Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jan 25:5:6.
doi: 10.3389/fnins.2011.00006. eCollection 2011.

Pelvic Nerve Injury Causes a Rapid Decrease in Expression of Choline Acetyltransferase and Upregulation of c-Jun and ATF-3 in a Distinct Population of Sacral Preganglionic Neurons

Christopher J Peddie  1 , Janet R Keast
Affiliations

Pelvic Nerve Injury Causes a Rapid Decrease in Expression of Choline Acetyltransferase and Upregulation of c-Jun and ATF-3 in a Distinct Population of Sacral Preganglionic Neurons

Christopher J Peddie et al. Front Neurosci. .

Abstract

Autonomic regulation of the urogenital organs is impaired by injuries sustained during pelvic surgery or compression of lumbosacral spinal nerves (e.g., cauda equina syndrome). To understand the impact of injury on both sympathetic and parasympathetic components of this nerve supply, we performed an experimental surgical and immunohistochemical study on adult male rats, where the structure of this complex part of the nervous system has been well defined. We performed unilateral transection of pelvic or hypogastric nerves and analyzed relevant regions of lumbar and sacral spinal cord, up to 4 weeks after injury. Expression of c-Jun, the neuronal injury marker activating transcription factor-3 (ATF-3), and choline acetyltransferase (ChAT) were examined. We found little evidence for chemical or structural changes in substantial numbers of functionally related but uninjured spinal neurons (e.g., in sacral preganglionic neurons after hypogastric nerve injury), failing to support the concept of compensatory events. The effects of injury were greatest in sacral cord, ipsilateral to pelvic nerve transection. Here, around half of all preganglionic neurons expressed c-Jun within 1 week of injury, and substantial ATF-3 expression also occurred, especially in neurons with complete loss of ChAT-immunoreactivity. There did not appear to be any death of retrogradely labeled neurons, in contrast to axotomy studies performed on other regions of spinal cord or sacral ventral root avulsion models. Each of the effects we observed occurred in only a subpopulation of preganglionic neurons at that spinal level, raising the possibility that distinct functional subgroups have different susceptibility to trauma-induced degeneration and potentially different regenerative abilities. Identification of the cellular basis of these differences may provide insights into organ-specific strategies for attenuating degeneration or promoting regeneration of these circuits after trauma.

