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Endotoxin, cerebral blood flow, amino acids and brain damage in young rabbits
Cerebral Blood Flow~ Amino Acids and Brain Damage in Young Rabbits
Endotoxin~
Masashi Ando, MD, Sachio Takashima, MD and Takashi Mito, MD
The effects of bacterial endotoxin (lipopolysaccharide; LPS) on the cerebral blood flow (CBF), amino acid levels and brain histology were studied in young rabbits. The CBP was slightly decreased in the cerebral cortex and markedly decreased in the cerebral white matter at 60 and 120 min after LPS administration. Histological examination revealed only slightly pyknotic neurons around small vessels at 24 hours, and multifocal necrosis in the deep cerebral cortex and white matter at 72 hours. Some amino acids were increased in the plasma and brain regions at 24 hours after LPS administration, most of which were essential amino acids. GABA in the cerebral white matter was decreased at 24 hours. At 72 hours, most non-essential and glucogenic amino acids were decreased. These results suggest that the brain histological changes are related mainly to hypoperfusion and vascular damage in the brain. The amino acid changes may also be related to inappropriate amino acid metabolism associated with brain cell damage. Key words: Endotoxin (lipopolysaccharide), cerebral blood flow, amino acid. Ando M, Takashima S, Mito T. Endotoxin, cerebral blood flow, amino acids and brain damage in young rabbits. Brain Dev 1988;10:365-70
Perinatal bacterial infections and endotoxemia may be important causes of subsequent brain damage in the developing brain [1-3]. Studies by Gilles et al [2] and Young et al [3] have shown that the administration of E coli lipopolysaccharide (LPS) to newborn animals produces brain injury, including periventricular leukomalacia. Previous studies suggested that LPS-induced hypotension causes the failure of cerebral autoregulation and selective impairment of the cerebral white matter, and histological changes, predominantly in the cerebral white matter, in the developing brain [3,4]. Many studies demonstrated the direct effects of LPS on cells in culture and on subcellular particles [5]. However, the primary cellular and molecular basis of the toxicity of LPS in the
From the Department of Pediatrics, National Matsue Hospital, Matsue (MA); Division of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo (ST); Division of Child Neurology, Institute of Neurological Science, Tottori University School of Medicine, Y onago (TM). Received for pUblication: May 23, 1988. Accepted for publication: July 30, 1988. Correspondence address: Dr. Masashi Ando, Department of Pediatrics, National Matsue Hospital, 483 Agenogicho, Matsue 690, Japan.
brain has not been definitely elucidated. Also, it has been controversial as to whether the histological changes are due to direct cellular damage or secondary concomitant pathogenetic factors. The free amino acids in the brain are maintained at steady levels, and play important roles as intermediates in energy metabolism, as candidates and precursors of neurotransmitters, and as precursors of proteins and peptides. However, there has been little comparative investigation of the effects of LPS on the amino acid metabolism in the developing brain as to energy metabolism. The goals of this study were to determine the effects of LPS on the CBF, amino acid levels and histology in the developing brain, and to evaluate the mechanism underlying histological changes (brain cell necrosis, leukomalacia). Another goal was to determine the characteristic changes in amino acids associated with endotoxemia in the plasma and brain.
MATERIALS AND METHODS Animals Young rabbits of both sexes aged 2 weeks after birth were used for the experiments. Twelve young rabbits were used for the CBF measurements. Eighteen young
rabbits were used for the amino acid measurements and histological examination. The young rabbits were weaned just before intravenous injection of LPS (10 mg/ kg) from Escherichia coli 055: B5 (Difco Lab). After LPS injection, the rabbits were fed ad libitum in a warm room for 24 or 72 hours (hr). CBF measurement The procedure used was similar to that previously described [6]. The CBF was measured by the H2 clearance method (Unique Medical Co). The electrodes were inserted into the parietal cortex and white matter through small holes made by craniotomy. Brain histological examination Six LPS-treated rabbits died before the experiments. The 12 young rabbits which survived were used for the experiments and another 10 LPS-untreated rabbits were used as controls. After a rabbit had been sacrificed at 24 or 72 hr after LPS administration, its brain was removed, and the right hemisphere was fixed in a 10% formalin solution and then cut into coronal sections. The tissue sections were mounted in paraffin and then stained with hematoxylin and eosin.
