The authors thank Tongalp Tezel, MD, for consultation regarding sT injection; Theresa Swayne, PhD, for consultation regarding SHG microscopy; and Jimmy Duong from the Design and Biostatistics Resource and the Biostatistical core facility of the Irving Institute at Columbia University Medical Center.
Supported in part by Research to Prevent Blindness and by National Institutes of Health Grants NCRR UL1RR024156, NEI P30 EY019007, NCI P30 CA013696, and NEI R01EY020495 (DCP). Columbia University owns related intellectual property: US issued patents no: 8,466,203 and no: 9,125,856. International patent pending: PCT/US2015/020276.
Images were collected in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant #P30 CA013696 (National Cancer Institute). The confocal microscope was purchased with NIH grant #S10 RR025686.

All procedures were performed using cadaveric rabbit eyes within intact outbred rabbit heads. All Institutional and National guidelines for the care and use of laboratory animals were followed.
1. Preparation of Solutions
<ol>
	<li>SMG preparation for TXL:
	<ol>
		<li>Prepare 1 mL of 0.2 M concentration of sodium bicarbonate solution (NaHCO3) solution using 0.0165g of NaHCO3 powder dissolved in 1 mL of the distilled water.</li>
		<li>Dissolve 0.1016 mg of powdered sodium hydroxymethylglycinate (SMG) in 1 mL of the distilled water to get a final concentration of 800 mM SMG. Adjust sodium bicarbonate solution to a final concentration of 0.1 M NaHCO3 and 400 mM SMG. Concentrations of SMG depending on the cross-linking effect desired. In the protocol described here we used 40, 100, and 400 mM SMG.</li>
	</ol>
	</li>
</ol>
2. SubTenon&#39;s Injection for TXL using SMG
<ol>
	<li>Fill two 1 mL insulin syringes (25G needles) with 400 &#181;L control and SMG solution, respectively.</li>
	<li>Place the rabbit&#39;s head in a profile plane with the help of a cushion. Styrofoam or a paper stack can be used to fix the head in an optimal position.</li>
	<li>Retract the eyelids with a pediatric eye speculum.</li>
	<li>Measure the initial intraocular pressure (IOP) using an applanation tonometry device.</li>
	<li>Mark the site of intended injection on the upper middle part of the limbus with a tissue marker.</li>
	<li>Retract the conjunctiva surrounding the site of injection with a conjunctival forceps (or any forceps with serrated round tip) and insert the needle through the conjunctiva, entering Tenon&#39;s capsule just slightly beyond the marked limbal site (i.e., 2-3 mm from the limbus). A small incision in the conjunctiva can also be made with iris scissors in order to facilitate the passage of the needle through Tenon&#39;s capsule.</li>
	<li>Once within Tenon&#39;s capsule, make sure that the needle is freely mobile by moving it side-to-side. During this time, the globe should not move. This confirms proper placement of the needle above the sclera in the sub-Tenon&#39;s (sT) space.</li>
	<li>Inject the solution from the syringe and discard the needle. Immediately following injection, the fluid will accumulate in the sT space creating an anterior bulge seen through the conjunctiva (i.e., chemosis).</li>
	<li>Repeat the IOP measurement in order to confirm that it did not change due to an inadvertent perforation of the globe.</li>
	<li>Remove the lid speculum, and perform digital massage through the closed eyelids for about 2-3 min.</li>
	<li>Leave the head for an incubation period of 3.5 h (room temperature = 18 &#176;C), prior to moving to the next step.</li>
</ol>
3. Tissue Preparation
<ol>
	<li>Retract the eyelids using the eyelid speculum in order to optimize access to the globe. Select the optimally sized speculum according to the size of the eye.</li>
	<li>Separate the conjunctiva surrounding the limbus. If it has already been incised near the site of injection, circumferentially expand the borders so it would contain an inoculum size of approximately 1x1 cm.</li>
	<li>Cut the extra-ocular muscles at their sites of scleral insertion.</li>
	<li>Elevate the eyeball with forceps, pushing it from the posterior side. This provides access to the posterior globe and will facilitate cutting of the optic nerve with ophthalmic artery and vein located near the posterior pole of the globe.</li>
	<li>Cut out the corneoscleral complex, with the outer border including the marked injection site. The stain should still be visible on the remaining part of the sclera.</li>
	<li>Remove the corpus vitreus and all layers attached to the inner side of the sclera by applying traction with tissue forceps. 
	NOTE: Further steps depend on the following procedures being performed: 4.-DSC analysis, 5.-SHG microscopy.</li>
</ol>
4. For Regional DSC Analysis
<ol>
	<li>For the treated eye: cut out four scleral sectors from the remaining scleral cup with the scissors so that the site of the injection is located in the upper sector and aligned centrally. Cut the remaining 3 sectors from both lateral sides (i.e., nasal and temporal), and the bottom. 
	NOTE: the numbering of the sectors (1-4) which are further divided into squares (1-16) is demonstrated in Figure 1A.</li>
	<li>Cut the scleral sectors (1-4) into smaller squares (1-16) of approximately 4 x 4 mm each. Sector 1 should be divided into 9 squares (make the exact site of injection an individual square [square 2]). Divide sectors 2 and 3 into 2 squares each (squares 10-11 and 12-13) and sector 4 into 3 squares (squares 14-16).</li>
