Phenyl N -tert-butylnitrone, a Free Radical Scavenger, Reduces Mechanical Allodynia in Chemotherapy-induced Neuropathic Pain in Rats

• ❖ Paclitaxel chemotherapy is often limited by painful neuropathy

• ❖ Free radicals may be involved in other types of neuropathic pain, but their role in chemotherapy-induced neuropathy has not been examined

• ❖ In rats, the free radical scavenger phenyl-N -tert-butylnitrone reduced hypersensitivity from paclitaxel but did not prevent it when coadministered

• ❖ Free radical scavengers may be useful in the treatment of paclitaxel-induced neuropathy

CHEMOTHERAPY-INDUCED peripheral neuropathy is a major side effect of many chemotherapeutic agents, including taxanes, platinum-based agents, and vinca alkaloids. Paclitaxel (Sigma, St. Louis, MO), one of the taxanes, is a widely used chemotherapeutic drug for the treatment of breast cancer, cervical cancer, ovarian cancer, nonsmall cell lung carcinomas, and Kaposi sarcoma.1,2It binds to β-tubulin of microtubules, thereby stabilizing microtubules and enhancing polymerization, thereby exerting its anticancer mechanism of action.3,4However, it produces peripheral neurotoxicity, which is characterized by pain, numbness, tingling, and burning sensation.5This peripheral neuropathic pain is a dose-limiting side effect. This is reported with paclitaxel monotherapy at the dose of greater or equal to 250 mg/m²body surface area in as many as 22–100% of patients6,7or as a combination therapy with cisplatin or vincristine. There are no effective treatment options for chemotherapy-induced neuropathic pain partly due to its unknown pathomechanisms.8 

Free radicals, including superoxide radical, hydrogen peroxide, nitric oxide, and nitroperoxide, have been implicated in many neurodegenerated diseases such as Alzheimer disease, amyotropic lateral sclerosis, and brain dysfunction due to injury or aging.8–11They easily react with nucleic acid, protein, and lipid and usually have short-half life. In normal conditions, they play a role in a number of biologic processes, including cell signaling process, and are removed by antioxidant systems such as catalase, superoxide dismutase, glutathione, and glutathione peroxidase. Thus, their levels are precisely controlled by antioxidant systems. However, in pathologic conditions, their levels increase due to either increased production or impaired antioxidant system.

Phenyl N -tert-butylnitrone (PBN) is a spin-trap reagent and a potent free radical scavenger. To date, PBN has been shown to have an analgesic effect in various animal models of pain including the spinal nerve ligation model, capsaicin-induced secondary hyperalgesia, formalin-induced pain, and zymosan-induced visceral pain.12–15Furthermore, other free radical scavengers, such as vitamin E and Tempol, produced significant analgesic effects in both neuropathic pain16–18and inflammatory pain models.19–21However, the effect of PBN on chemotherapy-induced neuropathic pain has not been reported.

Thus, the aims of this study were to determine: (1) whether PBN ameliorates paclitaxel-induced neuropathic pain in rats and (2) whether PBN prevents paclitaxel-induced neuropathic pain in rats.

Male adult Sprague-Dawley rats weighing 200–350 g (Harlan Sprague-Dawley Co., Houston, TX) were used in this study. The animals were housed in groups of two or three in plastic cages with soft bedding and free access to food and water under a 12/12-h light–dark cycle (dark cycle: 7:00 pm–7:00 am). All animals were acclimated in their cages for 1 week before any experiments were performed. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Miami (Miami, Florida) and were carried out in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.

Paclitaxel (Sigma) was dissolved in dimethyl sulfoxide at a concentration of 50 mg/ml and stored in a freezer (−60°C). The solution was mixed with an equal volume of Tween 80 and then diluted in sterile saline to a concentration of 2 mg/ml just before injection. Paclitaxel (2 mg/kg) was injected intraperitoneally on four alternate days (days 0, 2, 4, and 6; cumulative doses of 8 mg/kg) as previously described22to induce painful peripheral neuropathy. Control animals were injected with the same volume of vehicle (4% dimethyl sulfoxide and 4% Tween 80 in saline). The time course of the mechanical allodynia was measured before and at various time points after paclitaxel injection.

PBN (molecular weight, 177.24) was obtained from Sigma Chemical Company. It was dissolved in saline to two different concentrations, namely, (1) to a concentration of 10 mg/ml for the dose of 50 mg/kg and (2) to a concentration of 20 mg/ml for the dose of 100 mg/kg. Thus, all animals received a volume of 5 ml/kg.

