Neuroprotective Effect of Ramipril Is Mediated by AT2 in a Mouse MODEL of Paclitaxel-Induced Peripheral Neuropathy

Abstract

Paclitaxel (PTX)-induced peripheral neuropathy (PIPN) induces numerous symptoms affecting patient quality of life, leading to decreased doses or even to cessation of anticancer therapy. Previous studies have reported that a widely used drug, ramipril, improves neuroprotection in several rodent models of peripheral neuropathy. The protective role of the angiotensin II type 2 receptor (AT2) in the central and peripheral nervous systems is well-established. Here, we evaluate the effects of ramipril in the prevention of PIPN and the involvement of AT2 in this effect. Paclitaxel was administered in wild type or AT2-deficient mice on alternate days for 8 days, at a cumulative dose of 8 mg/kg (2 mg/kg per injection). Ramipril, PD123319 (an AT2 antagonist), or a combination of both were administered one day before PTX administration, and daily for the next twenty days. PTX-administered mice developed mechanical allodynia and showed a loss of sensory nerve fibers. Ramipril prevented the functional and morphological alterations in PTX mice. The preventive effect of ramipril against tactile allodynia was completely absent in AT2-deficient mice and was counteracted by PD123319 administration in wild type mice. Our work highlights the potential of ramipril as a novel preventive treatment for PIPN, and points to the involvement of AT2 in the neuroprotective role of ramipril in PIPN.

Keywords: Ramipril; angiotensin converting enzyme inhibitor; mouse model; neuropathic pain; paclitaxel-induced peripheral neuropathy.

Figure 1

Study design. D: day, DMSO: dimethylsulfoxide, DRG: dorsal root ganglion, IENF: intraepidermal nerve fibers, i.p.: intraperitoneal.

Figure 2

Motor coordination and heat nociception were not affected by PTX in wild type mice. (A) Motor coordination was evaluated by the rotarod test. The holding time reflects the capacity of mice to coordinate their movements in order to stay on the rod during acceleration (from 4 rpm to 40 rpm, 30 s). (B) Thermal nociception was evaluated by the hot plate test at 52 °C. n = 8 to 10. Black arrows indicate days on which PTX was administered. Ctrl: control, PTX: paclitaxel, RD: reference day, VEH: vehicle.

Figure 3

Ramipril prevents tactile allodynia induced by paclitaxel, this effect being counteracted by AT2 blockade, in wild type mice. Tactile sensitivity was evaluated by the von Frey filament test. n = 8 to 10. * p < 0.05, ** p < 0.01, *** p < 0.001 Ctrl-VEH vs. PTX-VEH. ^### p < 0.001, ^#### p < 0.0001 PTX-VEH vs. PTX-RAM. ^@ p < 0.05, ^@@ p < 0.01, ^@@@ p < 0.001 Ctrl-VEH vs. PTX-PD, ^$$ p < Ctrl-VEH vs. PTX-RAM+PD. ⁺⁺ p < 0.01 PTX-RAM vs. PTX-RAM+PD. Black arrows indicate days on which PTX was administered. AT2: angiotensin II type 2 receptor, Ctrl: control, PD: PD123319 (AT2 antagonist), PTX: paclitaxel, RAM: ramipril, RD: reference day, VEH: vehicle.

Figure 4

Ramipril did not show a neuroprotective effect in AT2-deficient (AT2KO) mice. Tactile sensitivity was evaluated by the von Frey filament test. n = 8 to 10. ** p < 0.01, *** p < 0.001, **** p < 0.0001 AT2KO-Ctrl-VEH vs. AT2KO-PTX-VEH. ^### p < 0.001, ^#### p < 0.0001 AT2KO-PTX-VEH vs. AT2KO-PTX-RAM. Black arrows indicate days on which PTX was administered. Ctrl: control, PD: PD123319, PTX: paclitaxel, RAM: ramipril, RD: reference day, VEH: vehicle.

Figure 5

Ramipril alleviates sensory neuron loss induced by PTX administration in DRG of wild type mice. Immunohistochemistry for PGP9.5 was performed on paw skin (A) and DRG sections. (B). Intraepidermal nerve fiber density was assessed. Three sections of paw skin were examined per mouse. n = 6 mice (C). Quantification of DRG neuron density was evaluated. Three DRG sections and three DRG per mice were counted. n = 6 per group (D). * p < 0.05, ** p < 0.01, vs. Ctrl-VEH. ^# p < 0.05, PTX-VEH vs. PTX-RAM. Ctrl: control, PTX: paclitaxel, RAM: ramipril, VEH: vehicle.

Figure 6

Ramipril alleviates PTX-induced loss of myelinated fibers in the sciatic nerve of wild type mice. (A–C) are representative images of sciatic nerve sections visualized by electron microscopy from the Ctrl-VEH, PTX-VEH and the PTX-RAM groups, respectively. (D) Quantification of myelinated fiber density. (E) Quantification of unmyelinated fiber density. n = 6 per group. * p < 0.05, ** p < 0.01, vs. Ctrl-VEH. ^# p < 0.05, PTX-VEH vs. PTX-RAM. Ctrl: control, PTX: paclitaxel, RAM: ramipril, VEH: vehicle.