The authors have no one to acknowledge.

The study was approved by the Ethics Committee Review Board of Peking University Third Hospital and conformed to the Declaration of Helsinki. The new method of surgery named Pentagram Suturing Anterior Chamber Plasty (PSACP) was approved by the medical technology clinical application management Committee Review Board of Peking University Third Hospital. All the patients undergoing PSACP had provided written informed consent before the surgery.
1. Preparations before PSACP
<ol>
	<li>Perform ophthalmic examinations for all the patients, including a visual acuity assessment, rebound tonometer, color slit lamp photography (Figure 1A,B), and ophthalmic ultrasound.</li>
	<li>Display the range of anterior synechia by anterior segment optical coherence tomography (ASOCT) and ultrasound biomicroscopy (UBM) to design the position of incisions (Figure 1C,D and Figure 2).</li>
	<li>Ensure adequate availability of 10-0 polypropylene sutures on a STC-6 needle, 29 G syringe, cohesive viscoelastic, and balanced salt solution (BSS) (see Table of Materials).</li>
	<li>Provide general anesthesia with 2% lidocaine hydrochloride injection (2.5 mL) (see Table of Materials).</li>
	<li>Place the patient in a supine position with routine disinfection, towel laying, film sticking, and palpebral fissure opening by an eye speculum.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Mark the five scleral puncture points 1.5 mm posterior to the limbus keeping away from the anti-glaucoma filtration bleb with 1% crystal violet solution. Ensure that the circular degree between the adjacent puncture points is 72&#176; (Figure 3).</li>
	<li>Remove the edematous epithelium of the cornea with a sclera tunnel knife. 
	&#8203;NOTE: Hypertonic glucose solution is recommended to reduce corneal stromal swelling.</li>
	<li>Make a limbus incision (1 mm), inject cohesive viscoelastic to separate the anterior synechia of the iris, and remove the fibrous membranes when necessary.</li>
	<li>Perform blunt separation for peripheral anterior synechia (PAS).</li>
	<li>Insert the 16 mm (STC-6) needle from the position 1 mm beside one of the puncture points (Figure 3) into the anterior chamber and retrieve across the surface of the iris within the bore of a 29 G syringe from another side puncture point with a circular distance of 144&#176; (Figure 3).</li>
	<li>When the 16 mm (STC-6) needle comes out from the anterior chamber, select the next inserting point 1 mm beside the retrieve point and 1.5 mm posterior to the limbus (Figure 3).</li>
	<li>Repeat steps 2.5-2.6 until the retention sutures in the anterior chamber form a pentagram-like barrier on the surface of the iris and pupil.</li>
	<li>Tie a surgical knot from the initial insert point and embed it into the superficial sclera under the bulbar conjunctiva.</li>
	<li>Perform the anterior chamber irrigation with BSS. Ensure removing all the viscoelastic, and then seal the incisions to watertight by a sterile irrigateor (see Table of Materials).</li>
</ol>