	<li>Assign a number to each square, as shown in Figure 1A, in order to localize the distance of the analyzed tissue from the location of the injected area.</li>
	<li>For the control eye: after dividing the tissue into four scleral sectors (similar to the treated tissue) cut out square pieces of tissue from the following locations: 3 squares from the top sector (sector 1), 1 from each side (sectors 2 and 3), and 1 from the bottom sector (sector 4).</li>
	<li>Scrape off the remaining retinal and choroidal layers and wash twice with fresh PBS each time, leaving the pieces submerged in solution for approximately 10 s at a time.</li>
</ol>
5. For SHG Imaging
<ol>
	<li>Cut the upper part of the sclera using scissors to create a 1&#160;x 1&#160;cm area with the site of injection aligned centrally.</li>
	<li>Scrape off the remaining retinal and choroid layers and wash twice with fresh PBS each time leaving the pieces in solution for approximately 10 s.</li>
	<li>Place the tissue in 1 mL tubes filled with PBS solution for transportation to the imaging facility. All the procedures, following the incubation time and beginning with the dissection of the eyeball should be performed within an hour.</li>
</ol>
6. Microscopy Protocol
NOTE: This protocol for imaging back-scattered SHG signal from collagen of sclera tissue is tailored for the laser scanning microscope.
<ol>
	<li>Microscopy set up
	<ol>
		<li>To maximize the signal and resolution when performing SHG microscopy use an objective lens optimize to transmit infrared light and with a high numerical aperture (NA). Our objective is Nikon Apo LWD 25x/NA1.1 water immersion.</li>
		<li>Adjust the correction collar of the lens to match the depth of the sample, in this case that is the thickness of the coverslip, 0.17 millimeters.</li>
		<li>Mount the 25x objective lens and add a generous amount of lubricating water-based gel to cover the imaging surface before mounting the sample. The water-based gel will not evaporate during the experiment and hence will maintain image quality.</li>
		<li>Place the scleral tissue from a 1 mL tube with PBS without drying between two 25-mm round coverslips (episcleral side down) providing maximal contact between the episclera and the coverslip surface. 
		NOTE: The tissue can also be placed uncovered on the coverslip. A good amount of PBS should keep the tissue hydrated during imaging. In this case, add the tissue piece and the PBS after assembling the cellchamber.</li>
		<li>Assemble the cell chamber by placing a 25-mm round coverslip, single or in a sandwich technique, on the bottom part of the chamber and screw the top part down in order to create a sealed round chamber. Do not screw down tightly when a top coverslip is used, in order to avoid artificially flattening and damage the tissue.</li>
		<li>Mount the cell chamber with the tissue sample on the microscope stage.</li>
		<li>Set the microscope for eye view with transmitted light on.</li>
		<li>Position the stage and adjust the height of the objective such that the lower surface of the sample is in focus, as determined by bright field inspection through the eye piece.</li>
		<li>Turn off all lights except the computer monitor and block as much light from the monitor as possible with aluminum foil sheets draped on the microscope stage. Minimizing any stray light reaching the detectors will ensure low-noise acquisition, as the GaAsP NDD detectors have high sensitivity.</li>
		<li>In the Ti Pad panel of the software, check that the lens definition is correct.</li>
		<li>In the A1 Compact GUI panel, choose the IR laser for imaging, select the NDD detectors and choose the DAPI channel that is equipped with a 400-450 nm bandpass filter.</li>
		<li>In the A1 MP GUI panel, set the wavelength of the infrared laser to 860 nm and open the shutter.</li>
		<li>Set laser scanning conditions in the A1 Compact GUI panel as follows. Select: (a) Galvano scanner, (b) Unidirectional scanning, (c) Pixel dwell time 6.2 &#181;s, (d) Frame size 1,024 x 1,024 pixels, (e) Line averaging 2x 
		NOTE: The Galvano scanner and unidirectional scanning ensures precise point by point alignment. A size of 1,024 x 1,024 for the full field of view translates into a pixel size of 0.5 &#956;m /pixel. Line averaging will reduce the shot noise in the image.</li>
		<li>Set imaging conditions in A1 Compact GUI panel by adjusting laser power and detector gain. Open the Look Up Table panel (LUTs) that displays a histogram of pixel intensity values in the current image. Turn on live imaging in &#8220;Find&#8221; mode and maximize the detected range of pixel values by adjusting laser power and detector gain. Avoid saturation. Typical values are 2.5% laser power, from a total of 2.35 W at 860 nm, and 100 HV (detector gain).</li>
		<li>NOTE: For this setup, the laser power measured with an internal power meter is 5.2 mW. Every time an experiment is performed, re-adjust the laser percentage such that the internal power measurement is constant at 5.2 mW between imaging sessions. Care should be taken when setting laser power. The Chameleon II laser is a 3 W laser at 800 nm and a 10% or higher power could potentially induce tissue damage.</li>
	</ol>
	</li>
	<li>Image acquisition
	<ol>
		<li>In preview mode, scan the tissue area using the XYZ Overview tool.</li>
		<li>Set the imaging to lower resolution (256 x 256 pixels and no line averaging) to speed up the acquisition of images in this mode.</li>
