The authors are solely responsible for the design and conduct of the presented study. Dr. Marbacher was supported by a grant from the Swiss National Science Foundation (PBSKP3-123454). The authors declare no conflict of interests. 
Author contributions to the study and manuscript preparation include the following. Conception and design: SM, JM, JF. Acquisition of data: SM, EA, JF. Analysis and interpretation of data: SM, JF, JM. Drafting the article: SM, JF, JM. Critically revising the article: JH, MN. Statistical analysis: SM, JF. Study supervision: JF, JH, MN.

NOTE: Male Wistar rats (mean body weight: 356 &#177; 44 g; 10-14 weeks old) were housed in the animal room at 22-24 &#176;C and twelve hour light/dark cycle with free access to pellet diet, regular tap water and also received humane care in conformity with institutional guidelines. The experiments were reviewed and approved by the Committee for Animal Welfare at the University of Helsinki, Finland. &#160;
NOTE: In the following demonstration our surgical method is as follows: Anesthetize the rat by weight-adapted subcutaneous injection of medetomidine hydrochloride (0.5 mg/kg) and intraperitoneal injection of ketamine hydrochloride (50 mg/kg). Test for the lack of a toe-pinch reflex to confirm that the rat is fully anesthetized. Apply eye ointment, clip the surgical site, and clean the skin with a suitable disinfectant, for example Chlorhexidine, either in alcohol or water. Wash hands, put on protective clothing, a head cover and facemask, and sterile surgical gloves. Have a surgical assistant aid in maintaining aseptic surgery conditions and to document the surgical characteristics (as listed in Table 1). Monitor the depth of the anesthesia every 15 min during surgery by following respiratory rate, heart rate, and reaction to noxious stimulation (toe pinch test). Subcutaneous injection of buprenorphine (0.03 mg/kg) was given for postoperative analgesia and repeated if necessary every 12 hr.
1. Hardware, Consumables, and Positioning
<ol>
	<li>Keep the small animal surgery room quiet, aseptic, and maintain the room temperature at 23 +/- 3 &#176;C. To perform the experimental aneurysm surgery the following minimal equipment is necessary:
	<ol>
		<li>Use a table top surgical microscope ideally equipped with an assistant scope and digital microscope camera. Use nonporous reusable operating surface and cleanable instrument surface to protect the laboratory bench.</li>
		<li>Prepare consumables such as skin disinfectants, physiological saline, smaller and larger gauze swabs and suture material including 9-0 micro-sutures, a 5-0 non-absorbable, and 3-0 absorbable suture.</li>
		<li>Use the following standard surgical instruments: Surgical scissors, tissue forceps, soft tissue spreader or self-retaining retractor, and two Mosquito surgical clamps.</li>
		<li>Use a basic microsurgical instrument set that includes: Curved microneedle holder, one curved and two straight microforceps, and a straight or curved microscissor.</li>
		<li>Keep the microsurgical instruments in a kidney dish filled with sterile saline to keep the instrument non-sticky and clean during surgery. The kidney dish is padded with rubber mat or a surgical glove to prevent damage to the tips of the micro-instruments. Ensure that all supplies are sterile and the procedure is performed with aseptic technique according to the current recommendations for survival surgery on laboratory animals8-10.</li>
		<li>In addition, use a vascular clip applicator and three atraumatic temporary vascular clamps. It is important that the clamps used have a low closing power to prevent injury to the very thin wall of the rat aorta. Also prepare a ruler with half millimeter scale bars, a small colored rubber pad, and a short blunt needle.</li>
		<li>Place the rats in a supine position, immobilize both front and hind paws with surgical tape without applying stretch or compression to the skin, and bend their back with a thick marker or cautery pen by placing it under the lumber region of the back. It is important to obtain as much lumbar spine lordosis as possible in order to improve retroperitoneal exposure and access to the infrarenal aorta which facilitates microsurgical anastomosis. This positioning is recommended for abdominal aorta aneurysm creation but not necessary for thoracic graft harvesting.</li>
