Name: Establishment of a Rat Model for Intrauterine Adhesions via Dual Injury: Curettage and Infection (Video) | JoVE Journal
Uploaded: 2025-10-03T15:00:00.000+00:00
Duration: 11 min 44 s
Description: Learn how to establish a reproducible rat model for intrauterine adhesions using mechanical curettage and lipopolysaccharide-induced infection. This method enables researchers to study IUA pathogenesis and evaluate therapeutic strategies effectively.

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5 minutes

The study was funded by the Guangdong Medical Research Fund (A2021132) and Shenzhen Maternity and Child Healthcare Hospital Research Fund (FYA2022006), China

All experimental procedures were approved by the Animal Ethics Committee of Guangdong Medical Laboratory Animal Center, China (C202307-2). The experiment was conducted in strict accordance with animal ethics standards. The experimental materials used in this protocol are listed in the Table of Materials. Conduct all procedures under strict aseptic conditions throughout the surgery.
1. Establishment of IUA models in rats
<ol>
	<li>Order female Sprague-Dawley (SD), 8-10 week-old, sexually mature, unpaired rats weighing 260-320 g.</li>
	<li>House rats in groups under specific-pathogen-free (SPF) conditions with a temperature of 21-25 &#176;C, humidity of 50-60%, a regular 12 h light: 12 h dark cycle before surgery, and free access to food and water.</li>
	<li>Acclimate rats to laboratory conditions for 1 week, then take daily smears of vaginal discharge from rats to determine their estrous cycle stages.Stain the slides with hematoxylin (Figure 1A-D). Randomly assign 12 rats with regular estrous cycles to the sham operation group (n = 6) or the dual injury group (n = 6). Perform surgery during the estrous period.</li>
	<li>Prepare LPS solution by dissolving LPS powder in phosphate-buffered saline (PBS) at a concentration of 6 mg/L. Before surgery, immerse sterile 4-0 surgical cotton sutures in the LPS solution for 24 h, then store them in a 4 &#176;C refrigerator to prepare LPS-impregnated sutures.</li>
	<li>Sterilize surgical instruments via autoclaving before surgery.</li>
	<li>Dual injury group procedures:
	<ol>
		<li>Weigh the rats. Prior to anesthesia induction, administer buprenorphine subcutaneously (0.05 mg/kg) as preoperative analgesia, 30 min before surgery. Then, anesthetize using isoflurane inhalation. Set the anesthesia induction concentration at 2-3% and the maintenance concentration at 1.5-2%.</li>
		<li>Assess anesthetic depth via toe pinch reflex; proceed only if no withdrawal response is observed. Place the rats in the supine position, and fix their limbs to the operating table.</li>
		<li>Shave a 4 cm x 6 cm area on the lower abdominal wall of the rats with a hair clipper. Disinfect the area with three alternating scrubs of 1% povidone-iodine and 70% ethanol, each applied in a circular motion from the incision center outward, and cover it with a sterile disposable surgical drape.</li>
		<li>Make a skin incision: Grasp the skin with tweezers and use a sterile scalpel blade to make a shallow vertical incision in the midline of the lower abdomen, 0.5 cm above the pubic symphysis, and approximately 4 cm in length.</li>
		<li>Expose the abdominal muscle, grasp its midline with a clamp, and carefully make a small incision into the peritoneal cavity. Then, insert the scissors into the opening and carefully cut in both directions to extend the incision to approximately 4 cm in length (Figure 2A). Take care to avoid inadvertently damaging the abdominal organs.</li>
		<li>Locate and expose the two uterine horns of the rat, which are situated above the pubic symphysis, behind the bladder, and between the intestines (Figure 2B).</li>
		<li>Make a 3 mm incision in the right uterine horn, 5 mm above the bifurcation, while avoiding blood vessels (Figure 3A).</li>
		<li>Insert a scraping spoon into the right horn through the uterine incision. Scrape the uterine cavity until it feels uneven and the inner wall becomes rough. Scratch approximately 30x in each direction (front, back, left, and right) of the cavity above the incision.</li>
		<li>When scratching along the direction of the uterine horn, apply force evenly to prevent tearing or perforating the uterine wall (Figure 3B).</li>
		<li>Straighten a 1/2-circle 6 x 24 surgical needle and thread an LPS-impregnated (6 mg/L) cotton suture through its eye.</li>
		<li>Insert the needle with LPS suture through the incision of the right uterine horn to the end of the cavity.</li>
		<li>Then, withdraw the needle, leaving the LPS suture inside the entire right uterine cavity with 3-4 cm of the suture length exposed above the bifurcation (Figure 4A,B). Leave the LPS suture in the uterine cavity for 48 h.</li>
		<li>Close the right horn incision intermittently with 7-0 absorbable sutures (Figure 5A).</li>
		<li>Treat the left uterine horn in the same manner (Figure 5B).</li>
		<li>Gently return both uterine horns to the abdominal cavity using forceps. Thoroughly wash the peritoneal cavity with sterile normal saline (0.9% sodium chloride) to remove any remaining endometrial debris.</li>
		<li>Close the abdominal incision.
		<ol>
			<li>Close the peritoneal cavity with continuous 4-0 absorbable sutures (Figure 6A).</li>
			<li>Inject 0.5% (50 mg/mL) cefazolin sodium solution (0.2 mL/100 g) into the abdominal wall muscle (Figure 6B) to prevent surgical site infections. 
