The major findings of our present study are: (1) etanercept injected systemically reduces motor and neurological deficits caused by FPI by day 3 after FPI; (2) the increased numbers of the co-localization of TNF-α and microglia specific marker cells are significantly and selectively higher in the ischemic cortex, white matter, hippocampus, and hypothalamus during FPI which can be attenuated by etanercept therapy; (3) overproduction of cerebral TNF-α in the ischemic cortex caused by FPI can be attenuated by etanercept; (4) by day 3 after FPI, neither the co-localization of TNF-α and astrocyte specific marker cells nor the TNF-α and neuron specific marker cells can be seem in the ischemic brain regions. Our data suggest that systemic administration of etanercept may improve outcome of FPI by attenuating the activation of microglial TNF-α in the ischemic brain. The hypothesis is in part supported by many investigations. For example, Li and Colleagues  has reported that TNF-α is significantly higher in the lesion boundary zone in the saline-treated rats by 3 days after FPI. Cerebral inflammation in response to trauma, stroke, and seizure is characterized by the activation of resident microglia [17–19]. Activated microglia proliferate, change morphology by assuming an amoeboid shape, increase phagocytosis, upregulate MHC class I molecules, and release cytokines [20, 21].
TNF-α transduces death- and survival-signaling through its cognate receptors TNFR1 and TNFR2 and is involved in the inflammatory response following TBI [22–25]. Increases in TNF-α and other cytokines have been reported in cerebrospinal fluid and plasma samples in TBI patients [2, 26–29]. Several groups [3, 5, 30–34] have reported increased TNF-α and other cytokine levels 1 h post-TBI, and peak levels 4 h post-TBI, after which, levels returned toward baseline. In Knoblach et al., , a secondary lesser increase at 72 h post-TBI was also reported. Furthermore, Holmin and Mathiesen  reported persistent elevations 4 days to 3 months after TBI, and Li et al.,  found that TNF-α and other cytokines were significantly higher in the lesion boundary zone in saline-treated TBI rats 3 days post-TBI. In our rat model, microglial overproduction of TNF-α in several ischemic brain regions 3 days after TBI was also reported. In particular, Knoblch et al.  reported peak levels of TNF-α very early after TBI (1–4 hours) with localization to neurons, whereas our present results showed the peak levels of TNF-α occurred at 72 hours and were localized to the microglial cells. Thus, it appears that the cellular sources of this early elevation of TNF-α may be time-dependent. However, the most important point is that this elevated post-TBI TNF-α production in brain tissues can be significantly attenuated by etanercept therapy.
Accumulated evidence shows that TNF-α and its receptor play an important role in the pathophysiology of TBI [22, 24, 25]. In contrast, some evidence suggests that TNF-α plays a neuroprotective role following TBI [35, 36]. Although TNF-α contributes to neuro-anatomical plasticity as well as an improvement of locomotor activity during recovery process , present data indicate that TNF-α is associated with the pathological effects as well as neurological motor deficits during acute phase after TBI. In the present study, despite the etanercept treatment there is still a robust TNF-α release post-injury. However, etanercept should be given only at acute phase [35, 36]. A greater dose of etanercept administered during recovery process would not improve outcome and even exacerbate the pathological effects of TBI.
Etanercept, when administered systemically at the dosage approved for its licensed indications (~50 mg/week in human), would not be expected to achieved therapeutic levels in the cerebrospinal fluid because of its high molecular weight . It should be mentioned that the etanercept doses used in the present set-up are far higher than the normal subcutaneous dose used for rheumatoid arthritis. Administration of a dose in humans might result in significant penetration of etanercept into the cerebrospinal fluid; particularly in an experimental setting such as TBI, in which the blood- cerebrospinal fluid barrier might be damaged.
The present study focuses mainly the effects of etanercept on microglial overproduction of TNF-α in the rat brain effected by TBI. Actually, in addition to TNF-α, TBI-induced increased levels of both interleukin-1β and interleukin-6 were all significantly reduced by etanercept treatment . Additionally, Iba1 stain was chosen as a marker of activated microglia. Stains for surveillance microglia would help clarify if etanercept interferes with microglia activation and/or interferes with chemokines production and subsequent migration of microglia to the contused/ischemic areas of the brain. Furthermore, it is possible that the beneficial effects of etanercept during TBI are a result of limiting macrophage recruitment in part .
It should be mentioned that in the present study, there appears to be quite a few instances of Iba1 and TNF-α labeling that do not coincide and the staining morphology is quite different in some instances (mor elongated Iba1 staining versus round/amoeboid TNF-α staining). Furthermore, Iba1 is not a particularly distinguishing cell-specific marker for microglia in the injured brain as infiltrating macrophages also express this antigen. In addition, although our present study showed that the sham-group displayed no evidence of damage 3 days post-FPI, Jones et al.  did display evidence of damage one month post-FPI. The discrepancy between our results and their findings could be due to time difference.
Finally, it should be mentioned that given that the sham animals in the present study did not receive injections on any treatment the authors might comment on the possible confound that the stress associated with the injections may have had on the TBI group versus sham.