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Analgesic Effects of Intrathecal Curcumin in the Rat Formalin Test

Background Curcumin has been reported to have anti-inflammatory, antioxidant, antiviral, antifungal, antitumor, and antinociceptive activity when administered systemically. We investigated the analgesic efficacy of intrathecal curcumin in a rat model of inflammatory pain. Methods Male Sprague Dawley rats were prepared for intrathecal catheterization. Pain was evoked by injection of formalin solution (5%, 50 µl) into the hind paw. Curcumin doses of 62.5, 125, 250, and 500 µg were delivered through an intrathecal catheter to examine the flinching responses. The ED50 values (half-maximal effective dose) with 95% confidence intervals of curcumin for both phases of the formalin test were calculated from the dose-response lines fitted by least-squares linear regression on a log scale. Results In rats with intrathecal administration of curcumin, the flinching responses were significantly decreased in both phases. The slope of the regression line was significantly different from zero only in phase 2, and the ED50 value (95% confidence interval) of curcumin was 511.4 µg (23.5-1126.5). There was no apparent abnormal behavior following the administration of curcumin. Conclusions Intrathecal administration of curcumin decreased inflammatory pain in rats, and further investigation to elucidate the precise mechanism of spinal action of curcumin is warranted.


INTRODUCTION
Curcumin (1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione) is a major bioactive component of turmeric, which has been used as a coloring agent in food. It has been used as an herbal medicine for centuries in India, China, Japan, and Korea. Recently, curcumin has received a great deal of attention for its anti-inflammatory, antioxidant, antiviral, antifungal, and antitumor actions [1][2][3][4]. In addition, curcumin has been reported to have antinociceptive activity when administered systemically. The analgesic effects of curcumin are thought to be exerted by suppression of nitrite, TNF-α, and capsaicininduced TRPV1 activity, and through the descending noradrenergic and serotonergic systems, [5][6][7]. Furthermore, curcumin has been shown to exhibit anti-inflammatory activity by means of inhibition of a number of different mediators including phospholipase, lipoxygenase, cyclooxygenase-2, leukotrienes, thromboxane, prostaglandins, and nitric oxide [1,[8][9][10]. As increased spinal cord expression of these substances is associated with pain [11][12][13], we hypothesized that intrathecal administration of curcumin may also produce antinociception.
However, neither the effect of intrathecally administered curcumin nor its spinal mechanism of action has been investigated. Therefore, the purpose of this study was to evaluate the analgesic efficacy of intrathecal curcumin in a rat model of inflammatory pain.

