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Early life vincristine fails to prime developing pain pathways
Neuroscience Letters 720 (2020) 134764
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Research article
Early life vincristine fails to prime developing pain pathways a,c
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Lauren M. Styczynski , Katie A. Schappacher , Mark L. Baccei * a b c
Medical Sciences Program, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267, USA Department of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati OH 45267, USA Pain Research Center, Department of Anesthesiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Neonatal Chemotherapy Nociception Rodent
Early life administration of vincristine (VNC), commonly used to treat pediatric leukemia, evokes peripheral neuropathy and mechanical pain hypersensitivity in rats that lasts into adolescence. However, the degree to which VNC-evoked neuropathic pain persists throughout adulthood has yet to be examined. It also remains unclear if pediatric VNC exposure can ‘prime’ developing nociceptive pathways and thereby exacerbate chronic pain following subsequent trauma later in life. To address these issues, rats received five total doses of VNC (60 μg/kg; or vehicle) on postnatal days (P) 11, 13, 17, 19 and 21 followed by a hindpaw surgical incision during adulthood. In addition, in order to model the clinical scenario where cancer relapse necessitates another round of chemotherapy, separate groups of rats that had been treated with VNC (or vehicle) as neonates were subsequently administered VNC as adults (five injections at 100 μg/kg). Intraepidermal nerve fiber density and baseline mechanical pain sensitivity were similar between the neonatal VNC and vehicle-treated littermate controls at 13–15 weeks of age, suggesting that the peripheral neuropathy, and resulting chronic pain, had resolved by adulthood. Importantly, there was no significant overall effect of early life VNC on the severity of post-operative pain following adult incision. Similarly, prior VNC exposure did not significantly influence the degree of mechanical pain hypersensitivity produced by adult VNC treatment. Collectively, these findings suggest that early life VNC administration does not increase the susceptibility to develop chronic pain as adults.
1. Introduction Vincristine (VNC) is a chemotherapeutic agent commonly used to treat acute lymphoblastic leukemia (ALL), the most prevalent cancer in the pediatric population. Chemotherapeutic agents including VNC frequently induce peripheral neuropathy and severe pain in both adults and children [1], often necessitating a dose reduction in the chosen cancer treatment [2]. Emerging evidence suggests that chemotherapyinduced neuropathic pain may involve mechanisms that are distinct from those underlying chronic pain arising from mechanical nerve injury [3]. In addition, it is becoming clear that the consequences of peripheral nerve damage for nociceptive processing, and pain sensitivity, can depend on the age at which the injury occurs [4,5]. With the goal of providing a means to explore the mechanisms by which early life chemotherapy evokes chronic pain in children and adolescents, our recent work established the first preclinical model of neonatal VNC-evoked neuropathic pain [6]. The administration of VNC between postnatal days (P) 11 and 21 in rats produced both static and dynamic mechanical pain hypersensitivity that emerged by P26 and persisted throughout adolescence. This was accompanied by a
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reduction in intraepidermal nerve fiber (IENF) density, which is a hallmark feature of chemotherapy-induced peripheral neuropathy [7]. Subsequent work demonstrated that early life VNC also increased the intrinsic excitability of both dorsal root ganglion (DRG) neurons and lamina I spino-parabrachial projection neurons during adolescence [8]. Nonetheless, one limitation of the above studies is that the duration of the chronic pain state, as well as the anatomical manifestations of peripheral neuropathy, elicited by neonatal VNC administration remains unknown since the prior studies did not extend the behavioral or immunohistochemical analyses beyond P56 [6]. As a result, the degree to which early life VNC evokes pain hypersensitivity throughout adulthood remains to be determined. The dynamic nature of developing nociceptive circuits may render them more susceptible to persistent reorganization following aberrant sensory input during early life. Indeed, neonatal injuries can evoke long-lasting changes in nociceptive signaling in the rodent central nervous system (CNS), which can lead to an exacerbation of pain hypersensitivity following a second injury later in life [9–11]. This has been linked to alterations in spinal neuroimmune interactions following early life tissue damage, as the activation of spinal microglia following
Corresponding author. E-mail address: [email protected] (M.L. Baccei).
https://doi.org/10.1016/j.neulet.2020.134764 Received 25 November 2019; Received in revised form 13 January 2020; Accepted 16 January 2020 Available online 17 January 2020 0304-3940/ © 2020 Elsevier B.V. All rights reserved.
Neuroscience Letters 720 (2020) 134764
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2.3. Response to mechanical stimuli
adult surgical injury is exaggerated when preceded by neonatal surgical injury [5,11,12]. However, whether early life VNC treatment might induce a similar ‘priming’ of developing pain pathways has yet to be investigated. Given the high survival rate of patients with ALL or other pediatric cancers [13–16], as well as the mounting evidence that survivors can be predisposed to adverse health outcomes throughout adulthood [16–18], it is important to better understand how adult pain sensitivity may be influenced by early life chemotherapy. Therefore, the goals of the present study were: (1) to determine if neonatal VNC-evoked neuropathic pain resolves during adulthood; and (2) to identify the potential long-term effects of early life VNC on the severity of chronic pain induced by subsequent injuries later in life. To address the second goal, we employed a well-established preclinical model of post-operative pain [11,19], as well as a second round of VNC administered during adulthood, in order to account for the subset of patients that require additional courses of chemotherapy due to cancer relapse [17,20]. Collectively, the results indicate that neuropathic pain evoked by neonatal VNC resolves by adulthood which is accompanied by a recovery of IENF density in the skin. Furthermore, we fail to observe any evidence that early life VNC increases the severity of adult chronic pain resulting from either surgical incision or additional chemotherapy, thereby providing further evidence that the ability of neonatal trauma to prime pain circuits likely depends on both the precise nature and timing of the early life injury.
