Nerve injury produced sensory abnormalities in all three dietary groups, lasting at least 14 days after surgery. Compared with intact rats, all three PSL-injured groups developed a significant tactile allodynia, because their withdrawal threshold to touch was significantly lower after nerve injury (PSL-RMH versus CON-RMH, P = 0.0004; PSL-CAS versus CON-CAS, P = 0.0002; PSL-BC versus CON-BC, P = 0.001) (Fig. 2). Diet significantly affected levels of tactile allodynia after PSL injury (P = 0.017). Consumption of RMH, a diet containing soy, produced significantly lower levels of tactile allodynia than diets devoid of soy (PSL-RMH versus PSL-CAS, P = 0.01; PSL-RMH versus PSL-BC, P = 0.039). Levels of allodynia to touch in rats fed diets devoid of soy were not significantly different (PSL-CAS versus PSL-BC, P = 0.9).
Compared with intact rats, all three PSL-injured groups developed a significant mechanical hyperalgesia, because their response duration to pinprick was significantly prolonged after nerve injury (PSL-RMH versus CON-RMH, P = 0.001; PSL-CAS versus CON-CAS, P = 0.0002; PSL-BC versus CON-BC, P = 0.01) (Fig. 3). Levels of mechanical hyperalgesia did not depend on the type of diet (P = 0.2).
Compared with intact rats, a significant heat allodynia developed in the two groups fed a diet devoid of soy (CAS and BC), because their withdrawal threshold to heat was significantly lower after nerve injury (PSL-CAS versus CON-CAS, P = 0.0003; PSL-BC versus CON-BC, P < 0.0001). However, consuming RMH had not changed the time needed to elicit paw withdrawal (PSL-RMH versus CON-RMH, P = 0.08) (Fig. 4). Diet had a significant effect on withdrawal threshold to heat after PSL injury (P < 0.0001). This effect was more robust in rats consuming diets devoid of soy compared with rats fed a diet containing soy (PSL-BC versus PSL-RMH, P = 0.0002; PSL-CAS versus PSL-RMH, P = 0.0006). Levels of heat allodynia in the two groups fed diets devoid of soy did not differ significantly (PSL-CAS versus PSL-BC, P = 0.5).
A significantly prolonged response duration to heat developed in all nerve-injured groups, compared with intact rats (PSL-RMH versus CON-RMH, P = 0.0005; PSL-CAS versus CON-CAS, P = 0.0001; PSL-BC versus CON-BC, P = 0.0004) (Fig. 5). The response duration did not depend on the type of diet (P = 0.3).
Of PSL-CAS and PSL-BC rats, 43% and 36%, respectively, expressed tactile allodynia on the contralateral intact hindpaw, compared with only 17% of PSL-RMH rats. In addition, 44% and 20% of PSL-BC and PSL-CAS rats, respectively, presented heat allodynia on the intact hindpaw, whereas none of the rats of the PSL-RMH group did so. Thus, consumption of soy-containing diet prevented development of allodynia on the mirror image side as well.
Surprisingly, very little is known about the effect of diet on chronic pain in animals (3,12) or humans (13). The main results of this experiment were that the type of diet rats consumed played an important role in neuropathic pain levels after partial denervation. Specifically, we found that a soy-containing diet significantly blunted tactile and heat allodynia after PSL injury, but not mechanical hyperalgesia. The analgesic properties of soy were not limited to a single laboratory because we reaffirmed its effect in a different laboratory and experimental design.
The only difference between our previous results in Jerusalem (2) and this study in Baltimore is that soy diet did not prevent the development of increased response duration to heat in the latter. This difference may be caused by the different rat strain used in the two studies (Sabra and Wistar, respectively). Genetic effects in the PSL and related models have been demonstrated in rats and mice (14–16). In addition, the differing results could be caused by different methods for production of heat (CO2 laser versus the Hargreaves method). Because in the latter method, rats withdraw from the stimulus at threshold, the level of sensitization it produces is lower compared with the CO2 laser.
