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Research Paper

Early life vincristine exposure evokes mechanical


pain hypersensitivity in the developing rat
Katie A. Schappachera,b, Lauren Styczynskia, Mark L. Bacceia,*

Abstract
Vincristine (VNC) is commonly used to treat pediatric cancers, including the most prevalent childhood malignancy, acute
lymphoblastic leukemia. Although clinical evidence suggests that VNC causes peripheral neuropathy in children, the degree to
which pediatric chemotherapeutic regimens influence pain sensitivity throughout life remains unclear, in part because of the lack of
an established animal model of chemotherapy-induced neuropathic pain during early life. Therefore, this study investigated the
effects of VNC exposure between postnatal days (P) 11 and 21 on mechanical and thermal pain sensitivity in the developing rat. Low
doses of VNC (15 or 30 mg/kg) failed to alter nociceptive withdrawal reflexes at any age examined compared with vehicle-injected
littermate controls. Meanwhile, high dose VNC (60 mg/kg) evoked mechanical hypersensitivity in both sexes beginning at P26 that
persisted until adulthood and included both static and dynamic mechanical allodynia. Hind paw withdrawal latencies to noxious heat
and cold were unaffected by high doses of VNC, suggesting a selective effect of neonatal VNC on mechanical pain sensitivity. Gross
and fine motor function appeared normal after VNC treatment, although a small decrease in weight gain was observed. The VNC
regimen also produced a significant decrease in intraepidermal nerve fiber density in the hind paw skin by P33. Overall, the present
results demonstrate that high-dose administration of VNC during the early postnatal period selectively evokes a mechanical
hypersensitivity that is slow to emerge during adolescence, providing further evidence that aberrant sensory input during early life
can have prolonged consequences for pain processing.
Keywords: Neonatal, Pain, Chemotherapy, Development, Pediatric, Nociception, Nerve injury, Neuropathic

1. Introduction early postnatal period,19,20,35 which influences their response to


Advances in the clinical treatment of pediatric cancers have tissue or nerve injury. For example, neonatal tissue damage can
produced a dramatic increase in survival rates.49 For example, evoke unique changes in nociceptive processing within both the
the 5-year survival rate for acute lymphoblastic leukemia (ALL), peripheral6,18,32 and central nervous systems.39–41,67 In addition,
the most common childhood malignancy with a peak onset the effects of peripheral nerve injury in the rodent are highly age
dependent,21,29,45 with the onset of neuropathic pain delayed in
between ages 2 and 3 years, has increased from 60% in 1975 to
;90% today.24,51 Current chemotherapy regimens for ALL the neonate because of a significant developmental shift in spinal
commonly include the vinca alkaloid derivative vincristine neuroimmune signaling toward a more proinflammatory pheno-
(VNC).38,56,60 Both adult and pediatric patients receiving type during adolescence.4,43
tubulin-binding drugs can experience symptoms of sensory and Although clinical evidence supports a link between early life
motor peripheral neuropathy including pain and paresthesia in the VNC exposure and pain hypersensitivity, the degree to which the
hands and feet, as well as a loss of deep tendon reflexes,28,37,73 severity of pain resulting from chemotherapy-induced peripheral
which often leads to dose reduction and negatively impacts the neuropathy (CIPN) changes with age remains unclear. Retro-
patients’ quality of life. spective studies include a wide range of ages at diagnosis and
Preclinical studies have clearly demonstrated that pain path- variations in the chosen chemotherapy agents and cumulative
dosage, making it difficult to rigorously assess the effects of VNC
ways are organized in a fundamentally different manner during the
on nociceptive processing at specific ages. Meanwhile, to our
knowledge, long-term prospective studies using quantitative
Sponsorships or competing interests that may be relevant to content are disclosed sensory testing approaches78 to longitudinally measure nocicep-
at the end of this article.
a
tive sensitivity in pediatric cancer survivors during adolescence or
Pain Research Center, Department of Anesthesiology, University of Cincinnati
adulthood have yet to be undertaken. An animal model could
College of Medicine, Cincinnati, OH, USA, b Departments of Pharmacology and Cell
Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH, USA yield valuable insight into the consequences of pediatric
*Corresponding author. Address: Pain Research Center, Department of Anesthe-
chemotherapeutic regimens for pain sensitivity throughout life.
siology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Although CIPN models have been well established in adult
Cincinnati, OH 45267, USA. E-mail address: mark.baccei@uc.edu (M. L. Baccei). rodents,1,3,46,72 the known developmental changes in pain
Supplemental digital content is available for this article. Direct URL citations appear circuits make it difficult to extrapolate the previous findings to
in the printed text and are provided in the HTML and PDF versions of this article on the pediatric population.
the journal’s Web site (www.painjournalonline.com). Therefore, this study investigated the effects of early life
PAIN 158 (2017) 1647–1655 exposure to VNC on mechanical and thermal pain sensitivity in
© 2017 International Association for the Study of Pain the developing rat, with the goal of establishing a preclinical
http://dx.doi.org/10.1097/j.pain.0000000000000953 model of pediatric CIPN. Our results demonstrate that VNC

