The study was supported by National Key Research and Development Program of China (grants 2021YFC2700701), the National Natural Science Foundation of China (grants 82088102, 82071731, 82171613, 8227034, 81601238), Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant 2019-I2M-5-064), the Science and Technology Commission of Shanghai Municipality (grants 21Y11907600), Shanghai Municipal Commission of Health and Family Planning (grant 20215Y0216), Collaborative Innovation Program of Shanghai Municipal Health Commission (grant 2020CXJQ01), Clinical Research Plan of Shanghai Hospital Development Center (grant SHDC2020CR1008A), Shanghai Clinical Research Center for Gynecological Diseases (grant 22MC1940200), Shanghai Urogenital System Diseases Research Center (grant 2022ZZ01012), Shanghai Frontiers Science Research Base of Reproduction and Development, The Science and Technology Commission of Quzhou Municipality (grant 2022K54), Open Fund Project of Key Laboratory of Reproductive Genetics, Ministry of Education, Zhejiang University (grant KY2022035), and Open Fund Project of Guangdong Academy of Medical Sciences (grant YKY-KF202202).

All animal experimental protocols in this study were approved by&#160;Animal Ethics Committee&#160;of Fudan University. Female C57BL/6J mice, aged 4 weeks, were used here. The details of the animals, reagents, and equipment used in this study are listed in the Table of Materials.
1. Preparation of the mini-pump
<ol>
	<li>Take the letrozole powder (50 mg) stored in the original packaging bottle, centrifuge the letrozole powder in the bottle at 300 x g for 5 s (at room temperature), then open the cap.</li>
	<li>Slowly add 625 &#181;L of DMSO to the letrozole powder with a pipette and mix well to obtain a stock solution at a concentration of 8 mg/100 &#181;L, totaling 625 &#181;L. 
	NOTE: If using a different package size of letrozole powder, please calculate the required volume of DMSO in advance to configure the stock solution.&#160;The letrozole powder, DMSO, and other related compounds must be of sterile grade. The pipette tips, tubes, and other related items should be sterile or sterilized before use.</li>
	<li>Count the number of required mice according to the experimental design.</li>
	<li>Draw up the total amount of letrozole stock solution required with a pipette, depending on the number of required mice, and inject it into the centrifuge tube slowly. 
	NOTE: 4-week-old mice for an 8-week exposure were selected to induce the PCOS mice model. The total dose of letrozole is 8 mg for each mouse, which requires two consecutive pumps to be implanted (one pump releases for 4 weeks), i.e., 4 mg/4 weeks per pump. As mentioned in step 1.2, the concentration of the letrozole stock solution is 8 mg/100 &#181;L. To prepare the pumps, 50 &#181;L of stock solution is required for each pump as calculated below: a total of 4 mg letrozole divided by 8 mg/100 &#181;L.</li>
	<li>Draw up an equal amount of PEG300, add it to the letrozole solution, and mix well (it can be vortexed appropriately).</li>
	<li>Prepare the control vehicle solution for the control group (50 &#181;L of DMSO + 50 &#181;L of PEG300 for each pump without letrozole).</li>
	<li>Prepare an adequate number of sterile centrifuge tubes according to the number of pumps required; one 15 mL centrifuge tube needs to be prepared for each pump.</li>
	<li>Draw saline with a syringe and add 5 mL of saline to each centrifuge tube. Then, cap the centrifuge tubes and place them on a tube rack for further use.</li>
	<li>Wear a new pair of sterile gloves, take out the mini-pumps in the desired quantity, and place them on a clean, sterile countertop or container.</li>
	<li>Gently pinch the cap that matches the pump body with the thumb and forefinger, and hold the pump body with the other hand.</li>
	<li>Slowly poke the filling port of the pump several times with the thin tube attached to the cap (Figure 1B). 
	NOTE: If not unclogged, fluid may not flow freely in the tube after filling.</li>
	<li>Take the needle that matches the pump and a 1 mL sterile syringe. Attach the needle to the syringe and make it tight (Figure 1C).</li>
	<li>Carefully and slowly draw up the final solution of letrozole configured above with the newly assembled syringe (Figure 1D). 
	NOTE: Aspiration of the letrozole solution needs to be done slowly and evenly. Rapid aspiration may generate air bubbles, which may affect the subsequent drug solution injection.</li>
	<li>Inject the letrozole solution into the mini-pump.
	<ol>
		<li>Carefully pinch a mini-pump with one hand so that it stands upright, and with the other hand, hold the aforementioned 1 mL syringe and slowly insert it into the very bottom of the pump (Figure 1E). 
		NOTE: If the needle is not inserted into the bottom of the pump, the liquid in the pump can easily bubble during injection, which will affect the pump&#39;s subsequent release of the drug.</li>
		<li>Then slowly inject the configured solution; the injection can be noticed when the opening of the pump slightly bubbles.</li>
		<li>Inject more slowly when the injection volume approaches 90 &#181;L. Once the pump is fully filled (about 100 &#181;L), withdraw the syringe.</li>
		<li>Carefully insert the cap into the pump body and make it tight; at this point, a small amount of solution will spill out. Carefully wipe it away.</li>