To examine the sedative and hyperalgesic effect of PBN, PBN (100 mg/kg) was injected intraperitoneally twice daily for 3 consecutive days starting on day 0 in normal rats.

The analgesic effect of PBN was evaluated when PBN was given as a single intraperitoneal injection in an established paclitaxel-induced neuropathic pain model in rats. Mechanical allodynia was measured after various doses of PBN by using the randomized Latin square design to minimize the number of rats.12,18,23Hence, treatment of paclitaxel was done on four alternate days (days 0, 2, 4, and 6) in six rats. Beginning on the 34th postpaclitaxel injection day, mechanical allodynia was measured, and rats were randomly divided into three groups (saline, PBN 50, and PBN 100). Rats in each group received an intraperitoneal injection of one of the two doses of PBN (50 or 100 mg/kg) or saline as a control. Then, behavioral tests were repeated at 0.5, 1, 2, 4, and 6 h postinjection. After wash-out periods of 3 or 4 days, all rats were then crossed over and got and the next injection, and behavior testing was repeated at the 39th and 42nd postpaclitaxel injection days. Thus, each rat received all three treatments (two doses of PBN and saline) in a random order by the end of the testing period.

The analgesic effects of PBN were determined when multiple intraperitoneal injections of PBN were given after paclitaxel-induced pain had developed. At the 35th postpaclitaxel injection day, 11 rats were divided into two groups, namely, PBN group and control group. Rats in the PBN group received PBN (100 mg/kg) twice daily at 12-h intervals for 3 consecutive days, and the control group received saline (5 ml/kg) twice daily for 3 consecutive days. Behavioral tests to determine mechanical allodynia were performed a day before the injections and the rest of the experimental period.

PBN (100 mg/kg, intraperitoneal) was given twice a day in two different paradigms: (1) starting with the first injection of paclitaxel for 8 days (days 0–7) and (2) starting 7 days after the first injection of paclitaxel for 9 days (days 7–15). Mechanical allodynia was measured repeatedly before and on experimental days 9, 11, 14, 16, 18, and 21 for experiment I or experimental days 2, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21, 23, 25, 28, 30, 32, 35, 37, and 39 for experiment II after each treatment of PBN.

Behavioral tests were conducted blindly so that the experimenter who conducted the tests did not know the nature of the experimental treatment. The behavioral tests measured were foot withdrawal thresholds (as indicator for mechanical allodynia) in response to mechanical stimuli applied to the left and right hind paws.24For each test, the animals were placed in a plastic chamber (8.5 × 8.5 × 28 cm) and habituated for at least 15 min. The chamber was placed on top of a mesh screen, so that mechanical stimuli could be administered to the plantar surface of the left and right hind paws. Thresholds were determined by the up–down method25by using a set of von Frey monofilaments (von Frey filament values: 3.65, 3.87, 4.10, 4.31, 4.52, 4.74, 4.92, and 5.16; equivalent to: 0.45, 0.74, 1.26, 2.04, 3.31, 5.50, 8.32, and 14.45 g values). A von Frey filament was applied perpendicularly to the most sensitive areas of the plantar surface at the center area of paw or the base of the third or fourth toes with sufficient force to bend the filament slightly for 3–4 s. An abrupt withdrawal of the foot during stimulation or immediately after stimulus removal was considered as a positive response. The first stimulus was always the 4.31 filament. When there was a positive response, the next lower filament was used, and when no response was observed, the next higher filament was applied. This testing pattern continued until responses to the sixth von Frey stimuli from the first change of response (either higher or lower than the first stimulus depending on whether the first response was negative or positive) were measured. The responses were then converted into a 50% threshold value using the formula: 50% threshold = 10^{(X  +kd  )}/10⁴, where X  is the value of the final von Frey hair used in log units, k  is the tabular value for the pattern of positive or negative responses, and d  is the mean differences between stimuli in log units (0.22). When positive or negative responses were still observed at the 3.65 or 5.16 filament, values of 0.3 or 18.6 g were assigned, respectively, by assuming a value of ±0.5 for k  in these cases.

The following assessments were made to see whether PBN or paclitaxel induces sedation, which interferes with posture and righting reflexes, as opposed to analgesia which does not.12,26 

Five-point scale for posture:

• 0, normal posture, rearing, and grooming;

• 1, moderate atonia and ataxia. Weight support, but no rearing;

• 2, weight support, but severe ataxia;

• 3, muscle tone but no weight support and only small purposive movements; and

• 4, flaccid atonia, fully immobilized with no attempts at movement.

