Abstract
Sleep restriction disrupts circadian organization and represents a behavioral condition with systemic consequences that extend beyond the central nervous system. In this study, we evaluated structural and cellular changes in the distal colon of rats exposed to sustained sleep restriction. Sixteen male Rattus norvegicus were assigned to a control group (n = 8) or a sleep-restricted group (n = 8) and subjected to a modified multiple-platform protocol for 21 days. Blood samples were collected for inflammatory assessment, and the distal colon was analyzed using histochemical and immunohistochemical approaches. Sleep-restricted rats exhibited reduced body mass gain and an increased proportion of polymorphonuclear leukocytes. In the distal colon, sleep restriction increased mucosal thickness and crypt depth, depleted goblet cells, and caused mast cell hyperplasia. It also produced quantitative and morphometric changes in neurons and enteric glial cells in the myenteric and submucosal plexuses. Notably, these alterations occurred in the absence of macroscopic mucosal lesions. Together, these findings demonstrate that sustained sleep restriction promotes coordinated remodeling of distal colonic histoarchitecture, epithelial composition, immune-related cells and enteric neuro-glial organization. Considering that sleep restriction disrupts circadian entrainment and alters behavioral patterns such as feeding, these results support the concept that intestinal remodeling represents a peripheral consequence of circadian disruption.
Keywords: Circadian disruption, Goblet cells, Mast cells, Myenteric plexus, Submucosal plexus, Neuro-glial remodeling
Highlights
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Sleep restriction lowers body mass gain and raises blood polymorphonuclear cells.
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Mucosal thickening and increased crypt depth occur without macroscopic lesions.
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Goblet cell depletion and mast cell hyperplasia alter distal colonic composition.
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Enteric neurons and glial cells show coordinated remodeling in both plexuses.
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Distal colonic changes reflect integrated sleep- and circadian-related disruption.
1. Introduction
The sleep-wake cycle underlies circadian rhythmicity, a fundamental process for maintaining psychological and biological homeostasis in mammals over an approximately 24-h period. Sleep is classically divided, based on polysomnographic criteria, into rapid eye movement (REM) and non-rapid eye movement (NREM) sleep. NREM sleep accounts for approximately 75–80% of total sleep time, whereas REM sleep represents 20–25% and is closely associated with dreaming and key cognitive processes, including learning and memory consolidation [1].
Sleep is an evolutionarily conserved behavioral state, and its disruption has consequences that extend beyond central nervous system function, influencing both physiological and systemic processes. Experimental evidence demonstrates that sleep deprivation alters behavioral responsiveness, attention and cognitive performance across different animal models [2], reflecting a broad impact on organismal function [3]. The magnitude and nature of these responses may vary with the experimental model and the specific sleep-restriction protocol. This indicates that circadian disruption produces varied but coordinated behavioral and physiological adaptations [3]. Together, these observations indicate that sleep restriction is not only a neurophysiological disturbance. It is a complex behavioral condition with effects on multiple systems [2,3].
Circadian rhythms are coordinated by a central pacemaker located in the suprachiasmatic nucleus of the hypothalamus, which integrates environmental cues to synchronize physiological processes [[4], [5], [6]]. Peripheral synchronizers also contribute to circadian regulation, with feeding behavior acting as a potent modulator of gastrointestinal motility, secretion and mucosal organization [7,8]. Disruption of sleep-wake patterns may therefore affect gastrointestinal physiology through combined alterations in central circadian control and peripheral entrainment.
Sleep deprivation and sleep restriction disrupt circadian organization and have been associated with dysfunctions of both the central nervous system (CNS) [9] and the enteric nervous system (ENS) [10]. Organized into interconnected ganglia within the myenteric and submucosal plexuses, the ENS regulates gastrointestinal motility, secretion, epithelial dynamics and immune interactions [11]. Experimental and clinical evidence indicates that stress-related states contribute to gastrointestinal disorders. These disorders are commonly associated with increased intestinal permeability and altered colonic motility. In these processes, corticotrophin-releasing factor and mucosal mast cells play key roles in neuro-immune communication [10,12,13].
In addition to its effects on stress-related pathways, sleep restriction is commonly associated with metabolic disturbances [9] and increased oxidative stress [14]. Reactive oxygen species generated during wakefulness are normally counterbalanced during sleep [15]. However, excessive accumulation of these species can induce lipid peroxidation, DNA damage and protein degradation, ultimately affecting multiple cell types [16,17], including enteric neurons [18].