			NOTE: This systemic prophylaxis targets potential extrauterine bacterial contamination while preserving the localized LPS-induced uterine injury, as LPS acts as an endotoxin rather than a live bacterial inoculum.</li>
			<li>Close the skin with continuous 4-0 absorbable sutures (Figure 6C).</li>
		</ol>
		</li>
		<li>Leave approximately 2 cm of the LPS suture protruding outside the skin. To prevent the rats from biting the remaining sutures, fix them to the skin with 4-0 absorbable sutures 1 cm from the abdominal incision (ensuring no interference with future suture removal) (Figure 7A). Trim excess suture material (Figure 7B). 
		NOTE: The suture is fixed 1 cm from the incision with absorbable sutures to prevent biting and for easy removal after 48 h. No postoperative infections are observed, as the suture&#39;s exteriorization aims to facilitate removal, not induce external infection.</li>
	</ol>
	</li>
	<li>Sham operation group procedures: Perform no uterine injury procedures when the uterine horns are exposed following laparotomy. Wash the peritoneal cavity with sterile normal saline. Inject 0.5% cefazolin sodium solution into the abdominal wall muscle to prevent infection. Close the peritoneal cavity and the skin with 4-0 absorbable sutures.</li>
	<li>Dry the rats with sterile gauze. Monitor animals closely during anesthesia recovery.</li>
	<li>Postoperatively, administer buprenorphine (0.05 mg/kg, SC) every 12 h for 3 days to manage pain. Monitor for signs of discomfort (e.g., reduced appetite, hunched posture).</li>
	<li>Two days after surgery, use forceps to gently extract the LPS sutures from the uterine cavities of rats in the dual injury group.</li>
	<li>Monitor rats for food intake, defecation, and overall health status daily. Weigh animals every 3 days to assess recovery.</li>
</ol>
2. Uterine horn collection
<ol>
	<li>Collect and examine uteri 7 and 14 days after the initial surgery in both groups, corresponding to approximately two and four regular estrous cycles, respectively.</li>
	<li>Resterilize surgical instruments via autoclaving and anesthetize rats by isoflurane inhalation.</li>
	<li>Reopen the abdominal cavity of rats.</li>
	<li>Remove both uterine horns and evaluate macroscopic characteristics: observe for swelling, congestion, luminal narrowing, surface roughness, or loss of elasticity. Check bilateral symmetry of the horns and inspect the uterine cavities for occlusion, stenosis, or effusion (Figure 8A-D).</li>
	<li>Randomly excise an appropriate amount of tissue from either horn and immerse it in a 10% formalin solution for histopathological analysis.</li>
	<li>Euthanize rats via overdose of isoflurane inhalation (approved by the Animal Ethics Committee) after uterine horn collection, as this is a non-survival surgery.</li>
</ol>
3. Evaluation of endometrial fibrosis
<ol>
	<li>Embed the uterine tissues in paraffin and section them at 4 &#181;m thickness using a microtome. Deparaffinize sections in xylene and dehydrate them in a graded series of ethanol solutions: 70%, 80%, 95%, and 100%.</li>
	<li>Hematoxylin and eosin (HE) staining and histomorphological analysis:
	<ol>
		<li>Randomly select three sections from each uterine horn and stain them with routine HE protocol16,17: deparaffinization in xylene, rehydration through a descending graded ethanol series (100%, 95%, 80%, 70%, and water), staining with hematoxylin, differentiation, bluing, staining with eosin, and finally dehydration through an ascending graded ethanol series (70%, 80%, 95%, and 100%), clearing in xylene, and mounting with a synthetic resin.</li>
		<li>Observe endometrial histomorphological changes in each group under an optical microscope. Conduct a single-blind histological evaluation of the sections by a single experienced pathologist.</li>
	</ol>
	</li>
	<li>Use Masson trichrome staining to assess the endometrial fibrosis.
	<ol>
		<li>Randomly select three sections from each uterine horn and stain them with Masson Trichrome stain16,17, carrying out deparaffinization in xylene and rehydration through a graded ethanol series (100%, 95%, 80%, 70%, and water), staining with Weigert&#39;s Iron Hematoxylin for nuclei, differentiation in acidic ethanol, bluing, staining with Ponceau-Acid Fuchsin solution for muscle fibers and cytoplasm, differentiation in phosphomolybdic acid solution, counterstaining with Aniline Blue or Light Green for collagen fibers, brief rinsing in weak acetic acid solution, and finally dehydration through an ascending graded ethanol series (95% and 100%), clearing in xylene, and mounting with a synthetic resin.</li>
		<li>Perform quantitative analysis of endometrial fibrosis using a whole-slide image analysis platform. Load the high-resolution whole slide image into the analysis platform. From the module list, select the Area Quantification brightfield module. Use the real-time tuning feature to adjust the parameter settings for accurately identifying and segmenting the blue collagen fibers. For the total endometrial area measurement, use the software&#39;s annotation tools to manually trace and define the total endometrial area.</li>
		<li>Calculate the ratio of fibrosis area of each section by the analysis system and use it to grade the endometrial fibrous hyperplasia. 
		Ratio of fibrosis area (%) = (total fibrosis area / total area of the endometrial interstitium and glands) &#215; 100</li>
	</ol>
	</li>
</ol>