Five-point scale for righting reflexes:

• 0, rat struggles when placed on its side, followed by rapid forceful righting;

• 1, moderate resistance when the rat is placed on its side, with rapid but not forceful righting;

• 2, no resistance to the rat being placed on its side, with effortful but ultimately successful righting;

• 3, unsuccessful righting; and

• 4, no movements.

Rats were scored immediately after each behavioral test in all experiments.

Data are presented as means ± SEMs and analyzed using the SigmaStat program (Systat Software, Inc., Chicago, IL). Statistical analyses were done using two-way repeated-measures analysis of variances with two-repeated factors followed by Tukey post hoc  test for the experiment of Latin square design or two-way repeated-measures analysis of variances with one-repeated time factor followed by Tukey post hoc  tests. In all cases, P < 0.05 was considered statistically significant.

Because sedation can increase mechanical threshold, all rats treated with paclitaxel or PBN were assessed for sedation throughout the experimental periods. All rats in this study scored 0 indicating that they were not sedated for both posture and righting reflexes, suggesting that increase in mechanical allodynia observed in PBN-treated animals was indeed the result of its analgesic effect.

Rats treated with paclitaxel or PBN showed normal gain of body weight compared with control groups (fig. 1). The baseline mechanical thresholds of all rats before paclitaxel injection was 18.6 g, which was the value as the maximal cutoff point (fig. 2). Paclitaxel decreased the mechanical threshold of both hind paws. Further, the mechanical threshold of both hind paws did not differ significantly from each other. Thus, we used the right hind paw as the site to measure mechanical threshold. After paclitaxel treatment (2 mg/kg) on days 0, 2, 4, and 6, mechanical thresholds began to decrease on days 7–10 and reached its lowest levels (0.8 ± 0.2 g) on days 12–16, and then plateaued for 45 days. Vehicle treatment (4% dimethyl sulfoxide and 4% Tween 80 in saline) did not change mechanical thresholds for 2 months. Furthermore, intraperitoneal injection of PBN (100 mg/kg, twice a day for 3 days starting day 0) in normal rats did not induce changes in mechanical thresholds (fig. 2).

Changes in mechanical thresholds in paclitaxel-induced neuropathic rats after intraperitoneal injections of PBN (50 and 100 mg/kg) were shown in figure 3. The 100 mg/kg of PBN significantly increased mechanical threshold up to normal (>10 g of mechanical threshold) at 2 h after injection and returned to baseline at 6 h (fig. 3).

To examine the prolonged analgesic effects of PBN, it was intraperitoneally injected twice daily at 12-h intervals for 3 days (a total of six doses) at the dose of 100 mg/kg during days 34–36 (fig. 4). The control group received an intraperitoneal injection of saline. Mechanical allodynia was measured once a day in the morning before injections of PBN. Repeated injections of PBN (100 mg/kg) significantly increased mechanical threshold at day 35 and maintained it for 4 days (fig. 4). These data indicate that repeated injections of PBN (100 mg/kg) produce analgesic effects in paclitaxel-induced pain behaviors without sedation.

To examine preventive analgesic effects, PBN (100 mg/kg) was given intraperitoneally twice daily starting either 1 h before the first paclitaxel injection (day 0) for 8 days (paradigm I) or on days 7 through 15 (for 9 days; paradigm II). The early treatment of PBN (paradigm I) did not affect the development of pain behaviors (fig. 5). However, the treatment of PBN starting on day 7 through day 15 completely prevented the development of mechanical allodynia for at least 30 days (fig. 6). Thus, PBN had potent preventive effects in paclitaxel-induced neuropathic pain in rats when given in the early phase of developing mechanical allodynia.

It is well known that oxidative stress plays an important role in the pathogenesis of degenerative diseases,27and neurodegenerative diseases are widely regarded as at least a partial consequence of reactive oxygen species (ROS) damage associated with increased levels of proinflammatory cytokines.9,28,29Moreover, ROS have been implicated in both neuropathic and inflammatory pain conditions because their production has been shown to be increased in injured peripheral nerve and inflamed tissue.30–33According to Kim et al. ,12a barrage of primary afferent injury discharges at the time of nerve injury followed by a steady flow of ectopic discharges into the spinal cord in the neuropathic condition may increase mitochondrial respiration and intracellular calcium and, consequently, lead to increased ROS production. The authors also speculate that it is also possible that excessive ROS builds up in glial cells, which then leaks to produce neuronal damage and dysfunction. Further, it has also been speculated that ROS triggers second messenger system as second messengers are involved in sensitization of dorsal horn neurons34,35and also possibly activates spinal glial cells, which in turn play an important role in chronic pain.36Further, ROS accumulation was also observed primarily in the mitochondria of dorsal horn neurons after capsaicin treatment in mice,19suggesting that increased mitochondrial ROS may be an important mechanism of capsaicin-induced hyperalgesia and central sensitization.