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	<li>Tsai, Y. -. Y., Tseng, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+trabeculectomy+and+vitrectomy+for+pseudophakic+malignant+glaucoma+and+extensive+peripheral+anterior+synechia-induced+secondary+glaucoma.">Combined trabeculectomy and vitrectomy for pseudophakic malignant glaucoma and extensive peripheral anterior synechia-induced secondary glaucoma.</a> Journal of Cataract &amp; Refractive Surgery. 30 (3), 715-717 (2004).</li><li>Lim, L. S., 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=Acute+primary+angle+closure:+configuration+of+the+drainage+angle+in+the+first+year+after+laser+peripheral+iridotomy.">Acute primary angle closure: configuration of the drainage angle in the first year after laser peripheral iridotomy.</a> Ophthalmology. 111 (8), 1470-1474 (2004).</li><li>Sawada, A., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+extent+of+preexisting+organic+angle+closure+and+long-term+outcome+after+laser+peripheral+iridotomy+in+eyes+with+primary+angle+closure.">Correlation between extent of preexisting organic angle closure and long-term outcome after laser peripheral iridotomy in eyes with primary angle closure.</a> Journal of Glaucoma. 21 (3), 174-179 (2012).</li><li>Ollerton, 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=Long-term+pathologic+follow-up+of+obsolete+design:+Choyce+Mark+VIII+anterior+chamber+intraocular+lens.">Long-term pathologic follow-up of obsolete design: Choyce Mark VIII anterior chamber intraocular lens.</a> Journal of Cataract &amp; Refractive Surgery. 38 (2), 368-372 (2012).</li><li>Latifi, G., 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=Effect+of+phacoemulsification+on+drainage+angle+status+in+angle+closure+eyes+with+or+without+extensive+peripheral+anterior+synechiae.">Effect of phacoemulsification on drainage angle status in angle closure eyes with or without extensive peripheral anterior synechiae.</a> European Journal of Ophthalmology. 23 (1), 70-79 (2013).</li><li>Narang, P., Agarwal, A., Kumar, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-pass+four-throw+pupilloplasty+for+angle-closure.">Single-pass four-throw pupilloplasty for angle-closure.</a> Indian Journal of Ophthalmology. 66 (1), 120-124 (2018).</li><li>Nakatani, S., Murakami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-year+outcome+of+Descemet+stripping+automated+endothelial+keratoplasty+for+bullous+keratopathy+after+argon+laser+iridotomy.">Three-year outcome of Descemet stripping automated endothelial keratoplasty for bullous keratopathy after argon laser iridotomy.</a> Cornea. 33 (8), 780-784 (2014).</li><li>Liarakos, V. S., 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=Endothelial+keratoplasty+for+bullous+keratopathy+in+eyes+with+an+anterior+chamber+intraocular+lens.">Endothelial keratoplasty for bullous keratopathy in eyes with an anterior chamber intraocular lens.</a> Journal of Cataract &amp; Refractive Surgery. 39 (12), 1835-1845 (2013).</li><li>Narang, P., Agarwal, A., Kumar, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-pass+4-throw+pupilloplasty+for+pre-descemet+endothelial+keratoplasty.">Single-pass 4-throw pupilloplasty for pre-descemet endothelial keratoplasty.</a> Cornea. 36 (12), 1580-1583 (2017).</li><li>Ozbek, Z., Kaynak, S., Zengin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transscleral+fixation+of+a+black+diaphragm+intraocular+lens+in+severely+traumatized+eyes+requiring+vitreoretinal+surgery.">Transscleral fixation of a black diaphragm intraocular lens in severely traumatized eyes requiring vitreoretinal surgery.</a> Journal of Cataract &amp; Refractive Surgery. 33 (8), 1494-1498 (2007).