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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39261674
72270
10.1038/s41598-024-72270-w
Article
Ex vivo electrical bioimpedance measurements and Cole modelling on the porcine colon and rectum
Jaimes-Morales S. A. samuel.jaimes34053@ucaldas.edu.co

Aguirre-Cardona V. E.
Gonzalez-Correa C. A.
https://ror.org/049n68p64 grid.7779.e 0000 0001 2290 6370 Research Group on Electrical Bio-Impedance (GruBIE), Universidad de Caldas, Manizales, Colombia
11 9 2024
11 9 2024
2024
14 212662 3 2024
5 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Different pathological changes in the large intestine wall, associated with the development of different chronic diseases, including colorectal cancer, could be reflected in electrical bioimpedance readings. Thickness and composition of the mucus bilayer covering it in the luminal side, abundance of bacteria of the intestinal microbiota, the permeability of the epithelium and inflammation are some of these. However, scientific literature on electrical passive properties of the large intestine is scarce. In this study, complex impedance measurements at 8 frequencies were carried out on 6 specimens of porcine colorectal tissue, within half ab hour post-mortem, obtained from a local abattoir. For 5 different distances, measured proximally from the border of the anus, 3 readings were taken at 3 different points with a tetrapolar probe. The results show 2 different dielectric dispersions in the α and β regions and it seems that there is a relationship between the values of resistivities and the thickness of the wall. Also, parameter values both for the Cole and the geometrical models are given. Another set of electrical bioimpedance readings was carried out in order to assess the effect of the mucus layer on electrical properties of the tissue. It seems that these layers are related to the low frequency dispersion. Finally, electrical passive properties of porcine colorectal tissue, reported in this work, give reference values and behaviour patterns that could be applied for further research in human medicine, based on bioimpedance measurements.

Subject terms

Dysbiosis
Gastrointestinal cancer
Electrodiagnosis
Biomedical engineering
http://dx.doi.org/10.13039/100022965 Ministerio de Ciencia, Tecnología e Innovación issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

The colon has been postulated as the key to wellbeing, health and disease, mainly due to the presence and role of the colonic microbiota1–3. Gonzalez-Correa et al.1 have suggested a common pathophysiological cascade for most chronic non-communicable diseases, including colorectal cancer (CRC), in which unhealthy lifestyles produce dysbiosis and this, in turn, translates into disruption of the colonic mucus bi-layer, an increase in epithelium permeability and, then, meta inflammation. All these changes alter the passive electrical properties of the colorectal wall, i.e., its electrical resistance4 and could be reflected by bioimpedance spectroscopy (EBIS) measurements in a similar way as in the skin5. So far, few publications are found in the scientific literature about the use of EBIS on the colon and rectum and even on other locations of the gut wall. The Research Group on Electrical Bioimpedance (GruBIE) at University of Caldas has carried out some bioimpedance studies in leporine colon tissue6, as well in human rectum tissue7,8, albeit with a device that only measured the real part of the impedance. In these studies, measurements were taken using a pencil like probe, as the one used in this study, in patients that underwent total colonoscopy and before it. The parameter ρ0 (resistivity at frequency 0), related to the extracellular liquids, was estimated, showing statistically significant differences between healthy and altered tissue, as well as with the presence or absence of cancer8. Some other studies have been focused in the evaluation of healthy and tumoral tissue in rats9 and humans10, finding that EBIS is sensitive to changes associated with the presence of cancer. Thus, EBIS has the potential to become a tool for detecting different colorectum conditions.

Other studies have put more emphasis on the development of technological tools for colorectal cancer detection and other anomalies of the gastrointestinal tract, carried out in human ex vivo11 and guinea pig12,13 tissue. These publications have shown promising results, establishing correlations between cancer and EBIS measurements, but reference values and the characteristics of healthy tissue are still lacking. The role of the mucus bilayer covering the large intestine and the intestinal microbiota that inhabits it have also, so far, not been considered. This knowledge is necessary for a better understanding of the physical phenomena that occur during the development of disease in relation to the passive electrical properties taking place in the affected tissues.