Mechanical sensitivity was assessed at: (1) 13–15 weeks of age; (2) 7 h and 2, 4, 7, 9, 11 and 14 days after hindpaw surgical incision at P90; or (3) during and up to 27 days after a second round of VNC treatment initiated at P90 by an experimenter blinded to the neonatal treatment group. Rats were placed into clear acrylic containers separated by black opaque dividers on a mesh flooring at room temperature and allowed to acclimate for at least 20 min. Calibrated von Frey hair monofilaments (filament numbers 12–16) were applied to the plantar hindpaw according to the Simplified Up and Down (SUDO) method [22]. To assess dynamic mechanical allodynia, a paintbrush was used to stimulate the lateral plantar region of the hindpaw in the heel to toe direction. This test was repeated on each paw three times at 10 s intervals and responses were graded on a scale of 0 (no response) to 3 (shaking and licking of the paw) as described previously [23]. Static mechanical hypersensitivity was measured using the pinprick test, in which a blunted needle was placed on the plantar surface of the hindpaw without penetration three times at one-minute intervals, and the percentage of withdrawals was calculated [23]. 2.4. Immunohistochemistry To evaluate the degree of recovery from neonatal VNC-evoked peripheral neuropathy, adult rats were euthanized at P90 with CO2, and skin from the plantar hindpaw was removed and placed in 4 % PFA. After 4 h, tissue was transferred to 30 % sucrose overnight, then frozen in Tissue Freezing Medium (TFM, General Data Healthcare; Cincinnati, OH), sectioned at 40 μm using a cryostat (Leica; Wetzlar, Germany) and processed as free-floating sections. Following a 1 h incubation in blocking solution (0.1 M phosphate-buffered saline containing 5 % normal donkey serum/0.3 % Triton X-100) at room temperature (RT), liquid was decanted and sections were incubated overnight at 4 °C with the primary antibodies: PGP9.5 (Dako; rabbit; 1:500), and collagen IV (Southern Biotech; goat; 1:500). The next day, primary antibody was decanted and sections were washed three times for 30 min each in wash buffer of 0.3 % PBS-TX. After the wash buffer was removed, secondary antibodies (donkey anti-rabbit Alexa 488 at 1:500 and donkey anti-goat Alexa 594 at 1:500; Thermofisher; Waltham, MA) were added for 30 min incubation at RT. The tissue was washed three times for 30 min each then mounted onto slides (Vectashield, Vector Laboratories; Burlingame, CA), cover-slipped, and stored at 4 °C until imaging.
2. Materials and methods 2.1. Animals and drug administration All animals in this study were treated in accordance with welfare guidelines established by the University of Cincinnati Institutional Animal Care and Use Committee. Male and female Sprague Dawley rats (n = 175; Harlan, Indianapolis, IN) were used in this study. Animals were randomly assigned to one of two groups: neonatal vehicle (n = 85) or neonatal vincristine (n = 90). During neonatal treatment, animals received 5 total intraperitoneal (i.p.) injections of 60 μg/kg VNC, or saline as a vehicle control, on postnatal days (P) 11, 13, 17, 19 and 21 as established in our previous work [6]. This protocol is known to evoke peripheral neuropathy and mechanical pain hypersensitivity by P26. Rats aged P1-P21 are referred to as neonates (pre-weaning), P22-P56 as adolescents (encompassing puberty) and > P56 as adults [21]. To explore the potential effects of early life VNC on the severity of post-operative pain during adulthood, a subset of adult rats (n = 31) which received either vehicle or VNC as neonates were further subdivided into the following experimental groups: (1) Neonatal Vehicle + Anesthesia only (n = 8); (2) Neonatal Vehicle + Incision (n = 6); (3) Neonatal VNC + Anesthesia only (n = 9); and (4) Neonatal VNC + Incision (n = 8). In addition, to investigate the effects of neonatal VNC on neuropathic pain evoked by a second VNC regimen later in life, a separate cohort of adult rats which had received either vehicle (n = 12) or VNC (n = 13) as neonates was administered VNC (100 μg/ kg) daily for one week beginning at P90.
2.5. Quantification of intraepidermal nerve fibers (IENFs) For IENF and Langerhans cell quantification, six plantar skin sections were selected per animal at random by an experimenter blinded to the treatment group. Images were obtained using an Olympus BX63 upright motorized fluorescence microscope applying a 20x objective and captured as z-stacks of optical sections at a z-separation of 1 μm. IENFs that crossed the collagen IV-stained dermal/epidermal junction into the epidermis were counted, and fibers that branched after crossing the basement membrane were recorded as a single fiber. For each animal, IENF density was calculated as the average number of fibers per mm of epidermis (IENF/mm). Langerhans cells were counted if they showed immunoreactivity for PGP9.5 and their cell body was visible. For each animal, the Langerhans cells were calculated as the average number of Langerhans cells per mm of epidermis (LC/mm).
2.2. Hindpaw incision As a model of postoperative pain, adult rats were anesthetized with isoflurane (2–3 % in oxygen), and a midline incision in the left hindpaw from mid-heel to the footpad was made through the skin and fascia to expose the plantaris muscle, which was then incised [19]. The wound was closed with 3-0 sutures (Ethicon; Cornelia, GA) and the rats were allowed to recover in their home cage. The wound fully healed by postoperative day 14.
2.6. Statistical analysis Baseline mechanical withdrawal thresholds in adulthood were compared between the neonatal VNC and vehicle-treated groups using two-way ANOVA with Drug and Sex as factors. The effects of early life VNC exposure on behavioral sensitivity to subsequent hindpaw incision were analyzed using Repeated Measure (RM) three-way analysis of 2
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variance (ANOVA) with Time (as the repeated measure), Injury (Incision vs. Anesthesia Only) and Drug (Neonatal VNC vs. Neonatal Vehicle) as factors, and Sidak post hoc tests for multiple comparisons (Prism 8.0; Graphpad Software; San Diego, CA). Meanwhile, the effects of neonatal VNC on the degree of neuropathic pain evoked by adult VNC administration were analyzed with RM two-way ANOVA with Time (as the repeated measure) and Drug (Neonatal VNC vs. Neonatal Vehicle) as factors, and Sidak post hoc tests for multiple comparisons. IENF cell counts and Langerhans cell counts were analyzed using the Mann-Whitney test. For all tests, the significance level was set at p < 0.05. n refers to the number of rats in a given group unless otherwise stated. All data are reported as mean + SEM. 3. Results 3.1. Early life vincristine (VNC) does not alter mechanical sensitivity during adulthood While our prior work clearly demonstrated that the administration of VNC during early life evoked mechanical pain hypersensitivity compared to saline-treated littermate controls that lasted into adolescence, the duration of VNC-evoked neuropathic pain remains unknown since the previous studies did not examine pain behaviors after P56 [6]. To determine if the chronic pain induced by neonatal VNC treatment lasts throughout adulthood, VNC (60 μg/kg; or saline) was administered via i.p. injections at P11, 13, 17, 19 and 21, and mechanical withdrawal thresholds were analyzed by using von Frey monofilaments at 13–15 weeks of age. In light of the accumulating evidence to support sex differences in the mechanisms by which peripheral nerve or tissue damage can trigger chronic pain [12,24,25], data from males and females were analyzed separately. The results indicate that the mechanical pain hypersensitivity induced by early life VNC had resolved by adulthood, as there were no significant differences in mechanical withdrawal thresholds at 13–15 weeks, regardless of sex (n = 19 in each group; Drug: F(1,108) = 0.227, p = 0.635; Sex: F(1,108) = 0.303, p = 0.583; two-way ANOVA; Fig. 1A). Given the lack of sex differences, data from male and female rats were pooled for the subsequent experiments described below. A hallmark feature of chemotherapy-induced peripheral neuropathy (CIPN) is a reduction in the density of intraepidermal nerve fibers [26–28], which was reproduced in our preclinical model of pediatric VNC-evoked neuropathic pain [6]. However, we observed no significant effect of neonatal VNC on intraepidermal nerve fiber (IENF) density (Fig. 1B) at the 13–15 week time point (n = 6–8 in each group; U = 17; p = 0.414; Mann-Whitney test; Fig. 1C), thus pointing to a restoration of peripheral innervation by adulthood. PGP9.5 immunoreactivity (IR) was observed in Langerhans cells (LCs), the resident immune cells of the skin. Notably, while our previous work showed that early life VNC increases the density of LCs exhibiting PGP9.5-IR during adolescence, the density in adulthood was similar between the groups receiving VNC and vehicle as neonates (n = 6–8 in each group; U = 20.5; p = 0.686; Mann-Whitney test; Fig. 1D), further suggesting recovery from the neonatal VNC exposure. 