Reliability of sucrose preference testing following short or no food and water deprivation-a Systematic Review and Meta-Analysis of rat models of chronic unpredictable stress.

The sucrose preference test is a popular test for anhedonia in the chronic unpredictable stress model of depression. Yet, the test does not always produce consistent results. Long food and water deprivation before the test, while often implemented, confounds the results by introducing unwanted drives in the form of hunger and thirst. We assessed the reliability of the test when only short or no fasting was used. We searched PubMed, Embase, and Web of Science for studies in rats exposed to chronic unpredictable stress that used no more than 6 h of food and/or water deprivation before the test. Sweet consumptions, for stressed and control/antidepressant-treated animals, in 132 studies were pooled using random effects models. We found a decrease in sweet consumption in stressed rats, compared to controls, that was halved when a non-caloric sweetener was used and significantly reduced when sucrose consumption was corrected for body weight. What is more, the length of food and water deprivation was found to confound the effect. The effect was reversed when the stressed rats were treated with antidepressants. Methodological strategies meant to control for recognized sources of bias when conducting the test were often missing, and so was a clear and complete report of essential study information. Our results indicate that not only is food and water deprivation before the test unnecessary, but not recommended. Even in absence of long fasting, we found evidence of an additional effect on sweet consumption that is unrelated to anhedonia. Without properly controlling for non-hedonic drivers of consumption, the test is unreliable as a proxy measure of anhedonia. Strengthening the methodological rigor and addressing the confounding effect of metabolic factors in the sucrose preference test prevents misleading conclusions that harm the translatability of the associated research and perpetuates the use of animals for little gain.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Hair glucocorticoids are not a historical marker of stress - Exploring the time-scale of corticosterone incorporation into hairs in a rat model.

Hair glucocorticoids are increasingly popular biomarkers, used across numerous research fields, and studied species, as a measure of stress. Although they are suggested to be a proxy of the average HPA axis activity spanning a period of weeks or months into the past, this theory has never been tested. In the present study, adrenalectomized rats with no endogenous (adrenal) glucocorticoid production were used to study how circulating glucocorticoid levels would be reflected in the glucocorticoid levels found in hair samples. By dosing the animals daily with high levels of corticosterone for seven days, while sampling hairs before, during, and after treatments, a timeline for glucocorticoid uptake into hairs was constructed. This kinetic profile was compared to two hypothetical models, and the theory that hair glucocorticoids are a record of historical stress had to be rejected. Corticosterone concentrations in hairs were found to increase within three hours of the first injection, the highest concentrations were found on the seventh day of treatments, and the decrease in concentrations post-treatment suggests rapid elimination. We speculate that hair glucocorticoid levels can only be used to characterize a stress-response for a few days following a postulated stressor. An updated model, where glucocorticoids diffuse into, along, and out of hairs needs to be adopted to reconcile the experimentally obtained data. The inescapable consequence of this updated model is that hair glucocorticoids become a marker of - and can only be used to study - recent, or ongoing, stress, as opposed to historical events, weeks or months in the past.

Effects of Transdermal Fentanyl Treatment on Acute Pain and Inflammation in Rats with Adjuvant-induced Monoarthritis.

Eliminating unnecessary pain is an important requirement of performing animal experimentation, including reducing and controlling pain of animals used in pain research. The goal of this study was to refine an adjuvant-induced monoarthritis model in rats by providing analgesia with a transdermal fentanyl solution (TFS). Male and female Sprague-Dawley rats, single- or pair-housed, were injected with 20 µL of complete Freund adjuvant (CFA) into the left ankle joint. CFA-injected rats treated with a single dose of transdermal fentanyl solution (0.33 or 1 mg/kg) were compared with an untreated CFA-injected group and sham groups that received either no treatment or TFS treatment (1 mg/kg) during 72 h. At the tested doses, TFS reduced mechanical hyperalgesia and improved the mobility, stance, rearing, and lameness scores at 6 h after CFA injection. Joint circumferences were not reduced by TFS treatment, and no significant differences were detected between the 2 doses of TFS, or between single- and pair-housed rats. Treatment with TFS did not appear to interfere with model development and characteristics. However, overall, the analgesic effect was transient, and several opioid-related side effects were observed.

Inhibition of P2X7 receptors by Lu AF27139 diminishes colonic hypersensitivity and CNS prostanoid levels in a rat model of visceral pain.

Visceral pain is a prominent feature of various gastrointestinal diseases. The P2X7 receptor is expressed by multiple cell types including dorsal root ganglion satellite glial cells, macrophages, and spinal microglia, all of which have been implicated in nociceptive sensitization. We have used the selective and CNS penetrant P2X7 receptor antagonist Lu AF27139 to explore this receptor's role in distinct rat models of inflammatory and visceral hypersensitivity. Rats injected with CFA in the hindpaw displayed a marked reduction in hindpaw mechanical threshold, which was dose-dependently reversed by Lu AF27139 (3-30 mg/kg, p.o.). In rats injected with TNBS in the proximal colon, the colorectal distension threshold measured distally was significantly lower than sham treated rats at 7 days post-injection (P < 0.001), indicative of a marked central sensitization. Colonic hypersensitivity was also reversed by Lu AF27139 (10-100 mg/kg) and by the κ-opioid receptor agonist U-50,488H (3 mg/kg, s.c.). Moreover, both Lu AF27139 and U-50,488H prevented a TNBS-induced increase in spinal and brain levels of PGE2 and LTB4, as well as an increase in brain levels of PGF2α and TXB2. Lu AF27139 was well tolerated as revealed by a lack of significant effect on rotarod motor function and coordination at all doses tested up to 300 mg/kg. Thus, P2X7 receptor antagonism is efficacious in a rat model of visceral pain, via a mechanism which potentially involves attenuation of microglial function within spinal and/or supraspinal pain circuits, albeit a peripheral site of action cannot be excluded.

P2X7 receptor-mediated release of microglial prostanoids and miRNAs correlates with reversal of neuropathic hypersensitivity in rats.

BACKGROUND: P2X7 receptor antagonists have potential for treating various central nervous system (CNS) diseases, including neuropathic pain, although none have been approved for clinical use. Reasons may include insufficient understanding of P2X7 receptor signalling in pain, and the lack of a corresponding preclinical mechanistic biomarker. METHODS: Lu AF27139 is a highly selective and potent small molecule antagonist at rat, mouse and human forms of the P2X7 receptor, with excellent pharmacokinetic and CNS permeability properties. In the current experiments, we probed the utility of previously characterized and novel signalling cascades exposed to Lu AF27139 using cultured microglia combined with release assays. Subsequently, we assessed the biomarker potential of identified candidate molecules in the rat chronic constriction injury (CCI) model of neuropathic pain; study design limitations precluded their assessment in spared nerve injury (SNI) rats. RESULTS: Lu AF27139 blocked several pain-relevant pathways downstream of P2X7 receptors in vitro. At brain and spinal cord receptor occupancy levels capable of functionally blocking P2X7 receptors, it diminished neuropathic hypersensitivity in SNI rats, and less potently in CCI rats. Although tissue levels of numerous molecules previously linked to neuropathic pain and P2X7 receptor function (e.g. IL-6, IL-1ß, cathepsin-S, 2-AG) were unaffected by CCI, Lu AF27139-mediated regulation of spinal PGE2 and miRNA (e.g. rno-miR-93-5p) levels increased by CCI aligned with its ability to diminish neuropathic hypersensitivity. CONCLUSIONS: We have identified a pain-relevant P2X7 receptor-regulated mechanism in neuropathic rats, which could hold promise as a translatable biomarker and by association enhance the clinical progression of P2X7 receptor antagonists in neuropathic pain. SIGNIFICANCE: Sub-optimal translation of preclinical molecules has hindered the clinical development of novel mechanism of action analgesics. We have undertaken a comprehensive in vitro analysis of migroglial signalling mechanisms recruited upon P2X7 receptor activation, a number of which were shown to be modulated by a selective P2X7 receptor antagonist in a well characterized animal model of neuropathic pain. Subject to further confirmation in other neuropathic models, this opens up the possibility to investigate their clinical utility as potential pain biomarkers in patients.

Effects of buprenorphine on model development in an adjuvant-induced monoarthritis rat model.

Complete Freund's adjuvant (CFA)-induced arthritis in rats is a common animal model for studying chronic inflammatory pain. However, modelling of the disease is associated with unnecessary pain and impaired animal wellbeing, particularly in the immediate post-induction phase. Few attempts have been made to counteract these adverse effects with analgesics. The present study investigated the effect of buprenorphine on animal welfare, pain-related behaviour and model-specific parameters during the disease progression in a rat model of CFA-induced monoarthritis. The aim was to reduce or eliminate unnecessary pain in this model, in order to improve animal welfare and to avoid suffering, without compromising the quality of the model. Twenty-four male Sprague Dawley rats were injected with 20 µl of CFA into the left tibio-tarsal joint to induce monoarthritis. Rats were treated with either buprenorphine or carprofen for 15 days during the disease development, and were compared to a saline-treated CFA-injected group or a negative control group. Measurements of welfare, pain-related behaviour and clinical model-specific parameters were collected. The study was terminated after 3 weeks, ending with a histopathologic analysis. Regardless of treatment, CFA-injected rats displayed mechanical hyperalgesia and developed severe histopathological changes associated with arthritis. However, no severe effects on general welfare were found at any time. Buprenorphine treatment reduced facial pain expression scores, improved mobility, stance and lameness scores and it did not supress the CFA-induced ankle swelling, contrary to carprofen. Although buprenorphine failed to demonstrate a robust analgesic effect on the mechanical hyperalgesia in this study, it did not interfere with the development of the intended pathology.

The influence of rat strain on the development of neuropathic pain and comorbid anxio-depressive behaviour after nerve injury.

Back-translating the clinical manifestations of human disease burden into animal models is increasingly recognized as an important facet of preclinical drug discovery. We hypothesized that inbred rat strains possessing stress hyper-reactive-, depressive- or anxiety-like phenotypes may possess more translational value than common outbred strains for modeling neuropathic pain. Rats (inbred: LEW, WKY, F344/ICO and F344/DU, outbred: Crl:SD) were exposed to Spared Nerve Injury (SNI) and evaluated routinely for 6 months on behaviours related to pain (von Frey stimulation and CatWalk-gait analysis), anxiety (elevated plus maze, EPM) and depression (sucrose preference test, SPT). Markers of stress reactivity together with spinal/brain opioid receptor expression were also measured. All strains variously developed mechanical allodynia after SNI with the exception of stress-hyporesponsive LEW rats, despite all strains displaying similar functional gait-deficits after injury. However, affective changes reflective of anxiety- and depressive-like behaviour were only observed for F344/DU in the EPM, and for Crl:SD in SPT. Although differences in stress reactivity and opioid receptor expression occurred, overall they were relatively unaffected by SNI. Thus, anxio-depressive behaviours did not develop in all strains after nerve injury, and correlated only modestly with degree of pain sensitivity or with genetic predisposition to stress and/or affective disturbances.

Stress sensitivity and cutaneous sensory thresholds before and after neuropathic injury in various inbred and outbred rat strains.

Chronic pain is associated with altered affective state, stress, anxiety and depression. Conversely, stress, anxiety and depression can all modulate pain perception. The relative link between these behavioural constructs in different inbred and outbred rat strains, known to be variously hypo/hyperresponsive to stress has not been determined. Hindpaw sensory thresholds to repeated mechanical (von Frey filament and electronic Randall Selitto) and thermal (Hargreaves, cold plate and hot plate) stimulation were routinely assessed over three weeks in non-injured male rats of the following strains; WKY, LEW, F344, Hsd:SD and Crl:SD. Thereafter, threshold responses to Spared Nerve Injury (SNI) were assessed using von Frey, pin prick and Hargreaves testing in the same strains over a three month period. Finally, anxiolytic efficacy of the benzodiazepine drug diazepam was assessed using the Elevated Plus Maze (EPM), as a surrogate index of functional plasticity of circuits involved in affective processing. Repeated nociceptive testing was associated with distinct strain-dependent changes in sensory thresholds in naïve rats; stress-hyporesponsive LEW rats presented with a mechanical/thermal hyperalgesia phenotype, whereas stress-hyperresponsive WKY rats presented with an unexpected heat/cold hypoalgesia phenotype. After SNI, LEW rats showed minimal signs of neuropathic sensitivity. Diazepam was anxiolytic in all tested strains with the exception of LEW rats reflecting distinct inherent affective processing only in this strain. The contribution of stress reactivity to nociceptive sensory profiles appears to vary in the absence or presence of neuropathic injury. Intriguingly, the functional responsiveness of affective state prior to injury may be a predisposing factor to developing chronic pain.

The analgesic efficacy of morphine varies with rat strain and experimental pain model: implications for target validation efforts in pain drug discovery.

