Authors would like to thank Mr. Pascal Lejeune for his technical support during all procedures.

The presented protocol is used for mares presented in the equine hospital and follows institutional animal care guidelines.
NOTE: Perform endometrial cysts photoablation before anestrus: heats observed in late July or August thus seem most reliable. Do not perform this procedure too late in the breeding season as it can limit the number of estruses available to treat a potential endometritis.
1. During the preceding estrus
<ol>
	<li>Perform a complete breeding soundness examination of the mare, including an endometrial biopsy1 (not described here) to ensure that the mare can potentially get pregnant after the procedure.</li>
	<li>In case of poor endometrial biopsy staging according to Kenney&#8217;s classification1, give the embryo transfer or intra cytoplasmic sperm injection (ICSI) program preference over endometrial cysts photoabalation.</li>
</ol>
2. During the preceding diestrus
<ol>
	<li>Once the rectum is empty, count, measure and map endometrial cysts of the mare in the uterus using trans-rectal ultrasonography guided by rectal palpation with a 7.5 or 5 MHz linear rectal probe and calipers on the screen (Figure 3) in a contention stock.</li>
	<li>Administer 250 &#181;g of cloprostenol intra-muscularly to induce estrus within 2 or 3 days after day 6&#8210;7 post-ovulation.</li>
	<li>On a daily basis, perform common routine ultrasonography and cervix palpation to observe estrus signs, including significant endometrial edema, antral follicle of 35 mm and softened and opened cervix.</li>
</ol>
3. Preparation when the mare is in heat
<ol>
	<li>Restrict access to the operation room to people wearing eye protection glasses.</li>
	<li>After emptying of the rectum, wrap the tail and clean the vulva aseptically using iodine-povidone.</li>
	<li>To ensure easy manipulation and comfort of the mare, administer detomidine (10 &#181;g/kg) and butorphanol (0.1 mg/kg) intravenously in the jugular vein with a 21 G needle.</li>
	<li>Per vagina, install the embryo collection catheter through the cervix and insufflate the balloon with 40 mL of air.</li>
	<li>Infuse the uterus with 1.5 L of sterile saline solution (0.9% NaCl)&#160;to inflate the uterus.</li>
	<li>Deflate the balloon and remove the catheter.</li>
	<li>Pass the endoscope aseptically through the cervix within a sterile sleeve.&#160;</li>
</ol>
4. Technique
<ol>
	<li>Connect the 600 &#181;m quartz fiber to a diode laser operating at 980 nm. Set the power at 20&#8210;25 W in continuous mode.</li>
	<li>Pass the flexible quartz fiber via the biopsy channel of a 10 mm diameter video-endoscope until 3&#8210;4 cm of free fiber can be identified on the screen. A minimum of 10 mm from the end of the fiber should be visible.</li>
	<li>Activate the laser to puncture the cyst ideally at its apex: a dedicated pedal is available on the device to keep hands free. In some cases, multiple punctures at various locations are necessary to achieve complete voiding of the cyst. The cystic fluid is passively drained out into the uterus.</li>
	<li>After the cyst lining has collapsed around the fiber, deploy the laser until the membrane shrinks.&#160;&#160;</li>
	<li>Perform transrectal ultrasonography of the uterus as many times as necessary during the procedure to confirm that endometrial cysts have disappeared.</li>
</ol>
5. Post-operative treatments
<ol>
	<li>Administer general antibiotic treatment: 22,000 UI/kg penicillin-procaine suspension, intra-muscularly, twice a day, for 3 days.</li>
	<li>Just after intervention, perform uterine lavage as described above (see steps 3.4-3.6).
	<ol>
		<li>Collect the liquid infused in the uterus before the endoscopic procedure through a sterile cuffed catheter.</li>
		<li>Flush the uterus with successive instillations of 1 L sterile isotonic solution (0.9% NaCl) until a clear liquid is collected.</li>
		<li>At the end of the procedure, administer 20 IU of oxytocin intravenously.</li>
		<li>Perform transrectal ultrasonography of the mare uterus the day after: intraluminal free liquid is frequently observed (see step 2.1).</li>
	</ol>
	</li>
	<li>As long as free intra-luminal fluid is observed by transrectal ultrasonography and until the end of the heat, perform uterine lavages daily with the procedure described above (steps 3.4-3.6). Sometimes, serum-like, limpid and yellow stained liquid is collected for several days. 
	NOTE: Giving repeated intra-muscular injections of oxytocin (20 IU) every 4 h or less when the mare is in estrus can avoid fluid accumulation and reduce the risk of bacterial contamination in the uterus.</li>
	<li>Perform trans-rectal uterine ultrasonography during the following diestrus to confirm disappearance of endometrial cysts and the absence of intraluminal free fluid. 
	NOTE:&#160;If free intraluminal fluid is observed, prostaglandin injection is recommended to induce a new estrus, to diagnose an eventual subsequent endometritis and to treat it.</li>
</ol>

