Dong-Fu Feng

Quantitative evaluation of microscopic injury with diffusion tensor imaging in a rat model of diffuse axonal injury

Diffuse axonal injury (DAI) is the predominant effect of severe traumatic brain injury and contributes significantly to neurological deficits. However, it is difficult to diagnose or characterize non‐invasively with conventional imaging. Our study provides significant validation of a visual and statistical diffusion tensor imaging (DTI) technique as compared with pathological and electron microscopic study in a rat DAI model at multiple predilection sites and time points following trauma. Two DTI parameters, fractional anisotropy (FA) and axial diffusivity (AD), were significantly reduced from 12 h to 5 days post‐trauma, corresponding to pathological axonal injury. At 7 days post‐trauma, FA remained decreased, whereas AD pseudo‐normalized and radial diffusivity increased. The temporal alterations in DTI parameters were observed in multiple predilection sites, and the extent of the changes in these parameters correlated significantly with the severity of histologically visualized axonal injury, as assessed by integrated optical density of immunochemically stained injured axons with quantitative stereology. Although anatomical T2‐weighted magnetic resonance images showed no abnormal signals in microscopic lesions, we detected and characterized axonal injury directly by DTI at each time point. These results demonstrate that DTI has significant potential as a non‐invasive tool with which to quantitatively diagnose and evaluate microstructural injury in the experimental and clinical assessment of DAI. This method can assist in accurate evaluation of the extent of axonal injury, detection of severe predilection foci, determination of approximate time of injury, and monitoring of the pathogenic condition at the early post‐injury stage.

Upregulation of SATB1 is associated with the development and progression of glioma

BackgroundSpecial AT-rich sequence-binding protein-1 (SATB1) has been reported to be expressed in several human cancers and may have malignant potential. This study was aimed at investigating the expression and potential role of SATB1 in human glioma.MethodThe relationship between SATB1 expression, clinicopathological parameters, Ki67 expression and MGMT promoter methylation status was evaluated, and the prognostic value of SATB1 expression in patients with gliomas was analyzed. SATB1-specific shRNA sequences were synthesized, and U251 cells were transfected with SATB1 RNAi plasmids. Expression of SATB1 mRNA and protein was investigated by RT-PCR and immunofluoresence staining and western blotting. The expression of c-Met, SLC22A18, caspase-3 and bcl-2 protein was determined by western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. The growth and angiogenesis of SATB1 low expressing U251 cells was measured in an in vivo xenograft model.ResultsOf 70 tumors, 44 (62.9%) were positive for SATB1 expression. SATB1 expression was significantly associated with a high histological grade and with poor survival in univariate and multivariate analyses. SATB1 expression was also positively correlated with Ki67 expression but negatively with MGMT promoter methylation in glioma tissues. SATB1 shRNA expression vectors could efficiently induce the expression of SLC22A18 protein, increase the caspase-3 protein, inhibit the expression of SATB1, c-Met and bcl-2 protein, the growth, invasion, metastasis and angiogenesis of U251 cells, and induce apoptosis in vitro. Furthermore, the tumor growth of U251 cells expressing SATB1 shRNA were inhibited in vivo, and immunohistochemical analyses of tumor sections revealed a decreased vessel density in the animals where shRNA against SATB1 were expressed.ConclusionsSATB1 may have an important role as a positive regulator of glioma development and progression, and that SATB1 might be a useful molecular marker for predicting the prognosis of glioma.

Biomarkers Associated With Diffuse Traumatic Axonal Injury: Exploring Pathogenesis, Early Diagnosis, and Prognosis

BACKGROUND Diffuse traumatic axonal injury (dTAI) is a significant pathologic feature of traumatic brain injury and is associated with substantial mortality and morbidity. It is still a challenge for clinicians to make an early diagnosis of dTAI and generate accurate prognosis and direct therapeutic decisions because most patients rapidly progress to coma after trauma and because specific neurologic symptoms and focal lesions detectable with current routine imaging techniques are absent. To address these issues, many investigations have sought to identify biomarkers of dTAI. METHODS This article is a review of the pertinent medical literature. RESULTS From the perspective of the pathophysiology of dTAI, we reviewed several biomarkers that are associated with structural damage and biochemical cascades in the secondary injury or repair response to traumatic brain injury. Although some biomarkers are not specific to dTAI, they are nevertheless useful in elucidating its pathogenesis, making early diagnosis possible, predicting outcomes, and providing candidate targets for novel therapeutic strategies. CONCLUSIONS The availability of biomarker data, clinical case histories, and radiologic information can improve our current ability to diagnose and monitor pathogenic conditions and predict outcomes in patients with dTAI.