<ol><li>Deans, R., Abbott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+intrauterine+adhesions%5BTitle%5D)+AND+%22J+Minim+Invasive+Gynecol%22%5BJournal%5D)">Review of intrauterine adhesions.</a> J Minim Invasive Gynecol. 17 (5), 555-569 (2010).</li>
<li>Yu, D., Wong, Y. M., Cheong, Y., Xia, E., Li, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Asherman+syndrome-one+century+later%5BTitle%5D)+AND+%22Fertil+Steril%22%5BJournal%5D)">Asherman syndrome-one century later.</a> Fertil Steril. 89 (4), 759-779 (2008).</li>
<li>Ang, C. J., Skokan, T. D., McKinley, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+regeneration+and+fibrosis+in+the+endometrium%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">Mechanisms of regeneration and fibrosis in the endometrium.</a> Annu Rev Cell Dev Biol. 39, 197-221 (2023).</li>
<li>Hooker, A., Fraenk, D., Brölmann, H., Huirne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+intrauterine+adhesions+after+termination+of+pregnancy%3A+A+systematic+review%5BTitle%5D)+AND+%22Eur+J+Contracept+Reprod+Health+Care%22%5BJournal%5D)">Prevalence of intrauterine adhesions after termination of pregnancy: A systematic review.</a> Eur J Contracept Reprod Health Care. 21 (4), 329-335 (2016).</li>
<li>Salzani, A., Yela, D. A., Gabiatti, J. R. E., Bedone, A. J., Monteiro, I. M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+uterine+synechia+after+abortion+evacuation+curettage%5BTitle%5D)+AND+%22Sao+Paulo+Med+J%22%5BJournal%5D)">Prevalence of uterine synechia after abortion evacuation curettage.</a> Sao Paulo Med J. 125 (5), 261-264 (2007).</li>
<li>Baradwan, S., Baradwan, A., Al-Jaroudi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+association+between+menstrual+cycle+pattern+and+hysteroscopic+March+classification+with+endometrial+thickness+among+infertile+women+with+Asherman+syndrome%5BTitle%5D)+AND+%22Medicine+%28Baltimore%29%22%5BJournal%5D)">The association between menstrual cycle pattern and hysteroscopic March classification with endometrial thickness among infertile women with Asherman syndrome.</a> Medicine (Baltimore). 97 (27), e11314(2018).</li>
<li>Ventolini, G., Zhang, M., Gruber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hysteroscopy+in+the+evaluation+of+patients+with+recurrent+pregnancy+loss%3A+A+cohort+study+in+a+primary+care+population%5BTitle%5D)+AND+%22Surg+Endosc%22%5BJournal%5D)">Hysteroscopy in the evaluation of patients with recurrent pregnancy loss: A cohort study in a primary care population.</a> Surg Endosc. 18 (12), 1782-1784 (2004).</li>
<li>Kim, S. W., Kim, Y. Y., Kim, H., Ku, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+closer+to+intrauterine+adhesive+pathology%5BTitle%5D)+AND+%22Ann+Transl+Med%22%5BJournal%5D)">Animal models closer to intrauterine adhesive pathology.</a> Ann Transl Med. 8 (18), 1125(2020).</li>
<li>Cai, G., Hou, Z., Sun, W., Li, P., Zhang, J., Yang, L., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+developments+in+biomaterial-based+hydrogel+as+the+delivery+system+for+repairing+endometrial+injury%5BTitle%5D)+AND+%22Front+Bioeng+Biotechnol%22%5BJournal%5D)">Recent developments in biomaterial-based hydrogel as the delivery system for repairing endometrial injury.</a> Front Bioeng Biotechnol. 10, 894252(2022).</li>
<li>Dreisler, E., Kjer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Asherman%27s+syndrome%3A+Current+perspectives+on+diagnosis+and+management%5BTitle%5D)+AND+%22Int+J+Womens+Health%22%5BJournal%5D)">Asherman's syndrome: Current perspectives on diagnosis and management.</a> Int J Womens Health. 11, 191-198 (2019).</li>
<li>Sylvie, C. A., Fadila, M., Catherine, R. B., Sabine, T., Hervé, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hysteroscopic+treatment+of+severe+Asherman%27s+syndrome+and+subsequent+fertility%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">Hysteroscopic treatment of severe Asherman's syndrome and subsequent fertility.</a> Hum Reprod. 14 (5), 1230-1233 (1999).</li>
<li>Roy, K. K., Baruah, J., Sharma, J. B., Kumar, S., Kachawa, G., Singh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproductive+outcome+following+hysteroscopic+adhesiolysis+in+patients+with+infertility+due+to+Asherman%27s+syndrome%5BTitle%5D)+AND+%22Arch+Gynecol+Obstet%22%5BJournal%5D)">Reproductive outcome following hysteroscopic adhesiolysis in patients with infertility due to Asherman's syndrome.</a> Arch Gynecol Obstet. 281 (2), 355-361 (2010).</li>
<li>Li, L., Shi, J., Zhang, Q. F., Yan, J., Yan, L. Y., Shen, F., Qiao, J., Feng, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+curettage+and+copper+wire+on+rabbit+endometrium%3A+A+novel+rabbit+model+of+endometrial+mechanical+injury%5BTitle%5D)+AND+%22Chin+Med+J+%28Engl%29%22%5BJournal%5D)">Effect of curettage and copper wire on rabbit endometrium: A novel rabbit model of endometrial mechanical injury.</a> Chin Med J (Engl). 121 (11), 1708-1713 (2011).</li>
<li>Liu, F., Zhu, Z. J., Li, P., He, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creation+of+a+female+rabbit+model+for+intrauterine+adhesions+using+mechanical+and+infectious+injury%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">Creation of a female rabbit model for intrauterine adhesions using mechanical and infectious injury.</a> J Surg Res. 183 (1), 296-303 (2013).</li>
<li>Wang, X., Quinn, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipopolysaccharide%3A+Biosynthetic+pathway+and+structure+modification%5BTitle%5D)+AND+%22Prog+Lipid+Res%22%5BJournal%5D)">Lipopolysaccharide: Biosynthetic pathway and structure modification.</a> Prog Lipid Res. 49 (2), 97-107 (2010).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Theory+and+practice+of+histological+techniques%22">Theory and practice of histological techniques</a>. Bancroft, J. D., Gamble, M. , 6th ed, Churchill Livingstone Elsevier. (2008).</li>
<li>Kiernan, J. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKiernan%2C+JA+%22Histological+and+histochemical+methods%3A+Theory+and+practice%22">Histological and histochemical methods: Theory and practice</a>. , 5th ed, Scion Publishing Ltd. (2015).</li>
<li>Brasted, M., White, C. A., Kennedy, T. G., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mimicking+the+events+of+menstruation+in+the+murine+uterus%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Mimicking the events of menstruation in the murine uterus.</a> Biol Reprod. 69 (4), 1273-1280 (2003).</li>
<li>Bannerman, D. D., Goldblum, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+effects+of+endotoxin+on+the+endothelium+barrier+function+and+injury%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">Direct effects of endotoxin on the endothelium barrier function and injury.</a> Lab Invest. 79 (10), 1181-1199 (1999).</li>
</ol>

