Microdissection of the Rodent Eye

Podsumowanie

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.

Streszczenie

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'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. 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.

Wprowadzenie

Ocular dissection is an important technique in ophthalmic research and has allowed investigators to access the segments of the eye for targeted studies. Previously, ocular researchers relied on the ocular tissue from diseased individuals for their studies. However, the progressively growing number of strains of ophthalmic rodent models¹ over the years has diminished the need for human ocular tissue. These mouse strains have permitted a deeper understanding of ocular disease and interventions. Yet, they have also generated a need for innovative techniques of ocular micro-dissection. The small size and limited area of operation severely constrain....

Protokół

This procedure was approved by the Institutional Animal Ethics Committee of the National Institute of Immunology, New Delhi. The serial reference number of the approval is IAEC#480/18. The experiments were performed in accordance with the regulation guidelines of the Committee for Control and Supervision of Experiments on Animals, Ministry of Fisheries, Animal Husbandry and Dairying, Government of India, under the supervision of a professional Veterinarian at the SAF, NII.

1. Preparation

1. Dilate the rodent eyes using a drop of 0.8% tropicamide and 5% phenylephrine ophthalmic solutions in each eye. Place the anima....

Wyniki

A wholemount of rd1 mouse eye/corneal tissue was prepared to study potential lymph-angiogenesis in the anterior/corneal tissue in a diseased state. The attached conjunctival tissue from the third eyelid acted as a positive control, since the cornea lacks lymphatic vessels. For the study, the corneal tissue was dissected with the conjunctiva and was fixed with 4% PFA, followed by permeabilization and blocking. The tissue was then stained with a primary antibody against the lymphatic endothelial marker (LYVE1)

Dyskusje

Ocular microdissection has been found to be a difficult task owing to the small size and spherical shape of the rodent eye, and the rodent eye requires innovative techniques for efficient handling⁸.

In the current demonstrated method, the enucleated mouse eyeball is obtained with the third eyelid attached for effective and easy handling. Using the third eyelid, the eyeball can be immobilized completely, which allows the dissection to proceed with ease and with minimal .......

Materiały

Name	Company	Catalog Number	Comments
Acetaminophen (Biocetamol)	EG Pharmaceuticals		No specific Catalog Number (Local Procurement)
Alkaline Phosphatase Kit (DEA)	Coral Clinical System, India		No specific Catalog Number (Local Procurement)
Automated analyser	Tulip, Alto Santracruz, India	Screen Maaster 3000	Biochemical analyser for liver functional test
Betadine (Povidon-Iodine Solution)	Win-Medicare;  India		No specific Catalog Number (Local Procurement)
Biological safety cabinet ( Class I)	Kartos international; India		No specific Catalog Number (Local Procurement)
Bright Field Microscope	Olympus, Japan	LX51
CBA/J inbred mice	The Jackson Laboratory	Stock No. 000654
Cefotaxime (Taxim)	AlKem ; India		cefotaxime sodium injection, No specific Catalog Number (Local Procurement)
Cell Strainer	Sigma ; US	CLS431752
Collagenase Type I	Gibco by Life Technologies	17100-017
Cotton Buds	Pure Swabs Pvt Ltd ; India		No specific Catalog Number (Local Procurement)
DPX Mountant	Sigma ; US	6522
Drape Sheet	JSD Surgicals, Delhi, India		No specific Catalog Number (Local Procurement)
Eosin Y solution, alcoholic	Sigma ; US	HT110132
Forceps	Major Surgicals; India		No specific Catalog Number (Local Procurement)
Gas Anesthesia System	Ugo Basile; Italy	211000
Glucose	Himedia, India	GRM077
Hair removing cream (Veet)	Reckitt Benckiser , India		No specific Catalog Number (Local Procurement)
Hematoxylin Solution, Mayer's	Sigma ; US	MHS16
Heparin sodium salt	Himedia; India	RM554
Hyaluronidase From Sheep Testes	Sigma ; US	H6254
I.V. Cannula (Plusflon)	Mediplus, India	Ref 1732411420
Insulin Syringes	BD ; US	REF 303060
Isoflurane ( Forane)	Asecia Queenborough	No B506	Inhalation Anaesthetic
Ketamine (Ketamax)	Troikaa Pharmaceuticals Ltd.		Ketamine hydrochloride IP, No specific Catalog Number (Local Procurement)
Meloxicam (Melonex)	Intas Pharmaceuticals Ltd; India		No specific Catalog Number (Local Procurement)
Micro needle holders straight & curved	Mercian ;  England	BS-13-8
Micro needle holders straight & curved	Mercian ;  England	BS-13-8
Microtome	Histo-Line Laboratories, Italy	MRS3500
Nylon Thread	Mighty ; India		No specific Catalog Number (Local Procurement)
Paraformaldehyde	Himedia; India	GRM 3660
Percoll	GE Healthcare	17-0891-01
Refresh Tears/Eyemist Gel	Allergan India Private Limited/Sun Pharma, India	P3060	No specific Catalog Number
RPMI	Himedia; India		No specific Catalog Number (Local Procurement)
Scalpel	Major Surgicals; India		No specific Catalog Number (Local Procurement)
Scissors	Major Surgicals; India		No specific Catalog Number (Local Procurement)
SGOT (ASAT) KIT	Coral Clinical System, India		No specific Catalog Number (Local Procurement)
SGPT (ALAT) KIT	Coral Clinical System, India		No specific Catalog Number (Local Procurement)
Shandon Cryotome E Cryostat	Thermo Electron Corporation ; US		No specific Catalog Number
Sucrose	Sigma ; US	S0389
Surgical Blade No. 22	La Medcare, India		No specific Catalog Number (Local Procurement)
Surgical Board	Locally made		No specific Catalog Number (Local Procurement)
Surgical White Tape	3M India ; India	1530-1	Micropore Surgical Tape
Sutures	Ethicon, Johnson & Johnson, India	NW 5047
Syringes (1ml, 26 G)	Dispo Van; India		No specific Catalog Number (Local Procurement)
Trimmer (Clipper)	Philips	NL9206AD-4 DRACHTEN QT9005
Weighing Machine	Braun		No specific Catalog Number (Local Procurement)
William's E Media	Himedia; India	AT125
Xylazine (Xylaxin)	Indian Immunologicals Limited		Sedative, Pre-Anaesthetic, Analgesic and muscle relaxant