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Published in final edited form as: Microvasc Res. 2024 Mar 21;154:104682. doi: 10.1016/j.mvr.2024.104682

Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature

Anamika Sharma 1,2, Dhiraj Kumar Gupta 1,2, Shivantika Bisen 1,2, Nikhlesh K Singh 1,2,*
PMCID: PMC11180566  NIHMSID: NIHMS1980483  PMID: 38521153

Abstract

Dysfunctional pericytes and disruption of adherens or tight junctions are related to many microvascular diseases, including diabetic retinopathy. In this context, visualizing retinal vascular architecture becomes essential for understanding retinal vascular disease pathophysiology. Although flat mounts provide a demonstration of the retinal blood vasculature, they often lack a clear view of microaneurysms and capillary architecture. Trypsin and elastase digestion are the two techniques for isolating retinal vasculatures in rats, mice, and other animal models. Our observations in the present study reveal that trypsin digestion impacts the association between pericytes and endothelial cells. In contrast, elastase digestion effectively preserves these features in the blood vessels. Furthermore, trypsin digestion disrupts endothelial adherens and tight junctions that elastase digestion does not. Therefore, elastase digestion emerges as a superior technique for isolating retinal vessels, which can be utilized to collect reliable and consistent data to comprehend the pathophysiology of disorders involving microvascular structures.

Keywords: Trypsin digestion, elastase digestion, retinal vasculature, pericytes, tight junction, adherens junction

1. Introduction

The International Diabetes Federation (IDF) predicts that around 463 million people have diabetes in 2019, and by 2045, that figure is predicted to climb to 745 million, demonstrating a concerning trend of rising prevalence (1). Globally, diabetic retinopathy (DR), a consequence of diabetes that damages the retina’s blood vessels, is the leading cause of vision loss and blindness (2). Diabetic retinopathy is classified into two stages based on vascular changes: early non-proliferative (NPDR), which includes progressive capillary blockage and degeneration, and proliferative diabetic retinopathy (PDR). Retinal microvascular abnormalities, including pericyte loss, thickening of the foundation membrane, formation of microaneurysms, and intraretinal microvascular abnormalities, are characteristics of the NPDR (3). PDR is the more advanced stage, marked by the neovascularization (growth of new blood vessels) on the retina’s surface. Consequently, tractional retinal detachment and fibrovascular alterations occur, resulting in blindness (4). A suitable animal model is needed to understand both these mechanisms and the genetic factors contributing to DR. Thus far, the disease’s advanced neovascular phase has not been developed in animal models of diabetic retinopathy (5,6). However, animal models are available to investigate the earlier stage of the retinopathy.

In order to analyze the mechanisms of diabetic retinopathy, it is necessary to visualize the intact vascular pattern and the intricate structure of the retinal blood vessels, which allows one to evaluate the vascular abnormalities, such as pericyte loss, microaneurysms, and capillary degeneration associated with DR (7,8). The introduction of retinal whole mount procedures helps preserve the vasculature and reveal capillary microaneurysms in DR; however, the vascular network isolated using digestion techniques provides a clear picture of capillary degeneration, allows for the accurate measurement of endothelial cell and pericyte counts, arteriolar diameters, and the development of acellular capillaries (9). Researchers employed enzymes like trypsin and elastases to digest the nonvascular components of the retina to analyze the vasculature of the retina (10). These enzymes have specific properties that allow for the isolation, manipulation, and analysis of retinal vessels. Trypsin is a protease enzyme that specifically cleaves peptide bonds at the carboxyl side of basic amino acids, such as lysine and arginine (11). Trypsin digestion involves careful removal of the neural tissue and other components while preserving the integrity of the vascular network. This technique allows researchers to study the structure and characteristics of retinal vessels in isolation. Although several researchers have raised technical difficulties with the trypsin digest process, none have offered a thorough and standardized procedure to overcome those difficulties (12–14). As a result, researchers have turned to using elastase as a substitute for trypsin. Laver et al. in 1993 introduced elastase as an alternative protease for the isolation of retinal vessels in the trypsin digest method (15). Elastases are enzymes that breakdown elastin, a protein found in the extracellular matrix of many tissues, including blood vessels (16). Although elastase is the sole pancreatic enzyme capable of digesting elastin, it is only one of numerous substrates for this enzyme. The entire amino acid sequence and three-dimensional structure of elastase revealed that it is highly homologous to other serine proteinases (17). Elastase activity is required for tissue remodeling and maintenance, and the same is valid for vessels.

