The authors have no acknowledgements.

1. Preparation:
<ol>
	<li>Obtain a tabletop stereo microscope, with a variable 3,5 x to 45x zoom, 200 mm working distance, 55 to 75 mm variable inter-pupillary distance and an accessory led ring light. Any similar microscope is adequate, it can be set up on a small table and is not extremely expensive.</li>
	<li>Gather and prepare a set of microsurgical instruments with two microsurgical pickups, one dissecting scissor, and one needle-driver; this is the minimum required, it must be taken into account that these are practice instruments, and high end quality instruments are not needed.</li>
	<li>Use 9/0 monofilament micro-suture for all the practices. Buy commercially available chicken thighs or wings and food colorant at the local supermarket.</li>
	<li>A 20 G angio-catheter is used for the patency tests on the chicken femoral arteries. For the Brachial arteries a thinner 27 G catheter is needed. These can be acquired over the internet at a local Pharmacy or with the instruments set. Buy a commercially available syringe to inject the food colorant.</li>
</ol>
2. Macroscopic dissection techniques:
<ol>
	<li>The approach to the femoral vessels is straightforward. For chicken thighs, perform a blunt dissection technique just medial to the femoral shaft, separate the femoral muscle from the bone and expose the femoral artery, vein and nerve. The nerve can be further dissected into two separate fascicles for different types of nerve repair practices.</li>
	<li>In order to access the chicken Brachial artery in the chicken wings, incise the skin longitudinally with a scissor, from the shoulder up to the tip of the wing. Observe the artery running between the biceps and triceps brachii.</li>
</ol>
3. Recommended training regimen:
<ol>
	<li>Training with the femoral artery. 
	NOTE: The first step in the training regimen is to practice with the chicken femoral artery.</li>
</ol>

<ol>
	<li>Place the piece under the microscope and start the microsurgical dissection. Free the femoral artery from the underlying tissues; and divide it horizontally. Perform an adventicectomy. Use the micro-pickups and micro-scissors to trim any adventicia overlying the arterial lumen. Rinse the inner lumen with regular tap water. Write down the starting time</li>
	<li>Perform an end to end anastomosis.
	<ol>
		<li>Start on the back wall of the artery on the side opposite to the operator. Place the first stitch; start on the left side if working horizontally, on the lower side if working vertically. Pass the needle from the outside of the artery towards the lumen and then from the lumen to the outside of the artery.</li>
		<li>Tie the stitch, perform a double knot first, and then two single throws alternating directions. Complete the whole back wall with the above described technique.</li>
		<li>Starting on the right hand side if you are working horizontally or the upper side if you are working vertically, place a single stitch from the outside to the lumen and then from the lumen to the outside. Tie the knot as described above. Complete the front wall repeating the procedure described in 3.1.2.2.</li>
		<li>Write down the final time this will allow you to keep a time record, so you can control your progression. 
		NOTE: The authors prefer a one way up technique; but any type of microsurgical suture can be rehearsed. It is recommendable that the trainee completes around 25 microsurgical anastomoses in this manner before he can try other clinical scenarios. These include vein grafting for arterial gaps, end to end nerve repairs, and the use of vein conduits for the treatment of nerve gaps. An important technique in microvascular surgery, that can be learned with this training method is the continuous or running suture, to perform this technique the trainee proceeds as 3.1.2.1 but the stich is not cut, continue to perform stitches as in 3.1.2.1 without cutting them until you complete the whole suture and tie it to the original stitch at the end.</li>
	</ol>
	</li>
</ol>

