Takamichi Yuguchi

Loss and apoptosis of smooth muscle cells in intracranial aneurysms studies with in situ DNA end labeling and antibody against single-stranded DNA

SummaryPathological specimens were collected from 14 unruptured and 13 ruptured aneurysms at the time of clipping and studied in order to assess the underlying mechanism of rupture by investigating degeneration of the aneurysmal wall and possible involvement of apoptosis. Immunohistochemistry with anti-actin antibody showed few smooth muscle cells in the ruptured aneurysms and replacement of the muscularis layer by a fibro-hyalin tissue. However, at least one layer of smooth muscle cells was clearly observed in the unruptured aneurysms. Thus, smooth muscle cells in the wall of the ruptured aneurysms were much more degenerated than those in the wall of unruptured aneurysms. In addition, unruptured aneurysms with an angiographically smooth wall showed well-layered positive staining for anti-smooth muscle actin antibody while those with irregular shapes rarely reacted. We found, for the first time, evidence of DNA fragmentation in the aneurysmal wall. Apoptotic bodies were detected by means of a terminal transferase (TdT)-mediated dUTP biotin nick end labelling technique (TUNEL) and an anti-single-stranded DNA antibody in 54% (7/13) of the ruptured aneurysms. In contrast, apoptotic bodies were found in only 7% (1/14) of the unruptured cases. These results suggest that apoptotic cell death might be involved in the rupture of aneurysms.

Traumatic brain injury induces biphasic upregulation of ApoE and ApoJ protein in rats

Apolipoproteins play an important role in cell repair and have been found to increase shortly after traumatic brain injury (TBI). In addition, apolipoproteins reduce amyloid‐β (Aβ) accumulation in models of Alzheimers disease. Considering that TBI induces progressive neurodegeneration including Aβ accumulation, we explored potential long‐term changes in the gene and protein expression of apolipoproteins E and J (ApoE and J) over 6 months after injury. Anesthetized male Sprague‐Dawley rats were subjected to parasagittal fluid‐percussion brain injury and their brains were evaluated at 2, 4, 7, 14 days, and 1 and 6 months after TBI. In situ hybridization, Western blot, and immunohistochemical analysis demonstrated that although there was a prolonged upregulation in both the gene expression and protein concentration of ApoE and J after injury, these responses were uncoupled. Upregulation of ApoE and J mRNA expression lasted from 4 days to 1 month after injury. In contrast, a biphasic increase in protein concentration and number of immunoreactive cells for ApoE and ApoJ was observed, initially peaking at 2 days (i.e., before increased mRNA expression), returning to baseline by 2 weeks and then gradually increasing through 6 months postinjury. In addition, ApoE and J were found to colocalize with Aβ accumulation in neurons and astrocytes at 1–6 months after injury. Collectively, these data suggest that ApoE and J play a role in the acute sequelae of brain trauma and reemerge long after the initial insult, potentially to modulate progressive neurodegenerative changes.

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.

Prostaglandin E1 Induces c-Fos and Myc Proteins and Protects Rat Hippocampal Cells against Hypoxic Injury

We investigated the effects of prostaglandin (PG) E1 on the hypoxic injury of fetal rat hippocampal cells. Primary hippocampal cell cultures (embryonic day 18) were established and maintained. After 72 h in culture, PGE1 was added to the serum-free medium at a final concentration of 10−5-10−9 M. Cultures were divided into two groups: The normoxia group was in culture for another 48 h, and the hypoxia group was exposed to 24 h of hypoxia followed by continuation of culture for another 24 h. As a quantitative measure of cell death, lactate dehydrogenase (LDH) activity was estimated in the culture medium. The LDH activity, released by the hypoxic insult, was significantly smaller with PGE1 treatment at 10−6, 10−7, and 10−8 M (p < 0.01) and 10−9 M (p < 0.05) compared with the control. No differences in the LDH activities were observed in the normoxia group. Glial culture was not affected by the hypoxia. Western blot analysis showed an increased induction of 62-kDa c-Fos and 58, 60, and 66 kDa Myc proteins in rat hippocampal cells with 10−7 M PGE1 treatment. We conclude that PGE1 at concentrations of 10−6-10−9 M protects rat hippocampal neurons against hypoxic insult.

Induction of Na^+/myo-inositol cotransporter mRNA after focal cerebral ischemia : Evidence for extensive osmotic stress in remote areas

Myo-inositol is one of the major organic osmolytes in the brain. It is accumulated into cells through an Na+/myo-inositol cotransporter (SMIT) that is regulated by extracellular tonicity. To investigate the role of SMIT in the brain after cerebral ischemia, we examined expression of SMIT mRNA in the rat brain after middle cerebral artery occlusion, which would reflect alteration of extracellular tonicity. The expression of SMIT mRNA was markedly increased 12 h after surgery in the cortex of the affected side and lasted until the second day. Increased expression was also found in the contralateral cingulate cortex. Up-regulated expression was found predominantly in the neurons in remote areas, although nonneuronal cells adjacent to the ischemic core also expressed this mRNA. These results suggest that cerebral ischemia causes extensive osmotic stress in brain and that the neuronal cells respond to this stress by increasing SMIT expression.

