Nerve Regeneration Restores Supraspinal Control of Bladder Function after Complete Spinal Cord Injury

PNG + aFGF + ChABC improves micturition patterns after complete SCI. Representative metabolic cage voiding patterns were from select groups (A) and quantification of the results (B, C) from each group at 3 and 6 months after SCI. In each step-like curve of the graph, the horizontal line represents individual micturition events, and the vertical lines are the volume of urine expelled (change from baseline). PNG + aFGF + ChABC animals showed higher frequencies and lower volumes per micturition than the other SCI groups. There results were significant when compared with all SCI groups (*p < 0.05), when compared with Tx-only, PNG, and aFGF + ChABC groups (^†p < 0.05), when compared with the Tx-only, PNG, aFGF + ChABC, PNG + ChABC, and PNG + aFGF group (^§p < 0.05), and when compared with 3 months with its own group (^ψp < 0.05).

PNG + aFGF + ChABC improves urodynamics and bladder/EUS coordination after complete SCI. A, Representative voiding and bladder pressure recordings (top 2 panels; * indicates micturition point) and EUS EMG activity (bottom 2 panels) were recorded for each group at 6 months after SCI. The two middle panels show magnified portions at the point at which micturition occurred. Animals that received PNG + aFGF + ChABC treatment had much reduced amplitudes and frequencies of non-voiding bladder contractions and much better coordination between EUS EMG bursting activity and bladder contractions during the micturition point. Quantification of the CMG results (B) and EUS EMG bursting patterns (C) during voiding shows that the triple-combination animals had the longest durations of EUS EMG bursting activity, better coordination between bladder contractions and EUS bursting activity during voiding, and the lowest firing rates of bursting when compared with animals that received alternative treatments. There results were significant when compared with all SCI groups (^†p < 0.05), when compared with Tx-only, PNG, and aFGF + ChABC groups (^ψp < 0.05), when compared with Tx-only, PNG, aFGF + ChABC, and PNG + ChABC groups (^§p < 0.05), and when compared with the Tx-only, PNG, aFGF + ChABC, PNG + ChABC, and PNG + aFGF group (^Øp < 0.05).

PNG + aFGF + ChABC treatment improves bladder morphology. A–N, The Tx-only, PNG, aFGF + ChABC, and PNG + ChABC groups show edematous connective tissue and the proliferation of urothelial layers. Both the PNG + aFGF and PNG + aFGF + ChABC groups show less tissue edema and less proliferation of the urothelial layers (arrow). Scale bars: A, C, E, G, I, K, M, 500 μm; B, D, F, H, J, L, N, 250 μm. O, PNG + aFGF + ChABC animals had the most significantly reduced bladder weight among all the SCI groups. The red color indicates muscle fibers, and the blue color indicates collagen. There results were significant when compared with all SCI groups (^†p < 0.05), when compared with Tx-only, PNG, aFGF + ChABC, and PNG + ChABC groups (^§p < 0.05), and when compared with the Tx-only, PNG, aFGF + ChABC, PNG + ChABC, and PNG + aFGF group (^Øp < 0.05).

Axonal regeneration by PNG + aFGF treatment. A, Representative confocal images of 5-HT immunostaining in the PNG + aFGF group showing several regenerated 5-HT fibers (red) growing into the caudal end of the spinal cord. The caudal spinal cord interface is demarcated by a white line. Scale bar, 200 μm. B, C, Higher-magnification images from A show identified 5-HT fibers (red, arrow) at different locations within the caudal end of the spinal cord. Scale bar, 40 μm. D, In the Tx-only group, 5-HT fibers (red) can be identified in the rostral cord (identified by GFAP, green) but not entering the lesion cavity. The rostral spinal cord interface is demarcated by a white line. Scale bar, 500 μm. * indicates the cavity formation in the rostral cord. E, Higher-magnification images from D show identified 5-HT fibers (red, arrow) at different locations within the rostral end of the spinal cord. Scale bar, 200 μm. F, Representative confocal images of TH immunostaining in the PNG + aFGF group showing several regenerated TH fibers (red) growing into the caudal end of the spinal cord. The caudal spinal cord interface is demarcated by a white line. Scale bar, 200 μm. G, H, Higher-magnification images from F show identified TH fibers (red, arrow) at different locations within the caudal end of the spinal cord. Scale bar, 40 μm. I, In the Tx-only group, TH fibers (red) can be identified at the rostral cord (identified by GFAP, green) but not entering the lesion cavity. The rostral spinal cord interface is demarcated by a white line. Scale bar, 500 μm. * indicates cavity formation in the rostral cord. J, Higher-magnification images from I show identified TH fibers (red, arrow) at different locations within the rostral end of the spinal cord. Scale bar, 200 μm.

Significant axonal regeneration with PNG + aFGF + ChABC treatment. The triple combination of ChABC with PNG and aFGF promotes regeneration of both 5-HT and TH fibers beyond the PNG/caudal spinal cord interface. A, Confocal images of a sagittal section of the spinal cord showing that regenerated 5-HT fibers (red, white arrow head) have grown beyond the PNG (GFAP-negative area) and penetrated deep into the CNS (identified by GFAP, green). The caudal spinal cord interface is demarcated by a white line. Scale bar, 300 μm. B–D, Higher-magnification images from A show identified 5-HT fibers (white arrows) at different locations within the caudal end of the spinal cord. Scale bar, 40 μm. Note in A the alignment of the astrocytes at a point at which axons reenter the cord. E, Confocal photomicrographs of a sagittal section of the spinal cord showing that TH fibers (red, white arrow head) have regenerated beyond the graft (GFAP negative area) and well into the CNS (identified by GFAP, green). The arrowheads indicate TH fibers crossing the PNG/caudal spinal cord interface (demarcated by a white line). Scale bar, 300 μm. F–H, Higher-magnification images from E show identified TH fibers (white arrows) at different distances beyond the PNG/caudal spinal cord interface. Scale bar, 40 μm. I, Confocal images from immunostaining reveal a close proximity between the regenerated fibers and synapsin (SYN) puncta in PNG + aFGF + ChABC-treated animals. Double-staining image indicates the colocalization of TH fibers and SYN (arrow). Scale bar, 70 mm. J, Regenerated 5-HT fibers with expansive terminal arborizations were identified within the lumbar cord as depicted in this representative transverse section. Scale bar, 200 μm. K, L, Higher-magnification images from J. Note the bouton structures on 5-HT fibers (white arrow). The interface between gray matter and white matter is demarcated by a white dashed line. GM, Gray matter; WM, white matter. Scale bar, 50 μm. M, N, TH fibers were also identified well into the lumbar cord as shown in this representative transverse section. Note the bouton structures on TH fibers (white arrow). The interface between gray matter and white matter is demarcated by a white dashed line. GM, Gray matter; WM, white matter. Scale bars: M, 200 μm; N, 50 μm. O, Quantitative analysis showed greater numbers and longer-distance regeneration of 5-HT fibers beyond the lesion in the PNG + aFGF + ChABC treatment group compared with the other SCI groups. P, Quantitative analysis showed more and longer-distance regeneration of TH fibers beyond the lesion in the PNG + aFGF + ChABC treatment group versus the other SCI groups. ^†p < 0.05 when compared with Tx-only, PNG, and aFGF + ChABC groups, *p < 0.05 when compared with Tx-only, PNG, aFGF + ChABC, and PNG + ChABC groups, and **p < 0.05 when compared with Tx-only, PNG, aFGF + ChABC, PNG + ChABC, and PNG + aFGF groups.

A few BDA-positive fibers grow beyond the graft in the PNG + aFGF animals. A, A low-magnification sagittal section shows, with the combination of BDA labeling (red) and GFAP staining (green), the overall anatomy of the PNG and spinal cord (left side is rostral and right side is caudal). Scale bar, 500 μm. B, High-magnification image shows a few BDA-labeled fibers (red) entering the caudal end of the spinal cord (GFAP-positive area) from PNG (GFAP-negative area). The interface of the PNG and spinal cord is demarcated by a white line. Scale bar, 100 μm.

Anterograde tracing to label regenerating axons in the PNG + aFGF + ChABC treatment group. A, These three low-magnification sagittal sections of confocal images show with BDA labeling (red) and GFAP staining (green) and the two combined, the overall anatomy of the PNG and spinal cord (left side is rostral and right side is caudal). Scale bar, 1000 μm. B, High-magnification confocal image shows the large number of BDA-labeled fibers (red) entering the PNG (GFAP negative area) from the rostral end of the spinal cord (GFAP-positive area). The interface of the PNG and spinal cord is demarcated by a white line. Scale bar, 100 μm. Significant numbers of BDA-labeled fibers were identified within the PNG (C) and penetrating deeply into the interface of the PNG with the caudal end of the spinal cord (D) (demarcated by a white line). Note the alignment of the GFAP-positive astrocyte processes. D′, An adjacent section to D showing more evidence of the BDA-labeled fibers (red and arrowheads) crossing the PNG/caudal spinal cord interface. E–G, The BDA-labeled fibers (red, white arrows) were identified at various locations continuing far caudally into the spinal cord. Scale bar, 50 μm. H, Quantitative analysis showed greater numbers and longer-distance penetration of BDA-labeled fibers beyond the lesion in the PNG + aFGF + ChABC treatment group than the other SCI groups. ^δp < 0.05 when compared with Tx-only and aFGF + ChABC groups, ^ψp < 0.05 when compared with Tx-only group, ^†p < 0.05 when compared with Tx-only, PNG, and aFGF + ChABC groups, *p < 0.05 when compared with Tx-only, PNG, aFGF + ChABC, and PNG + ChABC groups, and **p < 0.05 when compared with Tx-only, PNG, aFGF + ChABC, PNG + ChABC, and PNG + aFGF groups.

