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Article

DRG Explant Model for Understanding Mechanism of Oxaliplatin-Induced Peripheral Neuropathy and Identifying Potential Therapeutic Targets

by
Junwei Du
1,2,
Leland C. Sudlow
1,
Igor D. Luzhansky
1,2 and
Mikhail Y. Berezin
1,2,*
1
Mallinckrodt Institute of Radiology, School of Medicine, Washington University, St. Louis, MO 63110, USA
2
Institute of Materials Science & Engineering, Washington University, St. Louis, MO 63130, USA
*
Author to whom correspondence should be addressed.
Antioxidants 2026, 15(2), 230; https://doi.org/10.3390/antiox15020230
Submission received: 30 December 2025 / Revised: 29 January 2026 / Accepted: 5 February 2026 / Published: 10 February 2026
(This article belongs to the Special Issue Chronic Pain and Oxidative Stress)
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Abstract

Oxaliplatin-triggered chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating side effect of cancer treatment that limits the efficacy of chemotherapy and negatively impacts patients’ quality of life dramatically. To better understand the mechanisms of CIPN and to screen for potential therapeutic targets, it is critical to have reliable in vitro assays that effectively mirror the neuropathy in vivo. In this study, we established a dorsal root ganglia (DRG) explant model. This model displayed dose-dependent inhibition of neurite outgrowth in response to oxaliplatin, while oxalic acid exhibited no significant impact on the regrowth of DRG. The robustness of this assay was further demonstrated by the inhibition of OCT2 transporter, which facilitates oxaliplatin accumulation in neurons, largely restoring the neurite regrowth capacity. Using this model, we revealed that oxaliplatin triggered a substantial increase of oxidative stress in DRG. Notably, inhibition of TXNIP with verapamil reduced oxidative stress levels. Our results demonstrated the use of DRG explants as an efficient model to study the mechanisms of CIPN and screen for potential treatments.

Graphical Abstract

1. Introduction

Oxaliplatin, a third-generation platinum-based chemotherapy drug, is mostly used in the treatment of colorectal cancer, with recent broadening of its application to include lung, gastric, ovarian, and prostate cancers [1,2,3,4]. One of the most common side effects associated with oxaliplatin treatment is peripheral neuropathy, which develops in a “stocking and glove” pattern in patients’ feet and hands [5]. Symptoms of oxaliplatin-triggered CIPN can appear minutes to hours after treatment and are likely to persist and accumulate with repeating dosages, resulting in chronic symptoms lasting months or even years later. CIPN affects nearly every aspect of patients’ daily lives, compelling clinicians to lower the dose of cancer chemotherapy agents and consequently decreasing the therapeutic efficacy [6,7].
Oxaliplatin exerts its anti-tumor effects by damaging DNA that generates excess reactive oxygen species (ROS) leading to apoptosis in cancer cells [8,9]. While these mechanisms are crucial for the drug’s anti-cancer activity, they can also have unintended consequences on healthy cells [10], including those in the peripheral nervous system (PNS). The PNS, especially the dorsal root ganglia (DRG), appears to be highly susceptible to the chemo-induced oxidative stress as a result of lacking vascular barriers and an absence of lymphatic drainage [11]. Peripheral neurons are also highly vulnerable to oxidative stress [10,12] due to their high metabolic rate; their long axonal structures; and the presence of supporting myelin and satellite glial cells (SGCs), which can themselves be damaged by ROS. Once the neurons are damaged, they trigger a cascade of events that may result in symptoms like tingling, cold/hot allodynia, numbness, and eventually chronic pain, the hallmarks of CIPN [13,14,15].
Significant effort has been made to study how to mitigate CIPN side effects, including the use of antioxidants and other neuroprotective agents, without compromising the drug’s anti-cancer efficacy [16,17,18]. However, clinical trials targeting ROS have not yet demonstrated significant relief from the symptoms. This discrepancy reflects the complexities of CIPN and raises questions about the effectiveness of simply suppressing ROS with antioxidants as a therapeutic strategy that so far only provides mild to moderate pain relief [19,20].
Finding better treatments for CIPN may require different types of assays. Most of the published techniques used to understand the mechanism of CIPN rely on either cell cultures or live animals. Both of these traditional models have substantial limitations that impede the discovery of effective therapies. For example, neuronal cell cultures that are often used to study oxaliplatin-induced ROS [21,22] are too simplified as a model and do not replicate the complex microenvironment found in vivo. These models focused on isolated neurons do not take into account other types of cells such as SGCs, Schwann cells, or immune cells, which play significant roles in the pathogenesis of CIPN. Neuronal cultures also have limited lifespans, restricting the length of time over which experiments can be conducted, which may not be representative of chronic conditions like CIPN. Animal models, such as those based on wild-type mice treated with chemotherapy drugs, introduce a host of systemic variables such as metabolic changes [23], immune responses, and changes in the blood composition, which can confound results and make it challenging to isolate the specific impacts of a drug on CIPN. In addition, live animal models have low throughput and require a lengthy period to develop chronic CIPN.
Given these limitations, new assay types are needed for CIPN research that combine the throughput of the neuronal culture while having substantial relevant complexity such as in live animals. We envisioned that DRG that houses clusters of sensory neuron somata located along the spinal cord can be such a model. The DRG play a key role in transmitting sensory information and pain signals from the periphery to the central nervous system, and being isolated might be used as an alternative CIPN assay. These isolated DRG ex vivo models are increasingly being used for studying a variety of neurological diseases including CIPN and for screening potential drugs [24,25]. Unlike simplified cell culture models, DRG models retain the native arrangement and diversity of cell types, including neurons, SGC, and other supporting cells, providing a more biologically relevant environment. DRGs are also physiologically relevant and represent a natural and intact microenvironment that preserves the native cellular interactions and signaling pathways found in vivo. Compared with the live animal models, where systemic factors like immune response and metabolic rate can influence outcomes, extracted DRG models help eliminate these confounding variables and isolate the effect of the drug on sensory neurons.
We hypothesized that the neurotoxicity of oxaliplatin, and potentially other drugs that cause ROS neurotoxicity, may be quantified by using an in vitro model of extracted DRG. This assay can be also used for studying pathways and testing new molecules as potential drugs to prevent or treat CIPN. Specifically, we were interested in understanding the role of the thioredoxin interacting protein (TXNIP) mediated ROS inflammatory pathway as a trigger to CIPN that we have identified earlier in animal models [26]. To test this hypothesis, we extracted DRG from mice, measured neurite outgrowth, evaluated oxidative stress, and tested TXNIP inhibitors, aiming to understand the pathology of CIPN and to identify therapeutic targets.

