Effects of inhibition of Nav1.3, Nav1.7, and Nav1.8 channels on pain-related behavior in Speke's hinge-back tortoise (Kinixys spekii).

Comparative studies using reptiles as experimental animals in pain research could expand our knowledge on the evolution and adaptation of pain mechanisms. Currently, there are no data reported on the involvement of voltage-gated sodium ion channels on nociception in reptiles. The aim of this study was to investigate the involvement of Nav1.3, Nav1.7, and Nav1.8 ion channels in nociception in Speke's hinge-back tortoise. ICA 121341 (selective blocker for Nav1.1/Nav1.3), NAV 26 (selective blocker for Nav1.7), and A803467 (selective blocker for Nav1.8) were used to investigate the involvement of Nav1.3, Nav1.7, and Nav1.8, respectively. The chemicals were administered intracoelomically thirty minutes before the start of nociceptive tests. ICA 121341 did not cause a significant decrease in the time spent in pain-related behavior in all the nociceptive tests. NAV 26 and A8034667 caused a statistically significant decrease in the mean time spent in pain-related behavior in the formalin and capsaicin tests. Only A803467 caused a statistically significant increase in the mean latency to pain-related behavior in the hot plate test. NAV 26 and A803467 had no observable side effects. In conclusion, Nav1.7 and Nav1.8 are involved in the processing of chemically induced inflammatory pain in Speke's hinge back tortoise. In addition, Nav1.8 are also significantly involved in the development of thermal-induced pain-related behavior in this species of reptile. However, our results do not support the involvement of Nav1.3 on the development of chemical or thermal induced pain-related behavior in the Speke's hinge back tortoise.

Refinement of the motorised laminectomy-assisted rat spinal cord injury model by analgesic treatment.

Usage and reporting of analgesia in animal models of spinal cord injury (SCI) have been sparse and requires proper attention. The majority of experimental SCI research uses rats as an animal model. This study aimed to probe into the effects of some commonly used regimens with NSAIDs and opioids on well-being of the rats as well as on the functional outcome of the model. This eight-week study used forty-two female Wistar rats (Crl: WI), randomly and equally divided into 6 treatment groups, viz. I) tramadol (5mg/kg) and buprenorphine (0.05mg/kg); II) carprofen (5mg/kg) and buprenorphine (0.05mg/kg); III) carprofen (5mg/kg); IV) meloxicam (1mg/kg) and buprenorphine (0.05mg/kg); V) meloxicam (1mg/kg); and VI) no analgesia (0.5 ml sterile saline). Buprenorphine was administered twice daily whereas other treatments were given once daily for five days post-operatively. Injections were given subcutaneously. All animals underwent dental burr-assisted laminectomy at the T10-T11 vertebra level. A custom-built calibrated spring-loaded 200 kilodynes force deliverer was used to induce severe SCI. Weekly body weight scores, Rat Grimace Scale (RGS), and dark-phase home cage activity were used as markers for well-being. Weekly Basso Beattie and Bresnahan (BBB) scores served as markers for functionality together with Novel Object Recognition test (NOR) at week 8 and terminal histopathology using area of vacuolisation and live neuronal count from the ventral horns of spinal cord. It was concluded that the usage of analgesia improved animal wellbeing while having no effects on the functional aspects of the animal model in comparison to the animals that received no analgesics.

Harmonisation of education, training and continuing professional development for laboratory animal caretakers, technicians and technologists: Report of the FELASA-EFAT Working Group.

