Osamu Mimura

A possible linkage between AMP‐activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway

Background: The mammalian target of rapamycin (mTOR) regulates multiple cellular functions including translation in response to nutrients, especially amino acids. AMP‐activated protein kinase (AMPK) modulates metabolism in response to energy demand by responding to changes in AMP.

Molecular Mechanism and Role of Endothelial Monocyte Chemoattractant Protein-1 Induction by Vascular Endothelial Growth Factor

Objective—We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in vascular endothelial growth factor (VEGF)–induced angiogenesis and vascular permeability and the underlying molecular mechanism of VEGF-induced endothelial MCP-1 expression in vitro and in vivo. Methods and Results—We used an anti–MCP-1 neutralizing antibody for specific inhibition of MCP-1. VEGF increased tubule formation in the angiogenesis assay and vascular permeability in the Miles assay, and these effects were markedly inhibited by anti–MCP-1 antibody. Using a luciferase MCP-1 promoter-gene assay, we found that the activator protein-1 (AP-1) binding site of the MCP-1 promoter region contributes to the increase in MCP-1 promoter activity by VEGF. To specifically inhibit AP-1, we used recombinant adenovirus containing a dominant-negative c-Jun (Ad-DN-c-Jun). Ad-DN-c-Jun inhibited VEGF-induced endothelial MCP-1 mRNA expression and promoter activity in vitro. In vivo gene transfer of DN-c-Jun into rat carotid artery, with the hemagglutinating virus of the Japan liposome method, significantly blocked VEGF-induced MCP-1 and macrophage/monocyte (ED1) expression in endothelium. Conclusions—These results reveal that endothelial MCP-1 induced by VEGF seems to participate in angiogenesis, vascular leakage, or arteriosclerosis. AP-1 plays a critical role in the molecular mechanism underlying induction of MCP-1 by VEGF.

Contribution of IL-33 to induction and augmentation of experimental allergic conjunctivitis

IL-33, a member of the IL-1 family of cytokines, is the ligand for ST2 (IL-33Ralpha chain). IL-33 has the capacity to induce T(h)2 cytokine production from T(h)2 cells, mast cells and basophils, indicating that IL-33 has the potential to induce T(h)2 cytokine-mediated allergic inflammation of the eye. Thus, we tested the pathological role of IL-33 in allergic conjunctivitis (AC). As reported elsewhere, animals immunized with ragweed pollen (RW)/alum and boosted with RW/PBS developed AC promptly (within 15 min) and conjunctival eosinophilic inflammation after a delay (within 24 h) in response to eye drop challenge with RW. Furthermore, RW-immunized mice, when topically challenged with both RW and IL-33, developed more striking eosinophilia in their conjunctiva without exacerbation of the clinical AC score. This in vivo IL-33 treatment significantly increased the capacity of T cells in the cervical lymph nodes of RW-immunized mice to produce IL-4, IL-5 and IL-13 upon challenge with anti-CD3 and anti-CD28 antibodies in vitro. Furthermore, the infiltrating cells were largely eosinophils and a small proportion of CD4(+) T cells, both of which express ST2. We also found that even splenic eosinophils express ST2 and show increased expression in response to IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-33. Eosinophils, stimulated with IL-5 and/or GM-CSF, are responsive to IL-33, which induces production of IL-4 and chemokines. Finally, we showed that conjunctival tissues constitutively express biologically active IL-33, suggesting that IL-33 might play a crucial role in the induction and augmentation of AC.

Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.

We developed an in vivo electroporation method to introduce foreign genes into retinal ganglion cells (RGCs). After the intravitreous injection of the plasmid gene (20 μg), five electric pulses (6 V/cm, 100 ms duration) were each delivered twice with 5 min interval to the rat eye using a contact lens-type electrode (cathodal) attached to the cornea and a needle electrode (anodal) inserted to the middle of the forehead. The efficiency of the genetic introduction into RGCs and tissue damage to the eyeball was evaluated using a green fluorescent protein (GFP) gene, TUNEL and histological observation. DiI retrograde labeling revealed that 24.4±4.7% of all RGCs were electrointroduced with the GFP gene. TUNEL and histological analysis showed a few tissue damages in the cornea, lens and retina. To confirm whether this method can actually rescue damaged RGCs, glial cell line-derived neurotrophic factor (GDNF) was electrointroduced into RGCs after optic nerve transection. After the electrointroduction, a significant increase in the number of surviving RGCs was observed 2 and 4 weeks after the optic nerve transection, and the decrease of caspase 3 and 9 was detected by RT-PCR. These results suggest that this method may be useful for the delivery of genes into RGCs with simplicity and minimal tissue damage.