	</ol>
	</li>
	<li>Take the pump loaded with the drug, with the outlet facing upwards (the end with the cap), take the previously prepared centrifuge tube, and fully immerse the pump in sterile saline (Figure 1F). Then, cap the centrifuge tube and place it on the tube rack. 
	NOTE: Take care to avoid contaminating the pump throughout the process.</li>
	<li>After all the mini-pumps are made, place all the centrifuge tubes in a 37 &#176;C thermostat for 48 h.</li>
</ol>
2. Preparation for operation and anesthesia
<ol>
	<li>Prepare and sterilize all required instruments on the day of surgery, including forceps, hemostats, surgical scissors, needle holders, and 4-0 absorbable surgical sutures.</li>
	<li>Before using the isoflurane anesthesia machine (Figure 2B), carefully check whether the pipeline connection is correct and ensure the pipeline is intact and does not leak.</li>
	<li>Unscrew the filling sealing cap at the anesthesia machine&#39;s evaporator, then slowly add the anesthetic.</li>
	<li>Keep the anesthetic level between the upper and lower limits labeled on the anesthesia machine.</li>
	<li>Convert the three-way valve switch to ensure that the airflow from the anesthesia machine evaporator is connected to the anesthesia induction box.</li>
	<li>Open the air pump and the evaporator, then adjust the concentration of isoflurane for anesthesia induction (generally 3%-4%).</li>
	<li>Wait for about 1 min until the anesthetic fills the induction box, then put the mouse into the induction box and wait until complete anesthesia (this process takes about 2-3 min).</li>
	<li>Check that the mice are adequately anesthetized by gently tipping&#160;the box. If the mice are turned over and show no signs of returning to the recumbent position, anesthesia is induced.</li>
</ol>
3. Implantation of the letrozole mini-pump
<ol>
	<li>Put on a surgical mask and sterile gloves.</li>
	<li>Remove the pre-anesthetized mice from the induction box and apply the depilatory cream to the back of the mice, then wait for 1 min. Gently wipe off the depilatory cream and hair with wet gauze.</li>
	<li>At the same time, change the three-way valve switch to ensure that the airflow from the anesthesia machine evaporator is connected to the anesthesia mask and adjust the maintenance concentration (generally 2%).</li>
	<li>After dehairing, hold the mouse with the dominant hand and place its head/nose into the anesthesia mask.</li>
	<li>Check the depth of anesthesia by testing the pedal withdrawal reflex. 
	NOTE: All procedures involving anesthesia must be performed using gas filters fitted with activated charcoal tablets, and the procedure should be performed in a fume hood.</li>
	<li>Secure the mouse in the prone position on the operating plate and apply eye lubricant to both eyes.</li>
	<li>Apply povidone-iodine swabs three times to the exposed skin (Figure 2C). Then, inject meloxicam subcutaneously at a dose of 2 mg/kg.&#160;</li>
	<li>Confirm the anesthetic depth via a toe pinch again. Then, at the midline of the back of the neck, favoring the skin on the hairless side, pick up the skin with forceps and make an incision of approximately 0.5 cm laterally with a scalpel or surgical scissors (Figure 2D).</li>
	<li>Then open the epidermis underneath the incision and access it with a hemostat or needle holder to free the skin, making room for a mini-pump (roughly 2 cm away from the incision) (Figure 2E).</li>
	<li>Take the pump out of the saline in the centrifuge tube with sterile forceps, with the open end of the pump facing downward, and be ready to insert the pump subcutaneously in the mice (Figure 2F).</li>
	<li>Clamp up one side of the skin incision with forceps and push the pump into the subcutaneous space. There should be no resistance when entering the pump. Once inside, use the fingers to slightly and gently squeeze the pump inward as far as possible (Figure 1G).</li>
	<li>Suture the incision site carefully using 4-0 surgical sutures (Figure 2H). 
	NOTE: Take care not to damage the subcutaneous pump body when suturing.</li>
	<li>After suturing, disinfect the skin at the surgical site twice with iodophor swabs.</li>
</ol>
4. Animal recovery
<ol>
	<li>After surgery, gently transfer the mice from the mattress used during anesthesia to a dry, clean mattress. Ensure that the mice are in a warm and quiet environment.</li>
	<li>Closely monitor the conditions of the mice, such as the depth and frequency of their breaths.</li>
	<li>Observe the behavior of the mice as their mobility begins to return. Finally, ensure that they can stand and walk without any obvious abnormal behaviors such as dizziness or uncoordinated movements.</li>
	<li>After the mice have recovered, gently transfer them to pre-prepared cages. Place sterile jelly and food at the bottom of the cage for the mice to feed on.&#160; 
	NOTE: The stretching of the surgical site may prevent the mice from easily drinking and eating during the early postoperative period, so place jelly and food on the bottom of the cage.</li>
	<li>Inject meloxicam subcutaneously at a dose of 2 mg/kg (every 24 h for the first 3 days after surgery). After 8 weeks of letrozole treatment (i.e., two consecutive mini-pump implantations), test the mice models.</li>
</ol>

