Ketogenic Diet Alleviates Mechanical Allodynia in the Models of Inflammatory and Neuropathic Pain in Male Mice

Abstract

Inflammation is involved in the induction of chronic inflammatory and neuropathic pain. Moreover, the ketogenic diet, a high-fat, low-carbohydrate, and adequate protein diet, has an anti-inflammatory effect. Thus, we hypothesized that a ketogenic diet has a therapeutic effect on both types of chronic pain. In the present study, we investigated the effect of a ketogenic diet on mechanical allodynia, a chronic pain symptom, in formalin-induced chronic inflammatory pain and nerve injury-induced neuropathic pain models using adult male mice. Formalin injection into the hind paw induced mechanical allodynia in both the injected and intact hind paws, and the ketogenic diet alleviated mechanical allodynia in both hind paws. In addition, the ketogenic diet prevented formalin-induced edema. Furthermore, the diet alleviated mechanical allodynia induced by peripheral nerve injury. Thus, these findings indicate that a ketogenic diet has a therapeutic effect on chronic pain induced by inflammation and nerve injury.

INTRODUCTION

Pain that lasts for a long time is called chronic pain, which has several symptoms, such as allodynia, a pain sensation evoked by innocuous stimuli. There are two types of chronic pain: chronic inflammatory pain and chronic neuropathic pain.¹⁾ Long-lasting inflammation in the peripheral tissue causes plastic changes in the peripheral and central nervous systems and induces chronic inflammatory pain.²⁾ On the other hand, damage to the peripheral nerve alters neuronal function and causes neuropathic pain. Inflammatory mediators are released after tissue damage or nerve injury and alter neuronal functions in the peripheral and central nervous systems. Therefore, inflammation is critical for the induction of both types of chronic pain.²⁾

The carbohydrate-restricted ketogenic diet (KD) is a classic diet that has long been used to treat epilepsy.³⁾ The KD contains high fat, low carbohydrate, and an adequate amount of proteins. Instead of sugars, ketone bodies made from fats are used as an alternative energy source for the brain. Because ketone bodies suppress the production of reactive oxygen species, they exert anti-inflammatory effects.⁴⁾ Therefore, KD can alleviate the chronic pain induced by inflammation and nerve injury. A recent report demonstrated that KD alleviated complete Freund’s adjuvant (CFA)-induced inflammatory pain.⁵⁾ However, whether KD alleviates other types of chronic inflammatory and neuropathic pain remains unclear. It is well known that the formalin model is used as a chronic inflammatory pain model, and injection of formalin into the dorsal surface of the hind paw induces long-lasting inflammatory pain in the plantar surface of the hind paw.^6–8) The partial sciatic nerve ligation model is widely used as a neuropathic pain model, and partial ligation of the sciatic nerve induces persistent pain.⁹⁾ Thus, in the present study, we investigated the effects of KD on formalin-induced chronic inflammatory pain and nerve injury-induced chronic pain.

MATERIALS AND METHODS

The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, as prescribed by the Japanese Physiological Society. All animal experiments were approved by the Kitasato University Institutional Animal Care and Use Committee (Approval Number: EiKen20-11-3).

Animals

Adult male C57BL/6 mice were purchased from Japan SLC and housed in cages with ad libitum access to food and water. The room was maintained on a 12-h light/12-h dark cycle. The mice were divided into two groups and fed a control diet (CD) (CE-2, CLEA Japan Inc., Japan) or KD (5TJQ, TestDiet, U.S.A.) for 1 month. The KD consisted of 15.6% protein, 2.1% carbohydrate, and 82.3% fat by calories. The KD is slightly sticky and wet. After 4 weeks of CD or KD feeding, pain models were created. To induce inflammatory pain, 20 µL of formalin (2.5%, Sigma, St. Louis, MO, U.S.A.) was injected subcutaneously into the dorsal surface of the left hind paw. The control mice were injected with the same amount of saline into their hind paws. To induce neuropathic pain, a partial sciatic nerve ligation model was created under isoflurane anesthesia (1.5–3%). The left sciatic nerve was exposed, and one-third to one-half of the nerve diameter was ligated with a 9-0 suture. In the sham surgery group, the skin was cut, and the muscles were dissected, but the nerve was not ligated using sutures.

von Frey Test

The mice were placed in a transparent box on a mesh floor and acclimatized for 15 min before the von Frey test. The paw withdrawal threshold (the 50% withdrawal threshold) was determined using the up-down method¹⁰⁾ with von Frey filament bending force (North Coast Medical, Morgan Hill, CA, U.S.A.). The plantar surface of the hind paw was pressed with von Frey filaments (0.04 to 4.0 g) for 3–4 s, and slight buckling against the paw was defined as a positive response. We tested the next filament with a higher force if a mouse did not show a positive response. If a mouse showed a positive response, we tested the next filament with lower force. Four additional tests were examined when we observed the first change in response-pattern. The 50% threshold was calculated as previously reported.¹⁰⁾

Paw Volume Measurement

To measure the volume of the hind paws, a homemade paw volume examining device¹¹⁾ was used, and the volume of the hind paws was measured as the amount of water increased.

