of the total number: 5000-7000 cells have been counted for each sample. In addition the incidence of apoptosis has been quantified in the placentae of 19 pregnancies complicated by IUGR. Electron microscopy, fluorescence microscopy and TUNEL (terminal uracil nick-end labelling) staining have also been used to confirm the presence of apoptotic cells.
680
The Mode
of Cell Death
in the Developing
(1998),
Vol. 19
London,
UK
Brain
A. D. Edwards Department
of Paediatrics
and Neonatal
Medicine,
Royal Postgraduate
The pathogenesis of brain damage following hypoxicischaemic injury in the developing brain is complex and incompletely understood (Vannucci, 1990). However, noninvasive studies using 31P magnetic resonance spectroscopy (MRS) have shown that hypoxia-ischaemia followed by resuscitation leads not only to primary cellular injury during the period of the insult, but also to a delayed secondary injury 24 to 48 h later (Wyatt et al., 1989). The degree of later neurodevelopmental impairment is directly related to the severity of the secondary injury (Roth et al., 1992). Measurements using the ‘13Xe clearance technique or nearinfrared spectroscopy have shown that cerebral blood flow is increased, although the normal response to changes in carbon dioxide is lost (Pryds, 1991; Reynolds et al., 1991). Despite the high cerebral blood flow during this period, there is an accumulation of lactate within the brain, and there is a direct correlation between the severity of delayed cerebral injury and the concentration of lactate in the brain soon after birth (Hanrahan et al., 1996). In infants who go on to develop neurodevelopmental impairment cerebral lactate concentrations remain high for many weeks. Magnetic resonance imaging has also helped to demonstrate the process of brain injury. Soon after an insult, diffusion weighted images are abnormal, while conventional Tl and T2 images become abnormal some days later (Cowan et al., 1994). It is also clear that brain injury and repair is a prolonged process: the concentration of lactate remains high for many weeks in the brains of infants who develop neurodevelopmental impairment (Yue et al., 1996), and brain structure shows significant alterations, including both the development of atrophy distant from the original site of injury and in some cases accelerated growth in areas of focal infarction (Rutherford et al., 1996). The mechanisms of secondary injury are poorly understood, but probably involve cellular injury mediated by excess concentrations of excitatory neurotransmitters (Rotman and Olney, 1987), free radical formation and lipid peroxidation (Watson and Ginsberg, 1989), activation of immune mechanisms (Giulian et al., 1991), and removal of trophic factors which normally support cell survival (Raff, 1992). Recent studies have demonstrated that the mechanisms of programmed cell death are activated after hypoxia-ischaemia, and a considerable proportion of cells die not by necrosis but by apoptosis (Mehmet et al., 1994). Apoptosis was discovered in 1972 by Kerr, Wyllie and Currie who noted that many tumour cells underwent a different and unusual mode of death without membrane rupture and inflammation. It is now under-
Medical
School,
Hammersmith
Hospitals,
stood that these apoptotic cells were demonstrating a physiological cell death that is ubiquitous and essential to the development and survival of multicellular organisms (Raff, 1992). This mode of death involves the activation of an intrinsic, constitutive ‘death programme’ that can be triggered by multiple stimuli, including loss of trophic factors, activation of specific membrane receptors, viral infection and cellular injury resulting from insults such as hypoxia-ischaemia or ionizing radiation. Apoptotic death is distinct from necrosis, requiring time, energy, and in some cases, gene transcription and translation. In a pathological study of infants who either died after birth asphyxia or underwent a sudden unexpected infant death, between one and three-quarters of all dead cells had the morphology of apoptosis (Edwards et al., 1997). Experimental data are accumulating which demonstrate that interruption of the apoptotic cascade will ameliorate brain injury after hypoxia-ischaemia. Studies in rodents have shown that: overexpression of the anti-apoptotic gene bcl-2 (Martinou et al., 1994), attenuation of pro-apoptotic genes such as P53 (Crumrine, Thomas and Morgan, 1994), or the administration of survival factors such as insulin-like growth factor-l (Gluckman et al., 1992) ameliorates neural damage after hypoxia-ischaemia. As apoptosis is an important part of the human response to intrauterine cerebral injury, clinically applicable anti-apoptotic strategies, such as moderate brain cooling (Edwards et al., 1995), may be worth evaluating for their therapeutic potential.
REFERENCES Cowan F, Pennock JM, Hanrahan D, Manji K & Edwards AD (1994) Early detection of infarction and hypoxic-ischaemic encephalopathy in neonates using diffusion weighted magnetic resonance imaging Neuropediatria,
25, 172-115.
Crumrine RC, Thomas AL & Morgan PF (1994) Attenuation of ~53 expression protects against focal ischemic damage in transgenic mice. 3 Cereb Blood Flow Metab, 14, 887-891. Edwards AD, Yue X, Squier MV, Thoresen M, Cady EB, Penrice J, Cooper C, Wyatt JS, Reynolds EOR & Mehmet H (1995) Specific inhibition of apoptosis after cerebral hypoxia-ischaemia by moderate postinsult hypothermia. Biochem Biophys Res Commun, 217, 1193-l 199. Edwards AD, Yue X, Cox P, Hope PL, Azzopardi D, Squier MV & Mehmet H. (1997) Apoptosis in the brains of infants suffering intrauterine cerebral injury. Pediatric Research, 42, 684689. Giulian D, Johnson B, Krebs JF, George JK & Tapscott M (1991) Microglial mitogens are produced in the developing and injured mammalian brain. J Cell Biol, 112, 323-333. Gluckman PD, Klempt N, Guan J, Mallard C, Sirimanne E, Dragunow M. Klempt M. Singh K. Williams CE & Nikolics K (1992) A role for 16F-l in ihe rescue-of 6NS neurons following hypoxiclischemic injury. B&hem Biophys Res Commun, 182, 593-599.
