This work was supported by the Strategic Consulting Project of the Chinese Academy of Engineering: Strategic research on anti-aging effect of Traditional Chinese Medicine (Grant No.: 2022-XY-45), the Natural Science Foundation of Hebei Province in China (Grant No.:2022106065), S&#38;T Program of Hebei, China (Grant No.:22372502D) and High-level S&#38;T Innovation and Entrepreneurship Talent Project of Shijiazhuang, Hebei, China (Grant No.: 07202203).

The animal experiments were approved by the Committee on Animal Research and Ethics of the Hebei Yiling Medical Research Institute (approval number: N2020150). The 36 Sprague-Dawley (SD) female rats (3 months old, weighing 180-200 g) (see Table of Materials) were fed with normal food and clean water in the new drug evaluation center of Hebei Yiling Medical Research Institute and were exposed to artificial light for 12 h per day in rooms with controlled temperatures (20-26 &#176;C) and relative humidity (40%-70%).
1. Animal experiment
<ol>
	<li>Divide the rats into six groups randomly, a sham-operated group (SHAM group, 10 mL/kg/d saline), model group (OVX group, 10 mL/kg/d saline), progynova15 group (0.2 mg/kg/d), low-dose BZBS group (BZBSL group, 1 g/kg/d), middle-dose BZBS group (BZBSM group, 2 g/kg/d), and high-dose BZBS group (BZBSH group, 4 g/kg/d). 
	NOTE: The dose of the BZBSM group was determined from the recommended dosages for humans and calculated as follows: equivalent experimental dose for rats (mg/kg/d) = human dose (mg/kg)/body weight (60 kg) x 6.3.</li>
	<li>Employ 3% and 1.5% isoflurane for the induction and maintenance of rat anesthesia throughout the operation, respectively. 
	NOTE: If the rats do not blink and the pedal reflex is absent, the depth of anesthesia is confirmed and the maintenance of rat anesthesia is conducted, respectively.</li>
	<li>Remove the fur of the rat&#39;s abdomen with a sterile razor and disinfect the skin of the rat&#39;s abdomen using alternative rounds of ethanol and povidone-iodine 3 times with a sterile cotton ball. Apply local analgesia with a topical injection of 1.5 mg/kg bupivacaine after anesthesia. 
	NOTE: The disinfection method adopts a circular pattern centered around the incision and starting from the center outward.</li>
	<li>Perform a 2 cm midline incision on the abdomen of the rat with a sterile scalpel in the SHAM group and suture the midline incision using a 4-0 surgical suture. 
	NOTE: First, the internal muscle layer is sutured with absorbable suture, and the external/skin closure is sutured with monofilament. On the 10th day, use scissors to cut open the monofilament and use tweezers to extract the monofilament.</li>
	<li>For the rats in the other groups, isolate and expose the intact ovary using sterile forceps and ligate at the root of the ovary using a 4-0 surgical suture. Remove the intact ovary using sterile surgical scissors. Suture the midline incision using a 4-0 surgical suture. 
	NOTE: All postoperative rats were placed in isolation in 37 &#176;C containers until the rats were awake and able to move freely.</li>
	<li>After 4 months of intragastric administration of designated drugs (in step 1.1), first anesthetize the rat with 3% isoflurane, use hair removal cream to remove the fur from the back of the rat, and then disinfect the skin of the groin and entire lower limb using ethanol and povidone-iodine 3x with a sterile cotton ball. 
	NOTE: The drug was administered once a day with a 10 cm stainless steel gavage needle. The gavage dose was adjusted weekly according to the weight of the rats.</li>
	<li>Cut and peel the skin of the groin to expose the femoral artery using sterile surgical scissors and tweezers. Collect a blood sample from the femoral artery using a vacuum blood collection tube and then apply pressure to stop bleeding with a sterile cotton ball. Euthanize the rats with excessive inhalation of carbon dioxide.</li>
	<li>Continue to cut and peel the skin of the groin and the lower limb to expose the intact tibia using sterile surgical scissors and tweezers. After removing the tibia, wash it three times with sterile saline and continue to remove excess muscle tissue in saline with&#160;tweezers and ophthalmic scissors.</li>
	<li>Disinfect the back skin of the lumbar L4 using ethanol and povidone-iodine 3x with a sterile cotton ball. Use sterile surgical scissors and tweezers to cut the back skin and expose the lumbar L4.</li>
	<li>Sever the upper and lower vertebrae of the lumbar L4 with a rongeur and cut off the sacrum around the lumbar L4 with sterile surgical scissors. Wash the isolated lumbar L4 with saline three times and continue to remove excess muscle tissue in saline with tweezers and micro scissors.</li>
	<li>Store the tibia and L4 vertebra in 10% formalin tissue fluid (see Table of Materials) at room temperature for 3 days.</li>