The authors have no conflicts of interest to declare.

The pathogenesis and pathophysiological processes of IUAs remain incompletely understood, primarily due to ethical restrictions limiting the acquisition of uterine tissue from affected patients. This knowledge gap hinders progress in elucidating its mechanisms and developing targeted therapies. IUAs represent a global health challenge, severely compromising female fertility, reproductive health, and overall quality of life, thus underscoring the urgent need to investigate the biological mechanisms underlying IUA formation and identify strategies to mitigate endometrial fibrosis during adhesion development. Previous studies, such as those by Li et al.13 and Liu et al.14, established IUA models in rabbits using mechanical curettage combined with copper wire implantation or LPS suture placement. Rats, however, offer distinct advantages as research models due to their high genetic homology with humans, minimal inter-individual variability, cost-effectiveness, ease of husbandry, and suitability for large-scale experimentation-attributes that render them ideal for studying endometrial injury and repair.
Damage to the endometrial basalis layer, regardless of etiology, initiates a cascade of connective tissue hyperplasia, scar formation, and impaired neovascularization. Histopathologically, IUAs are characterized by dense fibrous tissue deposition within the uterine cavity, often accompanied by a small amount of residual or absent normal endometrium, atrophic glands, and reduced stromal vascularity3. Trauma and infection are widely acknowledged as the primary risk factors for IUAs2,3. Iatrogenic injuries from procedures like dilation and curettage, induced abortion, or transcervical resection of submucosal fibroids are leading causes, while secondary postoperative infections, endometritis, and puerperal infections further exacerbate the risk by promoting inflammatory-mediated tissue damage3. Other factors associated with IUAs include cesarean section, mechanical injuries, uterine artery embolization, and other ischemic injuries.
The existing methods of IUA animal models are mainly divided into physical injury, chemical injury, and combined injury. Physical methods include mechanical injury, thermal injury, short-term blocking of the ovarian artery, etc.; chemical injury includes intrauterine injection of phenol mucilage, 95% ethanol, and trichloroacetic acid; combined injury is mainly mechanical injury combined with intrauterine infection. Such methods can induce endometrial damage and adhesion formation, but they often deviate from the clinical pathogenesis of human IUAs. Concerns persist regarding the translational relevance of these models, as retained chemical or physical agents may interfere with subsequent histological and functional assessments. Herein, we addressed this limitation by leveraging the two primary clinical risk factors-trauma and infection-and adapted the methodology of Liu et al.14&#160;to develop a dual-injury rat model, systematically evaluating its efficacy in recapitulating human IUA pathology.
It is generally accepted that a valid injury model must satisfy two criteria: tissue damage induced by injury factors and incomplete self-repair following factor removal13. In this study, the dual injury method of mechanical and infection is simulated to create a model of uterine adhesion. First, the endometrium is mechanically injured by curettage. It has been reported that the endometrial interstitial area and epithelial cells can be completely restored to the undamaged state by 48 hours after physiological injury18. Thus, if curettage is insufficient during IUA modeling, the endometrial damage is relatively superficial, similar to physiological damage, and will cause the failure of the uterine adhesion animal model. Therefore, in this experiment, thorough curettage (approximately 120x in total for either horn) is required to damage the basalis layer of the endometrium and induce fibrotic scar hyperplasia to establish animal models of IUAs. Subsequent intrauterine LPS exposure exacerbated inflammation, promoting edema, leukocyte infiltration, and stromal fibrosis19. There is a consensus that infection may promote the further development of IUAs.
Secondary infection after intrauterine procedures, acute and chronic endometritis, and puerperal infection can all cause endometrial inflammation, inhibit endometrial repair, and even destroy normal endometrium, promoting endometrial fibrosis and resulting in IUA formation3. Histological analysis at 7 and 14 days post surgery demonstrated progressive pathological changes in the dual injury group: epithelial thinning or deficiency, glandular atrophy, interstitial congestion, necrotic debris, fibrous tissue hyperplasia, and fibrous adhesion leading to luminal occlusion. At 14 days post surgery, only a few endometrial epithelia have regenerated, and others are still mostly replaced by fibrous tissue. The histological changes of discontinuous endometrial epithelia, sparse glands, and interstitial fibrosis are basically consistent with the pathological features of clinical IUAs. The characteristic pathophysiological change in IUAs is massive hyperplasia of collagen fibers. Masson trichrome staining confirmed significantly increased collagen deposition, quantified by fibrosis area ratio, which validated the model&#39;s fidelity to human IUA pathophysiology.
Our findings demonstrate that the dual-injury approach establishes a stable, reproducible IUA model in rats, with procedural feasibility and consistent pathological features. This model provides a practical approach for investigating IUA pathogenesis and testing therapeutic strategies. However, while our model effectively incorporates trauma and infection -- two key risk factors -- it does not fully recapitulate the multifactorial etiology of human IUAs. Future studies integrating additional clinical variables (e.g., ischemia, hormonal fluctuations) may enhance translational relevance.
Clinically, current treatments for IUAs focus on restoring uterine anatomy, yet functional recovery -- particularly reproductive potential -- remains challenging, especially in moderate and severe cases. The persistent endometrial fibrosis and vascular deficit underlying IUAs represent major barriers to pregnancy, highlighting the need for innovative therapies targeting tissue repair and anti-fibrosis. Our animal model, by recapitulating human endometrial damage, serves as a critical tool for investigating cytokine-based interventions, biomaterial applications, and stem cell therapies -- avenues that may ultimately improve clinical outcomes for IUA patients.

Intrauterine adhesions (IUAs), the most common pathological condition resulting from endometrial injury, are characterized by partial or complete obliteration of the uterine cavity and replacement of endometrial tissue with fibrotic tissue. First formally described by Asherman in 1948, IUAs are thus clinically referred to as &#34;Asherman&#39;s syndrome&#34;.
IUAs most commonly arise secondary to surgical procedures that cause endometrial injury, such as curettage, induced abortion, cesarean section, and myomectomy1. It is generally believed that trauma and infection are the major risk factors for IUAs2,3. Although the exact incidence of IUAs remains unclear, the prevalence of IUAs following curettage has been reported to range from 21.2% to 37.6%4,5. Among infertile women and those with recurrent pregnancy loss, 4.6% and 21.8% have been diagnosed with IUAs, respectively6,7. Compared to other infertility causes, such as tubal factors, ovulatory dysfunction, and male factors, endometrial factors are uniquely challenging to address8.
Damage to the endometrial basalis layer hinders endometrial regeneration, concurrently reducing the number of endometrial epithelial cells and glands while increasing fibrotic tissue and collagen deposition. This pathological process leads to partial or complete obliteration of the uterine cavity, with or without involvement of the cervix of the uterus2. IUAs may result in hypomenorrhea, as well as adverse reproductive outcomes, including infertility, subfertility, recurrent miscarriage, and placenta accreta, which severely compromise women&#39;s reproductive health and overall well-being. As a critical public health issue affecting women of childbearing age, IUAs demand urgent attention9.
Hysteroscopy serves as the gold-standard approach for the investigation, assessment, and treatment of IUAs10. Currently, the primary treatment option for IUAs is transcervical resection of adhesions (TCRA), which refers to the resection of fibrotic tissue and restoration of the uterine cavity. Although this procedure can achieve anatomical restoration, the lack of endometrial regeneration and functional recovery leads to a high recurrence rate of IUAs and limited effectiveness in treating repeated embryonic implantation failure8. Multiple modalities have been applied to reduce the incidence and recurrence of IUAs, such as cyclic hormone therapy, intrauterine devices, Foley catheterization, balloon stenting, hyaluronic acid gel, and amnion graft. Nevertheless, their efficacy is restricted, particularly in patients with severe IUAs. Re-adhesion remains a significant challenge. It is reported that the postoperative recurrence rate of IUAs is up to 62.5%11, while the pregnancy rate is merely 33.3%12, indicating a poor prognosis.
Tissue engineering-based endometrial reconstruction holds substantial promise for the future, yet it remains suboptimal due to procedural complexity and high costs, including challenges in culturing three-dimensional endometrial tissues and organoids. Additionally, the absence of suitable IUA animal models hinders the exploration of endometrial damage-repair mechanisms, as well as in vivo evaluation of treatments. Consequently, there is an urgent need to develop an effective IUA animal model that accurately recapitulates the clinical injury features.Such a model would not only serve as a critical tool for dissecting IUA pathogenesis but also accelerate the translation of therapeutic strategies.
The selection of appropriate animal models for IUAs is critical for advancing research and addressing this clinical challenge. Female rabbits have been used to establish such models.For example, Li et al. simultaneously performed uterine curettage and copper wire implantation in female rabbits, demonstrating successful IUA model establishment 4 weeks post surgery13; Liu et al. similarly created rabbit IUA models through combined curettage and LPS suture placement14. Current endometrial injury models primarily utilize rats, mice, New Zealand rabbits, and some primates. Although primates share physiological, anatomical, and genetic similarities with humans, including comparable menstrual cycles, their use remains limited due to challenges in procurement, high costs, and intensive breeding requirements. Among rodents, rats offer distinct advantages: they exhibit significant genetic homology with humans, low inter-individual variability, and relatively larger uterine structures compared to mice. Additionally, their cost-effectiveness, ease of maintenance, and suitability for large-scale model development make rats ideal for studying endometrial injury and repair mechanisms.
The rat model described herein employs a dual-injury approach: curettage and infection, to simulate the pathological process of uterine synechiae formation. Mechanical injury is induced by curettage, while lipopolysaccharide (LPS), a key component of the Gram-negative bacterial cell wall and a potent endotoxin15, is used to elicit infectious inflammation, mimicking the clinical scenario where infection exacerbates tissue damage. As an established endotoxin, LPS triggers robust inflammatory responses and tissue injury, which are critical contributing factors in the progression of IUAs. Through this study, we aim to establish an ideal animal model for investigating the pathogenesis, regenerative mechanisms, and therapeutic strategies of this clinically challenging condition.

