Novel Translational Endpoints of Neuropathic Pain and Pain Relief

This thesis examined novel neuropathic pain endpoints for their reproducibility, reversibility and translatability to identify if they could improve how we measure neuropathic pain preclinically. Burrowing, an ethological behaviour that can be objectively measured in rodents was impaired in the streptozotocin (STZ) type-1 diabetic rat model of neuropathic pain. Burrowing deficits developed slower (from day 18) than measures of neuropathic pain using the classical evoked technique of von Frey paw withdrawal thresholds (from day 9). Only home caging an STZ diabetic rat with a control rat improved the STZ diabetic rat’s individual burrowing level. Whilst the first line neuropathic pain treatment pregabalin did not rescue burrowing deficits in STZ diabetic rats at 3, 10 or 30mg/kg doses. This was despite all three doses of pregabalin reversing paw withdrawal thresholds measurements of neuropathic pain. The burrowing deficits in the STZ diabetic rats up to day 18 were also not caused by changes in locomotor activity or gait. Ultimately this indicated that burrowing is a likely measure of the animal’s overall wellbeing that may prove useful as a secondary endpoint in neuropathic pain studies. The welfare of the STZ diabetic rats used in these and future studies was refined through the characterisation of a lower single 55mg/kg dose of anomer-equilibrated STZ. This lower single dose of STZ successfully produced mechanical allodynia that was reversed by pregabalin (10-30mg/kg) and a diabetic phenotype of hyperglycaemia (>16mmol/L), polydipsia and polyphagia. At the same time the STZ dose reduced the bodyweight loss and toxicity side effects of STZ administration, improving the welfare of these animals across three separate studies. It was identified that increased blood glucose levels correlated with increased weight loss as well as kidney and liver hypertrophy in these STZ diabetic rats. This is an important step to encourage researchers in this field to use insulin as a rescue measure to minimise excessive bodyweight loss and hyperglycaemic complications. Furthermore, whilst classical evoked measures of paw withdrawal threshold have limited translatability potential, a refinement to the current technique by omitting social isolation during preclinical testing was identified. Locomotor activity is another ethological behaviour that can be measured objectively in animals. In rats that developed neuropathic pain after chronic constrictive injury (CCI) surgery to the sciatic nerve of the hind paw, locomotor activity levels were decreased. Decreased locomotor activity was identified as early as 3 days after CCI surgery but was not identified in STZ diabetic rats within the first 3 weeks. The reduced amount of locomotor activity in CCI rats is reminiscent of the decrease in neuropathic pain patient’s activity levels. Locomotor activity levels and mechanical allodynia in CCI rats were rescued by 10mg/kg ketamine, a novel analgesic that is potentially more efficacious than currently available treatments such as pregabalin (10mg/kg) which reversed mechanical allodynia but not locomotor activity levels. Therefore, measuring home cage locomotor activity levels in CCI rats (but not STZ diabetic rats) could provide an objective and translatable neuropathic pain endpoint. Sleep disruption is a common comorbidity experienced by neuropathic pain patients that can lead to worse pain outcomes. Sleep fragmentation was identified in CCI rats through electroencephalogram (EEG) recordings by measuring the average length and number of sleep/wake bouts and particularly short periods of wakefulness. The development of sleep fragmentation in CCI rats was fully reversed by 10mg/kg ketamine and partially reversed by 10mg/kg pregabalin. These results were obtained whilst using an automated sleep/wake scoring algorithm that can greatly increase the efficiency and reduce the cost of preclinical EEG screening studies. However, in the oxaliplatin single dose model of transient chemotherapy induced peripheral neuropathy (CIPN), sleep fragmentation was not identified. This demonstrates that sleep disruption endpoints in CCI rats (but not CIPN rats) are sensitive to analgesic treatments, can be measured objectively and are translatable to human patients. Sleep/wake amounts and EEG power spectral measurements of pain were also assessed as potential neuropathic pain endpoints. CCI rats spent more time asleep and similar results were identified in the CIPN rats. The increased time CCI rats spent asleep was corrected by ketamine treatment but not by pregabalin. However, increased sleep is not seen in most preclinical neuropathic pain models or in human patients limiting the translatability of these findings. Similarly, EEG power changes were not consistent with other preclinical or clinical reports and were not reversible with analgesic treatments, limiting their potential use as preclinical neuropathic pain screening endpoints. The results of these studies can be used to further the research into the use of burrowing, locomotor activity and sleep/wake behaviours as preclinical neuropathic pain endpoints.