	<li>Pack the carcass in a special cadaver bag, and place it in an animal carcass storage cabinet.</li>
</ol>
2. Micro-CT imaging16
<ol>
	<li>Press the green power button to turn on the micro-CT scanner (see Table of Materials) and start the software to heat the x-ray source. 
	NOTE: To avoid scattering of the x-rays, move any metal objects near the imaging area before starting. Micro-CT examination of rat tibial tissue in step 1.11 with insufficient x-ray irradiation can affect the findings. The bone should be kept moist during the micro-CT scanning. The animal bed should be taken out while warming up. Make sure that the key is turned to the ON position on the SAFETY KEY in the lower left corner in front of the CT instrument.</li>
	<li>Create a database by clicking on a new one to build a file to save the data to obtain next.</li>
	<li>Set the data acquisition parameters in the software control window as follows: x-ray tube voltage (50 kV); CT x-ray tube current (100 &#181;A); live x-ray tube current (100 &#181;A); field of view (FOV) (18 mm); no gating technique; scanning technique (high resolution 4 min).</li>
	<li>Wash the tibia with saline three times and blot out excess water with filter paper. Wrap the tibia tissue on the bed with plastic film to fix its position. Adjust the tibia tissue to the center of the imaging field by rotating the button in the instrument.</li>
	<li>Close the instrument door and turn on the live mode. Press the capture button to view the tibial tissue.</li>
	<li>Start the scan by clicking the CT scan button. Click Yes to produce the image. 
	NOTE: Once the x-ray is turned on, the orange light at the top of the instrument will be illuminated and the sliding door will not open for the safety of the operator. When the scan is complete, a new window appears in the 2D Viewer software showing the reconstructed cross-axis, coronal plane, and sagittal plane. Click Paste to the drawing board to save.</li>
	<li>Take the tibia out of the animal bed. Open the Analyze 12.0 software and import the objective CT image by clicking File &#62; Load &#62; Process. Click the Image Calculator... and Region Pad options to display the SubRegion dialog box.
	<ol>
		<li>Click the Interactive option and select the bone in the 2 mm thickness area. Click Apply to select Change a Copy of the Loaded Volume. In the Analyze 12.0 screen, click the Apps option to select BMA.</li>
		<li>Click Segment Cortex and Segment Trabeculae, then click Save Final Object Map. Click Measure Bone to display the value of specific parameter indicators, including bone mineral density (BMD, g/cm3), bone volume fraction (bone tissue volume/tissue volume [BV/TV], %), number of bone trabeculae (Tb.N, 1/mm), bone trabecular thickness (TB.Th, &#181;m), and trabecular separation degree (Tb.Sp, &#181;m). 
		&#8203;NOTE: Micro-CT was not performed with L4 vertebrae.</li>
	</ol>
	</li>
</ol>
3. Hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>Take out the tibia and L4 vertebra samples of the rat in step 1.11 and store them in 20% formic acid solution for a 4 day decalcification. 
	NOTE: The 20% formic acid solution is prepared by mixing 2,400 mL of formaldehyde solution and 600 mL of formic acid solution.</li>
	<li>Put the processed tibia and L4 vertebra sample of the rat from step 3.1 in the embedding box and wash them with running water for more than 6 h.</li>
	<li>Dehydrate the samples with gradient concentration alcohol solutions (60% ethanol for 1 h, 70% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 2 h, and 100% ethanol for 2 h) using an automated tissue processor.</li>
	<li>Place the tissue specimens in xylene for 2 h to make them translucent. At the end of dehydration, place the permeabilized samples in paraffin wax at 60 &#176;C for 3 h and embed them in an automatic processor.</li>
	<li>Obtain the 4 &#181;m sections by using a rotary slicer. Place the sections in hematoxylin stain for 3-8 min and eosin stain for 1-3 min.</li>
	<li>Transfer the stained sections to pure alcohol and xylene, respectively. Seal and fix the stained sections with neutral gum for pathological examination under an optical microscope.</li>
	<li>Scan the slices with a slide scanner (see Table of Materials) at 40x magnification and use the viewing software to acquire the H&#38;E staining results of the entire tibia and L4 vertebra.</li>
</ol>
4. Immunohistochemistry
<ol>
	<li>Perform heat-induced epitope retrieval using the 4 &#181;m tibia sections obtained in step 3.5. Add the appropriate antigen repair buffer (citric acid buffer, pH = 6.0) to a microwaveable container. Place the carrier sheet into the microwaveable container and place the container in the microwave oven. Set the program to hold the antigen repair for 20 min at 98 &#176;C.</li>