<ol>
	<li>Kenney, R. M., Ganjam, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+pathological+changes+of+the+mare+uterus+and+ovary.">Selected pathological changes of the mare uterus and ovary.</a> Journal of Reproduction and Fertility. (23), 335-339 (1975).</li><li>Bracher, V., Mathias, S., Allen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Videoendoscopic+evaluation+of+the+mare's+uterus:+II.+Findings+in+subfertile+mares.">Videoendoscopic evaluation of the mare's uterus: II. Findings in subfertile mares.</a> Equine Veterinary Journal. 24 (4), 279-284 (1992).</li><li>Kaspar, B., Kahn, W., Laging, C., Leidl, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+cysts+in+the+mare.+1.+Post-mortem+studies:+occurrence+and+morphology.">Endometrial cysts in the mare. 1. Post-mortem studies: occurrence and morphology.</a> Tier&#228;rztliche Praxis. 15 (2), 161-166 (1987).</li><li>Leidl, W., Kaspar, B., Kahn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+cysts+in+the+mare.+2.+Clinical+studies:+occurrence+and+significance.">Endometrial cysts in the mare. 2. Clinical studies: occurrence and significance.</a> Tier&#228;rztliche Praxis. 15 (3), 281-289 (1987).</li><li>Mather, E. C., Refsal, K. R., Gustafsson, B. K., Seguin, B. E., Whitmore, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fibre-optic+techniques+in+clinical+diagnosis+and+visual+assessment+of+experimental+intrauterine+therapy+in+mares.">The use of fibre-optic techniques in clinical diagnosis and visual assessment of experimental intrauterine therapy in mares.</a> Journal of Reproduction and Fertility. (27), 293-297 (1979).</li><li>Stanton, M. E., Mc Kinnon, A. O., Squires, E. L., Vaala, W. E., Varner, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Equine Reproduction Vol. 2. , 2665-2668 (2011).</li><li>Bracher, V., Mathias, S., Allen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+chronic+degenerative+endometritis+(endometrosis)+on+placental+development+in+the+mare.">Influence of chronic degenerative endometritis (endometrosis) on placental development in the mare.</a> Equine Veterinary Journal. 28 (3), 180-188 (1996).</li><li>Tannus, R. J., Thun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+endometrial+cysts+on+conception+rate+of+mares.">Influence of endometrial cysts on conception rate of mares.</a> Zentralbl Veterinarmed A. 42 (4), 275-283 (1995).</li><li>Pipers, F. S., Zent, W., Holder, R., Asbury, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+as+an+adjunct+to+pregnancy+assessments+in+the+mare.">Ultrasonography as an adjunct to pregnancy assessments in the mare.</a> Journal of the American Veterinary Medical Association. 184 (3), 328-334 (1984).</li><li>LeBlanc, M., Mc Kinnon, A. O., Mc Kinnon, A. O., Squires, E. L., Vaala, W. E., Varner, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Equine Reproduction Vol. 2. , 2620-2642 (2011).</li><li>van Ittersum, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrosurgical+treatment+of+endometrial+cysts+in+the+mare.">The electrosurgical treatment of endometrial cysts in the mare.</a> Tijdschr Diergeneeskd. 124 (21), 630-633 (1999).</li><li>Rambags, B. P., Stout, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcervical+endoscope-guided+emptying+of+a+transmural+uterine+cyst+in+a+mare.">Transcervical endoscope-guided emptying of a transmural uterine cyst in a mare.</a> Vet Record. 156 (21), 679-682 (2005).</li><li>Blikslager, A. T., Tate, L. P., Weinstock, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neodymium:yttrium+aluminum+garnet+laser+irradiation+on+endometrium+and+on+endometrial+cysts+in+six+mares.">Effects of neodymium:yttrium aluminum garnet laser irradiation on endometrium and on endometrial cysts in six mares.</a> Veterinary Surgery. 22 (5), 351-356 (1993).</li><li>Bartmann, C. P., Stief, B., Schoon, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+injury+and+wound+healing+of+the+endometrium+subsequent+to+minimally+invasive+transendoscopic+use+of+Nd:YAG-laser-and+electrosurgery+in+horses.">Thermal injury and wound healing of the endometrium subsequent to minimally invasive transendoscopic use of Nd:YAG-laser-and electrosurgery in horses.</a> Dtsch Tierarztl Wochenschr. 110 (7), 271-280 (2003).</li><li>Conley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+reproductive+endocrinology+of+the+pregnant+and+parturient+mare.">Review of the reproductive endocrinology of the pregnant and parturient mare.</a> Theriogenology. 86 (1), 355-365 (2016).</li><li>Guzeloglu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+enzymes+and+receptors+of+leukotriene+pathway+genes+in+equine+endometrium+during+the+estrous+cycle+and+early+pregnancy.">Expression of enzymes and receptors of leukotriene pathway genes in equine endometrium during the estrous cycle and early pregnancy.</a> Theriogenology. 80 (2), 145-152 (2013).</li><li>Senger, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathways to pregnancy and parturition, 3rd edn. , (2015).</li></ol>

