This work was supported by National Eye Institute/NIH grant R01EY028242 (to K.G.), Research to Prevent Blindness/International Retinal Research Foundation Catalyst Award for Innovative Research Approaches for AMD (to K.G.), The Stephen Ryan Initiative for Macular Research (RIMR) Special Grant from W.M. Keck Foundation (to Doheny Eye Institute), and Ursula Mandel Fellowship and UCLA Graduate Council Diversity Fellowship (to I.S.T.). This work was also supported by an Unrestricted Grant from Research to Prevent Blindness, Inc. to the Department of Ophthalmology at UCLA.&#160;The content in this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Measurement of Stiffness in Mouse Retinal Capillaries and Subendothelial Matrix

<ol>
	<li>Duh, E. J., Sun, J. K., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy:+current+understanding,+mechanisms,+and+treatment+strategies.">Diabetic retinopathy: current understanding, mechanisms, and treatment strategies.</a> JCI Insight. 2 (14), e93751 (2017).</li><li>Roy, S., Kern, T. S., Song, B., Stuebe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+pathological+changes+in+the+diabetic+retina:+Implications+for+targeting+diabetic+retinopathy.">Mechanistic insights into pathological changes in the diabetic retina: Implications for targeting diabetic retinopathy.</a> Am J Pathol. 187 (1), 9-19 (2017).</li><li>Antonetti, D. A., Silva, P. S., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+the+molecular+and+cellular+pathology+of+diabetic+retinopathy.">Current understanding of the molecular and cellular pathology of diabetic retinopathy.</a> Nat Rev Endocrinol. 17 (4), 195-206 (2021).</li><li>Chandrakumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+retinal+vascular+inflammation+and+degeneration+in+diabetes.">Mechanical regulation of retinal vascular inflammation and degeneration in diabetes.</a> Diabetes. 73 (2), 280-291 (2024).</li><li>Chandrakumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subendothelial+matrix+stiffening+by+lysyl+oxidase+enhances+RAGE-mediated+retinal+endothelial+activation+in+diabetes.">Subendothelial matrix stiffening by lysyl oxidase enhances RAGE-mediated retinal endothelial activation in diabetes.</a> Diabetes. 72 (7), 973-985 (2023).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+stiffening+promotes+retinal+endothelial+activation+associated+with+diabetes.">Basement membrane stiffening promotes retinal endothelial activation associated with diabetes.</a> FASEB J. 30 (2), 601-611 (2016).</li><li>Wolfenson, H., Yang, B., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steps+in+Mechanotransduction+pathways+that+control+cell+morphology.">Steps in Mechanotransduction pathways that control cell morphology.</a> Annu Rev Physiol. 81, 585-605 (2019).</li><li>Pan, Z., Ghosh, K., Liu, Y., Clark, R. A., Rafailovich, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+stresses+and+translational+distortion+of+the+nucleus+during+fibroblast+migration+on+a+physiologically+relevant+ECM+mimic.">Traction stresses and translational distortion of the nucleus during fibroblast migration on a physiologically relevant ECM mimic.</a> Biophys J. 96 (10), 4286-4298 (2009).</li><li>Ghosh, K., Ingber, D. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence+increases+choroidal+endothelial+stiffness+and+susceptibility+to+complement+injury:+Implications+for+choriocapillaris+loss+in+AMD.">Senescence increases choroidal endothelial stiffness and susceptibility to complement injury: Implications for choriocapillaris loss in AMD.</a> Invest Ophthalmol Vis Sci. 57 (14), 5910-5918 (2016).</li><li>Cabrera, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell+stiffness+contributes+to+complement-mediated+injury+of+choroidal+endothelial+cells+in+a+monkey+model+of+early+age-related+macular+degeneration.">Increased cell stiffness contributes to complement-mediated injury of choroidal endothelial cells in a monkey model of early age-related macular degeneration.</a> J Pathol. 257 (3), 314-326 (2022).</li><li>Krieg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy-based+mechanobiology.">Atomic force microscopy-based mechanobiology.</a> Nat Rev Phys. 1, 41-57 (2019).</li><li>Chou, J. C., Rollins, S. D., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+digest+protocol+to+analyze+the+retinal+vasculature+of+a+mouse+model.">Trypsin digest protocol to analyze the retinal vasculature of a mouse model.</a> J Vis Exp. (76), e50489 (2013).</li><li>Beacham, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+extracellular+matrices+produced+by+cultured+and+primary+fibroblasts.">Preparation of extracellular matrices produced by cultured and primary fibroblasts.</a> Current protocols in cell biology. , (2007).</li><li>Bae, Y. H., Liu, S. L., Byfield, F. J., Janmey, P. A., Assoian, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+stiffness+of+ex+vivo+mouse+aortas+using+atomic+force+microscopy.">Measuring the stiffness of ex vivo mouse aortas using atomic force microscopy.</a> J Vis Exp. (116), e54630 (2016).</li><li>Huynh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+intimal+stiffening+enhances+endothelial+permeability+and+leukocyte+transmigration.">Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration.</a> Sci Transl Med. 3 (112), 112 (2011).</li><li>Sharma, A., Gupta, D. K., Bisen, S., Singh, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+trypsin+and+elastase+digestion+techniques+for+isolation+of+murine+retinal+vasculature.">Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature.</a> Microvasc Res. 154, 104682 (2024).</li><li>Chaqour, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy-based+measurements+of+retinal+microvessel+stiffness+in+mice+with+endothelial-specific+deletion+of+CCN1.">Atomic force microscopy-based measurements of retinal microvessel stiffness in mice with endothelial-specific deletion of CCN1.</a> Methods Mol Biol. 2582, 323-334 (2023).</li><li>Ashraf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+density+of+deep,+intermediate+and+superficial+vascular+plexuses+are+differentially+affected+by+diabetic+retinopathy+severity.">Vascular density of deep, intermediate and superficial vascular plexuses are differentially affected by diabetic retinopathy severity.</a> Invest Ophthalmol Vis Sci. 61 (10), 53 (2020).</li><li>Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+D:+an+Macrophage-derived+factor+mediating+increased+endothelial+cell+permeability+with+implications+for+alteration+of+the+blood-retinal+barrier+in+diabetic+retinopathy.">Cathepsin D: an Macrophage-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.</a> FASEB J. 30 (4), 1670-1682 (2016).</li></ol>

