Hypoxia-ischaemia and the developing brain: Hypotheses regarding the pathophysiology of fetal–neonatal brain damage

BJOG: An International Journal of Obstetrics and Gynaecology

Volumn 104, Issue 6, 1997, Pages 652-662

  Longo, Lawrence D ^a   Packianathan, Satyseelan ^a  

^a LOMA LINDA UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ACIDOSIS; BRAIN DEVELOPMENT; BRAIN HYPOXIA; BRAIN INJURY; BRAIN ISCHEMIA; FETUS; FETUS HYPOXIA; GENE EXPRESSION; HUMAN; HYPOTHESIS; INFANT; INTOXICATION; NERVE CELL LESION; NEWBORN; NONHUMAN; OXIDATIVE STRESS; PATHOPHYSIOLOGY; PLACENTA CIRCULATION; PRIORITY JOURNAL; REVIEW; SIGNAL TRANSDUCTION;

ACIDOSIS, LACTIC; ADENOSINE TRIPHOSPHATE; BRAIN; BRAIN CHEMISTRY; EMBRYONIC AND FETAL DEVELOPMENT; EXCITATORY AMINO ACIDS; FETAL HYPOXIA; GENE EXPRESSION REGULATION, DEVELOPMENTAL; HUMANS; REACTIVE OXYGEN SPECIES;

EID: 0030913176     PISSN: 14700328     EISSN: 14710528     Source Type: Journal    
DOI: 10.1111/j.1471-0528.1997.tb11974.x     Document Type: Review

References (122)

1. Del Toro J, PT Louis, Goddard Finegold J. Cerebrovascular regulation and neonatal brain injury. Pediatr Neurol 1991; 7: 3–12.
2. Kjellmer I. Mechanisms of perinatal brain damage. Ann Med 1991; 23: 675–679.
3. Siesjö BK. Pathophysiology and treatment of focal cerebral ischaemia: Part I. Pathophysiology. J Neurosurg 1992; 77: 169–184.
4. Siesjö BK. Pathophysiology and treatment of focal cerebral ischaemia: Part II. Mechanisms of damage and treatment. J Neurosurg 1992; 77: 337–354.
5. Vannucci RC. Experimental biology of cerebral hypoxia-ischaemia: relation to perinatal brain damage. Pediatr Res 1990; 27: 317–326.
6. Vannucci RC. Cerebral carbohydrate and energy metabolism in perinatal hypoxic/ischaemic brain damage. Brain Pathol 1992; 2: 229–234.
7. 2. Am J Physiol 1990; 68: 833–842.
8. Little WJ. Course of lectures on the deformities of the human frame. Lancet 1843–1844; 1: 5–7, 38–44; 70–74; 209–212; 230–233; 257–260; 290–293; 318–320; 346–349; 350–354.
9. Little WJ. On the Nature and Treatment of the Deformities of the Human Frame. Being a course of lectures delivered at the Royal Orthopaedic Hospital in 1843. London: Longman, Brown, Green, and Longmans, 1853.
10. Little WJ. On the influence of abnormal parturition, difficult labours, premature birth, and asphyxia neonatorum, on the mental and physical condition of the child, especially in relation to deformities. Trans Obstet Soc London 1861–1862: 293–344.
11. Osier W. The cerebral palsies of children: lectures I–V. Med News (Philadelphia) 1888; 53: 29–35, 57–66, 85–90, 113–116, 141–145.
12. Osler W. The Cerebral Palsies of Children. A clinical study from the Infirmary for Nervous Diseases. Philadelphia: P. Blakiston, 1889.
13. Freud S, Rie O. Klinische Studie ueber die halbseitige Zerebrallähmungen des Kindes. Kassowitz Beiträge 1891; old series: 3.
14. Freud S. Zur Kenntnis der cerebralen Diplegien des Kindesalters. (Im Anschluss an die Little'sche Krankheit). Leipzig: F. Deuticke, 1893.
15. Freud S. Die infantile Cerehrallähmung. Nothnagel's Specielle Pathologie und Therapie. Wien: A. Holder, 1897: Vol 9, Part 2, Section 2.