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administration during the early postnatal period evokes a selective of these rats undergoing additional behavioral tests of mechan-
increase in mechanical pain sensitivity that persists into adult- ical, heat, and cold sensitivity. To assess dynamic mechanical
hood. This work lays a foundation for the future exploration of the allodynia, animals were first placed individually into clear acrylic
neurobiological mechanisms underlying CIPN in children and containers separated by black opaque dividers and allowed to
adolescents. acclimate for at least 20 minutes. A paintbrush was used to
stimulate the lateral plantar region of the hind paw in the heel-to-
toe direction. Responses were recorded on a scale of 0 to 3 and
2. Methods
presented as an allodynia score, where 0 5 no response or
2.1. Subjects occasionally walking away; 1 5 lifting the stimulated paw toward
the body; 2 5 strong lateral lifting of the paw above the level of the
Male and female Sprague Dawley rats (n 5 108; Harlan,
body; and 3 5 shaking or licking the paw.17 To obtain the average
Indianapolis, IN) were used in this study. Animals were randomly
score for each animal, this test was repeated on each paw 3 times
assigned to one of 2 groups: vehicle (n 5 54) or VNC (n 5 54).
at 10-second intervals. For the pinprick test, a blunted needle was
During treatment, animals were weighed before each injection
pressed against the plantar surface of the hind paw without
and then once per week after treatment ended. Behavioral assays
penetration and repeated on each paw 3 times at 1-minute
were performed on separate days to avoid stress induced by
intervals.17 The total number of withdrawals was counted for
multiple tests, with a minimum of 24 hours between assays in
each rat and expressed as a percent response (based on
a given animal. All rats underwent a combination of tests for
a maximum of 6).
mechanical and thermal sensitivity as well as motor function,
although an individual animal was not subjected to all 7 behavioral
assays (von Frey hairs, paintbrush, pinprick, Hargreaves test; 2.3.2. Response to heat stimuli
cold plantar assay, rotarod, and foot fault test; see below) to again
Sensitivity to noxious heat was assessed using the Hargreaves
minimize the level of stress induced over the course of postnatal
test.25 Briefly, animals were first placed individually into clear
development. All experiments adhered to animal welfare guide-
acrylic containers separated by black opaque dividers on the
lines established by the University of Cincinnati Institutional
glass surface of the thermal testing apparatus (Model 336; IITC/
Animal Care and Use Committee.
Life Science Instruments, Woodland Hills, CA) and allowed to
acclimate for at least 20 minutes. A mobile radiant heat source
2.2. Drug administration was directed to the midplantar surface of the hind paw at 5-
minute intervals per rat, alternating paws, for a total of 6 trials. The
Preliminary dose–response studies consisted of 9 total intraper-
radiant heat beam was turned off automatically by a photocell
itoneal injections of VNC (15, 30, 45, or 60 mg/kg dissolved in
feedback sensor on paw withdrawal and the time was recorded.
sterile saline; Tocris) with a 4-day on, 2-day off, and 5-day on
To avoid potential tissue damage, the cutoff time was set to 20
schedule starting on postnatal day (P)11. Because daily
seconds. The mean withdrawal latency was calculated for each
administration of 45 or 60 mg/kg VNC led to death after the third
rat from the 6 trials. Withdrawal latencies were evaluated weekly
consecutive dose, we modified the dosing schedule to consist of
starting at P26 until P54.
5 total injections of 60 mg/kg VNC on P11, 13, 17, 19, and 21.3,72
Although drawing exact parallels between the age of rats and
humans is inherently difficult, previous work has estimated the 2.3.3. Response to cold stimuli
neurodevelopmental stage of a P7 rat as roughly equivalent to
The cold plantar assay was used to evaluate noxious cold
that of a human born at full term.13 P11 was chosen as a starting
sensitivity11 in a separate cohort of rats as was used in the tests
point in this study to (1) better reflect the clinical scenario in which
for noxious heat sensitivity. Briefly, animals were first placed
VNC is most commonly administered to children between the
individually into clear acrylic containers separated by black
ages of 2 and 3 rather than to newborns51 and (2) facilitate the
opaque dividers that were set on top of 3/160 borosilicate glass
comparison of our findings with those of previous studies of infant
(Stemmerich Inc, St. Louis, MO) and allowed to acclimate for 20
neuropathic pain which used mechanical trauma to the peripheral
minutes before testing. A dry ice pellet was applied to the hind
nerve at P10.43 In addition, the duration of VNC treatment may be
paw through the glass and the time until hind paw withdrawal was
relevant to the clinical observation that patients with ALL can
recorded at 5-minute intervals per rat, alternating paws, for a total
receive VNC at varying intervals over a period of several years.49
of 6 trials, and the mean withdrawal latency was calculated.
Littermates injected with equivalent volumes of sterile saline
Withdrawal latencies were evaluated weekly starting at P20 until
served as a control group.
approximately 7 weeks of age.

2.3. Behavioral testing


2.3.4. Evaluation of gross motor function
2.3.1. Response to mechanical stimuli
Animals were placed on an accelerating rotarod treadmill set at
Mechanical reflex withdrawal thresholds were measured before a constant speed (4 r.p.m.). The rod was then accelerated from
each injection, and then at weekly intervals until P54, by an a rate of 4 to 40 r.p.m. over a 10-minute period. The animal’s
investigator blinded to treatment group. Briefly, rats (P11-P47) performance was recorded as the latency to fall (in seconds) and
were lightly restrained on a flat surface and calibrated von Frey evaluated weekly starting after the last injection of VNC.
hair monofilaments were applied to the dorsal hind paw,71
whereas at P54, rats were placed individually into clear acrylic
2.3.5. Evaluation of fine motor function
containers separated by black opaque dividers and the von Frey
hair filaments applied to the plantar surface of the hind paw. The foot fault test was used for skilled locomotor assessment.44
Mechanical withdrawal thresholds were measured in all subjects On P20, animals were trained to cross a horizontal ladder with
according to the Simplified Up and Down method,8 with subsets irregular rung spacing (ranging from 1 to 2 cm) from a neutral