3.2. Neonatal VNC exposure does not significantly influence post-surgical pain in adulthood To begin investigating the degree to which early life chemotherapy can persistently alter the susceptibility to developing chronic pain in response to subsequent injury, VNC (or saline) was administered during the neonatal period as described above. At P90, rats received either a unilateral hindpaw incision [19,29], or anesthesia only as a control, and mechanical withdrawal thresholds were measured at various time points for up to 2 weeks post-injury. Importantly, while we observed a significant effect of incision on mechanical withdrawal thresholds as expected (n = 6–9 per group; Injury: F(1,27) = 238.5, p < 0.0001; RM
(caption on next page)
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Fig. 1. Peripheral neuropathy and chronic pain evoked by neonatal vincristine (VNC) resolves by adulthood. A: Plots of hindpaw mechanical withdrawal threshold (PWT) in adult rats treated with vehicle (VEH) or vincristine (VNC) as neonates. No significant differences were observed between the groups in either males or females (n = 19 in each group; Drug: F(1,108) = 0.227, p = 0.635; Sex: F(1,108) = 0.303, p = 0.583; two-way ANOVA). B: Representative images taken from vehicle-treated (top) and neonatal VNC-treated (bottom) adult rats illustrating PGP9.5-immunoreactive (green) Langerhans cells (clear arrowheads) and sensory fibers (white arrows) crossing the dermal-epidermal junction which was identified via immunostaining for collagen IV (red). C: The density of intraepidermal nerve fibers (IENFs) was similar between neonatal VNC-treated adult rats and VEHtreated littermate controls (n = 6–8 in each group; U = 17; p = 0.414; MannWhitney test). D: The density of PGP9.5-immunoreactive Langerhans cells (LCs) was also similar between groups during adulthood (n = 6–8 in each group; U = 20.5; p = 0.686; Mann-Whitney test).
three-way ANOVA; Fig. 2A), there was no overall effect of neonatal VNC treatment (Drug: F(1,27) = 0.913, p = 0.348) and no significant interaction between early life VNC and incision (Drug X Injury Interaction: F(1,27) = 0.269, p = 0.608). Dynamic mechanical allodynia was also examined before and up to 2 weeks after adult hindpaw incision using the paintbrush test as described previously [6,30]. The data suggest that incision during adulthood increased the sensitivity to innocuous mechanical stimuli (n = 6–8 per group; Injury: F(1,26) = 5.839, p = 0.023; RM three-way ANOVA; Fig. 2B), but did so independently of the neonatal exposure to VNC (Drug X Injury Interaction: F(1,26) = 0.002, p = 0.962), and there was no significant overall effect of neonatal VNC on mechanical allodynia during this period (Drug: F(1,26) = 1.0, p = 0.327). The pinprick test was used to investigate the degree to which neonatal VNC exposure primes spinal nociceptive circuits for static mechanical hyperalgesia following adult injury. The data suggest that incision in adulthood increased the sensitivity to noxious mechanical stimuli (n = 6–9 per group; Injury: F(1,26) = 13.17, p = 0.001; RM three-way ANOVA; Fig. 2C), but this was independent of neonatal exposure to VNC (Drug X Injury Interaction: F(1,26) = 0.255, p = 0.618) with no significant overall effect of VNC on mechanical sensitivity following adult incision (Drug: F(1,26) = 0.013, p = 0.911). 3.3. Neonatal VNC exposure does not significantly influence the severity of neuropathic pain evoked by chemotherapy during adulthood An estimated 10–25 % of ALL patients experience relapse which necessitates additional rounds of chemotherapy [31,32]. While the majority of relapses occur within 3 years of the initial diagnosis in childhood, relapses can also be observed more than a decade later [33]. Therefore, we also investigated whether neonatal VNC exposure influences mechanical hypersensitivity evoked by a second round of VNC treatment during adulthood. Neonatal VNC exposure did not influence the onset or magnitude of mechanical hypersensitivity resulting from adult VNC (n = 12–13 in each group; Neonatal Drug: F(1,23) = 0.133, p = 0.719; Time: F(8,184) = 12.74, p < 0.0001; Neonatal Drug X Time Interaction: F(8,184) = 1.193; p = 0.305; RM two-way ANOVA; Fig. 3). (caption on next page)
4. Discussion The present results suggest that mechanical hypersensitivity evoked by early life VNC treatment [6] resolves by adulthood. This was accompanied by a rescue of two hallmark features of chemotherapy-induced peripheral neuropathy (CIPN), namely a reduction in IENF density in the skin and the appearance of PGP9.5-immunoreactive (IR) Langerhans cells (LCs), thereby pointing to a recovery of peripheral nerve integrity by 13–15 weeks of age. Furthermore, the data indicate that the degree of post-surgical pain evoked by adult hindpaw incision is not significantly influenced by prior exposure to VNC during the
neonatal period (Fig. 2). Similarly, the magnitude and time course of neuropathic pain produced by adult VNC administration was unaffected by early life treatment with VNC (Fig. 3). Collectively, the results suggest that VNC chemotherapy delivered during early life does not lead to a persistent increase in the susceptibility to developing chronic pain during adulthood, although it is clear that additional injury models need to be investigated moving forward. Our prior work demonstrated that the administration of VNC during
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Fig. 2. Early life vincristine (VNC) administration does not exacerbate mechanical pain after adult surgical injury. A: Plot of paw withdrawal threshold (PWT) as a function of post-operative time in rats treated with either vehicle or VNC as neonates, followed by the administration of hindpaw surgical incision (or anesthesia only as a control) during adulthood. There was no overall effect of neonatal VNC treatment (Drug: F(1,27) = 0.913, p = 0.348) and no significant interaction between early life VNC and incision (Drug X Injury Interaction: F(1,27) = 0.269, p = 0.608). B: Withdrawal responses to innocuous brush were plotted as a function of time following adult hindpaw incision, demonstrating that while surgical injury did evoke mechanical allodynia (n = 6–8 per group; Injury: F(1,26) = 5.839, p = 0.023; RM three-way ANOVA), it did so to a similar degree in neonatal VNC-treated rats and vehicle-treated controls (Drug X Injury Interaction: F(1,26) = 0.002, p = 0.962). C: Adult rats treated with VNC or vehicle during early life also exhibited a similar magnitude of static mechanical hyperalgesia, as measured by the pinprick test (n = 6–9 per group; Injury: F(1,26) = 13.17, p = 0.001; Drug X Injury Interaction: F(1,26) = 0.255, p = 0.618; RM three-way ANOVA).
