Hideki Sakai

Spinal injuries in snowboarders: risk of jumping as an integral part of snowboarding

BACKGROUNDnThe purpose of this study was to clarify the occurrence rate and characteristics of spinal injuries caused by snowboarding that were sustained at the Okumino skiing area in Gifu Prefecture, Japan, from 1988 to 2000.nnnMETHODSnThis study was a retrospective review of 13,490 cases of snowboard- or ski-related injury treated at Sumi Memorial Hospital over this period.nnnRESULTSnA total of 7,188 patients sustained snowboard-related injuries, and 238 of these had spinal injuries caused by snowboarding (3.3%), whereas 6,302 patients sustained ski-related injuries, and 86 of these had spinal injuries caused by skiing (1.4%). Although there were no significant differences in the difficulty of slope, location of vertebral fracture, or spinal cord injury between snowboarders and skiers, the incidence of transverse process fractures was significantly higher in snowboard-related than in skiing-related injury (p < 0.05). In addition, there was a significantly higher incidence of spinal injury among beginner snowboarders than among beginner skiers (p = 0.04). Furthermore, intermediate or expert snowboarders were more likely to be injured because of jumping than beginners (p < 0.001), whereas about 70% of spinal injuries caused by skiing resulted from a simple fall.nnnCONCLUSIONnSpinal injuries sustained while snowboarding are increasing considerably in incidence and are characterized as complex injuries. We must educate young snowboarders of the risk of this sport, to prevent these serious injuries.

Changes in the Activity and mRNA Levels of Phospholipase D During Ceramide‐Induced Apoptosis in Rat C6 Glial Cells

Abstract: N‐Acetylsphingosine (C2‐ceramide), a membrane‐permeable analogue, induced apoptosis in C6 glial cells. Phase‐contrast micrographs showed that the round cells appeared 3 h after exposure to 25 µM C2‐ceramide and the number of floating cells increased time‐dependently. Staining with Hoechst 33258 dye showed condensed or fragmented nuclei in round cells at 12 h. DNA fragmentation was also observed by agarose gel electrophoresis at 12 h. To understand the mechanism underlying glial cell death induced by C2‐ceramide treatment, changes in phospholipase D (PLD) activity in response to guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and expression of mRNA levels of PLD isozymes were examined. In cell lysate, GTPγS‐dependent PLD activity was down‐regulated after ceramide treatment in a time‐dependent manner. In the in vitro PLD assay, membrane‐associated PLD activation in response to recombinant ADP‐ribosylation factor 1 was greatly suppressed. Furthermore, levels of rPLD1a and rPLD1b mRNAs were found to be down‐regulated, whereas the level of rPLD2 mRNA increased gradually, peaking at 3 h, followed by a slow decrease, as inferred by reverse transcription‐polymerase chain reaction. Decreases in GTPγS‐dependent PLD activity were well correlated with those in rPLD1a and rPLD1b mRNAs levels. Taken together, these data suggest that levels of PLD enzymes might be decreased by ceramide treatment.

Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor

Neuronal expression of brain-derived neurotrophic factor (BDNF) has been implicated in the mechanism of infarct tolerance (resistance to stroke) (H. Yanamoto et al., Infarct tolerance accompanied enhanced BDNF-like immunoreactivity in neuronal nuclei, submitted to Brain Res.), a process that takes more than 7 days following a preconditioning of repetitive cortical spreading depression (CSD). To investigate whether an elevated level of BDNF protein in the brain solely protects neurons against temporary focal ischemia, recombinant (r)BDNF was infused into the rat neocortex. Recombinant BDNF (or vehicle: saline) was administered into the left neocortex via an implanted osmotic minipump for 2.5, 7, 10 or 14 days pre-ischemia, during ischemia and for 2 days post-ischemia (8 microgram in total) in male Sprague-Dawley rats (n=6 each). Temporary focal ischemia was induced in the left middle cerebral artery (MCA) territory by three-vessel occlusion of bilateral common carotid arteries (CCAs) and MCA for 2 h, and the cerebral infarct volume was analyzed 2 days after ischemia using TTC staining. Regional cerebral blood flow (rCBF) of the left neocortex was monitored after 14 days of intracerebral administration of BDNF or vehicle (n=10 each). The distribution of BDNF following different periods of rBDNF or vehicle-infusion was analyzed using immunohistochemical techniques (n=5 each). In the groups treated with 8 microgram of rhBDNF for 7, 10, or 14 days pre-ischemia, there were significant reductions of neocortical infarct volume compared to in the control or vehicle-treated groups (p<0.05). In the rCBF study, there was no significant change after the infusion of 8 microgram rhBDNF for 14 days. In the histological study, a wide distribution of BDNF-like immunoreactivity in the neuronal nuclei in the ipsilateral neocortex was demonstrated after the infusion of 8 microgram rhBDNF for 14 days. The BDNF-like immunoreactivity in the neuronal nuclei was enhanced at the time that the resistance to stroke was achieved by direct intra-cerebral infusion of exogenous rBDNF. Elucidating the function of the BDNF-like protein located in the neuronal nuclei should reveal a new strategy for neuroprotection against ischemic brain attack in humans.

