Name: Mouse Models of Epididymitis Induced by Pathogen-Associated Molecular Patterns (Video) | JoVE Journal
Uploaded: 2025-12-12T13:50:00.000+00:00
Duration: 8 min 30 s
Description: Learn surgical injection models for inducing epididymitis in mice using interstitial and intravasal techniques. These methods enable region-specific study of inflammatory responses in reproductive tissues.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors thank Maria Ang&#233;lica Spadella and Rosa Maria dos Santos Sabatini, Mar&#237;lia Medical School, and the Department of Structural and Functional Cell Biology, Morphology sector, IBB/UNESP, for technical assistance in histopathological evaluation. Helio Kushima and Paulo Mioni, Department of Biophysics and Pharmacology, IBB/UNESP, for their technical assistance. Nat&#225;lia Calixto Miranda Santos and Beatriz Rezende Santos, Department of Biophysics and Pharmacology, IBB/UNESP, for their help with the shoot. Funded by S&#227;o Paulo Research Foundation (FAPESP, #2021/04746-3 and #2021/06718-7); in part by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES, #88887.657630/2021-00); Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPQ, #303616/2022-9; 311179/2016-9).

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institute of Health, USA) and National Council for the Control of Animal Experimentation (CONCEA, Brazil). Procedural approval was granted by the Ethics Committee for the Use of Experimental Animals (CEUA-IBB/UNESP, Brazil; protocol number: 6272050320; 2269040625). Adult C57BL/6 male mice (90-120 days) were used in these studies. The reagents and the equipment used in this study are listed in the Table of Materials.
1. Interstitial injection in the initial segment of the epididymis
<ol>
	<li>Mouse preoperative preparation
	<ol>
		<li>Induce general anesthesia with ketamine/xylazine (60/20 mg/kg, i.p.) (following institutionally approved protocols). When anesthetized, place the mouse in a supine position on the surgical field. 
		NOTE: Before incision, firmly pinch the toe to ensure the pedal reflex is absent. Inhaled anesthetics (e.g., isoflurane) can be used to replace ketamine/xylazine.</li>
		<li>Make the trichotomy of the mouse abdomen using the electric trimmer pen shape (Figure 2A).</li>
		<li>Perform abdominal asepsis with sterile cotton wet with chlorhexidine 0.25% (v/v) and alcohol.</li>
	</ol>
	</li>
	<li>Injection in the interstitial compartment of the initial segment
	<ol>
		<li>Using the scalpel size 15, gently make a ~4 mm vertical incision on the right side of the abdominal wall (Figure 2A). 
		NOTE: Be careful with the preputial gland.</li>
		<li>With the angled blunt-ended surgical forceps, gently separate the skin. Using the scalpel size 15, gently make a ~4 mm vertical incision on the peritoneum at the site of the first incision in the abdominal wall.</li>
		<li>Carefully lift the epididymis and testis for the abdominal space, pushing the right side of the scrotum. 
		NOTE: To facilitate the following procedures, use either the headband magnifier or a stereoscopic microscope.</li>
		<li>With the curve surgical forceps, carefully pull the epididymal white adipose tissue (eWAT) to localize the initial segment of the epididymis. Using the eWAT, gently expose the initial segment at the incision area (Figure 2A).</li>
		<li>Position the Hamilton syringe, preloaded with the injection solution, at an angle approximately 90&#176; parallel to segment #1 of the initial segment. Carefully insert the needle in the interstitial space with the needle bevel oriented down (Figure 2A and Supplementary Figure 1). 
		NOTE: Be careful not to puncture the epididymal duct; do not tilt the needle.</li>
		<li>Position the curve surgical forceps in the needle insert region and gently pinch the epididymal capsule and the needle. Slowly lower the syringe plunger until all the stimulus solution is expelled (Figure 2A). 
		NOTE: Maximum injection volume: 10 &#181;L.</li>
		<li>Keeping the curve surgical forceps in position, carefully remove the needle. After ~30 s, remove the surgery curve forceps.</li>
		<li>Using the eWAT, gently return the tissue to the abdominal cavity. 
		NOTE: Do not pinch the initial segment, only the eWAT.</li>
		<li>Localize the peritoneum and make two suture stitches with the needle holder and size 5/0 surgical suture (Supplementary Figure 1). 
		NOTE: Check that the incision in the peritoneum is closed; if necessary, make another stitch.</li>
		<li>Close the skin incision with two suture stitches with the needle holder and size 5/0 surgical sutures (Figure 2A).</li>
		<li>Repeat steps 1.2.1-1.2.10 to make the interstitial injection on the left side.</li>
