Ex Vivo Study of Colon Health, Contractility and Innervation in Male and Female Rats after Regular Exposure to Instant Cascara Beverage

Abstract

Instant Cascara (IC) is a sustainable beverage made from dried coffee cherry pulp, a by-product of coffee processing. It is rich in nutrients and bioactive compounds and has a high concentration of antioxidants. This study explored the impact of regular IC consumption on colonic motor function and innervation. Over a period of 4 weeks, male and female healthy rats were given drinking water containing 10 mg/mL of IC. Thereafter, colon samples were obtained to evaluate the longitudinal (LM) and circular (CM) smooth muscle contractile response to acetylcholine (ACh) and electrical field stimulation (EFS) in an organ bath, before and after atropine administration (10^-6 M). Histological and immunohistochemical analyses assessed colon damage, muscle thickness, and immunoreactivity to substance P (SP) and neuronal nitric oxide synthase (nNOS). ACh and EFS induced similar responses across groups, but the CM response to EFS was greater in females compared with males, despite their lower body weight. Atropine completely blocked the response to ACh but only partially antagonized the neural response to EFS, particularly that of CM in females treated with IC, which had a greater liquid intake than those exposed to water. However, in the myenteric ganglia, no statistically significant differences were observed in SP or nNOS. Our results suggest that regular IC exposure may enhance specific neural pathway functions, particularly in females, possibly due to their increased IC consumption.

Keywords: Instant Cascara; antioxidants; coffee by-products; colon contractility; muscarinic receptors; myenteric plexus; organ bath; rat; sex; substance P.

Figure 1

Experimental procedure. Male and female rats were given either water (control group) or IC beverage for a duration of 4 weeks. During the fourth week, animals were sacrificed to analyze the health of the colon wall using histological methods, colonic muscle strip contractility employing organ bath procedures, and its innervation using immunohistochemistry techniques.

Figure 2

Schematic representation of how the longitudinal and circular muscle strips were obtained. Once the colonic segment was removed from the rat, it was pinned on a Petri dish covered with Sylgard^® and filled with Krebs solution. A longitudinal cut was performed through the mesenteric border. Once stretched and pinned on the dish surface, the mucosa and submucosa layers were removed, and the circular (CM) and longitudinal (LM) muscle strips were obtained by cutting perpendicular or parallel to the longitudinal axis of the colon, respectively.

Figure 3

Experimental protocol of the organ bath experiments. Longitudinal and circular muscle strips were suspended in organ bath cups. Upper panel: After an initial 60 min stabilization period with 3 Krebs renewals, potassium chloride (KCl) was added at 50 mM to study the contractility of the strips. (A) After 2 Krebs renewals, the strips were electrically stimulated (EFS) at increasing frequencies (0.1–20 Hz) and posteriorly with acetylcholine (ACh) at increasing concentrations (10⁻⁸–10⁻⁵ M), Krebs was renewed two times before the next concentration was added. Lower panel (B): The same electrical and chemical stimulations were repeated in the presence of atropine (10⁻⁶ M), only this time just with ACh 10⁻⁵ M and without Krebs renewals. Finally, Krebs was renewed twice, and KCl (50 mM) was added to the organ bath.

Figure 4

Representative traces of colonic smooth muscle contractile responses and parameters measured. (A) Measurement of the amplitude of the phasic (PA) and tonic (TA) components of the contractions induced by ACh (and KCl). TA occurs after PA, as a plateau, generally below the value of PA. (B) Measurement of Amplitude 1 (A₁) and Amplitude 2 (A₂) of the contraction induced during and after electrical stimulation (EFS), respectively. Thick vertical arrow in A and thin double-head arrow in B represent stimulus (administration of ACh in (A), EFS duration in (B)).

Figure 5

The impact of Instant Cascara (IC) beverage regarding the microscopic features of the colon in male and female rats. Colonic damage (A) was evaluated by examining ten randomly selected fields per section at 40× magnification, using three distinct sections of colon tissue for each animal. In addition, the width of the muscle layer was measured (B). The images (C–F) show the muscle layer of the colon. During the fourth week of IC beverage administration, tissue samples were collected from animals across four experimental groups: Males—Control, Males—IC, Females—Control, and Females—IC. Each group consisted of six animals. The results are presented as mean ± SEM (standard error of the mean). Significant differences related to sex were observed, with p < 0.0001 indicated by #### for comparisons between Females—Control and Males—Control, and $$$$ for comparisons between Females—IC and Males—IC. Statistical analysis was conducted using one-way ANOVA with Bonferroni’s post hoc test. Bar: 100 μm.

Figure 6

Electrical field stimulation (EFS) was applied to longitudinal (LM) and circular (CM) muscle strips using 10 s pulse trains (0.3 ms) at frequencies of 0.1, 0.5, 1, 2, 5, 10, and 20 Hz. The following metrics were recorded: (A) maximum amplitude observed during stimulation (Amplitude 1) in LM; (B) maximum amplitude detected after stimulation (Amplitude 2) in LM; (C) Amplitude 1 in CM; (D) Amplitude 2 in CM. Data are reported as mean ± SEM, with each group consisting of 5–6 rats and 17–20 muscle strips for both LM and CM. Statistically significant differences related to sex are indicated by the following: # p < 0.05; ## p < 0.01; ### p < 0.001; #### p < 0.0001 for Male—Control vs. Female—Control comparisons; $ p < 0.05; $$$$ p < 0.0001 for Male—IC vs. Female—IC comparisons. Statistical significance was determined using two-way ANOVA followed by Bonferroni’s post hoc test.

Figure 7

The effects of electrical field stimulation (EFS) on longitudinal (LM) and circular (CM) muscle strips were evaluated under non-muscarinic conditions after atropine (10⁻⁶ M). EFS comprised 10 s pulse sequences (0.3 ms, 100 V) across frequencies of 0.1, 0.5, 1, 2, 5, 10, and 20 Hz. The following were recorded: (A) peak amplitude during stimulation (Amplitude 1) in LM; (B) peak amplitude post stimulation (Amplitude 2) in LM; (C) Amplitude 1 in CM; (D) Amplitude 2 in CM. Data are reported as mean ± SEM for each experimental condition, with 5–6 rats and 17–20 muscle strips per condition. Sex-related significant differences are indicated as follows: # p < 0.05; ## p < 0.01; ### p < 0.001 comparing Male—Control to Female—Control; $$ p < 0.01; $$$ p < 0.001 comparing Male—IC to Female—IC. Significant beverage-related differences are shown by + p < 0.05 for Females—IC versus Females—Control. Analysis was performed using two-way ANOVA with Bonferroni’s post hoc test.

Figure 8

The contractile responses of longitudinal (LM) and circular (CM) muscle strips to acetylcholine (ACh) stimulation were examined by exposing the samples to a range of ACh concentrations (10⁻⁸ to 10⁻⁵ M). The data illustrate the phasic (A) and tonic (B) responses in LM and the phasic (C) and tonic (D) responses in CM. Results are presented as mean ± SEM for each group, with six rats per group and 17–20 strips per group for both LM and CM. The analysis was conducted using two-way analysis of variance (ANOVA) with subsequent Bonferroni’s correction for multiple comparisons, and no significant differences were observed (p > 0.05).

Figure 9

Contractile responses of longitudinal (LM) and circular (CM) muscle strips to acetylcholine (ACh, 10⁻⁵ M) were evaluated both before (−) and after (+) atropine (10⁻⁶ M) administration. This setup was used to determine the role of muscarinic receptors in the ACh-induced contractions. The amplitude of the contractile responses, including both the phasic (PA) and tonic (TA) components, were compared for LM and CM under these conditions, specifically: (A) phasic response (PA) in LM; (B) tonic response (TA) in LM; (C) phasic response (PA) in CM; (D) tonic response (TA) in CM. Data were expressed as mean ± SEM for each group, with six rats and 17–20 strips per group for both LM and CM. Significant differences between atropine-treated and non-treated groups are indicated by ᵒᵒᵒᵒ p < 0.0001. Statistical evaluation was performed using two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test.

Figure 10

Impact of Instant Cascara (IC) beverage on the immunoreactivity of substance P (SP) within the colonic myenteric ganglia of male and female rats. Image (A) shows the myenteric ganglia of the colon, where SP is found. Images (B–E) show the myenteric ganglia immunoreactive to SP in the different experimental groups. The area immunoreactive to SP in the myenteric ganglia of the colon sections was quantified with the program Image J-Fiji (F). After a four-week period of administering the IC beverage, animals were sacrificed to evaluate various parameters across four experimental groups classified by sex and beverage type: Males—Control, Males—IC, Females—Control, and Females—IC. Results are expressed as mean ± SEM (standard error of the mean) for each group, with six animals per group. Statistical analysis was performed using a one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test, with no significant differences found (p > 0.05). Bar: 50 μm.

Figure 11

Whole-mount longitudinal muscle–myenteric plexus (LMMP) preparations were processed with the pan-neuronal marker HuC/D to detect all neurons (A) and neurons expressing neuronal nitric oxide synthase (nNOS), which is mainly present in myenteric neurons involved in inhibitory motor circuits (B). Using the program ImageJ-Fiji, the area occupied by the ganglia was measured (dashed lines (A)). Neurons immunoreactive for each of the mentioned markers were counted (A,B). Asterisk: extraganglionic neuron. White arrow: neuron positive for HuC/D and nNOS. Empty arrow: neuron positive for HuC/D and negative for nNOS. Scale bar: 100 µm.