Hindlimb Unloading in Rodents Induces Gastrointestinal Inflammation. Running head: Simulated Microgravity in Rodents Induces GI Inflammation

Hindlimb Unloading in Rodents Induces Gastrointestinal Inflammation. 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Running head: Simulated Microgravity in Rodents Induces GI Inflammation S. Anand Narayanan, Ramon Boudreux, Susan Bloomfield, Harry Hogan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8475703/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract The hindlimb unloading (HU) rodent model is used to simulate changes incurred in spaceflight due to microgravity. We have previously shown 2 weeks of rat HU impairs lymphatic function systemically ( 42 ). We also previously demonstrated diminished vasoconstrictor responses of both mesenteric arteries and veins ( 8 ). However, gut biological adaptations in response to spaceflight have been minimally investigated, a concern given gut biology’s involvement in maintaining overall biological health as well as dietary absorption; moreover, intestinal inflammation and gut immunological alterations have not been systemically measured in microgravity models before. Recently we characterized and observed rat gastrointestinal (GI) inflammation and mesenteric lymphatic vessel associated immune cells after 4 weeks of HU. Body weight and food intake differences were notable, with HU animals weighing less as well as having increased daily food intake ( p < 0.05 ). Colon histopathology indicated elevated damage in HU compared to controls ( p < 0.05 ). Fecal calprotectin (a clinical IBD marker of GI inflammation) was significantly increased at 2-weeks of HU and trended towards elevation at 4-weeks. Furthermore, we noted shifts in innate immune cell populations (CD163 + and MHCII + ) localized with mesenteric lymphatics. CD163 + cells increased in both numbers and localization with lymphatics after 4 weeks of HU ( p < 0.05 ). Conversely MHCII + immune cells were reduced in both total number and their association with lymphatics in HU, suggesting altered antigen presentation capacity ( p < 0.05) . These patterns are similar to our observations in models of gut inflammation. These findings may provide insight with adaptations astronauts may experience related with immune dysregulation, nutrient malabsorption, and GI adaptations. These observations also present an unrecognized inflammatory stress response that may explain physiological adaptations occurring between HU and space-flown rodent studies. Health sciences/Gastroenterology Biological sciences/Immunology Biological sciences/Physiology simulated microgravity lymphatics digestive system inflammation spaceflight analogues Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Spaceflight exerts multiple environmental stresses upon astronauts causing short- and long-term physiological adaptations that may interfere with the ability of crewmembers to perform in space for long durations ( 10 ). The physiological adaptations that occur resemble an accelerated aging phenotype that include fluid compartment shifts, lower body disuse, and aberrant catabolic processes ( 10 ). Ground-based models enable studying spaceflight adaptations by providing isolated, controllable environments for experiments that may not be feasible to do in space due to limited space, availability and cost ( 10 ). The hindlimb unloading (HU) rodent model is used to simulate microgravity-induced changes, though there are different adaptations with this model compared with space-flown animal data ( 78 , 79 ). Bone and muscle exhibit mechanosensitivity, with microgravity exposure leading to musculoskeletal atrophy and deconditioning ( 11 , 52 , 78 , 79 , 123 ). Microgravity-induced redistribution of pressures and flows across and within the cardiovascular system also cause changes with cardiovascular structure and function. Microgravity exposure leads to diminished venous pressures, baroreflex response, plasma volume, stroke volumes, while causing elevated oxygen uptake, heart rates, and ejection fractions leading to post-flight orthostatic intolerance ( 10 , 23 ). Extensive investigations of the effects of microgravity on other physiological processes and organ systems such as endocrine, renal, pulmonary, reproductive, etc. have also been investigated, highlighting how spaceflight leads to several systemic adaptations ( 10 ). Although it is known that the immune system plays an intrinsic role in the metabolic and physiological processes for of all these systems ( 72 , 75 , 82 , 101 – 104 ), it has not been considered to be a contributing factor to these physiological adaptations in the context of space physiology. Moreover, the HU model has been shown to induce skeletal muscle ( 33 , 94 , 111 ), bone ( 43 , 48 , 53 , 66 , 68 , 81 , 98 , 109 , 112 ), liver ( 90 , 100 ), and vascular ( 30 , 61 , 74 , 93 ) inflammation. In these contexts, investigators have looked at the inflammatory response in tissue beds to compare with what occurs in exercise, oxidative stress, long-term bed-rest, or other scenarios. However, the implications of HU-induced inflammation on bone, muscle, or other parenchymal tissues in the context of space life sciences has not been studied in great detail; thus, we aimed to investigate these adaptations as they relate with simulated microgravity induced-adaptations. Moreover, immunological alterations have been shown to occur in both space-flown and HU animals. Spaceflight induces altered cytokine production patterns, natural killer cell function, leukocyte distributions, monocyte/granulocyte function, T cell intracellular signaling, neuroendocrine responses, and leukocyte proliferation ( 3 , 15 , 16 , 24 , 26 , 27 , 45 – 47 , 57 – 59 , 60 , 64 , 69 , 76 , 82 , 86 , 99 , 101 – 104 ). The changes in the immune system and response with the HU model are different, observed to have a different response when compared to the spaceflight adaptations ( 78 , 79 ). Considering the complexity of immune dysfunction in both models, understanding the similarities and distinctions in the mechanisms involved may provide further insight into the immunological changes occurring in manned spaceflight as well as the immunological roles it plays in other spaceflight physiological adaptations. The lymphatics are part of the cardiovascular system comprising a network of vessels that act as the main transport path of fluid and other elements (proteins, antigens, cytokines, chemokines, immune cells, macromolecules) constituting lymph from the parenchymal tissues to the nodes via the afferent lymphatics and from the nodes back to the venous blood via the efferent lymphatics. The lymphatic pathway has a critical function in maintaining immunological function and response, and disruptions in this balance can lead to immune dysfunction and inflammation both locally and systemically. It is known that the lymphatic vasculature has altered functionality in response to HU, with systemically diminished capability for the lymphatics to transport fluid and immune cells ( 42 ). Furthermore, it is also known that intestinal inflammation and lymphatic dysfunction are tied together ( 23 , 26 , 125 ). Spaceflight experiments characterizing the lymphatics response in microgravity have yet to be accomplished. Chronic immune dysregulation has many associated pathologies, one of the most prominent being intestinal inflammation and inflammatory bowel disease (IBD). IBD is a broad-range of pathologies associated with chronic inflammation along any or all parts of the digestive tract. IBD pathogenesis has not been fully elucidated, but both genetic and lifestyle/environmental factors such as stress, increase the risk for developing IBD ( 2 , 22 , 35 , 37 , 38 , 57 , 62 , 77 , 84 , 117 ). Many inflammatory gastrointestinal (GI) pathologies are associated with other complications such as malnutrition, increased risks for colon cancer, bowel obstruction, ulcers, etc. It is also known that the lymphatic system is dysfunctional in IBD and is a major contributing factor of the pathology ( 2 , 23 , 26 , 54 , 57 , 89 , 117 , 118 ). Characterizations of the impact of spaceflight on GI changes in astronauts are predominantly anecdotal and quite limited ( 99 , 126 ). However, it is known that astronauts experience reduced appetites, decreased body weights, and negative energy balance with reduced dietary intake and other nutritional perturbations ( 76 , 99 , 126 ). With regards to animal data, it is known from limited HU studies that there appears to be GI structural changes as well as increased bacterial translocation ( 18 , 123 ). It is plausible these changes may occur due to lymphatic dysfunction, given its immunological regulatory role and the known HU-induced lymphatic dysfunction ( 13 , 42 ). Therefore the aim of this study was to determine if HU leads to development of inflammatory changes of the GI tract. Disease activity indices used in IBD models were measured and the gut tissue histopathology was analyzed to determine any HU-induced changes in GI structure. We observed a unique pathological response in HU animals that resembles stress-induced ileitis/colitis in both disease indices and histopathology. Fecal calprotectin levels, a clinical IBD marker, was also quantified in the HU animals. These data showed a significant elevation of fecal calprotectin compared to control animals, which corroborated the other disease activity indices measured. Because there is an intertwined relationship between lymphatic function and immunological responses, we also characterized the distribution of immune cell sub-populations associated with lymphatic vessels in rat mesenteric tissues after simulated microgravity exposure. Our findings provide evidence that HU induces an inflammatory phenotype in the gut analogous to what is seen in models of IBD. Methods Animals: Twelve adult male Sprague–Dawley rats (6 months old, ~ 400 g) were obtained from Harlan (Houston, TX) and individually housed in a climate-controlled room (23 +/- 2°C) with a 12-h light (0600–1800)–12-h dark cycle (1800–0600) in an animal care facility accredited by the AAALAC. Rats were provided standard rodent chow (Harlan Teklad 8604) and water ad libitum and assigned to groups by body mass (normalized distribution) to age-matched cage control and HU groups (n = 6 per group). HU animals underwent 28 days of hindlimb suspension, after which all animals were euthanized under anesthesia (Ketamine/DexaDomitor 3:2 cocktail, 0.3 mL per animal) and tissues harvested. HU animals were anesthetized before removal from tail suspension to prevent any weight bearing by the hindlimbs. At necropsy, the whole gut loop (duodenum, jejunum, ileum, mesentery, caecum, colon) was excised, washed briefly in PBS, and processed. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Texas A&M University. Hindlimb unloading: HU was achieved by tail suspension as previously described ( 78 , 79 ). Briefly the animal is suspended by a harness attached to the tail to remove weight-bearing loads from the hindlimbs of the animal. While the rat was under anesthesia (Atropine, 0.3 mL per animal, Ketamine/Dexdomitor 3:2 cocktail, 0.3 mL per animal), the tail was cleaned and dried thoroughly. A thin layer of adhesive (Amazing Goop, Eclectic Products, Los Angeles, CA, USA) was applied to the tail along the medial and lateral sides. A standard porous tape (Kendall, Mansfield, MA, USA), harness was pressed firmly to the glue and allowed to dry (~ 30 min). A paper clip was used to attach the animals tail harness to a swivel apparatus on a rod spanning the top of a 45x45x45 cm cage. The heights of the animal hindquarters were adjusted to prevent any contact of the hindlimbs with the cage floor, resulting in approximately a 30° head-down tilt. The forelimbs of the animal maintained contact with the cage floor allowing the rat full access to the entire cage. All animals were monitored twice daily for health, including assessment of tail integrity. Observations: Rodents were assessed daily throughout the 28 day HU time frame for various physical symptoms of stressed behavior (porphyrin secretion, raised fur, jittery, aggravated easily if touched, testicles withdrawn), as well as symptoms of GI-associated pathology (yellow, greasy rectal discharge/dyslipidemia, diarrhea, rectal prolapse and damage). This was complemented by daily measures of food intake and weekly of weight and fecal occult (ColoScreen®) scoring of disease activity indices (DAI). Occult blood scoring was based on the appearance of the stool and the degree of reactivity of the fecal blood test as follows; 0 = no color change and normal looking stool, 1 = faint blue streaking and normal looking stool, 2 = strong evenly distributed blue coloration and dark stool, 3 = strong very dark blue reaction and dark/black tar-like stool, 4 = frank red blood on surface of stool and near liquid black stool. At euthanasia the GI tract was evaluated for inflammatory injury including vascular hyperplasia, patchy injury, fibrosis, blood in the intestine, transmural injuries and perforations of the gut wall. Fecal Calprotectin Analysis: Fecal quantities between 1–5 grams were collected at baseline, 2, and 4 four weeks of the study from both cage controls and HU animals. Directly after collection, samples were stored at -80°C. Calprotectin in fecal extracts were analyzed using the S100A8/S100A9 Calprotectin ELISA kit (30-6936, ALPCO, Salem, NH). The technique for processing, running, and analyzing the assay and results were performed following manufacturer guidelines. Histopathology: Upon removing rat small and large intestinal loops, sections were cut from the terminal ileum and colon that were flushed of fecal matter, washed in DPBS and fixed in 4% paraformaldehyde for 2 hours at room temperature, washed in PBS briefly, and then subsequently placed in 70% ethanol to dehydrate overnight in 4°C. Sections were then further dehydrated via TAMU-HSC’s Shared Core Facilities Thermo-Scientific STP 120 Spin Tissue Processor, paraffinized via a Thermo Shandon Histocenter 3 Embedding tool, sectioned (6 µm) via microtome, and adhered to positively charged glass slides for staining via Hematoxylin and Eosin (H&E) staining solutions. Scoring was performed by trained investigators on a 0–4 scale (0 being normal, and 4 being severe damage or alteration) for epithelial cell loss, crypt loss, edema, and cellularity. Immunohistochemistry: The mesenteric loop was pinned out into Sylgard ® 184 silicone coated glass petri dishes submerged in PBS. The tissues were washed several times with PBS to remove excess debris and blood. Loops were subsequently fixed in 4% paraformaldehyde for 2 hours at room temperature, washed again in PBS, and then permeabilized with 0.01% Triton-X 100, PBS, at room temperature for 1hr. At this point, the mesenteric arcades were cut from the gut wall and blocked in 5% goat serum for 2 hours, after which tissues were incubated overnight in primary antibodies with either Mouse Anti-Rat CD163 (1:200 AbD Serotec MCA342GA) or Mouse Anti-Rat Major Histocompatibility Complex Class II (MHCII, 1:200, Santa Cruz, sc-53721). Appropriate secondary antibodies for MHCII (Alexa Fluor 488 Goat Anti-Mouse IgG2a, A-21131) and CD163 (Alexa Fluor 488 Goat Anti-Mouse IgG1, A-21121) were applied both at 1:200 for 2 hours at room temperature in the dark, and tissues then mounted on 25X60mm glass coverslips using ProLong Gold Antifade Reagent. Lymphatic vessels were determined by morphology (thin vessel wall, absence of red blood cells) and the presence of unique leaflet valves. Images were taken using confocal microscopy (Leica, AOBPS) at 20x magnification, and ImageJ64 v.1.48u was used to determine the distribution of immune cell populations from confocal average projections. Cells were considered to be interacting with a lymphatic vessel if they were within a 10um range or residing on/in the vessel. Maximal intensity projections representative of the average data are shown. Statistical Analysis : Statistical analyses were carried out with SAS JMP v9 for Mac OS X. All data were analyzed first by 1-way analysis of variance (ANOVA) with appropriate post-hoc Dunnett’s t-test to analyze differences between groups. Regression analyses were performed on fecal occult blood and calprotectin measures. The significance level was set at p < 0.05. Data are presented as means ± SE. Results Calprotectin Assay: A significant increase of fecal calprotectin levels were seen in HU animals compared to control animals at the two week time-point (199.4 ± 18.3 \(\:\frac{ng}{mL}\) 2-week HU vs. 91.7 ± 18.1 \(\:\frac{ng}{mL}\) , control). At the four week time-point, calprotectin levels in HU were much higher than the levels at two weeks of HU (784.73 ± 269.0 \(\:\frac{ng}{mL}\) ), but were not statistically different due to the high variability in the data (Fig. 1 ). Furthermore the HU calprotectin levels at 2 and 4-weeks were both elevated in comparison to their baseline values. We further characterized by regression the calprotectin levels over time with statistical differences seen in HU versus control (R 2 = 0.264, p < 0.0087, slopes: 0.0635 in control versus 0.395 in HU over time) providing support for calprotectin being a potential marker of GI inflammation in the HU model (Fig. 1 ). Figure 1 summarizes the calprotectin assay results. Histopathology: A significant increase in intestinal epithelial damage, crypt loss, and cellularity with minimal accounts of edema occurred in HU animals versus age-matched cage control (Fig. 1 ). Notably in the HU animal colons, significant portions exhibited increases in cellularity so severe that granulomas and/or tertiary lymphoid organs were commonly prevalent (Fig. 1 C and D). Regions in the HU animals’ colon showed degradation and/or complete erosion of the intestinal epithelial layer as well as significant loss of the lamina propria, with segments of colon having a perforated mucosal layer (Fig. 1 C and D). A few age matched control animals also experienced elevated histopathological scores specifically for epithelial damage and cellularity; this is most likely indicative of an isolation-induced stress as animals were single-housed for an extended period of time. The animal facility veterinarians found no signs of pathogens in sentinel control rats housed in the same room as these rats. Figure 1 summarizes the histopathology analysis. Gross Anatomical and Pathophysiological Observations: Rats experienced various physical symptoms immediately after HU with > 50% displaying porphyrin secretion around the eyes as well as stressed behavior (raised fur, jittery, aggravated easily if touched, testicles withdrawn) [Fig. 2 ]. Other symptoms developed later during suspension such as a yellow, greasy rectal discharge and weight loss suggest a failure of the intestine to absorb lipid content of the diet (Fig. 2 , 3 , 4 ). There were cases of bloody urination and diarrhea, which suggested gross inflammation was occurring. It took approximately 7–10 days for stress-related symptoms (raised hair, etc.) to abate; however there appeared to be an increase in the animals displaying yellow rectal discharge to almost 100% of the rats by day 10 (Fig. 2 , 3 ). Around day 10 rodent stools became a mixture of brown/blackish (tar-like consistency) material, suggesting increased blood stool content. At two weeks of HU, animals developed inflammation of the rectum (prolapse) in ~ 50% of the rats and all had brown/black stool and significant hair loss (Fig. 2 , 3 ). After three weeks of suspension, there were several cases (~ 25%) of severe inflammation around the rectum, with significant erythema and further development of rectal prolapse and alopecia (Fig. 2 , 3 ). During the fourth and final week, ~ 50% of the rats experienced significant hair-loss around the rectum, with erythema around the rectum, increased nociception, and rectal prolapse and apparent reduced defecation