Contractility of isolated colonic smooth muscle strips from rats treated with cancer chemotherapy: differential effects of cisplatin and vincristine

Abstract

Background: Certain antineoplastic drugs cause gastrointestinal disorders even after the end of treatment. Enteric neuropathy has been associated with some of these alterations. Our goal was to assess the impact of repeated treatment with cisplatin and vincristine on the contractility of circular and longitudinal muscle strips isolated from the rat colon.

Methods: Two cohorts of male rats were used: in cohort 1, rats received one intraperitoneal (ip) injection of saline or cisplatin (2 mg kg^-1 week^-1) on the first day of weeks 1-5; in cohort 2, rats received two cycles of five daily ip injections (Monday to Friday, weeks 1-2) of saline or vincristine (0.1 mg kg^-1 day^-1). Body weight and food and water intake were monitored throughout the study. One week after treatment, responses of colonic smooth muscle strips to acetylcholine (10^-9-10^-5 M) and electrical field stimulation (EFS, 0.1-20 Hz), before and after atropine (10^-6 M), were evaluated in an organ bath.

Results: Both drugs decreased body weight gain. Compared to saline, cisplatin significantly decreased responses of both longitudinal and circular smooth muscle strips to EFS, whereas vincristine tended to increase them, although in a non-significant manner. No differences were observed in the muscle response to acetylcholine. Atropine abolished the contractile responses induced by acetylcholine, although those induced by EFS were only partially reduced in the presence of atropine.

Conclusion: The findings suggest that although both drugs cause the development of enteric neuropathy, this seems to have a functional impact only in cisplatin-treated animals. Understanding the effects of chemotherapy on gastrointestinal motor function is vital for enhancing the quality of life of cancer patients.

Keywords: chemotherapy; cisplatin; colon contractility; organ bath; vincristine.

FIGURE 1

Experimental protocol to study the effects of cisplatin and vincristine in the rat. COHORT 1: Rats were intraperitoneally (ip) administered with saline (2.5 mL kg^–1) or cisplatin (2 mg kg^–1) for 5 consecutive weeks (weeks 1–5). Treatments were: saline (black, n = 7) and cisplatin (red, n = 9). COHORT 2: The rats were ip administered with saline (2.5 mL kg^–1) or vincristine (0.1 mg kg^–1) for 10 days (Monday to Friday, weeks 1–2). Treatments were: saline (black, n = 10) and vincristine (pink, n = 8). The in vitro study of colonic contractility (IVCC) was performed on week 6 (cohort 1) and week 3 (cohort 2).

FIGURE 2

Experimental protocol used to study the contractility of colonic strips in organ bath. (A) Experimental protocol of the in vitro study: after removal of mucosa and submucosa, muscle strips cut in the longitudinal and circular direction were suspended in organ baths. After an initial stabilization period (60 min) with 3 Krebs washes, 50 mM potassium chloride (KCl) was added to study the strips contractility. After 2 Krebs washes, the strips were subjected to electrical stimulation at increasing frequencies and then to chemical stimulation with acetylcholine (ACh) at increasing concentrations. After each ACh administration, Krebs was changed twice. The same electrical and chemical stimuli (but this time ACh was used only at 10^–5 M) were repeated, in the presence of atropine (10^–6 M). Finally, after a new Krebs renewal, 50 mM KCl was added to the organ bath, and, after two more Krebs renewals, the strips were removed and weighed. (B) Representative traces of the contractile responses. (B.a) KCl and ACh induced a similar biphasic response, with two components, whose amplitudes were recorded: the first one was short lasting and occurred immediately after administration (phasic, PA); the second one occurred after PA, as a plateau that lasted for 1–3 min (tonic, TA). (B.b) Electrical stimulation (EFS) induced a biphasic response, with two components, whose amplitudes were recorded: the first one occurred during EFS (A1) and the second one occurred once EFS had ended (A2).

FIGURE 3

Effect of cisplatin and vincristine on general health parameters in the rat. COHORT 1: body weight gain (A), food intake (C) and water intake (D) were measured in rats intraperitoneally administered with saline (2.5 mL kg^–1) or cisplatin (2 mg kg^–1) for 5 consecutive weeks (weeks 1–5). Treatments were: saline (black line/bar, n = 7), cisplatin (red line/bar, n = 9). COHORT 2: body weight gain (B), food intake (E) and water intake (F) were measured in rats intraperitoneally administered with saline (2.5 mL kg^–1) or vincristine (0.1 mg kg^–1) for 10 days (Monday to Friday, weeks 1–2). Treatments were: saline (black line/bar, n = 10) and vincristine (pink line/bar, n = 8). Food and water intake during treatment in both cohorts [from week 1 to weeks 5 (cohort 1) or 2 (cohort 2)] is shown. Data represent the mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. saline [two-way ANOVA followed by Tukey’s post-hoc test in (A,D)]; one-way ANOVA followed by Tukey’s post-hoc test in (B,C,E,F).

