Molecular alterations in the hippocampus after experimental subarachnoid hemorrhage

Patients with aneurysmal subarachnoid hemorrhage (SAH) frequently have deficits in learning and memory that may or may not be associated with detectable brain lesions. We examined mediators of long-term potentiation after SAH in rats to determine what processes might be involved. There was a reduction in synapses in the dendritic layer of the CA1 region on transmission electron microscopy as well as reduced colocalization of microtubule-associated protein 2 (MAP2) and synaptophysin. Immunohistochemistry showed reduced staining for GluR1 and calmodulin kinase 2 and increased staining for GluR2. Myelin basic protein staining was decreased as well. There was no detectable neuronal injury by Fluoro-Jade B, TUNEL, or activated caspase-3 staining. Vasospasm of the large arteries of the circle of Willis was mild to moderate in severity. Nitric oxide was increased and superoxide anion radical was decreased in hippocampal tissue. Cerebral blood flow, measured by magnetic resonance imaging, and cerebral glucose metabolism, measured by positron emission tomography, were no different in SAH compared with control groups. The results suggest that the etiology of loss of LTP after SAH is not cerebral ischemia but may be mediated by effects of subarachnoid blood such as oxidative stress and inflammation.     

Aging aggravates long-term renal ischemia-reperfusion injury in a rat model

Aim

Ischemia-reperfusion injury (IRI) has been considered as the major cause of acute kidney injury and can result in poor long-term graft function. Functional recovery after IRI is impaired in the elderly. In the present study, we aimed to compare kidney morphology, function, oxidative stress, inflammation, and development of renal fibrosis in young and aged rats after renal IRI.

Materials and methods

Rat models of warm renal IRI were established by clamping left pedicles for 45 min after right nephrectomy, then the clamp was removed, and kidneys were reperfused for up to 12 wk. Biochemical and histologic renal damage were assessed at 12 wk after reperfusion. The immunohistochemical staining of monocyte macrophage antigen-1 (ED-1) and transforming growth factor beta 1 (TGF-β1) and messenger RNA level of TGF-β1 in the kidney were analyzed.

Results

Renal IRI caused significant increases of malondialdehyde and 8-hydroxydeoxyguanosine levels and a decrease of superoxide dismutase activity in young and aged IRI rats; however, these changes were more obvious in the aged rats. IRI resulted in severe inflammation and tubulointerstitial fibrosis with decreased creatinine (Cr) clearance and increased histologic damage in aged rats compared with young rats. Moreover, we measured the ratio of Cr clearance between young and aged IRI rats. It demonstrated that aged IRI rats did have poor Cr clearance compared with the young IRI rats. ED-1 and TGF-β1 expression levels in the kidney were significantly higher in aged rats than in young rats after IRI.

Conclusion

Aged rats are more susceptible to IRI-induced renal failure, which may associate with the increased oxidative stress, increased histologic damage, and increased inflammation and tubulointerstitial fibrosis. Targeting oxidative stress and inflammatory response should improve the kidney recovery after IRI.     

Intranasal nerve growth factor bypasses the blood-brain barrier and affects spinal cord neurons in spinal cord injur y

The purpose of this work was to investigate whether, by intranasal administration, the nerve growth factor bypasses the blood-brain barrier and turns over the spinal cord neurons and if such therapeutic approach could be of value in the treatment of spinal cord injury. Adult Sprague-Dawley rats with intact and injured spinal cord received daily intranasal nerve growth factor administration in both nostrils for 1 day or for 3 consecutive weeks. We found an in-creased content of nerve growth factor and enhanced expression of nerve growth factor receptor in the spinal cord 24 hours after a single intranasal administration of nerve growth factor in healthy rats, while daily treatment for 3 weeks in a model of spinal cord injury improved the deifcits in locomotor behaviour and increased spinal content of both nerve growth factor and nerve growth factor receptors. These outcomes suggest that the intranasal nerve growth factor bypasses blood-brain barrier and affects spinal cord neurons in spinal cord injury. They also suggest exploiting the possible therapeutic role of intranasally delivered nerve growth factor for the neuroprotection of damaged spinal nerve cells.     

Green tea polyphenols protect spinal cord neurons against hydrogen peroxide-induced oxidative stress

Green tea polyphenols are strong antioxidants and can reduce free radical damage. To investigate their neuroprotective potential, we induced oxidative damage in spinal cord neurons using hydrogen peroxide, and applied different concentrations(50–200 μg/mL) of green tea polyphenol to the cell medium for 24 hours. Measurements of superoxide dismutase activity, malondialdehyde content, and expression of apoptosis-related genes and proteins revealed that green tea polyphenol effectively alleviated oxidative stress. Our results indicate that green tea polyphenols play a protective role in spinal cord neurons under oxidative stress.     

Hyperbaric oxygen therapy improves local microenvironment after spinal cord injury

Clinical studies have shown that hyperbaric oxygen therapy improves motor function in patients with spinal cord injury. In the present study, we explored the mechanisms associated with the recovery of neurological function after hyperbaric oxygen therapy in a rat model of spinal cord injury. We established an acute spinal cord injury model using a modification of the free-falling object method, and treated the animals with oxygen at 0.2 MPa for 45 minutes, 4 hours after injury. The treatment was administered four times per day, for 3 days. Compared with model rats that did not receive the treatment, rats exposed to hyperbaric oxygen had fewer apoptotic cells in spinal cord tissue, lower expression levels of aquaporin 4/9 mRNA and protein, and more NF-200 positive nerve fibers. Furthermore, they had smaller spinal cord cavities, rapid recovery of somatosensory and motor evoked potentials, and notably better recovery of hindlimb motor function than model rats. Our findings indicate that hyperbaric oxygen therapy reduces apoptosis, downregulates aquaporin 4/9 mRNA and protein expression in injured spinal cord tissue, improves the local microenvironment for nerve regeneration, and protects and repairs the spinal cord after injury.     

Temporal delimitation of the healing phases via monitoring of fracture callus stiffness in rats

The healing process consists of at least three phases: inflammatory, repair, and remodeling phase. Because callus stiffness correlates with the healing phases, it is suitable for evaluating the fracture healing process. Our aim was to develop a method which allows determination of callus stiffness in vivo, the healing time and the duration of the repair phase. The right femurs of 16 Wistar rats were osteotomized and stabilized with either more rigid or more flexible external fixation. Fixator deformation was measured using strain gauges during gait analysis. The strains were recalculated as the callus stiffness over the time course of healing, and the healing phases were identified based on stiffness thresholds. Our hypothesis was that stabilization with more flexible external fixation prolongs the repair phase, therefore resulting in an extended healing time. Confirming our hypothesis, the duration of the repair phase (rigid: approximately 15 days, flexible: approximately 41 days) and the healing time (rigid: approximately 27 days, flexible: approximately 62 days) were significantly longer for more flexible external fixation. Our method allows the quantitative detection of differences in the healing time and duration of the repair phase without multiple time‐point sacrifices, which reduces the number of animals in experimental studies. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1589–1595, 2014.     

大鼠髁突软骨细胞发育中相关信号通路的初步筛查

目的:通过建立的大鼠髁突软骨细胞发育过程中蛋白表达差异谱,分析可能参与的信号通路,为进一步认识髁突软骨生理性及病理性生长改建的信号调控机制提供相关信息。方法:收获出生后1、7、14、28 d共4组SD大鼠髁突软骨细胞,甲苯胺蓝染色、Ⅱ型胶原免疫组化染色鉴定软骨细胞。提取各组软骨细胞总蛋白,采用i TRAQ标记定量蛋白,2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(MALDI-TOF-TOF),获取出生后大鼠髁突软骨细胞发育过程中差异蛋白的表达谱,所得数据用MASCOT软件处理,筛选样本之间有意义的差异蛋白,运用GO法及David软件进行KEGG信号通路分析。结果 :共鉴定137种具有可信度表达的蛋白,有44种蛋白参与至少27条KEGG信号通路,其中ECM-受体相互作用、焦点粘连、actin骨架调节、Ca2+信号通路、血管平滑肌收缩、Gn RH信号通路、肌醇三磷酸代谢、磷脂酰肌醇信号系统以及核糖体等信号通路具有统计学意义。结论:在大鼠髁突软骨发育中,各信号通路蛋白在时间、空间上差异性表达,共同形成复杂的信号传递网络。     

FGF-9参与调控小鼠上颌突间充质细胞体外成骨分化的研究

目的:研究成纤维细胞生长因子9(FGF-9)对体外培养的小鼠上颌突间充质细胞成骨分化的调控作用。方法 :体外培养E12.5(胚胎第12.5天)的小鼠上颌突间充质细胞,取第1代细胞进行成骨诱导,实验组加入FGF-9人重组蛋白,诱导培养1周后通过q PCR、免疫荧光和茜素红染色检测其成骨能力。结果:体外培养的E12.5小鼠上颌突间充质细胞表达FGF9和FGFR3。成骨诱导可促进上颌突间充质细胞表达成骨标记物ALP、Runx2和OCN;加入FGF-9人重组蛋白可降低成骨标记物ALP、Runx2和OCN的表达,减少钙结节形成。结论:体外培养时,FGF9参与负调控上颌突间充质细胞的成骨分化。