</li><li>Aslam, S. A., Wong, S. C., Ficker, L. A., MacLaren, 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=Implantation+of+the+black+diaphragm+intraocular+lens+in+congenital+and+traumatic+aniridia.">Implantation of the black diaphragm intraocular lens in congenital and traumatic aniridia.</a> Ophthalmology. 115 (10), 1705-1712 (2008).</li><li>Li, J., Yuan, G., Ying, L., Yu, B., Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+implantation+of+black+diaphragm+intraocular+lens+in+traumatic+aniridia.">Modified implantation of black diaphragm intraocular lens in traumatic aniridia.</a> Journal of Cataract &amp; Refractive Surgery. 39 (6), 822-825 (2013).</li><li>Qiu, X., Ji, Y., Zheng, T., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+efficacy+and+complications+of+black+diaphragm+intraocular+lens+implantation+in+patients+with+traumatic+aniridia.">Long-term efficacy and complications of black diaphragm intraocular lens implantation in patients with traumatic aniridia.</a> British Journal of Ophthalmology. 99 (5), 659-664 (2015).</li><li>Qiu, X., Ji, Y., Zheng, T., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+and+complications+of+black+diaphragm+intra-ocular+lens+implantation+in+patients+with+congenital+aniridia.">The efficacy and complications of black diaphragm intra-ocular lens implantation in patients with congenital aniridia.</a> Acta Ophthalmologica. 94 (5), 340-344 (2016).</li><li>Chorgiewicz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transscleral+fixation+of+black+diaphragm+intraocular+lens+in+complete+aniridia+and+aphakia+due+to+posttraumatic+eye+rupture:+A+pilot+study.">Transscleral fixation of black diaphragm intraocular lens in complete aniridia and aphakia due to posttraumatic eye rupture: A pilot study.</a> Journal of Clinical Medicine. 8 (1), 46 (2019).</li><li>Gentile, R. C., Eliott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicone+oil+retention+sutures+in+aphakic+eyes+with+iris+loss.">Silicone oil retention sutures in aphakic eyes with iris loss.</a> Archives of Ophthalmology. 128 (12), 1596-1599 (2010).</li><li>Y&#252;ksel, K., 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=Silicone+oil+barrier+sutures+in+aphakic+eyes+with+iris+defects.">Silicone oil barrier sutures in aphakic eyes with iris defects.</a> Retina. 36 (6), 1222-1226 (2016).</li><li>Syed, R., Jusufbegovic, D., Schaal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+needle-free+minimally+invasive+surgical+technique+for+the+placement+of+silicone+oil+retention+sutures.">A needle-free minimally invasive surgical technique for the placement of silicone oil retention sutures.</a> Retina. 36 (5), 1032-1034 (2016).</li><li>Du, W., Chen, F., Zhu, J., Xie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+modified+silicone+oil+retention+sutures+in+traumatic+aphakic+eyes+with+iris+loss.">Application of modified silicone oil retention sutures in traumatic aphakic eyes with iris loss.</a> Retina. , (2019).</li><li>Peng, R. -. M., Hao, Y. -. S., Chen, H. -. J., Sun, Y. -. X., Hong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+keratoplasty:+the+use+of+viscoelastic+as+an+aid+in+reattaching+the+dislocated+graft+in+abnormally+structured+eyes.">Endothelial keratoplasty: the use of viscoelastic as an aid in reattaching the dislocated graft in abnormally structured eyes.</a> Ophthalmology. 116 (10), 1897-1900 (2009).</li><li>Busin, M., Madi, S., Santorum, P., Scorcia, V., Beltz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+descemet's+stripping+automated+endothelial+keratoplasty+with+the+microkeratome+double-pass+technique:+Two-year+outcomes.">Ultrathin descemet's stripping automated endothelial keratoplasty with the microkeratome double-pass technique: Two-year outcomes.</a> Ophthalmology. 120 (6), 1186-1194 (2013).</li></ol>