Differential expression of Rho family GTP-binding proteins and protein kinase C isozymes during C6 glial cell differentiation

The differential expression of Rho family of low molecular weight GTP-binding proteins and protein kinase C (PKC) isozymes were examined during differentiation of rat C6 glial cells to astrocytic phenotypes induced by dibutyryl cAMP (dbcAMP)/theophylline. The cells showed rapid and distinct morphological changes, resembling stellate astrocytes at 12 h after the treatment. The treated cells had a round cell body that extended several long processes each with a beaded appearance. In addition to morphological changes, Western blot analysis revealed that S-100 protein, known as a glial cell differentiation marker, increased and reached the maximal level (approximately 6-fold increase) at 24 h following the addition of dbcAMP. In the control experiments with cells cultured in the absence of serum but also without dbcAMP/theophylline, morphological changes were marginal and apparent increases of S-100 protein were not observed by Western blotting. In response to dbcAMP/theophylline treatment, RhoA showed increases in the mRNA level followed by the protein level, as inferred by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Rac1 and Cdc42 proteins were undetectable by Western blot analyses. In PKC isozymes, increases were observed in PKC beta 1, epsilon, and zeta by RT-PCR, and in beta 1 and epsilon by Western blotting. Among them, PKC epsilon showed the most distinct changes. Its mRNA level transiently increased from 3 to 6 h and then decreased even below the basal level at 18 h after the treatment. In contrast, Western blot analysis revealed that PKC epsilon gradually increased time-dependently to 24 h (approximately 6-fold increase), and remained elevated until 48 h. These results suggested that RhoA and PKC epsilon, and probably also PKC beta 1 and PKC zeta, were closely implicated in C6 cell differentiation.

Angiotensin I-converting enzyme gene polymorphism in intracranial saccular aneurysm individuals

A polymorphism in the angiotensin I-converting enzyme (ACE) gene has been associated with cerebrovascular diseases as a new potent risk factor. The purpose of this study was to investigate an association of the gene polymorphism with intracranial saccural aneurysmal patients. The study population consisted of 83 aneurysmal patients (age range 41-85 years) (the AN group) and 104 matched control subjects (age range 30-81 years) (the Control group). For detection of the ACE gene polymorphism, the standard PCR method was performed by using genomic DNA isolated from peripheral blood leukocytes. The PCR products were a 490-bp in the presence of the insertion (I) and a 190-bp fragment in the absence of the insertion (D). The ACE gene polymorphism was classified into three genotypes: I/I genotype (a 490-bp band); D/D genotype (a 190-bp band); or I/D genotype (both a 490-bp and a 190-bp band). The number of subjects with I/I, I/D, and D/D genotypes was 38, 40, and 5 in the AN group and 43, 45, and 16 in the Control group, respectively. The frequency of the D/D genotype in the AN group was significantly lower (5/83 = 0.06) than that in the Control group (16/104 = 0.15) (chi 2 = 4.06; p = 0.044). There was no significant difference between the genotype sof hypertensive patients and normotensive patients in the AN group. Thus, this present study suggests that genetic heterogeneity of the ACE gene may be correlated with the etiology of intracranial aneurysms.

Elevation of mRNA levels of tissue-type plasminogen activator and urokinase-type plasminogen activator in hippocampus and cerebral cortex following middle cerebral artery occlusion in rats

Abstract Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) have been used for thrombolitic therapy. In contrast, it is suggested that these compounds might be involved in neuronal cell damage. The activation and the function of tPA and uPA are less understood in ischemic brain tissue. Therefore, changes in tPA and uPA mRNA in rat brain tissue after MCA occlusion and in the neuronal cell line, PCI2 cells, during the hypoxic stimulation were examined. Permanent middle cerebral artery (MCA) occlusion was induced by advancing a filament into the internal carotid artery in 36 adult male Sprague-Dawley rats. The ischemic cerebral cortex and contralateral cortex of MCA area, and bilateral hippocampus were collected at 0 (controls), 1, 3, 6, 12 and 24 h after occlusion. Hypoxia was induced in PCI2 cells with a multigas incubator (set to 1% 02). The quantitative reverse transcription-polymerase chain reaction acted as a measurement of alteration in mRNA levels. The mRNA levels of tPA and uPA were significantly increased after MCA occlusion in the ischemic cerebral cortex. The magnitude of the increase in tPA and uPA mRNA in 24 h after occlusion was twice the value in sham-operated rat (0 h). The increases oftPA mRNA were time-dependent in insult and contralateral hippocampus. The increase ofuPA mRNA was also seen in the hippocampus bilaterally, although the increase was more significant on the ipsilateral side. In PCI2 cells, necrotic (~ 35%) and apoptotic cells (~65%) could be distinguished by hypoxic stimulus for 24 h, and the mRNA for tPA was significantly increased for 6 h-12h, while the mRNA for uPA was not detected at any point in the study. Our results suggest that focal ischemia might result in the activation of these proteases not only in the insult but also in the contralateral brain tissue. [Neurol Res 2000; 22: 413-419]

MOLECULAR CLONING OF A CDNA ENCODING A SERINE PROTEASE HOMOLOGOUS TO COMPLEMENT C1S PRECURSOR FROM RAT C6 GLIAL CELLS AND ITS EXPRESSION DURING GLIAL DIFFERENTIATION

A cDNA of rat C6 cells was cloned, which was considered to be involved in glial cell differentiation induced by dibutyryl cyclic AMP and theophylline. The cDNA fragment of the gene, termed r-gsp, was originally isolated by mRNA fingerprinting using arbitrarily primed polymerase chain reaction, and was homologous to complement C1s precursors of hamster and human. It encodes a protein of 694 amino acids containing a potential signal peptide, an epidermal growth factor-like domain surrounded by two complement C1r/C1s-related repeats, and a putative trypsin-type serine protease domain. Since the hamster and human C1s, and a protein encoded by r-gsp shared high similarity in primary structure, the r-gsp gene could encode a C1s counterpart of the rat. Messenger RNA expression of this gene was markedly increased during cyclic AMP-induced glial cell differentiation. Its expression profile was well correlated with those of glial fibrillary acidic protein (GFAP) and S100B, which are known as glial differentiation markers. It was, moreover, observed that the r-gsp expression in brain increased considerably after birth, like those of S100B and GFAP. The results presented here suggest that the rat C1s gene would be also implicated in glial differentiation besides the complement cascade.

Analysis of phospholipase C gene in patients with subarachnoid hemorrhage due to ruptured intracranial saccular aneurysm