Competent, confident and caring laboratory animal caretakers, technicians and technologists (LAS staff) are vital for good animal welfare, high-quality science and a secure Culture of Care. This requires high-quality education, training, supervision and continuing professional development (CPD) of LAS staff. However, there is a lack of harmonisation regarding how this education and training is conducted among European countries, and nor are there recommendations adapted to Directive 2010/63/EU. Therefore, FELASA and EFAT established a working group with the task of establishing recommendations for education, training and CPD for LAS staff. The working group established five different levels (LAS staff levels 0-4), defining the required level of competence and attitude, as well as suggesting educational requirements for reaching each level. Defining these levels should help to ensure that appropriate educational and CPD activities are in place, and to enable employers and LAS staff to determine the level and career stage attained. Furthermore, proper assessment of competencies and effective CPD schemes for all relevant staff should be established. Regulators should support this by setting standards for competence assessment and ensuring that they are consistently applied. In addition, establishments should involve the LAS staff in defining and developing the Culture of Care. The Animal Welfare Body should be involved and have oversight of education, training and CPD. These recommendations will contribute to harmonisation and increased quality of education, training and CPD, as well as provide clearer career pathways for LAS staff, helping to ensure high standards of animal welfare and science.

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.

Antinociceptive effects of nortriptyline and desipramine hydrochloride in Speke's hinge-back tortoise (Kinixys Spekii).

Some of the most commonly used analgesic drugs in animals are of questionable efficacy or present adverse side effects among the various species of reptiles. Tricyclic antidepressants have been demonstrated to have antinociceptive effects in several animal models of pain and could be a good alternative for use in reptiles. The aim of the study was to investigate the antinociceptive effects of nortriptyline and desipramine hydrochloride in Speke's hinge-back tortoise. A total of 24 animals weighing 600-1000 g were used for nociceptive tests, i.e., formalin, capsaicin, and hot plate tests. Drugs were administered intracoelomically 30 min before starting the tests. The time spent in nocifensive behavior and the associated observable effects during the tests were recorded. Only the highest dose of 40 mg/kg of nortriptyline hydrochloride caused statistically significant decrease in nocifensive behavior in both the formalin and the capsaicin test. Desipramine hydrochloride at doses of 20 and 40 mg/kg caused statistically significant decrease in nocifensive behavior in the formalin test. Also, desipramine hydrochloride at doses of 15, 20, and 60 mg/kg caused statistically significant decrease in nocifensive behavior in the capsaicin test. None of the doses used for both drugs had any statistically significant effect on nocifensive behavior in the hot plate test. The results show that nortriptyline and desipramine hydrochloride have significant antinociceptive effects in the chemical but not thermal inflammatory pain-related behavior in the Speke's hinge-back tortoise. The most common associated side effect following administration of the higher doses of either of the drugs is excessive salivation.

Effects of Multimodal Therapy, Blinding, and Multi-laboratory Protocol Conduct on the Robustness of the Rat Model of Adjuvant Induced Arthritis.

BACKGROUND/AIM: The aim of this study was to investigate the effects of multimodal therapy comprising buprenorphine (BUP) and indomethacin (IND) on key translational parameters in the rat adjuvant induced arthritis (AIA) model. Furthermore, we investigated the difference between visual assessment scores and histology scores generated by blinded and non-blinded assessors and the robustness and generalizability of results by conducting a multi-laboratory study. MATERIALS AND METHODS: The experiment was terminated on day 26 after 11 days (days 15-25) of voluntarily ingested buprenorphine and 7 days of gavage delivered indomethacin treatment (days 19-25). The treatment effects were assessed on the last day of the study, relying on body weight assessment, serum concentrations of α1- acid glycoprotein, and assessment of affected hind paws swelling, in-life and post mortem. RESULTS: Across two laboratories, the combined analgesic treatments had minimal effects on the measured model parameters indicating that multimodal treatment did not affect the translatability of the model. We found an improvement in clinical scores (a negative change in scores) in nearly all medicated animals when scored informed, whereas it was essentially 50:50 for the blinded scorings and no difference between the blinded and informed histological scoring. CONCLUSION: The present results support the use of more effective analgesic treatment regimens and the good practice recommendations advocating blinding as a mandatory practice in conduct of preclinical in vivo efficacy studies. In spite of minor differences between results obtained at the two sites, there was good agreement between them indicating robustness of the AIA model.

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.

