Name: second harmonic generation signals in rabbit sclera as a tool for evaluation of therapeutic tissue cross-linking (txl) for myopia
Uploaded: 2018-01-06T13:00:00
Description: 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

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 hydroxyme...

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 ...

Research

JoVE Journal

Medicine

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 ...

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 ...

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

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 ...

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 ...

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 ...

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

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 ...

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 ...

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 ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...