The current study compares the original trypsin digest approach to the elastase method for separating and preparing the retinal vasculature to evaluate microvascular complications resulting due to retinal vascular disorders. We looked at the effects of trypsin and elastase digestion techniques on endothelial cell adherens junction and tight junction integrity. We concluded that the elastase digestion technique for retinal vessel network isolation from murine retina maintains the retinal vascular architecture (i.e., the pericyte to endothelial cell ratio, adherens, and tight junction proteins’ distribution on the plasma membrane) compared to the trypsin digestion technique.

2. Methods

2.1. Reagents used for the experiments.

Anti-ZO1 (8193, dilution 1:100) antibodies were purchased from Cell signaling technologies (Beverly, MA). Anti-VE-Cadherin (SC-9989, dilution 1:100) antibodies were purchased from Santa Cruz Biotechnology (Dalla, Texas). Goat anti-rabbit Alexa Fluor 488 antibodies (A11008, dilution 1:250), goat anti-mouse Alexa Fluor 488 antibodies (A10667, dilution 1:250), 4% paraformaldehyde (J61899.AK), DPBS (14190-144), Isolectin GS-IB4 (121413), prolong gold antifade mounting medium (P36930), and 2.5% trypsin (15090-046) were purchased from ThermoFisher Scientific (Waltham, MA). Anti-NG2, Alexa Fluor 488 Conjugate Antibody (AB5320A4, dilution 1:100), and elastase (324682) were purchased from Sigma Aldrich. Histoprep (HC8001Gal), Permount (SP15-100), Safeclear (314-629), Slides (12-544-2), and Coverslip (12542B) were purchased from Fisher Scientific (Hampton, NH). Hematoxylin and Eosin Stain Kit (H-3502), and DAPI (H3570) were purchased from Vector Laboratories (Burlington, Ontario, Canada).

2.2. Mice.

The C57BL/6 mice were provided by Charles River Laboratories (Wilmington, MA, USA). The mice were grown in a 12-hour light/12-hour dark cycle and given unlimited access to food and drink. The animals were housed in the DLAR facility at Wayne State University in Detroit, Michigan’s DLAR animal facility. Male and female mice, ages 4–6 weeks, were employed in this study. Animals from the same litter were used to compare trypsin and elastase digestion methods. The Wayne State University’s Institutional Animal Care and Use Committee (IACUC), Detroit, Michigan approved every animal experiment.

2.3. Elastase digestion technique.

For isolation of retinal vascular network, we used the methods described by Veenstra et al (18) with some modifications. Briefly, mouse was euthanized, and eyes were enucleated followed by fixing it in 4% PFA for at least 1 week. The anterior half of the eye was cut off and discarded, and retina was isolated keeping the optic nerve head attached to the retina. Excess fixative is removed by rinsing the retina overnight in water and incubated the retina with elastase solution at 37°C for at least two hours. Even though the enzyme is inactive in these circumstances, it will seep into the tissue. A drop or two of the elastase solution [40 U/ml of elastase (#324682, Sigma Aldrich) in 100 mM sodium phosphate buffer (pH 6.5), with 150 mM sodium chloride and 5mM EDTA] is transferred with the retina, and it is then incubated at room temperature for at least 12 hours with 1 mL or more of the activating solution (Tris buffer pH 8.5). Elastase functions best at a pH of 8.5. The retina is then moved to a shallow plate, like the top of a petri dish filled with deionized water free of lint. The single hairbrushes were used to extract neuronal and glial tissue from the vascular tree under a dissecting microscope with side illumination. Remaining retina is taken out into a new dish with deionized water, and cleaned gently till the blood vessel network is visible and then transferred to a fresh dish of clean water. Then it is picked up with the help of inverted Pasteur Pipette and put it on the lint free clean slide. The slide is kept as such overnight for air dry so that vasculature sticks to the slide.

2.4. Trypsin digestion technique.

For isolation of retinal vascular network, we used the methods described by Veenstra et al (18) with some modifications. Briefly, retinas were fixed in 4% PFA, kept overnight under running water, and then it was kept in deionized water for shaking up to 4–6 hours to loosen the neural tissue from the vascular bed. The retina was then placed in trypsin solution which was tried at different concentrations ranging from 0.5% to 2.5% trypsin solution (#15090046, ThermoFisher Scientific, Waltham, MA) at 37°C in hot air oven at gentle shaking for overnight. Next day, the retina was cleaned under dissecting microscope. Though, tissue cleaning was not easy in this method like with elastase method.

2.5. Hematoxylin and Eosin (H&E) staining.

The H&E staining was performed using Hematoxylin and Eosin Stain Kit (H-3502) following manufacturer guidelines. Briefly, the slides with retinal vessels were washed with distilled water and stained with hematoxylin for 2–3 minutes. Thereafter, the slides were washed, immersed in the Bluing reagent for 15 sec, and then incubated with Eosin Y solution for 1 minute. The slides were then dehydrated, cleared with 2 Changes of Safeclear and slides were covered with a glass coverslip using Permount mounting medium.

2.6. Immunofluorescence staining.

The slides were permeabilized for 15 minutes with 0.25% Triton x100 before being probed with anti-NG2, anti-ZO1, or anti-VE-Cadherin antibodies in conjunction with isolectin B4 overnight at 4°C. The following day, the slides were washed and stained with Alexa Fluor 488-conjugated secondary antibodies for one hour. The slides were cleaned, stained with DAPI, and covered with a glass coverslip using glass fade mounting medium. The slides were examined under a Zeiss LSM800 confocal microscope, and images were acquired with the image analysis software Zen (Carl Zeiss Imaging Solutions GmbH).

2.7. Quantification of retinal pericytes and pericytes to endothelial ratio.

To quantify retinal pericytes, at least 18 photos of elastase and trypsin digests were collected (n=9 animals, 2 images per animal). The retinal vasculature near the optic nerve and the exterior boundary were excluded from the analysis. As previously reported, ECs and pericytes were classified based on size and shape. NIH ImageJ software was used to calculate the total number of pericytes and their ratio to endothelial cells in a 0.1 milimeter square area of the retina.

2.8. Statistical Analysis

To compare elastase and 2.5% trypsin digest techniques, the student’s t-test (GraphPad Prism 10; GraphPad Software, CA, USA) was used. The data is presented as Mean ± SD. P<0.05 is considered as significant.

3. Results

3.1. Elastase digestion was more effective than trypsin digestion in restoring retinal vascular architecture.

Vascular abnormalities increase with the frequency and severity of diabetes. In diabetic rodent models, retinal vascular abnormalities, and capillary loss, are assessed using elastase and trypsin digestion methods of retinal vessel isolation and histologic analysis. During our study, we obtained a flat mount of the whole murine retinal vasculature network and stained it with hematoxylin (H&E). The resulting flat mount of the mouse retinal vasculature obtained by the elastase digestion technique provides a detailed description of the vascular architecture, endothelial cells, pericytes, and associated structures, as well as the differentiation of endothelial cells and pericytes comprehensively (Fig. 1A). While the architecture was not vivid when isolated using the trypsin digestion procedure (Fig. 1B). In vessels isolated using the trypsin digestion procedure, the interaction between pericytes and endothelial cells is disrupted, and very few associations between endothelial cells and pericytes are visible (Fig. 1C). We also observed a significant decrease in number of retinal pericytes in retinal vasculature isolated using trypsin digestion technique (Fig. 1D). To further strengthen our observations that trypsin digestion disrupts endothelial and pericyte interactions, we stained the vessels with neural/glial antigen 2 (NG2), and isolectin B4. Isolectin B4 is an endothelial cell marker and NG2 is a membrane proteoglycan present in the plasma membrane of pericytes (18). It has also been observed that NG2 directly mediates communication between vascular endothelial cells and pericytes (19). The association between endothelial cells and pericytes were found to be intact in the vascular architecture isolated using elastases digestion technique (Fig. 2A). However, this association was disturbed in the vessels network isolated by the trypsin digestion technique (Fig. 2B). Trypsin digestion has proteolytically damaged the plasma membrane of pericytes within the artery walls, as seen by the intracellular staining of NG2.

Figure 1: Retinal Vascular network isolation with 2.5% trypsin and elastase digestion.

Figure 1:

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A, image showing the retinal vascular architecture isolated using elastase. B, Image displaying the retinal vascular architecture isolated using 2.5% trypsin. The right-hand column images are the higher magnification of the area selected. Red arrows show pericytes with round shapes and dark staining. Black arrows indicate endothelial cells with an oval shape and lighter staining. The bar graphs show the quantitative analysis of retinal images from 9 animals (n=9), expressed as Mean ± SD. Scale bar represents 50 μm in panel A & B.

Figure 2: The association of pericytes with ECs is preserved in retinal vessels isolated by elastase digestion.

Figure 2:

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A, image showing colocalization of NG2 with isolectin B4 in retinal vessels isolated using elastase. B, Image demonstrating disruption of NG2 association with isolectin B4 in retinal vessels isolated using 2.5% trypsin. Red arrows show pericytes. n = 6 eyes per group. Scale bar represents 20 μm in panel A and B upper row, and 2 μm in panel A and B lower row.

3.2. Vessels isolated using the trypsin digestion technique lacks intact endothelial adherens junctions within the vasculature.

Diabetes contributes to the breakdown of the blood-retinal barrier (BRB) through proteolytic degradation of vascular endothelial (VE)-cadherin. (20). VE-cadherin is a fundamental component of adherens junctions and regulates a range of endothelial cell functions such as morphogenesis, growth factor response, motility, and cell survival (21). The mouse retinal vasculature obtained by the elastase and trypsin digestion was stained with isolectin B4 and VE-cadherin. We observed an intact VE-cadherin on the plasma membrane of endothelial cells in the vessels isolated using elastase (Fig. 3A), suggesting that there is no modulation to endothelial adhesion strength and adherens junction stability. However, we observed VE-cadherin endocytosis in the vessels network isolated by the trypsin digestion technique (Fig. 3B), suggesting increased plasma membrane permeability and disrupted adherens junction.

Figure 3: Retinal vessels isolated with elastase digestion have intact adherens junctions.

Figure 3:

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A, the image shows VE-cadherin colocalization with isolectin B4 in retinal vessels isolated using elastase. B, image demonstrating disruption in VE-cadherin colocalization of with isolectin B4 in retinal vessels isolated with 2.5% trypsin. n = 6 eyes per group. Scale bar represents 20 μm in panel A and B upper row, and 2 μm in panel A and B lower row.

3.3. Trypsin digestion breaks down the endothelial tight junction connections within the vasculature.

The inner BRB made by the blood vessels of the retina control the flow of fluid and solutes in the retina through a well-developed tight junction. Claudins, occludin, and Zonula occludens-1 (ZO-1) are the most studied proteins associated with tight junctions. Therefore, we stained the vessels with isolectin B4 and ZO-1 to observe the effect of elastase and trypsin digestion on endothelial cell tight junctions’ disruption. We observed intact VE-cadherin on the plasma membrane of endothelial cells in the vessels isolated using elastase (Fig. 4A), whereas intracellular VE-cadherin staining was observed in trypsin-digested vasculature (Fig. 4B).

Figure 4: Retinal vessels isolated using elastase digestion showed intact tight junctions.

