Intensive Care Med (2001) 27: 1655±1660 DOI 10.1007/s001340101067
Naoto Adachi Baiping Lei Gautam Deshpande Frank J. Seyfried Ichiro Shimizu Takumi Nagaro Tatsuru Arai
Received: 9 February 2001 Final revision received: 9 February 2001 Accepted: 17 July 2001 Published online: 31 August 2001 Springer-Verlag 2001
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N. Adachi ( ) ´ B. Lei ´ G. Deshpande ´ F. J. Seyfried ´ I. Shimizu ´ T. Nagaro ´ T. Arai Department of Anaesthesiology and Resuscitology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791±0295, Japan E-mail:
[email protected] Phone: +81-89-9 60 53 83 Fax: +81-89-9 60 53 86
EXPERIM ENTAL
Uraemia suppresses central dopaminergic metabolism and impairs motor activity in rats
Abstract Objective: Uraemia often provokes various neurological disorders, such as mental changes, malperception, confusion, seizures and coma. Since changes in neurotransmissions induce neurological symptoms, we investigated changes in the monoamine metabolism and motor activity in uraemic rats. Design: Prospective, randomised, controlled animal study. Subjects: Male Wistar rats. Interventions: Acute renal failure was induced by occlusion of bilateral renal arteries for 60 min, and the motor activity and brain monoamine turnover were examined 48 h later. The brain monoamine turnover was evaluated by the depletion of norepinephrine (NE) and dopamine (DA) induced by a-methyl-p-tyrosine (a-MT), or the accumulation of 5-hydroxyindoleacetic
Introduction Acute renal failure, which occurs in patients with renal ischaemia, intoxication and infection, is often accompanied by various disturbances in the central nervous system (CNS), such as mental changes, malperception, confusion, seizures and coma [1, 2, 3]. These central disorders are referred to as uraemic encephalopathy. It also alters cognitive function and electrophysiology, as assessed by a series of neuropsychological tests, electroencephalogram and somatosensory-evoked potentials [4, 5]. However, the pathogenic mechanism responsible for uraemic encephalopathy is unclear, although changes in ratios of plasma concentrations of amino acids
acid (5-HIAA) induced by probenecid. Measurements and results: Marked damage in renal function was found in animals subjected to renal ischaemia 48 h after the operation. The motor activity of the uraemic rats was impaired. The turnover of DA in the striatum, mesencephalon and hypothalamus was decreased in these rats. The turnover of NE and 5-hydroxytryptamine (5-HT) was unchanged in all regions examined. Conclusions: Suppression of the central DA turnover appears to be involved in the impairment of motor activity in uraemic rats. Keywords Acute renal failure ´ Central nervous system ´ Dopamine ´ Norepinephrine ´ Rats ´ Serotonin
have been implicated as a mechanism [5, 6, 7, 8]. Aromatic amino acids are precursors of monoaminergic neurotransmitters and the central monoaminergic system is closely related to psychomotor activity, such as behaviour, mood, sleep and memory. In the present study, therefore, we determined the levels of monoamines and evaluated the turnover rates of monoaminergic systems in uraemic rats. Then, we correlated them to motor activity in uraemic encephalopathy.
