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* These authors contributed equally
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.
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.
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 seizures1,2,3,4,5,6,7. 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 epilepsies7, 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 human13,15, 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 characteristics16,17. 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 laryngospasm18,19,20. 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.
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
2. Surgical preparation (Figure 2)
3. Placement of the implant in the abdomen
4. Placement of the ECG leads
5. Preparation of the dorsal surface
6. Placement of EEG leads
7. Anesthesia recovery
8. Postoperative care and monitoring
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...
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, and the use of an esophageal stethoscope attachment for heart rate monitoring. However, all were ultimately abandoned for ...
The authors have nothing to disclose.
The authors are grateful for funding by NIH R61NS130070 to LLI.
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 administrationΒ |
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 supportΒ |
Dopper Gel - Aquasonic | PattersonΒ | 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 PTFEΒ | PattersonΒ | 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 | PattersonΒ | 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 animalΒ |
Sterile paint brush | N/A | N/A | To apply dental acrylicΒ |
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 | PattersonΒ | 07-802-2349 | To connect anesthesia circuit to face mask |
Telemetry implant | emka technologies | Β easyTEL+_M1_EETA_B_35 | |
Trocar | SAI | TRO-10-6 | To tunnel wires |
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