The authors have nothing to disclose.

AFM has been widely used to measure disease-associated changes in the stiffness of larger vessels, such as the aorta and arteries16. These findings have helped establish the role of endothelial mechanobiology in cardiovascular complications such as atherosclerosis17. Based on these findings, we have begun to investigate the previously unrecognized role of endothelial mechanobiology in the development of retinal microvascular lesions in early DR. Success in this pursuit, how...

Retinal capillaries are essential for maintaining retinal homeostasis and visual function. Indeed, their degeneration in early diabetes is strongly implicated in the development of vision-threatening complications of diabetic retinopathy (DR), a microvascular condition that affects nearly 40% of all individuals with diabetes1. Vascular inflammation contributes significantly to retinal capillary degeneration in DR. Past studies have demonstrated an important role for aberrant molecular and biochemical cues in diabetes-induced retinal vascular inflammation2,3. However, recent work has introduced a new paradigm for DR pathogenesis that identifies retinal capillary stiffening as a crucial yet previously unrecognized determinant of retinal vascular inflammation and degeneration4,5,6.
Specifically, the diabetes-induced increase in retinal capillary stiffness is caused by the upregulation of collagen crosslinking enzyme lysyl oxidase (LOX) in retinal endothelial cells (ECs), which stiffens the subendothelial matrix (basement membrane)4,5,6. Matrix stiffening, in turn, stiffens the overlying retinal ECs (due to mechanical reciprocity), thus leading to the overall increase in retinal capillary stiffness4. Crucially, this diabetes-induced retinal capillary stiffening alone can promote retinal EC activation and inflammation-mediated EC death. This mechanical regulation of retinal EC defects can be attributed to altered endothelial mechanotransduction, the process by which mechanical cues are converted into biochemical signals to produce a biological response7,8,9. Importantly, altered EC&#160;mechanical cues and subendothelial matrix structure have also been implicated in choroidal vascular degeneration associated with early age-related macular degeneration (AMD)10,11,12, which attests to the broader implications of vascular mechanobiology in degenerative retinal diseases.
Notably, retinal capillary stiffening occurs early on in diabetes, which coincides with the onset of retinal inflammation. Thus, the increase in retinal capillary stiffness may serve as both a therapeutic target and an early diagnostic marker for DR. To this end, it is important to obtain reliable and direct stiffness measurements of retinal capillaries and subendothelial matrix. This can be achieved by using an atomic force microscope (AFM), which offers a unique, sensitive, accurate, and reliable technique to directly measure the stiffness of cells, extracellular matrix, and tissues13. An AFM applies minute (nanoNewton-level) indentation force on the sample whose stiffness determines the extent to which the indenting AFM cantilever bends- the stiffer the sample, the more the cantilever bends, and vice versa. We have used AFM extensively to measure the stiffness of cultured endothelial cells, subendothelial matrices, and isolated mouse retinal capillaries4,5,6,11,12. These AFM stiffness measurements have helped identify endothelial mechanobiology as a key determinant of DR and AMD pathogenesis. To help broaden the scope of mechanobiology in vision research, here we provide a step-by-step guide on the use of AFM for stiffness measurements of isolated mouse retinal capillaries and subendothelial matrix.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Retinal Capillary Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m PVDF syringe filter</td>
			<td>Merck Millipore</td>
			<td>SLGVM33RS</td>
			<td>Low Protein Binding Durapore</td>
		</tr>
		<tr>
			<td>10X Dulbecco&#39;s Phosphate Buffered Saline without calcium % magnesium</td>
			<td>Corning</td>
			<td>20-031-CV</td>
			<td>Final concentration 1X, pH 7.4</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Falcon Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td>Rnase-Dnase-free, Nonpyrogenic</td>
		</tr>
		<tr>
			<td>20 mL Luer-Lok TIP syringe</td>
			<td>BD</td>
			<td>302830</td>
			<td></td>
		</tr>
		<tr>
			<td>5 3/4 inch Disposable Borosilicate Glass/Non-sterile Pasteur pipette</td>
			<td>FisherBrand</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>60x15 mm Tissue Culture Dish</td>
			<td>Falcon Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Falcon Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua-Hold 2 Pap - 13 mL Pen</td>
