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From the Departments of *Anesthesiology and Physiology; and Divisions of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.
Address correspondence and reprint requests to Yuki Sato, MD, PhD, Department of Anesthesiology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Address e-mail to aneyuki{at}jichi.ac.jp.
Abstract
BACKGROUND: The calcineurin inhibitor, cyclosporine, is widely used for preventing allograft rejection in organ transplantation. Systemically administered cyclosporine is prevented from entering into the brain by the action of P-glycoprotein, encoded by the multidrug resistant 1 (mdr1) gene. However, in many transplant recipients, cyclosporine administration causes postoperative neuropsychological side effects, such as confusion, depression, and anxiety. Recently, calcineurin-inhibitor-induced pain syndrome, characterized by severe pain in the lower limbs, has also been recognized in both organ and stem-cell transplantations.
METHODS: In the present study, we developed behavioral models in wild-type and mdr1a knockout mice to reveal whether peripheral or central cyclosporine alters pain reactions and hypnotic sensitivities. Cyclosporine's central actions can be better evaluated in mdr1a knockout mice that lack P-glycoprotein. After intraperitoneal administration of cyclosporine, we examined tail-flick latency in the tail immersion test, or duration of loss of righting reflex in response to pentobarbital and ketamine.
RESULTS: In wild-type mice, the highest dose of cyclosporine significantly prolonged the duration of loss of righting reflex in response to ketamine, but not to pentobarbital. On the other hand, the lower doses of cyclosporine significantly increased both pentobarbital- and ketamine-induced sleep durations in mdr1a knockout mice. Tail-flick latencies in the tail immersion test were significantly shortened in both wild-type and knockout mice by the administration of cyclosporine.
CONCLUSIONS: Our results suggest that centrally accumulated cyclosporine enhances the hypnotic response to pentobarbital and ketamine, but peripheral cyclosporine induces hyperalgesia.
Cyclosporine, a cyclic peptide of 11 aminoacids, is used as a first-line treatment for preventing allograft rejection (1), and acts selectively on the early activated T-lymphocytes to inhibit production of soluble proliferative factors, interleukin-2, and other cytokines (2). A large portion of systemically administered cyclosporine is prevented from concentrating in the brain by the action of P-glycoprotein, an adenosine triphosphate-dependent efflux pump transporter encoded by the multidrug resistant 1 (mdr1) gene (3,4). However, in many transplant recipients, cyclosporine administration causes postoperative neuropsychological side effects, such as confusion, depression, and anxiety. Furthermore, calcineurin-inhibitor-induced pain syndrome, characterized by severe pain in the lower limbs, has recently been recognized in the setting of organ and stem-cell transplantations (5–8). Although the underlying mechanisms of these side effects remain unclear (9–13), previous animal experiments demonstrated contradicting effects that cyclosporine increases not only sleeping time from barbiturate administration (14–16) but also isoflurane minimal alveolar concentration of anesthetic (which produces immobility in response to a noxious stimulus in 50% of subjects) (17). Because cyclosporine concentrations in the brain are 17 to 50-fold higher in mdr1a knockout mice than in wild-type mice after systemic administration (3,4), it is thus possible that cyclosporine may affect pain sensitivity at a lower dose of systemic administration in mdr1 knockout mice lacking P-glycoprotein than in wild-type mice. Here we examined the effects of cyclosporine on nociceptive sensitivity in mdr1a knockout and wild-type mice. Using these mice, we also investigated whether cyclosporine affects hypnotic responses induced by anesthetics.
METHODS
Animals
All experiments were performed in accordance with the Jichi Medical University Guide for Laboratory Animals. We obtained permission from the animal care committee to perform the study. Wild-type male Friends virus B mice (CLEA, Tokyo, Japan) and male mdr1a knockout mice (Taconic, NY) on a Friends virus B background weighing 25–35 g were used. These wild-type mice and knockout mice were maintained in a pathogen-free room in a controlled environment (22°C ± 2°C temperature, light/dark cycle [12 h each], light on at 07:00, 40%–60% humidity). During the experiments, each mouse was housed individually in its own cage, and food and water were provided ad libitum.
Drugs
Cyclosporine (Wako, Osaka, Japan) was dissolved in 95% ethanol at a concentration of 100 mg/mL, diluted in Intralipid (Otsuka, Tokyo, Japan) as a carrier (18) and injected intraperitoneally. To examine the hypnotic action of IV anesthetics, pentobarbital (Dainippon, Tokyo, Japan) or ketamine (Sankyo, Tokyo, Japan) diluted in saline was used.
Evaluation of Baseline Motor Function
To confirm whether motor function of knockout mice is not different from that of wild-type mice, we used the automated accelerating rotarod (Ohara, Tokyo, Japan) (19,20). Each mouse was placed on the rotating cylinder and the speed of the cylinder rotation was gradually accelerated from 4 to 40 revolutions per min over a 5-min period. Latency to fall from the rotarod was recorded (21).
Effect of Cyclosporine on Hypnotic Responses
Four hours after intraperitoneal injection of cyclosporine (10 and 60 mg/kg), pentobarbital (50 mg/kg), or ketamine (200 mg/kg) was administered intraperitoneally. In rodents, it has been demonstrated that the bioavailability of cyclosporine in the serum peaks at approximately 4 h after intraperitoneal administration (22). The brain concentration of peripherally injected [3H]cyclosporine was significantly increased 17-fold in mdr1a gene-deficient mice compared with those of normal mice, providing the in vivo evidence for the involvement of P-glycoprotein in transportation of cyclosporine (3,4,23,24).
Hypnotic responses were evaluated as the duration of loss of the righting reflex. As described previously (25), loss of righting reflex was defined as a failure of the mouse to right itself for at least 10 s after being placed on its back. Recovery from the loss of righting reflex was defined as having occurred when the mouse spontaneously righted itself (25). During anesthesia, the animals were kept warm on a plate heated to 38°C. The investigators were blinded to the animals and continuously observed the behavior of the animals during the experiments.
Measurement of Blood Plasma Concentration of Ketamine and Pentobarbital
Because cyclosporine is a potent inhibitor of P450s and both ketamine and pentobarbital are metabolized by P450, the effects of cyclosporine on the pharmacokinetics of pentobarbital and ketamine were measured. Mice were intraperitoneally injected with 60 mg/kg of cyclosporine or vehicle. Four hours later, pentobarbital or ketamine was administered intraperitoneally. Ten, 20, and 60 min after the administration of these anesthetics, trunk blood samples were obtained by decapitation. The plasma concentration of ketamine and pentobarbital was determined by high-performance liquid chromatography at Research Laboratories in Daiich-Sankyo Co. Ltd. and Sumika Chemical Analysis Service, Ltd., respectively.
Effect of Cyclosporine on Pain Reaction
The tail-immersion assay was used to study the pain response (18,26). Four hours after intraperitoneal injection of cyclosporine (1, 10, and 60 mg/kg), the tails of the mice were immersed 2 cm in water of 48°C and the latency time to a rapid tail flick was measured five separate times on each animal over a 15-min period allowing a 3–4 min recovery period between each trial. To prevent injury, the tail was removed from the water within 10 s if the animal did not respond. The times for each animal in each group were then averaged to yield a group mean and standard deviations for comparisons.
Statistics
Statistical analyses were performed using two- or one-way analysis of variance (ANOVA), or t-test. For multiple comparisons, the Tukey's method was used as a post hoc test. Data are expressed as the mean ± sd. A value of P < 0.05 was considered statistically significant.
RESULTS
Baseline Motor Function in Wild-Type and mdr1a Knockout Mice
To examine motor functions, we first performed the rotarod test and latencies to fall from the rotarod were measured. As shown in Figure 1, there were no significant differences between the wild-type and knockout mice by one-way ANOVA [F(1,74) = 1.63, P = 0.21]. These results indicate that knockout mice have no abnormal motor functions and behave normal in laboratory conditions.
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Cyclosporine Augments Hypnotic Responses to Pentobarbital and Ketamine
Without cyclosporine treatments, pentobarbital (Fig. 2) and ketamine (Fig. 3) induced duration of loss of righting reflex in mdr1a knockout mice were similar to those in wild-type mice (P = 0.94 and 0.31, respectively). With cyclosporine treatments, only 60 mg/kg of cyclosporine significantly prolonged the duration of the loss of the righting reflex after ketamine [F(2,35) = 21.96, P < 0.001 by one-way ANOVA] (Fig. 3) but not pentobarbital administration [F(2,32) = 0.99, P = 0.38 by one-way ANOVA] (Fig. 2) in wild-type mice. On the other hand, in mdr1a knockout mice, both 10 and 60 mg/kg of cyclosporine significantly increased pentobarbital- and ketamine-induced sleep times [F(2,29) = 37.05, P < 0.001, and F(2,32) = 9.42, P < 0.001 by one-way ANOVA, respectively] (Figs. 2 and 3). These results suggest that centrally accumulated cyclosporine might enhance the hypnotic response to ketamine and pentobarbital.
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Plasma Blood Concentrations of Ketamine and Pentobarbital in Cyclosporine-Treated Wild-Type Mice
To examine whether cyclosporine administration changes the pharmacokinetics of anesthetic drugs, plasma concentrations of pentobarbital and ketamine were measured in wild-type mice. As shown in Figure 4, there were no significant differences between concentrations of pentobarbital in cyclosporine-treated and untreated wild-type mice [F(1,31) = 0.06, P = 0.81 by one-way ANOVA] (n = 4 in each). On the other hand, cyclosporine-treated wild-type mice had significantly different concentrations of ketamine from untreated mice [F(1,31) = 7.42, P = 0.03 by one-way ANOVA] (n = 4 in each) (Fig. 5).
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Cyclosporine Enhances Pain Reactions
As shown in Figure 6, after administration of cyclosporine (10 and 60 mg/kg), tail-flick latencies in the tail immersion test were significantly shortened in both wild-type [F(3,63) = 13.18, P < 0.001] and knockout mice [F(3,68) = 15.00, P < 0.001] by one-way ANOVA. Two-way ANOVA showed no significant differences between wild-type and knockout mice [F(1,130) = 2.59, P = 0.11], indicating that cyclosporine induces hyperalgesia in both wild-type and mdr1a knockout mice.
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DISCUSSION
The entry of most drugs into the brain is strictly regulated by the blood–brain barrier. Major determinants of the permeation of drugs across the blood–brain barrier have long been thought to be lipophilicity and molecular weight (27,28). Anesthetic-related drugs are often characterized by moderate to high permeability across the blood–brain barrier due to their lipophilicity and intermediate molecular weight (29). This was confirmed using our animal models. As demonstrated in Figures 2 and 3, without cyclosporine treatment, pentobarbital- and ketamine-induced duration of loss of righting reflex in mdr1a knockout mice was similar to those in wild-type mice. These results strongly support that P-glycoprotein itself is not involved in the anesthetic action of pentobarbital and ketamine.
On the other hand, although anticancer drugs, such as vincristine, vinblastine, doxorubicin, and cyclosporine, are highly lipophilic, the apparent permeation of these drugs across the blood–brain barrier is unexpectedly low (27,28). P-glycoprotein, which is coded by the mdr1 acts as a membrane-active efflux system for these drugs (30). Therefore, mdr1a knockout mice lacking P-glycoprotein are useful animal tools for testing the central effects of cyclosporine, because cyclosporine could easily pass into their brains. As shown in Figures 2 and 3, a 60 mg/kg dose of cyclosporine changed the response to ketamine, but not pentobarbital, in wild-type mice. These findings could be explained by pharmacokinetic data showing that 60 mg/kg of cyclosporine increased the concentration of ketamine, but not pentobarbital (Figs. 4 and 5). On the other hand, in mdr1a knockout mice, both 10 and 60 mg/kg of cyclosporine significantly increased pentobarbital- and ketamine-induced sleep times. Although we could not exclude pharmacokinetic drug interactions between cyclosporine and ketamine or pentobarbital in these knockout mice, our results suggest that centrally accumulated cyclosporine enhances hypnotic responses to both ketamine and pentobarbital.
In the next experiment, we tested whether cyclosporine would modulate pain responses in wild-type and mdr1a knockout mice. There are several previous studies demonstrating pain sensitivities in mdr1a knockout mice. For example, Thompson et al. (31) found that morphine induced greater analgesia in P-glycoprotein knockout mice compared with that in wild-type mice, whereas Scott et al. (32) showed that systemic ondansetron increased pain sensitivity in P-glycoprotein knockout mice but had no effect in wild-type mice. As shown in Figure 6, we found that cyclosporine induces hyperalgesia; however, there were no significant differences between wild-type and knockout mice. Therefore, these results suggest that either cyclosporine has a peripheral effect rather than a central effect or that a sufficient quantity reaches the central nervous systems to have hyperalgesic action.
Consistent with our findings, cyclosporine was shown to attenuate the antinociceptive effects of morphine (18) by activation of a nitric oxide (NO) pathway (33). It has been demonstrated that cyclosporine reduces catalytic activity of neuronal NO synthase (nNOS) via inhibition of calcineurin-mediated dephosphorylation of nNOS and decreases the production of NO (27,28). nNOS is localized in the peripheral and central nervous systems, although the sites of cyclosporine remain unclear. On the other hand, desensitization of capsaicin-evoked currents is greatly reduced by cyclosporine in cultured dorsal root ganglion neurons (34). It is thus tempting to speculate that the direct action of cyclosporine on peripheral nerves for pain perception might be related to the pathogenesis of calcineurin-inhibitor-induced neuropathic pain in humans (35).
To conclude, the present study suggests the possibility that cyclosporine augments hypnotic responses centrally and induces hyperalgesia peripherally. Although the small amount of centrally located cyclosporine could be responsible for the hyperalgesic effect, cyclosporine might have different effects on the central and peripheral nervous systems.
Footnotes
Accepted for publication July 26, 2007.
Supported by the grant from Ministry of Education, Culture, Sports, Science, and Technology of Japan (18791104 to YS).
REFERENCES
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