Effects of Oxaliplatin Treatment on the Myenteric Plexus Innervation and Glia in the Murine Distal Colon

Abstract

Oxaliplatin (platinum-based chemotherapeutic agent) is a first-line treatment of colorectal malignancies; its use associates with peripheral neuropathies and gastrointestinal side effects. These gastrointestinal dysfunctions might be due to toxic effects of oxaliplatin on the intestinal innervation and glia. Male Balb/c mice received intraperitoneal injections of sterile water or oxaliplatin (3 mg/kg/d) triweekly for 2 weeks. Colon tissues were collected for immunohistochemical assessment at day 14. The density of sensory, adrenergic, and cholinergic nerve fibers labeled with calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and vesicular acetylcholine transporter (VAChT), respectively, was assessed within the myenteric plexus of the distal colon. The number and proportion of excitatory neurons immunoreactive (IR) against choline acetyltransferase (ChAT) were counted, and the density of glial subpopulations was determined by using antibodies specific for glial fibrillary acidic protein (GFAP) and s100β protein. Oxaliplatin treatment induced significant reduction of sensory and adrenergic innervations, as well as the total number and proportion of ChAT-IR neurons, and GFAP-IR glia, but increased s100β expression within the myenteric plexus of the distal colon. Treatment with oxaliplatin significantly alters nerve fibers and glial cells in the colonic myenteric plexus, which could contribute to long-term gastrointestinal side effects following chemotherapeutic treatment.

Keywords

colon glial cells myenteric neurons nerve fibers oxaliplatin

Introduction

Colorectal cancer is one of the leading causes of cancer-related death globally.^1,2 Treatment strategies for colorectal cancer include surgical resection for patients diagnosed at stages I–II and adjuvant chemotherapy for patients diagnosed at stages III–IV when metastasis to secondary locations has occurred.^3,4 Colorectal cancer is typically asymptomatic at the early stages, whereas weight loss, rectal bleeding, altered bowel habits, and abdominal pain can present at the later stages of disease progression.^5,6

Oxaliplatin is an effective chemotherapeutic agent used in the first-line treatment for colorectal cancer.⁷ Common side effects of oxaliplatin include peripheral sensory neuropathy of the extremities as well as gastrointestinal complications.⁸ Nausea, vomiting, constipation, and diarrhea are prominent symptoms experienced by patients undergoing anticancer chemotherapy.^8,9 These gastrointestinal side effects are the major causes for dose limitations and/or total cessation of anticancer treatment.^8,10 In severe cases, these gastrointestinal side effects can be life threatening and can result in the death of patients.^8,10,11 Current treatment options to alleviate these gastrointestinal symptoms also come with a plethora of adverse reactions. Antiemetic agents induce central nervous system effects (insomnia, twitching, tremor), cardiovascular (arrhythmia, heart failure), hepatic and renal complications; antidiarrheal agents induce abdominal pain, bloating, paralytic ileus, and anaphylaxis.^11–13

The conventional thought is that gastrointestinal symptoms are a result of damage to the intestinal mucosa.¹⁴ The high turnover rate of the intestinal epithelial cells, indeed, makes them attractive targets for cytotoxic drugs, and mucosal damage certainly plays a role in the acute stages of these symptoms.¹⁵ However, despite the rapid regeneration of the intestinal epithelial cells, the gastrointestinal complications can persist from months up to 10 years following anticancer chemotherapy.¹⁶ The persistence of gastrointestinal dysfunction is suggestive that chemotherapeutic agents may also induce damage to other systems regulating intestinal functions, including the peripheral nervous system innervating the gastrointestinal tract.¹⁷

