Hypothesis / aims of study
Spinal cord injury (SCI) can have detrimental consequences to the lower urinary tract (LUT) resulting in loss of voluntary bladder control, decreased bladder compliance, increased risk of infections and potentially damage to the upper urinary tract. Neurotrophins have been implicated to play a significant role in the development of neurogenic bladder overactivity (NDO), where nerve growth factor (NGF) has been implicated in the sensitization of C-fibre afferent pathways and detrusor overactivity following SCI. Interestingly, brain derived neurotrophic factor (BDNF) also plays a crucial role in the development of SCI-induced LUT dysfunction by counteracting spinal neural remodelling and inhibiting the emergence of NDO . Therefore, our aim was to determine the therapeutic potential of LM22B-10, a selective small molecule agonist of TrkB/C receptors, in ameliorating voiding dysfunction using a mouse model of incomplete spinal cord injury.
Study design, materials and methods
Spinal cord contusion surgery and functional assessments: Female C57Bl/6 mice were anesthetized using 1.5-2% isoflurane, a laminectomy performed and the spinal cord exposed between T9-T10 vertebrae. The exposed cord was subjected to severe contusion injury (75 kDy force; Infinite Horizon Impactor, Precision Instrument). Sham controls underwent laminectomy surgeries without contusion. The surgical wound was packed with haemostatic sponge and the muscle and skin sutured. After surgery, the animals had their bladders expressed twice a day by gentle abdominal compression and given daily prophylactic antibiotics and analgesics for up to one week. Mice were evaluated for hindlimb locomotion recovery using the Basso mouse scale  and two-hour urine spot tests at one, three, seven, 14, 21 and 28 days following injury. The effects of LM22B-10 were evaluated by subcutaneous implantation of osmotic pumps to deliver 5 mg/kg/day of drug or vehicle over a two-week period; pumps were implanted at 21 to 28 days post-SCI surgeries.
Immunofluorescence: Mice were anesthetized with 5% isoflurane, the urinary bladder dissected out and the mouse was transcardially perfused with 1 x tris-buffered saline (1xTBS) followed by 4% paraformaldehyde for dissection of the spinal cord. Bladders were fixed flat in a dissection dish with 4% PFA. Spinal cord and bladder tissues were stored overnight in 30% sucrose solution then embedded in optimal cutting medium for cryosectioning. Tissue section were washed in 1xTBS, blocked with 10% normal donkey serum + 0.1% Triton-X in 1xTBS and incubated overnight in primary antibodies at 4ºC. Sections were then incubated in appropriate fluorescent secondary antibodies and visualized using an Olympus IX62 fluorescent upright microscope.
Interpretation of results
These data demonstrate activation of TrkB/C signalling by LM22B-10 has a positive effect in ameliorating SCI-induced bladder overactivity in the mouse model. Our data showed contusion SCI resulted in increased TrkB expression at the site of injury that appeared to be associated with astrocytes or glial cells. Astrocytes selectively express a truncated TrkB isoform that when activated induces their proliferation, release of inflammatory cytokines and formation on the glial scar ; thus, hindering neural regeneration and in turn recovery of LUT function. It is hypothesized that LM22B-10 may elicits its beneficial effects by downregulating truncated TrkB expression on astrocytes and/or biased activation of regenerative signalling pathways.