1. Introduction
Bortezomib is a first-line chemotherapy drug used mainly for the treatment of multiple myeloma. Approximately 30% to 70% of patients with cancer who receive bortezomib (BTZ) develop bortezomib-induced peripheral neuropathy (BIPN). 1 , 2 , 40 , 46 , 47 Bortezomib-induced peripheral neuropathy symptoms include pain, numbness, paresthesia, and altered thermal sensitivity in a “stocking and glove distribution.” 40 , 46 , 47 Assessments of epidermal innervation suggest that patients with BIPN have reduced intraepidermal nerve fiber density (IENFD), consistent with a small-fiber neuropathy. 4 , 19 Currently, there are no successful treatment options for BIPN. However, duloxetine, gabapentin, tricyclic antidepressants, and topical creams are commonly prescribed to try to reduce symptomatic neuropathic pain. 47 The consequences of BIPN often lead to dose reduction or cessation of BTZ treatment, which both negatively affect patient quality of life and survival rates. 46 , 47 Although BIPN symptoms may resolve over time, symptoms can also be chronic and often irreversible. 8 , 47
Bortezomib is known to be mitotoxic and can increase glycolysis in DRG neurons, both of which contribute to the pathophysiology of BIPN. 34 , 47 , 48 The maintenance of neuronal metabolism and bioenergetics represents a finely regulated balance, where dysregulation can have detrimental effects on cellular processes. 18 , 20 Various studies have shown that abnormal glycolysis plays a role in the development of pain, 12 , 28 , 31 , 34 , 44 while glycolytic inhibition alleviates pain. 23 , 32 This highlights the role of metabolism and neuronal bioenergetics in pain, specifically in relation to glycolytic activity and signaling. Currently, there is little research regarding the effects of neuronal bioenergetics on axon degeneration and IENF density in peripheral neuropathy models.
Interest has grown in the role of ketones in pain modulation. 22 Circulating ketones are elevated when consuming a ketogenic diet (KD), and evidence has revealed that ketones alter neuronal metabolism by decreasing reactive oxygen species production, increasing oxidative phosphorylation, and modulating nicotinamide adenine dinucleotide levels. 9 , 15 , 16 , 36 A KD has numerous benefits for various neurological conditions and neurodegenerative diseases, 13 , 21 , 24 , 26 , 27 , 30 , 41 , 43 leading us to test whether a KD can be used as an adjunct therapy in BIPN. Previous work from our laboratory has shown that a KD can prevent and rescue fiber loss and mechanical allodynia in several mouse models of metabolic-driven peripheral neuropathy (DPN). 17 We believe that the benefits of a KD can be extended beyond DPN also to benefit BIPN. Our study aimed to explore the interplay among BTZ, ketones, and neuronal metabolism in peripheral neuropathy. Our data suggest that in mice, KD consumption prevents or normalizes key changes in behavior and sensory neuron metabolism induced by BTZ. These results extend the range of benefits for treating neuropathy and may provide new avenues for treating BIPN that involve neuronal bioenergetics.
2. Materials and methods
2.1. Animals and diets
All animal work was conducted following review and approval by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center. Eight-week-old C57/Bl6 #027 male mice were purchased from Charles River Laboratories and maintained on a 14:10 light:dark cycle in the animal research facility at the University of Kansas Medical Center. Mice were given ad libitum access to water and either a control rodent chow (TD.8604, Envigo, Madison, WI; 14% fat, 32% protein, and 54% carbohydrate by kcal) or a ketogenic diet (TD.96355; Envigo, 90.5% fat, 9.2% protein, and 0.3% carbohydrate by kcal). Mice fed a ketogenic diet were given fresh ketogenic food every 3 to 4 days. After baseline behavior measures, mice were randomized into 1 of 4 groups: bortezomib or vehicle-treated, and receiving either a standard chow diet or a ketogenic diet. The diet was initiated at least 7 hours before the first injection. Mice were given an intraperitoneal injection of either BTZ (0.2 mg/kg) or vehicle for 5 consecutive days. Afterward, the mice were monitored daily for 5 days during a washout period.
2.2. Drug administration
For in vivo studies, bortezomib (Tocris, Catalog No. 7282) was dissolved in 0.5% DMSO (Fisher Scientific), then diluted in 0.9% saline (Hospira, Lake Forest, IL), and administered through intraperitoneal (i.p.) injection using a 1-mL insulin syringe with a 28 G needle (BD; Catalog No. 329420). Vehicle solution was 0.9% saline (Hospira; Lake Forest, IL) with 0.5% DMSO. Mice received an i.p. injection per day of either vehicle or BTZ (0.2 mg/kg) for 5 consecutive days. For in vitro studies, BTZ (100 nM) was dissolved in 0.5% DMSO, then diluted into DMEM (Gibco; Catalog No.11885-084).
