Sprouting of substance P-expressing primary afferent central terminals and spinal micturition reflex NK1 receptor dependence after spinal cord injury

Abstract

The primary afferent neurotransmitter triggering the spinal micturition reflex after complete spinal cord injury (SCI) in the rat is unknown. Substance P detected immunohistochemically in the sacral parasympathetic nucleus was significantly higher in 12 SCI rats than in 12 spinally intact rats (P = 0.008), suggesting substance P as a plausible candidate for the primary afferent neurotransmitter. The effects of the tachykinin NK1 receptor antagonist L-733060 on the spinal micturition reflex were then determined by performing conscious cystometry in an additional 14 intact rats and 14 SCI rats with L-733060 (0.1-100 microg) administered intrathecally at L6-S1. L-733060 was without effect in intact rats, but blocked the spinal micturition reflex in 10 of 14 SCI rats and increased the intermicturition interval in 2 of 4 others at doses ranging from 10 to 100 microg. Both phasic and nonphasic voiding contractions, differentiated according to the presence of phasic external urethral sphincter (EUS) activity, were present in most SCI rats. Both types of contractions were blocked by high doses of L-733060. Interestingly, there was a relative decline in phasic voiding contractions at high doses as well as a decline in contraction amplitude in nonphasic voiding contractions. In other respects, cystometric variables were largely unaffected in either spinally intact or SCI rats. L-733060 did not affect tonic EUS activity at any dose except when the spinal micturition reflex was blocked and tonic activity was consequently lost. These experiments show that tachykinin action at spinal NK1 receptors plays a major role in the spinal micturition reflex in SCI rats.

Fig. 1.

Analysis of EUS-EMG waveforms. EUS-EMG and intravesical pressure (IVP) are shown for the 4 phases of a phasic voiding contraction, i.e., 1 showing phasic EUS-EMG activity (bursts and pauses) and high-frequency oscillations of IVP. Phasic duration (P_d) is the time during which periodic burst and pauses of EUS-EMG activity occur (phase 2). For IVP-EMG plots, mean rectified EUS-EMG activity was measured for each 2-mm-wide span around every multiple of 5 mmHg of IVP during phases 1 and 4. Approximations of such spans for multiples of 10 mmHg of IVP are indicated by gray shading. For nonvoiding contractions (NVCs) and nonphasic voiding contractions (npVCs), the rising and falling phases of IVP are considered to be phases 1 and 4 even though there is no phase 2 or 3.

Fig. 2.

Substance P and choline acetyltransferase (ChAT) in the L6 sacral parasympathetic nucleus. The spinal gray matter schematic (left; dorsal surface up) shows the approximate site from which the color pictures were taken. The color panels show 3 examples from intact rats (top) and 3 from spinal cord injury (SCI) rats (bottom) stained for substance P (red) and ChAT (green). ChAT-positive neurons are parasympathetic preganglionic neurons. All pictures were identically processed for substance P; ChAT was slightly altered from picture to picture for best definition of the parasympathetic preganglionic neurons. Right: total area positive for substance P (SP) per rat (mean ± SE). Difference is significant at P = 0.008.

Fig. 3.

NVCs, npVCs, and phasic voiding contractions (phVC) in an SCI rat. A: EUS-EMG activity, IVP, and micturition volume for the 3 types of contractions. *Onsets of detection of voiding. B: comparison of EUS-EMG activities for the 17 s centered around the peaks of the micturition contractions for the second npVC (npVC2) and the 2 phVCs (phVC1, phVC2). Arrows mark beginning of phasic EUS-EMG activity. C: two left-hand plots show that neither peak IVP nor micturition volume differ significantly between npVCs and phVCs. Right: plot shows the distribution of npVCs and phVCs among the 14 SCI rats; for the 4 rats with a mixture of npVCs and phVCs, a mean of 64.5% of all voiding contractions were phVCs (range 43.5–85.7%).

Fig. 4.

