Adult Sprague-Dawley male rats weighing 280–320 g were obtained from the Laboratory Animal Center of the Fourth Military Medical University. The animals had continuous access to food and water and were housed in cages in a room maintained at 20–22˚C with a 12 h light/12 h dark cycle. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University.

PD98059 and LY294002 (Cell Signaling Technology, Ozyme, France) were dissolved in DMSO and diluted in saline (1% final DMSO concentration). CHPG (Sigma, Saint Louis, MO, USA) was dissolved in saline. For the in vitro experiments, CHPG (1 mM), PD98059 (10 μM) or LY294002 (50 μM) was directly added into the culture medium 30 min before traumatic injury was induced. For the in vivo experiments, vehicle (1% DMSO in saline), CHPG (250 nM), PD98059 (5 nM) or LY294002 (15 nM) was injected in a volume of 5 μl into right lateral ventricle (anteroposterior, 0.8 mm; lateral, 1.5 mm; depth, 3.5 mm from bregma) 30 min before TBI.

Cortical neurons were cultured from Sprague-Dawley rats using a modified method that has been previously described (26). Briefly, cortical tissue was removed from embryos at 16–18 days, and maintained in PBS at 4˚C during dissection. Tissues were dissociated by 0.25% trypsin digestion for 15 min at 37˚C and gentle trituration. Neurons were resuspended and plated onto poly-D-Lysine-coated (50 μg/ml) 60 mm culture dishes at a density of 3×10⁵ cells/cm². The neurons were cultured in neurobasal medium (Gibco, Gaithersburg, MD, USA) containing 2% B27, 0.5 mM L-glutamine and 100 U/ml penicillin at 37˚C in a humidified 5% CO₂ incubator and half of the culture medium was changed every other day. Cultures were utilized for experiments at 8–10 days when more than 95% of cells were cortical neurons as determined by immunofluorescence staining of neurofilament 200 (data not shown).

Our in vitro trauma model was based somewhat on the mechanical injury model described previously (17,27). Briefly, each 60 mm dish confluent culture was manually scratched with a sterile plastic pipette tip following a 20×20-square grid (with 3 mm spacing between the lines). These cuts caused immediate death to cells directly under the blades, followed by progressive secondary injury of neurons at a distance from these cuts. After injury, the cultures were washed with PBS to remove cellular debris, and then incubated for further 24 h at 37˚C in a humidified 5% CO₂ incubator.

The release of LDH, a cytoplasmic enzyme released from neurons with ruined cell membranes, was used as a marker of neuronal damage and was assessed 24 h after traumatic injury. The amount of LDH released into the medium was measured using a diagnostic kit according to the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China). Pyruvate and reduced form of nicotinamide-adenine dinucleotide (NADH) were added into the medium samples from each group, and after 15 min of incubation at 37˚C, the reaction was stopped by adding 0.4 mol/l NaOH. The absorbance of the sample was read at 490 nm, and the results were expressed as a percentage of LDH release from the sample vs. the maximal value, which was determined by treating control cultures with 1% Triton X-100 for 60 min to lyse all cells.

Neuronal apoptosis was analyzed by staining the nuclear chromatin with Hoechst 33342 (Molecular Probes, USA). In brief, 24 h after TBI the culture medium was removed and neurons were washed with PBS. Hoechst 33342 (5 μg/ml) was added, and then neurons were maintained for 15 min at 37˚C in a CO₂ incubator. Finally, these labeled neurons were observed using a Leica fluorescence microscope (B-251, Berlin, Germany), and the number of apoptotic cells with nuclear condensation and fragmentation were counted. Apoptotic rate is presented as the percentage of the total number of neurons.

Traumatic brain injury in vivo was induced as previously described (28). In brief, rats were anesthetized using 2% isoflurane in oxygen and placed in the stereotaxic frame. A craniotomy was performed using a portable drill and a 5 mm trephine over the right parietotemporal cortex. The resulting bone flap was removed and the dura remained intact. To induce injury, a pneumatic piston impactor device with a 5 mm diameter and rounded tip (Biomedical Engineering Facility, Virginia Commonwealth University, Richmond, VA) was used to impact the brain at a depth of 2 mm for 250 ms. After injury, the bone flap was replaced and sealed with bone wax. Sham animals underwent similar anesthetic and surgical interventions, including craniotomy, but did not receive the TBI application. Core body temperature was continuously monitored with a rectal probe and maintained at 37˚C with a thermostatically controlled heating pad during surgery.

Apoptosis in brain sections was detected by the TUNEL assay, a method used to observe DNA strand breaks in nuclei. In brief, after being washed in Tris-HCl (pH 7.7) three times, sections were treated with proteinase K solution (20 μg/ml) for 10 min at room temperature to permeabilize tissues. Sections were then labelled with fluorescein TUNEL reagent mixture for 60 min at 37˚C according to the manufacturer's suggested protocol (Promega Corporation, Madison, USA). The reactions were terminated by immersing the sections in 2X SSC buffer (0.3 M NaCl, 30 mM Na₃C₆H₅O₇, pH 7.0) for 15 min at room temperature. After that, sections were examined by fluorescence microscopy and the number of TUNEL-positive (apoptotic) cells was counted in five fields in each section.

Lesion volume was measured 7 days after TBI. Rats were anesthetized with 4% isoflurane in oxygen and decapitated. The brains were rapidly removed and cooled in iced saline for 10 min. At each 500 μm interval, 30 μm sections were mounted on slides and stained with 0.2% cresyl violet solution (Sigma Chemical, St. Louis, MO) to visualize lesions. The areas of the lesions were integrated, and the results are presented as percentage of control.

For Western blot analysis, cortical neurons and tissue samples were homogenized in a lysis buffer containing protease inhibitor 1 mM PMSF and phosphatase inhibitors 10 mM glycerophosphate, 10 mM NaF and 0.3 mM Na₃Vo₄. The lysates were sonicated and centrifuged, and the protein concentration was determined using a BCA protein assay kit (Jiancheng Bioengineering Institute). Equivalent amounts of protein (40 μg/lane) were loaded and separated on 10% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk solution in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h, and then incubated overnight at 4˚C with the following primary antibody dilutions in TBST: anti-p-ERK1/2, ERK1/2, 1:800; p-Akt and Akt, 1:1,000 (Cell Signaling Technology, Danvers, MA). After that the membranes were washed and incubated with secondary antibody for 1 h at room temperature. The analysis software ImageJ was used to quantify the optical density of each band. The activation of Akt and ERK1/2 is presented as the ratio of phosphorylated kinase bands to the total kinase bands.

Statistical analysis was performed using SPSS 16.0, a statistical software package. All data are presented as mean ± SD. Statistical evaluation of the data was performed by one-way ANOVA followed by Student-Newman-Keuls test (SNK test) for comparison of differences between the two groups by ANOVA. A value of P<0.05 was considered statistically significant. All apoptosis measures were analyzed by observers that were blinded to treatment grouping.

To determine the potential protective effects of CHPG in an in vitro trauma model, LDH release and Hoechst 33342 staining were measured 24 h after mechanical injury (Fig. 1). In this model, injury to neuron cultures markedly increased LDH release, and this increase was attenuated by the addition of 1 mM CHPG. CHPG significantly reduced the neuronal apoptotic rate from 28.9±1.1% in the TBI group to 11.4±1.0% in the TBI+CHPG group (Fig. 1B and C). The results in the control group and the CHPG group were not apparently different, suggesting that CHPG in all concentrations used have no cytotoxicity.

To assess the efficacy of CHPG in an in vivo model of TBI, rats were randomly divided into the following four groups, the vehicle group (which received saline but did not undergo TBI application), the CHPG group (which was treated with CHPG but did not undergo TBI application), the TBI group (which underwent TBI application) and the TBI+CHPG group (which was treated with CHPG and underwent TBI application). Rats in the first two groups underwent similar surgical procedure, but TBI was not induced. There were no obvious TUNEL-positive cells in the vehicle group or the CHPG group (Fig. 2). However, 24 h after TBI the number of TUNEL-positive cells significantly increased (117.0±5.0 for the TBI group). Pre-treatment with 250 nM of CHPG attenuated this increase to 63±7.0 for the TBI+CHPG group. Cresyl violet staining, as a measure of cerebral lesion volume, was done 7 days after TBI, and the results demonstrated that the lesion volume of the TBI+CHPG group was significantly smaller than that of the TBI group (P<0.05).

To explore possible mechanisms of CHPG-induced neuroprotection, the expression levels of total and phosphorylated ERK and Akt, two pro-survival molecules downstream of mGluR5, were examined by Western blot analysis. CHPG induced an up-regulation (212±13% in vitro and 156±7% in vivo) of phosphor-ERK (p-ERK) after traumatic injury, but with no effect on control neurons or in the non-injured animals (Fig. 3). The expression of phsopho-Akt (p-Akt) was reduced following traumatic injury. CHPG treatment alone increased p-AKT levels (145±8% in vitro and 189±11% in vivo), an effect which was augmented following traumatic injury (178±9% in vitro and 289±10% in vivo).

To further elucidate the mechanism of neuroprotection by CHPG, two antagonists PD98059 and LY294002 were used in both in vitro and in vivo models of TBI to block ERK and Akt activation, respectively. CHPG reduced the TBI-induced LDH release and neuronal apoptosis, and these effects were diminished by application of either PD98059 or LY294002 (Fig. 4A and B). Similar results were obtained in the in vivo model of TBI. Specifically, pretreatment with either PD98059 or LY294002 significantly increased the number of TUNEL-positive cells and the lesion volume compared to the TBI+CHPG group (Fig. 4C and D), suggesting that neuroprotection was partially reversed. When PD98059 and LY294002 were used together, the CHPG-induced reduction of TBI-induced apoptosis both in vitro and in vivo was abolished (Fig. 4B and C). LDH release and lesion volume were increased following co-application of PD98059 and LY294002 as compared to the TBI+CHPG group, but still lower than TBI group (P<0.05), suggesting that CHPG-induced protection was attenuated, but not totally reversed.