In addition to the possible application of EBIS, in relation to human health, a better understanding of these phenomena could also be relevant to agriculture, for instance for the evaluation of the leaky gut syndrome in pigs, which is usually evaluated by means of either transepithelial electrical resistance (TEER) or fluorescein isothiocyanate-dextran (FITC-d)14. Both techniques require taking samples either of the epithelium or of blood, as well as different equipment. In contrast, EBIS is a non-invasive technique that can be used, in vivo and in real time, to evaluate tissue permeability, by establishing changes in the passive electrical properties of the intestinal wall, probably mostly associated with the parameter ρ0, related to the resistivity of the extracellular space.

In this study, we report ex vivo measurements (of both the real and the imaginary components of the bioimpedance) taken from colorectal tissue from six pig specimens, using eight frequencies (1, 2, 5, 10, 20, 50, 100 and 200 kHz), within half an hour post-mortem, as well as the calculated resistivity values. The main aim of the study was to obtain initial values for complex resistivity and behaviour patterns of the tissue, with readings orally taken from the anus at 5 distances (D1–D5: 5, 10, 15, 30 and 40 cm, or D1, D2, D3, D4 and D5, respectively), as well as to see if there are significant statistical differences between the readings taken at those distances. Another 3 specimens were used to evaluate the effect of the mucus bilayer on the bioimpedance measurements. It is worth mentioning that the mucosa coat of the pig´s rectum and its length are considered as a valid animal model for biomedical research with future human purposes15,16.

Material and methods

Samples

This study was approved by the Bioethics Committee of the Faculty of Health Sciences at the University of Caldas (minutes number 09, communication ID CBCS-035, from July the 12th 2022). EBIS measurements were taken within half an hour post-mortem, from 6 specimens obtained from the local abattoir, all of them pertaining to the domestic pig species (Sus scrofa domesticus), with weights around 110 kg, when standing, and approximate ages between 150 and 160 days, according to the information provided by the abattoir’s authorities. The sex of the animals was not taken in consideration. The animals were put down by electronarcosis using 240 V and a maximum current of 1.5 A, with ulterior bleeding. This procedure is not expected to alter the passive properties of the intestine as the current only flows through the animal´s head, making it insensible and unconscious17. The feces were removed by washing the samples with tap water, a task undertaken by the abattoir’s personnel. On each specimen, a longitudinal cut was performed to expose the luminal side of the intestinal wall and proceed to the EBIS measurements from three different points, at 5 different distances (D1–D5), orally measured from the distal border of the anus: D1 at 5, D2 at 10, D3 at 15, D4 at 30 and D5 at 40 cm, as shown in Fig. 1. In the specimens, a clear difference between the rectum (left) and the colon (right) can be detected with the naked eye, as well as the border between both tissues at approximately 19 cm, in accordance with what is reported in the literature15. Another three specimens were used to take readings at the same distances, alongside the central axis (points marked as P2 in Fig. 1) under three different consecutive conditions (C1–C3): C1, tissue without any treatment; C2, tissue after cleansing with a cotton swab, and C3, tissue after removal of the internal (luminal) layer, using a razor blade.Fig. 1 Distal portion of the large intestine (anus, rectum and segment of the distal colon) where the points for EBISreadings were selected. D1-D5 identify the 5 distances orally taken from the anus.

To assess the possible ischemia effect, bioimpedance readings were also carried out on 1 specimen for 22 min, at 15 cm orally from the anus. All samples were put on a previously disinfected piece of glass.

EBIS readings and thickness tissue measurements

The first part of this study includes a total of 270 EBIS spectra from 6 specimens (S1–S6) times 5 distances (D1–D5) times 3 points/distance (P1–P3) times 3 readings per point (R1–R3), all taken with a homemade phase sensitive Tissue Bioelectrical Impedance Meter (an evolution of BioZspectra-v118 to allow the measurements of 4 components of the impedance Z′,Z″,Z and φ), a device developed by Dintech and University of Caldas, Colombia. This device was set up to apply 100 peak to peak μA current at 8 frequencies: 1, 2, 5, 10, 20, 50, 100 and 200 kHz. The equipment is controlled by an interface developed in Processing 4.1.2. The probe was the same as the one used in other studies, namely a pencil-like, tetrapolar probe built in Sheffield, UK19 (Fig. 2). Measurements were taken from distance D5 to distance D1. The probe was placed on a mechanic base to guarantee that the same pressure was applied on the tissue along all reading and a good contact between the surface of the probe and the tissue was reached. Pressure was calculated as about 9.36 kPa, given by the weight of the probe (measured with an analytical balance by Bel Engineering, model M1003i) and its contact area.Fig. 2 Pencil-like probe: (a) photograph of the tip, showing the electrode configuration, (b) dimensions of the tip with distances in mm.