All authors are fully employed by Equine Clinic of Li&#232;ge University and have no conflicts of interest with any company trading one of the products mentioned above.

Hysteroscopy and laser photoablation of endometrial cysts is an easy procedure to reduce large intra-luminal cysts in the mare, as previously suggested14. Technically, this method is improved when performed on a mare in estrus. Inflating the uterus with sterile saline solution improved visibility when compared to air filling and continuous aspiration while performing the procedure.&#160; Small intra-parietal cysts (&#60;10 mm) are more difficult to reach and to treat. Moreover, treating these small cysts could lead to piercing of the endometrium and myometrium if laser treatment time is prolonged. However, they seldom interfere with the embryonic vesicle&#8217;s mobility and the early pregnancy diagnosis, due their localization and their size6.
Reducing numbers of intra-luminal large cysts under hysteroscopic control with the laser6 improves the mare&#8217;s annual fertility by reducing the interval between breeding and pregnancy confirmation. On day 14 post-ovulation, ultrasonographic presence of a ~14 mm diameter anechogenic embryo is used to confirm gestation, or, if it is not observed, leads to use the next heat to inseminate or breed again9,10. However, large and/or multiple endometrial cysts can delay this decision, because embryos may be hidden by endometrial cysts or confused with them6,9,10, thus limiting opportunities to get the mare pregnant, due to unexploited heats happening between two ultrasonographic controls. Hysteroscopy and laser ablation of cysts makes the day-14 pregnancy diagnosis possible again in mares that had large endometrial cysts, and thus maximizes the number of attempts, as recommended to manage difficult mares10. It also decreases the risks of an inadequate twin pregnancy diagnosis or to miss such a pregnancy.&#160;
Assessing a statistical effect of hysteroscopy and laser resection of endometrial cysts on observed fertility is very hazardous: several management factors can vary before and after surgery making comparison impossible. However, embryo migration between day 6.5 and day 16-17 could be mechanically impeded by large endometrial cysts1,4, and this incomplete uterine pregnancy recognition can lead to premature luteolysis and early pregnancy loss15,16,17. In this case of mechanical obstruction, photoablation of cysts can effectively decrease embryonic losses and improve the fertility of the mare.
However, laser resection of cysts cannot resolve the underlying causes of infertility such as endometritis or endometriosis. Response to the treatment can be disappointing when these conditions are present as they are more likely to be the cause of the poor fertility than the cysts themselves. Complete breeding soundness examination, including endometrial biopsy1,10, should be performed prior considering cyst ablation by laser in order to exclude the presence of endometrial pathologies and to define their prognosis. Any other condition compromising the mare&#8217;s fertility should be first treated, and alternatives such as embryo transfer or ICSI program should be proposed when applicable. Hysteroscopically guided endometrial cysts resection with laser is solely of interest when the endometrial cysts delay the pregnancy diagnosis or interferes with embryo migration and recognition.

