This work was funded by the Key R&#38;D projects of the Science and Technology Department of Sichuan Province (2020YFS0539).

The procedures for the care and use of animals were approved by the Ethics Committee of Mianyang Normal University, and all relevant institutional and governmental regulations concerning the ethical use of animals were strictly followed. For the present study, SPF rats (Kunming species, both male and female, weighing 180-220 g) were utilized. The animals were obtained from a commercial source (see Table of Materials). All animals were housed in a pathogen-free environment and provided ad libitum access to food.
1. Preparation of reagents
<ol>
	<li>Prepare a 5% sodium salicylate solution by weighing 2.5 g of sodium salicylate powder (see Table of Materials) with an electronic scale. Dissolve the powder in a small amount of distilled water and dilute it to a total volume of 50 mL.</li>
	<li>Prepare the decoction of D. caudatum (12.8 g/kg). Weigh 128 g of D. caudatum (see Table of Materials), place it into a round-bottom flask containing 1000 mL of laboratory distilled water, and concentrate it to a final volume of 100 mL.</li>
</ol>
2. Grouping and administration
<ol>
	<li>Divide the rats into different groups and intragastrically administer the required solutions. 
	NOTE: For the present study, 18 rats (9 males, 9 females) were divided into three groups: the control group (Group C), the model group (Group M), and the treatment group (Group T), with 6 rats in each group. The control group received saline solution, and the model group received a 5% sodium salicylate solution for 5 weeks at a dose of 10 mL/kg/day15. The treatment group was pretreated with a 5% sodium salicylate solution for 5 weeks at a dose of 10 mL/kg/day, followed by administration of the D. caudatum decoction at a dosage of 1 mL/100 g for 10 days15,16.</li>
	<li>Collect feces from the rats in dry and sterile microcentrifuge tubes using the tail-carrying method17 on the last day of the experiment and freeze them in a liquid nitrogen ultra-low-temperature freezer.</li>
	<li>After the end of sampling, sacrifice the rats by cervical dislocation (following institutionally approved protocols). Open the abdominal cavity with scissors to expose the abdominal aorta.
	<ol>
		<li>Insert the needle (23-25 G) almost parallel into the abdominal aorta, collect the serum, and preserve it in an ultra-low-temperature freezer. Take out the stomachs from the abdominal cavity16 and soak in a 10% formalin solution for preservation. 
		NOTE: In Group M, the chronic gastritis model is considered successful if the gastric mucosa is dilated and congested with regular glandular morphology15, while observing a small amount of inflammatory cell infiltration in the lamina propria.</li>
	</ol>
	</li>
</ol>
3. Pathological section of gastric wall
<ol>
	<li>Take the gastric wall samples from rats in each group for pathological sections. Subsequently, perform routine hematoxylin-eosin staining to observe and analyze the pathological changes18.</li>
</ol>
4. Determination of serum gastrointestinal hormones
<ol>
	<li>In the serum sample, detect the contents of gastrin (GAS) and malondialdehyde (MDA)19 using ELISA following the manufacturer&#39;s instructions (see Table of Materials). Utilize a microplate reader for the analysis.</li>
</ol>
5. Fecal total DNA extraction and PCR amplification and purification
<ol>
