Rho kinase inhibitor Y-27632 facilitates recovery from experimental peripheral neuropathy induced by anti-cancer drug cisplatin

Abstract

Chemotherapy drugs have neurotoxicity associated with treatment, which can become a dose-limiting problem when clinical presentation is severe. However, there is no effective therapy to circumvent the neurotoxicity of anti-cancer drug treatment. In this study, we utilized a newly designed mouse model of cisplatin-induced peripheral neuropathy to determine both the severity of neurotoxicity induced by drug treatment and the effectiveness of the Rho kinase inhibitor Y-27632 in post-treatment recovery. Sensory nerve conduction studies revealed a significant increase in mean distal (peak) latency with cisplatin treatment, indicating a deterioration of sensory nerve function. Also, hind paw touch sensitivity decreased steadily with increasing cumulative dose of cisplatin. Histological and immunohistochemical analyses of the sural nerve using neuronal marker protein gene product 9.5 (PGP 9.5) demonstrated abnormal nerve fiber morphology in cisplatin-treated mice. Remarkably, post-treatment with Y-27632 improved the sural nerve distal (peak) latency and sensory threshold to return to pre-treatment levels. Sural nerve histology worsened in the absence of Y-27632 during recovery. These studies suggest that Rho kinase inhibitor Y-27632 can initiate regeneration of damaged nerves following cisplatin treatment.

Figure 1. Sensory nerve action potentials (SNAPs) in cisplatin-treated mice

(A) Representation of sural nerve SNAPs from saline-treated mice (n=9) demonstrate a normal morphology. (B) SNAP morphology of cisplatin-treated mice (n=11) is altered when compared to saline-treatment values. Note: after final cisplatin treatments, three mice had no detectable sensory conductance. Here is the example of sural nerve SNAP from one mouse that showed recordable waveform. The gain is 20 microvolts (y axis) with a sweep of 10 milliseconds (x axis). Waveform marker representations: 1= onset latency, 2 = peak latency, 3 = second peak, and 4 = baseline. (C) Cisplatin-treated mice have a significantly increased mean distal latency (peak latency) when compared to the saline only treatment group. (D) The action potential amplitude of cisplatin-treated mice SNAP is decreased, but not statistically significant. p<0.001***.

Figure 2. Touch perception of cisplatin-treated mice progressively declines with increasing cumulative dose

Each animal was tested for mechanical (touch) stimulus perception using Von Frey/Semmes-Weinstein monofilaments. The results indicate a decrease in stimulus recognition with increasing dose of cisplatin, while saline-treated animals maintain a baseline level of touch perception. Values represent mean ± SEM. p<0.01**.

Figure 3. Histological analysis of sural nerve cross sections indicates nerve degeneration in cisplatin-treated mice

A. Protein gene product 9.5 (PGP 9.5) immunostaining was used to visualize nerve fibers (brown to black) in sciatic samples from both saline- (a, asterisk) and cisplatin (b, arrows)-treated animals. Luxol fast blue counterstaining was used to recognize myelin sheaths (a, arrows). Examples of cisplatin tissue samples demonstrated a narrowing of axons (b, arrows) when compared to saline controls. Scale bar: 50μm. B. Quantification of % of myelin covered nerve fibers in saline- or cisplatin-treated mouse sural nerves. Values represent mean ± SEM. p<0.05*.

Figure 4. Sural nerve SNAPs in mice recovering from cisplatin-induced peripheral neuropathy

Cisplatin-treated animals are allowed to recover for a one-month period while receiving either saline (n=5) or Y-27632 (n=5). (A) Animals receiving saline during recovery maintain an altered SNAP morphology a month after cessation of cisplatin treatment. (B) Y-27632 treatment during recovery returns SNAP morphology to that detected in normal animals. The gain is 20 microvolts with a sweep of 10 milliseconds. Waveform marker representations: 1= onset latency, 2 = peak latency, 3 = second peak, and 4 = baseline. (C). Animals receiving Y-27632 treatment were able to demonstrate normal mean distal latencies after the recovery period. Latency did not improve for the group treated with saline during recovery. P<0.05*

Figure 5. Y-27632-treated mice demonstrate recovery from cisplatin-induced touch sensation deficits

Cisplatin-treated mice underwent a one-month recovery period following drug treatment. During the recovery period, the animals were randomly selected to receive either saline or Y-27632. Mice treated with Y-27632 demonstrated an increase in functional recovery of touch sensation when compared to saline recovery group. Values represent mean ± SEM. P<0.01**

Figure 6. Histological analysis of sural nerve cross sections from mice recovering from cisplatin-induced peripheral neuropathy

PGP 9.5 immunostaining of nerve sections revealed nerve fiber bundles (brown-black) with luxol fast blue staining of myelin sheaths (blue). A. Examples of mice treated with saline (a) have a reduction in myelin staining and nerve fibers remain flattened (arrows) whereas Y-27632-treated animal nerve sections (b) have a more robust presence of myelin (arrows). Some normalization of the rounded nerve fiber bundle morphology is clearly visible (b, arrows). Scale bar: 50μm. B. Quantification of % of myelin covered nerve fibers in saline- or cisplatin-treated mouse sural nerves during recovery. Values represent mean ± SEM. p<0.05*.