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starting cage to reach their home cage. Testing consisted of 2 confocal optical sections at a thickness of 1 mm. Intraepidermal
trials per animal and was performed weekly starting on P21 until nerve fibers that crossed the collagen IV–stained dermal/
P37. Two independent experimenters blinded to the treatment epidermal junction into the epidermis were counted (as the
group rated each step on a 7-category scale based on the number of fibers per square millimeter of epidermis; IENF per
position and accuracy, as well as errors that occurred in paw square millimeter), and fibers that branched after crossing the
placement, according to the following scoring criteria: 0 5 total basement membrane were recorded as a single fiber. Langer-
miss; 1 5 deep slip; 2 5 slight slip; 3 5 replacement; 4 5 hans cells (LCs) that showed PGP9.5 immunoreactivity were
correction; 5 5 partial placement; and 6 5 correct placement. counted if the cell body was visible. For each section, a mean
The scores from the 2 experimenters were then averaged and IENF density and LC count was calculated from the 4 fields of
presented as percent accuracy. view by 2 independent experimenters blinded to treatment group,
and the results across experimenters were then averaged. For
each experimental group, 4 to 6 rats were analyzed in this
2.4. Immunohistochemistry manner.
At P25 and P33, animals were deeply anesthetized with sodium
pentobarbital and transcardially perfused with 0.1M phosphate- 2.6. Statistical analysis
buffered saline (PBS). The skin from the plantar surface of the hind
paw was removed and placed in 4% paraformaldehyde (PFA) for Behavioral data and IENF counts were analyzed using 2-way
4 hours, then transferred to 20% sucrose overnight at 4˚C. Tissue analysis of variance with Holm–Sidak post hoc tests (Prism;
was embedded in Tissue Freezing Medium (General Data GraphPad Software, La Jolla, CA). Langerhans cell counts were
Healthcare, Cincinnati, OH), sectioned at 40 mm using a cryostat compared between groups using the unpaired Student’s t test.
(Leica) and processed as free-floating sections. After a 1-hour For all tests, the significance level was set at P , 0.05. n refers to
incubation in blocking solution (0.1M PBS containing 5% normal the number of rats in a given group unless otherwise stated. All
donkey serum/0.3% Triton X-100) at room temperature (RT), data are reported as mean 6 SEM.
liquid was decanted and sections were incubated overnight at RT
with the primary antibodies: PGP9.5 (rabbit, 1:500; Dako) and 3. Results
collagen IV (goat, 1:500; Southern Biotech). The primary antibody
was then decanted and sections were washed 3 times for 30 3.1. Toxicity of vincristine administration in the
minutes each in wash buffer consisting of PBS 1 0.3% Triton X- developing rat
100. After the wash buffer was removed, the secondary Beginning at postnatal day (P) 11, rats were injected intraperito-
antibodies donkey anti-rabbit Alexa 488 (1:500) and donkey neally with VNC (15, 30, 45, or 60 mg/kg) or vehicle with a 4 day-
anti-goat Alexa 594 (1:500; Jackson Immunoresearch) were on, 2 day-off, 5 day-on schedule (see Methods). The daily
added for 30 minutes at RT. After incubation with the secondary administration of VNC at 45 or 60 mg/kg routinely led to death of
antibodies, the tissue was placed in wash buffer 3 times for 30 the animal after the third injection (data not shown). Therefore, to
minutes each then mounted onto slides, coverslipped, and explore the effects of high VNC doses on sensorimotor function in
stored at 4˚C until imaging. the developing rat, we modified the dosing protocol such that
VNC was administered at 60 mg/kg 5 times between P11 and
P21 (see Methods). At this dose, VNC caused a slight, but
2.5. Quantification of intraepidermal nerve fibers
significant, decrease in the rate of weight gain in both males
For intraepidermal nerve fiber (IENF) quantification, 3 plantar skin (F(8,433) 5 11.03, P , 0.0001, n 5 18-27; Fig. 1A) and females
sections were selected per animal and 4 fields of view per section (F(8,451) 5 7.40, P , 0.0001, n 5 14-27; Fig. 1B) beginning at 3
were chosen at random by an experimenter blinded to the weeks of age. Nonetheless, weight loss was never observed and
treatment group. Images were obtained using a Zeiss LSM 710 the animals were judged to be in good overall health. In support of
confocal microscope equipped with argon and helium–neon this notion, VNC administration (60 mg/kg) had no effect on gross
lasers applying a 320 objective and captured as z-stacks of motor function at any time point examined, as there were no

Figure 1. Vincristine (VNC) treatment significantly reduces weight gain in both sexes. (A) In male rats treated with VNC, there is a significant reduction in weight gain
starting at P21 which persisted until the end of testing (F(8,433) 5 11.03, P , 0.0001, n 5 18-27, 2-way ANOVA). (B) In females, VNC significantly attenuates weight
gain starting at P19 (F(8,451) 5 7.40, P , 0.0001, n 5 14-27, 2-way ANOVA; *P , 0.05; ***P , 0.001; ****P , 0.0001; Holm–Sidak post hoc analysis). Each rat
received one intraperitoneal injection (arrow) on P11, 13, 17, 19, and 21. ANOVA, analysis of variance.

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Figure 2. Neonatal vincristine administration does not impact gross or fine motor function in the developing rat. (A) Vincristine treatment does not significantly affect
gross motor function compared with vehicle-treated littermate controls on the rotarod test (F(1,192) 5 0.18, P 5 0.82, n 5 17, 2-way ANOVA). (B) Assessment of fine
motor function demonstrated no difference in performance on the foot fault test between vehicle and VNC treated animals (F(1,76) 5 0.08, P 5 0.78, n 5 10-16 in
each group, 2-way ANOVA). ANOVA, analysis of variance; VNC, vincristine.

differences in latency to fall on the rotarod test between VNC- Supp. Fig. 1, http://links.lww.com/PAIN/A420). Withdrawal
treated rats and vehicle-treated littermates (P 5 0.82; Fig. 2A). latencies to noxious heat were also unaltered by the administra-
Assessment of fine motor skills was also performed weekly using tion of VNC at these doses (F(8,70) 5 0.24, P 5 0.9819, n 5 7;
the foot fault test (see Methods). The high dose of VNC had no Supp. Fig. 2, http://links.lww.com/PAIN/A420).
impact on foot fault accuracy scores compared with vehicle- Meanwhile, early life exposure to VNC at 60 mg/kg resulted in
treated controls (P 5 0.78; Fig. 2B). hypersensitivity to mechanical stimuli that emerged after the
completion of the VNC regimen in both males (F(10,493) 5 3.42,
P 5 0.0002, n 5 18-27 in each group; Fig. 3A) and females
3.2. Vincristine administration during early life evokes (F(10,457) 5 6.35, P , 0.0001, n 5 14-27; Fig. 3B), as VNC-
delayed mechanical pain hypersensitivity
treated animals exhibited a significant decrease in mechanical
Mechanical withdrawal thresholds were measured at various withdrawal thresholds (;35%) beginning on P26 (ie, 5 days after
postnatal ages using von Frey hair filaments after the adminis- the final VNC injection) compared with vehicle-treated littermates
tration of VNC or vehicle solution between P11 and P21 (see (Fig. 3A, B). This persisted throughout the testing period, with
above). As described previously,29,66 mechanical thresholds mechanical withdrawal thresholds in the VNC groups remaining
steadily increased with age in males (F(10,493) 5 147.56, P , significantly lower than the vehicle groups at P54. Given that VNC
0.0001, n 5 18-27; Fig. 3A) and females (F(10,457) 5 113.70, P , evoked mechanical hypersensitivity with a similar onset and
0.0001, n 5 14-27; Fig. 3B) within both experimental groups. duration in males and females, data from both sexes were pooled
Low-dose administration of VNC (15 or 30 mg/kg) failed to for subsequent behavioral analyses.
produce behavioral signs of neuropathic pain at any stage of To investigate the degree to which VNC during the early
postnatal development examined, as mechanical withdrawal postnatal period induces dynamic mechanical allodynia in the
thresholds were similar between VNC-treated and littermate developing rat, the prevalence of hind paw withdrawals in
controls injected with vehicle (F(20,232) 5 1.15, P 5 0.2976, n 5 9; response to innocuous brush was examined at multiple ages

Figure 3. Neonatal VNC exposure results in static mechanical allodynia by P26 in both sexes. Vincristine treatment produces a delayed reduction in mechanical
withdrawal thresholds in response to von Frey hair (VFH) application in both (A) male and (B) female rats compared with vehicle controls. Male VFH: (F(10,493) 5
3.42, P 5 0.0002, n 5 18-27 in each group); Female VFH: (F(10,457) 5 6.35, P , 0.0001, n 5 14-27, 2-way ANOVA; *P , 0.05; **P , 0.01; ***P , 0.001; ****P ,
0.0001; Holm–Sidak post hoc analysis). Each rat received one intraperitoneal injection (arrow) on P11, 13, 17, 19, and 21. ANOVA, analysis of variance; VNC,
vincristine.