The gastrointestinal tract is innervated by extrinsic parasympathetic motor neurons, postganglionic sympathetic neurons, vagal and spinal sensory afferents, as well as the intrinsic enteric nervous system (ENS). Extrinsic and intrinsic innervation provide control of the gastrointestinal functions such as motility, secretion, absorption, and vascular tone.¹⁸ The ENS is an intrinsic and complex orchestration of neurons and glia located within the intestinal wall, which form ganglia and give rise to two major plexi: myenteric and submucosal.¹⁸ Calcitonin gene-related peptide (CGRP)-immunoreactive (IR) neurons facilitate mucus production and vasomotor tone in the gastrointestinal mucosa, and also play a role in motility reflexes. Adrenergic fibers identified by their immunoreactivity against tyrosine hydroxylase (TH) innervate the enteric ganglia and gastrointestinal smooth muscles, which can influence motility, secretion, and blood flow.¹⁹ Vesicular acetylcholine transporter (VAChT)-IR fibers label cholinergic axons, which contain acetylcholine in synaptic vesicles, important for excitatory neurotransmission within the gastrointestinal tract.^20,21

Furthermore, the ENS contains inhibitory and excitatory neurons that are identified through their expression of neuronal nitric oxide synthase (nNOS) or choline acetyltransferase (ChAT), respectively.¹⁸ These neurons play an important role in the regulation of gastrointestinal motility. Moreover, the ENS is rich in heterogeneous enteric glia, which can be identified by their expression of glial fibrillary acidic protein (GFAP) and s100β.²² Glial cells were once considered as supporting cells with respect to neuronal integrity and function. However, research has demonstrated that enteric glia play a role in neurotransmission, gastrointestinal motility, maintaining epithelial and mucosal integrity, and that they also have immunomodulatory functions.²² There are subpopulations of myenteric glia that express both GFAP and s100β, and others that are IR for only one.^23,24 Furthermore, these glial subpopulations are thought to differ in function and in various pathologies. A reduction in GFAP-IR glia is often observed in pathological states of inflammation and injury, whereas an increase in s100β expression is observed in traumatic brain and spinal cord injury.^24–26

Previous work has shown that the predecessor platinum-based agent cisplatin induces a reduction in CRGP-IR neurons within the rat myenteric plexus.²⁷ Furthermore, studies in mice have revealed that oxaliplatin causes a reduction in number of colonic myenteric neurons, which is associated with increased proportions of inhibitory nNOS-IR neurons, and dysmotility^28,29 We have previously demonstrated that oxaliplatin treatment induces enteric glial toxicity within the ileum.²³ However, no studies were done to determine whether oxaliplatin induces damage to sensory, sympathetic, and cholinergic nerve fibers supplying the myenteric plexus of the colon, and the effects on excitatory neuronal populations, as well as glial cells in the colon.

Damage to these parts of the myenteric plexus of the colon could be implicated in the multifaceted pathophysiology of oxaliplatin-induced dysmotility. Briefly, the aims of this study were to investigate the effects of oxaliplatin treatment on (1) density of GCRP and TH-IR fibers innervating the myenteric plexus, (2) total number of neurons and inhibitory/excitatory subpopulations, and (3) GFAP and s100β expression within the myenteric plexus of the colon.

Materials and Methods

Animals

Male Balb/c mice (5–8 weeks of age, weighing 18–25 g) obtained from the Animal Resource Centre (Australia) were used in this study. Mice had free access to food and water and were kept under a 12-hr light/dark cycle in a well-ventilated room at a constant temperature of 22C. Mice acclimatized for up to 1 week before the commencement of in vivo intraperitoneal injections. All procedures were approved by the Victoria University Animal Experimentation Ethics Committee and performed in accordance to the National Health and Medical Research Council “Code of Practice for the Care and Use of Animals for Scientific Purposes.”

In Vivo Intraperitoneal Injections

Mice were separated into 2 cohorts (n=26 in total): (1) vehicle-treated (sterile water) and (2) oxaliplatin-treated (3 mg/kg/d; Sigma-Aldrich; Castle Hill, NSW, Australia). All dosages were calculated per body surface area, and mice received a maximum volume of 200 µl/injection.^30,31 All mice received intraperitoneal injections with 26 gauge needles, 3 times a week for up to 14 days. Mice were culled via cervical dislocation 14 days after their first intraperitoneal injection, and the colons were harvested for ex vivo experiments.

IHC in Wholemount Preparations

Distal colon segments were cut along the mesenteric border, stretched maximally, and pinned to silicone-based petri dishes containing PBS and an L-type calcium channel blocker nicardipine (3 µM) to relax the smooth muscle. Tissues were incubated in Zamboni’s fixative (2% formaldehyde, 0.2% picric acid, and 0.1 M sodium phosphate buffer of pH 7.0) overnight at 4C. The following day, tissues were washed using 100% DMSO 3× for 10 min, followed by washing with PBS, 3× for 10 min. The mucosa, submucosa, and the circular muscle layers were dissected out. The remaining longitudinal muscle-myenteric plexus (LMMP) wholemount preparations were processed for IHC.

Wholemount preparations were incubated in a blocking solution comprised of PBS and 0.1% Triton X-100 (PBS-T; Sigma-Aldrich) and 10% normal donkey serum (Merck Millipore; Burlington, MA) for 1 hr at room temperature. Preparations were then washed with PBS-T, 3× for 10 min. Wholemount preparations were labeled with primary antibodies (Table 1) overnight at 4C, then washed with PBS-T, 3× for 10 min. Secondary antibodies (Table 1) were then incubated for 2 hr at room temperature, and then washed with PBS-T, 3× for 10 min. Wholemount preparations were mounted onto glass slides using an antifade mounting medium (DAKO; Kingsgrove, NSW, Australia). Negative controls were tested for all antibodies used.

Table 1.

Details on Primary and Secondary Antibodies Used in This Study.

Antibody	Species	Dilution	Source
Primary antibodies
CGRP	rabbit	1:2000	Abcam; Burlingame, CA
TH	sheep	1:1000	Abcam; Burlingame, CA
VAChT	rabbit	1:1000	Abcam; Burlingame, CA
B-Tubulin III	chicken	1:1000	Abcam; Burlingame, CA
ChAT	goat	1:500	Abcam; Burlingame, CA
GFAP	goat	1:500	Abcam; Burlingame, CA
s100β	rabbit	1:500	Abcam; Burlingame, CA
Secondary antibodies
Alexa Fluor 488	rabbit	1:200	Jackson ImmunoResearch Laboratories, Australia
	goat	1:500
	sheep	1:1000
Alexa Fluor 594	rabbit	1:200	Jackson ImmunoResearch Laboratories, West Grove, PA
Alexa Fluor 594	chicken		Jackson ImmunoResearch Laboratories, West Grove, PA

Abbreviations: CGRP, calcitonin gene-related peptide; TH, tyrosine hydroxylase; VAChT, vesicular acetylcholine transporter; ChAT, choline acetyltransferase; GFAP, glial fibrillary acidic protein.

Imaging and Analysis

Three-dimensional (1 μm step z-series) images of the distal colon wholemount preparations were taken using an Eclipse Ti confocal microscope (Nikon; Tokyo, Japan). All images were captured at identical laser strength and gain conditions. Excitation wavelengths were set to 559 nm for Alexa 594, and 473 nm for Alexa 488. The immunoreactivity of nerve fibers and glial cells was assessed by analyzing the density of fluorescent labeling/area (8 images/preparation taken at 40× magnification with a total area of 2 mm²). All images were captured at the same distance from the tissue edges, at identical acquisition exposure-time conditions, calibrated to standardized minimum baseline fluorescence. Minimum baseline fluorescence was determined from the sham-treated tissue, these acquisition settings were used as the acquisition exposure-time for all samples. All images were converted to binary, set to identical thresholds, and changes in fluorescence from baseline were measured as mean gray value using ImageJ software (National Institutes of Health; Bethesda, MD). Differences in fluorescence were measured using the ImageJ “Analyze → Measure” function, which gives % area of staining/image. The % area values were used to determine nerve fiber and glial cell densities. The number of neurons based on counting of their cell bodies was determined from 4 random images/preparation (20× magnification with a total area of 1 mm²) from each wholemount preparation using the ImageJ “Cell Counter” plug-in to ensure all neurons were counted only once. All images were coded and analyzed blindly.

Statistical Analysis

Statistical analysis of the data included a paired Student’s t-test or a one-way ANOVA followed by a Bonferroni’s post hoc test for multiple comparisons using GraphPad Prism v6.0 (GraphPad Software; San Diego, CA). The data are represented as mean ± standard error of the mean (SEM). Statistical significance was defined where p<0.05.

