Sensitization of capsaicin and icilin responses in oxaliplatin treated adult rat DRG neurons

Abstract

Background: Oxaliplatin chemotherapy induced neuropathy is a dose related cumulative toxicity that manifests as tingling, numbness, and chronic pain, compromising the quality of life and leading to discontinued chemotherapy. Patients report marked hypersensitivity to cold stimuli at early stages of treatment, when sensory testing reveals cold and heat hyperalgesia. This study examined the morphological and functional effects of oxaliplatin treatment in cultured adult rat DRG neurons.

Results: 48 hour exposure to oxaliplatin resulted in dose related reduction in neurite length, density, and number of neurons compared to vehicle treated controls, using Gap43 immunostaining. Neurons treated acutely with 20 μg/ml oxaliplatin showed significantly higher signal intensity for cyclic AMP immunofluorescence (160.5 ± 13 a.u., n = 3, P < 0.05), compared to controls (120.3 ± 4 a.u.). Calcium imaging showed significantly enhanced capsaicin (TRPV1 agonist), responses after acute 20 μg/ml oxaliplatin treatment where the second of paired capsaicin responses increased from 80.7 ± 0.6% without oxaliplatin, to 171.26 ± 29% with oxaliplatin, (n = 6 paired t test, P < 0.05); this was reduced to 81.42 ± 8.1% (P < 0.05), by pretreatment with the cannabinoid CB2 receptor agonist GW 833972. Chronic oxaliplatin treatment also resulted in dose related increases in capsaicin responses. Similarly, second responses to icilin (TRPA1/TRPM8 agonist), were enhanced after acute (143.85 ± 7%, P = 0.004, unpaired t test, n = 3), and chronic (119.7 ± 11.8%, P < 0.05, n = 3) oxaliplatin treatment, compared to control (85.3 ± 1.7%). Responses to the selective TRPM8 agonist WS-12 were not affected.

Conclusions: Oxaliplatin treatment induces TRP sensitization mediated by increased intracellular cAMP, which may cause neuronal damage. These effects may be mitigated by co-treatment with adenylyl cyclase inhibitors, like CB2 agonists, to alleviate the neurotoxic effects of oxaliplatin.

Figure 1

Phase contrast photomicrographs of cultured adult rat DRG neurons. A. without oxaliplatin; B. after 48 hour treatment with 5 μg/ml oxaliplatin, C. 20 μg/ml oxaliplatin and D. 50 μg/ml oxaliplatin, showing a dose-related loss of neurite integrity, loss of cell bodies, and accumulation of cell debris. Bar = 50 μm.

Figure 2

Gap 43 immunostaining in adult rat DRG neurons in vitro. A. without oxaliplatin treatment, DRG neurons are densely distributed and the cell bodies and neurites are intensely positive for Gap43 immunostaining (green), with long, robust and profusely branched neurites. B. Neurons treated with 5 μg/ml oxaliplatin have shorter, less dense neurites (double arrowheads), and reduced intensity of Gap43 immunostaining (double arrowheads). C. after 20 μg/ml and D. 50 μg/ml oxaliplatin treatment, there is a dose related loss of neurites (double arrowheads), cell bodies (single arrowheads) and loss of Gap43 immunostaining. Bar = 30 μm.

Figure 3

Morphological effects of oxaliplatin treated neurons. A. Dose-related decrease in maximum neurite length in oxaliplatin treated adult rat DRG neurons in vitro; 33% reduction after 5 μg/ml oxaliplatin, 42.7% reduction after 20 μg/ml, and 75% reduction after 50 μg/ml oxaliplatin treatment. B. Decrease in the percentage of Gap43 positive neurons bearing neurites (solid bars) and increased proportion of neurons without neurites (clear bars) after treatment with increasing oxaliplatin concentration. C. The number of neurons surviving after 48 hours oxaliplatin treatment, expressed as a percentage of vehicle treated neurons was significantly reduced after treatment with 5, 20 and 50 μg/ml oxaliplatin.

Figure 4

Increased cAMP signal intensity in oxaliplatin treated neurons. A. Immunofluorescence images of cAMP positive adult rat DRG neurons acutely treated with 8-bromo-cAMP (positive control), or oxaliplatin for 15 minutes, compared to vehicle treated neurons. Bar = 50 μm. B. Fluorescence signal intensity for cAMP immunostaining (arbitrary units), was increased in oxaliplatin treated adult rat DRG neurons compared to positive controls, and significantly higher than vehicle treated neurons.

Figure 5

Sensitization of capsaicin responses by acute oxaliplatin treatment: A. Representative trace showing baseline 340/380 intracellular ratio in a DRG neuron and its increase in response to addition of 200 nM capsaicin (arrow) to test for capsaicin sensitivity. B. Following washout of medium, a second stimulus of 1 μM capsaicin (arrow) after 30 minutes gave a smaller second response compared with the first response (in A), due to desensitization. C. Same as in Figure 5A, showing traces 1,2,3,4 from 4 individual neurons, where trace 1 shows increased 340/380 ratio in response to added 200 nM capsaicin (arrow), but not traces 2,3 or 4. D. Incubation with 20 μg/ml oxaliplatin for 10 minutes after the first stimulus and washout significantly enhanced the response to 1 μM capsaicin (arrow, trace 1) and evoked responses in previously unresponsive neurons (traces 2 and 4).

Figure 6

Sensitization of capsaicin responses after acute (A) and chronic (B) oxaliplatin treatment; Sensitization of responses to icilin (C), but not WS12 (D), after oxaliplatin treatment. A. Graph showing the average magnitude of desensitization (2^nd bar) normalised to the first response (1^st bar) in vehicle treated neurons. Acute oxaliplatin treatment resulted in significantly enhanced second responses (3^rd bar), compared to the first capsaicin response, that was reversed by preincubation with 5 μM CB2 agonist GW833972 (4^th bar). B. Chronic treatment with oxaliplatin for 48 hours also enhanced responses to 200 nM capsaicin (n.s. at 5 μg/ml oxaliplatin, but highly significant at 20 and 50 μg/ml oxaliplatin). C. Vehicle treated neurons responded to 1 μM icilin with calcium influx (1^st bar), and a smaller second response following washout (2^nd bar). Pretreatment with 20 μg/ml oxaliplatin before the second icilin stimulus significantly enhanced responses to 1 μM icilin (3^rd bar), and significantly higher than the second icilin response without oxaliplatin (P = 0.005). Similarly, chronic oxaliplatin treatment (20 μg/ml for 24 hours), also resulted in significantly enhanced second responses to 1 μM icilin compared to those without oxaliplatin (P < 0.05). D. Vehicle treated neurons responded to the TRPM8 specific ligand WS-12 (20 μM stimulus), with calcium influx (1^st bar). Following washout, a second 20 μM WS-12 stimulus resulted in a reduced response (2^nd bar), that was unaffected by the presence of oxaliplatin.