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Figure 4. Early life vincristine (VNC) produces dynamic mechanical allodynia and static mechanical hyperalgesia. (A) Plot of allodynia score of hind paw withdrawal
in response to a paintbrush stimulus as a function of postnatal age after neonatal VNC treatment. A significant main effect of VNC treatment was observed when
compared with vehicle-treated littermate controls (F(1,134) 5 8.32, P 5 0.0046, n 5 19-26 in each group, 2-way ANOVA; **P , 0.01; Holm–Sidak post hoc
analysis), suggesting the presence of dynamic mechanical allodynia after VNC. (B) Plot of frequency of hind paw withdrawal in response to a pinprick stimulus at
different ages after early VNC exposure. A significant main effect of VNC treatment was observed when compared with vehicle-treated littermate controls (F(1,97) 5
12.00, P 5 0.0008, n 5 13-23, 2-way ANOVA; ***P , 0.001; Holm–Sidak post hoc analysis). ANOVA, analysis of variance.

between P20 and P33. These time points were chosen to span treatment had no effect on the withdrawal latencies in response to
the age at which static mechanical allodynia emerged (as noxious cold stimuli at any time point (F(1,238) 5 0.01, P 5 0.92, n
measured by the administration of von Frey hairs). Importantly, 5 25; Fig. 5B), suggesting that early life VNC exposure selectively
VNC treatment significantly increased allodynia scores in re- increases mechanical pain sensitivity in the developing rat.
sponse to a paintbrush stimulus (F(1,134) 5 8.32, P 5 0.0046, n 5
19-26 in each group), which was most evident at P26 (P 5
3.4. Loss of intraepidermal nerve fibers after vincristine
0.0038; Holm–Sidak posttest for multiple comparisons; Fig. 4A).
treatment during early life
In addition, the frequency of hind paw withdrawal to pinprick
stimuli was significantly elevated by VNC exposure (F(1,97) 5 Previous studies in both humans and rats have demonstrated
12.00, P 5 0.0008, n 5 13-23; Fig. 4B), suggesting that VNC that VNC- and paclitaxel-induced painful peripheral neuropathies
treatment during early life also significantly increases the are accompanied by a reduction of IENF density corresponding to
sensitivity to noxious mechanical stimuli. the onset of mechanical hypersensitivity.5,9,10,47,57 Based on
these reports, we next sought to determine whether the onset of
mechanical allodynia was associated with changes in IENF
3.3. Early vincristine treatment does not alter sensitivity to innervation in the hind paw skin. Vincristine treatment caused
noxious thermal stimuli
a statistically significant reduction of IENF density (F(1,56) 5 5.616, P
Sensitivity to noxious radiant heat was examined weekly between 5 0.021, n 5 12-18 sections from 4 to 6 rats in each group; Fig.
P26 and P54 in rats treated with 60 mg/kg VNC (or vehicle), 6A–C). Although there was no difference in the number of IENFs
corresponding to a period during which a clear hypersensitivity to per square millimeter of the epidermal border between treatment
mechanical stimulation was observed (Figs. 3 and 4). There was groups at P25 (P 5 0.607, Holm–Sidak posttest), VNC-treated rats
no significant effect of neonatal VNC administration on mean exhibited significantly lower IENF counts relative to controls at P33
withdrawal latencies to noxious heat at any age examined (F(1,145) (P 5 0.047, Holm–Sidak posttest; Fig. 6C, right). At antibody
5 0.06, P 5 0.81, n 5 16 in each group; Fig. 5A). Similarly, VNC dilutions optimized for visualizing IENFs, PGP9.5 immunoreactivity

Figure 5. Vincristine (VNC) during the early postnatal period does not significantly affect withdrawal thresholds to noxious heat or cold. (A) Vincristine did not
significantly impact withdrawal latencies to noxious heat (F(1,145) 5 0.06, P 5 0.81, n 5 16, 2-way ANOVA). (B) Vincristine also failed to alter withdrawal latencies to
noxious cold stimuli (F(1,238) 5 0.01, P 5 0.92, n 5 25, 2-way ANOVA). ANOVA, analysis of variance.

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Figure 6. Neonatal vincristine (VNC) treatment leads to decreased epidermal innervation and the presence of PGP9.5-positive Langerhans cells at P33. Confocal
images of representative plantar skin sections immunostained for PGP9.5 (green) and collagen IV (red) from vehicle- and VNC-treated animals. On P33, VNC-
treated animals (B) had fewer PGP9.5-stained intraepidermal nerve fibers (IENFs) compared with those treated with saline (A). Arrows point to faintly stained
Langerhans cells. Scale bar 5 50 mm. (C) VNC leads to a reduction in PGP9.5-immunoreactive intraepidermal nerve fibers 2 weeks after the end of treatment
(F(1,56) 5 5.616, P 5 0.021, 2-way ANOVA, *P , 0.05; Holm–Sidak post hoc analysis). (D) VNC leads to an increased presence of PGP9.5-positive Langerhans
cells at P33 (*P 5 0.017, unpaired t test). ANOVA, analysis of variance.

was also observed in LCs (Fig. 6B), which are the resident immune was similar.1,3,46,62,63 Importantly, recent preclinical work has
cells of the skin.2,30,64 As previously reported in the adult rat,57 the clearly shown that peripheral nerve damage occurring before 3
reduction in IENF density after VNC treatment was accompanied weeks of age evokes neuropathic pain with a significant delay in
by an increase in the number of PGP9.5-positive LCs (P 5 0.017, onset relative to adult nerve injury,14,29,66 which has been
unpaired t test, n 5 12 sections from 4 rats in each group; Fig 6D). ascribed to a shift from an anti-inflammatory to proinflammatory
cytokine profile within the CNS which occurs during adoles-
cence.43 Interestingly, this delay appears to be unique to
4. Discussion
neuropathic pain, as neonatal rats exhibit robust inflammatory
The major goal of this study was to establish the first preclinical pain from the first days of life after the intraplantar injection of
model of pediatric chemotherapy-induced neuropathic pain carrageenan or complete Freund’s adjuvant.42,70
resulting from the administration of vinca alkaloid derivatives Nonetheless, it should be noted that unlike models involving
during early life. The results clearly demonstrate that neonatal a discrete physical injury to the peripheral nerve, the precise time
VNC exposure evokes mechanical pain hypersensitivity that point at which the neonatal VNC administration causes axonal
emerges after the conclusion of the treatment, persists into trauma is difficult to ascertain. Although we first observed
adulthood, and includes enhanced responses to both static and a reduction in IENF density at P33 (Fig. 6), it is possible that the
dynamic mechanical stimuli without accompanying changes in fibers entered an early phase of degeneration well before this age.
the sensitivity to noxious thermal stimulation or motor function. Future immunohistochemical or molecular analyses of neuronal
In humans, symptoms of VNC-induced peripheral neuropathy stress markers in the DRG,63 or electrophysiological measure-
are often persistent and can sometimes worsen after the ments of axonal conduction along primary afferent fibers,62,75 are
termination of chemotherapy or even emerge after drug needed to better pinpoint the onset of axonal dysfunction after
withdrawal.48,50 Interestingly, the mechanical hypersensitivity early life VNC exposure. In addition, given that symptoms of VNC-
observed after VNC administration in the neonatal rat (as induced peripheral neuropathy can depend on cumulative
measured using von Frey hairs) failed to completely resolve dose,16,23 we cannot exclude the possibility that VNC doses
during the period examined and emerged with a delay (;5 days) greater than 60 mg/kg could result in an earlier emergence of
relative to the cessation of the VNC regimen (Fig. 3). The mechanical hypersensitivity in the developing rat. Although our
increased responsiveness to a dynamic, innocuous mechanical dosage is lower than those used in clinical practice to achieve
stimulus (ie paintbrush; Fig. 4A) and a punctate, noxious antineoplastic activity,53 toxicity issues prevented the examina-
mechanical stimulus (ie, pinprick; Fig. 4B) also appeared after tion of higher VNC doses in our rodent model during this particular
the termination of the VNC treatment, with a slightly longer delay stage of development.
in onset observed for the hypersensitivity to pinprick. Meanwhile, As is the case in adults, mounting evidence points to important
previous studies of VNC-induced CIPN in adult rats have reported sex differences in the prevalence and magnitude of pediatric pain,
an earlier onset of mechanical allodynia (ie during the second with females often exhibiting greater pain sensitivity in both the
week of treatment) even at lower doses (50 mg/kg72) than used clinical and experimental settings.7,33,78 However, we observed
here, although the magnitude of the mechanical hypersensitivity no striking differences in the time course or magnitude of

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mechanical hypersensitivity between male and female littermates characterize the excitability of developing sensory neurons at
after early VNC exposure as measured by von Frey hair testing. various time points after neonatal VNC treatment.
Distinct mechanisms could nonetheless underlie CIPN-related It should also be noted that aberrant sensory input resulting
pain after neonatal VNC administration in males versus females. from injury during the early postnatal period can have prolonged
Indeed, recent work demonstrates that the key neuroimmune consequences for central nociceptive processing, including (but
interactions driving neuropathic pain are sexually dimorphic with not limited to) a disruption in the balance between excitatory and
microglia reportedly playing a prominent role in males but not inhibitory synaptic transmission and a facilitation of synaptic
females,58,59 although it should be noted that the importance of long-term potentiation (LTP) in the spinal dorsal horn,40,41
microglial activation for the induction of CIPN-induced neuro- elevated spinal neuroimmune responses,4,67 heightened opioi-
pathic pain has been questioned.77 Interestingly, the mecha- dergic tone in the brain,34,35 and changes in descending
nisms contributing to neuropathic pain may partly depend on the inhibitory pathways.68,76 This highlights the need for future
extent of peripheral nerve damage, as knockout of toll-like studies examining the effects of early life VNC exposure on the
receptor 4 signaling abolishes tactile allodynia selectively in males functional organization of developing pain networks within the
after spinal nerve ligation61 but prevents hypersensitivity in both CNS. Similarly, although mounting evidence from both
sexes after cisplatin-induced polyneuropathy.74 rodents20,39–41,54,66,68,71 and humans26,27,69 suggests that
In contrast to the marked mechanical hypersensitivity in the neonatal injuries can “prime” developing nociceptive pathways
aftermath of neonatal VNC administration, withdrawal latencies to such that a subsequent injury evokes an exacerbated level of
noxious heat or cold stimuli were not influenced by VNC treatment pain hypersensitivity, it remains to be determined whether
during early life. Previous work in adult rats has produced mixed neonatal VNC treatment modulates the behavioral response to
findings with regards to the degree of thermal hyperalgesia and an unrelated injury later in life. Finally, although this study
cold allodynia produced by VNC, with heat sensitivity reportedly investigated the effects of VNC administration to naive rats as
increased,1 decreased,3,63 or unchanged.46,72 Similarly, cold a means to isolate the effects of the drug itself, it will ultimately be
allodynia has been observed in response to VNC-induced critical to elucidate how neonatal VNC alters pain sensitivity and
neuropathy in some studies3 but not others.46,72 In humans, nociceptive circuitry when administered in the presence of ALL
sensory testing has revealed VNC-induced impairments in or other pediatric cancers to better mimic the clinical scenario.
temperature sensation. Adults with VNC-induced neuropathy are In conclusion, this work lays an essential foundation for future
more sensitive to noxious cold stimuli and have elevated warm investigations into the neurobiological underpinnings of
detection thresholds,16 whereas children are less likely to discern chemotherapy-induced neuropathic pain during early life.
differences in temperature throughout the first year of VNC
treatment when innocuous cold objects are applied to the skin.36,37
Unfortunately, to our knowledge, sensitivities to noxious cold or Conflict of interest statement
heat stimuli have not been evaluated in the pediatric population The authors have no conflict of interest to declare.
after VNC treatment. Therefore, the degree to which the neonatal This work was supported by the US National Institutes of
rat model fully recapitulates the clinical phenotype seen after Health grant NS080889 (M.L.B.).
pediatric VNC exposure remains unclear. For example, although
motor dysfunction and loss of deep tendon reflexes are observed
clinically in pediatric VNC-induced neuropathy,31,36,51,52,65 we did Acknowledgements
not observe alterations in either gross or fine motor skills in our The authors thank Mrs Elizabeth Serafin for her technical
experiments (Fig. 2). assistance and Drs Judith Strong and Temugin Berta for their
Damage to peripheral nerve endings and LC activation are constructive feedback regarding this manuscript.
implicated in chemoneuropathy and other neuropathic pain
conditions such as postherpetic neuralgia, diabetic neuropathy,
and type-I complex regional pain syndrome.2,12,57 Vincristine Appendix A. Supplemental Digital Content
administration during early life significantly decreased epidermal Supplemental digital content associated with this article can be
innervation and led to the appearance of PGP9.5-positive LCs found online at http://links.lww.com/PAIN/A420.
(Fig. 6), which have also been noted in adult rodent models of
CIPN at the onset of hypersensitivity to mechanical stimuli.10,57
Langerhans cells represent the population of epidermal dendritic Supplemental media
cells closely associated with IENF terminals.22,30,55 As the Video content associated with this article can be found online at
resident immune cells of the skin, LCs are responsible for antigen http://links.lww.com/PAIN/A421.
presentation, cytokine production, and proinflammatory receptor
expression.2,30,64 Intraepidermal nerve fibers have been shown to Article history:
regulate LC dynamics30 and, in turn, LCs can regulate cutaneous Received 17 March 2017
innervation.15 Although the functional significance of the potential Received in revised form 9 May 2017
interactions between IENFs and LCs in the context of CIPN Accepted 10 May 2017
remains unclear, it is possible that IENF loss may signal LC Available online 30 May 2017
activation and subsequent cytokine release, resulting in the
sensitization of the remaining nociceptive fibers or those in the
process of degenerating. Indeed, Ad and C fibers from adult rats References
with CIPN exhibit increased spontaneous activity.3,62,75 How- [1] Aley KO, Reichling DB, Levine JD. Vincristine hyperalgesia in the rat:
ever, recent work demonstrates that the subtypes of primary a model of painful vincristine neuropathy in humans. Neuroscience 1996;
73:259–265.
afferent neurons whose excitability is altered by peripheral [2] Asahina A, Hosoi J, Grabbe S, Granstein RD. Modulation of Langerhans
inflammation are dependent on the age at which the insult cell function by epidermal nerves. J Allergy Clin Immunol 1995;96(suppl
occurs.32 In this regard, it will ultimately be important to 6):1178–1182.