<table><tbody><tr><td>Absorbable suture 4-0</td><td>Goleden ring</td><td>R413</td><td></td></tr><tr><td>Absorbable suture 7-0</td><td>Goleden ring</td><td>R731</td><td></td></tr><tr><td>Acidic Ethanol</td><td>Macklin</td><td>A885191-100ml</td><td></td></tr><tr><td>Aniline Blue</td><td>Macklin</td><td>A787491-500g</td><td></td></tr><tr><td>Black braided silk suture 4-0&amp;nbsp;</td><td>Ethicon</td><td>ZJ3915</td><td></td></tr><tr><td>Buffered formalin 10%&amp;nbsp;</td><td>Acmec</td><td>AC11741</td><td></td></tr><tr><td>Calipers</td><td>Deli</td><td>NJ006-063-01</td><td></td></tr><tr><td>Cefazolin sodium</td><td>Aladdin</td><td>C336103-1g</td><td></td></tr><tr><td>Cotton swab</td><td>Milone</td><td>ML-10200</td><td></td></tr><tr><td>Eosin</td><td>Aladdin</td><td>E774785</td><td></td></tr><tr><td>Eppendorf Tubes 1.5 mL</td><td>Biosharp</td><td>BS-15-M</td><td></td></tr><tr><td>Eppendorf Tubes 50 mL</td><td>Oneshine</td><td>WS-LXG-45- 50ml</td><td></td></tr><tr><td>Ethanol 95%&amp;nbsp;</td><td>Macklin</td><td>E801077-500ml</td><td></td></tr><tr><td>Filter tips 100 &amp;mu;L&amp;nbsp;</td><td>Brand</td><td>6Y5883</td><td></td></tr><tr><td>Glass slide</td><td>Citotest</td><td>188105W</td><td></td></tr><tr><td>Hair clipper</td><td>Kerong</td><td>LS220621006</td><td></td></tr><tr><td>Hematoxylin</td><td>Beyotime</td><td>C0105S</td><td></td></tr><tr><td>Iodine disinfectant solution</td><td>Oneshine</td><td>annjet</td><td></td></tr><tr><td>Lipopolysaccharide</td><td>Beyotime</td><td>ST1470</td><td></td></tr><tr><td>Phosphomolybdic Acid Solution</td><td>Macklin</td><td>P822362-100ml</td><td></td></tr><tr><td>Pipettes 1mL&amp;nbsp;</td><td>Jet</td><td>GSP010001-ZX</td><td></td></tr><tr><td>Ponceau-Acid Fuchsin Solution</td><td>Macklin</td><td>A800390-100g</td><td></td></tr><tr><td>Powder free sterile gloves</td><td>Medicom</td><td>1144</td><td></td></tr><tr><td>Scraping spoon</td><td>Shengyan</td><td>899918163731PT</td><td></td></tr><tr><td>SlideViewer Software</td><td>3DHISTECH Ltd.&amp;nbsp;</td><td>V2.6.0.166179</td><td></td></tr><tr><td>Small serrated semi-curved forceps</td><td>Baoya</td><td>A2052</td><td></td></tr><tr><td>Small surgical scissors</td><td>Baoya</td><td>A2053</td><td></td></tr><tr><td>Small toothless forceps</td><td>Baoya</td><td>A2054</td><td></td></tr><tr><td>Standard light microscope</td><td>Leica</td><td>DM IL LED Fluo</td><td></td></tr><tr><td>Sterile gauze 10 x 10 cm</td><td>Biosharp</td><td>S-0121</td><td></td></tr><tr><td>Sterile phosphate buffered saline (PBS)</td><td>Beyotime</td><td>ST478</td><td></td></tr><tr><td>Sterile saline (0.9% sodium chloride)</td><td>Oneshine</td><td>J-SL-500ml</td><td></td></tr><tr><td>Sterile water</td><td>Meilunbio</td><td>PWL093-2</td><td></td></tr><tr><td>Surgical drape 45 x 45 cm&amp;nbsp;</td><td>Xiangbo</td><td>XBSSD40-50</td><td></td></tr><tr><td>Surgical suture needle 1/2 8 x 24&amp;nbsp;</td><td>Goleden ring</td><td>5569-18</td><td></td></tr><tr><td>Synthetic Resin</td><td>Macklin</td><td>N861409-500ml</td><td></td></tr><tr><td>Syringe 2 cc</td><td>Beyotime</td><td>FS802-30pcs</td><td></td></tr><tr><td>Weak Acetic Acid Solution</td><td>Ecotopbio</td><td>ED-8457-500ml</td><td></td></tr><tr><td>Weigert&#039;s Iron Hematoxylin</td><td>Acmec</td><td>AC11556-2*100ml</td><td></td></tr><tr><td>Xylene</td><td>Macklin</td><td>X823028-100ml</td><td></td></tr><tr><td>Halo Image Analysis Software</td><td>Indica Labs</td><td>v3.0.311.314</td><td></td></tr></tbody></table>

establishment of a rat model for intrauterine adhesions via dual injury: curettage and infection

Establishment of a Rat Model for Intrauterine Adhesions via Dual Injury: Curettage and Infection

Intrauterine adhesions (IUAs), the most common disorders of endometrial injury with incompletely understood etiology, profoundly disrupt female reproductive health. In severe cases, IUAs lead to a postoperative adhesion recurrence rate of 62.5% and a low pregnancy rate of 33.3%. Trauma and infection are recognized as major risk factors. Animal models facilitate a better understanding of the mechanisms underlying this complex disease. Here, we describe a cost-effective rat model mimicking human IUA pathogenesis through dual injury: mechanical endometrial curettage and infection. First, the endometrium was scraped ~30x in each direction (front, back, left, and right) with a curette. Second, a cotton suture impregnated with 6 mg/L lipopolysaccharide was placed in the uterine cavity for 48 h to induce inflammatory damage. Histological changes were evaluated at 7 and 14 days post surgery. Macroscopic examination revealed uterine atrophy and loss of elasticity, while histological analysis showed thinning or absence of the endometrium, decreased glandular number, narrowing or obliteration of the uterine cavity, and increased area of fibrosis. This dual-injury approach efficiently establishes a stable rat IUA model by day 14, recapitulating key clinical and pathological hallmarks of human IUAs.