Conference
681
Report
Hanrahan D, Sargentoni J, Azzopardi D, Manji K, Cowan F, Rutherford MA, Cox IJ, Bell JD, Bryant D & Edwards AD (1996) Cerebral metabolism within 18 hours of birth asphyxia: a proton magnetic resonance spectroscopy study. Pediarr Res, 39, W-590. Kerr JF, Wyllie AH & Currie AR (1972) Apoptosis, a basic biological phenomenon with wide-ranging implications in human tissue kinetics. Br3 Cancer, 36, 239-257. Martinou JC, Dubois Dauphin M, Staple JK, Rodriguez I, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S, Pietra C & Huarte J (1994) Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental &hernia. Neuron, 13, 1017-1030. Mehmet H, Yue X, Squier MV, Lorek A, Cady E, Penrice J, Sarraf C, Wylezinska M, Kirkbride V, Cooper C, Brown GC, Wyatt JS, Reynolds EOR & Edwards AD (1994) Increased apoptosis in the cingulate sulcus of newborn piglets following transient hypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult. Neurosci Lett, 181, 121-125. Pryds 0 (1991) Control of cerebral circulation in the high risk neonate. Ann Neural, 30, 321-329. Raff MC (1982) Social controls on cell survival and cell death. Narure, 356, 397-400. Reynolds EOR, McCormich DC, Roth SC, Edwards AD & Wyatt JS (1991) New non-invasive methods for the investigation of cerebral oxidative metabolism and haemodynamics in newborn infants. Ann Med, 23,681-686. Roth SC, Edwards AD, Cady EB, Delpy DT, Wyatt JS, Azzsopardi D, Baudin J, Townsend J, Stewart AL & Reynolds EOR (1992) Relation between cerebral oxidative metabolism following birth asphyxia and neurodevelopmental outcome and brain growth at one year. Dev Med Child Neural, 43, 285-295.
Apoptosis
P. N. Baker, Department
in the Human
S. C. Smith,
of Obstetrics
Rotman S & Olney JW (1987) Excitotoxicity and the NMDA receptor. TINS, 10, 299-302. Rutherford MA, Pennock JM, Schweiso J, Cowan F & Dubowitz L (1996) Hypoxic-ischaemic encephalopathy: early and late magnetic resonance imaging findings in relation to outcome. Arch Dis Child, 75, F145-F151. Vannucci RC (1990) Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage. Pediutr Rex, 27, 317-326. Watson BD & Ginsberg MD (1989) Ischemic injury in the brain. Role of oxygen radical-mediated processes. Ann NY Acad Sci, 559, 269-281. Wyatt JS, Edwards AD, Azzopardi D & Reynolds EOR (1989) Magnetic resonance and near infrared spectroscopy for investigation of perinatal hypoxic-ischaemic brain injury. Arch Dis Child, 64, 953-963. Yue X, Mehmet H, Penrice J, Cooper C, Cady E, Wyatt JS, Reynolds EOR, Edwards AD & Squier MV (1996) Apoptosis and necrosis in the newborn piglet brain following transient cerebral hypoxia-ischaemia. Neuropathol Appl Neurobiol, 22, 482-503.
DISCUSSION The following points were made. Magnesium is not protective against apoptosis. Steroids have not been fully evaluated, therefore there is conflicting interest. Antioxidants reduce apoptotic damage.
Placenta
E. Price and E. M. Symonds
and Gynaecology,
University
Hospital,
INTRODUCTION Apoptosis is associated with physiological or ‘programmed’ cell death, in contrast to necrosis which is associated with cell injury. It was first described in 1972 by Kerr et al. Apoptosis is involved in many physiological processes: normal tissue turnover, embryonic development, atrophy of tissues following removal of endocrine stimulus, etc. Defects in the process of apoptosis can lead to disease states (Savill, 1994). Apoptosis within the placenta has only recently been described (Smith, Baker and Symonds, 1997), and has not previously been quantified. Apoptotic cells are rapidly cleared (l-2 h), thus small changes in the absolute number of apoptotic cells are of great significance. There are no distinct features within the placentae of pregnancies complicated by intrauterine growth restriction (IUGR), but these placentae do tend to be smaller than those from normal pregnancies (Baker et al., 1995). Does an increase in the incidence of apoptosis account for this difference? The aims of this study were (1) to demonstrate apoptosis in the human placenta; (2) to quantify apoptosis throughout
Nottingham,
UK
pregnancy (see below); and (3) to investigate the role of apoptosis in the pathophysiology of IUGR.
METHODS We have quantified the incidence of apoptosis in 28 firsttrimester placental samples, and in the third-trimester placentae of 35 normal pregnancies. The percentage of apoptotic cells was determined by use of light microscopy. Paraffin-embedded sections were stained with haematoxylin and eosin. For each placenta, 50 high-power fields ( x 1000, oil immersion) were examined, and the number of apoptotic cells identified was expressed as a percentage of the total number: 5000-7000 cells have been counted for each sample. In addition the incidence of apoptosis has been quantified in the placentae of 19 pregnancies complicated by IUGR. Electron microscopy, fluorescence microscopy and TUNEL (terminal uracil nick-end labelling) staining have also been used to confirm the presence of apoptotic cells.