Investigation of TRPV1 loss-of-function phenotypes in TRPV1 Leu206Stop mice generated by N-ethyl-N-nitrosourea mutagenesis.

N-ethyl-N-nitrosourea (ENU) random mutagenesis was used to generate a mouse model for the analysis of the transient receptor potential vanilloid 1 (TRPV1) cation channel. A transversion from T→A in exon 4 led to a Leu206Stop mutation generating a loss-of-function mutant. The TRPV1 agonist capsaicin was used to analyze functional and nociceptive parameters in vitro and in vivo in TRPV1 Leu206Stop mice and congenic C3HeB/FeJ controls. Capsaicin-induced [Ca2+]i changes in small diameter DRG neurons were significantly diminished in TRPV1 Leu206Stop mice and administration of capsaicin induced neither hypothermia nor nocifensive behaviour in vivo. TRPV1 Leu206Stop mice were tested in the spinal nerve ligation of mononeuropathic pain and developed mechanical hypersensitivity two weeks after nerve injury. In the open field test, a significant increase in spontaneous locomotion was detected in TRPV1 Leu206Stop mice as compared to wildtype controls. TRPV1 knockout mice have been reported to carry a similar phenotype regarding capsaicin-evoked responses in vitro and in vivo. However, in contrast to TRPV1 Leu206Stop mice, TRPV1 knockout mice did not differ in spontaneous locomotion as compared to congenic C57BL/6 mice, suggesting subtle ENU-dependent or independent strain differences between TRPV1 Leu206Stop mice and their wildtype controls. In summary, these data revealed a target-related (i.e. capsaicin-evoked) phenotype of TRPV1 Leu206Stop mice closely resembling that of published TRPV1 knockout mice. However, since ENU-mutant mice are congenic with the mouse strain initially used in random mutagenesis, direct phenotypic comparison with the respective wildtype controls is possible, and the time-consuming backcrossing in lines with targeted mutations is avoided.

Limited potential of cebranopadol to produce opioid-type physical dependence in rodents.

Cebranopadol is a novel potent analgesic agonist at the nociceptin/orphanin FQ peptide (NOP) and classical opioid receptors. As NOP receptor activation has been shown to reduce side effects related to the activation of µ-opioid peptide (MOP) receptors, the present study evaluated opioid-type physical dependence produced by cebranopadol in mice and rats. In a naloxone-precipitated withdrawal assay in mice, a regimen of seven escalating doses of cebranopadol over 2 days produced only very limited physical dependence as evidenced by very little withdrawal symptoms (jumping) even at cebranopadol doses clearly exceeding the analgesic dose range. In contrast, mice showed clear withdrawal symptoms when treated with morphine within the analgesic dose range. In the rat, spontaneous withdrawal (by cessation of drug treatment; in terms of weight loss and behavioral score) was studied after 4-week subacute administration. Naloxone-precipitated withdrawal (in terms of weight loss and behavioral score) was studied in the same groups of rats after 1-week re-administration following the spontaneous withdrawal period. In both tests, cebranopadol-treated rats showed only few signs of withdrawal, while withdrawal effects in rats treated with morphine were clearly evident. These findings demonstrate a low potential of cebranopadol to produce opioid-type physical dependence in rodents. The prospect of this promising finding into the clinical setting remains to be established.

Hemostatic Efficacy and Safety of the Novel Medical Adhesive, MAR VIVO-107, in a Rabbit Liver Resection Model.

BACKGROUND: Topical hemostatic agents are useful when hepatic hemorrhage is difficult to control. The aim of this study was to evaluate the hemostatic efficacy and safety of a biodegradable polyurethane-based adhesive, MAR VIVO-107 (MAR), in comparison with a clinically used fibrin glue. METHODS: Thirty female New Zealand white rabbits were randomly assigned to 3 study groups as follows: MAR (n = 10), fibrin glue (n = 10), and saline groups (n = 10). After standardized partial liver resection was performed, each agent was immediately applied to the wound area. Bleeding time until hemostasis and blood loss were recorded. After 7 days, body weight, hematology parameters, and serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured. Simultaneously, the severity of intra-abdominal adhesion was evaluated. RESULTS: The mean bleeding time in the MAR (38 ± 10 s) and fibrin glue groups (65 ± 17 s) was significantly shorter than that in the saline group (186 ± 12 s). Similarly, the mean blood loss in the MAR (9 ± 3 g) and fibrin glue groups (9 ± 3 g) was significantly less than that in the saline group (23 ± 4 g). No significant differences in bleeding time and blood loss were found between the MAR and fibrin glue groups. The postoperative survival rate was 100% in all the groups. Body weight as well as hematological and serum biochemical values on day 7 were within the small and physiological range when compared with the preoperative baseline values, and significant differences were not detected among the MAR, fibrin glue, and saline groups. The severities of adhesion were similar between the 3 groups. CONCLUSION: Our data demonstrated that MAR was not inferior to fibrin glue in terms of hemostatic efficacy and safety.

