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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 OS^3,4. 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 diseases^5,6,7. 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 purposes^10,11. A gross examination of literatures found only few studies showing the whole intact ocular surface being demonstrated without detailed description of dissection protocols^12,13. 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.

Protokół

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).

Wyniki

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...

Dyskusje

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.

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Materiały

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