			<td>Scientific Device Laboratory</td>
			<td>9804-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade holder</td>
			<td>X-ACTO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Steel Surgical Blade #10</td>
			<td>Bard-Parker</td>
			<td>371110</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Wax</td>
			<td>Electron Microscopy Sciences</td>
			<td>50-949-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Am-scope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Millipore Sigma</td>
			<td>HT501128-4L</td>
			<td>Final concentration 5% (v/v)</td>
		</tr>
		<tr>
			<td>Kimwipes - wiper tissue</td>
			<td>Kimtech Science</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro spatula</td>
			<td>Fine Science Tools</td>
			<td>10089-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Lab Genius</td>
			<td>SK-O180</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO Economy #7 Stainless Steel 115mm&#160; Tweezer</td>
			<td>Ted Pella, Inc.</td>
			<td>5667</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>Nikon TS2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purifier Logic+ Class II, Type A2 Biosafety Cabinet</td>
			<td>Labconco</td>
			<td>302380001</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock microcentrifuge tubes 2 mL</td>
			<td>Eppendorf</td>
			<td>22363352</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>AmScope</td>
			<td>SM-3 Series Zoom Trinocular Stereomicroscope 3.5X-90X</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscopy slide - White tab - Pre-cleaned - 25x75x1.0 mm</td>
			<td>FisherBrand</td>
			<td>1255015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffer, 0.1M solution, pH 7.4 - Biotechnology Grade</td>
			<td>VWR</td>
			<td>E553-500ML</td>
			<td>pH 8 for trypsin solution</td>
		</tr>
		<tr>
			<td>Trypsin 1:250 powder Tissue Culture Grade</td>
			<td>VWR</td>
			<td>VWRV0458-25G</td>
			<td>10 % (w/v) trypsin solution</td>
		</tr>
		<tr>
			<td>Water Molecular Biology Grade</td>
			<td>Corning</td>
			<td>46-000-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Subendothelial Matrix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Corning</td>
			<td>20-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS with calcium and magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040-117</td>
			<td>pH 7.4</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>338818</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV antibody</td>
			<td>Novus Biologicals</td>
			<td>NBP1-26549</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>398136</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>Invitrogen-Thermo Fisher Scientific</td>
			<td>00-4958-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips (12mm)</td>
			<td>Fisher</td>
			<td>12-541-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Electron microscopy Sciences</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>Human retinal endothelial cells (HREC)</td>
			<td>Cell Systems Corp</td>
			<td>ACBRI 181</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB131 medium</td>
			<td>Corning</td>
			<td>15-100-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse retinal endothelial cells (mREC)</td>
			<td>Cell Biologics</td>
			<td>C57-6065</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco-Thermo Fisher Scientific</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM Measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 &#181;m Probe</td>
			<td>Bruker</td>
			<td>SAA-SPH-1UM</td>
			<td>A 19 micron tall hemispherical probe with 1 
			micron end radius, Spring constant 0.25N/m</td>
		</tr>
		<tr>
			<td>70 nm LC probe</td>
			<td>Bruker</td>
			<td>PFQNM-LC-V2</td>
			<td>A 19 micron tall hemispherical probe with 70nm end radius, 
			&#160;Spring constant 0.1N/m</td>
		</tr>
		<tr>
			<td>&#160;camera</td>
			<td>XCAM family</td>
			<td>Toupcam</td>
			<td>1080P HDMI</td>
		</tr>
		<tr>
			<td>Desktop to run the camera</td>
			<td>Asus</td>
			<td>Asus desktop</td>
			<td>Intel i5-6600 CPU , 8GB RAM</td>
		</tr>
		<tr>
			<td>Dish holder for coverslip</td>
			<td>Cellvis</td>
			<td>D29-14-1.5-N</td>
			<td>29mm glass bottom dish with 
			&#160;14 mm micro-well</td>
		</tr>
		<tr>
			<td>Nanowizard 4</td>
			<td>Bruker</td>
			<td>Nanowizard 4</td>
			<td>Bioscience atomic force microscope mounted on an optical microscope for sensitive measurement of the mechanostructural properties (stiffness and topography) of soft biological samples</td>
		</tr>
		<tr>
			<td>Phase contrast micrscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200</td>
			<td>Inverted microscope with 10X objective</td>
		</tr>
	</tbody>
</table>

