Implantation of Electroencephalogram and Electrocardiogram Telemetry Devices in Neonatal Rabbit Kits

Summary

Mechanisms of sudden unexpected death in epilepsy (SUDEP) are poorly understood and challenging to translate from current models. Transgenic rabbits may offer insights into these mechanisms. We describe a method for long-term, continuous electroencephalography and electrocardiography recordings in transgenic rabbit kits to evaluate serious events that may lead to death.

Abstract

Pathogenic variants in ion channel genes are associated with a high rate of sudden unexpected death in epilepsy (SUDEP). Mechanisms of SUDEP are poorly understood but may involve autonomic dysfunction and cardiac arrhythmias in addition to seizures. Some ion-channel genes are expressed in both the brain and the heart, potentially increasing the risk of SUDEP in patients with ion-channelopathies associated with epilepsy and cardiac arrhythmias. Transgenic rabbits expressing epilepsy variants provide a whole organism to study the complex physiology of SUDEP. Importantly, rabbits more closely replicate human cardiac physiology than do mouse models. However, rabbit models have additional health and anesthesia considerations when undergoing invasive monitoring procedures. We have developed a novel method to surgically implant a telemetry device for long-term simultaneous electroencephalogram (EEG) and electrocardiogram (ECG) monitoring in neonatal rabbit kits. Here, we demonstrate surgical methods to implant a telemetry device in P14 (weight range 175-250 g) kits with detailed attention to surgical approach, appropriate anesthesia and monitoring, and postoperative care, resulting in a low complication rate. This method allows for continuous monitoring of neural and cardiac electrophysiology during critical points in the development of cardiac arrhythmias, seizures, and potential SUDEP in rabbit models of genetic or acquired epilepsies.

Introduction

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of death in patients with epilepsy. Mechanisms of SUDEP are poorly understood but potentially involve autonomic dysfunction, apnea, and cardiac arrhythmias in addition to seizures^{1,2,3,4,5,6,7}. Patients with channelopathy-linked genetic epilepsies have among the highest rates of SUDEP. For example, SUDEP occurs in up to 20% of patients with variants in the voltage-gated sodium channel gene SCN1A⁸, the gene responsible for Dravet syndrome, a genetic epilepsy with onset in the first year of life. Many epilepsy-linked ion-channel genes are expressed in both the brain and the heart, with laboratory and clinical data suggesting that cardiac arrhythmias may be present in patients with channelopathy-linked genetic epilepsies^{7, 9,10,11,12}, potentially increasing their risk of SUDEP due to a seizure-induced fatal cardiac arrhythmia or simultaneous occurrence of seizures and arrhythmias. Evaluating SUDEP in the laboratory setting poses numerous challenges. From a cardiac viewpoint, cardiac action potentials in mice are very different than in humans¹³, and human iPSC-cardiac myocyte models¹⁴ cannot replicate the complexities of the whole organism. Transgenic rabbit models of genetic epilepsies provide an ideal system to study SUDEP, as rabbit cardiac physiology more closely replicates that of the human^13,15, while providing a whole organism to study complex pathophysiology. As SUDEP may occur as early as the first seizure, evaluating these animal models from an early time point is essential to understanding the onset of both seizures and cardiac arrhythmias. Video recording during the neonatal period is challenging, as rabbit kits are often still in the nest. Continuous electroencephalogram (EEG) or electrocardiogram (ECG) recording with a traditional wired system is not possible while kits are with the dam. Intermittent recording is unlikely to capture rare, terminal events associated with SUDEP. We have therefore turned to wireless implantable telemetry monitoring to provide long-term, continuous, simultaneous EEG and ECG recording in rabbit kits.

Keys to success in this protocol are appropriate anesthetic and postoperative support for these vulnerable animals. Rabbits are at a much higher risk of anesthetic death (1.39%-4.8%) compared to dogs and cats (0.17%-0.24%) due to unique anatomical and physiologic characteristics^16,17. The main contributors to this increased anesthetic risk include sub-optimal airway management and acute postoperative complications. Multiple factors contribute to the difficulty of intubation in rabbits, including a long, narrow mouth with a broad tongue, an acute angle between the mouth and larynx, dorsal displacement of the epiglottis, increased susceptibility to laryngeal trauma, and increased propensity to laryngospasm^18,19,20. After the immediate anesthetic episode, rabbits are at risk of developing life-threatening gastrointestinal stasis syndrome. This is a complex, multi-factorial problem, and anesthesia is postulated to be contributory via direct drug effects inhibiting gastric motility and/or secondary anorexia post-procedurally for any reason (unrelieved pain, nausea, etc.)²¹.

The unique physiology of rabbit neonates and infants compound the challenges associated with anesthesia and surgery. Rabbits have altricial young born with underdeveloped mechanisms for physiologic homeostasis and special anatomical considerations. Intravenous access and monitoring are difficult as most commercial products are not optimized for the small vascular size, high resting heart rate, and pigmented skin of Dutch-belted and New Zealand White cross rabbit kits. As cardiac output is essentially heart rate dependent in neonates²² and, in general, drug clearance by the renal or hepatic route is decreased compared to adults²³, considerations for appropriate drug selection and dosage are critical. The primary cause of anesthetic death in rabbits is thought to be secondary to respiratory depression and apnea. In addition to the airway management problems already discussed for all rabbits, neonates have a depressed respiratory drive in the face of hypoxemia and hypercapnia, making this already challenging aspect of anesthesia more risky²⁴.

In this protocol, we describe a successful method for EEG and ECG telemetry implant (Figure 1) in a neonatal rabbit model of epilepsy with a high surgical and anesthetic survival rate. This information will enable other researchers to tackle challenging neonatal rabbit models to advance research into epilepsy, cardiac arrhythmia, and related neurodevelopmental disorders.

Protocol

All work described was reviewed and approved by the University of Michigan Institutional Animal Care and Use Committee as part of an approved animal use protocol and is in line with relevant federal laws and guidelines, including the USDA Animal Welfare Act and NIH Public Health Service Policy. The University of Michigan is an AAALACi-accredited institution.

1. Animal preparation

1. Rough-shave kits (age P14-P19, weight >175 g) 1-2 days prior to the procedure to minimize anesthesia time on the day of surgery using clippers.
2. Sterilize all surgical tools and materials (autoclave or gas sterilization).
   NOTE: There are no formulations of dental cement where sterility is ensured in the manufacturing process. As such, use a new and in date container of dental cement for each surgical cohort and include this exception in the IACUC approved protocol.
3. Induce anesthesia with ketamine (10 mg/kg IM), buprenorphine HCl (0.01 mg/kg IM) 0.3 mg/mL diluted to 0.03 mg/mL, and sevoflurane and oxygen via mask anesthesia using a non-rebreathing circuit.
4. Place a 26 G ¾" intravenous (IV) catheter in the auricular vein (preferred) or cephalic vein and flush with heparinized saline 10 units/mL.
5. Shave the abdomen, chest, back, neck, and head as close to the skin as possible using a #40 or #50 blade.
6. Apply non-medicated lubricating ophthalmic ointment to prevent corneal ulceration.
7. Administer analgesics (carprofen 4 mg/kg SQ - diluted to 25 mg/mL) and peri-operative antibiotic (cefazolin 20 mg/kg IV diluted to 50 mg/mL). Re-administer antibiotics every 90-180 min of surgical time.

2. Surgical preparation (Figure 2)

1. Transfer the anesthetized kit to the operating table and place it supine on an infrared heating pad controlled via a rectal thermometer.
2. Place the nose and mouth in a custom 3D printed facemask connected with a swivel connector to a non-rebreathing Jackson-Rees circuit (0.5 L bag) and maintain on sevoflurane anesthesia (1.5%-7% to effect) with oxygen flow at 2 L/min.
3. Maintain anesthesia with sevoflurane and monitor the depth of anesthesia with a pulse oximeter on either the ear or paw and/or a Doppler on either the femoral artery or directly on the heart.
4. Adjust anesthesia throughout the procedure to maintain heart rate (HR) between 180-260, oxygen saturation >95%, and respiratory rate between 10-50 breaths per minute (direct visualization of excursions or movement of the re-breathing bag).
   NOTE: Have emergency drugs available (glycopyrrolate, epinephrine, doxapram).
5. Secure the kit to the face mask by gently taping the front limbs to the mask.
6. Position the kit in a slightly right-lateral position by loosely securing the left hind leg to the operating table.
7. Prepare the entire abdomen with a warmed surgical scrub alternating betadine and sterile saline.
   NOTE: An additional surgical solution can be used to finish the scrub if desired. The surgeon, wearing dedicated scrubs, hair bonnet, and shoe covers, will aseptically scrub and don sterile gown and gloves to perform the procedure under sterile conditions.
8. Place adhesive surgical towels on either side of the kit and cover it with a large surgical drape. Cut an appropriately sized hole to expose the abdomen and chest.
9. Open the implant onto the surgical field and place non-absorbable anchor sutures into each of the implant anchor holes. Leave the suture attached with a 5-6 cm tail (Figure 3A). Place the implant in a bowl of warmed sterile saline.