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Materials and methods Animals This study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan. All animals were cared for in compliance with the Principles of Laboratory Animal Care formulated by Ehime University School of Medicine. Male Wistar rats, weighing about 300 g each, were obtained from Charles River Laboratories (Yokohama, Japan). The animals were housed in a temperature-controlled room at 23 1 C. Food and water were provided ad libitum. Thirty-six animals were used for the monoamine turnover examination. Motor activity and renal function were estimated in twelve of them. Another set of twelve rats were subjected to measurements of the brain water content and arterial blood gas tensions. Surgery The rats were anaesthetised with 3 % isoflurane in balanced 50 % oxygen and 50 % nitrous oxide. Following a transverse abdominal incision, bilateral renal arteries and veins were clamped after injection of 50 U heparin into the tail vein. The surgical incision was sutured and the animals were allowed to recover from anaesthesia. They were anaesthetised again and blood flow was resumed 60 min after occlusion by removing the clips. Again the animals were allowed to recover from anaesthesia and were returned to their cages in a room maintained at a constant temperature. Food and water were provided ad libitum. For sham-operated animals, laparotomy was performed in a similar manner without clamping. Monoamine turnover Monoamine turnover was estimated based on the depletion of norepinephrine (NE) and dopamine (DA) induced by a-methylp-tyrosine (a-MT) or the accumulation of 5-hydroxyindoleacetic acid (5-HIAA) induced by probenecid [9, 10]. Thirty-six rats were divided into six groups: three ischaemic and three sham-operated groups. Forty-eight hours later, each pair of groups received aMT (250 mg/kg), probenecid (200 mg/kg) or saline. a-MT and probenecid were injected intraperitoneally 3 h and 90 min before decapitation, respectively. After isoflurane anaesthesia, the animals were decapitated, then the brains were rapidly removed, rinsed in saline, placed on ice and quickly dissected into the following six regions, according to the method of Glowinski and Iversen with a slight modification [11]. First, the whole hippocampus and striatum on both sides were dissected. The brain was cut from the rostral and caudal portions along the coronal planes at the optic chiasma and the caudal edge of the mamillary body. The dorsal portions of the cerebral cortex on both sides were cut from the rhinal fissure between these cut planes. Then, the mesencephalon, hypothalamus and medulla oblongata were dissected. Each tissue sample was homogenised in 1 ml of 0.4 mol/l perchloric acid containing 0.1 % L-cysteine. After being centrifuged, 10 ml of the supernatant was applied to a high-performance liquid chromatography (HPLC) system with electrochemical detection for determining the levels of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT) and 5-HIAA, according to the method of Magnusson et al. with a slight modification [12]. The HPLC system was composed of a pump equipped with a damper (EP-300, Eicom, Kyoto, Japan), an electrochemical detector (ECD-300, Eicom) with a graphite working electrode operated at 750 mV versus an Ag-AgCl reference electrode (RE-100, Ei-
com) and a reverse-phase column (MA-5ODS, 2.1 150 mm inside diameter, Eicom). The mobile phase was 0.1 mol/l citrate0.1 mol/l sodium acetate buffer (pH 3.9) containing 13 % methanol, 1.0 mmol/l sodium 1-octanesulfonate and 10 mmol/l disodium ethylenediaminetetraacetic acid. To determine the level of NE, a 10-ml portion of the remaining supernatant was applied to the HPLC system with the electrochemical detector operating at 450 mV and a reverse-phase column (CA-5ODS, 2.1 150 mm inside diameter, Eicom). The mobile phase was 0.1 mol/l sodium phosphate buffer (pH 6.0) containing 5 % methanol, 2.5 mmol/l sodium 1-octanesulfonate and 50 mmol/l disodium ethylenediaminetetraacetic acid. Motor activity The spontaneous motor activity was evaluated in a pair of ischaemic and sham-operated groups injected with saline. The activity was determined using five grades as follows: Grade 0: normal motor activity, startle response to sound and an airpuff, righting reflex present; Grade 1: mild lethargy, i.e., reduced spontaneous motor activity, normal responses and righting reflex; Grade 2: no spontaneous motor activity but some movement when aroused, responses and righting reflex present; Grade 3: motionless, great reluctance to move, responses reduced and righting reflex present; Grade 4: motionless, no responses and no righting reflex. When these animals were decapitated for the brain monoamine analysis, blood samples were collected and the plasma concentrations of urea nitrogen, creatinine and K+ were analysed by routine laboratory procedures. Brain water content Twelve rats were divided equally between an ischaemic and a sham-operate group and were operated. After 48 h, the rats were anaesthetised and the brains were rapidly removed in 100 % humidity. The whole brains were dried at 120 C for 24 h. The water content was calculated according to the equation: % water content = 100 (wet weight-dry weight)/wet weight. Before decapitation, arterial blood samples were collected and blood gas tensions were analysed. Drugs and chemicals Isoflurane was obtained from Abbott Laboratories (North Chicago, Ill., USA). a-MT hydrochloride methyl ester and probenecid were purchased from Sigma Chemical, (St. Louis, Mo., USA). aMT hydrochloride methyl ester was dissolved in saline. Probenecid was first dissolved in 0.15 mol/l NaOH, and the pH was reduced to 8.5. The doses of all drugs are expressed as the weight of the free bases. Statistical analysis The results from the turnover experiment were evaluated by analysis of variance with the Bonferroni adjustment in comparing each corresponding sham control. The data from the motor activity were evaluated with the Mann-Whitney test. The results of the blood analysis were evaluated by analysis of variance with the Bonferroni adjustment. The difference in the body weight was evaluated by repeated measures analysis of variance with the Bonferroni adjustment.
