1 Department of Clinical Science, Section of Obstetrics and Gynecology, Umeå Neurosteroid Research Center, Umeå University, SE 901 85 Umeå, Sweden. 2 Department of Pharmacology and Clinical Neuroscience, Pharmacology, Umeå University, SE 901 85 Umeå, Sweden
* Corresponding author: Umeå University Hospital, Obstetrics and Gynaecology, Umeå Neurosteroid Research Centre, Bldg 5B, 5th floor, SE 901 85 UMEÅ, Sweden. E-mail: di.zhu{at}obgyn.umu.se
Accepted for publication May 27, 2004.
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Abstract |
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Methods. Induction of acute tolerance to AlloP was studied in male rats using a threshold technique of deep anaesthesia. AlloP was infused at a dose rate of 4.0 mg kg1 min1. The infusion was stopped when a burst suppression of 1 s or more (the silent second, SS) occurred in the EEG. To maintain anaesthesia, the infusion was restarted when no SS had been seen in the EEG for 1 min. This interrupted targeted infusion towards an EEG end-point (SS) was continued until 30, 60 or 90 min of anaesthesia had been reached. At these times the rats were killed and AlloP concentrations in serum, muscle, fat and different brain regions were determined by radioimmunoassay.
Results. Maintenance dose rate (MDR) was calculated using 20-min intervals. During anaesthesia the MDR increased (P<0.001) from 0.67 (SEM 0.03) mg kg1 min1 (in the interval 1030 min) to 0.98 (0.04) mg kg1 min1 (in the interval 6585 min). After 60 min a slight increase in MDR was observed. After 90 min of anaesthesia the AlloP concentrations in the hippocampus and brainstem had increased by more than 50% compared with control values of 25.2 (1.13) and 52.7 (5.81) nmol g1 respectively, and after 60 min to around 40%. At 30 min no increase was seen in any brain region analysed.
Conclusions. Measurements in vivo and in vitro record acute tolerance to AlloP occurring with a delay.
Keywords: anaesthesia, depth ; anaesthetics i.v., allopregnanolone ; measurement techniques, radioimmunoassay ; model, rat ; pharmacology, tolerance
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Introduction |
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Acute tolerance is an alteration in sensitivity to a drug within the duration of a continuous exposure and studies of acute tolerance can reveal the tendency of AlloP to induce chronic tolerance.15 21 The electroencephalographic (EEG) threshold method measures the sensitivity of the brain to anaesthetic drugs.5 18 36 By comparing maintenance dose rates (MDR) needed to retain anaesthesia and by assessing brain concentrations after different periods of anaesthesia, acute tolerance can be revealed. In an earlier report tolerance was not induced by 10 min of anaesthesia.36 In the present study we reassess the importance of the duration of development of anaesthesia in acute tolerance to AlloP by testing anaesthesia periods of 3090 min.
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Methods |
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Drugs
Unless otherwise stated, chemicals used were of analytical quality and purchased from a local supplier. AlloP was purchased from Umecrine (Umeå, Sweden). AlloP was dissolved in 10% 2-hydroxypropyl-ß-cyclodextrin (ß-cyclodextrin; Sigma, St Louis, MO, USA) at a concentration of 3 mg ml1. The preparations were placed in a Bransonic 2210 ultrasonic bath for approximately 15 h and agitated occasionally. All steroids were dissolved in ß-cyclodextrin under visual inspection.
EEG threshold method
The anaesthetic effect of AlloP in rats was determined with an i.v. EEG threshold method.18 The AlloP solution was infused i.v. with a Sage Instruments Model 355 syringe pump (Orion Research, Cambridge, MA, USA) via a tail vein with a constant, optimal infusion rate of 4 mg kg1 min1. This optimal dose rate was defined as that which gave the lowest threshold dose when different dose rates were tested.5 18 36 Differences in body weight were compensated by small corresponding changes in infusion rate. The EEG was recorded continuously with a Mingograph EEG 10 (Siemens-Elema, Stockholm, Sweden), from subcutaneous stainless steel electrodes placed in a bifrontal configuration with a crocodile clip attached to one of the ears as a signal ground. This crocodile clip was not attached until the rat was under deep anaesthesia. The infusion was immediately stopped when the first burst suppression period of 1 s or more was recorded in the EEG (the silent second, SS). As illustrated in an earlier paper,18 the SS is easily detected against a background of an EEG, which at this stage consists mainly of high-amplitude potentials. The appearance of SS occurs at a deeper level of anaesthesia than the loss of righting reflex.18 The time to reach SS was recorded, and the amount of AlloP needed to induce the effect was calculated. This dose was considered to be the threshold dose. The threshold doses recorded in all groups are given in Fig. 1A.
