Cancer-related pain is extremely disruptive to a patient's quality of life and is one of the most difficult symptoms to treat. Bone cancer pain is the most common cancer-related pain (1–3). Bone metastases have been identified at autopsy in up to 90% of patients dying from prostate cancer (4,5) and 85% of those dying from breast or lung cancer (6). Bone metastasis-induced pain manifests as spontaneous pain, hyperalgesia and allodynia (7,8). Opioid treatments are fraught with side effects that markedly limit their utility (9,10), whereas nonsteroidal antiinflammatory drugs do not have good efficacy against bone cancer pain (6). Reportedly, 36%–50% of cancer patients suffer from pain severe enough to compromise their daily lives (11,12).
Acupuncture, a traditional therapeutic modality, has been used in China and other Asian countries for thousands of years to treat a variety of diseases and symptoms, including pain (13). Electroacupuncture (EA) has been investigated extensively in studies with normal (uninjured) animals (14). These studies should be extended into injured animal models that resemble clinical pathologic chronic pain. Chronic pain is associated with hyperexcitability of the central nervous system in relation to the transmission and modulation of the noxious messages that give rise to hyperalgesia (15).
Several studies demonstrate that EA has significant therapeutic effects on rat models of chronic inflammatory (16), neuropathic (17), and ankle sprain pain (18). Our study with an animal model of inflammatory pain showed that 10 Hz EA at acupoint Huantiao (GB30) significantly attenuates hyperalgesia (16). However, the effects of EA on bone cancer pain have not been scientifically investigated. Acupuncture has been used clinically to treat cancer-related pain, but the data on the efficacy of acupuncture/EA in cancer pain are ambiguous, and the mechanisms of the effects of acupuncture on such pain are not clear (19,20). The aim of the present study was to evaluate the efficacy and mechanisms of EA on bone cancer pain in an established rat model, produced by injecting prostate cells into the tibia cavity of the rat, causing paw hyperalgesia that closely mimics the human condition (21).
It has been demonstrated that interleukin-1β (IL-1β) is upregulated during bone cancer pain (21). Previous studies have also demonstrated that IL-1 receptor antagonist (IL-1ra) produces analgesic effects in rat models of neuropathic pain (22,23). Intrathecal (IT) IL-1β induces mechanical (24) and thermal hyperalgesia (25) and also enhances responses to C-fiber stimulation, wind-up phenomenon and postdischarge of wide-dynamic range neurons in the spinal dorsal horn of anesthetized rats (24). These data support a role for spinal cord IL-1β in the development of neuropathic pain. Whether IL-1β is involved in bone cancer-induced pain has not been investigated. Therefore, in this study we also investigated the role of IL-1β in bone cancer pain and the effects of EA on bone cancer-induced IL-1β expression, hypothesizing that EA significantly inhibits bone cancer-induced hyperalgesia and concomitantly suppresses IL-1β expression in the spinal cord.
Male Copenhagen rats weighing 200–220 g (Harlan Indianapolis, IN) were kept under controlled conditions (22°C ± 0.5°C, relative humidity 40%–60%, 7:00 am to 7:00 pm alternate light–dark cycles, food and water ad libitum). The animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine.
The study consisted of the following three experiments. Experiment 1: Effects of EA on hyperalgesia: bone cancer rats were divided into EA treatment (n = 7) and sham EA (n = 7) groups. Another group of sham cancer rats plus EA treatment (n = 6), in which vehicle (Hank's solution) was injected into the tibia, was used as a control. Thirty-minute EA treatment was given on Days 14–18, by which time cancer rats showed significant hyperalgesia in our previous study (21). Paw withdrawal latency (PWL) was determined at baseline and on Days 12, 15, and 18, 20 minutes after the EA treatment. The investigator performing the behavioral tests was blind to the treatment assignments. After behavioral testing, rats were deeply anesthetized and the lumbar4–5 spinal cord was removed. Experiment 2, Effect of EA on IL-1β expression in the spinal cord: The spinal cords from Experiment 1 were used to measure relative IL-1β and its mRNA levels using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Another group of sham cancer rats with no intervention (n = 5) was also used as a control. Experiment 3, Effects of Anakinra (also named Kineret), a recombinant, nonglycosolated version of human IL-1ra, on hyperalgesia: Catheter-indwelled cancer rats were divided into IL-1ra treatment (n = 6) and vehicle control (n = 6) groups. Anakinra (Amgen, Thousand Oaks, CA) blocks the biological activity of naturally occurring IL-1 by competitively inhibiting the binding of IL-1 to the IL-1 Type I receptor. The administration of IL-1ra (100 μg/2 μL per rat, IT) or saline was started 13 days postinoculation and continued daily until Day 18. PWL tests were conducted at baseline, 12 days and on 15 and 18 days 2 h postdrug.
