Ketogenic diet effects on inflammatory allodynia and ongoing pain in rodents

Ketogenic diets are very low carbohydrate, high fat, moderate protein diets used to treat medication-resistant epilepsy. Growing evidence suggests that one of the ketogenic diet’s main mechanisms of action is reducing inflammation. Here, we examined the diet’s effects on experimental inflammatory pain in rodent models. Young adult rats and mice were placed on the ketogenic diet or maintained on control diet. After 3–4 weeks on their respective diets, complete Freund’s adjuvant (CFA) was injected in one hindpaw to induce inflammation; the contralateral paw was used as the control. Tactile sensitivity (von Frey) and indicators of spontaneous pain were quantified before and after CFA injection. Ketogenic diet treatment significantly reduced tactile allodynia in both rats and mice, though with a species-specific time course. There was a strong trend to reduced spontaneous pain in rats but not mice. These data suggest that ketogenic diets or other ketogenic treatments might be useful treatments for conditions involving inflammatory pain.


Scientific Reports
| (2021) 11:725 | https://doi.org/10.1038/s41598-020-80727-x www.nature.com/scientificreports/ and to make animals docile for paw volume testing. All testing occurred during the lights-on period of the daily cycle. For rats, 100 µl complete Freund's adjuvant (CFA; Thermo Scientific, 0.5 mg/ml) was injected intraplantar into the right hindpaw with the needle tip as close to the center of the footpads as possible; for mice, injections were similar apart from reducing the dose of CFA to 20 μl. All CFA injections were performed in the morning to keep the four h time point well within the light cycle. Volumes of rat right hindpaws before CFA injection did not differ between CD-and KD-fed groups (p > 0. 50), such that the 100 µl dose of CFA can be considered equivalent in the two groups. Before tactile sensitivity testing, rats were habituated by being brought in their home cage into the testing room for 15 min, then placed on an elevated mesh stand in 20 × 20 × 12 cm acrylic enclosures (to minimize locomotion; IITC) for another 15 min; mice were habituated by being placed on the mesh stand in 10 × 10 × 12 cm acrylic enclosures (IITC) for 60 min. At various times before and after CFA injection, tactile sensitivity was measured with an electronic von Frey probe (IITC, Fig. 1). The rigid von Frey probe was applied alternately to each hindpaw until the animal either withdrew the paw or allowed it to be lifted by the probe; the maximum force on each trial was recorded. Three trials with no less than a 120 s intertrial interval occurred per hindpaw. If the animal began to take a step or otherwise shift its position during a trial, that trial was repeated.
In rats, spontaneous pain behavior was assessed 48 h prior to and 24 h after CFA injection. After a 15 min habituation to the testing room, animals were placed singly in 19 × 29 × 13 cm clear-bottom cages for 30 min and filmed vertically from below. Later, videos were watched for indications of spontaneous pain for 60 s every five minutes; videos were watched by two scorers who were independent and blind to dietary treatment and pre-versus post-CFA state. Epochs with grooming or locomotion were avoided. Position of each hindpaw was placed into the following categories: 0, normal weight bearing; 1, light weight bearing; 2, only paw edge touching floor; 3, paw nearly raised off floor; 4, paw completely raised; 5, licking raised paw, and time spent in each position was recorded. For each observed minute, a weighted pain score was calculated (t 1 + 2t 2 + … 5t 5 , in which t x is the time spent in category x) and averaged across the six observed minutes 39 . This test was not used in mice as preliminary experiments indicated that mouse paws were too small to reliably distinguish the categories.
In rodents, ongoing painful states interfere with ongoing behavior [40][41][42] . We assessed this effect in mice with marble burying [43][44][45] at the indicated times (Fig. 1). Animals were habituated to the testing room for 30 min, then placed individually in 19 × 29 × 13 cm cages for 30 min. These cages had a 3 × 5 array of 1.6 cm diameter black marbles placed on five cm of wood ship bedding. Photos were taken vertically from above before and after the session and analyzed with Photoshop. The quick selection tool was used to measure the total number of pixels associated with the black marbles, and the polygonal lasso tool was used to measure the number of pixels of the bedding field; the number of marble pixels was expressed as a percentage of the entire field's pixels. This calculation was performed on before and after pictures, with the comparative decrease in the marble percentage compared to before indicating the amount of burying. Data from two mice that demonstrated very little burying in the baseline test were excluded from analysis. The marble burying test was not used in rats as preliminary experiments indicated that rats did virtually no burying under our conditions.
Volumes of rat hindpaws were measured by water volume displacement in 25 ml graduated cylinders 24 h prior to and 50 h after CFA injection. At sacrifice of subjects, glucose and the ketone body β-hydroxybutyrate were measured in tail vein blood with Precision Xtra meters (Abbott). www.nature.com/scientificreports/ T-tests were used for CD versus KD comparisons, with significance indicated by pound signs. For comparisons of post-CFA time points to baseline time points, multiple Bonferroni comparisons were made, with significance indicated by asterisks. Comparisons were considered significant if p < 0.05. All data are presented as mean ± standard error.

Results
Plantar tactile sensitivity was low in baseline and not different between CD-and KD-fed rats in injected paws (CD 85.0 ± 7.0 g, KD 85.4 ± 10.6 g, p > 0.50) or uninjected paws (CD 95.6 ± 7.3 g, KD 80.8 ± 9.2 g, g > 0.20). All rats demonstrated strong allodynia of the injected hindpaw; however the magnitude of this effect was significantly smaller at 4 h post-injection in KD-fed animals (Fig. 2). This difference did not remain significant at the 48 h time point (Fig. 2). As expected, plantar tactile sensitivity did not change with diet or time in the uninjected hindpaw (Fig. 2).
Behaviors indicative of ongoing pain states related to the hindpaws such as licking and avoiding weight bearing were essentially absent pre-CFA, as expected (Fig. 3). After injection, such behaviors were present and directed to only the injected hindpaw in all rats; however there was a strong trend toward less such behavior in KD-fed rats (Fig. 3).
As expected 46 , CFA-induced hindpaw swelling was significantly reduced by KD treatment, measured by change in volume from baseline (Fig. 4). The uninjected hindpaw was unaffected (Fig. 4).
Plantar tactile sensitivity was low in baseline and not different between CD-and KD-fed mice in injected paws (CD 3.18 ± 0.63 g, KD 2.72 ± 0.47 g, p > 0.50) or uninjected paws (CD 2.84 ± 0.15 g, KD 2.46 ± 0.32 g, g > 0.30).). All mice demonstrated robust tactile allodynia of the CFA-injected hindpaw; however, in CD-fed mice significant allodynia remained out to the last time point assessed (7d), whereas in KD-fed mice allodynia was starting to reverse at 2d and tactile sensitivity was no longer different from baseline at 4d (Fig. 5). As expected, plantar tactile sensitivity did not change with diet or time in the uninjected hindpaw (Fig. 5).  For the injected paw, spontaneous pain was significantly higher compared to pre-injection for both groups (not indicated). However, there was a strong trend for spontaneous pain to be lower in ketogenic diet-treated rats. There were no effects regarding the uninjected paw. Control n = 14, ketogenic n = 12. # p = 0.059.  20). Marble burying in mice was strikingly reduced after CFA injection, likely indicating an ongoing pain state (Fig. 6). Burying behavior slowly recovered in both diet groups; and statistics indicated that there was no diet-related difference in recovery rate. Notably, at 4 and 7 days the KD group is burying at levels above baseline, albeit non-significantly, something not found in the CD group (Fig. 6). . Effects of dietary treatment on CFA-induced inflammatory paw swelling in rats. Paw volume was expressed as the difference between volume at 50 h after CFA injection and volume at baseline. There was significantly less swelling in rats fed the ketogenic diet. There was no effect in control paws. Control n = 14, ketogenic n = 10. # p < 0.05. Injected paws became hypersensitive after CFA injection, but the rate of recovery differed in the groups. Control diet-fed mice were still strongly hypersensitive at the last examined timepoint, whereas a gradual and complete recovery occurred in ketogenic diet-fed mice. There were no effects in the uninjected paw. Control n = 6, ketogenic n = 8. ***p < 0.001, **p < 0.05, ( * ) p = 0.071 compared to baseline. www.nature.com/scientificreports/ Ketogenic diet treatment strongly elevated blood ketone bodies in both species, reduced blood glucose in mice, and produced a trend for decreased mouse body mass (Table 1).

