Preparing a Rat Model to Assess Visual Pathway Integrity Using an Electrophysiology Setup

Abstract

Source: Nguyen, C. T. et al., Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats. J. Vis. Exp. (2016)

This video demonstrates the preparation of a rat model to study retinal health and visual pathway integrity. Visual evoked potential and electroretinography reference and active electrodes are connected to the rat, followed by aligning the platform with the rat in front of the Ganzfeld bowl for even retinal illumination and closing the Faraday cage to avoid external interference.

Protocol

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

1. Pre-implantation of Chronic visual evoked potential (VEP) Electrodes

NOTE: If concurrent electroretinogram (ERG) and VEP signals are to be collected animals must be surgically implanted with VEP electrodes at least 1 week prior to signal collection.

1. Sterilize the surgical bench prior to experimentation by cleaning with chlorhexidine (0.5% in 70% ethanol). Autoclave all surgical equipment before use. Cover the animal with a sterilized surgical drape. Ensure all experimenters wear surgical masks, gowns, and sterilized gloves.
2. Induce anesthesia with 3 - 3.5% isoflurane with O₂ at a flow rate of 3 L/min. Maintain anesthesia at 1.5% and 2 L/min throughout the surgery. Ensure sufficient depth of anesthesia by the absence of paw pinch reflex.
3. Apply 1% carboxymethylcellulose sodium on the cornea to prevent drying of the eyes.
4. Shave a 30 mm x 30 mm area over the forehead, posterior to the eyes and anterior to the ears.
5. Place the animal on a heat pad (37˚C) to maintain body temperature and stabilize animal's head with a stereotaxic frame.
6. Disinfect the shaved area with 10% povidone-iodine three times. For the area near the eye, avoid the use of alcohol-based antiseptics, consistent with the Standard of Practice set out by the Association of Surgical Technologists.
7. Make a median-sagittal incision on the head with a scalpel and from this excise a ~ 20 mm diameter circle of dermal tissue to expose the cranial bone.
8. Remove the underlying periosteum by scraping and drying with gauze to expose the coronal and sagittal cranial sutures.
9. Using a dental burr attached to a drill, trepan two holes (0.7 mm diameter, depth ~ 1 mm) through the skull on both hemispheres at the stereotaxic coordinates: 7 mm caudal to bregma, 3 mm lateral to midline.
10. Screw in stainless steel screws (diameter 0.7 mm, length 3 mm, sterilized with chlorhexidine) into the two pre-made holes up to a depth of ~ 1 mm (2 mm of screw exposed) to allow firm anchorage. This contacts the dura without damaging the underlying cortical tissue.
11. Prepare the surgical area for dental amalgam by drying the cranial bone with gauze, and retracting loose skin with two 3 - 0 sutures at ~ 4 and 8 o'clock.
12. Spread dental amalgam over the exposed skull to secure the screw electrodes (stainless steel screws described in step 1.10) in place. Ensure ~ 1.5 mm of the screws remain exposed for recording.
13. Remove the retraction sutures.
14. Inject 0.5% carprofen subcutaneously (5 mg/kg) for analgesia and saline (sodium chloride 0.9%, 1.5 ml) subcutaneously for fluid replacement.
15. Allow animals to recover in separate cages. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.
16. Do not return animal to the company of other animals until it has fully recovered from surgery (minimum 5 days).
17. Continue to administer 0.5% carprofen subcutaneously for analgesia (5 mg/kg) once a day for 4 days.
18. Record ERG and VEP 1 week following surgery.

2. ERG and VEP Recording 

1. Data collection preparation​
   1. Use computer software to simultaneously trigger stimulus and acquire data according to the settings recommended below.
      1. Amplify the signals (ERG: ×1,000, VEP: x10,000) with the gain internally set by an isolated pre-amplifier and amplifier, and with both eyes matched for impedance.
      2. Set the sampling rate for the ERG to 4 kHz over a 650 msec recording window (2,560 points), To do this, click the tab for "time base" in the data acquisition software (for name and version of software see Table for Materials), select "2,560" for Samples, and "500 ms" for time which will return a 650 msec recording window.
         1. Use the same method to set the sampling rate for the VEP to 10 kHz over a 250 msec epoch. Allow a 10 msec pre-stimulus baseline for both ERG and VEP recordings. To do this, click the "Setup" tab; select "Stimulator" to bring up a new dialogue window; in that window select "pulse" from the drop down list for "Mode"; and set the value for "delay" to "10 ms".
      3. Set ERG band-pass filtering to 0.3 - 1,000 Hz (- 3 dB). This is done by clicking "Bio Amplifier" in the data acquisition software. Then set the value for "High Pass" to "0.3Hz", and the value for "low pass" to "1 kHz".
      4. Using the abovementioned method in 2.1.1.3, set VEP band-pass settings to 0.1 - 100 Hz (- 3 dB) as recommended by the International Society for Clinical Electrophysiology of Vision (ISCEV) for human VEP recordings.

