Name: Ovariectomy and 17 and beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration (Video) | JoVE Journal
Uploaded: 2012-06-07T12:00:00.000+00:00
Duration: 6 min 51 s
Description: Learn ovariectomy and 17β-estradiol replacement techniques in rats and mice using silastic capsules and oral Nutella delivery. These methods enable stable hormone control for neuroscience and endocrinology research.

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5 minutes

The study was funded by Link&ouml;ping University and the County Council of &Ouml;sterg&ouml;tland.

1. Ovariectomy via the Dorsal Route
<ol>
	<li>Anesthetize the animal, for example using 1.5% isoflurane (4% for induction) in an oxygen/nitrous oxide 30%/70% mixture. Place the animal in prone position on a heating plate connected to a rectal thermometer. It is important to have a thin layer of isolation between the animal and the plate, and that the thermometer feed-back system keeps the body temperature within 37 ± 0.5 °C. Apply gel for eye protection and administer 5 mg Rimadyl/kg bodyweight diluted in saline subcutaneously on the lateral aspect of the animal's neck for postoperative pain relief.</li>
	<li>Shave a 4*4 cm (3*3 cm for mice) area cephally from the iliac crest. Thoroughly wash the shaved area with for example Jodopax before covering the animal with a 4*4 cm aperture surgery sheet.</li>
	<li>Make a 2-3 cm midline incision and bluntly dissect the skin from the underlying fascia. One cm lateral of the midline, make another incision through the fascia. Bluntly dissect laterally under this fascia as superficially as possible until reaching the abdominal cavity.</li>
	<li>Using tweezers, grab the adipose tissue that surrounds the ovary in the abdominal cavity and gently pull it out. Identify the ovary and ligate the uterine horns and vessels 0.5-1 cm proximally of this structure. Cut the ovary and ligated adipose tissue and put the remaining tissue back into the abdominal cavity. Make another incision in the contralateral fascia and repeat the procedure.</li>
	<li>Close the wounds, preferably using a monofilament suture, and let the animal awake from anesthesia. Place the animal in a heated cage (25-27 °C) during at least 2 hours after surgery. The animals should be housed separately the first days postoperatively. It is pivotal that the cage is frequently cleaned/replaced during the recovery phase. Twenty-four hours after surgery, inject the animals subcutaneously with another 5 mg Rimadyl/kg bodyweight diluted in saline.</li>
</ol>
2. Subcutaneous Administration of 17β-estradiol through Silastic Capsules
<ol>
	<li>Thoroughly mix the desired amount of 17β-estradiol in sesame oil. For physiological concentrations in rats and mice, we recommend 180 μg/mL and 18-36 μg/mL respectively. Placebo capsules are filled with pure sesame oil.</li>
	<li>Cut 3 cm (2 cm for mice) lengths of silastic tubing and 5 mm (3 mm for mice) lengths of wooden applicator sticks for plugging. Using forceps, squeeze one wooden plug into the silastic capsule before injecting the 17β-estradiol/sesame oil solution to fill the entire length. Gently cap the silastic capsule in the open end with another wooden plug (the plugs are supposed to remain permanently in the capsule). Incubate the capsules in the remaining 17β-estradiol solution overnight before implantation.</li>
	<li>Implantation of the capsule can be performed during the anesthesia used at ovariectomy in rats. For mice, however, a 14-day recovery period after ovariectomy is strongly recommended to minimize the risk of the capsule protruding through the fragile skin.</li>
	<li>After surgical preparations as in 1.1, or after closing the ovariectomy incision, shave and wash a 2*2 cm area on the dorsal aspect of the animal's neck. Make a 1 cm incision and bluntly dissect a subcutaneous pocket caudolaterally, with ample space for the capsule. Wipe the silastic capsule (that until this time-point has been incubated in the 17β-estradiol solution) and gently install it through the incision, which is subsequently closed by a suture. It is pivotal to make sure that the capsule strains the skin as little as possible, particularly in the area close to the wound. From production of capsules to the implantation, it is important that contamination is avoided.</li>
</ol>
3. Peroral Administration of 17β-estradiol via the Nut-cream Nutella
<ol>