Discussion
We found that treatment with a KD significantly ameliorated CFA-induced tactile allodynia in two model species, with more modest effects on indices of ongoing spontaneous pain. A KD-induced amelioration of induced inflammatory pain in rodents is consonant with the KD's reduction of inflammation itself produced by various insults in various tissues in rodents 19,28,31,[46][47][48][49][50][51][52][53] and patients [54][55][56] , and reducing pro-inflammatory cytokines 19,28,37,47,49,[57][58][59][60] and eicosanoids 55,58 while elevating anti-inflammatory cytokines 60,61 . These results may appear to contradict a large body of literature showing that high-fat diets promote inflammation 62 . However, this literature refers to diets such as the Western or standard American diet (SAD), high in fat but not low in carbohydrates. The metabolic response to dietary fats differs greatly depending on the presence of carbohydrates: the high-fat-plus-carbohydrate diet promotes fat storage, whereas the high fat, low-carbohydrate diet promotes fat metabolism 63 . Recently, KD feeding was shown to reverse the tactile allodynia produced in a mouse model of metabolic syndrome 64 (strikingly, the KD in this study had almost twice the fat level of the high-fat/moderate carbohydrate diet that induced metabolic syndrome). Metabolic syndrome-related allodynia relates to inflammation in the peripheral nervous system 65 , and as diabetic neuropathy is thought also to involve inflammation in the spinal cord 66 , beneficial effects of the KD against pain syndromes involving inflammation could be peripheral, central, or both. KD treatment, however, appears not to be equally effective in all neuropathic pain syndromes 67 possibly relating to involvement of inflammation.
Clinical work with KD and pain had a very early start, specifically regarding migraine 68,69 and this use may be undergoing a resurgence 70 . Notably, oxidative stress has been hypothesized to be the trigger of many types of migraines 71 . KD feeding effectively treats pain and other symptoms in inflammatory bowel syndrome 72 and Parkinson's disease 73 . Overall body pain is alleviated in overweight diabetic patients 74 , although it was not specified if the type of pain was neuropathic. Given the metabolic parallels between KD treatment and fasting, and the established efficacy of fasting against rheumatoid arthritis 75,76 , a KD could be particularly useful in this disorder. Existing studies suggest little clinical benefit 77,78 ; however, KD treatment in these studies lasted only seven days (to parallel a fasting treatment, which was itself effective). We have found that antinociceptive effects of the KD evolve over days to weeks 79 and others have found a similar pattern in reduced oxidative stress 18 , suggesting that longer treatment durations should be attempted in rheumatoid arthritis.
There were several differences between the results with rats and mice. Mice appeared to be more affected physiologically by ketogenic diet treatment, with lowered glucose and a trend to lower body mass (not occurring in rats) and a more than two-fold higher elevation in β-hydroxybutyrate than in rats. These are clearly species related. There were also differences in behavioral outcomes. The ketogenic diet improved tactile allodynia in rats at four hour post-injection, but not later, whereas beneficial effects on tactile allodynia in mice appeared in the two to four day range and persisted thereafter. Given relative species paw sizes and the currently used doses of CFA, the effective dose in mice is substantially higher, and so either or both dose and species could underlie these differences in diet responsiveness. Ongoing pain states were unaffected by diet in mice, with a trend to improvement in rats. Species differences, however, led us to use different tests for each species. The mouse marble burying test clearly showed that a state of ongoing pain reduced performance of this behavior. The lack of a ketogenic diet effect indicates that either that the diet does not improve spontaneous pain in this model in mice, or that marble burying is inappropriate behavior to assess these types of changes.
It is somewhat unclear which of the main metabolic actions of a KD (elevated ketone bodies, lowered and less variable glucose) leads to limiting inflammation and inflammatory pain. Certainly, chronically elevated glucose is undesirable: high fasting glucose and/or impaired glucose tolerance associate with elevated blood cytokines 80-85 , C-reactive protein [80][81][82]86 , oxidative markers 87 , circulating white blood cells 88 , and inflammatory response of white blood cells 89 . In fact, acute hyperglycemia elevates circulating cytokines through an oxidative mechanism 90 . On the other hand, elevated ketone bodies themselves seem to have beneficial effects: in vivo and in vitro studies show that β-hydroxybutyrate itself moderates the endoplasmic reticulum stress-induced inflammasome 91,92 and the NRLP3 inflammasome 49,60,[92][93][94] in various organs in a manner apparently unrelated to its use as a substrate for the tricarboxylic acid cycle 93 . In addition, free fatty acids from a KD might be directly beneficial by reducing mitochondrial production of reactive oxygen species 27 . Besides being anti-inflammatory, there are other possible mechanisms for a KD to limit pain 95 . Regardless of mechanism, this study and a growing body of evidence suggest that pain be added as a variable in more clinical studies of the KD generally, and specifically that more studies of KD treatment in clinical inflammatory pain syndromes is warranted. Table 1. Effect of diet treatment on physiological parameters. Physiological measures were taken after the end of behavioral testing. Control v. ketogenic ***p < 0.001, **p < 0.01, ( * ) p = 0.06. Rat: n = 12 CD, 14 KD; mouse: n = 6 CD, 8 KD.