with associated constipation (based on general stool quantity and observation) [Fig. 2 , 3 ]. Disease Activity Index: Increased gross indicators of GI inflammation were accompanied by significant weight loss in HU animals, despite their increased food consumption (Fig. 3 B and D). The weight loss was not due to differential starting body weights and suggests a difference in either nutritional or metabolic balance from the diet due to HU (Fig. 3 ). Furthermore, during tissue harvest, it was observed HU animals showed depletion of mesenteric fat stores, unique from what is typically seen in IBD, and gross signs of inflammation in the GI tract including increased vascular hyperplasia and networking and branching, patchy injury and blood in the intestine (Fig. 3 A). There were full transmural injuries as detected by near and complete perforations of the gut (Fig. 3 A). The injury does seem to self-limit to gut areas of high bacterial load (ileum, caecum, colon). These data matched temporally with the occurrence of increased fecal occult blood scoring and explained the observation of the development of blackish tar-like stools (Fig. 3 C). Immunohistochemistry: A shift in localization and activation of the mesenteric lymphatic associated immune cell populations was seen in HU animals compared to control. The number of CD163 + macrophages per 20X frame increased after HU (Fig. 4 A-C). Localization of CD163 + macrophage shifted from a normal distribution across the lymphatic vessel and associated mesentery to localization near a lymphatic vessel, leading us to believe that normally anti-inflammatory CD163 + cells either cannot suppress the inflammation occurring or have switched to an alternate activation state (Fig. 4 CD163 images and A-C). Activation seems plausible as the number of cells interacting with lymphatic vessels and their projections increased supporting a change towards this phenotype (investigator’s observations). There was an exceptional increase in total CD163 + cells and of cells in association with lymphatic vessels (Fig. 4 A-C). Additionally, the numbers of MHCII + macrophages that reside near or integrated into the wall of the lymphatics declined, suggesting either decreased expression of MHCII + on cells or decreased number of MHCII + expressing antigen presentation cells (Fig. 4 D-F). This has consequences not only for the immunological state of the rodents, but also for lymphatic function as the immune cell population near the vessel can alter lymphatic functionality and remodeling processes, drastically adapting the tissue environment. Discussion Though the HU model has been used to simulate the effects of microgravity for studies of various tissue compartments (bone, muscle, cardiovascular, etc.) and immunological alterations (lymph node, thymus, spleen), the effects of HU on the GI system have not been extensively studied. We systematically examined the gut for the appearance of gross inflammation. While we are not the first to observe changes in the GI tract of HU animals ( 18 , 123 ), we are the first to approach the GI inflammation seen in HU in the context of the pathogenesis of a disease. We found that the ileum, colon, and mesenteric tissue exhibit patterns of inflammation akin to rodent models of IBD ( 23 , 35 , 37 , 38 , 62 , 67 , 77 , 84 , 117 ). This may provide context for the impaired lymphatic function we previously reported, as well as the immunological alterations seen systemically in this model ( 42 ). The critical finding of this study was the presence of GI tissue damage and inflammation, including significant loss of epithelial continuity and crypt structure, as well as immune cell invasion and edema (Fig. 1 ). This is comparable to what we have seen during spaceflight, where we observed changes in the gut microstructure and immune status. Through this study, we have been able to identify the specifics of these microgravity driven adaptations, in particular identifying immune versus fluid shift driven adaptations. Additionally, there were significant shifts in the peri-lymphatic immune cell populations of the ileal mesentery that may provide context to the lymphatic dysfunction seen previously in this model ( 42 ) (Fig. 5). Intestinal injury indices included elevated histopathology scores, gross signs of inflammation around the rectum, weight loss, significant fecal occult blood scores, and elevated fecal calprotectin levels (Fig. 1 – 4 ). These metrics in the HU animals were analogous to inflammatory progression in classic IBD models ( 23 , 35 , 37 , 38 , 62 , 67 , 77 , 84 , 117 ). Histopathological analysis showed clear signs of intestinal epithelial and lamina propria loss of integrity, disruption of the colonic mucosal barrier, and increased immune cell infiltrates that formed granulomas (Fig. 1 ). The intestinal epithelial barrier is critical in preventing bacterial translocation, maintaining colonic function, and maintaining immunological balance; a breach in this barrier will cause significant GI functional disruption. Granulomas were shown to breach into the lamina propria and epithelial layers. This type of injury has significant implications on GI function. Slowing of intestinal motility and a prolongation of colonic transit time has been observed in acute (2-day) HU in rats, utilizing acetaminophen as a probe ( 40 ). The context of these observations under more long-term HU is not known, but combined with the profound degree of damage seen from our observations, the potential GI functional alterations may be significant and warrant further investigation.This is relevant as astronauts have been shown to develop gut permeability adaptations, though the mechanisms for this are unknown ( 126 , 128 , 129 ). Fecal calprotectin analysis provided strong objective corroboration of our histological and gross observations of GI inflammation (Fig. 1 ). Calprotectin is an abundant neutrophilic and monocytic protein that is released upon activation, and is elevated in both plasma and stool during intestinal inflammation ( 32 , 74 , 80 ). The fecal calprotectin assay is a clinical diagnostic marker for IBD, and provides clear evidence of GI inflammation in the rodents due to HU ( 29 , 62 , 67 ). Calprotectin levels were increased in HU animals at 2- and 4-weeks compared to control animals and calprotectin levels trended to increase over time of HU (Fig. 1 ). This indicates that GI inflammation was increasing in severity with time. Further analysis of this model is needed to determine if this inflammation will resolve if the animals are allowed to recover from HU, or if the inflammation becomes chronic or recurring. The number of GI investigations in the HU model is limited. One study using female ICR mice demonstrated that breaks in the terminal ileal epithelium, and an accumulation of E. coli lipopolysaccharide (LPS) in the ileal subepithelial region, after 4 days of HU ( 4 ). A chronic HU investigation using male Wistar rats suspended for 14- and 21-days found that expression and localization of the junctional proteins occludin and Zonula occludins-1 in the small intestinal mucosa were reduced ( 9 ). Indirect measures of intestinal permeability in those rats (serum diamine oxidase and D-lactate levels) increased in a time dependent manner. Other investigations have shown portal endotoxemia-induced liver damage by chronic HU with elevated circulating LBP, which is a morbidity seen in GI pathologies such as IBD ( 90 , 91 ). In our HU animals, the digestive tracts showed signs of vascular congestion, swelling, and injury along the gut wall (Fig. 4 ). There were also blood vascular changes in the associated mesentery (vascular hyperplasia and increased vessel networking). It has been shown that Wistar rats experiencing 15 days of HU had increased mesenteric vascular bed blood flow ( 30 ). Accompanying the elevated mesenteric arterial blood flow, protein levels of eNOS and iNOS were decreased in the mesenteric arterial vasculature. Thus, despite an impairment in NO synthesis, GI blood flow is elevated [6, 72, 74]. The venous side of the gut blood circulation has also been shown to be affected by HU. Isolated HU small mesenteric veins (21 days in Sprague-Dawley) are less responsive to norepinephrine ( 8 , 34 ). This adrenergic hyporesponsiveness occurs in a vascular bed where blood flow and arterial pressure changes should be minimal based on the hydrodynamic alterations but could be associated with changes in the natriuretic proteins observed in HU. Previously, we have shown elevations in serum atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) during HU, which are both known to also increase blood and lymphatic vessel permeability ( 50 , 97 ). The lymphatic vasculature appears to be impacted by HU in a manner that is not entirely dependent on the altered gravitational forces acting on the body, as suggested by the systemic impairment in cervical, thoracic, mesenteric, and femoral lymphatics ( 49 ). Two-week HU decreased stretch-activated myogenic stimulation and altered flow-mediated inhibition in all of the active lymph pumps to differing degrees regionally, but not in a manner consistent with changes in the hydrodynamic conditions ( 49 ). This suggests broad vascular adaptations in both the arteriovenous and lymphatic systems. We found that the normal complement of immune cells associated with the lymphatics was altered by HU. We have shown that lymphatic-associated immune cells can alter lymphatic transport function, this may explain both our previous findings and play a part in the phenomenon we are reporting here ( 13 , 17 , 22 , 23 ). However, the shift in the immune cell populations reported here differs from what we found in a rodent IBD model ( 23 ). In the IBD model there was a dramatic increase in the number of MHCII + immune cells around the vessels. Here we see a reduction in the number of MHCII + cells and an increase in the number of CD163 + cells (Fig. 5). These cells appear to be innate