Recording of Electroretinogram and Visual Evoked Potential in a Rat

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing Electrodes

Note: A three-channel transmitter is used for surgical implantation, which enables 2 electroretinogram (ERG) and 1 visual evoked potential (VEP) recording to be conducted simultaneously. The three active and three inactive electrodes need to be pre-fashioned into a ring shape before implantation in order to attach to the eye. For identification purposes, the manufacturer has enclosed active electrodes in half-white, half-colored plastic sheaths, while inactive electrodes are covered in full-colored sheaths. The ground electrode (clear plastic sheath) is left unaltered for all active and inactive electrodes to conduct steps 1.1, 1.2, 1.3, and 1.7.

1. Untwist the double-stranded stainless-steel electrode with two fine-tipped pliers.
2. Trim one of the stainless-steel strands (approximately 1 cm from the tip), leaving a single longer straight strand remaining to shape the ring electrode.
3. Fold the single stainless-steel strand back onto itself and twist, forming a smooth ring at the tip of the electrode.
4. For the ERG active electrodes, fashion this loop ~ 0.2 - 0.5 mm in diameter by twisting the base of the loop (for the purpose described here, shape two active electrodes in this way to record ERG from both eyes), and for the ERG inactive and VEP electrodes make the loop diameter ~ 0.8 mm diameter (in this example, do this for one active VEP electrode and all three inactive electrodes).
5. Hook the circular VEP active electrode around a stainless-steel screw (diameter 0.7 mm, length 3 mm) so the electrode rests against the screw head.
6. Hook the 3 inactive electrodes (2 ERG, 1 VEP) around a second stainless steel screw (diameter 0.7 mm, length 3 mm).
7. Pull the plastic sleeve forward over the sharp ends of the two stainless-steel strands to reduce irritation.
8. Sterilize the telemetry transmitters by soaking them in 2% glutaraldehyde for more than 10 hours at approximately 25 °C. Then, rinse the transmitter with sterile saline three times.