For example, hindpaw incision during the neonatal period increases the magnitude and duration of post-surgical pain following adult incision [11,12,43]. Importantly, clinical studies utilizing quantitative sensory testing (QST) techniques have reported that children with prior experience in a neonatal intensive care unit (NICU), where tissue damage is an inevitable part of their essential medical treatment [44–46], exhibit enhanced perceptual sensitization in response to prolonged nociceptive stimulation even a decade or more after leaving the hospital [47,48]. As a result, it may seem surprising that early life VNC did not influence the degree of mechanical pain hypersensitivity following hindpaw surgical injury administered during adulthood (Fig. 2). However, this could be explained by the fact that the VNC was administered at P11-21, which was selected given that ALL in children has a peak onset between the ages of two and three years. As a result, our dosing regimen may fall outside of the critical period to evoke long-term alterations in the functional organization of central nociceptive circuits, as prior studies have observed that the initial injury must occur within the first postnatal week in order to ‘prime’ pain behavioral responses [9,10,42]. Alternatively, it should be noted that spinal microglia have been implicated as a key mediator of the priming of developing nociceptive pathways after neonatal injury, at least in male rodents [11,12]. Meanwhile, the degree to which chemotherapy-evoked neuropathy is accompanied by the activation of spinal microglia has been controversial, with some studies reporting significant microglial responsivity [49–51] while others failed to detect significant changes in microglial activation [17,52–54]. It has also been clearly demonstrated that peripheral nerve damage in early life evokes a more muted neuroimmune response in the immature spinal dorsal horn compared to the adult [55,56], where an anti-inflammatory cytokine profile can dominate in the weeks after a neonatal injury [5]. This raises the interesting, as yet untested, possibility that a dampened activation of spinal microglia following neonatal VNC explains the absence of exaggerated post-surgical pain after adult hindpaw incision. Among all children with acute lymphoblastic leukemia (ALL), approximately 98 % attain remission, and 85 % of newly diagnosed ALL patients are expected to be long-term event-free survivors with 90 % surviving at 5 years [13,57–59]. Nonetheless, relapse can occur in some of these patients, which requires a second round of chemotherapy that can include VNC [60–63]. Therefore, we also examined whether pediatric VNC exacerbated the degree of neuropathic pain produced by VNC treatment later in life and did not find any persistent effects of prior VNC exposure (Fig. 3). Nevertheless, there are some remaining questions about how these preclinical findings would translate to the clinical setting. For example, the rats in the present study were exposed only to VNC, while pediatric patients are usually administered VNC in combination with other agents such as bortezomib, dexamethasone, prednisone, pegylated L-asparaginase or doxorubicin [61,63]. As a result, future studies should examine the potential consequences of combinatorial chemotherapeutic regimens delivered during early life on the susceptibility to persistent pain during adulthood. In conclusion, these findings demonstrate that neonatal VNC administration fails to ‘prime’ developing pain pathways, thereby suggesting that the potential adverse long-term consequences of early life chemotherapy for the overall quality of life in survivors of childhood cancers may not include an enhanced vulnerability to developing chronic pain after subsequent injury.
Fig. 3. The severity of neuropathic pain evoked by adult vincristine (VNC) does not depend on prior exposure to VNC during early life. Plot of hindpaw mechanical withdrawal threshold (PWT) before and after the administration of VNC to adult rats that had been previously treated with VNC or vehicle as neonates. The resulting mechanical hypersensitivity was similar between the two experimental groups (n = 12–13 in each group; Neonatal Drug: F(1,23) = 0.133, p = 0.719; Time: F(8,184) = 12.74, p < 0.0001; Neonatal Drug X Time Interaction: F(8,184) = 1.193; p = 0.305; RM two-way ANOVA).
early life (i.e. P11-21) produced a delayed onset hypersensitivity to static and dynamic mechanical stimuli that persisted throughout adolescence in both males and females, while no changes in the sensitivity to noxious heat or cold were detected [6]. In addition, a decrease in cutaneous IENF density and the appearance of PGP9.5-IR LCs were observed by P33 [6]. IENF reductions have been extensively reported in CIPN [7,26,34] as well as several other chronic pain states including peripheral neuropathy resulting from diabetes [35–37] and HIV [38], where IENF loss correlates with the severity of neuropathy and may occur in an axonal length-dependent manner [39]. Patients with smallfiber neuropathies can also reportedly exhibit an increased number of LCs in the epidermis, which may play a role in the development and/or maintenance of neuropathic pain [40,41]. Our current results suggest that while peripheral neuropathy and neuropathic pain evoked by neonatal VNC may persist into adolescence [6], they do not continue throughout adulthood, as evidenced by the similar mechanical withdrawal thresholds, IENF densities and immunoreactive LC counts between adult rats administered VNC during early life compared to vehicle-treated littermate controls. Given that neonatal VNC also evokes cell type-dependent alterations in the intrinsic membrane properties of both dorsal root ganglion (DRG) neurons and spinal dorsal horn neurons during adolescence [8], it would be of interest to determine if the documented hyperexcitability of medium- and large-diameter primary afferents and lamina I spinoparabrachial neurons also normalizes during adulthood. Growing evidence suggests that tissue damage during a critical period of early postnatal development can exacerbate the pain hypersensitivity evoked by subsequent injury during adulthood [9,10,42].