2. Materials and Methods

2.1. Animals

All animal experiments were conducted in compliance with Washington University Institutional Animal Studies Committee (Animal Welfare Assurance #D16-00245, animal protocol #24-0297, Approval date 13 November 2024). Male 8–12-week-old C57BL/6 mice from Charles River Laboratory were used in this study. Animals were housed with free access to water and food and maintained on a 12:12 h light/dark cycle with controlled temperature and humidity.

2.2. Extraction and DRG Preparation

Thoracic and lumbar DRGs from adult (8–12 weeks) healthy male C57BL/6 mice were aseptically removed from levels L1–L6 and transferred into ice-cold serum-free media (SFM), which was a customized DMEM-based media (ThermoFisher Scientific, Waltham, MA, USA, cat # A4192101) containing 1% BSA (MilliporeSigma, Burlington, MA, USA, cat # A7906), 50 µg/mL vitamin C (MilliporeSigma, cat # A4544), 3 g/L extra glucose (MilliporeSigma, cat # G8270), 1% L-glutamine (ThermoFisher, cat # 25030149), 1% insulin-transferrin-selenium (ThermoFisher, cat # 51300044), and 1% penicillin-streptomycin (ThermoFisher, cat # 15140122). The DRGs were trimmed to remove any fibers connecting to the ganglia. A stock solution of Matrigel-SFM mixture (1:1 vol) was prepared by mixing growth-factor-reduced Matrigel (Corning, Corning, NY, USA, cat # 354230) with SFM in a sterile Eppendorf tube until homogenized and kept on ice. Matrigel-SFM mixture (20 μL) was placed in each well of a 12-well plate (Corning, cat # 3513). DRGs were placed individually in each well into the Matrigel-SFM mixture and incubated in 5% CO2 at 37 °C for 50 min. Finally, 1.5 mL pre-warmed SFM was added to each well, and DRGs were incubated at 37 °C for further use.
Differences in sample size among experimental groups were due to the loss of some DRGs during experimental procedures. At the start of each experiment, all groups contained the same number of DRGs. Tissues were embedded in 50% Matrigel for attachment, and some DRGs were lost during the multiple staining and washing steps. As a result, the final sample sizes (n) differed slightly among groups. No data were intentionally excluded, and no outlier analyses were performed.

2.3. Fluorescent Imaging

Imaging was performed with Zeiss Cell Discover 7 confocal microscope (Zeiss, Jena, Germany). All images were captured using a 5× objective. The pixel time was set at 0.52 μs. Three laser wavelengths were used for immunochemistry staining: 561 nm for Alexa Fluor 555 with a detection wavelength range of 550–700 nm; 488 nm for Alexa Fluor 488 with a detection wavelength range of 400–550 nm; and 405 nm for DAPI, also with a detection range of 400–550 nm. For ROS imaging, the laser wavelength was 488 nm, and the detection range was 490–700 nm. The ROS level in the DRG was determined by dividing the fluorescence intensity within the DRG region of interest (ROI) by that of the ROI outside the DRG. The calculated value was then normalized to the control values at the time zero.

2.4. DRG Outgrowth Assay

Freshly excised isolated DRGs were placed on a Matrigel-SFM mixture (see above) at the bottom of individual wells of a 12-well plate and placed in an incubator with 5% CO2 at 37 °C. SFM media was replaced on the 3rd day. The outgrowth assay for the control untreated DRG was determined within 5 days. The DRG were periodically imaged with a bright-field inverted microscope to monitor the neurite outgrowth (Olympus CKX53, Olympus, Tokyo, Japan). Images of DRG neurites outgrowth were analyzed using the Sholl Analysis plugin in ImageJ (version 1.54). Briefly, the explant DRG was excluded by a circle enveloping it, and rings were created around it in 5 µm increments. The number of intersections of neurites crossing each ring were counted, and the neurite-occupied area was represented by the total number of intersections of all rings.

2.5. Treatment of DRG with Oxaliplatin

To investigate the effects of oxaliplatin on the DRG regrowth, the media for the oxaliplatin-treatment DRGs was replaced 48 h after plating with SFM mixed with different concentrations of oxaliplatin (in sterile water) and incubated for an additional 72 h. Oxaliplatin (1056313, Sanofi, Beijing, China) was diluted in sterilized water to create stock solutions at 1 mM. The stock solutions of oxaliplatin were mixed with culture media in the wells, resulting in final oxaliplatin concentrations of 10 µm, 50 µm, or 100 µM. The control group received an equal volume of sterilized water.