Retinal sensitivity and spatial summation in the foveal and parafoveal regions

Retinal sensitivity and the critical area of the spatial summation in the foveal and parafoveal regions were investigated by using a fundus-controlled perimeter to monitor accurately a subjects fundus picture and target position on the retina. The first experiment showed that retinal sensitivity and the critical area change continuously from fovea to periphery and that the diameter of the critical area is a linear function of retinal eccentricity. In the second experiment, this linear relationship was verified by another method.

Axonal regeneration induced by repetitive electrical stimulation of crushed optic nerve in adult rats

PurposeTo investigate whether electrical stimulation promoted axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush in adult rats.MethodsTranscorneal electrical stimulation (TES), which stimulates the retina with current from a corneal contact lens electrode, was used to stimulate the eye. TES was applied for 1 h immediately after ON crush. Axonal regeneration was determined by anterograde labeling of RGC axons. To examine whether the axonal regeneration was mediated by insulin-like growth factor 1 (IGF-1) receptors, an IGF-1 receptor antagonist, JB3, was injected intraperitoneally before each TES application. Immunostaining for IGF-1 was performed to examine the effects of TES. To test the survival-promoting effects of TES applied daily, the mean density of retrogradely labeled RGCs was determined on day 12 after ON crush.ResultsCompared with sham stimulation, the mean number of regenerating axons significantly increased at 250 μm distal from the lesion and increased IGF-1 immunoreactivity was observed in retinas treated daily with TES. Preinjection of an IGF-1 receptor antagonist significantly blocked axonal regeneration by TES applied daily. TES applied daily also markedly enhanced the survival of RGCs 12 days after ON crush.ConclusionTES applied daily promotes both axonal regeneration and survival of RGCs after ON crush.

Vasculogenesis in Experimental Stroke After Human Cerebral Endothelial Cell Transplantation

Background and Purpose— Despite the reported functional recovery in transplanted stroke models and patients, the mechanism of action underlying stem cell therapy remains not well understood. Here, we examined the role of stem cell–mediated vascular repair in stroke. Methods— Adult rats were exposed to transient occlusion of the middle cerebral artery and 3 hours later randomly stereotaxically transplantated with 100K, 200K, or 400K human cerebral endothelial cell 6 viable cells or vehicle. Animals underwent neurological examination and motor test up to day 7 after transplantation then euthanized for immunostaining against neuronal, vascular, and specific human antigens. A parallel in vitro study cocultured rat primary neuronal cells with human cerebral endothelial cell 6 under oxygen-glucose deprivation and treated with vascular endothelial growth factor (VEGF) and anti-VEGF. Results— Stroke animals that received vehicle infusion displayed typical occlusion of the middle cerebral artery–induced behavioral impairments that were dose-dependently reduced in transplanted stroke animals at days 3 and 7 after transplantation and accompanied by increased expression of host neuronal and vascular markers adjacent to the transplanted cells. Some transplanted cells showed a microvascular phenotype and juxtaposed to the host vasculature. Infarct volume in transplanted stroke animals was significantly smaller than vehicle-infused stroke animals. Moreover, rat neurons cocultured with human cerebral endothelial cell 6 or treated with VEGF exhibited significantly less oxygen-glucose deprivation–induced cell death that was blocked by anti-VEGF treatment. Conclusions— We found attenuation of behavioral and histological deficits coupled with robust vasculogenesis and neurogenesis in endothelial cell–transplanted stroke animals, suggesting that targeting vascular repair sets in motion a regenerative process in experimental stroke possibly via the VEGF pathway.

Cytokine-induced injury of the lacrimal and salivary glands.

Damages to the lacrimal and salivary glands that accompany various autoimmune diseases are categorized as secondary Sjögren syndrome. Cytokines and free radicals are thought to be responsible for the pathologic changes, but the precise mechanisms are not clear. We evaluated whether cytokines alone can cause the damages in these exocrine tissues, and whether gaseous molecules such as nitric oxide (NO) play a role in these injuries. Various knockout (KO) mice as well as wild-type mice were injected intraperitoneally (i.p.) with the proinflammatory cytokines, IL-12 and IL-18, singly or in combination. Concurrent administration of IL-12 and IL-18 to mice caused serious atrophy in the lacrimal and salivary glands, which was spared when each cytokine was singly administered. Microscopically, there were apparently no infiltrating cells; nonetheless, numerous apoptotic cells were observed in the epithelium, which was confirmed by DNA ladder formation on gel electrophoresis. Serum levels of IFN-&ggr; and NO2/NO3 were markedly elevated. Combined injections of IL-12 and IL-18 caused the same changes in Fas-deficient and Fas-ligand deficient mice, as well as in perforin-KO mice, but the same changes were not detected in inducible NO synthase-KO mice or IFN-&ggr; KO mice. Thus, the synergistic effect of IL-12 and IL-18 was dependent on production of IFN-&ggr; and NO, but independent of Fas/Fas ligand system and perforin-dependent cytotoxic T cells. IL-18 together with IL-12 caused destructive changes in the glandular tissues without apparent lymphocyte infiltration. It is suggested that these cytokines can mediate apoptosis in glandular epithelial cells and that the elevated NO production is responsible for the change.

Pathogenesis of transient high myopia after blunt eye trauma

OBJECTIVE To determine the pathogenesis of transient high myopia after blunt eye trauma. DESIGN Two observational case reports and literature review. METHODS Refraction was measured in two patients with an autorefractometer in the acute and convalescent stages after a blunt eye injury. The anterior chamber angle, the ciliary body, and the choroid were examined by ultrasound biomicroscopy (UBM) in the acute and convalescent stages. In one patient, the anterior chamber depth, lens thickness, and axial length were measured by A-scan ultrasonography in the acute and convalescent stages. MAIN OUTCOME MEASURES Comparison of the refraction, anterior chamber depth, lens thickness, axial length, and the UBM-determined appearance of the choroid and ciliary body during the acute stage with the values during the convalescent stages. RESULTS The first patient showed a myopic shift of -9.75 diopters (D) and an anterior chamber shallowing of 0.94 mm measured 3 days after trauma by an air bag inflation compared with the measurements at the convalescent phase. UBM showed an annular ciliochoroidal effusion with ciliary body edema, anterior rotation of the ciliary processes, and disappearance of the ciliary sulcus. Eleven days after the injury, these UBM findings normalized, and the myopia decreased to -0.75 D, 27 days after trauma. The second patient had a myopic shift of -8.9 D compared with the convalescent phase, immediately after blunt trauma by a firework. Seven days after the injury, UBM revealed a partial cyclodialysis in addition to findings similar to those in the first patient. Ten days after injury, a myopic shift (-4.75 D), anterior chamber shallowing (by 1.1 mm), and thickening of the crystalline lens (by 0.27 mm) were observed compared with the convalescent phase. Associated UBM findings confirmed the anterior shift of the lens-iris diaphragm. Seventeen days after trauma, the UBM findings, including the cyclodialysis, were normalized, and the myopia had decreased to -1.0 D. CONCLUSIONS Transient high myopia after blunt trauma is caused by anatomic changes in the ciliary body and crystalline lens. The anterior shift of the lens-iris diaphragm caused by ciliochoroidal effusion with ciliary body edema and thickening of the crystalline lens from blunt eye trauma are involved in traumatic high myopia.

Morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid of the rat.

The cornea is sensitive to nociceptive stimuli and receives dense sensory innervations from the trigeminal ganglion, which also innervates the upper eyelid. We investigated the morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid. We injected the retrograde tracer Fluoro-Gold (FG) into the cornea and the retrograde tracer cholera toxin subunit b (CTb) into the upper eyelid of the same animal. Less than 10% of the FG-labeled neurons were also labeled with CTb. The FG-labeled neurons were small (29.6+/-0.6microm), while the CTb-labeled neurons were large (36.1+/-0.5microm). We also characterized the neurons in the trigeminal ganglion with the retrograde tracer FG following its injection into the cornea or the upper eyelid, and immunohistochemical double-labeling with nociception-related neuronal markers, such as calcitonin gene-related peptides (CGRP), transient receptor potentiated vanilloid 1 (TRPV1), and substance P (SP). About 27% of the neurons innervating the cornea were double-labeled with CGRP, about 23% with TRPV1, and about 8% with SP. About 4% of the neurons innervating the upper eyelid were double-labeled for CGRP, about 11% for TRPV1, and 3% for SP. Thus, the percentages of double-labeled neurons for the neurons innervating the cornea were higher than those for the neurons innervating the upper eyelid. These results indicate that the cornea and the upper eyelid receive innervations mainly from different neurons of the trigeminal ganglia. The cornea is innervated by many characteristic sensory neurons containing nociception-related neuronal markers.