	<li>Remove the container and rinse the interior with cold tap water for 10 min. Block endogenous peroxidase activity with hydrogen peroxide (3% H2O2) (see Table of Materials), then incubate the slice for 15 min at room temperature.</li>
	<li>Wash the sections in phosphate-buffered saline (PBS) (see Table of Materials) three times, 5 min each.</li>
	<li>Put the sections into normal goat serum blocking solution (see Table of Materials), then incubate the samples in a moist chamber for 15 min at room temperature.</li>
	<li>Add 200 &#181;L of RANKL antibody (1:200) (see Table of Materials), OPG antibody (1:300) (see Table of Materials), and sclerostin antibody17 (1:200) (see Table of Materials) into the slice samples and incubate for one night at 4 &#176;C in the dark. 
	NOTE: Sclerostin is a negative regulator of bone growth. It is a secreted glycoprotein with a C-terminal cysteine knot-like domain and has a sequence similarity to the DAN family of bone morphogenetic protein antagonists17. This protein is not a member of the RANKL pathway.</li>
	<li>Add biotin-labeled goat anti-rabbit IgG (see Table of Materials) into the sections and incubate at room temperature for 30 min. Rinse the sections with PBS and use diaminobenzidine (DAB) (see Table of Materials) to stain the sections for 3-5 min at room temperature.</li>
	<li>Dye the sections with hematoxylin for 1-2 min. After dehydrating and drying the sections, seal them with neutral gum.</li>
	<li>Scan the sections at 40x magnification using a slide scanner (see Table of Materials) and capture digital images using an automatic microscope imaging system (see Table of Materials).</li>
	<li>Open Imagepro Plus software (see Table of Materials).
	<ol>
		<li>Click File to import the analysis image. Click Measure &#62; Calibration &#62; Intensity Calibration for optical density correction. Click the Measure, IOD, Count/Size, and Select Colors options one by one to display the Segmentation interface.</li>
		<li>Click the Histogram Based option to select HIS and click Load File. Click Count to generate the IOD value and Area value.</li>
	</ol>
	</li>
</ol>
5. Enzyme-linked immunosorbent assay (ELISA) protocol
<ol>
	<li>Ensure that the ELISA kit for detecting 17&#946;-estradiol includes the anti-17&#946;-estradiol IgG-coated microplate (12 x 8 wells), 15 mL of stop solution, 22 mL of 17&#946;-estradiol-HRP conjugate, 15 mL of 3,3&#39;,5,5&#39; tetramethylbenzidine (TMB) substrate solution, 50 mL of 10x washing solution, cover foils, a strip holder, the 17&#946;-estradiol control, and a different concentration gradient of 17&#946;-estradiol standard (see Table of Materials).</li>
	<li>Remove the 17&#946;-estradiol kit from the refrigerator and place all the reagents at room temperature (18-25 &#176;C).</li>
	<li>Reagent preparation: Dilute the 10x washing solution with deionized water to prepare a 1x washing solution. Mix 50 mL of 10x washing solution with 450 mL of deionized water to make 500 mL of 1x washing solution.</li>
	<li>Remove the serum sample from the refrigerator and thaw it at room temperature.</li>
	<li>Remove the excess microtiter strips from the plate frame, return them to the foil bag containing the desiccant pack, reseal them, and store at 4 &#176;C.</li>
	<li>Add 25 &#181;L of control, standard, or sample to each of their respective wells. Then, add 200 &#181;L of 17&#946;-estradiol-HRP conjugate to each well.</li>
	<li>Cover all the wells with foil and incubate the plate at 37 &#176;C for 2 h.</li>
	<li>Remove the foil and liquid from each well and wash the well three times with 300 &#181;L of 1x washing solution. 
	NOTE: Avoid overflowing of the reaction wells. Also, the soak time between each wash cycle should be &#62;5 s. At the end, carefully remove the remaining fluid by tapping tissue paper strips before the next step.</li>
	<li>Add 100 &#181;L of TMB substrate solution to each well and incubate in the dark for 30 min at room temperature.</li>
	<li>Add 100 &#181;L of stop solution to each well and gently shake the microtiter plate for 30 s. Measure the absorbance of the sample at 450 nm within 30 min.</li>
	<li>Draw the average absorbance of the standards against concentration. Plot the best-fit curve by the plotted points.</li>
	<li>Obtain the corresponding concentration values expressed in pg/mL by interpolating the values of the samples on the standard curve. 
	&#8203;NOTE: The other protocols for detecting serum Ca2+, phosphorus, SOST, TRACP-5b, and &#946;-CTX are the same as the 17&#946;-estradiol measurement, according to the manufacturer&#39;s instructions.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Data were presented as mean &#177; standard deviation. Multiple comparisons were performed by one-way ANOVA followed by Tukey&#39;s test. p &#60; 0.05 was considered statistically significant.</li>
</ol>

