Name: dissection, microct scanning and morphometric analyses of the baculum
Uploaded: 2017-03-19T15:00:00
Description: Watch this Scientific Journal Video about Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum at JoVE.com

Tim Daley and Andrew Smith provided many useful computational discussions during the early days; Tim Daley wrote the program rotate_translate_cylindrical necessary for Protocol 5. Computational resources were provided by the High Performance Computing Cluster at the University of Southern California. This work was supported by NIH grant #GM098536 (MDD).

All procedures and personnel were approved by the University of Southern California&#39;s Institute for Animal Care and Use Committee (IACUC), protocol #11394.
1. Baculum Dissection and Preparation
<ol>
	<li>Euthanize a sexually mature male mouse via carbon dioxide over-exposure, according to protocols set forth by the relevant Institutional Animal Care and Use Committee (IACUC).</li>
	<li>Lay the animal in the supine position, and protract the penis by applying pressure with thumbs lateral ...

<ol>
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The critical steps in the above protocol are 1) dissecting the bacula, 2) gathering the microCT images, 3) converting the microCT output to a flat file of x-y-z coordinates, 4) segmenting out each specimen&#39;s point cloud, 5) transforming each specimen to a standardized coordinate system, and 6) defining semi-landmarks. These steps are easily modified to accommodate different objects.
These methods can likely be applied to any object that is essentially &#34;rod-shaped&#34;, or at least not ...

The field of morphometrics includes a diversity of methods to quantify the size and shape of the biological form, a fundamental step in scientific inquiry1,2,3,4,5,6. Traditionally, the statistical analysis of size and shape begins by identifying landmarks on a biological structure, and then measuring linear distances, angles and ratios, which could be analyzed in a multivariate framework. Landmark-based Geometric Morphometrics is an approach that retains the spatial position of landmarks, preserving geometric information from data collection through analysis and visualization5. Generalized Procrustes Analysis (GPA) can be applied to remove variation in location, scale, and rotation of landmarks to produce an alignment between specimens that minimizes their squared differences - what remains is shape dissimilarity7.
An important concept of any morphometric analysis is homology, or the idea that one can reliably identify landmarks representing biologically meaningful and discrete features that correspond between specimens or structures. For example, human skulls have homologous processes, foramina, sutures, and ducts that can enable morphometric analyses. Unfortunately, the identification of corresponding landmarks is difficult across many biological structures, especially those with smooth surfaces or curves8,9,10.
We approach this problem below using computational geometry. The general workflow is to generate a three dimensional scan of the object that can be represented as a cloud of points, and then rotate and transform that point cloud so that all specimens are oriented on a common coordinate system. Then we mathematically define semi-landmarks from specific regions of the object. Discrete semi-landmarks placed on such regions are biologically arbitrary11. Conducting GPA and subsequent statistical analyses can produce undesirable artifacts8,12 because arbitrarily placed landmarks may not be biologically homologous. Therefore, we allow these semi-landmarks to mathematically &#34;slide&#34;. This procedure minimizes the potential difference between structures. As argued elsewhere the sliding algorithm used here is appropriate to quantify similar anatomical regions lacking easily identified corresponding landmarks3,6,8,10,11,12. These methods have their limitations13, but should be adaptable to objects of different size and shape.
Here, we illustrate how this method was applied in a recent study of the mouse baculum14, a bone in the penis that has been gained and lost multiple independent times during mammalian evolution15. We discuss the dissection and preparation of a specific bone, the baculum (Protocol 1), the generation of microCT images (Protocol 2), and the conversion of these images to a format that enables all downstream computational geometry (Protocols 3 and 4). After these steps, each specimen is represented by ~100K x-y-z coordinates. We then walk through a series of transformations that effectively align all specimens into a common orientation (Protocol 5), then define semi-landmarks from aligned specimens (Protocol 6). Protocols 1-4 should be similar regardless of the object being analyzed. Protocol 5 and Protocol 6 are specifically designed for a baculum, but it is our hope that by detailing these steps, investigators can imagine modifications that would be relevant for their object of interest. For example, modifications of these methods were applied to study whale pelvic bones and rib bones16.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting scissors</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">470106-338</td>
			<td style="width:167px;">Most sizes should work</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting Forceps, Fine Tip, Curved</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">82027-406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">1.7 mL microcentrifuge tube</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">87003-294</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Absolute Ethanol</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:79px;">CAS 64-17-5</td>
			<td>To be diluted to 70% for dissections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Floral Foam</td>
			<td style="width:287px;">Wholesale Floral</td>
			<td style="width:79px;">6002-48-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">uCT50 scanner&#160;</td>
			<td colspan="2">Scanco Medical AG, Bruttisellen, Switzerland</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection, microct scanning and morphometric analyses of the baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

The x-y-z coordinates of the semi-landmarks produced in Protocol 6 can be directly imported into any landmark-based geometric morphometrics analysis17. The computational pipeline above has been applied to study mouse bacula14, as well as whale pelvic and rib bones16. More details on the computational definition of semi-landmarks are presented here, in an attempt to help researchers visualize steps that might be modifi...

Watch this Scientific Journal Video about Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum at JoVE.com

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

The overall goal of this dissection and morphometric analysis is to illustrate the robustness of our morphometric methodology for studying structures with complex shapes like the mouse penis bone. We first had the idea for this study when we realized that existing methods of measuring baculum variation such as taking length and width dimensions lost all information about curvature and shape complexity, therefore we devised this method to quantify size and shape variation of any bone or structure of interest in three dimensions. The main advantage of this technique is that it applies modern morphometrics to structures without landmarks.This should advance the field by providing improved repeatable measurements of complex structures. Position a euthanized, sexually mature male mouse supine and protract the penis by applying thumb pressure lateral to the preputial opening. Once the penis emerges, extend the tissue through the prepuce as far as possible.Next, using scissors, cut the penile body proximal to the gland's penis where the baculum resides, detaching the penis. Then fully submerge the penis in about 200 microliters of tap water in a microcentrifuge tube. To soften the tissue, incubate it at about 50 degrees celsius for three to five days.Tissue softening reduces the change of the bone breaking during extraction. After incubation, use a dissection microscope and forceps to remove most of the tissue surrounding the baculum. After the gross dissection, gently squirt 70 percent ethanol to push off the remaining tissue and clean the bone.Then place the dissected baculum into a new microcentrifuge tube, and let the bone dry at room temperature with the cap left open. To begin, prepare the material in which the bone will be placed. Press a CT scan cylindrical holder into a brick of florist foam.Then extract the resulting cylinder of foam and cut slices of foam that are between two and five centimeters thick. Now push 10 to 15 dried bacula into the florist foam around the periphery of an individual slice to minimize interference during scanning. Take note of the precise orientation of the bone for future identification.Next, carefully place the bones in foam into the micro CT holder and load the holder into a micro CT 50 scanner. Set the micro CT 50 scanner as follows. Use 90 kilovolt pascals, a voxel size of 155 angstroms, a 5 milliliter aluminum filter, 750 projections per 180 degrees, and a 500 millisecond exposure time.Then proceed to scan the specimen. Baculum bones were dissected, prepared, and scanned using the described protocol. Many bones can be packed into one CT scan.The images were then processed using computational geometry as described in the text protocol. This imaging process should be applicable to the study of a wide array of bones and other subjects of interest. The analysis provides detailed information on the landmark features of the object and is highly automated.This process only required about 10 manual adjustments when analyzing 369 bacula. The exciting thing is that our methods could be applied to a wide variety of complex structures. These methods allowed us to quantify the size and shape of the baculum in three dimensions.

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