The authors have nothing to disclose.

Bullous keratopathy with extensive anterior synechia has been considered one of the most challenging clinical problems. Although well-controlled IOP with successful lens extraction and anterior vitrectomy surgery was performed, the iris lens diaphragm was still flabby with the existence of an atrophied and tension-free iris. Case 1 showed that even when PKP was completed, conventional anterior chamber plasty with synechia separation would only partially and temporarily form in the anterior chamber. The main reasons are: (1) the aqueous humor circulation would continuously push and squeeze the atrophied and tension-free iris forward; (2) the effect of &#34;roller&#34; formed in the process of inflammation or restoration would make the iris from PAS to extensive anterior synechia again; (3) the fibration would result in synechia from iris to the cornea.
Keratoplasty with iris-diaphragm IOL implantation was a valuable method to cure the bullous keratopathy with aniridia10,11,12,13,14. To solve such extensive anterior synechia of the iris, iris-diaphragm IOL implantation after removing the iris might work15. However, total iridectomy was a surgery with significant iatrogenic injury, and iris-diaphragm IOL comes at an unaffordable price in China. A new method of forming a stable anterior chamber without recurrent extensive anterior synechia was needed to be explored.
Gentile et al.16 first reported the &#34;# pattern&#34; retention sutures across the anterior chamber to simulate an iris diaphragm. They made the sutures act as a barrier between the silicone oil and aqueous, preventing silicone oil-corneal contact, and this has been effective over 3-6 months. To deal with an atrophied and tension-free iris, there must be enough line segments and points of crossing to prevent the peripheral iris or pupil from moving forward to the cornea. The &#34;# pattern&#34; retention sutures could work well theoretically. But it was a complicated operation with 20 suture segments and 16 points of crossing times of sclerocentesis, which prolonged the operation time and increased the risk of infection. Similarly, this problem was even more severe in the report of Kemal et al.17 with &#34;Chessboard pattern&#34; barrier sutures.
Syed et al.18 and Du et al.19 suggested that improved &#34;Z pattern&#34; retention sutures could prevent silicone oil from moving to the anterior chamber in aphakic eyes with iris loss for 3-6 months. However, the points of crossing within the improved &#34;Z pattern&#34; sutures were not enough to prevent anterior synechia of the iris. Moreover, theoretically, triangle or quadrilateral sutures would be even worse than the improved &#34;Z pattern&#34; sutures.
In the current study, PSACP was invented because pentagram was a magical pattern with 15 line segments and 10 points of crossing, especially drawing with one stroke. Therefore, PSACP could not only use one 10-0 prolene suture alone across the anterior surface of the iris, forming a barrier in front of the iris by the 15 suture segments from the pentagram but also prevent the pupil from moving forward to the cornea by the central five points of crossing and fix the suture to the surface or superficial sclera by the peripheral five points of crossing. With the help of PSACP, the anterior chamber was stable with iris fixation and almost without recurrency of anterior synechia for at least 12 weeks in Case 2. Finally, Case 2 received DSAEK, and the bullous keratopathy was cured.
To our knowledge, DSAEK was one of the best surgeries to cure bullous keratopathy with minimally invasive, time-saving, and fast recovery7,20,21. The operative procedure needed adequate space basis in the anterior chamber. In Case 3, some recurrent anterior synechia resulted in an unstable anterior chamber in 24 weeks post-PSACP, suggesting that the timing of DSAEK was recommended from 4-12 weeks post-PSACP. Furthermore, Case 3 had received DSAEK jet and had also been performed with conventional anterior chamber plasty several times. If DSAEK was performed again, it might bring in more severe inflammation, restoration or fibration, and uncontrolled IOP. Therefore, PKP was finally performed. However, retention sutures were broken accidentally in PKP, and PAS was formed rapidly in 1-week post PKP, implying that PSACP prevented the iris from moving toward the cornea in such a complicated case only to some extent. Care needs to be taken of these sutures during the PKP procedure.
The limitation of this study was that it was a pilot study with only three cases with stringent inclusion and exclusion criteria. The success and benefits of PSACP with subsequent DSAEK or PKP were shown in two instances from only one hospital. Further studies in multi-center with a bigger sample size might be helpful to improve the flow chart.
In summary, iris fixation via PSACP might provide an adequate space basis for DSAEK to help cure the bullous keratopathy caused by a vanished anterior chamber or extensive anterior synechia to some extent. PSACP and DSAEK or PKP might be an effective way to cure the bullous keratopathy with extensive anterior synechia of the iris.