2. Transmitter Implantation

1. Animal Preparation​
   1. Disinfect the surgical area prior to experimentation by cleaning it with 70% ethanol. Autoclave all surgical equipment before use and maintain equipment in chlorhexidine when not in use during surgery. Cover the animal with a surgical drape during surgery to maintain a sterile environment. Ensure all experimenters wear surgical masks, sterile gloves, and gowns.
   2. Induce anesthesia with 1.5 - 2% isoflurane at a flow rate of 3 L/min and maintained at 1.5 - 2 % at 2 L/min throughout the surgery. Confirm sufficient depth of anesthesia by the absence of a pedal reflex upon pinching the muscle between the toes.
   3. Shave a 40 mm x 30 mm area over the abdomen from above the groin to the sternum.
   4. Shave a 30 mm x 20 mm area over the forehead, posterior to the eyes and anterior to the ears.
   5. Disinfect the two shaved areas. For the forehead area disinfect with 10% povidone-iodine three times (avoid the use of alcohol-based antiseptics for area near the eye, being consistent with the Standard of Practice set out by the Association of Surgical Technologists). Over the abdomen, disinfect with 10% povidone-iodine and 70% ethanol.
   6. Apply 1 drop of proxymetacaine to the cornea for additional topical anesthesia.
   7. Apply 1 drop of carboxymethylcellulose sodium to the cornea to prevent drying of the eyes.
2. Surgical implantation
   1. Make a 10 mm incision on the head along the vertical midline between the ears with a surgical scalpel.
   2. Make a 5 mm incision on the abdomen through the skin layer along the midline below the sternum.
   3. Tunnel a 5 mm diameter cannula subcutaneously from the abdomen incision to the head incision.
   4. Feed the electrode wires (3 active and 3 inactive) of the transmitter through the cannula from the abdomen to the head.
   5. Leave the reference electrode with the transmitter base and cover the electrode tip with aseptic gauze.
   6. Cover the electrode tips (3 active and 3 inactive) with aseptic gauze.
   7. Secure the rat's head to a stereotaxic platform.
   8. Extend the forehead incision to 30mm in length with surgical scissors.
   9. Expose the surgical area by retracting loose skin with 2 sutures (3 - 0) at ~ 3 and 9 o'clock.
   10. Scrape off the periosteum overlying the skull using sterilized gauze to expose bregma, lambda, and midline sutures.
   11. Drill two holes through the skull at the VEP active (7 mm ventral to bregma, 3 mm lateral to midline) and inactive (5 mm rostral to bregma on the midline) stereotaxic coordinates.
   12. Attach VEP active and inactive electrodes with pre-attached stainless-steel screws (diameter 0.7 mm, length 3 mm) to the skull with a small screwdriver to ~ 1 mm in depth into the premade holes. This anchors the screw to the bone without damaging the underlying cortical tissue.
   13. To implant the ERG active electrodes, use an 8 - 0 suture to retract the upper eyelid temporarily.
   14. Insert a 16 to 21 G cannula subcutaneously from behind the eye through to the superior conjunctival fornix.
   15. Remove the guiding needle.
   16. Feed the active electrode through the shortened plastic catheter from the forehead towards the eye. Then, remove the plastic catheter.
   17. Use a temporary suture (8 - 0), which is threaded through the electrode loop, to prevent the electrode from retracting back into the tunnel.
   18. Make a 0.5 mm incision on the superior conjunctiva at 12 o'clock, 1 mm behind the limbus. Use blunt dissection to expose the underlying sclera.
   19. Implant an 8 - 0 or 9 - 0 suture immediately behind the limbus at half scleral thickness.
   20. Remove temporary suture from ERG active electrode.
   21. Anchor the ERG active electrode to the half scleral thickness suture by tying 3 consecutive knots, ensuring the electrode tip is situated close to the limbus.
   22. Close the conjunctival flap using 1 to 2 interrupted sutures (8 - 0 to 9 - 0). Ensure that the conjunctiva completely covers the ERG electrode to improve comfort.
   23. Remove the eyelid retracting suture.
   24. Repeat the procedure for the contralateral eye.
   25. Apply cyanoacrylate gel over the skull to secure all stainless screws and electrode wires. Ensure the ERG active electrodes are not pulled too tight before securing them to enable eye movements.
   26. Close the head wound using a non-absorbable 3 - 0 suture.
   27. Rotate the rodent to expose the abdominal area. Lengthen the abdominal dermal incision to 40 mm along the Linea alba with surgical scissors.
   28. Make a 35 mm incision through the inner muscle wall to expose the inner abdominal cavity.
   29. Using two sutures (3 - 0), the transmitter body is attached to the animal's right-hand side inner abdominal wall. Avoid contacting the liver.
   30. Loop the ground electrode and secure it in this shape with a suture (3 - 0). Then, place it free-floating in the abdominal cavity.
   31. Close the peritoneum using a continuous suture (3 - 0).
   32. Close the skin incision using interrupted sutures (3 - 0).
3. Post-operative care​
   1. Monitor the animal until it has regained sufficient consciousness to maintain sternal recumbency. House the animal singly following the surgery.
   2. Administer carprofen subcutaneously for analgesia (5 mg/kg) once a day for 4 days.
   3. Add prophylactic oral antibiotics (Enrofloxin, 5 mg/kg) to the drinking water for 7 days post-surgery.
   4. Apply an anti-inflammatory ointment to skin incision sites to reduce irritation for the first 7 days post-surgery.

3. Conduct ERG and VEP Recordings in Conscious Rats​

1. Dark adapt animal for 12 hours prior to ERG and VEP recordings.
2. Conduct all experimental manipulations under dim red illumination (17.4 cd.m-2, λmax = 600 nm)
3. Apply topical anesthesia (0.5% proxymetacaine) and dilating (0.5% tropicamide) drops to the cornea.
4. Guide the conscious rodent into a custom-made, clear restrainer.
   Note: The length of this plastic tube can be adjusted to accommodate different-sized rats, with the overall diameter fixed at 60 mm. The front end of the device is tapered to minimize head movement and contains perforations to enable normal breathing. This tapered front allows alignment and stabilization of the rat's head and eyes to the opening of the Ganzfeld sphere. Note that the rodent has been acclimatized to the restrainer (3 to 5 occasions) prior to surgery.
5. Place the rodent in front of the Ganzfeld bowl with the eyes aligned with the opening of the bowl.
6. Turn on the indwelling transmitter by passing a magnet within ~ 5 cm of it. Then, verify that the transmitter is on by checking the LED status light on the receiver base.
7. Collect signals over a range of luminous energies (i.e., -5.6 to 1.52 log cd.s.m²).