2.3. Blood measurements
Blood ketone levels were measured once using a hand-held monitor and B-hydroxybutyrate blood ketone strips (β-Ketone blood test strips, Abbott Laboratories, Chicago, IL; Precision Xtra, Abbott Laboratories). Animals were not fasted before blood ketone measurements.
2.4. Mechanical sensitivity (monofilament) testing
Mice were placed in 11 cm diameter × 19 cm height cylindrical plexiglass containers on top of a 9.4 × 10-cm wire platform and allowed to acclimate to the testing environment for 30 minutes. Evoked mechanical sensitivity was assessed using nylon monofilaments of graded bending forces applied using the up-down method to the plantar surface of each hind paw. Testing was conducted in duplicate, with a minimum of 5 minutes between tests, to assess the average force that evokes a withdrawal response.
2.5. Cold sensitivity (acetone evaporation) testing
Fifteen minutes after mechanical sensitivity testing, cold sensitivity was assessed using acetone (Fisher Scientific). In brief, a droplet of acetone was placed on the plantar surface of 1 hind paw using a 1-mL insulin syringe with a 28 G needle (BD; Catalog No. 329420). Training was conducted before experiments to ensure that the acetone droplet could be placed on the footpad without touching the needle to the hind paw. The number of behavioral responses to the acetone-treated foot was recorded for 1 minute. Responses included paw shaking, licking, biting, favoring, or attending. Assessment of sensitivity was performed over the course of 2 trials on each hind paw. Response numbers were averaged for each paw, statistically compared to ensure no difference between paws, and averaged for final analysis and presentation.
2.6. Intraepidermal nerve fiber density
Footpads were collected from all mice and postfixed in Zamboni fixative for 18 hours before incubation in 30% sucrose overnight. Footpads were cryopreserved in Optimal Cutting Temperature Compound (Sakura Tissue-Tek) and cut into 30-μm sections. Footpad sections were blocked for 4 hours in Superblock (ThermoFisher, Grand Island, NY), 1.5% Normal Donkey Serum, 0.5% Triton X-100 (Sigma), and 0.5% Porcine Gelatin at room temperature. Slides were incubated overnight with rabbit a-PGP9.5 (1:1000, UCHL1; ProteinTech; Rosemont, IL). Slides were incubated for 1 hour with 555-tagged donkey-a-rabbit secondary antibody (1:1000; Alexa Fluor). Stained slides were imaged using a Nikon Eclipse 90i microscope with a ×20 objective. Three images were captured per tissue section, for a total of 9 images per slide. Nikon Elements was used to measure the length of the dermis–epidermis junction. Intraepidermal nerve fiber density was quantified as the number of fibers crossing the junction per millimeter. The average IENFD for each mouse was used for statistical analyses.
2.7. Neurite outgrowth
Methods were based on Malin et al. 38 and modified as follows. Lumbar DRG neurons were harvested and dissociated into a single cell suspension. Cells were plated using the drop-plate method, in which a 40-μL drop is placed in the center of each well. Cells were incubated at room temperature for 1 to 2 hours and then fed with DMEM (Gibco; Catalog No. 11885-084) before being placed in a 37°C incubator. For in vivo studies, neurons were grown for 4 days and then fixed with 4% paraformaldehyde for 15 minutes. For in vitro studies, depending on the group, neurons received ketones (3 mM beta-hydroxybutyrate; Sigma-Aldrich and 3 mM acetoacetate; Sigma-Aldrich) 48 hours postplating and BTZ 96 hours postplating. Cells were fixed 144 hours postplating with 4% paraformaldehyde for 15 minutes. Immunocytochemistry was performed using Beta-III tubulin (1:1000; TUJ1; R&D Systems), Hoechst (1:2000; Invitrogen; Eugene, OR), and 488-tagged donkey-a-mouse secondary (1:2000, Alexa Fluor) stains. Plates were imaged, with 5 to 6 photographs per well and 3 to 4 wells per animal. Neurite outgrowth was quantified using ImageJ. A grid was superimposed on the well images. The number of somas producing neurites was counted, along with the number of square neurons (attached to a soma) that passed through. The average square-to-soma ratio and the total number of squares were calculated by averaging the results from each well and then averaging the well replicates to obtain a single standard value per animal.