	</ol>
	</li>
	<li>Postoperative care
	<ol>
		<li>Observe the mouse postoperatively to ensure appropriate recovery (check animal behavior, shivering, locomotion, and other signs of toxicity). Keep animals warm until they recover from surgery. It is expected that the mouse will move more slowly within the cage after surgery, returning to normal behavior after ~12-24 h.</li>
	</ol>
	</li>
</ol>
2. Intravasal injection in the cauda epididymidis
<ol>
	<li>Mouse preoperative preparation
	<ol>
		<li>Induce general anesthesia with ketamine/xylazine (60/20 mg/kg, i.p.). When anesthetized, place the mouse in a supine position on the surgical field. 
		NOTE: Before incision, firmly pinch the toe to ensure the pedal reflex is absent. Inhaled anesthetics (e.g., isoflurane) can be used to replace ketamine/xylazine.</li>
		<li>Place the index finger and thumb below the scrotum to check the position of the testes and epididymides. If necessary, carefully massage the sides of the abdomen toward the scrotum to position the testicles and epididymides in the scrotum.</li>
		<li>Make the trichotomy of the mouse scrotum using the electric trimmer pen shape (Figure 3A).</li>
		<li>Perform scrotum asepsis with sterile cotton wet with chlorhexidine 0.25% (v/v) and alcohol.</li>
	</ol>
	</li>
	<li>Retrograde injection in the luminal compartment of the vas deferens toward the cauda epididymidis 
	NOTE: To facilitate the following procedures, use either the headband magnifier or a stereoscopic microscope.
	<ol>
		<li>With the index finger and thumb below the scrotum, keeping the skin of the scrotum gently stretched, gently make a ~2 mm vertical incision on the medial portion of the scrotum with the scalpel size 15, cutting the skin, tunica dartos, and external spermatic fascia (Figure 3A).</li>
		<li>Using the index finger, gently move the left testis and epididymis back to the abdomen, keeping the right testis and epididymis in the scrotum.</li>
		<li>With the index finger and thumb below the right testis and epididymis, keeping the skin of the scrotum gently stretched, carefully open and close the angled blunt-ended surgical forceps on the internal spermatic fascia, until breaking the internal spermatic fascia and the vaginal tunic.</li>
		<li>Relieve pressure from the index finger and thumb. Through the opening in the vaginal tunic, insert the angled surgical forceps towards the base of the scrotum. Carefully pull the distal epididymal fat to localize the cauda epididymidis.</li>
		<li>Localize the vas deferens and position the straight surgical forceps below the vas deferens. In the vas deferens, position the bulldog tweezer clamp, keeping enough space for opening the straight surgical forceps that must be between the cauda epididymidis and the bulldog tweezer clamp (if necessary, limit the opening of the straight surgical forceps to avoid causing damage to the vas deferens) (Figure 3A).</li>
		<li>Position the Hamilton syringe, preloaded with the injection solution, at an angle of approximately 10-15&#176; parallel to the vas deferens and insert the needle bevel oriented up (Figure 3A, and Supplementary Figure 1).</li>
		<li>Squeeze the straight surgical forceps to loosen the vas deferens, and then slowly lower the syringe plunger until all the stimulus solution is expelled (Figure 3A). 
		NOTE: Maximum injection volume: 10 &#181;L.</li>
		<li>Remove the straight surgical forceps. Position the curved surgical forceps in the needle insert region, pinch gently, and carefully remove the needle.</li>
		<li>After ~30 s, remove the surgery curve forceps and the bulldog tweezer clamp. Return gently the vas deferens to the scrotum.</li>
		<li>Localize the vaginal tunic and make one suture stitch with the needle holder and size 5/0 surgical suture (Supplementary Figure 1). 
		NOTE: Check that the incision in the vaginal tunic is closed; if necessary, make another stitch.</li>
		<li>Using the index finger, gently move the right testis and epididymis back to the abdomen and position the left testis and epididymis in the scrotum.</li>
		<li>Repeat the steps 2.2.3-2.2.10 to make the intravasal injection on the left side.</li>
		<li>Close the skin incision with two suture stitches with the needle holder and size 5/0 surgical sutures (Figure 3A).</li>
	</ol>
	</li>
	<li>Postoperative care
	<ol>
		<li>Observe the mouse postoperatively to ensure appropriate recovery (check animal behavior, shivering, locomotion, and other signs of toxicity). Keep animals warm until they recover from surgery. It is expected that the mouse will move more slowly within the cage after surgery, returning to normal behavior after ~12-24 h.</li>
	</ol>
	</li>
</ol>

