This study examined the effect of scES at the L3 spinal level on urinary bladder and EUS function in both uninjured and T9 spinally transected female rats. The absence of all supraspinal inputs to the lumbosacral circuits controlling LUT function after an anatomically complete transection maximizes the known neuroplasticity24–27 and subsequent potential changes in scES effects post-SCI relative to intact controls. Physiological parameters were collected by performing terminal urodynamic procedures in urethane anesthetized rats while applying different combinations of scES with varying intensity and frequency. The results of this mapping study show the effect of scES at the L3 level is highly dependent on the phenotypic urodynamic function prior to stimulation (I.e., presence of partial EUS bursting, DSD, or overflow incontinence), demonstrating the importance of the spinal circuits controlling voiding reflexes at this level 28,29 in both uninjured and after complete chronic SCI 24–27
The activity of the EUS in rats has been extensively studied 30–32 and shown to be crucial for efficient micturition due to the pumping action of the rhythmic contractions. Indeed, as seen with the typical example in Fig. 4D, scES at high frequencies and intensities results in phasic activation (with a similar frequency to scES trains) but disrupts the normal pattern of EUS bursting. Such a disruption negatively impacts the voided volume, and presumably the voiding efficiency (as estimated by the in-out ratio, Fig. 3A and 3D). These results highlight the importance of normal EUS bursting activity for efficient voiding in rats.
Intensity and frequency dependent increases in the ICI were found (Fig. 3B) indicating that certain combinations of scES parameters cause an inactivation of the normal bladder reflexes and therefore elongate the ICI. These data are consistent with previous studies which show that tonic activation of EUS, by scES at L3 level in uninjured animals33. Because recurrent inhibition of the sacral parasympathetic neurons has a positive relationship with increasing frequencies of stimulation12, scES is likely changing the ICI by modulating these parasympathetic pathways to promote higher bladder capacities.
Bursting activity in the EUS has been shown to be modulated by L3 scES33. Data from the current study shows a frequency dependent increase in the duration of burst activity (Fig. 3C) occurred during L3 scES. Previous data also shows that a higher in/out ratio is a consequence of longer EUS bursting periods23. The frequency dependent increase of the in-out ratio in this study is consistent with those observations (Fig. 2D).
Noteworthy is the observation of subgroups of animals showing different urodynamic outcomes post-transection which cannot be explained by the SCI itself (histological verification of an anatomically complete transection was conducted). The subgroups observed in this study are likely due to variable neuroplastic changes below the level of injury. Different functional outcomes in the chronic stage post-SCI could be due to a number of factors, including differences in post-injury bladder management, urinary tract infections, the presence of bladder stones, or other idiopathic post-injury factors, which can lead to structural changes in both the detrusor muscle and neural control5,18,24,34,35.
The three described patterns of urination following SCI have been described previously, but a clear understanding of the changes underlying these patterns is still an open question 15,16,24,30,31,36,37. An added complication is that the previously described neural circuits controlling LUT function can vary and literature descriptions are sometimes contradictory 38. Furthermore, the same level and grade of SCI can lead to different urinary complications in human patients17,39,40 indicating a more thorough understanding of the changes that occur after SCI is needed. In consideration of the above factors, we hypothesize that the neural plasticity occurring in the lumbo-sacral circuitry after SCI is not uniform and therefore can cause functional differences in individuals that have the same SCI injury characteristics. Thus, targeting these circuitries with a therapeutic intervention such as scES would require some degree of initial mapping to optimize functional outcomes across individuals. Furthermore, the surgical placement of the bladder catheter and/or EUS electrodes, could also impact urodynamic performance in SCI animals 23,41.
As seen in the first subgroup of SCI rats where the EUS bursting is still present after spinal cord complete transection, scES at 5Hz, and intensities above 100µA, triggers an immediate and effective void (Fig. 5B). As previously shown in spinally intact animals, electrical stimulation of Barrington’s nucleus triggers bladder contractions and urination 42,43 through direct connections from the PMC to the sacral sympathetic neurons controlling the urinary bladder via the dorsolateral funiculus (DLF) 44. Additionally, direct stimulation of the LSCC at L3 level causes bursting activity of the EUS 45. Therefore, scES in this study is likely producing the observed effects in the first subgroup of animals (Bursting) by activation of the L3 LSCC structures 10,46,47 where it is possible that the PMC projections modulate the bursting activity under normal conditions. Interestingly, the DLF is one of the principal targets for the pain-relieving effect of scES 48 by activation of the endogenous opioid system which has also been shown to modulate urinary bladder activity 49,50.