2.6. Treatment of DRG with Verapamil and Cimetidine

Verapamil (V4629 -1g, MiliporeSigma) was dissolved in sterilized water to make a stock solution of 100 mM. Further series dilutions in water were made to create concentrations of 1 mM, 100 μM, and 10 μM. For treatment, 10 μL of drug solution was added per 1 mL of control or oxaliplatin-treated culture media in the DRG plating wells to make the final concentration of verapamil 10 μM, 1 μM, or 0.1 μM. Control groups were treated with an equal volume of water.
Cimetidine (C4522-5G, MiliporeSigma) was dissolved in DMSO, followed by addition of equal volumes of sterile water to make stock solutions of 100 mM, 10 mM, 1 mM, and 100 μM. For treatment, 10 μL of drug solution was added per 1 mL of control or oxaliplatin-treated culture media in the DRG plating wells to make the final concentration of cimetidine 1 μM, 10 μM, 100 μM, or 1 μM. Control groups received an equal volume of 50% DMSO, resulting in a final DMSO concentration of 0.5%.
Oxalic acid (75688-50G, MiliporeSigma) was dissolved in sterile water to create a 1 mM stock. Oxalic acid stock was added to media to a final concentration of 100 µM.
For all experiments, drugs were diluted in either water or 50% DMSO and applied to the media by adding an equal volume of drug solution to achieve the indicated final concentrations (1:100 dilution). Correspondingly, control groups received an equal volume of the appropriate vehicle (water or 50% DMSO).

2.7. Immunochemistry of DRG

After treatment with oxaliplatin or other drugs, DRGs were fixed in 10% neutral buffered formalin (NBF) for 30 min at room temperature. Following additional incubation with a 1X PBS buffer containing 0.1% Triton-X (PBST), DRGs were blocked with 10% donkey serum (MilliporeSigma, cat # D9663) in 0.1% PBST. DRG explants were then incubated with the primary antibody against β-III tubulin (MilliporeSigma, cat # MAB1637, 1:500) overnight at 4 °C, followed by incubation with Alexa Fluor 555 (ThermoFisher, cat # A31570, 1:1000) and 488 (ThermoFisher, cat # A32814, 1:1000)-conjugated secondary antibody for 1 h at room temperature. Nuclei were stained with DAPI (ThermoFisher, cat # 62248) for 20 min. The entire DRGs were scanned with a Zeiss Cell Discover 7 confocal microscope under 5× objective to generate 16 images that were stitched together to capture the whole DRG and surrounding axons.

2.8. DRG ROS Imaging

CM-H2DCFDA (ThermoFisher, cat #C6827) or CellRox Green (ThermoFisher, cat #C10444) dissolved in DMSO were added to pre-warmed to 37 °C HBSS buffer at a final concentration of 10 µM. DRGs were then incubated with the dye at 37 °C for 40 min in the incubator. Then, the buffer was removed, and DRGs were washed with HBSS three times and recovered in the SFM for 10 min. Fluorescences of untreated DRGs were determined as a baseline and recorded periodically within 120 min. To investigate the effects of oxaliplatin and OCT2 inhibitor on ROS, after CM-H2DCFDA loading and washing, DRGs were exposed to 100 μM oxaliplatin, mixtures of 100 μM oxaliplatin and 1000 μM cimetidine, or 0.5% DMSO (control). For testing effects of TXNIP inhibition on ROS, after CM-H2DCFDA loading and washing, DRGs were treated with 100 μM oxaliplatin, 100 μM oxaliplatin combined with 1 μM verapamil, and sterile water. Fluorescence intensity was monitored every 10 min with a Zeiss Cell Discover 7 confocal microscope for 120 min under 5x objective with no stitching to capture the whole DRG.

2.9. Statistics and Data Analysis

The values were then processed in Prism 9 for statistical analysis using a Student t-test or one-way ANOVA test. Details of the analysis are included in the caption of each figure. Data are expressed in mean ± SD.

3. Results

3.1. Growth Media and ECM Scaffolding Induces Stable and Uniform Distribution of Neurite Outgrowth

Under our standard growth media conditions (Matrigel-SFM, 37 °C, 5% CO2), DRGs showed stable and evenly distributed neurite outgrowth. Bright-field images of DRG (Supplemental, Figure S1) outgrowth were difficult to quantify due to the lack of contrast between the background and small fibers in bright-field views. To overcome this limitation, we used fluorescent labeling against the neuronal marker β-III tubulin and employed high-resolution fluorescence microscopy (see Methods) with stitching capabilities. This imaging method allowed us to capture the entire DRG outgrowth with single neurite resolution. High-resolution fluorescent imaging data were further quantified using Sholl analysis, which is commonly used to quantify regrowth patterns in the DRG [27,28]. This computational technique measures the neurite extensions’ complexity and extent, as illustrated in Figure 1A. The method involves placing concentric circles at regular 5 µm intervals around the entire DRG [29]. The number of times neurites intersect these circles is counted, and the data are represented as a Sholl profile (Figure 1B). On this profile, the x-axis indicates the distance from the soma (i.e., the radius of the circles), and the y-axis shows the number of intersections at each radius. The example of Sholl analysis for the DRG is shown in Figure 1A,B.

3.2. Oxaliplatin Induces Decreases in DRG Neurite Outgrowth in a Dose-Dependent Pattern

The addition of oxaliplatin to the SFM culture media had a profound and dose-dependent effect on the DRG outgrowth, as shown in Figure 1. The media with a 50 μM level of oxaliplatin yielded inhibition of almost half (48%) of the neurite outgrowth, and 100 μM oxaliplatin inhibited more than 70% of neurites compared with the control group during antibody staining against bIII-tubulin.