<ol><li>Hinton, B., Robaire, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Epididymis%5BTitle%5D)+AND+%22Knobil+and+Neill%27s+Physiology+of+Reproduction+Volume+1%22%5BJournal%5D)">The Epididymis.</a> Knobil and Neill's Physiology of Reproduction Volume 1. 1, 691-771 (2015).</li>
<li>Avellar, M. C. W., Hinton, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epididymis%5BTitle%5D)+AND+%22Encyclopedia+of+Endocrine+Diseases%22%5BJournal%5D)">Epididymis.</a> Encyclopedia of Endocrine Diseases. , 807-813 (2019).</li>
<li>Johnston, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mouse+epididymal+transcriptome%3A+Transcriptional+profiling+of+segmental+gene+expression+in+the+epididymis%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">The mouse epididymal transcriptome: Transcriptional profiling of segmental gene expression in the epididymis.</a> Biol Reprod. 73 (3), 404-413 (2005).</li>
<li>Fijak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infectious%2C+inflammatory+and+%27Autoimmune%27+male+factor+infertility%3A+How+do+rodent+models+inform+clinical+practice%5BTitle%5D)+AND+%22Hum+Reprod+Update%22%5BJournal%5D)">Infectious, inflammatory and 'Autoimmune' male factor infertility: How do rodent models inform clinical practice.</a> Hum Reprod Update. 24 (4), 416-441 (2018).</li>
<li>Rodrigues, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+toll-like+receptor+4+%28TLR4%29+by+in+vivo+and+in+vitro+exposure+of+rat+epididymis+to+lipopolysaccharide+from+Escherichia+coli%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Activation of toll-like receptor 4 (TLR4) by in vivo and in vitro exposure of rat epididymis to lipopolysaccharide from Escherichia coli.</a> Biol Reprod. 79 (6), 1135-1147 (2008).</li>
<li>Palladino, M. A., Johnson, T. A., Gupta, R., Chapman, J. L., Ojha, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Members+of+the+toll-like+receptor+family+of+innate+immunity+pattern-recognition+receptors+are+abundant+in+the+male+rat+reproductive+tract%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Members of the toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract.</a> Biol Reprod. 76 (6), 958-964 (2007).</li>
<li>Pleuger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+regional+distribution+of+resident+immune+cells+shapes+distinct+immunological+environments+along+the+murine+epididymis%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis.</a> eLife. 11, e82193(2022).</li>
<li>Battistone, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Region-specific+transcriptomic+and+functional+signatures+of+mononuclear+phagocytes+in+the+epididymis%5BTitle%5D)+AND+%22Mol+Hum+Reprod%22%5BJournal%5D)">Region-specific transcriptomic and functional signatures of mononuclear phagocytes in the epididymis.</a> Mol Hum Reprod. 26 (1), 14-29 (2020).</li>
<li>Mendelsohn, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+initial+segment+to+cauda%3A+A+regional+characterization+of+mouse+epididymal+CD11c%2B+mononuclear+phagocytes+based+on+immune+phenotype+and+function%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">From initial segment to cauda: A regional characterization of mouse epididymal CD11c+ mononuclear phagocytes based on immune phenotype and function.</a> Am J Physiol Cell Physiol. 319 (6), C997-C1010 (2020).</li>
<li>Pleuger, C., Silva, E. J. R., Pilatz, A., Bhushan, S., Meinhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+immune+response+to+infection+and+acute+inflammation+along+the+epididymis%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Differential immune response to infection and acute inflammation along the epididymis.</a> Front Immunol. 11, 3125(2020).</li>
<li>Avellar, M. C. W., Ribeiro, C. M., Dias-da-Silva, M. R., Silva, E. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+search+of+new+paradigms+for+epididymal+health+and+disease%3A+Innate+immunity%2C+inflammatory+mediators%2C+and+steroid+hormones%5BTitle%5D)+AND+%22Andrology%22%5BJournal%5D)">In search of new paradigms for epididymal health and disease: Innate immunity, inflammatory mediators, and steroid hormones.</a> Andrology. 7 (5), 690-702 (2019).</li>
<li>Michel, V., Pilatz, A., Hedger, M. P., Meinhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epididymitis%3A+Revelations+at+the+convergence+of+clinical+and+basic+sciences%5BTitle%5D)+AND+%22Asian+J+Androl%22%5BJournal%5D)">Epididymitis: Revelations at the convergence of clinical and basic sciences.</a> Asian J Androl. 17 (5), 756-763 (2015).</li>
