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 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.
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 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.
All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal and Care Use Committees (IACUC; protocol number ARC-2020-030) at the University of California, Los Angeles (OLAW institution animal welfare assurance number A3196-01). The following protocol has been performed using retinal capillaries isolated from adult (20-week-old) male C57BL/6J mice weighing ~25 g (diabetic mice) and ~32 g (nondiabetic mice; Jackson Laboratory).
1. Isolation of mouse retinal capillaries for AFM stiffness measurement (Days 1-4)
NOTE: This protocol, reported in a recent study4, details the enucleation and mild fixation of the mouse eye, retinal isolation, and trypsin digestion, and subsequent mounting of the resultant retinal vasculature on microscopy slides for AFM stiffness measurement.
2. Obtaining subendothelial matrix from retinal microvascular endothelial cell (REC) cultures (Days 1-17)
NOTE: This protocol, adapted from Beacham et al.15 and reported in recent studies4,5,6, describes REC culture on modified glass coverslips, followed by decellularization to obtain subendothelial matrix for subsequent AFM stiffness measurement.
3. AFM stiffness measurement
NOTE: This protocol, adapted from a standard AFM user manual and reported in recent studies4,5, details the acquisition and analysis of stiffness data from retinal capillaries and subendothelial matrix using an AFM and data analysis software. Although the steps outlined below are based on a specific model of AFM (see Table of Materials), the underlying principles are generally applicable to all AFM models.
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 °C prior to vessel isolation. This mild fixation protocol with reduced formalin concentration, low fixation temperature, limited fixation time, and lack of corneal puncture was developed to minimize any potential crosslinking/stiffening artifacts caused by chemical fixation. As shown in Figure 2B,C, this relatively mild fixation ensures that the isolated retinal vasculature is structurally robust and sufficiently durable for the AFM measurement. In contrast, retinal vessels isolated from unfixed eyes (using the hypotonic method)4 or briefly fixed eyes (for 8 h) become fragmented or collapse, thereby making them unsuitable for AFM measurement (Figure 2B).
Retinal subendothelial matrix
Vascular stiffness reflects the combined stiffness of vascular cells and the basement membrane (subendothelial matrix)4. Since cells adapt to matrix stiffness by undergoing a similar change in their own stiffness, a process termed mechanical reciprocity9, subendothelial matrix stiffness, becomes an important determinant of the overall vascular stiffness. For matrix stiffness measurement, it is important to obtain a homogeneously dense subendothelial matrix. For human retinal ECs grown in ascorbic acid-supplemented culture medium, this usually takes 10-15 days (Figure 3A)4,5,6. This difference in culture period may arise from lot-to-lot differences in commercially available primary retinal ECs. Further, we generally find that commercially available retinal ECs from C57BL/6 mice deposit a denser matrix when compared with primary human retinal EC culture, thus indicating species-specific differences. As shown in Figure 3A, phase contrast images only provide a gross view of the matrix at a macro scale. However, the finer nano-to-micro-scale fibrillar structure becomes apparent in high-resolution confocal fluorescence images of the matrix immunolabeled with antibodies against matrix structural proteins collagen IV and fibronectin (Figure 3B). It should be noted that these matrix proteins also provide instructive cues to endothelial cells by binding to specific integrin receptors9.
AFM stiffness measurement
The selection of appropriate cantilever stiffness (spring constant, k) and probe dimension is essential for reliable and sensitive measurements. These parameters should be chosen to match the stiffness and dimensions of the biological sample. After the selected cantilever has been mounted on the AFM, the infrared laser beam must be focused on the tip of the cantilever, and the reflected laser spot must be centered on the photodetector. This laser alignment ensures precise and sensitive detection of cantilever deflection and, consequently, stiffness measurement. An AFM stiffness measurement begins with the z-piezo moving the cantilever vertically down toward the sample. There is no cantilever deflection at this time, which produces a flat baseline of the approach curve (Figure 4A). As the cantilever probe contacts and indents the sample, the cantilever bends, causing laser deflection on the photodetector, which is depicted by the vertical deflection of the approach curve. After applying a preset indentation force (setpoint force) on the sample, the cantilever retracts to the starting position (Z target height) away from the sample. The deflected retraction curve is then fitted to the Hertz/Sneddon model to calculate the sample's Young's modulus (stiffness).