The Aachen Minipig: Phenotype, Genotype, Hematological and Biochemical Characterization, and Comparison to the Göttingen Minipig.

BACKGROUND: The pig is one of the most frequently used large animal models for biomedical research, especially in the field of translational research and surgical models. While standard livestock breeds are used in short-term and acute studies, minipig breeds are the preferred breeds in long-term and chronic studies due to their limited growth and body weight. OBJECTIVE: In consideration of the 3R principle (refinement, reduction, replacement) and the increasing demand, the aim of this study was to generate a new, robust, non-specific-pathogen-free minipig breed, the Aachen minipig. METHODS: Phenotype, genotype, and hematological as well as clinical chemistry parameters were characterized, and reference values of the Aachen minipig were generated and compared to the values in the commonly used Göttingen minipig. Organ weights of the heart, kidney, liver, lung, spleen, and brain were determined using a laboratory balance. Blood samples were collected for hematology and clinical chemistry. Assessment of genetic diversity was performed by microsatellite markers. Nasal swabs were collected from 11 individual minipigs representing 6 races for DNA extraction. DNA was quantified and the identity and origin of the Aachen minipigs at the genomic level was determined by microsatellites. RESULTS: The Aachen minipig established here is based on the Mini-LEWE breed and consists of the Vietnamese potbelly pig, the Schwäbisch Hällisch Landpig, the German Landrace, and the Minnesota minipig. Relative organ weights (lung, heart, kidneys, brain), hematology (hemoglobin, hematocrit, platelet count, mean corpuscular hemoglobin concentration, segmented neutrophils, lymphocytes, eosinophils, basophils), and clinical chemistry parameters (sodium, calcium, chloride, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, creatine kinase) of the Aachen minipigs and the Göttingen minipigs were not significantly different. Significant differences where only seen in relative organ weights (liver, spleen), hematology (red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, banded neutrophils, monocytes), and clinical chemistry parameters (inorganic phosphorus, potassium, glucose, cholesterol, albumin, amylase). CONCLUSION: The Aachen minipig is a suitable model for research due to its similarity to other minipig breeds, especially the Göttingen minipig. The reference values established in this study may be used for the comparison of scientific data and encourage the use of the Aachen minipig as an animal model for biomedical research.

Cebranopadol: a novel potent analgesic nociceptin/orphanin FQ peptide and opioid receptor agonist.

Cebranopadol (trans-6'-fluoro-4',9'-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1'(3'H)-pyrano[3,4-b]indol]-4-amine) is a novel analgesic nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonist [Ki (nM)/EC50 (nM)/relative efficacy (%): human NOP receptor 0.9/13.0/89; human mu-opioid peptide (MOP) receptor 0.7/1.2/104; human kappa-opioid peptide receptor 2.6/17/67; human delta-opioid peptide receptor 18/110/105]. Cebranopadol exhibits highly potent and efficacious antinociceptive and antihypersensitive effects in several rat models of acute and chronic pain (tail-flick, rheumatoid arthritis, bone cancer, spinal nerve ligation, diabetic neuropathy) with ED50 values of 0.5-5.6 µg/kg after intravenous and 25.1 µg/kg after oral administration. In comparison with selective MOP receptor agonists, cebranopadol was more potent in models of chronic neuropathic than acute nociceptive pain. Cebranopadol's duration of action is long (up to 7 hours after intravenous 12 µg/kg; >9 hours after oral 55 µg/kg in the rat tail-flick test). The antihypersensitive activity of cebranopadol in the spinal nerve ligation model was partially reversed by pretreatment with the selective NOP receptor antagonist J-113397[1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] or the opioid receptor antagonist naloxone, indicating that both NOP and opioid receptor agonism are involved in this activity. Development of analgesic tolerance in the chronic constriction injury model was clearly delayed compared with that from an equianalgesic dose of morphine (complete tolerance on day 26 versus day 11, respectively). Unlike morphine, cebranopadol did not disrupt motor coordination and respiration at doses within and exceeding the analgesic dose range. Cebranopadol, by its combination of agonism at NOP and opioid receptors, affords highly potent and efficacious analgesia in various pain models with a favorable side effect profile.

Characterisation of tramadol, morphine and tapentadol in an acute pain model in Beagle dogs.

OBJECTIVE: To evaluate the analgesic potential of the centrally acting analgesics tramadol, morphine and the novel analgesic tapentadol in a pre-clinical research model of acute nociceptive pain, the tail-flick model in dogs. STUDY DESIGN: Prospective part-randomized pre-clinical research trial. ANIMALS: Fifteen male Beagle dogs (HsdCpb:DOBE), aged 12-15 months. METHODS: On different occasions separated by at least 1 week, dogs received intravenous (IV) administrations of tramadol (6.81, 10.0 mg kg(-1) ), tapentadol (2.15, 4.64, 6.81 mg kg(-1) ) or morphine (0.464, 0.681, 1.0 mg kg(-1) ) with subsequent measurement of tail withdrawal latencies from a thermal stimulus (for each treatment n = 5). Blood samples were collected immediately after the pharmacodynamic measurements of tramadol to determine pharmacokinetics and the active metabolite O-demethyltramadol (M1). RESULTS: Tapentadol and morphine induced dose-dependent antinociception with ED50-values of 4.3 mg kg(-1) and 0.71 mg kg(-1) , respectively. In contrast, tramadol did not induce antinociception at any dose tested. Measurements of the serum levels of tramadol and the M1 metabolite revealed only marginal amounts of the M1 metabolite, which explains the absence of the antinociceptive effect of tramadol in this experimental pain model in dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Different breeds of dogs might not or only poorly respond to treatment with tramadol due to low metabolism of the drug. Tapentadol and morphine which act directly on µ-opioid receptors without the need for metabolic activation are demonstrated to induce potent antinociception in the experimental model used and should also provide a reliable pain management in the clinical situation. The non-opioid mechanisms of tramadol do not provide antinociception in this experimental setting. This contrasts to many clinical situations described in the literature, where tramadol appears to provide useful analgesia in dogs for post-operative pain relief and in more chronically pain states.

Refinement in Post-Operative Care for Orthopaedic Models: Implementing a Sheep Walking Cast (SWC) for Effective Tibial Fracture Management.

In the healthcare system, lower leg fractures remain relevant, incurring costs related to surgical treatment, hospitalization, and rehabilitation. The duration of treatment may vary depending on the individual case and its severity. Casting as a post-surgical fracture treatment is a common method in human and experimental veterinary medicine. Despite the high importance of sheep in preclinical testing materials for osteosynthesis, there is no standardised cast system ensuring proper stabilisation and functionality of hind limbs during the healing of tibia fractures or defects. Existing treatment approaches for tibial osteosynthesis in laboratory animal science include sling hanging, external fixators, or former Achilles tendon incision. These methods restrict animal movement for 4-6 weeks, limit species-typical behaviour, and impact social interactions. Our pilot study introduces a Standardised Walking Cast (SWC) for sheep, enabling immediate physiological movement post surgery. Seven Rhone sheep (female, 63.5 kg ± 6.45 kg) each with a single tibia defect (6 mm mechanical drilled defect) underwent SWC application for 4 weeks after plate osteosynthesis. The animals bore weight on their operated leg from day one, exhibiting slight lameness (grade 1-2 out of 5). Individual step lengths showed good uniformity (average deviation: 0.89 cm). Group housing successfully started on day three after surgery. Weekly X-rays and cast changes ensured proper placement, depicting the healing process. This study demonstrates the feasibility of using an SWC for up to 72 kg of body weight without sling hanging via ceiling mounting or external fixation techniques. Allowing species-typical movement and social behaviour can significantly improve the physiological behaviour of sheep in experiments, contributing to refinement.

Investigation of TRPV1 loss-of-function phenotypes in transgenic shRNA expressing and knockout mice.

The function of the transient receptor potential vanilloid 1 (TRPV1) cation channel was analyzed with RNA interference technologies and compared to TRPV1 knockout mice. Expression of shRNAs targeting TRPV1 in transgenic (tg) mice was proven by RNase protection assays, and TRPV1 downregulation was confirmed by reduced expression of TRPV1 mRNA and lack of receptor agonist binding in spinal cord membranes. Unexpectedly, TRPV3 mRNA expression was upregulated in shRNAtg but downregulated in knockout mice. Capsaicin-induced [Ca(2+)](i) changes in small diameter DRG neurons were significantly diminished in TRPV1 shRNAtg mice, and administration of capsaicin hardly induced hypothermia or nocifensive behaviour in vivo. Likewise, sensitivity towards noxious heat was reduced. Interestingly, spinal nerve injured TRPV1 knockout but not shRNAtg animals developed mechanical allodynia and hypersensitivity. The present study provides further evidence for the relevance of TRPV1 in neuropathic pain and characterizes RNA interference as valuable technique for drug target validation in pain research.

Tramadol has a better potency ratio relative to morphine in neuropathic than in nociceptive pain models.

BACKGROUND AND OBJECTIVE: Treatment of neuropathic pain remains a challenge and the role of various analgesics in this setting is still debated. The effects of tramadol, an atypically acting analgesic with a combined opioid and monoaminergic mechanism of action, and morphine, a prototypical opioid, were tested in rat models of neuropathic and nociceptive pain. METHODS: Cold allodynia and mechanical hypersensitivity, symptoms of neuropathic pain, were studied in rat models of mononeuropathic pain. Cold allodynia was analyzed in the chronic constriction injury (CCI) model and mechanical hypersensitivity was analyzed in the spinal nerve ligation (SNL) model. Heat-induced rat tail-flick latencies were determined as measure for nociceptive pain. RESULTS: Cold allodynia and mechanical hypersensitivity were strongly attenuated with similar absolute potency after intravenous administration of tramadol and morphine. The doses of drug that were calculated to result in 50% pain inhibition (ED(50)) for tramadol and morphine were 2.1 and 0.9 mg/kg, respectively, in CCI rats and 4.3 and 3.7 mg/kg, respectively, in SNL rats. In the tail-flick assay of acute nociception, the potency of the two drugs differed markedly, as seen by ED(50) values of 5.5 and 0.7 mg/kg intravenously for tramadol and morphine, respectively. Accordingly, the analgesic potency ratio (ED(50) tramadol/ED(50) morphine) of both compounds differed in neuropathic (potency ratio 2.3 in CCI and 1.2 in SNL) and nociceptive pain models (potency ratio 7.8), suggesting a relative increase in potency of tramadol in neuropathic pain compared with nociceptive pain. CONCLUSION: The results of this study are consistent with clinical data supporting the efficacy of opioids in neuropathic pain conditions, and furthermore suggest an additional contribution of the monoaminergic mechanism of tramadol in the treatment of neuropathic pain states.

Novel synthetic adhesive as an effective alternative to Fibrin based adhesives.

AIM: To compare a novel, fully synthetic, polyurethane based glue (MAR-1) to fibrin sealant in a partial liver resection rat model. METHODS: After 50% resection of the lateral left liver lobe in male Wistar rats (n = 7/group/time point), MAR-1, Fibrin or NaCl was applied. After 14, 21 and 90 postoperative days, sealant degradation, intra-abdominal adhesions were scored, and histological examination of liver tissue was performed. RESULTS: (Mean ± SEM) (MAR-1 vs Fibrin vs NaCl). Bleeding mass was significantly higher in NaCl (3.36 ± 0.51 g) compared to MAR-1 (1.44 ± 0.40 g) and Fibrin (1.16 ± 0.32 g). At 14 and 90 d, bleeding time was significantly lower in MAR-1 (6.00 ± 0.9 s; 13.57 ± 3.22 s) and Fibrin (3.00 ± 0.44 s; 22.2 ± 9.75 s) compared to NaCl (158.16 ± 11.36 s; 127.5 ± 23.3 s). ALT levels were significantly higher in MAR-1 (27.66 ± 1 U/L) compared to Fibrin (24.16 ± 0.98 U/L) and NaCl (23.85 ± 0.80 U/L). Intrabdominal adhesions were significantly lower in MAR-1 (11.22% ± 5.5%) compared to NaCl (58.57% ± 11.83%). Degradation of the glue was observed and MAR-1 showed almost no traces of glue in the abdominal cavity as compared to the Fibrin (10% ± 5% 14 d; 7% ± 3% 21 d). Survival showed no significant differences between the groups. CONCLUSION: Compared to Fibrin, MAR-1 showed similar hemostatic properties, no adverse effects, and is biocompatible. Further studies on adhesion strength and biodegradability of synthetic sealants are warranted.