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Table 1 Arterial blood gas tensions and plasma concentrations of urea nitrogen, creatinine and K+ pH PaCO2 (mmHg) PaO2 (mmHg) Base excess (mmol/1) Urea nitrogen (mg/dl) Creatinine (mg/dl) K+ (mmol/1)
Sham
Ischaemia
7.490 0.044 37 3 133 31 4.6 1.9 17 3 0.5 0.1 5.8 0.4
7.344 0.038a 28 2a 112 21 ±9.4 1.7a 288 44a 8.4 1.3a 9.8 1.8a
Each value is the mean SD of six animals a p < 0.001 compared with each corresponding value in the sham group
Results No animal in either group died during the experimental period. There were no differences in body weight between the two groups at operation (sham 291 31 g vs ischaemia 301 36 g; mean SD; n = 18 each). The extent of the decrease in body weight after 48 h tended to be greater in the uraemic group than in the sham-operated group, but the effect was not significant (sham 281 26 g vs ischaemia 273 37 g). The pH and base excess values were markedly decreased by 60-min ischaemia (Table 1). A decrease in the PaCO2 level was also observed in the rats subjected to renal ischaemia, whereas the PaO2 level did not change. The plasma concentrations of urea nitrogen, creatinine and K+ were much greater in the ischaemic rats than in the sham-operated controls. The brain concentrations of NE and DA in each region examined did not differ between the two groups 48 h after the ischaemic event (Table 2). The concentraTable 2 Effect of acute renal failure on brain monoamines and their metabolites (ng/g) (NE norepinephrine, DA dopamine, DOPAC 3,4-dihydroxyphenylacetic acid, 5-HT 5-hydroxytryptamine, 5-HIAA 5-hydroxyindoleacetic acid)
Discussion In the present study, renal ischaemia for 60 min induced acute, severe renal damage and provoked marked alter-
NE Cerebral cortex Sham Ischaemia
Mesencephalon Sham Ischaemia Hypothalamus Sham Ischaemia Medulla oblongata Sham Ischaemia
DA
DOPAC
220+21 227+16
Striatum Sham Ischaemia Hippocampus Sham Ischaemia
Each value is the mean SD of six animals
tion of the striatal DOPAC, a metabolite of DA, also showed no change. As for the concentration of 5-HT, there were no remarkable differences between the two groups in any region. Likewise, the concentration of 5HIAA, a metabolite of 5-HT, was not changed by renal ischaemia in any region. The administration of a-MT decreased the concentrations of NE and DA in all brain regions examined in the sham-operated rats (Tables 3 and 4). While the a-MT treatment decreased the NE content in the ischaemic rats to an extent similar to that of sham-operated rats, the a-MT-induced depletion of DA in ischaemic rats was significantly inhibited in the striatum, mesencephalon and hypothalamus. The administration of probenecid increased the brain concentration of 5-HIAA in all regions in both the sham-operated and the ischaemic groups, and there were no differences in the extent of the accumulation of 5-HIAA after the treatment with probenecid between the two groups in any of the regions examined (Table 5). Normal motor activity in the sham group was preserved after 48 h (Fig. 1). However, in animals subjected to renal ischaemia, motor activity was markedly disturbed and most animals were motionless (p = 0.0019, U = 0.000). The brain water content in the sham and ischaemic groups was 78.14 0.23 % and 78.18 0.23 %, respectively (mean SD; n = 6 each), and there were no differences between the groups.