Statistical Analysis

Data are presented as the mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed using Origin PRO 8.6 J (OriginLab, Northampton, MA, U.S.A.). Data from paw volume were analyzed using one-way ANOVA, followed by Bonferroni’s test. Differences were considered statistically significant at p < 0.05. Data from the von Frey test were analyzed using one-way ANOVA, followed by Bonferroni’s test.

RESULTS AND DISCUSSION

First, we conducted the von Frey test to examine the effect of KD on formalin-induced mechanical allodynia (Fig. 1A). One week after formalin injection into the dorsal part of the hind paws in CD-fed mice, paw withdrawal thresholds of the formalin-injected paws and contralateral paws decreased, as previously reported⁷⁾ (Figs. 1B, C). In contrast, the thresholds of saline-injected and contralateral paws in CD-fed mice did not change a week after injection (Figs. 1B, C). KD significantly increased the mechanical thresholds of both formalin-injected and contralateral hind paws compared with CD-fed mice (Figs. 1B, C). Furthermore, the mechanical thresholds of saline-injected hind paws and contralateral hind paws in KD-fed mice were consistent with those of CD-fed mice (Figs. 1B, C). These results suggest that KD alleviated mechanical allodynia in both contralateral and ipsilateral areas of the formalin-injected hind paws. Next, we examined the effect of the dietary switch from CD to KD. The CD was switched to KD just after formalin injection (Fig. 1A). This dietary switch from CD to KD did not alter mechanical thresholds in both formalin-injected (Figs. 1B, C) and contralateral paw compared with CD-fed mice (Figs. 1D, E). In addition, the effect of the dietary switch from KD to CD on the mechanical threshold was also tested. The KD was switched to CD at 3 d after formalin injection (Fig. 1A). One week after formalin injection, the dietary switch from KD to CD did not increase the mechanical threshold in the formalin-injected paw compared with CD-fed mice (Figs. 1B, C), while the dietary switch increased the mechanical threshold in the contralateral paw compared with CD-fed mice (Figs. 1D, E). These results suggest that although only pretreatment of KD has a slight analgesic effect in the contralateral hind paw, it is not enough to attenuate mechanical allodynia in the formalin-injected paw. Thus, pretreatment and continuous treatment of KD are critical for the analgesic effect of KD in formalin-induced inflammatory pain.

Fig. 1. Ketogenic Diet Alleviated Mechanical Allodynia Induced by Formalin Injection

(A) The diagram of experiments. CD, control diet; KD, ketogenic diet; VF, von Frey test. (B) Paw withdrawal thresholds to von Frey filaments in saline or formalin-injected hind paws before (Pre) and one week (1W) after injection (Saline + CD, n = 10; Formalin + CD, n = 13; Saline + KD, n = 12; Formalin + KD, n = 8; Formalin + CD-KD, n = 8; Formalin + KD-CD, n = 9). (C) Bar graphs represent paw withdrawal thresholds one week after injection in (B). (D) Paw withdrawal thresholds to von Frey filaments in hind paws contralateral to saline- or formalin-injected side (Saline + CD, n = 10; Formalin + CD, n = 13; Saline + KD, n = 12; Formalin + KD, n = 8; Formalin + CD-KD, n = 8; Formalin + KD-CD, n = 9). (E) Bar graphs represent paw withdrawal thresholds one week after injection in (D). Data represent the mean ± S.E.M. * p < 0.05 vs. Formalin + CD. ^† p < 0.05 vs. Saline + CD, ^# p < 0.05 vs. Saline + KD. ^& p < 0.05 vs. Formalin + KD.