-0-
CBF
-A-
ml/100gm/min 70
Amino acid and glucose measurements At 24 or 72 hr after LPS administration, blood was collected by intracardiac puncture with a heparinized syringe under ether anesthesia. Then the blood was centrifuged at 1,600 xg for 15 min at 4°C. The plasma was deproteinized in an equal volume of 2.5% sulfosalicylic acid, followed by centrifugation at 1,600 xg for 15 min at 4°C. The supernatant was analyzed with a Hitachi amino acid analyzer (Model 835). The brain was not perfused with any solution. After the brain had been removed, the left hemisphere was separated into the frontal and parietal cerebral cortices, cerebral white matter, diencephalon, midbrain, pons, medulla oblongata arid cerebellum. Each brain tissue specimen was weighed and then homogenized with a teflon homogenizer in ice-cold 1.5% sulfosalicylic acid. The homogenate was centrifuged at 1,600 xg for 15 min at 4°C. The supernatant was analyzed with the amino acid analyzer. The plasma glucose concentration was determined, using the plasma residue, by the glucose oxidative method (POD). Statistical analysis Each experiment was performed 5 times. The results are
Cerebra 1 cortex Cerebral white matter
+LPS 10mg/kg
60
50 ~o
30
20 10
L..--r-.---r---y------.....--......._ .---,--. _ Before
120
5 30 -- -60- - minutes
24
72
hours
Fig 1 Cerebral blood flow changes in the cerebral cortex and white matter of 2-week-old rabbits with LPS administration. The values are means ± SD. *indicate the significant differences (p < 0.05; Student's t-test) between the CBF before and after LPS administration.
366 Brain & Development, Vol 10, No 6,1988
Fig 2 Vesicular enlargement of astrocyte nuclei and karyorrhexis of cells in the deep cerebral cortex and white matter of the parietal region of a 2-week-old rabbit at 72 hr after LPS administration. H & E stains, x 150.
Table I Effects of LPS on amino acids in brain regions and plasma at 24 hr after LPS administration Brain region
Amino acid
FC
PC
WM
DC
MB
Ps
MO
Cm
Plasma
Ile Leu Val Met Thr Lys His
+ 43* + 58* + 91** + 80* + 163** + 50** +162*
+ 25* + 50* + 83** +125* +136** + 47** +108*
+ 13 + 31 + 62** +100* +121* + 25* +138*
+ 43 + 58 + 91 ** +167* +152* + 17 +185**
+ 43* + 58* + 92** +100** +163* + 33* + 148**
+ 67 + 64 + 83** +133* +154* + 23 +150**
+ 50 + 64* +118** +133* +163* + 31* + 182**
+ 57 + 67* + 80** +167** +167* + 32 + 143**
+ 50** + 72** + 64** + 85** +154** + 26 +102**
Glu Gin Asp GABA
+ 4 + 28*
+ 3 + 19
+ 9 + 43**
-
-
4
-
9
- 18**
+ 11 + 52** + 9 + 6
+ 10 + 44** + 8
-
+ 14 + 49** + 8 - 10
+ + + +
18* 38* 16 13
+ 3 + 46** + 1 + 5
+ 22** + 42 + 35** UN
-
5
+ 8 + 18 - 9 - 17 - 23
+ + + -
+ 21
+ 24 + 23*
o
- 17 - 18
34 33* 8 17 10
+ 35 + 32
20 17
+ + + -
- 25 - 10
+ 5 + 42 + 28** - 18 - 9
+ 13 + 36
+ 16 + 38
+ 4 + 32
7 4
+ +
7 9
+ 13 + 14
+ 113** + 58
Ser Gly Ala
am
+ 3 + 34* 1 - 29
+ 21
-
5
o
- 25 - 13
7 + 44
+ 42
Arg Tau Cysth
2 4
-
7
4
29 21
4
+ 22 + 30
6
+
o
+ +
-
4
The data are % changes in representative amino acid concentrations in brain regions and plasma in LPS-treated rabbits compared to the LPS-untreated control rabbits. Asterisks indicate the significant differences (* p < 0.05, ** P < 0.01) between the LPS-treated and LPS-untreated groups. Standard abbreviations for amino acids are used. FC: frontal cortex, PC: parietal cortex, WM: cerebral white matter, DC: diencephalon, MB: midbrain, Ps: pons, MO: medulla oblongata, Cm: cerebellum, UN: undetectable.
Table 2 Effects of LPS on amino acids in brain regions and plasma at 72 hr after LPS administration Brain region
Amino acid
FC
PC
WM
Ile Leu Val Met Thr Lys His
+ 14 + 20 + 17
+ 14 + 8 + 15
+ 18 + 17
+ 91** + 60** - 48**
+ 88** + 47** - 46**
Glu Gin Asp GABA
-
11* 39** 26** 15*
- 12** - 41 ** - 21*
-
Ser Gly Ala
Arg
-
44* 23* 22* 50** 37**
-
Tau Cysth
- 13 + 59**
am
o
o
DC
MB
Ps
o
+ 17 + 9
+ 75 + 10
+ 93** + 60** - 43**
+ + + + -
20 18 25 79** 62** 48**
9
-
12** 32** 19* 12
-
13* 39** 21* 16
-
43* 24* 20* 57** 41 **
-
36* 17 20* 50* 41 **
-
36* 12 21* 50* 35**
-
o
8 + 70**
o
+ 4 + 53**
+ 10 + 50*
+ + + -
o
8 25 78** 60** 52**
MO
Cm
o
+ 17 + 10
+ 10 + 8
o o
o
Plasma + + + + +
46 70 40 29 28
6
- 33 + 109** + 42** - 60**
+ 105** + 42** - 50**
+ 35* + 48** - 52**
-
7
-
17** 44** 28** 16
-
13* 40** 20** 14**
-
18** 44** 21** 11
+ 19 - 40* + 3 UN
39* 15* 20* 50** 41 **
-
52** 21 ** 32** 60** 50**
-
47** 9* 23* 60** 39**
-
57** 18** 32** 53** 40**
-
- 15** - 43** - 20*
1 + 26
+ 10 + 34
+ 7 + 22
+ 8 + 36
- 34*
62** 50** 57** 59** 55**
+105** + 67
The data are % changes in representative amino acid concentrations in brain regions and plasma in LPS-treated rabbits compared to the LPS-untreated control rabbits. Asterisks indicate the significant differences (*p < 0.05, **p < 0.01) between the LPS-treated and LPS-untreated groups.
Ando et al: Endotoxin and developing brain 367
expressed as the means ± SD. The values were compared with those in the case of LPS-untreated young rabbits as controls. Statistical analysis was performed by Student's t-test.
vs 160 ± 17 mg/ dl, LPS-untreated group). It tended to be decreased at 72 hr (117 ± 35 mg/dl vs 161 ± 10 mg/dl, LPS-untreated group), but there was no significant difference.
RESULTS
DISCUSSION
CBF Immediately after intravenous LPS injection, the CBF was decreased by approximately 10 ml/l00 gm body weight/min in the cerebral cortex and white matter. The CBF was markedly decreased in the cerebral white matter at 60 and 120 min after LPS administration, compared with that before LPS injection. Then the CBF improved gradually, as seen at 24 and 72 hr (Fig 1). Histology Histological examination of the brain at 24 hr after LPS administration revealed only slightly pyknotic neurons around small vessels in the gray matter of the cerebral hemispheres. However, at 72 hr, the brain showed multifocal necrosis with spongy tissue, eosinophilic neurons, slight glial activation and karyorrhexis, predominantly in the deep cerebral cortex and white matter (Fig 2). Amino acids At 24 hr after LPS administration, some amino acids were increased in both the plasma and most brain regions. They included threonine, valine, methionine, histidine, phenylalanine, isoleucine and leucine. Though glutamine, glycine and lysine showed no changes in the plasma, they were increased in some brain regions. Alanine was increased only in the plasma. GAB A was significantly decreased only in the cerebral white matter (Table 1). At 72 hr after LPS administration, for most of the amino acids, which were increased at 24 hr, no significant differences were observed between the brain and plasma of the experimental groups and those of the LPS-untreated control groups. However, threonine and lysine were increased in all brain regions, despite no significant changes in the plasma. Histidine was decreased in both the brain and plasma. Glutamate, aspartate, glutamine, serine, glycine, alanine, histidine and arginine were decreased in most of the brain regions. Cystathionine was increased in the cerebral cortex, white matter and diencephalon. Table 2 shows the % changes in the concentrations of representative amino acids in brain regions and the plasma, compared with those in the LPS-untreated groups. Plasma glucose The plasma glucose concentration was significantly decreased at 24 hr after LPS administration (107 ± 19 mg/dl
368 Brain & Development, VallO, No 6, 1988
Although the pathogenesis of LPS-induced brain injury, including perinatal leukomalacia, remains uncertain, it appears to be multifactorial in origin. The possible pathogenetic factors for brain injury associated with LPS may be cerebral hypoperfusion, immature vascularization, premyelination glial cells, impaired cerebral vascular permeability and autoregulation in the developing brain [4, 7, 8]. It may also be related to the direct cellular effect of LPS associated with damage to the endothelium and the escape of LPS into the brain [9, 10] or may be related to the stimulation of the release by lymphocytes, monocytes and macrophages of numerous factors (prostaglandins, interferons and cytokines) [11]. The present study focused on the effects of LPS administration on the CBF, amino acids and brain histology, as candidates of pathogenetic factors. LPS administration caused a transient decrease of the CBF in the cerebral white matter and histological changes around small vessels at 24 hr after LPS administration_ These findings support that there is a close relationship between histological changes, hypoperfusion and vascular damage associated with increased vascular permeability [12, 13]. On the other hand, at 24 hr after LPS administration, histological changes were predominantly observed in the deep cerebral cortex and white matter. These findings support that these areas may exhibit selective vulnerability associated with regional disturbance in the CBF. There has been little comparative investigation of the effect of LPS on amino acids in the brain. Jeppsson et al [13] showed a decrease in some amino acids in the plasma and an increase in most neutral amino acids in the brain in a rat model of abdominal sepsis. Our results for young rabbits were different from those of their study. The changes in amino acids were different between at 24 and 72 hr after LPS administration, and among anatomical brain regions. The pattern of changes also differed among the amino acids. At 24 hr after LPS administration, some amino acids were increased in both the plasma and brain regions, most of which were essential amino acids. The increases in these amino acids in the plasma may be due to increased net breakdown of proteins into amino acids [14], increased flux of amino acids from the muscle and stimulated hepatic gluconeogenesis. The essential amino acids in the plasma may cross the blood brain barrier by means of diffusion and carrier-mediated transport [15]. At 72 hr, however, most of the essential amino acids that were
increased at 24 hr had returned to normal levels. The effect of endotoxemia on these amino acids seemed to be transient. Most of the decreased amino acids in the brain regions at 72 hr after LPS administration were nonessential, glucogenic amino acids. These amino acids, especially glutamate and related amino acids, are mainly regulated in mitochondria through the citric acid cycle and GABA shunt in the brain. These amino acid changes may suggest inappropriate amino acid metabolism related to the citric acid cycle in most brain regions at 72 hr. And this may be caused by mitochondrial dysfunction associated with cell damage in the brain_ Hypoglycemia is often seen in endotoxemia [3, 16, 17], and causes amino acid changes in the brain [18]. In our experiment, the plasma glucose concentration was significantly decreased at 24 hr after LPS administration. However, the decrease was not so marked as that found in previous investigations [16, 17]. In addition, at 24 hr, the changes in amino acids in the brain were different from those observed in investigations on hypoglycemia [18, 19]. On the other hand, those at 72 hr were not significantly different between the two groups. Therefore, it is difficult to ascribe the changes in these nonessential, glucogenic amino acids in the brain only to hypoglycemia. Cerebral hypoperfusion appears to be poorly related to the amino acid changes in the brain, because our results showed different amino acid patterns from those in the case of cerebral ischemia reported in the literature [20, 21]. Although changes in most amino acids were seen in most brain regions as well as the cerebral white matter, GABA was decreased only in the cerebral white matter at 24 hr after LPS administration. This may be related to the decreased CBF in the cerebral white matter, because GABA may act as a vascular dilator for vascular smooth muscle of cerebral arteries, and thus play an important role as to the CBF [22,23]. Another possible mechanism affecting amino acid metabolism is the release of vasoactive substances that may alter cellular metabolism and may affect amino acid metabolism in the brain. In conclusion, LPS affects the CBF, amino acid concentrations and histology in the developing brain. Our results suggest that brain histological changes are related to transient hypoperfusion and vascular damage in the brain. The changes in brain amino acids differ with the time after LPS administration, among amino acid groups, and among brain regions. The increases in brain essential amino acids observed at 24 hr may be mainly associated with increases in them in the plasma, alterations in blood-brain permeability and/or transport activity for these amino acids. On the other hand,
the decreases in non-essential, glucogenic amino acids in the brain observed at 72 hr may reflect inappropriate amino acid metabolism associated with brian cell damage. ACKNOWLEDGMENTS This study was supported by grants from the NCNMMD and for Maternal and Child Health Research from the Ministry of Health and Welfare of Japan. REFERENCES 1. Ornoy A, Altshuler G. Maternal endotoxemia, fetal anomalies, and central nervous system damage: a rat model of a human problem. Am J Obstet Gynecol 1976;124:196204. 2. Gilles FH, Averill DR Jr, Kerr CS. Neonatal endotoxin encephalopathy. Ann Neurol 1977;2:49-56. 3. Young RSK, Yagel SK, Towfighi J. Systemic and neuropathologic effects of E coli endotoxin in neonatal dogs. Pediatr Res 1983;17:349-53. 4. Young RSK, Hernandez MJ, Yagel SK. Selective reduction of blood flow to white matter during hypotension in newborn dogs: a possible mechanism of periventricular leukomalacia. Ann Neurol 1982;12:445-8. 5. Bradley SG. Cellular and molecular mechanisms of action of bacterial endotoxins. Ann Rev Microbiol 1979;33: 67-94. 6. Takashima S, Ando Y, Takeshita K. Hypoxic-ischemic brain damage and cerebral blood flow changes in young rabbits. Brain Dev (Tokyo) 1986;8:274-7. 7. Takashima S, Tanaka K. Development of cerebrovascular architecture and its relationship to periventricular leukomalacia. Arch Neurol 1978;35:11-6. 8. Takashima S, Becker LE, Nishimura M, Tanaka J. Developmental changes of glial fibrillary acidic protein and myelin basic protein in perinatal leukomalacia: relationship to a predisposing factor. Brain Dev (Tokyo) 1984;6:44450. 9. Clawson CC, Hartmann JF, Vernier RL. Electron microscopy of the effect of Gram-negative endotoxin on the bloodbrain barrier. Comp Neurol 1966;127:183-98. 10. Reidy MA, Schwartz SM. Endothelial injury and regeneration. IV. Endotoxin: a nondenuding injury to aortic endothelium. Lab Invest 1983;48:25-34. 11. Morrison DC, Ulevitch RJ. The effects of bacterial endotoxins on host mediation systems. Am J Pathol 1978;93: 527-617. 12. du Moulin GC, Paterson 0, Hedley-Whyte J, Broitman SA. E coli peritonitis and bacteremia cause increased blood-brain barrier permeability. Brain Res 1985;340:261-8. 13. Jeppsson B, Freund HR, Gimmon Z, James JH, Meyenfeldt MF, Fischer JE. Blood-brain barrier derangement in sepsis: cause of septic encephalopathy? Am J Surg 1981;141: 136-42. 14. Freund HR, Ryan JA Jr, Fischer lE. Amino acid derangements in patients with sepsis: treatment with branched chain amino acid rich infusions. Ann Surg 1978;188: 423-30. 15. Oja SS, Korpi ER. Amino acid transport. In: Lajtha A, ed. Handbook of neurochemistry. 2nd ed. Vo15. New York: Plenum, 1984:311-37. 16. Filkins JP, Cornell RP. Depression of hepatic gluconeogenesis and the hypoglycemia of endotoxin shock. Am J Physiol
Ando et al: Endotoxin and developing brain 369
1974;227 :778-81. 17. Wichterman KA, Chaudray IH, Baue AE. Studies of peripheral glucose uptake during sepsis. Arch Surg 1979;114: 740-5. 18. Massieu GH, Ortega BG, Syrquin A, Tuena M. Free amino acids in brain and liver of deoxypyridoxine-treated mice subjected to insulin shoc\<. J Neurochem 1962;9:143-51. 19. Engelsen B, Westerberg E, Fonnum F, Wieloch T. Effect of insulin-induced hypoglycemia on the concentrations of glutamate and related amino acids and energy metabolites in the intact and decorticated rat neostriatum. J Neurochem 1986;47: 1634-41. 20. Erecinska M, Nelson D, Wilson DF, Silver IA. Neurotransmitter amino acids in the CNS. I: Regional changes in amino
370 Brain & Development, Valla, No 6,1988
acid levels in rat brain duri!!!, i~chemia and reperfusion. Brain Res 1984;304:9-22. 21. Lust WD, Mrsulja BB, Mrsulja BJ, Passonneau lV, Klatzo l. Putative neurotransmitters and cyclic nucleotides in prolonged ischemia of the cerebral cortex. Brain Res 1975;98: 394-9. 22. Fujiwara M, Muramatsu I, Shibata S. Gamma-aminobutyric acid receptor on vascular smooth muscle of dog cerebral arteries. BrJ PharmacoI1975;55:561-2. 23. Taniyama K, Shirakawa l, Hosoda K, Sano I, Takeda T, Tanaka C. GABAA receptor-mediated inhibition of serotonin-induced concentration of the isolated smooth muscle cells from cerebral blood vessels of guinea pig. Jpn J Pharmacal (Kyoto) 1987;43:299.
Endotoxin~
Masashi Ando, MD, Sachio Takashima, MD and Takashi Mito, MD
The effects of bacterial endotoxin (lipopolysaccharide; LPS) on the cerebral blood flow (CBF), amino acid levels and brain histology were studied in young rabbits. The CBP was slightly decreased in the cerebral cortex and markedly decreased in the cerebral white matter at 60 and 120 min after LPS administration. Histological examination revealed only slightly pyknotic neurons around small vessels at 24 hours, and multifocal necrosis in the deep cerebral cortex and white matter at 72 hours. Some amino acids were increased in the plasma and brain regions at 24 hours after LPS administration, most of which were essential amino acids. GABA in the cerebral white matter was decreased at 24 hours. At 72 hours, most non-essential and glucogenic amino acids were decreased. These results suggest that the brain histological changes are related mainly to hypoperfusion and vascular damage in the brain. The amino acid changes may also be related to inappropriate amino acid metabolism associated with brain cell damage. Key words: Endotoxin (lipopolysaccharide), cerebral blood flow, amino acid. Ando M, Takashima S, Mito T. Endotoxin, cerebral blood flow, amino acids and brain damage in young rabbits. Brain Dev 1988;10:365-70
Perinatal bacterial infections and endotoxemia may be important causes of subsequent brain damage in the developing brain [1-3]. Studies by Gilles et al [2] and Young et al [3] have shown that the administration of E coli lipopolysaccharide (LPS) to newborn animals produces brain injury, including periventricular leukomalacia. Previous studies suggested that LPS-induced hypotension causes the failure of cerebral autoregulation and selective impairment of the cerebral white matter, and histological changes, predominantly in the cerebral white matter, in the developing brain [3,4]. Many studies demonstrated the direct effects of LPS on cells in culture and on subcellular particles [5]. However, the primary cellular and molecular basis of the toxicity of LPS in the
From the Department of Pediatrics, National Matsue Hospital, Matsue (MA); Division of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo (ST); Division of Child Neurology, Institute of Neurological Science, Tottori University School of Medicine, Y onago (TM). Received for pUblication: May 23, 1988. Accepted for publication: July 30, 1988. Correspondence address: Dr. Masashi Ando, Department of Pediatrics, National Matsue Hospital, 483 Agenogicho, Matsue 690, Japan.
brain has not been definitely elucidated. Also, it has been controversial as to whether the histological changes are due to direct cellular damage or secondary concomitant pathogenetic factors. The free amino acids in the brain are maintained at steady levels, and play important roles as intermediates in energy metabolism, as candidates and precursors of neurotransmitters, and as precursors of proteins and peptides. However, there has been little comparative investigation of the effects of LPS on the amino acid metabolism in the developing brain as to energy metabolism. The goals of this study were to determine the effects of LPS on the CBF, amino acid levels and histology in the developing brain, and to evaluate the mechanism underlying histological changes (brain cell necrosis, leukomalacia). Another goal was to determine the characteristic changes in amino acids associated with endotoxemia in the plasma and brain.
MATERIALS AND METHODS Animals Young rabbits of both sexes aged 2 weeks after birth were used for the experiments. Twelve young rabbits were used for the CBF measurements. Eighteen young
rabbits were used for the amino acid measurements and histological examination. The young rabbits were weaned just before intravenous injection of LPS (10 mg/ kg) from Escherichia coli 055: B5 (Difco Lab). After LPS injection, the rabbits were fed ad libitum in a warm room for 24 or 72 hours (hr). CBF measurement The procedure used was similar to that previously described [6]. The CBF was measured by the H2 clearance method (Unique Medical Co). The electrodes were inserted into the parietal cortex and white matter through small holes made by craniotomy. Brain histological examination Six LPS-treated rabbits died before the experiments. The 12 young rabbits which survived were used for the experiments and another 10 LPS-untreated rabbits were used as controls. After a rabbit had been sacrificed at 24 or 72 hr after LPS administration, its brain was removed, and the right hemisphere was fixed in a 10% formalin solution and then cut into coronal sections. The tissue sections were mounted in paraffin and then stained with hematoxylin and eosin.
-0-
CBF
-A-
ml/100gm/min 70
Amino acid and glucose measurements At 24 or 72 hr after LPS administration, blood was collected by intracardiac puncture with a heparinized syringe under ether anesthesia. Then the blood was centrifuged at 1,600 xg for 15 min at 4°C. The plasma was deproteinized in an equal volume of 2.5% sulfosalicylic acid, followed by centrifugation at 1,600 xg for 15 min at 4°C. The supernatant was analyzed with a Hitachi amino acid analyzer (Model 835). The brain was not perfused with any solution. After the brain had been removed, the left hemisphere was separated into the frontal and parietal cerebral cortices, cerebral white matter, diencephalon, midbrain, pons, medulla oblongata arid cerebellum. Each brain tissue specimen was weighed and then homogenized with a teflon homogenizer in ice-cold 1.5% sulfosalicylic acid. The homogenate was centrifuged at 1,600 xg for 15 min at 4°C. The supernatant was analyzed with the amino acid analyzer. The plasma glucose concentration was determined, using the plasma residue, by the glucose oxidative method (POD). Statistical analysis Each experiment was performed 5 times. The results are
Cerebra 1 cortex Cerebral white matter
+LPS 10mg/kg
60
50 ~o
30
20 10
L..--r-.---r---y------.....--......._ .---,--. _ Before
120
5 30 -- -60- - minutes
24
72
hours
Fig 1 Cerebral blood flow changes in the cerebral cortex and white matter of 2-week-old rabbits with LPS administration. The values are means ± SD. *indicate the significant differences (p < 0.05; Student's t-test) between the CBF before and after LPS administration.
366 Brain & Development, Vol 10, No 6,1988
Fig 2 Vesicular enlargement of astrocyte nuclei and karyorrhexis of cells in the deep cerebral cortex and white matter of the parietal region of a 2-week-old rabbit at 72 hr after LPS administration. H & E stains, x 150.
Table I Effects of LPS on amino acids in brain regions and plasma at 24 hr after LPS administration Brain region
Amino acid
FC
PC
WM
DC
MB
Ps
MO
Cm
Plasma
Ile Leu Val Met Thr Lys His
+ 43* + 58* + 91** + 80* + 163** + 50** +162*
+ 25* + 50* + 83** +125* +136** + 47** +108*
+ 13 + 31 + 62** +100* +121* + 25* +138*
+ 43 + 58 + 91 ** +167* +152* + 17 +185**
+ 43* + 58* + 92** +100** +163* + 33* + 148**
+ 67 + 64 + 83** +133* +154* + 23 +150**
+ 50 + 64* +118** +133* +163* + 31* + 182**
+ 57 + 67* + 80** +167** +167* + 32 + 143**
+ 50** + 72** + 64** + 85** +154** + 26 +102**
Glu Gin Asp GABA
+ 4 + 28*
+ 3 + 19
+ 9 + 43**
-
-
4
-
9
- 18**
+ 11 + 52** + 9 + 6
+ 10 + 44** + 8
-
+ 14 + 49** + 8 - 10
+ + + +
18* 38* 16 13
+ 3 + 46** + 1 + 5
+ 22** + 42 + 35** UN
-
5
+ 8 + 18 - 9 - 17 - 23
+ + + -
+ 21
+ 24 + 23*
o
- 17 - 18
34 33* 8 17 10
+ 35 + 32
20 17
+ + + -
- 25 - 10
+ 5 + 42 + 28** - 18 - 9
+ 13 + 36
+ 16 + 38
+ 4 + 32
7 4
+ +
7 9
+ 13 + 14
+ 113** + 58
Ser Gly Ala
am
+ 3 + 34* 1 - 29
+ 21
-
5
o
- 25 - 13
7 + 44
+ 42
Arg Tau Cysth
2 4
-
7
4
29 21
4
+ 22 + 30
6
+
o
+ +
-
4
The data are % changes in representative amino acid concentrations in brain regions and plasma in LPS-treated rabbits compared to the LPS-untreated control rabbits. Asterisks indicate the significant differences (* p < 0.05, ** P < 0.01) between the LPS-treated and LPS-untreated groups. Standard abbreviations for amino acids are used. FC: frontal cortex, PC: parietal cortex, WM: cerebral white matter, DC: diencephalon, MB: midbrain, Ps: pons, MO: medulla oblongata, Cm: cerebellum, UN: undetectable.
Table 2 Effects of LPS on amino acids in brain regions and plasma at 72 hr after LPS administration Brain region
Amino acid
FC
PC
WM
Ile Leu Val Met Thr Lys His
+ 14 + 20 + 17
+ 14 + 8 + 15
+ 18 + 17
+ 91** + 60** - 48**
+ 88** + 47** - 46**
Glu Gin Asp GABA
-
11* 39** 26** 15*
- 12** - 41 ** - 21*
-
Ser Gly Ala
Arg
-
44* 23* 22* 50** 37**
-
Tau Cysth
- 13 + 59**
am
o
o
DC
MB
Ps
o
+ 17 + 9
+ 75 + 10
+ 93** + 60** - 43**
+ + + + -
20 18 25 79** 62** 48**
9
-
12** 32** 19* 12
-
13* 39** 21* 16
-
43* 24* 20* 57** 41 **
-
36* 17 20* 50* 41 **
-
36* 12 21* 50* 35**
-
o
8 + 70**
o
+ 4 + 53**
+ 10 + 50*
+ + + -
o
8 25 78** 60** 52**
MO
Cm
o
+ 17 + 10
+ 10 + 8
o o
o
Plasma + + + + +
46 70 40 29 28
6
- 33 + 109** + 42** - 60**
+ 105** + 42** - 50**
+ 35* + 48** - 52**
-
7
-
17** 44** 28** 16
-
13* 40** 20** 14**
-
18** 44** 21** 11
+ 19 - 40* + 3 UN
39* 15* 20* 50** 41 **
-
52** 21 ** 32** 60** 50**
-
47** 9* 23* 60** 39**
-
57** 18** 32** 53** 40**
-
- 15** - 43** - 20*
1 + 26
+ 10 + 34
+ 7 + 22
+ 8 + 36
- 34*
62** 50** 57** 59** 55**
+105** + 67
The data are % changes in representative amino acid concentrations in brain regions and plasma in LPS-treated rabbits compared to the LPS-untreated control rabbits. Asterisks indicate the significant differences (*p < 0.05, **p < 0.01) between the LPS-treated and LPS-untreated groups.
Ando et al: Endotoxin and developing brain 367
expressed as the means ± SD. The values were compared with those in the case of LPS-untreated young rabbits as controls. Statistical analysis was performed by Student's t-test.
vs 160 ± 17 mg/ dl, LPS-untreated group). It tended to be decreased at 72 hr (117 ± 35 mg/dl vs 161 ± 10 mg/dl, LPS-untreated group), but there was no significant difference.
RESULTS
DISCUSSION
CBF Immediately after intravenous LPS injection, the CBF was decreased by approximately 10 ml/l00 gm body weight/min in the cerebral cortex and white matter. The CBF was markedly decreased in the cerebral white matter at 60 and 120 min after LPS administration, compared with that before LPS injection. Then the CBF improved gradually, as seen at 24 and 72 hr (Fig 1). Histology Histological examination of the brain at 24 hr after LPS administration revealed only slightly pyknotic neurons around small vessels in the gray matter of the cerebral hemispheres. However, at 72 hr, the brain showed multifocal necrosis with spongy tissue, eosinophilic neurons, slight glial activation and karyorrhexis, predominantly in the deep cerebral cortex and white matter (Fig 2). Amino acids At 24 hr after LPS administration, some amino acids were increased in both the plasma and most brain regions. They included threonine, valine, methionine, histidine, phenylalanine, isoleucine and leucine. Though glutamine, glycine and lysine showed no changes in the plasma, they were increased in some brain regions. Alanine was increased only in the plasma. GAB A was significantly decreased only in the cerebral white matter (Table 1). At 72 hr after LPS administration, for most of the amino acids, which were increased at 24 hr, no significant differences were observed between the brain and plasma of the experimental groups and those of the LPS-untreated control groups. However, threonine and lysine were increased in all brain regions, despite no significant changes in the plasma. Histidine was decreased in both the brain and plasma. Glutamate, aspartate, glutamine, serine, glycine, alanine, histidine and arginine were decreased in most of the brain regions. Cystathionine was increased in the cerebral cortex, white matter and diencephalon. Table 2 shows the % changes in the concentrations of representative amino acids in brain regions and the plasma, compared with those in the LPS-untreated groups. Plasma glucose The plasma glucose concentration was significantly decreased at 24 hr after LPS administration (107 ± 19 mg/dl
368 Brain & Development, VallO, No 6, 1988
Although the pathogenesis of LPS-induced brain injury, including perinatal leukomalacia, remains uncertain, it appears to be multifactorial in origin. The possible pathogenetic factors for brain injury associated with LPS may be cerebral hypoperfusion, immature vascularization, premyelination glial cells, impaired cerebral vascular permeability and autoregulation in the developing brain [4, 7, 8]. It may also be related to the direct cellular effect of LPS associated with damage to the endothelium and the escape of LPS into the brain [9, 10] or may be related to the stimulation of the release by lymphocytes, monocytes and macrophages of numerous factors (prostaglandins, interferons and cytokines) [11]. The present study focused on the effects of LPS administration on the CBF, amino acids and brain histology, as candidates of pathogenetic factors. LPS administration caused a transient decrease of the CBF in the cerebral white matter and histological changes around small vessels at 24 hr after LPS administration_ These findings support that there is a close relationship between histological changes, hypoperfusion and vascular damage associated with increased vascular permeability [12, 13]. On the other hand, at 24 hr after LPS administration, histological changes were predominantly observed in the deep cerebral cortex and white matter. These findings support that these areas may exhibit selective vulnerability associated with regional disturbance in the CBF. There has been little comparative investigation of the effect of LPS on amino acids in the brain. Jeppsson et al [13] showed a decrease in some amino acids in the plasma and an increase in most neutral amino acids in the brain in a rat model of abdominal sepsis. Our results for young rabbits were different from those of their study. The changes in amino acids were different between at 24 and 72 hr after LPS administration, and among anatomical brain regions. The pattern of changes also differed among the amino acids. At 24 hr after LPS administration, some amino acids were increased in both the plasma and brain regions, most of which were essential amino acids. The increases in these amino acids in the plasma may be due to increased net breakdown of proteins into amino acids [14], increased flux of amino acids from the muscle and stimulated hepatic gluconeogenesis. The essential amino acids in the plasma may cross the blood brain barrier by means of diffusion and carrier-mediated transport [15]. At 72 hr, however, most of the essential amino acids that were
increased at 24 hr had returned to normal levels. The effect of endotoxemia on these amino acids seemed to be transient. Most of the decreased amino acids in the brain regions at 72 hr after LPS administration were nonessential, glucogenic amino acids. These amino acids, especially glutamate and related amino acids, are mainly regulated in mitochondria through the citric acid cycle and GABA shunt in the brain. These amino acid changes may suggest inappropriate amino acid metabolism related to the citric acid cycle in most brain regions at 72 hr. And this may be caused by mitochondrial dysfunction associated with cell damage in the brain_ Hypoglycemia is often seen in endotoxemia [3, 16, 17], and causes amino acid changes in the brain [18]. In our experiment, the plasma glucose concentration was significantly decreased at 24 hr after LPS administration. However, the decrease was not so marked as that found in previous investigations [16, 17]. In addition, at 24 hr, the changes in amino acids in the brain were different from those observed in investigations on hypoglycemia [18, 19]. On the other hand, those at 72 hr were not significantly different between the two groups. Therefore, it is difficult to ascribe the changes in these nonessential, glucogenic amino acids in the brain only to hypoglycemia. Cerebral hypoperfusion appears to be poorly related to the amino acid changes in the brain, because our results showed different amino acid patterns from those in the case of cerebral ischemia reported in the literature [20, 21]. Although changes in most amino acids were seen in most brain regions as well as the cerebral white matter, GABA was decreased only in the cerebral white matter at 24 hr after LPS administration. This may be related to the decreased CBF in the cerebral white matter, because GABA may act as a vascular dilator for vascular smooth muscle of cerebral arteries, and thus play an important role as to the CBF [22,23]. Another possible mechanism affecting amino acid metabolism is the release of vasoactive substances that may alter cellular metabolism and may affect amino acid metabolism in the brain. In conclusion, LPS affects the CBF, amino acid concentrations and histology in the developing brain. Our results suggest that brain histological changes are related to transient hypoperfusion and vascular damage in the brain. The changes in brain amino acids differ with the time after LPS administration, among amino acid groups, and among brain regions. The increases in brain essential amino acids observed at 24 hr may be mainly associated with increases in them in the plasma, alterations in blood-brain permeability and/or transport activity for these amino acids. On the other hand,
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