<li>Nicholson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+epididymo-orchitis+in+primary+care%3A+Results+from+a+large+UK+primary+care+database%5BTitle%5D)+AND+%22Br+J+Gen+Pract%22%5BJournal%5D)">Management of epididymo-orchitis in primary care: Results from a large UK primary care database.</a> Br J Gen Pract. 60 (579), e407-e422 (2010).</li>
<li>Pilatz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+epididymitis+revisited%3A+Impact+of+molecular+diagnostics+on+etiology+and+contemporary+guideline+recommendations%5BTitle%5D)+AND+%22Eur+Urol%22%5BJournal%5D)">Acute epididymitis revisited: Impact of molecular diagnostics on etiology and contemporary guideline recommendations.</a> Eur Urol. 68 (3), 428-435 (2015).</li>
<li>Lang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+functional+integrity+of+spermatozoa+is+compromised+as+a+consequence+of+acute+uropathogenic+E.+coli-associated+epididymitis%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Structural and functional integrity of spermatozoa is compromised as a consequence of acute uropathogenic E. coli-associated epididymitis.</a> Biol Reprod. 89 (59), 1-10 (2013).</li>
<li>Rusz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+urogenital+infections+and+inflammation+on+semen+quality+and+male+fertility%5BTitle%5D)+AND+%22World+J+Urol%22%5BJournal%5D)">Influence of urogenital infections and inflammation on semen quality and male fertility.</a> World J Urol. 30 (1), 23-30 (2012).</li>
<li>Schuppe, H. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Urogenital+infection+as+a+risk+factor+for+male+infertility%5BTitle%5D)+AND+%22Dtsch+Arztebl+Int%22%5BJournal%5D)">Urogenital infection as a risk factor for male infertility.</a> Dtsch Arztebl Int. 114 (19), 339-346 (2017).</li>
<li>Andrade, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipopolysaccharide-induced+epididymitis+modifies+the+transcriptional+profile+of+Wfdc+genes+in+mice%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Lipopolysaccharide-induced epididymitis modifies the transcriptional profile of Wfdc genes in mice.</a> Biol Reprod. 104 (1), 144-158 (2021).</li>
<li>Andrade, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+modulation+of+toll-like+receptor+signaling+pathway+genes+in+acute+epididymitis+in+mice%5BTitle%5D)+AND+%22Andrology%22%5BJournal%5D)">Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice.</a> Andrology. 12 (5), 1024-1037 (2024).</li>
<li>Silva, E. J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipopolysaccharide+and+lipotheicoic+acid+differentially+modulate+epididymal+cytokine+and+chemokine+profiles+and+sperm+parameters+in+experimental+acute+epididymitis%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Lipopolysaccharide and lipotheicoic acid differentially modulate epididymal cytokine and chemokine profiles and sperm parameters in experimental acute epididymitis.</a> Sci Rep. 8 (1), 103(2018).</li>
<li>Cheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-Like+receptors+4+and+5+cooperatively+initiate+the+innate+immune+responses+to+uropathogenic+Escherichia+coli+infection+in+mouse+epididymal+epithelial+cells%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Toll-Like receptors 4 and 5 cooperatively initiate the innate immune responses to uropathogenic Escherichia coli infection in mouse epididymal epithelial cells.</a> Biol Reprod. 94 (3), 58(2016).</li>
<li>Stammler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epididymitis%3A+Ascending+infection+restricted+by+segmental+boundaries%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">Epididymitis: Ascending infection restricted by segmental boundaries.</a> Hum Reprod. 30 (7), 1557-1565 (2015).</li>
<li>Turner, T. T., Bomgardner, D., Jacobs, J. P., Nguyen, Q. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+segmentation+of+the+epididymal+interstitium+with+segmented+tubule+function+in+rats+and+mice%5BTitle%5D)+AND+%22Reproduction%22%5BJournal%5D)">Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice.</a> Reproduction. 125 (6), 871-878 (2003).</li>
<li>Barrachina, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CX3CR1+deficiency+leads+to+impairment+of+immune+surveillance+in+the+epididymis%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">CX3CR1 deficiency leads to impairment of immune surveillance in the epididymis.</a> Cell Mol Life Sci. 80 (1), 15(2022).</li>
<li>Klein, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+tissue-specific+damage+caused+by+bacterial+epididymo-orchitis+in+the+mouse%5BTitle%5D)+AND+%22Mol+Hum+Reprod%22%5BJournal%5D)">Differential tissue-specific damage caused by bacterial epididymo-orchitis in the mouse.</a> Mol Hum Reprod. 26 (4), 215-227 (2020).</li>
</ol>