3.3. Oxalate Does Not Affect DRG Regrowth

One subject of debate is the role of oxalate in the development of CIPN. Oxalate not only is a key component of oxaliplatin’s structure—serving as a ligand that chelates Pt2+—but is also a major metabolite during oxaliplatin treatment. Several studies have suggested that the strong chelating properties of oxalates could bind Ca2+ ions in neurons, thereby affecting the function of voltage-dependent sodium channels [30,31]. This alterative model has been proposed as a primary factor in the acute neurotoxicity induced by oxaliplatin. To examine the role of oxalate, we performed DRG outgrowth assays in its presence at concentrations equivalent to those of oxaliplatin. We observed no statistically significant differences between DRGs treated with oxalate at comparable doses of oxaliplatin (100 μM) and control ones (Figure 1E,F). These findings suggest that oxalate alone has a minimal effect on DRG neurite outgrowth, challenging the hypothesis that oxalate is a key factor in CIPN [7,32,33,34].

3.4. Inhibition of OCT2 Preserves DRG Neurite Outgrowth

Having demonstrated the negative impact of oxaliplatin on DRG neurite regrowth, we further validated the assay’s utility by inhibiting oxaliplatin uptake into the SGCs. Recent studies in mouse models have shown that the organic cation transporter 2 (OCT2) mediates the uptake of oxaliplatin into SGCs, initiating neuropathy symptoms. Blocking this transporter with an OCT2 inhibitor cimetidine [35] has been previously shown to minimize these symptoms [36,37]. To investigate whether OCT2 inhibition could protect neurite outgrowth in the presence of oxaliplatin, DRG explants were co-cultured with a high dose of oxaliplatin (100 μM) and varying concentrations of cimetidine. We observed a significant increase in the area occupied by neurites in the DRG explants treated with both cimetidine and oxaliplatin compared with those treated solely with oxaliplatin (Figure 2). The protective effects became statistically significant at a cimetidine concentration of around 100 μM, with an EC50 value of approximately 159.6 μM. These results are in line with the published data on the neuroprotective properties of cimetidine in oxaliplatin-treated mice, further validating the assay’s relevance to animal studies.

3.5. Oxaliplatin Leads to Oxidative Stress in the DRG Explant

To investigate whether ROS can be seen in the DRG, we added activatable fluorescent ROS sensors CM-H2DCFDA or CellRox into the media. In this assay, the DRG was extracted, placed in a well as per the Extraction and DRG preparation methods section, and allowed to regrow for two days, followed by ROS dye treatment. Fluorescence intensity of the dye in the presence of DRG was rather low and slightly decreased over the duration of the experiment (120 min) when normalized to the background and to the intensity right after addition of the dye (Figure 3). In contrast, addition of oxaliplatin substantially increased the fluorescence intensity of the DRG. We observed steady increase of ROS relative to the background when the DRG was exposed to 100 μM oxaliplatin for 120 min. Furthermore, the inhibition of OCT2 via cimetidine almost completely prevented the oxaliplatin-induced oxidative stress with the average normalized intensity in the DRG very similar to the control DRG with no oxaliplatin added. These findings suggested the DRG explant as a viable model for investigating the role of ROS in oxaliplatin-mediated DRG tissue damage.
The strong fluorescence signal observed around the DRG at timepoint 0 likely results from interaction of the dye with the surrounding ECM matrix from Matrigel. For quantitative analysis, we specifically selected the area of interest corresponding to the DRG body and excluded the surrounding Matrigel. Within the DRG body, the fluorescence signal decreased by approximately 20% over 120 min. The visually larger difference in Figure 3A is likely due to the background signal from the Matrigel rather than changes in the DRG itself.
The apparent differences in DRG shape between the control and the “Oxa before” images reflect natural variability rather than treatment effects. DRGs from different spinal levels can have slightly different sizes and shapes, and minor changes may also occur during trimming and preparation. The DRGs shown in the panel are representative DRG morphologies, and we did not observe significant changes in the shape of DRG body before and after oxaliplatin treatment.

3.6. Inhibition of TXNIP Through Verapamil Decreases ROS Level Induced by Oxaliplatin

Encouraged by the establishment of both neurite outgrowth and ROS assay as metrics of tissue damage and oxidative stress, we evaluated the potential neuroprotective effects of inhibiting TXNIP with verapamil. Verapamil, a clinically approved calcium channel inhibitor, has been previously shown to attenuate neuropathy induced by diabetes through blocking of TXNIP, a regulator of intracellular ROS in both the cytosol and mitochondria by interacting and inhibiting thioredoxin [38] (see the Section 4). For that, DRG explants were cultured in presence of a high level of oxaliplatin and variable dosages of verapamil. We found 1 μM verapamil resulted in an approximately 70% enhancement in neurite outgrowth compared with the DRGs treated with oxaliplatin (Figure 4A,B) with almost complete suppression of the ROS (Figure 4C,D).
Verapamil was unable to completely restore the DRG neurite outgrowth to control levels of outgrowth even at a higher dosage, possibly because of its own potential neurotoxic effect [39] (Supplemental, Figure S2). Moreover, DRGs co-cultured with verapamil and oxaliplatin demonstrated significantly lower ROS [39] compared with oxaliplatin-only DRGs, pointing to the protection effect of verapamil on the DRG (Figure 4C,D). These data indicate while verapamil was not able to entirely block oxaliplatin anti-regrowth effect, it substantially mitigated oxaliplatin-induced oxidative stress, suggesting targeting TXNIP might offer a promising approach to attenuate the inflammatory effects of oxaliplatin on DRG.
The apparent difference in baseline fluorescence between Figure 3A and Figure 4C arises from the use of different ROS probes. In Figure 3A, CM-H2DCFDA, a commonly used, cell-permeable probe for intracellular ROS, was utilized. In Figure 4C, CellROX Green, a newer-generation ROS probe with higher photostability and lower background, was used. Both probes show the same trends in ROS changes for control and oxaliplatin-treated groups, providing confidence in the robustness of our findings.