immune cells, presumably macrophages. CD163 + macrophages are generally classified as alternatively activated, and maintain tissue homeostasis by clearance and removal of cellular debris during remodeling ( 36 , 73 ). MHCII + antigen presenting cells have been previously shown to reside within the prenodal lymphatic wall in great abundance and possess morphology with extensive cell extensions similar to the interdigitating dendritic cells of the lymph node ( 13 , 23 ). The reduction of the relative abundance of these cells associated with the lymphatics after HU suggests that there may be impaired antigen presentation by this sub-population of immune cells, which may result in a reduced or dysfunctional immune responses to events in the gut. High numbers of CD163 + cells were present in mesenteric tissue of HU animals and associated to a higher degree with lymphatic vessels of HU animals, suggesting there is chronic remodeling and inflammation in the mesentery, centered around the lymphatic vessels. This is similar to findings in the mucosa of IBD-patients, which show high numbers of CD163 + macrophages, contributing to the resolution of inflammation and injury ( 116 ). Immune dysregulation has been observed by some to be associated with HU ( 4 , 5 , 6 , 9 , 33 , 39 , 69 , 120 ). In addition, some studies examining the effect of HU on the response to a pathogen challenge has shown that HU animals experienced compromised resistance to pathogens and delays in immunoglobulin production ( 4 , 5 , 6 , 9 , 33 , 39 , 69 , 120 )). Others have shown enhanced innate immune responses, but compromised adaptive immunity. It is uncertain if there is a global immune dysfunction, if there are specific immunological components that are impaired, or if HU animals are more susceptible to pathogens ( 4 , 9 , 39 , 69 ). Impaired lymphatic function could contribute to an immunological dysregulation due to reduced trafficking of antigens, cytokines, and antigen presenting cells from the parenchyma to the lymph node ( 13 ).There have been reports of changes with the astronaut microbiome, of which, these observations may explain for, in terms of changes with the gut and immune structure and function ( 126 – 129 ). While this is the first report of gross GI inflammation and injury in the HU model, there is even less known about this phenomenon in space flight. Enhancing our understanding of GI adaptations to the space flight environment, especially given the paucity of data available, is paramount since it is the site for internalization and incorporation of food, water, and nutrients. The documented immunological shifts after long-duration spaceflight are consistent with the possible development of inflammation comparable to what we report here ( 24 ). This is critical to assess in animal models so that we can appropriately translate findings to spaceflight adaptations and risks that astronauts will face during long-term missions. Therefore, it is necessary to carefully compare the effects of HU and spaceflight on the immunology and physiology of the digestive tract, to determine if the changes we have seen here in the HU model are also occurring in space. Declarations Author Contribution S. A. Narayanan and W. Cromer conceptualized the work, collected data, analyzed the data, and drafted and revised the manuscript. R. Boudreux supervised the study. S. A. Bloomfield, D. C. Zawieja, H. Hogan, interpreted the data and revised the manuscript; and all authors reviewed and approved the final version of the manuscript. Acknowledgement The authors would like to acknowledge the contributions of the following individuals: Zawieja SD (for contributing scientifically), Brezicha JE and Lenfest SE contributing with the study. 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Anand Narayanan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYHAD5gMfwPQB4rWwJc4gVQuPIXFa+Gc3P/vws80uj392z8fGn20Mcnw3EvBrkbhzzHhmb1tyscSdsxubedsYjCUJaWG4kWDMwNvGnNhwI3f7Y8Y2hsQNhLTI30j/zPi3rT5x/o2chyCH1RPUYnAjx5iZt+0w0PAcxgagwxIMCGkxvJFTzCxz7njixhtphs085yQMZ555gF+L3I30zYxvyqoT591Iftj4o8xGnu84AVvAgJENzpQgQjkY/CFW4SgYBaNgFIxIAABiBExJAL1WsQAAAABJRU5ErkJggg==","orcid":"","institution":"Florida State University","correspondingAuthor":true,"prefix":"","firstName":"S.","middleName":"Anand","lastName":"Narayanan","suffix":""},{"id":588263212,"identity":"8816e65b-67bc-4d15-9013-baedaa1c7f93","order_by":1,"name":"Ramon Boudreux","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Ramon","middleName":"","lastName":"Boudreux","suffix":""},{"id":588263213,"identity":"ef9b55f2-5363-49e0-862e-be1c70cd71a9","order_by":2,"name":"Susan Bloomfield","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"","lastName":"Bloomfield","suffix":""},{"id":588263214,"identity":"0def3042-77b3-4a08-bb3f-cedcfc2d9fb0","order_by":3,"name":"Harry Hogan","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Harry","middleName":"","lastName":"Hogan","suffix":""},{"id":588263215,"identity":"6a39e994-686f-4b8b-8ba8-e6533793851c","order_by":4,"name":"David Zawieja","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Zawieja","suffix":""},{"id":588263216,"identity":"3cce58d1-4656-4814-a53d-5f0784ab974f","order_by":5,"name":"Walter Cromer","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Walter","middleName":"","lastName":"Cromer","suffix":""}],"badges":[],"createdAt":"2025-12-29 20:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8475703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8475703/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102503840,"identity":"2bfdceaa-1b14-44d3-9204-0a95a93d9162","added_by":"auto","created_at":"2026-02-12 11:06:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1490390,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology and calprotectin scores. A \u0026amp; B: Cage Control, C \u0026amp; D: HU. The HU score was statistically significant from cage control, max score is 16 (p \u0026lt; 0.05). Fecal calprotectin changes compared at each time-point (baseline, 2-, 4-weeks) between HU and control animals, as well as linear correlation plot over time of calprotectin levels.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8475703/v1/fa60d00c6b2f8ac038a7aad8.png"},{"id":102503843,"identity":"ab9bd7d8-724a-4615-bdff-6194e163fff4","added_by":"auto","created_at":"2026-02-12 11:06:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":321100,"visible":true,"origin":"","legend":"\u003cp\u003eTime-line of symptoms in HU animals during the 28 day study. The Y-axis represents percentage of total animals that experience a particular symptom (alopecia, diarrhea, rectal prolapse/inflammation, or stressed behavior).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8475703/v1/b44271a5f1de565923c2de35.png"},{"id":102503841,"identity":"bc9120ad-1a2a-482b-bbd0-46bcef502e98","added_by":"auto","created_at":"2026-02-12 11:06:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":359633,"visible":true,"origin":"","legend":"\u003cp\u003eDisease activity indices (A and C), daily food intake (B) and body weight differences (D). Panel A illustrates visual differences in ileal small intestinal and associated mesentery loops for control (above) and HU (below) animals. Note discoloration of HU small intestinal wall, vascular hyperplasia, and reduced mesenteric adipose levels.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8475703/v1/9d1d52ba5b17064e1f4c041a.png"},{"id":102503842,"identity":"b62fee10-c71e-47e5-a79a-c3395fcb9eec","added_by":"auto","created_at":"2026-02-12 11:06:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212817,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry for MHCII (M1, pro-inflammatory marker) and CD163 (M2, anti-inflammatory marker) expressing immune cells, p \u0026lt; 0.05 for significance. Interfacing with vessel represents an immune cell within 5 μm of the lymphatic vessel or residing on/in the vessel (outlined by the hash-marks). Cells in the periphery are the cells within the field of view outside the 10 μm range of a lymphatic vessel. Total cell number are all positive cells in the field of view. HU data is shown as a fold change difference normalized to the control values. Blue datasets denote control animals and red denotes HU.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8475703/v1/7173ab9578bfd02d4149a284.png"},{"id":102746349,"identity":"ef329598-b502-49b0-9f25-e53669bbbd30","added_by":"auto","created_at":"2026-02-16 08:56:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2918291,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8475703/v1/58c1b28c-d4e1-4fc9-b134-a59b63020c88.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hindlimb Unloading in Rodents Induces Gastrointestinal Inflammation. Running head: Simulated Microgravity in Rodents Induces GI Inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpaceflight exerts multiple environmental stresses upon astronauts causing short- and long-term physiological adaptations that may interfere with the ability of crewmembers to perform in space for long durations (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The physiological adaptations that occur resemble an accelerated aging phenotype that include fluid compartment shifts, lower body disuse, and aberrant catabolic processes (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Ground-based models enable studying spaceflight adaptations by providing isolated, controllable environments for experiments that may not be feasible to do in space due to limited space, availability and cost (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The hindlimb unloading (HU) rodent model is used to simulate microgravity-induced changes, though there are different adaptations with this model compared with space-flown animal data (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBone and muscle exhibit mechanosensitivity, with microgravity exposure leading to musculoskeletal atrophy and deconditioning (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e). Microgravity-induced redistribution of pressures and flows across and within the cardiovascular system also cause changes with cardiovascular structure and function. Microgravity exposure leads to diminished venous pressures, baroreflex response, plasma volume, stroke volumes, while causing elevated oxygen uptake, heart rates, and ejection fractions leading to post-flight orthostatic intolerance (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Extensive investigations of the effects of microgravity on other physiological processes and organ systems such as endocrine, renal, pulmonary, reproductive, etc. have also been investigated, highlighting how spaceflight leads to several systemic adaptations (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Although it is known that the immune system plays an intrinsic role in the metabolic and physiological processes for of all these systems (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan additionalcitationids=\"CR102 CR103\" citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e), it has not been considered to be a contributing factor to these physiological adaptations in the context of space physiology.