Rats were prepared for IT injection under pentobarbital sodium anesthesia (45 mg/kg, IP) as before (26). Detailed procedures for implanting cancer cells into tibia have been described previously (21). The rats were tested for PWL by a previously described method (27,28). Mean PWL was established by averaging the latency of four tests with a 5-minute interval between each test.
The EA treatment procedure was the same as reported in our previous study (16). Ten Hz EA, which in previous studies showed significant antihyperalgesic effects in the rat inflammation model (16,26), were used in the present study. The equivalent of human acupoint GB30 on the rat's hindlimbs was treated bilaterally. In humans, GB30 is located at the junction of the lateral 1/3 and medial 2/3 of the distance between the greater trochanter and the hiatus of the sacrum (13). The comparable landmarks were used to locate GB30. GB30 was chosen based on traditional Chinese medicine meridian theory (29), its successful use in our previous studies, and its use in studies by others (16,30,31).
The animals were gently handled for 30 min each day and habituated to the plastic chamber for 2–3 days. After cleaning the skin with alcohol swabs, two acupuncture needles (gauge # 32, 0.5 in. in length) were swiftly inserted approximately one-half inch deep into each hindlimb of the rat bilaterally at GB30 by one investigator while another gently held the animal. The needles were stabilized with adhesive tape (16). EA was delivered by a stimulator (Electrostimulator 8-C, Pantheon Research Inc., Huntington Beach, CA) via two electrodes at 10 Hz, 2 mA, 0.4 ms pulse width for 30 min on Days 14–18 postsurgery. One end of electrode was soldered to the needle handles in advance, and another end was connected to the output channel of the electrostimulator. A symmetrical biphasic wave was delivered to the electrodes so that the electrode was alternately positive and negative and the bilateral GB30 was stimulated alternately. To minimize discomfort, stimulation intensity was gradually increased over a period of 2 min to 2 mA, which we have found to be the maximum level that can be tolerated by unrestrained rats. Mild muscle twitching was observed. During EA treatment, each rat was placed under an inverted clear plastic chamber (approximately 5 in. × 8 in. × 11 in.) but was neither restrained nor given any anesthetic. The animals remained awake and still during treatment and gave no observable signs of distress. For sham control, acupuncture needles were inserted bilaterally into GB30 without electrical stimulation or manual needle manipulation. Sham EA showed little antihyperalgesia in our previous study (16), making it an appropriate control for nonspecific needling effects. The sham-treated and EA-treated animals were handled identically.
