Fracture-induced pain-like behaviours in a femoral fracture mouse model.

This study is the first comprehensive characterisation of the pain phenotype after fracture using both evoked and naturalistic behaviours in adult male and ovariectomised female mice. It also shows that an anti-nerve growth factor (NGF) therapy could be considered to reduce pain after fracture surgery. INTRODUCTION: Bone fractures are common due to the ageing population and very painful even after healing. The phenotype of this pain is still poorly understood. We aimed to characterise it in a femoral fracture model in mice. METHODS: We employed both adult male, and female ovariectomised (OVX) mice to mimic osteoporotic fractures. Mice underwent a unilateral femoral fracture maintained by an external fixator or a sham surgery. Pain behaviours, including mechanical and thermal sensitivity, weight bearing and LABORAS, were measured from baseline to 6 weeks after fracture. The effect on pain of an antibody against nerve growth factor (anti-NGF) was assessed. Changes in nerve density at the fracture callus were analysed by immunohistochemistry. RESULTS: Following surgery, all groups exhibited high levels of invoked nociception. Mechanical and thermal hyperalgesia were observed from 1 week after surgery, with nociceptive sensitization in the fracture group maintained for the 6 weeks, whereas it resolved in the sham group after 3 weeks. OVX induced reduction in pain thresholds, which was maintained after fracture. The frequency of naturalistic behaviours did not change between groups. Anti-NGF administered before and weekly after surgery alleviated fracture-induced mechanical nociception. The density of nerve fibres in the fracture callus was similar in all groups 6 weeks after surgery. CONCLUSIONS: Fractures in rodent models are highly painful in both sexes. This pain-like phenotype is prolonged and should be routinely considered in fracture healing studies as it can affect the study outcome. The anti-NGF alleviates fracture-induced mechanical pain.

Osteoarthritis-related nociceptive behaviour following mechanical joint loading correlates with cartilage damage.

OBJECTIVE: In osteoarthritis (OA), the pain-structure relationship remains complex and poorly understood. Here, we used the mechanical joint loading (MJL) model of OA to investigate both knee pathology and nociceptive behaviour. DESIGN: MJL was used to induce OA in the right knees of 12-week-old male C57BL/6 mice (40 cycles, 9N, 3x/week for 2 weeks). Mechanical sensitivity thresholds and weight-bearing ratios were measured before loading and at weeks one, three and six post-loading. At these time points, separate groups of loaded and non-loaded mice (n = 12/group) were sacrificed, joints collected, and fur corticosterone levels measured. µCT analyses of subchondral bone integrity was performed before joint sections were prepared for nerve quantification, cartilage or synovium grading (scoring system from 0 to 6). RESULTS: Loaded mice showed increased mechanical hypersensitivity paired with altered weight-bearing. Initial ipsilateral cartilage lesions 1-week post-loading (1.8 ± 0.4) had worsened at weeks three (3.0 ± 0.6, CI = -1.8-0.6) and six (2.8 ± 0.4, CI = -1.6-0.4). This increase in lesion severity correlated with mechanical hypersensitivity development (correlation; 0.729, P = 0.0071). Loaded mice displayed increased synovitis (3.6 ± 0.5) compared to non-loaded mice (1.5 ± 0.5, CI = -2.2-0.3) 1-week post-loading which returned to normal by weeks three and six. Similarly, corticosterone levels were only increased at week one post-loading (0.21 ± 0.04 ng/mg) compared to non-loaded controls (0.14 ± 0.01 ng/mg, CI = -1.8-0.1). Subchondral bone integrity and nerve volume remained unchanged. CONCLUSIONS: Our data indicates that although the loading induces an initial stress reaction and local inflammation, these processes are not directly responsible for the nociceptive phenotype observed. Instead, MJL-induced allodynia is mainly associated with OA-like progression of cartilage lesions.

Genomic, transcriptional and mutational analysis of the mouse microphthalmia locus.

Mouse microphthalmia transcription factor (Mitf) mutations affect the development of four cell types: melanocytes, mast cells, osteoclasts, and pigmented epithelial cells of the eye. The mutations are phenotypically diverse and can be arranged in an allelic series. In humans, MITF mutations cause Waardenburg syndrome type 2A (WS2A) and Tietz syndrome, autosomal dominant disorders resulting in deafness and hypopigmentation. Mitf mice thus represent an important model system for the study of human disease. Here we report the complete exon/intron structure of the mouse Mitf gene and show it to be similar to the human gene. We also found that the mouse gene is transcriptionally complex and is capable of generating at least 13 different Mitf isoforms. Some of these isoforms are missing important functional domains of the protein, suggesting that they might play an inhibitory role in Mitf function and signal transduction. In addition, we determined the molecular basis for six microphthalmia mutations. Two of the mutations are reported for the first time here (Mitf(mi-enu198) and Mitf(mi-x39)), while the others (Mitf(mi-ws), Mitf(mi-bws), Mitf(mi-ew), and Mitf(mi-di)) have been described but the molecular basis for the mutation not determined. When analyzed in terms of the genomic and transcriptional data presented here, it is apparent that these mutations result from RNA processing or transcriptional defects. Interestingly, three of the mutations (Mitf(mi-x39), Mitf(mi-bws), and Mitf(mi-ws)) produce proteins that are missing important functional domains of the protein identified in in vitro studies, further confirming a biological role for these domains in the whole animal.

Mutations determining generalized resistance to aminoglycoside antibiotics in Escherichia coli.

Mutations conferring resistance to low levels of kanamycin in Escherichia coli have been mapped at 3 locations: the unc locus (min. 83), a locus we have designated kanA (MIN. 72), close to strA (rpsL), and a locus at min. 86.5 previously discovered by Plate (1976) that we have designated ecfB. The unc and ecfB mutations are associated with defects in energy metabolism, while mutations at kanA may be in the gene coding for ribosomal protein S12 (rpsL). The three types of mutations cause cross resistance to a number of different aminoglycoside antibiotics and the effects of the mutations are cumulative in combination.

Deletions of ribosomal protein genes in Escherichia coli merodiploids heterozygous for resistance to streptomycin and spectinomycin.

In a merodiploid strain of Escherichia coli heterozygous for the ribosomal protein genes spc and str, deletions were observed preventing the expression of either gene but permitting the expression of the other. This suggests that the spc and str genes are in separate transcriptional units.