Experimental models of sleep restriction have been employed to investigate gastrointestinal outcomes. Everson and Szabo reported hyperphagia accompanied by weight loss and reduced adiposity under sleep restriction, consistent with a state of energy imbalance [19]. Ibarra-Coronado et al. demonstrated an acute inflammatory response in the gastric mucosa of sleep-deprived rats, including increased eosinophils, mast cells and goblet cells in the duodenum [20]. More recently, Triplett et al. showed that chronic sleep restriction alters intestinal morphology in a region-dependent manner, increasing villus distribution in the cecum and proximal colon and crypt depth in the distal ileum [21]. Despite these advances, relatively few studies have addressed the effects of sleep restriction on colonic histoarchitecture, epithelial cellular composition and mucosal immune cells, particularly in the distal colon.
Chronic sleep disruption is known to induce neuronal alterations in the central nervous system [22]; however, its effects on the enteric nervous system under comparable experimental conditions remain poorly characterized. In particular, the extent to which sleep restriction drives structural remodeling in the distal colon, integrating epithelial, immune and neuro-glial components, is still insufficiently understood. In this study, we investigated structural and cellular changes in the distal colon of rats subjected to sustained sleep restriction, with emphasis on mucosal histoarchitecture, epithelial composition, mast cells, and enteric neuro-glial organization within the myenteric and submucosal plexuses. By addressing these components, we aimed to characterize intestinal remodeling as a peripheral outcome of sustained sleep- and circadian-related disruption during the peripubertal period. We focused on the peripubertal window (postnatal days 40–61) because the developing ENS remains plastic and susceptible to early-life environmental and metabolic challenges [23]. In addition, this same peripubertal sleep-restriction model has been shown to disturb systemic homeostasis in rats [24,25], providing a suitable window to test whether circadian- and sleep-related disruption reshapes the distal colon.
2. Material and methods
Ethical approval
All procedures were approved by the COMMISSION ON ETHICAL CONDUCT IN THE USE OF ANIMALS at State University of Londrina - UEL (CEUA/UEL no. 3467.2014.86) and were conducted and reported in accordance with the ARRIVE 2.0 guidelines (Essential 10 and Recommended items) [26].
2.1. Experimental design
Sixteen peripubertal male Wistar rats (Rattus norvegicus), aged 30 days and weighing approximately 124.7 ± 2.45 g (mean ± standard error), were used. Animals were obtained from the Central Animal Experimental Station of the State University of Londrina (UEL) and acclimatised for 10 days in the vivarium of the Laboratory of Toxicology and Metabolic Reproductive Disorders prior to the experimental period. Rats were randomly assigned to two experimental groups (n = 8 per group) and studied for 21 consecutive days, from postnatal day (PND) 40 to PND 61, as previously described [24].
Sleep restriction was induced using the modified multiple-platform method [27]. Briefly, eight rats were housed together in a water tank (140 × 60 × 30 cm) containing 20 circular platforms (6.5 cm in diameter), with the water level maintained approximately 3 cm below the upper surface of the platforms. The number of platforms exceeded the number of animals, allowing locomotion between platforms. During paradoxical (REM) sleep, muscle atonia caused animals to lose postural control and contact the water, resulting in arousal. Rats in the sleep-restricted group remained in the tank for 18 h per day, from 16:00 to 10:00 the following day. Subsequently, animals were returned to standard polypropylene cages (43 × 30 × 15 cm) for a 6-h recovery period (10:00–16:00), during which sleep was allowed, as previously reported [28]. Control animals were housed in standard cages throughout the experimental period. The 18-h window (16:00–10:00) covered the entire light phase, when rodents concentrate their rest, and extended into the early dark phase. In the modified multiple-platform method, sleep is interrupted whenever paradoxical (REM)-associated muscle atonia occurs. This restricts sleep across the whole circadian cycle, not only during the active period of the animals [27,28].
Both experimental groups had free access to standard laboratory chow and filtered water throughout the study. However, due to differences in housing conditions between groups, the present protocol does not allow complete exclusion of potential differences in feeding behaviour or feeding timing during the sleep restriction period. Body mass was recorded regularly during the experimental period. The percentage loss of total body mass was determined using the following formula: % loss of body mass = (ibm-fbm + gbm)/(ibm + gbm) x 100. Where: ibm = the initial body mass of the rats in the sleep-restricted group. fbm = the final body mass of the rats in the sleep-restricted group. gbm = indicates the mean body mass gain in the control group (adapted from Ref. [18]).