\u003c/p\u003e \u003cp\u003eMoreover, the HU model has been shown to induce skeletal muscle (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e), bone (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e), liver (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e), and vascular (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e) inflammation. In these contexts, investigators have looked at the inflammatory response in tissue beds to compare with what occurs in exercise, oxidative stress, long-term bed-rest, or other scenarios. However, the implications of HU-induced inflammation on bone, muscle, or other parenchymal tissues in the context of space life sciences has not been studied in great detail; thus, we aimed to investigate these adaptations as they relate with simulated microgravity induced-adaptations.\u003c/p\u003e \u003cp\u003eMoreover, immunological alterations have been shown to occur in both space-flown and HU animals. Spaceflight induces altered cytokine production patterns, natural killer cell function, leukocyte distributions, monocyte/granulocyte function, T cell intracellular signaling, neuroendocrine responses, and leukocyte proliferation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan additionalcitationids=\"CR102 CR103\" citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e). The changes in the immune system and response with the HU model are different, observed to have a different response when compared to the spaceflight adaptations (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). Considering the complexity of immune dysfunction in both models, understanding the similarities and distinctions in the mechanisms involved may provide further insight into the immunological changes occurring in manned spaceflight as well as the immunological roles it plays in other spaceflight physiological adaptations.\u003c/p\u003e \u003cp\u003eThe lymphatics are part of the cardiovascular system comprising a network of vessels that act as the main transport path of fluid and other elements (proteins, antigens, cytokines, chemokines, immune cells, macromolecules) constituting lymph from the parenchymal tissues to the nodes via the afferent lymphatics and from the nodes back to the venous blood via the efferent lymphatics. The lymphatic pathway has a critical function in maintaining immunological function and response, and disruptions in this balance can lead to immune dysfunction and inflammation both locally and systemically. It is known that the lymphatic vasculature has altered functionality in response to HU, with systemically diminished capability for the lymphatics to transport fluid and immune cells (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Furthermore, it is also known that intestinal inflammation and lymphatic dysfunction are tied together (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e). Spaceflight experiments characterizing the lymphatics response in microgravity have yet to be accomplished.\u003c/p\u003e \u003cp\u003eChronic immune dysregulation has many associated pathologies, one of the most prominent being intestinal inflammation and inflammatory bowel disease (IBD). IBD is a broad-range of pathologies associated with chronic inflammation along any or all parts of the digestive tract. IBD pathogenesis has not been fully elucidated, but both genetic and lifestyle/environmental factors such as stress, increase the risk for developing IBD (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e). Many inflammatory gastrointestinal (GI) pathologies are associated with other complications such as malnutrition, increased risks for colon cancer, bowel obstruction, ulcers, etc. It is also known that the lymphatic system is dysfunctional in IBD and is a major contributing factor of the pathology (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e, \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e, \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCharacterizations of the impact of spaceflight on GI changes in astronauts are predominantly anecdotal and quite limited (\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e). However, it is known that astronauts experience reduced appetites, decreased body weights, and negative energy balance with reduced dietary intake and other nutritional perturbations (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e). With regards to animal data, it is known from limited HU studies that there appears to be GI structural changes as well as increased bacterial translocation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e). It is plausible these changes may occur due to lymphatic dysfunction, given its immunological regulatory role and the known HU-induced lymphatic dysfunction (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore the aim of this study was to determine if HU leads to development of inflammatory changes of the GI tract. Disease activity indices used in IBD models were measured and the gut tissue histopathology was analyzed to determine any HU-induced changes in GI structure. We observed a unique pathological response in HU animals that resembles stress-induced ileitis/colitis in both disease indices and histopathology. Fecal calprotectin levels, a clinical IBD marker, was also quantified in the HU animals. These data showed a significant elevation of fecal calprotectin compared to control animals, which corroborated the other disease activity indices measured. Because there is an intertwined relationship between lymphatic function and immunological responses, we also characterized the distribution of immune cell sub-populations associated with lymphatic vessels in rat mesenteric tissues after simulated microgravity exposure. Our findings provide evidence that HU induces an inflammatory phenotype in the gut analogous to what is seen in models of IBD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals:\u003c/h2\u003e \u003cp\u003eTwelve adult male Sprague\u0026ndash;Dawley rats (6 months old, ~\u0026thinsp;400 g) were obtained from Harlan (Houston, TX) and individually housed in a climate-controlled room (23 +/- 2\u0026deg;C) with a 12-h light (0600\u0026ndash;1800)\u0026ndash;12-h dark cycle (1800\u0026ndash;0600) in an animal care facility accredited by the AAALAC. Rats were provided standard rodent chow (Harlan Teklad 8604) and water \u003cem\u003ead libitum\u003c/em\u003e and assigned to groups by body mass (normalized distribution) to age-matched cage control and HU groups (n\u0026thinsp;=\u0026thinsp;6 per group). HU animals underwent 28 days of hindlimb suspension, after which all animals were euthanized under anesthesia (Ketamine/DexaDomitor 3:2 cocktail, 0.3 mL per animal) and tissues harvested. HU animals were anesthetized before removal from tail suspension to prevent any weight bearing by the hindlimbs. At necropsy, the whole gut loop (duodenum, jejunum, ileum, mesentery, caecum, colon) was excised, washed briefly in PBS, and processed. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Texas A\u0026amp;M University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHindlimb unloading:\u003c/h3\u003e\n\u003cp\u003eHU was achieved by tail suspension as previously described (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). Briefly the animal is suspended by a harness attached to the tail to remove weight-bearing loads from the hindlimbs of the animal. While the rat was under anesthesia (Atropine, 0.3 mL per animal, Ketamine/Dexdomitor 3:2 cocktail, 0.3 mL per animal), the tail was cleaned and dried thoroughly. A thin layer of adhesive (Amazing Goop, Eclectic Products, Los Angeles, CA, USA) was applied to the tail along the medial and lateral sides. A standard porous tape (Kendall, Mansfield, MA, USA), harness was pressed firmly to the glue and allowed to dry (~\u0026thinsp;30 min). A paper clip was used to attach the animals tail harness to a swivel apparatus on a rod spanning the top of a 45x45x45 cm cage. The heights of the animal hindquarters were adjusted to prevent any contact of the hindlimbs with the cage floor, resulting in approximately a 30\u0026deg; head-down tilt. The forelimbs of the animal maintained contact with the cage floor allowing the rat full access to the entire cage. All animals were monitored twice daily for health, including assessment of tail integrity.