Decreased Expression of Intestinal P-Glycoprotein Increase the Analgesic Effects of Oral Morphine in a Streptozotocin-Induced Diabetic Mouse Model
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE Received; June 2, 2011 Published online; August 30, 2011 Accepted; August 12, 2011 doi; 10.2133/dmpk.DMPK-11-RG-051
Decreased expression of intestinal P-glycoprotein increase the analgesic effects of oral morphine in a streptozotocin-induced diabetic mouse model
Ayaka Nawa1, Wakako Fujita-Hamabe1, Shiroh Kishioka2, and Shogo Tokuyama1＊
1 Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin
University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japan
2 Department of Pharmacology, Wakayama Medical University, 811-1 Kimiidera,
Wakayama 641-8059, Japan
1
Copyright C 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
Running title page:
P-gp increase the analgesic effects of oral morphine
*Correspondence to:
Shogo Tokuyama Ph. D.
Department of Clinical Pharmacy, School of Pharmaceutical Sciences
Kobe Gakuin University
1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japan
Tel./Fax: +81-78-974-4780
Email: [email protected]
The number of text pages; 31
The number of figures; 7
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SUMMARY
Morphine is one of the strongest analgesics and commonly used for the treatment of chronic pain. The pharmacokinetic properties of morphine are, in part, modulated by
P-glycoprotein (P-gp). We have previously reported that intestinal P-gp expression levels are influenced via the activation of inducible nitric oxide synthase (iNOS) in streptozotocin (STZ)-induced diabetic mice. Herein, we examined the analgesic effects of orally administrated morphine and its pharmacokinetic properties under diabetic conditions, specifically we focusing on the involvement of intestinal P-gp in a type 1 diabetic mouse model. We assessed the analgesic effect of morphine using the tail-flick test. Serum and brain morphine levels were determined on a HPLC-ECD system. Oral morphine analgesic effects, and serum and brain morphine content were significantly increased nine days after STZ administration. The increase in the analgesic effects of morphine, as well as serum and brain morphine content, was suppressed by aminoguanidine, a specific iNOS inhibitor. Conversely, there were no changes in the analgesic effects obtained with subcutaneous morphine in STZ-treated mice. In conclusions, our findings suggest that the analgesic effects of oral morphine are dependent on intestinal P-gp expression, and that may be one of the problems against obtaining stable pharmacological effects of morphine in diabetic patients.
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Key words
P-glycoprotein, diabetes, intestine, inducible nitric oxide synthase, morphine
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Introduction
Opioids are the strongest analgesics available and commonly used for the treatment of chronic pain in the palliative care of cancer patients (1). However, the severe side effects associated with opioids use, such as vomiting, respiratory, constipation and drowsiness, may outweigh their therapeutic benefits (1). Thus, it is critical that the pharmacokinetics and/or pharmacodynamics of opioids are appropriately regulated. It was previously reported that the pharmacokinetics and pharmacodynamics of opioids may be regulated not only by opioid receptor sensitivity, but also other factors, such as metabolizing enzymes (e.g. UDP-glucuronyltransferase) and drug efflux transporters (2).
P-glycoprotein (P-gp), a drug efflux pumps that transports a large number of drugs, including opioids, is expressed in numerous tissues, such as the blood-brain barrier (BBB), intestines and liver (3-5). It has been reported that opioid disposition and their analgesic effects were significantly enhanced in a P-gp knockout mouse (6).
Furthermore, many reports demonstrate that functional differences in P-gp may result in differences in the analgesic effects produced by opioids (7). These findings indicate that the analgesic effects of opioids are highly dependent on the functional differences in
P-gp. Interestingly, the majority of research in the area has focused on P-gp function
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within the BBB, and only a few reports have focused on intestinal P-gp. Given that the current guidelines of World Health Organization (WHO) recommend to orally administer opioids in the palliative care setting (1), and that the absorptive processes of the small intestine influence the pharmacological effects of orally administered drugs, the present study focused on the influence of intestinal P-gp on the analgesic effects of opioids.