2.2. Euthanasia and sample collection
After the experimental period, the rats were weighed and then anaesthetised with halothane vapour to obtain peripheral blood via the retro-orbital plexus. Global leukocyte counting was performed in a Neubauer chamber and differential counting was performed in blood smears stained with May-Grunwald-Giemsa [29]. The rats were then euthanised by deepening the anaesthesia with halothane vapour; a vertical laparotomy was performed to remove and measure the distal colon.
2.3. Scoring of macroscopic changes in the distal colon
A 4-cm segment of the distal colon of each rat was opened at the mesocolic margin and fixed on a Styrofoam plate with the mucosa facing upward. The colonic mucosa was then washed with saline (0.9%) and examined under a stereomicroscope with transillumination (Olympus SZ 61, Tokyo, Japan) to assess macroscopic changes. The criteria used and the assigned score are shown in Table 1 [30,31].
Table 1.
Stereomicroscopic evaluation.
| Category | Criterion and Definition | Score |
|---|---|---|
| Macroscopic changes in colonic mucosa |
Absence of inflamed areas. | 0 |
| Localized hyperemia without ulceration. | 1 | |
| Linear ulcerations without significant inflammation and mild hyperemia. | 2 | |
| Ulcerations without necrosis (crusts) and 2 to 4 cm of inflammation. | 3 | |
| Crusted ulcerations, megacolon, and 2 to 4 cm of inflammation. | 4 | |
| Crusted ulcerations, megacolon, stenosis, and inflamed areas of more than 4 cm. | 5 |
2.4. Histological processing
A 1-cm ring of the distal colon of each rat was fixed in Bouin and subjected to routine histology to obtain 4 μm semi-serial cross-sections. The techniques performed and their objectives are shown in Fig. 1. Samples for the different analyses were taken from adjacent segments of the distal colon, collected sequentially from a fixed anatomical reference. The segment used for each technique is indicated in Fig. 1. The labels 1 cm, 2 cm and 3 cm denote the portions processed for histology and histochemistry and for whole-mount preparations.
Fig. 1.
Schematic representation of the analyses performed in the present study with the control group and the sleep-restricted group. Haematoxylin and eosin (HE); PAS (Periodic Acid-Schiff); AB (Alcian blue).
2.5. Histomorphometric analyses of the distal colonic wall
All subsequent analyses were performed with Image Pro-Plus 3.0.1 software (Media Cybernetics, Silver Spring, MD, USA) using images (1 image/quadrant in 4 sections) on a trinocular light microscope (Olympus BX50, Minato, Ku, Japan) coupled to a high-resolution camera (3CCD Pro series). Images taken with a 20× objective on slides stained with haematoxylin and eosin (HE) were used to measure the total wall thickness, mucosa, depth and width of the crypts. Sixty-four measurements of each parameter were taken on 4 sections of each rat along the circumference of the distal colon [29].
2.6. Quantification of type I, type III, total collagen fibres, and soluble collagen
Type I and type III collagen fibres stained with Picro Sirius Red were quantified in the submucosa and mucosa. For the quantification of collagen types, 16 images per rat were acquired with a 20× objective and a high-resolution camera (Roper Scientific Photometrics®) attached to the microscope and transferred to a microcomputer using image-pro plus software (Media Cybernetics, MD, USA). Polarizing filters (Olympus U-POT Japan) were used to measure type I and III collagen fibres (μm2). Another 16 images were acquired without polarizer to measure total fibrillar collagen (μm2) [32].
Total soluble collagen was measured in whole tissue homogenates by the Sirius Red reagent based-assay [33]. Results are expressed as μg collagen/mg wet tissue.
2.7. Quantification of intraepithelial lymphocytes, goblet cells, and mast cells
Intraepithelial lymphocytes (IELs) present in a range of 2560 consecutive epithelial cells in the mucosa of the distal colon of each rat were counted on slides stained with HE. Quantification was performed using a light microscope (in 100× objective). Results were expressed as the percentage of IELs per 100 epithelial cells [34].
Goblet cells among the 2560 epithelial cells of each rat were counted in four sections, which were then divided into four quadrants. The counting was performed under a light microscope (in 40× objective). The percentage of goblet cells/100 epithelial cells from slides stained with the techniques PAS+, AB pH 1.0+, and AB pH 2.5+ was then calculated [34].
Mast cells stained with toluidine blue were quantified using a light microscope (in 100× objective). One hundred random microscope fields were quantified for each rat, totaling 9.96 mm2 of colonic mucosa examined. Results were expressed in mm2 [29].