CRediT authorship contribution statement Lauren M. Styczynski: Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Katie A. Schappacher: Conceptualization, Methodology. Mark L. Baccei: Conceptualization, Writing - original draft, Writing review & editing, Supervision, Project administration, Funding acquisition. 5
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Acknowledgements
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Sengupta, The laboratory rat: relating its age with human’s, Int. J. Prev. Med. 4 (6) (2013) 624–630. [22] R.P. Bonin, C. Bories, Y. De Koninck, A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments, Mol. Pain 10 (2014) 26. [23] B. Duan, et al., Identification of spinal circuits transmitting and gating mechanical pain, Cell 159 (6) (2014) 1417–1432. [24] R.E. Sorge, et al., Different immune cells mediate mechanical pain hypersensitivity in male and female mice, Nat. Neurosci. 18 (8) (2015) 1081–10813. [25] S. Megat, et al., A critical role for dopamine D5 receptors in pain chronicity in male mice, J. Neurosci. 38 (2) (2018) 379–397. [26] C. Siau, W. Xiao, G.J. Bennett, Paclitaxel- and vincristine-evoked painful peripheral neuropathies: loss of epidermal innervation and activation of Langerhans cells, Exp. Neurol. 201 (2) (2006) 507–514. [27] S.T. Hsieh, et al., Epidermal denervation and its effects on keratinocytes and Langerhans cells, J. Neurocytol. 25 (9) (1996) 513–524. [28] W. Grisold, G. Cavaletti, A.J. Windebank, Peripheral neuropathies from chemotherapeutics and targeted agents: diagnosis, treatment, and prevention, Neuro Oncol 14 (Suppl 4) (2012) iv45–54. [29] J.L. LaPrairie, A.Z. Murphy, Long-term impact of neonatal injury in male and female rats: sex differences, mechanisms and clinical implications, Front. Neuroendocrinol. 31 (2) (2010) 193–202. [30] D. Bowsher, Dynamic mechanical allodynia in neuropathic pain, Pain 116 (1–2) (2005) 164–165. [31] J.M. Chessells, Relapsed lymphoblastic leukaemia in children: a continuing
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Early life vincristine fails to prime developing pain pathways a,c
b,c
T
c,
Lauren M. Styczynski , Katie A. Schappacher , Mark L. Baccei * a b c
Medical Sciences Program, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267, USA Department of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati OH 45267, USA Pain Research Center, Department of Anesthesiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Neonatal Chemotherapy Nociception Rodent
Early life administration of vincristine (VNC), commonly used to treat pediatric leukemia, evokes peripheral neuropathy and mechanical pain hypersensitivity in rats that lasts into adolescence. However, the degree to which VNC-evoked neuropathic pain persists throughout adulthood has yet to be examined. It also remains unclear if pediatric VNC exposure can ‘prime’ developing nociceptive pathways and thereby exacerbate chronic pain following subsequent trauma later in life. To address these issues, rats received five total doses of VNC (60 μg/kg; or vehicle) on postnatal days (P) 11, 13, 17, 19 and 21 followed by a hindpaw surgical incision during adulthood. In addition, in order to model the clinical scenario where cancer relapse necessitates another round of chemotherapy, separate groups of rats that had been treated with VNC (or vehicle) as neonates were subsequently administered VNC as adults (five injections at 100 μg/kg). Intraepidermal nerve fiber density and baseline mechanical pain sensitivity were similar between the neonatal VNC and vehicle-treated littermate controls at 13–15 weeks of age, suggesting that the peripheral neuropathy, and resulting chronic pain, had resolved by adulthood. Importantly, there was no significant overall effect of early life VNC on the severity of post-operative pain following adult incision. Similarly, prior VNC exposure did not significantly influence the degree of mechanical pain hypersensitivity produced by adult VNC treatment. Collectively, these findings suggest that early life VNC administration does not increase the susceptibility to develop chronic pain as adults.
1. Introduction Vincristine (VNC) is a chemotherapeutic agent commonly used to treat acute lymphoblastic leukemia (ALL), the most prevalent cancer in the pediatric population. Chemotherapeutic agents including VNC frequently induce peripheral neuropathy and severe pain in both adults and children [1], often necessitating a dose reduction in the chosen cancer treatment [2]. Emerging evidence suggests that chemotherapyinduced neuropathic pain may involve mechanisms that are distinct from those underlying chronic pain arising from mechanical nerve injury [3]. In addition, it is becoming clear that the consequences of peripheral nerve damage for nociceptive processing, and pain sensitivity, can depend on the age at which the injury occurs [4,5]. With the goal of providing a means to explore the mechanisms by which early life chemotherapy evokes chronic pain in children and adolescents, our recent work established the first preclinical model of neonatal VNC-evoked neuropathic pain [6]. The administration of VNC between postnatal days (P) 11 and 21 in rats produced both static and dynamic mechanical pain hypersensitivity that emerged by P26 and persisted throughout adolescence. This was accompanied by a
⁎
reduction in intraepidermal nerve fiber (IENF) density, which is a hallmark feature of chemotherapy-induced peripheral neuropathy [7]. Subsequent work demonstrated that early life VNC also increased the intrinsic excitability of both dorsal root ganglion (DRG) neurons and lamina I spino-parabrachial projection neurons during adolescence [8]. Nonetheless, one limitation of the above studies is that the duration of the chronic pain state, as well as the anatomical manifestations of peripheral neuropathy, elicited by neonatal VNC administration remains unknown since the prior studies did not extend the behavioral or immunohistochemical analyses beyond P56 [6]. As a result, the degree to which early life VNC evokes pain hypersensitivity throughout adulthood remains to be determined. The dynamic nature of developing nociceptive circuits may render them more susceptible to persistent reorganization following aberrant sensory input during early life. Indeed, neonatal injuries can evoke long-lasting changes in nociceptive signaling in the rodent central nervous system (CNS), which can lead to an exacerbation of pain hypersensitivity following a second injury later in life [9–11]. This has been linked to alterations in spinal neuroimmune interactions following early life tissue damage, as the activation of spinal microglia following
Corresponding author. E-mail address: [email protected] (M.L. Baccei).
https://doi.org/10.1016/j.neulet.2020.134764 Received 25 November 2019; Received in revised form 13 January 2020; Accepted 16 January 2020 Available online 17 January 2020 0304-3940/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Response to mechanical stimuli
adult surgical injury is exaggerated when preceded by neonatal surgical injury [5,11,12]. However, whether early life VNC treatment might induce a similar ‘priming’ of developing pain pathways has yet to be investigated. Given the high survival rate of patients with ALL or other pediatric cancers [13–16], as well as the mounting evidence that survivors can be predisposed to adverse health outcomes throughout adulthood [16–18], it is important to better understand how adult pain sensitivity may be influenced by early life chemotherapy. Therefore, the goals of the present study were: (1) to determine if neonatal VNC-evoked neuropathic pain resolves during adulthood; and (2) to identify the potential long-term effects of early life VNC on the severity of chronic pain induced by subsequent injuries later in life. To address the second goal, we employed a well-established preclinical model of post-operative pain [11,19], as well as a second round of VNC administered during adulthood, in order to account for the subset of patients that require additional courses of chemotherapy due to cancer relapse [17,20]. Collectively, the results indicate that neuropathic pain evoked by neonatal VNC resolves by adulthood which is accompanied by a recovery of IENF density in the skin. Furthermore, we fail to observe any evidence that early life VNC increases the severity of adult chronic pain resulting from either surgical incision or additional chemotherapy, thereby providing further evidence that the ability of neonatal trauma to prime pain circuits likely depends on both the precise nature and timing of the early life injury.