	<li>Perform microbial DNA extraction utilizing the commercially available DNA isolation kit (see Table of Materials) and determine the concentration and purity using UV-Vis spectrophotometer16. Subsequently, assess DNA integrity through 1% agarose gel electrophoresis18.</li>
	<li>Amplify the bacteria 16S rRNA gene with primers 338F (5&#8242;-ACT CCT ACG GGA GGC AGC AG-3&#8242;) and 806R (5&#8242;-GAC TAC HVG GGG GTW TCT AT-3&#8242;) using a thermocycler PCR system17. The primers are obtained from commercial sources (see Table of Materials). 
	NOTE: Follow the PCR program: denaturation (3 min, 95 &#176;C), followed by 27 cycles (30 s, 95 &#176;C; 30 s, 55 &#176;C; 45 s, 72 &#176;C), and a final extension (10 min, 72 &#176;C). Conduct PCR reactions with the following components: 4 &#181;L of 5&#215; DNA polymerase buffer, 2 &#181;L of 2.5 mM dNTPs, 0.8 &#181;L of each primer (5 &#181;M), 0.4 &#181;L of DNA polymerase, and 10 ng of template DNA (see Table of Materials).</li>
	<li>Extract, purify, and quantify the PCR products sequentially using 2% agarose gel, commercially available DNA gel extraction kit, and fluorometer, respectively17.</li>
</ol>
6. Sequencing
NOTE: Step 6 and step 7 are performed following previously published methods20,21.
<ol>
	<li>Utilize the Illumina platform for sequencing. 
	NOTE: One end of the DNA fragment complements the base of the connector embedded in the chip, securing it onto the chip. The other end randomly complements the base of another adjacent buried joint, forming a &#34;bridge.&#34;</li>
	<li>Perform PCR amplification to generate DNA clusters. Linearize DNA amplifiers into single strands.</li>
	<li>Introduce an altered DNA polymerase alongside deoxyribonucleotide triphosphates (dNTPs) featuring four distinct fluorescent markers. Synthesize only one base per cycle.</li>
	<li>Utilize a laser to scan the surface of the reaction plate and determine the nucleotide types polymerized during the initial reaction of each template sequence.</li>
	<li>Chemically cleave the &#34;fluorescence group&#34; and &#34;termination group&#34; to reestablish the 3&#39; sticky end. Subsequently, advance to polymerizing the second nucleotide.</li>
	<li>Obtain the sequence of template DNA fragments by analyzing the fluorescence signal results collected in each round.</li>
</ol>
7. Processing of sequencing data
<ol>
	<li>Initially, trim the reads of raw fastq data at any site with a score &#60; Q20 and eliminate reads with uncertain bases. Additionally, permit two mismatches in precisely matched primers. Subsequently, merge the sequences with an overlap &#62;10 bp.</li>
	<li>Cluster operational taxonomic units (OTUs) with 97% similarity using UPARSE, and remove chimeric sequences with UCHIME20,21.</li>
	<li>Employ the RDP Classifier algorithm to analyze the taxonomy of each 16S rRNA gene sequence against the Silva (SSU123) 16S rRNA database, using a confidence threshold of 70%.</li>
	<li>Perform Alpha diversity analysis and Beta diversity analysis using Mothur and Qiime, respectively (see Table of Materials).</li>
</ol>
8. Statistical analysis
<ol>
	<li>Analyze the data in this study using statistical analysis software (see Table of Materials). Employ one-way analysis of variance (ANOVA) for comparisons among multiple groups and utilize the least-significant-difference (LSD) test for comparisons between two groups.</li>
	<li>Consider P &#60; 0.05 as statistically significant in all comparisons. P &#60; 0.01 is regarded as extremely significant.</li>
</ol>