From the representative force indentation measurement shown in Figure 4, it is clear that the approach and retraction curves obtained from a retinal capillary isolated from a diabetic mouse (Figure 4B) are substantially steeper than those obtained from a nondiabetic mouse (Figure 4A). The steeper slope of force indentation curves indicates greater cantilever deflection caused by higher sample resistance to force indentation, which reflects higher sample stiffness13. Indeed, subsequent data analysis of multiple force indentation curves revealed that mouse retinal capillaries become significantly stiffer in diabetes4. It should also be noted that contact between the cantilever probe and biological samples often causes nonspecific surface adhesion, which leads to negative cantilever deflection during retraction, as seen from the extension of the retraction curve beyond the baseline (Figure 4B). Further, biological samples like cells, matrix, and blood vessels are viscoelastic by nature and thus may undergo some permanent deformation and/or change in apparent stiffness following force indentation. If so, this will be reflected in the misalignment of approach and retraction curves (hysteresis). Indeed, comparing Figure 4A and Figure 4B confirms the expected trend where force indentation of softer capillaries in nondiabetic mice produces greater hysteresis than that seen in their stiffer counterparts. As previously reported5,6, stiffness (Young's modulus) of subendothelial matrices obtained from retinal EC cultures is also calculated in the aforementioned manner.
Figure 1: Schematic illustration of the incision cut made on a mouse eye for retinal isolation. Using tweezers to hold the optic nerve, a scalpel is used to make a full incision posterior to the limbus to separate the mouse retina from the lens and anterior chamber. The purple dashed line shows the location of the vertical incision. This schematic was drawn using a scientific image and illustration software. Please click here to view a larger version of this figure.
Figure 2: Protocol optimization for isolation of intact mouse retinal vessels for AFM measurement. (A) The stereoscope image shows an intact retina isolated from a mildly fixed mouse eye prior to the capillary isolation steps. Scale bar: 2 mm. (B) Representative phase contrast images at 4x magnification show mouse retinal vessels obtained using the different isolation methods. Comparing the structural integrity and durability of the isolated vessels, trypsin digestion of retinas from enucleated eyes fixed in 5% formalin for 24 h at 4 °C (red box) was found to yield the most suitable retinal vasculature for AFM stiffness measurement. Retinal vessels isolated using this method exhibited a clear vascular network that spread uniformly along the glass surface. Scale bar: 500 µm. (C) High magnification (20x) view of an intact retinal capillary network, similar to that shown in (B), confirms the high structural integrity of capillaries obtained from mildly fixed eyes. Scale bar: 100 µm. This figure has been modified from4. Please click here to view a larger version of this figure.
Figure 3: Decellularized matrices obtained from primary human and mouse REC cultures. (A) Representative phase contrast images at 20x magnification showing subendothelial matrix aggregates on glass coverslips following decellularization of 10-day (10 d) or 15-day (15 d) cultures of human or mouse RECs. Scale bar: 100 µm. (B) Representative high magnification (100x) confocal fluorescence images of decellularized matrices obtained from 15 d human REC cultures and labeled with anti-collagen IV and anti-fibronectin antibodies reveal a dense nanofibrillar collagen IV and fibronectin matrix. Scale bar: 20 µm. Please click here to view a larger version of this figure.
Figure 4: Force distance curves from AFM stiffness measurement of mouse retinal capillaries. Line graphs indicate a representative approach (light color) and retraction (dark color) curve from a single force indentation measurement at one location of a mouse retinal capillary isolated from a (A) nondiabetic or (B) diabetic mouse. The force curves obtained using an SAA-SPH 1 µm radius hemispherical cantilever probe, plot the relationship between cantilever-sample distance (controlled by the z-piezo) and the applied vertical force that causes cantilever deflection. (A) The yellow arrow indicates the z-piezo-driven cantilever approach towards the sample, the white arrow indicates the contact point where the cantilever probe makes contact with the sample, and the green arrow indicates the cantilever deflection up to a setpoint (peak) indentation force (*). (B) Both approach and retraction curves obtained from a retinal capillary isolated from diabetic mice exhibit a markedly steeper slope than those from their nondiabetic counterparts (shown in A), which indicates higher capillary stiffness in diabetic mice. The arrowhead indicates a dip in the retraction curve below the baseline, which reflects the negative deflection of the cantilever probe caused by adhesion between the probe and sample during indentation. Please click here to view a larger version of this figure.