The second part of the study consists of a total of 135 readings: 3 specimens (S1–S3), times 5 distances (D1–D5), times 3 readings (R1–R3), times 3 different conditions (as above mentioned, C1–C3), with the purpose of determining how the removal of the wall mucus alters the electrical properties of the tissue. For evaluation of the ischemia process, measurements were carried out with the same electrical set-up but with a gastric probe20 located in the lumen of tissue, without cutting it and making sure that a good contact was reached between the electrodes and the tissue under examination. Figure 3 shows the probe, kindly provided by the company Alandra Medical (Mexico DF-Mexico) for research purposes.Fig. 3 Gastric probe: (a) the whole piece, (b) tip showing the electrode configuration. This probe was kindly provided for research purposes by the company Alandra Medical (Mexico DF-Mexico).

After the EBIS readings were taken, the thickness of the tissue at each distance was also measured with a Truper digital calliper (Mexico City, Mexico). For this purpose, strips approximately 1 cm wide at the above-mentioned distances were cut and placed between two glass microscopy slides, total thickness measured and the value corresponding to the thickness of the two slides subtracted. Thickness values for all specimens are presented in Table S1.1 of the supporting material.

Calibration of the probe

When four electrode EBIS measurements are taken, deviations due to parasitic effects of the measuring probe, its coupling with the equipment and some other error sources are produced21, and, therefore, calibration of the system was carried out. For this purpose, 7 electrolytic solutions of different concentrations of NaCl in deionized water were used as a reference pattern, given their electrical properties, and covering impedance values in the range of those obtained from the measured tissues. The electrical conductivity of these solutions was measured with a Handylab LF 12 conductivity meter from Schott (Mainz-Germany), and their impedance spectra with the pencil-like probe and the BioZspectra v. 1.0. In Fig. S2.1 and Table S2.1 of supporting information, we show the spectra of these solutions and the electrical measurement values used as reference for calibration and conversion. From each solution, 80 ml were prepared.

Calibration of magnitude

The first step was to check the errors in the impedance magnitude for frequencies of 1, 5, 10, 20, 50, 100 and 200 kHz compared with their corresponding values at 2 kHz, to establish which frequencies should be calibrated. From here, it was found that, for frequencies of 1, 5 and 10 kHz, errors were less than 1%, except for 10 kHz with solution 1, with an error of 1.21%. For frequencies 20, 50, 100 y 200 kHz, errors were larger than 1% in most of cases (Table S2.2). For this reason, the calibration process was performed for these 4 latter frequencies. This process consisted of finding adjustment functions for each of the frequencies to be calibrated, plotting the data at 2 kHz (used as reference value), against the data at each frequency. Once the data was plotted, the trend line was found, as the mathematical expression that best fitted to the obtained behaviour. From these functions, the raw data was adjusted to obtain the calibrated magnitude. Impedance magnitudes at 2 kHz were used as reference values for the calibration, given that it is at low frequencies where the system shows smaller deviations, except for 1 kHz, and, therefore, it was not considered.

Calibration of phase angle

As it can be seen in Fig. S2.1b, there is a positive correlation between phase angle and magnitude in the impedance at higher frequencies. For this reason, the calibration was carried out as follows:The phase shift angle was corrected for 50, 100 and 200 kHz;

The remaining frequencies were not calibrated as the values of the phase shift were less than 1°.

Considering that the phase angle of the solutions ought to be 0°, trend lines and mathematical functions of phase shift against magnitude were established, in order to subtract this shift and, in this way, obtain a value for the calibrated phase angle.

Conversion to resistivity

Resistivity values were obtained from the conductivity measured for each solution. For each frequency, resistivity vs. calibrated impedance magnitude were plotted and the respective trend lines were obtained in the form of ρ=kZ+b where ρ is the magnitude of the electrical resistivity, Z is the impedance magnitude, k and b are calibration constants.

Statistical analysis

We first tested if there were significant statistical differences between: (a) the 3 readings, (b) the 3 points, and (c) the 5 distances. For this purpose, we used the Kruskal Wallis test for data with no normality and homoscedasticity (with Bonferroni post hoc) and Brown Forsythe for data with non-normality and heteroscedasticity (with Tamhane’s T2 post hoc). The distribution was tested by the Kolmogorov–Smirnov test and the homoscedasticity was tested with the Levene test. A p value < 0.05 was accepted as statistically significant. To explore a correlation between tissue thickness and EBIS readings, Pearson's rank correlation coefficient was applied. The analysis was carried out using SPSS v26. The post hoc statistical power of the comparative analyses was calculated with G-power software.

Spectra parameterization

Circle parameters of the data (values of x and y for the centre in the Cartesian plane and length of the radius, which will be designated as h, k, and r, respectively) were obtained by means of a 3 point (3P) approach22,23. With the geometrical parameters, two more variables were included: maximal reactance (Xcmax) and maximal phase angle (φmax), i.e., those corresponding to the characteristic frequency. From the geometrical values, the four Cole model’s parameters were also calculated as follows: resistivity at zero frequency or ρ0, was found from the interception of the circle with the real axis on the right side (i.e., ρ0 = (h + (r2 − k2)0.5); resistivity at infinite frequency or ρ∞, from the interception of the circle with the real axis on the left side (i.e., ρ∞ = (h − (r2 − k2)0.5); alpha (α) from the normalized angle formed between the radius touching ρ∞ and the abscise to the right side of this point, as per Ayllon et al.24, and, finally, relaxation time or τ was calculated from the Cole function evaluated for the three other calculated parameters and their respective impedance values, which has been reported by González-Correa et al.23.

Results

Calibration of the probe

The calibrated readings for the 7 solutions are shown in Fig. 4, both for magnitude and phase angle and using the adjustment functions. As can be seen, the calibration shows a horizontal behaviour for the magnitude and shifts lower than ± 0.5° for the phase angle (Table 1 shows the mean values). From these values, it is now possible to calculate the calibrated real and imaginary parts of the readings.Fig. 4 Calibrated impedance values for the readings obtained with the 7 solutions: (a) magnitude, (b) phase angle.

Table 1 Mean (µ) and standard deviation (σ) of the calibrated impedance magnitude.

	µ	σ	
S1	1139.38	5.18	
S2	785.28	1.28	
S3	474.18	1.35	
S4	387.20	2.02	
S5	207.33	1.26	
S6	95.55	0.60	
S7	54.99	0.35	

All functions obtained in the whole calibration process and their respective plots are shown as a Supporting information document, sections S3 to S5.

Statistical analysis

Firstly, the real and the imaginary parts of the 270 spectra (8 frequencies) were considered, to see if there were statistically significant differences between readings R1, R2 and R3. For this purpose, the Kruskal Wallis test was used, obtaining p values of 0.902 for the real part and 0.940 for the imaginary part, showing that R1, R2 and R3 are statistically equal. Therefore, we proceeded to average the 3 readings for each point, obtaining 90 spectra for the analysis.

For these 90 spectra, the Brown Forsythe test was used to see if there were statistically significant differences between the means of readings corresponding to each of the 3 points P1, P2 and P3. This gave p values of 0.055 for the real part and 0.131 for the imaginary part. Therefore, we averaged the mean values obtained for the three points, in order to have a mean value representing an approximate complex tissue impedance for each of the five distances (D1–D5) considered in the study, 30 spectra remaining. Figure 5 shows box and whisker plots for the latter spectra (8 frequencies): (a) real part of the resistivity, (b) imaginary part of the resistivity.Fig. 5 Box and whisker plots representing the median and quartiles for the real (ρ′) and the imaginary (ρ″) parts of the complex resistivity, for all frequencies. Letters indicate difference or equality between distances (p < 0.05), where a ≠ b ≠ c.