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Since the interval between infusions during the long periods of anaesthesia varied between individual rats, cumulative doses were calculated with intervals of 5 min (Fig. 1BD). These cumulative doses were used to calculate MDRs (mg kg1 min1) where differences in cumulative doses between two time points in individual rats were divided by the time interval used. In the present experiments a 20-min interval was used since several infusions had been performed during that time, which gave an adequate certainty.
When we designed the present study, we performed a pilot study with an anaesthesia duration of 60 min. This pilot study is presented as experiment 1. A control group of nine rats were killed at the first SS. One rat was excluded because of technical difficulties. Group 60 min, in which the rats were killed after 60 min of anaesthesia, consisted of eight rats. Two rats were excluded because of technical difficulties. After dissection, the AlloP concentrations were determined in different parts of the brain.
The pilot study was followed up in experiment 2, in which the effects of 30 and 90 min of anaesthesia were investigated. A control group of nine rats were killed at the first SS, in which one rat was excluded because of technical difficulties. Group 30 min, in which anaesthesia lasted 30 min, consisted of nine rats. Group 90 min, in which anaesthesia lasted 90 min consisted of nine rats. Two rats were excluded from the last two groups because of subcutaneous infusions. After dissection, only the right half of the brain was used to determine AlloP concentrations.
Tissue sample preparation
The rats were killed by decapitation immediately at the end of the predetermined last infusion. Trunk blood was collected and the brain was dissected immediately into the following parts: cerebellum, cortex, hippocampus, brainstem (medulla oblongata and midbrain) and striatum, largely according to Glowinski and Iversen.12 The major blood vessels on the surface of the brain hemispheres were carefully removed when dissecting the brain. A macroscopic autopsy was always carried out. Abdominal fat tissue from the retroperitoneal area and part of the psoas muscle tissue were removed from each rat. After weighing, the tissue was frozen at 70°C until analysis. All the samples were extracted later with 99.5% ethanol for 7 days at +4°C. The recovery of steroids in this procedure has previously been shown to be 100%.8
Celite chromatography and steroid assay
AlloP in tissue and serum extracts were separated with celite chromatography, as described by Bäckström and colleagues1 and verified by Corpéchot and colleagues.7 The recovery rate of AlloP from celite chromatography was determined in every assay using the [3H]allopregnanolone tracer as an indicator of recovery. Recovery of AlloP was 85%. The concentration of AlloP in brain tissue extracts was measured by radioimmunoassay. Radioactive steroid tracer [9,11,12-3H(N)]-5-pregnan-3
-ol-20-one was purchased from NEN Life Science Products (Boston, MA, USA). AlloP antiserum was raised against 3
-hydroxy-20-oxo-5
-pregnan-11
-yl carboxymethyl ether coupled to bovine serum albumin. The antiserum was kindly provided by Dr Robert Purdy (San Diego, CA, USA). Cross-reactivity has been tested earlier26 30 and was highly specific. The sensitivity of both assays was 25 pg, with an intra-assay coefficient of variation of 7% and inter-assay coefficient of variation of 8%. The intra-assay coefficient of variation is calculated from duplicate values in the assay.
Data analysis
A two-way ANOVA, followed ad hoc by a least significant difference test, was used to test the significance of differences for possible pairs in all series. When assessing the maintenance dose rates at different time intervals, the significance of differences was tested with paired Student's t-test for independent samples. Linear parametric correlations (r) and regression coefficients (b) were calculated with conventional methods. All statistical calculations were performed using SPSS statistical software (Chicago, IL, USA). A value of P<0.05 in the two-tailed test was taken to represent significant differences. Non-significant differences are indicated by NS. All results are presented as means with one standard error of mean (SEM); n denotes the number of animals in each test and d.f. is the number of degrees of freedom.