\u003c/p\u003e\n\u003ch3\u003eObservations:\u003c/h3\u003e\n\u003cp\u003eRodents were assessed daily throughout the 28 day HU time frame for various physical symptoms of stressed behavior (porphyrin secretion, raised fur, jittery, aggravated easily if touched, testicles withdrawn), as well as symptoms of GI-associated pathology (yellow, greasy rectal discharge/dyslipidemia, diarrhea, rectal prolapse and damage). This was complemented by daily measures of food intake and weekly of weight and fecal occult (ColoScreen\u0026reg;) scoring of disease activity indices (DAI). Occult blood scoring was based on the appearance of the stool and the degree of reactivity of the fecal blood test as follows; 0\u0026thinsp;=\u0026thinsp;no color change and normal looking stool, 1\u0026thinsp;=\u0026thinsp;faint blue streaking and normal looking stool, 2\u0026thinsp;=\u0026thinsp;strong evenly distributed blue coloration and dark stool, 3\u0026thinsp;=\u0026thinsp;strong very dark blue reaction and dark/black tar-like stool, 4\u0026thinsp;=\u0026thinsp;frank red blood on surface of stool and near liquid black stool. At euthanasia the GI tract was evaluated for inflammatory injury including vascular hyperplasia, patchy injury, fibrosis, blood in the intestine, transmural injuries and perforations of the gut wall.\u003c/p\u003e\n\u003ch3\u003eFecal Calprotectin Analysis:\u003c/h3\u003e\n\u003cp\u003eFecal quantities between 1\u0026ndash;5 grams were collected at baseline, 2, and 4 four weeks of the study from both cage controls and HU animals. Directly after collection, samples were stored at -80\u0026deg;C. Calprotectin in fecal extracts were analyzed using the S100A8/S100A9 Calprotectin ELISA kit (30-6936, ALPCO, Salem, NH). The technique for processing, running, and analyzing the assay and results were performed following manufacturer guidelines.\u003c/p\u003e\n\u003ch3\u003eHistopathology:\u003c/h3\u003e\n\u003cp\u003eUpon removing rat small and large intestinal loops, sections were cut from the terminal ileum and colon that were flushed of fecal matter, washed in DPBS and fixed in 4% paraformaldehyde for 2 hours at room temperature, washed in PBS briefly, and then subsequently placed in 70% ethanol to dehydrate overnight in 4\u0026deg;C. Sections were then further dehydrated via TAMU-HSC\u0026rsquo;s Shared Core Facilities Thermo-Scientific STP 120 Spin Tissue Processor, paraffinized via a Thermo Shandon Histocenter 3 Embedding tool, sectioned (6 \u0026micro;m) via microtome, and adhered to positively charged glass slides for staining via Hematoxylin and Eosin (H\u0026amp;E) staining solutions. Scoring was performed by trained investigators on a 0\u0026ndash;4 scale (0 being normal, and 4 being severe damage or alteration) for epithelial cell loss, crypt loss, edema, and cellularity.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry:\u003c/h2\u003e \u003cp\u003eThe mesenteric loop was pinned out into Sylgard \u0026reg; 184 silicone coated glass petri dishes submerged in PBS. The tissues were washed several times with PBS to remove excess debris and blood. Loops were subsequently fixed in 4% paraformaldehyde for 2 hours at room temperature, washed again in PBS, and then permeabilized with 0.01% Triton-X 100, PBS, at room temperature for 1hr. At this point, the mesenteric arcades were cut from the gut wall and blocked in 5% goat serum for 2 hours, after which tissues were incubated overnight in primary antibodies with either Mouse Anti-Rat CD163 (1:200 AbD Serotec MCA342GA) or Mouse Anti-Rat Major Histocompatibility Complex Class II (MHCII, 1:200, Santa Cruz, sc-53721). Appropriate secondary antibodies for MHCII (Alexa Fluor 488 Goat Anti-Mouse IgG2a, A-21131) and CD163 (Alexa Fluor 488 Goat Anti-Mouse IgG1, A-21121) were applied both at 1:200 for 2 hours at room temperature in the dark, and tissues then mounted on 25X60mm glass coverslips using ProLong Gold Antifade Reagent. Lymphatic vessels were determined by morphology (thin vessel wall, absence of red blood cells) and the presence of unique leaflet valves. Images were taken using confocal microscopy (Leica, AOBPS) at 20x magnification, and ImageJ64 v.1.48u was used to determine the distribution of immune cell populations from confocal average projections. Cells were considered to be interacting with a lymphatic vessel if they were within a 10um range or residing on/in the vessel. Maximal intensity projections representative of the average data are shown.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eStatistical analyses were carried out with SAS JMP v9 for Mac OS X. All data were analyzed first by 1-way analysis of variance (ANOVA) with appropriate \u003cem\u003epost-hoc\u003c/em\u003e Dunnett\u0026rsquo;s t-test to analyze differences between groups. Regression analyses were performed on fecal occult blood and calprotectin measures. The significance level was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCalprotectin Assay:\u003c/h2\u003e \u003cp\u003eA significant increase of fecal calprotectin levels were seen in HU animals compared to control animals at the two week time-point (199.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18.3 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{ng}{mL}\\)\u003c/span\u003e\u003c/span\u003e 2-week HU vs. 91.7\u0026thinsp;\u0026plusmn;\u0026thinsp;18.1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{ng}{mL}\\)\u003c/span\u003e\u003c/span\u003e, control). At the four week time-point, calprotectin levels in HU were much higher than the levels at two weeks of HU (784.73\u0026thinsp;\u0026plusmn;\u0026thinsp;269.0 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{ng}{mL}\\)\u003c/span\u003e\u003c/span\u003e), but were not statistically different due to the high variability in the data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore the HU calprotectin levels at 2 and 4-weeks were both elevated in comparison to their baseline values. We further characterized by regression the calprotectin levels over time with statistical differences seen in HU versus control (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.264, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0087, slopes: 0.0635 in control versus 0.395 in HU over time) providing support for calprotectin being a potential marker of GI inflammation in the HU model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the calprotectin assay results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology:\u003c/h2\u003e \u003cp\u003eA significant increase in intestinal epithelial damage, crypt loss, and cellularity with minimal accounts of edema occurred in HU animals versus age-matched cage control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably in the HU animal colons, significant portions exhibited increases in cellularity so severe that granulomas and/or tertiary lymphoid organs were commonly prevalent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). Regions in the HU animals\u0026rsquo; colon showed degradation and/or complete erosion of the intestinal epithelial layer as well as significant loss of the lamina propria, with segments of colon having a perforated mucosal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). A few age matched control animals also experienced elevated histopathological scores specifically for epithelial damage and cellularity; this is most likely indicative of an isolation-induced stress as animals were single-housed for an extended period of time. The animal facility veterinarians found no signs of pathogens in sentinel control rats housed in the same room as these rats. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the histopathology analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGross Anatomical and Pathophysiological Observations:\u003c/h2\u003e \u003cp\u003eRats experienced various physical symptoms immediately after HU with \u0026gt;\u0026thinsp;50% displaying porphyrin secretion around the eyes as well as stressed behavior (raised fur, jittery, aggravated easily if touched, testicles withdrawn) [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]. Other symptoms developed later during suspension such as a yellow, greasy rectal discharge and weight loss suggest a failure of the intestine to absorb lipid content of the diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There were cases of bloody urination and diarrhea, which suggested gross inflammation was occurring. It took approximately 7\u0026ndash;10 days for stress-related symptoms (raised hair, etc.) to abate; however there appeared to be an increase in the animals displaying yellow rectal discharge to almost 100% of the rats by day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Around day 10 rodent stools became a mixture of brown/blackish (tar-like consistency) material, suggesting increased blood stool content. At two weeks of HU, animals developed inflammation of the rectum (prolapse) in ~\u0026thinsp;50% of the rats and all had brown/black stool and significant hair loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After three weeks of suspension, there were several cases (~\u0026thinsp;25%) of severe inflammation around the rectum, with significant erythema and further development of rectal prolapse and alopecia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). During the fourth and final week, ~\u0026thinsp;50% of the rats experienced significant hair-loss around the rectum, with erythema around the rectum, increased nociception, and rectal prolapse and apparent reduced defecation with associated constipation (based on general stool quantity and observation) [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDisease Activity Index:\u003c/h2\u003e \u003cp\u003eIncreased gross indicators of GI inflammation were accompanied by significant weight loss in HU animals, despite their increased food consumption (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and D). The weight loss was not due to differential starting body weights and suggests a difference in either nutritional or metabolic balance from the diet due to HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, during tissue harvest, it was observed HU animals showed depletion of mesenteric fat stores, unique from what is typically seen in IBD, and gross signs of inflammation in the GI tract including increased vascular hyperplasia and networking and branching, patchy