For the statistical analysis related to distance the Brown Forsythe comparison test and Tamhane’s T2 post hoc were used, which showed that resistivity values (real as well as imaginary parts) for points at 30 and 40 cm are equal to each other but different from those at 5, 10 and 15 cm, i.e., electrical resistivity readings from the rectum seem to be different (lower) than those taken from the distal portion of the colon (higher). Interestingly, the real part of the resistivity for D1 (5 cm) was also statistically different from those at the distances D2 and D3 (10 and 15 cm). This can also be appreciated in Fig. 5.

The post hoc power test for the comparisons between distances was 0.9999, for the real part of the impedance, and 0.9296, for the imaginary part. For comparisons between readings and points, the power is less than 0.8. Details of the performed calculation are shown in Table S6.1 in the supporting files.

Figure 6 shows Wessel diagrams for the means of the complex resistivity at the five different distances. They clearly show that there are two dispersions, which we call dispersion LFD (low frequencies dispersion) and dispersion HFD (high frequencies dispersion). In this figure, the geometrical models for both dispersions, calculated with the 3P method22,23, are also shown. For LFD, the first three points were used, while, for HFD, the point with the maximal reactance and the closer point on both sides of it were used.Fig. 6 Wessel diagrams of the mean spectra at the five distances for two dispersions: LFD (left) and HFD (right). Blue lines represent fitting models obtained by the 3P method.

Spectra parameterization

Table 2 gives the values for all parameters and variables calculated with the 3P method22,23 for the mean readings of both dispersions at the five different distances. Table 2 Parameters and variables calculated for the mean readings of both dispersions at the five distances.

Distance (cm)	5	10	15	30	40	
Thickness (mm)	3.97	5.31	4.85	3.28	3.22	
Parameters for the first dispersion (lower frequencies)	
 h1 (Ω cm)	886.44	695.35	750.65	1152.67	1212.38	
 k1 (Ω cm)	 − 88.03	 − 34.13	 − 69.08	 − 127.07	 − 84.47	
 r1 (Ω cm)	151.42	80.56	124.88	225.94	180.8	
 Xc1max (Ω cm)	63.39	46.43	55.8	98.87	96.33	
 φmax1 (°)	4.09	3.82	4.25	4.90	4.54	
 ρ01 (Ω cm)	1009.64	768.33	854.68	1339.49	1372.23	
 ρ∞1 (Ω-cm)	763.24	622.37	646.62	965.85	1052.53	
 α1	0.61	0.72	0.63	0.62	0.69	
 τ1 (μs)	47.95	44.30	41.50	60.40	54.90	
 fc1 (kHz)	3.319	3.593	3.835	2.635	2.899	
Parameters for the second dispersion (higher frequencies)	
 h2 (Ω cm)	758.42	568.52	610.42	941.49	1025.15	
 k2 (Ω cm)	 − 138.11	 − 57.81	 − 76.45	 − 123.12	 − 124.31	
 r2 (Ω cm)	217.72	130.66	158.16	242.83	238.37	
 Xc2max (Ω cm)	79.61	72.85	81.71	119.71	114.06	
 φmax2 (°)	4.78	4.67	5.22	5.99	5.37	
 ρ02 (Ω cm)	926.73	685.69	748.88	1150.79	1228.54	
 ρ∞2 (Ω-cm)	590.11	451.35	471.96	732.19	821.77	
 α2	0.56	0.71	0.68	0.66	0.65	
 τ2 (μs)	3.21	1.65	1.90	3.05	3.90	
 fc2 (kHz)	49.581	96.458	83.766	52.182	40.809	
*Subindex: 1 indicates LFD and 2 indicates HFD.

The correlations between the thickness of the tissue and the resistivity at the characteristic frequency fc for both dispersions are shown in Fig. 7. These values were calculated from the geometrical parameters h and k (coordinates of the centre) and r (radio) of the adjusted semicircle obtained with the 3P method for each dispersion. The point (h, r + k) is the geometrical locus of the resistivity at the characteristic frequency.Fig. 7 Correlations between thickness and resistivity of the tissue at the characteristic frequency (h as the real part, and r + k as the imaginary part): (a) data from LFD, and (b) data from HFD. In both figures blue represents the real part of the resistivity (ρ′), while orange is the imaginary part. (ρ″).