2.8. Extracellular acidification rate and mitochondrial oxygen consumption rate
Lumbar DRG neurons were harvested and dissociated into a single cell suspension. Cells were plated in a poly- d -lysine–coated 96-well plate at 100 μL/well and fed with an additional 100 μL of media (either standard media or ketone media with 3 mM beta-hydroxybutyrate and 3 mm acetoacetate) after 2 hours. The following day, cells were analyzed for extracellular acidification rate and oxygen consumption rate using a Seahorse XFe96 analyzer (Agilent Technologies). Unbuffered DMEM containing 4 mM glutamine, pyruvate, and 5 mM glucose was added to plated cells, and ketones (as described previously) were added to the medium for the respective KD groups. The following substrates were injected in succession to measure ECAR and OCR: (1) 2 µM Oligomycin/Hoechst; (2) 0.5 µM Carbonyl Cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP); (3) 1 µM each Rotenone/Antimycin A; (4) 10 mM 2DG. Cells were counted for Hoechst stain in a Cytation 1 plate reader to normalize measures to cell counts.
2.9. Statistics and data analysis
All statistical analyses were performed using GraphPad Prism 10.5. All data are presented as mean ± standard error of the mean. All data collected over time were analyzed using a 2-way analysis of variance (ANOVA) with repeated measures. All other analyses were performed using a 1-way or 2-way ANOVA, or a Pearson correlation, as appropriate. Data were analyzed post hoc by Tukey Honest Significant Difference or pairwise t test, as indicated. Assumptions of normal distribution and homogeneity of variance were confirmed by the Shapiro–Wilk test.
3. Results
3.1. Mouse responses to bortezomib and ketogenic diet
Ketogenic diet was administered to respective groups on day 1 (Fig. 1 A). Vehicle-treated KD-fed mice did not have significant weight loss over the course of the experiment (Fig. 1 B). The weight of BTZ-treated KD-fed mice was significantly different compared with vehicle-treated chow-fed animals on days 3 to 5, and significantly different from vehicle-treated KD-fed animals on days 4 and 5 (Fig. 1 B, 2-way ANOVA with repeated measures, time: P < 0.0001, group × time: P < 0.0001). However, all mice were still within a healthy weight range for their age, sex, and strain. A blood sample for beta-hydroxybutyrate was collected on day 10 to confirm that KD-fed animals had entered ketosis. KD-fed animals had significant increases in blood ketone levels (Fig. 1 C, 2-way ANOVA, diet: P < 0.0001).
3.2. Ketogenic diet mitigates bortezomib-induced peripheral neuropathy symptomology
Baseline sensory behavioral testing (mechanical sensitivity and cold sensitivity) was measured on day 0 (Fig. 1 A). Bortezomib treatment and KD administration began on day 1. Figure 2 A illustrates mechanical sensitivity testing using von Frey nylon monofilaments. Bortezomib-treated chow-fed mice developed mechanical allodynia by day 3, which continued for the remainder of the experiment (Fig. 2 B; 2-way ANOVA with repeated measures, group: P < 0.0001, group × time: P < 0.0001, time: P < 0.0001). A KD prevented the development of mechanical allodynia in BTZ-treated mice, maintaining mechanical thresholds similar to vehicle-treated, chow-fed controls. Figure 2 C depicts the change from baseline, further highlighting differences in mechanical sensitivity thresholds between groups (2-way ANOVA with repeated measures; group: P = 0.0005).
Assessment of cold sensitivity revealed no difference between the vehicle-treated chow-fed group compared with the vehicle-treated KD-fed group (Fig. 2 D, E, 2-way ANOVA with repeated measures, group: P < 0.0001, group × time: P < 0.0001, time: P < 0.0001). Bortezomib-treated groups had a significant increase in frequency of nociceptive behaviors (biting, licking, and favoring) when compared with vehicle-treated groups on days 3 and 6 (Fig. 2 E, 2-way ANOVA with repeated measures, group: P < 0.0001, group × time: P < 0.0001, time: P < 0.0001). On day 9, BTZ-treated chow-fed mice showed an increase in the frequency of nociceptive responses. They remained significantly different from vehicle-treated groups (Fig. 2 E). There were also fewer responses in the BTZ-treated KD-fed group on day 9 during the washout period and were not different from the vehicle-treated chow group but were different from vehicle-treated KD-fed mice (Fig. 2 E). Figure 2 F depicts the change from baseline, revealing that BTZ-treated chow-fed animals had a significant change from their baseline behaviors, along with BTZ-treated KD-fed animals (2-way ANOVA with repeated measures, group: P < 0.0001, group × time: P < 0.0001, time: P = 0.0082). However, behavioral changes in the BTZ-treated KD-fed mice were normalized by day 9.
3.3. Ketogenic diet protects against the development of bortezomib-induced peripheral neuropathy
Because BIPN causes fiber loss in patients 47 and rodents, 6 we tested whether a KD affects BIPN-induced reductions in IENFD (Fig. 3 A). Bortezomib-treated chow-fed animals had a significant decrease in IENFD when compared with all groups (Fig. 3 B, 2-way ANOVA, diet: P < 0.0001, drug × diet interaction: P < 0.0115). Vehicle-treated KD-fed animals had a significant increase in total square to cell body ratio from the harvested DRG compared with both chow-fed groups (Fig. 3 C, D, 2-way ANOVA, diet: P < 0.0008). Bortezomib-treated KD-fed animals also had a significant increase in total square to cell body ratio when compared with vehicle-treated chow-fed animals. However, when comparing total squares alone, there was no significant difference between groups (Fig. 3 D, 2-way ANOVA, P < 0.05).