Keywords: cauda equina syndrome; inferior hypogastric plexus; micturition; regeneration; spinal cord injury; spinal nerves; sprouting.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Immunoreactivity for c-Jun in lumbar and sacral preganglionic neurons after axotomy. The proportion of FluoroGold (FG)-positive neurons expressing c-Jun was recorded in horizontal sacral (L6–S1) and lumbar (L1–L2) spinal cord sections 1, 2, or 4 weeks after nerve transection and in naïve controls. (A) In the sacral intermediolateral column (IML), the proportion of FG-positive neurons expressing c-Jun was significantly increased ipsilateral to injury (main effect of side: F1,9 = 159.78, P < 0.001) and there was also a significant interaction between side and time (F2,9 = 5.06, P = 0.034), with the largest effect of injury apparent at 1 week. There was no effect contralateral to injury, when compared with naïve controls. (B) FG and c-Jun immunolabeling within the sacral IML, ipsilateral to pelvic nerve transection, 1 week after injury. Boxed region provides higher magnification images of c-Jun-positive nuclei within FG-positive neurons. (C) FG and c-Jun immunolabeling within the sacral IML, contralateral to pelvic nerve transection, 1 week after injury. (D,E) Higher magnification images of FG and c-Jun labeling in sacral IML, 2 and 4 weeks after pelvic nerve transection, respectively. (F,G) No effect of hypogastric nerve transection was detected in the sacral (F) or lumbar (G) IML, either in the context of side or relative to naïve controls. (H,I) Examples of FG and c-Jun labeling in the central autonomic area (CAA), 2 and 4 weeks after hypogastric nerve transection, respectively. (J) There was a significant increase in proportion of FG neurons expressing c-Jun within the CAA after hypogastric (HGN) but not pelvic nerve (PVN) transection, with this effect occurring 1 week after injury; asterisk indicates a significant difference from naïve controls (F6,27 = 4.37, P = 0.033). (K) There was a small but significant increase in the prevalence of c-Jun-positive FG neurons in the ipsilateral lumbar IML after pelvic nerve transection (main effect of side: F1,9 = 14.427, P = 0.004) and there was also a significant interaction between side and time (F2,9 = 5.664, P = 0.026), with the largest effect of injury occurring at 1 week. There was no effect contralateral to injury, when compared with naïve controls. (L) FG and c-Jun labeling within the lumbar IML, ipsilateral to hypogastric nerve transection, 1 week after injury; boxed region provides a higher magnification image to demonstrate a c-Jun-positive nucleus within a FG-positive neuron and an adjacent-Jun-negative neuron. (M) As per (L), contralateral to hypogastric nerve transection. (N) FG and c-Jun labeling within the CAA, 1 week after hypogastric nerve injury; boxed region shows c-Jun-positive nuclei within FG-positive neurons at higher magnification. Bars represent mean ± SEM, with n = 4 for all groups. Main effects and interactions identified by two-way ANOVA (repeated measures) indicated on relevant plots. Comparisons with naïve controls made with Dunnett's test. Scale bar: 100 μm (B,C), 40 μm (boxes for B,C; D,E,H,I), 120 μm (L,M), and 175 μm (N); 30 μm (boxes for L–N).
Figure 2
Figure 2
Effects of axotomy on choline acetyltransferase (ChAT) immunoreactivity in preganglionic neurons. The proportion of c-Jun-positive neurons immunoreactive for ChAT was recorded in horizontal sacral (L6–S1) and lumbar (L1–L2) spinal cord sectionsone, 2, or 4 weeks after nerve transection and in naïve controls. The proportion of ChAT-positive neurons expressing c-Jun was also analyzed. (A,B) Low power images illustrating the distribution of ChAT-positive neurons within sacral (A) and lumbar (B) cord, 2 weeks after pelvic or hypogastric nerve transection, respectively. (C,D) Higher magnification images showing c-Jun-positive nuclei in sacral intermediolateral column (IML), 1 week after pelvic nerve transection (C, ipsilateral; D, contralateral). In (C), some of the neurons with c-Jun-positive nuclei are ChAT-negative (arrows). In (D), intense c-Jun immunoreactivity is absent, but some dim labeling is indicated (arrow). (E) A c-Jun-positive nucleus within a ChAT-positive neuron (arrow) in the lumbar IML contralateral to hypogastric nerve transection, 1 week after injury. (F) A c-Jun-positive nucleus within a ChAT-negative neuron (arrow) in the lumbar central autonomic area (CAA), 1 week after hypogastric nerve injury. (G) In the sacral IML, a significant increase in the proportion of ChAT neurons expressing c-Jun was detected ipsilateral to pelvic nerve transection (main effect of side: F1,12 = 94.144, P < 0.001) but there was no significant interaction between side and time. There was no effect contralateral to injury, when compared with naïve controls. (H) Ipsilateral to hypogastric nerve transection, the proportion of ChAT neurons expressing c-Jun in the lumbar IML may have been increased relative to the contralateral side, but this did not quite reach statistical significance (main effect of side: F1,12 = 4.75, P = 0.05). There was no significant effect contralateral to injury, when compared with naïve controls. (I) No consistent effect was detected within the CAA after either pelvic (PVN) or hypogastric (HGN) nerve transection. (J) A significant reduction in the proportion of c-Jun-positive neurons expressing ChAT was recorded in the sacral IML after pelvic nerve transection (main effect of side: F1,12 = 8.871, P = 0.012), but there was no interaction with time. There was no effect contralateral to injury, when compared with naïve controls. (K) There was no significant change in the proportion of c-Jun positive neurons expressing ChAT, ipsilateral to hypogastric nerve transection. There was no effect contralateral to injury, when compared with naïve controls. (L) No effect of PVN or HGN was detected in the CAA compared to naïve controls. Bars represent mean ± SEM, with n = 5 for all groups. Main effects identified by two-way ANOVA (repeated measures) indicated on relevant plots. Comparisons with naïve controls made with Dunnett's test. Scale bar: 200 μm (A,B), 50 μm (C–F).
Figure 3
Figure 3
