Regional differences in the rheological properties of mucus

Due to the inter-animal and intra-sectional variations in the obtained volume of the mucus samples, it was not possible to obtain six replicates for all sections. For the distal jejunum section 2, only two rats had enough mucus in this section to analyze the rheological profile, while for the other sections, sufficient amounts of mucus were obtained from 4–6 rats (Fig. S1). In general, there seem to be a trend of less mucus in the distal jejunum section 2, as this was also prevalent in the rats used for the quantitative analyses.

As seen in Fig. 1, the mucus of the small intestine showed non-Newtonian shear-thinning properties with a plateau in the lower shear region followed by a shear thinning region. The rheological profiles for the individual rats in Fig. S1.

Fig. 1

Viscosity as a function of shear rate of mucus from each of the eight sections of the rat small intestine, i.e., duodenum ( ), proximal jejunum section 1 ( ) and 2 ( ), mid jejunum section 1 ( ) and 2 ( ), distal jejunum section 1 ( ) and 2 ( ), and ileum ( ). Data are depicted as mean ± SEM (n = 2–6). The small window is a zoom of the lower shear rate region. The black arrows indicate the onset of the shear-thinning in the duodenum and proximal jejunum section 1, while the grey arrow indicates the area of onset for the other sections

The flow curves from the mucus samples obtained from the different sections of the small intestine can be divided into two groups according to their viscoelastic behavior at low shear rates, i.e., the flow curves of the duodenum and proximal jejunum section 1 displayed a higher viscosity than the other six sections (proximal jejunum 2 to ileum). This difference is more easily observed in the zoom of the lower shear rate region in Fig. 1 and upon a more detailed analysis of the flow curves, in which regional differences are observed for the zero rate viscosity and at the onset of the shear-thinning region (Fig. 2A-B). The mucus from the duodenum and proximal jejunum section 1 tended to have higher zero-shear viscosity (~ 9.4 · 105 Pa·s) than mucus from the sections from mid jejunum 1 and onwards (~ 1.5 · 105 Pa·s), albeit the difference was not statistically significant (Fig. 2A). Additionally, the mucus from the duodenum and proximal jejunum section 1 had significantly lower onsets of the shear-thinning region at a shear rate of ~ 0.01 s−1 compared to the onset in the range of 0.04–0.07 s−1 observed for the mucus of the other six sections (p < 0.05) (Figs. 1, 2B). There was no difference in the slopes of the shear-thinning region (shear rates 0.03 – 10 s-1) when comparing the mucus collected from the different sections of the small intestine (Fig. 2C). Similarly, there was no difference in the apparent viscosity of the mucus from various sections at the shear rate of 0.46 s-1 (Fig. 2D), which is the closest measuring point to the reported physiological shear rate near the duodenal wall in the rat (0.21–0.41 s-1) [24].

Fig. 2

Rheological information from flow curves of the mucus from each of the eight sections of the rat small intestine: zero-shear rate viscosity (η0) (A), shear rate at the onset of shear-thinning (B), slope of the shear-thinning region (shear rates 0.03 – 10 s−1) (C), and apparent viscosity ηa at shear rate 0.46 s-1 (D). Data are depicted as mean ± SEM (n = 2–6). Statistically different values are marked with an asterisk when p < 0.05 according to a one-way ANOVA

The mucus layer of the small intestine proved to be a viscoelastic gel with solid-like properties as all sections had a higher G′ than G″ in the tested frequency range (Fig. 3A, B). This property is also evident from tan δ depicted in Fig. 3C, in which all sections showed solid-like behavior with tan δ < 1. As was the case with the shear flow viscosity, the moduli of the mucus from the first two sections, the duodenum and proximal jejunum section 1, were higher than the moduli of the mucus from the other six sections (Fig. 3A, B). Furthermore, tan δ was significantly lower for the first two sections at ~ 0.3 compared to the last five sections with tan δ ~ 0.6 (p < 0.05) (Fig. 3C). While still predominantly solid-like, as tan δ < 1, the mucus layer of the mid jejunum and onwards trended towards a less solid-like structure compared to the mucus of the duodenum and proximal jejunum section 1. An increase in tan δ might indicate a less rigid gel network in the lower sections of the small intestine compared to the upper sections, as has previously been observed in studies of porcine gastric mucus and mucoadhesive polymers [25].