RT-PCR was used to determine the effects of EA on IL-1β mRNA expression. The lumbar4–5 spinal cord was removed and separated ipsilaterally and contralaterally. Our previous study (21) showed that IL-1β was upregulated in the ipsilateral spinal cord of the bone cancer pain model, so the ipsilateral cord was used in the RT-PCR study. Total RNAs were extracted using the RNeasy Mini kit (QIAGEN sciences, Germantown MD), and the first strand of cDNA was synthesized with SuperScript RNase H Reverse Transcriptase (Invitrogen, Carlsbad, CA) at 42°C for 1 h. Multiplex PCR of IL-1β and glyceraldehyde phosphate dehydrogenase (GAPDH) was performed to measure the relative level of IL-1β mRNA (32). Each PCR reaction mixture (0.1 mL) contains cDNA reversely transcribed from 200 ng of total RNA, 10× QIAGEN PCR buffer providing a final concentration of 1.5 mM MgCl2, 4 deoxynucleoside triphosphates (0.2 mM each), 2.5 U of HotstarTaq DNA Polymerase (QIAGEN), and 0.5 μM of each of the 5′ and 3′ IL-1β sequence-specific primers (5′-GCACCTTCTTTTCCTTCATC-3′/5′-CTGATGTAC-CAGTTGGGGAA-3′). To reduce substrate competition, 0.2 μM of each of the 5′ and 3′GAPDH sequence-specific target primers (5′- TGAAGGTCGGTGTGAACGGATTT- GGC-3′/5′-CATGTAGGCCATGAGGTCCACCAC-3′)were added to the reaction mixture after the first 10 cycles of PCR reaction, and then additional 26 cycles of amplification were performed (33). The temperature cycle (Roche, Branchburg, NJ) was 95°C/15 min (initial denaturing), 94°C/0.5 min (denaturing), 54°C/0.5 min, 72°C/1 min (extension) and a final 10-min extension at 72°C. The PCR products, 448 bp for IL-1β and 983bp for GAPDH, were electrophoresed on a 3% ethidium bromide-stained agarose gel, which was photographed using a gel documentation system (DigiGenius Syst. DG1T, SYNGENE, Frederick, MD). The positive PCR bands were purified (Wizard DNA Clean-Up kit, Promega, Madison, WI) and sequenced, and the resulting sequences were identical to the targeted cDNA sequences. The raw data from four individual RT-PCR analyses of each group were used for statistical analysis. Mock RT-PCR reaction controls included the omission of reverse transcriptase, primers, or template. No specific PCR product was found in these reactions.
After the behavioral test, rats (n = 4) were deeply anesthetized with sodium pentobarbital (60 mg/kg, IP) and perfused transcardially with 4% paraformaldehyde (Sigma, St. Louis, MO) in 0.1 M phosphate buffer at pH 7.4. The lumbar 4–5 spinal cord was cut at 30 μm and stained as before (21). The stained sections were analyzed under a Nikon microscope for distribution of IL-1β -immunoreactive cells within the spinal dorsal horn. Five sections were randomly selected from each animal for cell counting. IL-1β-immunoreactive cells were counted in laminae I–II, V–VI, and X of each selected section, averaged separately for sections of each rat, and then averaged for the group.
Data from the thermal hyperalgesia tests were analyzed using analysis of variance (ANOVA) with repeated measures followed by post hoc Scheffé's multiple comparisons (Statistical Analysis System). Data from the immunohistochemistry and RT-PCR studies were analyzed using between-subject ANOVA followed by Scheffé's multiple comparisons. P < 0.05 was set as the level of statistical significance.
Figure 1A shows the effect of EA on PWL in bone cancer rats. Before prostate cancer cell inoculation of the tibia, there were no significant differences in overall mean baseline PWL to noxious thermal stimuli between the two groups of rats (10.66 ± 0.23 vs 10.76 ± 0.19 s). A repeated-measures ANOVA revealed the main effects of EA treatment (F = 28.87, P < 0.0001) and time (F = 10.69, P < 0.0001) as well as the interactions between the EA treatment and time (F = 4.72, P < 0.01). Post hoc means comparisons revealed that cancer cell inoculation of the tibia induced a significant (P < 0.05) decrease in PWL on Days 15 (7.02 ± 0.35 s) and 18 (7.20 ± 0.30 s) after inoculation in ipsilateral hindpaws compared to contralateral hindpaws, which remained at the preinjection level (data not shown). The EA treatment significantly (P < 0.05) increased PWL of ipsilateral hindpaws on Day 15 (9.18 ± 0.64 s) and 18 (9.19 ± 0.40 s) compared to sham EA. These analytical data indicate that bone cancer induced significant and progressive ipsilateral thermal hyperalgesia and that EA significantly alleviated this hyperalgesia. Regarding contralateral PWL, EA did not significantly increase the PWL of contralateral hindpaws compared to baseline (data not shown). Figure 1B shows the effect of EA on PWL in sham bone cancer rats. The ipsilateral and contralateral PWLs were 10.11 ± 0.66 s and 10.49 ± 0.75 s at baseline, respectively, and were 9.60 ± 0.53 s and 10.08 ± 0.54 s on Day 18 after surgery. There were no significant differences between PWLs before EA treatment, at baseline and on Day 12, and PWLs after EA treatment on Day 15 and 18 (Fig. 1B). These data demonstrated that sham injection caused no hyperalgesia and EA treatment did not significantly raise the pain threshold in sham cancer rats (Fig. 1B).