16. Longo LD, Ashwal S. William Osier, Sigmund Freud and the evolution of ideas concerning cerebral palsy. J Hist Neurosci 1993; 2: 255–282.
17. Hagberg B, Hagberg G, Glow I. The changing panorama of cerebral palsy in Sweden. Acta Paediatr Scand 1984; 73: 433–440.
18. Hagberg B, Hagberg G, Glow I, von Wendt L. The changing panorama of cerebral palsy in Sweden: V. Acta Paediatr Scand 1989; 78: 283–290.
19. Paneth N, Stark RI. Cerebral palsy and mental retardation in relation to indicators of perinatal asphyxia. Am J Obstet Gynecol 1983; 147: 960–966.
20. Paul RH, Yonekura ML, Cantrell CJ, Turkel S, Pavlova Z, Sipos L. Fetal injury prior to labor: does it happen Am J Obstet Gynecol 1986; 154: 1187–1193.
21. Hossman K-A. Glutamate-mediated injury in focal cerebral ischemia: The excitotoxin hypothesis revised. Brain Pathol 1994; 4: 23–36.
22. Paschen W. Molecular mechanisms of selective vulnerability of the brain to ischaemia. Circ MetabCerveau 1989; 6: 115–139.
23. Obrenovitch TP. The ischaemic penumbra: twenty years on. Cerebrovasc Brain Metabol Rev 1995; 7: 297–323.
24. Boyle R. New pneumatical experiments about respiration. Phil Trans R Soc 1670; 5: 2011–2031.
25. Himwich HE, Alexander FAD, Fazekas JF. Tolerance of the newborn to hypoxia and anoxia. Am J Physiol 1941; 133: 327.
26. Himwich HE, Bernstein AO, Herrlich H, Fazekas JF. Mechanisms for the maintenance of life in the newborn during anoxia. Am J Physiol 1942: 135: 387–391.
27. Duffy TE, Kohle S, Vannucci RC. Carbohydrate and energy metabolism in perinatal rat brain: Relation to survival in anoxia. J Neurochem 1975; 24: 271–276.
28. Thurston JH, McDougal Jr DB. Effect of ischaemia on metabolism of the brain of the newborn mouse. Am J Physiol 1969; 216: 348–352.
29. Cohn HE, Sacks EJ, Heymann MA, Rudolph AM. Cardiovascular responses to hypoxemia and acidemia in fetal lambs. Am J Obstet Gynecol 1974; 120: 817–824.
30. Peeters LLH, Sheldon RE, Jones Jr MD, Makowski EL, Meschia G. Blood flow to fetal organs as a function of arterial oxygen content. Am J Obstet Gynecol 1979; 135: 637–646.
31. Ashwal S, Majcher JS, Vain N, Longo LD. Patterns of fetal lamb regional cerebral blood flow during and after prolonged hypoxia. PediatrRes 1980; 14: 1104–1110.
32. Ashwal S, Majcher JS, Longo LD. Patterns of fetal lamb regional cerebral blood flow during and after prolonged hypoxia: studies during the post hypoxic recovery period. Am J Obstet Gynecol 1981; 139: 365–372.
33. Ashwal S, Dale PS, Longo LD. Regional cerebral blood flow: studies in fetal lamb during hypoxia, hypercapnia, acidosis, and hypotension. Pediatr Res 1984; 18: 1309–1316.
34. Nylund L, Lagercrantz H, Lunell NO. Catecholamines in fetal blood during birth in man. J Dev Physiol 1979; 1: 427–430.
35. Irestedt L, Lagercrantz H, Belfrage P. Causes and consequences of maternal and fetal sympathoadrenal activation during parturition. Ada Obstet Gynecol Scand 1984; 118: 111–115.
36. Ionides SP, Weiss MO, Angelopoulos M, Myers TF, Handa RJ. Plasma beta-endorphin concentrations and analgesia-muscle relaxation in the newborn infant supported by mechanical ventilation. Brain Res 1995; 689: 183–188.
37. Stark RI, Wardlaw SL, Daniel SS et al. Vasopressin secretion induced by hypoxia in sheep: developmental changes and relationship to beta-endorphin release. Am J Obstet Gynecol 1982; 144: 560–568.