Materiais

Name	Company	Catalog Number	Comments
Bioamplifier	ADInstruments	ML 135	Amplifies ERG and VEP signals
Carboxymethylcellulose sodium 1.0%	Allergan	CAS 0009000-11-7	Maintain corneal hydration during surgery
Carprofen 0.5%	Pfizer Animal Health Group	CAS 53716-49-7	Post-surgery analgesia, given with injectable saline for fluid replenishment
Chlorhexadine 0.5%	Orion Laboratories	27411, 80085	Disinfection of surgical instrument
Cyanoacrylate gel activator	RS components	473-439	Quickly dries cyanoacrylate gel
Cyanocrylate gel	RS components	473-423	Fix stainless screws to skull
Dental burr	Storz Instruments, Bausch and Lomb	E0824A	Miniature drill head of ~0.7mm diameter for making a small hole in the skull over each hemisphere to implant VEP screws
Drill	Bosch	Dremel 300 series	Automatic drill for trepanning
Enrofloxin	Troy Laboratories		Prophylactic antibiotic post surgey
Ganzfeld integrating sphere	Photometric Solutions International		Custom designed light stimulator: 36 mm diameter, 13 cm aperture size
Gauze swabs	Multigate Medical Products Pty Ltd	57-100B	Dries surgical incision and exposed skull surface during surgery
Isoflurane 99.9%	Abbott Australasia Pty Ltd	CAS 26675-46-7	Proprietory Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery
Kenacomb ointment	Aspen Pharma Pty Ltd		To reduce skin irritation and itching after surgery
Luxeon LEDs	Phillips Lighting Co.		For light stimulation, twenty 5 watt and one 1 watt LEDs, controlled by Scope software
Needle (macrosurgery)	World Precision Instruments	501959	for suturing abdominal and head surgery, used with 3-0 suture, eye needle, cutting edge 5/16 circle Size 1, 15mm
Needle holder (macrosurgery)	World Precision Instruments	500224	To hold needle during abdominal and head surgery
Needle holder (microsurgery)	World Precision Instruments	555419NT	To hold needle during ocular surgery
Optiva catheter	Smiths Medical International LTD	16 or 21 G	Guide corneal active electrodes from skull to conjunctiva
Povidone iodine 10%	Sanofi-Aventis	CAS 25655-41-8	Proprietory name: Betadine, Antiseptic to prepare the shaved skin for surgery 10%, 500 mL
Powerlab data acquisition system	ADInstruments	ML 785	Acquire signal from telemetry transmitter, paired to telemetry data converter
Proxymetacaine 0.5%	Alcon Laboratories	CAS 5875-06-9	Topical ocular analgesia
Restrainer	cutom made		Front of the restrainer is tapered to minimize head movement, length can be adjusted to accommodate different rat length, overall diameter is 60 mm.
Scapel blade	R.G. Medical Supplies	SNSM0206	For surgical incision
Scissors (macrosurgery)	World Precision Instruments	501225	for cutting tissue on the abodmen and forhead
Scissors (microsurgery)	World Precision Instruments	501232	To dissect the conjunctiva for electrode attachment
Scope Software	ADInstruments	version 3.7.6	Simultaneously triggers the stimulus via the ADI Powerlab system and collects data
Shaver	Oster	Golden A5	Shave fur from surgical areas
Stainless streel screws	MicroFasteners	L001.003CS304	0.7 mm shaft diameter, 3 mm in length
Stereotaxic frame	David Kopf	Model 900	A small animal stereotaxic instrument for locating the implantation landmarks on the skull
Surgical drape	Vital Medical Supplies	GM29-612EE	Ensure sterile enviornment during surgery
Suture (macrosurgery)	Ninbo medical needles	3-0	for suturing abdominal and head surgery, sterile silk braided, 60cm
Suture needle (microsurgery)	Ninbo medical needles	8-0 or 9-0	for ocular surgery including, suturing electrode to sclera and closing conjunctival wound, nylon suture, 3/8 circle 1×5, 30cm
Telemetry data converter	DataSciences International	R08	allows telemetry signal to interface with data collection software
Telemetry Data Exchange Matrix	DataSciences International		Gathers data from transmitters, pair with receiver
Telemetry data receiver	DataSciences International	RPC-1	Receives telemetry data from transmitter
Telemetry transmitter	DataSciences International	F50-EEE	3 channel telemetry transmitter
Tropicamide 0.5%	Alcon Laboratories		Iris dilation
Tweezers (macrosurgery)	World Precision Instruments	500092	Manipulate tissues during abdominal and head surgery
Tweezers (microsurgery)	World Precision Instruments	500342	Manipulate tissues during ocular surgery