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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+delivery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+causes+of+preterm+birth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+inflammatory+pathways+for+the+prevention+of+preterm+birth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+spontaneous+preterm+birth:+from+underlying+mechanisms+to+predictive+and+preventive+interventions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+parturition+biology+from+genetically+altered+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+simultaneous+measurement+of+mouse+systolic+arterial+blood+pressure+by+radiotelemetry+and+tail-cuff+methods.+American+journal+of+physiology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bilateral+pelvic+neurectomy+on+cervical+ripening+in+pregnant+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+intrauterine+pressure+by+telemetry:+a+new+approach+for+studying+parturition+in+mouse+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+modulates+the+onset+of+murine+parturition+by+competing+ovarian+and+uterine+effects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inwardly+rectifying+K++channel+KIR7.1+controls+uterine+excitability+throughout+pregnancy.">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.

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			<td style="width:207px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Surgical Glue</td>
			<td style="width:105px;">3M Vetbond</td>
			<td style="width:147px;">1469SB</td>
			<td>Tissue Adhesive--stored in Drierite</td>
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			<td height="21" style="height:21px;width:245px;">Absorbable 6-0 Sutures</td>
			<td style="width:105px;">Ethicon</td>
			<td style="width:147px;">J212H</td>
			<td>Vicryl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Non-absorbable 6-0 Sutures</td>
			<td style="width:105px;">Ethicon</td>
			<td style="width:147px;">8706H</td>
			<td>Prolene</td>
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			<td height="63" style="height:63px;">Water Jacket Blanket + Heating Pad</td>
			<td style="width:105px;">Gaymer</td>
			<td style="width:147px;">T/Pump PN 11184-000 Blanket-66N111CC</td>
			<td>Specialized for rodents</td>
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			<td height="21" style="height:21px;width:245px;">Bead sterilizer</td>
			<td style="width:105px;">Keller</td>
			<td style="width:147px;">Z378577</td>
			<td>Steri 250 Sterilizer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Disecting Microscope</td>
			<td style="width:105px;">Nikon</td>
			<td style="width:147px;">SMZ754</td>
			<td>Fibre optic gooseneck external light source</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:245px;">Sterile Surgical Gloves--Latex&#160;</td>
			<td style="width:105px;">Cardinal Health Triflex</td>
			<td style="width:147px;">2D7253</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">PhysioTel PA-C10 Pressure Transmitter</td>
			<td style="width:105px;">&#160;Data Sciences International</td>
			<td style="width:147px;">270-0135-001</td>
			<td style="width:207px;">TA11PA-C10</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Telemeter Reciever</td>
			<td style="width:105px;">&#160;Data Sciences International</td>
			<td style="width:147px;">272-6001-001</td>
			<td style="width:207px;">RPC-1</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Dataquest ART 4.3.2 Analysis Platinum</td>
			<td style="width:105px;">&#160;Data Sciences International</td>
			<td style="width:147px;">271-0147-141</td>
			<td>Analysis software</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Diet Recovery Gel</td>
			<td style="width:105px;">Clear H2O</td>
			<td style="width:147px;">72-01-5022</td>
			<td>Purified Soft Diet for Rodents</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tergazyme</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td>Z273287</td>
			<td>Enzyme detergent</td>
		</tr>
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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&#8217;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 ...

Watch this Scientific Journal Video about Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo at JoVE.com

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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

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. ...

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

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9.6K 次观看.  Washington University in St. Louis. This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy. 

视频: Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...