2.8. Morphological evaluation of the myenteric and submucosal plexuses
At the end of the experimental period, the distal colon was removed, gently rinsed with 0.1 M phosphate-buffered saline (PBS; pH 7.4), and a 3 cm segment was selected for analysis. Tissues were fixed by immersion in 4% paraformaldehyde for 3 h at room temperature, opened longitudinally along the mesenteric border, washed in PBS, and stored in PBS containing 0.08% sodium azide at 4°C.
Whole-mount preparations of the enteric plexuses were obtained under a stereomicroscope. For the myenteric plexus, the mucosal and submucosal layers were removed, preserving the longitudinal and circular muscle layers. For the submucosal plexus, the mucosa was gently scraped and the muscular tunica was dissected, leaving the submucosal intact. Preparations were mounted manually in a relaxed state, without controlled longitudinal stretching. Due to the nature of the procedure, complete standardization of tissue tension and longitudinal positioning along the distal colon could not be ensured. Whole mounts were permeabilized and blocked in PBS containing 0.5% Triton X-100, 20% donkey serum, and 2% bovine serum albumin, then incubated for 48 h with the primary antibodies listed in Table 2. After washing, tissues were incubated for 2 h with Alexa Fluor-conjugated secondary antibodies, mounted with ProLong Gold Antifade, and stored at 4°C [34,35].
Table 2.
Primary and secondary antibodies used for the immunofluorescence technique.
| Antibody | Species | Dilution | Manufacturer | Code |
|---|---|---|---|---|
| anti-HuC/HuD | Mouse | 1:400 | Molecular Probes®, Invitrogen | A21271 |
| anti-S-100β | Rabbit | 1:400 | Sigma-Aldrich, Merck | S2644 |
| Alexa Fluor® 488 anti-mouse | Donkey | 1:500 | Molecular Probes®, Invitrogen | A21202 |
| Alexa Fluor® 546 anti-rabbit | Goat | 1:500 | Molecular Probes®, Invitrogen | A11010 |
Neurons and enteric glial cells were quantified using an Olympus FSX-100 fluorescence microscope at ×20 magnification. For each animal and plexus, 32 non-overlapping fields were analyzed under identical acquisition settings. Cell counts are presented as apparent cellular density per microscopic field, and neuronal soma area was measured in 100 neurons per rat using Image-Pro Plus software [35]. Each microscopic field corresponded to a constant area of 0.2433 mm2 under the ×20 objective. Identical framing and acquisition parameters were applied to every sample. Counts are therefore directly comparable and are reported as apparent cellular density per field.
2.9. Statistical analysis
Tests were defined according to the data distribution; the D'Agostino-Pearson test checked the data distribution. Data that had a normal distribution were presented as mean ± standard error or mean ± standard deviation; data with free distribution were presented as median and interquartile range. Comparison between groups was performed with student's t-test or Mann Whitney test. The significance level was 5% (Zar JH., 1999).
3. Results
3.1. Sleep restriction conditions are associated with reduced weight gain and increased polymorphonuclear cells
Rats exposed to sleep restriction conditions gained less body mass compared with the control group (p < 0.05). With respect to circulating leukocytes, an increase of approximately 89% in polymorphonuclear cells (PMN) was observed in the sleep-restricted group relative to controls (p < 0.05; Table 3). Macroscopic examination of the distal colon revealed no visible differences between groups, and the mucosal lesion score was zero in both conditions (p > 0.05).
Table 3.
Analysis of body mass and blood leukocytes in peripubertal rats submitted to sleep restriction.
| Groups | Body mass |
Leukocytes (mm3 of blood x 1000) |
|||||
|---|---|---|---|---|---|---|---|
| Initial mass (g) | Final mass (g) | Body mass gain (%) | Body mass loss (%) | Global count | MNN | PMN | |
| CG | 124.3 ± 2.9 | 238.4 ± 4.9 | 91.7 | - | 8.7 ± 0.9 | 7.4 ± 0.8 | 1.4 ± 0.3 |
| SR | 125.1 ± 1.8 | 202.2 ± 7.4* | 61.6 * | 15.4 | 8.5 ± 2.2 | 5.8 ± 1.6 | 2.6 ± 0.7 * |
% loss of body mass = (ibm-fbm + gbm)/(ibm + gbm) x 100. Values were presented as mean ± standard error. CG: control group; SR: Sleep restriction; MNN: mononuclear; PMN: polymorphonuclear; Values were presented as mean ± standard deviation. *p < 0.05 compared to control by student's t-test.
