A Non-Invasive Method for Monitoring Osteogenesis and Osseointegration Using Near-Infrared Fluorescent Imaging: A Model of Maxilla Implantation in Rats.

Currently, computed tomography and conventional X-ray radiography usually generate a micro-artifact around metal implants. This metal artifact frequently causes false positive or negative diagnoses of bone maturation or pathological peri-implantitis around implants. In an attempt to repair the artifacts, a highly specific nanoprobe, an osteogenic biomarker, and nano-Au-Pamidronate were designed to monitor the osteogenesis. In total, 12 Sprague Dawley rats were included in the study and could be chategorized in 3 groups: 4 rats in the X-ray and CT group, 4 rats in the NIRF group, and 4 rats in the sham group. A titanium alloy screw was implanted in the anterior hard palate. The X-ray, CT, and NIRF images were taken 28 days after implantation. The X-ray showed that the tissue surrounded the implant tightly; however, a gap of metal artifacts was noted around the interface between dental implants and palatal bone. Compared to the CT image, a fluorescence image was noted around the implant site in the NIRF group. Furthermore, the histological implant-bone tissue also exhibited a significant NIRF signal. In conclusion, this novel NIRF molecular imaging system precisely identifies the image loss caused by metal artifacts and can be applied to monitoring bone maturation around orthopedic implants. In addition, by observing the new bone formation, a new principle and timetable for an implant osseointegrated with bone can be established and a new type of implant fixture or surface treatment can be evaluated using this system.

Protective multi­target effects of DL­3­n­butylphthalide combined with 3­methyl­1­phenyl­2­pyrazolin­5­one in mice with ischemic stroke.

DL­3­n­butylphthalide (NBP) and 3­methyl­1- phenyl­2­pyrazolin­5­one (edaravone) are acknowledged neuroprotective agents that protect against ischemic stroke. However, the underlying mechanisms of a combination therapy with NBP and edaravone have not yet been fully clarified. The aim of the present study was to explore whether the co­administration of NBP and edaravone had multi­target protective effects on the neurovascular unit (NVU) of mice affected by ischemic stroke. Male C57BL/6 mice were randomly divided into the following three groups: i) Sham operation control, ii) middle cerebral artery occlusion (MCAO) and reperfusion, iii) and MCAO/reperfusion with the co­administration of NBP (40 mg/kg) and edaravone (6 mg/kg) delivered via intraperitoneal injection at 0 and 4 h after reperfusion (NBP + edaravone). After ischemia and reperfusion, infarct volumes and neurological deficits were evaluated. The immunoreactivity of the NVU, comprising neurons, endothelial cells and astrocytes, was determined using immunofluorescence staining of neuronal nuclei (NeuN), platelet and endothelial cell adhesion molecule 1 (CD31) and glial fibrillary acidic protein (GFAP). Western blotting was used to detect the expression levels of apoptosis­related proteins. The infarct volume, neurological function scores and cell damage were increased in the MCAO group compared with the sham operation group. Furthermore, the MCAO mice had reduced NeuN and CD31 expression and increased GFAP expression compared with the sham group. By contrast, the NBP + edaravone group exhibited reduced cell damage and consequently lower infarct volume and neurological deficit scores compared with the MCAO group. The NBP + edaravone group exhibited increased NeuN and CD31 expression and decreased GFAP expression compared with the MCAO group. Furthermore, the expression levels of Bax and cleaved caspase­3 in the NBP + edaravone group were decreased significantly compared with the MCAO group, while the expression levels of Bcl­2 and mitochondrial cytochrome c were increased. In conclusion, the results of the present study demonstrated that NBP and edaravone effectively prevented ischemic stroke damage with multi­target protective effects. In addition, NBP + edaravone may be a promising combination therapy for ischemic stroke.

MPP+ inhibits mGluR1/5-mediated long-term depression in mouse hippocampus by calpain activation.

Neurotoxins are harmful to nervous system and cause either neuronal cell death or impairment of synaptic activity, which contributes to Parkinson's disease or other neuronal disorders. Hippocampal synaptic plasticity was proposed as a cellular model for memory processing. In this study, we reported a novel effect of neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), on metabotropic glutamate receptor 1/5 agonist, 3,5-dihydroxyphenylglycine (DHPG)-induced hippocampal synaptic plasticity, and MPP+ incubation blocked DHPG-induced hippocampal long-term depression (LTD) in Schaffer collateral-CA1 synapses. Our further findings indicated that, this blockage was reversed by pre-application of calpain inhibitor III, but not by cathepsin inhibitors. Biochemical analysis showed that MPP+ treatment stimulated calpain activation, displayed by spectrin breakdown. Interestingly, the level and activity of protein tyrosine phosphatase 1B (PTP1B) were reduced after MPP+ incubation and the decrease of PTP1B was prohibited by calpain inhibitor III. In addition, PTP1B inhibitor also blocked DHPG-induced LTD, mimicking the effect of MPP+. In summary, our data implicated that MPP+ activated calpain-dependent PTP1B degradation, which subsequently impaired hippocampal LTD. This novel effect of MPP+ might partially explain the impairment of memory processing in the pathogenesis of PD.

GPR30 Activation Contributes to the Puerarin-Mediated Neuroprotection in MPP+-Induced SH-SY5Y Cell Death.