All experimental rats recovered uneventfully postoperatively and survived through the study duration. Surgical incisions healed within 7 days without signs of infection.
Gross morphological observations of uterine horns: 
In normal rats, uteri exhibit a Y-shaped bicornuate structure with pink coloration, soft texture, regular contour, uniform wall thickness, and no evidence of hemorrhage or congestion (Figure 2B). During surgery, mechanical scraping of the uterine cavity induced immediate morphological changes: the uterine wall showed pronounced swelling, congestion, dark red discoloration, and occasional petechial hemorrhages on the horn surfaces (Figure 5).
At 7 days after surgery, the bilateral uterine horns in the sham operation group remain symmetrical, soft, and elastic, with no evidence of localized stiffness or fluid accumulation (Figure 8A). By contrast, the bilateral horns in the dual injury group exhibit mild rigidity, uneven uterine wall thickness, focal stiffness, and luminal stenosis (Figure 8B). At 14 days after surgery, the bilateral uterine horns maintain morphology comparable to presurgical baselines in the sham operation group, with intact symmetry and texture (Figure 8C). In the dual injury group, however, both horns display contracture, pale coloration, reduced elasticity, and irregular wall thickness. Localized stiffness and luminal narrowing were exacerbated, accompanied by occasional segmental dilation and hydropic changes (Figure 8D).
Histological change at 7 days post surgery: 
Microscopically, the normal rat uterus consists of four layers: the mucosal layer, the submucosal layer, the muscular layer, and the serous layer. The mucosal layer consists of a densely packed single layer of columnar epithelial cells, with a submucosa rich in endometrial glands. Endometrial interstitium contains blood vessels without edema, congestion, or hemorrhage, and collagen fibers are sparsely arranged in an orderly pattern. In Masson trichrome staining, the mucosa, submucosa, glands, blood vessels, and muscle fibers stain red, with a relatively regular luminal shape. Normal endometrial stromal cells surround glands and contain sparse, light-blue, fusiform collagen fibers arranged neatly. The stroma exhibits a star-shaped, radial architecture with occasional patchy sparsity. The inner stroma is rich in blood vessels and lymphatic vessels.
Day 7 postoperative findings: In the sham operation group, under HE and Masson trichrome staining, the endometrial epithelium, glandular architecture, and uterine lumen morphology closely resemble normal uterine structures. The endometrial surface exhibits a wavy contour, and the glands are round to elliptical in shape. The stroma demonstrates no evidence of edema, congestion, or hemorrhage, and collagen fibers remain sparsely distributed with loose organization (Figure 9A,B). In the dual injury group, under HE staining, the uterine cavity exhibits narrowing, partial occlusion, or filling with necrotic debris and leukocytes. The single-layered columnar epithelium is thinned or absent, replaced by flat or low-columnar epithelial cells. Endometrial glands are reduced in number or absent. Interstitial collagen fibers are increased and disorganized, accompanied by stromal edema, leukocyte infiltration, capillary hyperemia or occlusion, partial atresia of capillary lumens, and a small number of newly formed capillaries (Figure 9C). Some cases demonstrate intrauterine effusion or focal loss of the muscular layer. Under Masson trichrome staining, the uterine cavity shows shrinkage with an irregular shape or occlusion, and evident intrauterine hemorrhage. The number of endometrial glands decreases, with a relative reduction in the red-stained epithelial area. Conversely, the blue-stained fibrous tissue in the stroma increases in quantity, exhibits a disorganized arrangement, and appears darker in staining intensity. The stroma shows infiltration of numerous inflammatory cells and edematous changes (Figure 9D).
Histological change at 14 days post surgery: 
In the sham operation group, under HE and Masson trichrome staining,the endometrium of rats shows no significant changes, with morphology consistent with that observed at 7 days post surgery. Structural integrity of the uterine cavity is maintained, lacking notable alterations in epithelial or stromal architecture (Figure 10A,B). In the dual injury group, under HE staining,glandular and epithelial regeneration is nearly absent. Instead, extensive collagen deposition and interstitial fibrosis develop, leading to IUAs and near-complete or complete cavity occlusion. Increased inflammatory cell infiltration and/or partial muscular layer defect or uterine effusion can also be seen (Figure 10C). Under Masson trichrome staining, normal columnar epithelia and glands are replaced by fibrous tissue, and IUAs develop, which are manifested by a large number of atrophied glands, surrounded by a mass of collagen fibers. The interstitial fibrous tissue increases significantly, with collagen appearing denser and darker compared to the 7 day postsurgery stage (Figure 10D).
Quantitative analysis: At 14 days post surgery, the endometrial fibrosis area in the dual injury group is 23,72,781.22 ± 6,18,367.29 µm2, significantly larger than the sham operation group's 7,32,486.33 ± 3,29,410.32 µm2 (Table 1).Correspondingly, the fibrosis area ratio is 33.19 ± 18.32% in the dual injury group and 13.38 ± 3.22% in the sham operation group, with a statistically significant difference (P &lt; 0.01) (Figure 11). The IUA modeling success rate in the dual injury group is 100% (6/6), confirming consistent induction of pathological fibrosis and adhesion formation.
<img alt="Cell morphology, stained, microscopic image; samples labeled A-D; scale: 50 µm; biology analysis." class="xfigimg" src="/files/ftp_upload/67937/67937fig1.jpg" data-altupdatedat="1762425657000" data-alt="Figure 1"> 
Figure 1: Smears of vaginal discharge. (A) Proestrus; (B) Estrus; (C) Metestrus; (D) Diestrus Scale bars = 50 µm. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical incision process, measuring exposure depth, includes tissue handling in clinical setup." class="xfigimg" src="/files/ftp_upload/67937/67937fig2.jpg" data-altupdatedat="1762425657000" data-alt="Figure 2"> 
Figure 2: Laparotomy and uterine horn exposure. (A) Laparotomy. (B) Exposure of the bilateral uterine horns in a rat. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical exploration diagram showing gastrointestinal structure with tool manipulation for educational use." class="xfigimg" src="/files/ftp_upload/67937/67937fig3.jpg" data-altupdatedat="1762425657000" data-alt="Figure 3"> 
Figure 3: Endometrial scratching of each uterine horn. (A) Make an incision in the right uterine horn (red arrow). (B) Scratch the endometrium. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical suturing process; two-step closure technique; medical practice; tissue approximation." class="xfigimg" src="/files/ftp_upload/67937/67937fig4.jpg" data-altupdatedat="1762425657000" data-alt="Figure 4"> 
