Epigallocatechin Gallate Preferentially Inhibits O6-Methylguanine DNA-Methyltransferase Expression in Glioblastoma Cells Rather than in Nontumor Glial Cells.

OBJECTIVE: O6-methylguanine (O6-meG) DNA-methyltransferase (MGMT) is a main regulator of temozolomide (TMZ) resistance in glioblastomas. Some MGMT inhibitors have been studied in clinical trials but with very little success, because their inhibiting effects were not tumor-selective, and often cause severe toxicity in normal tissues in the presence of TMZ. The goal of this study is to explore whether Epigallocatechin gallate (EGCG), a natural small molecule, could preferentially modulate MGMT in glioblastoma cells. METHODS: Two MGMT-positive glioblastoma cell lines (GBM-XD and T98G) and one nontumor glial cell culture (GliaX) were included in this study. The MGMT promoter methylation status, mRNA abundance, and protein levels were determined before and after EGCG treatment. The mechanisms were characterized. RESULTS: EGCG substantially suppressed mRNA and protein expression of MGMT, and reversed TMZ resistance in MGMT-positive GBM-XD and T98G cells via the WNT/ß-catenin pathway. EGCG prevented ß-catenin translocation into the nucleus and might directly inhibit the transcription factors TCF1 and LEF1. Meanwhile, EGCG enhanced the MGMT expression in the nontumor glial cells, through inhibition of the DNMT1 and demethylation of MGMT promoter. CONCLUSIONS: EGCG preferentially inhibits MGMT and enhances TMZ cytotoxicity in glioblastoma cells rather than in nontumor glial cells.

FM19G11 inhibits O6 -methylguanine DNA-methyltransferase expression under both hypoxic and normoxic conditions.

FM19G11 is a small molecular agent that inhibits hypoxia-inducible factor-1-alpha (HIF-1α) and other signaling pathways. In this study, we characterized the modulating effects of FM19G11 on O6 -methylguanine DNA-methyltransferase (MGMT), the main regulator of temozolomide (TMZ) resistance in glioblastomas. This study included 2 MGMT-positive cell lines (GBM-XD and T98G). MGMT promoter methylation status, mRNA abundance, and protein levels were determined before and after FM19G11 treatment, and the roles of various signaling pathways were characterized. Under hypoxic conditions, MGMT mRNA and protein levels were significantly downregulated by FM19G11 via the HIF-1α pathway in both GBM-XD and T98G cells. In normoxic culture, T98G cells were strongly positive for MGMT, and MGMT expression was substantially downregulated by FM19G11 via the NF-κB pathway. In addition, TMZ resistance was reversed by treatment with FM19G11. Meanwhile, FM19G11 has no cytotoxicity at its effective dose. FM19G11 could potentially be used to counteract TMZ resistance in MGMT-positive glioblastomas.

Postoperative pneumocephalus increases the recurrence rate of chronic subdural hematoma.

OBJECTIVES: Pneumocephalus is a common operative complication of chronic subdural hematoma. This study is to analyze the relationship between postoperative pneumocephalus and the recurrence and surgical outcomes. PATIENTS AND METHODS: This is a retrospective case-cohort study, including a pneumocephalus group (n = 46) and a control group (n = 181). Their recurrence rates, CT attenuation values, hospital stay, healing time and the neurological status were recorded and analyzed. RESULTS: The pneumocephalus group had a recurrence rate of 32.6%, significantly higher than the control (17.7%). In addition, the pneumocephalus group had a higher rate of postoperative epilepsy (21.7% vs 3.3%), longer hospital stay (11.5 ±â€¯2.8 vs 7.8 ±â€¯1.2 days), longer healing time (10.8 ±â€¯5.4 vs 6.5 ±â€¯2.3 months), and worse neurological scores than the control. CONCLUSION: Pneumocephalus increases the recurrence rate of chronic subdural hematoma, and it not only prolongs the hospital stay and healing time, but also leads to deterioration of the neurological status.

Microvascular Decompression for Trigeminal Neuralgia: Zone Exploration and Decompression Techniques.

OBJECTIVE: The aim of this study is to introduce zone exploration of the trigeminal nerve and decompression techniques for different types of vasculars. METHODS: The trigeminal nerve was sectioned into 5 zones. Zone 1, 2, 3, 4 was located at the rostral, caudal, ventral, and dorsal part of the nerve root entry zone (REZ) respectively, and zone 5 was located at the distal of the nerve root. This study contained 86 patients with trigeminal neuralgia underwent microvascular decompression. Every zone was exposed through preoperative imaging. During the operation, offending vessels were explored from zone 1 to zone 5, and different decompression techniques were used for different types of vessels. RESULTS: Through zone exploration, the sensitivity of preoperative imaging was 96.5% and specificity was 100%. Location of the neurovascular conflict was in the zone 1 in 53.5% of the patients, zone 2 in 32.6%, zone 3 in 45.3%, zone 4 in 29.1%, and zone 5 in 34.9%. In total, 2 zones were both involved in 59.3%, and 3 zones were involved in 18.6%. All offending arteries were moved away and interposed with Teflon sponge. Offending veins of 11 patients were too small to interpose, and coagulated and cut was adopted. The other offending veins were interposed with wet gelatin and Teflon sponge, respectively. CONCLUSIONS: Zone exploration is helpful in finding offending vessels and adequate decompression can be achieved by choosing different methods according to different types of offending vessels.

Management of vessels passing through the facial nerve in the treatment of hemifacial spasm.

BACKGROUND: In hemifacial spasm, it is extremely rare to find a vessel passing through the facial nerve. In this study, we present our experience of the surgical treatment of four such patients. METHODS: From January 2010 to Match 2015, we treated 2,576 hemifacial spasm patients with microvascular decompression in our department. Of these, four had an intraneural vessel. Intraoperative findings and treatment were recorded, and postoperative outcomes were analyzed. RESULTS: In three patients, the intraneural vessel was the anterior inferior cerebellar artery, which we wrapped with small pieces of wet gelatin and Teflon sponge. A small vein found in the fourth patient was treated with facial nerve combing. Complete decompression was achieved and abnormal muscle response disappeared. Three patients got an excellent result and one patient got a good result. One patient had postoperative facial paralysis, which improved over 10 months of follow-up. CONCLUSION: If an artery passes through the facial nerve, it can be decompressed by wrapping the vessel with wet gelatin and Teflon sponge. If a vein passes through the facial nerve, combing can be used. Intraoperative abnormal muscle response monitoring is very helpful in achieving complete decompression.

Nimodipine-mediated re-myelination after facial nerve crush injury in rats.

This study aimed to investigate the mechanism of nimodipine-mediated neural repair after facial nerve crush injury in rats. Adult Sprague-Dawley rats were divided into three groups: healthy controls, surgery alone, and surgery plus nimodipine. A facial nerve crush injury model was constructed. Immediately after surgery, the rats in the surgery plus nimodipine group were administered nimodipine, 6 mg/kg/day, for a variable numbers of days. The animals underwent electromyography (EMG) before surgery and at 3, 10, or 20 days after surgery. After sacrifice, nerve samples were stained with hematoxylin and eosin (H&E) and luxol fast blue. The EMG at 20 days revealed an apparent recovery of eletroconductivity, with the surgery plus nimodipine group having a higher amplitude and shorter latency time than the surgery only group. H&E staining showed that at 20 days, the rats treated with nimodipine had an obvious recovery of myelination and reduction in the number of infiltrating cells, suggesting less inflammation, compared with the rats in the surgery only group. Luxol fast blue staining was relatively even in the surgery plus nimodipine group, indicating a protective effect against injury-induced demyelination. Staining for S100 calcium-binding protein B (S-100ß) was not evident in the surgery alone group, but was evident in the surgery plus nimodipine group, indicating that nimodipine reversed the damage of the crush injury. After a facial nerve crush injury, treatment with nimodipine for 20 days reduced the nerve injury by mediating remyelination by Schwann cells. The protective effect of nimodipine may include a reduction of inflammation and an increase in calcium-binding S-100ß protein.

The mechanism of hemifacial spasm: a new understanding of the offending artery.

Although neurovascular confliction was believed to be the cause of hemifacial spasm (HFS), the mechanism of the disorder remains unclear to date. Current theories, merely focusing on the facial nerve, have failed to explain the clinical phenomenon of immediate relief following a successful microvascular decompression surgery (MVD). With the experience of thousands of microvascular decompression surgeries and preliminary investigations, we have learned that the offending artery may play a more important role than the effect of merely mechanical compression in the pathogenesis of the disease. We believe that the attrition of neurovascular interface is the essence of the etiology, and the substance of the disease is emersion of ectopic action potentials from the demyelinated facial nerve fibers, which were triggered by the sympathetic endings from the offending artery wall. In this paper, we put forward evidence to support this hypothesis, both logically and theoretically.

The strategy of microvascular decompression for hemifacial spasm: how to decide the endpoint of an MVD surgery.

OBJECTIVE: Microvascular decompression (MVD) has become the standard treatment for hemifacial spasm. As not all patients get complete relief, this strategy is still controversial. The study aimed to figure out how to tell the proper endpoint to the surgery. METHODS: A series of 356 consecutive patients with hemifacial spasm were enrolled in this study. All patients fell into two groups according to the period they presented. Two different criteria (simple criterion vs. complex criterion) to end an operation were applied respectively. The intra-operative finding, results and complications of these two groups were compared. The advantage of the complex criterion was analyzed. RESULTS: The group which used complex criterion got better results than the group which used simple criterion. The complex criterion which combines full-length evidence, vascular evidence and electrophysiological evidence proved to be reliable to tell the proper endpoint to the surgery. CONCLUSION: MVD operations can be ended only after the full-length evidence, vascular evidence and electrophysiological evidence are all present.

The role of autonomic nervous system in the pathophysiology of hemifacial spasm.

OBJECTIVES: Despite the vascular compression of the seventh cranial nerve has been verified by the microvascular decompression surgery as the cause of hemifacial spasm (HFS), the mechanism of the disease is still unknown. We believe that the autonomic nervous system in adventitia of the offending artery may contribute to the HFS. To prove our hypothesis, we performed an experiment in SD rats. METHODS: Moller's HFS model was adopted and the abnormal muscle response (AMR) wave was electrophysiologically monitored. With randomization, some HFS rats underwent exclusion of the offending artery or removal of the ipsilateral superior cervical ganglion. Some HFS rats with negative AMR following exclusion of the offending artery were dripped with norepinephrine onto the neurovascular conflict site. RESULTS: With exclusion of the offending artery, AMR disappeared in 14 (70%) of the 20 HFS rats, while in three (30%) of the 10 from sham operation group (P<0·05). With ganglionectomy, AMR disappeared in 12 (75%) of the 16 HFS rats, while in two (25%) of the eight from the sham operation group (P<0·05). With norepinephrine drip, AMR reappeared in four (67%) of the six from those offending-artery-excluded HFS rats, while in zero of the six from normal-saline-dripped group (P<0·05). DISCUSSION: The neurotransmitter releasing from the autonomic nervous endings in the worn adventitia of the offending artery may induce an ectopia action potential in those demyelinated facial nerve fibers expanding to the neuromuscular conjunction and trigger an attack of HFS.

[The immunogenicity of mouse neural stem cells].

AIM: To explore the immunogenicity of mouse neural stem cells (NSCs), thus help to solve the problem of immunologic rejection in NSCs transplantation. METHODS: (1) Thirty C57BL/6 mice were assigned randomly into two groups, then the two groups were immuned by intraperitoneal injection respectively with BALB/c mice's NSCs or hepatic cells (as control). Afterwards one-way mixed lymphocyte culture was performed with BALB/c mice's NSCs or hepatic cells and immuned C57BL/6 mice's T lymphocytes. Immune response of T lymphocyte was detected by liquid scintillation counter. (2) The MHC-I and MHC-II molecules of BALB/c mice NSCs were stained with FITC- and PE-labeled antibody respectively. Then the expression of MHC-I and MHC-II were detected by flow cytometry. RESULTS: As shown by liquid scintillation counter, the cpm value of NSCs was 16592.8 +/- 2865.3, and that of the hepatic cells was 27815.0 +/- 2416.3 (P<0.01). About (87.55 +/- 3.65)% NSCs expressed neither MHC-I nor MHC-II, but there were only about (27.45 +/- 1.86)% hepatocytes showed both MHC-I and MYC-II negative (P<0.01). CONCLUSION: The mouse NSCs show weaker immunogenicity and their immunogenicity is significantly weaker than their hepatocytes.

Surgery for posttraumatic syringomyelia: a retrospective study of seven patients.

OBJECTIVE: To analyze retrospectively the clinical symptoms, signs, radiological findings and results of treatment of posttraumatic syringomyelia. METHODS: The data of 7 patients with posttraumatic syringomyelia confirmed by computerized tomography (CT) and magnetic resonance imaging (MRI) in our hospital between 1999 and 2004 were reviewed retrospectively. The patients underwent decompressive laminectomy or syringo-subarachnoid (S-S) shunting with microsurgery. Long-term follow-up was available (range: 13-65 months). RESULTS: The major clinical manifestations of posttraumatic syringomyelia usually included the onset of increasing signs and the development of new symptoms after an apparently stable period. The clinical symptoms included pain, sensory disturbance, weakness, and problems in autonomic nerves. Syrinx existed merely at the cervical level in 4 cases and extended downward to the thoracic levels in the other 3 cases. One case underwent decompressive laminectomy, 6 cases were treated by S-S shunting. During the early postoperative period, all the patients showed an improvement of symptoms of syrinx without major complication or death. The decreased size or collapse of the syrinx was demonstrated by postoperative MRI. CONCLUSIONS: Posttraumatic syringomyelia is a disabling sequela of spinal cord injury, developing months to years after spinal injury. MRI is the standard diagnostic technique for syringomyelia. The patients with posttraumatic syringomyelia combined with progressive neurological deterioration should be treated with operations. S-S shunting procedure is effective in some patients with posttraumatic syringomyelia. Decompressive procedure may be an alternative primary surgical treatment for patients with kyphosis and cord compression.

A novel method for culturing neural stem cells.

The standard culture method for neural stem cells cannot prevent the attachment of neurospheres, which eventually result in differentiation. This study developed a new method for long-term neural stem cell cultivation. In the antiattachment group, neural stem cells were cultured in flasks coated with 1.5% agarose gel. As a control, cells were cultured in plastic flasks. The 5-bromine-deoxyuridine incorporation assay was used to determine the S-phase labeling index of both groups. The methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to determine the total cell vitality. After a 3-mo culture, the spontaneous differentiation of stem cells was studied using immunocytochemistry for neuroepithelial stem cell protein. We found that neural stem cells grew rapidly in the antiattachment flasks. There was no statistically significant difference between the two groups in terms of the S-phase labeling index or MTT assay. When cultured for 3 mo in vitro, many more cells differentiated in the control than in the antiattachment group (32.05 vs. 0.64%, P < 0.01). Moreover, the neural stem cells in the antiattachment group remained multipotent. Therefore, flasks coated with agarose gel are suitable for long-term neural stem cell culture.

Transplantation of neural stem cells into the traumatized brain induces lymphocyte infiltration.

OBJECTIVE: This study examined the lymphocyte infiltration induced by neural stem cell grafts in the traumatized brain. METHODS: Sixty Sprague-Dawley rats were assigned randomly to transplantation (n = 30) or control (n = 30) groups, and each rat was subjected to brain contusion. The neural stem cells derived from Wistar rats were transplanted into the lesion of the transplantation group, and saline was injected instead into the controls. Local lymphocyte infiltration was studied using haematoxylin and eosin staining, immunohistochemistry and flow cytometry. The immunogenicity of neural stem cells was evaluated using MHC-I expression. RESULTS: About 6.57 +/- 0.44% of the neural stem cells expressed MHC-I. In the transplantation group, histological examination and immunohistochemistry revealed significant lymphocyte infiltration in the contusion. The ratio of CD4(+) lymphocytes to total cells in the lesions was 13.28 +/- 1.60% in the transplantation group and 0.41 +/- 0.12% in the controls (p < 0.01). Likewise, the ratio of CD8(+) lymphocytes to total cells was 5.11 +/- 1.03% in the transplantation group and 0.57 +/- 0.26% in the controls (p < 0.01). CONCLUSIONS: Neural stem cells possess immunogenicity and can induce lymphocyte infiltration when transplanted into a traumatised brain. Our findings imply that immunosuppressive treatment is necessary following neural stem cell transplantation.

Bcl-2 gene therapy for apoptosis following traumatic brain injury.

OBJECTIVE: To investigate the therapeutic effect of Bcl-2 fusion protein on apoptosis in brain following traumatic brain injury. METHODS: Bcl-2 gene was cloned by RT-PCR. Bcl-2 and EGFP genes were linked together and inserted into pAdeno-X vector. This recombinant vector was packaged into infectious adenovirus in HEK293 cells. Ninety Wistar rats were assigned randomly into experimental group (n=45) and control group (n=45). All rats were subjected to traumatic brain injury. Then recombinant adenovirus (for experimental group) or saline (for control group) was injected into the traumatic brain. The expression of Bcl-2 fusion protein was investigated by Western blotting, immunohistochemistry and fluorescence microscopy. Apoptosis in the injured brain was studied by TUNEL. Animals' behavior capacity was evaluated by tiltboard test. RESULTS: In the experimental group, many fluorescent cells were found around the traumatic locus, which were also proven to be Bcl-2 positive by immunohistochemistry. On the contrary, few Bcl-2 positive cells and no fluorescent cell were detected in the control group. Bcl-2 expression of experimental group was much higher than that of control group, which was illustrated by Western blotting. The apoptosis index of experimental group was 0.027+/-0.005, and that of control group was 0.141+/-0.025 (P < 0.01). Two weeks after injury, animals of the experimental group behaved better than those of the control group. CONCLUSIONS: A recombinant adenovirus vector expressing Bcl-2 fusion protein has been constructed. Bcl-2 fusion protein can suppress apoptosis and promote cell survival. Moreover, the behavior recovery of the injured animal is promoted. Bcl-2 fusion protein provides a way to track the target cells in vivo.