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W., Huang, L. <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+sclerostin+on+the+functions+and+related+mechanisms+of+cementoblasts+under+mechanical+stress.">Effect of sclerostin on the functions and related mechanisms of cementoblasts under mechanical stress.</a> West China Journal of Stomatology. 37 (2), 162-167 (2019).</li><li>Wang, 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=Therapeutic+potential+and+outlook+of+alternative+medicine+for+osteoporosis.">Therapeutic potential and outlook of alternative medicine for osteoporosis.</a> Current Drug Targets. 18 (9), 1051-1068 (2017).</li></ol>

The authors have nothing to disclose.

After ovariectomy, estrogen levels decreased sharply due to the continuous reduction of cancellous bone and increased bone turnover. Essentially, decreased estrogen secretion after menopause could cause significant bone loss18, leading to Ca2+ loss in ovariectomized rats19,20. It has been reported that taking estrogen can help the metaphyseal fracture recovery of ovariectomized rats, and estrogen replacement therapy can also be ...

Postmenopausal osteoporosis (PMOP) is a skeletal system disease caused by inadequate ovarian function, decreased estrogen, and enhanced osteoclast activity1, characterized by low bone mass, microarchitectural degeneration of the bone tissue, and damage of the bone trabecula. PMOP prone to cause fractures and can have serious effects on patients' quality of life. Osteoporosis affects about 200 million people worldwide2; about 40% of postmenopausal women suffer from osteoporosis3, and fractures occur in 33% of patients with PMOP4. The reduction of estrogen could lead to a relatively enhanced osteoclast activity5 and declined bone mass when bone resorption exceeds bone formation. Thus, osteoclast activation is usually considered a sign of bone loss6. Physiologically, RANKL/OPG serves as an important pathway involving bone remodeling by regulating osteoclast activation to promote bone resorption7. Meanwhile, estrogen can also mediate osteoclast formation and function by regulating RANKL/OPG signaling8.
Over recent years, traditional medicine has been increasingly used to treat different diseases with less adverse reactions and better prognosis9,10. Bazi Bushen capsule (BZBS), a traditional Chinese medicine, could be an alternative approach to prevent and treat PMOP. It is composed of Cuscuta, Fructus lycii, Fructus schisandrae, Fructus cnidii, Fructus rosae laevigatae (Cherokee rose fruit), Raspberry, Semen Allii Tuberosi, Toosendan fructus, Herba epimedii, Morinda officinalis, Herba cistanches, Rehmannia glutinosa, Medicinal cyathula root, Ginseng, Pilose antler, and Hippocampus Kelloggi (Table 1), containing 11 kinds of phytoestrogen with hormone-like effects. A previous study has confirmed that BZBS can delay the formation of atheromatous plaque through an estrogen-like effect11, which is similar to treating osteoporosis with estrogen12. However, the underlying mechanisms of BZBS in the prevention and treatment of osteoporosis caused by the deficiency of estrogen are unclear. Therefore, the present study aimed to verify whether BZBS has a bone-protective effect on ovariectomy-induced rat osteoporosis13,14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Proteintech Group, Inc.</td>
			<td>23408-1AP-100</td>
			<td></td>
		</tr>
		<tr>
			<td>17-Beta-Estradiol ELISA kits</td>
			<td>Proteintech Group, Inc.</td>
			<td>21933-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>3%H2O2</td>
			<td>ShanDong LIRCON Medical Technology Incorporated Company</td>
			<td>20221027</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-osteoprotegerin antibody</td>
			<td>Abcam</td>
			<td>ab203061</td>
			<td></td>
		</tr>
		<tr>
			<td>Bazibushen capsules</td>
			<td>Shijiazhuang Yiling Pharmaceutical Co. Ltd.</td>
			<td>XB2103001</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>ab207995</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>C004-2-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9018</td>
			<td></td>
		</tr>
		<tr>
			<td>Estradiol valerate tablets</td>
			<td>Bayer AG</td>
			<td>156A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene 1580R centrifuge</td>
			<td>GENE Co. Ltd.&#160;</td>
			<td>GZ422515090077</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-Pro Plus</td>
			<td>Media Cybernetics</td>
			<td>IPP 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DM6000B Microscope (fully automated upright microscope system)</td>
			<td>Leica</td>
			<td>361715</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum Blocking Solution</td>
			<td>Vector Laboratories</td>
			<td>S-1000-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>C006-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Servicebio, Wuhan, China</td>
			<td>CR10201M</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum GX2 microCT Imaging System</td>
			<td>PerkinElmer</td>
			<td>CLS149276</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL rabbit polyclonal antibody</td>
			<td>Elabscience Biotechnology Co. Ltd.</td>
			<td>E-EL-R3032-96T</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat SOST (Sclerostin) ELISA kit</td>
			<td>Elabscience Biotechnology Co. Ltd.</td>
			<td>E-EL-R1405c-96T</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat TRACP-5b (Tartrate Resistant Acid Phosphatase 5b) ELISA kit</td>
			<td>Abcam</td>
			<td>ab108667</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat &#946;-CTx (Beta Crosslaps) ELISA kit</td>
			<td>Elabscience Biotechnology Co., Ltd.</td>
			<td>E-EL-R0939c-96T</td>
			<td></td>
		</tr>
		<tr>
			<td>Sclerostin rabbit polyclonal antibody</td>
			<td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td>
			<td>SCXK(JING)2016-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide scanner</td>
			<td>Hamamatsu Photonics K.K.</td>
			<td>Nano Zoomer-SQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley female rats</td>
			<td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td>
			<td>ZLI-9381</td>
			<td></td>
		</tr>
	</tbody>
</table>

