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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

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 .......

Ujawnienia

The authors have nothing to disclose.

Podziękowania

Dr. Alaknanda Mishra, Department of Cell Biology and Human Anatomy, University of California Davis, USA, trained us in this method at the National Institute of Immunology, New Delhi. This work was supported by the core grant received from the Department of Biotechnology, Government of India to the National Institute of Immunology, New Delhi. P.S. was granted a research fellowship by the Department of Biotechnology.

....

Materiały

NameCompanyCatalog NumberComments
Acetaminophen (Biocetamol)EG PharmaceuticalsNo specific Catalog Number (Local Procurement)
Alkaline Phosphatase Kit (DEA)Coral Clinical System, IndiaNo specific Catalog Number (Local Procurement)
Automated analyserTulip, Alto Santracruz, IndiaScreen Maaster 3000Biochemical analyser for liver functional test
Betadine (Povidon-Iodine Solution)Win-Medicare;  IndiaNo specific Catalog Number (Local Procurement)
Biological safety cabinet ( Class I)Kartos international; IndiaNo specific Catalog Number (Local Procurement)
Bright Field MicroscopeOlympus, JapanLX51
CBA/J inbred miceThe Jackson LaboratoryStock No. 000654
Cefotaxime (Taxim)AlKem ; Indiacefotaxime sodium injection, No specific Catalog Number (Local Procurement)
Cell StrainerSigma ; USCLS431752
Collagenase Type IGibco by Life Technologies17100-017
Cotton BudsPure Swabs Pvt Ltd ; IndiaNo specific Catalog Number (Local Procurement)
DPX MountantSigma ; US6522
Drape SheetJSD Surgicals, Delhi, IndiaNo specific Catalog Number (Local Procurement)
Eosin Y solution, alcoholicSigma ; USHT110132
ForcepsMajor Surgicals; IndiaNo specific Catalog Number (Local Procurement)
Gas Anesthesia SystemUgo Basile; Italy211000
GlucoseHimedia, IndiaGRM077
Hair removing cream (Veet)Reckitt Benckiser , IndiaNo specific Catalog Number (Local Procurement)
Hematoxylin Solution, Mayer'sSigma ; USMHS16
Heparin sodium saltHimedia; IndiaRM554
Hyaluronidase From Sheep TestesSigma ; USH6254
I.V. Cannula (Plusflon)Mediplus, IndiaRef 1732411420
Insulin SyringesBD ; USREF 303060
Isoflurane ( Forane)Asecia QueenboroughNo B506Inhalation Anaesthetic
Ketamine (Ketamax)Troikaa Pharmaceuticals Ltd.Ketamine hydrochloride IP, No specific Catalog Number (Local Procurement)
Meloxicam (Melonex)Intas Pharmaceuticals Ltd; IndiaNo specific Catalog Number (Local Procurement)
Micro needle holders straight & curvedMercian ;  EnglandBS-13-8
Micro needle holders straight &
curved
Mercian ;  EnglandBS-13-8
MicrotomeHisto-Line Laboratories, ItalyMRS3500
Nylon ThreadMighty ; IndiaNo specific Catalog Number (Local Procurement)
ParaformaldehydeHimedia; IndiaGRM 3660
PercollGE Healthcare17-0891-01
Refresh Tears/Eyemist GelAllergan India Private Limited/Sun Pharma, IndiaP3060No specific Catalog Number
RPMIHimedia; IndiaNo specific Catalog Number (Local Procurement)
ScalpelMajor Surgicals; IndiaNo specific Catalog Number (Local Procurement)
ScissorsMajor Surgicals; IndiaNo specific Catalog Number (Local Procurement)
SGOT (ASAT) KITCoral Clinical System, IndiaNo specific Catalog Number (Local Procurement)
SGPT (ALAT) KITCoral Clinical System, IndiaNo specific Catalog Number (Local Procurement)
Shandon Cryotome E CryostatThermo Electron Corporation ; USNo specific Catalog Number
SucroseSigma ; USS0389
Surgical Blade No. 22La Medcare, IndiaNo specific Catalog Number (Local Procurement)
Surgical BoardLocally madeNo specific Catalog Number (Local Procurement)
Surgical White Tape3M India ; India1530-1Micropore Surgical Tape
SuturesEthicon, Johnson & Johnson, IndiaNW 5047
Syringes (1ml, 26 G)Dispo Van; IndiaNo specific Catalog Number (Local Procurement)
Trimmer (Clipper)PhilipsNL9206AD-4 DRACHTEN QT9005
Weighing MachineBraunNo specific Catalog Number (Local Procurement)
William's E MediaHimedia; IndiaAT125
Xylazine (Xylaxin)Indian Immunologicals LimitedSedative, Pre-Anaesthetic, Analgesic and muscle relaxant

Odniesienia

  1. Choi, Y., et al. Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice. Experimental and Molecular Medicine. 50 (8), 1-9 (2018).
  2. Sutherland, C., et al.

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MicrodissectionRodent EyeOcular ResearchNictitating MembraneNeural RetinaRPE LayerEnucleationEye DissectionOphthalmic Rodent ModelsEye ImmobilizationCorneal LimbusPosterior Eye CupAnterior Eye Cup

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