3. Placement of the implant in the abdomen

1. After ensuring adequate anesthesia, make a 3 cm incision through the skin along the linea alba with a scalpel approximately 1-2 cm caudal to the last rib.
2. Make a careful incision through the muscle to open the peritoneal cavity.
3. Place the implant into the cranial portion of the abdominal cavity and position it to the left of the incision.
4. Use a trocar to tunnel the negative ECG wire out of the peritoneal cavity and skin approximately 2 cm to the right of the incision. Tunnel the remaining 3 wires 3-4 cm to the left of the incision to allow the implant to sit comfortably within the peritoneal cavity.
5. Secure the implant with the anchor sutures to the ventral wall of the peritoneal cavity, ensuring no bowel entrapment (Figure 3B).
6. Close the abdominal wall with an absorbable suture in a continuous pattern.
7. Close the skin incision with a non-absorbable suture in an interrupted pattern.

4. Placement of the ECG leads

1. Tunnel the negative ECG lead subcutaneously to the right upper chest at the level of the first rib.
2. Bluntly dissect a subcutaneous pocket to loosely coil approximately 10 cm of wire.
   NOTE: Tight coils under the skin may lead to skin erosion and wire exposure.
3. Cut the excess wire and create a loop with the exposed wire by tying the end to the insulated wire with a non-absorbable suture.
4. Secure the loop to the muscle with 2 non-absorbable sutures.
5. Close the peritoneal muscle on the right around the wire with 1-2 absorbable sutures in an interrupted pattern.
6. Close the skin at the right upper chest and right abdomen with 2-3 non-absorbable sutures in an interrupted pattern.
7. Tunnel the positive ECG lead to the left lower rib and repeat the above steps to secure it to the muscle and close the incision.
8. Tunnel the EEG leads subcutaneously to the left lateral side as far as possible in the surgical field.
9. Add 1 absorbable suture to the peritoneal muscle on the left around the emerging wires. Close the skin with non-absorbable suture in an interrupted pattern.
10. Wrap the exposed EEG wires with sterile aluminum foil.

5. Preparation of the dorsal surface

1. A non-sterile assistant will then remove the sterile drape and leg tie.
2. Turn the kit into the prone position (Figure 2B) while ensuring the face mask stays securely in place by rotating using the swivel connector between the face mask and circuit. Adjust pulse-ox and/or doppler monitors as needed to ensure continuous anesthetic monitoring.
3. Prepare the surgical field with a betadine scrub to include the head, neck and entire back with care to scrub around the region of the exiting wires on the left side.
4. The surgeon will then place a sterile adhesive towel under the left side while the aluminum foil packet of wires is held by a non-sterile assistant.
5. Sterilely and gently remove the wires from the aluminum packet and place them on the sterile field. Finish draping with sterile towels.
6. Cover with a sterile drape and cut a window large enough to expose the entire sterile field.