estrogen-like effect of bazi bushen capsule in ovariectomized rats

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17&#946;-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, &#946;-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

The bone microstructure of the tibia was evaluated by micro-CT scanning 
The treatment of ovariectomized rats with BZBS significantly reduced OVX-induced trabecular structural changes. As shown in the reconstructed micro-CT images of the right tibia (Figure 1A,B), trabecular bone in the OVX group showed a significant decrease in BMD (Figure 1C), BV/TV (Figure 1D), Tb.Th (Fi...

Watch this Scientific Journal Video about Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats at JoVE.com

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

Presented here is a protocol to show that Bazi Bushen capsule (BZBS) can regulate the RANKL/OPG signaling pathway in the ovariectomized rodent model through its estrogen-like effect.

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley ...

estrogen-like-effect-of-bazi-bushen-capsule-in-ovariectomized-rats

Research

JoVE Journal

Medicine

775 Views.  Hebei Medical University. Presented here is a protocol to show that Bazi Bushen capsule (BZBS) can regulate the RANKL/OPG signaling pathway in the ovariectomized rodent model through its estrogen-like effect.

Video: Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

Roux-en-Y Gastric Bypass Operation in Rats

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12.
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Histological Quantification of Chronic Myocardial Infarct in Rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

One-anastomosis Gastric Bypass (OAGB) in Rats

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical model, longitudinal studies can be carried out to provide an improved understanding of the mechanisms underlying the outcomes seen after bariatric surgery in humans. For this purpose, rats are operated on through a laparotomy under general anesthesia with isoflurane. First, the surgeon creates a long and tubular gastric pouch: after greater curve and hiatal dissection, the nonglandular stomach is stapled and removed. Then, the remaining stomach is also stapled in order to create a gastric tube and exclude the antrum of the stomach. After that, the surgeon performs a single end-to-side gastrojejunostomy 35 cm from the duodenojejunal angle. This limb length has been chosen in order to reproduce the same ratio between the biliopancreatic limb (BPL) and common limb (CL) length as in human bariatric surgery. The operation ends by aponeurotic and cutaneous closure. The early postoperative management consists of subcutaneous hydration, an intramuscular prophylactic antibiotic injection, a parietal injection of xylocaine, the administration of painkillers, and a progressive reintroduction of diet.

One-anastomosis Gastric Bypass OAGB in Rats

This protocol is for the performance of one-anastomosis gastric bypass (OAGB) on rat. The operator performs a long and tubular-stapled gastric pouch followed by hand-sewn anastomosis. For this model, the operator reproduces the same ratio between biliopancreatic and common limb in humans; therefore, the biliopancreatic limb measures 35 cm.

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...