Pathophysiology of the Colonic Enteric Nervous System in Male and Female Rats Following Spinal Cord Injury
Restricted (Penn State Only)
- Author:
- Werner, Claire
- Graduate Program:
- Anatomy (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 16, 2023
- Committee Members:
- Gregory Yochum, Outside Unit & Field Member
Andras Hajnal, Major Field Member
Gregory Holmes, Chair & Dissertation Advisor
Prashant Nighot, Major Field Member
Patricia Mclaughlin, Program Head/Chair
Patricia Mclaughlin, Major Field Member - Keywords:
- spinal cord injury
neurogenic bowel
prokinetic
sex differences - Abstract:
- Most markedly, Spinal Cord Injury (SCI) frequently results in a loss of motor function to the lower limbs, however, individuals with SCI suffer a wide range of comorbidities that greatly impact their quality of life (QOL). With nearly 18 thousand new cases of SCI each year, understanding the physiological challenges of these individuals is important for them and their caretakers. One of the most prevalent and debilitating comorbidities is dysregulation of the gastrointestinal (GI) tract; ranging from delayed gastric emptying to loss of bowel control. The latter, is often classified as neurogenic bowel (NB) which manifests as chronic slow-transit constipation and impaction that often requires time consuming and invasive bowel management strategies. Unfortunately, frequently NB treatment has poor patient satisfaction and long-term effectiveness. A key component facilitating colonic motility is the Autonomic Nervous System (ANS), including its subcomponent, the Enteric Nervous System (ENS), which is positioned intrinsic to the GI tract. Interestingly, the ENS is not directly damaged by the SCI, yet the GI tract remains dysregulated following injury. Although regulated by the parasympathetic and sympathetic division of the ANS, the ENS can function independently. Within the smooth muscle of the colon, a variety of ENS plexuses contribute to both motor and secretory functions. One critical ENS interface is the Interstitial Cells of Cajal (ICC), which have named subtypes based on their location, function, and morphology. One ICC subtype, the myenteric plexus, is positioned between the distal colon smooth muscle layers and it plays a role in motor function crucial to peristalsis. Peristalsis, or the propulsion of luminal contents through the GI tract, is particularly disrupted in the distal colon, further contributing to the symptoms of NB. The ICC act as the intermediate pacemaker between smooth muscle cells and nerves largely through calcium currents to generate integrated motor responses. The cholinergic, muscarinic (M) receptor family includes five subtypes, all of which are seven transmembrane-spanning GPCRs (G protein-coupled receptors), responding to the neurotransmitter acetylcholine (ACh). The even numbered subtypes signal through adenylyl cyclase (AC) inhibition, whereas odd numbered subtypes signal through phospholipase C (PLC) activation. Three subtypes of the M receptors are present within the colon: M1, M2, and M3. The M2 and M3 contribute more directly to smooth muscle contraction, due to their localization being primarily on colonic smooth muscle (Harrington, Peck, et al., 2010). M1 receptors are primarily localized to the myenteric ganglia to facilitate ACh release (Harrington, Peck, et al., 2010). Regardless, M2 and M3 receptors have a small population present on the myenteric ganglia, thereby contributing in part to neurotransmitter release onto the smooth muscle cells. Conversely, a negligible population of M1 are present on the smooth muscle cells, thereby providing minimal contribution to activation of the pacemaker current compared to M2 and M3 subtypes. In the human population, generally M2 outnumber M3 with a 4:1 or 3:1 ratio in the smooth muscle, though M3 is considered to play a more significant role in smooth muscle contraction (Eglen, 2001; Harrington, Peck, et al., 2010; Uchiyama & Chess-Williams, 2004). M2 subtype functional consequences are less understood, but generally are thought to inhibit smooth muscle relaxation by acting to inhibit AC, thereby potentiating the action of M3 (Uchiyama & Chess-Williams, 2004). GI smooth muscle contraction is primarily the result of cholinergic-mediated excitatory neurotransmission and our laboratory has demonstrated that cholinergic-mediated smooth muscle depolarization potentials are diminished in the GI tract following experimental T3 SCI in male Wistar rats (White et al., 2020). Building upon this knowledge and review of the literature, this dissertation will test the following overarching hypothesis: following SCI, NB dysfunction is caused by decreased neuromuscular transmission mediated by pathophysiological remodeling to distal colon ICC and muscarinic receptors. The first set of experiments consisted of follow-up experimentation to access sex differences in enteric neuromuscular transmission following