Spermidine promotes retinal ganglion cell survival and optic nerve regeneration in adult mice following optic nerve injury.

Spermidine acts as an endogenous free radical scavenger and inhibits the action of reactive oxygen species. In this study, we examined the effects of spermidine on retinal ganglion cell (RGC) death in a mouse model of optic nerve injury (ONI). Daily ingestion of spermidine reduced RGC death following ONI and sequential in vivo retinal imaging revealed that spermidine effectively prevented retinal degeneration. Apoptosis signal-regulating kinase-1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase kinase kinase and has an important role in ONI-induced RGC apoptosis. We demonstrated that spermidine suppresses ONI-induced activation of the ASK1-p38 mitogen-activated protein kinase pathway. Moreover, production of chemokines important for microglia recruitment was decreased with spermidine treatment and, consequently, accumulation of retinal microglia is reduced. In addition, the ONI-induced expression of inducible nitric oxide synthase in the retina was inhibited with spermidine treatment, particularly in microglia. Furthermore, daily spermidine intake enhanced optic nerve regeneration in vivo. Our findings indicate that spermidine stimulates neuroprotection as well as neuroregeneration, and may be useful for treatment of various neurodegenerative diseases including glaucoma.

Dock3 protects myelin in the cuprizone model for demyelination.

Dedicator of cytokinesis 3 (Dock3) belongs to an atypical family of the guanine nucleotide exchange factors. It is predominantly expressed in the neural tissues and causes cellular morphological changes by activating the small GTPase Rac1. We previously reported that Dock3 overexpression protects retinal ganglion cells from excitotoxic cell death. Oligodendrocytes are the myelinating cells of axons in the central nervous system and these cells are damaged in demyelinating disorders including multiple sclerosis (MS) and optic neuritis. In this study, we examined if Dock3 is expressed in oligodendrocytes and if increasing Dock3 signals can suppress demyelination in a cuprizone-induced demyelination model, an animal model of MS. We demonstrate that Dock3 is expressed in oligodendrocytes and Dock3 overexpression protects myelin in the corpus callosum following cuprizone treatment. Furthermore, we show that cuprizone demyelinates optic nerves and the extent of demyelination is ameliorated in mice overexpressing Dock3. Cuprizone treatment impairs visual function, which was demonstrated by multifocal electroretinograms, an established non-invasive method, and Dock3 overexpression prevented this effect. In mice overexpressing Dock3, Erk activation is increased, suggesting this may at least partly explain the observed protective effects. Our findings suggest that Dock3 may be a therapeutic target for demyelinating disorders including optic neuritis.Cell Death and Disease (2014) 5, e1395; doi:10.1038/cddis.2014.357; published online 28 August 2014.

Renin-angiotensin system regulates neurodegeneration in a mouse model of normal tension glaucoma.

Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs). In the mammalian retina, excitatory amino acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs, and the loss of EAAC1 leads to RGC degeneration without elevated intraocular pressure (IOP). In the present study, we found that expressions of angiotensin II type 1 receptor (AT1-R) and Toll-like receptor 4 (TLR4) are increased in RGCs and retinal Müller glia in EAAC1-deficient (KO) mice. The orally active AT1-R antagonist candesartan suppressed TLR4 and lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expressions in the EAAC1 KO mouse retina. Sequential in vivo retinal imaging and electrophysiological analysis revealed that treatment with candesartan was effective for RGC protection in EAAC1 KO mice without affecting IOP. In cultured Müller glia, candesartan suppressed LPS-induced iNOS production by inhibiting the TLR4-apoptosis signal-regulating kinase 1 pathway. These results suggest that the renin-angiotensin system is involved in the innate immune responses in both neural and glial cells, which accelerate neural cell death. Our findings raise intriguing possibilities for the management of glaucoma by utilizing widely prescribed drugs for the treatment of high blood pressure, in combination with conventional treatments to lower IOP.

Brimonidine prevents neurodegeneration in a mouse model of normal tension glaucoma.

Glaucoma is one of the leading causes of irreversible blindness that is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs). In the mammalian retina, excitatory amino-acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs, and the loss of EAAC1 leads to RGC degeneration without elevated intraocular pressure (IOP). Brimonidine (BMD) is an α2-adrenergic receptor agonist and it is commonly used in a form of eye drops to lower IOP in glaucoma patients. Recent studies have suggested that BMD has direct protective effects on RGCs involving IOP-independent mechanisms, but it is still controversial. In the present study, we examined the effects of BMD in EAAC1-deficient (KO) mice, an animal model of normal tension glaucoma. BMD caused a small decrease in IOP, but sequential in vivo retinal imaging and electrophysiological analysis revealed that treatment with BMD was highly effective for RGC protection in EAAC1 KO mice. BMD suppressed the phosphorylation of the N-methyl-D-aspartate receptor 2B (NR2B) subunit in RGCs in EAAC1 KO mice. Furthermore, in cultured Müller glia, BMD stimulated the production of several neurotrophic factors that enhance RGC survival. These results suggest that, in addition to lowering IOP, BMD prevents glaucomatous retinal degeneration by stimulating multiple pathways including glia-neuron interactions.