Ischemia effect

Average time for the experiments was 16.20 (± 4.11) min, with a duration for each distance of 2.18 (± 0.55) min. For this reason, consecutive readings were carried out during the first 22 min, and their behaviour for 1 kHz and 200 kHz is presented in Fig. 8. Table 3 shows, in percent terms, the impedance increases in this time lapse. It is worth mentioning that, during all experiments with porcine tissue, including these, no peristaltic movements were observed due to possible post mortem hyperactivity.Fig. 8 Effects due to ischemia seen on impedance levels at low and high frequency.

Table 3 Percent increase in impedance levels due to ischemia process at low and high frequency.

1 kHz	200 kHz	
Z′	Z″	Z′	Z″	
10.87	21.67	3.75	18.38	

Effect of removal of the intestinal mucus

Results obtained after removal of the intestinal mucus are presented in Fig. 9, where it can be observed that the intervals given by the standard deviations for the magnitude overlap the 5 distances considered (5–30 cm), while, at 40 cm, impedances of the untreated tissue are higher. At all distances, the concavity looking upwards in the magnitude at low frequencies are diminished after remotion of the mucus layer.Fig. 9 Means (μ) (continuous lines) and standard deviations intervals (μ+σ;μ-σ) (dashed lines) of EBIS spectra before and after mucus removal. Blue: (C1) tissue without any treatment; yellow: (C2) tissue after cleansing with a cotton swab; red (C3) tissue after removal of the internal layer using a razor blade.

Additionally, the phase at lower frequencies is notoriously diminished, to the point that the interval given by the standard deviations for the normal tissue does not overlap with those of treatments C2 and C3. Meanwhile, at higher frequencies the phases are much more similar between them.

Once differences in the phase angle were observed, a comparative analysis of the three groups were performed, using the already mentioned statistical tests in “Statistical analysis” section. It could, then, be established that there are statistically significant differences between tissue without treatment (C1) and tissue with the mucus removed (C2 and C3), phase angle being larger in the C1. Additionally, phases for C2 and C3 treatments are statistically equal. Table S7.1 shows a resume of the statistical results.

Discussion

It seems that electrical resistivity readings taken from the distal part of the colon show higher values, both for the real and the imaginary parts, when compared to those obtained from the rectum. These differences seem to be associated with the thickness of their respective walls, with that of the colon being thinner than that of the rectum (Table 2). This behaviour is the same as shown by Mulett-Vásquez et al.6 in leporine colonic tissue. Taking into account the geometry of the pencil-like probe, as well as the results presented by González-Correa et al.19, one could say that approximately 90% of the information obtained with our measurements comes from a maximal depth of about 1.36 mm. If this value is compared with the minimal thickness of the samples, which was 2.1 mm (Table S1.1), the observed effects could not be produced by the area of influence of the probe.

According to the parameters of the Cole model, the real part of the electrical resistivity was in the range of 732.19 to 1372.23 Ω-cm for the colon and 451.35 to 1009.64 Ω-cm for the rectum. This range of values is similar to that reported in leporine colonic tissue (600 to 900 Ω-cm)6 and bigger than in vivo human tissue (310 to 420 Ω-cm7 and 120 to 520 Ω-cm8). This difference could be explained by the in vivo tissue having perfusion and because it is surrounded by other structures such as muscle, fascia, and visceral peritoneum. Additionally, another aspect to consider, when comparing readings taken with probes using a superficial contact, is the pressure applied to the probes, which affects impedance values, as pressure alters the volume of the tissue underneath them and a redistribution of the extracellular liquid11. For this reason, readings were taken with a calculated pressure of 9.36 Pa, which ensures a good contact25, but is below 20 kPa, a value reported as the upper limit to take EBIS readings without altering the resistivity of the colon tissue being examined in the distal human colon ex vivo11.