Free radicals are derivatives of molecular oxygen and nitrogen and consist of superoxide, hydroxyl radical, hydrogen peroxide, and peroxynitrite.37These molecules are ubiquitously present in the body and participate in many normal cellular processes including ion transport, transcription, neurotransmission, neuromodulation, and immune responses.37Sources of free radicals are both mitochondrial oxidative metabolisms to produce adenosine triphosphate and several enzymes such as xanthine oxidae, phospholipase A2, cytochrome P450, monoamine oxidase, and tyrosine hydroxylase. They are normally removed by antioxidant systems including superoxide dismutase, catalase, glutathione, glutathione peroxidase, ascorbate, and α-tocopherol. Thus, their levels are precisely controlled by antioxidant systems. However, in pathologic conditions, levels of free radicals may increase due to the increased production or decreased antioxidants level.38,39 

Conversely, antioxidants produce analgesia in both neuropathic12and inflammation pain.13,21However, the underlying mechanism by which ROS reduction alleviates pain remains unclear. Nonetheless, several investigators tried to address this using various neuropathic pain models. In the chronic constriction injury model of neuropathic pain in rats, systemic injection of antioxidants reduced heat hyperalgesia, suggesting that free radicals induced heat hyperalesia.16,30Others reported that systemic ROS scavengers ameliorate the behavioral signs of mechanical allodynia in the spinal nerve ligation model of neuropathic pain.12Further, in the capsaicin-induced pain model, an intradermal injection of capsaicin produced primary and secondary hyperalgesia as a result of peripheral sensitization and central sensitization, respectively.40Most importantly, ROS scavengers were observed to reduce pain in capsaicin-induced secondary hyperalgesia, suggesting ROS involvement in the spinal cord.13 

The molecular mechanisms underlying paclitaxel-induced neuronal damage is a complex phenomenon that needs to be defined. However, Selimovic et al.  addressed this by studying the mechanism of apoptosis in human melanoma cell lines A375 treated with taxol. The authors found that the apoptotic neuronal damage was the result of activation of apoptosis signal-regulated kinase, c-jun NH2-terminal kinase, p38 mitogen-activated protein kinase, and extracellular-regulated kinase together with the down-regulation of uncoupling protein 2.41They also reported that taxol-induced ROS enhanced DNA-binding activity of the transcription factors activator protein-1, activating transcription factor-2 and Ets LiKe gene1, release of cytochrome c , and cleavage of caspases-9 and -3 and poly (ADP-ribose) polymerase. Further, the authors showed that pretreatment of melanoma cells with the c-jun NH-terminal kinase inhibitor2or the p38 inhibitor blocked taxol-induced uncoupling protein 2 down-regulation, ROS generation, and apoptosis. However, extracellular-regulated kinase inhibitor had no such effect. Their conclusion was that taxol-induced mitochondrial stress occurs through the activation of c-jun NH2-terminal kinase and p38 pathways and suggested a novel role for uncoupling protein 2 in the modulation of taxol-induced apoptosis of melanoma cells.

Conversely, PBN has been shown to have a neuroprotective property. Tsuji et al.  42examined the effects of PBN on the p38 mitogen-activated protein kinase pathway and heat shock proteins and reported that the intraperitoneal administration of PBN before ischemia–reperfusion enhanced the activation of extracellular-regulated kinase, suppressed the activation of stress activated protein kinase/c-jun NH2-terminal kinase and p38, and increased the expression of heat shock protein 27 and heat shock protein70 in vivo . Their results indicate that PBN, which has a radical-scavenging property, protects against delayed neuronal death by regulating the p38 mitogen-activated protein kinase signaling pathway and induction of a tolerant state by upregulating heat shock protein in the brain. However, whether this phenomenon applies using our model of injury is yet to be seen.

When PBN is injected intraperitoneally in rats, it rapidly penetrates all organs including brain, spinal cord, and liver within 20 min, and is subsequently excreted in urine and has a half-life of approximately 134 min.43Thus, the sites of action of PBN are organs including both central nervous systems (brain and spinal cord) and peripheral nervous systems (sensory nerves, dorsal root ganglia, and nerve endings).