Injection of formalin into the dorsal side of the hind paws causes edema owing to the inflammatory effects of formalin.¹²⁾ To examine the anti-inflammatory effect of KD, we assessed the volume of hind paws 1 week after formalin or saline injection into the hind paws (Fig. 1A). The volume of the formalin-injected hind paws in the CD-fed mice was larger than that of the saline-injected hind paws in the CD-fed mice (Saline + CD, 163.8 ± 9.44 µL, n = 8; Formalin + CD, 283.8 ± 21.5 µL, n = 8; p < 0.05; Fig. 2A). In contrast, the volume of formalin-injected hind paws in the KD-fed mice was significantly smaller than that of the formalin-injected hind paws in the CD-fed mice (Formalin + KD, 226.9 ± 9.61 µL, n = 16, p < 0.05; Fig. 2A). As for the volume of the hind paws contralateral to formalin or saline injection, there were no significant differences among all groups (Saline + CD, 168.8 ± 8.75 µL, n = 8; Formalin + CD, 165.0 ± 8.45 µL, n = 8; Saline + KD, 175.0 ± 10.7 µL, n = 8; Formalin + KD, 160.6 ± 12.9 µL, n = 16; Fig. 2B). These results suggest that KD can inhibit formalin-induced inflammation and edema.

Fig. 2. Ketogenic Diet Reduced Paw Edema Induced by Formalin Injection

(A) The volume of saline- or formalin-injected hind paws (Saline + CD, n = 8; Formalin + CD, n = 8; Saline + KD, n = 8; Formalin + KD, n = 16). (B) The volume of hind paws contralateral to saline- or formalin injection (Saline + CD, n = 8; Formalin + CD, n = 8; Saline + KD, n = 8; Formalin + KD, n = 16). * p < 0.05. Data represent the mean ± S.E.M.

Finally, we examined the effect of KD on mechanical allodynia induced by partial sciatic nerve ligation (Fig. 3A). The mechanical thresholds of the injured paws gradually lowered until 3 d after nerve injury, and the lowered thresholds persisted for 3 weeks in the CD-fed mice. In contrast, KD significantly increased the mechanical thresholds of injured paws compared to the CD-fed mice (Figs. 3B, C). Mechanical thresholds of paws with sham surgery in the CD- and KD-fed mice did not change for 3 weeks after surgery (Figs. 3B, C). Moreover, the mechanical thresholds of paws contralateral to nerve injury or sham surgery did not change for 3 weeks after surgery (Figs. 3D, E). These results suggest that KD can alleviate nerve injury-induced mechanical allodynia. Next, we examined the effect of the dietary switch from CD to KD. The CD was switched to KD just after sciatic nerve ligation. This dietary switch did not alleviate mechanical allodynia in both paws (Figs. 3D, E). In addition, the effect of the dietary switch from KD to CD on the mechanical threshold was tested. The KD was switched to CD one week after nerve injury (Fig. 3A). The dietary switch from KD to CD did not change mechanical thresholds in injured and contralateral paws compared with CD-fed mice at 3 weeks after nerve injury (Figs. 3B–E). Thus, these results suggest that four weeks of pretreatment and continuous treatment of KD are necessary to show the analgesic effect of KD in nerve injury-induced neuropathic pain.

Fig. 3. Ketogenic Diet Alleviated Mechanical Allodynia Induced by Partial Sciatic Nerve Ligation (PSL)

(A) The diagram of experiments. CD, control diet; KD, ketogenic diet; VF, von Frey test. (B) Paw withdrawal thresholds to von Frey filaments in injured hind paws (Sham + CD n = 9; PSL + CD, n = 11; Sham + KD, n = 8; PSL + KD, n = 17; PSL + CD-KD, n = 8; PSL + KD-CD, n = 10). (C) Bar graphs represent paw withdrawal thresholds 3 weeks after nerve injury in (B). (D) Paw withdrawal thresholds to von Frey filaments in contralateral hind paws to injury (Sham + CD, n = 9; PSL + CD, n = 11; Sham + KD, n = 8; PSL + KD, n = 17; PSL + CD-KD, n = 8; PSL + KD-CD, n = 10). (E) Bar graphs represent paw withdrawal thresholds 3 weeks after nerve injury in (D). Data represent the mean ± S.E.M. * p < 0.05 vs. PSL + CD. ^† p < 0.05 vs. Sham + CD, ^# p < 0.05 vs. Sham + KD. ^& p < 0.05 vs. PSL + KD.

In the present study, we demonstrated the anti-inflammatory effects of KD on formalin-induced edema. Injection of formalin into the hind paw causes inflammation and, subsequently, edema. Reactive oxygen species have critical roles in inflammation¹³⁾ and a high concentration of reactive oxygen species causes oxidative damage to lipids, proteins, and DNA, which in turn causes cell damage.¹⁴⁾ Ketone bodies, generated from fats contained in KD, can scavenge reactive oxygen species and inhibit oxidative stress. In addition, ketone bodies reduce the production of reactive oxygen species,¹⁵⁾ and KD reduces the amount of reactive oxygen species.¹⁶⁾ Therefore, KD can exert antioxidant and anti-inflammatory effects. Thus, the anti-inflammatory effect of KD observed in this study may be due to the antioxidative effect of ketone bodies.