We conducted correlations between the levels of IENFD and mechanical sensitivity (Fig. 3 E), and IENFD and cold sensitivity (Fig. 3 F). The results identified a significant correlation between IENFD and mechanical sensitivity (r = 0.4544, P < 0.0037) and cold sensitivity (r = −0.5819, P < 0.0001), suggesting that reduced IENFD correlates with a worse behavioral change. Finally, we tested whether there is a relationship between in vivo blood ketone levels and in vitro DRG neurite outgrowth (Fig. 3 G). Results showed a significant correlation between the level of circulating ketones in vivo and neurite outgrowth of DRG in vitro (r = 0.5987, P < 0.0025). This revealed that ketone levels can influence features of DRG axons, even when cultured in vitro, suggesting that DRG neurons exhibit a metabolic memory in response to elevated ketone levels.
3.4. Bortezomib-induced metabolic alterations countered by ketogenic diet
Previous studies have shown that BTZ increases glycolysis in neurons. 34 We measured the ECAR and OCR of DRG neurons after in vivo administration of drugs and diets. Results showed that baseline ECAR for all groups was not significantly different (Fig. 4 A, 2-way ANOVA with repeated measures, group: P = 0.0085, group × time: P < 0.0001, time: P < 0.0001). However, BTZ-treated chow-fed animals had a significant increase in their DRG neurons' maximum glycolytic capacity (Fig. 4 B, 2-way ANOVA, diet: P = 0.0005, drug: P = 0.0194) and spare glycolytic capacity when compared with all other groups (Fig. 4 B, 2-way ANOVA, diet: P = 0.0002, drug: P = 0.0069). There were no significant differences for baseline mitochondrial respiration during OCR measure (Fig. 4 C, 2-way ANOVA with repeated measures, P < 0.05), but there was a significant difference between BTZ-treated chow-fed animals compared with vehicle-treated chow-fed animals after FCCP injection (Fig. 4 C, 2-way ANOVA with repeated measures, time: P < 0.0001). When looking at specific mitochondrial respiration measures, there were no significant differences between any groups (Fig. 4 D, 2-way ANOVA, P < 0.05).
3.5. Ketones protect against bortezomib-induced neurite degeneration
We conducted a short-term course to identify whether exogenously provided ketones could prevent BTZ-induced neurite degeneration in vitro (Fig. 5 A, B). Results showed that BTZ-treated DRG neurons exhibited a significant decrease in total square to cell body ratio (Fig. 5 C, one-way ANOVA, group: P < 0.0001). In addition, when examining total squares, the ketone-treated group showed a significantly higher value than all other groups, including control cells (Fig. 5 D, one-way ANOVA, group: P < 0.0001). Finally, the BTZ–ketone-treated group was significantly greater than the BTZ-treated group but remained significantly lower than the control and ketone-treated groups (Fig. 5 D; 1-way ANOVA, group: P < 0.0001).
4. Discussion
Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a substantial barrier when treating patients with cancer with chemotherapy drugs, and new treatment avenues are needed to lessen the neuropathy side effects induced by chemotherapy agents. Bortezomib is an excellent example, as the treatment of multiple myeloma using BTZ is strongly affected by BIPN. Here, we demonstrated that KD and ketones can prevent the development of BIPN symptoms, IENF loss, and neurite degeneration in a short-term, rapid BIPN development course. We also confirm that BTZ increases glycolysis, 34 which may be linked to the development of nociception and pain symptomology observed in BIPN. Our data suggest that KD consumption prevents this increase in glycolysis, indicating that this bioenergetic change plays a role in neuronal activation in neuropathy. These bioenergetic corrections induced in sensory neurons by a KD add to the growing understanding that neuronal metabolism plays a central role in neuropathy. Another study conducted in a rat model of paclitaxel-induced peripheral neuropathy showed that PPARγ, which is important for lipid metabolism, is implicated in the mechanism underlying the benefits of ketones. 49 Their work also highlighted that a KD reduced proinflammatory cytokine expression within the TLR4-NFκB pathway and significantly enriched oxidative phosphorylation pathways among differently expressed genes. This study aligns with our view that metabolism plays a key role in the benefits of a KD for neurons, and these results add to the growing body of evidence for the range of neuropathic conditions corrected by a ketogenic diet.