MATERIALS AND METHODS
The experimental protocol was approved by the Institutional Animal Care and Use Committee, Chonnam National University. Experiments were conducted using adult male Sprague Dawley rats weighing 250-300 g. The rats were housed in individual cages in a temperaturecontrolled room (22 ± 0.5 o C) with a 12/12-h light/dark cycle. Food and water were freely available. Intrathecal catheterization was performed for drug administration as described previously [14]. Briefly, the rats were anesthetized with sevoflurane under spontaneous respiration, and placed in a stereotaxic head holder. A polyethylene-10 catheter was inserted through an incision in the atlantooccipital membrane. The catheter was advanced caudally 8.5 cm from the incision site to the lumbar enlargement of the spinal cord. The external end of the catheter was tunneled subcutaneously to exit at the top of the head and plugged with a piece of steel wire, and the skin was closed using 3-0 silk suture. The rats were closely monitored and euthanized through a volatile anesthetic overdose if motor abnormalities appeared. Normal rats were kept in individual cages, and a period of not less than 5 days was allowed for each rat to recover from intrathecal catheterization. Rats showing apparently normal behavior and weight gain were assigned to the experiment.
Curcumin ([1E,6E]-1,7-Bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione, Tocris Cookson Ltd., Bristol, UK) was dissolved in 100% dimethyl sulfoxide (DMSO). The drug was administered intrathecally in a 10 μl solution volume using a hand-driven gear-operated syringe pump, followed by an additional 10 μl of vehicle solution to flush the catheter. To examine the analgesic efficacy of curcumin in the formalin test, curcumin (62.5, 125, 250, 500 μg) was administered intrathecally to 5 rats in each group 10 min prior to the formalin test. Doses of curcumin were determined by the maximum solubility of the drug and for approximately equal spacing on the log scale.
On the day of the experiment, rats were acclimatized in a restraint cylinder for 15-20 min, and were randomly allocated into drug-treatment groups, each receiving one of the doses of curcumin, or the control group, receiving DMSO only. Each rat was used only once and the researcher who tested the drugs was blind to the drug administered to each animal. The formalin test was performed by subcutaneously injecting 50 μl of 5% formalin solution into the plantar surface of the left hind paw using a 30 gauge needle. The formalin injection evoked a characteristic spontaneous flinching behavior, and thus the pain behavior was quantified by periodically counting the number of flinches of the injected paw. The number of flinches was counted for 1 min periods from 1 to 2 min and 5 to 6 min, and every 5 min thereafter, up to 60 min. The observed responses appeared biphasically and were divided into phase 1 (0-9 min) and phase 2 (10-60 min). After the entire observation period, the rats were euthanized with an overdose of volatile anesthetic.
The behavioral effects of curcumin were evaluated in an additional group of rats (n = 5). Motor function was assessed for 60 min after intrathecal administration of 500 μg curcumin. The righting reflex was examined by placing the rat horizontally with its back on the table, which normally produces an immediate coordinated twist-  ing of the body to achieve an upright position. The placing/stepping reflex was evoked by drawing the dorsum of either hind paw across the edge of the table, a stimulus which causes normal rats to try to put their paws forward into a position for walking. Pinna and corneal reflexes were evaluated by stimulating the ear canal or the cornea with a paper string. Normal rats spontaneously jerked their heads or blinked, respectively.
Data are expressed as mean ± SEM. The time-response data of the behavior evoked by formalin are presented as the number of flinches. The dose-response data are presented as a percentage of the control for each phase: % of control = Total number of flinches with drug in phase 1 (2) × 100% Total number of flinches of control in phase 1 (2) Dose-response data were analyzed using the Kruskal-Wallis test and Mann-Whitney U test. The ED50 values (half-maximal effective dose) with 95% confidence intervals of curcumin for both phases of the formalin test were calculated from the dose-response lines fitted by leastsquares linear regression on a log scale [15]. A test to determine whether the effect is dose-dependent was made from the F distribution as previously described [15]. A value of P ＜ 0.05 was considered to indicate statistical significance.

RESULTS
Subcutaneous injection of formalin into the hind paw produced a biphasic flinching response, with an early (phase 1) response lasting 5-10 min, and after a quiescent interval of 5-10 min, a subsequent late (phase 2) response up to 60 min. Fig. 1 shows the time course and dose-response data of intrathecal curcumin, administered 10 min before formalin injection, in the formalin test. In the control group with intrathecal injection of DMSO, the total number of flinches was (mean ± SEM) 28 ± 5 during phase 1 and 220 ± 15 during phase 2. Fig. 2A and 2B show the dose-response curves of intrathecal curcumin on flinching response during both phases of the formalin test. In rats with intrathecal administration of curcumin, the number of flinching responses was significantly decreased to 35-38% of the control group during phase 1 of the formalin test (P ＜ 0.05), but the extent of change was not statistically different over the range of administered doses (P ＞ 0.05, determined by Kruskal-Wallis test, Fig. 2A). During phase 2, intrathecal delivery of curcumin suppressed the flinching response up to 49% of control, and the extent of change was statistically different over the range of doses (P ＜ 0.05, determined by Kruskal-Wallis test, Fig. 2B).
The calculated F values for linear regression of analgesia on log dose of curcumin were 0.036 and 5.902, for phases 1 and 2, respectively. The F value of phase 2 exceeds the tabular F = 4.41 based on the degrees of freedom for this study (1,18) for P ＜ 0.05. These data indicate that the slope of the regression line was significantly different from zero only in phase 2 and that the curcumin-produced analgesia was dose-dependent in phase 2 but not phase 1 of the formalin test. Consequently the ED50 value (95% confidence interval) of curcumin was calculated only for phase 2 and was 511.4 μg (23.5-1,126.5).
There was no apparent abnormal behavior in the rats following the administration of curcumin.