3.2. Sleep restriction conditions are associated with histomorphometric changes in the distal colon wall
Animals subjected to sleep restriction conditions exhibited significant histomorphometric alterations in the distal colon wall. These included an increase of approximately 9% in mucosal thickness and a 32% increase in crypt depth, resulting in an overall increase in total wall thickness of the distal colon (p < 0.05; Table 4). No significant differences were observed between groups in collagen fibers or soluble collagen content in the distal colon (p > 0.05; Table 4, Supplementary Figure (Fig. S1)).
Table 4.
Histomorphometric analysis of the distal colon wall and the area occupied by collagen fibres in rats subjected to sleep restriction.
| Parameters | CG | SR |
|---|---|---|
| Total thickness (μm) | 615.6 ± 135.7 | 648.2 ± 135.5* |
| Mucosa (μm) | 362.5 ± 102.1 | 395.4 ± 99.2* |
| Crypt depth (μm) | 73.4 ± 43.5 | 97.1 ± 37.2* |
| Crypt width (μm) | 39.1 ± 12.9 | 34.7 ± 10.3 |
| Total collagen fibres (/μm2) | 125.2 ± 45.2 | 129.5 ± 44.1 |
| Collagen fibres type I (/μm2) | 43.7 ± 8.3 | 43.6 ± 16.5 |
| Collagen fibres type III (/μm2) | 22.1 ± 2.8 | 23.7 ± 3.4 |
| Soluble collagen (μg collagen/mg tissue) | 0.46 ± 0.13 | 0.55 ± 0.15 |
Values were presented as mean ± standard deviation; *p < 0.05 compared to control by student's t-test. CG: control group; SR: Sleep restriction.
3.3. Sleep restriction conditions are associated with goblet cell depletion and mast cell hyperplasia in the distal colon
Although the distribution of intraepithelial lymphocytes (IELs) was not altered, changes in epithelial cellular composition were observed in the distal colon of rats exposed to sleep restriction conditions. The proportion of goblet cells was reduced across all histochemical stains examined. Goblet cells stained with PAS, Alcian blue pH 1.0, and Alcian blue pH 2.5 decreased by 57.55%, 44.62%, and 44.78%, respectively, compared with controls (p < 0.05) (Fig. S1). In contrast, mast cell numbers in the colonic mucosa were increased by approximately 61.77% in rats exposed to sleep restriction conditions relative to controls (p < 0.05; Table 5).
Table 5.
Quantification of intraepithelial lymphocytes, goblet cells and mast cells in the distal colonic mucosa of rats subjected to sleep restriction.
| Cells | Technique | CG | SR |
|---|---|---|---|
| Intraepithelial lymphocytes (%) | HE | 11.2 ± 5.4 | 12.5 ± 5.3 |
| Goblet cells (%) | PAS | 10.6 ± 1.9 | 4.5 ± 1.4* |
| AB pH 1.0 | 6.5 ± 2.4 | 3.6 ± 1.4* | |
| AB pH 2.5 | 6.7 ± 2.3 | 3.7 ± 1.4* | |
| Mast cells (/mm2) | Toluidine blue | 468.8 ± 78.7 | 758.4 ± 178.8* |
Values were presented as mean ± standard deviation; *p < 0.05 compared to control by student's t-test. HE: haematoxylin and eosin; PAS: Periodic Acid-Schiff; AB: Alcian-blue. CG: control group; SR: Sleep restriction.
3.4. Sleep restriction conditions are associated with changes in the myenteric and submucosal plexuses of the distal colon
Quantitative and morphometric differences in neurons and enteric glial cells were observed in both the myenteric and submucosal plexuses of the distal colon in rats exposed to sleep restriction conditions.
In the myenteric plexus, the number of neurons per microscopic field increased by 19%, and the number of neurons per ganglion increased by 32% compared with the control group (p < 0.05). No significant difference was observed in the neuronal area profile between groups (p > 0.05) (Fig. 2A–C).
Fig. 2.
Morphometric and quantitative analysis of the myenteric plexus of the distal colon in the control group (CG) and the sleep-restricted group (SR). (A) Neurons per microscopic field; (B) neurons per ganglion; (C) neuronal profile area (μm2); (D) glial cells per microscopic field; (E) glial cells per ganglion; (F) glia-to-neuron ratio. (G) Representative immunofluorescence photomicrographs of the myenteric plexus (HuC/HuD (or HuC/D), S-100β and merge); the group and the marker are indicated in each image. Data are presented as mean ± standard deviation. Groups were compared using Student's t-test; *p < 0.05; **p < 0.01; ***p < 0.001 compared with CG. N = 8. Scale bar: 50 μm.
The number of enteric glial cells per microscopic field did not differ between groups; however, the number of enteric glial cells per ganglion increased by 13% in rats exposed to sleep restriction conditions (p < 0.05). Consequently, the ratio of enteric glial cells to neurons decreased by 14% in the myenteric plexus relative to controls (p < 0.05) (Fig. 2D–F).
In the submucosal plexus, the number of neurons per microscopic field increased by 35%, and the number of neurons per ganglion increased by 37% in rats exposed to sleep restriction conditions compared with controls (p < 0.05). In addition, neuronal area profile increased by 22% relative to the control group (p < 0.05) (Fig. 3A–C).
Fig. 3.
Morphometric and quantitative analysis of the submucosal plexus of the distal colon in the control group (CG) and the sleep-restricted group (SR). (A) Neurons per microscopic field; (B) neurons per ganglion; (C) neuronal profile area (μm2); (D) glial cells per microscopic field; (E) glial cells per ganglion; (F) glia-to-neuron ratio. (G) Representative immunofluorescence photomicrographs of the submucosal plexus (HuC/HuD (or HuC/D), S-100β and merge); the group and the marker are indicated in each image. Data are presented as mean ± standard deviation. Groups were compared using Student's t-test; *p < 0.05; **p < 0.01; ***p < 0.001 compared with CG. N = 8. Scale bar: 50 μm.
Regarding enteric glial cells in the submucosal plexus, the number of cells per microscopic field increased by 56%, and the number per ganglion increased by 51% in rats exposed to sleep restriction conditions (p < 0.05). The ratio of enteric glial cells to neurons in the submucosal plexus did not differ between groups (p > 0.05) (Fig. 3D–F).
4. Discussion
In the present study, rats exposed to a sleep restriction protocol exhibited systemic and distal colonic changes. Systemically, sleep-restricted animals showed reduced body mass gain and an increased proportion of polymorphonuclear cells (PMN). Locally, the distal colon showed histomorphometric remodeling (increased mucosal thickness and crypt depth), marked changes in epithelial cellular composition (goblet cell depletion) and mucosal immune-related cells (mast cell hyperplasia), together with quantitative and morphometric alterations in neurons and enteric glial cells in the myenteric and submucosal plexuses. Importantly, these findings were observed in the absence of macroscopic colonic lesions or a detectable mucosal injury score, indicating that substantial tissue-level remodeling can occur without overt gross damage.
Sleep-restricted rats gained less weight than controls, consistent with prior reports of reduced weight gain or weight loss under sleep deprivation/restriction despite increased food intake [14,19]. This dissociation between food intake and body mass shows that the effects of sustained sleep restriction are not necessarily linear. They may result from combined changes in energy expenditure, metabolic efficiency and stress-related neuroendocrine signals [14,19]. In addition, because the multiple-platform protocol imposes a distinct housing environment during a major part of the light-dark cycle, differences in feeding behavior and/or feeding timing between the sleep-restricted and control groups cannot be excluded. As feeding is a strong peripheral synchronizer of gastrointestinal circadian rhythms, this limitation must be considered when interpreting both systemic and intestinal outcomes. Therefore, the present results should be interpreted as consequences observed under sleep restriction conditions, potentially integrating sleep loss, circadian disruption and associated behavioral changes [24,25].
The increased proportion of PMN suggests a systemic shift toward innate immune activation. Sleep deprivation has been associated with reduced melatonin and imbalances in pro- and anti-inflammatory cytokines [36], and experimental sleep deprivation has been linked to inflammatory responses in the gastrointestinal mucosa [20]. In the current study, although PMN levels were elevated, no macroscopic lesions were detected in the distal colon, indicating that systemic inflammatory changes and local tissue remodeling may occur without grossly visible mucosal injury. The rise of about 89% in circulating polymorphonuclear cells may represent a state of systemic immune priming. Together with the loss of neutral and acidic mucins, which weakens the mucus barrier, this could predispose the mucosa to neutrophil infiltration if barrier function fails. No overt infiltrate was detected under the present conditions.
Histomorphometric analysis showed increased mucosal thickness and increased crypt depth in the distal colon of sleep-restricted rats. Comparable segment-specific changes have been reported in models of chronic sleep restriction, with increases in crypt depth and other morphological parameters in selected intestinal regions [21].The interpretation of these findings, however, requires caution. While increased crypt depth is often discussed in the context of altered epithelial dynamics, the present study did not directly measure proliferation, apoptosis, migration or shedding. Moreover, a simple attribution to “increased epithelial turnover due to tissue damage” is not mandatory, because increased turnover would be expected to reduce mucosal thickness if net cell loss exceeded production. Instead, the combined increase in mucosal thickness and crypt depth is compatible with alternative, non-mutually exclusive scenarios: reduced epithelial loss (decreased shedding), increased net colonocyte production with maintained senescence rates, or altered crypt histoarchitecture and packing density. Crypts are functional units driven by stem cell activity and vertical migration of progeny along the crypt axis [[37], [38], [39]], and future studies incorporating direct measurements of epithelial kinetics are needed to distinguish among these possibilities.
Collagen content (fibers I and III types and soluble collagen) did not differ between groups, suggesting that, within the timeframe of this protocol, the remodeling detected in the colonic wall was not accompanied by measurable collagen accumulation. At 21 days of sustained sleep restriction, these changes most likely reflect early, dynamic cellular and neuro-glial rearrangement rather than fibrillar collagen deposition or established fibrosis. This interpretation is consistent with evidence that intestinal extracellular-matrix remodeling can precede overt, clinically measurable disease [40], and it helps explain the preserved organ integrity and the macroscopic lesion score of zero.
A robust depletion of goblet cells was observed, affecting neutral mucins (PAS+) and acidic mucins (Alcian blue at pH 1.0 and pH 2.5). Goblet cells are central for mucin production and mucus barrier formation [41], and changes in mucin-producing populations can have functional consequences for barrier integrity and mucosal ecology. Previous work reported different goblet cell responses under sleep deprivation[20], which supports the view that epithelial responses may be segment-dependent. In the present study, goblet cell depletion occurred without macroscopic lesions, indicating that epithelial composition can shift substantially before gross damage becomes evident. The concurrent increase in crypt depth and depletion of histochemically detectable goblet cells may reflect two non-mutually exclusive mechanisms. The first is a shift in epithelial lineage differentiation along the expanded crypt axis. The second is accelerated discharge of mucin granules driven by the co-occurring mast-cell hyperplasia; its mediators, such as histamine and proteases, can promote goblet-cell degranulation [42].
In parallel, mast cell numbers increased markedly in the colonic mucosa. Mast cells are strategically positioned at mucosal interfaces and contribute to immune surveillance, inflammation and neuro-immune communication [42,43]. Similar mast cell expansion has been reported in the duodenum under sleep restriction [20], supporting the notion that mast cells are responsive to stress-related physiological states. Mast cells can release mediators relevant to mucosal homeostasis and sensory-motor regulation, including histamine, heparin and serotonin (5-HT) [42]. Psychological stress has been linked to mast cell activation and increased intestinal permeability, including pathways involving corticotrophin-releasing factor [13], and has been associated with symptom exacerbation in functional and inflammatory bowel disorders [[44], [45], [46]]. While the present study does not assess mediator release or permeability directly, the observed mast cell hyperplasia, combined with epithelial compositional changes, is consistent with a shift toward a mucosal milieu with altered barrier and immune features. The multiple-platform environment also engages neuroendocrine stress pathways. Activation of the hypothalamic-pituitary-adrenal axis and release of corticotrophin-releasing factor and corticosterone can induce mucosal mast-cell hyperplasia, degranulation and increased epithelial permeability, independently of sleep loss [13,46]. The present design cannot fully separate sleep restriction from this stress component. We therefore regard it as an intrinsic feature of the model rather than a separable variable.
Considering the neuronal-glial-epithelial unit [47] and functional coupling between mucosal immune cells and enteric nerve fibers [10,43,48], we examined neurons (HuC/HuD) and enteric glial cells (S-100β) in the myenteric and submucosal plexuses. We observed increases in neuronal counts per microscopic field and per ganglion in both plexuses, together with plexus-specific changes in glial counts and glia-to-neuron ratios. These results indicate substantial remodeling of enteric neural and glial architecture under sleep restriction conditions. A comparable sensitivity of the enteric glial compartment to early-life challenges has been described in a rat model. In that model, a perinatal high-fat diet increased myenteric glial density in the upper gastrointestinal tract and reduced nitrergic and total myenteric neurons, before the onset of obesity or systemic inflammation [23]. The shared feature is an early rise in enteric glial density that precedes overt disease. The neuronal direction, however, was opposite: an apparent increase in the distal colon in the present study, and neuronal loss in the upper gut in that model. This indicates that enteric neuro-glial remodeling is stressor- and region-specific, and it supports interpreting our neuronal findings as changes in apparent density and ganglionic composition rather than as absolute neuronal gain.
However, interpretation of these quantitative outcomes must remain aligned with the methodological constraints inherent to whole-mount preparations. Because whole mounts are sensitive to tissue tension, stretching and regional sampling along the longitudinal axis, the present results should be interpreted primarily as changes in apparent cellular density and ganglionic cellular composition rather than definitive evidence of absolute changes in total neuron or glial numbers. Moreover, the study does not evaluate proliferation, differentiation, apoptosis, or neuronal subtype composition. We suggest that sleep restriction conditions were associated with marked enteric neuro-glial remodeling that plausibly relates to the observed epithelial and immune changes, but the directionality, mediators and functional consequences require targeted investigation. In particular, the per-ganglion measure is less sensitive to longitudinal stretching than density per unit area, and it showed the same direction of change. A slight macroscopic contraction of the sleep-restricted colon, which is plausible under sympathetic activation, could still contribute to the higher apparent densities. For this reason, absolute neuron or glial numbers were not claimed.
The present findings therefore support the concept that sustained sleep restriction conditions, likely involving combined sleep loss, circadian misalignment and behavioral changes, are associated with systemic immune shifts and segment-specific remodeling of distal colonic histoarchitecture, epithelial composition and enteric neuro-glial organization.
Finally, rats exposed to sleep restriction exhibited systemic and distal colonic alterations. Systemically, reduced body mass gain and increased polymorphonuclear cells were observed. At the local level, the distal colon underwent coordinated remodeling involving mucosal architecture, epithelial cellular composition, immune-related cells and enteric neuro-glial elements. Together, these findings demonstrate that sustained sleep restriction during the peripubertal period induces integrated changes in body development, inflammatory profile and intestinal histoarchitecture. Importantly, these alterations support the concept that intestinal remodeling represents a peripheral consequence of circadian disruption. These results reinforce the close interplay between sleep, circadian regulation and gastrointestinal organization, providing a structural basis for understanding how sleep-related disturbances impact peripheral systems.
The main contribution of this study is to show that sustained peripubertal sleep restriction is associated with epithelial, immune, and enteric neuro-glial remodeling in the distal colon, even in the absence of macroscopic lesions. These findings support the colon as a peripheral target of sleep and circadian disruption. However, some limitations should be acknowledged. First, the use of a single peripubertal age window limits extrapolation to adult animals. Second, the multiple-platform model combines sleep restriction with environmental stress. Finally, complementary biochemical, immunological, and functional analyses, as well as the assessment of neuronal and glial subpopulations, were not included in the present study and will be important to confirm and expand these findings.
CRediT authorship contribution statement
Adriano Araujo Ferreira: Methodology, Investigation, Funding acquisition, Formal analysis, Data curation. Maria Jose Pastre: Methodology, Investigation. Aline Rosa Trevizan: Methodology. Lainy Leiny de Lima: Methodology. Amanda Gubert Alves dos Santos: Methodology. Gessilda Alcantara Nogueira-Melo: Methodology, Investigation. Gláucia Eloisa Munhoz de Lion Siervo: Methodology, Investigation, Data curation, Conceptualization. Glaura Scantamburlo Alves Fernandes: Writing – original draft, Visualization, Validation, Data curation, Conceptualization. Marcelo Biondaro Gois: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Conceptualization. Débora de Mello Gonçales Sant’Ana: Writing – review & editing, Writing – original draft, Supervision, Conceptualization.
Data availability
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors for its execution. However, the authors Pastre, M.J.; de Lima, L.L.; dos Santos, A.G.A.; and Lion-Siervo, G.E.M. were supported by fellowships from the Coordination for the Improvement of Higher Education Personnel (CAPES, Code 001) and the National Council for Scientific and Technological Development (CNPq), Brazil.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors are grateful for the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES 001), the National Council for Scientific and Technological Development (CNPq 445631/2024-4, Pró-Amazônia, coordinated by the corresponding author), the Graduate Program in Biosciences and Pathophysiology, the Department of Morphological Sciences and the neurogastroenterology research group at the State University of Maringá and the Department of General Biology at the State University of Londrina.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrep.2026.102812.
Appendix A. Supplementary data
The following is the Supplementary data to this article.
Data availability
Data will be made available on request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Data will be made available on request.