BACKGROUND: Translating efficacy of analgesic drugs from animal models to humans remains challenging. Reasons are multifaceted, but lack of sufficiently rigorous preclinical study design criteria and phenotypically relevant models may be partly responsible. To begin to address this fundamental issue, we assessed the analgesic efficacy of morphine in three inbred rat strains (selected based on stress reactivity and affective/pain phenotypes), and outbred Sprague Dawley (SD) rats supplied from two vendors. METHODS: Sensitivity to morphine (0.3-6.0 mg/kg, s.c.) was evaluated in the hot plate test of acute thermal nociception, the Complete Freund's Adjuvant (CFA) model of inflammatory-induced mechanical hyperalgesia, and in a locomotor motility assay in male rats from the following strains; Lewis (LEW), Fischer (F344), Wistar Kyoto (WKY), and SD's from Envigo and Charles River. RESULTS: F344 and SD rats were similarly sensitive to morphine in hot plate and CFA-induced inflammatory hyperalgesia (Minimum Effective Dose (MED) = 3.0 mg/kg). WKY rats developed a less robust mechanical hypersensitivity after CFA injection, and were less sensitive to morphine in both pain tests (MED = 6.0 mg/kg). LEW rats were completely insensitive to morphine in the hot plate test, in contrast to the reversal of CFA-induced hyperalgesia (MED = 3.0 mg/kg). All strains exhibited a dose-dependent reduction in locomotor activity at 3.0-6.0 mg/kg. CONCLUSION: Sensory phenotyping in response to acute thermal and inflammatory-induced pain, and sensitivity to morphine in various inbred and outbred rat strains indicates that different pathophysiological mechanisms are engaged after injury. This could have profound implications for translating preclinical drug discovery efforts into pain patients. SIGNIFICANCE: The choice of rat strain used in preclinical pain research can profoundly affect the outcome of experiments in relation to (a) nociceptive threshold responses, and (b) efficacy to analgesic treatment, in assays of acute and tonic inflammatory nociceptive pain.

Is there a reasonable excuse for not providing post-operative analgesia when using animal models of peripheral neuropathic pain for research purposes?

INTRODUCTION: The induction of neuropathic pain-like behaviors in rodents often requires surgical intervention. This engages acute nociceptive signaling events that contribute to pain and stress post-operatively that from a welfare perspective demands peri-operative analgesic treatment. However, a large number of researchers avoid providing such care based largely on anecdotal opinions that it might interfere with model pathophysiology in the longer term. OBJECTIVES: To investigate effects of various peri-operative analgesic regimens encapsulating different mechanisms and duration of action, on the development of post-operative stress/welfare and pain-like behaviors in the Spared Nerve Injury (SNI)-model of neuropathic pain. METHODS: Starting on the day of surgery, male Sprague-Dawley rats were administered either vehicle (s.c.), carprofen (5.0mg/kg, s.c.), buprenorphine (0.1mg/kg s.c. or 1.0mg/kg p.o. in Nutella®), lidocaine/bupivacaine mixture (local irrigation) or a combination of all analgesics, with coverage from a single administration, and up to 72 hours. Post-operative stress and recovery were assessed using welfare parameters, bodyweight, food-consumption, and fecal corticosterone, and hindpaw mechanical allodynia was tested for assessing development of neuropathic pain for 28 days. RESULTS: None of the analgesic regimes compromised the development of mechanical allodynia. Unexpectedly, the combined treatment with 0.1mg/kg s.c. buprenorphine and carprofen for 72 hours and local irrigation with lidocaine/bupivacaine, caused severe adverse effects with peritonitis. This was not observed when the combination included a lower dose of buprenorphine (0.05mg/kg, s.c.), or when buprenorphine was administered alone (0.1mg/kg s.c. or 1.0mg/kg p.o.) for 72 hours. An elevated rate of wound dehiscence was observed especially in the combined treatment groups, underlining the need for balanced analgesia. Repeated buprenorphine injections had positive effects on body weight the first day after surgery, but depressive effects on food intake and body weight later during the first week. CONCLUSION: Post-operative analgesia does not appear to affect established neuropathic hypersensitivity outcome in the SNI model.

Antinociceptive effects of voluntarily ingested buprenorphine in the hot-plate test in laboratory rats.

Researchers performing experiments on animals should always strive towards the refinement of experiments, minimization of stress and provision of better animal welfare. An adequate analgesic strategy is important to improve post-operative recovery and welfare in laboratory rats and mice. In addition, it is desirable to provide post-operative analgesia using methods that are minimally invasive and stressful. This study investigated the antinociceptive effects of orally administered buprenorphine ingested in Nutella® in comparison with subcutaneous buprenorphine administration. By exposing the animal to a thermal stimulus using a hot plate, significant antinociceptive effects of voluntarily ingested buprenorphine administered in Nutella® were demonstrated. This was evident at doses of 1.0 mg/kg 60 and 120 min post administration ( P < 0.01), although antinociceptive effects were not as marked as with subcutaneous administration, and had a later onset. It is advised to administer the oral formulation of buprenorphine in Nutella® in a 10-fold higher dose, as well as approximately 60 min earlier, than with the more commonly employed subcutaneous route of administration.