Inhibition of mammalian target of rapamycin (mTOR) improves neurobehavioral deficit and modulates inflammatory response after traumatic brain injury

Traumatic brain injury (TBI) induce primary and secondary damage on endothelium and brain parenchyma, leading neurons die rapidly by necrosis. The mammalian target of rapamycin signalling pathway (mTOR) mediates many aspects of cell growth and regeneration and is up-regulated after moderate to severe traumatic brain injury (TBI). The significance of this increased signalling event for recovery of brain function is presently unclear, here we used two different selective inhibitors of mTOR activity to explore the functional role of mTOR inhibition in an validated model of TBI, the controlled cortical impact injury (CCI). We treated animals withKU0063794, a dual mTORC1 and mTORC2 inhibitor, and with rapamycin a well-known inhibitor of mTOR, 1 and 4 hours after TBI. Our results demonstrated that mTOR inhibitors, especially KU0063794, significantly improve motor and cognitive recovery after TBI as well as reduce lesion volumes. Moreover we observed that mTOR inhibitors treatment ameliorate the neuroinflammation associated to TBI and showed that this acute treatment significantly diminished the extent of neuronal death, astrogliosis and apoptotic process after trauma. Our findings suggest that the neuronal mTORC1/2 activity after TBI is deleterious to brain function and acute intervention with selective mTORC1/2 inhibitor may represent an effective therapeutic strategy to improve recovery after brain trauma.


Introduction
Traumatic brain injury (TBI) is a serious public health problem affecting several people worldwide.
Even mild, closed-head trauma to the brain can lead to temporary or permanent neurological symptoms, including epileptic seizures, behavioural changes, and impaired motor and cognitive function. It is worth mentioning that neurological, behavioural and craniotomy (n = 20); TBI + vehicle: mice were subjected to CCI and vehicle (DMSO) was administered at 1 after craniotomy(n = 20); TBI + Rapamycin (1 mg/kg): mice were subjected to CCI and Rapamycin (1 mg/kg in 10% DMSO) was administered orally 1 h and 4 h after craniotomy(n = 20); TBI + KU0063794 (1 mg/kg): mice were subjected to CCI and KU0063794 (1 mg/kg in 10% DMSO) was administered intraperitoneally i.p. 1 h and 4 h after craniotomy (n = 20).
Sham + Rapamycin (1 mg/kg): mice were subjected to the surgical procedures as above group (anaesthesia and craniotomy) except that the impact tip was not applied and Rapamycin (1 mg/kg) was administered at 1 after craniotomy(n = 20).
Sham + KU0063794 (1 mg/kg): mice were subjected to the surgical procedures as above group (anaesthesia and craniotomy) except that the impact tip was not applied and KU0063794 (1 mg/kg) was administered at 1 after craniotomy(n = 20).
As describe below mice (n = 20 from each group and 10 for each technique) were sacrificed at 24 h after TBI to evaluate the various parameters.

Behavioural testing
In another set of experiment, all animals were subjected to behavioural tests. All behavioural testing was conducted during the light cycle phase and in enclosed behaviour rooms (50-55 dB ambient noise) within the housing room. The mice were placed in behaviour rooms 5 min for 2 days for acclimation prior to the onset of behavioural testing.
The behavioural tests were conducted by three different reliable expert observers blinded to the injury status of the animals. Tests are described below:

Rotarod test
The rotarod treadmill (Accuscan, Inc., Columbus, OH, USA) provided a motor balance and coordination assessment. This test was performed as previously described (16). Each animal was placed in a neutral position on a cylinder (1 cm diameter for mice), then the rod was rotated with the speed accelerated linearly from 0 to 24 rpm within 60 s, and the time spent on the rotarod was recorded automatically. The maximum score given to an animal was fixed to 60. For testing, animals were given three trials and the average score was used as the individual rotarod score.

Elevated Biased Swing Test
The EBST provided a motor asymmetry parameter and involved handling the animal by its tail and recording the direction of the biased body swings. The EBST consisted of 20 trials with the number of swings ipsilateral and contralateral to the injured hemisphere recorded and expressed in percentage to determine the biased swing activity. This analysis was performed as previous described (16).
Tissue processing and histology A qualified histopathologist evaluated coronal sections of 7-µm thickness from the perilesional brain area of each animal. Damaged neurons were counted and the histopathologic changes of the gray matter were scored on a six-point scale (22): 0, no lesion observed; 1, gray matter contained one to five eosinophilic neurons; 2, gray matter contained five to 10 eosinophilic neurons; 3, gray matter contained more than 10 eosinophilic neurons; 4, small infarction (less than one third of the gray matter area); 5, moderate infarction (one third to one half of the gray matter area); 6, large infarction (more than half of the gray matter area). The scores from all the sections of each brain were averaged to give a final score for individual mice. All the histological studies were performed in a blinded fashion.

