Here we report that EODF improves functional recovery after a thoracic spinal cord contusion injury of moderate severity in rats. Both EODF starting 3 weeks before the injury, or more importantly, starting it after the injury, were effective. The pre-EODF and post-EODF groups reached significantly higher scores on the RI-BBB for open-field locomotion when compared to the ad libitum
control group, but also the pair-fed group. The pair-fed group consumed the same total amount of calories offered daily. In addition, on several parameters measured with the CatWalk gait analysis device, both EODF feeding regimens elicited improved functional recovery. Hence, EODF has prophylactic and therapeutic potential after thoracic contusion SCI, and these findings are consistent with our previous findings with pre-EODF and post-EODF in a cervical SCI model, the dorsolateral funiculus crush (Plunet et al., 2008
While both EODF groups had better functional recovery than the two control groups (ad libitum
and pair-fed), the pre-EODF group (starting the diet 3 weeks prior to injury) improved functional recovery to a greater extent than the post-EODF group (). This observation is consistent with our two separate EODF studies after cervical SCI, although they were not performed at the same time and therefore cannot be directly compared (Plunet et al., 2008
). One can speculate that cellular pathways for neuroprotection have been already activated by 3 weeks of pre-EODF, and that this provides benefits during the early secondary injury phase. In contrast, this activation of EODF-induced effects may occur too slowly to influence the early cascades of secondary death and inflammation if the diet is started after injury. The clinical significance of our pre-EODF data is that such a regimen could conceivably be implemented prior to elective interventions that put the spinal cord at risk, such as surgery for deformities or tumors. Additionally, EODF started after injury, which would have wider clinical applications, also improved functional recovery after SCI.
Summary of Behavioral Effects Observed with Different Dietary Strategies after Thoracic Contusion Injury Compared to the ad Libitum Control Group
Daily caloric restriction (pair-fed group), in contrast to EODF, did not improve functional outcomes after thoracic contusion injury, and the pair-fed rats showed similar behavioral performance as the ad libitum
group. This differential outcome between the CR and EODF groups is consistent with previous research in mice showing increased neuroprotective efficacy of pre-EODF compared to caloric restriction after kainate-induced brain injury (Anson et al., 2003
). This suggests that similar levels of calorie restriction do not produce the same behavioral outcome, and the specific timing of food/fasting can make a substantial difference in the effects. Anson and coworkers suggested that differences in trophic factors may play a role. It is unknown how a change in metabolism and gene expression elicited by 24
h of fasting, followed by a feasting and free access to food for 24
h, contributes to the increased functional recovery seen after SCI. We propose that the 24-h fasting period increases blood ketone levels, which could exert neuroprotective effects. Such an increase in ketones is not seen on a caloric-restriction regimen (Anson et al., 2003
), such as the pair-fed control feeding regimen. This hypothesis is supported by recent data from our laboratory revealing that a ketogenic diet, which also increases ketone levels, after cervical hemi-contusion injuries in rats elicits robust improvements in behavioral outcomes (Streijger et al., in submission). In addition, it is conceivable that 24
h of feasting (eating ~140–160% of normal intake) contributes to the functional recovery by altering fundamental metabolic pathways, such as stimulation of the mTOR pathway, as has recently been reported in the brains of EODF animals, but not in similarly caloric-restricted animals that were fed every day (Martin et al., 2008
). Increased levels of mTOR have recently been shown to enhance the regenerative capacity of corticospinal axons (Liu et al., 2010
). Further research is necessary to clarify the relative contributions of these possible fasting and/or feasting effects.
In light of our previous findings of a reduced lesion size after a cervical SCI in both pre-EODF and post-EODF groups (Plunet et al., 2008
), we examined here if similar neuroprotection is seen in our thoracic injury model. Despite functional improvements in both EODF groups after thoracic SCI, we did not observe any differences in the amount of spared white or gray matter between the groups. Of note, in our cervical model EODF rescued mainly gray matter, and gray matter is typically entirely destroyed after thoracic contusions. However, there was a trend toward improved gray matter rescue at a distance of 2000–1600
μm from the epicenter. It is conceivable that the more complete and larger injury seen after thoracic contusion, compared to the unilateral cervical crush injury, masks any of the differences in neuroprotection; in other words, an equally small rim of rescued tissue would be more evident and easier to detect in a small lesion. Hence a small neuroprotective effect of EODF might have gone undetected in this more severe thoracic contusion model.
EODF is known to alter the expression of a large number of proteins that could account for increased plasticity (Mattson et al., 2003
), and indeed EODF increased axonal plasticity as measured by long-term changes in synaptic efficiency, coinciding with increases in NMDA receptor subunit NR2B, and sprouting of spared corticospinal axons (Fontan-Lozano et al., 2007
; Plunet et al., 2008
). The delayed functional recovery (50 days) in the both EODF groups would be consistent with enhanced neural plasticity being the major mechanistic player. In the cervical injury model we observed increased sprouting of the intact corticospinal tract (Plunet et al., 2008
). In the present study we quantified serotonergic axons, as this system plays an important role in limb coordination (Jordan et al., 2008
; Schmidt and Jordan, 2000
), and hence is often studied in thoracic SCI models. While the several parameters of SERT sprouting data correlated with functional recovery, we did not detect group differences among our multiple groups using conservative ANOVA statistics. These results suggest that additional fiber systems might be playing a role, and that individual fiber systems may only show subtle changes.
These results also raise the possibility that changes at the cortical level could have occurred that contributed to the change in functional recovery observed in the EODF groups. The potential cortical level changes could have occurred at a structural level and/or at a molecular level. Since EODF can have systemic affects, including in the brain, this could provide additional mechanisms for beneficial outcomes. Both calorie restriction and EODF have been reported to improve learning/memory in older animals, though there are other researchers that found no improvement (Bond et al., 1989
; Beatty et al., 1987
; Idrobo et al., 1987
; Pitsikas and Algeri, 1992
). Improved learning/memory in younger animals, such as those used in our experiment, is not established. In our study we did not specifically examine any cortical structural plasticity changes induced by EODF that could account for our behavioral results. An additional possibility is that a cortical learning effect may account for some of these improvements. However, we think that this is unlikely for several reasons. The behavior with the highest potential for learning, the skilled ladder crossing test, revealed no differences among the groups. The other two tests, open-field, and CatWalk, involve arguably less skilled locomotor movements (i.e., walking/running). While there is likely a component of re-learning after SCI for these motor patterns, these are more likely “hard wired” in comparison to a more complex task such as crossing an irregular ladder with specific foot placements on the slim rungs. Additionally, the equally calorie-restricted pair-fed group did not show any improvement in motor recovery after SCI, and if memory/learning was playing a role we would have expected similar improvements in motor recovery in this group, since the level of calorie restriction these animals were on is similar to what is reported to improve memory/learning, at least in older animals. Overall, this suggests that improved memory/learning did not play a major role in the motor recovery seen after SCI in this experiment.