This study showed for the first time that post-injury salidroside treatment significantly attenuated brain tissue damage and long-term behavioral deficits following TBI in mice, and decreased neuronal vulnerability to stretch-induced injury in vitro. Salidroside treatment also suppressed apoptosis and preserved mitochondrial integrity. These beneficial effects were associated with increased Akt phosphorylation and mitochondrial Bcl-2/Bax ratio. Co-administration of a specific PI3K inhibitor reversed the reduction of brain tissue loss by salidroside treatment, indicating that its therapeutic action is at least partly mediated by the PI3K/Akt signaling pathway.
We found that single dose salidroside treatment attenuated CCI-induced functional deficits in mice as early as 1 day post-injury and promoted > < sustained improvement of motor-sensory functions over 4-week observation period. This protective effect was associated with a decrease in neuronal damage and apoptotic cell death at day 1, and a reduction in contusion volume and brain tissue loss at day 28. The long-term protective effects in preserving tissue integrity with salidroside treatment is possibly due to a reduction in the number of degenerated neurons detected during the acute phase. Alternatively, some injured but viable neurons may shrink in response to loss of their terminal projections following early axonal injury, and contribute to cerebral atrophy and neurological dysfunctions at the chronic phase 
. Indeed, increased spacing among remaining neurons occurred in head injury patients with a more severe outcome 
. Volumetric magnetic resonance imaging (MRI) studies of chronic TBI provides clinical observation for the decrease in volume of grey matter, which is associated with impaired behavioral outcomes in long-term observation 
. We noted that long-term preservation of residual tissue was associated with better performances in behavior tasks following salidroside treatment. Our findings suggest that salidroside effectively ameliorated the pathological events leading to post-traumatic deficits during the first 24 h, which consequently led to a better prolonged recovery of neurological function.
We observed that salidroside attenuated brain water content in CCI mice, providing evidence for the effect of salidroside on brain edema after TBI. Cerebral edema has been reported to be one of the major factors leading to the high mortality and morbidity associated with patients with TBI 
. Cation accumulation is detected in cells undergoing apoptosis, and is induced by apoptotic signaling 
. This accumulation drives the influx of water into cells and induces cellular swelling, causing cytotoxic edema 
. In addition, apoptosis enhances inflammation as immune cells are activated by apoptotic cells to clean them up. The cerebral inflammatory response may further damage the microvascular endothelium and lead to blood-brain barrier (BBB) disruption and vasogenic edema 
. Hence, the anti-edematous effect of salidroside observed in our study is likely to be related to the inhibition of apoptosis.
We found that p-Akt Ser 473 and Thr 308 were constitutively expressed in sham-injured brains, but were diminished 1 day post-injury as apoptotic signatures arose. Both p-Akt Ser 473 and Thr 308 were predominantly localized in neurons post-injury, but rarely found in astrocytes or microglia. These results suggest that the expression of Akt phosphorylation is related to neuronal cell survival after TBI. Previous studies have demonstrated that p-Akt levels are time- and region-dependent after TBI 
. This difference may reflect the degree of neuronal damage. Indeed, p-Akt Ser 473 was decreased in the injured cortex of mice after CCI, whereas it was transiently increased in the peri-lesional cortex adjacent to the impact site 
. This regional difference in Akt activation was also observed following experimental cerebral ischemia 
. We showed that salidroside treatment increased cortical p-Akt levels and LY294002, a specific PI3K inhibitor, abolished salidroside-induced protection against tissue loss, indicating that the Akt signaling pathway was necessary for the salidroside-induced neuroprotection in this study. Consistent with our findings, several preclinical studies, using various neuroprotective agents, such as growth factors 
, estrogen 
, and statins 
, have reported Akt activation as a potential therapeutic target following TBI. Although pretreated LY294002 reduced p-Akt Thr 308 levels, the p-Akt Ser 473 levels remained unchanged at 1 day post-injury. A reason for this result could be the short half-
life of LY294002. Since LY294002 was administered 30 min before CCI, the decreased Akt phosphorylation could have been transient (i.e., several hours after treatment).
An unexpected finding of our study was that levels of the downstream targets of Akt kinase activity, p-Bad and p-FOXO1, remained unchanged after salidroside treatment at 1 and 3 days post-injury. Our results are consistent with previous findings in experimental stroke that showed that phosphorylation levels of factors downstream of Akt, such as Bad 
and GSK3β 
, do not increase together with p-Akt; rather, both of them decrease when p-Akt Ser 473 increases. One possible explanation for these disparities is that Bad and FOXO1 phosphorylation and inhibition could be induced via other signaling pathways, such as MEK/MAPK 
and SGK3 
. Thus, the levels of p-Bad and p-FOXO1 that we measured might may have resulted from activation of other upstream kinases at time points earlier than day 1.
We showed that administration of salidroside decreased cytosolic levels of mitochondrial inner proteins CytoC and Smac/DIABLO and reduced cleavage of caspase-3. This result indicates that salidroside reduced apoptosis at least partially via the mitochondria-dependent endogenous pathway. Mitochondrial integrity is highly controlled, primarily through interactions between pro- and anti-apoptotic members of the Bcl-2 protein family, such as Bcl-2 and Bax 
. Mitochondrial permeabilization needs Bax activation and its translocation from cytosol onto the mitochondrial surface 
Our data showed that salidroside treatment attenuated the mitochondrial Bcl-2/Bax ratio, but the ratio remained unchanged in whole cytosol lysate. These findings confirm previous in vitro
findings that salidroside modulates the balance of apoptosis-regulating proteins and preserves mitochondrial integrity and further demonstrate that this beneficial effect is functional in mouse TBI. However, the beneficial effects of salidroside may be in part attributed to the prevention of free radical and oxidant formation, since salidroside exerts potent antioxidant effects in vitro
. Further investigations are needed to clarify the anti-oxidative mechanism underlying the salidroside-mediated neuroprotection in TBI.
We observed that a single post-injury injection of 20 mg/kg salidroside protected mice against TBI but did not cause kidney or liver damage. Our present results are consistent with previous reports on animal models of focal cerebral ischemia 
and Alzheimer’s disease 
. Nevertheless, previous studies applied prophylactic treatment and only tested the efficacy of salidroside by morphological changes or at a fairly early time point (1 day) after treatment. Our results showing that salidroside administered at 6 h post-injury also exerts a neuroprotective effect is particularly important because it may mean that salidroside could be used clinically for treating TBI. The pharmacokinetic profile of salidroside has not been well established. The mean elimination half-life (t1/2
) of salidroside following intravenous or oral administration was around 0.5 h and 1.1 h, respectively, in rats 
. However, the pharmacokinetics in brain tissue or after intraperitoneal administration has not yet been reported. We found that both SALD 20 and SALD 50 were neuroprotective but the SALD50 was less effective than the SALD 20 group. Our results were consistent with those of previous findings showing reduced effectiveness of a higher dose of Rhodiola. rosea
extract on cognition function of rats 
. Since salidroside has been demonstrated to enhance immune responses and stimulate cytokine productions both in human peripheral blood lymphocytes 
and in mice 
, one possibility is that the enhancement of inflammation by salidroside overcomes its anti-apoptotic effect when a higher dose is administered. The reduced effectiveness of a higher dose of salidroside may also be attributed to its sedative effects on the central nervous system 
In conclusion, the present study showed that post-injury salidroside administration improved long-term functional and histological outcomes following TBI. This improvement was associated with preservation of mitochondrial integrity and increased Akt activation, and pharmacological PI3K/Akt inhibition reversed the protective effect. Our results suggest that the neuroprotective effects of SALD following TBI are mediated, at least in part, through activation of the PI3K/Akt signaling pathway.