Endometrial cysts in mares were histopathologically described in the seventies by Kenney1. In the late eighties, the use of hysteroscopy and ultrasonography led to their clinical description and helped to understand their consequences on fertility2,3,4,5.
In the early stages, endometrial cysts have been associated with a degeneration of the endometrium now called endometrosis1,3,4,6,7,8 and it is currently accepted that small (&#60;1 cm) cysts are related to endometrial glands fibrosis when larger cysts are related to lymphatic dilatation6. These large cysts are the most likely to disturb equine embryo migration between day 6.5 and day 17, thus decreasing the fertility by impairing maternal recognition1,3,4,6,8. Endometrial cysts may also interfere with early pregnancy diagnosis, especially if they are round and have a size similar to that of an embryonic vesicle3,4,6,9 (see Figure 1). Thus, they can potentially increase the interval between two inseminations by delaying first embryo ultrasonography and twin gestation assessment (see Figure 2). Endometrial cysts may also interfere mechanically with fluid collection during uterine lavage performed to treat endometritis or to collect the embryo. Consequently, endometrial cysts have effects on observed mare&#8217;s fertility, with potential economic consequences in breeding farms.
Besides attempts to treat the underlying endometrosis10, endoscopically-guided focal treatments of cysts have been proposed. Electrosurgical cauterization has been described, but heat exposure could injure the endometrium around the cyst11. Aspiration of the cyst is only efficient if the secreting structure is no more active at the time of the puncture12. Laser irradiation with a 980 nm diode laser with a contact fiber seems to show smaller subsequent endometrial scars6,13. Improvement of laser devices and procedures6 now allows us to propose this safe and efficient technique in equine clinical practice. However, mare owners must be informed about the limits of this option. Photoablation of the cysts will increase embryo recognition in some mares, will advance the pregnancy diagnosis in some cases and could limit the negative mechanical effect of endometrial cysts on uterine lavage performed to treat endometritis or to collect embryos. However, it will never treat the concomitant underlying endometriosis or endometritis.
This report aims to describe an efficient procedure to perform hysteroscopy-guided diode laser cyst ablation in mares. Preparation and procedures will be practically described for a clinical application.

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="663">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Butorphanol</td>
			<td style="width:121px;">Animedica</td>
			<td style="width:161px;">Morphasol 10 mg/mL</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Detomidine</td>
			<td style="width:121px;">Dechra</td>
			<td style="width:161px;">Domidine 10 mg/mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Endoscope</td>
			<td style="width:121px;">Olympus</td>
			<td style="width:161px;">Vet 170-10 SN2012729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Endoscope Vid&#233;o Tower</td>
			<td style="width:121px;">Olympus</td>
			<td style="width:161px;">CLE-E &#38; CV-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Laser device</td>
			<td style="width:121px;">Wuhan Giga Optronics</td>
			<td style="width:161px;">Velas II-60B GA19-V3186</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">NaCl 0.9%</td>
			<td style="width:121px;">Baxter</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">P&#233;nicilline-proca&#239;ne</td>
			<td style="width:121px;">Kela</td>
			<td style="width:161px;">PENI-Kel 300.00 UI/ml</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Ultrasonography</td>
			<td style="width:121px;">Aloka</td>
			<td style="width:161px;">Aloka 500 &#38; 5mHz transrectal probe</td>
			<td></td>
		</tr>
	</tbody>
</table>

endoscopy guided photoablation of endometrial cysts using a 980 nm laser with a contact fiber in mares

In mares, endometrial cysts are associated with endometriosis and can cause maternal recognition failure or compromise and delay pregnancy diagnoses. Historical treatments were invasive and had adverse effects on the endometrium. Hysteroscopically guided laser therapy is easy and effective for endometrial cysts resection, with no deleterious effects for the endometrium.
A 110 cm long and 1.0 cm wide endoscope is sterilely introduced in the uterus through the open cervix of an estrous mare after vulvar cleaning. The uterus is slowly infused with less than 1 L of physiologic solution and the laser fiber is inserted in the biopsy canal of the endoscope. Cysts are then cauterized with the 980 nm diode laser with a contact fiber set at 20&#8210;2 5W in continuous mode. Each cyst is punctured&#160;until complete voiding of the cyst and shrinking of the cyst wall around the fiber. Uterine lavages with sterile saline solution are performed directly after the surgery and for one or two days as non-inflammatory fluid can be observed.
This procedure is easy and quickly performed, with no obvious deleterious effects. Cysts resection makes ultrasound pregnancy diagnosis easier and, in some cases, could restore proper embryo migration in the uterine horns between day 6.5 and 17. However, this treatment does not improve the underlying histological lesions related to endometriosis. These considerations should be clearly expressed to the breeder before this procedure.