Polycystic Ovary Syndrome Induction in Mice Through Controlled Letrozole Release 

<ol>
	<li>Dapas, M., Dunaif, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+a+syndrome:+Genomic+insights+into+PCOS+causal+mechanisms+and+classification.">Deconstructing a syndrome: Genomic insights into PCOS causal mechanisms and classification.</a> Endocr Rev. 43 (6), 927-965 (2022).</li><li>Teede, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-m&#252;llerian+hormone+in+PCOS:+A+review+informing+international+guidelines.">Anti-m&#252;llerian hormone in PCOS: A review informing international guidelines.</a> Trends Endocrinol Metab. 30 (7), 467-478 (2019).</li><li>Joham, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+ovary+syndrome.">Polycystic ovary syndrome.</a> Lancet Diabetes Endocrinol. 10 (9), 668-680 (2022).</li><li>Pan, J. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+expression+and+DNA+methylation+of+lipid+metabolism+genes+in+PCOS:+A+new+insight+into+its+pathogenesis.">Aberrant expression and DNA methylation of lipid metabolism genes in PCOS: A new insight into its pathogenesis.</a> Clin Epigenetics. 10, 6 (2018).</li><li>Gao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitriol+attenuates+cardiac+remodeling+and+dysfunction+in+a+murine+model+of+polycystic+ovary+syndrome.">Calcitriol attenuates cardiac remodeling and dysfunction in a murine model of polycystic ovary syndrome.</a> Endocrine. 52 (2), 363-373 (2016).</li><li>Gao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+ovary+syndrome+fuels+cardiovascular+inflammation+and+aggravates+ischemic+cardiac+injury.">Polycystic ovary syndrome fuels cardiovascular inflammation and aggravates ischemic cardiac injury.</a> Circulation. 148 (24), 1958-1973 (2023).</li><li>Rosenfield, R. L., Ehrmann, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+polycystic+ovary+syndrome+(PCOS):+The+hypothesis+of+PCOS+as+functional+ovarian+hyperandrogenism+revisited.">The pathogenesis of polycystic ovary syndrome (PCOS): The hypothesis of PCOS as functional ovarian hyperandrogenism revisited.</a> Endocr Rev. 37 (5), 467-520 (2016).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+the+androgen+receptor+in+polycystic+ovary+syndrome.">Alternative splicing of the androgen receptor in polycystic ovary syndrome.</a> Proc Natl Acad Sci U S A. 112 (15), 4743-4748 (2015).</li><li>Noroozzadeh, M., Behboudi-Gandevani, S., Zadeh-Vakili, A., Ramezani Tehrani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone-induced+rat+model+of+polycystic+ovary+syndrome:+A+systematic+review.">Hormone-induced rat model of polycystic ovary syndrome: A systematic review.</a> Life Sci. 191, 259-272 (2017).</li><li>Tyndall, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+androgen+treatment+during+foetal+and/or+neonatal+life+on+ovarian+function+in+prepubertal+and+adult+rats.">Effect of androgen treatment during foetal and/or neonatal life on ovarian function in prepubertal and adult rats.</a> Reproduction. 143 (1), 21-33 (2012).</li><li>Jang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oriental+medicine+Kyung-ok-ko+prevents+and+alleviates+dehydroepiandrosterone-induced+polycystic+ovarian+syndrome+in+rats.">Oriental medicine Kyung-ok-ko prevents and alleviates dehydroepiandrosterone-induced polycystic ovarian syndrome in rats.</a> PLoS One. 9 (2), e87623 (2014).</li><li>Brawer, J. R., Munoz, M., Farookhi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+polycystic+ovarian+condition+(PCO)+in+the+estradiol+valerate-treated+rat.">Development of the polycystic ovarian condition (PCO) in the estradiol valerate-treated rat.</a> Biol Reprod. 35 (3), 647-655 (1986).</li><li>Mirabolghasemi, G., Kamyab, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+the+uterine+tissue+in+rats+with+polycystic+ovary+syndrome+induced+by+estradiol+valerate.">Changes of the uterine tissue in rats with polycystic ovary syndrome induced by estradiol valerate.</a> Int J Fertil Steril. 11 (1), 47-55 (2017).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amitriptyline+plays+important+roles+in+modifying+the+ovarian+morphology+and+improving+its+functions+in+rats+with+estradiol+valerate-induced+polycystic+ovary.">Amitriptyline plays important roles in modifying the ovarian morphology and improving its functions in rats with estradiol valerate-induced polycystic ovary.</a> Arch Pharm Res. 42 (4), 344-358 (2019).</li><li>Mamounas, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+letrozole+after+aromatase+inhibitor-based+therapy+in+postmenopausal+breast+cancer+(NRG+oncology/NSABP+B-42):+A+randomised,+double-blind,+placebo-controlled,+phase+3+trial.">Use of letrozole after aromatase inhibitor-based therapy in postmenopausal breast cancer (NRG oncology/NSABP B-42): A randomised, double-blind, placebo-controlled, phase 3 trial.</a> Lancet Oncol. 20 (1), 88-99 (2019).</li><li>Deveci, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+evaluation+of+rat+larynx+in+experimental+polycystic+ovary+syndrome+model.">Histological evaluation of rat larynx in experimental polycystic ovary syndrome model.</a> Eur Arch Otorhinolaryngol. 269 (8), 1945-1950 (2012).</li><li>Du, D. F., Li, X. L., Fang, F., Du, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+anti-m&#252;llerian+hormone+in+letrozole+rat+model+of+polycystic+ovary+syndrome.">Expression of anti-m&#252;llerian hormone in letrozole rat model of polycystic ovary syndrome.</a> Gynecol Endocrinol. 30 (12), 885-889 (2014).</li><li>Kelley, S. T., Skarra, D. V., Rivera, A. J., Thackray, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+microbiome+is+altered+in+a+letrozole-induced+mouse+model+of+polycystic+ovary+syndrome.">The gut microbiome is altered in a letrozole-induced mouse model of polycystic ovary syndrome.</a> PLoS One. 11 (1), e0146509 (2016).</li><li>Arroyo, P., Ho, B. S., Sau, L., Kelley, S. T., Thackray, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Letrozole+treatment+of+pubertal+female+mice+results+in+activational+effects+on+reproduction,+metabolism+and+the+gut+microbiome.">Letrozole treatment of pubertal female mice results in activational effects on reproduction, metabolism and the gut microbiome.</a> PLoS One. 14 (9), e0223274 (2019).</li><li>Pyun, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetragonia+tetragonioides+(pall.)+Kuntze+regulates+androgen+production+in+a+letrozole-induced+polycystic+ovary+syndrome+model.">Tetragonia tetragonioides (pall.) Kuntze regulates androgen production in a letrozole-induced polycystic ovary syndrome model.</a> Molecules. 23 (5), 1173 (2018).</li><li>Patel, R., Shah, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+sensitizers+modulate+GNRH+receptor+expression+in+PCOS+rats.">Insulin sensitizers modulate GNRH receptor expression in PCOS rats.</a> Arch Med Res. 49 (3), 154-163 (2018).</li><li>Chaudhari, N., Dawalbhakta, M., Nampoothiri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GNRH+dysregulation+in+polycystic+ovarian+syndrome+(PCOS)+is+a+manifestation+of+an+altered+neurotransmitter+profile.">GNRH dysregulation in polycystic ovarian syndrome (PCOS) is a manifestation of an altered neurotransmitter profile.</a> Reprod Biol Endocrinol. 16 (1), 37 (2018).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+Routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: Routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li><li>Lee, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Welsh+onion+root+(Allium+fistulosum)+restores+ovarian+functions+from+letrozole+induced-polycystic+ovary+syndrome.">Welsh onion root (Allium fistulosum) restores ovarian functions from letrozole induced-polycystic ovary syndrome.</a> Nutrients. 10 (10), 1430 (2018).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+medicine+(mahuang-tang)+improves+ovarian+dysfunction+and+the+regulation+of+steroidogenic+genes+in+letrozole-induced+PCOS+rats.">Traditional medicine (mahuang-tang) improves ovarian dysfunction and the regulation of steroidogenic genes in letrozole-induced PCOS rats.</a> J Ethnopharmacol. 248, 112300 (2020).</li><li>Corrie, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+updates+on+animal+models+for+understanding+the+etiopathogenesis+of+polycystic+ovarian+syndrome.">Recent updates on animal models for understanding the etiopathogenesis of polycystic ovarian syndrome.</a> Life Sci. 280, 119753 (2021).</li><li>Corbin, C. J., Trant, J. M., Walters, K. W., Conley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+testosterone+metabolism+associated+with+the+evolution+of+placental+and+gonadal+isozymes+of+porcine+aromatase+cytochrome+p450.">Changes in testosterone metabolism associated with the evolution of placental and gonadal isozymes of porcine aromatase cytochrome p450.</a> Endocrinology. 140 (11), 5202-5210 (1999).</li><li>Ryan, G. E., Malik, S., Mellon, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiandrogen+treatment+ameliorates+reproductive+and+metabolic+phenotypes+in+the+letrozole-induced+mouse+model+of+PCOS.">Antiandrogen treatment ameliorates reproductive and metabolic phenotypes in the letrozole-induced mouse model of PCOS.</a> Endocrinology. 159 (4), 1734-1747 (2018).</li><li>Bhattarai, P., Rijal, S., Bhattarai, J. P., Cho, D. H., Han, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+neurotransmission+on+gonadotropin-releasing+hormone+neurons+in+letrozole-induced+polycystic+ovary+syndrome:+A+mouse+model.">Suppression of neurotransmission on gonadotropin-releasing hormone neurons in letrozole-induced polycystic ovary syndrome: A mouse model.</a> Front Endocrinol (Lausanne). 13, 1059255 (2022).</li><li>Annie, L., Gurusubramanian, G., Roy, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+visfatin+by+fk866+mitigates+pathogenesis+of+cystic+ovary+in+letrozole-induced+hyperandrogenised+mice.">Inhibition of visfatin by fk866 mitigates pathogenesis of cystic ovary in letrozole-induced hyperandrogenised mice.</a> Life Sci. 276, 119409 (2021).</li><li>Mcdonald, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+blood+profile+of+C57BL/6+mice+exposed+to+chronic+unpredictable+stress.">Early blood profile of C57BL/6 mice exposed to chronic unpredictable stress.</a> Front Psychiatry. 10, 230 (2019).</li><li>Torres, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+a+healthy+gut+microbiome+protects+against+reproductive+and+metabolic+dysregulation+in+a+PCOS+mouse+model.">Exposure to a healthy gut microbiome protects against reproductive and metabolic dysregulation in a PCOS mouse model.</a> Endocrinology. 160 (5), 1193-1204 (2019).</li></ol>

The authors have nothing to disclose.

This report demonstrates a simple protocol for inducing PCOS in mice using easily accessible materials. The mouse PCOS model is essential for exploring PCOS mechanisms and screening drugs26. Of the available methods for inducing PCOS models in mice, letrozole induction is one of the most commonly used. The use of letrozole can develop and maintain a hyperandrogenic condition by inhibiting the conversion of testosterone to estradiol or by increasing testosterone synthesis27....

Polycystic ovary syndrome (PCOS) is one of the most common conditions among women of reproductive age1. It affects up to 18% of women globally and is the leading cause of female infertility worldwide2,3. PCOS is characterized by a series of interrelated reproductive abnormalities, including disturbed gonadotropin secretion, chronic anovulation, increased androgen production, and polycystic ovarian morphology4. In addition to gynecological disorders, PCOS also increases the risk of cardiovascular diseases5,6. Despite decades of research, the etiology of PCOS remains unclear7,8.
To gain better insights into the pathogenesis of PCOS and develop novel therapies, the creation of animal models that closely mimic human physiology is of tremendous importance9. Currently reported rodent models of PCOS include those induced by treatments with testosterone, letrozole, and estradiol valerate, among others. Testosterone induces hyperandrogenemia and is more commonly used as a PCOS inducer in rats10. DHEA has been used to induce PCOS in rodents, increasing testosterone levels, LH/FSH (luteinizing hormone/follicle-stimulating hormone) ratios, and causing irregular estrous cycles11. Estradiol valerate (EV) is a long-acting estrogen, and studies using this method show that levels of sex steroid hormones and gonadotropins vary depending on the dose of EV administered12,13,14. Letrozole is a nonsteroidal aromatase inhibitor15. Letrozole treatment induces non-cyclic estrus, increases ovarian weight, body weight, enlarges adipocytes, maximizes follicle development, and elevates testosterone levels in rats16,17. Letrozole-treated mice exhibit increased numbers of sinus follicles and hemorrhagic cysts, as well as elevated concentrations of LH, FSH, estradiol, and progesterone18,19.
Currently, the main methods for letrozole-induced PCOS modeling involve oral administration and subcutaneous injection, both of which require repeated daily dosing20,21,22. These methods are time-consuming and labor-intensive, and the repetitive administration likely causes significant stress to the animals23. Although some studies use letrozole pellets24,25, these products need to be customized and are expensive. This report describes a technique for inducing PCOS in mice using mini-pumps. This method is simple, time-saving, and uses surgical tools and equipment that are readily available in most laboratories.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Syringe</td>
			<td >Bofeng Biotech</td>
			<td >BD300841</td>
			<td >1ML (sterile)</td>
		</tr>
		<tr >
			<td >Centrifugation tube</td>
			<td>Biological Hope</td>
			<td>1850-K</td>
			<td>15ML (sterile)</td>
		</tr>
		<tr >
			<td >Pipette</td>
			<td >Eppendorf</td>
			<td >3123000268-A</td>
			<td>100&#956;L-1000&#956;L (sterile)</td>
		</tr>
		<tr >
			<td >Pipette</td>
			<td >TopPette</td>
			<td >7010101008</td>
			<td>10&#956;L-100&#956;L (sterile)</td>
		</tr>
		<tr >
			<td >Letrozole powder</td>
			<td >Sigma</td>
			<td >L6545-50MG</td>
			<td>Primary acting drugs (sterile)</td>
		</tr>
		<tr >
			<td >PEG(Poly(ethylene glycol))</td>
			<td >Solarbio</td>
			<td >P8250</td>
			<td>Used for dissolution (sterile)</td>
		</tr>
		<tr >
			<td >Sterile Physiological Saline Solution</td>
			<td >Biosharp</td>
			<td >BL158A</td>
			<td>Mini-pump storage</td>
		</tr>
		<tr >
			<td >Osmotic Pumps</td>
			<td >ALZET</td>
			<td >1004</td>
			<td>Letrozole storage and sustained release (sterile)</td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide</td>
			<td >Biosharp</td>
			<td >BS087</td>
			<td>Used for dissolution (sterile)</td>
		</tr>
		<tr >
			<td >Small Animal Anesthesia Machine</td>
			<td >RWD&#160;</td>
			<td >R500IP</td>
			<td>Used for anesthesia</td>
		</tr>
		<tr >
			<td >Isoflurane&#160;</td>
			<td>RWD</td>
			<td>20071302</td>
			<td>Used for anesthesia</td>
		</tr>
		<tr >
			<td >Hemostats</td>
			<td>Biosharp</td>
			<td>BS-HF-S-125</td>
			<td>Surgical instrument</td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td >Biosharp</td>
			<td >BS-SOR-S-100P</td>
			<td>Surgical instrument</td>
		</tr>
		<tr >
			<td >Needle holder</td>
			<td>ShangHaiJZ</td>
			<td>J32010</td>
			<td>Surgical instrument</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >RWD&#160;</td>
			<td>F12028</td>
			<td>Surgical instrument</td>
		</tr>
		<tr >
			<td >Needle and the 4-0 absorbable suture</td>
			<td >JINGHUAN</td>
			<td >CR413</td>
			<td>Surgical instrument</td>
		</tr>
		<tr >
			<td >Povidone-iodine swabs</td>
			<td >SingleLady</td>
			<td>GB26368-2010</td>
			<td>Skin disinfection</td>
		</tr>
		<tr >
			<td >Depilatory cream</td>
			<td >ZIKER BIOTECHNOLOGY</td>
			<td >ZK-L2701&#160;</td>
			<td>Depilation agent for laboratory animals</td>
		</tr>
		<tr >
			<td >Nitrile Gloves</td>
			<td >Biosharp</td>
			<td >BC040-L</td>
			<td>Used for aseptic operation</td>
		</tr>
		<tr >
			<td >Sterile gauze</td>
			<td >ZHENDE</td>
			<td >BA69087</td>
			<td>Used for wiping liquids</td>
		</tr>
		<tr >
			<td >C57BL/6J Mice</td>
			<td >Shanghai Model Organisms Center</td>
			<td >N/A</td>
			<td>Age: 4 weeks</td>
		</tr>
		<tr >
			<td >Pet Eye Lubricant</td>
			<td >BAITE</td>
			<td >SHF021153</td>
			<td>Used for mouse eye lubricant</td>
		</tr>
		<tr >
			<td >Meloxicam Injection</td>
			<td >MEIDAJIA</td>
			<td>R21064-21</td>
			<td>Used for mouse analgesia (sterile)</td>
		</tr>
	</tbody>
</table>

a modified technique for inducing polycystic ovary syndrome in mice

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of PCOS and for related drug development. Letrozole-induced mouse models replicate the metabolic and reproductive phenotypes of patients with PCOS. The traditional method of letrozole treatment in PCOS mice requires daily dosing over a certain period, which can be labor-intensive and cause significant stress to the mice. This study describes a simple and effective method for inducing PCOS in mice by implanting a controlled letrozole-releasing mini-pump. A mini-pump capable of stable, continuous release of a quantitative amount of letrozole was fabricated and implanted subcutaneously in mice under anesthesia. This study demonstrated that the mouse model successfully mimicked PCOS features after letrozole mini-pump implantation. The materials and equipment used in this study are readily available to most laboratories, requiring no special customization. Collectively, this article provides a unique, easy-to-perform method for inducing PCOS in mice.

The experimental protocol and some critical steps are shown in Figure 1 and Figure 2. Serum testosterone levels are displayed in Figure 3A. Letrozole mini-pump-treated mice (hereafter referred to as LTZ mice) exhibited significantly elevated serum testosterone levels compared to female control mice. Meanwhile, histological analysis of ovaries showed that LTZ mice showed polycystic ovaries with a significant reduction in corpora lute...

Author Spotlight: Investigating the Mechanisms and Inducing Models of Polycystic Ovary Syndrome

This protocol introduces a technique for inducing polycystic ovary syndrome (PCOS) models in mice through controlled letrozole release using mini-pumps. Under adequate anesthesia, the mini-pump was implanted subcutaneously, and PCOS was successfully induced in the mice after a certain period of the mini-pump release.

A Modified Technique for Inducing Polycystic Ovary Syndrome in Mice

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of ...

a-modified-technique-for-inducing-polycystic-ovary-syndrome-in-mice

Research

JoVE Journal

Medicine

783 Views.  Fudan University. This protocol introduces a technique for inducing polycystic ovary syndrome (PCOS) models in mice through controlled letrozole release using mini-pumps. Under adequate anesthesia, the mini-pump was implanted subcutaneously, and PCOS was successfully induced in the mice after a certain period of the mini-pump release.

Video: Author Spotlight: Investigating the Mechanisms and Inducing Models of Polycystic Ovary Syndrome

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options.

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...