Desmodium caudatum Decoction's Impact on Intestinal Microbiota of Rats

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The authors have nothing to disclose.

D. caudatum, a commonly used folk medicine by the Qiang nationality12, has shown significant efficacy in treating gastrointestinal diseases. With the advancement of modern pharmacological research, the imbalance of flora resulting from gastrointestinal microecology imbalance is identified as a key factor in acute and chronic gastrointestinal diseases22,23. Certain microorganisms in the intestinal tract play a crucial role in maintaining dynamic balance in the body, and hormones in the serum reflect the physiological and pathological state of the body. Therefore, the study focuses on intestinal flora and gastrointestinal hormones.
16S rRNA high-throughput gene sequencing is a primary method for detecting intestinal microorganisms. It enables the detection of microorganism types and functions in collected samples, allowing for accurate and quantitative analysis of each species of intestinal microorganisms. In recent years, 16S rRNA high-throughput gene sequencing has rapidly developed and been widely used for microbial diversity analysis in various ecological environments24,25,26.
The effects of D. caudatum on the intestinal flora of rats with gastritis were investigated through a detailed comparison of gut bacterial communities in three groups based on Illumina-based 16S rRNA gene sequencing. Modern scientific research indicates that Lactobacillus, norank_f__Muribaculaceae, Romboutsia, and Bifidobacterium are probiotics, each with specific health benefits such as reducing the risk of multiple malignant tumors, producing vitamin K with anti-inflammatory potential, promoting polysaccharide metabolism, and improving diarrhea and constipation27,28,29. Clostridium_sensu_stricto_1, a pathogenic bacterium in intestinal injury, can cause inflammation and bacteremia and is closely linked to blood and gastrointestinal tumors30. Chronic gastritis and atrophic gastritis contribute significantly to gastric tumors31.
Beta analysis indicates significant changes in the contents of Lactobacillus, norank_f__Muribaculaceae, Romboutsia, Bifidobacterium, and Clostridium_sensu_stricto_1 after treatment. The contents of Lactobacillus, norank_f__Muribaculaceae, and Romboutsia increased significantly, while Bifidobacterium and Clostridium_sensu_stricto_1 decreased significantly after D. caudatum treatment. Additionally, the community barplot suggests that the community composition of Group T is more similar to that of Group C. These results indicate that D. caudatum could improve the prevalence of gastritis in rats. However, the specific mechanisms behind the reduction in the content of the probiotic Bifidobacterium by D. caudatum require further research.
The ELISA kit method possesses high sensitivity, strong specificity, simplicity in operation, and suitability for small sample sizes32. In this study, it was utilized to detect the contents of malondialdehyde (MDA) and gastrin (GAS) in rat serum. MDA reflects the degree of lipid peroxidation, indirectly indicating the extent of cell damage. In rats with gastritis, the MDA level was significantly higher than that in normal rats, suggesting damage to gastric parietal cells. D. caudatum treatment significantly decreased the MDA level, indicating its potential in treating gastritis. Conversely, D. caudatum increased the reduced GAS level, contributing to the repair or treatment of gastritis.
However, there are limitations to both 16S rRNA high-throughput gene sequencing and the ELISA kit method. In 16S rRNA high-throughput gene sequencing, low resolution may make it challenging to distinguish strains or genera with highly similar sequences, leading to difficulties in classification at higher taxonomic levels such as genus, family, or phylum33. For the ELISA kit method, sample pretreatment steps like dilution and washing are usually required, introducing potential errors and variability34.
This study suggests that D. caudatum may treat gastritis by regulating gastrointestinal hormones and intestinal flora, providing insights into its broad clinical applications. However, future research should delve into other gastrointestinal hormones, the mechanisms of regulating intestinal flora, and the effective chemicals involved.