Variations in the EUS-EMG activity in SCI phasic voiding contractions. *Onset of detection of voiding. A: entire phasic portion of spinally intact rat EUS-EMG. Frequency of bursts is higher at the beginning and end of phasic activity; pauses are often quite long in between. B: part of a long stretch of EUS-EMG activity in an SCI rat; frequency of bursts is much higher than for the spinally intact rat in A. C: short stretch of EUS-EMG activity in an SCI rat. Pauses are almost obliterated, which is not uncommon in an SCI rat. D: long stretch of EUS-EMG activity in an SCI rat (note change in time base) in which multiple periods of bursting occurred associated with repeated onset of voiding. E and F: long stretch in EUS-EMG activity in an SCI rat, where phasic EUS-EMG activity was not associated with voiding. For comparison, the segment in F from the beginning of the recording in E is on the same time base as A–C.

Fig. 5.

Effects of L-733060 on reflex voiding in spinally intact and SCI rats. A: spinally intact rat; administration of 100 μg L-733060 is without effect. B: SCI rat; administration of the same dose of L-733060 blocks the spinal micturition reflex. C: L-733060 causes a decrease in the %phVCs among all voiding contractions in SCI rats, which becomes significant at 100 μg.

Fig. 6.

Filling cystometry dose-response curves for intrathecal L-733060 in intact and SCI rats. Separate values at left, which are not connected to other points, are vehicle values; bars indicate SEs. *Statistically significant differences for a given contraction type between the marked response and vehicle; other statistical information is presented in the text.

Fig. 7.

Free-running cystometry dose-response curves for effects of intrathecal L-733060 on phVCs in intact and SCI rats. Separate values at left, which are not connected to other points, are vehicle values; bars indicate SEs. *Statistically significant differences for a given contraction type between the marked response and vehicle; other statistical information is presented in the text.

Fig. 8.

IVP-EMG relationships. A: IVP is plotted against mean rectified EUS-EMG activity across the 8 SCI rats that exhibited npVCs following vehicle. The plot rises to a peak around 40 mmHg, then falls. Knowing that EUS-EMG amplitude varies from rat to rat, at least in part due to variations in the separation between the electrodes and the EUS, we replotted the data in B. Here, the peak value of rectified EUS-EMG within each rat is used to normalize the rectified EUS-EMG values. This results in a plot that is fairly linear but which, except at pressures over 40 mmHg, seems to level off after 25 mmHg or so. Because all 8 rats participate in the generation of the mean up to 25 mmHg but only 2 participated at the higher pressures, we separately plotted the mean values from the rats with the 3 lowest and 2 highest peak IVPs. The result, shown C, is 2 fairly linear plots with similar slopes but different intercepts and ranges.

Fig. 9.

Effect of L-733060 on relationship between IVP and tonic mean rectified EUS-EMG activity in intact phVCs (A), SCI phVCs (B), and SCI npVCs (C). The dips in several curves in A (arrows), most pronounced at ∼20–25 mmHg, are all from phase 1 of the micturition cycle, and occur because the only rat having opening pressures this high also exhibited very little EUS-EMG activity during phase 1 but had pronounced activity during phase 4. The anomalous portion of the 10 μg curve in B arises because, whereas 10 rats contribute data to the curves at 15 mmHg, only 1 rat (and only 1 contraction) contributed data to this high-pressure part of the curve. For the anomalous portion of the 100 μg curve in B, again only 1 rat contributed to data at 25 mmHg and above. With such anomalies accounted for, there appears to be little difference between the curves for intact phVCs, SCI phVCs, and SCI npVCs (other than the lower peak pressure for intact phVCs as per Fig. 6D), and there is no indication that L-733060 affects tonic EUS-EMG activity.

Fig. 10.

Volume of manually expressed urine on each morning following spinal cord transection for SCI rats that did or did not exhibit phasic EUS-EMG activity during cystometry before L-733060 was administered. A total of 22 SCI rats are included (11 nonphasic, 11 phasic), some of which are from another study but which were identically treated. The difference is surprisingly minor.