4.1. Ketogenic diet is well-tolerated by animals and induces ketosis
The KD is sustainable and maintainable, but the logistical burden and the diet's palatability can be onerous for some individuals. 3 Our analysis suggests that the mice tolerated the KD, as BTZ-treated KD-fed mice weighed significantly less than vehicle-treated KD-fed mice; all mice remained within a healthy weight range throughout the study. Both KD-fed groups had a significant increase in blood ketone levels, showing that they had entered a state of ketosis.
4.2. Ketogenic diet prevents bortezomib-induced mechanical and cold hypersensitivity
Bortezomib treatment is known to cause nociception in animals 7 , 14 , 45 and painful BIPN in humans. 47 Previous studies have shown that BTZ-treated animals do not typically develop thermal sensitivity but instead develop cold sensitivity, 7 , 14 leading us to measure cold sensitivity as well. Here, BTZ induced mechanical hypersensitivity in chow-fed animals, which persisted throughout the study; however, it did not affect KD-fed mice. Interestingly, BTZ induced cold sensitivity in both chow-fed and KD-fed animals at varying degrees. Ketogenic diet–fed animals did recover from cold sensitivity during the washout period, whereas chow-fed mice's sensitivity continued to climb. It is plausible that KD prevented mechanical allodynia but only mitigated cold sensitivity, given the different fiber types and mechanisms at play. These differences among neuronal phenotypes should be further explored.
4.3. Bortezomib causes fiber loss, but a ketogenic diet protects against fiber loss
It has previously been shown that BTZ reduces IENFD levels. 6 Previous studies in our laboratory have shown that a KD can prevent reductions in IENFD in a mouse model of DPN. Here, our results showed that BTZ induces significant fiber loss within a short period of time, consistent with our previous studies on type 1 diabetes, type 2 diabetes, and high-fat diet-induced IENFD reductions. 10 , 17 Importantly, BTZ-induced fiber loss can be prevented by consuming a KD. In addition, KD-fed animals have a heightened ability to grow neurites when harvested from KD-fed mice and placed in vitro. 10 Although the mechanism is unknown, this strongly suggests that a KD can have lasting effects through improvements in metabolic memory, and experiments need to address the mechanisms underlying metabolic memory and how long they persist. This will be an essential feature for clinical translational use of a KD for CIPN.
4.4. Ketogenic diet inhibits bortezomib-induced metabolic changes within dorsal root ganglia neurons
This study in C57BL/6 mice showed that BTZ administration increased the ECAR in DRG neurons. These results are consistent with a similar study using BTZ to induce pain in ICR mice that first reported glycolytic changes. 34 Our results strongly support this previous finding, showing that BTZ significantly increased the maximum and spare glycolytic capacities in DRG neurons compared with all other groups. This suggests that BTZ may alter the energetic phenotype of neurons toward a glycolytic state, which could contribute to the nociception observed in mice after BTZ administration. There is further evidence suggesting that unregulated glycolysis can lead to aberrant neuronal functioning and “glycolytic pain,” 22 , 28 , 31 and that inhibiting excess glycolysis can reduce various pain phenotypes. 23 , 32 , 39 Our results reveal that the BTZ-induced increase in glycolysis can be prevented by a KD, suggesting that one consequence of a KD is to modulate bioenergetics to prevent increases in glycolysis. This is consistent with the view that ketones can be directly used by neuronal mitochondria, bypassing the need for pyruvate-generated adenyl cyclase from glycolysis. 22 , 33 , 35 Our results did not show changes in OCR in response to ketones or BTZ. However, the inhibition of glycolysis by ketones may be a beneficial mechanism, as acetoacetate has been shown to inhibit glucose uptake and downstream glycolytic events. 37 Moreover, glutamine added to the media can be metabolized by mitochondria, potentially masking differences in OCR. Future studies should test how glutamine in the media affects neuronal bioenergetics. In addition, our cultures were not pure neurons, and future studies should investigate the impact of non-neuronal cells. Finally, we recognize that in vivo elevations in ketones may have led to metabolic adaptations in cells in vitro, thereby affecting the results. Overall, this relationship between glycolysis and mitochondrial oxidative phosphorylation merits further study, as it seems to play a crucial role in sensory neuron function, neuropathy, and pain. 22 , 33 , 35
4.5. Ketones promote growth and prevent bortezomib-induced neurite degeneration
It is known that BTZ causes neurite degeneration in vitro, 42 but there is little research surrounding preventing BTZ-induced neurite degeneration. Previous work from our laboratory has demonstrated that ketones can promote neurite outgrowth in vitro. 10 Here, the addition of ketones in vitro 2 days before BTZ significantly mitigated neurite loss, supporting the effect on IENFD in vivo and suggesting that ketones may protect neurites by altering their bioenergetics.