Results

Oxaliplatin Treatment Causes a Reduction in Sensory and Adrenergic Innervation of the Myenteric Plexus

To determine whether oxaliplatin treatment induced changes in the sensory, adrenergic, and cholinergic fibers within the myenteric plexus, processes were labeled with antibodies against CGRP, TH, and VAChT. Neuronal fiber densities were quantified within a total of 2 mm² area in wholemount LMMP preparations of the colon (n=4/group).

Oxaliplatin treatment caused a significant reduction in the density of CGRP-IR fibers in the colon (4.2 ± 0.6, *p<0.05) when compared with the vehicle-treated group (6.4 ± 0.4; Fig. 1A–C). Furthermore, a significant reduction in TH-IR fibers was also observed in the colon from oxaliplatin-treated mice (2.11 ± 0.2, **p<0.01) when compared with the vehicle-treated group (3.5 ± 0.2; Fig. 2A–C). No significant differences in the density of VAChT-IR fibers were observed following oxaliplatin treatment (4.9 ± 0.1) when compared with the vehicle-treated cohort (5.4 ± 0.4; Fig. 3A–C).

Figure 1.

Oxaliplatin treatment induces a reduction in CGRP-IR fiber density in the myenteric plexus of the distal colon. Nerve fibers within the myenteric plexus were labeled with anti-CGRP antibody in wholemount preparations of the colon at day 14 posttreatment (A–B′). A significant reduction in CGRP-IR nerve fiber density was observed in the myenteric plexus of the colon following oxaliplatin treatment when compared with the vehicle-treated cohort (C). A, B = 40× magnification, scale bar = 50 µm; A′–B′ = 100× magnification, scale bar = 10 µm. Abbreviations: CGRP, calcitonin gene-related peptide; IR, immunoreactive. *p<0.05; n=4/group.

Figure 2.

Oxaliplatin treatment induces a reduction in TH-IR fiber density in the myenteric plexus of the distal colon. Nerve fibers within the myenteric plexus were labeled with anti-TH antibody in wholemount preparations of the colon at day 14 posttreatment (A–B′). A significant reduction in TH-IR nerve fiber density was observed in the myenteric plexus of the colon following oxaliplatin treatment when compared with the vehicle-treated cohort (C). A, B = 40× magnification, scale bar = 50 µm; A′–B′ = 100× magnification, scale bar = 10 µm. Abbreviations: TH, tyrosine hydroxylase; IR, immunoreactive. **p<0.01; n=4/group.

Figure 3.

VAChT-IR fiber density in the myenteric plexus of the distal colon is not affected by oxaliplatin treatment. Nerve fibers within the myenteric plexus were labeled with anti-VAChT antibody in wholemount preparations of the colon at day 14 posttreatment (A–B′). No significant differences in VAChT-IR nerve fiber density were observed in the myenteric plexus of the colon following oxaliplatin treatment when compared with the vehicle-treated cohort (C). A, B = 40× magnification, scale bar = 50 µm; A′–B′ = 100× magnification, scale bar = 10 µm. Abbreviations: VAChT, vesicular acetylcholine transporter; IR, immunoreactive. n=4/group.

Oxaliplatin Treatment Causes a Reduction in Excitatory Motor Neurons Within the Myenteric Plexus of the Colon

To determine any effects of oxaliplatin on excitatory motor neurons, the myenteric plexus was labeled with β-Tubulin III and ChAT. The total number of ChAT-IR neurons was counted within 1 mm² area from each wholemount preparation (n=4/group). Oxaliplatin treatment induced a significant reduction in the total number of ChAT-IR neurons within the myenteric plexus (178 ± 10; *p<0.05) when compared with vehicle-treated cohort (300 ± 35; Fig. 4A–C). Furthermore, oxaliplatin treatment significantly reduced the proportion of ChAT-IR neurons (16% ± 1.3%; *p<0.05) when compared with the vehicle-treated cohort (23% ± 1.6%; Fig. 4D).

Figure 4.

Oxaliplatin treatment induces a reduction in the total number and proportion of ChAT-IR neurons within the colon myenteric plexus. Wholemount preparations of the myenteric plexus labeled with β-Tubulin III (red) and ChAT (green; A–B″). Oxaliplatin treatment induced a significant reduction in the total number of ChAT-IR neurons within the myenteric plexus when compared with vehicle-treated cohort (C). Furthermore, oxaliplatin significantly reduced the proportion of ChAT-IR neurons when compared with the vehicle-treated group (D). A–B″ = 40× magnification, scale bar = 50 µm. Abbreviations: ChAT, choline acetyltransferase; IR, immunoreactive. *p<0.05; n=4/group.

Oxaliplatin Treatment Differentially Affects Myenteric Glial Cell Populations

To determine any effects of oxaliplatin on glial cells, the myenteric plexus was labeled with GFAP or s100β. A total of 8 images/animal (total area of 2 mm²) was analyzed from each wholemount preparation (n=4/group). Oxaliplatin treatment caused a significant reduction in the density of GFAP-IR glia (6.1 ± 0.5; **p<0.01) when compared with the vehicle-treated cohort (9.8 ± 0.6; Fig. 5A–C). Conversely, oxaliplatin treatment caused a significant increase in the density of s100β-IR glia (8.8 ± 0.1; **p<0.01) when compared with the vehicle-treated group (7.7 ± 0.2; Fig. 6A–C).

Figure 5.

Oxaliplatin treatment induces a reduction in GFAP-IR glia within the colon myenteric plexus. Wholemount preparation of the myenteric plexus was labeled with GFAP (A–B′). Oxaliplatin treatment induced a significant reduction in the density of GFAP-IR glial cells within the myenteric plexus when compared with vehicle-treated cohort (C). A–B are 20x magnification, scale bar = 100 µm; A’–B’ are 40x magnification, scale bar = 50 µm. Abbreviations: GFAP, glial fibrillary acidic protein; IR, immunoreactive. **p<0.01; n=4/group.

Figure 6.

Oxaliplatin treatment causes an increase in s100β-IR glia within the colon myenteric plexus. Wholemount preparation of the myenteric plexus was labeled with s100β (A–B′). Oxaliplatin treatment induced a significant increase in the density of s100β-IR glial cells within the myenteric plexus when compared with vehicle-treated cohort (C). A–B are 20x magnification, scale bar = 100 µm; A’–B’ are 40x magnification, scale bar = 50 µm. Abbreviation: IR, immunoreactive. **p<0.01; n=4/group.

Discussion

The results of this study demonstrate that oxaliplatin treatment significantly alters innervation of the colon myenteric plexus, which may be implicated in the manifestation and persistence of gastrointestinal dysmotility. Our previous studies have shown that oxaliplatin-treated mice fail to gain weight when compared with their vehicle-treated counterparts and that their gastrointestinal transit and motility are significantly disturbed.^28,29 Furthermore, we have also found that oxaliplatin-treated mice demonstrate signs of chronic constipation²⁹ and pica (unpublished observations).

Our present work revealed that oxaliplatin significantly reduces CGRP-IR fiber density within the myenteric plexus. Anti-CGRP antibodies label afferent fibers from sensory ganglia.³² It should be noted that sensory innervations to the myenteric plexus are not exclusively extrinsic as CGRP-IR sensory neurons can also be found within the ENS, however, they are not easily identifiable given that labeling is weak/punctuate, especially in mouse tissues. Thus, the majority of studies focus on investigating changes to nerve fiber densities.^33–36 In this study, we have not differentiated between extrinsic and intrinsic CGRP-IR fibers in the colon. Rigorous examination of α-CGRP and β-CGRP fibers, alongside anterograde/retrograde labeling of sensory fibers in the colon after chemotherapy, should be further conducted. Platinum-based drug toxicity to CGRP-IR neurons in the dorsal root ganglia (DRG) and peripheral nerve fibers supplying the extremities (peripheral sensory neuropathy) has been investigated previously.^37–39 The chronic form of peripheral sensory neuropathy is thought to result from the accumulation of the platinum-based chemotherapeutic agents, inducing changes in nerve conduction potentials, soma size, and nuclear morphology, and, ultimately, neuronal death.^{37,38,40–42} A linear relationship between a cumulative dose of cisplatin and an increase in histopathological toxicity within the DRG is reported, suggesting that the platinum is being retained in active and toxic forms.³⁷ Our present findings are in agreement with a study that showed a reduction in CGRP-IR fibers and myenteric neurons in the rat colon following cisplatin administration.²⁷ In the gut, CGRP released from sensory nerve fibers innervating the gastrointestinal tract plays an important protective role. CGRP facilitates mucus production and controls blood flow in the gastrointestinal mucosa; and, thus, the loss of CGRP-IR sensory fibers and/or sensory neuron dysfunction can impair mucosal protection.⁴³ CGRP-IR fibers projecting to the mucosa can also mediate local intrinsic reflexes following mucosal stimulation.³² The reduction in these nerve fibers may still impact colonic motility through diminished or abolished reflex activity.

Treatment with oxaliplatin induced a reduction in TH expressing nerve fibers within the myenteric plexus of the colon. The anti-TH antibody was used to identify adrenergic fibers and neurons.^20,44 The majority of TH-IR fibers are from an extrinsic origin (sympathetic neurons within the paravertebral ganglia and the celiac-mesenteric ganglia). TH-IR neurons have been reported in only a very small proportion of myenteric neurons in the ileum of adult Balb/c mice (less than 0.5%), and are most frequently observed in the upper digestive tract such as the esophagus and in the submucosal plexus.^20,45,46 Similar to previous studies,^47,48 we have not found TH-IR cell bodies in the myenteric plexus of the colon, thus, have analyzed the fibers exclusively. Furthermore, a study conducted by Lucas et al. investigated the effects of the platinum-based drug, cisplatin, on TH-IR fibers in murine bone marrow. In agreement with our study, their research showed a significant reduction in TH-IR following platinum-based chemotherapy.⁴⁹ Sympathetic adrenergic fibers innervating gastrointestinal smooth muscles and enteric ganglia in the colon can modulate motility, secretion, blood flow, and immune system activation.¹⁹ The platinum-based agents cisplatin and oxaliplatin both induce gastrointestinal dysmotility, which is characteristic of chronic constipation.^27,28 The release of norepinephrine by sympathetic TH-IR fibers functions to inhibit gastrointestinal motility. Thus, it does not appear that the reduction in TH innervation has major implications regarding the contractility of the gut, given that motility is still slowed down following oxaliplatin treatment. However, a reduction in sympathetic noradrenergic innervation of the colon could still impact secretion, blood flow, and gastrointestinal immunity, which needs to be further investigated. Furthermore, functional changes in the activity of the remaining sympathetic fibers after oxaliplatin treatment should be analyzed.

Moreover, we did not observe any differences in VAChT-IR fibers within the myenteric plexus of the colon following oxaliplatin treatment. The anti-VAChT antibody labels cholinergic axons containing acetylcholine in synaptic vesicles,^20,21 and we did not discriminate between extrinsic or intrinsic projections throughout the myenteric plexus. Our results correlate well with the electrophysiological studies investigating evoked junction potentials in smooth muscle cells of the colon following oxaliplatin treatment, which showed normal excitatory junction potentials that are mediated by acetylcholine.²⁹