Bodyweight, locomotion, and behavioral responses of the naked mole rat (Heterocephalus glaber) to lipopolysaccharide administration.

The naked mole rat has unique biologic characteristics that include atypical inflammatory responses. Lipopolysaccharide induces inflammation which triggers brain centers controlling feeding, and behavior to result in "sick animal behavior". We characterized the bodyweight, locomotor, and other behavioral responses of this rodent to lipopolysaccharide administration. Lipopolysaccharide caused weight losses, which were not prevented by TAK 242. In the open field test, lipopolysaccharide did not depress locomotion, while urination, defecation, and activity freezing were rare. The animals exhibited walling but not rearing and fast backward movements that were unaffected by lipopolysaccharide. Failure to depress locomotion suggests either a unique immunity-brain crosstalk or motor responses/centers that tolerate depressive effects of inflammation. The absence of activity freezing and rarity of urination and defecation suggests that novel environments or lipopolysaccharide do not induce anxiety, or that anxiety is expressed differently in the animal. The absence of rearing could be due to the design of the animal's locomotor apparatus while fast backward movement could be a mechanism for quick escape from threats in the tunnels of their habitat. Our results elucidate the unique biology of this rodent, which elicits interest in the animal as a model for inflammatory research, although the findings require mechanistic corroborations.

Antidepressant Fluoxetine Does Not Appear to Interfere With Key Translational Parameters in the Rat Adjuvant-induced Arthritis Model.

BACKGROUND/AIM: This study aimed to investigate the analgesic effects of fluoxetine on Lewis rats of both sexes in the adjuvant-induced arthritis (AIA) rat model. In humans, chronic pain syndromes typical of rheumatoid arthritis (RA) co-exist with depression which is often treated with fluoxetine antidepressant known to have antinociceptive effects. MATERIALS AND METHODS: The experiment was terminated on day 26, after seven days of oral treatment (days 19-25) with fluoxetine and indomethacin. The effects of treatments were assessed on the final day of the study through measuring body weight, serum concentrations of a1-acid glycoprotein, visual arthritis assessment and post mortem histopathology assessment. RESULTS: Statistically significant difference was determined in the body weight of male subjects, with indomethacin-treated animals putting on significantly more weight than the vehicle and fluoxetine-treated counterparts. No differences were found between the different treatment groups in other study assessments. CONCLUSION: The present study did not provide support for analgesic effects of fluoxetine aimed at reducing the severity of the AIA model.

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.

Refinement of the spinal cord injury rat model and validation of its applicability as a model for memory loss and chronic pain.

BACKGROUND: Laminectomy produces trauma in spinal cord injury (SCI) animal models resulting in impinging artefacts and welfare issues. Mechanizing laminectomy using a dental burr assisted (DBA) technique to reduce the impact of conventionally performed laminectomy on animal welfare without any alterations in the outcome of the model was previously demonstrated. However, further validation was necessary to establish it as an alternative in developing SCI rats as a model of chronic pain and memory loss. NOVEL METHOD: DBA technique was employed to perform laminectomy at T10-T11 vertebrae in rats undergoing contusion SCI as a model of chronic pain and memory loss. In a 56-day study, 24 female Wistar rats (Crl: WI) were assigned randomly to four equal groups: conventionally laminectomised, DBA laminectomised, conventionally laminectomised with SCI and DBA laminectomised with SCI. RESULTS: The study revealed DBA technique to cause less surgical bleeding (p = 0.001), lower Rat Grimace Scale (p = 0.0006); resulted in better body weight changes (p = 0.0002 on Day 7 and p = 0.0108 on Day 28) and dark phase activity (p = .0.0014 on Day 1; p = 0.0422 on Day 56). Different techniques did not differ in Basso Beattie Bresnahan score, novel object recognition, mechanical allodynia, number of surviving neurons and the area of vacuolation- indicating that the new method doesn't affect the validity of the model. COMPARISON WITH EXISTING METHODS: In comparison with the conventional technique, motorised laminectomy can be a valid tool that evokes lesser pain and ensures higher well-being in rats modelled for chronic pain and memory loss. CONCLUSIONS: The intended outcome from the model is not influenced by techniques whereas the DBA-technique is a refined alternative to the conventional method in achieving better welfare in SCI studies.