Copyright Ó 2017 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
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K.A. Schappacher et al. 158 (2017) 1647–1655 PAIN®

[3] Authier N, Gillet J-P, Fialip J, Eschalier A, Coudore F. A new animal model of Vincristine treatment of advanced cancer: a cooperative study of 392
vincristine-induced nociceptive peripheral neuropathy. Neurotoxicology cases. Cancer Res 1973;33:1258–1264.
2003;24:797–805. [29] Howard RF, Walker SM, Mota MP, Fitzgerald M. The ontogeny of
[4] Beggs S, Currie G, Salter MW, Fitzgerald M, Walker SM. Priming of adult neuropathic pain: postnatal onset of mechanical allodynia in rat spared
pain responses by neonatal pain experience: maintenance by central nerve injury (SNI) and chronic constriction injury (CCI) models. PAIN 2005;
neuroimmune activity. Brain 2012;135:404–417. 115:382–389.
[5] Bennett GJ, Liu GK, Xiao WH, Jin HW, Siau C. Terminal arbor [30] Hsieh ST, Choi S, Lin WM, Chang YC, McArthur JC, Griffin JW. Epidermal
degeneration–a novel lesion produced by the antineoplastic agent denervation and its effects on keratinocytes and Langerhans cells.
paclitaxel. Eur J Neurosci 2011;33:1667–1676. J Neurocytol 1996;25:513–524.
[6] Boada MD, Gutierrez S, Giffear K, Eisenach JC, Ririe DG. Skin incision- [31] Jain P, Gulati S, Seth R, Bakhshi S, Toteja GS, Pandey RM. Vincristine-
induced receptive field responses of mechanosensitive peripheral induced neuropathy in childhood ALL (acute lymphoblastic leukemia)
neurons are developmentally regulated in the rat. J Neurophysiol 2012; survivors: prevalence and electrophysiological characteristics. J Child
108:1122–1129. Neurol 2014;29:932–937.
[7] Boerner KE, Birnie KA, Caes L, Schinkel M, Chambers CT. Sex [32] Jankowski MP, Ross JL, Weber JD, Lee FB, Shank AT, Hudgins RC. Age-
differences in experimental pain among healthy children: a systematic dependent sensitization of cutaneous nociceptors during developmental
review and meta-analysis. PAIN 2014;155:983–993. inflammation. Mol Pain 2014;10:34.
[8] Bonin RP, Bories C, De Koninck Y. A simplified up-down method (SUDO) [33] King S, Chambers CT, Huguet A, MacNevin RC, McGrath PJ, Parker L,
for measuring mechanical nociception in rodents using von Frey MacDonald AJ. The epidemiology of chronic pain in children and
filaments. Mol Pain 2014;10:26. adolescents revisited: a systematic review. PAIN 2011;152:2729–2738.
[9] Boyette-Davis JA, Cata JP, Driver LC, Novy DM, Bruel BM, Mooring DL, [34] LaPrairie J, Murphy AZ. Neonatal injury alters adult pain sensitivity by
Wendelschafer-Crabb G, Kennedy WR, Dougherty PM. Persistent increasing opioid tone in the periaqueductal gray. Front Behav Neurosci
chemoneuropathy in patients receiving the plant alkaloids paclitaxel and 2009;3:31.
vincristine. Cancer Chemother Pharmacol 2013;71:619–626. [35] LaPrairie JL, Murphy AZ. Long-term impact of neonatal injury in male and
[10] Boyette-Davis J, Xin W, Zhang H, Dougherty PM. Intraepidermal nerve female rats: sex differences, mechanisms and clinical implications. Front
fiber loss corresponds to the development of Taxol-induced hyperalgesia Neuroendocrinol 2010;31:193–202.
and can be prevented by treatment with minocycline. PAIN 2011;152: [36] Lavoie Smith EM, Li L, Chiang C, Thomas K, Hutchinson RJ, Wells EM,
308–313. Ho RH, Skiles J, Chakraborty A, Bridges CM, Renbarger J. Patterns and
[11] Brenner DS, Golden JP, Gereau RW. A novel behavioral assay for severity of vincristine induced peripheral neuropathy in children with acute
measuring cold sensation in mice. PLoS One 2012;7:e39765. lymphoblastic leukemia. J Peripher Nerv Syst 2015;20:37–46.
[12] Casanova-Molla J, Morales M, Planas-Rigol E, Bosch A, Calvo M, [37] Lavoie Smith EM, Li L, Hutchinson RJ, Ho R, Burnette WB, Wells E,
Grau-Junyent JM, Valls-Sole J. Epidermal Langerhans cells in small Bridges C, Renbarger J. Measuring vincristine-induced peripheral
fiber neuropathies. PAIN 2012;153:982–989. neuropathy in children with acute lymphoblastic leukemia. Cancer Nurs
[13] Clancy B, Finlay BL, Darlington RB, Anand KJS. Extrapolating brain 2013;36:E49–E60.
development from experimental species to humans. Neurotoxicology [38] LeClerc JM, Billett AL, Gelber RD, Dalton V, Tarbell N, Lipton JM, Barr R,
2007;28:931–937. Clavell LA, Asselin B, Hurwitz C, Schorin M, Lipshultz SE, Declerck L,
[14] Costigan M, Scholz J, Woolf CJ. Neuropathic pain: a maladaptive Silverman LB, Cohen HJ, Sallan SE. Treatment of childhood acute
response of the nervous system to damage. Annu Rev Neurosci 2009;32: lymphoblastic leukemia: results of Dana-Farber ALL Consortium Protocol
1–32. 87-01. J Clin Oncol 2002;20:237–246.
[15] Doss AL, Smith PG. Langerhans cells regulate cutaneous innervation [39] Li J, Baccei ML. Neonatal tissue injury reduces the intrinsic excitability of
density and mechanical sensitivity in mouse footpad. Neurosci Lett 2014; adult mouse superficial dorsal horn neurons. Neuroscience 2014;256:
578:55–60. 392–402.
[16] Dougherty PM, Cata JP, Burton AW, Vu K, Weng H-RR. Dysfunction in [40] Li J, Baccei ML. Neonatal tissue damage promotes spike timing-
multiple primary afferent fiber subtypes revealed by quantitative sensory dependent synaptic long-term potentiation in adult spinal projection
testing in patients with chronic vincristine-induced pain. J Pain Symptom neurons. J Neurosci 2016;36:5405–5416.
Manage 2007;33:166–179. [41] Li J, Blankenship ML, Baccei ML. Deficits in glycinergic inhibition within
[17] Duan B, Cheng L, Bourane S, Britz O, Padilla C, Garcia-Campmany L, adult spinal nociceptive circuits after neonatal tissue damage. PAIN 2013;
Krashes M, Knowlton W, Velasquez T, Ren X, Ross SE, Lowell BB, Wang 154:1129–1139.
Y, Goulding M, Ma Q. Identification of spinal circuits transmitting and [42] Marsh D, Dickenson A, Hatch D, Fitzgerald M. Epidural opioid analgesia in
gating mechanical pain. Cell 2014;159:1417–1432. infant rats II: responses to carrageenan and capsaicin. PAIN 1999;82:
[18] Fitzgerald M. Cutaneous primary afferent properties in the hind limb of the 33–38.
neonatal rat. J Physiol 1987;383:79–92. [43] McKelvey R, Berta T, Old E, Ji R-R, Fitzgerald M. Neuropathic pain is
[19] Fitzgerald M. The development of nociceptive circuits. Nat Rev Neurosci constitutively suppressed in early life by anti-inflammatory neuroimmune
2005;6:507–520. regulation. J Neurosci 2015;35:457–466.
[20] Fitzgerald M, Jennings E. The postnatal development of spinal sensory [44] Metz GA, Whishaw IQ. Cortical and subcortical lesions impair skilled
processing. Proc Natl Acad Sci U S A 1999;96:7719–7722. walking in the ladder rung walking test: a new task to evaluate fore- and
[21] Fitzgerald M, McKelvey R. Nerve injury and neuropathic pain—a question hindlimb stepping, placing, and co-ordination. J Neurosci Methods 2002;
of age. Exp Neurol 2016;275(pt 2):296–302. 115:169–179.
[22] Gaudillere A, Misery L, Souchier C, Claudy A, Schmitt D. Intimate [45] Moss A, Beggs S, Vega-Avelaira D, Costigan M, Hathway GJ, Salter MW,
associations between PGP9-5-positive nerve fibres and Langerhans Fitzgerald M. Spinal microglia and neuropathic pain in young rats. PAIN
cells. Br J Dermatol 1996;135:343–344. 2007;128:215–224.
[23] Grisold W, Cavaletti G, Windebank AJ. Peripheral neuropathies from [46] Nozaki-Taguchi N, Chaplan SR, Higuera ES, Ajakwe RC, Yaksh TL.
chemotherapeutics and targeted agents: diagnosis, treatment, and Vincristine-induced allodynia in the rat. PAIN 2001;93:69–76.
prevention. Neuro Oncol 2012;14(suppl 4):iv45–iv54. [47] Peleshok JC, Ribeiro-da-Silva A. Delayed reinnervation by
[24] Gurney JG, Severson RK, Davis S, Robison LL. Incidence of cancer in nonpeptidergic nociceptive afferents of the glabrous skin of the rat
children in the United States. Sex-, race-, and 1-year age-specific rates hindpaw in a neuropathic pain model. J Comp Neurol 2011;519:49–63.
by histologic type. Cancer 1995;75:2186–2195. [48] Postma TJ, Benard BA, Huijgens PC, Ossenkoppele GJ, Heimans JJ.
[25] Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive Long-term effects of vincristine on the peripheral nervous system.
method for measuring thermal nociception in cutaneous hyperalgesia. J Neurooncol 1993;15:23–27.
PAIN 1988;32:77–88. [49] Pui C-H, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J
[26] Hermann C, Hohmeister J, Demirakca S, Zohsel K, Flor H. Long-term Med 2006;354:166–178.
alteration of pain sensitivity in school-aged children with early pain [50] Quasthoff S, Hartung HP. Chemotherapy-induced peripheral
experiences. PAIN 2006;125:278–285. neuropathy. J Neurol 2002;249:9–17.
[27] Hohmeister J, Kroll A, Wollgarten-Hadamek I, Zohsel K, Demirakça S, [51] Ramchandren S, Leonard M, Mody RJ, Donohue JE, Moyer J,
Flor H, Hermann C. Cerebral processing of pain in school-aged children Hutchinson R, Gurney JG. Peripheral neuropathy in survivors of
with neonatal nociceptive input: an exploratory fMRI study. PAIN 2010; childhood acute lymphoblastic leukemia. J Peripher Nerv Syst 2009;14:
150:257–267. 184–189.
[28] Holland JF, Scharlau C, Gailani S, Krant MJ, Olson KB, Horton J, Shnider [52] Reinders-Messelink HA, Schoemaker MM, Hofte M, Göeken LNH,
BI, Lynch JJ, Owens A, Carbone PP, Colsky J, Grob D, Miller SP, Hall TC. Kingma A, van den Briel MM, Kamps WA. Fine motor and handwriting