	</ol>
	</li>
</ol>
2. Graft Harvesting
<ol>
	<li>Under general anesthesia, open the thoracic cavity (start graft ischemia time). Apply a noxious toe pinch to confirm that the rat is unresponsive before proceeding with the following steps. Cut through the midventral abdominal wall, identify the diaphragm just above the liver, and cut the connective tissue at the bottom of diaphragm to allow access to the rib cage. With large scissors, blunt side down, cut through ribs just one centimeter left and right of the rib cage midline and open up the thoracic cavity. The lungs are mobilized to the right side of the heart. Sacrifice the rats by overdosing with intracardiac injection of ketamine hydrochloride.Pulmonary trunk, left subclavian vein, left cranial vena cava, and the azygous vein interfere with the harvest of the proximal thoracic aorta.</li>
	<li>Caudal to the prominent veins there is a good entry point to start the dissection of the descending thoracic aorta using micro-scissors and microforceps.</li>
	<li>Trace the thoracic aorta back from the dorsal wall of the thorax upwards to the aortic arch by gentle blunt retraction and dissection with the Mosquito surgical clamp.</li>
	<li>Clamp and then cut the veins with scissors. Maintain the clamps on the veins and use as a retractor to expose the underlying aortic arch.</li>
	<li>Place a non-absorbable 6-0 silk ligature just above the first intercostal artery leaving the aorta.</li>
	<li>Cut the descending aorta just below the left subclavian artery and then below the ligature. Trimming can be done in order to get a perpendicular standardized aneurysm geometry, or if needed, to get a specific angle between the axis of the aneurysm and the aorta. Measure the graft in its width and length. 
	NOTE: Harvested grafts can either be immediately transplanted into recipient rats or further processed to achieve decellularization of the graft wall. Decellularized grafts can be stored at -4 degree Celsius until re-implantation at a later date (Figure 1). The decellularization of the aneurysm wall has been shown to predispose the aneurysm to enlarge11.</li>
</ol>
3. Graft Decellularization
<ol style="margin-left: 40px;">
	<li>Freeze donor grafts in phosphate buffered saline at -4 &#176;C.</li>
	<li>The next day, thaw the grafts, rinse with purified and deionized water at room temperature, and incubate for 10 hr at 37 &#176;C in 0.1% sodium dodecyl sulfate.</li>
	<li>Finally, wash the sodium dodecyl sulfate-treated grafts three times with gentle agitation, refreeze in phosphate buffered saline, and keep at -4 &#176;C until use.</li>
</ol>
4. Aneurysm Creation
<ol style="margin-left: 40px;">
	<li>Dissection of the Abdominal Aorta
	<ol>
		<li>After the animal has been anaesthetized, clip the fur from the surgical site and clean the skin with a suitable disinfectant. Test for the lack of a toe-pinch response prior to skin incision. Start the incision 1cm proximal to the genitals in midventral position (start operation time). Carefully separate the skin from the underlying muscles. End dissection one to two centimeters below the sternum.</li>
		<li>Carefully but firmly pull up the underlying abdominal muscles to avoid damage to the underlying organs. Extend the longitudinal cut along the linea alba upwards to the xiphoid process and end in caudal direction at the level of the bladder.</li>
		<li>Gently apply pressure to the bladder to empty it in order to facilitate access to the retroperitoneal space.</li>
		<li>Move the small intestines and the prominent cecum to the right or the left. Identify the large intestine namely the descending colon on the left bottom of the abdominal cavity.</li>
		<li>Cut the ligament in between the small intestine and the descending colon in cranial direction to allow wider exposure of the dorsal body wall. Place a self-retaining retractor to hold the bowels apart.</li>
		<li>The ideal location of end-to-side aneurysm anastomosis is found at the level between the renal and iliolumbar veins. The abdominal aorta lies retroperitoneal embedded in fat tissue. During dissection pay particular attention to the paired almost transparent ureters and testicular vessels.</li>
		<li>If further retraction of intestines is needed, use larger gauze swaps. The lumbar spine lordosis induced during positioning by placing a cautery pen or similar object under the lower back of the rat, reduces significantly the need of bowel retraction.</li>
		<li>The ventral surface of the dorsal body wall is covered with a thin parietal peritoneum. Once this is opened, visualize the aorta just underneath. During careful sharp and blunt dissection of the abdominal aorta from adjacent large veins grasp only the adventitia to avoid damage to the vessel wall.</li>
		<li>Infrequently, small lumbar arteries arise as segmental vessel from the dorsal surface of the abdominal aorta and interfere with preparation. Ligation and cutting of the vessel is needed to avoid retrograde oozing during aneurysm suturing. Use of curved micro-forceps can facilitate ligature placement in the depth.</li>
	</ol>
	</li>
	<li>End-to-side Anastomosis
	<ol>
		<li>Put a colored rubber pad underneath the abdominal aorta and upholster it with a small gauze swab. Remove loose connective tissue and adventitia at the level of the planned anastomosis site.</li>
		<li>Clamp the abdominal aorta distal to the anastomosis first, then proximal (start aortic clamping time). This ensures a firm filling of the vessel and facilitates subsequent arteriotomy.</li>
		<li>Perform the arteriotomy using either straight or curved micro-scissors. A micro-forceps holds up a very small piece of the vessel wall to cut out an elliptical shape.</li>
		<li>Flush the artery thoroughly with saline in both directions using a blunt tipped needle.</li>
		<li>Place the first two sutures of this end-to-side anastomosis at the proximal and distal end of the arteriotomy (start anastomosis time). Avoid grasping of the vessel wall with the micro-forceps whenever possible. Make sure that each suture is placed through all layers of the vessel wall.</li>
		<li>Perform suturing either as continuous or interrupted sutures. If interrupted suturing is chosen then place the back side nine o`clock suture first. Subsequent sutures can be spaced apart starting adjacent to the very first suture. Grasp the adventitia carefully. Avoid any squeezing/grasping of the intima.</li>
		<li>When the back wall is finished, check the endoluminal part of the anastomosis for misplaced sutures. Perform the same procedures in the same order on the front side. Ensure that the first of a total of three knots per suture is firm but not too tight.</li>
	</ol>
	</li>
	<li>Hemostasis and Closure
	<ol>
		<li>After the end to side anastomosis is completed (end anastomosis time), rinse the site with saline and remove the distal clamp first to allow for backflow (end aortic clamping time and end graft ischemia time).</li>
		<li>If obvious bleeding occurs from backflow an extra stitch may be needed (start time of hemostasis). In case of minor oozing, achieve hemostasis with gentle pressure over the bleeding site using a small piece of a gauze swab.</li>
		<li>Remove the proximal vascular clamp, rinse the anastomosis site once more, and cut off the remaining ends of the ligature at the aneurysm dome.</li>
		<li>Confirm patency of the aneurysm by observation of volume increase of the aneurysm during peak arterial pulse wave. Assess distal abdominal artery patency through the direct &#8220;milking test&#8221;.</li>
		<li>Remove the plastic sheet and the small gauze swab underneath. The pulsating blood whirl within the created aneurysm is clearly visible.</li>
		<li>Cover suture lines around the anastomosis with small pieces of adipose tissue or Spongostan for additional hemostasis if small oozing is still present (end time of hemostasis).</li>
		<li>Remove the soft-tissue spreader and gauze swabs. Place the small intestines, the cecum, and the fat mass back in their correct position.</li>
		<li>Close the midline abdominal muscles using 5-0, 4-0, or 3-0 resorbable or non-absorbable sutures followed by skin wound closure using continuous suture technique with 3-0 resorbable polyfilament suture (end operation time). Note: In our experience rats tolerated resorbable skin sutures better than non-absorbable monofilament sutures.</li>
	</ol>
	</li>
</ol>

<ol>
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AJNR.</a> American journal of neuroradiology. 32, 772-777 (2011).</li><li>Frosen, J., 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=Contribution+of+mural+and+bone+marrow-derived+neointimal+cells+to+thrombus+organization+and+wall+remodeling+in+a+microsurgical+murine+saccular+aneurysm+model.">Contribution of mural and bone marrow-derived neointimal cells to thrombus organization and wall remodeling in a microsurgical murine saccular aneurysm model.</a> Neurosurgery. 58, 936-944 (2006).</li><li>Marjamaa, J., 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=Mice+with+a+deletion+in+the+first+intron+of+the+Col1a1+gene+develop+dissection+and+rupture+of+aorta+in+the+absence+of+aneurysms:+high-resolution+magnetic+resonance+imaging.">Mice with a deletion in the first intron of the Col1a1 gene develop dissection and rupture of aorta in the absence of aneurysms: high-resolution magnetic resonance imaging.</a> at 4.7 T, of the aorta and cerebral arteries. 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The authors have no financial or commercial interest in any of the drugs, materials, or equipment used.

Progress in our understanding of the complex biology of saccular cerebral artery aneurysm depends on analysis of epidemiological and clinical data, complemented by laboratory studies on patient samples and experimental work in animal models3,12,13.
Small animals such as the rat are inherently associated with lower costs of experiments and housing, and reduced need of specialized equipment. An average total operation time of less than 60 minutes for microsurgical creation of a sidewa...

Rupture of a saccular cerebral artery aneurysm causes life threatening hemorrhage leading to stroke, permanent neurological damage, or death. Rupture can be prevented by either microsurgical clipping or endovascular aneurysm occlusion. A medical treatment to prevent aneurysm growth and rupture has not yet been established.
Experimental models for saccular aneurysms are needed to study the biology of arterial aneurysms and for the testing of novel therapeutic devices and strategies. For these purposes, several different models in different species have been developed and published1. Larger aneurysm models in pigs, dogs, and rabbits are preferably used to test endovascular innovations in complex aneurysm architecture1,2. Murine aneurysm models, on the other hand, allow testing research questions in genetically modified species3,4 and facilitate clarification of aneurysm biology at cellular and molecular level far better than larger species1. Although endovascular trans-carotid and trans-iliac device deployment is limited to bigger rats (&#62;400-500 g) and stents smaller than 2.0 mm and 1.5 mm in diameter5,6, stents can also be placed through direct insertion into the abdominal aortic segment harbouring the experimental aneurysms. Previous work using the rat microsurgical abdominal aortic sidewall aneurysm model demonstrated its feasibility in testing novel embolic devices and its use in studying the molecular basis of aneurysm biology3,7.
Many of the currently published experimental saccular aneurysm models require expensive equipment, special environments (e.g. sterile operation rooms with fluoroscopy capabilities), interventional radiology competence, or use of expensive species. These requirements limit the widespread use of these models, and lead to the use of different models in different laboratories, which makes data comparison and meta-analysis difficult, if not impossible. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various labs in order to get comparable results from different institutions. For this purpose, we created the rat aorta sidewall saccular arterial aneurysm model.
The aim of this report is to present step-by-step procedural instructions, information on necessary equipment, and to discuss important anatomical and surgical characteristics for successful microsurgical creation of abdominal aortic sidewall aneurysms in the rat.

<table border="0" cellpadding="0" cellspacing="0" width="752">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Material/ Equipment</td>
			<td style="width:236px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Medetomidine</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td>Any genericon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ketamin</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td>Any genericon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Buprenorphine</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td>Any genericon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Phosphate buffered saline</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium dodecyl sulfate (0.1%)</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">3-0 resorbable suture</td>
			<td style="width:236px;">Ethicon Inc., USA</td>
			<td><a name="RANGE!C7">VCP824G</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">5-0 non absorbable suture</td>
			<td style="width:236px;">Ethicon Inc., USA</td>
			<td>8618G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">6-0 non absorbable silk suture</td>
			<td style="width:236px;">B. Braun, Germany</td>
			<td style="width:119px;">C0761060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">9-0 nylon micro suture</td>
			<td style="width:236px;">B. Braun, Germany</td>
			<td style="width:119px;">G1118471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Spongostan</td>
			<td style="width:236px;">Ethicon Inc., USA</td>
			<td style="width:119px;">MS0002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Operation microscope</td>
			<td style="width:236px;">Leica , Germany</td>
			<td style="width:119px;">M651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Digital microscope camera</td>
			<td style="width:236px;">Sony, Japan</td>
			<td style="width:119px;">SSC-DC58AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Standard surgical instruments</td>
			<td style="width:236px;">B. Braun, Germany</td>
			<td style="width:119px;">Multiple</td>
			<td>See protocol 1.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Microsurgical instruments</td>
			<td style="width:236px;">B. Braun, Germany</td>
			<td style="width:119px;">Multiple</td>
			<td>See protocol 1.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Vascular clip applicator</td>
			<td style="width:236px;">B. Braun, Germany</td>
			<td style="width:119px;">FT495T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Temporary vascular clamps</td>
			<td style="width:236px;">B. Braun, Germany</td>
			<td style="width:119px;">FT250T</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Graph Pad Prism statistical software&#160;</td>
			<td style="width:236px;">GraphPad Software, San Diego, California, USA</td>
			<td style="width:119px;"></td>
			<td>V 6.02 for Windows</td>
		</tr>
	</tbody>
</table>

the helsinki rat microsurgical sidewall aneurysm model

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced into clinical practice. Furthermore, experimental models are needed to elucidate the complex aneurysm biology leading to rupture of saccular aneurysms.
Several different kinds of experimental models for saccular aneurysms have been established in different species. Many of them, however, require special skills, expensive equipment, or special environments, which limits their widespread use. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various institutions.
The microsurgical rat abdominal aortic sidewall aneurysm model combines the possibility to study both novel endovascular treatment strategies and the molecular basis of aneurysm biology in a standardized and inexpensive manner. Standardized grafts by means of shape, size, and geometry are harvested from a donor rat&#39;s descending thoracic aorta and then transplanted to a syngenic recipient rat. The aneurysms are sutured end-to-side with continuous or interrupted 9-0 nylon sutures to the infrarenal abdominal aorta.
We present step-by-step procedural instructions, information on necessary equipment, and discuss important anatomical and surgical details for successful microsurgical creation of an abdominal aortic sidewall aneurysm in the rat.

A pilot series comprised 14 rats. Subsequently a total of 84 animals were operated according to the presented protocol for several research projects between March and September 2012. Additional 29 animals served as donors for arterial saccular grafts. The remaining experiments were performed using pre-treated grafts harvested and stored from previous experiments using rats of the same gender, strain, weight, and age.
Body weight, overall operation time, aortic clamping time, time for anastomos...

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The Helsinki Rat Microsurgical Sidewall Aneurysm Model

Microsurgical sidewall aneurysms in rats are created by end-to-side anastomosis of an aortic graft to the abdominal aorta. We present step-by-step instructions and discuss anatomical and surgical details for successful experimental saccular aneurysm creation.

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced ...

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12.2K Views.  University of Helsinki, Helsinki, Finland. Microsurgical sidewall aneurysms in rats are created by end-to-side anastomosis of an aortic graft to the abdominal aorta. We present step-by-step instructions and discuss anatomical and surgical details for successful experimental saccular aneurysm creation.

Video: The Helsinki Rat Microsurgical Sidewall Aneurysm Model

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2 To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8 Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11 
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12 The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13 These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16 Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.

An optimized technique for the microsurgical creation of arterial bifurcation aneurysms mimicking bifurcation human cerebral aneurysms is described. A venous pouch is sutured into an artificially created true bifurcation of both common carotid arteries. Facilitated microsurgical techniques and aggressive postoperative anticoagulation and analgesia lead to minimized morbidity rates and high aneurysm patency rates.

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...