The relative levels of spinal IL-1β mRNA were significantly (P < 0.05) higher in cancer rats given sham EA treatment than in sham cancer rats. This suggests that bone cancer induced IL-1β mRNA upregulation. The levels of spinal IL-1β mRNA were significantly (P < 0.05) lower in cancer rats given EA treatment than in cancer rats given sham EA. This indicates that EA treatment significantly (P < 0.05) inhibited bone cancer-induced IL-1β mRNA transcription compared to sham EA (Fig. 2). At the protein level, it was found that the number of IL-1β-labeled cells was similar in the ipsilateral and contralateral spinal cord in sham cancer rats. Ipsilateral IL-1β-labeled cells numbered 34.4 ± 2.6 per section in laminae I–II, 34 ± 2.2 in V–VI, and 26.5 ± 1.6 in lamina X. IL-1β immunoreactive staining in sham cancer rats plus EA was the same as that in sham cancer rats alone. However, IL-1β immunoreactive staining in the ipsilateral spinal laminae I–II (49.2 ± 1.2 per section), V–VI (74.6 ± 3.8), and X (25.8 ± 1.9) was significantly increased in cancer rats given sham EA treatment compared to that of sham cancer rats (Fig. 3). EA treatment in cancer rats decreased the number of IL-1β-labeled cells significantly (P < 0.05) in laminae V–VI (59.0 ± 2.8), slightly in I–II (42.0 ± 1.7 per section), and had no effects on L-1β in lamina X (26.8 ± 1.5) compared to that of control rats. The data suggest that EA inhibited IL-1β synthesis more powerfully in deep laminae than in superficial laminae in the spinal dorsal horn. The number of IL-1β-labeled cells observed on the contralateral side of the spinal cord was the same in cancer and control rats. Control sections showed no labeling.
Figure 4 shows the effect of IL-1ra on PWL in bone cancer rats. Before prostate cancer cell inoculation of the tibia, there were no significant differences in overall mean baseline PWL to noxious heat stimuli among the groups of rats or between PWL of the left and right hindpaws [ANOVA, F(3,20) = 0.19; P = 0.89]. A 2 × 2 × 4 repeated-measures ANOVA revealed the main effects of IL-1ra treatment (F(1,74) = 4.47, P < 0.05), laterality (F(1,74) = 28.70, P < 0.0001) and time (F(3,74) = 7.12, P < 0.0003) as well as the interaction between laterality and time (F(3,88) = 5.99, P = 0.001). Post hoc means comparisons revealed that IL-ra treatment significantly (P < 0.05) attenuated ipsilateral hyperalgesia compared to vehicle control, increasing the ipsilateral PWL from 7.6 ± 0.33 s to 9.13 ± 0.80 s at Day 15 and from 7.39 ± 0.64 s to 9.89 ± 0.55 s at Day 18. It had no effect on the PWL of contralateral hindpaws. These analytical data indicate that IL-1ra significantly alleviated the hyperalgesia of the ipsilateral paws but did not alter the thermal pain threshold of the contralateral hindpaws (Fig. 4).
