Validation of a Preclinical Spinal Safety Model

Abstract

Background Preclinical studies demonstrate increased neuroapoptosis after general anesthesia in early life. Neuraxial techniques may minimize potential risks, but there has been no systematic evaluation of spinal analgesic safety in developmental models. We aimed to validate a preclinical model for evaluating dose-dependent efficacy, spinal cord toxicity, and long term function following intrathecal morphine in the neonatal rat. Methods Lumbar intrathecal injections were performed in anesthetized rats aged postnatal day (P)3, 10 and 21. The relationship between injectate volume and segmental spread was assessed post mortem and by in-vivo imaging. To determine the antinociceptive dose, mechanical withdrawal thresholds were measured at baseline and 30 minutes following intrathecal morphine. To evaluate toxicity, doses up to the maximum tolerated were administered, and spinal cord histopathology, apoptosis and glial response were evaluated 1 and 7 days following P3 or P21 injection. Sensory thresholds and gait analysis were evaluated at P35. Results Intrathecal injection can be reliably performed at all postnatal ages and injectate volume influences segmental spread. Intrathecal morphine produced spinally-mediated analgesia at all ages with lower dose requirements in younger pups. High dose intrathecal morphine did not produce signs of spinal cord toxicity or alter long-term function. Conclusions The therapeutic ratio for intrathecal morphine (toxic dose / antinociceptive dose) was at least 300 at P3, and at least 20 at P21 (latter doses limited by side effects). This data provides relative efficacy and safety data for comparison with other analgesic preparations and contributes supporting evidence for the validity of this preclinical neonatal safety model.

Intrathecal Clonidine in the Neonatal Rat

“…Percutaneous intrathecal injections were made at the low lumbar level (intervertebral space L4–5 or L5–L6) with a 30-gauge needle perpendicular to the skin, connected to a micro-injector and 50 µl Hamilton syringe. Based on our previous work (44), an injectate volume of 0.5 µl per gram bodyweight was used as this provides a uniform spread across lumbar and low thoracic segments in rat pups of all current age (and corresponding weight) groups. Following preparation of different concentrations of clonidine, an independent colleague coded samples of drug and saline, to ensure the experimenter (SMW) was unaware of treatment group during behavioral testing.…”

“…In this model, morphine had a high therapeutic ratio, i.e. toxicity was not seen at 300 times the analgesic dose in neonatal rats (44). In contrast, intrathecal ketamine increased apoptosis and glial reactivity in the spinal cord and produced persistent changes in gait and mechanical threshold in the same dose range as analgesia, i.e.…”

“…Accordingly, it is important when evaluating novel neuraxial therapeutics for neonates and infants, to define the potential effects of the agent with respect to the developing spinal cord. We have recently developed a model for assessing spinal toxicity in the neonatal rat that includes evaluation of: i) dose-dependent analgesic effects; ii) histopathology, neuronal apoptosis and glial reactivity in the spinal cord at doses up to the maximum tolerated; and iii) functional outcomes (43,44). Calculation of a therapeutic ratio (toxic dose/analgesic dose) allows comparison of the effects of different drugs at different postnatal ages.…”

General Anesthetics in Pediatric Anesthesia: Influences on the Developing Brain

“…There is not much literature looking specifically at the apoptotic potential of opioids in the developing brain. There is a report of ketamine but not morphine being associated with neuro-apoptosis in the rat pup spinal cord[48, 49]. There are several reports of neuro-apoptosis in the developing brain of animals exposed to nociceptive stimuli, which was attenuated by ketamine leading researchers to believe that pain is a potent cause of neuro-apoptosis possibly mediated by the Il-1b family of inflammatory proteins[29, 60].…”

Current and Future Issues in the development of spinal agents for the management of pain

“…The current concerns over the effects of general anesthetics on neural development in the young population has increased the focus on the neuraxial route. Yet, there has been a paucity of data to indicate the relative safety of neuraxial anesthetics in the same population (see [196,[493][494][495][496]. Given issues of pathway development and synaptic connectivity, assessment of the effects of neuraxial agents on concurrent and future spinal function must be considered in further application of analgesics and analgesic agents to this space [491].…”