5. Limitations
We focused on a short time course to identify alterations in sensory behaviors, IENFD, and cellular bioenergetics. This paradigm does not reflect the duration of time patients experience when receiving BTZ. Future studies should expand the time course to identify changes in nociception and cellular bioenergetics, and examine repeated BTZ dosing regimens or exogenous ketones to translate these effects better. In addition, a KD administered to rodents is believed to be protein-deficient, which may result in increased signaling pathways related to protein deficiency that act independently of ketone-mediated effects. 29 Further studies could focus on ketone body metabolism and the impact of inhibition on it. Finally, as a starting point, we only conducted these studies in male mice. There seems to be no sex difference in the response of humans or rodents to BTZ treatment, 5 , 11 and additional studies in female mice may be warranted. Recent work has shown that there are sex differences in KD efficacy for weight loss in humans, concluding that these differences may be attributed to factors such as sex hormones, neurotransmitters, genetics, immunity, and gut microbiota. 25 Finally, to avoid contraindications, patients should consult their doctors before starting a KD to ensure proper education and maintenance.
6. Conclusion
We report that BTZ can induce rapid distal axon degeneration, and that a KD can prevent reductions in IENFD, mechanical allodynia, and a neuronal glycolytic shift in BTZ-treated mice. In addition, ketones can prevent BTZ-induced neurite degeneration in vitro. Our study underscores the benefits of a KD in a BIPN model and highlights bioenergetic changes that may provide benefits in neuropathy. Future studies could focus on a rescue intervention for BIPN with KD, or on a more intensive prevention paradigm in which KD is administered earlier. Finally, further exploration regarding the metabolic alterations occurring with DRG neurons as a result of drug and diet administration should be pursued.
Disclosures
The authors have no conflicts of interest to declare.
Acknowledgements
Supported by the National Institutes of Health R01NS043314 (DEW), R01NS144393 (DEW), and the Kansas INBRE P20GM1033418 (DEW). Data Availability: All experimental data generated or analyzed in this study are included in the article and are available from the corresponding author upon request. No data were generated through publicly accessible sources. Figures were created with a licensed version of BioRender.com .
Footnotes
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Contributor Information
Lana L. Heslop, Email: lheslop2@kumc.edu.
Trent K. Madden, Email: tmadden3@kumc.edu.
Gentry Totta-Griese, Email: ggriese@kumc.edu.
Sarah J. Crowards, Email: scrowards@kumc.edu.
Will Hauser, Email: whauser2@kumc.edu.
Janelle Ryals, Email: jryals@kumc.edu.
Kyle M. Baumbauer, Email: kbaumbauer@kumc.edu.
Heather M. Wilkins, Email: hwilkins@kumc.edu.
References
[1] Argyriou AA, Iconomou G, Kalofonos HP. Bortezomib-induced peripheral neuropathy in multiple myeloma: a comprehensive review of the literature. Blood 2008;112:1593–9.
[2] Badros A, Goloubeva O, Dalal JS, Can I, Thompson J, Rapoport AP, Heyman M, Akpek G, Fenton RG. Neurotoxicity of bortezomib therapy in multiple myeloma: a single-center experience and review of the literature. Cancer 2007;110:1042–9.
[3] Batch JT, Lamsal SP, Adkins M, Sultan S, Ramirez MN. Advantages and disadvantages of the ketogenic diet: a review article. Cureus 2020;12:e9639.
[4] Boyette-Davis JA, Cata JP, Zhang H, Driver LC, Wendelschafer-Crabb G, Kennedy WR, Dougherty PM. Follow-up psychophysical studies in bortezomib-related chemoneuropathy patients. J Pain 2011;12:1017–24.
[5] Bruna J, Alberti P, Calls-Cobos A, Caillaud M, Damaj MI, Navarro X. Methods for in vivo studies in rodents of chemotherapy induced peripheral neuropathy. Exp Neurol 2020;325:113154.
[6] Bruna J, Udina E, Ale A, Vilches JJ, Vynckier A, Monbaliu J, Silverman L, Navarro X. Neurophysiological, histological and immunohistochemical characterization of bortezomib-induced neuropathy in mice. Exp Neurol 2010;223:599–608.
[7] Carozzi VA, Renn CL, Bardini M, Fazio G, Chiorazzi A, Meregalli C, Oggioni N, Shanks K, Quartu M, Serra MP, Sala B, Cavaletti G, Dorsey SG. Bortezomib-induced painful peripheral neuropathy: an electrophysiological, behavioral, morphological and mechanistic study in the mouse. PLoS One 2013;8:e72995.
[8] Cavaletti G, Jakubowiak AJ. Peripheral neuropathy during bortezomib treatment of multiple myeloma: a review of recent studies. Leuk Lymphoma 2010;51:1178–87.
[9] Cooper MA, McCoin C, Pei D, Thyfault JP, Koestler D, Wright DE. Reduced mitochondrial reactive oxygen species production in peripheral nerves of mice fed a ketogenic diet. Exp Physiol 2018;103:1206–12.
[10] Cooper MA, Menta BW, Perez-Sanchez C, Jack MM, Khan ZW, Ryals JM, Winter M, Wright DE. A ketogenic diet reduces metabolic syndrome-induced allodynia and promotes peripheral nerve growth in mice. Exp Neurol 2018;306:149–57.
[11] Corso A, Mangiacavalli S, Varettoni M, Pascutto C, Zappasodi P, Lazzarino M. Bortezomib-induced peripheral neuropathy in multiple myeloma: a comparison between previously treated and untreated patients. Leuk Res 2010;34:471–4.
[12] Deng YT, Ma L, Mei Y, Wang JS, Bai XH, Zheng XJ, Ren JX, Zhong D, Zhou BL, Dan J, Li X, Gao YJ, Yu L, Yan M, Jiang BC. Amphiregulin contributes to neuropathic pain by enhancing glycolysis that stimulates histone lactylation in sensory neurons. Sci Signal 2025;18:eadr9397.
[13] Dowis K, Banga S. The potential health benefits of the ketogenic diet: a narrative review. Nutrients 2021;13:1654.
[14] Duggett NA, Flatters SJL. Characterization of a rat model of bortezomib‐induced painful neuropathy. Br J Pharmacol 2017;174:4812–25.
[15] Elamin M, Ruskin DN, Masino SA, Sacchetti P. Ketogenic diet modulates NAD(+)-dependent enzymes and reduces DNA damage in hippocampus. Front Cell Neurosci 2018;12:263.
[16] Elamin M, Ruskin DN, Sacchetti P, Masino SA. A unifying mechanism of ketogenic diet action: the multiple roles of nicotinamide adenine dinucleotide. Epilepsy Res 2020;167:106469.
[17] Enders J, Swanson MT, Ryals J, Wright DE. A ketogenic diet reduces mechanical allodynia and improves epidermal innervation in diabetic mice. PAIN 2022;163:682–9.
[18] Garcia-Garcia FJ, Monistrol-Mula A, Cardellach F, Garrabou G. Nutrition, bioenergetics, and metabolic syndrome. Nutrients 2020;12:2785.
[19] Giannoccaro MP, Donadio V, Gomis Perez C, Borsini W, Di Stasi V, Liguori R. Somatic and autonomic small fiber neuropathy induced by bortezomib therapy: an immunofluorescence study. Neurol Sci 2011;32:361–3.
[20] Hees JT, Harbauer AB. Metabolic regulation of mitochondrial protein biogenesis from a neuronal perspective. Biomolecules 2022;12:1595.
[21] Henderson ST, Vogel JL, Barr LJ, Garvin F, Jones JJ, Costantini LC. Study of the ketogenic agent AC-1202 in mild to moderate alzheimer's disease: a randomized, double-blind, placebo-controlled, multicenter trial. Nutr Metab (Lond) 2009;6:31.
[22] Heslop LL, Wright DE. Modulation of pain by ketones: a mini-review. Am J Physiol Cell Physiol 2025;329:C31–C37.
[23] Hua T, Kong E, Zhang H, Lu J, Huang K, Ding R, Wang H, Li J, Han C, Yuan H. PRMT6 deficiency or inhibition alleviates neuropathic pain by decreasing glycolysis and inflammation in microglia. Brain Behav Immun 2024;118:101–14.
[24] Jiang Z, Yin X, Wang M, Chen T, Wang Y, Gao Z, Wang Z. Effects of ketogenic diet on neuroinflammation in neurodegenerative diseases. Aging Dis 2022;13:1146–65.
[25] Jiao Y, Chen X, Liu L, Lu Y, Gao M, Wang Q, Chi X, Mo Y. Sex differences in ketogenic diet: are men more likely than women to lose weight? Front Nutr 2025;12:1600927.
[26] Keith L, Seo CA, Rowsemitt C, Pfeffer M, Wahi M, Staggs M, Dudek J, Gower B, Carmody M. Ketogenic diet as a potential intervention for lipedema. Med Hypotheses 2021;146:110435.
[27] Ko A, Kwon HE, Kim HD. Updates on the ketogenic diet therapy for pediatric epilepsy. Biomed J 2022;45:19–26.
[28] Kong E, Li Y, Ma P, Zhang Y, Ding R, Hua T, Yang M, Yuan H. Lyn-mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn. J Cell Mol Med 2023;27:1664–81.
[29] Laeger T, Henagan TM, Albarado DC, Redman LM, Bray GA, Noland RC, Munzberg H, Hutson SM, Gettys TW, Schwartz MW, Morrison CD. FGF21 is an endocrine signal of protein restriction. J Clin Invest 2014;124:3913–22.