Dock3 attenuates neural cell death due to NMDA neurotoxicity and oxidative stress in a mouse model of normal tension glaucoma.

Dedicator of cytokinesis 3 (Dock3), a new member of the guanine nucleotide exchange factors for the small GTPase Rac1, promotes axon regeneration following optic nerve injury. In the present study, we found that Dock3 directly binds to the intracellular C-terminus domain of NR2B, an N-methyl-D-aspartate (NMDA) receptor subunit. In transgenic mice overexpressing Dock3 (Dock3 Tg), NR2B expression in the retina was significantly decreased and NMDA-induced retinal degeneration was ameliorated. In addition, overexpression of Dock3 protected retinal ganglion cells (RGCs) from oxidative stress. We previously reported that glutamate/aspartate transporter (GLAST) is a major glutamate transporter in the retina, and RGC degeneration due to glutamate neurotoxicity and oxidative stress is observed in GLAST-deficient (KO) mice. In GLAST KO mice, the NR2B phosphorylation rate in the retina was significantly higher compared with Dock3 Tg:GLAST KO mice. Consistently, glaucomatous retinal degeneration was significantly improved in GLAST KO:Dock3 Tg mice compared with GLAST KO mice. These results suggest that Dock3 overexpression prevents glaucomatous retinal degeneration by suppressing both NR2B-mediated glutamate neurotoxicity and oxidative stress, and identifies Dock3 signaling as a potential therapeutic target for both neuroprotection and axonal regeneration.

Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury.

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced RGC apoptosis. In this study, we found that ONI-induced p38 activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor and inducible nitric oxide synthase in microglia were decreased when the ASK1-p38 pathway was blocked. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 pathways could be beneficial in the treatment of ONI.

ASK1 deficiency attenuates neural cell death in GLAST-deficient mice, a model of normal tension glaucoma.

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced retinal ganglion cell (RGC) apoptosis. In the mammalian retina, glutamate/aspartate transporter (GLAST) is a major glutamate transporter, and the loss of GLAST leads to optic nerve degeneration similar to normal tension glaucoma (NTG). In GLAST⁻(/)⁻ mice, the glutathione level in the retina is decreased, suggesting the involvement of oxidative stress in NTG pathogenesis. To test this hypothesis, we examined the histology and visual function of GLAST(+/)⁻:ASK1⁻(/)⁻ and GLAST⁻(/)⁻:ASK1⁻(/)⁻ mice by multifocal electroretinograms. ASK1 deficiency protected RGCs and decreased the number of degenerating axons in the optic nerve. Consistent with this finding, visual function was significantly improved in GLAST(+/)⁻:ASK1⁻(/)⁻ and GLAST⁻(/)⁻:ASK1⁻(/)⁻ mice compared with GLAST(+/)⁻ and GLAST⁻(/)⁻ mice, respectively. The loss of ASK1 had no effects on the production of glutathione or malondialdehyde in the retina or on the intraocular pressure. Tumor necrosis factor (TNF)-induced activation of p38 MAPK and the production of inducible nitric oxide synthase were suppressed in ASK1-deficient Müller glial cells. In addition, TNF-induced cell death was suppressed in ASK1-deficient RGCs. These results suggest that ASK1 activation is involved in NTG-like pathology in both neural and glial cells and that interrupting ASK1-dependent pathways could be beneficial in the treatment of glaucoma, including NTG.

Production of methyl-oxonium ion and its complexes in the core-excited (HC(O)OCH3)n clusters: H/H+ transfer from the alpha carbonyl.

Dissociation of free methyl-formate (MF), HC(O)OCH3, and its clusters (MF)n, (HC(O)OCH3)n, induced by core-level excitation was studied near the oxygen K edge by time-of-flight fragment-mass spectroscopy. Besides the protonated clusters, (MF)nH+ with n < or = 15, we identified the production for another series of (MF)mCH3OH2+ with m < or = 14 as well as methyl-oxonium ion, CH3OH2+, characteristic of hydrogen transfer reactions in the cationic clusters. Here; specifically labeled methyl-formate-d (MFD), DC(O)OCH3 was also used to examine the core-excited dissociation mechanisms. Deuterium-labeled experiments indicated that MFD+ with low internal energies, partially generated after the core excitation, produces CH3OD+ via a site-specific deuterium transfer from the alpha carbonyl in the molecular cation and that CH3OD2+ can be formed via the successive transfer of another deuterium from the neighbor molecule in the clusters. The deuteron (proton) transfer was also found to take place preferentially from the alpha carbonyl of the neighbor molecule for the production of deuteronated (MFD)nD+, (protonated (MF)nH+), clusters. The minimal energy requirement paths were examined for dimer (MF)2+ cation to support the present dissociation mechanisms of core-excited (MF)n clusters using ab initio molecular-orbital calculations.