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, however, relies on the accurate measurement of diabetes-induced changes in retinal capillary stiffness. Recent studies have revealed that similar to large vessels, stiffness of retinal capillaries and retinal EC-secreted subendothelial matrix can also be directly, accurately, and reliably measured using AFM4,5,6.
This approach involves the isolation of mouse retinal capillaries from mildly fixed eyes using trypsin digestion. Based on experience, this mild fixation step is necessary as capillaries isolated from unfixed eyes (using the hypotonic method) are fragile and become fragmented, rendering them unsuitable for AFM stiffness measurement4. Conversely, stronger fixation and the ensuing need for extensive trypsin digestion, which has been reported in other retinal trypsin digestion protocol18, may also compromise capillary structural integrity, as judged by the disruption of tight junctions. Thus, the mild capillary fixation and gentle trypsin digestion employed here help reduce stiffness artifacts arising from excessive formalin-induced crosslinking and ensure the structural integrity of the capillaries for reliable AFM stiffness measurement.
As an alternative approach, a recent study reported AFM stiffness measurement of retinal capillaries from lightly fixed retinal flat mounts19. By performing AFM force indentations on retinal capillaries within the intact retina, this approach enables in situ stiffness measurement. However, these capillary stiffness measurements likely include the stiffness of the inner limiting membrane. Further, this approach is restricted to the measurement of only superficial capillaries that are accessible on a retinal flat mount while leaving out the deeper-lying capillary plexus that is specifically affected in early DR20. These issues can be addressed using this approach, which extracts vessels cleanly (devoid of residual retinal tissue) from all retinal layers. We are currently optimizing this protocol to isolate choroidal vessels from animal models of early AMD. That said, we realize that the mild formalin fixation employed in the protocol may cause a crosslinking-associated stiffening artifact in AFM measurements. Therefore, it would be prudent to interpret the vascular stiffness values obtained using this approach more in terms of relative changes in stiffness (between different experimental groups) rather than absolute stiffness.
In contrast to retinal capillaries that require mild fixation, the subendothelial matrix obtained from retinal EC cultures can be assessed without any modification. This is largely due to the robustness of the deposited matrix, which results from a combination of several factors, including the addition of ascorbic acid in culture medium (which enhances collagen synthesis15), chemical modification of glass coverslips that prevents matrix detachment during decellularization, and prolonged duration of culture (10-15 days). Importantly, we have shown that the hyperglycemia-induced increase in subendothelial matrix stiffness in vitro is consistent with the diabetes-induced increase in retinal capillary stiffness seen in vivo4. This is not surprising as an increase in matrix stiffness is expected to increase the stiffness of overlying retinal ECs (due to mechanical reciprocity) that, together, increase the overall capillary stiffness. Indeed, we recently showed that retinal ECs become stiffer when grown under diabetic conditions4, likely via an increase in Rho/ROCK-dependent actin cytoskeletal tension21. These findings also imply that the choroidal EC stiffening seen in the rhesus monkey model of early AMD reflects the increased stiffness of choroidal subendothelial matrix and intact vessels12, an idea that is being tested in ongoing studies. Overall, the fact that stiffness alterations in the unfixed retinal subendothelial matrix and ECs mirror the trend seen in mildly fixed intact retinal vessels testifies to the validity of the mild fixation approach.
Direct stiffness measurement of retinal capillaries from animal models of DR not only helps establish endothelial mechanobiology as a novel therapeutic target for DR management, but it also provides a strong rationale to develop new sensitive imaging techniques for non-invasive assessment of retinal capillary stiffness in DR patients in the future. Such noninvasive techniques could potentially detect subtle changes in blood flow that are expected to arise from capillary stiffening, similar to the changes in arterial pulse wave velocity that are commonly detected in diabetes-induced cardiovascular diseases. AFM stiffness measurements of retinal capillaries isolated from post-mortem human eyes from diabetic donors will provide important proof-of-concept in this regard. Given that retinal capillary stiffness increases early on in the (streptozotocin) mouse model of type 1 diabetes4, successful use of AFM in validating stiffness-measuring imaging modalities may lead to the identification of retinal capillary stiffness as a clinical biomarker for early DR pathogenesis.
The authors have nothing to disclose.
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. 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.
