Name: Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo (Video) | JoVE Journal
Uploaded: 2015-04-06T15:00:00.000+00:00
Duration: 7 min 3 s
Description: Learn intrauterine telemetry to measure contractile pressure in pregnant mice in vivo. This method enables real-time assessment of uterine pressure patterns for reproductive physiology research.

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This work was supported by funding from the March of Dimes FY12-133 (S.K.E.) and the National Institutes of Health R01HD037831 (S.K.E.). We would like to thank Dr. Deborah Frank for critical review of the manuscript.

NOTE: All animal procedures complied with guidelines for the care and use of animals set forth by the National Institutes of Health. All protocols were approved by the Animal Studies Committee at Washington University in St. Louis. 
NOTE: Specific materials and equipment for this protocol are listed Materials and Equipment table.
1. Timed Breeding of Female Mice
<ol>
	<li>Breed adult females between two and six months of age in a 2 hr time window with males. Specific breeding hr are not needed as long as they are kept consistent for the entirety of the experiment.</li>
	<li>Confirm pregnancy by the presence of a copulatory plug. Designate the day and hr on which the plug is first seen as day post-coitus 0 (dpc0). Remember the specific hour when starting pressure recording.</li>
	<li>Perform surgeries on dpc8&#8211;13. The exact dpc will depend on the mouse strain.</li>
	<li>To determine if a mouse is pregnant before surgery, perform ultrasound imaging of the abdomen with a 40 MHz linear array probe coupled to a imaging system before administering anesthesia. This will allow visualization of gestational sacs. If an ultrasound system is not available, use the weight of the mouse. 
	NOTE: A weight gain of at least 2 g between dpc0 and dpc8 and distension of the abdomen is a good indicator of pregnancy.</li>
</ol>
2. Telemetry Surgery Prep
<ol>
	<li>Use an autoclave to sterilize instruments, gauze, cotton swabs, surgical drapes, and pipette tips within 24 hr before surgery.</li>
	<li>Clean surgery surface with 70% ethanol.</li>
	<li>Turn on heating pad (specialized for rodents) and bead sterilizer.</li>
</ol>
3. Telemetry Surgery
<ol>
	<li>For anesthesia, use 4-5% isoflurane with an oxygen flow rate of 1-2 L/min.</li>
	<li>Wait until the mouse shows signs of anesthetization and then inject subcutaneously with buprenorphine (analgesic:&#160;0.1 ml per 20 g).</li>
	<li>Shave the abdomen of the mouse from the bottom of the ribs down to the bladder.</li>
	<li>Bring the mouse to the surgery table on pre-warmed heating pad, and cover the mouse&#8217;s eyes with ophthalmic ointment&#160;to prevent drying.</li>
	<li>Begin sterile technique. Put on sterile gloves and keep the wrapper as a sterile surface on which to place the iodine and ethanol scrubs.</li>
	<li>Remove the telemeter from the package or saline and use sterile forceps to place it on sterile gauze.
	<ol>
		<li>Use the syringe (provided by the telemeter supplier) to fill the catheter tip with gel.&#160; Gently squeeze the catheter creating enough negative pressure to draw the gel into the catheter.</li>
	</ol>
	</li>
	<li>Place a sterile tip on the pipette. Pipette 2 &#181;l of surgical glue and set the pipette on a sterile surface.</li>
	<li>To clean the surgical site, swab the area with iodine and then with 70% ethanol.</li>
	<li>Cut a hole in the surgical drape just large enough for the area needed for the incision and place over the mouse.</li>
	<li>Perform a foot pinch (pedal response) to ensure the mouse is completely anesthetized before making an incision.</li>
	<li>Make a small vertical midline incision. Next, make a similar incision through the underlying body wall muscle.</li>
	<li>Gently pull the uterus out of the body cavity and locate the uterine horn containing the most pups that are viable.
	<ol>
		<li>Optional - For addition of miRNA, inject viral vector into muscle layer of uterus before insertion of telemeter.</li>
