Silencing gut pain without pain killers flinders CommsBio
. Following removal of DRG, the wound was irrigated with 0.5% Bupivicaine and muscle closed with individual 5.0 polyglycholic acid absorbable suture . Skin was closed with 6.0 Nylon non-absorbable suture and the site cleaned with 0.5% chlorhexidine and 70% alcohol swab. Prior to withdrawal of anaesthesia, animals were administered a second s.c. dose of 0.05 mg/kg buprenorphine and s.c. antibiotics—100 mg/kg Ampicillin and 10 mg/kg Baytril .
For ex vivo and immunohistochemical studies, mice were euthanized using isoflurane inhalation overdose, followed by cervical dislocation.The identical procedure for sham mice was used as for DRG lesion mice, except that DRGs were not physically exposed in sham mice, nor lesions made to the dorsal nerve roots. An incision was made along the dorsal midline and skeletal muscles retracted to expose the vertebral column.
VMRs to electrical stimulation of rectum and bladder 6 months after surgery were performed by anaesthetising with pentobarbital sodium ; depth of anaesthesia was assessed by lack of response to hindlimb or tail pinch. Electromyographic electrodes were placed into the left external oblique muscle and a reference electrode placed in the quadriceps muscle of the opposing leg. EMG recordings were acquired at 20 kHz and recorded on a PC running LabChart 7 Pro software and high pass filtered .
Slides were viewed with an Olympus IX71 epifluorescence microscope using appropriate laser wavelengths. Images were captured as TIFF files at ×4 magnification with a CoolSNAP™ camera and AnalySIS Image 5.0 computer software . Background autofluorescence in each image was normalised using the
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