Extensive anterior synechia is usually observed in the eyes of patients with malignant glaucoma or primary angle-closure glaucoma after laser peripheral iridotomy or iridoplasty and after anterior segment surgery1,2,3,4,5,6. This can cause gradual reduction of the anterior chamber leading to visual dysfunction. Once extensive anterior synechia forms, the aqueous humor can enter the cornea due to endothelium decompensation, leading to bullous keratopathy7. Although a well-controlled intraocular pressure (IOP) has been obtained after lens extraction and anterior vitrectomy, the iris lens diaphragm is still soft due to atrophied and tension-free iris8. Therefore, conventional anterior chamber plasty can only temporarily form the anterior chamber9. In cases where anterior synechia is extensive, the anterior chamber disappears again and adds difficulty to the subsequent keratoplasty surgery.
Keratoplasty with iris-diaphragm intraocular lens (IOL) implantation was reported as an effective way to cure the bullous keratopathy with aniridia10,11,12,13,14. Therefore, it may be helpful to treat eyes with bullous keratopathy and extensive anterior synechia after removing the atrophied and tension-free iris. However, this idea is possibly bringing significant iatrogenic injury now and not feasible in China due to unaffordable price of such IOL.
A recent study suggested that retention or barrier sutures could prevent silicone oil from moving into the anterior chamber in aphakic eyes with iris loss15,16,17,18. Sutures enhance the surface tension of silicone oil in such situations. Therefore, the current study was conducted to investigate whether these retention or barrier sutures could improve atrophy&#39;s stress and make the iris tension-free by preventing the iris and iris lens diaphragm from approaching the cornea. The presented study was performed to explore an effective way of doing anterior chamber plasty before keratoplasty to cure the bullous keratopathy with extensive anterior synechia of the iris. The current surgery method was named &#34;Pentagram Suturing Anterior Chamber Plasty&#34; (PSACP) with a core process of iris fixation via external pentagram suturing.
Malignant glaucoma patients with bullous keratopathy and extensive anterior synechia who already underwent lens extraction, anterior vitrectomy, IOL implantation, and had a well-controlled IOP were included for the current study. Willingness to receive further keratoplasty to cure bullous keratopathy was also considered an inclusion criterion. Exclusion criteria include (1) eyes with no light perception, (2) IOP &#8805; 35 mmHg under anti-glaucoma medications, (3) eyes with infection, acute inflammation,aniridia or chronic uveitis, (4) eyes with retinal detachment, choroidal detachment, or ciliary detachment, (5) patients with uncontrolled hypertension, cardiovascular disease, cerebrovascular disease, or diabetes mellitus, (6) patients with a severe hemorrhagic tendency or mental disorders.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% crystal violet solution</td>
			<td>HEBEI JINZHONG PHARMACEUTIAL CO., LTD. Hebei, PRC</td>
			<td>200709</td>
			<td></td>
		</tr>
		<tr>
			<td>10-0 prolene sutures on an STC-6 needle</td>
			<td>Alcon Laboratories, Inc.,Sinking Spring, PA</td>
			<td>13M4AT</td>
			<td></td>
		</tr>
		<tr>
			<td>2% lidocaine hydrochloride injection</td>
			<td>TIANSHENG PHARMACEUTIAL GROUP CO., LTD. Hubei, PRC</td>
			<td>42021839</td>
			<td></td>
		</tr>
		<tr>
			<td>29-gauge syringe</td>
			<td>Becton, Dickinson and Company, NE 68949</td>
			<td>160404</td>
			<td></td>
		</tr>
		<tr>
			<td>Anterior segment optical coherence tomography</td>
			<td>Carl Zeiss Meditec, Inc., Dublin, CA</td>
			<td>1000-1238</td>
			<td></td>
		</tr>
		<tr>
			<td>Balanced salt solution</td>
			<td>Alcon Laboratories,Inc., Fort Worth,TX</td>
			<td>7950191</td>
			<td></td>
		</tr>
		<tr>
			<td>Cohesive viscoelastic</td>
			<td>Bausch+Lomb Inc., Shandong, CN</td>
			<td>6.92409E+12</td>
			<td></td>
		</tr>
		<tr>
			<td>Color slit lamp photography</td>
			<td>HAAG-STREIT AG, Gartenstadtstrasse 10, 3098 Koeniz, Switzerland</td>
			<td>6543-2012</td>
			<td></td>
		</tr>
		<tr>
			<td>Rebound tonometer (iCare, Type TA01i)</td>
			<td>Tiolat Oy, Finland</td>
			<td>44</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile irrigator for single use</td>
			<td>SHANDONG WEIGAO GROUP MEDICAL POLYMER CO., LIMITED</td>
			<td>(01)06932992101486</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound biomicroscopy</td>
			<td>Paradigm Medical Industries, Salt Lake City, UT</td>
			<td>5290-2012</td>
			<td></td>
		</tr>
	</tbody>
</table>

iris fixation via external pentagram suturing

Extensive anterior synechia of the iris can result in the gradual disappearance of the anterior chamber. It is one of the most common outcomes of both oculopathy and complicated post anterior segment surgery. This can impact visual function and lead to bullous keratopathy, making it one of the most complex clinical problems. Conventional anterior chamber plasty will partially create the anterior chamber, but the anterior chamber disappears again in some cases. The main reasons are: (1) the iris lens diaphragm is loose with atrophied and tension-free iris so that the aqueous humor circulation will push and squeeze the iris forward; (2) the effect of "roller" formed in the process of inflammation or restoration will change the iris structure from peripheral anterior synechia (PAS) to extensive anterior synechia again; (3) the fibration will result in synechia from iris to the cornea. In such cases, corneal endothelium deficiency can't stop the aqueous humor from entering the cornea. This results in persistent corneal edema post-conventional anterior chamber plasty, resulting in progressive rubbing and lachrymation. Therefore, anterior chamber plasty is not the first choice for patients without surgical indications of Descemet's stripping automated endothelial keratoplasty (DSAEK). However, this can be performed in patients with surgical indications who plan to receive DSAEK. A unique method of iris fixation via external pentagram suturing anterior chamber plasty (PSACP) is described here. The present technique is also compared with the conventional anterior chamber plasty. PSACP and DSAEK might be an effective way to cure the bullous keratopathy with extensive anterior synechia of the iris and disappeared anterior chamber.

Representative results from three bullous keratopathy patients with extensive anterior synechia who had a well-controlled IOP after lens extraction, anterior vitrectomy, and IOL implantation to cure malignant glaucoma are described in the study. In these cases, one received conventional anterior chamber plasty with synechia separation and penetrating keratoplasty (PKP), the other two received PSACP first, and then received Descemet&#39;s stripping automated endothelial keratoplasty (DSAEK) or PKP. Postop reaction and pigment release of all three cases were mild and faded within 4 weeks under routine anti-infection and anti-inflammatory eye drops. The prognosis of these three patients was compared on different days after 360&#176; PAS appearance with the ASOCT (1-day post-operation) or UBM (12 weeks or 24 weeks post-operation) after the anterior chamber plasty surgery.
Case 1 
A 68-year-old female was diagnosed with bullous keratopathy and extensive anterior synechia in her right eye from IOP-well-controlled (21 mmHg with four kinds of anti-glaucoma eye drops) malignant glaucoma. She already had lens extraction, anterior vitrectomy, and IOL implantation. Her right eye&#39;s best-corrected visual acuity (BCVA) was 20/4000. The iris&#39;s extensive anterior synechia was shown with ASOCT and UBM (Figure 1 and Figure 2).
She received anterior chamber plasty with synechia separation and penetrating keratoplasty (PKP) in her right eye. One-day post-surgery (Figure 4), she showed no PAS with the ASOCT (Figure 4C), and BCVA improved to 20/1000 with elevated IOP (32 mmHg). However, 12 weeks after surgery, she showed 360&#176; PAS with the UBM (Figure 4D), and BCVA decreased to 20/1600. At that moment, IOP was uncontrolled over 25 mmHg, and the bullous keratopathy reoccurred.
Case 2 
A 75-year-old female was diagnosed with bullous keratopathy and extensive anterior synechia in her left eye from IOP-well-controlled (18 mmHg with three kinds of anti-glaucoma eye drops) malignant glaucoma, which already had lens extraction, anterior vitrectomy, and IOL implantation. BCVA of her left eye was 20/1600. The disappearance of the anterior chamber was observed with ASOCT and UBM (Figure 5).
She received PSACP, where pentagram sutures were placed as a barrier in front of the anterior surface of the iris (Figure 6). No PAS was seen with ASOCT or UBM from day 1 till 12 weeks post-surgery (Figure 7). Then, she received DSAEK. On 24 weeks post-PSACP, no corneal edema was observed with ASOCT, and one clock hour PAS was demonstrated with UBM (Figure 8), with BCVA 20/66. IOP remained normal without any medication post-surgery.
Case 3 
A 69-year-old female was diagnosed with bullous keratopathy and extensive anterior synechia in her right eye from IOP-well-controlled (12 mmHg with four kinds of anti-glaucoma eye drops) malignant glaucoma, which was post lens extraction, anterior vitrectomy, IOL implantation, and DSAEK. The BCVA of her right eye was 20/1600. Extensive PAS was shown with ASOCT and UBM (Figure 9).
She received PSACP and showed 2 clock hours PAS with ASOCT on day 1 post-surgery, and she missed the 12-week-follow-up. As she showed 6 clock hours PAS with UBM in 24 weeks post-surgery (Figure 10), she received PKP. During PKP, the suture was broken accidentally and then removed. On 1-week post PKP, she showed mild edema in the cornea graft and 6 clock hours PAS with the ASOCT (Figure 11) with improved BCVA 20/400. IOP remained normal without any medication post-surgery. Unfortunately, she did not follow up.
Comparisons 
BCVA, IOP, cornea or corneal graft, anterior synechia of iris were found in these three patients preoperationally and post-operationally (Table 1 and Table 2). The prognosis of Case 2 and Case 3 was significantly better than in Case 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63256/63256fig01.jpg" /> 
Figure 1: Preoperative examinations for Case 1. (A) Anterior segment photography. (B) Color slit-lamp photography. (C) Anterior segment optical coherence tomography (ASOCT). (D) Ultrasound biomicroscopy (UBM). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig01large.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/63256/63256fig02.jpg" /> 
Figure 2: Assessment of anterior synechia of iris for Case 1.&#160;(A) Anterior segment optical coherence tomography. (B) Ultrasound biomicroscopy. Red circle: anterior synechia of the iris. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig02large.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/63256/63256fig03.jpg" /> 
Figure 3: Schematic representation of the procedure of PSACP for Case 3. (A) Black dashed circle: pupil; black circle: limbus; purple dashed circle: the mark of scleral puncture point; numbers and red arrow: suturing sequence and direction, both ends of the red arrow represent the exact puncture points; X: the surgical knot. (B) Red arrow: suturing position and direction. PSACP = Pentagram Suturing Anterior Chamber Plasty. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig03large.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/63256/63256fig04.jpg" /> 
Figure 4: Postoperative examinations for Case 1. (A) Color slit lamp photography on day 1 post-operation. (B) Color slit lamp photography in 12 weeks post-operation. (C) Anterior segment optical coherence tomography (ASOCT) on day 1 post-operation. (D) Ultrasound biomicroscopy (UBM) in 12 weeks post-operation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63256/63256fig05.jpg" /> 
Figure 5: Preoperative examinations for Case 2. (A) Anterior segment photography. (B) Color slit-lamp photography. (C) Anterior segment optical coherence tomography (ASOCT). (D) Ultrasound biomicroscopy (UBM). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63256/63256fig06.jpg" /> 
Figure 6: Diagrammatic sketches of PSACP in operation for Case 2. (A) Color photography for sutures. (B) Blueline: pentagram sutures. (C) Anterior segment optical coherence tomography (ASOCT) on day 1 post-operation, red circle: sutures. (D) Black dashed circle: pupil; black circle: limbus; red arrow: suturing direction; X: the surgical knot. PSACP = Pentagram Suturing Anterior Chamber Plasty. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63256/63256fig07.jpg" /> 
Figure 7: Postoperative examinations for Case 2. (A) Color slit lamp photography on day 1 post-operation. (B) Color slit lamp photography in 12 weeks post-operation. (C) Anterior segment optical coherence tomography (ASOCT) on day 1 post-operation. (D) Ultrasound biomicroscopy (UBM) in 12 weeks post-operation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63256/63256fig08.jpg" /> 
Figure 8: Postoperative examinations for Case 2 in 24 weeks post PSACP. (A) Color slit lamp photography, yellow arrow: sutures in the anterior chamber. (B) Color slit lamp photography, blue line: pentagram sutures. (C) Anterior segment optical coherence tomography (ASOCT) showed the cornea and endothelium graft. (D) Ultrasound biomicroscopy (UBM). PSACP = Pentagram Suturing Anterior Chamber Plasty. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63256/63256fig09.jpg" /> 
Figure 9: Preoperative examinations for Case 3. (A) Anterior segment photography. (B) Color slit-lamp photography. (C) Anterior segment optical coherence tomography (ASOCT). (D) Ultrasound biomicroscopy (UBM). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/63256/63256fig10.jpg" /> 
Figure 10: Postoperative examinations for Case 3. (A) Color slit lamp photography on day 1 post-operation. (B) Color slit lamp photography in 24 weeks post-operation. (C) Anterior segment optical coherence tomography (ASOCT) on day 1 post-operation. (D) Ultrasound biomicroscopy (UBM) in 24 weeks post-operation. Red circle: sutures. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/63256/63256fig11.jpg" /> 
Figure 11: Postoperative examinations for Case 3 in 25 weeks post PSACP. (A) Anterior segment photography in 1 week post PKP. (B) Color slit lamp photography in 1 week post PKP. (C) Anterior segment optical coherence tomography (ASOCT) showed the cornea and graft. (D) Anterior segment optical coherence tomography (ASOCT) showed anterior synechia of the iris. PSACP = Pentagram Suturing Anterior Chamber Plasty. PKP = penetrating keratoplasty. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63256/63256fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Case</td>