	</ol>
	</li>
	<li>Make a small incision at the tip of the uterine horn with 3 mm cutting edge spring scissors.</li>
	<li>Thread the catheter, without squeezing, between the uterine wall and fetal sacs. Insert the catheter past several sacs to reduce its accidental removal. Make sure the fetal sacs are not disturbed by the insertion.</li>
	<li>Pipette the surgical glue to the site of telemeter insertion to adhere the catheter to the uterine horn and prevent the catheter from sliding out of the uterine horn. Wait a few seconds for the glue to become rigid, and then carefully place the uterus back inside the body cavity.</li>
	<li>Place the telemeter in the body cavity on the opposite side of the catheter insertion site.</li>
	<li>Suture the body cavity closed with 6.0 absorbable sutures. First, do a purse stitch and then three to four individual stitches.</li>
	<li>Suture the skin by using the same technique, but with 6-0 non-absorbable suture.</li>
	<li>Swab dibucaine (topical analgesic) ointment over the incision site.</li>
	<li>Inject 0.3-0.4 ml of sterile saline subcutaneously to help rehydrate the animal.</li>
	<li>Monitor the animal after surgery until it is fully awake. Do not place the mouse back in its cage until it is able to freely hold its head above the table as certain types of bedding can suffocate a heavily anesthetized mouse. For a mouse that has been anesthetized for 30-45 min, one should expect the mouse to be fully awake by 1 hr and 15 min.</li>
	<li>If performing additional surgeries, wipe instruments with 70% ethanol and bead sterilize before reuse.</li>
</ol>
4. Post-surgery Recovery
<ol>
	<li>Monitor daily post-surgery progress in a log. Catalog weight gain and loss and watch for lethargy, bleeding, or incomplete closure of the surgical incision.
	<ol>
		<li>Feed the mouse softened mouse chow (~the consistency of applesauce) or specialized recovery diet food.</li>
		<li>Examine the sutures and make sure they are not pulled out, but try not to handle the mouse excessively.</li>
	</ol>
	</li>
</ol>
5. Telemetry Recording
<ol>
	<li>On the day of interest (at least a two to three days after the mouse has recovered from surgery), place a magnet near the mouse to turn on the telemeter. Put the mouse cage on the receiver. The light on the receiver should turn on if the telemeter is activated.</li>
	<li>Calibrate brand new telemeters with manufacturer specific calibrations provided. Reused telemeter calibration specifications will be kept in software.</li>
	<li>Within the software, start recording by right clicking on &#8220;animal&#8221; and click &#8220;start sampling, continuous,&#8221; which allows sampling at rates of up to 500 Hz. Make sure the software is set to save AND trace. 
	NOTE: When the sampling mouse icon turns green, pressures are being recorded. Disturbing the mouse during delivery can interrupt or alter labor, so observe cautiously.</li>
	<li>After delivery or time period of interest, stop recording by right clicking on &#8220;animal&#8221; and selecting &#8220;stop recordings, all&#8221;. Use a magnet to turn off telemeter to save battery life, as the telemeter battery will typically only last for 1.5 months of continuous recording.</li>
	<li>Euthanize the mouse by CO2 overdose and recover the telemeter. Pups are euthanized by decapitation.&#160;</li>
</ol>
6. Telemeter Sterilization
<ol>
	<li>Place the telemeter in a solution of 1% enzyme detergent in deionized water overnight at room temperature.</li>
	<li>Rinse the telemeter in deionized water and remove all tissue and glue, being careful not to squeeze the catheter.</li>
	<li>Incubate the telemeter in 2% glutaraldehyde for 24 hr at room temperature to sterilize.</li>
	<li>Rinse the telemeter with sterile saline and store it in sterile saline until next implantation or to be sent back to manufacturer to be refurbished. Telemeters should be refurbished if tissue is located inside the catheter after sterilization. Typically telemeters can be used 4-5 times before needing to be refurbished.</li>
</ol>

<ol><li>Slattery, M. M., Morrison, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preterm+delivery%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Preterm delivery.</a> Lancet. 360, 1489-1497 (2002).</li>
<li>Goldenberg, R. L., Culhane, J. F., Iams, J. D., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+and+causes+of+preterm+birth%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Epidemiology and causes of preterm birth.</a> Lancet. 371, 75-84 (2008).</li>
<li>Keelan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+inhibition+of+inflammatory+pathways+for+the+prevention+of+preterm+birth%5BTitle%5D)+AND+%22Journal+Of+Reproductive+Immunology%22%5BJournal%5D)">Pharmacological inhibition of inflammatory pathways for the prevention of preterm birth.</a> Journal Of Reproductive Immunology. 88, 176-184 (2011).</li>
<li>Voltolini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+spontaneous+preterm+birth%3A+from+underlying+mechanisms+to+predictive+and+preventive+interventions%5BTitle%5D)+AND+%22Reproductive+Sciences%22%5BJournal%5D)">Understanding spontaneous preterm birth: from underlying mechanisms to predictive and preventive interventions.</a> Reproductive Sciences. 20, 1274-1292 (2013).</li>
<li>Ratajczak, C. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+parturition+biology+from+genetically+altered+mice%5BTitle%5D)+AND+%22Pediatric+research%22%5BJournal%5D)">Insights into parturition biology from genetically altered mice.</a> Pediatric research. 64, 581-589 (2008).</li>
<li>Rajakumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Placental+HIF-1+alpha%2C+HIF-2+alpha%2C+membrane+and+soluble+VEGF+receptor-1+proteins+are+not+increased+in+normotensive+pregnancies+complicated+by+late-onset+intrauterine+growth+restriction%5BTitle%5D)+AND+%22American+Journal+Of+Physiology.+Regulatory%2C+Integrative+And+Comparative+Physiology%22%5BJournal%5D)">Placental HIF-1 alpha, HIF-2 alpha, membrane and soluble VEGF receptor-1 proteins are not increased in normotensive pregnancies complicated by late-onset intrauterine growth restriction.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 293, (2007).</li>
<li>Whitesall, S. E., Hoff, J. B., Vollmer, A. P., D'Alecy, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+simultaneous+measurement+of+mouse+systolic+arterial+blood+pressure+by+radiotelemetry+and+tail-cuff+methods.+American+journal+of+physiology%5BTitle%5D)+AND+%22Heart+And+Circulatory+Physiology%22%5BJournal%5D)">Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. American journal of physiology.</a> Heart And Circulatory Physiology. 286, H2408-H2415 (2004).</li>
<li>Mackay, L. B., Shi, L. B., Maul, H. ., Maner, W. L., Garfield, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+bilateral+pelvic+neurectomy+on+cervical+ripening+in+pregnant+rats%5BTitle%5D)+AND+%22Journal+Of+Perinatal+Medicine%22%5BJournal%5D)">The effect of bilateral pelvic neurectomy on cervical ripening in pregnant rats.</a> Journal Of Perinatal Medicine. 37, 263-269 (2009).</li>
<li>Pierce, S. L., Kutschke, W., Cabeza, R., England, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+measurement+of+intrauterine+pressure+by+telemetry%3A+a+new+approach+for+studying+parturition+in+mouse+models%5BTitle%5D)+AND+%22Physiol+Genomics%22%5BJournal%5D)">In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models.</a> Physiol Genomics. 42, 310-316 (2010).</li>
<li>Imamura, T., Luedke, C. E., Vogt, S. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxytocin+modulates+the+onset+of+murine+parturition+by+competing+ovarian+and+uterine+effects%5BTitle%5D)+AND+%22American+Journal+Of+Physiology.+Regulatory%2C+Integrative+And+Comparative+Physiology%22%5BJournal%5D)">Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 279, R1061-R1067 (2000).</li>
<li>McCloskey, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+inwardly+rectifying+K%2B+channel+KIR7.1+controls+uterine+excitability+throughout+pregnancy%5BTitle%5D)+AND+%22EMBO+Mol+Med%22%5BJournal%5D)">The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.</a> EMBO Mol Med. 6 (9), 1161-1174 (2014).</li>
</ol>