Chronic gastritis (CG), one of the most common clinical diseases, is characterized by chronic and persistent inflammatory changes in the gastric mucosa epithelium, which is frequently and repeatedly attacked by various pathogenic factors1. The incidence rate of CG ranks first among all types of stomach diseases, accounting for 40% to 60% of the outpatient service rate in the Department of Gastroenterology2. Moreover, the incidence rate generally increases with age, especially in individuals who are middle-aged and older3. Undoubtedly, CG significantly reduces people&#39;s quality of life, emphasizing the critical need to discover new therapeutic agents.
Numerous studies have reported that the occurrence and development of CG are linked to the secretion of gastrointestinal hormones, such as gastrin (GAS)4,5. GAS, a common gastrointestinal peptide hormone in the digestive tract, stimulates cells to secrete gastric acid by promoting the release of histamine from eosinophils. Additionally, it improves the nutrition and blood supply of the gastric mucosa, promoting the proliferation of gastric mucosa and parietal cells6. Therefore, gastrin can be utilized as an indicator to evaluate the development level of CG. Furthermore, lipid peroxidation products triggered by reactive oxides can activate inflammatory cells, leading to CG. Malondialdehyde (MDA), a lipid peroxidation marker, is a commonly used indicator to measure the degree of oxidative stress. It reflects the level of free radicals in the gastric mucosa to a certain extent. The MDA level can indicate the attack of unsaturated fatty acids in the local gastric mucosa caused by free radicals7,8.
Intestinal microecology consists of millions of microorganisms residing in the host gut, playing a vital role in maintaining host health and regulating host immunity. A healthy intestinal flora, characterized by high richness, diversity, and stable microbiota function, acts as a protective barrier against the invasion of pathogenic microorganisms by participating in metabolism. Disturbances in the intestinal flora make individuals more susceptible to acute and chronic gastrointestinal diseases9,10. In recent years, microbiota therapy for gastrointestinal diseases has progressed rapidly and demonstrated significant efficacy11. In summary, the consideration of intestinal flora is crucial in understanding and addressing gastrointestinal diseases.
As an indispensable component of the treasure trove of traditional Chinese medicine, folk medicinal materials hold great significance for clinical practice and the modernization of national medicine. The roots and the entire parts of Desmodium caudatum (Thunb.) DC. has been widely used for relieving stomach discomfort in the Beichuan Qiang area of Mianyang City for an extended period. Some articles indicate the scientific basis for treating gastrointestinal diseases with D. caudatum, citing its effects such as hemostasis, anti-oxidation, and gastrointestinal protection12,13,14. However, due to the lack of in-depth research, no clinical pharmacodynamics mechanism has been established. This article aims to study the effects of D. caudatum in treating gastritis based on gastrointestinal hormones and intestinal flora, providing a basis for its rational clinical application.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alpha diversity analysis</td>
			<td>Mothur 1.30.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AxyPrep deoxyribonucleic acid (DNA) gel extraction kit&#160;</td>
			<td>Axygen Biosciences&#160;</td>
			<td>AP-GX-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta diversity analysis</td>
			<td>Qiime 1.9.1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic refrigerator</td>
			<td>Forma-86C ULT freezer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desmodium caudatum</td>
			<td>The Traditional Chinese Medicine Hospital of Beichuan Qiang Autonomous County</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E.Z.N.A. soil kit</td>
			<td>Omega Bio-tek</td>
			<td>D5625-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina MiSeq Platform</td>
			<td>Illumina Miseq PE300/NovaSeq PE250</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiskan Spectrum</td>
			<td>spectraMax i3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OTU clustering</td>
			<td>Uparse 7.0.1090</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OTU statistics</td>
			<td>Usearch 7.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR instrument</td>
			<td>TransGen AP221-02</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR instrument</td>
			<td>ABI GeneAmp 9700</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiFluor-ST double-stranded DNA (dsDNA) system</td>
			<td>Promega Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sequence classification annotation</td>
			<td>RDP Classifier 2.11</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium salicylate</td>
			<td>Sichuan Xilong Chemical Co., Ltd</td>
			<td>54-21-7</td>
			<td></td>
		</tr>
		<tr>
			<td>SPF rats</td>
			<td>Chengdu Dashuo Experimental Animal Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS 18.0</td>
			<td>IBM</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

effects of desmodium caudatum on gastrointestinal hormones and intestinal flora in rats with gastritis

In order to preliminarily explore the effects of Desmodium caudatum on gastritis and intestinal flora in rats, a chronic gastritis rat model was established using the classic sodium salicylate method. Eighteen SPF rats were divided into three groups: the control group (Group C), the model group (Group M), and the treatment group (Group T). Pathological sections of the gastric wall were taken from rats in each group. Furthermore, the concentrations of gastrin and malondialdehyde in the serum of rats in each group were determined by ELISA. Additionally, the effects of D. caudatum on the intestinal flora of rats with gastritis were explored through a detailed comparison of gut bacterial communities in the three groups, employing Illumina-based 16S rRNA gene sequencing. The results indicated that D. caudatum decoction could reduce the malondialdehyde content and increase the gastrin content. Moreover, D. caudatum decoction was found to enhance the diversity and abundance of intestinal flora, exerting a positive impact on the treatment of gastritis by regulating and restoring the intestinal flora.