Figure 4: Insertion of an LPS-impregnated thread into each uterine horn. (A) Insert an LPS thread with a straightened needle through the incision. (B) Insert the LPS thread to the end of the cavity. Abbreviation: LPS = lipopolysaccharide. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical stitching technique; close-up, ruler measurement of incision on blue surgical drape." class="xfigimg" src="/files/ftp_upload/67937/67937fig5.jpg" data-altupdatedat="1762425657000" data-alt="Figure 5"> 
Figure 5: Closure of the uterine incision with LPS thread left in both uterine cavities. (A) Suture the left uterine incision with 7-0 absorbable sutures intermittently. (B) Leave LPS-impregnated threads in both uterine cavities. Abbreviation: LPS = lipopolysaccharide. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical wound closure process, three-step sequence, suturing technique, medical procedure." class="xfigimg" src="/files/ftp_upload/67937/67937fig6.jpg" data-altupdatedat="1762425657000" data-alt="Figure 6"> 
Figure 6: Abdominal cavity closure and skin suturing. (A) Closure of the abdominal cavity. (B) Injection of 0.5% cefazolin sodium solution into the abdominal wall muscle. (C) Skin suturing with a 2-cm-long LPS thread left external to the abdominal wall. Abbreviation: LPS = lipopolysaccharide. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical wound healing comparison, two stitches, postoperative suture technique, medical study result." class="xfigimg" src="/files/ftp_upload/67937/67937fig7.jpg" data-altupdatedat="1762425657000" data-alt="Figure 7"> 
Figure 7: Fixation of LPS threads to the abdominal wall. (A) Securing the two LPS threads with 4-0 absorbable sutures, each 1.0 cm from the abdominal incision. (B) Trimming of excess LPS thread length. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tissue morphology comparison with ruler scale; each sample labeled A-D; analysis of structural changes." class="xfigimg" src="/files/ftp_upload/67937/67937fig8.jpg" data-altupdatedat="1762425657000" data-alt="Figure 8"> 
Figure 8: Gross morphology of uterine horns. (A) At 7 days post surgery, bilateral uterine horns in the sham operation group exhibit symmetry, soft texture, and elasticity, with no evidence of local stiffness or fluid accumulation. (B) In the dual injury group at 7 days post surgery, uterine horns are slightly rigid, characterized by uneven wall thickness, focal stiffness, and luminal stenosis. (C) By 14 days post surgery, the sham operation group's uterine horns maintain pre-surgical morphological integrity without discernible changes. (D) In the dual injury group at 14 days post-surgery, both horns are contracted, pale, and hypoelastic, with uneven thickness, focal rigidity, and luminal narrowing. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Histological cross-sections of arteries, staining results, microscopic analysis of tissue differentiation." class="xfigimg" src="/files/ftp_upload/67937/67937fig9.jpg" data-altupdatedat="1762425657000" data-alt="Figure 9"> 
Figure 9: Histological staining of uterine horns at 7 days post surgery. (A) HE staining (sham operation group): The endometrial surface displays a wavy contour, with round-to-elliptical glands. The stroma lacks edema, congestion, or hemorrhage, and collagen fibers exhibit a sparse, organized arrangement. (B) Masson trichrome staining (sham operation group): The endometrial architecture is intact, with red-stained mucosa, submucosa, glands, blood vessels, and muscle fibers. The lumen maintains a regular shape. Stromal cells appear light blue, fusiform, and neatly arranged, with abundant vascular and lymphatic structures in the inner stroma. (C) HE staining (dual injury group): The uterine cavity exhibits narrowing, partial occlusion, or filling with necrotic debris and leukocytes. The single-layer columnar epithelium is thinned or absent, with reduced or absent glands. Interstitial collagen fibers are increased and disorganized, accompanied by leukocyte infiltration, focal capillary hyperemia, and occlusion. (D) Masson trichrome staining (dual injury group): The uterine cavity is contracted with an irregular shape or occlusion, showing intra-cavity hemorrhage. Endometrial glands are reduced in number, with a relative decrease in the red-stained epithelial area. Conversely, blue-stained stromal fibrous tissue increases in quantity, exhibits a disorganized arrangement, and has a darker staining intensity. The stroma demonstrates extensive inflammatory cell infiltration and edematous changes. The insets show zoomed-in images of the regions. Scale bars = 500 µm. Abbreviation: HE = hematoxylin-eosin. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Histological stain analysis, four tissue cross-sections, microscopy images, cellular structure study." class="xfigimg" src="/files/ftp_upload/67937/67937fig10.jpg" data-altupdatedat="1762425657000" data-alt="Figure 10"> 
Figure 10: Histological staining of uteri at 14 days post surgery. (A) HE staining (sham operation group): Microscopically, the endometrium shows no significant changes, consistent with the 7-day post-surgery uterine cavity morphology. (B) Masson trichrome staining (sham operation group): Endometrial morphology remains comparable to the 7 day postsurgery state, with preserved structural integrity. (C) HE staining (dual injury group): Glandular and epithelial regeneration is nearly absent, replaced by extensive collagen deposition and interstitial fibrosis. Intrauterine adhesions lead to near-complete or complete luminal occlusion. (D) Masson trichrome staining (dual injury group): Normal columnar epithelium and glands are replaced by fibrous tissue, with intrauterine adhesions characterized by atrophied glands surrounded by dense collagen fibers. Interstitial fibrous tissue increases significantly, with collagen appearing denser and darker than at 7 days post surgery. The insets show zoomed-in images of the regions. Scale bars = 500 µm. Abbreviation: HE = hematoxylin-eosin. <a href="https://www.jove.com/files/ftp_upload/67937/67937fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graph comparing fibrosis ratios; dual injury vs. sham operation; statistical difference noted." class="xfigimg" src="/files/ftp_upload/67937/67937fig11.jpg" data-altupdatedat="1762425657000" data-alt="Figure 11"> 
Figure 11: Endometrial fibrosis area due to injury. The area ratio of endometrial fibrosis in the dual injury group is significantly higher than that in the sham operation group (P &lt; 0.01). <a href="https://www.jove.com/files/ftp_upload/67937/67937fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Fibrosis area data. Fibrosis area in the dual injury group and sham operation group at 14 days post surgery. <a href="https://www.jove.com/files/ftp_upload/67937/67937_Table%201.xlsx" target="_blank">Please click here to download this Table.</a>

Intrauterine adhesions (IUAs), the most common pathological consequence of endometrial injury, severely impair reproductive function. Trauma and infection are widely recognized as key risk factors for IUA development. We have successfully established a reproducible rat model of IUAs by combining two critical injury methods: mechanical curettage and infectious induction with lipopolysaccharide.

Intrauterine adhesions (IUAs), the most common disorders of endometrial injury with incompletely understood etiology, profoundly disrupt female ...

Intrauterine adhesions, IUAs, the most common pathological consequence of endometrial injury severely impair reproductive function. Tumor and infection are widely recognized as key risk factor for IUA development. We have successfully established a reproducible rat model of IUAs by integrating two critical pathogenic mechanisms, curettage and infection.House female SD rats that are 8 to 10 weeks old, sexually mature, and unpaired in groups under SPF conditions. Take daily vaginal discharge smear from rats to determine their as estrous cycle stage. Slides are stained with hematoxylin.Perform surgery during the estrous period. Before surgery, dissolve lipopolysaccharide, LPS, powder in PBS at a concentration of six milligram per liter. Immerse sterile 4/0 surgical cutting sutures in the LPS solution for 24 hours.Then, store them in a four degrees centigrade refrigerator to prepare LPS-impregnated sutures. Prior to anesthesia induction, administer buprenorphine subcutaneously as pre-operative analgesia. Then, anesthetize the rat by isoflurane inhalation.Assess anesthetic depth via toe pinch reflex. Proceed only if no withdrawal response is observed. Shave a four centimeter plus six centimeter area on the lower abdominal wall of the rat with a hair clipper.Disinfect the area with three alternating scrubs of 1%povidone-iodine and 70%ethanol and cover it with a sterile disposable surgical drape. Make a skin incision in the midline of the lower abdomen above the pubic symphysis. Expose the abdominal muscle.Carefully make a small incision into the peritoneal cavity and extend the incision. Take care to avoid inadvertently damaging the abdominal organs. Rotate and expose the two uterine horns situated above the pubic symphysis behind the bladder.Make a three millimeter incision in the right uterine horns five millimeter above the bifurcation while avoiding blood vessels. Insert a scraping spoon into the right horn through the uterine incision. Scrape the uterine cavity until it feels uneven and the inner wall become rough.Scratch approximately 30 times in each direction, front, back, left, and right. When scratching around the direction of the uterine horn, apply force evenly to prevent tearing or perforation the uterine wall. Insert a straightened surgical needle with LPS-impregnated suture through the incision of the right uterine horn to the end of the cavity.Then, withdraw the needle, leaving the LPS suture inside the entire right uterine cavity with three to four centimeters of suture length exposed above the bifurcation. Leave the LPS suture in the uterine cavity for 48 hours. Close the right horn incision intermittently with 7/0 absorbable sutures.Treat the right uterine horn in the same manner. Thoroughly wash the peritoneal cavity with sterile normal saline to remove any remaining endometrial debris. Close the peritoneal cavity with continuous 4/0 absorbable sutures.Inject 0.5%cefazolin sodium solution into the abdominal wall muscle to prevent surgical site infection. Close the skin with continuous 4/0 absorbable sutures. Leave approximately two centimeter of the LPS suture protruding outside the skin.To prevent the rats from biting the remaining sutures, fix them to the skin with 4/0 absorbable suture one centimeter from the abdominal incision. Trim excess suture material. Perform no uterine injury procedures when the uterine horns are exposed following laparotomy.Wash the peritoneal cavity with sterile normal saline. Close the peritoneal cavity and the skin with 4/0 absorbable sutures. Two days after surgery, use forceps to gentle eject the LPS sutures from the uterine cavity of rats in the dual injury group.Collect and examine uteri 7 and 14 days after the initial surgery in both groups, corresponding to approximately two and four regular estrous cycles respectively. Reopen the abdominal cavity after anesthetize the rat with isoflurane. Remove both uterine horns and evaluate macro characteristics.Observe for swelling, congestion, luminal narrowing, surface roughness, or loss of elasticity. Check bilateral symmetry of the horns and inspect uterine cavities for occlusions, stenosis, or effusion. Euthanize rats via overdose of isoflurane inhalation, approved by the Animal Ethics Committee, after uterine horn collection as this a non-survival surgery.Gross morphological observation of uterine horns. At seven days after surgery, the bilateral uterine horns in the sham operation group remain symmetrical, sharp, and elastic with no evidence of localized stiffness or fluid accumulation. By contrast, the bilateral horns in the dual injury group exhibit mild rigidity, uneven uterine wall thickness, focal stiffness, and ruminal stenosis.At 14 days after surgery, the bilateral uterine horns maintained morphology comparable to presurgical baseline in the sham operation group, with intact symmetry and texture. In the dual injury group, however, both horns display contracture, pale coloration, reduce elasticity, and irregular wall thickness. Localized stiffness and luminal narrowing were exacerbated.Histological change at seven days post-surgery. In the sham operation group, under HE and Masson trichrome staining, the endometrial epithelium, glandular architecture, and uterine lumen morphology closely resembled normal uterine structures. Endometrial surface exhibit a wave contour and the glands are round to elliptical in shape.The stroma demonstrates no evidence of edema, congestion, or hemorrhage, and collagen fibers remain sparsely distributed with loose organization. In the dual injury group, under HE staining, the uterine cavity exhibits narrowing, partial occlusion, or filling with necrotic debris and leukocytes. The single layer columnar epithelium is thin or absent, replaced by flat or row-columnar epithelial cells.Endometrial glands are reduced in number or absent. Under Masson trichrome staining, the number of endometrial gland decreases with the relative reduction in the epithelial area. Conversely, the blue-stained fibrous in the stroma increases in quantity, exhibit disorganized arrangement, and appears darker in staining intensity.Histological change at 14 days post-surgery. In the sham operation group under HE and Masson trichrome staining, the endometrium of rats shows no significant changes with morphology consistent with that observed at seven days post-surgery. In the dual injury group, under HE staining, glandular and epithelial regeneration is nearly absent.Instead, extensive collagen depositioned and interstitial fibrosis developed, leading to intrauterine adhesions and near-complete or complete cavity occlusion. Under Masson trichrome staining, normal columnar epithelia and glands are replaced by fibrous tissue, and intrauterine adhesions developed which are manifested by a large number of atrophic glands and surrounded by a mass of collagen fibers. The interstitial fibrous tissue increases significantly, with collagen appearing denser and darker compared to the seven day post-surgery stage.At 14 days post-surgery, the area ratio of endometrial fibrosis in the dual injury group is significantly higher than that in the sham operation group. Our findings demonstrate that the dual injury approach establish a stable, reproducible IUA model in rats with procedural feasibility and consistent pathological features. This model provides a practical approach for investigating IUA pathogenesis and testing therapeutic strategies.

establishment-rat-model-for-intrauterine-adhesions-via-dual-injury

Research

JoVE Journal

Medicine

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Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

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Use of a Rat Model to Study Ventral Abdominal Hernia Repair

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Creation of Abdominal Adhesions in Mice

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Development of a Uterosacral Ligament Suspension Rat Model

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Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

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Mouse Model of Surgical Uterine Injury and Subsequent Pregnancy Outcomes

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A Translational Surgical Porcine Model for Postoperative Intra-Abdominal Adhesion Formation

Learn how to establish a reproducible rat model for intrauterine adhesions using mechanical curettage and lipopolysaccharide-induced infection. This method enables researchers to study IUA pathogenesis and evaluate therapeutic strategies effectively.

Establishment of a Rat Model for Intrauterine Adhesions via Dual Injury: Curettage and Infection

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