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Cancer pain is a significant issue, reaching global proportions. Recognizing the enormity of cancer pain, the International Society for the Study of Pain named 2008-2009 the Global Year Against Cancer Pain. 1.6 million Americans were diagnosed with cancer in 2013 (American Cancer Society), many of whom underwent chemotherapy. Paclitaxel, a commonly prescribed chemotherapy for solid tumor and lymphoid cancers, is associated with a painful, dose-limiting neuropathy that can persist long after adjuvant therapy is completed (Dougherty et al., 2004; Marupudi et al., 2007). Approximately 20% of all patients undergoing a paclitaxel protocol require dose reductions of upwards to 25%; an additional 6% have premature cessation of treatment secondary to neuropathy and neuropathic pain (Mielke et al., 2003). Despite these alarming numbers, little is known about the risk and resilience factors in the precipitation and perpetuation of paclitaxel-induced neuropathic pain. The purpose of this study was to determine whether there were sex- or hypothalamic-pituitary-adrenal (HPA) axis responsivity differences in paclitaxel-induced mechanical hypersensitivity.
Methods:
This reverse-translational, bedside to bench study employed 82 adult, male and female rats (n= 29 Sprague Dawley, n = 26 Lewis, n = 27 Fischer 344 [F344]). The in-bred F344 and Lewis rats were employed due to their HPA axis hyper- and hypo-responsivity, respectively (Sternberg et al., 1992). Rats were entered into the study at 14-16 weeks. Rats were maintained on a 12:12-hr light:dark cycle (lights on at 0800), in an environmental temperature of 23o C (+ 2o), with standard rat chow and water available ad libitum.
Baseline blood withdrawal per tail tip clip was performed during the latter half of the dark phase; no less than 48 hours later, rats were video recorded undergoing the novel stress apparatus Elevated Plus Maze (EPM) for 5 minutes. This testing was conducted during the latter half of the dark phase in a room illuminated with dim red lighting. At the conclusion of EPM testing, rats were returned to the vivarium. Post EPM blood withdrawal was conducted 30 minutes after removal from the EPM apparatus. All blood was collected in a heparinized tube, centrifuged, aliquoted and stored at -80o C. Corticosterone levels were measured in duplicate using corticosterone Enzyme Immunoassay kits from Enzo Life Sciences (Plymouth Meeting, PA).
von Frey Hair testing (VFH) was used to operationalize mechanical hypersensitivity. VFH filaments, also used to measure neuropathy in humans, are a series of small monofilaments of incrementally increasing bending force, ranging from 0.45 to 16.69 gms. The 8-middle VFH filaments were used in this study. Rats sat in small plexiboxes, atop wire mesh. The middle VFH was applied to the plantar hindpaw, between footpads, with force applied to bend it. If a brisk paw withdrawal resulted, the next lower VFH was applied next; in the absence of response, the next higher VFH was applied. This resulted in 4-9 perturbations to each foot. 50% paw withdrawal threshold (PWT), the measure of mechanical hypersensitivity, was calculated according to the methods of Dixon (1980). To be eligible for study entry, rats had to exhibit bilateral 50% PWT of 10 grams. Rats underwent VFH testing daily throughout the protocol, during the latter half of the light phase.
Paclitaxel (Henry Schein) was diluted in 0.9% bacteriostatic saline, just prior to administration, to a concentration of 1 mg/kg; vehicle-injected rats were injected with 0.9% bacteriostatic saline. Rats were weighed on the evening prior to injections. Rats were injected every other day for 7 days (days 1, 3, 5, 7); the next 7 days (8 – 14) were drug free. Injections were completed before lights on. This 14-day cycle was repeated twice (days 15 – 28 and 29 – 42) to model a clinical chemotherapy protocol.
50% PWT data were log transformed to meet normality assumption. Two Repeated Measures ANOVA models were run: (1) to determine the accumulative (post 4, 8, and 12 mg/kg) effects of paclitaxel, in addition to sex and rat strain on mechanical hypersensitivity, and (2) to determine the sustained (7 days after 4, 8, and 12 mg/kg) effects of paclitaxel, in addition to sex and rat strain on mechanical hypersensitivity. Linear regression was run to determine whether HPA axis responsivity was a predictor of paclitaxel induced mechanical hypersensitivity.