The neuroprotective action of puerarin in Parkinson's disease (PD) models has been well investigated. However, the mechanisms involved in protection have not been completely understood. G protein-coupled receptor 30 (GPR30) is a G protein-coupled estrogen receptor and considered a potential target in the neuroprotection against PD. In this study, we investigated whether puerarin prevented against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death via GPR30. Our results showed that the GPR30 agonist, G1, exhibited puerarin-mediated neuroprotection against MPP+-induced cell death of SH-SY5Y cells. This protective action was reversed by the GPR30 antagonist. Moreover, a time- and concentration-dependent effect of puerarin on GPR30 expression was verified at the protein level but not at the mRNA level. Further, we showed that an mTor-dependent new GPR30 synthesis contributed to the protection conferred by puerarin. Finally, glial cell line-derived neurotrophic factor (GDNF) levels were enhanced by puerarin and G1 in both control and MPP+-lesioned cells via GPR30. Taken together, our data strongly suggest that puerarin prevents MPP+-induced cell death via facilitating GPR30 expression and GDNF release.

Curcumin rescues aging-related loss of hippocampal synapse input specificity of long term potentiation in mice.

Curcumin has neuroprotective effect and could enhance memory. However, the mechanisms underlying the protection of curcumin on aging-related memory decline are not well understood. In this study, high frequency stimulation (HFS)-induced long term potentiation (LTP) was evaluated by a cellular model of memory formation. A two-input stimulation paradigm was used to record the potentiation as well as synapse input specificity. The data suggested that an N-Methyl-D-aspartate receptors (NMDAR) -dependent LTP was inducible in adult hippocampal slices with a characteristic of synapse input specificity. It also indicated that aging resulted in a reduction in LTP but more importantly a loss of synaptic input specificity. The reason behind the above conclusions is that LTP induction is more dependent on the calcium channel. This is due to a switch of the dependence of LTP induction to voltage-dependent calcium channel (VDCC) compared to NMDA receptors. Curcumin administration recovers input specificity by re-establishing NMDA receptor dependence of induction. In addition, curcumin administration ameliorated aging-related increase of brain thiobarbituric acid-reactive substances and elevated aging-related decrease of glutathione in hippocampus. It is then concluded that curcumin modulates hippocampal redox status and restores aging-related loss of synapse input specificity of HFS-induced LTP by switching VDCC calcium source into NMDA receptor-dependent one.

[Protective effects of puerarin against 1-methyl-4-phenylpyridinium-induced mitochondrial apoptotic death in differentiated SH-SY5Y cells].

It is well known that puerarin possesses protective activity on neurodegenerative diseases. However, the exact path way involved in the protective effect of puerarin on MPP+ -induced cell death is unclear. In this study, we focused on mitochondria im pairment in the apoptotic process of MPP+ -elicited SH-SY5Y cells and detected the protection of puerarin. As evidenced by Trypan blue assay, the cell viability was significantly decreased by 1 mmol x L(-1) MPP+, but reversed by different concentrations puerarin pre treatment. Flow cytometer analysis revealed that MPP+ -induced SH-SY5Y cells apoptosis and arrested the cells in G2/M phase, where as puerarin pretreatment concentration dependently reversed the apoptosis ratio. In addition to the apoptosis ratio, 50.0 micromol x L(-1) puerarin pretreatment even altered the MPP+ -induced G2/M phase arrest. JC-1 assay suggested that MPP+ significantly opened MMP of the SH-SYSY cells; pretreatment with puerarin attenuated the deterioration of the MMP. Both ELISA and Western blotting showed that puerarin prevented the release of cytochrome c from the mitochondrial interior to the cystol elicited by MPP+. DNA ladder showed that typical DNA ladder was present in the MPP+ -induced SH-SY5Y cells. Additionally, MPP+ enhanced caspase-9 and caspase-3 ac tivity, respectively, while not caspase-8. However,the enhancement was concentration dependently blocked by puerarin pretreatment. Taken together, puerarin can modulate mitochondrial membrane potential and inhibit the cytochrome c releasing-caspase cascade to pre vent MPP+ -induced cell injury.

Involvement of ubiquitin proteasome system in protective mechanisms of Puerarin to MPP(+)-elicited apoptosis.

It has been well documented that dysfunction of ubiquitin proteasome system (UPS) in the neuron exacerbated the Parkinson's disease (PD). However, whether or not UPS is involved in the protective effect of Puerarin on 1-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine (MPP(+))-elicited cell death is yet to be elucidated. In this study, treatment of SH-SY5Y cells with 1mM MPP(+)-elicited a characteristic apoptotic cell death and pretreatment with Puerarin protected cells against MPP(+)-induced apoptosis as evidenced by promoting cell viability, improving morphological changes and reducing apoptotic rate. To further explore the potential protective mechanism of Puerarin in MPP(+)-induced SH-SY5Y cell death, UPS activity, mitochondria-dependent apoptosis and caspase-3 activity were measured. Puerarin pretreatment attenuated MPP(+)-induced dysfunction of protease activity, thereby reducing accumulation of ubiquitin-conjugated proteins. Meanwhile, caspase-3 activity was remarkably attenuated by Puerarin. In addition, the ratio of bcl-2/bax was increased by Puerarin in comparison with MPP(+)-treated group. Taken together, these results suggest that Puerarin could protect MPP(+)-induced SH-SY5Y cells from apoptosis by regulating the function of UPS.