Mechanical sensitivity was assessed at: (1) 13–15 weeks of age; (2) 7 h and 2, 4, 7, 9, 11 and 14 days after hindpaw surgical incision at P90; or (3) during and up to 27 days after a second round of VNC treatment initiated at P90 by an experimenter blinded to the neonatal treatment group. Rats were placed into clear acrylic containers separated by black opaque dividers on a mesh flooring at room temperature and allowed to acclimate for at least 20 min. Calibrated von Frey hair monofilaments (filament numbers 12–16) were applied to the plantar hindpaw according to the Simplified Up and Down (SUDO) method [22]. To assess dynamic mechanical allodynia, a paintbrush was used to stimulate the lateral plantar region of the hindpaw in the heel to toe direction. This test was repeated on each paw three times at 10 s intervals and responses were graded on a scale of 0 (no response) to 3 (shaking and licking of the paw) as described previously [23]. Static mechanical hypersensitivity was measured using the pinprick test, in which a blunted needle was placed on the plantar surface of the hindpaw without penetration three times at one-minute intervals, and the percentage of withdrawals was calculated [23]. 2.4. Immunohistochemistry To evaluate the degree of recovery from neonatal VNC-evoked peripheral neuropathy, adult rats were euthanized at P90 with CO2, and skin from the plantar hindpaw was removed and placed in 4 % PFA. After 4 h, tissue was transferred to 30 % sucrose overnight, then frozen in Tissue Freezing Medium (TFM, General Data Healthcare; Cincinnati, OH), sectioned at 40 μm using a cryostat (Leica; Wetzlar, Germany) and processed as free-floating sections. Following a 1 h incubation in blocking solution (0.1 M phosphate-buffered saline containing 5 % normal donkey serum/0.3 % Triton X-100) at room temperature (RT), liquid was decanted and sections were incubated overnight at 4 °C with the primary antibodies: PGP9.5 (Dako; rabbit; 1:500), and collagen IV (Southern Biotech; goat; 1:500). The next day, primary antibody was decanted and sections were washed three times for 30 min each in wash buffer of 0.3 % PBS-TX. After the wash buffer was removed, secondary antibodies (donkey anti-rabbit Alexa 488 at 1:500 and donkey anti-goat Alexa 594 at 1:500; Thermofisher; Waltham, MA) were added for 30 min incubation at RT. The tissue was washed three times for 30 min each then mounted onto slides (Vectashield, Vector Laboratories; Burlingame, CA), cover-slipped, and stored at 4 °C until imaging.
2. Materials and methods 2.1. Animals and drug administration All animals in this study were treated in accordance with welfare guidelines established by the University of Cincinnati Institutional Animal Care and Use Committee. Male and female Sprague Dawley rats (n = 175; Harlan, Indianapolis, IN) were used in this study. Animals were randomly assigned to one of two groups: neonatal vehicle (n = 85) or neonatal vincristine (n = 90). During neonatal treatment, animals received 5 total intraperitoneal (i.p.) injections of 60 μg/kg VNC, or saline as a vehicle control, on postnatal days (P) 11, 13, 17, 19 and 21 as established in our previous work [6]. This protocol is known to evoke peripheral neuropathy and mechanical pain hypersensitivity by P26. Rats aged P1-P21 are referred to as neonates (pre-weaning), P22-P56 as adolescents (encompassing puberty) and > P56 as adults [21]. To explore the potential effects of early life VNC on the severity of post-operative pain during adulthood, a subset of adult rats (n = 31) which received either vehicle or VNC as neonates were further subdivided into the following experimental groups: (1) Neonatal Vehicle + Anesthesia only (n = 8); (2) Neonatal Vehicle + Incision (n = 6); (3) Neonatal VNC + Anesthesia only (n = 9); and (4) Neonatal VNC + Incision (n = 8). In addition, to investigate the effects of neonatal VNC on neuropathic pain evoked by a second VNC regimen later in life, a separate cohort of adult rats which had received either vehicle (n = 12) or VNC (n = 13) as neonates was administered VNC (100 μg/ kg) daily for one week beginning at P90.
2.5. Quantification of intraepidermal nerve fibers (IENFs) For IENF and Langerhans cell quantification, six plantar skin sections were selected per animal at random by an experimenter blinded to the treatment group. Images were obtained using an Olympus BX63 upright motorized fluorescence microscope applying a 20x objective and captured as z-stacks of optical sections at a z-separation of 1 μm. IENFs that crossed the collagen IV-stained dermal/epidermal junction into the epidermis were counted, and fibers that branched after crossing the basement membrane were recorded as a single fiber. For each animal, IENF density was calculated as the average number of fibers per mm of epidermis (IENF/mm). Langerhans cells were counted if they showed immunoreactivity for PGP9.5 and their cell body was visible. For each animal, the Langerhans cells were calculated as the average number of Langerhans cells per mm of epidermis (LC/mm).
2.2. Hindpaw incision As a model of postoperative pain, adult rats were anesthetized with isoflurane (2–3 % in oxygen), and a midline incision in the left hindpaw from mid-heel to the footpad was made through the skin and fascia to expose the plantaris muscle, which was then incised [19]. The wound was closed with 3-0 sutures (Ethicon; Cornelia, GA) and the rats were allowed to recover in their home cage. The wound fully healed by postoperative day 14.