		<li>Capture 5 x 5, 3 x 3, or single fields of view to cover the entire surface of the tissue. At each location, before the overview capture, turn on the live &#34;Scan&#34; mode and bring the tissue into focus. Note that different regions of the tissue will have slightly different positions in the axial direction.</li>
		<li>Find a flat area where the collagen fibers are seen in the entire field of view and double-click that position in the overview tool to move the stage to that particular location.</li>
		<li>Turn on live &#34;Scan&#34; mode, adjust the Z position of the objective such that the bottom plane is in focus and, in the Ti Pad, use the Z drive to move the optical plane 10-15 &#956;m above this bottom layer.</li>
		<li>Acquire an image at high resolution with 1,024 x 1,024 pixels and 2x line averaging, using the &#34;Capture&#34; button.</li>
		<li>Save the location in the XYZ Overview using the &#34;+&#34; button. This ensures that the same area of tissue is not recaptured.</li>
		<li>For each piece of tissue capture 10 images of non-overlapping fields of view.</li>
	</ol>
	</li>
</ol>
7. DSC Protocol
Note:&#160;Proceed to this step as soon as tissue preparation is complete, for regional DSC analysis, or after tissue imaging when SHGM is performed.
<ol>
	<li>Prepare DSC pans, weighed and labeled. 
	NOTE: This step should be done before tissue dissection in order to minimize tissue desiccation.</li>
	<li>Dry each scleral square with an absorbent tissue and lay it flat on the bottom of a DSC pan using toothed forceps.</li>
	<li>Weigh the pan with the tissue inside and lid crimped and covered to obtain the tissue wet weight (mass of the samples should be in the range of 5 to 11 mg). 
	Note: Seal each pan using the crimper before proceeding to the next tissue sample. The pans are hermetically sealed, preventing any water loss prior to thermal analysis.</li>
	<li>Once the sample is crimped, place it on its designated location on the DSC tray. There should be 6 samples for the control and 16 for the treated eye.</li>
	<li>Create a method using instrument managing software, specifying the weight of the tissue, and run the thermal analysis using the following parameters: Temperature range of 40 to 80 &#176;C, Heating rate: 1 &#176;C/min, Heat flow: 17.37 mW, Gas (N2) flow: 19.8 mL/min, Gas pressure: 2.2 bar.</li>
	<li>Once completed, analyze the data for each sample by extracting the transition temperature peak at which thermal denaturation occurs using the instrument managing software.</li>
</ol>
8. Image Analysis
<ol>
	<li>SHG Signal
	<ol>
		<li>Select at least 5-10 of the most representative images from each treatment and its control, such that the area of the image is occupied by mostly collagen fibers.</li>
		<li>Upload each image in ImageJ software and measure the average pixel intensity by selecting Analyze&#62; Measure for the active image.</li>
		<li>The values extracted are reported as the mean pixel intensity and can also be shown by plotting the histogram of intensities by selecting from the menu Analyze &#62; Histogram.</li>
		<li>Using an Excel sheet, create a table to document all measured data accordingly to the sample ID.</li>
		<li>Calculate the mean and standard deviation of pixel intensity for each treatment and control condition.</li>
		<li>Using student&#39;s t-test, compare differences for all pairwise comparisons of concentrations (i.e., 40 mM SMG vs. 0 and 400 mM SMG vs. 0). [P&#8804;0.05].</li>
	</ol>
	</li>
	<li>Waviness
	<ol>
		<li>Select an image that displays collagen fibers. At least 10 images per sample should be analyzed (including a control sample for each concentration - at least 40 in total).</li>
		<li>Open ImageJ&#62;Plugins&#62; NeuronJ. NeuronJ requires prior installation.</li>
		<li>Upload all imagesby dragging into an opened NeuronJ window.</li>
		<li>Create tracing lines along the fibers, following the contour of the fibril with the mouse (pen drawing tablets could be used), click M to measure the distance of total fiber length.</li>
		<li>Select &#34;Option&#34; to draw a tangential straight line and connect the beginning and the end of the previously drawn fiber contour. Now click M to measure the end-to-end length.</li>
		<li>Repeat the same procedure on at least 10 fibrils per image.</li>
		<li>Collect those two measurements from each of 10 fibrils and input the data into an excel spreadsheet, expressing total fiber length (contour) and end-to-end length (straight connecting line) as Length [curve] and Length [linear], respectively.</li>
		<li>Calculate the waviness index (W) using the formula: W= Length [curve]/ Length [linear].</li>
		<li>Calculate the % of waviness comparing data from the images of treated samples(SMG) with the images from control samples using the formula: (W[SMG] - 1) / ( W[control] - 1 )</li>
		<li>Perform a pairwise t-test for waviness index (W) in order to determine statistical differences (p-values) of collagen fiber morphology between different treatment conditions and control.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Conducted experiments have shown evidence supporting the use of SHG signal microscopy as a method for evaluation of collagen cross-linking effects in sclera, raising the future possibility of using this technique as a monitoring tool for cross-linking treatments that target collagen proteins. Of note, an instrument already is in clinical use that can potentially capture this SHG signal. Although this instrument was primarily designed for imaging skin human dermis, it has been used successfully to image cornea and sclera<...