2. Electrode preparation

1. Custom-make the ERG active/inactive and VEP active/inactive electrodes by attaching silver wire or an alligator clip to an electrode lead, respectively. Commercially obtain ground electrode.
2. For the 4 custom-made electrodes, cut the male end from the electrode lead extension. Remove 1 cm of the outer polytetrafluoroethylene insulation coating with a scalpel blade ensuring the inner wire is not damaged.
3. Pre-fashion the ERG inactive electrodes by cutting a 70 mm length of silver wire (0.3 mm thickness) and forming a loop ~ 8 mm in diameter to encircle the rat eye. Prepare a uniform circle by shaping the loop on a 1 ml pipette tip.
4. Pre-fashion VEP inactive electrodes by cutting a 70 mm length of silver and forming an ellipse ~ 8 mm in lengthwise diameter to hook onto rat incisors.
5. Pre-fashion the ERG active electrodes by cutting a 30 mm length of silver wire and forming a small loop to gently contact the rat cornea (~ 1-2 mm in diameter)
6. Securely attach electrodes (2 ERG active, 2 ERG inactive, 1 VEP inactive) to the electrode lead by entwining the silver with the exposed inner wire.
7. Insulate excess exposed metal with masking tape to reduce photovoltaic artifacts.
8. On the ERG inactive electrodes stick a small piece of hook-and-loop fastener (~ 5 mm × 20 mm) to the masking tape to enable stable attachment to the rodent neck strap.
9. Attach the alligator clip to the inner wire of the electrode leads to make the VEP active electrodes.
10. Prior to recordings, electroplate the exposed surfaces of the silver wires (i.e., the inactive ring and active tip) with chloride using a 9 V DC source for 20 sec to improve signal conduction.
    1. To do this, immerse the silver tip of the ERG electrode wire (acting as the anode of a primary cell) into normal saline; and connect the other end of this electrode wire to the positive terminal of a 9 V battery.
    2. Connect another wire (the cathode) to the negative terminal of the battery, and immerse the other end into saline as well. Disconnect after 20 sec and observe the silver tip of the ERG electrode wire to be coated evenly in white color.
       NOTE: Prepare new ERG electrodes for each experimental session (~ up to 8 hr) to ensure patency of the chloride coating.

3. Animal preparation

1. Dark-adapt the animals overnight (≥ 8 hr) prior to recordings in a light-tight room. Ensure maximal dark adaptation by turning off room lights, and closing all doors and blinds. Minimize light leakage by placing light-proof materials around junctions of doors/windows and placing computer screens outside thick black curtains.
2. Conduct animal preparation in a dark room with the aid of dim red light-emitting diode (LED; 17.4 cd.m^-2, λ_max = 600 nm) to sustain rod sensitivity.         
3. Anesthetize the rat by injecting ketamine/xylazine (60: 5 mg/kg) intramuscularly. Confirm sufficient depth of anesthesia by the absence of a paw pinch reflex.
4. To maintain sedation, administer a further dose of anesthesia (50% of initial dose) after 50 min if necessary.
5. For additional topical anesthesia apply one drop of 0.5% proxymetacaine to each eye, and blink off excess fluid.
6. For pupil dilation apply one drop of 0.5% tropicamide to each eye, then dry off excess fluid.