Effects of axotomy on choline acetyltransferase (ChAT) immunoreactivity in retrogradely labeled preganglionic neurons. The proportion of FluoroGold (FG)-positive neurons with ChAT-immunoreactivity was recorded in horizontal sacral (L6–S1) and lumbar (L1–L2) spinal cord sections 1, 2, or 4 weeks after nerve transection and in naïve controls. (A) In the sacral intermediolateral column (IML), the proportion of ChAT-negative FG neurons was significantly increased ipsilateral to pelvic nerve transection (main effect of side: F1,9 = 194.87, P < 0.001) and there was also a significant interaction between side and time (F2,9 = 9.521, P = 0.006), with the largest effect of injury occurring at 1 week. There was no effect contralateral to injury, when compared with naïve controls. (B) Within the lumbar IML, there was no difference in the proportion of ChAT-negative FG neurons between sides, but relative to naïve controls there was an increase contralateral to injury at 1 week (F3,15 = 11.62, P = 0.007). Together this infers that there was also an increase in the ipsilateral at 1 week, relative to naïve controls. (C) One week after hypogastric nerve (HGN) transection, an increase in the proportion of ChAT-negative FG neurons was detected in the central autonomic area (CAA), compared to naïve controls (F5,27 = 2.3, P = 0.033). No effect was seen at any other time or after pelvic nerve (PVN) transection. (D–F) Images of sacral IML ipsilateral to pelvic nerve transection, showing examples of ChAT-negative FG neurons at 1 (D), 2 (E), and 4 (F) weeks after injury. (G–I) Images of lumbar cord showing examples of ChAT-negative FG neurons, 1 week after hypogastric nerve transection, within the IML both ipsilateral (G) and contralateral (H) to injury, and within the CAA (I). Bars represent mean ± SEM, with n = 4 for all groups. Main effects and interactions identified by two-way ANOVA (repeated measures) indicated on relevant plots. Comparisons with naïve controls made with Dunnett's test. In B and C, asterisk indicates a significant difference from naïve controls, with P values as indicated above. Scale bar: 20 μm (D–I).
Figure 4
Figure 4
Choline acetyltransferase (ChAT) immunoreactivity and soma size of sacral preganglionic neurons after pelvic nerve transection. (A) Intensity of ChAT-immunofluorescence (0–256 grayscale, 8-bit images) in the sacral intermediolateral column (IML) was significantly reduced ipsilateral to pelvic nerve transection (main effect of side: F1,19 = 88.76, P < 0.001). There was also a significant interaction between side and time (F2,19 = 5.04, P = 0.018), with the effect being largest at 1 week. (B,C) Image montages of sacral IML immunolabeled for ChAT, 1 (B) or 2 (C) weeks after pelvic nerve transection, each showing a reduction in labeling intensity ipsilateral to injury. (D) Area of soma profiles from ChAT-positive preganglionic neurons in the sacral IML were reduced ipsilateral to injury (main effect of side: F1,12 = 37.922, P < 0.001) but this was unaffected by time. One week after injury the somata on the contralateral side were slightly smaller than naïve controls (F3,19 = 4.853, P = 0.025) and, because the ipsilateral neurons are smaller than contralateral neurons, the former are also smaller than naïve controls. Main effects and interactions identified by two-way ANOVA (repeated measures) indicated on relevant plots. Comparisons with naïve controls made with Dunnett's test. Bars represent mean ± SEM, with n = 5 for soma size analyses and 4–9 for intensity measurements. Scale bar: 300 μm (B,C).
Figure 5
Figure 5
Activating transcription factor-3 expression in axotomized lumbar and sacral preganglionic neurons. The proportion of choline acetyltransferase (ChAT)-positive neurons expressing activating transcription factor-3 (ATF-3) was recorded in horizontal sacral (L6–S1) and lumbar (L1–L2) spinal cord sections at 1, 2, and 4 weeks after nerve transection and in naïve controls. (A) In the sacral intermediolateral column (IML), some ChAT-positive neurons expressed ATF-3 after pelvic nerve transection; no ATF-3 expression was detected contralateral to nerve injury at any time point, or in naïve controls. (B) In the lumbar IML a small proportion of ChAT-positive neurons expressed ATF-3 after hypogastric nerve transection; this was detected bilaterally but there was no difference between sides. No ATF-3 was detected in lumbar IML of naïve controls or after pelvic nerve transection. (C) In the central autonomic area (CAA), a minority of ChAT-positive neurons expressed ATF-3 after hypogastric nerve transection, peaking at 1 week, and declining over the following weeks. No ATF-3 expression was detected within the CAA in naïve controls or after pelvic nerve transection. (D–F) All ATF-3-positive nuclei were found within FluoroGold (FG)-positive neurons, with representative images from sacral (D) and lumbar IML (E) and CAA (F) horizontal spinal cord sections 1 week after injury. Arrows highlight ATF-3-positive FG neurons across each series. (G) Ipsilateral and contralateral sides from the same horizontal section of sacral spinal cord labeled for ChAT and ATF-3, 1 week after pelvic nerve transection. ATF-3-positive nuclei are located in the IML ipsilateral to injury, and are almost exclusively within weakly ChAT-positive, or ChAT-negative neurons. The contralateral side displays no ATF-3 immunoreactivity. (H) Both ipsilateral and contralateral to a hypogastric nerve transection, examples of ATF-3 immunoreactivity in weakly ChAT-positive neurons. Potential changes in the ipsilateral side were examined by two-way ANOVA (repeated measures). Bars represent mean ± SEM, with n = 4 for all groups. Scale bars: 50 μm (D–F), 100 μm (G), and 50 μm (H).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anderson C. R., Edwards S. L. (1994). Intraperitoneal injections of Fluorogold reliably labels all sympathetic preganglionic neurons in the rat. J. Neurosci. Methods 53, 137–141 - PubMed
    1. Armstrong D. M., Brady R., Hersh L. B., Hayes R. C., Wiley R. G. (1991). Expression of choline acetyltransferase and nerve growth factor receptor within hypoglossal motoneurons following nerve injury. J. Comp. Neurol. 304, 596–607 - PubMed
    1. Averill S., Michael G. J., Shortland P. J., Leavesley R. C., King V. R., Bradbury E. J., McMahon S. B., Priestley J. V. (2004). NGF and GDNF ameliorate the increase in ATF3 expression which occurs in dorsal root ganglion cells in response to peripheral nerve injury. Eur. J. Neurosci. 19, 1437–1445 - PubMed
    1. Barber R. P., Phelps P. E., Houser C. R., Crawford G. D., Salvaterra P. M., Vaughn J. E. (1984). The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study. J. Comp. Neurol. 229, 329–346 - PubMed
    1. Dail W. G., Evan A. P. (1978). Effects of chronic deafferentation on adrenergic ganglion cells and small intensely fluorescent cells. J. Neurocytol. 7, 25–3710.1007/BF01213458 - DOI - PubMed