Progressive myopia is postulated to be treatable through non-enzymatic scleral cross-linking (photochemical and/or chemical), which makes sense given that blocking collagen enzymatic cross-linking can increase experimental form deprivation (FD)-induced myopia1. Elsheikh and Phillips2 recently discussed the feasibility and potential of using standard ultraviolet-A irradiation (UVA)-riboflavin mediated photochemical cross-linking (also known as the Dresden protocol), abbreviated here as (riboflavin CXL) for posterior scleral stabilization to halt axial elongation in myopia. This photochemical method has been successfully used for treating destabilization of the anterior globe surface (i.e., the bulging cornea) seen in keratoconus and post-LASIK keratectasia. However, application of this CXL protocol for the sclera is hindered by issues related to difficulties in accessing the posterior sclera with an ultraviolet (UV) light source, as well as the need to modify a much greater tissue surface area. That being said, the CXL approach has been used to halt axial elongation in visually form deprived rabbits (by tarsorrhaphy), although multiple regions of posterior sclera required multiple separate irradiation zones in that study3. By contrast, injection of a chemical stabilizing agent (i.e., cross-linking agent) via the sT space could represent a simpler way to modify the posterior sclera, avoiding the need for introducing a UV light source. This injection technique is well known as a useful way of inducing ocular anesthesia during ophthalmologic procedures such as cataract surgery4,5,6. Wollensak7 has described previously the use of an sT injection using glyceraldehyde (a chemical cross-linking agent similar in concept to the formaldehyde releasing agents (FARs) described in this study) to stiffen the rabbit sclera and genipin has been shown to limit axial length in FD guinea pigs8,9. These investigators have demonstrated a clear advantage of using a soluble chemical agent over the photochemical CXL technique. Thus, scleral cross-linking using an injectable chemical agent of some type, including the FARs (i.e., TXL)10, could provide a feasible treatment method to halt the progression of scleral elongation seen in myopia.
In the protocols presented here, we use a chemical cross-linking solution of sodium hydroxymethylglycinate (SMG), delivered via sT injection to the sclera of cadaveric rabbit eyes. We have implemented similar protocols previously for topical chemical cross-linking in the cornea. Notably in those previously reported studies, concentration dependent cross-linking effects could be obtained using SMG, with an effect range spanning well above that achievable with photochemical CXL as determined by thermal denaturation analysis11.
Here we describe protocols to assess the cross-linking effect of SMG delivered via sT injections to scleral tissue, thermal denaturation using Differential Scanning Calorimetry (DSC), and Second Harmonic Generation Microscopy (SHGM).
Using differential scanning calorimetry (DSC), also known as thermal analysis, a thermal denaturation transition is measured, which for scleral tissue is predominately guided by the properties of the fibrillar collagens, since they constitute the bulk majority of protein. This method evaluates the stability of collagen molecular structure and the cross-linked bonds that stabilize the collagen fibrils, the principal tertiary protein structure. During heating in the DSC, a critical transition temperature is achieved that results in denaturation of the collagen molecule, resulting in uncoiling of the triple helix, a process that forms what is commonly known as gelatin. This thermal denaturation disrupts hydrogen bonds along the collagen molecule and can be shifted to higher temperatures through induced cross-linking methods12,13. This method has been used for many decades, particularly in the biomaterials industry and for processes that include leather-making. However, this method requires extraction of the sclera tissue and therefore can only be useful as an ex vivo technique.
Second-harmonic generation microscopy (SHGM) is based on the non-linear optical properties of particular materials, with non-centrosymmetric molecular environments. In such materials, intense light, for example light produced by lasers, generates SHG signals, in which the incident light is doubled in frequency. Biological materials that are known to create SHG signals are collagen, microtubules, and muscle myosin. For example, collagen excited with an infrared light of 860 nm wavelength will emit an SHG signal in the visible range with 430 nm wavelength. Second harmonic generation (SHG) signal imaging is a promising method for evaluating therapeutic collagen cross-linking. It has been known for more than 30 years that collagen fibrils in tissues emit SHG signals14. However, only recently could high resolution images be obtained15 in a variety of tissues, including tendon16, skin, cartilage17, blood vessels18, and in collagen gels19.
Based on this knowledge, this study evaluates the SHG signal changes induced in the sclera through SMG chemically induced cross-linking of collagen. The results indicate that SMG modification of the sclera increases the SHG signals produced from tissue collagen fiber bundles (the higher order quaternary structure comprised of collagen fibrils) and also produces a structural morphologic change in the collagen fiber network, reflected in fiber bundle &#34;straightening.&#34;

<table border="0" cellpadding="0" cellspacing="0" width="1441">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:245px;">MILLI-Q SYNTHESIS A10 120V</td>
			<td style="width:289px;">EMD Millipore, Massachusetts, USA</td>
			<td style="width:111px;"></td>
			<td style="width:796px;">Double distilled, deionized water. - protocol step 1.1.1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium hydroxymethylglycinate&#160;</td>
			<td>Tyger Chemicals Scientific, Inc. Ewing, NJ, USA</td>
			<td></td>
			<td>Crosslinking reagent - protocol step 1.1.2</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Injection needle with luer-lock syringe</td>
			<td>BD Eclipse, NJ, USA</td>
			<td></td>
			<td>Syringe for sub tenon injection. - protocol step 2.1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rabbit head</td>
			<td>La Granja poultry</td>
			<td>Outbred</td>
			<td>Rabbit head separated and delivered within 1 hour postmortem. - protocol step 2.2</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tono-pen&#160;</td>
			<td>Reichter Technologies Depew, NY</td>
			<td></td>
			<td>IOP measurements - protocol step 2.4</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DSC 6000 Autosampler</td>
			<td>Perkin-Elmer Waltham, MA, USA</td>
			<td></td>
			<td>Thermal denaturation analyzer - protocol step 7.4</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pyris software&#160;</td>
			<td>Perkin-Elmer, Waltham, MA, USA</td>
			<td>Ver 11.0</td>
			<td>&#160;protocol step 7.5</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CFI75 Apochromat LWD 25X/1.10 W MP</td>
			<td>Nikon Instruments, Melville, NY, USA</td>
			<td></td>
			<td>A water immersionn objective with high IR transmittance with a working distance of 2.0 mm - protocol step 8.1.1.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">GenTeal&#160;</td>
			<td>Alcon, Fort Worth, TX&#160;</td>
			<td>B000URVDQ8</td>
			<td>Water-based gel used as objective immersion medium instead of water to prevent evaporation - 8.1.1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Chameleon Vision II&#160;</td>
			<td>Coherent, Santa Clara,CA, USA</td>
			<td></td>
			<td>Ti:Sapphire pulsed laser with a 140 fs pulse width at 80 MHz and a tunable range from 680 nm to 1080 nm. - protocol step 8.1.11</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">AttoFluor cell chamber</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>A7816</td>
			<td>Fixation of the cover slip - protocol step 8.1.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">25-mm round coverslips, #1.5</td>
			<td>Neuvitro Corporation, Vancouver, WA, USA</td>
			<td>GG-25-1.5</td>
			<td>protocol step 8.1.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Eclipse Ti-E</td>
			<td>Nikon Instruments, Melville, NY, USA</td>
			<td></td>
			<td>protocol step 8.1.4.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Non-descanned (NDD) GaAsP detector</td>
			<td>Nikon Instruments, Melville, NY, USA</td>
			<td></td>
			<td>Equipped with a 400-450 nm band pass filter - protocol step 8.1.7</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">A1R-MP laser scanning system</td>
			<td>Nikon Instruments, Melville, NY, USA</td>
			<td></td>
			<td>Compatible with infrared (IR) multi-photon excitation. - protocol step 8.1.8</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NIS Elements software</td>
			<td>Nikon Instruments, Melville, NY, USA</td>
			<td>Ver 4.3</td>
			<td>refered to as &#34;software&#34; in the text - protocol step 8.1.9</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fiji/ImageJ</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>&#160;protocol step 9.1.2</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">NeuronJ</td>
			<td style="width:289px;">Eric Meijering, Erasmus University Medical Center, Rotterdam, The Netherlands</td>
			<td></td>
			<td>https://imagescience.org/meijering/software/neuronj/, for protocol step 9.2.2</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microsoft Excel&#160;</td>
			<td>Microsoft Corporation, Redmond, WA, USA</td>
			<td>Ver 14</td>
			<td>protocol step 9.2.8</td>
		</tr>
	</tbody>
</table>

