We want to thank the animal laboratory of Shandong Provincial Hospital for technical support. This work was supported by the National Natural Science Foundation of China (NSFC 82101645), the Natural Science Foundation of Shandong Province, China (ZR2020QH088), and the Science and Technology Support Plan for Youth Innovation of Colleges in Shandong Province (2021KJ051).

The following protocol complied with all institutional ethical guidelines regarding the use of research animals and was approved by the Animal Ethics Committee at Shandong Provincial Hospital, China. All surgical manipulations were performed under deep anesthesia, and the animals did not experience pain at any stage during the procedure.
1. Pre-operation preparation
<ol>
	<li>Instrument sterilization
	<ol>
		<li>Steam-sterilize surgical instruments in an autoclave (121&#176;C for 15 min) prior to the surgery. Prepare sufficient disposable sutures and needles.</li>
	</ol>
	</li>
	<li>Surgery platform setup
	<ol>
		<li>Perform the surgery in a room dedicated to surgical procedures. Assign a bench area of at least 60 cm x 60 cm for the operation. Clean the surface of the area with 75% alcohol and cover with a disposable medical towel, and then disinfect it with ultraviolet radiation 30 min in advance (Figure 1A).</li>
	</ol>
	</li>
	<li>Animal preparation
	<ol>
		<li>House all animals in a temperature-controlled room (20-25 &#176;C) with a 12 h light, 12 h dark cycle. Acclimate 8-week-old female C57BL/6 mice to the housing facility for 1 week before surgery.</li>
		<li>Weigh mice before the surgery. Administer all 9-week-old female mice with general anesthesia by intraperitoneal injection of Tribromoethanol (280&#160;mg/kg), to achieve painlessness at any stage during the procedure. Inject meloxicam (2 mg/kg) subcutaneously, about 1 h before an operation to relieve pain.</li>
		<li>Apply eye ointment to prevent corneal dryness during the surgery.</li>
		<li>Apply hair removal lotion on the back using a clean cotton swab. Let the lotion sit on a mouse for 3-5 min, then remove the hair using gauze and cotton swabs. Repeat this step until all hair has been removed from the back of the mouse.</li>
		<li>Use gauze and cotton swabs to clean the skin with 75% alcohol. Fix the mouse on the surgery platform back up using a rubber strip or cotton rope (Figure 1B) and apply iodophor solution to clean the back. 
		NOTE: Confirm the depth of anesthesia via a toe-pinch before Ovariectomy.</li>
	</ol>
	</li>
</ol>
2. Ovariectomy
NOTE: Tribromoethanol can be maintained for approximately 30 min, ensuring the surgery is completed as much as possible.
<ol>
	<li>Make a ~1.0 cm dorsal incision longitudinally from the thigh base upwards using a disposable scalpel, ensuring that only the skin and subcutaneous fascia are incised and avoiding cutting into the posterior peritoneum at this time.</li>
	<li>Pull the incision to the left, and a white fat pad can be seen. Cut ~0.5 cm along the white fat pad to expose the intraperitoneal cavity using micro-forceps and scissors.</li>
	<li>After cutting the posterior peritoneum, slowly and gently remove the white fat pad from the intraperitoneal cavity with micro-forceps. Immediately moisten the white fat tissue with 0.9% sterile saline outside the soaked gauze. Keep exposed tissue always moistened while outside the abdominal cavity.</li>
	<li>A pink granular substance, namely the ovary, is attached to the lower part of the white fat pad (Figure 2A). The ovaries are connected to a slender duct, namely the uterus. Use 5-0 absorbable sutures to ligate the ovarian end of the uterus and remove the left ovary (Figure 2B).</li>
	<li>When removing an ovary, preserve the surrounding fatty tissue as much as possible. Avoid direct contact between surgical instruments and the ovaries and prevent intraperitoneal implantation of ovarian tissue.</li>
	<li>Carefully place the white fat pad back in the intraperitoneal cavity. Perform a simple intermittent suture on the posterior peritoneum with a 5-0 absorbable suture (Figure 2C). After the suture is complete, clean any bleeding with 0.9% sterile saline-soaked gauze.</li>
	<li>Pull the skin incision to the right and remove the right ovary using the same method.</li>
	<li>Perform an intermittent suture with 4-0 non-absorbable sutures and clean any bleeding with 0.9% sterile saline-soaked gauze (Figure 2D).</li>
	<li>Clean the wound with an iodophor solution after completing both sutures. Intraperitoneally inject broad-spectrum antibiotics.</li>
</ol>
3. Post-operation observation
<ol>
	<li>Move the mice to a 37 &#176;C constant temperature blanket after the surgery. Until the mice can move freely, keep the animals in their individual cage. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Inject meloxicam (2 mg/kg) subcutaneously 24 h after the operation to relieve pain.</li>
	<li>Monitor the mice daily to ensure that the surgical wound is healing properly without any signs of complications (dehiscence) present.</li>
</ol>
4. Estradiol supplementation
<ol>
	<li>Prepare feed supplemented with estradiol valerate. Use 2.6 mg beta-estradiol 17-valerate supplemented per 1 kg of feed.</li>
	<li>At 3 days after the completion of the surgery, feed the mice with estradiol valerate.</li>
</ol>
5. FSH injection
<ol>
	<li>Prepare recombinant human FSH solution. Dissolve recombinant human FSH powder for injection with 0.9% sterile saline to 100 IU/mL.</li>
	<li>Group mice according to experimental plans and give solvent or different doses of recombinant FSH via intraperitoneal injection for 2 weeks. According to the biological activity of recombinant FSH, use the injection dose of FSH in mice equivalent to the serum FSH level in women during the menopausal transition period. 
	NOTE: Based on different treatments, ovariectomized estrogen-supplemented mice were randomly divided into three groups, solvent (N.S.) group receiving 100 &#181;L/day solvent, low-dose FSH (L-FSH) group receiving 15 IU/kg body weight per day and high-dose FSH (H-FSH) group receiving 30 IU/kg body weight per day.</li>
</ol>

Developing an Ovariectomized Mouse Model with Elevated FSH Level

<ol>
	<li>Harlow, S. D., 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=Executive+summary+of+the+Stages+of+Reproductive+Aging+Workshop+++10:+addressing+the+unfinished+agenda+of+staging+reproductive+aging.">Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging.</a> Journal of Clinical Endocrinology &amp; Metabolism. 97 (4), 1159-1168 (2012).</li><li>Ulloa-Aguirre, A., Zari&#241;&#225;n, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Follitropin+Receptor:+Matching+Structure+and+Function.">The Follitropin Receptor: Matching Structure and Function.</a> Molecular Pharmacology. 90 (5), 596-608 (2016).</li><li>Franks, S., Stark, J., Hardy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle+dynamics+and+anovulation+in+polycystic+ovary+syndrome.">Follicle dynamics and anovulation in polycystic ovary syndrome.</a> Human Reproduction Update. 14 (4), 367-378 (2008).</li><li>Guo, Y., 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=Blocking+FSH+inhibits+hepatic+cholesterol+biosynthesis+and+reduces+serum+cholesterol.">Blocking FSH inhibits hepatic cholesterol biosynthesis and reduces serum cholesterol.</a> Cell Research. 29 (2), 151-166 (2019).</li><li>Xiong, 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=FSH+blockade+improves+cognition+in+mice+with+Alzheimer's+disease.">FSH blockade improves cognition in mice with Alzheimer's disease.</a> Nature. 603 (7901), 470-476 (2022).</li><li>Sun, 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=FSH+Directly+Regulates+Bone+Mass.">FSH Directly Regulates Bone Mass.</a> Cell. 125 (2), 247-260 (2006).</li><li>Liu, X. 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=FSH+regulates+fat+accumulation+and+redistribution+in+aging+through+the+G&#945;i/Ca(2+)/CREB+pathway.">FSH regulates fat accumulation and redistribution in aging through the G&#945;i/Ca(2+)/CREB pathway.</a> Aging Cell. 14 (3), 409-420 (2015).</li><li>Maclellan, R. A., 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=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Vascular+Anomalies.">Expression of Follicle-Stimulating Hormone Receptor in Vascular Anomalies.</a> Plastic and Reconstructive Surgery. 133 (3), 344e-351en (2014).</li><li>Liu, 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=Blocking+FSH+induces+thermogenic+adipose+tissue+and+reduces+body+fat.">Blocking FSH induces thermogenic adipose tissue and reduces body fat.</a> Nature. 546 (7656), 107-112 (2017).</li><li>Ji, Y., 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=Epitope-specific+monoclonal+antibodies+to+FSH&#946;+increase+bone+mass.">Epitope-specific monoclonal antibodies to FSH&#946; increase bone mass.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (9), 2192-2197 (2018).</li><li>El Khoudary, S. R., 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=Trajectories+of+estradiol+and+follicle-stimulating+hormone+over+the+menopause+transition+and+early+markers+of+atherosclerosis+after+menopause.">Trajectories of estradiol and follicle-stimulating hormone over the menopause transition and early markers of atherosclerosis after menopause.</a> European Journal of Preventive Cardiology. 23 (7), 694-703 (2016).</li><li>Radu, A., 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=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Tumor+Blood+Vessels.">Expression of Follicle-Stimulating Hormone Receptor in Tumor Blood Vessels.</a> The New England Journal of Medicine. 363 (17), 1621-1630 (2010).</li><li>Sowers, M. R., 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=Endogenous+hormones+and+bone+turnover+markers+in+pre-+and+perimenopausal+women:+SWAN.">Endogenous hormones and bone turnover markers in pre- and perimenopausal women: SWAN.</a> Osteoporosis International. 14 (3), 191-197 (2003).</li><li>Kristensen, V. N., Kure, E. H., Erikstein, B., Harada, N., B&#248;rresen-Dale, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+and+environmental+estrogen-like+compounds.">Genetic susceptibility and environmental estrogen-like compounds.</a> Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 482 (1), 77-82 (2001).</li></ol>

The authors have nothing to disclose.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes. Levels of FSH rise during the menopausal transition1. Emerging studies have revealed that FSH in various extragonadal tissues and organs is critical in the pathogenesis of multiple diseases, including dyslipidemia4, Alzheimer&#39;s disease5, osteoporosis9,10, atherosclerosis11, obesity9, and cancer12. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. The OVF mouse model mimics the early stage of menopause transition with relatively stable estrogen and rising FSH levels and is particularly suitable for studies to explore the extragonadal actions of FSH.
In this method, ovariectomy was made using a single dorsal back incision, approximately 1 cm from the thigh base upwards (Figure 1B). The skin was cut almost together with the dorsal muscles using sharp dissecting scissors, and the peritoneal cavity was thus accessed. After the operation, the muscle incision required no suturing, and the skin wound was closed bilaterally with one catgut suture (Figure 2). The operation is technically easier, less time-consuming, and less harmful for female mice as compared to other methods used.
Some details that should be attended to during the surgery procedure. First, all surgical procedures should be kept clean and as sterile as possible to reduce the risk of postoperative infection. Second, because the ovarian tissue is very fragile, surgical instruments cannot contact the ovaries directly during ovariectomy, to avoid intraperitoneal implantation. Third, after the surgery, the mice were moved to a 37 &#176;C constant temperature blanket during recovery to prevent postoperative hypothermia leading to death.
A previous study has proved that endogenous estrogen is synthesized in the ovarian theca cells of premenopausal women or adipose stromal cells of the breast of postmenopausal women and in minor quantities in peripheral tissue14. The serum estrogen dropped sharply for ovariectomized mice but cannot be eliminated (Figure 3B). However, endogenous estrogen synthesized in extragonadal tissue does not affect the stability of estrogen levels in the OVF model (Figure 4B).
There are some limitations in the OVF model. Once the surgical operation is not careful and leads to ovarian intraperitoneal implantation, it may lead to model failure. In this case, the serum estrogen does not drop sharply and fluctuates during different stages of the estrous cycle. After exogenous administration of estrogen and FSH, it takes approximately 1 week for the body to reach equilibrium. Thus, pathological changes of the OVF model that occur within 1week cannot indicate the effects of FSH.
In conclusion, the OVF model has the advantages of being stable, low-cost, and easy to operate. The systemic effects of high-level FSH can be observed after intraperitoneal injection of FSH; that is, the OVF model is suitable for studies that explore the extragonadal actions of FSH. However, requirements for model surgery and intraperitoneal injection procedures are quite high. If funding is sufficient, specific knockout models are the best choice.

Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition (the term menopausal transition was defined in 2011 at stages of reproductive aging workshop (STRAW) + 10 system)1. It is during the menopausal transition, a period characterized by rising FSH levels and relatively stable estrogen1, thatwomen experience menstrual cycle changes and significant physiological changes involving various cells and tissues. These changes can seriously affect the quality of life and health of women. Exploring the effects of FSH may improve women's quality of life and health.
FSH is secreted from gonadotrope cells in the anterior pituitary and is critical in controlling gonadal function and reproduction2. The function of FSH is mediated through the FSH receptor (FSHR), which belongs to the G protein-coupled receptor (GPCR)3. FSHR is generally expressed in gonads, namely, the ovary and testis. It has been proved that FSHR is universally expressed in multiple extragonadal cells and tissues, including liver4, hippocampus5, osteoclasts6, adipocytes7, and endothelial cells8. Emerging studies have revealed extra gonadal actions of FSH and its potential clinical relevance in dyslipidemia4, Alzheimer's disease5, osteoporosis9,10, atherosclerosis11, obesity9, and cancer12. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important in exploring the actions of FSH alone.
In the protocol, we introduced the procedure for establishing a mouse model with relatively stable estrogen and rising FSH levels13. The mouse model mimics the menopause transition by ovariectomized surgery and then supplemented with estradiol valerate and recombinant FSH. As the ovariectomized mice were supplemented with exogenous estrogen to maintain similar estrogen levels with the sham-operated mice, the levels of endogenous FSH were stable due to estrogen feedback at the pituitary gland. In this condition, it could control the FSH levels by administering exogenous FSH without altering the estrogen levels. Thus, the OVF mouse model can exclude the influence of estrogen and observe the extragonadal physiological and pathological effects of FSH. We believe the detailed and visualized procedure is useful for researchers to establish the OVF mouse model in their laboratory and apply it to investigate physiological and pathological changes during the menopause transition as needed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>beta-estradiol 17-valerate</td>
			<td>Macklin</td>
			<td>E829824</td>
			<td></td>
		</tr>
		<tr>
			<td>Estradiol sensitive ELISA</td>
			<td>Demeditec</td>
			<td>DE4399</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin Staining Solution</td>
			<td>Beyotime</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Aladdin</td>
			<td>M129228</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human Follicle-stimulating hormone</td>
			<td>Merck Serono</td>
			<td>N19Z8803G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>Aliphatic name: 2,2,2-Tribromoethanol</td>
		</tr>
	</tbody>
</table>

exploring independent effects of follicle-stimulating hormone in vivo in a mouse model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

The OVF mouse model mimics the early stage of menopause transition with relatively stable estrogen and rising FSH levels13. First, for ovary removal surgery, 9-week-old female C57BL/6 mice were administered general anesthesia and subjected to either a sham operation (Sham) or bilateral ovariectomy (OVX). As smear images of Papanicolaou stained cells clearly identified the proestrus, estrus, metestrus, and diestrus stages of the estrous cycle, the OVX mice lost the estrous cycle (Figure 3A), and the ELISA method showed a significant decrease in serum estradiol (E2) levels (Figure 3B). Second, the OVX mice were supplemented with beta-estradiol 17-valerate (OVX + E2) to maintain serum estrogen at the same level as the Sham group. Third, the OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65665/65665fig01.jpg" /> 
Figure 1. Surgical environment and mouse posture. (A) A bench area of at least 60 cm x 60 cm for the operation. Clean the surface of the area with 75% alcohol and cover it with a disposable medical towel, and then disinfect with ultraviolet radiation 30 min in advance. (B) Fix the mouse on the surgery platform back up using a rubber strip or cotton rope. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65665/65665fig02.jpg" /> 
Figure 2. Key steps of surgical operation. (A) Ovarian position, (B) ovariectomy, (C) suture peritoneum, and (D) suture skin incision. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65665/65665fig03.jpg" /> 
Figure 3. Vaginal cytology. Vaginal cytology represents stages of the estrous cycle and endogenous estrogen in the ovariectomized mice (OVX) and the sham-operated ones (Sham; n = 12 for the Sham group; n = 10 per OVX groups). (A) Vaginal cytology represents stages of the estrous cycle according to the relative presence of leukocytes, cornified epithelial cells, and nucleated epithelial cells. Stages of the estrous include proestrus, the predominance of nucleated epithelial cells; estrus, the predominance of enucleated cornified cells; metestrus, the presence of leukocytes, and cornified and nucleated epithelial cells; diestrus, the predominance of leucocytes. Scale bar = 100 &#181;m. (B) Endogenous estrogen in the ovariectomized mice (OVX) and the sham-operated ones (Sham). Data are shown as the mean &#177; SEM. Student&#39;s t-test is used for statistical analysis. ***p&#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65665/65665fig04.jpg" /> 
Figure 4. OVF model and serum hormone levels. (A) Flow-chart OVF model. (B) ELISA analysis of serum estrogen (E2) and FSH concentrations. Data are represented as the mean &#177; SEM. One-way ANOVA was used for statistical analysis. * p&#60; 0.05 and ** p&#60; 0.01. This figure has been modified from4. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

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1.6K Views.  The Affiliated Hospital of Qingdao University. Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

Video: Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

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We will show how to record flash responses from single mouse cones using a suction electrode.

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

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Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

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The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

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Single-cell Suction Recordings from Mouse Cone Photoreceptors

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Quantitation of Endothelial Cell Adhesiveness In Vitro

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging