Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

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Summary

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

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Guo, D., Liu, C. Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry. J. Vis. Exp. (152), e60086, doi:10.3791/60086 (2019).

Abstract

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

Introduction

The ocular surface consists of a continuous sheet of regionalized epithelium including palpebral conjunctiva, bulbar conjunctiva and cornea1. Many glandular structures are associated with the ocular surface epithelium and together generate a layer of tear film protecting the cornea surface from drying and environmental invasions2. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). Both genetic factors and environmental irritants or their interactions contribute to pathological alterations of the OS3,4. Accordingly, a variety of genetically-engineered and physically or chemically-induced animal models have been used for studying disease processes of the human OS.

The structure and function of the mouse OS is similar to that of humans in many ways. The tear film components secreted by the ocular glands are also similar between mice and humans. A wealth of studies has been conducted using mouse models for elucidating mechanisms of human OS diseases5,6,7. In many occasions, it is critical to analyze global instead of local molecular changes of the OS to gain comprehensive information under the same experimental treatment. Therefore, sample preparation with good integrity is needed to ensure each part of the OS to be analyzed simultaneously.

The mouse OS tightly associates with the eyeball that was embedded in the eye socket/orbit (a bony cup made of several different skull bones) and connects to it through thin connective tissues. There exist tremendous challenges for dissecting the whole ocular surface without damaging the palpebral or bulbar conjunctiva. These challenges descend from: (i) the OS is soft and thin and affixed to physically separate yet movable eyelids and eyeball, therefore vulnerable for distortion and damage; and (ii) the limited space between the orbital bones and eyeball restrict the dissection operations. The challenges are much greater for the adult mouse. In the embryonic mouse, the orbital bone ossification is not complete and surrounding tissues are relative loose8. The head can be removed and bisected, and then directly subjected to paraformaldehyde (PFA) fixation and embedding9. By contrast, the postnatal and adult mouse orbital bones are fully ossified with thick surrounding tissues, making the penetration of fixatives less efficient. Furthermore, the orbital/skull bones are hard and brittle, easily broken when sectioning them in the soft embedding compounds such as paraffin. The broken pieces of bones will unanimously tear the nearby tissues resulting in inferior tissue morphology.

Many published studies often showed partial ocular surface, which may be sufficient for their particular research purposes10,11. A gross examination of literatures found only few studies showing the whole intact ocular surface being demonstrated without detailed description of dissection protocols12,13. In this protocol, we detailed a dissection method to obtain integral postnatal ocular surface, in which orbital and skull bones were orderly removed, leaving untouched ocular surface together with the eyeball and eyelids, minimizing the physical damages. We further examined the OS histology and performed immunohistochemistry using the tissue sections prepared with this protocol.

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Protocol

All procedures involving the use of mice were approved by the Animal Care and Use Committee, Zhongshan Ophthalmic Center, and adhered to ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Dissection of eyeball with intact ocular surface and eyelids

  1. Dissect the head.
    1. Euthanize postnatal day 10 (P10) and P28 mice (see the Table of Materials for mouse strains) by cervical dislocation, cut the head off the neck by a pair of sharp scissors.
    2. Bisect head with a pair of straight scissors along the sagittal midline beginning at the interparietal bone rostrally to the nasal (Figure 1A,B).
      NOTE: The skull bone hardens with mouse aging, be careful to keep the scissors cutting along the midline as much as possible.
    3. Place each half of the bisected head in a clean Petri dish, cut off the jaw and tongue first using sharp scissors. Free the optic nerve by cutting it off the brain prior to the optic chiasm location, and remove all brain tissues including olfactory bulb attached to the skull wall.
    4. Now the remaining tissues shall have all skull/orbital bones associated with the eyeball (Figure 1C,D). Wash the remaining part with PBS to clean blood and hair debris.
    5. Prefix tissues with 4% paraformaldehyde (PFA, in phosphate-buffered saline [PBS], pH 7.4) in a 50 mL conical tube at room temperature (RT) with rotation. The approximate fixation time is as follows: ~10 min for P0 to P7; ~20 min for P8 to P28; and ~30 min for P29 and older.
      NOTE: The prefixation offers tissue rigidity making the ensuing dissection easier. Additionally, prefixation avoids leaving fresh tissue unfixed too long before dissection is complete.
      CAUTION: PFA is hazardous and must be handled with care.
  2. Remove skull/orbital bones.
    1. After prefixation, quickly wash the dissected head in PBS three times to further eliminate the broken hairs and fixatives in order to proceed with further dissection.
      CAUTION: Exposure to fixatives during dissection can be injurious to health.
    2. Place the tissue in 10 cm Petri dish, cut the skull into three parts along the planes indicated in Figure 1D,E. The eyeball in the socket is hidden in the middle part of the skull under ethmoid, frontal and maxillary bones (Figure 1D,E).
    3. Use two pairs of No. 4 straight forceps to peel off the ethmoid bone to fully expose the frontal and maxillary bones (Figure 1F).
    4. Geographically divide the skull surface (including maxillary and the frontal bones) into 4 areas (Figure 1F). Insert the tip of curved forceps horizontally into each area to remove the maxillary and frontal bones sequentially (Figure 1F).
      NOTE: The maxillary bones cannot all be removed at one time. Patiently dissect them piece by piece. The frontal bone is directly connected to the eyeball through soft connective tissues. Take caution when removing it from the eyeball to prevent stretching the eyeball and damaging the conjunctiva.
    5. After removal of partial maxillary bone and all frontal bone, the underlying lacrimal and jugal bones would be exposed (Figure 1G). Remove the two bones and associated subcutaneous muscles and fats surrounding the eyeball, trim the eyeball with attached eyelids and skin into a small square-shape block to reduce tissue volume and facilitate orienting the tissue when embedding (Figure 1H).
    6. Place the eyeball and associated tissues back into 4% PFA and continue to fix overnight at 4 °C. The fixed tissue can be preserved for at least one month at 4 °C in PBS with addition of 0.02% NaN3. Alternatively, proceed to histological analysis immediately.
      NOTE: If the tissue needs to be stored for longer periods, it can readily be stored in 70% ethanol.

2. Histological analysis

  1. Paraffin section and hematoxylin and eosin (H&E) staining
    1. Follow standard protocol described elsewhere9 to perform paraffin embedding and sectioning.
  2. Immunohistochemistry (IHC)
    1. Dewax the paraffin sections with xylene and rehydrate the sections through alcohol series (100%, 95%, 80%) into distilled water (dH2O).
    2. Perform antigen retrieval by microwave treatment of the tissue slides in 0.01 M citric acid buffer (3 g of trisodium citrate, 0.4 g citric acid per 1 L double distilled H2O) in a glass slide jar with low power (120 W). Energy should be intermittently delivered for total 5-10 min with each interval lasting about 2 min.
      NOTE: High-power microwave or consistent heating would lead to detachment of tissue sections from the slide.
    3. Pick out the slides from glass jar, carefully wipe off residual liquid surrounding each section using facial tissues and place them onto the slide rack in a histology box. Draw a square around each section with a waterproof histological pen. Place 100 µL of blocking buffer (0.1% triton X-100 and 10% donkey serum in 1x PBS) onto each square, and incubate for 30 min at RT.
    4. Carefully remove the blocking solution with vacuum, add the primary antibody (see the Table of Materials) with desired dilution in blocking buffer, and continue to incubate the slides at 4 °C for 24 h or longer.
      NOTE: The concentration for each primary antibody used for IHC varies, and needs to be tested out in pilot experiments.
    5. Wash tissue slides with PBST (0.1% Triton in PBS) three times, for 10 min each. Repeat step 2.2.4 using the secondary antibody (see Table of Materials) together with 4',6-diamidino-2-phenylindole (DAPI) replacing the blocking buffer. Continue to incubate for at least 4 h or longer at room temperature (RT).
    6. Remove the secondary antibody, wash tissue sections with PBST solution three times, for 10 min each, then wash with clean PBS for another 5 min.
    7. Wipe off the residual PBS surrounding tissue sections, mount coverslips on sections with mounting medium. Perform fluorescent microscopy to obtain images (see the Table of Materials).

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Representative Results

The major skull bones viewed from different perspectives were illustrated in Figure 1A-E, with colors denoting different bones. We used four-week old animal for demonstration of the dissection processes. Following dissection steps 1.1.1-1.1.3 and a short prefixation (step 1.1.4), the eyeball with associated facial bones are demonstrated in Figure 1E. Further trimming to remove anterior (nasal and premaxillary) and posterior (e.g., parietal) as well as ethmoid bones generated tissue block with mainly maxillary and frontal bones atop the eyeball (Figure 1F). Sequential removal of these bones according to designated areas in Figure 1F exposed underlying lacrimal and jugal bones as well as Harderin gland (Figure 1G). The Harderin gland attached to the eyeball served as a landmark (Figure 1G), and should be kept in place at all times. Isolation of the eyeball was completed by removal of all bones and surrounding fats and muscle tissues (Figure 1H).

After paraffin embedding, the eyeball was sectioned and H&E-stained. Representative images are shown in Figure 2. The relative intact positions of eyelids, corneal and conjunctival surface were visualized in one section (Figure 2A), parts of which were magnified in Figure 2B,C. The lens morphology is hard to maintain (Figure 2A) because of its rigidity and fragility. We applied this dissection method to other postnatal ages and showed an example of H&E histology of P10 eyeball section (Figure 3). In general, the younger the mouse is, the better the eyeball morphology is preserved. Immunostained paraffin sections from serial postnatal ages are shown in Figure 4, with keratin 12 (K12) (Figure 4A-F) and keratin 14 (K14) (Figure 4G-L) staining the cornea and the whole OS epithelium, respectively.

Figure 1
Figure 1: Illustration of the eyeball dissection in 4-week old adult mouse. (A-D) The schematic diagram of the skull composition viewed from top (A), bottom (B), lateral (C) and middle plane (D), respectively. Dashed lines in (A) and (B) indicate bisected planes. (E) Bisected-half skull at 4-week old age that exactly matches (D). (F) Medial-plane view of dissected tissue between the two transverse planes in (D) and (E). The eyeball (dashed circle) was mainly embedded underneath the frontal (Fron) and maxillary (Max). Colored dashed lines geographically divide the bisected plane into 4 areas, in which bones were roughly removed as per the order or numbers. Note that the ethmoid (Eth) bone has been removed. (G) After removal of frontal bone and partial maxillary, Harderin gland (Har) and jugal (Jug) and lacrimal (La) bones were exposed. Circle dashed lines indicate the eyeball position. (H) Isolated eyeball viewed from inside after dissection completed. Arrow points to optic nerve (On). E = Eye, Na = Nasal, Pre = Premaxilla, Max = Maxillary, La = Lacrimal, Fron = Frontal, Ali = Alisphenoid, Jug = Jugal, Pal = Palatine, Ptery = Pterygoid, Squ = Squamosal, Par = Parietal, Eth = Ethmoid. Please click here to view a larger version of this figure.

Figure 2
Figure 2: H&E histology of the ocular surface tissue at postnatal 4-week old. (A) Eyelid, conjunctiva and cornea are visualized in one tissue section. Boxed areas of "b" and "c" were magnified in (B) and (C), respectively. (B) The cornea epithelium, stroma and endothelium. (C) The conjunctiva with goblet cells. ce = cornea epithelium, st = stroma, ed = endothelium, gc = goblet cell. Please click here to view a larger version of this figure.

Figure 3
Figure 3: H&E histology of the ocular surface tissue at postnatal day 10. (A) Intact eyeball at P10. Boxed areas of "b" and "c" were magnified in (B) and (C), respectively. (B) The cornea. (C) The conjunctiva. Ce = cornea epithelium, st = stroma, ed = endothelium, cj = conjunctiva. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Immunostaining of the ocular surface from P5 to P15. Square-boxed area in each panel was magnified in their right. (A-F) Keratin 12 was specifically expressed in the cornea epithelium (arrows). (G-L) The keratin 14 expressed in the ocular surface. ac = anterior chamber, pc = palpebral conjunctiva. This figure was modified from a previous publication Guo et al.14. Please click here to view a larger version of this figure.

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Discussion

One critical reminder for preparation of the intact eyeball is that all orbital bones must be removed completely, especially the juga and lacrimal bones, which are small and located near the bottom of eye socket. Any left-over bones may complicate the ensuing histology. In case a tiny piece of bone was not completely removed from dissection by accident, it may be picked out from the embedding paraffin block using a pair of sharptweezers. The hole left behind should be filled with melted paraffin after this operation.