The authors declare that they have no conflicts of interest.

Here, two distinct surgical injection procedures for the induction of region-specific epididymitis in mice are presented. They are used to perform experimental models of epididymitis specifically targeting the initial segment via interstitial injection or the cauda epididymidis via retrograde intravasal injection. The detailed and visual guide increases the clarity and accessibility of these protocols, seeking to enhance the reproducibility of these methods.
The diameter of t...

The epididymis is a mucosal male reproductive organ composed of a single, highly convoluted tubule that connects the efferent ducts to the vas deferens. The epididymis is essential for sperm maturation (acquisition of sperm motility and fertilizing ability), concentration, transport, storage, and protection until the ejaculation1,2. Classically, the rodent epididymis has four anatomical regions: the initial segment, caput, corpus, and cauda1 (Figure 1). Furthermore, each region of the epididymis can be subdivided into additional segments separated by interstitial connective septa, indicating the presence of 10 segments in the mouse epididymis3 (Figure 1).
The immunobiology of the epididymis is an expanding field of investigation due to the association between immune dysregulation and male infertility4. Studies in the last two decades have unraveled that the epididymis is populated by different components of the immune system, such as resident immune cell types (e.g., mononuclear phagocytes and lymphocytes) and pathogen-recognition receptors [PRRs, e.g., Toll-like receptors (TLRs) and NOD-like receptors] expressed on several non-immune and immune cell types, which together create dynamic region-specific epididymal microenvironments5,6,7,8,9. These epididymal immune components play a crucial role in the dual ability of the epididymis to coordinate immune tolerance towards immunogenic spermatozoa while rapidly triggering immune responses to ascending urethral infections and other harmful stimuli4,10. Factors affecting this intricate immune balance in the epididymis warrant further investigation since they can be detrimental to epididymal function and, thus, may negatively impact male fertility.
In this context, inflammatory diseases of the epididymis, collectively known as epididymitis, are highly prevalent in men of all ages and are important etiological factors in male subfertility and infertility4,11,12. Epididymitis is the fifth most common male urological diagnosis in men between 18 and 50 years old, with ~600,000 cases per year in the US alone4,13. Ascending canalicular infections with urogenital pathogens (e.g., Escherichia coli) or sexually transmitted diseases (e.g., Chlamydia trachomatis) are the most common cause of epididymitis4,14. Most epididymitis patients experience pain in the cauda epididymidis but less frequently in the caput region, underscoring the clinical implications associated with the ability of the epididymis to trigger region-specific responses to inflammatory stimuli10. Epididymitis can cause profound deterioration in seminal parameters (e.g., sperm concentration and motility), together with pronounced leukocytospermia in the acute phase of the disease4,12,15. Notably, a substantial cohort of epididymitis patients suffers from persistent oligozoospermia or azoospermia, despite successful pharmacological therapy and symptom remission16,17. The mechanisms underlying the long-term impact of epididymitis outcomes on sperm quality remain poorly understood.