The authors have nothing to disclose.

Investigations of myometrial contractility have relied on ex vivo measurements of muscle tension. This methodology can be useful for early-stage testing of newly developed uterotonic drugs that cannot be administered to live animals. However, in vivo approaches are necessary to longitudinally understand the progression of labor. The in vivo measurements serve several purposes. First, they enable capture of a complete picture of changes in uterine contractile pattern over the duration of pregnan...

Preterm birth is the leading cause of perinatal morbidity and mortality in developed countries; it is responsible for 50% of perinatal morbidity and 75% of perinatal mortality1,2. Preterm labor is multifaceted and can be idiopathic. Although much research has emerged on the molecular and electrical pathways transforming the myometrium from a quiescent tissue into a contractile one, the exact pathophysiology of preterm labor remains elusive. Endocrine, inflammatory, and gene regulation have all been linked to preterm labor3,4. However, ethical concerns limit the ability to conduct research on the mechanisms of preterm labor in humans.
Given these limitations, many researchers have turned to mice as a model system with which to study the physiology of parturition. Mice have short gestations lasting approximately three weeks and can be easily genetically manipulated. Additionally, multiple genetic mouse models have been developed to determine the signaling pathways that are essential for labor5. Despite key differences between mouse and human parturition, mice and humans share many of the same mechanisms that are essential for labor, including intrauterine inflammation and infection6. Thus, mice serve as an invaluable tool for examining uterine activity. To date, the gold standard to measure uterine contractility in mice has been ex vivo isometric tension recordings; however, this is limited to one gestational time point per experiment and requires removal of the uterus from its physiological environment. Another important factor is the large number of animals required for these investigations. Lastly, this methodology does not allow for longitudinal studies examining induction of labor and preterm pathophysiology.
Recent advancements made in radiometric telemetry devices used to study arterial pressure changes7 in mice and intrauterine pressure in rats8 begged the question to whether the same technology could be used to study changes in intrauterine pressure in mice during pregnancy. After initial troubleshooting, a method was successfully developed to measure labor induction and progression in a mouse. This in vivo approach can measure the transition from the low-pressure state of the quiescent uterus to the high-pressure state indicative of forceful labor contractions. This method has also been able to detect significant pressure differences between transgenic mice with compromised parturition and wild type mice, demonstrating the physiological impact of gene expression9. This protocol provides a real-time, in vivo method to study mouse uterine pressure during pregnancy.

<table><tbody><tr><td>Surgical Glue</td><td>3M Vetbond</td><td>1469SB</td><td>Tissue Adhesive--stored in Drierite</td></tr><tr><td>Absorbable 6-0 Sutures</td><td>Ethicon</td><td>J212H</td><td>Vicryl</td></tr><tr><td>Non-absorbable 6-0 Sutures</td><td>Ethicon</td><td>8706H</td><td>Prolene</td></tr><tr><td>Water Jacket Blanket + Heating Pad</td><td>Gaymer</td><td>T/Pump PN 11184-000 Blanket-66N111CC</td><td>Specialized for rodents</td></tr><tr><td>Bead sterilizer</td><td>Keller</td><td>Z378577</td><td>Steri 250 Sterilizer</td></tr><tr><td>Disecting Microscope</td><td>Nikon</td><td>SMZ754</td><td>Fibre optic gooseneck external light source</td></tr><tr><td>Sterile Surgical Gloves--Latex&amp;nbsp;</td><td>Cardinal Health Triflex</td><td>2D7253</td><td></td></tr><tr><td>PhysioTel PA-C10 Pressure Transmitter</td><td>&amp;nbsp;Data Sciences International</td><td>270-0135-001</td><td>TA11PA-C10</td></tr><tr><td>Telemeter Reciever</td><td>&amp;nbsp;Data Sciences International</td><td>272-6001-001</td><td>RPC-1</td></tr><tr><td>Dataquest ART 4.3.2 Analysis Platinum</td><td>&amp;nbsp;Data Sciences International</td><td>271-0147-141</td><td>Analysis software</td></tr><tr><td>Diet Recovery Gel</td><td>Clear H2O</td><td>72-01-5022</td><td>Purified Soft Diet for Rodents</td></tr><tr><td>Tergazyme</td><td>Sigma-Aldrich</td><td>Z273287</td><td>Enzyme detergent</td></tr></tbody></table>

intrauterine telemetry to measure mouse contractile pressure in vivo

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Real-time in vivo intrauterine pressures can be recorded by using a telemetric acquisition system. Pressure sampling paired with simultaneous video recording of the mouse was used to capture the exact time of each pup’s delivery and correlate delivery time to the intrauterine pressure of the mother. All the data points can then be plotted throughout the recording to generate a pressure-versus-time plot (Figure 1A). This plot shows the points during gestation when the intrauterine pressure change...