This study is designed to determine whether patients with aneurysmal subarachnoid hemorrhage have mutations in the phospholipase C-delta 1 (PLC-delta 1) gene, which was identified as a gene responsible for hypertension in spontaneously hypertensive rats. Seventy-two cases (31 male and 41 female) with intracranial saccular aneurysms were analyzed. The mean age was 60.1 +/- 11.5 years (mean +/- SD) (range 24-85 years). There were 35 patients (48.6%) with hypertension, 5 (6.9%) with diabetes mellitus, 12 (16.7%) with hyperlipidemia, 8 (11.1%) with ischemic heart disease, and 25 (34.7%) who were active smokers. The location of aneurysm was distributed as follows: 33 (33%) were at anterior cerebral artery, 23 (23%) were at middle cerebral artery, 28 (28%) were at internal carotid artery, and 16 (16%) were at vertebro-basilar artery. Six patients (8.3%) had a family history of intracranial aneurysms. There were 20 patients (27.8%) with multiple aneurysms, and 8 patients (11.1%) with a large or giant aneurysm. The four regions of PLC-delta 1 gene (bases 1099-1271, 1254-1401, 1343-1481, and 1882-2023) where genetic mutations were found in spontaneously hypertensive rats, were screened by PCR-SSCP analysis and their nucleotide sequences of all patients were determined. However, no mutations were detected in all patients. These results suggest that mutations of PLC-delta 1 gene previously implicated in hypertensive factor in rats may not be the case with human patients and therefore may be poorly related with aneurysmal subarachnoid hemorrhage.

The significance of the expression of tumor suppressor gene DCC in human gliomas.

Deleted in colorectal carcinoma (DCC) gene has been as a candidate of tumor suppressor genes, has been identified recently and is thought to relate to the metastatic potential in some cancers. We examined the gene in 60 human gliomas (26 glioblastomas multiforme (GBMs), 16 anaplastic astrocytomas (AAs), 6 low grade astrocytomas (LGAs) of WHO Grade II, and 11 recurrent gliomas) and A172 human GBM cell line by reverse transcription polymerase chain reaction (RT-PCR). Twenty (77%) GBMs, 11 (69%) AAs, and 1 (17%) LGA revealed the reduced or absent DCC expression. Reduced DCC expression was also shown in 10 (91%) recurrent gliomas. Furthermore, in 5 cases with both primary and recurrent GBM, the DCC expressions of all recurrent tumors were lower than those of primary tumors. No significant correlation between DCC expression and Mib-1 labeling index was confirmed. The survival rate of patients without reduced DCC expression was significantly superior to that of patients with reduced DCC expression in overall malignant astrocytic tumors. In GBM and AA separately, DCC expression also tended to correlate with patients prognosis.These results suggest that reduced DCC expression is an important marker in tumor malignancy and recurrence in astrocytic tumors and that may be a useful prognostic factor in patients with malignant astrocytic tumors.

Clinical trial of external beam-radiotherapy combined with daily administration of low-dose cisplatin for supratentorial glioblastoma multiforme – A pilot study

This trial tested the assumed efficacy and safety of externalbeam-radiotherapy combined with daily administration of low dose cisplatin(CDDP) (ERCLC therapy) for patients with glioblastoma multiforme (GBM).Thirty adult patients with supratentorial GBM received daily postoperativetreatment with low dose intravenous CDDP (4–6 mg/m2) administered30 minutes before external irradiation. In 10 patients, intraoperativeradiotherapy (IORT) following surgery was given prior to ERCLC therapy.Tumor response on MRI, interval to tumor progression, survival, andtoxicities were analyzed. None of the patients showed a tumor response toERCLC therapy. Overall, the median time to tumor progression was 6 monthswith a 1-year tumor progression-free rate of 26.7% and a 2-year rateof 0%. The median survival time was 15 months with a 1-year survivalrate of 69.9% and a 2-year rate of 31.5%. The survival rate ofpatients with IORT was better than that of those without IORT, however,there was no significant difference. Anorexia associated with nauseaoccurred in 70% and general fatigue in 10.0%. Leukopenia andthrombocytopenia occurred in 26.7% and 33.3%, respectively.However, none of the patients had to be withdrawn from therapy due to thesetoxicities. Other toxicities were not observed. This clinical study showedthat daily administration of low dose CDDP did not enhance tumor response toirradiation for GBM on MRI. Regarding toxicity, however, ERCLC therapy waswell tolerated. Although this trial did not provide sufficient data todetermine whether ERCLC therapy was effective for GBM due to the smallnumber of patients, additional clinical trials of this therapy may bewarranted because that the survival rate in this study was equal to thebetter results recently reported for newly diagnosed GBM.