Hypothesis / aims of study
Lower urinary tract dysfunction (LUTD) is a common complication of spinal cord injury (SCI) and a major cause of death. It has been reported that nerve growth factor (NGF) is a crucial mediator involved in the emergence of LUTD after SCI. Previous studies demonstrated that neutralization of NGF can reduce non-voiding contractions (NVCs), improve voiding efficiency and restore the hyperexcitability of bladder afferent neurons in SCI mice. Na+ current is a dominant factor of neuronal excitability. There are nine subtypes of Nav channels found throughout the body (Nav1.1-1.9), and Nav 1.6-1.9 channels were detected in isolated muscle afferent neurons. Previous studies demonstrated that Nav channels existed in bladder afferent neurons and a transition from tetrodotoxin(TTX)-resistant to TTX-sensitive of Na+ current was found after SCI. Here, we aim to investigate the effect of NGF neutralization on Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury.
Study design, materials and methods
Thirty-sixth female C57/BL6 mice were randomly divided into three groups: spinally intact (SI) group, SCI group and SCI+NGF Ab group. SCI was conducted by transection at the Th8/9 level. In SCI+NGF Ab group, anti-NGF antibodies (10 μg•kg-1 per hour) were administered subcutaneously for 2 weeks by osmotic pump. Bladder was emptied by pressing twice a day for SCI mice. Bladder afferent neurons were labelled with Fluoro-gold (FG), injected into the bladder wall three weeks after SCI. Four weeks after SCI, The cystometrograms (CMGs) of all group were recorded to evaluate the effect of NGF on bladder function. Besides, L6-S1 dorsal root ganglion (DRG) neurons were dissociated and whole cell patch clamp recordings were performed on FG-labelled neurons. Action potential (AP) and Na+ current were recorded before and after TTX intervention. L6-S1 DRGs were harvested for immunofluorescence (IF) staining of Nav1.7 and Nav1.8 subtypes.
The results of CMG showed the bladder overactivity, performed as increased non-voiding contractions (NVCs), in SCI mice. NGF neutralizing treatment could significantly reduce the number of NVCs and improve the voiding efficiency. The whole-cell patch clamp recordings showed that the excitability of bladder afferent DRG neurons was significantly increased in SCI mice, performed as lower threshold of AP and multiple firing pattern. Besides, TTX could partly inhibit AP and Na+ current of bladder afferent neuron in SI mice, which are almost completely inhibited in SCI mice. (Figure 1) Specifically, the data showed increased total Na+ current, TTX-Sensitive Na+ current and decreased TTX-Resistant Na+ current after SCI. (Figure 2) These changes of physiological properties can be partially reversed by NGF-antibody treatment. IF staining showed that Nav1.7 was significantly increased and Nav1.8 was significantly decreased in bladder afferent neurons after SCI, which can be restored by NGF-antibody treatment.
Interpretation of results
These results indicate that (1) Increased level of NGF can induce bladder overactivity after SCI, and NGF neutralizing treatment is effective for restoring the bladder function; (2) SCI can induce a transition of Na+ channels from TTX-resistant to TTX-sensitive subtypes, which is associated with the increased level of NGF and can be restored by NGF neutralizing treatment. (3) SCI can induce increased expression of Nav1.7 (TTX-sensitive) and decreased expression of Nav1.8 (TTX-resistant), which can be restored by NGF neutralizing treatment. Thus, it is assumed that increased level of NGF after SCI can mediate Na+ channel plasticity of bladder afferent neurons that TTX-resistant subtypes shift to TTX-sensitive subtypes, and result in bladder overactivity.