Retrograde anatomical tracing indicated the neuronal populations that have regenerated axons below the lesion site in the PNG + aFGF + ChABC treatment group. A, FG-labeled cells were identified in the pontine micturition center, including Barrington's nucleus (arrow) and D region (arrowhead). Scale bar, 800 μm. B, FG-labeled cells were identified in both raphe nuclei (arrow) and the reticular formation (arrowhead). Scale bar, 400 μm. C, The retrogradely FG-labeled neurons (arrow) could be identified in locus ceruleus. Scale bar, 250 μm. D, Quantification of the FG-labeled cells in the retrograde tracing study. Me, Mesencephalic nucleus of trigeminal nerve; DTN, dorsal tegmental nucleus; 4V, fourth ventricle; VCA, ventral cochlear nucleus, anterior; Py and PT, pyramidal tract. *p < 0.05 when compared with Tx-only, PNG, and aFGF + ChABC groups, and **p < 0.05 when compared with Tx-only, PNG, aFGF + ChABC, PNG + ChABC, and PNG + aFGF groups.

Spinal cord re-transection eliminates or reduces the improvements in bladder function after regeneration. A, Raw data analysis of a representative PNG + aFGF + ChABC-treated animal at 2 weeks after T5 spinal cord re-transection. The micturition patterns revert to lower frequencies of voiding and higher volumes per micturition. The PNG + aFGF group also increased volume per micturition after re-transection. Tx-only, PNG, and aFGF + ChABC animals did not change. ^†p < 0.05 when compared with the before treatment. B, After re-transection, the PNG + aFGF + ChABC group developed increased amplitudes and frequencies of non-voiding bladder contractions. Coordination between bladder contractions and EUS EMG bursting activity during voiding period was eliminated (* indicates micturition point).

CMG and EUS EMG analysis after spinal cord re-transection. A, There was no significant difference in CMG parameters after spinal cord re-transection in the Tx-only, PNG, and aFGF + ChABC groups. The PNG + ChABC group showed increased times to first void and decreased voiding intervals after re-transection. The PNG + aFGF group showed significant increases in residual volume and decreases in voiding intervals after re-transection. The PNG + aFGF + ChABC group showed greatly increased times to first void, increased maximal voiding pressures, increased residual volumes, decreased voiding efficiency, and decreased voiding intervals after re-transection. B, Spinal cord re-transection does not alter EUS EMG parameters in Tx-only, PNG, aFGF + ChABC, PNG + ChABC, and PNG + aFGF groups. The PNG + aFGF + ChABC group showed elimination of the reacquired, patterned EUS bursting activity, decreased EUS bursting lengths, and no coordination between bladder contractions with EUS bursting after spinal cord re-transection. *p < 0.05 when compared with the before treatment.

Pharmacological manipulations eliminate or reduce the improvements in bladder function after regeneration. A, Raw data of a representative PNG + aFGF + ChABC-treated animal at 3 d after methysergide application. The micturition patterns returned to lower frequencies of voiding, but there was no difference in volume per micturition. B, Coordination between bladder contractions and EUS EMG bursting activity during voiding was diminished, but no significant differences occurred in the amplitudes of non-voiding contractions. C, Raw data of a representative PNG + aFGF + ChABC-treated animal at 3 d after α-methyl-dl-tyrosine application, the micturition patterns returned to lower frequencies of voiding and higher volumes per micturition. D, Amplitudes of non-voiding bladder contractions increased, and coordination between bladder contractions and EUS EMG bursting activity during the voiding period was eliminated. ^†p < 0.05 when compared with the before treatment.

CMG and EUS EMG analysis after methysergide application. A, There is no significant difference in CMG parameters after methysergide application in the Tx-only, PNG, and aFGF + ChABC groups. The PNG + ChABC and PNG + aFGF groups showed significantly decreased voiding intervals after methysergide application. The PNG + aFGF + ChABC group showed significantly increased time to first void and decreased voiding intervals. B, Methysergide application did not alter EUS EMG parameters in Tx-only, PNG, aFGF + ChABC, and PNG + ChABC groups. EUS bursting length was reduced in PNG + aFGF group after methysergide application. The PNG + aFGF + ChABC group showed elimination of EUS bursting activity and no coordination between bladder contractions with EUS bursting after methysergide application. *p < 0.05 when compared with the before treatment.