Retinal Digest Preparation: A Method to Study Diabetic Retinopathy

Abstract

Retinal digestion is a commonly used method for studying experimental diabetic retinopathy in animal models. The method allows to assess qualitatively and quantitatively the morphology of the retinal vasculature, including characteristics of endothelial cells and pericytes. The digestion method uses the enzyme trypsin and enables the precise evaluation of venolar and arteriolar diameters, endothelial cell and pericyte numbers, and the formation of acellular capillaries.

1 Introduction

The characteristics of diabetic retinopathy are (1) increased vascular permeability, (2) loss of pericytes, and (3) acellular capillary formation. Pericyte loss begins at 2 months of diabetes and increases (in diabetic rat retina stronger than in mouse retina) with disease duration (1). Persistent hyperglycemia induces the additional loss of endothelial cells which causes capillaries to occlude. The formation of these acellular capillaries is the critical quantitative lesion that represents the most typical alteration in a diabetic retina, and the origin of retinal ischemia. Acellular capillaries start to become increased in specific strains of diabetic rats and mice at 4 months, but are most commonly analyzed at 6 months of disease duration (1–3). However, rodents may differ in their propensity to develop acellular capillaries, most likely because of genetic differences (2).

The technique to display the morphology and morphometry of the retinal vasculature is the retinal digest preparation, by which retinal cells are digested away using trypsin leaving the vasculature behind. Originally, different digest mixtures were used for the digest of rodent retinae, i.e., a combination of pepsin and trypsin (4). Pepsin’s digestion properties are usually faster and stronger compared with trypsin. However, the advantage of pepsin is also the biggest disadvantage as vessels can be overdigested when a combination is used. On the other hand, using a combination of pepsin/trypsin may result in only partially digested retinae when exposure and concentrations are underused. Additionally, companies providing both chemicals have improved purification and activity of enzyme preparations. The retina contains little trypsin-resistant collagen and is therefore very easy to digest (5). We therefore developed a digestion protocol using trypsin only, in particular, for rat and mouse retinae. After culture dish exposure of the retina to the digestion solution, it is transferred to a glass slide and neuroglial cells are eliminated physical forces (dropping distilled water on the retina). The remaining vasculature is visualized using PAS staining, based on the abundance of mucopolysaccharides in the vessel walls of the retina.

2 Materials

Instruments

Stereomicroscope (×1 and ×2 magnification), cold light supply, incubator (37°C), electric pump, flexible infusion tube, culture dishes (35  ×  10 mm and 60  ×  15 mm), 5 mL syringe, 22G and 25G needles, fine forceps (e.g., Dumont No. 3 or No. 5), VANNAS scissors, fine spatula, aspirator, liquid blocker (e.g., Pap Pen), glass cuvettes, uncoated glass slides, and coverslips (Fig. 1).

Fig. 1

Needed instruments for retina isolation. From left to right: Aspirator, spatula, scissors, two forceps.

2.1 Fixation

Caution! Formalin is toxic and it is necessary to work with a hood! 4% Formalin (phosphate buffered saline (PBS)): fill 100 mL PBS 10× (from company) in a 1 L graduated glass cylinder, add 100 mL of 37% formalin and fill up to 1 L with Aqua bidest.

The fixation solution can be stored at room temperature in a glass bottle indefinitely.

2.2 Retina Isolation

For the isolation of the retina, PBS 1× is needed. Dilute 10× PBS 1:10 with Aqua bidest.

2.3 Retina Digestion

• 1. Aqua bidest.

• 2. 0.2 M Tris–HCl, pH 7.45: dissolve 24.22 g Tris base in 500 mL Aqua bidest. Adjust pH to 7.45 with HCl, then fill up to 1 L with Aqua bidest, and check pH again. The solution can be stored at room temperature (see Note 1).

• 3. 3% Trypsin in 0.2 M Tris–HCl, pH 7.45. Prepare the solution fresh on the day of digestion.

2.4 PAS Staining

• 1. Aqua bidest.

• 2. Schiff’s fuchsine-sulfite reagent (ready-to-use). Repetitive use (up to three times) possible.

• 3. 1% Periodic acid: for 200 mL final reagent, dissolve 2 g periodic acid in 20 mL Aqua bidest and fill up to 200 mL with 96% ethanol (see Note 2).

  The solution is stored at 4°C and is used up to three times.

• 4. Mayer’s hemalum solution 1:2 diluted with Aqua bidest. Store the solution at 4°C and filtrate (folded filters) it before use. Solution is used three to five times.

• 5. 70, 80, 96, and 100% ethanol.

• 6. Xylene.

• 7. Mounting medium based on xylene, e.g., Eukitt^® or Depex^®.

3 Methods

3.1 Fixation

After enucleation, eyes are fixed in 4% formalin for a minimum of 2 days at room temperature (see Note 3).

3.2 Retina Isolation

• 1. Wash the eye once in 1× PBS, and place the eye in a 35 mm culture dish under a stereomicroscope. The bulb should be covered with 1× PBS to avoid drying out during the retina isolation procedure (see Note 4).

• 2. Fix the eye with the forceps and cut with the scissors along the ora serrata. The cutting line is light gray in mice and rats with dark fur and white in albino mice and rats. It marks the border between retina and ciliary body (Fig. 2) (see Note 5).

• 3. Remove the cornea and the ciliary body. Occasionally, the lens will adhere to the eye cup. Remove the lens carefully using forceps, and allow the vitreous to become removed (see Note 6). Remaining parts of the ciliary body at the edge of the eye cup ought to be removed because retina may adhere and could be destroyed during subsequent preparations (Fig. 3).

• 4. Insert the small spatula between pigment epithelium and retina and split carefully around the eye cup, until the entire retina is removable. Dissect retina from optic nerve using the spatula as a scalpel. Caution! The retina is fragile! (Fig. 4) (see Note 7).

• 5. The retina is immersed in 4% formalin until digestion. From this step onward, use the aspirator to avoid rupture of the retina (Fig. 5) (see Note 8).

Fig. 2

Fixation and incision of the eye ball.

Fig. 3

Parts of the eye at various stages of preparation: (a) rear view of the cornea after dissection from the posterior globe. Upper arrow: iris, lower arrow: ciliary body. (b) Lens and vitreous after dissection. Upper arrow: lens, lower arrow: vitreous/membrane. (c) Posterior globe after dissection. Upper arrow: pigment epithelium, lower arrow: retinal surface.

Fig. 4

Spatula between retinal pigment epithelium (below  ) and retina (above  ).

Fig. 5

How to use the aspirator: aspire the retina for transfer.

3.3 Retinal Digestion

• 1. Fill a 35 mm culture dish with aqua bidest and place the retina using the aspirator. Discard aqua bidest to remove formalin. Measures of precaution to keep the retina in the dish are summarized in Note 9. Refill the dish with aqua bidest. The retina should be entirely covered. Incubate at 37°C for 1 h.

• 2. Substitute the Aqua bidest with 3% Trypsin and put the dish back to 37°C. Trypsin exposure at this step is highly variable due to species, strain, and disease conditions. Usually, rat retina digestion occurs faster than mouse retina digestion.

• 3. Hourly inspection of the digestion progress under the stereomicroscope is mandatory! After 1–2 h, the vitreous/membrane (if still in place) scales off. Careful removal at the optic nerve disc using scissors is essential (see Note 6). At this point, “shamrock” incision is performed for flattening of the retina during on-glass-slide preparation (see Note 10). Incubation at 37°C progresses thereafter.

• 4. While waiting for the right time point, prepare your instruments:

  Connect the infusion tube on one side with a pump, preferably with a glass bottle for waste disposal interconnected, and with a 22G needle for aspiration on the other side. Fill fresh aqua bidest in a beaker, charge the 5 mL syringe, and attach the 25G needle.

  Needles are reshaped for better handling (Fig. 7).

• 5. The adequate time point for transfer from the digestion bath to the glass slide is given, when you observe the following changes:

  • (a) The cell composition of the retina changes and tissue fragments can be located at the bottom of the dish.

    or

  • (b) The retina is flattening (see Note 11).

• 6. Prepare the glass slide by applying two barrier lines with the PAP pen on each short side and place the slide on a 60 mm culture dish under the stereomicroscope (Fig. 6) (see Note 12).

• 7. Cover the slide with aqua bidest using the syringe, transfer the retina to the slide with an upside down orientation using the aspirator (photoreceptor layer up, ganglion cell layer down) (see Note 13).

• 8. Clear the vasculature from the cells through dropping aqua bidest with the syringe on the retina, while eliminating the disintegrating neuroglial cells away at the same time through water aspiration (see Note 14). This step must be carefully monitored under the stereomicroscope (Fig. 7).

  The first layer that disintegrates is the photoreceptor layer. Usually, it can be removed in large fragments (Fig. 8a). Other retinal layers cells disintegrate (Fig. 8b–d) (see Note 15).

  With progressing digestion, the retina becomes adhesive to the glass slide and to dissecting instruments. Thus, it is strongly recommended to avoid contact of the retinal digests with forceps or needles! Any other material such as dust or hair also easily contaminates the preparations.

• 9. Wash digests repetitively! Remaining cell(s) aggregates may reattach to the isolated vasculature and cause artifacts during the drying process, even when distant to the sample.

  Sometimes, parts of the retina resist to digestions (Figs. 8d and 9c) In this case, leave the undigested parts as injury or destruction of the retinal digest may occur.

• 10. Eliminate as much solution as possible by aspiration and air-dry the vasculature while carefully monitoring the specimen under a normal microscope. The four leaves must be completely spread (they should not fold) (Fig. 9a, b) (see Note 16).

Fig. 6

(a) Arrows: PAP pen marks. (b) Equipment: glass slide on 60 mm culture dish.

Fig. 7

Right side  : syringe for dropping. Left side  : needle on infusion tube for sucking.

Fig. 8

(a) Photoreceptor layer disappear. (b, c) Smaller parts leaving the vasculature. (d) Cleared vasculature with one small indigestible part (top left  ).

Fig. 9

Risk of producing artifacts (a) Normal leave. (b) Folded leave. (c) Leave with artifacts.

3.4 PAS Staining

Fixation following the digestion procedure is unnecessary. Perform staining using glass cuvettes.

• 1. 1% Periodic acid: 15 min.

• 2. Wash briefly in Aqua bidest.

• 3. Schiff’s fuchsine-sulfite reagent: 15 min.

• 4. Tap water (no Aqua bidest) until digests turn pink (∼2 min).

  At this step, lukewarm tap water is used instead of flowing water.

• 5. Wash briefly in Aqua bidest.

• 6. Mayer’s hemalum solution: 30 s (fresh solution) to 2 min (often used solution).

• 7. Tap water (no Aqua bidest) until digests turn blue (∼2 min).

  Again, lukewarm tap water is used instead of flowing water.

• 8. Wash briefly in Aqua bidest.

• 9. 70% Ethanol: 1 min.

• 10. 80% Ethanol: 1 min.

• 11. 96% Ethanol: 5 min.

• 12. 100% Ethanol: 5 min.

• 13. Xylene 5 min.

• 14. Xylene 5 min.

• 15. Mounting medium.

3.5 Quantification

We use a microscope and Cell-F software (Olympus Opticals, Hamburg, Germany).

For the quantification of acellular capillaries, we use an integration ocular and count segments of acellular capillaries in ten randomly selected fields within the intermediate circular segment of the retina (see Fig. 10 for localization).

Fig. 10

Area of interest in which acellular capillaries are determined.

The cell numbers are normalized to square millimeter of capillary area (AC/mm² cap. area) (Fig. 11).

Fig. 11

Retinal digest preparation of diabetic rat. Arrows indicate acellular capillaries.

Pericytes and endothelial cells are identified by shape of the nuclei and their localization in relation to the capillaries (Fig. 12) (6).

Fig. 12

Retinal digest preparation of a normal rat retina. Arrows  : Pericytes with round shape and dark staining. Arrowheads: Endothelial cells with oval shape and lighter staining.

Cells are counted in ten randomly selected areas in a circular area of the intermediate third of the retina under ×400 magnification. The cell numbers are calculated relative to the retinal capillary area and expressed as numbers per square millimeter of capillary area (cells/mm² cap. area) (7).

4 Notes

• 1. It is possible to use Tris–HCl instead of Tris base and adjust the pH with NaOH. The solution sometimes forms a precipitate, which can be ignored. Exclude bacterial or fungal contamination!

• 2. The periodic acid must be completely dissolved in Aqua bidest before adding 96% ethanol.

• 3. The minimum fixation time is 48 h. There is no maximum fixation time, since eyes which were stored for months in 4% formalin are still digestible and give good morphological results. Adjusting in the digestion period may be necessary. Eyes frozen in liquid nitrogen and stored at −80°C are transferred to 4% formalin and can be digested after 48 h.

• 4. PBS immersion is only necessary when inexperienced and slow in manipulating.

• 5. Never hold the entire eye between forceps. Inadequate pressure will cause impression of the lens onto the retina and subsequent damage or destruction! Use extraocular structures to hold the eye.

• 6. After fixation, the vitreous appears as a fine membrane. Occasionally, the vitreous is removed together with the lens. If not, there are two alternatives:

  • (a) The vitreous can be removed by soft traction; the risk of loosing the larger vessels is high if traction is too strong. Central adhesion (optic disc) can be released by careful incision. Larger defects increase the risk of total destruction during subsequent manipulation. However, if separation in the periphery between vitreous and retina is unsuccessful, go immediately to step (b).

  • (b) Let the vitreous come off during the digestion procedure (see step 3 in 3.3 digestion).

    In mouse eyes, the lens “falls out” by itself and the vitreous cannot be identified. It is thus impossible to remove it before digestion. In rat eyes, the lens usually adheres to the corneal part and the vitreous can be removed or it adheres to the lens.

    Removal of the vitreous is mandatory because vessel remnants may potentially interfere with the morphometry procedure.

• 7. Residual large pigment epithelium remnants can be removed by careful use of forceps. Small pieces will come off during the digestion procedure.

• 8. Never use forceps to transfer the retina! Always use the aspirator (see Figs. 1 and 5)!

• 9. While removing dispensable solution from the culture dish, make sure that the retina sticks to the rim. For waste disposal, use a beaker, so that the inadvertent misplaced retinae can be rescued with the aspirator.

• 10. Incisions should be limited to two-third of the retina’ radius towards the disc. Otherwise, the risk of destruction is high during the digestion procedure.

• 11. From our experiences rat retinas need 2–6 h and mouse retinas 4–10 h. If there is nothing happening after 12 h, then a digestion is not possible.

• 12. Use 60 mm culture dish as slide carrier to avoid the loss of the retinal sample. When the retina is rinsed on the slide, it occasionally flows off. Rescue is possible if it flows into the culture dish, but impossible if it would be inserted between glass slide and microscope plate.

• 13. If digestion is too extensive at this point, the retina will completely disintegrate.

• 14. Be careful to use gentle drops rather than high flow to avoid holes in the vasculature.

  When using a minipump to remove waste solution around the digest, avoid close contact.

• 15. If only the photoreceptors can be loosened, but no other layers, transfer the retina back to trypsin, incubate it for 1 h and try again. As long as there is no part with “free” vessels, the procedure can be repeated.

• 16. Retinal leaves tend to move towards the retinal center. The vasculature will fold and be insufficiently spread quantitative analysis (e.g., see Fig. 9b). If the vessel net has dried out, correction is no longer possible. If the vessel net is still wet, you can try to spread it again with Aqua bidest.