second harmonic generation signals in rabbit sclera as a tool for evaluation of therapeutic tissue cross-linking (txl) for myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Thermal denaturation temperature (Tm) as an assay method to evaluate TXL cross-linking effect: A total of 16 pairs of rabbit eyes were used in these experiments for the TXL procedure.As an initial part of this study, the localization of cross-linking effect induced by a single injection of SMG cross-linking agent via sT space in the cadaveric rabbit head was evaluated. This type of experiment has relevance to the clinical treatment of patients, since injections in more th...

Watch this Scientific Journal Video about Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia at JoVE.com

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

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JoVE Journal

Medicine

7.7K Views.  Columbia University College of Physicians and Surgeons. This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using second harmonic generation imaging and differential scanning calorimetry.

Video: Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized placebo-controlled crossover clinical evaluation, we assessed&#160;the effectiveness of a dedicated dietary supplement to improve chronic halitosis in 32 dogs of different breeds and ages. This protocol describes how to evalute the presence of oral volatile suphur compunds, e.g. methyl mercaptan, hydrogen sulfide and dimethyl sulfide, by means of a portable gas chromatograph device coupled with a syringe, which was used to collect the breath, and a dedicated software, which allows the operator to monitor each compound concentration during each measurement, in a relatively short time (8 min).
A significant modification of halitosis parameters was observed after 30 days since the beginning of treatment (p &#60;0.05), while a long-lasting effect was still observed even 20 days after the suspension of the treatment. Portable gas chromatograph, which is also widely used in clinical practice, can be therefore used to confirm&#160;and control halitosis in humans and animals. Even though human and animal species present some differences,&#160;this innovative and alternative therapy for halitosis management might be extended to human clinical practice as an adjuvant dietary approach.

We describe a simple approach for diagnosis of halitosis in dogs as well as a dietary approach for its management. This protocol may be extended to the management of halitosis in humans in the near future.

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...