Design and in vivo evaluation of a microparticulate depot formulation of buprenorphine for veterinary use

Buprenorphine is a frequently used analgetic agent in veterinary medicine. A major drawback, however, is the short duration of action requiring several daily administrations. We therefore designed a poly-lactic-co-glycolic acid (PLGA) based microparticulate drug formulation for sustained parenteral drug release. Particles were designed to allow for a fast onset of action and a duration of the analgesic effect of at least two days in laboratory mice. Microparticles were produced using a solvent evaporation technique. Release rate was dependent on polymer type and particle size. Spherical particles used for subsequent animal studies had a mean size of 50 µm and contained 4.5% of buprenorphine. Drug release was characterized by an initial burst release of 30% followed by complete release over seven days. In vivo pharmacokinetic experiments in female C57BL/6 J mice confirmed prolonged exposure in plasma and brain tissue and correlated with the pharmacological effect in the hot plate assay or after minor abdominal surgery. No adverse side effects with respect to food and water intake, body weight, local tolerability, or nesting behavior were observed. Our formulation is an attractive alternative to established immediate release formulations. A use for prolonged pain management in laboratory animals is proposed

Administration of Tramadol or Buprenorphine via the drinking water for post-operative analgesia in a mouse-osteotomy model

Adequate analgesia is essential whenever pain might occur in animal experiments. Unfortunately, the selection of suitable analgesics for mice in bone-linked models is limited. Here, we evaluated two analgesics - Tramadol [0.1 mg/ml (Tlow) vs. 1 mg/ml (Thigh)] and Buprenorphine (Bup; 0.009 mg/ml) - after a pre-surgical injection of Buprenorphine, in a mouse-osteotomy model. The aim of this study was to verify the efficacy of these opioids in alleviating pain-related behaviors, to provide evidence for adequate dosages and to examine potential side effects. High concentrations of Tramadol affected water intake, drinking frequency, food intake and body weight negatively in the first 2-3 days post-osteotomy, while home cage activity was comparable between all groups. General wellbeing parameters were strongly influenced by anesthesia and analgesics. Model-specific pain parameters did not indicate more effective pain relief at high concentrations of Tramadol. In addition, ex vivo high-resolution micro computed tomography (µCT) analysis and histology analyzing bone healing outcomes showed no differences between analgesic groups with respect to newly formed mineralized bone, cartilage and vessels. Our results show that high concentrations of Tramadol do not improve pain relief compared to low dosage Tramadol and Buprenorphine, but rather negatively affect animal wellbeing

Lidocaine and bupivacaine as part of multimodal pain management in a C57BL/6J laparotomy mouse model

While the use of local anesthesia as part of multimodal pain management is common practice in human and veterinarian surgery, these drugs are not applied routinely in rodent surgery. Several recommendations on the use of local anesthesia exist, but systematic studies on their efficacy and side effects are lacking. In the present study, male and female C57BL/6J mice were subjected to a sham vasectomy or a sham embryo transfer, respectively. We tested whether a mixture of subcutaneously injected Lidocaine and Bupivacaine in combination with systemic Paracetamol applied via drinking water results in superior pain relief when compared to treatment with local anesthesia or Paracetamol alone. We applied a combination of methods to assess behavioral, emotional, and physiological changes indicative of pain. Voluntary Paracetamol intake via drinking water reached the target dosage of 200 mg/kg in most animals. Local anesthesia did not lead to obvious side effects such as irregular wound healing or systemic disorders. No relevant sex differences were detected in our study. Sevoflurane anesthesia and surgery affected physiological and behavioral measurements. Surprisingly, Paracetamol treatment alone significantly increased the Mouse Grimace Scale. Taken together, mice treated with a combination of local anesthesia and systemic analgesia did not show fewer signs of post-surgical pain or improved recovery compared to animals treated with either local anesthesia or Paracetamol

Analysis of Pain and Analgesia Protocols in Acute Cerulein-Induced Pancreatitis in Male C57BL/6 Mice