4. ERG and VEP electrode positioning

1. Place the animal on the ERG platform in front of the Ganzfeld bowl situated in the Faraday cage. Avoid using an electrical heating pad, as it can introduce electrical noise into the electrophysiological recordings.
   NOTE: The platform is attached to a circulated heated water platform to maintain body temperature.
2. Secure the animal to the platform with a strip of hook-and-loop fastener placed firmly but not tightly around the nape.
3. Hook the inactive VEP electrode around the bottom incisors of the anesthetized rat.
4. Position the ERG inactive electrodes by encircling the scleral ring non-invasively around the eye's equator. Stabilize this by attaching electrodes to the hook-and-loop fastener strip around the nape. Repeat for the contralateral eye.
5. Fasten VEP active electrodes by attaching alligator clips to stainless-steel screws pre-implanted on the skull.
6. Place a small drop of 1% carboxymethylcellulose sodium on cornea prior to placement of the ERG active electrode to improve signal quality.
   NOTE: The viscose fluid also helps maintain corneal hydration throughout experimentation to minimize the formation of desiccation-type cataract in rodents.
7. Place a small drop of 1% carboxymethylcellulose sodium on the lower incisors to improve contact of the VEP inactive electrode and thus signal quality.
8. Position the ERG active electrodes to lightly touch the central corneal surface using a micromanipulator attached to a custom-built stereotaxic arm.
9. Insert 2 - 5 mm of the ground needle electrode (stainless steel) subcutaneously into the tail.
10. If necessary, dry any excess fluid from the inferior eyelid prior to recording to improve signal quality.
11. Slide platform closer to the Ganzfeld bowl ensuring the animal's eyes align with the opening of the bowl to enable even illumination of both retinas (see step 2.4.1).
12. Close the Faraday cage to reduce extraneous noise.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alligator clip	Generic brand	HM3022	Stainless steel 26 mm clip for connecting VEP screw electrodes to cables
Bioamplifier	ADInstruments	ML 135	For amplifying ERG and VEP signals
Carboxymethylcellulose sodium 1.0%	Allergan	CAS 0009000-11-7	Viscous fluid for improving signal quality of the active ERG electrode
Carprofen 0.5%	Pfizer Animal Health Group	CAS 53716-49-7	Proprietary name: Rimadyl injectable (50 mg/mL). For post-surgery analgesia, diluted to 0.5% (5 mg/mL) in normal saline
Chlorhexadine 0.5%	Orion Laboratories	27411, 80085	For disinfecting surgical instruments
Circulating water bath	Lauda-Königshoffen	MGW Lauda	For maintaining body temperature of the anesthetized animal during surgery and electrophysiological recordings
Dental amalgam	DeguDent GmbH	64020024	For encasing the electrode-skull assembly to make it more robust
Dental burr	Storz Instruments, Bausch and Lomb	#E0824A	A 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	An automatic drill for trepanning
Electrode lead	Grass Telefactor	F-E2-30	Platinum cables for connecting silver wire electrodes to the amplifier
Faraday Cage	custom-made		Ensures light proof to maintain dark adaptation. Encloses the Ganzfeld setup to improve signal to noise ratio
Gauze swabs	Multigate Medical Products Pty Ltd	57-100B	For drying the surgical incision and exposed skull surface during surgery
Ganzfeld integrating sphere	Photometric Solutions International		Custom designed light stimulator: 36 mm diameter, 13 cm aperture size
Velcro	VELCRO Australia Pty Ltd	VELCRO Brand Reusable Wrap	Hook-and-loop fastener to secure the electrodes and the animal on the recording platform
Isoflurane 99.9%	Abbott Australasia Pty Ltd	CAS 26675-46-7	Proprietary Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery
Ketamine	Troy Laboratories	Ilium Ketamil	Proprietary name: Ketamil Injection, Brand: Ilium. Pharmaceutical-grade anesthetic for electrophysiological recording
Luxeon LEDs	Phillips Lighting Co.		For light stimulation twenty 5 watt and one 1 watt LEDs.
Micromanipulator	Harvard Apparatus	BS4 50-2625	Holds the ERG active electrode during recordings
Needle electrode	Grass Telefactor	F-E2-30	Subcutaneously inserted in the tail to serve as the ground electrode for both the ERG and VEP
Phenylephrine 2.5% minims	Bausch and Lomb	CAS 61-76-7	Instilled with Tropicamide to achieve maximal dilation for ERG recording
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	Controls the LEDs
Proxymetacaine 0.5%	Alcon Laboratories	CAS 5875-06-9	For corneal anaesthesia during ERG recordings
Saline solution	Gelflex		Non-injectable, for electroplating silver wire electrodes
Scope Software	ADInstruments	version 3.7.6	Simultaneously triggers the stimulus via the Powerlab system and collects data
Silver (fine round wire)	A&E metal	0.3 mm	Used to make active and inactive ERG electrodes, and the inactive VEP electrode
Stainless streel screws	MicroFasterners		0.7 mm shaft diameter, 3 mm in length to be implanted over the primary visual cortex and serve as the active VEP electrodes
Stereotaxic frame	David Kopf	Model 900	A small animal stereotaxic instrument for locating the primary visual cortices according to Paxinos & Watson's 2007 rat brain atlas coordinates
Surgical blade	Swann-Morton Ltd.	206	For incising the area of skin overlaying the primary visual cortex to implant the VEP electrodes
Suture	Shanghai Pudong Jinhuan Medical Products Co.,Ltd		3-0 silk braided suture non-absorbable, for skin retraction during VEP electrode implantation surgery
Tobramycine eye ointment 0.3%	Alcon Laboratories	CAS 32986-56-4	Proprietary name: Tobrex. Prophylactic antibiotic ointment applied around the skin wound after surgery
Tropicamide 0.5%	Alcon Laboratories	CAS 1508-75-4	Proprietary name: 0.5% Mydriacyl eye drop, Instilled to achieve mydriasis for ERG recording
Xylazine	Troy Laboratories	Ilium Xylazil-100	Pharmaceutical-grade anesthetic for electrophysiological recording
Pipette tip	Eppendorf Pty Ltd	0030 073.169	Eppendorf epTIPS 100 - 5000 mL, for custom-made electrodes
Lethabarb Euthanazia Injection	Virbac (Australia) Pty Ltd	LETHA450	325 mg/mL pentobarbital sodium for rapid euthanazia
Microsoft Office Excel	Microsoft	version 2010	spreadsheet software for data analysis