6. Placement of EEG leads

1. Make a 3 cm incision through the scalp at the midline to expose the skull.
2. Use a trocar to subcutaneously tunnel the EEG leads from the left side to the skull.
3. Clean and scrape the periosteum from the exposed parietal bones using a scalpel.
4. Insert a handheld drill into a sterile ultrasound cover. Insert a 1.0 mm drill burr into the drill.
5. Drill bilateral burr holes into the parietal bones approximately 0.5 cm anterior to lambda and 0.5 cm lateral to the sagittal suture.
   NOTE: Caution with the amount of pressure placed on the dorsal skull as this can occlude the ventral airway, so respiratory monitoring is key at this point in the procedure. A steady or significant drop in SpO₂ and heart rate may indicate respiratory occlusion (bradycardia secondary to apnea) and should prompt immediate assessment and action.
6. Use fine forceps to place a screw into the burr hole. Use the screwdriver to insert approximately halfway so the screw tip remains epidural, ~1-1.5 mm (Figure 3C).
7. Bluntly dissect a subcutaneous pocket along the back of the neck to loosely coil approximately 10 cm of wire.
8. Cut the excess wire. Strip the insulation from the tip and stretch the wire.
9. Create a loop at the end of the exposed wire by tying a knot, keeping a small loop. Place the loop over the screw and tighten the screw to the skull, ensuring that the wire is touching the screw. Place the ground wire on the left and the recording wire on the right.
10. Assess the telemetry signals on the analysis software for fidelity once all wires are in place. The EEG signal will appear at a low amplitude while the kit is sedated.
11. Secure the screws and wires to the skull with dental acrylic and allow it to harden completely.
12. Close the skin with a non-absorbable suture on the head and the left flank.
13. Inject bupivacaine (maximum dose 2 mg/kg of 5 mg/mL diluted to 2.5 mg/mL) subcutaneously at each incision. Cover each incision with a small amount of skin glue administered using a tuberculin syringe if necessary for secure skin closure.

7. Anesthesia recovery

1. Turn off sevoflurane anesthesia and provide only oxygen for at least 5 min while removing the remaining tape, draping, and anesthetic monitoring.
2. Check blood glucose levels using a glucometer and administer warmed subcutaneous fluids at 10% of body weight (kg).
3. Once the animal is reactive to a painful stimulus (toe pinch), move to a recovery incubator set at 37-38 °C.
   NOTE: Often, the kit's temperature will significantly drop during this transfer. It may be beneficial to return the kit to the biofeedback infrared heating pad or provide additional supplemental heat.
4. Monitor visually continuously and record rectal temperature, pulse oximeter readings, heart rate, and respiratory rate every 10-15 min.
5. Once the animal is consistently ambulatory and alert, remove the intravenous catheter and apply pressure to the site until the bleeding stops.

8. Postoperative care and monitoring

1. Return kit to the dam and litter mates. Ensure nesting material and supplemental nutrition (Table of Materials) are available in the cage to aid in thermoregulation and recovery.
2. Check on the kit daily for 7-10 days following the surgery, weighing daily and providing supplemental nutrition in the cage.
3. For the first 2 days post-recovery (D1 and D2), give additional analgesics every 24 h (carprofen 4 mg/kg SQ - diluted to 5 mg/mL) and subcutaneous fluids (5-7 mL).
4. For the first 3 days post-recovery (D1, D2, and D3), check on the kit twice per day, evaluating for evidence of pain, ambulation, incisional appearance, and hydration. Once per day during this time period, take the kit's temperature to ensure no evidence of infection and appropriate thermoregulation.
5. Remove sutures if incisions appropriately heal at 7-10 days.

Results

The successful outcome of this project required the development of multiple parameters in the implant procedure and recording protocol. Implant surgery was attempted or performed on 16 rabbit kits, with 14 successfully surviving the procedure. Of those, 12 survived to the experimental endpoint. Reasons for intraoperative or postoperative death are highlighted in Table 1, along with procedure modifications that allowed for future success in achieving the experimental endpoint. The most common operative co...