Surgeries were performed on mares having cysts with diameters over 15 mm. However, if other smaller cysts were also present, photoablation of the other cysts was attempted. Generally, these large cysts were at the bifurcation or the basis of the horns. However, some of them were present in the body of the uterus and were equally treated.&#160;
In our experience, intraluminal cysts are very easy to localize with the endoscope. Moreover, performing this procedure in saline solution increases the visibility inside the uterus: it avoids emission of vapors, foam production due to endometrial mucus and lymphatic liquid mixing.&#160; Manual closure of the cervix while maintaining and guiding the endoscope helps to keep the liquid inside. Trans-rectal manipulation of the endoscope in the uterus can sometimes help to guide the endoscope into the desired horn. Thereafter, laser intervention is quite easy and quick, with a maximum treatment time of 5 min per cyst, depending on the size and on presence of inner walls or loculations that may require multiple laser activations for the same structure. The direct contact decreased the overall temperature in the uterus. Parietal cysts were more difficult to identify, because of their localization and their smaller size (see Figure 4). In some cases, it was impossible to visualize (with the endoscope) the parietal endometrial cysts previously identified by transrectal ultrasonography. However, only the smallest parietal cysts were impossible to treat, but they do not interfere with the embryonic vesicle&#8217;s movements and are not easily confused with day-14 embryos, because of their size and localization. No direct surgical complications have been observed after the performed procedures.
Liquid accumulation was frequently observed by ultrasonography for several days after surgery. However, the collected liquid was clear and yellow and no inflammatory cells were observed on endometrial swabs. We attributed this feature to lymphatic fluid accumulation in the uterus after the opening of the cysts. Routine lavage procedures and repeated oxytocin boluses were sufficient to treat this condition before the end of estrus.
Delay between breeding and pregnancy diagnosis was shortened by decreasing the number of large size cysts. In a mare, the diagnosis was made possible again at day 14, whereas before treatment, it was only possible after day 24 or day 32 when cardiac activity or embryo migration could be respectively assessed. While laser treatment of endometrial cysts has obvious advantages, concluding about its direct effect on fertility would be dangerous, because of the many factors impacting fertility and even the birth of a foal the following season would not allow a statistically valid conclusion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61569/61569fig1.jpg" /> 
Figure 1: Day 14 embryonic vesicle (~14 mm diameter). Normal appearance of an embryo observed on early pregnancy diagnosis. <a href="https://www.jove.com/files/ftp_upload/61569/61569fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61569/61569fig2.jpg" /> 
Figure 2: Day 14 embryonic vesicle and endometrial cyst. The embryo (above) is attached to the endometrial cyst (below). <a href="https://www.jove.com/files/ftp_upload/61569/61569fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61569/61569fig3.jpg" /> 
Figure 3: Endometrial cysts in diestrus. Multiple cysts in the bifurcation of the horns of a non-pregnant mare (measure scale on the right side is in centimeters). <a href="https://www.jove.com/files/ftp_upload/61569/61569fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61569/61569fig4.jpg" /> 
Figure 4: Small parietal endometrial cyst. The mare is non-pregnant and in diestrus (scale bar is on the left side is in centimeters). <a href="https://www.jove.com/files/ftp_upload/61569/61569fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Endoscopy Guided Photoablation of Endometrial Cysts using a 980 nm Laser with a Contact Fiber in Mares at JoVE.com

Endoscopy Guided Photoablation of Endometrial Cysts using a 980 nm Laser with a Contact Fiber in Mares

The aim of this report is to describe the photoablation of endometrial cysts in the mare using an endoscopic laser device, and to discuss the interest and limitations of this new approach to deal with low fertility issues.

