DevNeurosci 1990;12:116-125
© 1990 S. Karger A G, Basel 0378-5866/90/0122-0116 S 2.75/0
Brain Damage and Recovery in Hyperphenylalaninémie Rats R. Burria, J.-M. Matthieub, M. Vandeveldec, F. Lazeyrasd, S. Posse0, N. Herschkowitza a Department of Pediatrics,c Institute of Animal Neurology, d NMR-Unit of the Medical Faculty, University of Berne, and b Department of Pediatrics, University Medical Center, Lausanne, Switzerland
Abstract. Rats were made hyperphenylalaninémie by injecting a mixture of alphamethylphenylalanine and phenylalanine. Brain development was measured by biochemical, histological and 31-P nuclear magnetic resonance (NMR) methods. In 17-day-old hyper phenylalaninémie rats, brain myelinogenesis was disturbed. Compared to controls, test animals had lower body weights, brain weights, cerebrosides, sulfatides, myelin basic protein (MBP) and reduced cerebroside sulfotransferase (CST) and 2'3'-cyclic nucleotide-3'-phosphohydrolase (CNP) activities. No changes were found in total proteins, total lipids, total phospholipids, phosphatidylethanolamine and phosphorylethanolamine. In the brain of 17-day-old hyperphenylalaninémie rats no changes in phosphomonoesters, phosphodiester and phosphocreatine were found using in vivo 31-P NMR spectroscopy. Because body weights of hyperphenylalaninémie rats were significantly lower than those of controls, we compared them with undernourished rats. Undernourished rats had lower body weights, brain weights and CNP activity. No other changes were found. Therefore, we conclude that hyperphenylalaninemia per se and not undernutrition affected myelinogenesis in test ani mals. After treatment was discontinued, test rats recovered completely within 6 weeks with regard to biochemical and histological measurements; at 59 days they had normal body weights, cerebrosides, sulfatides, MBP, total proteins, total lipids, total phospholipids, phosphatidylethanolamine, phosphorylethanolamine and normal CST and CNP activities. Brain weights were significantly reduced.
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 4:31:16 AM
Key Words. Brain development • Hyperphenylalaninemia • Cerebroside sulfotransferase • 2'3'-Cyclic nucleotide-3'-phosphohydrolase • Brain lipids • 31-P nuclear magnetic resonance • Rat
Introduction In phenylketonuric (PKU) patients he patic phenylalanine hydroxylase is nearly ab sent. Brain damage resulting from this dis turbed metabolism can be prevented to a large extent by providing a phenylalanine poor diet until adolescence. As an animal model for this disease hyperphenylalaniné mie rats are obtained by injecting phenylala nine (Phe) and alpha-methylphenylalanine (alpha-MP), an inhibitor of the phenylala nine hydroxylase [12, 15]. Daily injections of these two compounds lead to plasma Phe concentrations in the rat which are compar able to plasma Phe concentrations in un treated PKU patients [12]. To get the most severe effects on brain development, these injections have to be given between postnatal day 3 and 17, the time period during which the rat brain is most vulnerable to metabolic disturbances [15, 16, 18]. There is an extensive literature about changes in brain development of hyper phenylalaninémie rats [33]. Different drugs block rat Phe hydroxylase. One of these drugs, /?-chlorophenylalanine, has severe side effects, especially on brain levels of bio genic amines [18]. The use of alpha-MP in combination with Phe is now well estab lished. The following effects are found in injected animals: Loss of body weight [16, 18], microencephaly, decrease in myelin in the rat forebrain and spinal cord, reduced protein content in myelin [18, 29], smaller number of neurons (Purkinje and/or gran ule cells) in the cerebellum, thinner individ ual neuronal layers [16], and reduced size and density of synapses in rat neocortex [20]. No changes are seen in brain water content, proteins, RNA concentration [16]. Different experimental procedures have
117
revealed a disturbed myelinogenesis in hy perphenylalaninémie animals: (1) Hyper phenylalaninémie rabbits have a reduced in corporation of radioactive acetate into brain total lipids and specifically into cerebrosides of myelin [2]. (2) Reduced CNP activities are measured in the brains of pups of hyper phenylalaninémie rats [14]. (3) In mice, high Phe levels reduce galactosulfatide synthesis in vivo and in cell culture [28]. (4) Myelin basic protein (MBP), the marker protein of brain myelin, is reduced in hyperphenylala ninémie rats [11, 18, 29], It is important to know whether brain damage caused by hyperphenylalaninemia is reversible or permanent. Newborn pups from hyperphenylalaninémie rats with histo logically confirmed brain damage, recover during their first 12 days of life [25]. How ever, Hirano et al. [14] showed in a similar experiment that 15-day-old rats have re duced brain 2'3'-cyclic nucleotide-3'-phosphohydrolase (CNP) activity but recover be tween day 15 and 30. In the present study, we measured the brain damage in the rat using the alpha-MP/ Phe model. Myelination was investigated by measuring in treated, 17-day-old rats the en zymes cerebroside sulfotransferase (CST) and CNP [7, 10, 30, 32, 34]. Galactocerebrosides and galactosulfatides, which are mainly formed in brain tissue during the time of rapid myelinogenesis [17, 34], were deter mined. Other groups of alpha-MP/Phetreated animals were allowed to recover for 6 weeks after treatment was discontinued. In vivo nuclear magnetic resonance (NMR) spectroscopy can be used to follow brain development by measuring brain com pounds containing phosphorus, such as phosphomonoesters (PME), phosphodiesters (PDE), phosphocreatine (PCr) and
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 4:31:16 AM
Brain Damage and Recovery
Burri/Matthieu/Vandevelde/I.azeyras/Posse/Herschkowitz
118
Table 1. Brain damage and recovery in hyperphenylalaninémie rats Body weight g
Brain weight mg
Proteins mg/g brain
CST pm ol/h/g brain
CNP mm ol/h/g brain
Cer. mg/g brain
Controls 17 days n
40.0 ±2.0 12
958.8 ±49.4 12
104.3 ±6.1 11
348.6 ± 108.6 12
8.20 ±1.20 12
0.62±0.14 12
Test 17 days n p
25.0±4.3 12
© 1990 S. Karger A G, Basel 0378-5866/90/0122-0116 S 2.75/0
Brain Damage and Recovery in Hyperphenylalaninémie Rats R. Burria, J.-M. Matthieub, M. Vandeveldec, F. Lazeyrasd, S. Posse0, N. Herschkowitza a Department of Pediatrics,c Institute of Animal Neurology, d NMR-Unit of the Medical Faculty, University of Berne, and b Department of Pediatrics, University Medical Center, Lausanne, Switzerland
Abstract. Rats were made hyperphenylalaninémie by injecting a mixture of alphamethylphenylalanine and phenylalanine. Brain development was measured by biochemical, histological and 31-P nuclear magnetic resonance (NMR) methods. In 17-day-old hyper phenylalaninémie rats, brain myelinogenesis was disturbed. Compared to controls, test animals had lower body weights, brain weights, cerebrosides, sulfatides, myelin basic protein (MBP) and reduced cerebroside sulfotransferase (CST) and 2'3'-cyclic nucleotide-3'-phosphohydrolase (CNP) activities. No changes were found in total proteins, total lipids, total phospholipids, phosphatidylethanolamine and phosphorylethanolamine. In the brain of 17-day-old hyperphenylalaninémie rats no changes in phosphomonoesters, phosphodiester and phosphocreatine were found using in vivo 31-P NMR spectroscopy. Because body weights of hyperphenylalaninémie rats were significantly lower than those of controls, we compared them with undernourished rats. Undernourished rats had lower body weights, brain weights and CNP activity. No other changes were found. Therefore, we conclude that hyperphenylalaninemia per se and not undernutrition affected myelinogenesis in test ani mals. After treatment was discontinued, test rats recovered completely within 6 weeks with regard to biochemical and histological measurements; at 59 days they had normal body weights, cerebrosides, sulfatides, MBP, total proteins, total lipids, total phospholipids, phosphatidylethanolamine, phosphorylethanolamine and normal CST and CNP activities. Brain weights were significantly reduced.
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 4:31:16 AM
Key Words. Brain development • Hyperphenylalaninemia • Cerebroside sulfotransferase • 2'3'-Cyclic nucleotide-3'-phosphohydrolase • Brain lipids • 31-P nuclear magnetic resonance • Rat
Introduction In phenylketonuric (PKU) patients he patic phenylalanine hydroxylase is nearly ab sent. Brain damage resulting from this dis turbed metabolism can be prevented to a large extent by providing a phenylalanine poor diet until adolescence. As an animal model for this disease hyperphenylalaniné mie rats are obtained by injecting phenylala nine (Phe) and alpha-methylphenylalanine (alpha-MP), an inhibitor of the phenylala nine hydroxylase [12, 15]. Daily injections of these two compounds lead to plasma Phe concentrations in the rat which are compar able to plasma Phe concentrations in un treated PKU patients [12]. To get the most severe effects on brain development, these injections have to be given between postnatal day 3 and 17, the time period during which the rat brain is most vulnerable to metabolic disturbances [15, 16, 18]. There is an extensive literature about changes in brain development of hyper phenylalaninémie rats [33]. Different drugs block rat Phe hydroxylase. One of these drugs, /?-chlorophenylalanine, has severe side effects, especially on brain levels of bio genic amines [18]. The use of alpha-MP in combination with Phe is now well estab lished. The following effects are found in injected animals: Loss of body weight [16, 18], microencephaly, decrease in myelin in the rat forebrain and spinal cord, reduced protein content in myelin [18, 29], smaller number of neurons (Purkinje and/or gran ule cells) in the cerebellum, thinner individ ual neuronal layers [16], and reduced size and density of synapses in rat neocortex [20]. No changes are seen in brain water content, proteins, RNA concentration [16]. Different experimental procedures have
117
revealed a disturbed myelinogenesis in hy perphenylalaninémie animals: (1) Hyper phenylalaninémie rabbits have a reduced in corporation of radioactive acetate into brain total lipids and specifically into cerebrosides of myelin [2]. (2) Reduced CNP activities are measured in the brains of pups of hyper phenylalaninémie rats [14]. (3) In mice, high Phe levels reduce galactosulfatide synthesis in vivo and in cell culture [28]. (4) Myelin basic protein (MBP), the marker protein of brain myelin, is reduced in hyperphenylala ninémie rats [11, 18, 29], It is important to know whether brain damage caused by hyperphenylalaninemia is reversible or permanent. Newborn pups from hyperphenylalaninémie rats with histo logically confirmed brain damage, recover during their first 12 days of life [25]. How ever, Hirano et al. [14] showed in a similar experiment that 15-day-old rats have re duced brain 2'3'-cyclic nucleotide-3'-phosphohydrolase (CNP) activity but recover be tween day 15 and 30. In the present study, we measured the brain damage in the rat using the alpha-MP/ Phe model. Myelination was investigated by measuring in treated, 17-day-old rats the en zymes cerebroside sulfotransferase (CST) and CNP [7, 10, 30, 32, 34]. Galactocerebrosides and galactosulfatides, which are mainly formed in brain tissue during the time of rapid myelinogenesis [17, 34], were deter mined. Other groups of alpha-MP/Phetreated animals were allowed to recover for 6 weeks after treatment was discontinued. In vivo nuclear magnetic resonance (NMR) spectroscopy can be used to follow brain development by measuring brain com pounds containing phosphorus, such as phosphomonoesters (PME), phosphodiesters (PDE), phosphocreatine (PCr) and
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 4:31:16 AM
Brain Damage and Recovery
Burri/Matthieu/Vandevelde/I.azeyras/Posse/Herschkowitz
118
Table 1. Brain damage and recovery in hyperphenylalaninémie rats Body weight g
Brain weight mg
Proteins mg/g brain
CST pm ol/h/g brain
CNP mm ol/h/g brain
Cer. mg/g brain
Controls 17 days n
40.0 ±2.0 12
958.8 ±49.4 12
104.3 ±6.1 11
348.6 ± 108.6 12
8.20 ±1.20 12
0.62±0.14 12
Test 17 days n p
25.0±4.3 12