Discussion

The protocol described for anesthetic induction, monitoring, and support balances research needs for surgical approach and ease with gold standards of veterinary care. Prior to the laboratory adopting the described protocol as standard procedure, several other potential refinements were trialed, including dorsal subcutaneous implant placement, the use of an endotracheal tube or laryngeal mask airway (LMA), and the use of an esophageal stethoscope attachment for heart rate monitoring. However, all were ultimately abandone...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 inch elastic wrap - Coban or Vetwrap	3M	https://www.3m.com/3M/en_US/p/d/b00003186/
4-0 PDS monofilament suture	Ethicon	https://www.jnjmedtech.com/en-US/company/ethicon/all-products
5-0 Ethilon nylon suture	Ethicon	https://www.jnjmedtech.com/en-US/company/ethicon/all-products
Acquisition computer	Dell	https://www.dell.com/en-us
Adhesive surgical towels	N/A	N/A
Anesthesia circuit - Jackson-Reevs with 0.5 L rebreathing bag	JorVet	J0248GA
Betadine scrub	N/A	N/A
Bupivicaine (0.5%)	N/A	N/A	Diluted to 2.5 mg/mL prior to administration
Buprenorphine (0.3 mg/mL)	N/A	N/A	Diluted to 0.03 mg/mL prior to administration
Burr - 1.00 mm	Cell Point Scientific	60-1000	to drill skull
Cafazolin (1 g lypholized)	N/A	N/A	Diluted to 50 mg/mL
Carprofen (50 mg/mL)	MWI Veterinary		Diluted to 25 mg/mL prior to administration
Cotton tipped applicators	N/A	N/A
Custom 3-D printed face mask	N/A	https://www.thingiverse.com/thing:923725
Dental acrylic	N/A	N/A
Diet Gel Criticare	Clear H2O	72-05-5042	Nutritional support
Dopper Gel - Aquasonic	Patterson	07-890-5542
Doppler - Vet-Dop2	Patterson	07-888-8986
Doxapram (20 mg/mL)	MWI Veterinary	N/A	Emergency only
Dumont #5 Fine Forceps	Fine Science Tools	11254-20	For holding screws
Duraprep	3M	8630	Final skin prep
ecgAuto data analysis software	emka technologies	N/A
Epinephrine (1:1000)	MWI Veterinary	N/A	Emergency only
Gauze	N/A	N/A
Glucometer ipet Pro	MWI Veterinary	63867	Monitor if poor recovery
Glycopyrrolate (0.2 mg/mL)	MWI Veterinary	N/A	Emergency only
Gram scale	N/A	N/A
Hemostats	Fine Science Tools	13008-12	Hold wire loops while tying the loop in place
Ideal Micro-drill	Cell Point Scientific	67-1204	To drill skull
Incubator	DRE-veterinary (Infantia - NB1)	N/A
Induction box	VetEquip	941444
Infared heating pad - RightTemp Jr	Kent Scientific Corporation	RT-0502
IOX2 data acquisition software	emka technologies	N/A
IV Catheter - Covidein Monoject 26 G, 3/4 inch PTFE	Patterson	07-836-8494
ketamine (100 mg/mL)	MWI Veterinary	N/A
Medical tape	N/A	N/A
Narrow Pattern Forceps - Straight/12 cm	Fine Science Tools	11002-12
Neonatal stethescope	Ultrascope	N/A
Olsen-Hegar Needle holder with scissors - 12 cm	Fine Science Tools	12002-12	For suturing
Ophthalmic ointment Puralube	MWI Veterinary	N/A	Administered to both eyes during anesthesia
Opthalmic Lubricant - Paralube Vet	Patterson	07-888-2572
Pulse oximeter (AccuWave Portable )	Patterson	07-892-9128	For prep and recovery; reads HR up to 400
Pulse oximeter (SDI - Vet/Ox plus 4700)	Heska	N/A	Intra-operative; no longer producted
Receiver	emka technologies	N/A	1 receiver for every 4 telemetry implants
Rectal thermometer	N/A	N/A
Scalpel	Fine Science Tools	10003-12
Scissors	Fine Science Tools	14002-12	To cut drape
Screw driver - 1.0 mm	N/A	N/A	From mini-screwdriver set for electronics
Screws 00-96 x 3/32 (2.4 mm)	Protech International	8L0X3905202F
Sevoflurane	MWI Veterinary		Maintenance anesthesia
Sevoflurane vaporizer and anesthesia machine	N/A	N/A
Skin glue, Gluture	MWI Veterinary	34207	Apply sparingly with syringe
Small scissors	Fine Science Tools	14084-08
Sterile aluminum foil	N/A	N/A	To wrap wires prior to rotating animal
Sterile paint brush	N/A	N/A	To apply dental acrylic
Sterile Saline	N/A	N/A
Sterile surgical gloves	N/A	N/A
Sterile ultrasound cover	N/A	N/A	To cover the drill
Sterile Water	N/A	N/A	For cefazolin reconstitution
Surgical blade no. 15	N/A	N/A
Surgical drape	N/A	N/A
Surgical gown	N/A	N/A
Swivel connector - Jorgensen Labs	Patterson	07-802-2349	To connect anesthesia circuit to face mask
Telemetry implant	emka technologies	easyTEL+_M1_EETA_B_35
Trocar	SAI	TRO-10-6	To tunnel wires