Selective small molecule TrkB/C agonist, LM22B-10, improves voiding function in spinal cord contused mice

Ikeda Y1, Zabbarova I1, Getchell S1, Kozlowski M1, Birder L1, Kanai A1

Research Type

Basic Science / Translational

Abstract Category

Neurourology

Abstract 211
Neurourology and Interventions
Scientific Podium Short Oral Session 10
Wednesday 4th September 2019
15:30 - 15:37
Hall G3
Animal Study Basic Science Spinal Cord Injury Voiding Dysfunction Pre-Clinical testing
1.University of Pittsburgh
Presenter
Y

Youko Ikeda

Links

Abstract

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 [1].  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 [2] 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.
Results
Following SCI, urine spot tests showed that injured mice exhibited decreased voided volume and increased urinary frequency (presented as multiple small urine spots) compared to sham controls (Figure 1A versus 1B).  A two-week treatment with LM22B-10 normalized the voiding pattern of SCI mice to that comparable with sham operated mice (Figure 1C versus 1A). There did not appear to be a significant improvement in the hindlimb locomotion recovery in LM22B-10 treated mice compared to vehicle treated group (not shown).  Chronic SCI resulted in the extensive deposition of collagen fibers at the site of injury (Gomori's trichrome staining, Figure 2A-C).  Expression of TrkB receptors was increased in the spinal cord around the injury site and appeared to be predominantly associated with growth associated protein-43 (GAP-43) or glial fibrillary acidic protein (GFAP; not shown) positive cells surrounding the fibrotic glial scar (Figure 2B, D and F).  SCI-induced upregulation of TrkB receptors in the spinal cord was ameliorated by LM22B-10 treatment (Figure 2F versus 2D).
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 [3]; 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.
Concluding message
Our data demonstrate that a two-week administration of the TrkB/C agonist, LM22B-10, improved voiding function in chronic SCI mice that was associated with decreased expression of TrkB at the injury site.  Modulation of neurotrophin signalling with small molecules could represent a novel treatment for SCI-induced LUT dysfunction.
Figure 1 Figure 1. Urine spot tests from sham control and spinal cord injured mice with and without LM22B-10 treatment.
Figure 2 Figure 2. Histological and immunofluorescence images of T8-T10 spinal cord from control and spinal cord injured mice with and without LM22B-10 treatment.
References
  1. Frias B et al., J Neurosci., 35(5):2146-60, 2015
  2. Basso et al., J Neurotrauma, 23(5):635-59, 2006
  3. Matyas J et al., J Neursci., 37(14):3956-3971, 2017
Disclosures
<span class="text-strong">Funding</span> Awards from NIH/NIDDK; DK098361 (Kanai and Drake), P01 DK093424 (Kanai) and Department of defense SC170171 (Kanai and Ikeda) <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> mouse <span class="text-strong">Ethics Committee</span> University of Pittsburgh Institutional Animal Care and Use Committee