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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell+and+tissue+development:+implications+for+tissue+engineering.">Micromechanical control of cell and tissue development: implications for tissue engineering.</a> Adv Drug Deliv Rev. 59 (13), 1306-1318 (2007).</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=Aberrant+cell+and+basement+membrane+architecture+contribute+to+sidestream+smoke-induced+choroidal+endothelial+dysfunction.">Aberrant cell and basement membrane architecture contribute to sidestream smoke-induced choroidal endothelial dysfunction.</a> Invest Ophthalmol Vis Sci. 55 (5), 3140-3147 (2014).</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=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

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

388 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.