Additional caution should be exercised when handling the lens. The broken pieces of lens are usually scattered through the whole section. This will considerably affect microscopy examination and image analysis. Attempts to remove lens would damage either the ocular surface (if the operation was conducted from facial side) or the retina (if the operation was from the optic disc side of the retina). An alternative way to reduce the negative impact of the presence of lens tissue without dissection is to change sectioning directions. For instance, to save the ocular surface morphology, sectioning of the paraffin block should be performed from anterior of the eyeball to the posterior, otherwise, from the opposite direction.

A useful note to make for the PFA-fixed paraffin section is that antigen retrieval is usually necessary for immunostaining such as using K12 and K14 antibodies. However, there are many exceptions that antigen retrieval would generate non-specific signals increasing the background staining. Thus, one should be cautious when using antigen retrieval technology, and should test antibodies in pilot experiments if possible, beforehand.

In general, there are two methods for isolation of the eyeball from the skull/orbital bones: (i) directly dissect the eyeball from facial side; and (ii) remove the inside skull/orbital bones to free the eyeball. The challenge for the first method is that eyeball sinks deep into the orbital socket with a narrow space in between for dissection. Furthermore, the movable eyeball would stretch the conjunctiva epithelium at any given point when the dissection tools touch it, creating unintentional damages. By contrast, the second method begins with dissection of skull/orbital bones, which are farther from the eyeball and associated ocular surface tissue, thus reducing the risk of damaging the conjunctiva. Moreover, there are no spatial constrictions for performing dissections. Even though the first method can work if great cautions were taken, the second one is definitely easier and better.

In summary, we have described a method for isolation of mouse eyeball with intact eyelid and associated ocular surface from the postnatal mice. The described protocol is suitable for isolation of the eyeball with intact eyelid and associated ocular tissues in postnatal mice. The protocol is especially useful when integrity of the OS or entire ocular tissues is required. We have used this method to prepare tissue samples for examination of keratin markers in OS under normal and pathological conditions. We conclude that the ocular tissues from such preparations are particularly helpful for looking at differential regulations of a protein in different parts of the OS on a single section.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

The authors thank Prof. Rong Ju for critical reading of the manuscript, and all lab members for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (NSFC: 31571077; Beijing, China), the Guangzhou City Sciences and Technologies Innovation Project (201707020009; Guangzhou, Guangdong Province, China), "100 People Plan" from Sun Yat-sen University (8300-18821104; Guangzhou, Guangdong Province, China), and research funding from State Key Laboratory of Ophthalmology at Zhongshan Ophthalmic Center (303060202400339; Guangzhou, Guangdong Province, China).

Materials

Name Company Catalog Number Comments
1× Phosphate buffered saline (PBS) Transgen Biotech FG701-01
50ml centrifuge tube Corning 430829
Adhesive microscope slides Various
Alexa Fluor 488 Phalloidin Invitrogen/Life Technologies A12379 Suggested concentration 1:500 - 1,000
Alexa Fluor 568 Phalloidin Invitrogen/Life Technologies A12380 Suggested concentration 1:500 - 1,000
Anti-K12 antibody Abcam ab124975 Suggested concentration 1:1,000
Anti-K14 antibody Abcam ab7800 Suggested concentration 1:800
Citric acid Various
Cover slide Various
Curved forceps World Precision Instruments 14127
Dissecting microscope. Olmpus SZ61
Ethyl alcohol Various
Fluorescent Microscope Zeiss AxioImager.Z2
Fluoromount-G Mounting media SouthernBiotech 0100-01
Micro dissecting scissors-straight blade World Precision Instruments 503242
Microwave ovens Galanz P70D20TL-D4
Mouse strains C57/BL6 and Sv129 mixed
No.4 straight forceps World Precision Instruments 501978-6
Normal Goat Serum Various
Paraformaldehyde (PFA) Various Prepare a 4% solution in 1× PBS and filter with 0.45μm filter membrane
Tissue culture dish Various
Trisodium citrate Various
Triton X-100 Sigma-Aldrich SLBW6818

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References

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