Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit
Summary
Transcutaneous intratracheal injection allows for effective intrapulmonary drug delivery during spontaneous respiration. Single and multiple injections are well tolerated with no effect on survival. The technique is simple to perform and can examine the effect of substances on lung development and the prevention of lung injury in newborn rabbits.
Abstract
Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and minimizing systemic drug exposure. The transcutaneous technique is simple and allows for the IT delivery of substances to the lung of prematurely born rabbits shortly after birth. Newborn pups are anesthetized with inhaled Isoflurane before being placed in a supine position with the neck extended. The larynx is identified and stabilized before transcutaneous placement of a 26-gauge (G) catheter into the trachea. Following catheterization of the trachea, a 30 G blunt needle attached to a Hamilton syringe is introduced into the IT catheter and is used for delivering a precise volume into the trachea during spontaneous respiration. After the IT injection is completed, the needle and catheter are withdrawn, and the pup is allowed to recover from anesthesia. Transcutaneous IT injection delivers a large proportion of the injected substance to the lung, with the majority remaining in the lung 3 hours after the intervention. The injections are well tolerated from the day of birth and can be repeated for multiple consecutive days without influencing survival. This technique can be used to investigate the effect of pharmaceutical agents on lung development and in the prevention of neonatal lung injury in preterm rabbits.
Introduction
Chronic neonatal lung disease (CNLD) following premature birth continues to occur in a significant number of infants1. Improved modern neonatal care has significantly increased survival and decreased the majority of significant complications following preterm birth. While neurological, gastrointestinal, and ophthalmological complications have decreased, respiratory complications remain largely unchanged over the past 2 decades with nearly one in two infants born before 28-week gestation developing lung disease.
Prematurity, inflammation, oxidative damage, and ventilator-associated injury all play a role in the pathophysiology of CNLD and poor respiratory outcomes following preterm birth2,3,4. Despite the significant advancements of modern neonatal care, limited effective therapy is available to treat or prevent the development of CNLD5,6.
New approaches and interventions are required to develop therapy to prevent and treat CNLD. Intrapulmonary drug delivery is an attractive intervention to deliver drugs to the lung and could alter the course of respiratory disease in neonates. Intrapulmonary drug therapy has the benefit of direct delivery of active agents to the lung, thereby minimizing accumulation of the drug in off-target organs7,8, potentially limiting systemic side effects. Despite over 2 decades of intrapulmonary surfactant replacement, no additional intrapulmonary drugs have been validated to improve neonatal respiratory outcomes. Recently, budesonide-surfactant combination therapy has been described to improve pulmonary outcomes following preterm birth in mechanically ventilated infants9,10. However, much remains unknown on the functional and structural effects of IT drug therapy, few new therapies have been identified, and the value of intratracheal drug delivery in the neonatal period remains uncertain. Animal models are required to identify potential drugs and aid the development of much needed therapy for CNLD.
Animal studies examining newborn lung disease are most commonly performed in small animal models such as rats and mice11,12,13. The rabbit has the additional advantage of preterm delivery to more closely mimic the structure and function of the immature human lung14. A limitation of the preterm rabbit is the difficulty of accessing the airway to allow the delivery of intrapulmonary interventions. While adult rabbit and rodent models allow trans-oral endotracheal intubation, these techniques are difficult in newborn pups due to their small size and the unique anatomy of the upper airway15,16. Alternative approaches are required to allow access to the trachea for the delivery of drugs in newborn rabbit pups.
In this manuscript, we describe the use of a transcutaneous needle tracheostomy to allow tracheal intubation and drug delivery.
Protocol
Representative Results
Discussion
Several critical steps should be followed to successfully perform IT injection. When performed correctly, the transcutaneous IT injection method allows for effective and reliable intrapulmonary drug delivery in the preterm rabbit. Temperature control is important as the newborn pups easily become hypothermic, which can negatively influence survival. Prior to placing the pups in the induction chamber, temperature control should be ensured to maintain normothermic conditions. A heating matt placed under the induction chamb…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by a C2 grant from KU Leuven (C24/18/101) and a research grant from the Research Foundation – Flanders (FWO G0C4419N). A.G. is supported by the Erasmus+ Programme of the European Commission (2013-0040). Y.R. is holder of an FWO-SB fellowship (Research Foundation – Flanders, 1S71619N). None of the funding bodies were involved in the design of the study and in the collection, analysis, and interpretation of data.
Materials
| Anesthesia | |||
| Heating matt to prevent cooling during anesthesia | 1 | ||
| Isoflurane vaporizer with oxygen supply | 1 | ||
| Isoflurane (Iso-Vet; 1000 mg/g) | Dechra Veterinary Products NV, Belgium | 2% at 2 liters/minute | |
| Plexiglas induction chamber with exhaust and scavenger | In house built | 1 | |
| Positioning for injection | |||
| Mounting stage | In-house built (made out of styrofoam to allow flexible positioning | 1 | |
| Nose cone connected to anesthetic circuit | 1 | ||
| Scavenger system | 1 | ||
| Tape to restrain limbs | Any | 1 roll | |
| Intratracheal injection | |||
| Allis tissue forceps | 1 | ||
| 19-mm-long 26-gauge catheter | BD Biosciences | 391349 | 1 |
| Hamilton syringe (10µl with 20 mm blunt 30-gauge needle | Hamilton Company | 7638-01 | 1 |
| Pharmaceutical substance of choice | as per protocol | ||
| Saline (0.9% NaCl) | 5 µl per animal | ||
| Animal housing | |||
| Humidity- and temperature-controlled incubator | Okolab Srl. Custom built cage incubator. Alternatively, in-house built cage incubators can be used |
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