2.6. Statistical analysis Baseline mechanical withdrawal thresholds in adulthood were compared between the neonatal VNC and vehicle-treated groups using two-way ANOVA with Drug and Sex as factors. The effects of early life VNC exposure on behavioral sensitivity to subsequent hindpaw incision were analyzed using Repeated Measure (RM) three-way analysis of 2
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variance (ANOVA) with Time (as the repeated measure), Injury (Incision vs. Anesthesia Only) and Drug (Neonatal VNC vs. Neonatal Vehicle) as factors, and Sidak post hoc tests for multiple comparisons (Prism 8.0; Graphpad Software; San Diego, CA). Meanwhile, the effects of neonatal VNC on the degree of neuropathic pain evoked by adult VNC administration were analyzed with RM two-way ANOVA with Time (as the repeated measure) and Drug (Neonatal VNC vs. Neonatal Vehicle) as factors, and Sidak post hoc tests for multiple comparisons. IENF cell counts and Langerhans cell counts were analyzed using the Mann-Whitney test. For all tests, the significance level was set at p < 0.05. n refers to the number of rats in a given group unless otherwise stated. All data are reported as mean + SEM. 3. Results 3.1. Early life vincristine (VNC) does not alter mechanical sensitivity during adulthood While our prior work clearly demonstrated that the administration of VNC during early life evoked mechanical pain hypersensitivity compared to saline-treated littermate controls that lasted into adolescence, the duration of VNC-evoked neuropathic pain remains unknown since the previous studies did not examine pain behaviors after P56 [6]. To determine if the chronic pain induced by neonatal VNC treatment lasts throughout adulthood, VNC (60 μg/kg; or saline) was administered via i.p. injections at P11, 13, 17, 19 and 21, and mechanical withdrawal thresholds were analyzed by using von Frey monofilaments at 13–15 weeks of age. In light of the accumulating evidence to support sex differences in the mechanisms by which peripheral nerve or tissue damage can trigger chronic pain [12,24,25], data from males and females were analyzed separately. The results indicate that the mechanical pain hypersensitivity induced by early life VNC had resolved by adulthood, as there were no significant differences in mechanical withdrawal thresholds at 13–15 weeks, regardless of sex (n = 19 in each group; Drug: F(1,108) = 0.227, p = 0.635; Sex: F(1,108) = 0.303, p = 0.583; two-way ANOVA; Fig. 1A). Given the lack of sex differences, data from male and female rats were pooled for the subsequent experiments described below. A hallmark feature of chemotherapy-induced peripheral neuropathy (CIPN) is a reduction in the density of intraepidermal nerve fibers [26–28], which was reproduced in our preclinical model of pediatric VNC-evoked neuropathic pain [6]. However, we observed no significant effect of neonatal VNC on intraepidermal nerve fiber (IENF) density (Fig. 1B) at the 13–15 week time point (n = 6–8 in each group; U = 17; p = 0.414; Mann-Whitney test; Fig. 1C), thus pointing to a restoration of peripheral innervation by adulthood. PGP9.5 immunoreactivity (IR) was observed in Langerhans cells (LCs), the resident immune cells of the skin. Notably, while our previous work showed that early life VNC increases the density of LCs exhibiting PGP9.5-IR during adolescence, the density in adulthood was similar between the groups receiving VNC and vehicle as neonates (n = 6–8 in each group; U = 20.5; p = 0.686; Mann-Whitney test; Fig. 1D), further suggesting recovery from the neonatal VNC exposure. 3.2. Neonatal VNC exposure does not significantly influence post-surgical pain in adulthood To begin investigating the degree to which early life chemotherapy can persistently alter the susceptibility to developing chronic pain in response to subsequent injury, VNC (or saline) was administered during the neonatal period as described above. At P90, rats received either a unilateral hindpaw incision [19,29], or anesthesia only as a control, and mechanical withdrawal thresholds were measured at various time points for up to 2 weeks post-injury. Importantly, while we observed a significant effect of incision on mechanical withdrawal thresholds as expected (n = 6–9 per group; Injury: F(1,27) = 238.5, p < 0.0001; RM
(caption on next page)
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Fig. 1. Peripheral neuropathy and chronic pain evoked by neonatal vincristine (VNC) resolves by adulthood. A: Plots of hindpaw mechanical withdrawal threshold (PWT) in adult rats treated with vehicle (VEH) or vincristine (VNC) as neonates. No significant differences were observed between the groups in either males or females (n = 19 in each group; Drug: F(1,108) = 0.227, p = 0.635; Sex: F(1,108) = 0.303, p = 0.583; two-way ANOVA). B: Representative images taken from vehicle-treated (top) and neonatal VNC-treated (bottom) adult rats illustrating PGP9.5-immunoreactive (green) Langerhans cells (clear arrowheads) and sensory fibers (white arrows) crossing the dermal-epidermal junction which was identified via immunostaining for collagen IV (red). C: The density of intraepidermal nerve fibers (IENFs) was similar between neonatal VNC-treated adult rats and VEHtreated littermate controls (n = 6–8 in each group; U = 17; p = 0.414; MannWhitney test). D: The density of PGP9.5-immunoreactive Langerhans cells (LCs) was also similar between groups during adulthood (n = 6–8 in each group; U = 20.5; p = 0.686; Mann-Whitney test).
three-way ANOVA; Fig. 2A), there was no overall effect of neonatal VNC treatment (Drug: F(1,27) = 0.913, p = 0.348) and no significant interaction between early life VNC and incision (Drug X Injury Interaction: F(1,27) = 0.269, p = 0.608). Dynamic mechanical allodynia was also examined before and up to 2 weeks after adult hindpaw incision using the paintbrush test as described previously [6,30]. The data suggest that incision during adulthood increased the sensitivity to innocuous mechanical stimuli (n = 6–8 per group; Injury: F(1,26) = 5.839, p = 0.023; RM three-way ANOVA; Fig. 2B), but did so independently of the neonatal exposure to VNC (Drug X Injury Interaction: F(1,26) = 0.002, p = 0.962), and there was no significant overall effect of neonatal VNC on mechanical allodynia during this period (Drug: F(1,26) = 1.0, p = 0.327). The pinprick test was used to investigate the degree to which neonatal VNC exposure primes spinal nociceptive circuits for static mechanical hyperalgesia following adult injury. The data suggest that incision in adulthood increased the sensitivity to noxious mechanical stimuli (n = 6–9 per group; Injury: F(1,26) = 13.17, p = 0.001; RM three-way ANOVA; Fig. 2C), but this was independent of neonatal exposure to VNC (Drug X Injury Interaction: F(1,26) = 0.255, p = 0.618) with no significant overall effect of VNC on mechanical sensitivity following adult incision (Drug: F(1,26) = 0.013, p = 0.911). 3.3. Neonatal VNC exposure does not significantly influence the severity of neuropathic pain evoked by chemotherapy during adulthood An estimated 10–25 % of ALL patients experience relapse which necessitates additional rounds of chemotherapy [31,32]. While the majority of relapses occur within 3 years of the initial diagnosis in childhood, relapses can also be observed more than a decade later [33]. Therefore, we also investigated whether neonatal VNC exposure influences mechanical hypersensitivity evoked by a second round of VNC treatment during adulthood. Neonatal VNC exposure did not influence the onset or magnitude of mechanical hypersensitivity resulting from adult VNC (n = 12–13 in each group; Neonatal Drug: F(1,23) = 0.133, p = 0.719; Time: F(8,184) = 12.74, p < 0.0001; Neonatal Drug X Time Interaction: F(8,184) = 1.193; p = 0.305; RM two-way ANOVA; Fig. 3). (caption on next page)
4. Discussion The present results suggest that mechanical hypersensitivity evoked by early life VNC treatment [6] resolves by adulthood. This was accompanied by a rescue of two hallmark features of chemotherapy-induced peripheral neuropathy (CIPN), namely a reduction in IENF density in the skin and the appearance of PGP9.5-immunoreactive (IR) Langerhans cells (LCs), thereby pointing to a recovery of peripheral nerve integrity by 13–15 weeks of age. Furthermore, the data indicate that the degree of post-surgical pain evoked by adult hindpaw incision is not significantly influenced by prior exposure to VNC during the
neonatal period (Fig. 2). Similarly, the magnitude and time course of neuropathic pain produced by adult VNC administration was unaffected by early life treatment with VNC (Fig. 3). Collectively, the results suggest that VNC chemotherapy delivered during early life does not lead to a persistent increase in the susceptibility to developing chronic pain during adulthood, although it is clear that additional injury models need to be investigated moving forward. Our prior work demonstrated that the administration of VNC during
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Fig. 2. Early life vincristine (VNC) administration does not exacerbate mechanical pain after adult surgical injury. A: Plot of paw withdrawal threshold (PWT) as a function of post-operative time in rats treated with either vehicle or VNC as neonates, followed by the administration of hindpaw surgical incision (or anesthesia only as a control) during adulthood. There was no overall effect of neonatal VNC treatment (Drug: F(1,27) = 0.913, p = 0.348) and no significant interaction between early life VNC and incision (Drug X Injury Interaction: F(1,27) = 0.269, p = 0.608). B: Withdrawal responses to innocuous brush were plotted as a function of time following adult hindpaw incision, demonstrating that while surgical injury did evoke mechanical allodynia (n = 6–8 per group; Injury: F(1,26) = 5.839, p = 0.023; RM three-way ANOVA), it did so to a similar degree in neonatal VNC-treated rats and vehicle-treated controls (Drug X Injury Interaction: F(1,26) = 0.002, p = 0.962). C: Adult rats treated with VNC or vehicle during early life also exhibited a similar magnitude of static mechanical hyperalgesia, as measured by the pinprick test (n = 6–9 per group; Injury: F(1,26) = 13.17, p = 0.001; Drug X Injury Interaction: F(1,26) = 0.255, p = 0.618; RM three-way ANOVA).
