Minocycline Prevents the Development of Mechanical Allodynia in Mouse Models of Vincristine-Induced Peripheral Neuropathy

Abstract

Vincristine is an antineoplastic substance that is part of many chemotherapy regimens, used especially for the treatment of a variety of pediatric cancers including leukemias and brain tumors. Unfortunately, many vincristine-treated patients develop peripheral neuropathy, a side effect characterized by sensory, motoric, and autonomic symptoms. The sensory symptoms include pain, in particular hypersensitivity to light touch, as well as loss of sensory discrimination to detect vibration and touch. The symptoms of vincristine-induced neuropathy are only poorly controlled by currently available analgesics and therefore often necessitate dose reductions or even cessation of treatment. The aim of this study was to identify new therapeutic targets for the treatment of vincristine-induced peripheral neuropathy (VIPN) by combining behavioral experiments, histology, and pharmacology after vincristine treatment. Local intraplantar injection of vincristine into the hind paw caused dose- and time-dependent mechanical hypersensitivity that developed into mechanical hyposensitivity at high doses, and lead to a pronounced, dose-dependent infiltration of immune cells at the site of injection. Importantly, administration of minocycline effectively prevented the development of mechanical hypersensitivity and infiltration of immune cells in mouse models of vincristine induce peripheral neuropathy (VIPN) based on intraperitoneal or intraplantar administration of vincristine. Similarly, Toll-like receptor 4 knockout mice showed diminished vincristine-induced mechanical hypersensitivity and immune cell infiltration, while treatment with the anti-inflammatory meloxicam had no effect. These results provide evidence for the involvement of Toll-like receptor 4 in the development of VIPN and suggest that minocycline and/or direct Toll-like receptor 4 antagonists may be an effective preventative treatment for patients receiving vincristine.

Keywords: chemotherapy; mechanical allodynia; minocycline; mouse models; neuro-inflammation; neuropathic pain; toll-like receptor 4; vincristine.

FIGURE 1

Schedule of vincristine injections and behavioral measurements. Vincristine (<10 μg i.pl. or 0.5 mg/kg i.p.) or vehicle (5% glucose i.pl. or PBS i.p.) (open square, gray square) were administered for five consecutive days, with 2 days break, followed by another five consecutive injections. The highest vincristine dose (10 μg i.pl.) (black square) was administered for four consecutive days, followed by 5 days break and another two injections. Minocycline (25 mg/kg i.p.) (open triangle) was administered once daily, 3 days before vincristine administration, and then with each vincristine injection. Arrows indicate the time points of behavioral assessments, which were always performed before the next vincristine injection with the exception of day 0, where behavioral assessment was performed 3 h after the first vincristine injection.

FIGURE 2

Behavioral characterization of a novel mouse model of vincristine-induced peripheral neuropathy (VIPN). Single daily injection of vincristine (i.pl; 10 μl solution containing 10 μg or i.p.; 10 μl/g solution containing 0.5 mg/kg) or vehicle (i.pl; 10 μl 5% glucose or i.p.; 10 μl/g PBS) were administered using the schedule in Figure 1. Dotted lines indicate baseline values. (A) Mechanical paw withdrawal threshold (PWT) following vincristine administration, measured using an electronic von Frey instrument (MouseMet, TopCat Metrology. (B) Thermal PWT following vincristine administration assessed using MouseMet Thermal (TopCat Metrology). (C) Paw thickness 30 min after injection was assessed using a digital Vernier caliper (Kincrome, VIC, Australia). (D) Ipsilateral paw print area expressed in percent of the contralateral paw following vincristine administration was assessed using the CatwalkXT platform (Noldus Information Technology, Netherlands). Print area is the average of paw contact area with the glass platform (in cm²). Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons test; the experimental groups (vincristine i.pl. or i.p.) were compared to the respective vehicle control group (i.pl., 5% glucose or i.p., PBS). No significant differences between the sexes were detected. All data are shown as mean ± SEM; n = 6 for all groups (3 females and 3 males). ^∗p <0.05.

FIGURE 3

Local vincristine administration leads to infiltration of leukocytes. Representative pictures (H&E staining) showing histopathological changes of the epidermis, dermis, hypodermis and adjacent areas of the plantar glabrous skin of the injected hind paw after administration of vincristine (i.pl.,10 μg or 100 ng vincristine) or vehicle (i.pl., 5% glucose) after 24 h (A) or 7 days (B). Injections were performed as indicated in Figure 1. Magnification 200 x; scale bar (100 μm). Black arrow, leukocytes; open arrow, arteriolar necrosis; green arrow, edema; blue arrow, dermal necrosis; double arrow, nerve vacuolization.

FIGURE 4

Minocycline prevents the development of vincristine-induced mechanical allodynia in multiple mouse models. (A,B) Minocycline (i.p., 25 mg/kg) prevented the development of mechanical hypersensitivity following local [i.pl., 100 ng; (A)] and systemic [i.p., 0.5 mg/kg; (B)] administration of vincristine (PWT: paw withdrawal threshold). (C) Treatment with minocycline (i.p., 25 mg/kg) had a small but significant (^∗p < 0.05) effect on paw swelling following administration of vincristine (100 ng, i.pl.). (D) The vincristine-induced decrease in paw withdrawal threshold (i.pl., 100 ng) was significantly increased (^∗p < 0.05 compared with wild-type controls) in Tlr4^-/- animals at day 2. (E) Vincristine-induced paw swelling (i.pl., 100 ng) was significantly decreased (^∗p < 0.05 compared with wild-type controls) in Tlr4^-/- animals at 3 h and day 4. Dotted line represents baseline values. Minocycline was administered as described in Figure 1. (F) Treatment with gabapentin (100 mg/kg i.p.) but not the anti-inflammatory meloxicam (5 mg/kg i.p.) reversed mechanical allodynia elicited by local administration of vincristine (100 ng, i.pl.) ^#p < 0.05 compared with baseline; n.s., p > 0.05 compared with vehicle. All data are shown as mean ± SEM; n = 6 for all groups (3 females and 3 males) (^∗p < 0.05 compared with vehicle). Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons test.

FIGURE 5

Minocycline prevents infiltration of leukocytes following local administration of vincristine. Representative pictures (H&E staining) showing histopathological changes of the epidermis, dermis, hypodermis, and adjacent areas of the plantar glabrous skin of the injected hind paw after administration of vincristine (i.pl., 100 ng vincristine) and PBS (A) or co-treatment with minocycline (i.p., 25 mg/kg) (B) in C57BL/6J mice or Tlr4^−/− mice (C) treated with vincristine only (i.pl., 100 ng) after 7 days. Injections were performed as indicated in Figure 1. Magnification 200×; scale bar (100 μm). Black arrow, leukocytes; green arrow, edema.