DISCUSSION
Formalin-induced nociception consists of two different nociceptive states, and distinct mechanisms underlie the two phases of behavioral response. Acute nociception (phase 1) is followed by the facilitated state (phase 2). The phase 1 response appears to result from the immediate and intensive increase in activity of the primary afferent fibers induced by formalin, reflecting an acute pain. On the other hand, the phase 2 response may result from the activation of wide dynamic range neurons in the dorsal horn, with a continuous low level of activity in the primary afferent fibers [16]. Therefore, phase 2 represents a facilitated state which appears to be a prominent and intensified pain state in spite of a decreased level of afferent input. This pain model has been utilized as a tool for observing the effects of various antinociceptive agents on these two types of pain at once.
In the present study, we evaluated the analgesic effect of curcumin at the spinal level in a rat model of in-flammatory pain. Intrathecally administered curcumin reduced the flinching response evoked by formalin injection during both phases. Intrathecal curcumin also produced no behavioral abnormalities. These findings suggest that curcumin possesses a spinal mechanism of action, and present the possibility of curcumin as a novel analgesic agent to be delivered in the spinal cord.
Previous reports have demonstrated analgesic effects of systemically administered curcumin in rodent models of diabetic neuropathic pain [5], formalin-induced orofacial pain [17], capsaicin-induced thermal hyperalgesia [6], and chronic constriction injury-induced neuropathic pain [7]. The mechanisms underlying the analgesic action of systemically administered curcumin have been suggested to be associated with suppression of brain nitrite and serum tumor necrosis factor α levels [5], antagonistic effects on the transient receptor potential vanilloid 1 channel [6], and the descending monoamine system coupled with opioid receptors [7]. Although the spinal analgesic effect of curcumin has not been studied, the results of the study of Zhao et al. [7] are consistent with the current study. Intrathecal but not intracerebroventricular injection of adrenergic or serotonergic antagonists blocked the antinociceptive effect of curcumin [7]. As curcumin can pass through the blood-brain barrier [18], systemically administered curcumin could have enhanced spinal serotonergic or adrenergic transmission, which might also contribute to the antinociception produced by intrathecal administration in the current study.
The spinal cord represents a novel site of investigation for pain research, positioned optimally for regulation of pain both in respect to convenient external access for drug delivery and relative isolation from other parts of the central nervous system. Application of spinal delivery of analgesic agents in animal studies has contributed to advances in understanding of the basic science of pain and the analgesic mechanisms of experimental drugs [19]. Furthermore, the establishment of intrathecal drug administration methods in humans has provided a new pain relief option for patients suffering from chronic intractable pain refractory to systemically administered analgesics [20]. As the spinal cord neurons are not known to regenerate, development of novel compounds for spinal delivery must pass particularly strict screening tests before trials in humans [19]. Although curcumin has been shown to be safe in general, it has been demonstrated to cause some gastric irritation in humans [21] and hepatotoxicity in rodents at high doses [22,23]. The findings in the current study may therefore raise the possibility of curcumin as a novel analgesic for spinal delivery, reducing the adverse side effects of systemic administration and thus providing therapeutic benefit in some patients, and further investigation is warranted to elucidate its precise mechanism of spinal action.
There are some limitations to this study. The mechanisms of the observed curcumin-produced analgesia were not elucidated and remain to be further examined. In addition, the differential effects of intrathecal curcumin on the acute and facilitated states should be explored in a future study.
In conclusion, intrathecal administration of curcumin decreased inflammatory pain in rats, and further investigation is warranted to elucidate the precise mechanism of spinal action of curcumin.