Fig. 3

Comparison of the storage modulus G′ (A) and loss modulus G″ (B) from a frequency sweep of each of the eight sections of the rat small intestine, i.e., duodenum ( ), proximal jejunum section 1 ( ) and 2 ( ), mid jejunum section 1 ( ) and 2 ( ), distal jejunum section 1 ( ) and 2 ( ), and ileum ( ). The tan δ in the different sections of the rat small intestine at an angular frequency of 1 rad/s (C) was compared using a one-way ANOVA. Statistically different values are marked with an asterisk when p < 0.05. Data are depicted as mean ± SEM (n = 2–6)

The flow curves obtained in the current study are comparable to the flow curves presented in a study by Zahm et al., in which gastric and duodenal mucus obtained from fasted Wistar rats was studied [9]. Although the previous study observed no zero-shear plateau, possibly due to analysis at higher shear rates (1·10-2—5·101 s-1), the viscosity of the duodenal mucus was in a similar range as reported in the current study (starting at 104 – 106 Pa·s). The slope of the shear-thinning region reported by Zahm et al. was, however, less steep at -0.82 to -0.93 log(Pa·s)/log(s-1) [9] when compared to the current study reporting a mean slope of -1.39 ± 0.04 log(Pa·s)/log(s-1) (Fig. 2C). Whether this difference is caused by experimental settings, or strain differences, is unclear. Boegh et al. studied the rheological behavior of porcine small intestinal mucus using analytical conditions similar to those used in the current study [7] and did not observe a zero-shear viscosity plateau, but a linear shear-thinning region starting at a comparatively lower viscosity of ~ 103 Pa·s [7]. Canine mucus has been shown to have an even lower viscosity, starting the linear shear-thinning at a viscosity of ~ 102 Pa·s [8]. It has to be noted that the rat mucus were analyzed at 25 °C (in the current study and in Zahm et al. [9]), whereas the porcine and canine mucus were analyzed at 37 °C [7, 8]. While the difference in experimental temperature would influence the results, it is not likely that this has caused the large differences observed (from 104 – 106 Pa·s in the rat to ~ 103 Pa·s in pigs and ~ 102 Pa·s in dogs), and the primary cause is thus expected to be differences in the rheological properties of the small intestinal mucus layer in the different species.

Similar to the rheological flow curves, the G′ of rat intestinal mucus (G′ at ~ 103-105 Pa; Fig. 3A) was generally higher than what has been reported in pigs (G′ at ~ 101–102 Pa [6, 26,27,28]) and dogs (G′ ~ 101-102 Pa [8]). While the tan δ of porcine duodenal mucus was lower than the rat duodenal mucus (0.15-0.16 [11, 28] vs. 0.3), studies have observed an increase in tan δ to 0.3–0.6 for porcine small intestinal mucus [10, 27, 28], which is similar to what was observed in the current study (Fig. 3C). Although the moduli values of rats are higher than those of other species, the tan δ seem to change in a similar pattern along the small intestine, suggesting that they have a similar gel network.

Regional differences in mucus pH and concentrations of proteins and endogenous surfactants

The mean values for the rat mucus pH and concentrations of proteins and endogenous surfactants are depicted in Fig. 4. The boxplots in Fig. S2 show the values of the individual rats.

Fig. 4

Mucus characteristics exhibiting regional differences in pH (A) and concentrations of proteins (B), bile salts (C), polar lipids (D), and neutral lipids (E) in the mucus of the eight sections of the rat small intestine. Data depicted as mean ± SEM (n = 4–8). Statistically different values are marked with an asterisk when p < 0.05 according to a one-way ANOVA

A recent study by Klitgaard et al determined the regional differences in luminal pH and concentrations of endogenous surfactants in the rat small intestine [20]. The median placement of each of the four luminal incisions in the small intestine, made in the previous study, were paired with the corresponding mucus section in the current study, allowing a direct comparison between luminal and mucus values. Specifically, the previous study’s luminal incisions in the duodenum, proximal jejunum, mid jejunum, and ileum correspond to the mucus sections termed duodenum, proximal jejunum 1, mid jejunum 2, and ileum in the current study, respectively.

Mucus pH

In Fig. 4A, it is evident that the pH in the mucus layer was almost constant at ~ 6.5 from the duodenum to the distal jejunum 1, after which it steadily increased until reaching pH 7.5 ± 0.1 in the ileum. Only a few other studies have previously determined the pH of the mucus layer of the rat small intestine, often designated as the unstirred water layer, to which the mucus layer is indistinguishable. In agreement, Högerle and Winne studied the pH of the mucus layer during perfusion of different buffer systems in the proximal jejunum of fasted Wistar rats and found that the pH was largely stable at ~ 6.6 [29]. Lucas et al. and Daniel et al. both measured the pH of the mucus layer ex vivo from fed Wistar rats and found it to be pH 5.5–6.3 [30] and 6.8 ± 0.2 [31], respectively. Although there were differences in measurement methods, rat strains, and the prandial state of the rats, the values of the previous studies correlate very well with those of the present study.