9183 1499 9696 1637
2531 636 2748 408
393 29 383 25
5-HT
5-HIAA
154 48 139+20
276+30 282 53
308 62 271 67
665 153 744 133
167 37 179 35
384 92 453 81
668 82 694 26
566 112 561 141
159 24 165 35
603 104 531 142
855 104 940 160
1824 641 1660 206
645 203 726 95
209+50 271 100
352 94 350 115
416 114 371 98
635 100 601 155
720 87 907 150
518 20 519 35
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Table 3 Effect of acute renal failure on brain monoamine turnover in the rat brain with regard to norepinephrine (NE norepinephrine, a-MT a-methyl-p-tyrosine)
Table 5 Effect of acute renal failure on brain monoamine turnover in the rat brain with regard to 5-hydroxytryptamine (5HIAA 5-hydroxyindoleacetic acid)
Norepinephrine turnover
5-Hydroxytryptamine turnover
NE levels (ng/g) Control
% Decrease
5-HIAA levels (ng/g)
a-MT
Control
Probenecid
% Increase
Cerebral cortex Sham Ischaemia
220 21 227 16
106 16 90 9
51.8 60.4
Cerebral cortex Sham Ischaemia
276 30 282 53
501 121 600 207
81.5 112.8
Hippocampus Sham Ischaemia
393 29 383 25
242 58 256 57
38.4 33.2
Striatum Sham Ischaemia
665 153 744 133
881 284 926 136
32.5 24.5
Mesencophalon Sham Ischaemia
668 82 694 26
359 78 426 61
46.3 38.6
Hippocampus Sham Ischaemia
384 92 453 81
612 228 646 248
59.4 42.6
1824 641 1660 206
1006 118 1128 107
44.8 32.0
855 104 940 160
1379 277 1399 315
61.3 48.8
518 20 519 35
288 32 303 40
44.4 41.6
416 114 371 98
537 321 500 147
29.1 34.8
720 87 907 150
1506 319 1679 480
109.2 85.1
Hypothalamus Sham Ischaemia Medulla oblongata Sham Ischaemia
Saline or a-MT (250 mg/kg) was injected intraperitoneally 48 h after 60 min renal ischaemia and the brain NE concentration was determined Each value is the mean SD of six animals
Table 4 Effect of acute renal failure on brain monoamine turnover in the rat brain with regard to dopamine (DA dopamine, aMT a-methyl-p-tyrosine) Dopamine turnover DA levels (ng/g) Striaturn Sham Ischaemia Mesencephalon Sham Ischaemia Hypothalamus Sham Ischaemia
% Decrease
Control
a-MT
9183 1499 9696 1637
3724 467 5566 996a
59.4 42.6
566 112 561 141
145 68 351 186a
74.4 37.4
645 203 726 95
157 78 298 92a
75.7 59.0
Saline or a-MT (250 mg/kg) was injected intraperitoneally 48 h after 60 min renal ischaemia and the brain DA concentration was determined Each value is the mean SD of six animals a p < 0.05 compared with each corresponding value in the sham group
ations in both neurochemical and behavioural variables 48 h after the operation. To estimate the metabolism of the brain catecholaminergic system, we applied the enzyme-inhibition technique using a-MT, which inhibits the hydroxylation step from tyrosine to dopa, the rate-limiting step in the
Mesencephalon Sham Ischaemia Hypothalamus Sham Ischaemia Medulla oblongata Sham Ischaemia
Saline or probenecid (200 mg/kg) was injected intraperitoneally 48 h after 60 min renal ischaemia and the brain 5-HIAA concentrations were determined Each value is the mean SD of six animals
synthesis of catecholamines [9]. The extent of the aMT-induced decrease in DA was suppressed in uraemic rats in all regions examined, suggesting a decrease in the DA turnover in uraemia. On the other hand, the synthesis of NE in uraemic rats was depressed by a-MT to an extent similar to that in sham-operated rats. The turnover of NE, therefore, may not be affected by acute renal failure. The change in the turnover of catecholamines seems to occur only in the dopaminergic system. In another study that examined the turnover of DA and NE in uraemic rats subjected to bilateral ligation of the ureters, the levels of both catecholamines in the uraemic group were lower than those in the control group [13]. However, the turnover of the catecholamines may not have been assessed explicitly in this study, since the turnover rate can be evaluated when the static levels without the a-MT treatment are equal [9]. The decrease in the turnover rate seems to be obvious only in DA. Most neuroleptic agents used in psychiatry are known to diminish spontaneous motor activity in both experimental animals and humans. The agents induce characteristic cataleptic immobility that allows the animals to be placed in abnormal postures. The animal appears to be indifferent to most stimuli, although it continues to withdraw from those that are noxious and painful. A similar phenomenon was observed in the cur-