Human acute epididymitis samples are rare, since epididymal biopsies are contraindicated due to the risk of uncontrolled dissemination of pathogens through the puncture, which can lead to sepsis and organ damage, and sperm leakage to the interstitial compartment due to duct rupture4. Thus, animal models of epididymitis represent relevant tools to investigate the disease mechanisms, allowing the evaluation of inflammatory responses and outcomes4,12. In this regard, rodents (rats and mice) represent the most common model organisms used to study epididymitis in a controlled experimental setting4,12. Different rodent models of bacterial epididymitis have been established based on the type of pathogen (e.g., uropathogenic E. coli and C. trachomatis), infection routes (e.g., retrograde canalicular injection and interstitial injection), local of infection (e.g., initial segment and cauda epididymidis), and temporal post-injection course (e.g., hours to days)4,12.
The use of pathogen-associated molecular patterns (PAMPs) represents a valuable tool to study epididymitis, since it allows the investigation of pathophysiological mechanisms of the disease triggered by different molecules associated with distinct pathogens (e.g., bacteria, viruses, and fungi) through the activation of specific PRRs, such as TLRs and signaling pathways5,18,19,20,21. For instance, PAMPs derived from the cell wall of Gram-negative and Gram-positive bacteria, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), respectively, have been used to elicit epididymitis through multiple administration routes, including intravenous, intraperitoneal, or via direct injection into the epididymal regions10. Under these conditions, both LPS and LTA have been shown to induce inflammatory responses in the epididymis in rats and mice, associated with modulation of a distinct subset of inflammatory mediators and immune cell recruitment in a region- and temporal-specific manner19,20. In particular, the region-specific administration of PAMPs in the interstitial compartment of the initial segment or in the luminal compartment of the vas deferens toward the cauda epididymidis of mice recently emerged as relevant models to investigate the proximal and distal response of the epididymis to different inflammatory stimuli, thus providing a framework for the understanding of the different immune environments of the organ and their contributions to disease outcomes4,18,19.
Here, two different experimental protocols are presented for inducing direct inflammatory stimuli in distinct regions of the mouse epididymis, namely the initial segment and the cauda epididymidis, using interstitial and retrograde intravasal injection routes, respectively. Combined, these different approaches permit a regional delivery of PAMPs and the evaluation of opposite regions of the epididymis during inflammation.

<table><tbody><tr><td>Angled blunt-ended surgical forceps&amp;nbsp;</td><td>Bonther</td><td>xtycga1012</td><td></td></tr><tr><td>Angled surgical forceps</td><td>Bonther</td><td>pwghvv1012</td><td></td></tr><tr><td>Blue Evans solution</td><td>Sigma-Aldrich</td><td>E2129</td><td></td></tr><tr><td>Bulldog tweezer clamp&amp;nbsp;</td><td>Bonther</td><td>jtkpqs1012</td><td></td></tr><tr><td>Chlorhexidine</td><td>Rioqu&amp;iacute;mica</td><td>Riohex 0.25%</td><td></td></tr><tr><td>Curve surgical forceps</td><td>Bonther</td><td>506e6e1012</td><td></td></tr><tr><td>Electric trimmer pen shape</td><td>-</td><td>-</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton</td><td>80400</td><td></td></tr><tr><td>Headband magnifier&amp;nbsp;</td><td>OptiVISOR</td><td>-</td><td></td></tr><tr><td>Ketamine</td><td>Venco Animal Health</td><td>Ketalex</td><td></td></tr><tr><td>LPS&amp;nbsp; from E. coli 055:B55</td><td>Invivogen</td><td>Tlrl-pb5lps</td><td></td></tr><tr><td>LTA from Staphylococcus aureus</td><td>Invivogen</td><td>Tlrl-pslta</td><td></td></tr><tr><td>Needle holder</td><td>Bonther</td><td>8pihbf1012</td><td></td></tr><tr><td>Size 5/0 surgical suture</td><td>Shalon Medical</td><td>n550cti20</td><td></td></tr><tr><td>Sterile saline</td><td>-</td><td>-</td><td></td></tr><tr><td>Straight surgical forceps</td><td>Bonther</td><td>ejaiuz1012</td><td></td></tr><tr><td>Xylazine</td><td>Syntec</td><td>Xilazin</td><td></td></tr></tbody></table>

