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), &#34;100 People Plan&#34; 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).

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
<ol>
	<li>Dissect the head.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Remove skull/orbital bones.
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Use two pairs of No. 4 straight forceps to peel off the ethmoid bone to fully expose the frontal and maxillary bones (Figure 1F).</li>
		<li>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.</li>
		<li>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).</li>
		<li>Place the eyeball and associated tissues back into 4% PFA and continue to fix overnight at 4 &#176;C. The fixed tissue can be preserved for at least one month at 4 &#176;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.</li>
	</ol>
	</li>
</ol>
2. Histological analysis
<ol>
	<li>Paraffin section and hematoxylin and eosin (H&#38;E) staining
	<ol>
		<li>Follow standard protocol described elsewhere9 to perform paraffin embedding and sectioning.</li>
	</ol>
	</li>
	<li>Immunohistochemistry (IHC)
	<ol>
		<li>Dewax the paraffin sections with xylene and rehydrate the sections through alcohol series (100%, 95%, 80%) into distilled water (dH2O).</li>
		<li>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.</li>
		<li>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 &#181;L 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.</li>
		<li>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 &#176;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.</li>
		<li>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&#39;,6-diamidino-2-phenylindole (DAPI) replacing the blocking buffer. Continue to incubate for at least 4 h or longer at room temperature (RT).</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>

<ol><li>Swamynathan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+surface+development+and+gene+expression%5BTitle%5D)+AND+%22Journal+of+Ophthalmology%22%5BJournal%5D)">Ocular surface development and gene expression.</a> Journal of Ophthalmology. 2013, (2013).</li>
<li>Arita, R., Fukuoka, S., Morishige, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+Morphology+of+the+Lipid+Layer+of+the+Tear+Film%5BTitle%5D)+AND+%22Cornea%22%5BJournal%5D)">Functional Morphology of the Lipid Layer of the Tear Film.</a> Cornea. 36 Suppl 1, S60-S66 (2017).</li>
<li>Guo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+surface+pathogenesis+associated+with+precocious+eyelid+opening+and+necrotic+autologous+tissue+in+mouse+with+disruption+of+Prickle+1+gene%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.</a> Experimental Eye Research. 180, 208-225 (2018).</li>
<li>Knop, E., Knop, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomy+and+immunology+of+the+ocular+surface%5BTitle%5D)+AND+%22Chemical+Immunology+and+Allergy%22%5BJournal%5D)">Anatomy and immunology of the ocular surface.</a> Chemical Immunology and Allergy. 92, 36-49 (2007).</li>
<li>Mizoguchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+surface+alkali+injury+damages+meibomian+glands+in+mice%5BTitle%5D)+AND+%22The+Ocular+Surface%22%5BJournal%5D)">Ocular surface alkali injury damages meibomian glands in mice.</a> The Ocular Surface. 15 (4), 713-722 (2017).</li>
<li>Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Goblet+cells+of+the+conjunctiva%3A+A+review+of+recent+findings%5BTitle%5D)+AND+%22Progress+in+Retinal+and+Eye+Research%22%5BJournal%5D)">Goblet cells of the conjunctiva: A review of recent findings.</a> Progress in Retinal and Eye Research. 54, 49-63 (2016).</li>
<li>Nowell, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+inflammation+imposes+aberrant+cell+fate+in+regenerating+epithelia+through+mechanotransduction%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction.</a> Nature Cell Biology. 18 (2), 168-180 (2016).</li>
<li>Nagata, M., Ohashi, Y., Ozawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+histochemical+study+of+the+development+of+premaxilla+and+maxilla+during+secondary+palate+formation+in+the+mouse+embryo%5BTitle%5D)+AND+%22Archives+of+Histology+and+Cytology%22%5BJournal%5D)">A histochemical study of the development of premaxilla and maxilla during secondary palate formation in the mouse embryo.</a> Archives of Histology and Cytology. 54 (3), 267-278 (1991).</li>
<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+MEK+kinase+1+in+TGF-beta%2Factivin-induced+epithelium+movement+and+embryonic+eyelid+closure%5BTitle%5D)+AND+%22The+Embo+Journal%22%5BJournal%5D)">A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure.</a> The Embo Journal. 22 (17), 4443-4454 (2003).</li>
<li>Sharov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noggin+overexpression+inhibits+eyelid+opening+by+altering+epidermal+apoptosis+and+differentiation%5BTitle%5D)+AND+%22The+Embo+Journal%22%5BJournal%5D)">Noggin overexpression inhibits eyelid opening by altering epidermal apoptosis and differentiation.</a> The Embo Journal. 22 (12), 2992-3003 (2003).</li>
<li>Setala, N. L., Metso, J., Jauhiainen, M., Sajantila, A., Holopainen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dry+eye+symptoms+are+increased+in+mice+deficient+in+phospholipid+transfer+protein+%28PLTP%29%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">Dry eye symptoms are increased in mice deficient in phospholipid transfer protein (PLTP).</a> The American Journal of Pathology. 178 (5), 2058-2065 (2011).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mastermind-like+transcriptional+co-activator-mediated+Notch+signaling+is+indispensable+for+maintaining+conjunctival+epithelial+identity%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity.</a> Development. 140 (3), 594-605 (2013).</li>
<li>McCauley, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGFbeta+signaling+inhibits+goblet+cell+differentiation+via+SPDEF+in+conjunctival+epithelium%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">TGFbeta signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium.</a> Development. 141 (23), 4628-4639 (2014).</li>
<li>Guo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+surface+pathogenesis+associated+with+precocious+eyelid+opening+and+necrotic+autologous+tissue+in+mouse+with+disruption+of+Prickle+1+gene%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.</a> Experimental Eye Research. 180, 208-225 (2019).</li>
</ol>

The authors have nothing to disclose.

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....

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.

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

isolation of intact eyeball to obtain integral ocular surface tissue for histological examination and immunohistochemistry

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.

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...

Watch this Scientific Journal Video about Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry at JoVE.com

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

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

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

isolation-intact-eyeball-to-obtain-integral-ocular-surface-tissue-for

Research

JoVE Journal

Biology

8.4K Views.  Sun Yat-sen University. This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

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

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Medicine

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Neuroscience

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Intravitreal Injections in the Ovine Eye

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. 
We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

Intravitreal injections were performed in the sheep eye with the aim of delivering viral-mediated gene therapy to the retina.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or ...

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

Obtaining High-Quality Cryosections of Whole Rabbit Eye

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, M&#252;ller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse M&#252;ller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, M&#252;ller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for M&#252;ller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn&#39;t be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

This manuscript describes a detailed protocol for isolating retinal glial M&#252;ller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of M&#252;ller cells.

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...

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

Summary

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