Intrathecal Clonidine in the Neonatal Rat

Abstract

BACKGROUND Neuraxial clonidine is utilized for peri-operative analgesia in children of all ages. Preclinical studies in the postnatal rat allow comparison of the relative toxicity and safety of spinal analgesics throughout postnatal development. METHODS Rat pups aged 3, 7 or 21 postnatal (P) days were briefly anesthetized for intrathecal injections of saline or clonidine. At each age, the maximum tolerated, anti-nociceptive (increased hindlimb mechanical withdrawal threshold) and anti-hyperalgesic (hindpaw carrageenan inflammation) doses were determined. Lumbar spinal cord sections were assessed for apoptosis and cell death (histology, activated caspase-3 immunohistochemistry, Fluoro-Jade C staining), histopathology (haematoxylin and eosin staining), and increased glial reactivity (microglial and astrocytic markers). P3 intrathecal ketamine sections served as positive controls. In additional groups, thermal latency and mechanical withdrawal threshold were measured at P35. RESULTS Intrathecal clonidine produces age- and dose-dependent analgesia in rat pups. Maximal doses of clonidine did not alter the degree or distribution of apoptosis or increase glial reactivity in the neonatal spinal cord. No spinal histopathology was seen 1 or 7 days following injection at any age. Intrathecal clonidine did not produce persistent changes in reflex sensitivity to mechanical or thermal stimuli at P35. CONCLUSIONS Intrathecal clonidine in the postnatal rat did not produce signs of spinal cord toxicity, even at doses much greater than required for analgesia. The therapeutic ratio (maximum tolerated dose/anti-hyperalgesic dose) was >300 at P3, >30 at P7, and >10 at P21. These data provide additional information to inform the clinical choice of spinal analgesic agent in early life.

Evaluation of Spinal Toxicity and Long-term Spinal Reflex Function after Intrathecal Levobupivaciane in the Neonatal Rat

“…Spinal cord function was assessed at 5 weeks of age (P35) by evaluating hindlimb withdrawal reflex responses to mechanical and thermal stimuli, with the experimenter blinded to initial treatment group. 29 Following habituation on an elevated mesh platform, the mechanical stimulus (electronic von Frey device; Dynamic Plantar Aesthesiometer, Ugo Basile, Comerio, Italy) was applied to the mid-plantar surface of the hindpaw. The threshold was averaged from 3 measures of the force (0 to 50 grams; ramp 20 grams/sec) required to produce hind-limb withdrawal.…”

“…Transverse sections of lumbosacral spinal cord (7 and 14 µm) were cut using a cryostat (Leica CM 1800, San Marcos, CA), mounted on slides (Fisher Superfrost Plus, Fisher Scientific, Houston, TX), and stored at −30°C. Using our previously described protocols, 27–29 we assessed: histopathology with hematoxylin and eosin staining 24hrs and 7 days post-injection; apoptosis with activated caspase-3 immunohistochemistry and Fluoro-Jade C staining at 24 hours; and glial reactivity by staining with microglial (ionized calcium binding adapter molecule 1; Iba-1) and astrocytic (glial fibrillary acidic protein) markers 7 days following injection. For histopathological evaluations of the nerve roots, cauda equina was cut from the spinal cord 24 hours and 7 days following injection and transferred into 2.5% glutaraldehyde in 0.1M PB.…”

“…Compared with the same dose of racemic bupivacaine, levobupivacaine has an improved systemic toxicity profile after intravenous administration 39 and reduced neurotoxicity following intrathecal administration in some, 23 but not all, 40 adult animal studies. Our developmental model of spinal toxicity had sufficient sensitivity to demonstrate a relatively wide safety margin following morphine 27 and clonidine, 29 but adverse outcomes following ketamine, 28 and will now be the basis for further evaluation of local anesthetic toxicity in the neonatal spinal cord.…”

“…26 Prolonged general anesthesia, but not intrathecal bupivacaine, at P7 increases apoptosis in both cortex and spinal cord, but neither intervention produced long-term motor deficits. 20 As our previous work demonstrated higher baseline apoptosis in the cord at P3/4, predominantly in the dorsal horn, 27–29 we have included a younger age group and evaluation of sensory outcomes in our toxicicty studies. As different local anesthetics produce varying degrees of apoptosis in neuronal cell cultures, 30,31 evaluation of additional preparations in in-vivo developmental models will further define the safety profile of spinal anesthesia in early life.…”

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].…”