Hypothesis / aims of study
Spinal cord injury leads to loss of voluntary control over bladder function followed by the emergence of neurogenic detrusor overactivity (NDO), characterized by strong involuntary detrusor contractions that result in urinary incontinence. NDO results from neuroplastic changes occurring in the lumbosacral cord, particularly from growth of the central processes of bladder afferents, which does not occur in non-lesioned conditions. The fine mechanisms regulating this abnormal sprouting are still unresolved but may reflect changes in the expression and effects of repulsive cues that block axonal growth. These repulsive cues include myelin associated inhibitors (MAIs), such as Nogo-A, and astrocyte-derived chondroiting sulfate proteoglycans (CSPGs), as phosphacan. In the CNS, these repulsive proteins prevent regrowth of damaged axons that express their specific receptors by inducing growth cone collapse. It is unclear if these repulsive cues are present and regulated in the lumbosacral cord following thoracic SCI and if bladder afferents respond to these proteins adjusting growth accordingly, as measured with GAP-43 expression, an established marker of sprouting of bladder afferents [1].
Study design, materials and methods
Adult female Wistar rats were used and submitted to largely incomplete spinal cord transection (SCT) at T8/T9 thoracic spinal segments T8/T9 [1, 2]. Animals were divided into three experimental groups (n=8/group). One group of female rats was used as control and not submitted to spinal lesion. Another two groups of rats were submitted to spinal cord injury and left to recover for 7 days (n=8) or 28 days (n=8). Seven (7 dpi) or 28 days post-injury (28 dpi), all animals underwent 1-hour cystometry under urethane-anaesthesia, followed by spinal cord tissue collection. Lumbosacral spinal cords (L5-S1) were processed for Western Blotting and Immunohistochemistry and expression and distribution of GAP-43, Phosphacan and Nogo-A were determined.
Results
Spinal intact animals had the normal pattern of bladder contractions. In contrast, at 7 days post-SCT dpi, bladder reflex contractions were abolished, reflecting spinal shock (p≤0.0001 compared with spinal intact). At 28 dpi, NDO was evident in all animals, with a significant increase in frequency and amplitude of detrusor contractions (p≤0.0001 versus 7 dpi animals).
Western blot analysis of lumbosacral tissue showed a significant increase of Nogo- A and Phosphacan expression at 7 dpi (p≤0.05 versus spinal intact), the expression of which was reduced at 28 dpi. Preliminary tests found a time-dependent increase in GAP43 expression, as suggested by other studies [1, 3].
Immunostaining was used to determine the spinal distribution of Nogo-A, Phosphacan and GAP43. In spinal intact animals, Nogo-A was mostly present in the dorsolateral funiculus. At 7 dpi, Nogo-A the intensity of the immunostaining was stronger in comparison with spinal intact animals. Twenty-eight days post-SCT, location and intensity of Nogo-A immunoreaction was similar to that observed in spinal intact rats. In spinal intact animals, Phosphacan was expressed in the superficial dorsal horn (medial and lateral), the dorsolateral and ventral funiculus and in some motoneurons of the ventral horn. Following SCT, specifically 7 dpi, staining intensity increased, returning to values similar to spinal intact animals at 28 dpi. GAP-43 was present in the superficial dorsal horn (where bladder afferents are known to terminate), dorsal commissure and corticospinal tract and the dorsolateral funiculus in spinal intact animals. GAP-43 expression increased 7 days and 28 days post-injury in identical regions of the lumbosacral spinal cord, except for the dorsal corticospinal tract, where expression was practically abolished by SCT. It was possible to observe lack of co-expression of GAP43 in Phosphacan and Nogo-A positive spinal areas.
Interpretation of results
Results suggest that, following thoracic SCT, changes in the expression of growth-repellent proteins, particularly Phosphacan and Nogo-A, occur at distant sites from the lesion, particularly the lumbosacral cord. Increased levels of these repulsive cues were accompanied by sprouting of bladder afferents, as shown by GAP43 expression, and may be linked to NDO development.
Concluding message
This study demonstrates that changes in the expression of repulsive cues, typically observed at lesioned tissue and subsequent scar, are not restricted to the injury but extend caudally, affecting the lumbosacral cord. Thus, response to injury is not restricted to the scar but is a widespread event impacting general recovery and development of SCI-induced pathophysiological processes, as NDO emergence. Though it is unclear if sensory neurons recognize centrally expressed growth-repellent cues, observations here suggest that bladder afferents may express receptors for Phosphacan and Nogo-A, a matter to be clarified in ongoing studies.