	<li>Thoroughly dissolve 17β-estradiol in sesame oil, and then mix the sesame oil with the Nutella. We recommend that each daily portion contains 28 μg 17β-estradiol, 5 μL sesame oil and 1 g Nutella per kilogram body weight for rats and 1.12 μg 17β-estradiol, 0.312 μL sesame oil and 60 mg Nutella for each 30-g mouse. Placebo nut-cream is prepared identically except without the 17β-estradiol.</li>
	<li>Train the animals to eat hormone-free nut-cream for five days prior to the experiment. The first three days of training, the rats should optimally be trained in groups (approximately 0.5 g Nutella to 2-5 animals), in their home cages. On the fourth and fifth days of training, the animals are put in separate cages before receiving the daily portion to resemble the experimental situation.</li>
	<li>During the experiment, the animals are put in separate cages for every feeding occasion, after which the Nutella portion is served on small ceramic tiles. If training has been performed as above, our experience is that more than 95% of rats and mice accept the nut-cream, and once fully habituated, most of the animals consume it within seconds.</li>
</ol>
4. Representative Results
Physiological serum 17β-estradiol concentrations, which the current methods aim to reproduce, are approximately 5-140 pg/mL for rats and 5-35 pg/mL for mice4,6. The subcutaneous silastic capsules according to the instructions above, when administered to 300 g female Sprague-Dawley rats, produce slowly decreasing 17β-estradiol serum concentrations from around 40 pg/mL day 2 to 10 pg/mL day 35. However, immediately after administration, a serum concentration peak around 1000 pg/mL can be expected, falling to the physiological range within 8 hours (Figure 1A)4,5. The silastic capsules for mice described above with 36 μg 17β-estradiol/mL, when administered to 25 g female C57BL/6 mice, render serum concentrations that slowly decrease from approximately 90 pg/mL day 2 to 25 pg/mL day 35. This is probably preceded by a similar peak as was found in the rats, although this has not been investigated (Figure 1B)6.
The peroral Nutella method with the dosages described above, when administered to 300 g female Sprague-Dawley rats, renders physiological levels of 17β-estradiol with daily fluctuations between 10-70 pg/mL (Figure 2A)4. For mice using the above dosage, the daily levels fluctuate between approximately 20 and 120 pg/mL (Figure 2B)6.
<img alt="Graph comparing 17β-estradiol levels over time in rats and mice with E2/mL capsules." src="/files/ftp_upload/4013/4013fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Stylized graphs presenting 17β-estradiol concentrations in rats (A; n=10 in time-points up to 24 h, n=15 in later time-points)4,5 and mice (B; n=10)6 after capsule implantation. The silastic capsules produce a short initial peak of serum concentrations up to 1000 pg/mL, but decrease within hours to levels below 100 pg/mL. For the coming 4-5 weeks, the capsules yield stable, although steadily decreasing, serum concentrations. The shaded areas correspond to physiological 17β-estradiol serum concentrations in intact female rats and mice respectively. Even though not visually demonstrated in the graphs, there is inter-individual variability in the 17β-estradiol concentrations, rendering coefficients of variation from 27 to 183 % in rats and from 28 to 115 % in mice with the silastic capsule method (very low mean concentrations often generate high coefficients of variation).
<img alt="Time-concentration graph, 17β-estradiol levels in rats and mice post-oral E2 dose over 96 hours." src="/files/ftp_upload/4013/4013fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Stylized graphs presenting 17β-estradiol concentrations in rats (A; n=15)4 and mice (B; n=10)6 as a consequence of daily hormone administration in Nutella. The peroral Nutella method renders daily peaks, however not by far as high as produced by subcutaneous or intravenous methods. This mimics the pharmacokinetics of human hormone replacement therapy, and the peroral treatment can proceed for as long as needed, even if the example above only present an initial 96 hours. The shaded areas correspond to physiological 17β-estradiol serum concentrations in intact female rats and mice respectively. Even though not visually demonstrated in the graphs, there is inter-individual variability in the 17β-estradiol concentrations, rendering coefficients of variation from 40 to 132 % in rats and from 13 to 219 % in mice with the Nutella method (very low mean concentrations often generate high coefficients of variation).

<ol><li>Strom, J. O., Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dose-related+neuroprotective+versus+neurodamaging+effects+of+estrogens+in+rat+cerebral+ischemia%3A+a+systematic+analysis%5BTitle%5D)+AND+%22J.+Cereb.+Blood+Flow+Metab%22%5BJournal%5D)">Dose-related neuroprotective versus neurodamaging effects of estrogens in rat cerebral ischemia: a systematic analysis.</a> J. Cereb. Blood Flow Metab. 29, 1359-1372 (2009).</li>
<li>Strom, J. O., Theodorsson, E., Holm, L., Theodorsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+methods+for+administering+17beta-estradiol+to+ovariectomized+rats+result+in+opposite+effects+on+ischemic+brain+damage%5BTitle%5D)+AND+%22BMC+Neurosci%22%5BJournal%5D)">Different methods for administering 17beta-estradiol to ovariectomized rats result in opposite effects on ischemic brain damage.</a> BMC Neurosci. 11, 1471-2202 (2010).</li>
<li>Strom, J. O., Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hormesis+and+Female+Sex+Hormones%5BTitle%5D)+AND+%22Pharmaceuticals%22%5BJournal%5D)">Hormesis and Female Sex Hormones.</a> Pharmaceuticals. 4, 15(2011).</li>
<li>Isaksson, I. M., Theodorsson, A., Theodorsson, E., Strom, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+17beta-estradiol+administration+to+rats%5BTitle%5D)+AND+%22Scandinavian+journal+of+clinical+and+laboratory+investigation%22%5BJournal%5D)">Methods for 17beta-estradiol administration to rats.</a> Scandinavian journal of clinical and laboratory investigation. , (2011).</li>
<li>Strom, J. O., Theodorsson, E., Theodorsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Order+of+magnitude+differences+between+methods+for+maintaining+physiological+17beta-oestradiol+concentrations+in+ovariectomized+rats%5BTitle%5D)+AND+%22Scand.+J.+Clin.+Lab+Invest%22%5BJournal%5D)">Order of magnitude differences between methods for maintaining physiological 17beta-oestradiol concentrations in ovariectomized rats.</a> Scand. J. Clin. Lab Invest. 68, 814-822 (2008).</li>
<li>Ingberg, E., Theodorsson, A., Theodorsson, E., Strom, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+long-term+17beta-estradiol+administration+to+mice%5BTitle%5D)+AND+%22General+and+comparative+endocrinology%22%5BJournal%5D)">Methods for long-term 17beta-estradiol administration to mice.</a> General and comparative endocrinology. , (2011).</li>
<li>Kalliokoski, O., Jacobsen, K. R., Hau, J., Abelson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+concentrations+of+buprenorphine+after+oral+and+parenteral+administration+in+male+mice%5BTitle%5D)+AND+%22Vet.+J%22%5BJournal%5D)">Serum concentrations of buprenorphine after oral and parenteral administration in male mice.</a> Vet. J. 187, 251-254 (2011).</li>
<li>Simpkins, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogens+may+reduce+mortality+and+ischemic+damage+caused+by+middle+cerebral+artery+occlusion+in+the+female+rat%5BTitle%5D)+AND+%22J.+Neurosurg%22%5BJournal%5D)">Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat.</a> J. Neurosurg. 87, 724-730 (1997).</li>
<li>Wise, P. M., Dubal, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estradiol+protects+against+ischemic+brain+injury+in+middle-aged+rats%5BTitle%5D)+AND+%22Biol.+Reprod%22%5BJournal%5D)">Estradiol protects against ischemic brain injury in middle-aged rats.</a> Biol. Reprod. 63, 982-985 (2000).</li>
<li>Saleh, T. M., Cribb, A. E., Connell, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen-induced+recovery+of+autonomic+function+after+middle+cerebral+artery+occlusion+in+male+rats%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Regul.+Integr.+Comp.+Physiol%22%5BJournal%5D)">Estrogen-induced recovery of autonomic function after middle cerebral artery occlusion in male rats.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, 1531-1539 (2001).</li>
<li>Bingham, D., Macrae, I. M., Carswell, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detrimental+effects+of+17beta-oestradiol+after+permanent+middle+cerebral+artery+occlusion%5BTitle%5D)+AND+%22J.+Cereb.+Blood+Flow+Metab%22%5BJournal%5D)">Detrimental effects of 17beta-oestradiol after permanent middle cerebral artery occlusion.</a> J. Cereb. Blood Flow Metab. 25, 414-420 (2005).</li>
<li>Carswell, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+effects+of+17beta-estradiol+upon+stroke+damage+in+stroke+prone+and+normotensive+rats%5BTitle%5D)+AND+%22J.+Cereb.+Blood+Flow+Metab%22%5BJournal%5D)">Differential effects of 17beta-estradiol upon stroke damage in stroke prone and normotensive rats.</a> J. Cereb. Blood Flow Metab. 24, 298-304 (2004).</li>
<li>Yong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((17beta-estradiol+potentiates+ischemia-reperfusion+injury+in+diabetic+ovariectomized+female+rats%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">17beta-estradiol potentiates ischemia-reperfusion injury in diabetic ovariectomized female rats.</a> Brain Res. 1054, 192-199 (2005).</li>
<li>Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estradiol+increases+brain+lesions+in+the+cortex+and+lateral+striatum+after+transient+occlusion+of+the+middle+cerebral+artery+in+rats%3A+no+effect+of+ischemia+on+galanin+in+the+stroke+area+but+decreased+levels+in+the+hippocampus%5BTitle%5D)+AND+%22Peptides%22%5BJournal%5D)">Estradiol increases brain lesions in the cortex and lateral striatum after transient occlusion of the middle cerebral artery in rats: no effect of ischemia on galanin in the stroke area but decreased levels in the hippocampus.</a> Peptides. 26, 2257-2264 (2005).</li>
<li>Levin-Allerhand, J. A., Sokol, K., Smith, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safe+and+effective+method+for+chronic+17beta-estradiol+administration+to+mice%5BTitle%5D)+AND+%22Contemp.+Top+Lab+Anim.+Sci%22%5BJournal%5D)">Safe and effective method for chronic 17beta-estradiol administration to mice.</a> Contemp. Top Lab Anim. Sci. 42, 33-35 (2003).</li>
</ol>

