Norihiro Ohba

Biphasic expression of activating transcription factor-3 in neurons after cerebral infarction.

It has been demonstrated that some of immediate early genes such as c-Jun are induced immediately and transiently following focal cerebral ischemia. Here we newly characterize the activating transcription factor (ATF)-3 as a focal ischemia associated immediate early gene. Using in situ hybridization and immunohistochemistry, we compared the expression profile of ATF-3 with those of ATF-2 and c-Jun after middle cerebral artery (MCA) occlusion. Focal cerebral ischemia induced two temporal and spatial patterns of ATF-3 expression. Early and transient induction of ATF-3 mRNA was observed in the core and margins of the cortex immediately after MCA occlusion. Late-onset and prolonged expression of ATF-3 mRNA and its protein were specifically identified in the peri-infarct cortex and thalamus where neurons survive at least 1 month. The expression profiles of ATF-3 and c-Jun were virtually similar, but c-Jun expression was also observed in other regions of the brain in control rats. Expression of ATF-2 was ubiquitously seen in neuronal cells throughout the brain in normal rats, but was suppressed in ischemic regions. Double immunohistochemical labeling revealed concurrent expression of ATF-3 and phospho-c-Jun in neurons. We conclude that the transcription factor ATF-3 is a suitable marker of neurons subjected to ischemic insult directly and indirectly, and that cooperative works of ATF-3 and c-Jun may be crucial triggers of various transcriptional responses to the ischemic insult.

Transgenic mouse overexpressing the Akt reduced the volume of infarct area after middle cerebral artery occlusion

Transgenic mouse lines expressing the active form Akt gene under the control of the damage-induced neuronal endopeptidase (DINE) promoter were made from three different founder mice, and its neuroprotective potential against ischemic brain damage was investigated. Twenty-four hours after middle cerebral artery occlusion, two DINE-Akt-transgenic mouse lines displayed reductions of the infarcted area by 35% compared to the wild-type littermate. RT-PCR assays showed a high level of transgene in response to ischemic brain damage in these lines. These results suggest that the DINE promoter is a useful promoter, which responds to neuronal insults and that the Akt-induced neuroprotective effect against ischemic damage is potent in vivo.

Expression of damage-induced neuronal endopeptidase (DINE) mRNA in peri-infarct cortical and thalamic neurons following middle cerebral artery occlusion.

Damage‐induced neuronal endopeptidase (DINE) is a unique nerve‐injury associated molecule, which was recently identified in a peripheral nerve injury model. The aim of this study was to determine the expression profiles and distribution of DINE in adult rats after middle cerebral artery (MCA) occlusion. Focal cerebral ischemia induced late‐onset and prolonged expression of DINE mRNA in the peri‐infarct cortex and specific nuclei of thalamus. Double labeling using immunohistochemistry and in situ hybridization revealed that DINE mRNA was exclusively expressed in cells that were positive to a neuronal marker NeuN. Previously established knowledge on neuroanatomical fiber connection suggests that DINE mRNA was expressed in areas projecting their axons to or through the core region of the infarction. This unique expression profile was similar to that of activating transcription factor‐3 (ATF‐3), which is a marker of nerve‐injured neuron. More than 98% of ATF‐3 immunoreactive neurons simultaneously expressed DINE mRNA, suggesting that DINE expression is observed in injured neurons of CNS as well as PNS. Since DINE expression promotes antioxidant activity, our results suggest that DINE may act as a neuroprotective molecule in neurons under ischemic insult.

Targeted gene therapy toward astrocytoma using a Cre/loxP-based adenovirus system

The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.

Vesicular acetylcholine transporter can be a morphological marker for the reinnervation to muscle of regenerating motor axons.

This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), which packages acetylcholine into synaptic vesicles, can be used as a marker for regenerating motor axon terminal. We examined motor axon regeneration in the tongue after hypoglossal nerve axotomy, using an anterograde tracer biotin-dextran (BD), retrograde tracer Fluoro-Gold (FG), electron microscopic (EM) observation, and VAChT immunocytochemistry. BD study demonstrated that outgrowth of thin regenerating axons into the frontal area of the tongue was firstly observed at 14 post-operative days, and presynaptic formation of neuromuscular junction (NMJ) was observed from 21 post-operative days. Under electron microscopic observation, reconstruction of new NMJs was observed within the interval between 21 and 28 days. VAChT-immunoreactive nerve terminals disappeared by 3 days after axotomy, slightly appeared at 14 post-operative days, and thereafter gradually increased in number from 21 to 28 post-operative days. The re-expression of VAChT positive presynaptic terminal was almost the same as those obtained in BD, FG and EM studies. Regenerating axons tip in the crush model of the hypoglossal nerve exhibited prominent VAChT immunoreactivity in growing tip of regenerating axons. These indicate that VAChT is an excellent morphological indicator for regenerating nerve terminals of motor neurons.

The p53-independent nuclear translocation of Cyclin G1 in degenerating neurons by ischemic and traumatic insults

Cyclin G1 (CG1) was identified as a p53-transactivated target gene, and yet its physiological and pathological roles have been unclear. Here, we demonstrate that CG1 is translocated from cytoplasm to the nuclei of neurons in response to variety of injuries. In the normal matured rodent brain, CG1 immunoreactivity was hardly observed; however, some brain injuries exhibited intense CG1 immunoreactivity in the nuclei of the damaged neurons. Transient common carotid artery occlusion (CCAO) in the gerbil showed strong CG1-like immunoreactivity in the hippocampal CA1 neurons, and permanent middle cerebral artery occlusion (MCAO) in the mouse showed strong CG1-like immunoreactivity in the nuclei of neurons located in the ischemic brain regions. TUNEL staining did not exactly overlap with the CG1-positive cells, but overlapped highly with Fluoro-Jade B staining, a degeneration marker. Brain trauma caused by knife cut, cold injury, and kinate injection also showed CG1 accumulation in the neuronal nuclei located near the injury site. These observations were obtained in p53-deficient mice as well, suggesting that the accumulation of CG1 in the injured neurons is p53-independent. A similar nuclear translocation of endogenous CG1 was confirmed in a primary culture of cortical neurons when a toxic level of N-methyl-D-aspartate (NMDA) was applied. These results demonstrate that nuclear translocation of CG1 from cytoplasmic region occurs in damaged and degenerating neurons in a p53-independent manner, and the CG1 nuclear staining could be a good marker for the neurons received fatal damages.

Cutaneous ciliated cyst on the cheek in a male

A 53‐year‐old man presented with a 2‐year history of a painless mass on the right cheek. On physical examination, the lump was a solitary, well‐demarcated, mobile lesion. There was no abnormality in the surrounding skin. Ultrasonography demonstrated a single, cystic, well‐defined, subcutaneous cyst, measuring 20 mm in diameter. Laboratory tests, including sex hormones, were within the normal range. The tumor was resected surgically under local anesthesia. The cyst had a slightly yellow wall and contained clear, watery fluid. The patient had no other medical problems and no history of other skin disease.

Clinical results of OK-432 injection therapy for ganglions.

We performed a study of intralesional OK‐432 injection therapy for the non‐surgical treatment of ganglions. OK‐432 is an agent made from penicillin‐killed and lyophilized preparations of a low‐virulence strain of group A streptococcus pyogenes, which has been developed as an immune‐augmentation agent for cancer therapy. Derived from an extract of bacterial culture it, induces an immunological response and causes local inflammation and subsequent tissue shrinkage following intralesional injection. After skin anesthesia and aspiration of the ganglion contents, OK‐432 was injected into the ganglion cavity. When the ganglion recurred, this procedure was repeated usually up to a total of three times. Clinical evaluations six months after the last injection were: 56.6% complete cure, 35.3% incomplete cure with size reduced, 7.5% no change. Complications were as follows. There were no cases of shock. High fever was seen in five patients (9.4%); two patients suffered a high fever up to 39.0°C for one day, and the others had fevers from 1 to 3 days. Thirty‐two patients (60.4%) complained of local swelling that persisted for 1 to 7 days, and 11 (20.8%) complained of continuous pain for 1 to 3 days. Intralesional OK‐432 injection therapy is thought to be a safe and convenient alternative to surgical treatment.

Improvement of the radial forearm donor site by compression with hydrocolloid dressing and adhesive sponge

Conclusions. With our method, general improvement is obtained as compared with traditional split-thickness skin grafting of the radial forearm flap donor site. As our method is simple and easy, the same results can be obtained wherever and by whomever it is performed. Objective. The radial forearm flap is associated with complications of graft take and a poor aesthetic appearance despite its usefulness in reconstructing the oral cavity and oropharynx. We describe a simple technique for improvement of the radial forearm donor site. Patients and methods. We studied 12 patients who underwent reconstruction with radial forearm free flaps following resection of oral or oropharyngeal tumors. We covered the donor site defect using traditional split-thickness skin grafts and performed aftercare with a hydrocolloid dressing and an adhesive sponge to retain moisture and apply compression. After the treatment series, color matching, texture matching, depressive deformity, and hypertrophic scar were evaluated. Results. The results of comprehensive evaluation of the two patients with premature discontinuation of compression were good. One patient was assigned only 1 point for hypertrophic scar, and another only 1 point for color match. The evaluation of the other 10 patients was excellent.

Mucous-flap method for cleft-lip revision using transverse everted full-length lower-lip flap.

In patients who had undergone the first surgery for cleft lip and in whom the volume of tissue was smaller for the upper lip than for the lower lip, transfer of tissue from the lower lip using a full-length mucous flap allowed the tissue volume of the upper lip to be increased and external appearance of the lips to be improved. The subjects of this study were 6 patients who underwent this surgery between February and September 2001 and were followed for up to 3 years postoperatively. This surgery can be performed under topical anesthesia, without necessitating restriction on mouth opening and oral ingestion. Furthermore, it allows easy adjustment of the tissue volume in both upper and lower lips. This operative procedure is recommended for cases of cleft lip where surgical treatment has been performed before and the tissue volume is smaller in the upper prolabium than in the vermilion.