The present study demonstrates that EA significantly attenuates bone cancer-induced thermal hyperalgesia in rats. EA had no significant effect on PWLs of sham cancer rats or of contralateral hindpaws of cancer rats. These data indicate that EA at 10 Hz, 2 mA, which is much lower than the EA intensity used in a previous study (34), had little antinociceptive effect in normal rats, but had a significant antihyperalgesic effect in bone cancer rats. Clinical case reports suggest that acupuncture provides significant pain relief to cancer patients (35–38). EA treatment significantly relieved pain in 68%–83% of cancer patients with bone metastasis and markedly reduced the analgesic requirement (38). However, because of problems in methodological design, such as the lack of an appropriate control (35–38) and the absence of patient blinding (39), the clinical evidence regarding the efficacy of acupuncture/EA in cancer pain has been ambiguous. Our data, produced using both treatment and control groups, clearly demonstrate that EA provides significant relief for bone cancer-induced pain.
Opioid treatments for bone cancer pain produce side effects, and both physicians and patients have concerns regarding the risks of dependency and subsequent drug addiction (40). Because acupuncture produces few side effects, the present study suggests that EA treatment has the potential to greatly advance the management of bone cancer pain.
The data also show that IT IL-1ra significantly inhibits bone cancer-induced thermal hyperalgesia. A previous study demonstrated that IL-1β was upregulated in a neuropathic pain rat model (41) and that IT IL-1ra produced an antipain effect in such a model (22). Since IL-1ra is a prototypic antagonist of the IL-1 Type I receptor, through which IL-1β exerts its biological effects, these data support roles for spinal cord IL-1β in neuropathic pain. Our previous study (21) demonstrated that spinal IL-1β is upregulated during bone cancer pain, which suggests that it is involved in bone cancer-induced pain perception. This is further evidenced by the present study's finding that IT IL-1ra significantly inhibited bone cancer pain, which indicates that IL-1β is involved in the spinal transmission and processing of noxious inputs from the peripheral cancer area and facilitates bone cancer hyperalgesia. IL-1ra treatment did not alter PWL in the contralateral hindpaws. This suggests that IL-1β may not be involved in pain perception under uninjured conditions. It should also be noted that our research shows that EA treatment did not raise the thermal pain threshold in sham cancer rats or in the contralateral hindpaws of bone cancer rats.
The RT-PCR study demonstrates that EA significantly suppresses spinal IL-1β mRNA transcription during bone cancer pain. Immunohistochemistry also demonstrates that EA significantly inhibits IL-1β synthesis in the spinal cord. Consistent with the behavioral test, EA did not affect the baseline levels of spinal IL-1β in sham cancer rats or in the contralateral spinal cords of bone cancer rats. Our previous study (21) showed that astrocytes produce IL-1β during bone cancer pain, so the upregulated IL-1β mRNA may be produced by astrocytes. Because our IL-1ra behavioral data indicate that IL-1β facilitates bone cancer-induced hyperalgesia, the EA inhibition of IL-1β expression may contribute, at least in part, to the EA alleviation of bone cancer-induced hyperalgesia. However, we do not exclude the fact that other chemicals, such as opioids, are involved in EA-antihyperalgesia (26). Our previous study demonstrated that EA produces an antihyperalgesic effect in an inflammatory pain rat model by activating the endorphin/ endomorphin (for μ receptors) and enkephalin (for Δ receptors) systems at the spinal level (26). The release of substance P (SP), a key neuropeptide for the transmission of noxious inputs, is also blocked by activation of μ and Δ opioid receptors (42). Further, acupuncture inhibits a tooth pulp stimulation-evoked increase in the release of immunoreactive SP. (43) Because SP is responsible for activating glial cells during pain (44), EA may reduce the release of neurotransmitters (e.g., SP) and inhibit IL-1β expression during pain through the release of endogenous spinal opioids. How opioids and cytokines interact during EA will be clarified in our future studies.
In conclusion, the present study demonstrates, in rats, that EA significantly attenuates bone cancer-induced hyperalgesia, which, at least in part, is mediated by EA suppression of IL-1β expression.
We thank Dr. Lyn Lowry for her editorial support.
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