			<td>Kind of surgery</td>
			<td colspan="2">BCVA</td>
			<td colspan="2">IOP (mmHg)</td>
			<td colspan="2">Kind of anti-glaucoma eye drops</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Pre-op.</td>
			<td>Postop.</td>
			<td>Pre-op.</td>
			<td>Postop.*</td>
			<td>Pre-op.</td>
			<td>Postop.</td>
		</tr>
		<tr>
			<td>1</td>
			<td>CACP+PKP</td>
			<td>20/4000</td>
			<td>20/1600</td>
			<td>21</td>
			<td>25</td>
			<td>4</td>
			<td>3</td>
		</tr>
		<tr>
			<td>2</td>
			<td>PSACP, DSAEK</td>
			<td>20/1600</td>
			<td>20/66</td>
			<td>18</td>
			<td>13</td>
			<td>3</td>
			<td>0</td>
		</tr>
		<tr>
			<td>3</td>
			<td>PSACP, PKP</td>
			<td>20/1600</td>
			<td>20/400</td>
			<td>12</td>
			<td>14</td>
			<td>3</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Visual acuity, intraocular pressure, and medications of the presented cases. BCVA = best-corrected visual acuity; IOP = intraocular pressure; Pre-op. = pre-operational; Postop. = post-operational; CACP = conventional anterior chamber plasty; PKP = penetrating keratoplasty; PSACP = Pentagram Suturing Anterior Chamber Plasty; DSAEK = Descemet&#39;s stripping automated endothelial keratoplasty.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Case</td>
			<td>Kind of surgery</td>
			<td colspan="2">Cornea/Corneal graft edema</td>
			<td colspan="2">Anterior synechia of iris (ASOCT)</td>
			<td colspan="2">Anterior synechia of iris (UBM)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Pre-op.</td>
			<td>Postop.</td>
			<td>Pre-op.</td>
			<td>Postop.*</td>
			<td>Pre-op.</td>
			<td>Postop.</td>
		</tr>
		<tr>
			<td>1</td>
			<td>CACP+PKP</td>
			<td>Yes</td>
			<td>No</td>
			<td>8CH</td>
			<td>No</td>
			<td>9CH</td>
			<td>9CH</td>
		</tr>
		<tr>
			<td>2</td>
			<td>PSACP, DSAEK</td>
			<td>Yes</td>
			<td>No</td>
			<td>12CH</td>
			<td>No</td>
			<td>12CH</td>
			<td>1CH</td>
		</tr>
		<tr>
			<td>3</td>
			<td>PSACP, PKP</td>
			<td>Yes</td>
			<td>No</td>
			<td>12CH</td>
			<td>2CH</td>
			<td>12CH</td>
			<td>6CH#</td>
		</tr>
	</tbody>
</table>
Table 2: Clinical characteristics of cornea and iris of the presented cases. ASOCT = anterior segment optical coherence tomography; UBM = Ultrasound biomicroscopy; Pre-op. = preoperational; Postop. = post operational; CACP = conventional anterior chamber plasty; PKP = penetrating keratoplasty; CH = clock hour; PSACP = Pentagram Suturing Anterior Chamber Plasty; DSAEK = Descemet&#39;s stripping automated endothelial keratoplasty. * = 1 day post PSACP. # = 24 weeks post PSACP.

Watch this Scientific Journal Video about Iris Fixation via External Pentagram Suturing at JoVE.com

Iris Fixation via External Pentagram Suturing

The present protocol describes how a 10-0 polypropylene suture crosses the anterior surface of the iris, forming a pentagram to prevent both iris and pupil from moving toward the cornea. This can be combined with subsequent keratoplasty to cure bullous keratopathy with extensive anterior synechia of the iris.

Iris Fixation via External Pentagram Suturing

Extensive anterior synechia of the iris can result in the gradual disappearance of the anterior chamber. It is one of the most common outcomes of both ...

iris-fixation-via-external-pentagram-suturing

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

1.2K Views.  Peking University Third Hospital. The present protocol describes how a 10-0 polypropylene suture crosses the anterior surface of the iris, forming a pentagram to prevent both iris and pupil from moving toward the cornea. This can be combined with subsequent keratoplasty to cure bullous keratopathy with extensive anterior synechia of the iris.

Video: Iris Fixation via External Pentagram Suturing

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

Iris Fixation via External Pentagram Suturing

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Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

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