When represented as Wessel plots, two dispersions are clearly distinguishable, a feature which is also present in the gastric reading reported by Beltran et al.26. Similar behaviours are also seen in the research reported by Pathiraja et al.10 (human tissue, ex vivo), Mulett-Vásquez et al.7 (human tissue, in vivo) and the dielectric properties data provided for different biological tissues in “Foundation for Research on Information Technologies in Society (IT’IS, under Dielectric Properties, IT’IS Foundation)27. These values are based on measurements taken on ovine colon tissue published by Gabriel et al.28, where two curvatures in the response of the electrical impedance magnitude in the intestine can be seen: one with the concavity pointing upwards at low frequencies (LFD) and another with the concavity pointing downwards at higher frequencies (HFD). These two dispersions could probably be explained by the complex structure of the colorectal wall, especially due to the presence of the mucus bilayer on top of the epithelium and the mucosa, as a whole. This behaviour is present in healthy tissue, but it is more evident as the ischemic process advances26, while, when evaluating cancer ex vivo, both dispersions are more prominent in samples taken from patients who have responded well to chemotherapy10.

In relation to the layer structure, it is worth mentioning that the most internal (luminal) layer of the large intestine wall is a loose mucus layer inhabited by the microbiota, mainly consisting of trillions of bacteria with a large species diversity, most of them with either a round or rod-like form (cocci and bacilli, respectively). This layer is formed from the subjacent firm layer of mucus by an endogenous proteolytic process29. Therefore, we could think of this system as a suspension of small spheres that could produce a dispersion in the α region, due to a counter ion effect, in which a lateral displacement of the electrical charges on the bacterial surface is present (see Martinsen and Grimnes, Chapter. 2 pp. 34 and Chapter. 3 pp 84 and 9030 and Abasi et al.31). This would, therefore, be represented in the results obtained at low frequencies, where the characteristic frequency is about 14.07 and 26.81 times smaller than those of the second dispersion (Table 2). In fact, the characteristic frequencies found for LFD are in the range of the α dispersion of (10 Hz–10 kHz31). This hypothesis would be supported by the results obtained when the more internal (luminal) mucus layer was removed, where a decrease in both the upwards concavity of the magnitude as well as in the phase at lower frequencies can be observed (Fig. 9). This could indicate a partial loss of the LFD, which cannot be totally removed, as the basal layer is firmly attached to the epithelium and it is not possible to gain access to the crypts with the razor blade, without damaging the epithelium and the rest of the tissue. In terms of electrical behaviour, the partial removal of the mucus decreases the capacitive property of the tissue wall, mainly at lower frequencies, possibly due to the loss of luminal tissue, where the microbiota is present. Wessel diagrams of the measurements taken at each distance, for the three treatments, are presented in Fig. S8.1, where it can be seen how the ratios between the right and left dispersions (LFD and HFD, respectively) are notoriously reduced at all distances, after performing treatments C2 and C3.

On the other side, the innermost mucus layer, is mainly made up of glycoproteins29 and is almost devoid of bacteria, which means that a possible dispersion there could be due to protein polarization and that it ought to appear at much larger frequencies than those used in this study32. For this reason, the effect of this more inner mucus layer on the electrical behaviour of the tissue would be purely resistive. Beyond the mucus bilayer, the actual cell structures appear (mucosa, submucosa, muscular propria and serosa), which could explain the second dispersion and is probably due to polarization of epithelium, and the presence of tight junctions and cell membranes (Martinsen and Grimnes, Chapter. 3 pp. 9030 and Abasi et al.31). This would be reflected in our results where fc2 is in the β region, between 10 kHz and 10 MHz31 (Table 2). However, it is possible that the predominant effect is due to the epithelium, since the tight junctions make this structure highly resistive in addition to the proximity of the electrodes used for the readings. According to Kassanos et al.33, for an interelectrode separation of 0.75 mm, as it is the case with our probe, 10% of the information corresponds to the mucus layer and 90% to the tissue itself (75% mucosa, 10% submucosa and 5% for the remaining layers). In Fig. 10, we present a possible circuit for modelling the electrical properties of the tissue layers, taking the above-mentioned hypothesis into account. However, given the complexity of the tissue and the shape of the probe, readings are the result of in series and in parallel combinations of the different components of the tissue.Fig. 10 Possible circuit model for the layer structure of the colorectal tissue. ρeom: resistivity of outer mucus; ρiom: resistivity of intracellular space of bacteria; QMom: constant phase element for model counter ion effect; ρim: resistivity of inner mucus; ρi: resistivity of intracellular space; ρe: resistivity of extracellular space and QM: constant phase element for model cellular membrane.

In relation to tissue behaviour during the first 22 min of ischemia, an increase in the impedance level is observed, both at 1 and 200 kHz. This behaviour is possibly due to cell inflammation, consequent to closure of ionic channels, which, in turn, reduces the space in the tight junctions and, therefore, the electrical conductance, while the capacitive property of the cell membranes is increased34. This is evident in the readings, where the capacitance increases much more than resistance (21.67% y 10.87% at 1 kHz and 22 min, respectively). This phenomenon would not be perceptible in the analysis of the readings and the different points, as readings at each distance takes about 2.18 min, in average, that is a tenth of the time that the whole experiment takes. On the other hand, the analysis related to the measurements at the different distances shows that it is affected as the total mean time of duration is 16.20 min. Nevertheless, given the fact that the first measurements are taken in the colon, it is expected that, from 40 cm (D5) towards 5 cm (D1), impedance values be progressively higher, and, in this way, overestimating the values in the rectum. In other words, were ischemia not present, the differences between the readings from colon and rectum would probably be more prominent than those presented in this study. The penetration depth of this measuring probe can be estimated as 4.2 mm, considering a separation of 6 mm between the current electrodes and that they are in a Wenner configuration38. This depth is smaller than the average thickness of the tissue 15 cm aboral from the anus (Table S1.1), so that the measured impedance covers practically all tissue layers. Besides, it must be mentioned that the obtained spectra present low noise (Fig. S9.1), which allows to state that there was good contact between the electrodes and the tissue.

Finally, it is important to highlight that the results from the measurements with electrolytic solutions mainly show parasite capacitive effects, with an increment in the phase at higher frequencies (Fig. S2.1). These effects are related to the capacitances associated with the measuring cables, along with those of the measuring equipment and those produced between the samples and the measuring system, as it has been reported in the literature 359. In order to guarantee the reliability of the measurements, a calibration process was carried out, accounting for those deviations. For this purpose, saline solutions were used as standards as, in the interval of frequencies used in this study, they have a constant magnitude and phase 0 (Martinsen & Grimnes 2008, Chapter. 3 pp. 66 30)0. This approach can even be used for the calibration of systems that use very high frequencies 361. On the other side, deviations at lower frequencies were not present (Fig. S2.1), indicating that electrode polarization was mitigated by the use of a 4-electrode configuration. Additionally, the calculation of the resistivities allows an analysis based on the electrical characteristics of the tissue, eliminating the dependence on the electrodes and probe geometry. This allows the comparison of results of studies carried out under different conditions and enriches the bioimpedance research by establishing a common language.

Conclusions

We have shown the electrical resistivity behaviour of porcine colon and rectum tissue, giving what could be considered as initial and provisional reference values and behaviour patterns for further research. The pattern of two dispersions was shown and analysed, which is probably related to the layer structure of this tissue. This is relevant, because the scientific literature on electrical passive properties of the large intestine is scarce and limited, mainly because it only studied the real part of the bioimpedance spectrum. Given the results, a Cole model with two dispersions is proposed, one of them attributed to the luminal mucus layer and a second one related to the tissue layer structure. The finding that removal of mucus layer influences mainly the first dispersion shows that EBIS could detect it, which could be relevant for future investigations related to the health status of the intestinal microbiota. Further studies could be focused on in vivo measurements on tissue of porcine and human tissue, for applications like the development of new technologies for screening and diagnosis of colorectal diseases.

Supplementary Information

Supplementary Information 1.

Supplementary Information 2.

Supplementary Information 3.

Supplementary Information 4.

Supplementary Information 5.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72270-w.

Acknowledgements

To the local abattoir of Manizales-Caldas, for their help in providing the pig colorectal samples used in this study.

Author contributions

S.A.J.M.: Formal analysis, interpretation of data, writing review and editing. V.E.A.C.: Acquisition of data, investigation, validation, visualization, supervision. C.A.G.C.: Conceptualization, methodology, writing original draft, writing review and editing.

Funding

SA Jaimes-Morales would like to thank MinCiencias-Colombia (Colombian Ministry for Science, Technology and Innovation) for his PhD scholarship.

Data availability

The datasets supporting this article have been uploaded as part of the Supplementary Material.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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