injury and blood in the intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There were full transmural injuries as detected by near and complete perforations of the gut (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The injury does seem to self-limit to gut areas of high bacterial load (ileum, caecum, colon). These data matched temporally with the occurrence of increased fecal occult blood scoring and explained the observation of the development of blackish tar-like stools (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry:\u003c/h2\u003e \u003cp\u003eA shift in localization and activation of the mesenteric lymphatic associated immune cell populations was seen in HU animals compared to control. The number of CD163\u003csup\u003e+\u003c/sup\u003e macrophages per 20X frame increased after HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Localization of CD163\u003csup\u003e+\u003c/sup\u003e macrophage shifted from a normal distribution across the lymphatic vessel and associated mesentery to localization near a lymphatic vessel, leading us to believe that normally anti-inflammatory CD163\u003csup\u003e+\u003c/sup\u003e cells either cannot suppress the inflammation occurring or have switched to an alternate activation state (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e CD163 images and A-C). Activation seems plausible as the number of cells interacting with lymphatic vessels and their projections increased supporting a change towards this phenotype (investigator\u0026rsquo;s observations). There was an exceptional increase in total CD163\u003csup\u003e+\u003c/sup\u003e cells and of cells in association with lymphatic vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Additionally, the numbers of MHCII\u003csup\u003e+\u003c/sup\u003e macrophages that reside near or integrated into the wall of the lymphatics declined, suggesting either decreased expression of MHCII\u003csup\u003e+\u003c/sup\u003e on cells or decreased number of MHCII\u003csup\u003e+\u003c/sup\u003e expressing antigen presentation cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). This has consequences not only for the immunological state of the rodents, but also for lymphatic function as the immune cell population near the vessel can alter lymphatic functionality and remodeling processes, drastically adapting the tissue environment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThough the HU model has been used to simulate the effects of microgravity for studies of various tissue compartments (bone, muscle, cardiovascular, etc.) and immunological alterations (lymph node, thymus, spleen), the effects of HU on the GI system have not been extensively studied. We systematically examined the gut for the appearance of gross inflammation. While we are not the first to observe changes in the GI tract of HU animals (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e), we are the first to approach the GI inflammation seen in HU in the context of the pathogenesis of a disease. We found that the ileum, colon, and mesenteric tissue exhibit patterns of inflammation akin to rodent models of IBD (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e). This may provide context for the impaired lymphatic function we previously reported, as well as the immunological alterations seen systemically in this model (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe critical finding of this study was the presence of GI tissue damage and inflammation, including significant loss of epithelial continuity and crypt structure, as well as immune cell invasion and edema (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is comparable to what we have seen during spaceflight, where we observed changes in the gut microstructure and immune status. Through this study, we have been able to identify the specifics of these microgravity driven adaptations, in particular identifying immune versus fluid shift driven adaptations. Additionally, there were significant shifts in the peri-lymphatic immune cell populations of the ileal mesentery that may provide context to the lymphatic dysfunction seen previously in this model (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) (Fig.\u0026nbsp;5). Intestinal injury indices included elevated histopathology scores, gross signs of inflammation around the rectum, weight loss, significant fecal occult blood scores, and elevated fecal calprotectin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These metrics in the HU animals were analogous to inflammatory progression in classic IBD models (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistopathological analysis showed clear signs of intestinal epithelial and lamina propria loss of integrity, disruption of the colonic mucosal barrier, and increased immune cell infiltrates that formed granulomas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The intestinal epithelial barrier is critical in preventing bacterial translocation, maintaining colonic function, and maintaining immunological balance; a breach in this barrier will cause significant GI functional disruption. Granulomas were shown to breach into the lamina propria and epithelial layers. This type of injury has significant implications on GI function. Slowing of intestinal motility and a prolongation of colonic transit time has been observed in acute (2-day) HU in rats, utilizing acetaminophen as a probe (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The context of these observations under more long-term HU is not known, but combined with the profound degree of damage seen from our observations, the potential GI functional alterations may be significant and warrant further investigation.This is relevant as astronauts have been shown to develop gut permeability adaptations, though the mechanisms for this are unknown (\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e, \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e, \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFecal calprotectin analysis provided strong objective corroboration of our histological and gross observations of GI inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Calprotectin is an abundant neutrophilic and monocytic protein that is released upon activation, and is elevated in both plasma and stool during intestinal inflammation (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). The fecal calprotectin assay is a clinical diagnostic marker for IBD, and provides clear evidence of GI inflammation in the rodents due to HU (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). Calprotectin levels were increased in HU animals at 2- and 4-weeks compared to control animals and calprotectin levels trended to increase over time of HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This indicates that GI inflammation was increasing in severity with time. Further analysis of this model is needed to determine if this inflammation will resolve if the animals are allowed to recover from HU, or if the inflammation becomes chronic or recurring.\u003c/p\u003e \u003cp\u003eThe number of GI investigations in the HU model is limited. One study using female ICR mice demonstrated that breaks in the terminal ileal epithelium, and an accumulation of \u003cem\u003eE. coli\u003c/em\u003e lipopolysaccharide (LPS) in the ileal subepithelial region, after 4 days of HU (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). A chronic HU investigation using male Wistar rats suspended for 14- and 21-days found that expression and localization of the junctional proteins occludin and Zonula occludins-1 in the small intestinal mucosa were reduced (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Indirect measures of intestinal permeability in those rats (serum diamine oxidase and D-lactate levels) increased in a time dependent manner. Other investigations have shown portal endotoxemia-induced liver damage by chronic HU with elevated circulating LBP, which is a morbidity seen in GI pathologies such as IBD (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our HU animals, the digestive tracts showed signs of vascular congestion, swelling, and injury along the gut wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There were also blood vascular changes in the associated mesentery (vascular hyperplasia and increased vessel networking). It has been shown that Wistar rats experiencing 15 days of HU had increased mesenteric vascular bed blood flow (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Accompanying the elevated mesenteric arterial blood flow, protein levels of eNOS and iNOS were decreased in the mesenteric arterial vasculature. Thus, despite an impairment in NO synthesis, GI blood flow is elevated [6, 72, 74]. The venous side of the gut blood circulation has also been shown to be affected by HU. Isolated HU small mesenteric veins (21 days in Sprague-Dawley) are less responsive to norepinephrine (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). This adrenergic hyporesponsiveness occurs in a vascular bed where blood flow and arterial pressure changes should be minimal based on the hydrodynamic alterations but could be associated with changes in the natriuretic proteins observed in HU. Previously, we have shown elevations in serum atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) during HU, which are both known to also increase blood and lymphatic vessel permeability (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe lymphatic vasculature appears to be impacted by HU in a manner that is not entirely dependent on the altered gravitational forces acting on the body, as suggested by the systemic impairment in cervical, thoracic, mesenteric, and femoral lymphatics (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Two-week HU decreased stretch-activated myogenic stimulation and altered flow-mediated inhibition in all of the active lymph pumps to differing degrees regionally, but not in a manner consistent with changes in the hydrodynamic conditions (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). This suggests broad vascular adaptations in both the arteriovenous and lymphatic systems.