P-gp expression and activity levels are affected by certain pathophysiological conditions, such as cancer, diabetes or ischemic injury (5, 6, 8). We have previously reported that ileal P-gp expression levels are transiently decreased via the activation of inducible nitric oxide synthase (iNOS) in a streptozotocin (STZ)-induced type 1 diabetic mouse model. These changes may affect the pharmacokinetics and pharmacodynamics of orally administered opioids. Additionally, diabetic patients are susceptible to developing cancer (9), and thus it is important to investigate alterations in the pharmacokinetic and pharmacodynamic properties of opioids in the diabetic condition.
Herein, we examined the analgesic effects of orally administrated morphine, and its pharmacokinetic alterations in the diabetic condition. Specifically, we focused on the involvement of intestinal P-gp in a type 1 diabetic mouse model.
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METHODS
Animals
Male ddY mice (Japan SLC Inc., Shizuoka, Japan) (4-5 week-old) were provided with food and water ad libitum, and housed in an animal room that was maintained at
24C and 55 ± 5% humidity with a 12 h light/dark cycle (light phase 8:00-20:00). All procedures were conducted in accordance with the Guiding Principles for the Care and
Use of Laboratory Animals, adopted by the Japanese Pharmacological Society.
Additionally, all experiments were approved by the ethical committee for animals of
Kobe Gakuin University (approval number: A 060601-11).
Mice were injected with streptozotocin (STZ; Sigma, MO., USA) (230 mg/kg, i.p.) dissolved in citrate buffer (pH 4.28). On the ninth and 15th day after STZ administration, mice with non-fasting blood glucose levels above 400 mg/dL were used in the study. Control mice were injected with citrate buffer (0.1 mL/ 10 g).
Drug administration
Morphine (Takeda, Co., ltd., Osaka, Japan; 30 mg/kg) or the vehicle (water) was provided orally to mice. Additionally, morphine was administered subcutaneously (1-10 mg/kg) or intravenously (0.5-5 mg/kg). Aminoguanidine (AG; Sigma, MO., USA) (1
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mg/mL), a specific iNOS inhibitor, was added to drinking water, and provided to mice ad libitum for nine days immediately after STZ administration.
Tail-Flick Test
The morphine analgesia against noxious thermal stimuli was assessed with the tail-flick test. Mice were gently held with the tail positioned in the tail-flick apparatus
(MK-330B; Muromachi Kikai Co., Ltd., Tokyo, Japan) for radiant thermal stimulation of the dorsal surface of the tail. The intensity of the thermal stimulus was adjusted to cause the animal to flick its tail within 3 to 4 s as the base line of the tail-flick latency.
The tail-flick latency was measured before and 30, 60 and 90 min after morphine administration. The cut off time was set at 10 s to minimize tissue damage. The area under the curve (AUC) value for the morphine analgesia on each mouse was calculated for the relevant experiments.
Morphine content
Experiments were preformed as previously described by Shimizu et al. (10).
Briefly, blood and brain samples were collected 15 and 30 min after morphine administration. Serum was separated by centrifugation (3000 rpm for 10 min at 4℃).
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The brain was homogenized in 1 ml of pure water by sonication (20 s). The mixture of
sample (100 l of serum or brain homogenate) and 40% K2HPO4 solution (1 ml) was shaken with 5 ml of ethylacetate for 20 min and then centrifuged at 2000-x g for 5 min at 4℃. The organic layer was collected, and the aqueous layer was re-extracted with 5 min of ethylacetate. Then, the morphine in the organic layer was extracted with 1 ml of
1 M acetic acid, and a 0.9 ml portion of the aqueous layer was lyophilized. The samples were dissolved in 200 l of 0.01 M HCl, and 20 l was analyzed by the HPLC-ECD system. The condition of the HPLC-ECD system were as follows: column, Eicompak
MA-ODS (Eicom, Kyoto, Japan); mobile phase, 0.1 M citric acetate buffer (pH 3.9)/ methanol (82/18) containing 3 mg/l of EDTA and 150 mg/l of sodium octane sulfonate; flow rate, 1 ml/min; detector, ECD-100 (Eicom) 750 mV Ag/ AgCl; temperature, 25 ℃.
Preparation of membrane fractions from intestinal mucosa
Experiments were preformed as previously described by Kageyama et al. (11) with some modifications. Briefly, the ileal mucosa membrane was obtained from STZ or citrate buffer-treated (control) mice. After homogenization (400 rpm, 20 strokes) in homogenizing buffer, the homogenate was centrifuged at 3,000-x g for 10 min at 4°C.
The supernatant was then further centrifuged at 15,000-x g for 15 min at 4°C. Residual
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membrane fractions were resuspended with lysis buffer. Protein concentrations were measured using the Lowry method (DC Protein Assay kit II, Bio-Rad, CA, USA). The membrane fraction was used for P-gp expression quantification.
Western blot analysis for intestinal P-gp expression
Western blot analysis was carried out as previously described (12). Briefly, proteins extracted from the ileal mucosal membrane fraction (50 μg/lane) were separated by electrophoresis on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, and then electrophoretically transferred onto a nitrocellulose membrane. After blocking in blocking buffer containing Tris-buffered saline (TBS, pH 7.6), 0.1% Tween20 and 5% blocking agent (GE Healthcare UK Ltd, Bucks, England), the membrane was incubated with primary antibodies for P-gp (mAb C219, 1:200 dilution; Calbiochem, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 6C5, 1:20,000 dilution; Chemicon, CA, USA). The membrane was then incubated with an
HRP-conjugated anti-mouse secondary antibody (1:2,000 dilution; Kirkegaad & Perry
Laboratories, MD, USA). Immunoreactive bands were visualized using Light Capture
(ATTO, Tokyo, Japan) with an enhanced chemiluminescent substrate for horseradish peroxidase detection (ECL Western Blotting system, GE Healthcare UK Ltd, Bucks,
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England). Signal intensity of the immunoreactive bands was determined by using specialized software (CS-Analyzer version 3.0, ATTO, Tokyo, Japan).
Statistical analysis
Data are expressed as means with ± SEM. Statistical significance was assessed with an unpaired Student’s t-test or one way analysis of variance (ANOVA) followed by
Scheffe’s test. Differences were regarded as statistically significant when the p value was less than 0.05.
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RESULTS
Intestinal P-gp expression levels and analgesic effects of oral morphine in
STZ-treated mice
Ileal P-gp expression levels were significantly lower in STZ-treated mice compared to control mice (Fig. 1A). On the other hand, tail-flick latency following oral morphine administration was significantly greater in STZ-treated versus control mice
(Fig. 1B). Additionally, the analgesic effects of morphine, as determined by the area
under the curve (AUC0-90), were significantly greater in STZ-treated mice than in control mice (Fig. 1C).
Serum morphine concentrations and brain morphine content in STZ-treated mice following oral morphine administration
Serum morphine concentrations, 30 min after oral morphine administration, were significantly greater in STZ-treated mice than in control mice (Fig. 2A). Similarly, brain morphine content was also significantly higher in STZ-treated mice (Fig. 2B). However, brain/plasma ratio was not different between control and STZ-treated mice (Fig. 2C).
The analgesic effects of subcutaneous morphine, and the serum morphine
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concentrations and brain morphine content in STZ-treated mice
The analgesic effects of subcutaneous morphine (1-10 mg/kg) in STZ-treated mice were not significantly different from control mice (Fig. 3A). Additionally, serum morphine concentrations and brain morphine content were not significantly different between control and STZ-treated mice 30 min after subcutaneous administration of morphine (5 mg/kg) (Fig. 3B and C).
Dose-dependent changes in serum and brain morphine levels in STZ-treated mice following intravenous morphine administration
There were dose-dependent changes in both serum and brain morphine levels following intravenous administration of morphine in both control and STZ-treated mice
(Fig. 4). Furthermore, there were not differences in the slopes of the dose-response curves between control and STZ-treated mice.
Effects of an iNOS specific inhibitor on ileal P-gp expression levels and oral morphine analgesia in STZ-treated mice
The significant decrease in P-gp expression levels, which is apparent nine days after STZ administration, was suppressed by AG, a selective iNOS inhibitor. However,
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AG had no effects on P-gp expression levels in control mice (Fig. 5A).
Furthermore, the significant increase in the analgesic effects of oral morphine were suppressed by AG in STZ-treated mice. However, AG had no effects on analgesia induced by oral morphine in control mice (Fig. 5B). Interestingly, AG treatment did not alter the analgesic effects of subcutaneous morphine in STZ-treated mice (Fig. 5C).
Effects of an iNOS specific inhibitor on serum and brain morphine levels in
STZ-treated mice after oral morphine administration
The increase in serum morphine concentrations and brain morphine content 30 min after oral morphine administration was suppressed by AG in STZ-treated mice (Fig.
6).
The analgesic effects of oral morphine, and serum and brain morphine levels 15 days after STZ treatment
On the 15th day of STZ treatment, ileal P-gp expression levels were not significantly different from control mice (Fig. 7A), and the analgesic effects of oral morphine were similar in both control and STZ-treated mice (Fig.7B). Additionally, serum morphine concentrations and brain morphine content 30 min after oral morphine
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administration were not significantly different between control and STZ-treated mice
(Fig. 7C and D).
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DISCUSSION
Although numerous drugs, including morphine, are available in the oral dosage form, the bioavailability of orally administered drugs is lower than that of subcutaneous or intravenous forms due to the first pass effect within the intestines and liver (13). In the present study, it was found that the analgesic effects of oral morphine were significantly altered in STZ-treated mice. Given that the analgesic effects of oral morphine appear to be coupled to intestinal P-gp expression levels, then alterations in the analgesic effects of oral morphine may be due to changes in intestinal morphine-absorptive processes. Indeed, serum morphine concentrations were significantly increased after nine days of STZ treatment, and when intestinal P-gp was decreased. These findings suggest that intestinal absorption of morphine was enhanced in the diabetic condition, where the drug efflux activity of P-gp was down-regulated.
Although there are other types of drug-sensitive transporters (2, 14), only a few reports indicate morphine as their substrate. Thus, P-gp appears to be a key regulator of morphine’s pharmacological effects.
After nine days of STZ treatment, morphine brain content and analgesic effects were significantly increased following oral morphine administration. Since the analgesic effects of morphine are dependent on the binding of morphine to its receptor
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within the central nervous system, it may be that the enhancement of morphine analgesia in STZ-treated mice was a result of the BBB breakdown in the diabetic condition (15). Indeed, it has been reported that P-gp expression and activity levels within the BBB were decreased in the diabetic condition (16). However, our results clearly demonstrate that the analgesic effect, serum concentration and brain content of morphine following its subcutaneous administration (i.e. bypassing the intestinal absorption process) were not different in STZ-treated versus control mice. Moreover, the dose-dependent increase in serum concentrations and brain content of morphine following intravenous administration of morphine in STZ-treated mice did not differ from control mice. Thus, it appears that the BBB does not breakdown on the ninth day of STZ administration. In fact, after oral morphine administration, brain/plasma ratio of morphine contents was not different between control and STZ-treated mice. This finding strongly support the hypothesis that the altered intestinal absorption of morphine may increase morphine brain content and serum morphine concentrations, and thereby enhance its analgesic effects in STZ-treated mice.
Contrary to our present study, it was previously reported that the analgesic effects of subcutaneous or oral morphine was reduced in animals with diabetes-induced hyperalgesia (17, 18). As observed in these previous studies (17. 18), diabetes-induced
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hyperalgesia may also be present in our diabetic mouse model. In the present study, however, the enhancement of oral morphine analgesia associated with the decrease in intestinal P-gp expression levels after nine days of STZ treatment may mask the diabetes-induced hyperalgesia. On the other hand, since morphine levels following subcutaneous administration did not differ between the STZ-treated and control group, then the diabetes-induced decrease in subcutaneous morphine analgesia could be observed when estimated using more appropriate experimental methods for estimating hyperalgesia, such as the tactile mechanical or weak thermal stimuli.
With respect to the mechanism for the altered intestinal P-gp levels in the diabetic condition, we have previously reported the involvement of NOS (12, 19). In particular, intestinal NOS activity gradually and continuously increased after STZ administration accompanying with the increase of blood glucose levels (12). Additionally, when mice were administered NOR5, a known NO donor, to reproduce the increment of NO levels in intestine, intestinal P-gp expression levels were significantly decreased by NOR5 administration, clearly suggests that NOS activation is essential for the significant decrease of intestinal P-gp expression levels. Importantly, it is reported that “high glucose stress” per se could increase NOS activity in vitro (20).
As is well known, NOS is classified into three types of isoform as neuronal NOS
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(nNOS), endothelial NOS (eNOS) and iNOS (21). In our previous study, we have already reported that activation of iNOS plays an important role in decreasing intestinal
P-gp levels in STZ-treated mice (12). Thus, in the present study, we used AG to inhibit decreases in intestinal P-gp expression levels. Since AG has anti-glycation effects (22), it may also affect the diabetic state. However, in our previous study, AG did not affect blood glucose levels (12). Despite that AG was previously reported to attenuate the development of morphine tolerance and dependence (23), it did not affect the analgesic effects of morphine (23), as was shown in the present study. Thus, AG treatment appears to completely suppress the enhancement of morphine analgesia in the diabetic state. It is hypothesized that the analgesic effects of oral morphine are dependent on alterations in intestinal P-gp expression. Although the precise mechanism for alteration of intestinal
P-gp expression via iNOS activation under diabetic condition has not been fully understood in this study, it is expected that both the transcriptional and post-translational regulation may be involved in the mechanisms. For example, several transcription factors, such as NF-B, YB-1 and PXR that could bind elements of mdr1 promoter, might be repressed under NOS activation. In the aspect of post-translational regulation, we now hypothesize the involvement of ubiquitin-proteasome system that could be activated by NOS-mediated pathway (24) in the degradation of P-gp.
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Furthermore, following 15 days of STZ treatment, ileal P-gp expression levels, oral morphine analgesic effects, and serum and brain morphine levels had recovered back to control levels. Interestingly, our previous study clearly demonstrated that this recovery of ileal P-gp expression levels also mediated by NOS activation (19). In particular, when mice were administered L-NAME, a non-selective NOS inhibitor, only during 9th to 15th day after STZ administration, the recovery of P-gp expression levels observed on the 15th day after STZ administration was completely suppressed by
L-NAME (19). These findings suggest that NOS is involved not only in decrease but also in recovery of intestinal P-gp expression levels observed on 9th and 15th day of
STZ-administration, respectively. Noteworthy, it is reported that the effect of NO on the
P-gp function and expression levels was different between short-term and long-term exposure to NO in vitro. Specifically, short-term exposure to NO in rat brain capillary endothelial cells decreased the P-gp function through the PKC activation, while long-term exposure to NO increased the function and expression levels of P-gp (25).
In conclusions, our results clearly indicate that the analgesic effects of oral morphine are significantly enhanced by the iNOS-mediated transient decrease in intestinal P-gp expression. Furthermore, expression levels of P-gp appear to fluctuate throughout the developmental course of diabetes. Unfortunately, there are few
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descriptions of clinical evidence for the changes of pharmacokinetics of P-gp substrate drugs in diabetic patients. As we showed here, the significant changes in pharmacokinetics and pharmacodynamics of oral morphine under diabetic condition was appeared to be period- or severity-dependent. This may make it hard to get clear and critical evidence in clinical field. Our study will provide a caution for the hidden pharmacological changes of P-gp substrate drugs in diabetic patients during advancement of clinical stage. In summary, the alteration of intestinal P-gp expression levels under diabetic condition may be one of the problems against obtaining stable pharmacological effects of morphine in diabetic patients.
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Acknowledgments
This study was, in part, supported by grants-in-aid and special coordination funds from the ‘Academic Frontier’ Project, Cooperative Research Center of Life Sciences.
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Figure Legends:
Fig. 1: Intestinal P-gp expression levels and the analgesic effect of oral morphine in
STZ-treated mice.
Typical western blot image (A), effects of oral morphine analgesia (B and C).
Mice were treated with morphine (30 mg/kg, per os (p.o.)). The cut-off time for the tail flick experiments was set to 10 s to prevent inducing injury to the tail. Data are presented as mean ± SEM. Control (Water) n=10, Control (Morphine) n=10, STZ
(Water) n=9, and STZ (Morphine) n=10, *p < 0.05, **p < 0.01 vs. STZ (Water), #p <
0.05 vs. Control (Morphine), Scheffe’s test.
Fig. 2: Serum morphine concentrations and brain morphine content in
STZ-treated mice following oral morphine administration.