Intracerebroventricular administration of histamine H3 receptor antagonists decreases seizures in rat models of epilepsia.

The effects of histamine H3 antagonists on amygdaloid kindled and maximal electroshock seizures in rats were studied to determine their potential as new antiepileptic drugs. Under pentobarbital anesthesia, rats were fixed to a stereotaxic apparatus and a stainless steel guide cannula for drug administration was implanted into the lateral ventricle. In amygdaloid kindled seizures, electrodes were implanted into the right amygdala and electroencephalogram was recorded bipolarly; stimulation was applied bipolarly every day by a constant current stimulator and continued until a generalized convulsion was obtained. In the maximal electroshock (MES) seizure test, electroconvulsion was induced by stimulating animals through ear-clip electrodes, and the durations of tonic and clonic seizures were measured. Thioperamide, clobenpropit, iodophenpropit, VUF5514, VUF5515 and VUF4929 caused a dose-dependent inhibition of both seizure stage and afterdischarge (AD) duration of amygdaloid kindled seizures. The duration of tonic seizure induced by MES was also inhibited by H3 antagonists, but the duration of clonic seizures were unchanged. Among the H3 antagonists tested, clobenpropit and iodophenpropit were somewhat more potent than the other drugs on amygdaloid kindled seizures and MES seizures, respectively. These results indicate that some H3 antagonists may be useful as antiepileptic drugs, especially for secondary generalized seizures and/or tonic-clonic seizures in humans.

Activation of nuclear factor-kappa B in the conjunctiva with the epithelial scraping of the mouse cornea and human epidemic keratoconjunctivitis.

AIM: To examine the expression of p65, one of nuclear factor-kappa B (NF-kappa B), in the conjunctival epithelium of the C57Bl6 mouse and a patient with epidemic keratoconjunctivitis (EKC). METHODS: Normal and epithelial scraped cornea obtained 6 hours after the injury were processed for paraffin section. Samples of a normal and an EKC conjunctival epithelium were obtained using impression cytology. Both samples were analysed by immunocytochemistry using anti-p65 antibody. RESULTS: Immunocytochemistry with the anti-NF-kappa B p65 antibody revealed that p65 was localised in the cytoplasm of the conjunctival epithelium in the C57Bl6 mouse without the treatment. Six hours after the scraping of the cornea, p65 protein was expressed in the nuclei of the conjunctival epithelium. p65 was localised in the cytoplasm of the conjunctival epithelium in the human normal eye. p65 protein was expressed in the nuclei of the conjunctival epithelial cells in the EKC patient. CONCLUSION: These findings suggest that NF-kappa B was activated in the conjunctiva in the epithelial scraping of the mouse cornea and in human EKC.

Potential role of glial cell line-derived neurotrophic factor receptors in Müller glial cells during light-induced retinal degeneration.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and their receptors (GFRalpha1, GFRalpha2 and Ret) play an important role in the survival of neurons in the central and peripheral nervous system. For example, GDNF as well as other trophic factors promotes photoreceptor survival during retinal degeneration. Recent studies have proposed that part of neurotophic rescue of photoreceptors may be indirect, mediated by interaction of the neurotrophic factors with other cell types, that in turn release secondary factors that act directly on photoreceptors. In the present study, we examined the GDNF receptor expression in control and light-damaged retina, and found that GFRalpha2 protein is upregulated in retina-specific Müller glial cells during photoreceptor degeneration. We also examined the effect of GDNF or NTN on cultured Müller cells. Exogenous GDNF increased brain-derived neurotrophic factor, basic fibroblast growth factor and GDNF, but not NTN mRNA production. On the other hand, NTN increased NTN, but not GDNF mRNA production in cultured Müller cells. These observations suggest that GDNF, NTN and their receptors are involved in the regulation of trophic factor production in retinal glial cells, and that functional glia-neuron network may utilize GDNF family for the protection of neural cells during retinal degeneration.

NF-kappaB in epiretinal membranes after human diabetic retinopathy.