38. Gidday JM, Fitzgibbons JC, Shah AR, Park TS. Neuroprotection from ischaemic brain injury by hypoxic preconditioning in the neonatal rat. Neurosci Lett 1994; 168: 221–224.
39. Chopp M, Chen H, Ho KL et al. Transient hypothermia protects against subsequent forebrain ischaemic cell damage in the rat. Neurology 1989; 39: 1396–1398.
40. Terplan KL. Histopathologic brain changes in 1152 cases of the perinatal and early infancy period. Biol Neonate 1967; 11: 348–366.
41. Goddard-Finegold J, Mizrahi EM. Understanding and preventing perinatal, intracerebral, peri- and intraventricular hemorrhage. J Child Neurol 1987; 2: 170–185.
42. Philip AGS, Allan WC, Tito AM, Wheeler LR. Intraventricular hemorrhage in preterm infants: declining incidence in the 1980s. Pediatrics 1989; 84: 797–801.
43. Volpe JJ. Brain injury in the premature infant—current concepts of pathogenesis and prevention. Biol Neonate 1992; 62: 231–242.
44. Blennow M, Hagberg H, Ingvar M, Zeman J, Wang Y-S, Lagercrantz H. Neurochemical and biophysical assessment of neonatal hypoxicischemic encephalopathy. Semin Perinatol 1994; 18: 30–35.
45. Calame A, Fawer CL, Claeys V, Arrazola L, Ducret S, Jaunin L. Neurodevelopmental outcome and school performance of very-low-birth-weight infants at eight years of age. Eur J Pediatr 1986; 145: 461–466.
46. Fawer CL, Diebold P, Calame A. Periventricular leucomalacia and neurodevelopmental outcome in preterm infants. Arch Dis Child 1987; 62: 30–36.
47. Pharoah PO, Cooke T, Rosenbloom I, Cooke RW. Trends in birth prevalence of cerebral palsy. Arch Dis Child 1987; 62: 379–384.
48. Pharoah PO, Cooke T, Rosenbloom L, Cooke RW. Effects of birth weight, gestational age, and maternal obstetric history on birth prevalence of cerebral palsy. Arch Dis Child 1987; 62: 1035–1040.
49. Stanley FJ, Watson L. Trends in perinatal mortality and cerebral palsy in Western Australia, 1967 to 1985. BMJ 1992; 304: 1658–1662.
50. Petterson B, Nelson K, Watson L, Stanley F. Twins, triplets and cerebral palsy in Western Australia in the 1980s. BMJ 1993; 307: 1239–1243.
51. Bhushan V, Paneth N, Kiely JL. Impact of improved survival of very low birth weight infants on recent secular trends in the prevalence of cerebral palsy. Pediatrics 1993; 91: 1094–1100.
52. Volpe JJ. Brain injury in the premature infant—current concepts. Prev Med 1994; 23: 638–645.
53. Bejar R, Wozniak P, Allard M et al. Antenatal origin of neurologic damage in newborn infants. I. Preterm infants. Am J Obstet Gynecol 1988; 159: 357–363.
54. Grant A, O'Brien N, Joy M-T, Hennessy E, MacDonald D. Cerebral palsy among children born during the Dublin randomized trial of intrapartum monitoring. Lancet 1989; 2: 1233–1235.
55. Clapp JF III, Mann LI, Peress NS, Szeto HH. Neuropathology in the chronic fetal lamb preparation: Structure-function correlates under different environmental conditions. Am J Obstet Gynecol 1981; 141: 973–986.
56. Clapp JF III, Peress NS, Wesley M, Mann LI. Brain damage after intermittent partial cord occlusion in the chronically instrumented fetal lamb. Am J Obstet Gynecol 1988; 159: 504–509.
57. De Haan HH, Van Reempts JLH, Vles JSH, de Haan J, Hasaart THM. Effects of asphyxia on the fetal lamb brain. Am J Obstet Gynecol 1993; 169: 1493–1501.
58. Windle WF, Becker RF, Weil A. Alterations in brain structure after asphyxiation at birth: an experimental study in the guinea pig. JNeuropathol ExpNeurol 1944; 3: 224–238.