An exploratory investigation of glucocorticoids, personality and survival rates in wild and rehabilitated hedgehogs (Erinaceus europaeus) in Denmark.

BACKGROUND: The European population of hedgehogs (Erinaceus europaeus) is declining. It is therefore essential to optimise conservation initiatives such as the rehabilitation of sick, injured and orphaned hedgehogs. Wild animals placed in captivity may be prone to chronic stress, potentially causing negative health effects. Therefore, the effects of these rehabilitation efforts should consequently be evaluated. Furthermore, hand-raising orphaned hedgehogs is a laborious and costly task, and it is therefore relevant to document whether they have equal post release survival rates compared to their wild conspecifics. The objectives of this research were therefore to conduct an exploratory study of glucocorticoid levels in hedgehogs from different backgrounds and compare the post release survival of translocated, rehabilitated and wild, juvenile hedgehogs as well as the possible effect on survival of differences in shy or bold behaviour (personality) exhibited by individuals. RESULTS: We measured glucocorticoid levels in 43 wild-caught (n = 18) and rehabilitated (n = 25) hedgehogs and compared the post release survival and spatial behaviour of 18 translocated juvenile hedgehogs (eight hand-raised and ten wild) until hibernation. The possible effect on survival of differences in shy or bold behaviour (personality) exhibited by 17 juvenile individuals (seven hand-raised and ten wild) was also examined. Rehabilitated individuals and females had higher levels of faecal corticosterone metabolites compared to wild individuals and males, respectively. Rehabilitated individuals showed higher levels of saliva corticosterone than wild. The personality tests labelled 13 individuals as shy and 11 as bold. Post release survival was 57% for rehabilitated and 50% for wild individuals. Neither background nor personality affected post release survival. Home range measures were 3.54 and 4.85 ha. Mean dispersal length from the release sites was 217 ± 100 m. CONCLUSION: The higher levels of corticosterone observed in rehabilitated compared to wild hedgehogs calls for consideration of the duration of admission to wildlife rehabilitation centres to reduce stress levels in the patients. Hand-raised juveniles appear to have the same prospects as wild, and personality does not seem to affect post release survival in hedgehogs, indicating that hand-raising of orphaned juvenile hedgehogs is a relevant contribution to the conservation of this species.

Modulation of nociception by amitriptyline hydrochloride in the Speke's hinge-back tortoise (Kiniskys spekii).

BACKGROUND: There are limited studies on the utilization of analgesics in testudines. Management of pain in reptiles is by use of analgesics generally used in other vertebrate species. Evidently, some analgesics considered to be generally effective in reptiles are not effective in certain reptile species. OBJECTIVE: The purpose of this study was to examine the effect of amitriptyline hydrochloride on nociceptive behaviour in Speke's hinge-back tortoise. METHODS: Twenty-four adult Speke-hinged tortoises weighing 500-700 g were used. The effects of amitriptyline hydrochloride on nociception were evaluated using the formalin, capsaicin and hot plate nociceptive tests. Amitriptyline was administered intracoelomically at doses of 0.5, 1.0 and 3.0 mg/kg. RESULTS: The higher doses of amitriptyline hydrochloride caused an increase in nociceptive behaviour (time spent in hindlimb withdrawal) on the formalin and capsaicin nociceptive tests, suggesting a potentiating effect. However, the doses used had no significant change in nociceptive behaviour on withdrawal response in the hot plate test. CONCLUSIONS: The study showed that amitriptyline hydrochloride which is widely used in management of neuropathic pain potentiates nociceptive effects in the formalin and capsaicin nociceptive tests in the Speke's hinge-back tortoise. The hot plate test, which previously has not been reported in these animals, gave results not in line with the other tests and therefore more testing and validation of the test is required. Amitriptyline modulates chemical and thermal pain differently.