The results observed in the DSD group show the capacity of scES to rhythmically activate the EUS (Fig. 6B). The presence of a nuclear region, adjacent to the central canal within the gray matter, at the L3-L4 spinal segment that is responsible for controlling the phasic contractile activity of the EUS during voiding has been confirmed via physiologic, pharmacologic, molecular, and trans-neuronal tracing techniques 20,21,45,51. Additionally, it has been shown that scES at L3-L4 segments can selectively modulate the activity of EUS, inactivating the tonic activity45, or triggering the phasic contraction52 by changing the characteristics of the electrical stimuli. Therefore, the scES applied in this study is likely changing LUT function by modulating the activity of LSCC in the DSD group.
Although the scES induced activation of the EUS resulted in a decrease in voided volume in this group of animals (Fig. 8B), the temporal matching between the EUS electrical activity, the HFO’s, and the releasing of a urine drop with each EUS activation (Fig. 6D) demonstrates the effectiveness of this scES-induced EUS activity. Intermittent activation of the EUS during a bladder contraction has been used successfully to treat DSD in people living with SCI via sacral anterior root stimulation 53. In a similar way, the relaxation of EUS while the detrusor is still contracting explains the flow of urine during stimulation in this study. Importantly, intermittence of urine flow due to the rhythmic opening-closure of the EUS is the typical pattern of micturition in rats 32,38,54. Therefore, these data support the hypothesis that physiologically relevant timing of frequency and duration of the scES pulses (mimicking the natural frequency, active-inactive ratio, and duration of the bursting activity) can improve the voided volume in SCI animals. Indeed, studies have shown reduced voiding efficiency from frequencies and durations of stimulation that alter the naturally occurring bursting of the EUS 30,55, which could be due to incomplete relaxation of EUS during stimulation, impairing urine flow.
The finding that some but not all female rats exhibited overflow incontinence upon reaching capacity (continuous fluid expulsion at an elevated detrusor pressure) is consistent with what was observed in our previous studies 15,16 which found a higher prevalence in males versus females. This group of animals showed a mixed response to L3 scES, which consisted of bladder contraction (similarly to the burst subgroup) and the stimulus-dependent activation of the EUS (as with the DSD subgroup). Stimulation at L3 in this group triggered a massive expulsion of urine (Fig. 7B), which is consistent with scES at different spinal levels in our previous studies15,16 whereby the interconnected network within the lower cord 21,45,51 is activated. Additionally, the stimulus-dependent bursting generated by the activation of the EUS motoneurons can explain the high voided volumes collected from these animals. The poor performance of the urinary bladder prior to scES makes this finding more relevant as it shows that the modulation of L3 centers, mimicking the physiological rhythmical intermittence of the EUS during the voiding phase of the cycle, can improve the voiding performance in SCI animals which could have enormous translational potential for the use of scES at the L3 level to treat humans with SCI-induced LUT dysfunction. Preliminary evidence from such studies by our group in humans supports this potential (unpublished observations).
Taken together, the results of this study demonstrate that scES at the L3 spinal level can modulate the activity of both the bladder and EUS in intact and SCI rats. This further highlights the L3 segment as a promising target for the treatment of DSD in SCI patients using scES. These data also show the importance of different frequencies and intensities of scES in the response to stimulation as demonstrated by changes in EUS bursting and the impact on voiding efficiency. Additionally, the identification of three distinct phenotypes of LUT dysfunction (partial burst, DSD and overflow incontinence) after complete transection, and specific responses to L3 scES of each subgroup, highlights the complexity of neuroplasticity during SCI recovery and reinforces the importance of a patient-centered evaluation of post-injury complications. Note that larger groups will be necessary in future studies as the post-study division of 7 SCI rats into different functional outcome sub-types reduced power (important trends revealed but not to the level of significance due to N’s of 1 and 2 in two of the subgroups). Importantly, the current findings illustrate the need for further studies of urinary complications in a variety of clinical scenarios to fully understand the complex autonomic changes that occur after SCI. Furthermore, the ability of L3 scES to activate the EUS, and generate artificial bursting activity, holds great potential for improving effective emptying for people living with SCI.