59. Windle WF, Jacobson UN, Robert De Marirez De Arellano MI, Combs CM. Structural and functional sequelae of asphyxia neonatorum in monkeys (Macaca mulatta). Res Publ Assoc Research Nervous Mental Disease 1962; 39: 169–182.
60. Windle WF. Asphyxia at birth, a major factor in mental retardation: Suggestions for prevention based on experiments in monkeys. In: Zubin J, Jervis GA, editors. Psychopathology of Mental Development. New York: Grune & Stratton, 1967: 140–147.
61. Raju TNK. Some animal models for the study of perinatal asphyxia. Biol Neonate 1992; 62: 202–214.
62. Fazekas JF, Alexander FAD, Himwich HE. Tolerance of the newborn to anoxia. Am J Physiol 1941; 134: 281–287.
63. Mallard EC, Gunn AJ, Williams CE, Johnston B, Gluckman PD. Transient umbilical cord occlusion causes hippocampal damage in the fetal sheep. Am J Obstet Gynecol 1992; 167: 1423–1430.
64. Allen WC. The IVH complex of lesions: Cerebrovascular injury in the preterm infant. In: Bodensteiner JB, editor. Pediatric Neurology. New York: WB Saunders, 1990: 529–553.
65. Brann AW, Schwartz JF. Birth injury. In: Fanaroff AA, Martin RJ, editors. Neonatal-Perinatal Medicine: Diseases of the Fetus and Infant. St Louis: CV Mosby, 1987: 506–520.
66. Gilad GM, Gilad VH. Polyamines can protect against ischaemia induce nerve cell death in gerbil forebrain. Exp Neurol 1991; 111: 349–355.
67. Longo LD, Packianathan S, McQueary JA, Byus CV, Cain CD. Acute hypoxia increases omithine decarboxylase activity and polyamine concentrations in fetal rat brain. Proc Natl Acad Sci USA 1993; 90: 692–696.
68. Packianathan S, Cain CD, Stagg RB, Longo LD. Ornithine decarboxylase activity in fetal and newborn rat brain: responses to hypoxic- and carbon monoxide-hypoxia. Dev Brain Res 1993; 75: 131–140.
69. Packianathan S, Liwnicz BH, Cain CD, Longo LD. Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices. Brain Res 1995; 668: 61–71.
70. Cannon WB. The Wisdom of the Body. New York: Norton & Co, 1932.
71. Dawes GS, Jacobson HN, Mott JC, Shelley HJ. Some observations in foetal and newborn rhesus monkeys. J Physiol 1960; 152: 271–298.
72. Dawes GS, Jacobson HN, Mott JC, Shelley HJ, Stafford A. The treatment of asphyxiated mature foetal lambs and rhesus monkeys with intravenous glucose and sodium carbonate. J Physiol 1963; 169: 167–184.
73. Dawes GS, Hibbard E Windle, WF. The effect of alkali and glucose infusion on permanent brain damage in rhesus monkeys asphyxiated at birth. J Pediatr 1964; 65: 801–806.
74. +ion-activities, brain volume and metabolism for recovery after prolonged cerebral ischemia. In: Harper AM, Jennett WB, Miller JD et al, editors. Blood Flow and Metabolism in the Brain. Edinburgh: Churchill Livingstone, 1975.
75. Yager JY, Brucklacher RM, Vannucci RC. Paradoxical mitochondrial oxidation in perinatal hypoxic-ischaemic brain damage. Brain Res 1996; 712: 230–238.
76. Giffard RG, Monyer H, Christine DW, Choi DW. Acidosis reduces NMDA receptor activation, glutamate neurotoxicity, and oxygen-glucose deprivation neuronal injury in cortical cultures. Brain Res 1990; 506: 339–342.
77. Levine RL. Ischaemia: from acidosis to oxidation. FASEB J 1993; 7: 1242–1246.
78. Duffy TE, Vannucci RC. Perinatal brain metabolism: Effects of anoxia and ischemia. In Cerebral Vascular Diseases. Whisnant JP, Sandok BA, editors. New York: Grune & Stratton, 1975: 231–235.