Compared to the luminal pH in the rat small intestine [20], the mucus pH was significantly lower in both the duodenum (pH 6.53 ± 0.07 vs 6.92 ± 0.06 [20], (p = 0.006)) and the ileum (pH 7.47 ± 0.10 vs 7.87 ± 0.09 [20] (p = 0.006)). However, there were no significant differences between the mucus and luminal values for the proximal and mid jejunal pH.

Mucus protein concentrations

The protein concentration in the mucus layer decreased throughout the small intestine, starting at 56.4 ± 4.1 mg/mL in the duodenum and ending at 24.6 ± 2.4 mg/mL in the ileum (Fig. 4B). This decrease was statistically significant when comparing the protein concentration of the first two sections with that of each of the last five sections (p < 0.02). The change in protein concentration might, in part, be due to differences in mucin concentrations, as the colorimetric quantification kit would also react to the protein backbone of the mucins and not just non-mucin proteins such as serum albumin present in the sample. Previous studies have reported 31.8-50 mg/mL mucin in the duodenum and 20-30 mg/mL in the small intestine of pigs [4, 11, 32], indicating a decrease in overall mucin concentration. These studies have, however, used different methods of quantification and treatment of the mucus samples, making a comparison to the current study difficult. In a recent study on Wistar rats, the expression of mucin subtype Muc5ac was shown to drastically decrease from the duodenum to the jejunum [33], which has also been observed in pigs [6] and dogs [8]. However, how much the mucins in the rat mucus contributed to the total protein concentration in the mucus is unknown, and further studies are needed to assess the concentration of mucin in rat intestinal mucus. Recently, Mortensen et al. determined the total protein concentration of porcine mucus from the proximal jejunum, using a similar protein assay kit as the one used in the current study, and found a protein concentration of 126 ± 18 mg/mL [26]. Therefore, by comparison to this recent study and approximation to the others studies on pig mucus [5, 26], it seems that there might be a lower concentration of proteins in the mucus from fasted rats.

Mucus bile salt concentrations

The individual bile salts identified in the mucus of the small intestine of the rat were tauro-β-muricholic acid, taurocholic acid, glycocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, and cholic acid, with tauro-beta-muricholic acid and taurocholic acid being the most prominent (Fig. S3A). The total concentration of bile salts was 16.0 ± 2.2 mM in the duodenal mucus and gradually increased until peaking in the second section of the mid jejunum (55.1 ± 9.5 mM), after which it steadily decreased until the ileum (22.0 ± 8.1 mM) (Fig. 4C). Curiously, some sections in some rats contained no bile salts (Fig. S2C), even though the neighboring sections in the same rat had bile salts, and the other endogenous surfactants were recovered from the mucus of that section. This observation contributed to the high inter-individual variability observed for the bile salt concentrations in the mucus layer (Figs. 4 and S2C).

Although no significant difference in mucus bile salt concentrations was observed between the different sections of the small intestine (Fig. 4C), there is a clear pattern that follows the same tendency as the luminal bile salt concentrations [20]; i.e., increasing bile salt concentrations until the mid-jejunum and then a decrease, presumably caused by active bile salt reabsorption in the latter half of the small intestine [34]. The concentrations of bile salts in the mucus layer were significantly lower in the proximal jejunum (20.7 ± 3.5 mM) and mid jejunum (55.1 ± 9.5 mM) when compared to the luminal values (100.0 ± 10.2 mM and 123.4 ± 6.6 mM [20]). In the duodenum and ileum there were no significant differences between the mucus and luminal values. However, albeit it was not significantly different, there was a trend towards higher concentrations of bile salts in the mucus layer of the ileum (22.0 ± 8.1 mM) when compared to the luminal fluids (4.3 ± 2.2 mM [20]).

This is the first time the bile salt concentration in the mucus layer of the rat small intestine has been quantified. Since the rat lacks a gallbladder, bile salts and other endogenous surfactants are excreted to the small intestine at a continuous rate, which is likely the cause of the high endogenous concentrations of bile salts in the small intestine, including the mucus layer [35].

