Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice

Abstract

Polyneuropathy is a frequent and potentially severe side effect of clinical tumor chemotherapy. The goal of this study was to characterize paclitaxel-, cisplatin-, vincristine- and bortezomib-induced neuropathy in C57BL/6 mice with a comparative approach. The phenotype of the animals was evaluated at four time points with behavioral and electrophysiological tests, followed by histology. Treatment protocols used in this study were well tolerated and induced a sensory and predominantly axonal polyneuropathy. Behavioral testing revealed normal motor coordination, whereas all mice receiving verum treatment developed mechanical allodynia and distinct gait alterations. Electrophysiological evaluation showed a significant decrease of the caudal sensory nerve action potential amplitude for all cytostatic agents and a moderate reduction of nerve conduction velocity for cisplatin and paclitaxel. This finding was confirmed by histological analysis of the sciatic nerve which showed predominantly axonal damage: Paclitaxel and vincristine affected mostly large myelinated fibers, bortezomib small myelinated fibers and cisplatin damaged all types of myelinated fibers to a similar degree. Neuropathic symptoms developed faster in paclitaxel and vincristine treated animals compared to cisplatin and bortezomib treatment. The animal models in this study can be used to elucidate pathomechanisms underlying chemotherapy-induced polyneuropathy and for the development of novel therapeutic and preventative strategies.

Figure 1. Behavioral observations in animals receiving cytostatic drugs.

(A) Cisplatin-treated animals were up to 17 ± 2% lighter after the last injection than the control group, while no significant weight change was observed for bortezomib, paclitaxel or vincristine treatment. (B) The open field test revealed only moderate changes in activity patterns at the early and no significant changes at the late time point. Paclitaxel- and cisplatin-treated animals traveled longer distances, while bortezomib- and vincristine (the latter not significantly) treated animals moved less. (C) Motor performance assessed with the rotarod test was comparable in verum- and vehicle-injected animals. (D) All treatments led to mechanical allodynia with a significantly reduced mechanical withdrawal threshold in the hind paws compared to vehicle-injected animals. (E) Allodynia developed faster in PTX and VIN treated mice. Gait analysis with the catwalk method showed that animals receiving cytostatic drugs develop distinct gait alterations: The stance phase (F) and the duty cycle (G) decrease. The parameter duty cycle expresses the stance phase as a percentage of the entire step cycle (stand + swing). While paclitaxel, cisplatin and vincristine induced most prominent gait alterations of the hind paws, bortezomib affected forepaws more than hind paws. The data for bortezomib in (F + G) is presented for forepaws which is highlighted with a hash (^#). Superscript numbers indicate the number of animals used in each group (One CIS treated mouse died after the early time point; in Figure F + G different time points are separated by /).* p < 0.05; compared to the substance specific vehicle control group at the same time point.

Figure 2. Electrophysiological measurements in CIPN models.

(A) Representative serial electrophysiological measurements of the caudal sensory nerve in a mouse undergoing paclitaxel treatment. Compared to the baseline (solid line), the sensory nerve action potential (SNAP) amplitude is significantly reduced at the middle (dotted line) and late time point (dashed line). At the middle time point the potential is also shifted to the right, indicating a reduced nerve conduction velocity. (B) All animals receiving verum injections developed a significantly reduced SNAP amplitude of the caudal nerve which developed faster in PTX and VIN treated mice (C). (D) Nerve conduction velocity was only affected in paclitaxel- and cisplatin-treated animals. Superscript numbers indicate the number of animals used in each group (One CIS treated mouse died after the early time point). * p < 0.05; ** p < 0.01; compared to the substance specific vehicle control group at the same time point.

Figure 3. Histological analysis of sciatic nerves.

Representative micrographs of sciatic nerves from (A) paclitaxel-, (B) vincristine-, (C) control-, (D) cisplatin- and (E) bortezomib-treated animals. Degenerating axons are marked with an arrowhead. (F) Analysis of axon diameter distribution reveals that in paclitaxel- and vincristine-treated animals the histogram shifts to the left, suggesting a loss of bigger myelinated axons, while (G) cisplatin affected all myelinated fibers similarly and bortezomib led to a shift to the right, indicating a loss of smaller myelinated axons. (H) Mean axon diameter is reduced in paclitaxel- and vincristine-treated animals and increased in bortezomib-treated animals. (I) Distribution of myelin sheath thickness is similar in treated and control animals, underlining the predominantly axonal damage in animals suffering from chemotherapy-induced neuropathy. (J) Mean g-ratio is significantly increased in cisplatin treated animals indicating mild hypomyelination. (K) All cytostatic drugs lead to a significant reduction of myelinated fibre density. Superscript numbers indicate the number of animals used in each group.* p < 0.05; compared to the pooled control group.