<ol start="2">
	<li>Training with the Brachial artery
	<ol>
		<li>Place the piece under the microscope. Complete the procedure as described above for the femoral artery. 
		NOTE: The only difference is the size. The Brachial artery is much smaller than the femoral artery and the anastomosis is considerably more challenging.</li>
	</ol>
	</li>
	<li>Patency tests
	<ol>
		<li>Cross a smooth 20 or 27 G angio-catheter through the anastomosis, to check for obstructions caused by stitches suturing both vessel walls together.</li>
		<li>Apply a syringe with commercial food colorant to the end of the angio-catheter, and perfuse dye into the artery to test for jet leakage</li>
		<li>Open the artery longitudinally to visualize the stitches, their parallelism and the amount of wall taken.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+events+on+development+of+experimental+microsurgical+organ+transplantation.">Historical events on development of experimental microsurgical organ transplantation.</a> Yonsei Med J. 45 (6), 1115-1120 (2004).</li><li>Shurey, S., Akelina, Y., Legagneux, J., Malzone, G., Jiga, L., Ghanem, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+model+in+microsurgery+education:+classical+exercises+and+new+horizons.">The rat model in microsurgery education: classical exercises and new horizons.</a> Arch Plast Surg. 41 (3), 201-208 (2014).</li><li>Olabe, J., Olabe, J., Sancho, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cadaveric+infusi&#243;n+model+for+neurosurgical+training.">Human cadaveric infusi&#243;n model for neurosurgical training.</a> Surg Neurol. 72, 700-702 (2009).</li><li>Peled, I. J., Kaplan, H. Y., Wexler, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsilicone+anastomoses.">Microsilicone anastomoses.</a> Ann Plast Surg. 10 (4), 331-332 (1983).</li><li>Gluer, N. M., Rao, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canniesburn+&#8220;ever-ready&#8221;+model+to+practise+microsurgery.">Canniesburn &#8220;ever-ready&#8221; model to practise microsurgery.</a> Br J Plast Surg. 43 (3), 381-382 (1990).</li><li>Hino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Training+in+microvascular+surgery+using+a+Chicken+wing+artery.">Training in microvascular surgery using a Chicken wing artery.</a> Neurosurgery. 52 (6), 1495-1498 (2003).</li><li>Marsh, D. J., Norton, S. E., Mok, J., Patel, H. D., Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+training+the+Chicken+thigh+model.">Microsurgical training the Chicken thigh model.</a> Ann Plast Surg. 59 (3), 355-356 (2007).</li><li>Jeong, H. S., Moon, M. S., Kim, H. S., Lee, H. K., Yi, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+training+with+fresh+chicken+legs+and+their+histological+characteristics.">Microsurgical training with fresh chicken legs and their histological characteristics.</a> Ann Plast Surg. 70 (1), 57-61 (2013).</li><li>Couceiro, J., Ozyurekoglu, T., Sanders, S., Tien, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+training+regimen+with+nonliving+chicken+models.">Microsurgical training regimen with nonliving chicken models.</a> Microsurgery. 33 (3), 251-252 (2013).</li><li>Phoon, A. F., Gumley, G. J., Rtshiladze, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+training+using+a+pulsatile+membrane+pump+and+chicken+thigh:+A+new,+realistic,+practical,+nonliving+educational+model.">Microsurgical training using a pulsatile membrane pump and chicken thigh: A new, realistic, practical, nonliving educational model.</a> Plast Reconstr Surg. 126 (5), 278e-279e (2010).</li><li>Zhou, Y., Gu, X., Xiang, J., Qian, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+suture+versus+cuff+anastomosis+in+mouse+cervical+cardiac+transplant.">A comparative study on suture versus cuff anastomosis in mouse cervical cardiac transplant.</a> Exp Clin Transplant. 8 (3), 245-249 (2010).</li></ol>

The authors have nothing to disclose.

The clinical practice of microsurgery requires a continuous practice and a high level of expertise; it&#8217;s applications include free flaps, replantation and revascularization procedures, intracranial &#8211; extracranial anastomoses for neurosurgical procedures, peripheral nerve surgery and others. In spite of the wide variety of procedures that require microsurgical techniques, some of them are seldom performed in many centers. The surgeons therefore need sometimes to maintain their skill level rehearsing the surgeries on different practice models. 
The most widely spread training model is currently the rat 1,2; it is considered by many to be the &#8220;gold standard&#8221; in microsurgical instruction. Rats are indeed a very valuable tool in the teaching and learning of microsurgery. They provide a very similar environment to that of the clinical situation. This approach however exhibits some intrinsic limitations. The use of living animals presents an ethical dilemma; and is generally subjected to institutional review board approval. Furthermore they require anesthesia, and dedicated technicians and facilities. All These factors imply important economic costs; this restricts training on living animals to specific teaching units, experimental surgery departments and hospitals with similar resources. 
The use of nonliving animal models described in this protocol, has the advantage of not raising any ethical concerns; it does not require institutional review board approval. Economically it compares favorably to living animal training. The required equipment is limited and can be acquired over the internet for a reasonable price; the author&#8217;s tabletop stereomicroscope was bought for around 450 United States Dollars (USD) and the set of practice microsurgical instruments was purchased for around 150 USD. The author uses microsurgical sutures provided by his sponsoring institution; the price of sutures varies greatly and high quality sutures are not needed, a box of twelve sutures can be bought for around 70 USD on the internet. A microsurgical training station can be set up on a very limited area at home or at work; Jeong et al.8 Placed their microscope at the residents&#8217; office; whilst we9 used an available microscope close to the hand surgery fellows&#8217; quarters. 
The most critical point in the application of this protocol is the need for the trainee to be constant and to stay motivated with his training, it is therefore very important that he starts training with the bigger vessels first and then proceed to the smaller chicken wing vessels to avoid frustration. The limitations of training with nonliving models involve primarily the lack of bleeding and the inexistence of hemodynamic phenomena like vasospasm and clotting. The advantages and disadvantages of this type of training are summarized on Table 1. The three tests detailed in section three are aimed to help the trainee check the vessel&#8217;s patency in the absence of the earlier mentioned phenomena; they provide information on the quality of the trainee&#8217;s suture, leaking, suture misalignment, and lumen obstruction caused by stitches suturing the front and back wall together. 
The protocol can potentially be improved by the addition of a peristaltic pump10 to simulate vessel flow or by flooding the field with liquid to simulate superficial tension. It is the authors opinion that this would result in the production of an even more realistic training environment and this should be the direction of future research. 
Another possible field for development is the application of this training method to help the trainee learn more complex procedures, such as the cuff anastomosis technique11 before it&#8217;s actual application on animal research. The authors have however no experience with such techniques at present time. 
In conclusion the protocol described in the present manuscript allows a determined trainee to setup a small practice station and use it to learn, perfect and maintain microsurgical skills progressively and sustainably; without the need for special facilities and minimizing animal deaths.

An ample array of training models have been described for the learning and maintenance of both basic and advanced microsurgical skills. These include living animals1,2, human cadaveric specimens3, inert models4,5 and non-living animal models6-9.The Living animal models, specifically those using rats have been extensively used in the teaching of microsurgery 1,2; and they are considered the current gold standard of training. In spite of their remarkable instructive value, when an intensive or prolonged training is required; economical and ethical concerns can hinder their practical usage. 
Cadaveric models3 offer the opportunity to practice in an environment similar to that of the actual clinical situation; they are unfortunately restricted to anatomy labs and similar facilities, a microscope must be available at the lab as well; these models are therefore not widely available. Inert models such as rubber tubes, or gloves4,5 are cheap, and easily accessible, the required equipment for their application is minimal. The resemblance to a clinical situation is tenuous however; and their application is generally limited to the introductory stages of training, before the trainee starts practicing on rats. To the best of our knowledge Hino6 was the first to propose the use of non-living chicken for the development and maintenance of clinical microsurgical skills. His model is based on the extraction and subsequent usage of the chicken brachial artery from commercial chicken wings. This approach does not need institution review board approval, rodent anaesthesia, laboratory technicians or postoperative care. 
Following Hino&#180;s description of the chicken wing artery model numerous other nonliving animal models were published. The use of commercial chicken thighs was first suggested by Marsh et al.7 who reported the use of the femoral artery, vein and nerve for didactic purposes. This particular model expands the possibilities of training including structures different from the arteries; it permits the trainee to perform a variety of complex procedures such as vein grafts, nerve grafts or fascicular repairs. Jeong et al.8 Further studied Marsh&#8217; chicken thigh model and its applications to the teaching of resident doctors. The authors analysed chicken tissue samples histologically and found that it presented similarities to human tissues. The time to perform an anastomosis by the trainee was significantly reduced by practice in their study. They concluded that &#8220;the femoral neurovascular bundles of chicken are an appropriate and effective model for teaching and practicing microsurgery&#8221;. In 2013 my colleagues and I9 published a microsurgical training regimen that combined both the chicken thigh and the chicken wing models; this approach benefits from the versatility of the chicken thighs and from the limited size of the chicken wing artery which can represent more of a challenge than the femoral vessels for the more advanced trainees. 
On the current manuscript we describe step by step the process of setting up a microsurgical practice station; the materials required, how to dissect the neurovascular structures of each model and how to effectively apply the training regimen.

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:243px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:187px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereomicroscope</td>
			<td style="width:243px;">Amscope</td>
			<td style="width:119px;">SM-1BX</td>
			<td>Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Led light</td>
			<td style="width:243px;">Amscope</td>
			<td style="width:119px;">LED-80S-YA</td>
			<td>illumination system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microsurgical instruments set</td>
			<td style="width:243px;">Chen hen microsurgical instruments</td>
			<td style="width:119px;">HC- X803-1</td>
			<td>Microsurgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsurgical sutures</td>
			<td style="width:243px;">S/T microsurgical</td>
			<td style="width:119px;">9/0 Suture</td>
			<td>Sutures</td>
		</tr>
	</tbody>
</table>

step by step: microsurgical training method combining two nonliving animal models

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly laboratory rats. This method although extremely valuable can be economically demanding both for the surgeon and the sponsoring institution; it also requires special training facilities that may not always be available or accessible. Furthermore ethical concerns can limit the use of living animals for training purposes. Alternative training methods, such as inert tubes and gloves have not gained popularity among surgeons since they do not offer an experience similar to that of a clinical situation. Non-living animal models include the use of chicken thighs and wings; they offer a practice experience that resembles a clinical situation to a considerable extent. This type of training is relatively cheap and easily available. The microscope and instruments required can be acquired over the internet, and the chicken pieces can be bought at the local supermarket.
This approach allows a motivated trainee to rehearse different types of surgical techniques several times at a reasonable expense, helping to develop or maintain his surgical expertise if more complex facilities are not available. On the current manuscript we describe how to setup a small practice station, how to dissect the specimens, and how to practice both with the chicken thighs and with the chicken wings in a progressive fashion. This approach takes advantage on the versatility of the chicken thigh model and the small size of the chicken wing Brachial artery.

The authors have chronometered their times with the present training method and have seen it to significantly reduce the anastomosis times for a femoral artery at the end of the third week. The trainee can control the improvements on his technique by checking his times, his patency percentages and the alignment of his stitches. The system described in this manuscript is depicted in Figure 1 it includes a tabletop microscope and a set of microsurgical instruments. The macroscopic dissections are shown in Figures 2 and 3. An example of a femoral artery microsurgical anastomosis is shown in Figure 4. Figure 5 shows a Brachial artery microsurgical anastomosis. Figure 6 shows a femoral artery repair with a vein graft. The patency tests are shown in Figure 7.

<img alt="Figure 1" src="/files/ftp_upload/52625/52625fig1.jpg" /> 
Figure 1: The corresponding author&#8217;s own microsurgical practice station. It includes a tabletop microscope and a set of training microsurgical instruments. It can be set up on a very limited space at the office or at home.
<img alt="Figure 2" src="/files/ftp_upload/52625/52625fig2.jpg" /> 
Figure 2 A: A macroscopic dissection of a chicken thigh, The femoral vessels and sciatic nerve can be easily exposed running a finger just medial to the femoral shaft. The horizontal arrow marks the femoral head. The dissection plane is marked by the vertical arrow just parallel to the femoral shaft B: The femoral artery (a) vein (v) and sciatic nerve (n) are shown here in detail.
<img alt="Figure 3" src="/files/ftp_upload/52625/52625fig3.jpg" /> 
Figure 3 A and B: A macroscopic dissection of a chicken wing. The brachial artery runs between the Biceps and Triceps Brachii. The artery is marked with an arrow on both photographs it is quite superficial and the approach is simple; it can be achieved with commercial cooking scissors.
<img alt="Figure 4" src="/files/ftp_upload/52625/52625fig4.jpg" /> 
Figure 4: Microscopic image of a femoral artery end to end suture, here a one way up technique with single stitches was performed. Any other type of suture can be practiced at the trainee&#8217;s discretion.
<img alt="Figure 5" src="/files/ftp_upload/52625/52625fig5.jpg" /> 
Figure 5: End to end suture of a chicken Brachial artery. This arteries are much smaller than the femoral ones and the procedure is much more technically demanding.
<img alt="Figure 6" src="/files/ftp_upload/52625/52625fig6.jpg" /> 
Figure 6: A femoral artery repair with a vein graft harvested from the femoral vein. The chicken thigh model is very versatile allowing for different types of practice exercises.
<img alt="Figure 7" src="/files/ftp_upload/52625/52625fig7.jpg" /> 
Figure 7: The patency tests. A: An angio-catheter of the appropriate diameter is crossed through the anastomosis to check for obstructions caused by stiches suturing both of the vessel&#8217;s walls. B: Dye is perfused through the anastomosis to test for leakage. C: The anastomosis is opened longitudinally to visualize the suture alignment.

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Step By Step: Microsurgical training method combining two nonliving animal models

Here we describe how to set up a small microsurgical practice station and a simple and inexpensive method for the training of microsurgery with non-living animal models.

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly ...

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15.1K Views.  POVISA Hospital. Here we describe how to set up a small microsurgical practice station and a simple and inexpensive method for the training of microsurgery with non-living animal models. 

Video: Step By Step: Microsurgical training method combining two nonliving animal models

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

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 ...

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.

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 ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Combining Augmented Reality and 3D Printing to Display Patient Models on a Smartphone

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) printing (3DP) opens new possibilities in clinical applications. Although these technologies have grown exponentially in recent years, their adoption by physicians is still limited, since they require extensive knowledge of engineering and software development. Therefore, the purpose of this protocol is to describe a step-by-step methodology enabling inexperienced users to create a smartphone app, which combines AR and 3DP for the visualization of anatomical 3D models of patients with a 3D-printed reference marker. The protocol describes how to create 3D virtual models of a patient&rsquo;s anatomy derived from 3D medical images. It then explains how to perform positioning of the 3D models with respect to marker references. Also provided are instructions for how to 3D print the required tools and models. Finally, steps to deploy the app are provided. The protocol is based on free and multi-platform software and can be applied to any medical imaging modality or patient. An alternative approach is described to provide automatic registration between a 3D-printed model created from a patient&rsquo;s anatomy and the projected holograms. As an example, a clinical case of a patient suffering from distal leg sarcoma is provided to illustrate the methodology. It is expected that this protocol will accelerate the adoption of AR and 3DP technologies by medical professionals.

Presented here is a method to design an augmented reality smartphone application for the visualization of anatomical three-dimensional models of patients using a 3D-printed reference marker.

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) ...