Pancreatitis is known to be painful in humans and companion animals. However, the extent of pain in experimental mouse models of acute pancreatitis is unknown. Consequently, the severity classification of acute pancreatitis in mice is controversially discussed and standardized pain management is missing. In this study, we investigated acute Cerulein-induced pancreatitis with pain-specific and well-being orientated parameters to detect its impact on mice. Male C57BL/6J male mice were injected with Cerulein; animals that received saline injections served as control group. The animals were observed for weight change and water intake. To assess pain, behaviors like stretch-and-press and reduced rearing, the Mouse Grimace Scale, and von Frey hypersensitivity were assessed. Fecal corticosterone metabolites and burrowing behavior were assessed to detect changes in the animal’s well-being. Pancreatitis severity was evaluated with amylase and lipase in the blood and pancreas histology. To investigate whether different analgesics can alleviate signs of pain, and if they influence pancreas inflammation, animals received Buprenorphine, Paracetamol in combination with Tramadol, or Metamizole in the drinking water. The calculated intake of these analgesics via drinking reached values stated to be efficient for pain alleviation. While pancreatitis did not seem to be painful, we detected acute pain from Cerulein injections that could not be alleviated by analgesics. The number of inflammatory cells in the pancreas did not differ with the analgesic administered. In conclusion: (1) Cerulein injections appear to be acutely painful but pain could not be alleviated by the tested analgesics, (2) acute pancreatitis induced by our protocol did not induce obvious signs of pain, (3) analgesic substances had no detectable influence on inflammation. Nevertheless, protocols inducing more severe or even chronic pancreatitis might evoke more pain and analgesic treatment might become imperative. Considering our results, we recommend the use of Buprenorphine via drinking water in these protocols. Further studies to search for efficient analgesics that can alleviate the acute pain induced by Cerulein injections are needed

Measuring endogenous corticosterone in laboratory mice - a mapping review, meta-analysis, and open source database

Evaluating stress in laboratory animals is a key principle in animal welfare. Measuring corticosterone is a common method to assess stress in laboratory mice. There are, however, numerous methods to measure glucocorticoids with differences in sample matrix (e.g., plasma, urine) and quantification techniques (e.g., enzyme immunoassay or radioimmunoassay). Here, the authors present a mapping review and a searchable database, giving a complete overview of all studies mea­suring endogenous corticosterone in mice up to February 2018. For each study, information was recorded regarding mouse strain and sex; corticosterone sample matrix and quantification technique; and whether the study covered the research theme animal welfare, neuroscience, stress, inflammation, or pain (the themes of specific interest in our con­sortium). Using all database entries for the year 2012, an exploratory meta-regression was performed to determine the effect of predictors on basal corticosterone concentrations. Seventy-five studies were included using the predictors sex, time-since-lights-on, sample matrix, quantification technique, age of the mice, and type of control. Sex, time-since-lights-on, and type of control significantly affected basal corticosterone concentrations. The resulting database can be used, inter alia, for preventing unnecessary duplication of experiments, identifying knowledge gaps, and standardizing or heterogenizing methodologies. These results will help plan more efficient and valid experiments in the future and can answer new questions in silico using meta-analyses

Measuring Endogenous Corticosterone in Laboratory Mice - a Mapping Review, Meta-Analysis, and Open Source Database

Evaluating stress in laboratory animals is a key principle in animal welfare. Measuring corticosterone is a common method to assess stress in laboratory mice. There are, however, numerous methods to measure glucocorticoids with differences in sample matrix (e.g., plasma, urine) and quantification techniques (e.g., enzyme immunoassay or radioimmunoassay). Here, the authors present a mapping review and a searchable database, giving a complete overview of all studies measuring endogenous corticosterone in mice up to February 2018. For each study, information was recorded regarding mouse strain and sex; corticosterone sample matrix and quantification technique; and whether the study covered the research theme animal welfare, neuroscience, stress, inflammation, or pain (the themes of specific interest in our consortium). Using all database entries for the year 2012, an exploratory meta-regression was performed to determine the effect of predictors on basal corticosterone concentrations. Seventy-five studies were included using the predictors sex, time-since-lights-on, sample matrix, quantification technique, age of the mice, and type of control. Sex, time-since-lights-on, and type of control significantly affected basal corticosterone concentrations. The resulting database can be used, inter alia, for preventing unnecessary duplication of experiments, identifying knowledge gaps, and standardizing or heterogenizing methodologies. These results will help plan more efficient and valid experiments in the future and can answer new questions in silico using meta-analyses

Evaluierung des Schmerzmanagements in einem Maus-Osteotomie-Modell – Integration eines Refinement Ansatzes in eine grundlagenwissenschaftliche Studie

Tramadol applied via the drinking water is a commonly used analgesia in the mouse osteotomy model. Another opioid that can be used is buprenorphine. The recommendation for tramadol in the drinking water was increased 40-fold by the GV-SOLAS from 2010 to 2015. A recommendation on buprenorphine is given for injection but not for the application in the drinking water. Nevertheless, some standard operating procedures are found on buprenorphine applied via the drinking water. Model-specific recommendations on pain management in the mouse osteotomy model are not available. In the current study, three pain management protocols, two dosages of tramadol and buprenorphine applied via the drinking water in the mouse osteotomy model were tested. This refinement project was integrated into a basic research study. The aim of this project was to provide researchers with a specific recommendation on pain treatment in bone-linked mouse models. The three pain management protocols (tramadol 0.1 mg/ml, tramadol 1 mg/ml and buprenorphine 0.009 mg/ml in the drinking water) were evaluated for feasibility, analgesic efficacy and impact on the bone healing in the mouse osteotomy model. The feasibility was tested with the assessment of body weight, food and water intake, serum level of tramadol and M1 as well as stress measurements. The stress measurements consisted of the analysis of fecal corticosterone metabolites as a parameter for short-term stress and the histological assessment of adrenal glands as a measurement for long-term stress. To check for possible side effects of the different medications, the livers were histologically assessed. With the help of parameters such as a clinical score, nest complexity, explorative behavior and an activity assessment the animals were screened for behavior indicating pain or reduced wellbeing. Model-specific pain parameters like the limp score, dragging score, flinching, guarding, grooming time of the operated limb and rear up time were applied. These behavioral and model-specific tests are used to evaluate the analgesic efficiency of tramadol and buprenorphine. To grade the impact of the different pain management protocols on the animal model parameters of bone healing were assessed. These included in vitro μCT and histology of the osteotomized bone after euthanasia. During the refinement project, more questions came up regarding the pre-emptive injection of buprenorphine. Therefore, a study on the impact of pre-emptive injected buprenorphine on bleeding during the surgery was conducted. The animals used in the study drank after the procedure with more drinking events in the night compared to the day. A reduced wellbeing in animals treated with the tramadol dosage of 1 mg/ml was found. Also seen in the body weight loss, reduced food intake and drinking events, the clinical scoring and nest complexity. Short stress was visible on day one in all animals, but there were no signs of histological changes in the adrenal glands. The used clinical scoring was not specific to pain, but rather displayed a reduced wellbeing in Thigh animals. In the explorative behavior Tlow animals showed the fastest recovery. Operated animals had lowest activity with no clear differences between the treatment groups. The pain-specific parameters showed no better pain relief in the high tramadol group. No impact of the changed pain management protocol on the bone healing and therefore on the main read-out of the mouse osteotomy model was seen. One animal was euthanized prior to the end of the study which was not related to the analgesic treatment but rather due to a preexisting potential metabolic disorder. Tramadol of the low dosage showed serum concentrations that can be assumed sufficient to relief pain compared to human values. The time point of pre-emptive buprenorphine injection influenced the bleeding during surgery, specifically an injection 1h before the surgery increased bleeding. Additionally, buprenorphine may alter wound healing. After successfully conducting this integrated refinement study, more projects of this kind can be recommended to gain knowledge of possible refinement benefits in specific animal models. Tramadol and buprenorphine in the drinking water is an efficient route of applying analgesia in the MOMo. The concern of a reduced water intake was not confirmed in tramadol of the low dosage and in buprenorphine. The use of tramadol in the dosage of 1 mg/ml is not necessary in the MOMo. It offers no extra benefit in pain reduction but rather reduces wellbeing in the animals. Tramadol in the dosage of 0.1 mg/ml or buprenorphine in the drinking water are a sufficient method to relief pain in the MOMo.Tramadol wird im Maus-Osteotomie-Model häufig als Schmerzmittel im Trinkwasser verwendet. Ein weiteres häufig angewendetes Opioid in der Schmerzbehandlung in der Versuchstierkunde ist Buprenorphin. Die Empfehlung der GV-SOLAS für Tramadol im Trinkwasser wurde von 2010 bis 2015 um ein vierzigfaches erhöht. Eine Empfehlung für die Injektion von Buprenorphin ist vorhanden, die Empfehlung für eine Gabe über das Trinkwasser gibt es nicht. Standardarbeitsanweisungen zu Buprenorphin im Trinkwasser sind vorhanden während modell-spezifische Empfehlungen zum Schmerzmanagement im Maus-Osteotomie-Modell fehlen. In dieser Studie wurden drei Schmerzmanagement Protokolle, zwei Dosierungen von Tramadol und Buprenorphin im Trinkwasser im Maus-Osteotomie-Modell untersucht. Dieses Refinement Projekt war in eine grundlagenwissenschaftliche Studie eingebettet, die das Maus-Osteotomie-Modell benutzte. Ziel dieses Projekts war es, Wissenschaftlern spezifische Empfehlungen zum Schmerzmanagement im Maus-Osteotomie-Modell zur Verfügung zu stellen. Drei Schmerzmanagement Protokolle (Tramadol 0.1 mg/ml, Tramadol 1 mg/ml und Buprenorphin im Trinkwasser) wurden unter dem Aspekt der Umsetzbarkeit, analgetischer Wirksamkeit und Einfluss auf die Knochenheilung im Maus-Osteotomie-Modell evaluiert. Die Realisierbarkeit wurde mit der Messung des Körpergewichts, Futter- und Wasseraufnahme und Messungen zur Stressbelastung bewertet. Diese bestanden aus einer Analyse der fäkalen Corticosteronmetaboliten als Parameter für Kurzzeitstress und der histologischen Untersuchung der Nebennieren als Parameter für Langzeitstress. Zusätzlich wurden die Serumkonzentration von Tramadol und M1 gemessen und die Lebern von operierten Mäusen nach der Euthanasie histologisch untersucht. Mit Hilfe von einem klinischen Score, der Nestkomplexität, des explorativen Verhaltens und einer Aktivitätsmessung wurden die Tiere auf Verhalten getestet, welches auf Schmerz oder reduziertes Wohlbefinden hinweist. Modell-spezifische Schmerzparameter wie der Limp Score und Dragging Score, Flinching, Guarding, Grooming und das Aufrichten auf beide Beine wurden angewandt. Diese Verhaltens- und Modell-spezifischen Tests dienten dazu die analgetische Wirksamkeit von Tramadol und Buprenorphin zu evaluieren. Um den Einfluss der verschiedenen Protokolle auf das Tiermodell zu untersuchen, werden Knochenheilungsparameter genutzt, die auch in der zugrundeliegenden Studie angewandt wurden. Diese beinhielten die Mikro-Computertomographie und die histologische Untersuchung der osteotomierten Beine nach der Euthanasie. Während der Studie kamen neue Fragen auf, was zu zusätzlichen Untersuchungen führte. Eine Studie zum Einfluss von Buprenorphin, injiziert als präemptives Schmerzmittel, auf Blutungen während der Operation wurde durchgeführt. Die Tiere in dieser Studie tranken zuverlässig nach der Operation, beziehungsweise Anästhesie und Gabe von Analgesie über das Trinkwasser. Während der Nacht war die Anzahl der Trinkevents höher als am Tag. Die Tiere mit Tramadol in der Dosis 1 mg/ml zeigten ein reduziertes Wohlbefinden, was sich im reduzierten Körpergewicht, der reduzierten Futteraufnahme, niedrigeren Wasseraufnahmefrequenzen, klinischen Scores und geringerer Nestkomplexität wiederspiegelte. Kurzzeitstress war sichtbar in allen Gruppen am Tag 1. Es gab keine Hinweise auf histologische Veränderungen in den Nebennieren. Der benutzte klinische Score war nicht schmerzspezifisch und wies vielmehr auf ein reduziertes Wohlbefinden in den Tieren mit einer hohen Tramadoldosis hin. Im explorativen Verhalten zeigten operierte Mäuse mit Tramadol in der niedrigen Dosis die schnellste Erholung. Operierte Tiere hatten die längsten Ruhezeiten, mit keinen deutlichen Unterschieden zwischen den einzelnen Behandlungsgruppen. Die schmerzspezifischen Parameter wiesen darauf hin, dass die hohe Tramadoldosis nicht zu einer verbesserten Schmerzausschaltung führt. Das geänderte Schmerzmanagement hatte keine Auswirkungen auf die Knochenheilung und damit auch nicht auf das hauptsächliche Auswertungskriterium des Maus-Osteotomie-Modells. Ein Tier wurde vorzeitig euthanasiert, was nicht in Verbindung mit der Behandlung stand, sondern vermutlich durch eine bereits bestehende Stoffwechselerkrankung hervorgerufen wurde. Verglichen mit der humanen effektiven Serumkonzentration, stellte die Serumkonzentration von Mäusen bei einer Behandlung mit der niedrigen Tramadoldosis vermutlich eine ausreichende Schmerzausschaltung sicher. Der Zeitpunkt einer präemptiven Buprenorphininjektion beeinflusste die Stärke und das Auftreten von Blutungen während der Operation. Zusätzlich veränderte Buprenorphin möglicherweise die Wundheilung. Nachdem diese integrierte Refinement Studie im Maus-Osteotomie-Modell erfolgreich zu Ende gebracht wurde, sollten Studien dieser Art vermehrt durchgeführt werden, um mögliche Vorteile von Refinementmethoden in spezifischen Tiermodellen aufzuzeigen. Die Gabe von Tramadol und Buprenorphin über das Trinkwasser ist eine effiziente Art des Schmerzmanagements im Maus-Osteotomie-Modell. Die Befürchtung eines reduzierten Trinkverhaltens konnte für die niedrige Tramadoldosis und Buprenorphin nicht bestätigt werden. Die erhöhte Tramadoldosis von 1 mg/ml ist im Maus-Osteotomie-Modell nicht notwendig. Die erhöhte Dosis führt zu keiner erhöhten Schmerzreduktion, sondern eher zu einem reduzierten Wohlbefinden der Tiere. Die niedrigere Tramadoldosis von 0,1 mg/ml und Buprenorphin im Trinkwasser führen zu einer ausreichenden Schmerzausschaltung im Maus-Osteotomie-Modell

Design and in vivo evaluation of a microparticulate depot formulation of buprenorphine for veterinary use

Buprenorphine is a frequently used analgetic agent in veterinary medicine. A major drawback, however, is the short duration of action requiring several daily administrations. We therefore designed a poly-lactic-co-glycolic acid (PLGA) based microparticulate drug formulation for sustained parenteral drug release. Particles were designed to allow for a fast onset of action and a duration of the analgesic effect of at least two days in laboratory mice. Microparticles were produced using a solvent evaporation technique. Release rate was dependent on polymer type and particle size. Spherical particles used for subsequent animal studies had a mean size of 50 µm and contained 4.5% of buprenorphine. Drug release was characterized by an initial burst release of 30% followed by complete release over seven days. In vivo pharmacokinetic experiments in female C57BL/6 J mice confirmed prolonged exposure in plasma and brain tissue and correlated with the pharmacological effect in the hot plate assay or after minor abdominal surgery. No adverse side effects with respect to food and water intake, body weight, local tolerability, or nesting behavior were observed. Our formulation is an attractive alternative to established immediate release formulations. A use for prolonged pain management in laboratory animals is proposed

Administration of Tramadol or Buprenorphine via the drinking water for post-operative analgesia in a mouse-osteotomy model

Adequate analgesia is essential whenever pain might occur in animal experiments. Unfortunately, the selection of suitable analgesics for mice in bone-linked models is limited. Here, we evaluated two analgesics – Tramadol [0.1 mg/ml (Tlow) vs. 1 mg/ml (Thigh)] and Buprenorphine (Bup; 0.009 mg/ml) – after a pre-surgical injection of Buprenorphine, in a mouse-osteotomy model. The aim of this study was to verify the efficacy of these opioids in alleviating pain-related behaviors, to provide evidence for adequate dosages and to examine potential side effects. High concentrations of Tramadol affected water intake, drinking frequency, food intake and body weight negatively in the first 2–3 days post-osteotomy, while home cage activity was comparable between all groups. General wellbeing parameters were strongly influenced by anesthesia and analgesics. Model-specific pain parameters did not indicate more effective pain relief at high concentrations of Tramadol. In addition, ex vivo high-resolution micro computed tomography (µCT) analysis and histology analyzing bone healing outcomes showed no differences between analgesic groups with respect to newly formed mineralized bone, cartilage and vessels. Our results show that high concentrations of Tramadol do not improve pain relief compared to low dosage Tramadol and Buprenorphine, but rather negatively affect animal wellbeing

Systematic review on the reporting accuracy of experimental details in publications using mouse femoral fracture models

The outcomes of animal experiments can be influenced by a variety of factors. Thus, precise reporting is necessary to provide reliable and reproducible data. Initiatives such as the ARRIVE guidelines have been enrolled during the last decade to provide a road map for sufficient reporting. To understand the sophisticated process of bone regeneration and to develop new therapeutic strategies, small rodents, especially mice, are frequently used in bone healing research. Since many factors might influence the results from those studies, we performed a systematic literature search from 2010 to 2019 to identify studies involving mouse femoral fracture models (stable fixation) and evaluated the reporting of general and model-specific experimental details. 254 pre-selected publications were systematically analyzed, showing a high reporting accuracy for the used mouse strain, the age or developmental stage and sex of mice as well as model-specific information on fixation methods and fracturing procedures. However, reporting was more often insufficient in terms of mouse substrains and genetic backgrounds of genetically modified mice, body weight, hygiene monitoring/immune status of the animal, anesthesia, and analgesia. Consistent and reliable reporting of experimental variables in mouse fracture surgeries will improve scientific quality, enhance animal welfare, and foster translation into the clinic