79. Choi DW. Calcium: still center-stage in hypoxic/ischaemic neuronal death. Trends Neurosci 1995; 18: 58–60.
80. Morley P, Hogan MJ, Hakim AM. Calcium-mediated mechanisms of ischemic injury and protection. Brain Pathol 1994; 4: 37–47.
81. Siesjö BK. Calcium in the brain under physiological and pathological conditions. Eur Neural 1990; 30 Suppl 2: 3–9.
82. Harman AW, Maxwell MJ. An evaluation of the role of calcium in cell injury. Annu Rev Pharmacol Toxicol 1995; 35: 129–144.
83. Jörgensen MB, Diemer N-H. Selective neuron loss after cerebral ischemia in the rat: Possible role of transmitter glutamate. Acta Neural Scand 1982; 66: 536–546.
84. Greenamyer JT, Penney JB, Young AB, Hudson C, Silverstein F, Johnston MV. Evidence for transient perinatal glutamatergic innervation of globus pallidus. J Neurosci 1987; 7: 1022–1030.
85. Hagberg H, Lehmann A, Sandberg M, Nyström, Jacobson I, Hamberger A. Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb Blood Flow Metab 1985; 5: 413–419.
86. Barks JD, Silverstein FS. Excitatory amino acids contribute to the pathogenesis of perinatal hypoxic/ischaemic brain injury. Brain Pathol 1992; 2: 235–243.
87. Hagberg H. Hypoxic-ischaemic damage in the neonatal brain: excitatory amino acids. Dev Pharmacol Ther 1992; 18: 139–144.
88. Hagberg H, Gilland E, Diemer NH, Andine P. Hypoxia-ischaemia in the neonatal rat brain: histopathology after post-treatment with NMDA and non-NMDA receptor antagonists. Biol Neonate 1994; 66: 205–213.
89. Hattori H, Morin AM, Schwartz PH, Fujikawa DG, Wasterlain CG. Posthypoxic treatment with MK-801 reduces hypoxic/ischaemic damage in the neonatal rat. Neurology 1989; 39: 713–718.
90. Hattori H, Waterlain CG. Hypothermia does not explain MK-801 neuroprotection in a rat model of neonatal hypoxic/ischaemic encephalopathy. Neurology 1991; 41: 330.
91. McDonald JW, Silverstein S, Johnson MW. MK-801 protects the neonatal brain from hypoxic/ischaemic damage. Eur J Pharmacol 1987; 140: 359–361.
92. Gunn AJ, Williams CE, Bennet L, Cook CJ, Gluckman PD. Perinatal cerebral asphyxia: pharmacological intervention. Fetal Ther 1988; 3: 98–107.
93. Andiné P, Lehmann A, Ellrén K et al. The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotection. Neurosci Lett 1988; 90: 208–212.
94. LeBlanc MH, Vig V, Smith B, Parker CC, Evans OB, Smith EE. MK-801 does not protect against hypoxic/ischaemic brain injury in piglets. Stroke 1991; 22: 1270–1275.
95. Cotman CW, Bridges RJ, Taube JS, Clark AS, Geddes JW, Monaghan DT. The role of the NMDA receptor in central nervous system plasticity and pathology. J NIH Res 1989; 1: 65–74.
96. Bernert H, Turski L. Traumatic brain damage prevented by the non-N-methyl-D-aspartate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline. Proc Natl Acad Sci USA 1996; 93: 5235–5240.
97. Gluckman PD, Williams CE. Is the cure worse than the disease? Caveats in the move from laboratory to clinic. Dev Med Child Neurol 1992; 34: 1010–1021.
98. 2adrenergic receptors in fetal rat heart: receptors in search of a function. J Dev Physiol 1992; 17: 183–187.
99. Chemtob S, Beharry K, Rex J, Varma DR, Aranda JV. Prostanoids determine the range of cerebral blood flow autoregulation of new-bom piglets. Stroke 1990; 21: 777–784.
100. 2 and indomethacin prevent impairment of post-ischemic brain reperfusion in the dog. Stroke 1979; 10: 629–637.
101. Lipton SA, Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 1994; 330: 613–624.
102. Lipton SA, Stamler JS. Actions of redox-related congeners of nitric oxide at the NMDA receptor. Neuropharmacology 1994; 33: 1229–1233.
103. Beckman JS. The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J Dev Physiol 1991; 15: 53–59.
104. Smith DS. Free radical scavengers and protection against ischemic brain damage. In: Schurr A, Rigor BM, editors. Cerebral Ischemia and Resuscitation. Boca Raton, Florida: CRC Press, 1990: 373–388.
105. Khan AU, Di Mascio P, Medeiros MHG, Wilson T. Spermine and spermidine protection of plasmid DNA against single strand-breaks induced by singlet oxygen. Proc Natl Acad Sci USA 1992; 89: 11428–11430.
106. Khan AU, Mei Y-H, Wilson T. A proposed function for spermine and spermidine: Protection of replicating DNA against damage by singlet oxygen. Proc Natl Acad Sci USA 1992; 89: 11426–11427.
107. Paschen W, Schmidt-Kastner R, Hallmayer J, Djuricic B. Polyamines in cerebral ischaemia. Neurochem Pathol 1988; 9: 1–20.
108. Paschen W. Polyamine metabolism in different pathological states of the brain. Mol Chem Neuropathol 1992; 16: 241–271.
109. Saito N, Kawai K, Nowak TS Jr. Reexpression of developmentally regulated MAP2c mRNA after ischemia: colocalization with hsp72 mRNA in vulnerable neurons. J Cerebr Blood Flow Metab 1994; 15: 205–215.
110. An G, Lin T-N, Liu J-S, Xue J-J, He Y-Y, Hsu CY. Expression of c-fos and c-jun family genes after focal cerebral ischaemia. Ann Neurol 1993; 33: 457–464.
111. Gunn AJ, Dragunow M, Faull RLM, Gluckman PD. Effects of hypoxia-ischaemia and seizures on neuronal and glial-like c-fos protein levels in the infant rat. Brain Res 1990; 531: 105–116.
112. Gubits RM, Burke RE, Casey-McIntosh G, Bandele A, Munell F. Immediate early gene induction after neonatal hypoxia-ischaemia. Brain Res (Molec Brain Res) 1993; 18: 228–238.
113. Herrera DG, Figueiredo BF, Cuello AC. Differential regulation of c-fos expression after cortical brain injury during development. Dev Brain Res 1993; 76: 79–85.
114. Munell F, Burke RE, Bandele A, Gubits RM. Localization of c-fos, c-jun, and hsp70 mRNA expression in brain after neonatal hypoxia-ischaemia. Dev Brain Res 1994; 77: 111–121.
115. Dwyer BE, Nishimura RN, Brown IR. Synthesis of the major inducible heat shock protein in rat hippocampus after neonatal hypoxia-ischaemia. Exp Neurol 1989; 104: 28–31.
116. Ferriero DM, Soberano HQ, Simon RP, Sharp FR. Hypoxia-ischaemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain. Dev Brain Res 1990; 53: 145–150.
117. Blumenfeld KS, Welsch FA, Harris VA, Pesenson MA. Regional expression of c-fos and heat shock protein-70 mRNA following hypoxia-ischaemia in immature rat brain. J Cereb Blood Flow Metabol 1992; 12: 987–995.
118. Satoh J, Kim SU. HSP72 induction by heat stress in human neurons and glial cells in culture. Brain Res 1994; 653: 243–250.
119. Soriano MA, Tortosa A, Planas AM, Rodriguez-Farré E, Ferrer I. Induction of HSP70 mRNA and the HSP70 protein in the hippocampus of the developing gerbil following transient forebrain ischaemia. Brain Res 1994; 653: 191–198.
120. Murphy S, Song D, Pastuszko A, Wilson DF, Welsh FA. Hypoxia induces heat-shock protein-72 mRNA in the cerebral white matter of neonatal piglets. J Cereb Blood Flow Metab 1995; 15 Suppl 1: S288.
121. Kato M, Mizuguchi M, Takashima S. Developmental changes of heat shock protein 73 in human brain. Dev Brain Res 1995; 86: 180–186.
122. Vannucci SJ, Seaman LB, Vannucci RC. Effects of hypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature rat brain. J Cereb Blood Flow Metab 1996; 16: 77–81.