4. Discussion

4.1. Oxaliplatin Has a Strong Impact on DRG Neurite Outgrowth

DRG neurons inherently possess a distinct regenerative capacity. When exposed to an appropriate environment or substrate, they are naturally programmed to extend axons in the process known as outgrowth. To facilitate the complete DRG outgrowth, we used Matrigel as a scaffold to which the DRG could attach. Matrigel is a viscous liquid derived from Engelbreth-Holm-Swarm mouse sarcoma that primarily comprises extracellular matrix (ECM). To minimize the role of growth factors, we selected Matrigel with low levels of growth factors. Isolated DRG cultured on Matrigel benefited from the matrix’s unique properties that promote axon outgrowth [40]. Occasionally, a DRG failed to attach to the Matrigel and consequently failed to show any neurite outgrowth. By offering a scaffold, Matrigel replicates the in vivo extracellular environment [41], supporting cell adhesion, survival, and growth.
We observed that oxaliplatin significantly reduced neurite outgrowth of DRG explants in vitro in a dose-dependent manner. The growth of DRG neurons in vivo is carefully regulated, while in vitro the regrowth is less constrained. In the presence of toxic elements, the regrowth can be slowed or completely stopped. Almost complete (ca. 90%) reduction in the normal regrowth of the intact DRG was achieved with 100 μM level of oxaliplatin after 72 h in line with the DRG neuronal cell culture studies, in which reduced cell viability was observed with concentrations of oxaliplatin higher than 10 µM [21,42].
The reduction of regrowth apparently was a result of the intact oxaliplatin molecule or its product of hydrolysis and not due to free oxalate. It has been suggested that dissociated oxalate ligand, which is usually displaced during the activation of the drug, would mediate the acute neuropathy through chelating of Ca2+ and Mg2+ ions in the nerve tissue [31,43]. In our DRG explant assay, oxalate alone did not affect neurite regrowth at concentrations equivalent to oxaliplatin, suggesting that free oxalate is unlikely to be a primary driver of chronic neurotoxicity.

4.2. Inhibition of OCT2 Restores DRG Outgrowth in the Presence of Oxaliplatin

The accumulation of oxaliplatin in the DRG cells has been shown to be facilitated by OCT2-expressing SGCs [36,44] that provide structural support to neurons and regulate substance trafficking to and from the primary neurons [45,46]. It has been observed that blocking OCT2 with different blockers, such as dasatinib [36] and cimetidine [35], suppresses the buildup of oxaliplatin in the DRG. Using the outgrowth assay, we have observed that blocking OCT2 with cimetidine, even with high concentrations of oxaliplatin, completely restores the outgrowth of DRG to almost a normal level (Figure 2). The measured EC50 = 159.6 µM for this inhibition was relatively high, requiring large doses of cimetidine (would be almost 10 times compared with oxaliplatin) to restore the DRG regrowth, suggesting the need for more effective inhibitors to mitigate oxaliplatin toxic effects.

4.3. Oxaliplatin Induces ROS Burst That Is Suppressed by TXNIP Inhibitor

While neurite outgrowth assay is established as an effective assay for assessing oxaliplatin toxicity, it is primarily based on measuring morphological changes. Herein, we extended the use of the DRG explant model to explore the response to oxidative stress. A recent study demonstrated that oxaliplatin could trigger an increase in ROS production within DRG neurons [21]. Herein we used activatable fluorescent ROS sensors CM-H2DCFDA or CellRox. CM-H2DCFDA is a widely used, cell-permeable probe designed to measure intracellular levels of ROS. CellRox Green is a newer generation of ROS probes with the same mechanism of action but with higher photostability according to the manufacturer. We hypothesize that oxaliplatin-induced oxidative stress engages a TXNIP-mediated pathway that limits thioredoxin availability, thereby amplifying ROS and inflammatory signaling. Despite studies conducted in cells to demonstrate the ROS production after treatment with oxaliplatin [21], direct evidence of this process on the extracted DRG has not been demonstrated. Addition of oxaliplatin to this media led to the rapid increase of fluorescence from an ROS sensing fluorophore within a few minutes and continued to grow for at least two hours (Figure 3). Predictably, the inhibition of OCT2 by cimetidine prevented oxaliplatin entry into the DRG cells and suppressed ROS production.
The cellular level of ROS in many cell types is highly controlled by a thioredoxin system that removes intracellular ROS [47,48]. This thioredoxin system is composed of thioredoxins TRX1 and TRX2 proteins (collectively, TRX) located in the cytosol and mitochondria [49], thioredoxin interacting protein (TXNIP), and other molecules.
TXNIP is an endogenous inhibitor of thioredoxin. Under normal physiological conditions with low levels of ROS, TXNIP and TRX are bound through the formation of intermolecular disulfide bonds [50], schematically shown in Figure 5. When exposed to ROS, TXNIP detaches from TRX, allowing TRX to perform its antioxidant roles [51].
Under a stronger level of stress, the expression of TXNIP, as we have observed in the DRG of mice treated with oxaliplatin [26], can be upregulated leading to less active TRX activity and an increase of ROS [52]. Consequently, ROS increases in the cytosol and mitochondria, initiating damage to proteins, damage to DNA, and mitochondrial distress.
Given the potential connection between neuropathic pain and oxidative stress/inflammation, we investigated whether inhibiting the TXNIP’s activity with a known TXNIP inhibitor verapamil [53] suppresses the negative role of oxaliplatin on DRG. Our results showed moderate but statistically significant improvement of the neurite outgrowth (Figure 4A) and suppression of the ROS in the DRG (Figure 4B) with even a small level of verapamil (ratio verapamil to oxaliplatin 1:100). This finding is consistent with the recent literature where verapamil was used for other similar pathologies, such as diabetic neuropathy [38,54] and rheumatoid arthritis [55].
The presented ex vivo DRG models might overlook certain systemic elements, such as sex-dependent effects, circulatory hormones, or immunological interactions, that could be substantial factors in CIPN manifestations [56]. Despite the convenience and speed of the DRG model, we will ultimately need to proceed to animal studies, including studies in both male and female mice.