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Fig. 1 Effect of acute renal failure on spontaneous motor activity. Activity was determined using five grades, as follows: Grade 0, normal; Grade 1, mild lethargy; Grade 2, no spontaneous motor activity; Grade 3, motionless with righting reflex; Grade 4, motionless without righting reflex. *p < 0.01 compared with the sham group
rent study. Spontaneous motor activity was markedly decreased in uraemic rats, although cataleptic immobility was not evaluated in this study. Since these effects of neuroleptic agents are mostly based on their ability to antagonise the actions of DA as a neurotransmitter in the basal ganglia and limbic portions in the CNS [14], the impairment of spontaneous motor activity in uraemic rats may be attributed to the decreased turnover of the dopaminergic system. Restless legs syndrome and periodic limb movement during sleep are sleep disorders that are prevalent and distressing to uraemic patients [15]. Although the pathophysiology of the symptom is still unsolved, Ldopa and DA agonists have been recommended for the treatment [16]. The agents have been reported to improve both restless legs syndrome and periodic limb movement during sleep [17, 18]. Considering these clinical findings, the depression of the central DA turnover may be a cause of neurological disorders in uraemia. Uraemic encephalopathy has been suggested to be associated with an imbalance in amino acid levels in the brain and plasma [5, 6, 7, 8], since aromatic amino acids, such as phenylalanine, tyrosine and tryptophan, are the direct precursors of monoamines. However, it seems to be difficult to explain changes in monoaminergic activity caused by alterations in amino acid levels. In other animal models of uraemia, the concentration of DA was decreased in the brain [19, 20], whereas brain concentrations of phenylalanine and tyrosine have been shown to be increased in most studies [7, 21]. Likewise, in our previous studies that examined the metabol-
ic encephalopathy caused by acute hepatic failure or sepsis, there was no clear involvement between changes in the plasma concentrations of amino acids and the turnover rates of the central monoaminergic systems [22, 23]. Furthermore, since DA and NE are synthesised from the same precursor, tyrosine, the decrease in the turnover rate of only DA cannot be explained by the altered amino acid patterns alone. This suggests the presence of a mechanism regulating catecholamine metabolism. Changes in the activity of the enzyme synthesising NE from DA are a possible mechanism. We evaluated the turnover rate of 5-HT by examining the probenecid-induced 5-HIAA accumulation in the brain. Probenecid blocks the transfer of 5-HIAA from the brain to plasma, and the rate at which 5HIAA accumulates equals the rate of 5-HIAA formation from 5-HT [10]. The increased amounts of 5HIAA after treatment with probenecid were similar for the two groups in all regions. Therefore, the turnover rate of 5-HT may not be affected by acute renal failure. In contrast, there are studies that have shown increases in the brain concentration of 5-HT and the turnover rate of 5-HT in uraemia [13, 24]. The different animal models may have caused the difference in 5-HT metabolism. Since most studies that showed changes in the central 5-HT were performed using chronic uraemic animals [13, 20, 24], 5-HT metabolism might be facilitated in a late stage of renal failure. In the rats subjected to renal ischaemia, severe metabolic acidosis was observed in blood analysis, and the plasma concentrations of urea nitrogen and K+ were also increased. Since several factors, such as anaemia, acidosis and undetermined molecules, have been suggested to contribute to neurological disorders in uraemia [5], these changes in blood analysis may be contributing factors as well as the suppression of the brain DA turnover. However, the brain water content did not increase in the present animal model of acute renal failure. Further, cerebral oedema has been shown to be uncertain in the brains of patients with chronic renal failure, although brain osmolarity is increased [5]. Therefore, it is unlikely that cerebral oedema affected the neurological signs in the present study. In acute renal failure, the excitatory neurotransmission by DA is depressed in the brain. Although the detailed neurochemical mechanisms responsible for uraemic encephalopathy are unconfirmed, changes in DA metabolism may be at least involved.
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