Quantification of lesion volume
The animals were anesthetized with ketamine, decapitated and their brains carefully removed. The brains were cut into 5 coronal slices of 2 mm thickness by using a McIlwain tissue chopper (Campdem instruments LTD). Slices were incubated in 2% solution of 2,3,5triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, Saint Louis, Missouri, USA) in Phosphate-buffered (0.1 mol/l) saline (PBS; pH 7.4) at 37 °C for 30 min and immersion fixed in 10% buffered formalin solution. Infracted area and volume was calculated from digital images (Canon 4×, Canon Inc., China) and ImageJ software 36. To account for brain edema, the lesioned areas were corrected by subtracting the area of the contralateral hemisphere area from the ipsilateral hemisphere 37. The corrected total lesion volume was estimated by summing the lesioned area in every slice and multiplying it by slice thickness (2 mm). Lesion volume and area were measured on coronal brain slices for a total of three slices per animal.

Immunohistochemistry
Tissue segments containing the lesion (1 cm on each side of the lesion) were fixed in 10% (w/v) buffered formaldehyde 24 h after TBI and sliced in 7-µm sections for paraffinembedding previously described (15). After deparaffinization, endogenous peroxidase was quenched with 0.30% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: anti-GFAP The immunohistochemical images were collected by Zeiss microscope using Axio Vision software. For graphic representation of densitometric analyses, we measured the intensity of positive staining (brown staining) by computer-assisted color image analysis (Leica QWin V3, UK). The percentage area of immunoreactivity (determined by the number of positive pixels) was expressed as percent of total tissue area (red staining) as seen previously (17).

Immunofluorescence staining
After deparaffinization and rehydration, detection of NEU-N was carried out after boiling in 0.1 M citrate buffer for one minute as described previously (18). Non-specific adsorption was minimized by incubating the section in 2% (volume/ volume (vol/vol)) normal goat serum in PBS for 20 minutes. Sections were incubated with mouse anti-Neun-N (1:100, vol/vol EMD Millipore) antibody in a humidified oxygen and nitrogen chamber for over night at 37 °C. Sections were incubated with secondary antibody Fluorescein isothiocyanate (FITC)-conjugated anti-mouse Alexa Fluor-488 antibody (1:2,000 vol/vol Molecular Probes, Monza, Italy) for one hour at 37 °C. For nuclear staining, 2 µg/ml 4′,6′diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS was added. To verify the binding specificity for used antibodies, control slices were incubated with only primary antibody or secondary antibody. In these controls no positive staining was detected Sections were observed at 20X magnification using a Leica DM2000 microscope (Leica, Milan, Italy). Optical sections of fluorescence specimens were obtained using a HeNe laser (543 nm), an ultraviolet laser (361 to 365 nm) and an argon laser (458 nm) at a oneminute, two seconds scanning speed with up to eight averages; 1.5 µm sections were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly labeled pixels and applying settings that allowed clear visualization of structural details, while keeping the highest pixel intensities close to 200. The same settings were used for all images obtained from the other samples that had been processed in parallel. Digital images were cropped and figure montages prepared using Adobe Photoshop 7.0 (Adobe Systems; Palo Alto, California, United States). Cell counting analysis was made on rostro-caudal brain slices for a total of three slices per animal (n = 10 for each group).

Materials
Rapamycin and KU0063794 was obtained by Tocris Bioscience (98%). All other chemicals were of the highest commercial grade available. All stock solutions were made in nonpyrogenic saline (0.9% NaCl, Baxter, Milan, Italy) or 10% dimethyl sulfoxide (DMSO).
Except otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd(Milan, Italy). All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter,Italy) or 10% dimethyl sulfoxide.

Statistical evaluation
All values in the figures and text are expressed as mean ± standard error of the mean (SEM) of N number of animals. In those experiments involving histology or immunohistochemistry, the pictures exhibited are representative of at least three experiments performed on different days. Results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p-value < 0.05 was considered significant. For one-way ANOVA statistic test, a single "F" value indicated as variation between sample means/variation within the samples was shown.

