This study revealed that restricted feeding regimen used prior to injury potently alleviated the deleterious effects of secondary injury by suppressing microglial activation, TNF-α and caspase-3 induction and neuronal cell death following cortical stab injury in rats.
In the normal central nervous system (CNS), microglial cells are highly ramified, with an elaborate tertiary and quaternary branch structure 
. The highly branched resting microglia provides the brain with a dynamic and efficient surveillance system. Virtually any CNS pathology or damage will lead to their activation and loss of the resting phenotype 
. Although microglial activation represents an integral part of the CNS response to injury, it is still not clear whether activated microglia promote neuronal survival, or whether these cells further exacerbate the extent of neuronal damage. While some findings imply a supportive role for microglial cells in the induction of neuroplastic changes after ischemia 
, a large body of data has rather convincingly shown that microglia possess neurotoxic properties [reviewed in 21 and 22]. This was supported by the fact that immunosuppressive strategies result in an inhibition of microglial activation and neuroprotection after acute traumatic or ischemic brain or spinal cord injury 
Results presented in this study show that, even though a total number of microglial cells around the site of lesion increase significantly in both AL and CR group, morphology of these cells is strikingly different. Namely, the majority of microglial cells seen early after injury in AL animals displayed highly activated morphology with large round cell bodies, whereas in the group of animals exposed to CR prior to injury, the microglial cells surrounding the lesion site maintained ramified morphology during the entire recovery period. While the study of Lee et al. 
showed that dietary restrictions decreased the number of newly generated microglia following kainate-induced brain lesions, the present study is, to the best of our knowledge, the first to demonstrate that CR, prior to mechanical trauma to the brain, has led to suppression of microglial activation following injury. This result is in good keeping with previous findings which indicate that microglial activation and recruitment, but not proliferation, mediate neurodegeneration following injury 
The activated microglia plays a pivotal role in the inflammatory response that lasts hours to days following TBI. Activated microglial cells are the main source of proinflammatory cytokines within the CNS 
. One of the predominant cytokines secreted by the microglial cells is TNF-α 
. It has been shown that the TNF-α expression and secretion are rapidly increased in neurons up to 4 hrs following excitotoxic events at the synapse 
. TNF-α induces proliferation of surrounding microglial cells and stimulates their activation. Importantly, microglial cells continue to express TNF-α as part of the maintenance and amplification of the inflammatory cascade 2–5 days following injury 
. Accordingly, the present study revealed that strong TNF-α induction occurred in the injured cortex of AL animals on the second day following injury. This correlates with the uppermost number of highly activated microglial cells detected in the overall microglial population. Therewithal, in the animals maintained on caloric restriction the abolishment of TNF-α protein induction was accompanied by a minimal microglial activation rate. Given that the TNF-α represents one of the main pro-inflammatory cytokines involved in initiation and expansion of secondary injury, abolishment of TNF-α protein expression following injury may reduce the extent of secondary injury.
Inflammatory processes following trauma ultimately lead to neuronal degeneration and apoptosis 
. Active caspase-3 represents a key executor of apoptosis 
, and a major underlying factor responsible for apoptotic cell death following CNS injury 
. As in other CNS injury paradigms [reviewed in 31], strong induction of active caspase-3 together with numerous degenerating neurons was observed in the injured cortex early after stab injury. Results presented in this study undoubtedly showed that CR represents very potent neuroprotective factor, since it completely abolished the induction of active caspase-3 and neurodegeneration caused by injury. Considering that outcome following injury is directly related to the number of lost neurons, neuronal cell death represents a major issue associated with TBI in the clinic. Thus, many strategies have been developed in an attempt to minimize neuronal cell loss following brain injury. Caloric restriction proved to be neuroprotective by preventing neurons from secondary cell death after mechanical injury.
Most of the studies on the anti-inflammatory and anti-apoptotic mechanisms of CR were focused on the prevention of stroke and other cardiovascular diseases in aging and obesity or in slowing aging processes 
. Some recent studies shown that prophylactic CR suppresses systemic inflammation in spontaneously hypertensive rats, and led to a delay in the onset of stroke 
. However, data concerning effects of CR on processes of secondary injury following stroke, mechanical or some other type of injury, are lacking. Our study is to the best of our knowledge the first to show that prophylactic CR suppresses injury-induced microglial activation, active caspase-3 induction and neuronal cell death in the injured rat cortex, consistent with the inhibitory effect of fasting on ischemia-induced increases of TNF-α 
. Even though the exact mechanisms of neuroprotective properties of CR remain unknown, it is tempting to speculate that CR might be capable of mimicking the immunosuppressive action of drugs in reducing damage following brain trauma.
Although the paradigm of pre-injury caloric restriction may appear as a treatment with limited clinical relevance, recent data showed that CR is more effective in improving functional recovery if applied pre- than post-injury 
. It is likely that cellular pathways for neuroprotection have been already activated by pre-injury CR (days to months before injury), and that this provides benefits during the early secondary post-injury phase. Therefore, if the diet is applied after TBI, we can assume that beneficial CR-induced effects will occur too slowly to influence the early cascades of secondary injury (hours to days).
In conclusion, our data provide evidence that CR ameliorates secondary injury after CSI by repressing microglial activation, TNF-α production, caspase-3 activation and neuronal cell death. These results, together with our previous report 
strongly suggest that prophylactic CR, as a part of a preventive life style could lead to better outcomes following brain injury.