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.

Imiquimod does not elicit inflammatory responses in the skin of the naked mole rat (Heterocephalus glaber).

OBJECTIVE: Naked mole rat (Heterocephalus glaber) has recently attracted interest in biomedical research due to its exceptional longevity, cancer resistance and tolerance to potentially harmful conditions or stimuli. Given its unique attributes, this study was designed to characterize inflammatory skin reactions of this animal to topical application of imiquimod, a toll-like receptor 7 and 8 agonist that triggers psoriasis-like skin reaction. RESULTS: Imiquimod did not cause the expected psoriasis-like skin changes. There was no epidermal thickening and a straight epidermo-dermal boundary was maintained. There was no parakeratosis and the granular layer of epidermis was well formed. In the dermis, there was no leukocyte infiltration. This points to an exceptional nature of inflammatory/immune responses of this animal, but the mechanism could not be explained by our results. Naked mole rat could be a valuable negative model for studying psoriasis and other inflammatory skin conditions but as a prerequisite, there is need for further investigations to establish the mechanisms behind its lack of response to imiquimod.

Hypophosphatemic Hypovitaminosis D Induces Osteomalacia in the Adult Female Rat.

Osteomalacia is a bone-demineralizing disease of adulthood, often caused by hypovitaminosis D. Current animal models of the disease mimic osteomalacia as a consequence of gastric bypass or toxic exposure to metals, but a relevant model of diet-induced osteomalacia is lacking. For that purpose, 7-month-old female Sprague Dawley rats were randomly assigned into 2 weight-stratified groups and maintained for 4 months on synthetic diets containing negligible or normal levels of vitamin D. The dietary regimen resulted in vitamin D deficiency as measured by 25-hydroxyvitamin D serum levels; however, hypovitaminosis D per se did not affect biomarkers of calcium metabolism and bone turnover, nor did it result in increased osteoid. Thus, vitamin D depletion through the diet was found to be insufficient to induce an osteomalacia-like phenotype in the adult rat. After 4 months, the phosphate content of the vitamin D-depleted diet had decreased to 0.16% (calcium:phosphorus ratio of 5.85), resulting in an osteomalacic-like condition (trabecular osteoid surface/bone surface constituted 33%; CI, 26-40). The diet change also affected both metabolic and bone turnover biomarkers, including significantly suppressing serum fibroblast growth factor 23. Furthermore, decreased dietary phosphate in a vitamin D-depleted diet led to microarchitectural changes of trabecular and cortical bone, lower bone mass density, lower bone mass content and decreased bone strength, all indicating reduced bone quality. Taken together, our results show that osteomalacia can be induced in the adult female rat by depleting vitamin D and lowering phosphate content in the diet.

Comparison of sublingual, facial and retro-bulbar blood sampling in mice in relation to animal welfare and blood quality.

INTRODUCTION: Repeated blood sampling is a common procedure in laboratory mice, but at present it is unknown which technique has the least impact on the animals when large or repeated blood samples are required. Retro-bulbar sinus puncture is a reliable technique but has been shown to cause many changes in the animals, why sublingual and facial vein puncture have been suggested as suitable alternatives. This study investigated 1) which of the three blood sampling techniques had the least impact on nest building activity, level of faecal corticosterone metabolites, body weight, fur status, and macroscopic changes, 2) whether the blood sampling techniques gave rise to variation in blood quality between blood samples, and 3) whether sublingual and facial vein puncture should be performed with or without anaesthesia in female C57BL/6 mice. METHOD: Three hundred and sixty C57BL/6 female mice divided into five batches were included in the study and randomized to a short (blood sampling on Day 8, 9 and 10) or a long protocol (blood sampling on Day 8, 15 and 22). Each protocol consisted of six identical groups: sublingual vein puncture (SVP), sublingual vein puncture in isoflurane (SVPiso), facial vein puncture (FVP), facial vein puncture in isoflurane (FVPiso), retro-bulbar sinus puncture (RBP), and a control group (CONTROL) with only scruffing being performed. At baseline (Day 2) nest building activity (NBA) was assessed and faecal pellets collected for evaluation of faecal corticosterone metabolites (FCM). The day after each blood sampling day NBA and FCM were reassessed. RESULTS AND CONCLUSION: None of the blood sampling techniques proved to be superior to the others in any of the measured parameters. Finally, sublingual and facial vein puncture performed under anaesthesia gave rise to variation in the quality of the blood. A refinement of all three techniques are therefore warranted.