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Results |
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The properties of the present system are further evaluated in Figure 2 by using the calculated dose rate of AlloP needed to maintain anaesthesia (MDR, mg kg1 min1). The difference in cumulative dose determined at 20-min intervals revealed a significant increase in MDR with regard to anaesthesia time in both experiments (paired t-test). In group 60 min of experiment 1 the MDR at the beginning of anaesthesia (MDR1030 min=0.70 (0.03) mg kg1 min1) was significantly different from that at the end of anaesthesia (MDR3555 min=0.79 (0.04) mg kg1 min1; t=4.27, d.f.=7, P<0.01). In group 90 min of experiment 2 the MDR at the beginning of anaesthesia (MDR1030 min=0.67 (0.03) mg kg1 min1) was not significantly different from that in the middle of anaesthesia (MDR3555 min=0.78 (0.04) mg kg1 min1; t=1.96, d.f.=8, P<0.10), while the MDR at the end of anaesthesia (MDR6585 min=0.98 (0.04) mg kg1 min1) was significantly increased when compared with MDR1030 min and MDR3555 min (t=9.42, df=8, P<0.001 and t=3.56, d.f.=8, P<0.01, respectively). Furthermore, in experiment 2 there was no significant difference between the MDR1030 in the group which had only 30 min of anaesthesia and the corresponding MDR in the group which had had 90 min of anaesthesia. Thus it is clear that acute tolerance, recorded as the increase in threshold dose needed to maintain SS, is evident after 90 min of anaesthesia. The induction of acute tolerance can also be traced in vivo after 60 min of anaesthesia.
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Relationship between in vivo and in vitro measurements
In experiment 2 there were some interesting correlations between variables from the group killed after 30 min of anaesthesia. Both MDR1030 min and AlloP concentrations in the hippocampus were positively correlated with the dose of AlloP required to induce the first SS (r=0.78, n=9, b=0.04, P<0.05 and r=0. 81, b=1.53, n=9, P<0.01, respectively).
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Discussion |
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In interpreting experiments on acute tolerance, it is important to investigate the influence of the duration of exposure to the drug. In the present experiments the development of acute tolerance to AlloP can be determined by changes in drug concentration and measurements of MDR. No sign of tolerance development was obtained after 30 min anaesthesia, as the concentrations of AlloP in the brain were not changed compared with the concentrations found at induction. Furthermore, a high correlation was noted between MDR1030 min and the threshold dose of AlloP at induction in the same rats, which indicates that no change had occurred in AlloP sensitivity measured in vivo at 30 min. After 60 min of anaesthesia, brain concentrations showed a significant increase in AlloP in the hippocampus (+42%) and brainstem (+38%). The corresponding MDR3555 min increased by only 13%, but was significantly different from the MDR1030 min. After 60 min of anaesthesia in experiment 2, there was no corresponding significant difference between MDR3555 min and MDR1030 min. Thus, the AlloP tissue concentrations at 60 min gave a clearer indication of acute tolerance than the MDR3555 min. This suggests that the changes in AlloP concentrations had occurred close to the end of 60 min of anaesthesia. After 90 min of anaesthesia, well-defined acute tolerance was recorded both in vitro and in vivo. Consequently, these results show that functional acute tolerance to AlloP develops, but its expression seems to be delayed to around 60 min of anaesthesia.
The AlloP concentrations in the hippocampus and brainstem increased significantly with duration of anaesthesia. In the cortex, cerebellum and striatum the concentration remained unchanged when the duration of anaesthesia was prolonged. This indicates that of the brain regions investigated, the hippocampus and brainstem seem to be the most interesting in relation to the induction of acute tolerance. This conclusion is supported by parallel changes in in vitro concentrations and in in vivo measurements of MDR at the different durations of anaesthesia. Earlier results also indicate that the hippocampus and brainstem are involved in the induction of anaesthesia with AlloP.36 The effect of different dose rates on the threshold doses and corresponding brain concentrations of AlloP was investigated. At the anaesthesia criterion, the concentrations of AlloP should, in critical brain regions, remain unchanged despite changes in dose rate, and this occurred in the hippocampus and brainstem but not in serum, cortex, striatum, cerebellum and muscle.36 In addition, there are other studies indicating that AlloP acts in the hippocampus. AlloP is known to decrease excitability in hippocampal slices at very low concentrations.34 Intraperitoneal administration of AlloP at doses regarded as medium (15 mg kg1) and high (17.5 mg kg1) produced significant reductions in the spontaneous firing rate of hippocampal pyramidal neurons in adult male rats, which lasted at least 60 min. Low doses (10 mg kg1) had no effect.31 Recent findings14 that AlloP treatment degrades spatial learning in rats tested in the Morris water maze also point to the hippocampus. A latency of 8 min between AlloP (2 mg kg1) injection and testing gave a positive response but the corresponding test with a latency of 20 min had no effect. Furthermore, the hippocampus was the region of the brain where the largest difference in AlloP concentration between these two test occasions was found.14 Significant increases in progesterone and pregnandione concentrations in the hippocampus were noted in progesterone-induced anaesthesia, and it was apparent that more pronounced metabolism of progesterone occurs in the hippocampus than in other brain regions.2 In mice, however, progesterone-reduced muscimol binding in the nucleus accumbens and the nucleus caudatus caused a marginal change in the gyrus dentatus but no changes in CA3 of the hippocampus and three regions of the cortex. Because these changes occurred in the GABAA system, they were probably mainly induced by the metabolite AlloP.8 Information on effects in which the brainstem is involved is less abundant. Frye and Vongher11 showed that local injections of three AlloP synthesis inhibitors into the ventral tegmental area reduced the concentration of AlloP and also reduced the lordosis response in hormone-primed females. These responses seemed to be specific, as no similar responses were obtained in other midbrain areas tested.