The authors have nothing to disclose.

The administration modes presented above have in earlier studies proved superior to commercial slow-release pellets and injections in terms of producing physiological serum concentrations of 17&beta;-estradiol. While the slow-release pellets produce very high and prolonged serum concentration peaks, the injections cause extreme, daily hormone fluctuations and require repeated stressful handling of the animals 4-6. The importance of choosing adequate administration modes is highlighted by recent studies indicat...

<table><tbody><tr><td>Rimadyl, 50 mg/mL</td><td>Pfizer Pharma GmbH</td><td>462986</td><td>Postoperative analgesia after ovariectomy.</td></tr><tr><td>Lubrithal eye gel</td><td>Leo Laboratories</td><td></td><td>Eye protection during surgery.</td></tr><tr><td>Jodopax vet</td><td>Pharmaxim</td><td></td><td>Surgery disinfection.</td></tr><tr><td>Vicryl 4-0 suture</td><td>Johnson &amp;amp; Johnson</td><td></td><td>Wound suture and ovary ligation.</td></tr><tr><td>17&amp;#946;-estradiol, &amp;gt;98%</td><td>Sigma-Aldrich</td><td>E8875-1G</td><td>In sesame oil for silastic capsules and Nutella.</td></tr><tr><td>Sesame oil</td><td>Sigma-Aldrich</td><td>S3547-1L</td><td>Dissolving the 17&amp;#946;-estradiol.</td></tr><tr><td>Silastic tubing, ID/OD: 1.575/3.175 mm</td><td>Dow Corning</td><td>508-008</td><td>Production of silastic capsules.</td></tr><tr><td>Wooden applicator stick</td><td>Selefatrade</td><td>12-0783</td><td>Capping of silastic capsules.</td></tr><tr><td>Nutella</td><td>Ferrero</td><td></td><td>Medium for peroral 17&amp;#946;-estradiol.</td></tr></tbody></table>

ovariectomy and 17&beta;-estradiol replacement in rats and mice: a visual demonstration

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

The overall goal of this procedure is to gain strict control over the otherwise fluctuating serum estrogen concentrations in female rats and mice. This is accomplished by first removing the ovaries by the dorsal root. Next cel elastic capsules and a Nutella mixture are prepared.Finally, a capsule is inserted subcutaneously, or the Nutella mixture is administered orally. Ultimately, results can be obtained using radioimmunoassay that shows stable or fluctuating serum 17 beta estradiol concentrations for the capsule and Nutella methods respectively. The main advantage of these techniques over existing methods like slow release pellets, is that they have been proven to produce physiological concentrations of estradiol for prolonged periods of time.This method can help answer key questions in the estrogen field, such as differences in effects between administration, roots and doses. After anesthetizing the animal, place it in a prone position on a heating plate connected to a rectal thermometer. Apply IGEL and for postoperative relief on the lateral aspect of the animal's Neck subcutaneously.Administer five milligram per kilogram IL in saline anteriorly from the iliac crest shaver four by four centimeter area. Then disinfect the area with an antiseptic before covering the animal with a four by four centimeter aperture surgery sheet. Make a two to three centimeter midline incision and bluntly dissect the skin from the under underlying facia, one centimeter lateral of the midline.Make another incision through the fascia. Then as superficially as possible, bluntly dissect laterally under this fascia until the abdominal cavity is reached. Using tweezers, grab the adipose tissue that surrounds the ovary in the abdominal cavity and gently pull it out.Identify the ovary and ligate the uterine horns and vessels. 0.5 to one centimeter proximally to this structure. Cut out the ovary and ligated adipose tissue and put the remaining tissue back into the abdominal cavity.Make another incision in the contralateral fascia and remove the other ovary. Close the wounds, and if not immediately administering 17 beta estradiol. Let the animal awake from anesthesia.Place the animal in a heated cage for at least two hours after surgery. Housed the rat separately for the first few days postoperatively. 24 hours after surgery subcutaneously.Inject the animals with another five milligram per kilogram IL to administer 17 beta estradiol through scholastic capsules. Thoroughly mix the desired amount in sesame oil. Cut three centimeter lengths of scholastic tubing and five millimeter lengths of wooden applicator sticks for plugging Using forceps.Squeeze one wooden plug into the scholastic capsule before injecting the 17 beta estradiol sesame oil solution. To fill the entire length, gently cap the scholastic capsule on the open end with another wooden plug. Incubate the capsules in remaining 17 beta estradiol solution overnight before implantation.After closing the ectomy incisions, shave and disinfect a two by two centimeter square area on the dorsal aspect of the rat's neck. Make a one centimeter incision and cord laterally and bluntly. Dissect a subcutaneous pocket making ample space for the capsule.Wipe off the scholastic capsule and gently insert it through the incision. Then close the incision with a suture. Ensure that the capsule strains the skin as little as possible, particularly around the wound.To use the nut cream Nutella as a medium of estrogen administration thoroughly dissolve 17 beta estradiol in sesame oil, and then mix the sesame oil with the nut cream. Prepare a placebo nut cream without the 17 beta estradiol for five days prior to the experiment. Train the rats to eat hormone free nut cream on days one through three.Train the animals in groups on the fourth and fifth days. Feed them the nut cream in separate cages throughout the experiments. Place the animals in separate cages when they are fed the nut cream physiological serum.17 beta estradiol concentrations, which the current methods aim to reproduce are approximately five to 140 picograms per milliliter for rats, and five to 35 picograms per milliliter for mice. As shown here. When administered to 300 gram female spread doy rats.The subcutaneous elastic capsules produce slowly decreasing 17 beta estradiol serum concentrations from around 40 picograms per milliliter on day two to 10 picograms per milliliter by day 35. However, immediately after administration, a serum concentration peak of approximately 1000 picogram per milliliter can be expected falling to the physiological range within eight hours when the capsules were administered to 25 gram female. C 57 black six mice.Serum concentration slowly decreased from approximately 90 picograms per milliliter on day two to 25 picograms per milliliter on day 35 when administered via the nut cream to 300 grams. Spra do rats. Physiological levels of 17 beta estradiol fluctuated daily between 10 to 70 picograms per milliliter in mice.The 17 beta estradiol levels fluctuated between 20 and 120 picograms per milliliter Once mastered, the technique of ectomy and capsule implantation can be done in 10 minutes if performed properly. While attempting this procedure, it's important to remember to strictly adhere to hygienic guidelines.

ovariectomy-17-estradiol-replacement-rats-mice-visual

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Learn ovariectomy and 17β-estradiol replacement techniques in rats and mice using silastic capsules and oral Nutella delivery. These methods enable stable hormone control for neuroscience and endocrinology research.

Ovariectomy and 17β-estradiol Replacement in Rats and Mice: A Visual Demonstration

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