FIGURE 4

Phasic response of longitudinal and circular muscle response to the initial potassium chloride administration in cohorts 1 (cisplatin) and 2 (vincristine). Longitudinal and circular strips from rat colon were exposed to 50 mM potassium chloride (KCl). Graphs (A,A’) represent the weight of circular (CM) and longitudinal (LM) muscle strips of both cohorts. Graphs (B,B’) represent the phasic component of the contractile response in LM and CM of both cohorts. The results show an average of the tension exerted (expressed in g) by the muscle strips in the organ bath. Graphs (C,C’) represent the phasic component of the contractile response in LM and CM after normalization to the weight of the corresponding muscle strips of both cohorts. COHORT 1: The rats were intraperitoneally administered with saline (2.5 mL kg^–1) or cisplatin (2 mg kg^–1) for 5 consecutive weeks (weeks 1–5). Treatments were: saline (black, n = 7) and cisplatin (red, n = 9). COHORT 2: The rats were intraperitoneally administered with saline (2.5 mL kg^–1) or vincristine (0.1 mg kg^–1) for 10 days (Monday to Friday, weeks 1–2). Treatments were: saline (black, n = 10) and vincristine (pink, n = 8). Data represent the mean ± SEM. ^$p < 0.05, ^$$p < 0.01 vs. cohort 1 (one-way ANOVA followed by Tukey’s post-hoc test).

FIGURE 5

Longitudinal and circular muscle response to chemical stimulation with acetylcholine in cohort 1: cisplatin. The preparations were exposed to increasing concentrations of acetylcholine (ACh: 10^–8–10^–5 M). Atropine was administered at 10^–6 M and thereafter, the effect of ACh at 10^–5 M was recorded (arrow). Graphs represent the phasic (A) and tonic (A’) component of the contractile response in longitudinal muscle and the phasic (B) and tonic (B’) component of the contractile response in circular muscle. Rats were intraperitoneally administered with saline (2.5 mL kg^–1) or cisplatin (2 mg kg^–1) for 5 consecutive weeks (weeks 1–5). Treatments were: saline (black line, n = 7) and cisplatin (red line, n = 9). Data represent the mean ± SEM.

FIGURE 6

Longitudinal and circular muscle response to chemical stimulation with acetylcholine in cohort 2: vincristine. The preparations were exposed to increasing concentrations of acetylcholine (ACh: 10^–8–10^–5 M). Atropine was administered at 10^–6 M and thereafter, the effect of ACh at 10^–5 M was recorded. Graphs represent the phasic (A) and tonic (A’) component of the contractile response in longitudinal muscle and the phasic (B) and tonic (B’) component of the contractile response in circular muscle. Rats were intraperitoneally administered with saline (2.5 mL kg^–1) or vincristine (0.1 mg kg^–1) for 10 days (Monday to Friday, weeks 1–2). Treatments were: saline (black line, n = 10) and vincristine (red line, n = 8).

FIGURE 7

Longitudinal and circular muscle response to electrical field stimulation (EFS) in cohort 1: cisplatin. Longitudinal (LM) and circular (CM) muscle strips were electrically stimulated, in absence and presence of atropine (10^–6M), with 10 s trains of pulses (0.3 ms, 100 V) at frequencies of 0.1–20 Hz. Amplitudes normalized to the phasic response to KCl 50 mM are represented. (A,A’) Maximum amplitude obtained for each frequency in LM, during (Amplitude 1, A1) and after (Amplitude 2, A2) electrical stimulation. (B,B’) Maximum amplitude obtained for each frequency in CM during (A1) and after (A2) electrical stimulation. Rats were intraperitoneally administered with saline (2.5 mL kg^–1) or cisplatin (2 mg kg^–1) for 5 consecutive weeks (weeks 1–5). Treatments were: saline (black line, n = 7), saline + atropine (black dotted line, n = 7), cisplatin (red line, n = 9), and cisplatin + atropine (red dotted line, n = 9). Data represent the mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. saline; ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. cisplatin (two-way ANOVA followed by Tukey’s post-hoc test).

FIGURE 8

Longitudinal and circular muscle response to electrical field stimulation (EFS) in cohort 2: vincristine. Longitudinal (LM) and circular (CM) muscle strips were electrically stimulated, in absence and presence of atropine (10^–6 M), with 10 s trains of pulses (0.3 ms, 100 V) at frequencies of 0.1–20 Hz. Amplitudes normalized to the phasic response to KCl 50 mM are represented. (A,A’) Maximum amplitude obtained for each frequency in LM, during (Amplitude 1, A1) and after (Amplitude 2, A2) electrical stimulation. (B,B’) Maximum amplitude obtained for each frequency in CM during (A1) and after (A2) electrical stimulation. Rats were intraperitoneally administered with saline (2.5 mL kg^–1) or vincristine (0.1 mg kg^–1) for 10 days (Monday to Friday, weeks 1–2). Treatments were: saline (black line, n = 10), saline + atropine (black dotted line, n = 10), vincristine (pink line, n = 8) and vincristine + atropine (pink dotted line, n = 8). *p < 0.05, **p < 0.01, ****p < 0.0001 vs. saline; ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001 vs. vincristine (two-way ANOVA followed by Tukey’s post-hoc test).