A comparison of serum and brain concentrations of AlloP indicates a specific kinetic situation in the present experiments. At 60 min (Figs 3 and 4) the concentration in the hippocampus and brainstem had increased without a primary increase in the serum. No similar increase was found in the other brain regions. At 90 min (Figs 3 and 4) the increases in the hippocampal and brainstem concentrations were slightly larger, but there was a corresponding increase in the serum concentration. In this case there were no changes in concentrations in the other brain regions. These results indicate that specific uptake is involved in the distribution of AlloP in the hippocampus and brainstem.
The mechanism of this specific uptake was not investigated in the present work and we can therefore only speculate about the reasons. However, differences in uptake could result from a high number of high-affinity AlloP-binding sites in the hippocampus and brainstem, increased concentrations in the cell membrane or active transport from the blood to these regions. One hypothesis is that AlloP binds to a high-affinity binding site on the GABAA receptor.24 No such binding site has so far been identified. However, different studies demonstrate that the interaction of AlloP with the GABAA receptor is dependent on the subunit composition and that the subunit composition varies between brain regions. In addition, there are changes in the subunit composition caused by AlloP exposure. Increased binding of AlloP to a modified GABAA receptor could explain the increased concentrations of AlloP in the hippocampus and brainstem described in the present paper. The delay of around 60 min in the induction of acute tolerance to AlloP could be long enough for the reorganization of the GABAA receptors in some specific brain regions. The possibility that neurones regulate the postsynaptic GABAA receptors by trafficking is another way to account for the neuronal changes involved in the development of the acute tolerance.16 These issues certainly merit further investigation.
An alternative explanation for the increased AlloP concentration in the hippocampus and brainstem could be high lipid solubility of AlloP. Increased uptake in the membrane could depend on the fluidity of the plasma membrane. This is a well-known explanation of the anaesthetic activity of lipophilic drugs;13 23 it has been questioned but not refuted. This possibility cannot be disregarded when dealing with AlloP, as it has been shown in neurones from the hippocampus that cholesterol enrichment of the neuronal membrane can reduce the effect of AlloP on GABA-induced currents.29 It is possible that the concentrations of AlloP observed in certain brain regions under maintenance of anaesthesia could change the membrane in a cholesterol-like way. This could decrease the potency of AlloP on the GABAA receptor but so far it cannot explain the specific increases in AlloP concentrations in the hippocampus and brainstem.
Data on the concentrations of AlloP in fat and muscle show that redistribution to fat, but not to muscle, is the important component in the elimination of AlloP from the brain. After 90 min of anaesthesia, data (Fig. 5) show that there was still no limit to the capacity of fat to accumulate AlloP and also that there was no increase in redistribution. Thus, it is unlikely that the high concentrations obtained in the hippocampus and brainstem can be explained by decreased transport out of relevant cells. Turnover cannot be determined, but a substantial amount of AlloP must have accumulated in the fairly large fat depot, indicating that the AlloP in the brain was easily accessible to the blood when the concentration in serum was reduced during the intervals between infusions. This supports the hypothesis that AlloP in the brain is bound to sites with affinity that is relatively low but higher than that existing in blood, as accumulation compared with blood concentrations had occurred. The increase in AlloP in the hippocampus and brainstem may thus be due to increased binding at some neurosteroid sites on the GABAA receptor, where changes in receptor structure and/or number could determine binding capacity.
In earlier experiments using the anaesthesia threshold method, acute tolerance to hexobarbital4 32 33 and propofol20 was recorded with the threshold technique. With hexobarbital, maximum acute tolerance had been established after 60 min, seen as a 3545% increase in concentrations in serum, cortex and brainstem. These rats were older than those used in the present experiment. With propofol, 60 min of anaesthesia did not induce any measurable acute tolerance in 30-day-old rats, but in 460-day-old rats acute tolerance with significantly increased concentrations (2050%) were recorded in serum, hippocampus, striatum, brainstem and cerebellum. No increase was seen in the cortex. A comparison with the present results using AlloP indicates that age could be a critical factor. However, there might also be specificity in the brain regions involved in the induction of acute tolerance by different anaesthetic agents. This needs further investigation.
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Acknowledgments |
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References |
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