AIMS/HYPOTHESIS: Formation of epiretinal membranes (ERMs) in the posterior fundus results in progressive deterioration of vision. ERMs have been associated with numerous clinical conditions including proliferative diabetic retinopathy (PDR), but its pathogenic mechanisms are still unknown. This study was conducted to examine whether or not nuclear factor kappa B (NF-kappaB), a transcription factor that can be activated by various pathological conditions, is involved in the formation of ERMs after PDR. METHODS: ERM samples were obtained by vitrectomy from 22 cases with PDR aged 56+/-11 years with 18+/-10 years of diabetes and 15 cases with idiopathic ERM. They were processed for reverse transcription-polymerase chain reaction (RT-PCR) analysis. In addition, 5 ERM samples from PDR patients aged 51+/-16 years with 15+/-6 years of diabetes were processed for immunohistochemical analysis. RESULTS: NF-kappaB mRNA expression levels were higher (20 out of 22 cases vs. 9 out of 15 subjects in idiopathic ERM, p<0.05) in PDR subjects. Immunohistochemical analysis showed NF-kappaB protein expression in all the 5 ERMs derived from PDR patients, and that region was partially double-labelled with interleukin-8 (IL-8) and von Willebrand factor (vWF). CONCLUSIONS/INTERPRETATION: These results suggest a possibility that NF-kappaB is involved in the formation of ERMs after PDR, especially for the development of vascular endothelial cell component.

Proliferation in the posterior region of the lens of c-maf-/- mice.

PURPOSE: To examine the involvement of the c-maf gene in the proliferation of the lens cells. METHODS: Eyes of the E13 and E18 stages of the wild-type and c-maf-/- mice were analyzed by BrdU incorporation assay, TUNEL assay and immunocytochemistry using a anti-P27(KIP1) and a anti-P57(KIP2) antibody. RESULTS: In the E13 and E18 c-maf mutant lens, BrdU-positive cells were detected at the posterior region of the lens. Cell-cycle inhibitor P27(KIP1) and P57(KIP2) were expressed in the equatorial and posterior region of the lens of both wild-type and c-maf-/- lenses. CONCLUSION: These results suggest that the expression of c-maf is required for differentiation and cell cycle arrest of lens fiber cells. It is also suggested that P27(KIP1) and P57(KIP2) were not involved in the continued proliferation of posterior region of the c-maf-/- lens.

N-acetylated-alpha-linked-acidic dipeptidase inhibitor has a neuroprotective effect on mouse retinal ganglion cells after pressure-induced ischemia.

Excessive glutamate receptor activation is thought to be involved in the retinal ganglion cell (RGC) death after ischemic injury. In this study, we examined the effect of 2-PMPA (2-(phosphonomethyl)pentanedioic acid) on RGC survival in an ischemia-reperfusion model using C57BL/6 mouse eyes. 2-PMPA is a NAALADase (N-acetylated-alpha-linked-acidic dipeptidase) inhibitor, an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. 100mg/kg 2-PMPA were given with intraperitoneal injections 30 min before ischemia followed per hour injection for 3h. 2-PMPA increased surviving RGCs as well as retinal thickness after pressure-induced retinal ischemia. In addition, neuroprotection afforded by 2-PMPA was greater than that of N-methyl-D-aspartate receptor blocker. These data indicate that NAALADase inhibition may be useful in retinal disorders in which excessive amino acid transmission is pathogenic.

Biomechanics and classification of the cartilaginous structures to project the nasal tip.

The present paper is the long-term conclusion of our preliminary presentation at the 1992 ISAPS Congress (Guadalajara, Mexico). This is the result of 29 cadaver dissections of different ages and both sexes. We have observed that length, thickness, and resistance correlate with the possibility to project the nasal tip with the mere structure of the alar cartilage and its medial crurae. We also demonstrated the existence and antagonistic action of Pitanguy's ligament as well as the depressing ligament to project the nasal tip. In this study, besides focusing on classifying the medial crus according to its thickness, length, and resistance, which is already different from any previous classification, we also focused on its surgical utility, and its interaction with other anatomic elements, to achieve the desired projection. Clinically, we present a 12-year experience with 1653 cases operated under this premise.

Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration.

Prolonged or high-intensity exposure to visible light leads to photoreceptor cell death. In this study, we demonstrate a novel pathway of light-induced photoreceptor apoptosis involving the low-affinity neurotrophin receptor p75 (p75NTR). Retinal degeneration upregulated both p75NTR and the high-affinity neurotrophin receptor TrkC in different parts of Müller glial cells. Exogenous neurotrophin-3 (NT-3) increased, but nerve growth factor (NGF) decreased basic fibroblast growth factor (bFGF) production in Müller cells, which can directly rescue photoreceptor apoptosis. Blockade of p75NTR prevented bFGF reduction and resulted in both structural and functional photoreceptor survival in vivo. Furthermore, the absence of p75NTR significantly prevented light-induced photoreceptor apoptosis. These observations implicate glial cells in the determination of neural cell survival, and suggest functional glial-neuronal cell interactions as new therapeutic targets for neurodegeneration.

Severe optic disc edema without hydrocephalus in neurofibromatosis 2.

A 26-year-old man who had neurofibromatosis type-2 with symptoms of unexplained optic disc edema is reported. Magnetic resonance imaging (MRI) revealed bilateral acoustic schwannomas. