Refinement of analgesia following thoracotomy and experimental myocardial infarction using the Mouse Grimace Scale

New Findings What is the central question of this study? There is an ethical imperative to optimize analgesia protocols for laboratory animals, but this is impeded by our inability to recognize pain reliably. We examined whether the Mouse Grimace Scale (MGS) provides benefits over a standard welfare scoring system for identifying a low level of pain in the frequently used murine surgical model of myocardial infarction. What is the main finding and its importance? Low-level pain, responsive to analgesia, was detected by MGS but not standard methods. In this model, most of the pain is attributable to the thoracotomy, excepted in mice with very large infarcts. This approach represents a model for assessing postsurgical analgesia in rodents. The Mouse Grimace Scale (MGS) was developed for assessing pain severity, but the general applicability to complex postsurgical pain has not been established. We sought to determine whether the MGS provides benefits over and above a standard welfare scoring system for identifying pain in mice following experimental myocardial infarction. Female C57BL/6J mice (n = 60), anaesthetized with isoflurane, were subjected to thoracotomy with ligation of a coronary artery or sham procedure. A single s.c. dose of buprenorphine (1.1 mg kg−1) was given at the time of surgery and pain assessed at 24 h by MGS and a procedure-specific welfare scoring system. In some animals, a second dose of 0.6 mg kg−1 buprenorphine was given and pain assessment repeated after 30 min. The MGS was scored from multiple photographs by two independent blinded observers with good correlation (r = 0.98). Using the average MGS score of both observers, we identified a subset of mice with low scores that were not considered to be in pain by the welfare scoring system or by single observer MGS. These mice showed a significant improvement with additional analgesia, suggesting that this low-level pain is real. Pain attributable to the myocardial injury, as opposed to thoracotomy, persisted at 24 h only in mice with large infarcts >40%. In conclusion, the use of a multi-observer, post hoc version of the MGS is a sensitive tool to assess the efficacy of postsurgical analgesic protocols. Following surgical induction of myocardial infarction, we identified a significant proportion of mice that were in low-level pain at 24 h that were not identified by other assessment methods

Impaired cardiac contractile function in arginine:glycine amidinotransferase knockout mice devoid of creatine is rescued by homoarginine but not creatine

Aims: Creatine buffers cellular adenosine triphosphate (ATP) via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:glycine amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine (HA) synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesized that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality. Methods and results: Withdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ∼2.7%/day; however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naïve to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naïve AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy, and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalization of myocardial creatine. AGAT-/- mice had low plasma HA and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that HA is necessary for normal cardiac function. Conclusions: Our findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that HA deficiency can impair cardiac function, which may explain why low HA is an independent risk factor for multiple cardiovascular diseases

An Aged Canid with Behavioral Deficits Exhibits Blood and Cerebrospinal Fluid Amyloid Beta Oligomers

Many of the molecular and pathological features associated with human Alzheimer disease (AD) are mirrored in the naturally occurring age-associated neuropathology in the canine species. In aged dogs with declining learned behaviour and memory the severity of cognitive dysfunction parallels the progressive build up and location of Aβ in the brain. The main aim of this work was to study the biological behaviour of soluble oligomers isolated from an aged dog with cognitive dysfunction through investigating their interaction with a human cell line and synthetic Aβ peptides. We report that soluble oligomers were specifically detected in the dog’s blood and cerebrospinal fluid via anti-oligomer- and anti-Aβ specific binders. Importantly, our results reveal the potent neurotoxic effects of the dog’s cerebrospinal fluid on cell viability and the seeding efficiency of the cerebrospinal fluid-borne soluble oligomers on the thermodynamic activity and the aggregation kinetics of synthetic human Aβ. The value of further characterising the naturally occurring Alzheimer-like neuropathology in dogs using genetic and molecular tools is discussed

Effect of acute oxidative stress on myocardial creatine kinase (CK) isoform activities: mitochondrial (Mito-CK), myofibrilar (CK-MM), minor cytosolic isoforms (CK-MB, CK-BB): Effect of 0.5 µM H₂O₂ on CK isoenzyme activities of (A) GAMT WT (n = 3) and KO (n = 6) (B) CrT-WT (n = 3) and OE (n = 4).

<p>Effect of doxorubicin on CK isoenzyme activities of (<b>C</b>) GAMT WT (n = 6) and KO (n = 7) and (<b>D</b>) CrT-WT (n = 4) and OE (n = 5). Enzyme activities given in IU/mg protein normalized to WT values. Data mean±S.E.M. Comparison with wild-type for that strain by Student’s t-test.</p

Graded effect of varying doses of H₂O₂ in Krebs-Henseleit buffer on functional arrest time (time for LVDP to reduce to 0 mmHg) in isolated perfused mouse hearts (n = 3/group) (A).

<p>(<b>B</b>) Protective effect of catalase against H₂O₂–induced function deterioration. *P<0.01 catalase treated (n = 4) <i>vs</i>. H₂O₂ treated group (n = 4). (<b>C</b>) Protective effect of dexrazoxane on <i>ex-vivo</i> cardiac function against doxorubicin (Dox) challenge. *P<0.05 <i>vs</i>. dexrazoxane treated hearts (n = 5); Dox (n = 3). LVDP-left ventricular developed pressure. Data mean±S.E.M. P value is for the effect of the genotype by ANOVA with Bonferroni’s correction.</p

Effect of exogenous creatine supplementation (500 µmol/L) on cardiac function during 70 min doxorubicin (Dox) exposure (n = 8, C57BL/6J; male).

<p>RPP- rate pressure product. Data mean±S.E.M.</p

Effect of 0.5 µM H₂O₂ on cardiac function during 30 min exposure in isolated perfused GAMT KO hearts and wild-type (WT) controls (A).

<p>GAMT KO (n = 4) with the age and sex matched corresponding wild-type (GAMT WT) mice (n = 4; female). (<b>B</b>) Effect of 0.5 µM H₂O₂ on cardiac function during 30 min exposure in isolated perfused Cr-T overexpressing (CrT-OE) hearts (n = 7; 7 female) and age matched wild-type (CrT-WT) controls (n = 4; female). RPP- rate pressure product. *P<0.05 vs WT. Data mean±S.E.M. P value is for the effect of the genotype by ANOVA with Bonferroni’s correction.</p