5. Conclusions

Our study established the DRG explant model as a reliable method for studying oxidative stress in CIPN. Using quantitative imaging of the DRG regrowth and oxidative stress levels, we demonstrated that oxaliplatin induces dose-dependent suppression of neurite regrowth and activation of ROS in intact DRG tissue.
Inhibition of oxaliplatin uptake via OCT2 transporter fully restored neurite outgrowth and prevented ROS accumulation, validating the assay. Furthermore, pharmacological inhibition of TXNIP with verapamil reduced oxidative stress and partially rescued neurite outgrowth supporting a TXNIP-based hypothesis of redox dysregulation.
Compared with traditional dissociated neuronal cultures and in vivo animal models, the DRG explant system preserves native cellular architecture and multicellular interactions at the same time allowing exact control over the drug exposure. Given that a mouse harbors over 31 DRG pairs, the model is particularly suitable for higher-throughput drug screenings for CIPN (compared with live mice), expediting research while reducing the use of live animals. This combination of factors makes the model particularly suitable for mechanistic studies and screening of candidate compounds targeting CIPN.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antiox15020230/s1: Figure S1. Image of the regrown DRG under bright field mode. Due to low contrast between the background and small fibers, neurites are difficult to quantify; Figure S2. Dose-response of verapamil on the DRG regrowth. Total number of intersections of DRG explants treated with 100 μM oxaliplatin and different dosages of verapamil. One-way ANOVA with Dunnett’s T3 multiple comparisons test. Data are expressed as the mean ± SD. * p < 0.05.

Author Contributions

Conceptualization, M.Y.B. and J.D.; methodology, J.D. and I.D.L.; data curation, J.D.; writing—original draft preparation, J.D.; writing—review and editing, L.C.S. and M.Y.B.; supervision and funding acquisition, M.Y.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by NIH/NCI R01CA208623 (MB), R21CA269099 (MB), NIH/NINDS R21NS135646 (MB), and NIH/NINDS 1R01NS139461 (MB).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Washington University School of Medicine (protocol 24-0297 and 13 November 2024).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

Imaging results were performed in part using Washington University Center for Cellular Imaging (WUCCI) supported by Washington University School of Medicine, The Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813), and the Foundation for Barnes-Jewish Hospital (3770 and 4642). Imaging results were also obtained in the Optical Spectroscopy Core (Washington University).

Conflicts of Interest

M.B. is the founder and owner of the company HSpeQ LLC that licensed IDCubePro software from Washington University and a consultant for Daxor Inc and Sarya LLC. These companies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results and did not contribute financially to this work.

Abbreviations

The following abbreviations are used in this manuscript (in alphabetical order):
ANOVAAnalysis of variance
BSABovine serum albumin
CIPNChemotherapy-induced peripheral neuropathy
CM-H2DCFDA5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate
DAPI4′,6-diamidino-2-phenylindole
DMEMDulbecco’s Modified Eagle Medium
DRGDorsal root ganglia
DMSODimethyl sulfoxide
ECMExtracellular matrix
EC50Half-maximal effective concentration
HBSSHanks’ Balanced Salt Solution
NBFNeutral buffered formalin
NLRP3NOD-Like Receptor Family Pyrin Domain Containing 3
OCT2Organic Cation Transporter 2
PBSTPhosphate-buffered saline with Triton-X
PNSPeripheral nervous system
ROIRegion of interest
ROSReactive oxygen species
SFMSerum-free medium
SGCSatellite glial cell
TRXThioredoxin
TRX1Thioredoxin-1
TXNIPThioredoxin interacting protein