Copyright Ó 2017 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
September 2017
· Volume 158
· Number 9 www.painjournalonline.com 1655

problems after treatment for childhood acute lymphoblastic leukemia. cell-like cell line XS52: further support for a functional relationship
Med Pediatr Oncol 1996;27:551–555. between Langerhans cells and epidermal nerves. J Invest Dermatol 1997;
[53] Reiter A, Schrappe M, Ludwig WD, Hiddemann W, Sauter S, Henze G, 109:586–591.
Zimmermann M, Lampert F, Havers W, Niethammer D, Odenwald E, [65] Vainionpää L, Kovala T, Tolonen U, Lanning M. Vincristine therapy for
Ritter J, Mann G, Welte K, Gadner H, Riehm H. Chemotherapy in 998 children with acute lymphoblastic leukemia impairs conduction in the
unselected childhood acute lymphoblastic leukemia patients. Results entire peripheral nerve. Pediatr Neurol 1995;13:314–318.
and conclusions of the multicenter trial ALL-BFM 86. Blood 1994;84: [66] Vega-Avelaira D, McKelvey R, Hathway G, Fitzgerald M. The emergence
3122–3133. of adolescent onset pain hypersensitivity following neonatal nerve injury.
[54] Ren K, Anseloni V, Zou SPP, Wade EB, Novikova SI, Ennis M, Traub RJ, Mol Pain 2012;8:30.
Gold MS, Dubner R, Lidow MS. Characterization of basal and re- [67] Walker SM, Beggs S, Baccei ML. Persistent changes in peripheral and
inflammation-associated long-term alteration in pain responsivity following spinal nociceptive processing after early tissue injury. Exp Neurol 2016;
short-lasting neonatal local inflammatory insult. PAIN 2004;110:588–596. 275(pt 2):253–260.
[55] Rice FL, Kinnman E, Aldskogius H, Johansson O, Arvidsson J. The [68] Walker SM, Fitzgerald M. Surgical injury in the neonatal rat alters the adult
innervation of the mystacial pad of the rat as revealed by PGP 9.5 pattern of descending modulation from the rostroventral medulla.
immunofluorescence. J Comp Neurol 1993;337:366–385. Anesthesiology 2015;122:1391–1400.
[56] Schorin MA, Blattner S, Gelber RD, Tarbell NJ, Donnelly M, Dalton V, [69] Walker SM, Franck LS, Fitzgerald M, Myles J, Stocks J, Marlow N. Long-
Cohen HJ, Sallan SE. Treatment of childhood acute lymphoblastic term impact of neonatal intensive care and surgery on somatosensory
leukemia: results of Dana-Farber cancer institute/children’s hospital perception in children born extremely preterm. PAIN 2009;141:79–87.
acute lymphoblastic leukemia consortium protocol 85-01. J Clin Oncol [70] Walker SM, Meredith-Middleton J, Cooke-Yarborough C, Fitzgerald M.
1994;12:740–747. Neonatal inflammation and primary afferent terminal plasticity in the rat
[57] Siau C, Xiao W, Bennett GJ. Paclitaxel-and vincristine-evoked painful dorsal horn. PAIN 2003;105:185–195.
peripheral neuropathies: loss of epidermal innervation and activation of [71] Walker SM, Tochiki KK, Fitzgerald M. Hindpaw incision in early life
Langerhans cells. Exp Neurol 2006;201:507–514. increases the hyperalgesic response to repeat surgical injury: critical
[58] Sorge RE, LaCroix-Fralish ML, Tuttle AH, Sotocinal SG, Austin JS, Ritchie period and dependence on initial afferent activity. PAIN 2009;147:
J, Chanda ML, Graham AC, Topham L, Beggs S, Salter MW, Mogil JS. 99–106.
Spinal cord Toll-like receptor 4 mediates inflammatory and neuropathic [72] Weng HRR, Cordella JV, Dougherty PM. Changes in sensory processing
hypersensitivity in male but not female mice. J Neurosci 2011;31: in the spinal dorsal horn accompany vincristine-induced hyperalgesia and
15450–15454. allodynia. PAIN 2003;103:131–138.
[59] Sorge RE, Mapplebeck JC, Rosen S, Beggs S, Taves S, Alexander JK, [73] Windebank AJ, Grisold W. Chemotherapy-induced neuropathy.
Martin LJ, Austin JS, Sotocinal SG, Chen D, Yang M, Shi XQ, Huang H, J Peripher Nerv Syst 2008;13:27–46.
Pillon NJ, Bilan PJ, Tu Y, Klip A, Ji RR, Zhang J, Salter MW, Mogil JS. [74] Woller SA, Corr M, Yaksh TL. Differences in cisplatin-induced mechanical
Different immune cells mediate mechanical pain hypersensitivity in male allodynia in male and female mice. Eur J Pain 2015;19:1476–1485.
and female mice. Nat Neurosci 2015;18:1081–1083. [75] Xiao WH, Bennett GJ. Chemotherapy-evoked neuropathic pain:
[60] Stanulla M, Schrappe M. Treatment of childhood acute lymphoblastic abnormal spontaneous discharge in A-fiber and C-fiber primary afferent
leukemia. Semin Hematol 2009;46:52–63. neurons and its suppression by acetyl-L- carnitine. PAIN 2008;135:
[61] Stokes JA, Cheung J, Eddinger K, Corr M, Yaksh TL. Toll-like receptor 262–270.
signaling adapter proteins govern spread of neuropathic pain and recovery [76] Zhang YH, Wang XM, Ennis M. Effects of neonatal inflammation on
following nerve injury in male mice. J Neuroinflammation 2013;10:148. descending modulation from the rostroventromedial medulla. Brain Res
[62] Tanner KD, Reichling DB, Levine JD. Nociceptor hyper-responsiveness Bull 2010;83:16–22.
during vincristine-induced painful peripheral neuropathy in the rat. [77] Zheng FY, Xiao WH, Bennett GJ. The response of spinal microglia to
J Neurosci 1998;18:6480–6491. chemotherapy-evoked painful peripheral neuropathies is distinct from
[63] Thibault K, Rivals I, M’Dahoma S, Dubacq S, Pezet S, Calvino B. that evoked by traumatic nerve injuries. Neuroscience 2011;176:
Structural and molecular alterations of primary afferent fibres in the spinal 447–454.
dorsal horn in vincristine-induced neuropathy in rat. J Mol Neurosci 2013; [78] Zohsel K, Hohmeister J, Oelkers-Ax R, Flor H, Hermann C. Quantitative
51:880–892. sensory testing in children with migraine: preliminary evidence for
[64] Torii H, Yan Z, Hosoi J, Granstein RD. Expression of neurotrophic factors enhanced sensitivity to painful stimuli especially in girls. PAIN 2006;123:
and neuropeptide receptors by Langerhans cells and the Langerhans 10–18.

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