isolation of mouse retinal capillaries and subendothelial matrix for stiffness measurement using atomic force microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

Author Spotlight: Investigating Vascular Stiffness and Inflammation in Early Diabetic Retinopathy

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy, which is a major vision-threatening microvascular complication of diabetes. Our lab is investigating whether and how mechanical cues in the form of vascular stiffness can contribute to these abnormal retinal vascular changes in early diabetes. There's a growing interest in developing ways to prevent diabetic retinopathy at the early stage with a particular focus on inhibiting retinal inflammation that occurs early on in diabetes and contributes to retinal neurovascular dysfunction.Our recent work indicates that in addition to genetic and biochemical factors, mechanical cues such as vascular stiffness can promote retinal inflammation in early diabetes. Our protocol will allow researchers to isolate intact retinal vessels and subendothelial matrix for subsequent stiffness measurements using atomic force microscope. These measurements will help determine the role of vascular stiffness and endothelial mechanobiology in the development of vascular defects associated with diabetic retinopathy and macular degeneration.By applying minute nano two picton level interation forces, atomic force microscopy offers a sensitive, accurate, and reliable technique to directly measure the stiffness of soft biological samples such as blood vessels, cells, and accessible metrics. To our knowledge, such gentle measurements are uniquely possible using an atomic force microscope. Our recent work has shown that retinal capillaries become stiffer in diabetes, which leads to retinal vascular inflammation and degeneration.A deeper understanding of the mechanical regulation of the diabetic retinopathy as the potential to identify microbiology based anti-inflammatory targets for more effective therapies in the future.

Mouse retinal capillaries 
AFM stiffness measurement of isolated retinal capillaries involves sample handling steps that could potentially damage their mechanostructural integrity. To prevent this and thereby ensure the feasibility, reliability, and reproducibility of AFM measurements, the enucleated eyes are fixed in 5% formalin overnight at 4 &#176;C prior to vessel isolation. This mild fixation protocol with reduced formalin concentration, low fixation temperature, limited fixation time, and lack...

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that ...

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Research

JoVE Journal

Biology

Isolation of Retinal Capillaries from a Mouse Eye for Atomic Force Microscope Stiffness Measurement

To begin, place a formal and fixed mouse eye on a piece of wax paper under a dissecting microscope. With a pair of micro forceps, hold the remnants of the muscle and optic nerve. Then orient the eye so that the cornea faces one side.Now use a surgical blade to make a one to two millimeter long incision behind and parallel to the limbus. Apply a downward force with the blade while holding the eye in place until the anterior segment separates from the posterior end. Then discard the anterior segment and lens.Transfer the posterior segment into a six centimeter dish filled with PBS. Use a micros spatula to scoop out the retina while simultaneously holding the optic nerve with micro forceps. To rinse the retina, transfer it into a well of a 12-well plate that has six wells filled with double distilled water.Use a P1000 pipette to carefully pipette water up and down adjacent to the retina while blowing the water to cause gentle agitation. Next, use an inverted glass pipette to transfer the retina to the second well. To digest the retina, transfer the rinsed retina into trypsin solution in a 12-well plate with an inverted glass pipette.Center a trypsin-soaked P200 pipette tip on the optic nerve. Then gently pipette the entire vascular network up and down to dissociate the nonvascular tissue. Now, use an inverted glass pipette pre rinsed in trypsin to transfer the retinal vasculature into a well containing double distilled water in a six-well plate.Swirl the plate, then pipette the vasculature up and down with an inverted trypsin-rinsed glass pipette to remove any residual nonvascular tissue. Carefully transfer the retinal vasculature to the center of the marked region on a charged surface microscopy slide. Then use a P200 pipette to carefully remove the excess water from one edge.Place the slide in a biosafety cabinet close to the front side to let the residual water evaporate. When the vasculature is almost dry, transfer it under a phase contrast microscope. Use a P200 pipette to slowly add double distilled water on one side of the marked region to carefully rehydrate the vasculature.Fixation of the isolated retinal vasculature resulted in structurally robust and sufficiently durable tissue suitable for AFM measurements. In contrast, the retinal vessels isolated from unfixed or briefly fixed eyes became fragmented and collapsed.