In agreement with other reports, heat pain sensitivity of intact rats was significantly affected by diet. Increased amounts of dietary sucrose reportedly attenuated heat pain (6,7,17) and paw inflammation (8), an effect putatively mediated by endogenous opioids (18). However, in this experiment, RMH and casein diets contained equal concentrations of carbohydrates (67.5% and 65%, respectively), yet PSL-injured rats fed these diets differed significantly in their tactile and heat allodynia. Moreover, BC diet contained 15%–17% fewer carbohydrates than CAS or RMH, but levels of allodynia and hyperalgesia in rats fed BC did not differ significantly from CAS. Thus, carbohydrates did not affect the reduction in tactile and heat threshold in the two soy-free groups.
Increasing fat content in the diet attenuates acute heat pain (5). It is unlikely, however, that in this experiment, dietary fat content affected levels of CNSD. The CAS and BC diets differed both in their fatty acids concentration and composition. Yet similar levels of CNSD developed in these two groups after nerve injury. Moreover, CAS and RMH contained similar fat levels but expressed significantly different levels of allodynia.
Consumption of highly palatable foods increases calorie intake (19) and attenuates responses of rodents to acute pain (6,7). In this experiment, rats fed BC consumed significantly more calories than rats fed the RMH or CAS diets. It is not clear whether the increased calorie consumption of rats fed the BC diet was caused by its increased palatability or to its being an unbalanced diet. Nevertheless, similar to previous studies, the withdrawal threshold to noxious heat of intact BC rats was significantly higher compared with the other two groups (Fig. 4) (6,7). Higher calorie intake had no effect, however, on the development of the PSL model, because BC and CAS rats, significantly differing in their calorie intake, developed similar neuropathic pain levels.
Younger rats tend to develop more pain in other models of neuropathy, arguably because of lower body weight [reviewed by Zeltser and Seltzer (20) and Bennett et al. (21)]. This was never examined, however, in the PSL model. BC-fed rats gained significantly less weight than the other two groups, but their levels of CNSD after PSL injury were not significantly different than CAS-fed rats. Thus, body weight in Wistar rats is not associated with levels of neuropathic disorders in this model.
It is likely that soy is the dietary ingredient responsible for suppression of CNSD in the PSL model. RMH and CAS diets differed mainly in the protein source, i.e., soy protein in RMH versus casein protein in CAS. Nevertheless, these diets were not identical, suggesting that other ingredients could suppress CNSD in this model. However, when rats were fed identical diets differing only in their protein, i.e., soy versus casein, soy-based diet was associated with suppressed levels of allodynia and hyperalgesia (2). Of the various ingredients in soy protein that could prevent the development of CNSD, phytoestrogens and certain amino acids are the most plausible candidates. Phytoestrogens are plant compounds possessing estrogenic properties (22). Because estrogens may augment antinociception (23), soy phytoestrogens could mediate the suppression of CNSD after PSL injury. Our recent results in Wistar rats partially support this suggestion (unpublished data). However, the correlation between dietary and plasma levels of amino acids in rats fed the RMH, CAS, and BC diets—and CNSD in these rats—was not significant for any amino acid (unpublished data). Thus, a possible role for certain amino acids in the suppression of CNSD in the PSL model has yet to be found.
We conclude that the effect of diet on the development of CNSD in the PSL model was evident across two different laboratories, rat strains, heat-producing stimulators, diet types, diet providers, and vivaria. Food vendors supply rat chows standardized for the relative content of basic nutritional ingredients, but not for the source and type of these ingredients. Therefore, standardized, artificial balanced diets are recommended for rat experiments that use the PSL model, and probably for other pain models. Neuropathic pain in humans is only partially understood and many times is refractory to treatment. Future studies will show whether dietary manipulation can alleviate pain in humans with these syndromes.
We are grateful to Marshall Devor for commenting on this manuscript. This project was supported in part by the US-Israel Binational Science Foundation 98203, NIH grant 26363, and the Blaustein Foundation for Pain Research.
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