Figure 4:

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A, the image shows ZO-1 colocalization with isolectin B4 in retinal vessels isolated using elastase. B, Image demonstrating disruption in ZO-1 colocalization with isolectin B4 in retinal vessels isolated using 2.5% trypsin solution. n = 6 eyes per group. Scale bar represents 20 μm in panel A and B upper row, and 2 μm in panel A and B lower row.

3.4. Lowering the trypsin concentration is not effective in vasculature isolation from the retina.

A disrupted interaction between vascular endothelial cells and pericytes was seen, along with disruption to the tight and adherens junctions of vascular ECs extracted using the 2.5% trypsin digestion procedure. These findings led us to infer that lowering trypsin concentrations could aid in the restoration of junctional integrity and pericyte interaction. As a result, we attempted to isolate the retinal vessels with 0.5% and 1% trypsin solutions. We discovered that 0.5% and 1% trypsin solutions could not digest the retina, and we could not get a flat mount of the entire retinal vasculature network (Fig. 5A & B). As a result, 0.5% and 1% trypsin concentrations were insufficient for clearly isolating the entire retinal vascular network.

Figure 5: Retinal digestion with 0.5% or 1% trypsin is ineffective in vasculature isolation.

Figure 5:

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A & B, image of undigested retina using 0.5% and 1% trypsin, respectively. The images in the right-hand column show the selected area at a higher magnification. n = 3 eyes per group. Scale bar represents 750 μm in panel A and B left column, and 150 μm in panel A and B right column.

Discussion

Retinal blood vessels are the primary sources of blood supply in the retina. The cellular structure in retinal vessels is essential to understanding retinal growth, visual processes, disease progression, and treatment (22). Analyzing the retinal vasculature is crucial for understanding several retinal conditions, such as DR and retinopathy of prematurity (ROP). Early detection of vascular anomalies in the retinal vasculature, such as degenerated capillaries, increased vessel diameter, and pericyte loss, is critical since these blood vessel changes can result in irreversible vision loss and blindness (23,24). This study compared and evaluated the trypsin and elastase digestion methods for separating and examining the retinal vasculature from mouse models. We assessed the isolated vessels using both techniques, analyzed for ECs and pericytes association, and investigated the impact of both methods on the tight junction and endothelial proteins. Our findings unequivocally demonstrate that, compared to trypsin digestion, the elastase technique of vessel isolation from the retina is more resilient in preserving the integrity of the vasculature network with intact adherens and tight junction.

The dysregulation of microvasculature contributes to many ocular conditions (25). Endothelial and perivascular cells interact to form the structure of retinal vessels. Endothelial cells line the inside of blood vessels, while perivascular cells, such as pericytes, mural cells, or smooth muscle cells, line the outside of blood vessels (26). The diabetic retinopathy is associated with microangiopathies, such as degeneration of pericytes and proliferation of endothelial cells (27). Therefore, isolation of vascular network with intact pericytes and EC association is important to compare the pathologies of diabetic retinopathy. We investigated the retinal vessels isolated from trypsin and elastase digestion at a cellular level by H&E staining and confocal microscopy imaging. In vessels isolated using the trypsin digestion procedure, the interaction between pericytes and endothelial cells is disrupted, and very few associations between endothelial cells and pericytes are visible. Neural/glial antigen 2 (NG2) is one of the molecular markers that have been utilized to detect perivascular cells in mammals (26). Furthermore, it has been noted that NG2 directly mediates communication between vascular ECs and pericytes (28). We stained the arteries with NG2 and isolectin B4 to corroborate our findings that trypsin digestion alters endothelial and pericyte interactions. In the vascular architecture that was isolated using the elastase digestion procedure, it was discovered that the relationship between endothelial cells and pericytes remained intact. Nevertheless, the trypsin digestion method disrupted this relationship in the isolated vessel network. The intracellular staining of NG2 indicates that the plasma membrane of pericytes within the artery walls has been proteolytically disrupted by trypsin digestion.

The blood-retinal barrier (BRB) integrity has been widely acknowledged as a vital component of visual perception, with disruption resulting in a variety of retinal disorders (29). BRB disruption causes macular edema, which in turn leads to lipid exudates and intraretinal fluid buildup (30). The adherens and tight junction between endothelial cells constitute the inner BRB (31,32). ZO-1 (zonula occludens-1) and VE-cadherin (vascular endothelial cadherin) are two important proteins involved in the formation and maintenance of cell-cell junctions between endothelial cells (33,34). These proteins play crucial roles in regulating endothelial barrier function and maintaining the integrity of blood vessel walls. In this study, we observed that the vasculature isolated using elastase has the presence of VE-cadherin and ZO-1 protein at the junctions of retinal vessels, suggesting the presence of intact adherens and tight junctions. However, the vessel network isolated using the trypsin digestion technique did not show the presence of ZO-1 and VE-Cadherin at cellular junctions, suggesting vascular network isolated using trypsin digestion technique has disrupted adherens, and tight junctions. To isolate the retinal vasculature, researchers have typically utilized commercial-grade powdered trypsin (2.5% to 3.0% w/v solution in 100 mM Tris buffer, pH 7.8). While the trypsin digesting process has proven beneficial and effective, the outcomes have been inconsistent. It is challenging to acquire high-quality retinal vascular mounts from mice, rats, and monkeys using this technique, even in the best circumstances (35–37). These findings infer that the vascular network isolated by the trypsin digestion technique could lead to unambiguous analysis of pathologic changes, which could result in misleading interpretations of experimental results. The earliest vessel lesions of diabetic retinopathy occur in the capillaries of the retina, and isolating the intact vessel networks is essential for understanding the pathologic changes of diabetic retinopathy. Loss of tight junction and adherens junction proteins from retinal digests of experimental animals because of inadequate procedures could bias the data and have profound consequences.

Our observations provide evidence that it’s difficult to isolate an intact and clean vascular bed from mice retinas using 0.5% and 1% trypsin digestion technique. Our experience using 2.5% trypsin digestion technique, although reasonably successful, often resulted in preparations affecting the expression and localization of ZO-1 and VE-cadherin. As mentioned earlier, trypsin digestion can disrupt cell-cell interactions and the integrity of cellular structures, which may have implications for the localization and expression of these proteins. The elastase-based digestion technique has consistently provided mounts of the entire mice retinal vasculature which can be used for meaningful comparisons between control and experimental retinas regarding disease-related changes.

Limitations of the study

The conclusions drawn from this study must be considered within certain constraints. The experiments used trypsin obtained from ThermoFisher Scientific (#15090046) and elastase (#324682) from Sigma-Aldrich. Alternative sources of trypsin and elastase were not assessed. Therefore, the outcomes presented are specific to the trypsin and elastase utilized in our experiments, and results may differ if other sources of these enzymes are employ

Highlights.

  • Visualizing retinal vascular architecture is essential for studying various ocular diseases, and trypsin and elastase digestion are the two widely used techniques for isolating retinal vasculatures.

  • Trypsin digestion technique disrupts the association between pericytes and endothelial cells, while elastase digestion effectively preserves this association.

  • Trypsin digestion disrupts VE-cadherin localization to plasma membrane, which disrupts adherens junctions, whereas elastase maintains adherens junctions integrity.

  • Endothelial tight junctions are disrupted by trypsin digestion, but not by elastase digestion.

Acknowledgements

The present research work was supported by National Institutes of Health grants (EY029709 to NKS), and P30EY04068 (LDH, PI of Core Grant), and an unrestricted grant from Research to Prevent Blindness. The Microscopy, Imaging and Cytometry Resources Core is supported in part by NIH Center grant P30 CA22453 to the Karmanos Cancer Institute and R50 CA251068-01 to Kamiar Moin, Wayne State University.

Footnotes

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Declaration of competing interest

The authors declare no competing interests.

Data availability

Data will be made available on request.

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