For example, hindpaw incision during the neonatal period increases the magnitude and duration of post-surgical pain following adult incision [11,12,43]. Importantly, clinical studies utilizing quantitative sensory testing (QST) techniques have reported that children with prior experience in a neonatal intensive care unit (NICU), where tissue damage is an inevitable part of their essential medical treatment [44–46], exhibit enhanced perceptual sensitization in response to prolonged nociceptive stimulation even a decade or more after leaving the hospital [47,48]. As a result, it may seem surprising that early life VNC did not influence the degree of mechanical pain hypersensitivity following hindpaw surgical injury administered during adulthood (Fig. 2). However, this could be explained by the fact that the VNC was administered at P11-21, which was selected given that ALL in children has a peak onset between the ages of two and three years. As a result, our dosing regimen may fall outside of the critical period to evoke long-term alterations in the functional organization of central nociceptive circuits, as prior studies have observed that the initial injury must occur within the first postnatal week in order to ‘prime’ pain behavioral responses [9,10,42]. Alternatively, it should be noted that spinal microglia have been implicated as a key mediator of the priming of developing nociceptive pathways after neonatal injury, at least in male rodents [11,12]. Meanwhile, the degree to which chemotherapy-evoked neuropathy is accompanied by the activation of spinal microglia has been controversial, with some studies reporting significant microglial responsivity [49–51] while others failed to detect significant changes in microglial activation [17,52–54]. It has also been clearly demonstrated that peripheral nerve damage in early life evokes a more muted neuroimmune response in the immature spinal dorsal horn compared to the adult [55,56], where an anti-inflammatory cytokine profile can dominate in the weeks after a neonatal injury [5]. This raises the interesting, as yet untested, possibility that a dampened activation of spinal microglia following neonatal VNC explains the absence of exaggerated post-surgical pain after adult hindpaw incision. Among all children with acute lymphoblastic leukemia (ALL), approximately 98 % attain remission, and 85 % of newly diagnosed ALL patients are expected to be long-term event-free survivors with 90 % surviving at 5 years [13,57–59]. Nonetheless, relapse can occur in some of these patients, which requires a second round of chemotherapy that can include VNC [60–63]. Therefore, we also examined whether pediatric VNC exacerbated the degree of neuropathic pain produced by VNC treatment later in life and did not find any persistent effects of prior VNC exposure (Fig. 3). Nevertheless, there are some remaining questions about how these preclinical findings would translate to the clinical setting. For example, the rats in the present study were exposed only to VNC, while pediatric patients are usually administered VNC in combination with other agents such as bortezomib, dexamethasone, prednisone, pegylated L-asparaginase or doxorubicin [61,63]. As a result, future studies should examine the potential consequences of combinatorial chemotherapeutic regimens delivered during early life on the susceptibility to persistent pain during adulthood. In conclusion, these findings demonstrate that neonatal VNC administration fails to ‘prime’ developing pain pathways, thereby suggesting that the potential adverse long-term consequences of early life chemotherapy for the overall quality of life in survivors of childhood cancers may not include an enhanced vulnerability to developing chronic pain after subsequent injury.
Fig. 3. The severity of neuropathic pain evoked by adult vincristine (VNC) does not depend on prior exposure to VNC during early life. Plot of hindpaw mechanical withdrawal threshold (PWT) before and after the administration of VNC to adult rats that had been previously treated with VNC or vehicle as neonates. The resulting mechanical hypersensitivity was similar between the two experimental groups (n = 12–13 in each group; Neonatal Drug: F(1,23) = 0.133, p = 0.719; Time: F(8,184) = 12.74, p < 0.0001; Neonatal Drug X Time Interaction: F(8,184) = 1.193; p = 0.305; RM two-way ANOVA).
early life (i.e. P11-21) produced a delayed onset hypersensitivity to static and dynamic mechanical stimuli that persisted throughout adolescence in both males and females, while no changes in the sensitivity to noxious heat or cold were detected [6]. In addition, a decrease in cutaneous IENF density and the appearance of PGP9.5-IR LCs were observed by P33 [6]. IENF reductions have been extensively reported in CIPN [7,26,34] as well as several other chronic pain states including peripheral neuropathy resulting from diabetes [35–37] and HIV [38], where IENF loss correlates with the severity of neuropathy and may occur in an axonal length-dependent manner [39]. Patients with smallfiber neuropathies can also reportedly exhibit an increased number of LCs in the epidermis, which may play a role in the development and/or maintenance of neuropathic pain [40,41]. Our current results suggest that while peripheral neuropathy and neuropathic pain evoked by neonatal VNC may persist into adolescence [6], they do not continue throughout adulthood, as evidenced by the similar mechanical withdrawal thresholds, IENF densities and immunoreactive LC counts between adult rats administered VNC during early life compared to vehicle-treated littermate controls. Given that neonatal VNC also evokes cell type-dependent alterations in the intrinsic membrane properties of both dorsal root ganglion (DRG) neurons and spinal dorsal horn neurons during adolescence [8], it would be of interest to determine if the documented hyperexcitability of medium- and large-diameter primary afferents and lamina I spinoparabrachial neurons also normalizes during adulthood. Growing evidence suggests that tissue damage during a critical period of early postnatal development can exacerbate the pain hypersensitivity evoked by subsequent injury during adulthood [9,10,42].
CRediT authorship contribution statement Lauren M. Styczynski: Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Katie A. Schappacher: Conceptualization, Methodology. Mark L. Baccei: Conceptualization, Writing - original draft, Writing review & editing, Supervision, Project administration, Funding acquisition. 5
Neuroscience Letters 720 (2020) 134764
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Acknowledgements
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