KU0063794 treatment reduced the severity of brain trauma and the infarct outcome
To evaluate the effects of mTOR inhibitors on brain infarctions in the TBI, we performed TTC staining. An increased necrotic tissue area was observed in TBI mice compared to Also, to consider the correlation between neurological deficit and motor function in the setting of TBI, we performed EBST and rotarod test. Mice subjected to CCI showed a range of impairments in locomotor tasks as showed in Fig. 1D and 1E. KU0063794 treatment more efficacy than Rapamycin group, improved latency compared to TBI group ( Fig. 1D and E respectively).

Neuronal injury examination after TBI
To evaluate the contusion areas after TBI, the sections obtained from each group were stained with hematoxylin and eosin (H&E) staining. Histological examination revealed a significant tissue disorganization and white matter alteration in the brain parenchyma of TBI mice compared to sham animals ( Fig. 2A  Moreover, NF-κBp65 nuclear translocation was significantly increased after TBI, compared with sham group (Fig. 4B,B1); instead KU0063794 treatment considerably decreased the levels of NF-κBp65 more effectively than Rapamycin (Fig. 4B,B1).

KU0063794 and Rapamycin attenuates inflammatory response induced by astrogliosis and microgliosis
Thus to evaluate the anti-inflammatory and neuroprotective effect of KU0063794 and Rapamycin treatments, we performed immunohistochemistry staining for TNFα and IL-1β.
We demonstrated that both TNFα and IL-1β levels were significantly increased after TBI ( Fig. 5B and F respectively, see densitometry analysis I and J respectively) compared to control group ( Fig. 5A and E respectively, see densitometry analysis I and J respectively).
Meanwhile, KU0063794 and Rapamycin treatments significantly attenuated TNFα ( Fig. 5C and D respectively, see densitometry analysis I and J respectively) and IL-1β ( Fig. 5G and H respectively, see densitometry analysis I and J respectively) production, stimulated by TBI-induced microgliosis, with a great trend of protection with KU0063794 treatment.
Microgliosis and astrogliosis, meant as astrocytes and microglia activation, are a key component of the pathological onset and progression of TBI. Thus we evaluated, by immunohistochemistry staining, the expression of Iba1 and GFAP, as a marker of microglial and astrocyte activation respectively. A substantial increase in GFAP and Iba1 expressions ( Fig. 6B and F respectively, see densitometry analysis I and J) were found in mice subject to TBI compared to sham animal ( Fig. 6A and E respectively, see densitometry analysis I and J respectively). Whereas astrogliosis and microgliosis were significantly attenuated by KU0063794 ( Fig. 6D and H respectively, see densitometry analysis I and J respectively) and Rapamycin treatments ( Fig. 6C and G respectively, see densitometry analysis I and J respectively), with an higher trend of protection was observed after KU0063794 treatment. KU0063794 and Rapamycin modulates COX2 and iNOS expression in the brain after TBI ROS-induced lipid peroxidation is the most studied mechanism of oxidative damage in TBI.
A major enzymatic pathways in lipid peroxidation involved activation of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) that we evaluated by western blot analysis. A substantial increase in COX2 and iNOS expression was observed in the brain from mice obtained at 24 h after TBI (Fig. 7C,C1 and D,D1) while KU0063794 treatment significantly reduced both expression more then the treatment with Rapamycin (Fig. 7C,C1 and D,D1).
Effects of KU0063794 and Rapamycin on apoptosis pathway in the brain after TBI To test whether brain damage was associated with apoptosis, the role of Bax and Bcl-2, a pro and anti-apoptotic factors respectively, was investigated by immunohistochemical staining. The expression of Bax was substantially increased in the brain subjected to TBI However, the use of rapamycin has limitations and warrant caution in the interpretation of results; for instance, the drug is recognised to confer nonspecific inhibition of other kinase complexes, such as mTORC2 and generate substantial side effects especially when treatment is long-drawn-out.
Thus, considered that an early intervention post-TBI could suppress neuronal mTORC activation reducing not only neuronal damage but also prevent glial dysfunction at later stages, we performed a CCI model of TBI that reproduces motor deficits and neuron loss that are evinced after TBI and we evaluated a neuroprotective effects of highly specific small-molecule inhibitor of mTOR kinase Ku0063794.
Ku0063794 inhibits both mTORC1 and mTORC2 thought phosphorylation of S6K1 and 4E-BP1, which are downstream substrates of mTORC1, and Akt phosphorylation on Ser473, which is the target of mTORC2 (26). We supposed that the strategies to target both mTORC1 and mTORC2 may produce better responses after TBI as well as we wonder that KU0063794, has less toxicity of Rapamycin and permit a clear interpretation of data.
Thus, in our work we evaluated the effect of Ku0063794 in the control of the inflammatory process associated to TBI, as in the activation of NF-κB pathway, in the modulation of astrogliosis and microgliosis as well as in the control of pro-inflammatory cytokines production.