Src Family Kinase Inhibitor PP1 Reduces Secondary Damage after Spinal Cord Compression in Rats

The synthetic pyrazolopyrimidine, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) is a novel, potent, and selective inhibitor of Src family tyrosine kinases. Vascular permeability appears to be mediated by vascular endothelial growth factor (VEGF), which requires the activation of downstream Src family kinases to exert its function. This study investigates the effects of PP1 on vascular permeability and inflammatory response in a rat spinal cord compression model. Ten minutes after compression, PP1 (PP1 group) or the vehicle only (control group) was administered. On days 1, 3, and 7 after compression, the spinal cords were removed and examined histopathologically to determine the expression of VEGF and the extent of edema and inflammation. The dryweight method was used to measure the water content of the spinal cords. The mRNA levels of tumor necrosis factor a (TNFalpha) and interleukin 1beta (IL-1beta), which is related to inflammatory responses, were measured with a real-time polymerase chain reaction (RT-PCR) system 6 h after compression. Although VEGF expression was similar in both groups, the extent of contusional lesion in the PP1 group was reduced by approximately 35% on day 3. Moreover, the water content on days 1, 3, and 7 was significantly reduced and macrophage infiltration on days 3 and 7 was dramatically reduced in the PP1 group. TNF and IL-1beta mRNA expression in the PP1 group were also significantly reduced. These results indicate that PP1 reduces secondary damage after spinal cord injury.

Expression of Growth Inhibitory Factor mRNA After Focal Ischemia in Rat Brain

Growth inhibitory factor (GIF) is a small protein belonging to the metallothionein family that has the capacity to inhibit neuronal survival and neurite formation in vitro. This study was conducted to investigate the role of GIF in the brain afflicted with ischemic injury. We used the in situ hybridization technique and Northern blot analysis to study the changes in GIF messenger RNA (mRNA) expression in a rat focal ischemia model. On the first day, the expression tended to decrease in the hemisphere ipsilateral to the injury. It returned to normal levels on the second day except for the central area of the middle cerebral artery (MCA) territory. On the third and fourth day, the expression increased diffusely in the hemisphere of the affected side, including the subcortical area. Two weeks after ischemia, the GIF mRNA expression increased again but only in the pert-infarcted area. Down-regulation of GIF on the first day in the cortex ipsilateral to the infarction might promote neurite sprouting. The subsequent increase in GIF mRNA expression on the third and fourth day might be a symptom of neurons attempting to inhibit excessive neurite outgrowth, or to protect themselves against toxicity caused by oxygen radicals. The later increase in the limited area around the infarction may be related to astroglial reaction. Growth inhibitory factor may play an important role in regulating the central nervous system after ischemic insults.

The morphological and neurochemical effects of diffuse brain injury on rat central noradrenergic system

Abstract The central noradrenergic system is widely distributed throughout the brain and is closely related to spontaneous motility and level of consciousness. The study presented here evaluated the morphological as well as neurochemical effects of diffuse brain injury on the central noradrenergic system in rat. Adult male Sprague–Dawley rats were subjected to impact–acceleration brain injury produced with a weight-drop device. Morphological changes in locus coeruleus (LC) neurons were examined by using immuno-histochemistry for dopamine-β-hydroxylase, and norepinephrine (NE) turnover in the cerebral cortex was measured by high performance liquid chromatography with electrochemical detection. The size of LC neurons increased by 11% 24 h after injury but had decreased by 27% seven days after injury. Axons of noradrenergic neurons were swollen 24 h and 48 h after injury but the swelling had dwindled in seven days. NE turnover was significantly reduced seven days after injury and remained at a low level until eight weeks after injury. These results suggest that focal impairment of axonal transport due to diffuse brain injury causes cellular changes in LC and that the neurochemical effect of injury on the central noradrenargic system lasts over an extended period of time. Chronic suppression of NE turnover may explain the sustained behavioral and psychological abnormalites observed in a clinical situation.

Changes in glutamate/aspartate transporter (GLAST/GluT-1) mRNA expression following facial nerve transection

Expression of glutamate/aspartate transporter (GLAST/GluT-1) was investigated in the axotomized facial nucleus by in-situ hybridization. Hybridization signals for GLAST mRNA were almost undetectable in the facial nucleus of sham-operated animals. However, the hybridization signals were seen from 3 days after facial nerve transection onward in the nucleus of the affected side. These signals lasted at least 5 weeks. Microautoradiograms showed that small non-neuronal cells in the ipsilateral facial nucleus expressed signals of GLAST mRNA after axotomy. These findings suggest that non-neuronal cells, presumably astrocytes, may protect axotomized motor neurons against glutamate toxicity via up-regulation of GLAST in the facial nucleus.

Posterior microendoscopic surgical approach for the degenerative cervical spine

Abstract We describe the advantages and clarify the technical key points of a microendoscopic, minimally invasive technique to the posterior surgical approach for cervical degenerative disease. The authors studied the microendoscopic posterior approach using the METRx system in both cadaver models and in clinical cases. This new technique needs only a small surgical route thus reducing damage to the paraspinous muscles. Moreover, this technique provides a clear view of the operating points, because of the oblique view angle of the endoscope. This technique is feasible for not only radiculopathy but also myelopathy caused by segmental canal stenosis. Posterior cervical decompression with this system is technically feasible and should be beneficial for reducing post-operative morbidity and spine deformity. This report deals with cases of cervical radiculopathy and segmental canal stenosis operated on with this system as well as the key points of this surgical procedure.