In mares, endometrial cysts are associated with endometriosis and can cause maternal recognition failure or compromise and delay pregnancy diagnoses. ...

endoscopy-guided-photoablation-endometrial-cysts-using-980-nm-laser

Universidad San Sebastian

Research

JoVE Journal

Medicine

4.2K Views.  Liège University. The aim of this report is to describe the photoablation of endometrial cysts in the mare using an endoscopic laser device, and to discuss the interest and limitations of this new approach to deal with low fertility issues.

Video: Endoscopy Guided Photoablation of Endometrial Cysts using a 980 nm Laser with a Contact Fiber in Mares

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

A Mobile Outside-in Technique of Transforaminal Lumbar Endoscopy for Lumbar Disc Herniations

Percutaneous endoscopic transforaminal lumbar discectomy (PETLD) has now become a standard of care for the management of lumbar disc disease. There are two techniques for the introduction of a working cannula with respect to disc&#8212;outside-in and inside-out. The aim of this prospective study is to describe the technical aspects of a novel mobile outside-in method in dealing with different types of disc prolapse. A total of 184 consecutive patients with unilateral lower limb radiculopathy due to lumbar disc prolapse were operated on with the mobile outside-in technique of PETLD. Their clinical outcomes were evaluated based on the type of disc prolapse they had, a visual analog scale (VAS) leg pain score, the Oswestry Disability Index (ODI), and the Macnab criteria. The completeness of the decompression was documented with a postoperative magnetic resonance imaging. The mean age of the patients was 50 &#177; 16 years and the male/female ratio was 2:1. The mean follow-up was 19 &#177; 6 months. A total of 190 lumbar levels were operated on (L1&#8211;L2: n = 4, L2&#8211;L3: n = 17, L3&#8211;L4: n = 27, L4&#8211;5: n = 123, and L5&#8211;S1: n = 19). Divided into types, the patient distribution was central: n = 14, paracentral: n = 74, foraminal: n = 28, far lateral: n = 13, superior-migrated: n = 8, inferior migrated: n = 38, and high canal compromise: n = 9. The mean operative time was 35 &#177; 12 (25 - 56) min and the mean hospital stay was 1.2 &#177; 0.5 (1&#8211;3) days. The VAS score for leg pain improved from 7.5 &#177; 1 to 1.7 &#177; 0.9. The ODI improved from 70 &#177; 8.3 to 23 &#177; 5. According to the Macnab criteria, 75 patients (40.8%) had excellent results, 104 patients (56.5%) had good results, and 5 patients (2.7%) had fair results. Recurrence (including early and late) was seen in 15 out of the 190 levels that were operated on (7.89%). This article presents a novel outside-in approach that relies on a precise landing within the foramen in a mobile manner and does not solely depend upon the enlargement of the foramen. It is more versatile in application and useful in the management of all types of disc prolapse, even in severe canal compromise and high migration.

Here we present a protocol of a novel outside-in technique of transforaminal endoscopic discectomy for lumbar disc herniations. The technical aspects of the technique, the wide indications of use, and the results of the treatment in 184 patients are described in detail.

Percutaneous endoscopic transforaminal lumbar discectomy (PETLD) has now become a standard of care for the management of lumbar disc disease. There are ...

Enucleation of the Prostate for the Treatment of Benign Prostatic Hyperplasia Using a 980 nm Diode Laser

In the aging male population, the occurrence of lower urinary tract symptoms (LUTS) caused by benign prostatic hyperplasia (BPH) is a common problem. Here, we introduce a new technique called 980 nm diode laser enucleation (DiLEP) to treat BPH1. Diode lasers can absorb both water and hemoglobin at the same time, so they are good for cutting and hemostasis2. The diode laser was approved by the FDA in 2007, and has been used in the treatment of BPH because of its effective cutting and hemostasis effect3. DiLEP presents several advantages over other techniques, such as TURP, HoLEP, and PVP. During the procedure, we define the boundary of a high-volume prostate and separate it into three lobes with a diode laser by burning two rings and one groove (like a Cupid&#39;s arrow). Compared to other procedures, mDiLEP has fewer intraoperative complications, a shorter learning curve, and achieves more tissue resection.

Here, we present a protocol for modified 980 nm diode laser enucleation to treat large volume benign prostatic hyperplasia.

In the aging male population, the occurrence of lower urinary tract symptoms (LUTS) caused by benign prostatic hyperplasia (BPH) is a common problem. ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...