Retinal Capillary Isolation | |||
0.22 µm PVDF syringe filter | Merck Millipore | SLGVM33RS | Low Protein Binding Durapore |
10X Dulbecco's Phosphate Buffered Saline without calcium % magnesium | Corning | 20-031-CV | Final concentration 1X, pH 7.4 |
12-well plate | Falcon Corning | 353043 | |
15 mL centrifuge tube | Corning | 430791 | Rnase-Dnase-free, Nonpyrogenic |
20 mL Luer-Lok TIP syringe | BD | 302830 | |
5 3/4 inch Disposable Borosilicate Glass/Non-sterile Pasteur pipette | FisherBrand | 13-678-20A | |
60×15 mm Tissue Culture Dish | Falcon Corning | 353002 | |
6-well plate | Falcon Corning | 353046 | |
Aqua-Hold 2 Pap – 13 mL Pen | Scientific Device Laboratory | 9804-02 | |
Blade holder | X-ACTO | ||
Carbon Steel Surgical Blade #10 | Bard-Parker | 371110 | |
Dental Wax | Electron Microscopy Sciences | 50-949-027 | |
Dissecting microscope | Am-scope | ||
Formalin solution, neutral buffered, 10% | Millipore Sigma | HT501128-4L | Final concentration 5% (v/v) |
Kimwipes – wiper tissue | Kimtech Science | 34133 | |
Micro spatula | Fine Science Tools | 10089-11 | |
Orbital Shaker | Lab Genius | SK-O180 | |
PELCO Economy #7 Stainless Steel 115mm Tweezer | Ted Pella, Inc. | 5667 | |
Phase contrast microscope | Nikon TS2 | ||
Purifier Logic+ Class II, Type A2 Biosafety Cabinet | Labconco | 302380001 | |
Safe-Lock microcentrifuge tubes 2 mL | Eppendorf | 22363352 | |
Stereoscope | AmScope | SM-3 Series Zoom Trinocular Stereomicroscope 3.5X-90X | |
Superfrost Plus microscopy slide – White tab – Pre-cleaned – 25x75x1.0 mm | FisherBrand | 1255015 | |
Tris Buffer, 0.1M solution, pH 7.4 – Biotechnology Grade | VWR | E553-500ML | pH 8 for trypsin solution |
Trypsin 1:250 powder Tissue Culture Grade | VWR | VWRV0458-25G | 10 % (w/v) trypsin solution |
Water Molecular Biology Grade | Corning | 46-000-CM | |
Subendothelial Matrix | |||
10X PBS | Corning | 20-031-CV | |
1X PBS with calcium and magnesium | Thermo Fisher Scientific | 14040-117 | pH 7.4 |
Ammonium hydroxide | Sigma-Aldrich | 338818 | |
Ascorbic Acid | Sigma-Aldrich | A4034 | |
Collagen IV antibody | Novus Biologicals | NBP1-26549 | |
DNase I | Qiagen | 79254 | |
Ethanolamine | Sigma-Aldrich | 398136 | |
Fibronectin antibody | Sigma-Aldrich | F6140 | |
Fluoromount | Invitrogen-Thermo Fisher Scientific | 00-4958-02 | |
Gelatin | Sigma-Aldrich | G1890 | |
Glass coverslips (12mm) | Fisher | 12-541-000 | |
Glutaraldehyde | Electron microscopy Sciences | 16220 | |
Human retinal endothelial cells (HREC) | Cell Systems Corp | ACBRI 181 | |
MCDB131 medium | Corning | 15-100-CV | |
Mouse retinal endothelial cells (mREC) | Cell Biologics | C57-6065 | |
Triton X-100 | Thermo Fisher Scientific | BP151-100 | |
Trypsin | Gibco-Thermo Fisher Scientific | 25200-056 | |
AFM Measurement | |||
1 µm Probe | Bruker | SAA-SPH-1UM | A 19 micron tall hemispherical probe with 1 micron end radius, Spring constant 0.25N/m |
70 nm LC probe | Bruker | PFQNM-LC-V2 | A 19 micron tall hemispherical probe with 70nm end radius, Spring constant 0.1N/m |
camera | XCAM family | Toupcam | 1080P HDMI |
Desktop to run the camera | Asus | Asus desktop | Intel i5-6600 CPU , 8GB RAM |
Dish holder for coverslip | Cellvis | D29-14-1.5-N | 29mm glass bottom dish with 14 mm micro-well |
Nanowizard 4 | Bruker | Nanowizard 4 | Bioscience atomic force microscope mounted on an optical microscope for sensitive measurement of the mechanostructural properties (stiffness and topography) of soft biological samples |
Phase contrast micrscope | Zeiss | Axiovert 200 | Inverted microscope with 10X objective |