The anti-inflammatory effects of KD are involved in its therapeutic effects on various diseases, such as Alzheimer’s disease,⁴⁾ Parkinson’s disease,¹⁷⁾ and epilepsy.³⁾ As for chronic inflammatory pain, KD alleviated mechanical allodynia induced by CFA.⁵⁾ In addition, we demonstrated that feeding with KD for 4 weeks could prevent the induction of allodynia induced by formalin injection, which is another model of chronic inflammatory pain.^6,7) Since KD prevented the edema induced by formalin owing to its anti-inflammatory effect, the analgesic effect of KD may be induced by inhibiting inflammation. The mechanisms of chronic pain induced by inflammation may differ from inflammatory pain models. In the CFA model, some reports demonstrate that inflammation does not activate glial cells, such as microglia in the spinal cord.^18,19) On the other hand, in the formalin-induced chronic inflammatory pain model, inflammation causes activation of microglia and astrocytes in the spinal cord.^6,7) Activated glial cells release various cytokines that modulate neuronal function and cause chronic pain.²⁰⁾ Moreover, inhibition of glial function in the spinal cord suppresses pain.²¹⁾ Thus, KD may alleviate chronic pain by suppressing inflammation and inhibiting cytokine secretion by activated glial cells in the spinal cord.

KD also suppressed allodynia in the hind paw contralateral to the formalin injection (Fig. 1). Formalin administration to the dorsal hind paw induces allodynia not only on the formalin-injected side but also on the opposite side.⁷⁾ Allodynia in the hind paws contralateral to formalin injection may be induced by increased inflammatory mediators and activated glial cells in the spinal cord contralateral to formalin injection.²²⁾ Therefore, inhibition of inflammation by KD may suppress the generation of inflammatory mediators and activation of glial cells in the spinal cord contralateral to the injection, thereby suppressing pain behavior in the contralateral hind paws.

As shown in Fig. 3, KD suppressed the mechanical allodynia caused by peripheral nerve injury. In the chronic pain model, mechanical allodynia is induced by neuronal hyperactivity in the peripheral and central nervous systems.^23,24) The KD can inhibit these neuronal hyperactivities through at least three possible mechanisms. The first mechanism is the anti-inflammatory effect of KD. When peripheral nerves are injured, infiltration and activation of immune cells result in pro-inflammatory cytokine release in the dorsal root ganglion and spinal cord. This influx of immune cells acts on neurons in the dorsal root ganglion and neurons and glia in the spinal cord, which induces chronic pain.^25,26) The anti-inflammatory effects of the KD may suppress the facilitation of peripheral and spinally mediated pain. The second possible mechanism is the inhibitory effect of ketone bodies, generated from fats, on neuronal hyperactivities. Ketone bodies can activate K_ATP channels causing subsequent hyperpolarization of the membrane potential, leading to the inhibition of neuronal hyperactivity.²⁷⁾ The final possible mechanism is the increase in the amount of inhibitory neurotransmitters. The KD alters the composition of gut microbiota, which in turn increases the amount of inhibitory neurotransmitters in the brain, which can attenuate neuronal activities.²⁸⁾ Because of these mechanisms, the KD may inhibit neuronal hyperactivities in the peripheral and central nervous systems, resulting in attenuation of chronic pain induced by peripheral nerve injury. Further studies are needed to clarify the precise underlying mechanisms of the effect of KD on chronic pain.

Recent accumulation of evidence suggests that the mechanisms of chronic pain differ depending on sex.²⁹⁾ As for inflammatory pain, intrathecal injection of lipopolysaccharide causes allodynia in only male mice but not in female mice.²⁹⁾ In addition, in the mouse model of neuropathic pain, inhibition of spinal microglial activation with a P2X inhibitor suppressed allodynia in males but not in females.²⁹⁾ In the present study, we used only male mice. In male mice, sciatic nerve injury- and formalin-induced allodynia were suppressed by KD treatment. However, because the mechanism of chronic pain is different between male and female mice, the effects of KD on allodynia may differ between male and female mice. Therefore, whether KD has an analgesic effect on female mice is unclear. Further research is required on the analgesic effect of KD in female mice.

In summary, we found that the KD can reduce inflammation and alleviate inflammation-induced and nerve injury-induced chronic pain. By establishing the usefulness of KD in the treatment of chronic pain, the diet can be used in combination with existing medication to provide a more effective treatment. Thus, this study indicates that KD can be a new adjunct therapeutic strategy for chronic pain in addition to treatment with medications.

Acknowledgments

We thank Chizuru Handa and Yukiko Sato for their technical support.

Conflict of Interest

The authors declare no conflict of interest.

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