The results of the pathological section of the stomach wall are depicted in Figure 1. In comparison to Group C, Group M exhibited mild gastric wall atrophy and mild inflammation. However, when compared to Group M, Group T showed no evident inflammation, intestinal metaplasia, or atrophy. This suggests that D. caudatum decoction can effectively improve gastritis.
The serum gastrointestinal hormone assay results are presented in Figure 2. The content of malondialdehyde (MDA) was significantly higher in Group M than in Group C. After treatment with D. caudatum decoction, it was significantly reduced. Regarding the content of gastrin (GAS), Group M showed significantly lower levels than Group C, but Group T exhibited a significant increase. These results indicate that D. caudatum decoction may regulate the level of gastrointestinal hormones to treat gastritis.
Alpha diversity analysis, as shown in Table 1 and Figure 3, reveals a significant difference between the communities of Group M and Group T. The number of bacteria in Group C and Group T is higher than that in Group M, suggesting that the number and types of intestinal bacteria in rats gradually tend to a normal level after treatment.
Species composition analysis 
According to Figure 4A, the community barplot illustrates that Lactobacillus and norank_f__Muribaculaceae account for the largest proportion in Group C. Group M contains a higher abundance of Clostridium_sensu_stricto_1 and some Helicobacter. The community composition of Group T is more similar to that of Group C, with important components being Lactobacillus and Romboutsia. Additionally, the Spearman correlation heatmap (Figure 4B) combined with the circos diagram (Figure 4C) reveals significant differences in flora composition between Group C and Group M, with distinct variations in dominant flora. After treatment, Group T tends to return to a normal flora state. Furthermore, Figure 5 indicates extreme differences in community composition between Group M and Group C, with significant changes occurring in the structure and composition of intestinal flora in rats with gastritis. After treatment, there are similarities in flora composition between Group C and Group T, as well as some similarities between Group T and Group M. These results indicate that D. caudatum decoction could restore the intestinal flora of rats with gastritis to a normal or close-to-normal state.
Species difference analysis 
From the multi-species comparison column chart in Figure 6A, it can be observed that Norank_f_Muribaculaceae is abundant in Group C and significantly differs from Group M and Group T. After treatment, the number of Norank_f_Muribaculaceae in Group T shows an increasing trend. Additionally, Clostridium_sensu_stricto_1, abundant in Group M, exhibits resilience in harsh environments. After treatment, the number decreases significantly, suggesting that D. caudatum decoction improves the survival environment of intestinal flora to some extent. On the other hand, the LEfSe multi-level species hierarchy tree analysis in Figure 6B indicates 44 species with significant differences between groups. Norank_f_muribaculaceae, Clostridium_sensu_stricto_1, and Romboutsia were found to be abundant in Group C, Group M, and Group T, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65744/65744fig01.jpg" /> 
Figure 1: The results of the pathological section of the stomach wall. (A) Pathological section of the stomach in Group C. (B) Pathological section of the stomach in Group M. (C) Pathological section of the stomach in Group T. <a href="https://www.jove.com/files/ftp_upload/65744/65744fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65744/65744fig02.jpg" /> 
Figure 2: The contents of serum gastrointestinal hormones. (A) Bar analysis chart of MDA (malondialdehyde) determination results. (B) Bar analysis chart of GAS determination results. *P &#60; 0.05, **P &#60; 0.01. <a href="https://www.jove.com/files/ftp_upload/65744/65744fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65744/65744fig03.jpg" /> 
Figure 3: Column gram of diversity index T. Red bar represents Group C, blue represents Group T, while green represents Group M.*P &#60; 0.05, **P &#60; 0.01. <a href="https://www.jove.com/files/ftp_upload/65744/65744fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65744/65744fig04v2.jpg" /> 
Figure 4: Community composition analysis. (A) Community histogram analysis. (B) Community heatmap analysis on the level of Genus. (C) Circos diagram analysis of relationship between samples and species. <a href="https://www.jove.com/files/ftp_upload/65744/65744fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65744/65744fig05.jpg" /> 
Figure 5: PLS-DA analysis on the level of Genus. Red represents Group C, blue represents Group T, and green represents Group M. <a href="https://www.jove.com/files/ftp_upload/65744/65744fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65744/65744fig06.jpg" /> 
Figure 6: Species difference analysis. (A) Kruskal-Wallis H test bar plot. (B) Tree map of LefSe multi-level species. Red represents Group C, blue represents Group T, and green represents Group M. <a href="https://www.jove.com/files/ftp_upload/65744/65744fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group</td>
			<td>Mean value</td>
			<td>Standard deviation</td>
		</tr>
		<tr>
			<td>C</td>
			<td>3.5201</td>
			<td>0.63641</td>
		</tr>
		<tr>
			<td>M</td>
			<td>3.2755</td>
			<td>0.28494</td>
		</tr>
		<tr>
			<td>T</td>
			<td>3.5388</td>
			<td>0.29945</td>
		</tr>
	</tbody>
</table>
Table 1: Alpha diversity index results.

Watch this Scientific Journal Video about Author Spotlight: Exploring the Mysteries of Sichuan's Herbal Medicine in Chinese Medicine Research at JoVE.com

Author Spotlight: Exploring the Mysteries of Sichuan's Herbal Medicine in Chinese Medicine Research

The present protocol describes the method for analyzing intestinal flora using Illumina-based 16S rRNA gene sequencing and provides a framework for evaluating the effectiveness of herbal decoctions.

Effects of Desmodium caudatum on Gastrointestinal Hormones and Intestinal Flora in Rats with Gastritis

In order to preliminarily explore the effects of Desmodium caudatum on gastritis and intestinal flora in rats, a chronic gastritis rat model was ...

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403 Views.  Mianyang Normal University. The present protocol describes the method for analyzing intestinal flora using Illumina-based 16S rRNA gene sequencing and provides a framework for evaluating the effectiveness of herbal decoctions.

Video: Author Spotlight: Exploring the Mysteries of Sichuan's Herbal Medicine in Chinese Medicine Research

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Exergaming in Older People Living with HIV Improves Balance, Mobility and Ameliorates Some Aspects of Frailty

Approximately 1.2 million people in the United States live with HIV infection. Medical advancements have increased the life expectancy and this cohort is aging. HIV-positive individuals have a high incidence of frailty (~20%) characterized by depression and sedentary behavior. Exercise would be healthy, but due to the frail status of many HIV-positive individuals, conventional exercise is too taxing. The aim of this study was to evaluate the effectiveness and acceptability of a novel game-based training program (exergame) in ameliorating some aspects of frailty in HIV-infected individuals. Ten older people living with HIV were enrolled in an exergame intervention. Patients performed balance exercises such as weight shifting, ankle reaching, and obstacle crossing. Real-time visual/audio lower-extremity joint motion feedback was provided using wearable sensors to assist feedback and encourage subjects to accurately execute each exercise task. Patients trained twice a week for 45 min for 6 weeks. Changes in balance, gait, psychosocial parameters and quality of life parameters were assessed at the beginning, midterm and at conclusion of the training program. Ten patients completed the study and their results analyzed. The mean age was 57.2 &#177; 9.2 years. The participants showed a significant reduction in center of mass sway (78.2%, p = .045) during the semi-tandem balance stance with eyes closed and showed a significant increase in gait speed during a dual task motor-cognitive assessment (9.3%, p = .048) with an increase in stride velocity of over 0.1 m/sec. A significant reduction in reported pain occurred (43.5%, p = .041). Preliminary results of this exergame intervention show promise in improving balance and mobility while requiring older people living with HIV to be more active. The exergame can be continued at home and may have long term as well as short-term benefits for ameliorating frailty associated with HIV infection.

Persons infected with HIV are often frail, depressed and live a sedentary lifestyle for which conventional exercise is too taxing. Here, we present an exercise protocol that ameliorates aspects of frailty in HIV-infected persons. An exergame integrating cognitive control was developed using biosensors that measured balance, weight-shifting and obstacle crossing.

Approximately 1.2 million people in the United States live with HIV infection. Medical advancements have increased the life expectancy and this cohort is ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.