\u003c/p\u003e \u003cp\u003eWe found that the normal complement of immune cells associated with the lymphatics was altered by HU. We have shown that lymphatic-associated immune cells can alter lymphatic transport function, this may explain both our previous findings and play a part in the phenomenon we are reporting here (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, the shift in the immune cell populations reported here differs from what we found in a rodent IBD model (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In the IBD model there was a dramatic increase in the number of MHCII\u003csup\u003e+\u003c/sup\u003e immune cells around the vessels. Here we see a reduction in the number of MHCII\u003csup\u003e+\u003c/sup\u003e cells and an increase in the number of CD163\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;5). These cells appear to be innate immune cells, presumably macrophages. CD163\u003csup\u003e+\u003c/sup\u003e macrophages are generally classified as alternatively activated, and maintain tissue homeostasis by clearance and removal of cellular debris during remodeling (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). MHCII\u003csup\u003e+\u003c/sup\u003e antigen presenting cells have been previously shown to reside within the prenodal lymphatic wall in great abundance and possess morphology with extensive cell extensions similar to the interdigitating dendritic cells of the lymph node (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The reduction of the relative abundance of these cells associated with the lymphatics after HU suggests that there may be impaired antigen presentation by this sub-population of immune cells, which may result in a reduced or dysfunctional immune responses to events in the gut. High numbers of CD163\u003csup\u003e+\u003c/sup\u003e cells were present in mesenteric tissue of HU animals and associated to a higher degree with lymphatic vessels of HU animals, suggesting there is chronic remodeling and inflammation in the mesentery, centered around the lymphatic vessels. This is similar to findings in the mucosa of IBD-patients, which show high numbers of CD163\u003csup\u003e+\u003c/sup\u003e macrophages, contributing to the resolution of inflammation and injury (\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmune dysregulation has been observed by some to be associated with HU (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e). In addition, some studies examining the effect of HU on the response to a pathogen challenge has shown that HU animals experienced compromised resistance to pathogens and delays in immunoglobulin production (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e)). Others have shown enhanced innate immune responses, but compromised adaptive immunity. It is uncertain if there is a global immune dysfunction, if there are specific immunological components that are impaired, or if HU animals are more susceptible to pathogens (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Impaired lymphatic function could contribute to an immunological dysregulation due to reduced trafficking of antigens, cytokines, and antigen presenting cells from the parenchyma to the lymph node (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).There have been reports of changes with the astronaut microbiome, of which, these observations may explain for, in terms of changes with the gut and immune structure and function (\u003cspan additionalcitationids=\"CR127 CR128\" citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile this is the first report of gross GI inflammation and injury in the HU model, there is even less known about this phenomenon in space flight. Enhancing our understanding of GI adaptations to the space flight environment, especially given the paucity of data available, is paramount since it is the site for internalization and incorporation of food, water, and nutrients. The documented immunological shifts after long-duration spaceflight are consistent with the possible development of inflammation comparable to what we report here (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). This is critical to assess in animal models so that we can appropriately translate findings to spaceflight adaptations and risks that astronauts will face during long-term missions. Therefore, it is necessary to carefully compare the effects of HU and spaceflight on the immunology and physiology of the digestive tract, to determine if the changes we have seen here in the HU model are also occurring in space.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS. A. Narayanan and W. Cromer conceptualized the work, collected data, analyzed the data, and drafted and revised the manuscript. R. Boudreux supervised the study. S. A. Bloomfield, D. C. Zawieja, H. Hogan, interpreted the data and revised the manuscript; and all authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to acknowledge the contributions of the following individuals: Zawieja SD (for contributing scientifically), Brezicha JE and Lenfest SE contributing with the study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors generated data from their study as part of their analysis and study findings.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlexander JS, Chaitanya GV, Grisham MB, Boktor M. Emerging roles of lymphatics in inflammatory bowel disease. \u003cem\u003eAnn N Y Acad Sci\u0026nbsp;\u003c/em\u003e1207 Suppl 1: E75-85, 2010.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAlexander JS, Ganta VC, Jordan PA, Witte MH. 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Effects of spaceflight on the human gastrointestinal tract microbiome. Journal of the Indian Institute of Science. 2023 Jul;103(3):761-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"simulated microgravity, lymphatics, digestive system, inflammation, spaceflight analogues","lastPublishedDoi":"10.21203/rs.3.rs-8475703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8475703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe hindlimb unloading (HU) rodent model is used to simulate changes incurred in spaceflight due to microgravity. We have previously shown 2 weeks of rat HU impairs lymphatic function systemically (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). We also previously demonstrated diminished vasoconstrictor responses of both mesenteric arteries and veins (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, gut biological adaptations in response to spaceflight have been minimally investigated, a concern given gut biology\u0026rsquo;s involvement in maintaining overall biological health as well as dietary absorption; moreover, intestinal inflammation and gut immunological alterations have not been systemically measured in microgravity models before. Recently we characterized and observed rat gastrointestinal (GI) inflammation and mesenteric lymphatic vessel associated immune cells after 4 weeks of HU. Body weight and food intake differences were notable, with HU animals weighing less as well as having increased daily food intake (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Colon histopathology indicated elevated damage in HU compared to controls (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Fecal calprotectin (a clinical IBD marker of GI inflammation) was significantly increased at 2-weeks of HU and trended towards elevation at 4-weeks. Furthermore, we noted shifts in innate immune cell populations (CD163\u003csup\u003e+\u003c/sup\u003e and MHCII\u003csup\u003e+\u003c/sup\u003e) localized with mesenteric lymphatics. CD163\u003csup\u003e+\u003c/sup\u003e cells increased in both numbers and localization with lymphatics after 4 weeks of HU (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Conversely MHCII\u003csup\u003e+\u003c/sup\u003e immune cells were reduced in both total number and their association with lymphatics in HU, suggesting altered antigen presentation capacity (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/em\u003e. These patterns are similar to our observations in models of gut inflammation. These findings may provide insight with adaptations astronauts may experience related with immune dysregulation, nutrient malabsorption, and GI adaptations. These observations also present an unrecognized inflammatory stress response that may explain physiological adaptations occurring between HU and space-flown rodent studies.\u003c/p\u003e","manuscriptTitle":"Hindlimb Unloading in Rodents Induces Gastrointestinal Inflammation. Running head: Simulated Microgravity in Rodents Induces GI Inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-12 11:06:30","doi":"10.21203/rs.3.rs-8475703/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-28T13:00:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13583956831315697387733649142831299859","date":"2026-03-12T13:40:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T06:20:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T06:20:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T06:19:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Microgravity","date":"2025-12-29T20:08:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aaf15e0c-6cde-48ec-925b-e198bb5c9c35","owner":[],"postedDate":"February 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62582164,"name":"Health sciences/Gastroenterology"},{"id":62582165,"name":"Biological sciences/Immunology"},{"id":62582166,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-02-12T11:06:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-12 11:06:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8475703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8475703","identity":"rs-8475703","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}