Correlation of low SLC22A18 expression with poor prognosis in patients with glioma.

We investigated the expression of the putative tumor suppressor SLC22A18 to evaluate it as a prognostic marker in glioma patients. Immunohistochemical and Western blot analyses of clinical tissue samples obtained from 120 patients with glioma were performed. Low expression of SLC22A18 was observed in 71.7% of patients. Loss of SLC22A18 expression in glioma was significantly related to pathological grade (p = 0.003). High pathological grade (World Health Organization III-IV) was correlated with negative (low or absent) expression of SLC22A18, which was correlated with a significantly shorter overall patient survival than in those with positive (high) expression (p = 0.007). Multivariate Cox regression analysis indicated that SLC22A18 expression level is an independent survival prognostic factor for patients with glioma (p = 0.011). Western blotting analysis confirmed decreased expression of SLC22A18 in glioma tissues compared with adjacent brain tissues. This study suggests that SLC22A18 functions as a tumor suppressor in glioma and represents a candidate biomarker for long-term survival in this disease.

Promoter methylation and downregulation of SLC22A18 are associated with the development and progression of human glioma

BackgroundDownregulation of the putative tumor suppressor gene SLC22A18 has been reported in a number of human cancers. The aim of this study was to investigate the relationship between SLC22A18 downregulation, promoter methylation and the development and progression of human glioma.MethodSLC22A18 expression and promoter methylation was examined in human gliomas and the adjacent normal tissues. U251 glioma cells stably overexpressing SLC22A18 were generated to investigate the effect of SLC22A18 on cell growth and adherence in vitro using the methyl thiazole tetrazolium assay. Apoptosis was quantified using flow cytometry and the growth of SLC22A18 overexpressing U251 cells was measured in an in viv o xenograft model.ResultsSLC22A18 protein expression is significantly decreased in human gliomas compared to the adjacent normal brain tissues. SLC22A18 protein expression is significantly lower in gliomas which recurred within six months after surgery than gliomas which did not recur within six months. SLC22A18 promoter methylation was detected in 50% of the gliomas, but not in the adjacent normal tissues of any patient. SLC22A18 expression was significantly decreased in gliomas with SLC22A18 promoter methylation, compared to gliomas without methylation. The SLC22A18 promoter is methylated in U251 cells and treatment with the demethylating agent 5-aza-2-deoxycytidine increased SLC22A18 expression and reduced cell proliferation. Stable overexpression of SLC22A18 inhibited growth and adherence, induced apoptosis in vitro and reduced in vivo tumor growth of U251 cells.ConclusionSLC22A18 downregulation via promoter methylation is associated with the development and progression of glioma, suggesting that SLC22A18 is an important tumor suppressor in glioma.

Hydroxyapatite nanoparticles inhibit the growth of human glioma cells in vitro and in vivo

Hydroxyapatite nanoparticles (nano-HAPs) have been reported to exhibit antitumor effects on various human cancers, but the effects of nano-HAPs on human glioma cells remain unclear. The aim of this study was to explore the inhibitory effect of nano-HAPs on the growth of human glioma U251 and SHG44 cells in vitro and in vivo. Nano-HAPs could inhibit the growth of U251 and SHG44 cells in a dose- and time-dependent manner, according to methyl thiazoletetrazolium assay and flow cytometry. Treated with 120 mg/L and 240 mg/L nano-HAPs for 48 hours, typical apoptotic morphological changes were noted under Hoechst staining and transmission electron microscopy. The tumor growth of cells was inhibited after the injection in vivo, and the related side effects significantly decreased in the nano-HAP-and-drug combination group. Because of the function of nano-HAPs, the expression of c-Met, SATB1, Ki-67, and bcl-2 protein decreased, and the expression of SLC22A18 and caspase-3 protein decreased noticeably. The findings indicate that nano-HAPs have an evident inhibitory action and induce apoptosis of human glioma cells in vitro and in vivo. In a drug combination, they can significantly reduce the adverse reaction related to the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).

c-Met-targeted RNA interference inhibits growth and metastasis of glioma U251 cells in vitro

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. c-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, invasion, and angiogenesis. The present study was designed to determine the role of c-Met in growth and metastasis of glioma U251 cells using RNA interference (RNAi) technology in vitro. We constructed three kinds of shRNA expression vectors aiming at the c-Met gene, then transfected them into glioma U251 cells by lipofectamineTM 2000. The level of c-Met mRNA was investigated by real-time polymerse chain reaction (RT-PCR). The protein expression of c-Met was observed by immunofluoresence staining and western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. We got three kinds of c-Met specific shRNA expression vectors which could efficiently inhibit the growth and metastasis of U251 cells and the expression of c-Met in U251 cells. RT-PCR, immunofluoresence staining and western blotting showed that inhibition rate for c-Met expression was up to 90%, 79% and 85%, respectively. The expression of c-Met can be inhibited by RNA interference in U251 cells, which can inhibit the growth and metastasis of U251 cell and induce cell apoptosis. These results indicate that RNAi of c-Met can be an effective antiangiogenic strategy for glioma.

Diffuse axonal injury induced by simultaneous moderate linear and angular head accelerations in rats

Diffuse axonal injury (DAI) is one of the most common and important pathologic features of human traumatic brain injury (TBI), accounting for high mortality and development of persistent post-traumatic neurologic sequelae. Although a relatively high number of therapies have been shown to be effective in experimental models, there are currently few treatments that are effective for improving the prognosis of clinical DAI. A major reason is the failure of current models to validly reproduce the pathophysiological characteristics observed after clinical DAI. In the present study, we employed a specially designed, highly controllable model to induce a sudden rotation in the coronal plane (75 degrees rotation at 1.6x10(4) degrees/s) combined with lateral translation (1.57 cm displacement at 3.4x10(2) cm/s) to the rats head. We were interested in discovering whether the combined accelerations could reproduce the pathophysiological changes analogous to those seen in human DAI. The axonal injury as assessed with amyloid protein precursor (APP) as a marker was consistently present in all injured rats. The commonly injured brain regions included the subcortical regions, deep white matter, corpus callosum and brain stem. The evolution of APP accumulations in brain sections depicted the detailed progression of axonal pathology. Ultrastructural studies gave further insights into the presence and progression of axonal injury. All injured rats exhibited transient physiological dysfunction, as well as immediate and dramatic neurological impairment that still persisted at 14 days after injury. These results suggest that this model reproduced the major pathophysiological changes analogous to those observed after severe clinical TBI and provides an attractive vehicle for experimental brain injury research.

Detection of white matter lesions in the acute stage of diffuse axonal injury predicts long-term cognitive impairments: a clinical diffusion tensor imaging study.

BACKGROUND White matter disruption is known to contribute to neurocognitive deficits after diffuse axonal injury (DAI). This study evaluated the relationship between white matter integrity using diffusion tensor imaging in the early stage and cognitions in the chronic stage. METHODS Diffusion tensor imaging was performed in 15 patients with DAI within 7 days of injury and in 15 patients in the control group. Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were calculated within regions of interest, including the posterior limb of the internal capsule, uncinate fasciculus (UF), anterior corona radiate (ACR), superior longitudinal fasciculus (SLF), inferior longitudinal fasciculus (ILF), genu of the corpus callosum, body of the corpus callosum, and splenium of the corpus callosum and cingulum bundle (CB). The patients with DAI and the patients in the control group also underwent neuropsychological testing during the chronic stage after DAI. RESULTS The region-of-interest analysis showed significantly reduced FA and AD values in all nine regions within 7 days of injury as well as increased MD values in the corpus callosum among patients in the DAI group. The patients demonstrated significantly poorer performance on the working memory tests and attention test. In patients, working memory function was positively correlated with the AD value in the UF and with the FA value in the CB, UF, SLF, and ILF. Working memory function was inversely correlated with the RD value in the CB, SLF, and ILF and with the MD value in the SLF and ILF. In addition, the attention function demonstrated a positive correlation with the RD value in the ACR, SLF, and ILF and with the MD value in the ACR, SLF, and ILF. In addition, attention was inversely correlated with the FA values for the posterior limb of the internal capsule, ACR, SLF, and ILF. CONCLUSION The results indicated that the presence of white matter changes during the early stage of DAI may be helpful for predicting cognitive dysfunction over the long term. LEVEL OF EVIDENCE Prognostic study, level III.

Standardizing optic nerve crushes with an aneurysm clip.

Abstract Objectives: Despite the widespread use of optic nerve injury models to simulate central nervous system injury, model protocols vary from laboratory to laboratory, making it difficult to directly compare findings between studies. Methods: To standardize the optic nerve crush injury model, the commercially available Yasargil aneurysm clip, which provides a consistent clamping force, was used to produce a crush injury to the rat optic nerve. Histology was verified with hematoxylin–eosin. The number of retinal ganglion cells (RGCs) was counted by fluorescent gold dye labeling. Results: Following nerve crush injury, the density of RGCs was substantially reduced in the aneurysm clip-operated group relative to the normal and sham-operated groups, and no discernable difference was noted between the latter two control groups. Discussion: The present findings suggest that Yasargil aneurysm clip effectively produces permanent injury to the optic nerve with evidence from retrograde tracing of RGCs and may provide a standard technique for optic nerve crush studies.