SCI. Our previously published data in male rats demonstrated decreased cholinergic-mediated excitatory and nitrergic-mediated inhibitory neurotransmission through assessment of junction potentials of the distal colon by smooth muscle cell electrophysiology recordings upon electrical field stimulation. The purinergic-mediated inhibitory neurotransmission remained unaffected after injury. Additionally, immunohistochemical labeling of excitatory (choline acetyltransferase; ChAT) and inhibitory (nitric oxide synthase; nNOS) neurons from male rats presented with decreased immunoreactivity at 3 days and 3 weeks post-injury (when the animals have stabilized), after SCI. Our present data demonstrates highly variable female colonic contractility in both frequency and amplitude. When compared to the previously reported male data, generally the females present with greater proximal colon levels of contractility, but less distal colon contractility. Female nitrergic- and purinergic-mediated inhibitory neuromuscular transmission are both reduced at 3 weeks following SCI, but no change in seen in the excitatory response. When comparing sexes, females have greater loss of inhibitory neuromuscular transmission following SCI and males have greater loss of excitatory neuromuscular transmission following SCI. This differential remodeling suggests that NB treatment should take sex into consideration, particularly regarding pharmacotherapeutics. The second set of experiments disproved our hypothesis that distal colon ICC populations are reduced following experimental T3 SCI. To the contrary, our data reveals that experimental SCI provokes pronounced levels of ICC plasticity. The ICC cell subtypes were analyzed following immunohistochemistry with the application of ICC-specific marker receptor tyrosine kinase (c-Kit) on wholemount sections of male and female rat distal colon. The myenteric plexus ICC (ICC-MY) exhibited increased cell counts 3 days following SCI in male rats, but this increase was delayed in females until 3 weeks after SCI. Additionally, ICC-MY total primary arborization length increased significantly in male rats at 3-Day, 3-Week, and 6-Week timepoints, whereas this did not occur in females until 6-Week timepoints post-SCI. The circular muscle ICC (ICC-CM) did not demonstrate post-SCI changes. These data suggest that hypertrophy of ICC-MY may reflect changes in the neural inputs onto these cells following SCI. Lastly, the third set of experiments utilized electrophysiological recordings of male distal colon smooth muscle cells to investigate the effectiveness of a prokinetic drug (Bethanechol) to quantify cell excitation. Bethanechol is a direct cholinergic agonist, acting on the muscarinic receptors. During electrophysiology experimentation, baseline cell membrane potentials were recorded prior to the application of increasing concentrations (5M and 25M) of Bethanechol to access dose dependent cell excitability. In 3-Day and 3-Week control recordings, there was a significant dose dependent response excitation of smooth muscle cells. However, for SCI distal colon tissue, this dose dependent response was not significant and nearly absent at the 3-Week timepoint post-injury. Additional distal colon smooth muscle tissue was harvested and fixed for wholemount immunohistochemistry of the M2 and M3 receptors. During imaging acquisition, myenteric ganglion were located and imaged for each receptor subtype. Receptor quantification within a standardized size region of interest (ROI), revealed that the M2 receptors demonstrate remodeling at 3 weeks following T3-SCI. There was no change in the quantity of M3 receptors, but M2 were shown to decrease in number in our 3-Week injured animal model. Additionally, this causes a shift in the ratio of receptor subtypes present in the distal colon. In our control animal tissue, M2:M3 are on average present at a 3:2 ratio, whereas in SCI animals demonstrate a near 1:1 relationship. Collectively, the electrophysiology and immunohistochemical findings suggest that the downregulation of M2 receptors following SCI contributes to decreased dose dependent response to muscarinic agonist, Bethanechol. This data provides some insight to why prokinetic drugs for treating NB are often ineffective for the SCI population. Consistently, individuals with SCI state that symptoms related to their bladder and bowel are most important to effectively treat. Currently, bowel related treatment is lacking because the mechanism for NB has yet to be elucidated. The results of this dissertation contribute to the understanding of the mechanism of colonic dysmotility after SCI. These findings also provide direction for future experimentation in order to further our understanding of NB. Collectively, this data and subsequent future directions can assist in the development of therapeutic targets and treatment for individuals with SCI experiencing NB to improve overall QOL.