Mucus polar lipid concentrations

The total polar lipid concentration was highest in the mucus of the upper half of the small intestine (~ 15 mM) and decreased to ~ 5 mM in the latter half, from the second mid jejunal section and onwards (Fig. 4D). The most prominent polar lipids were lysophosphatidylcholine and phosphatidylcholine, with a minor contribution to the total polar lipid concentration from phosphatidylethanolamine and barely any contribution from sphingomyelin (Fig. S3B). In the upper half of the small intestine, the contribution to the total polar lipid concentration was almost evenly divided between lysophosphatidylcholine and phosphatidylcholine. There was, however, a steady and gradual decrease in the concentration of lysophosphatidylcholine, that was highest in the duodenum (7.9 ± 1.7 mM) and barely detectable in the last section of the small intestine (0.1 ± 0.1 mM) (Fig. S3B).

Compared to the luminal values [20], there was a significantly higher concentration of polar lipids in the mucus layer of the duodenum (17.4 ± 2.2 mM vs. 7.3 ± 1.1 mM [20] (p = 0.002)). However, the polar lipid concentration in the following proximal jejunum section was significantly lower in the mucus layer compared to that of the lumen (17.6 ± 2.1 mM vs. 24.9 ± 3.6 mM [20] (p = 0.049)). There were no significant difference between the mucus and luminal fluid concentrations of polar lipids in the lower half of the small intestine.

In porcine mucus from the proximal small intestine, Larhed et al. determined that lipids contributed to 37% (w/w) of the dry weight [5]. The authors further determined that 5.1% (w/w) of the total amount of lipids recovered were polar lipids, primarily consisting of lysophosphatidylcholine (2.7% (w/w)) and to a lesser degree of phosphatidylethanolamine (0.9% (w/w)), phosphatidylcholine (0.8% (w/w)), and sphingomyelin (0.7% (w/w)), whereas the remaining lipids were neutral lipids [5].

Mucus neutral lipid concentrations

The neutral lipids present in the intestinal mucus layer of rats were identified as linoleic acid, oleic acid, palmitic acid, stearic acid, and cholesterol, with linoleic acid as the main contributor to the total neutral lipid concentration (Fig. S3C). There was a steady, gradual decrease in the concentration of neutral lipids throughout the small intestine, starting at 37.8 ± 1.6 mM in the mucus of the duodenum and ending at 10.7 ± 1.1 mM in the ileum (Fig. 4E). The observed decrease in total neutral lipid concentration (Fig. 4E) seemed to be dominated primarily by the concentration of linoleic acid, as the concentrations of the other neutral lipids only had a slight decrease from ~ 5 mM in the duodenum to ~ 3 mM in the ileum (Fig. S3C).

In comparison to the luminal concentrations in the rat, the neutral lipid concentrations in the mucus layer were significantly higher than those of the lumen in the duodenum (37.8 ± 1.6 mM vs 15.1 ± 1.2 mM [20] (p < 0.0001)), proximal jejunum (30.4 ± 3.4 mM vs 11.3 ± 0.5 mM [20] (p < 0.0001)), and mid jejunum (21.7 ± 2.0 mM vs 8.3 ± 1.9 mM [20], (p = 0.0005)). Though trending towards higher values, the mucus concentrations in the ileum were not significantly higher than the luminal concentrations (10.7 ± 1.1 mM vs 4.6 ± 1.4 mM [20]).

Larhed et al. described a similarly high amount of linoleic acid in the porcine mucus layer in the proximal small intestine. Of the lipids determined by Larhed et al, the majority were designated as neutral lipids with 24% (w/w) linoleic acid, while other free fatty acids each contributed with 13-17.7% (w/w) and cholesterol with 12% of the total lipid content [5]. The composition of neutral lipids was like those of the current study in rat mucus, albeit the rat had a slightly higher ratio of linoleic acid (Fig. S3C). Overall, the ratio of neutral to polar lipids reported for pigs are higher than those determined in the current study in the rat small intestinal mucus, which could be due to higher polar lipid concentrations in the rat luminal fluids [5, 20, 36].

Relation between rheological properties and concentrations of proteins and lipids

There seems to be an integral interplay between the endogenous concentrations of proteins and the rheological properties of the small intestinal mucus layer of the rat. The observed regional differences in the rheological properties of the mucus layer coincided with the regional differences in mucus protein concentration, i.e., the first two sections of the small intestine displaying a more viscous mucus (Figs. 1, 3) were also the sections containing the highest amount of proteins (Fig. 4B). Similarly, the remaining sections that showed similar rheological profiles had simil