mouse models of epididymitis induced by pathogen-associated molecular patterns

Mouse Models of Epididymitis Induced by Pathogen-Associated Molecular Patterns

Epididymitis is a highly prevalent disease in the outpatient urologic clinic and a relevant factor in male infertility. Bacterial infections, including sexually transmitted bacteria and common uropathogens, represent the most common etiological factors of epididymitis. Nevertheless, the pathophysiological mechanisms underlying the onset, natural history, and outcomes of bacterial epididymitis remain poorly understood. In this regard, rodent models are valuable tools for investigating the mechanistic responses of the epididymis when challenged with various inflammatory or infectious stimuli. Studies in rats and mice showed that bacterial-derived pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from Gram-negative and Gram-positive bacteria, respectively, trigger acute inflammation in the epididymis. The severity of these inflammatory responses differs according to the epididymal region affected and is associated with poor sperm parameters. Herein, two distinct surgical approaches are described in detail for inducing epididymitis in mice through the injection of PAMPs in the interstitial compartment of the initial segment or the luminal compartment of the vas deferens toward the cauda epididymidis, thus allowing investigation of the inflammatory responses of the epididymal proximal and distal regions. It is expected that these experimental models of epididymitis will enable future studies aimed at advancing the understanding of the mechanisms governing the epididymal responses to invading pathogens, opening novel ways for therapeutic interventions to mitigate the repercussions of epididymitis on male fertility.

Here, two distinct surgical injection models for inflammatory stimuli in the initial segment (interstitial injection) and cauda epididymidis (intravasal injection) in mice are described. The former triggers inflammatory responses in the proximal epididymis, whereas the latter triggers them in the distal epididymis.
The interstitial injection is performed through an incision in the abdomen, making use of the possibility of moving...

Here, two distinct surgical injection models are described for inducing inflammatory stimuli in the initial segment (interstitial injection) and cauda epididymidis (intravasal injection) of mice. These methods enable studies of the region-specific responses in the different regions of the epididymis.

Epididymitis is a highly prevalent disease in the outpatient urologic clinic and a relevant factor in male infertility. Bacterial infections, including ...

mouse-models-epididymitis-induced-pathogen-associated-molecular

Research

JoVE Journal

Immunology and Infection

Source: Conover, M. S., et al., J. Establishment and Characterization of UTI and CAUTI in a Mouse Model. J. Vis. Exp. (2015)This video demonstrates the procedure for assembling and implanting a transurethral catheter in a female mouse, followed by bacterial inoculation to establish catheter-associated urinary tract infection and investigate biofilm-mediated host-pathogen interactions.

Catheter-Associated Urinary Tract Infection in a Mouse Model

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work’s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Establishment and Characterization of UTI and CAUTI in a Mouse Model

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm – neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Preterm birth (delivery &lt; 37 weeks) is an urgent global health issue with suboptimal prevention and treatment options. We present a mouse model of ascending vaginal bacterial infection-induced preterm birth and outline how to analyze the resulting neonatal morbidity and mortality.

Modeling Ascending Vaginal Infection, Preterm Birth, and Neonatal Morbidity in Mice

This protocol describes the generation of a murine prostate organoid-based model grown in 2D. It includes organoid dissociation, cell seeding, differentiation, epithelial integrity assessment, and in-vitro infection with uropathogenic Escherichia coli. The model provides apical accessibility and is suitable for studying host-pathogen interactions, overcoming limitations of traditional 3D organoid systems.

Generation of a Mouse Prostate Organoid-Based Model for Studying Host-Pathogen Interactions

Learn surgical injection models for inducing epididymitis in mice using interstitial and intravasal techniques. These methods enable region-specific study of inflammatory responses in reproductive tissues.

Mouse Models of Epididymitis Induced by Pathogen-Associated Molecular Patterns

Explore More Videos