Even though it is not the subject of this study to investigate the site of action of PBN, we think it is important to address this by presenting the findings of other authors using a different model of injury, namely, spinal nerve ligation model. Given the fact that ectopic discharges originating from the injured peripheral nerve or dorsal root ganglia cells drive central sensitization that underlies neuropathic pain behavior44–46and the fact that systemic PBN (100 mg/kg), which ameliorated the neuropathic pain behaviors, did not reduce the ectopic discharges suggests that the site of action of PBN might not be these peripheral locations.12Specifically, we reported that PBN, ROS scavenger, did not change the rate of ectopic discharges originating from the dorsal root ganglia or peripheral nerve, and intrathecal injection produced a strong analgesic effect, suggesting that the major site of action was the spinal cord. Furthermore, we reported that a systemic administration of the spin-trap reagents PBN and 5,5-dimethyl-pyrroline-N -oxide had the greatest analgesic effect, intrathecal administration had almost as much of an effect, and intraventricular administration had a significant but much smaller effect, and the interpretation of our results was that the reagents worked primarily at spinal levels and somewhat at supraspinal levels.12Again, whether this is true for chemotherapy-induced neuropathic pain is to be determined.

Our study shows that PBN, a free radical scavenger, prevented and ameliorated pain behavior as measured by mechanical allodynia in the paclitaxel-induced neuropathic pain in rats. Further, PBN did not induce sedations at the doses we used, so that the behavioral changes are interpreted as analgesia. Our results also show that the time point of administering PBN is critical in preventing pain behaviors, namely, (1) systemic administration of PBN starting on day 0 through day 7 (before the induction of mechanical allodynia) did not show the preventive effect; (2) however, systemic administration of PBN starting on day 7 through day 15 (during the development of mechanical allodynia) completely prevented the development of pain behaviors; and (3) systemic single or repeated administration of PBN after day 15 (after the induction of pain behaviors) only temporarily ameliorated pain behaviors (mechanical allodynia).

It is well known that paclitaxel-induced neuropathic pain is a dose-limiting side effect, and its mechanism is not fully understood. In this model, nonsteroidal antiinflammatory drugs have little or no effect, and opioids have an analgesic effect only when given at high doses.47Conversely, anticonvulsants, antidepressants, and sodium channel blockers have effect only after repeated administrations.47Recently, Wolf et al.  8reviewed the analgesic effects of intravenous calcium and magnesium treatment, vitamin E, glutamine, glutathione, N -acetylcysteine, oxcarbazepine, and xaliproden in clinical and preclinical studies and concluded that currently no drugs have been proven to prevent chemotherapy-induced neuropathy. In this study, PBN had a potent analgesic effect when given before mechanical allodynia was established. It also had a significant mechanical allodynia-reducing effect when given after mechanical allodynia was established. This indicates that free radical scavengers are potential candidates for the treatment of chemotherapy-induced neuropathic pain.

It is quite interesting to note that repeated injections of PBN on days 7 through 15 completely prevented the development of paclitaxel-induced neuropathic pain in rats. However, when PBN was given on days 0 through 8, this effect was not observed. This suggested that the period between days 7 and 15 is critical in the development of paclitaxel-induced neuropathic pain.

In this study, PBN had potent analgesic effects in both established and developing neuropathic pain. PBN has been reported as an analgesic agent in pain models including the spinal nerve ligation, capsaicin-induced secondary hyperalgesia, formalin-induced pain, and zymosan-induced visceral pain.12–15In those models, it has been reported that the mechanisms of action of PBN is not only to be a free radical scavenger or antioxidant but also to suppress the genes induced by proinflammatory cytokines or other mediators.48Also, other free radical scavengers, such as vitamin E and Tempol, produced significant analgesic effects in the aforementioned pain models,16–21and thus, it was suggested that PBN had analgesic effects mainly via  its antioxidant activities.

In conclusion, systemic administration of PBN, a free radical scavenger, ameliorated the marked mechanical allodynia that developed in paclitaxel-induced neuropathic pain in rats. This analgesia can be prolonged by repeated injections with no side effects such as sedation. Furthermore, we were able to show that chemotherapy-induced neuropathic pain could be prevented when PBN was administered on days 7 through 15. We, thus, conclude that free radical scavengers might be useful in alleviating or preventing chemotherapy-induced neuropathic pain in a clinical setting.

The authors thank Kwan-Je Lee, Ph.D., Professor, Department of Statistics, Dongguk University, Seoul, Korea, for his statistical advice.