Serum morphine concentrations (A), brain morphine contents (B), brain/plasma ratio (C). Blood and brains were harvested from control and STZ-treated mice 30 min after morphine administration (30 mg/kg, p.o.). Data are presented as mean ± SEM.
Control n=15, STZ n=21, p < 0.05, p < 0.01 vs. Control, unpaired Student’s t-test.
Fig. 3: The analgesic effects of subcutaneous morphine, and serum morphine
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concentrations and brain morphine content in STZ-treated mice.
Effects of subcutaneous morphine analgesia (A), serum morphine concentration
(B), brain morphine contents (C). Mice were treated with morphine (1-10 mg/kg, subcutaneous (s.c.)). The cut-off time for the tail flick experiments was set to 10 s to prevent inducing injury to the tail. Blood and brain samples were harvested from mice
30 min after morphine administration (5 mg/kg, s.c.). Data are presented as mean ±
SEM. (A): Control (1 mg/kg) n=7; Control (3 mg/kg) n=7; Control (5 mg/kg) n=9;
Control (10 mg/kg) n=8; STZ (1 mg/kg) n=6; STZ (3 mg/kg) n=7; STZ (5 mg/kg) n=11; and STZ (10 mg/kg) n=9. (B and C): Control n=6; and STZ n=9.
Fig. 4: Dose-dependent changes in serum and brain morphine levels in
STZ-treated mice following intravenous morphine administration.
Mice were treated with morphine (0.5-5 mg/kg, intravenous (i.v.)), and after 15 minutes, blood and brain samples were harvested. Data are presented as mean ± SEM.
Control (0.5 mg/kg) n=15; Control (1 mg/kg) n=14; Control (2.5 mg/kg) n=12; Control
(5 mg/kg) n=13; STZ (0.5 mg/kg) n=9; STZ (1 mg/kg) n=4; STZ (2.5 mg/kg) n=5; and
STZ (5 mg/kg) n=3.
29 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
Fig. 5: Effects of an iNOS specific inhibitor on ileal P-gp expression levels and analgesic effects of oral morphine analgesia in STZ-treated mice.
Typical western blot image (A), effects of oral morphine analgesia (B), subcutaneous morphine analgesia (C). Mice were treated with morphine (30 mg/kg, p.o.). The cut-off time for the tail flick experiments was set to 10 s to prevent inducing injury to the tail. Aminoguanidine (AG; 1 mg/mL) was provided in the drinking water for 9 days. Data are presented as the mean ± SEM. (B): Control (Water/Water) n=8;
Control (Morphine/Water) n=10; Control (Morphine/AG) n=13; STZ (Water/Water) n=6; STZ (Morphine/Water) n=11; STZ (Morphine/AG) n=12. (C): Saline (SAL)
(Water) n=5; SAL (AG) n=4; Morphine (Water) n=5; and Morphine (AG) n=4.
Fig. 6: Effects of an iNOS specific inhibitor on serum and brain morphine levels in
STZ-treated mice following oral morphine administration.
Serum morphine concentration (A), brain morphine contents (B). Blood and brain samples were harvested 30 min after morphine administration (30 mg/kg, p.o.).
Aminoguanidine (AG; 1 mg/mL) was provided in the drinking water for 9 days. Data are presented as mean ± SEM. Control n = 14; STZ (Water) n=14; and STZ (AG) n=19,
**p < 0.01 vs. Control, p < 0.05 vs. STZ (Water), Scheffe’s test.
30 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
Fig. 7: The analgesic effects of morphine, and serum morphine concentrations and brain morphine content after oral morphine administration 15 days after STZ administration.
Typical western blot image (A), effects of oral morphine analgesia (B), serum morphine concentration (C), brain morphine contents (D). Mice were treated morphine
(30 mg/kg, p.o.). The cut off time was set at 10 s in the test to avoid injury to the tail.
Blood and brain were harvested 30 min after morphine administration (30 mg/kg, p.o.).
Data are presented as mean ± SEM. (B): Control (Water) n=5; Control (Morphine) n=7;
STZ (Water) n=7; and STZ (Morphine) n=14. (C and D): Control mice, n=11; and
STZ-treated mice, n=14.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Fig. 7
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