Balancing Nociceptive Activity in the Developing Spinal Cord

CONTEXT: In the UK, 2.5% of all newborn babies need treatment in intensive care after birth, and babies born preterm (7.7% of all UK births) are more likely to need intensive care for prolonged periods. As part of treatment, these babies may be exposed to a range of pain stimuli ranging from brief but frequent procedural interventions such as blood tests, through to major surgical procedures to treat complications or correct birth defects (eg. congenital heart abnormalities). Even the youngest babies respond to painful stimuli, but as the nervous system is not fully mature, the response to pain, surgical injury and different analgesics can vary from that seen in older children and adults. Different treatments are available to reduce pain, but we need more evidence to confirm which are the safest and most effective for these vulnerable patients. Many years following neonatal surgery or intensive care, changes can still be measured in children: sensitivity to temperature and pressure is different; they are more sensitive to painful stimuli; and they need more analgesia if further surgery is needed.

In order to understand how pain and injury in early life produce persistent changes in pain sensitivity, we are evaluating the impact of surgical injury (plantar hindpaw incision) in newborn rats. This incision produces longterm changes in sensory processing and an increased sensitivity to repeat surgery in later life that mirrors the changes we have seen in children. As almost one third of post-surgical patients treated by our Acute Pain Service at Great Ormond Street Hospital are undergoing surgery for at least the second time, determining why the developing nervous system responds in this way will help us identify children at risk of increased pain sensitivity and improve their treatment. As our laboratory studies show that changes in sensitivity are permanent, learning how to prevent these effects has an impact that extends beyond the healthcare of children and could help explain why, despite having the same operation, some adults develop more severe and prolonged pain.

AIMS AND OBJECTIVES: The spinal cord is an important site for modulating pain inputs, but pain signaling here is not fully mature at birth, and sensitivity can be altered by high levels of pain input in early life. Based on our previous work, we propose that factors contributing to the longterm increased pain sensitivity include; i) effects on the sensitivity of nerve cells (neurons) and circuits within the spinal cord; ii) an excessive loss of nerve cells that would inhibit pain signaling at later stages; or iii) interactions with microglial cells which which can retain a long-term "memory" of this early injury and communicate with neurons to alter their level of sensitivity.

Objective 1. How widely distributed to other parts of the body is the increased pain sensitivity following neonatal surgery?
Objective 2. Does increased death of nerve cells (neuronal apoptosis) in the neonatal spinal cord increase pain sensitivity? Can these long-term changes be blocked by different types of analgesia?
Objective 3. Do spinal microglia contribute to the increased apoptosis in the neonatal cord and subsequent pain hypersensitivity in adulthood?

POTENTIAL APPLICATIONS AND BENEFITS: Results from these experiments will help us in two ways:
i) identifying patients at risk of increased pain sensitivity so we can adjust their treatment accordingly. Improved understanding of how surgical injury in early life alters pain sensitivity, and if these changes are generalized from surgery at different body sites, will determine which groups are at risk of persistent post-surgical pain throughout later life and into adulthood.
ii) determine which analgesics are both safe to use in neonates and are most effective at preventing long-term changes in sensitivity. Demonstrating beneficial effects by reducing microglial reactivity has the potential to lead to new treatments.

Plantar hindpaw incision in the first postnatal week, but not at older ages, is associated with an enhanced response to subsequent incision. Factors in the spinal cord that contribute to developmentally-regulated alterations in nociceptive sensitivity include: activity-dependent changes in neuronal circuitry; neuronal apoptosis; and microglial reactivity.

To assess the long-term effects of neonatal surgical injury, neuronal and microglial responses to adult incision will be compared in animals with prior injury, and in age and gender-matched controls. Outcomes will include:
i) behavioural and electrophysiological measures of spinal reflex sensitivity to allow quantification of the response to both threshold and suprathreshold stimuli;
ii) in vivo extracellular recordings in the spinal cord to directly quantify dorsal horn neuronal sensitivity; and
iii) complementary tissue analysis and functional measures: tissue analysis and evaluation of the degree and distribution of activation by MAPK phosphorylation (pERK in neurons and p-p38 in microglia); plus dose-dependent effects of inhibitors of phosphorylation on behavioural hyperalgesia.

The ability of mechanistically distinct neonatal analgesics (morphine or ketamine vs saline) to balance the effects of neonatal injury will be assessed by effects on:
i) acute hyperalgesia, apoptosis, and microglial reactivity;
ii) long-term hyperalgesic and microglial response to subsequent adult incision (in rats and mice).

Microglial contributions to enhanced sensitivity will be assessed by:
i) in vivo imaging for detailed temporal and spatial changes in neonatal and adult mice with microglial-specific fractalkine receptor CX3CR1 locus replaced with the gene encoding green fluorescent protein;
ii) effects of neonatal microglial inhibition (minocycline) or ablation (intrathecal Mac1-saporin).

In the UK, 2.5% of all newborn babies need intensive care, and preterm birth has become a public health priority due to its high prevalence (7.7% of UK births) and cost of care (Blencowe et al. Lancet 2012). The Department of Health Report of the Children and Young People's Health Outcomes Forum (www.dh.gov.uk/health/files/2012/07/CYP-report.pdf) recognized that management of pain is an important health care outcome for children and parents.

TRANSLATIONAL IMPACT AND IMPROVED HEALTH OUTCOMES. This proposal has translational clinical relevance from two different perspectives. Firstly, understanding how the developing nervous system responds to injury and analgesia is essential for informing clinical choice of the most effective and developmentally appropriate analgesic and anaesthetic interventions. Secondly, relevance extends beyond the care of neonates and infants, as our laboratory work has shown that repeat surgery at all times to adulthood is associated with an increased degree and duration of hyperalgesia. One third of patients managed by the PI with the Acute Pain Service at Great Ormond Street Hospital are undergoing major surgery for at least the second time. Persistent post-surgical pain occurs in 10-50% of adults, but much of the individual variability in response remains to be explained. We propose that pain and injury in early life are important risk factors for persistent post-operative pain in later life, and improving management will impact on both acute and long-term pain sensitivity and well-being. The PI contributes to evidence-based acute pain management clinical practice guidelines that have been endorsed by national and international professional bodies. These regularly updated, free to download documents are widely accessible to both health care providers and the public (http://www.fpm.anzca.edu.au/resources/books-and-publications/publications-1/Acute%20Pain%20-%20final%20version.pdf; http://www.apagbi.org.uk/publications/apa-guidelines), and include recent research related to pain mechanisms and analgesic pharmacodynamics. The importance of neonatal and infant pain management is recognized as a research priority as there is limited high quality clinical evidence to guide clinical choice of the safest and most effective analgesic interventions at this age.

PHARMACEUTICAL INDUSTRY: Mechanism-based analgesic targets, particularly neuroimmune interactions and the development of more specific drugs, are of substantial interest to the pharmaceutical industry. Our proposed work will provide proof of concept evidence for potential neonatal preventive strategies and also identify targets for minimizing persistent postoperative pain in later life.

CHARITIES AND PUBLIC INTEREST: Pain is one of the most feared consequences of hospitalization for children and the safe and effective administration of anaesthetics and analgesics for young children is of major public interest. The PI has promoted paediatric pain research with the Great Ormond Street Hospital Children's Charity, which competitively funds peer-reviewed research both locally and nationally. The PI contributed to the Association of Paediatric Anaesthetists of Great Britain Policy Statement and Parental Information regarding anaesthesia and potential developmental effects (http://www.apagbi.org.uk/). The charity SmartTots (www.smarttots.org/) also provides information and funds research related to anaesthesia in early life (in partnership with the US Food and Drug Administration, IARS and multiple stakeholders), and our work has previously been presented in a SmartTots session at the IARS (International Anesthesia Research Society) Annual Meeting. Results from the current proposal will allow us to better inform parents about the impact of surgery and analgesia in early life, and also direct future research.