The early phase of damage usually occurs within minutes or 24 h following impact and it is directly associated to tissue damage, neurological dysfunction attributed to rapid cell death resulted in extensive dendritic degeneration and synapse reduction. Histological evaluation demonstrated that treatment with Ku0063794 determinate a reduction of the lesion area and showed a minor morphological modification that are visible following TBI.
Moreover, to assess the neurodegeneration occurring at an early stage following TBI, we evaluated by immunofluorescence staining a marker of mature neurons NeuN.
We observed that the expression of NeuN in the cortex and in the hippocampus was significantly decreased in mice subject to TBI; whereas the treatment with rapamycin and more efficacy with KU0063794 increased the number of NeuN positive cells, confirming the beneficial effect of mTOR inhibition on the loss of neuronal cells.
Following TBI, the inflammatory condition is a typical response that occurs to the adult mammalian CNS. It is know that some inflammatory mediators are locally released after injury and interact to control the cellular changes that occur in TBI. In particular, reactive gliosis is initiated in the surrounding neural tissue and spreads along the edges of the wound by the proliferation and migration of glial cells, this extended microglial activation at the focal site of injury becomes detrimental over time (27). Microglia are rapidly activated increasing in cell numbers at the site of the insult and produce inflammatory mediators such as pro-inflammatory cytokines that leads to actiavtion of astroglial and neovascularisation at trauma sites. Thus to better recognise if the mTOR inhibition could modulate the inflammatory process involved in TBI we evaluated the role of mTOR inhibitors in the control of the inflammatory pathway NF-κB as well as in decreasing microglia and astrocytes activation.
Our results clearly demonstrated that rapamycin and significantly better KU0063794, reduced the translocation of NF-κB in to the nucleus, translocation that is considerably increased in TBI group. NF-κB activation during TBI and the consequent translocation in the nucleus determinate the activation and the production of inflammatory factors such as pro-inflammatory cytokines. Thus, treatment with mTOR inhibitors along with NF-κB modulation had the capacity to decrease the amount of inflammatory cytokines, such as TNF-α and IL1-β. Therefore, pro-inflammatory cytokines are synthesized and secreted by astrocytes and microglia; consequently we investigate the role of mTOR inhibition in modulating astrogliosis and microgliosis by immunostaining for GFAP and IBA1 respectively markers for astrocytes and microglia activation. Accordingly we observed that rapamycin and much more KU0063794 significantly reduced astrocytes and microglia activation.
Once secreted, these pro-inflammatory cytokines can bind specific receptors to increase the amount of iNOS and COX2, as well as they can act as molecular inducers of programmed cell death or apoptosis (28).
We evaluated that mTOR inhibition regulate the expression of iNOS and COX2 that are significantly increased after TBI.
Moreover, an ensuing event associated with inflammation after TBI is the secondary cell death process of apoptosis (29). It is generally recognised that one mechanism underlying apoptotic cell death in TBI is a shift in the balance between pro-and anti-apoptotic factors towards the expression of proteins that promote cell death (30). Therefore, in the present study we also observed the role of mTOR signalling on cell death through the modulation of pro-and anti-apoptotic factors such as BAX and Bcl2 and in particular we observed that the treatment with mTOR inhibitors significantly reduced BAX expression and restored Bcl2 levels as control levels.
Thus, various inflammatory mediators play a pivotal role in produces systemic tissue damage following acute TBI and limiting the influx of inflammatory cells to the site of injury is a valuable approach to modulate the extent and distribution of inflammatory factors expressed in the injured CNS. Moreover, identifying the signalling pathway that could sustain microglia preserving their regenerative function after injury versus the predominating inflammatory activity, will provide an homeostatic mechanisms in maintaining a healthy brain.
Thereby, here we identify that mTOR activation, in hippocampal neurons, drives to cognitive dysfunction, neuronal damage, widespread astrogliosis and microgliosis. In particular, early intervention with mTOR inhibitors is considerably beneficial to limit tissue damage and improve functional recovery; especially we defined that inhibition of both mTORC1 and mTORC2 resulted more efficacy in reducing microglia and macrophage activation and significantly improving brain function.
In conclusion, a fuller understanding of the above pathophysiological processes will undoubtedly help to develop early diagnosis and potential therapeutic strategies and decrease the mortality rate for the TBI patients.

Declarations
Ethics approval and consent to participate:

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests