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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Create PCOS-like Mouse Model
NOTE: The mice used in this study were a mixed background (C57/B6, CD1, 129Sv) and were maintained with food and water&#160;ad libitum&#160;in a 14/10 h light/dark cycle at 24 &#176;C.
<ol>
	<li>DHT Pellet Preparation
	<ol>
		<li>Autoclave silastic tubing to sterilize. Cut silastic tubes to a length of 15 mm with a razor blade.</li>
		<li>Seal one side by injecting medical adhesive silicone into the tube with a 20 G&#160;blunted needle attached to a 3 mL syringe. Plunger should be withdrawn from the syringe and then adhesive inserted into the reservoir. The plunger is reinserted and adhesive pushed down until it begins to emerge from the needle. The blunted needle is made by cutting the sharp tip side of 20 G needle with any strong scissors. The length of silicone in the tube should be more than 2 mm to allow for post-production trimming.</li>
		<li>Dry overnight; check for air bubbles on the sealed side. Those with no air bubbles are used for DHT pellets. Others can be used for no-DHT control pellets.</li>
		<li>Wear gloves, mask, goggles and lab coat before making DHT-pellet in a hood to avoid DHT exposure to skin.</li>
		<li>Pour DHT powder into a plastic weigh boat and press the open side of the adhesive capped tubes (produced in 1.1.1) into the DHT powder.</li>
		<li>DHT powder can be tamped down with a large paper clip that is straightened. Continue until DHT reaches a height of 4 mm (or desired length). Check the length of DHT with a ruler, and seal the open side with silicone, dry overnight.</li>
		<li>Cut each sealed side to make the length of silicone 2 mm long. The total length of pellet will be 8 mm.</li>
		<li>Seal both side of empty silastic tubing as control (no-DHT) pellet.</li>
		<li>Keep pellets in a 50 mL conical tube wrapped with foil (to prevent light exposure) at room temperature until use. The DHT pellets maintain full efficacy for at least 3 months of storage.</li>
	</ol>
	</li>
	<li>DHT Insertion and Replacement
	<ol>
		<li>Up to 20 DHT pellets or control pellets are submerged separately in a 50 mL conical tube with 30 mL sterile 0.9% saline for 24 h at 37&#160;&#176;C for equilibration just before insertion.</li>
		<li>Prior to surgery, the surfaces and gloves are disinfected with clidox and the work surfaces are covered with clean plastic-backed absorbent paper (surgical pad).&#160;All instruments that come in contact with animals are decontaminated prior to entering the animal facility by autoclaving.&#160; Instruments will be allowed to dry and cool prior to use.</li>
		<li>2-month-old female mice are used (4&#8211;5 mice/cage). Mice&#160;are injected intraperitoneally with Xylazine (3.5 mg/kg bw)&#160;and Ketamine (78.8 mg/kg bw) using an insulin syringe. Calculations for mixing and dosing anesthesia are in&#160;Table 1.&#160;</li>
		<li>After adequate anesthesia is achieved, as measured by loss of toe reflex and slowed breathing, the mouse will be prepped for surgery.</li>
		<li>Disinfect the skin of the area with betadine using sterile gauze and clean with 70% ethanol. &#160;The area will again be painted with betadine.</li>
		<li>Cut a hole around 5 mm length with scissors under the skin near the neck. Using a 10 G trochar, make a small tunnel (15 mm) in the rostral direction. The pellet is inserted dorsally with the trochar.</li>
		<li>The opening is then sealed with surgical adhesive. Manually approximate the wound edges with forceps and gentle brushing strokes to apply a thin film of liquid adhesive to the approximated wound edges. Buildup 3 thin layers of adhesive to ensure the adhesive is evenly distributed over the wound. The adhesive should extend 1 cm on each side of the apposed wound edges. Suture or surgical clips can also be used to close the hole. Put mice back into cage, individually housed, with a heat pad for recovery. Replace pellet every 4 weeks to maintain a constant level of androgen exposure. The original pellet will be removed and new pellet will be inserted as described above, in a similar position.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ketamine 
			
			(100 mg/mL)
			</td>
			<td>Xylazine
			
			(100 mg/mL)
			</td>
			<td>Saline
			
			(0.9%)
			</td>
		</tr>
		<tr>
			<td>175 &#956;L</td>
			<td>15 &#956;L</td>
			<td>810 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Intraperitoneal injection of working solution: 0.1 mL/20g body weight</td>
		</tr>
	</tbody>
</table>
Table 1. Ketamine/xylazine cocktail.

<table><tbody><tr><td>Crystalline 5&amp;alpha;-DHT powder&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A8380-1G</td><td></td></tr><tr><td>Dow Corning Silastic tubing</td><td>Fisher Scientific</td><td>11-189-15D</td><td>0.04in/1mm inner diameter x 0.085in/2.15mm outer diameter</td></tr><tr><td>Medical adhesive silicone</td><td>Factor II, InC.</td><td>&amp;nbsp;A-100</td><td></td></tr><tr><td>Goggles, lab coats, gloves and masks</td><td></td><td></td><td></td></tr><tr><td>3 mL Syring&amp;nbsp;</td><td>Becton, Dickinson and Company (BD)</td><td>30985</td><td></td></tr><tr><td>Attached needle: 20G</td><td>BD</td><td>305176</td><td></td></tr><tr><td>Ruler: any length than 10 cm with milimeter scale.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Vet one AnnSeA LA, MWI, Boise</td><td>NDC13985-704-10</td><td>100 mg/mL</td></tr><tr><td>Ketamine Hydrochloride</td><td>Hospira, Inc</td><td>NDC 0409-2051-05</td><td>100 mg/mL</td></tr><tr><td>Surgical staple</td><td>AutoClip&amp;reg; System, Fine Science Tool</td><td>12020-00</td><td></td></tr><tr><td>Insulin syringe</td><td>BD</td><td>329461</td><td>1/2 CC, low dose U-100 insulin syringe</td></tr><tr><td>Trocar&amp;nbsp;</td><td>Innovative Research of America</td><td>MP-182</td><td></td></tr><tr><td>Razor blade</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Clidox</td><td>Fisher Scientific</td><td>NC0089321</td><td></td></tr><tr><td>Surgical underpad</td><td>Fisher Scientific</td><td>50587953</td><td>Andwin Scientific&amp;nbsp;56616018</td></tr><tr><td>Betadine Antiseptic Solution</td><td>Walgreens</td><td></td><td></td></tr><tr><td>3M Vetbond (n-butyl cyanoacrylate)</td><td>3M Science. Applied to Life</td><td></td><td></td></tr></tbody></table>

subcutaneous dihydrotestosterone (dht) treatment: mouse model of polycystic ovary syndrome (pcos)

Source: Xue, P., et al. <a href="https://www.jove.com/video/58379/a-hyperandrogenic-mouse-model-to-study-polycystic-ovary-syndrome">A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome</a>.&#160;J. Vis. Exp. (2018).
This article describes a way to prepare and insert dihydrotestosterone (DHT) pellets to model Polycystic Ovary Syndrome (PCOS) in mice. The example protocol demonstrates the procedure in mice used for reproductive and metabolic studies. &#160;&#160;

Subcutaneous Dihydrotestosterone (DHT) Treatment: Mouse Model of Polycystic Ovary Syndrome (PCOS)

Source: Xue, P., et al. A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome.&#160;J. Vis. Exp. (2018).
This article describes a way to ...

First, to prepare the pellet, inject medical adhesive silicone down one end of a pre-cut piece of silicone tubing. Next, pack dihydrotestosterone or DHT crystal powder tightly inside the tubing. Then, use medical adhesive silicone to seal the other end of the tubing. Pellets should have equal amounts of silicone at each end with no bubbles. Place pellets in saline solution before inserting them in a female mouse.
On an anesthetized mouse, use a cotton swab with disinfectant to clean the shaved surgical area. Make a small incision through the skin near the neck. Use a trochar to make a tunnel under the skin in the direction of the mouse&#39;s nose. Insert the pellet into the tunnel. Lastly, seal the incision with surgical adhesive. The pellet will release a steady dose of DHT and is replaced every 4 weeks to ensure prolonged exposure to the drug.

subcutaneous-dihydrotestosterone-dht-treatment-mouse-model-polycystic

Research

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Adult

1.6K Views. Źródło: Xue, P., et al. Hiperandrogeniczny model myszy do badania zespołu policystycznych jajników. J. Vis. Exp. (2018). Ten artykuł opisuje sposób przygotowania i wstawiania granulek dihydrotestosteronu (DHT) do modelowania zespołu policystycznych jajników (PCOS) u myszy. Przykładowy protokół demonstruje procedurę na myszach wykorzystywanych do badań reprodukcyjnych i metabolicznych.    

Video: Leczenie podskórnego dihydrotestosteronu (DHT): mysi model zespołu policystycznych jajników (PCOS) - Concept

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

JoVE Journal

Cancer Research

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Developmental Biology

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. ...

Subcutaneous Dihydrotestosterone (DHT) Treatment: Mouse Model of Polycystic Ovary Syndrome (PCOS)

Transkrypcja

Subcutaneous Dihydrotestosterone (DHT) Treatment: Mouse Model of Polycystic Ovary Syndrome (PCOS)