Collection of Subendothelial Matrix from Retinal Microvascular Endothelial Cell (REC) Cultures for Atomic Force Microscope (AFM) Stiffness Measurement

To begin, rinse glutaraldehyde cross-linked gelatin coated cover slips in PBS containing calcium magnesium about five times on an orbital shaker. During the first three rinses, lift the cover slips using a sterile tweezer to allow thorough rinsing of the glutaraldehyde. Next, replace the culture medium of mouse RECs with fresh medium supplemented with sterile, filtered ascorbic acid.After 15 days of treatment, rinse the cells with calcium magnesium free PBS. Incubate the cells in 250 microliters of warm decellularization buffer for two to three minutes. Confirm the decellularization of the cells under a phase contrast microscope.Gently pipette out the buffer without disturbing the REC secreted subendothelial matrix. Then add 0.5 milliliters of PBS into each well. After removing the PBS, add 200 microliters of RNase free DNase one.Incubate the mixture at 37 degrees Celsius for 30 minutes to remove all cellular debris. Next, view the Macroscale fibrous subendothelial matrix under a phase contrast microscope. Then use a confocal microscope to view the finer nanoscale matrix fibers at 100x magnification.Subendothelial matrix aggregates were observed on the glass cover slips following decellularization of 10 day or 15 day cultures of human or mouse RECs. Confocal microscopy of the decellularized matrices showed a dense nano fibrillar collagen-IV and fibronectin matrix.

Stiffness Measurements of Retinal Capillaries and Subendothelial Matrix Using Atomic Force Microscopy (AFM)

To begin, fix the cantilever holder of an AFM onto the cantilever changing stand. Use watchmaker forceps to mount the selected cantilever on the holder. Tighten the screw on the holder to secure the cantilever.Place and lock the cantilever holder on the AFM head that is resting on the stand. Use the step motor function in the AFM software to withdraw the AFM head to the highest point. and set that position as 0.0.Carefully lift the AFM head from its stand, then mount it on the sample stage by placing the legs in their respective slots. To calibrate the cantilever, use the step motor function to bring it close to the sample surface. Then click on Approach in the Contact Mode for Spectroscopy window.Bring down the cantilever in small increments of 15 micrometers. Press Acquire to capture a force curve. Then select the force curve, open it in the Calibration Manager window and select Contact-Based Mode in the Method section.Now use the select fit range function and select the cantilever retraction curve for a linear curve fit. Next, check the Sensitivity check box to convert the force unit from volt to Newton. Lift the cantilever by 100 to 200 micrometers in the liquid and select the thermal noise function Again, click on Select Fit Range and fit the thermal noise bell curve with a Lawrence Curve.After curve fitting, choose the Spring Constant K box. Confirm that the spring constant is close to the manufacturer's value and note it down for future reference. Next place the slide mounted sample of rodent eye retinal vessels or subendothelial matrix on the AFM stage and visualize the retinal vessels.Select Contact Mode for Spectroscopy in the software's Experiment section. Then set the set point force to 0.5 nano-Newtons and click Approach. Use the stage screws on the AFM stage to carefully position the cantilever probe at a desired location on the sample.Click Approach to position the cantilever closer to the sample, and then click Acquire to capture the force curves. Finally, save all force curves for analysis. The approach and retraction curves obtained from a retinal capillary isolated from a diabetic mouse were steeper than those obtained from a non-diabetic mouse.

375 Views.  University of California, Los Angeles.  We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

Measurement of Stiffness in Mouse Retinal Capillaries and Subendothelial Matrix

Summary