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.

A novel technique to develop thoracic spinal laminectomy and a methodology to assess the functionality and welfare of the contusion spinal cord injury (SCI) rat model.

This study reports the advantage of a novel technique employing a motorised dental burr to assist laminectomy over the conventional manual technique at T10-T11 vertebra level in a rat model of spinal cord injury. Twenty-four female rats were randomly assigned to four groups: (1) conventionally laminectomised, (2) dental burr assisted laminectomised, (3) conventionally laminectomised with spinal cord contusion and (4) dental burr assisted laminectomised with spinal cord contusion. Basso Beattie Bresnahan (BBB) score, postoperative body weights, rat grimace scale (RGS), open cage activity and rearing was studied at 1, 7, 14, 21 and 28 days postoperatively, and area of spinal tissue affected was evaluated histologically. Laminectomised and spinal cord injured rats from dental burr groups showed significantly more weight gain and less weight loss respectively in comparison with respective conventionally laminectomised groups at various time points. Significantly higher RGS score was noticed in conventionally laminectomised animals on Day 1 in comparison to burr assisted laminectomy and presence of pain was evident until Day 7 in the conventionally spinal cord injured group. BBB score did not differ between techniques, whereas laminectomy groups showed more resting time than spinal injury groups. High rearing score was significantly higher in groups which underwent dental burr assisted technique at various time points with respect to their conventional counterparts. This study suggests that the use of dental burr assisted technique to perform laminectomy will bring refinement by producing less pain, aiding in better recovery, removing procedural artefacts without affecting the outcome of the model.

Clinical, Physiologic, and Behavioral Evaluation of Permanently Catheterized NMRI Mice.

Vascular catheterization is becoming a popular technique in laboratory rodents, facilitating repetitive blood sampling and infusion in individual animals. In mice, catheterization is complicated by their small body size, which may increase the risk of postoperative complications that may both threaten catheter longevity and animal welfare. Less obvious complications to a permanent catheter may include subclinical infection, visceral tissue damage from disseminating microthrombi released from the catheter, and distress from being isolated from conspecifics and other experimental stressors. Such complications may go unnoticed and may affect animal welfare as well as confound research outcomes. This study investigated the implications of long-term arterial catheterization in NMRI mice by evaluating clinical, physiologic and behavioral parameters. Body weight and food and water consumptions were monitored during the study period. Fecal corticosterone metabolites were quantified as biomarkers of stress, and nucleic acid metabolites (8-oxo-7,8-dihydro-2'-deoxyguanisine and 8-oxo-7,8-dihydroguanosine) as biomarkers of oxidative damage. Behavioral dysfunction was studied by scoring animal welfare and nest building. Catheters were placed the right common carotid artery of mice; catheterized mice were compared with sham-operated and nonsurgical control mice. Except for an increase in the body weight of catheterized mice during the experimental period, clinical parameters (body weight and food and water consumptions) did not differ between groups. Physiologic parameters (oxidized nucleic acid metabolites and fecal corticosterone metabolites) were higher in control mice during the first week of experimentation compared with the end of study but did not differ between groups. Likewise, catheterization had no effect on behavioral parameters (nest building and animal welfare assessment). Long-term arterial catheterization of mice had no detectable implications on animal welfare in this study.