We have previously shown that oxaliplatin treatment causes a significant reduction in the number of myenteric neurons (β-Tubulin III⁺) as well as inhibitory (nNOS⁺) neurons in the mouse colon.²⁹ However, the proportion of nNOS neurons relative to the total number of neurons revealed a significant increase in the proportion of this inhibitory population following oxaliplatin treatment.²⁹ There are only a few studies that have investigated the effects of platinum-based chemotherapeutic agents (cisplatin and oxaliplatin) on the rat and mouse ENS.^27,28,50 These studies have demonstrated that platinum-based agents cause a significant reduction (25–30%) in the total number of myenteric neurons. The loss of myenteric neurons has been associated with colonic dysmotility following chemotherapeutic administration of 5-fluorouracil, an antimetabolite typically used in conjunction with oxaliplatin for the treatment of colorectal malignancies.⁵¹ It is apparent that the ENS, and specifically the myenteric neurons, are particularly sensitive to anticancer agents, despite their postmitotic nature. It is possible that oxaliplatin could accumulate within the ENS just as it does within other parts of the nervous system, however, this remains to be investigated. In our study, oxaliplatin treatment caused a significant reduction in the number of ChAT neurons within the myenteric plexus of the colon. ChAT-IR neurons utilize acetylcholine as their primary neurotransmitter, which excites interstitial cells of Cajal (ICC), and, in turn, stimulates muscle contraction and gastrointestinal motility.⁵² Our unpublished data have shown that oxaliplatin treatment also affects ICCs, and, therefore, this may also contribute to altered gastrointestinal motility. Furthermore, our previous work has demonstrated that gastrointestinal motility is decreased following oxaliplatin treatment.²⁹ This suggests that nNOS neurons maintain functional integrity over excitatory neurons expressing ChAT to some degree. We have previously shown that oxaliplatin treatment results in mitochondrial superoxide production and protein nitrosylation within the myenteric plexus, as well as increased expression of inducible nitric oxide synthase (iNOS) within the LMMP.²⁹ The nNOS neurons generally show a greater capacity to deal with damaging stimuli, including oxidative stress. NO production by nNOS neurons is presumed to have protective effects against intestinal ischemia/reperfusion injury in mice.⁵³ In addition, nNOS neurons have also demonstrated the ability to withstand NMDA and NO-mediated neurotoxicity.⁵⁴ ChAT-IR neurons, in contrast, are particularly sensitive to oxidative stress.^55,56 The ChAT enzyme contains an unusually high number of reactive cysteine thiol groups, which are principally vulnerable to oxidative and/or nitrosative modifications.^57,58 Both oxidative and nitrosative stress can result in altered structure and function of ChAT, which could greatly impact acetylcholine synthesis and inhibit cholinergic transmission at presynaptic terminals.⁵⁸ The efficacy of antioxidants in restoring cholinergic transmission and essentially gastrointestinal motility following oxaliplatin should be researched.

Our study also revealed that oxaliplatin treatment causes differential effects on colonic myenteric glial cells. Our results show that oxaliplatin treatment causes a reduction in GFAP-IR myenteric glia, with a concurrent increase in s100β expression. These findings are in agreement with a previous study demonstrating a reduction and increase in GFAP and s100β expression, respectively, within the ileal myenteric plexus following oxaliplatin treatment.²³ GFAP-IR glial cells function to promote cell survival and neuronal regeneration.^59,60 A reduction in GFAP expression has also been correlated with glial cell damage in a mouse model of enteric glial disruption, and in various pathological conditions associated with neuronal damage, inflammation, and type I diabetes.^23,61–64 Furthermore, an increase in s100β expression is commonly observed in chronic neuropathological conditions, and elevated levels of this protein in cerebrospinal fluid are used as a biomarker for brain damage.⁶⁵ The s100β over expression by glial cells has been correlated with increases in iNOS and NO, which can potentiate cell damage and death of other glial subpopulations as well as neurons.^66,67

In summary, oxaliplatin treatment significantly alters innervation of the colon myenteric plexus. Changes in nerve fiber densities, myenteric plexus cellularity, as well as alterations in inhibitory and excitatory motor neuron populations, can impact gastrointestinal motility functions. Furthermore, the differential effects of oxaliplatin treatment on enteric glial populations can exacerbate myenteric neurotoxicity within the murine colon. Further studies concerning the mechanisms underlying myenteric plexus toxicity following oxaliplatin treatment are crucial. Such studies could lead to novel neuroprotective strategies with the aims to advance treatment efficacy and tolerance through minimizing gastrointestinal dysfunction.

Footnotes

Competing Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author Contributions

All authors have contributed to this article as follows: VS conducted experiments, analyzed data, and wrote the manuscript; RMM and SM conducted experiments and revised the manuscript; KN developed study conception and design, provided supervision and funding for the study, and revised the manuscript; and all authors have read and approved the manuscript as submitted.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Victoria University Research Support grant (KN), College of Health and Biomedicine (VS), Victoria University.

ORCID iD

K Nurgali

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