This study was approved by the Johns Hopkins Animal Care and Use Committee, Protocol # RA08M267.
Results:
There were no significant strain differences in baseline corticosterone level. Following the 5 minute novel stressor EPM, Lewis rats exhibited significantly reduced corticosterone response compared to F344 (p < 0.001) and Sprague Dawley rats (p < 0.02). Female rats exhibited significantly greater corticosterone response to stress (p < 0.05). Neither baseline corticosterone, nor post-stress corticosterone levels significantly predicted 50% PWT.
The accumulative effects of PAC resulted in significant reductions in 50% PWT (F(1,80) = 50.77, p < 0.001) and there was a significant strain by drug interaction (F(2,79) = 4.75, p < 0.05), with Sprague Dawley rats demonstrating greater PAC-induced mechanical hypersensitivity than Lewis or F344 rats.
The sustained effects of PAC resulted in significant reductions in 50% PWT (F(1,80) = 9.53, p < 0.01), and there were significant interaction effects including sex by strain (F(2,79) = 3.34, p < 0.05), with Sprague Dawley male and Lewis female rats exhibiting significantly reduced 50% PWT compared to their strain counterparts; and strain by drug (F(2,79) = 7.35, p < 0.01), with PAC-injected Sprague Dawley rats (both sexes pooled) exhibiting significantly reduced 50% PWT compared to both in-bred rat strains. PAC-injected F344 rats did not exhibit sustained effects secondary to paclitaxel and exhibited 50% PWT that were not statistically different from their VEH-injected counterparts.
Conclusions:
To our knowledge, this is the first rodent study of paclitaxel-induced mechanical hypersensitivity to employ a paclitaxel paradigm that modeled a clinical chemotherapy protocol that a person with cancer might undergo. Compared to baseline, PAC-injected rats exhibited significantly reduced 50% PWT at the completion of each active drug phase. Compared to the in-bred F344 and Lewis rats, known for their HPA axis hyper- and hypo-responsivity, respectively, the out-bred Sprague Dawley rats exhibited significant, sustained, adverse effects on 50% PWT that were maximized 7 days after receipt of 12 mg/kg cumulative. HPA axis responsivity was not a significant predictor of mechanical hypersensitivity. The a priori hypothesis was that female F344 rats would exhibit significantly greater PAC-induced mechanical hypersensitivity, but our data did not support this hypothesis. The F344 rats nearly returned to baseline 50% PWT levels during the recovery week of each paclitaxel cycle, demonstrating the ability to recover from the adverse effects of the paclitaxel. Work in other labs suggests that the F344 rat’s hyper-responsive HPA axis with its resultant enhanced glucocorticoid and the influence of corticotrophin-releasing hormone on immune response may contribute to the resilience against certain types of pain (Webster et al., 2002), or possibly due to upregulation of brain-derived neurotrophic factor mRNA in the dorsal root ganglia (Herradon et al., 2007).
Paclitaxel receipt is associated with a painful and debilitating neuropathy that can begin early in treatment or weeks after treatment cessation, and it may last for many months. This painful and dose-limiting peripheral neuropathy can adversely affect a cancer patient’s ability to perform activities of daily living, engage in usual roles, and ultimately worsen their quality of life (Bakitas, 2007; Bezjak et al., 2004). Nurses are well-positioned to educate patients and families on the early signs and symptoms of paclitaxel-induced neuropathic pain, as well as assess patients at each chemotherapy appointment. As the prevalence of cancer survivors grows, it is imperative that we better understand the long term pain consequences conferred by paclitaxel therapy and learn the means through which paclitaxel-induced pain onset can be averted or delayed, and/or pain severity reduced. If these findings hold in humans, future research can be conducted to determine: (1) Mechanisms that may confer resilience against chemotherapy-induced neuropathic pain, and (2) Whether therapies targeted at altering HPA axis responsivity show promise in attenuating paclitaxel-induced neuropathic pain.
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