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

The overall goal of this procedure is to measure the intrauterine pressure of a pregnant mouse in vivo. This is accomplished by first locating the uterine horn containing the highest number of viable pups. The day of surgery will depend on your model and may require troubleshooting.The catheter of a small TER is then threaded into the horn and the TER is activated. Ultimately, the intrauterine placement of the TER allows assessment of the real time contractual pressure patterns of the pregnant animal. This method can help answer key questions in the reproductive field, such as which proteins and signaling molecules are responsible for the induction and contractile states of pregnancy.Visual demonstration of this method is critical as there's little room for catheter insertion deviation when threading the catheter into the pregnant uterus without leading to an increased risk of fetal demise. Before beginning the procedure, clean the surgery surface with 70%ethanol and turn on the heating pad and bead sterilizer. Next, shave the abdomen of an anesthetized mouse from the bladder to the bottom of the ribs and place the mouse on the heating pad.The nozzle the mouse is placed under is for anesthetization, not respiration. The mouse is more quickly sedated, went on its abdomen than went on its back. Pull on a pair of sterile gloves.Place the packaging under the mouse as your sterile surgical surface using sterile forceps. Next, place the telemeter on sterile gauze. Then gently squeeze the catheter of the TER syringe to draw gel into the tip of the catheter.Load a pipette with two microliters of surgical glue and place the pipette on a sterile surface. Then swab the surgical area on the animal sequentially with iodine and 70%ethanol. Place a fenestrated drape over the mouse.And then after confirming complete anesthetization by toe pinch, make a small vertical midline incision in the skin, followed by a similar incision through the underlying body wall muscle. Now gently pull the uterus out of the body cavity and locate the uterine horn containing the highest number of viable pups. Use three millimeter cutting edge spring scissors to make a small incision at the top of the uterine horn.Then thread the catheter without squeezing between the uterine wall and the fetal sax. Inserting the catheter past several sacks to reduce its accidental removal. When the catheter is in place, dispense the surgical glue onto the site of insertion to adhere the catheter to the uterine horn.And wait a few seconds for the glue to become rigid. Then carefully place the uterus back inside the body cavity and place the TER on the opposite side of the insertion site. Now use a six zero absorbable suture to make a purse stitch, followed by three to four individual stitches to close the body cavity, suture the skin by the same technique, but with a six zero non-absorbable suture.Then swab a topical analgesic ointment over the incision site and inject 0.3 to 0.4 milliliters of sterile saline subcutaneously to help rehydrate the animal. Monitor the mouse after surgery until it is fully awake. Placing the animal back in its cage when it is able to freely hold its head above the table.Monitor the daily post-surgery progress in a log and feed the mouse specialized recovery diet food as well. Examine the sutures to make sure they are not pulled out. Taken care not to handle the mouse excessively at least two to three days.After the surgery, place a magnet near the animal to turn on the TER and place the mouse cage on the receiver. The light on the receiver will turn on if the TER is activated. Calibrate a brand new TER with a provided manufacturer specific calibrations, and then within the TER software right click on animal.Then click start sampling continuous to allow sampling at rates of up to 500 hertz. Taking care that the software is set to save and trace in this experiment. A real-time in vivo intrauterine pressure sampling was paired with the simultaneous video recording of the mouse to capture the exact time of each pup's delivery, allowing correlation of the delivery time to the intrauterine pressure of the mother.Additionally, a moving average of the intrauterine pressure was also plotted to more easily visualize the sustained increases in intrauterine pressure characteristic of the contractions associated with the labor process. Intrauterine telemetry can also be used to test in vivo responses to pharmacological agents during pregnancy, for example, for early drug testing. Prior to clinical trials.In this representative experiment, the TER was implanted at the same time as a micro osmotic pump delivering a steady rate of oxytocin. Note the differences observed in the recordings between nonpregnant and pregnant mice treated with continuous oxytocin. By injecting micro RNA expressing lentil viral vectors into the myometrium during the TER implantation, the uterine specific knockdown of a gene of interest can be examined.For example, as seen in these representative tracings administration of scrambled or cure 7.1 micro RNA demonstrates that knocking down expression of the potassium channel cure 7.1 causes intrauterine pressure to increase While attempting the surgical procedure. It's important to remember not to force or squeeze the catheter over the TER.

intrauterine-telemetry-to-measure-mouse-contractile-pressure-in-vivo

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JoVE Journal

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Learn intrauterine telemetry to measure contractile pressure in pregnant mice in vivo. This method enables real-time assessment of uterine pressure patterns for reproductive physiology research.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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