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Figure 1. Oxaliplatin-induced dose-dependent reduction in neurite outgrowth in DRG explants. (A,B) Neurite outgrowth was quantified with Sholl analysis. Representative images of the Sholl mask (A) and the Sholl profile, The color in the Sholl Mask is generated by the algorithm to represent the complexity of neurites. Warm color (red, orange, yellow) indicates a higher number of intersections, and cooler colors (green, blue) indicate lower intersections. (B) displayed the counts of intersections per sampling shell against the distance from the center (generated in ImageJ). The neurite-occupied area can be represented by the total number of intersections. (C) β-III tubulin staining of DRG explant after 72 h treatment with 0, 10, 50, and 100 μM oxaliplatin. (D) Oxaliplatin suppressed neurite outgrowth in a dose-dependent manner. Statistical significance was detected at concentrations of 50 and 100 μM oxaliplatin. One-way ANOVA with Dunnett’s multiple comparisons test. *** p < 0.001, **** p < 0.0001. (E) β-III tubulin staining of DRG after 0 and 100 μM oxalic acid treatment. (F) Oxalic acid did not affect the neurite outgrowth of DRG explants. Student t-test. Scale bar: 200 μm. Zeiss Cell Discover 7, obj 5×, stitched images. Data are presented as mean ± SD.
Figure 1. Oxaliplatin-induced dose-dependent reduction in neurite outgrowth in DRG explants. (A,B) Neurite outgrowth was quantified with Sholl analysis. Representative images of the Sholl mask (A) and the Sholl profile, The color in the Sholl Mask is generated by the algorithm to represent the complexity of neurites. Warm color (red, orange, yellow) indicates a higher number of intersections, and cooler colors (green, blue) indicate lower intersections. (B) displayed the counts of intersections per sampling shell against the distance from the center (generated in ImageJ). The neurite-occupied area can be represented by the total number of intersections. (C) β-III tubulin staining of DRG explant after 72 h treatment with 0, 10, 50, and 100 μM oxaliplatin. (D) Oxaliplatin suppressed neurite outgrowth in a dose-dependent manner. Statistical significance was detected at concentrations of 50 and 100 μM oxaliplatin. One-way ANOVA with Dunnett’s multiple comparisons test. *** p < 0.001, **** p < 0.0001. (E) β-III tubulin staining of DRG after 0 and 100 μM oxalic acid treatment. (F) Oxalic acid did not affect the neurite outgrowth of DRG explants. Student t-test. Scale bar: 200 μm. Zeiss Cell Discover 7, obj 5×, stitched images. Data are presented as mean ± SD.
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Figure 2. Inhibition of OCT2 preserves neurite outgrowth under oxaliplatin treatment. (A) β-III tubulin staining of DRG explants under DMSO (control) and 100 μM oxaliplatin treatment for 72 h. (B) The 1000 μM cimetidine led to significant improvement of neurite outgrowth under oxaliplatin. There was no significance between DRGs with no drug treatment and those treated with 100 μM oxaliplatin in presence of 1000 μM cimetidine. One-way ANOVA with Dunnett’s T3 multiple comparisons test. **** p < 0.0001. (C) β-III tubulin staining of DRGs co-cultured with 100 μM oxaliplatin and varying concentrations of cimetidine: 1 μM, 10 μM, 100 μM, and 1000 μM. (D) Cimetidine improved neurite outgrowth in a dose-dependent pattern. The estimated concentration associated with 50% neurite regrowth (EC50) is ~159.6 μM. Scale bar: 200 μm. Zeiss Cell Discover 7, obj 5×, 16 stitched images. Data are presented in mean ± SD. Cimetidine: 1 μM, 10 μM, 100 μM, and 1000 μM.
Figure 2. Inhibition of OCT2 preserves neurite outgrowth under oxaliplatin treatment. (A) β-III tubulin staining of DRG explants under DMSO (control) and 100 μM oxaliplatin treatment for 72 h. (B) The 1000 μM cimetidine led to significant improvement of neurite outgrowth under oxaliplatin. There was no significance between DRGs with no drug treatment and those treated with 100 μM oxaliplatin in presence of 1000 μM cimetidine. One-way ANOVA with Dunnett’s T3 multiple comparisons test. **** p < 0.0001. (C) β-III tubulin staining of DRGs co-cultured with 100 μM oxaliplatin and varying concentrations of cimetidine: 1 μM, 10 μM, 100 μM, and 1000 μM. (D) Cimetidine improved neurite outgrowth in a dose-dependent pattern. The estimated concentration associated with 50% neurite regrowth (EC50) is ~159.6 μM. Scale bar: 200 μm. Zeiss Cell Discover 7, obj 5×, 16 stitched images. Data are presented in mean ± SD. Cimetidine: 1 μM, 10 μM, 100 μM, and 1000 μM.
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Figure 3. Oxaliplatin led to prolonged increasing of oxidative stress in DRG. (A) Representative CM-H2DCFDA images of DRG explants treated with DMSO (control), oxaliplatin, and cimetidine (1000 μM) with oxaliplatin (100 μM) at two timepoints: immediately after drug loading (before, time zero) and 120 min after. (B) Quantification of CM-H2DCFDA fluorescence signal revealed oxaliplatin induced increased oxidative stress throughout the experimental period. Inhibition of OCT2 by cimetidine prevented the burst of ROS generation. Data from the DRG ROI were divided by the intensity of the solution ROI and then normalized to the time point 0. Scale bar: 200 μm. Data are presented as mean ± SD. N = 3. Zeiss Cell Discover 7, obj 5×.
Figure 3. Oxaliplatin led to prolonged increasing of oxidative stress in DRG. (A) Representative CM-H2DCFDA images of DRG explants treated with DMSO (control), oxaliplatin, and cimetidine (1000 μM) with oxaliplatin (100 μM) at two timepoints: immediately after drug loading (before, time zero) and 120 min after. (B) Quantification of CM-H2DCFDA fluorescence signal revealed oxaliplatin induced increased oxidative stress throughout the experimental period. Inhibition of OCT2 by cimetidine prevented the burst of ROS generation. Data from the DRG ROI were divided by the intensity of the solution ROI and then normalized to the time point 0. Scale bar: 200 μm. Data are presented as mean ± SD. N = 3. Zeiss Cell Discover 7, obj 5×.