[30] Li Q, Liang J, Fu N, Han Y, Qin J. A ketogenic diet and the treatment of autism spectrum disorder. Front Pediatr 2021;9:650624.
[31] Li Y, Kong E, Ding R, Chu R, Lu J, Deng M, Hua T, Yang M, Wang H, Chen D, Song H, Wei H, Zhang P, Han C, Yuan H. Hyperglycemia-induced Sirt3 downregulation increases microglial aerobic glycolysis and inflammation in diabetic neuropathic pain pathogenesis. CNS Neurosci Ther 2024;30:e14913.
[32] Liu P, Chen T, Tan F, Tian J, Zheng L, Deng Y, Chen J, Chi X. Dexmedetomidine alleviated neuropathic pain in dorsal root ganglion neurons by inhibition of anaerobic glycolysis activity and enhancement of ROS tolerance. Biosci Rep 2020;40:BSR20191994.
[33] Llorente-Folch I, Dussmann H, Watters O, Connolly NMC, Prehn JHM. Ketone body beta-hydroxybutyrate (BHB) preserves mitochondrial bioenergetics. Sci Rep 2023;13:19664.
[34] Ludman T, Melemedjian OK. Bortezomib-induced aerobic glycolysis contributes to chemotherapy-induced painful peripheral neuropathy. Mol Pain 2019;15:1744806919837429.
[35] Lutas A, Yellen G. The ketogenic diet: metabolic influences on brain excitability and epilepsy. Trends Neurosciences 2013;36:32–40.
[36] Maalouf M, Sullivan PG, Davis L, Kim DY, Rho JM. Ketones inhibit mitochondrial production of reactive oxygen species production following glutamate excitotoxicity by increasing NADH oxidation. Neuroscience 2007;145:256–64.
[37] Maizels EZ, Ruderman NB, Goodman MN, Lau D. Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat. Biochem J 1977;162:557–68.
[38] Malin SA, Davis BM, Molliver DC. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc 2007;2:152–60.
[39] Masino SA, Ruskin DN. Ketogenic diets and pain. J Child Neurol 2013;28:993–1001.
[40] Peng L, Ye X, Zhou Y, Zhang J, Zhao Q. Meta-analysis of incidence and risk of peripheral neuropathy associated with intravenous bortezomib. Support Care Cancer 2015;23:2813–24.
[41] Pietrzak D, Kasperek K, Rekawek P, Piatkowska-Chmiel I. The therapeutic role of ketogenic diet in neurological disorders. Nutrients 2022;14:1952.
[42] Sprague JM, Yekkirala AS, Singh B, Tochitsky I, Stephens M, Viramontes O, Ivanis J, Biscola NP, Havton LA, Woolf CJ, Latremoliere A. Bortezomib-induced neuropathy is in part mediated by the sensitization of TRPV1 channels. Commun Biol 2023;6:1228.
[43] Zhang H, Tao Y, Leng SX. Ketogenic diet: an effective treatment approach for neurodegenerative diseases. Curr Neuropharmacol 2022;20:2303–19.
[44] Xie H, Li J, Lian N, Xie M, Wu M, Tang K, Kang Y, Lu P, Li T. Defective branched-chain amino acid catabolism in dorsal root ganglia contributes to mechanical pain. EMBO Rep 2023;24:e56958.
[45] Yamamoto S, Kawashiri T, Higuchi H, Tsutsumi K, Ushio S, Kaname T, Shirahama M, Egashira N. Behavioral and pharmacological characteristics of bortezomib-induced peripheral neuropathy in rats. J Pharmacol Sci 2015;129:43–50.
[46] Yan W, Wu Z, Zhang Y, Hong D, Dong X, Liu L, Rao Y, Huang L, Zhang X, Wu J. The molecular and cellular insight into the toxicology of bortezomib-induced peripheral neuropathy. Biomed Pharmacother 2021;142:112068.
[47] Yang Y, Zhao B, Lan H, Sun J, Wei G. Bortezomib-induced peripheral neuropathy: clinical features, molecular basis, and therapeutic approach. Crit Rev Oncol Hematol 2024;197:104353.
[48] Zheng H, Xiao WH, Bennett GJ. Mitotoxicity and bortezomib-induced chronic painful peripheral neuropathy. Exp Neurol 2012;238:225–34.
[49] Zhong S, Zhou Z, Lin X, Liu F, Liu C, Liu Z, Deng W, Zhang X, Chang H, Zhao C. Ketogenic diet prevents paclitaxel-induced neuropathic nociception through activation of PPARgamma signalling pathway and inhibition of neuroinflammation in rat dorsal root ganglion. Eur J Neurosci 2021;54:5341–56.