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Figure 4. Inhibition of TXNIP through verapamil partially restored neurite outgrowth and decreased ROS level induced by oxaliplatin. (A) Representative images and quantification of the total number of intersections of DRG under treatment of vehicle (control), 100 μM oxaliplatin (OXA), and 100 μM oxaliplatin combined with 1 μM verapamil (OXA + VER). (B) Quantification of the total number of intersections of DRG under the above treatments. Verapamil resulted in an approximately 70% enhancement in neurite outgrowth compared with the oxaliplatin-only DRGs. One-way ANOVA with Dunnett’s T3 multiple comparisons test. ** p < 0.01, *** p < 0.001. (C) Representative images of 2 h short-term ROS detection for DRG explants treated with DMSO (control), oxaliplatin (100 μM), and oxaliplatin (100 μM) combined with verapamil (1 μM) at two timepoints: right after drug loading (time zero) and 120 min. The ROS level in the DRG was determined by dividing the fluorescence intensity within the DRG ROI by that of the ROI outside the DRG. This value was then normalized to the baseline (time 0). (D) Quantification of fluorescence signal throughout the experimental period. Verapamil prevented ROS burst in DRG under oxaliplatin treatment. N = 3–4. CellRox Green was used for ROS detection. Scale bar: 100 μm. Data are expressed as the mean ± SD.
Figure 4. Inhibition of TXNIP through verapamil partially restored neurite outgrowth and decreased ROS level induced by oxaliplatin. (A) Representative images and quantification of the total number of intersections of DRG under treatment of vehicle (control), 100 μM oxaliplatin (OXA), and 100 μM oxaliplatin combined with 1 μM verapamil (OXA + VER). (B) Quantification of the total number of intersections of DRG under the above treatments. Verapamil resulted in an approximately 70% enhancement in neurite outgrowth compared with the oxaliplatin-only DRGs. One-way ANOVA with Dunnett’s T3 multiple comparisons test. ** p < 0.01, *** p < 0.001. (C) Representative images of 2 h short-term ROS detection for DRG explants treated with DMSO (control), oxaliplatin (100 μM), and oxaliplatin (100 μM) combined with verapamil (1 μM) at two timepoints: right after drug loading (time zero) and 120 min. The ROS level in the DRG was determined by dividing the fluorescence intensity within the DRG ROI by that of the ROI outside the DRG. This value was then normalized to the baseline (time 0). (D) Quantification of fluorescence signal throughout the experimental period. Verapamil prevented ROS burst in DRG under oxaliplatin treatment. N = 3–4. CellRox Green was used for ROS detection. Scale bar: 100 μm. Data are expressed as the mean ± SD.
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Figure 5. Suggested role of TXNIP in regulating ROS and inflammatory burden in DRG cells. Under normal conditions, cytosolic TRX1 and mitochondrial TRX2 suppress ROS via either direct reaction with ROS or by reducing oxidized proteins. This process is dynamically controlled by TXNIP in both cytosol and mitochondria. At higher level of stress, TXNIP is overexpressed, and TRX become less available through direct interaction between TXNIP and TRX blocking. Overproduced TXNIP shuttles to the cytosol and mitochondria where it binds TRX1 and TRX2, respectively, inhibiting the ROS elimination process. Consequently, ROS levels increase, initiating mitochondrial distress. In addition, overexpressed TXNIP binds NLRP3 and triggers the formation of inflammasome NLRP3 leading to inflammatory response and neuropathic pain. (Created in BioRender. Berezin, M. (2026) https://BioRender.com/m6xvv1p, accessed on 28 January 2026).
Figure 5. Suggested role of TXNIP in regulating ROS and inflammatory burden in DRG cells. Under normal conditions, cytosolic TRX1 and mitochondrial TRX2 suppress ROS via either direct reaction with ROS or by reducing oxidized proteins. This process is dynamically controlled by TXNIP in both cytosol and mitochondria. At higher level of stress, TXNIP is overexpressed, and TRX become less available through direct interaction between TXNIP and TRX blocking. Overproduced TXNIP shuttles to the cytosol and mitochondria where it binds TRX1 and TRX2, respectively, inhibiting the ROS elimination process. Consequently, ROS levels increase, initiating mitochondrial distress. In addition, overexpressed TXNIP binds NLRP3 and triggers the formation of inflammasome NLRP3 leading to inflammatory response and neuropathic pain. (Created in BioRender. Berezin, M. (2026) https://BioRender.com/m6xvv1p, accessed on 28 January 2026).
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Du, J.; Sudlow, L.C.; Luzhansky, I.D.; Berezin, M.Y. DRG Explant Model for Understanding Mechanism of Oxaliplatin-Induced Peripheral Neuropathy and Identifying Potential Therapeutic Targets. Antioxidants 2026, 15, 230. https://doi.org/10.3390/antiox15020230

AMA Style

Du J, Sudlow LC, Luzhansky ID, Berezin MY. DRG Explant Model for Understanding Mechanism of Oxaliplatin-Induced Peripheral Neuropathy and Identifying Potential Therapeutic Targets. Antioxidants. 2026; 15(2):230. https://doi.org/10.3390/antiox15020230

Chicago/Turabian Style

Du, Junwei, Leland C. Sudlow, Igor D. Luzhansky, and Mikhail Y. Berezin. 2026. "DRG Explant Model for Understanding Mechanism of Oxaliplatin-Induced Peripheral Neuropathy and Identifying Potential Therapeutic Targets" Antioxidants 15, no. 2: 230. https://doi.org/10.3390/antiox15020230

APA Style

Du, J., Sudlow, L. C., Luzhansky, I. D., & Berezin, M. Y. (2026). DRG Explant Model for Understanding Mechanism of Oxaliplatin-Induced Peripheral Neuropathy and Identifying Potential Therapeutic Targets. Antioxidants, 15(2), 230. https://doi.org/10.3390/antiox15020230

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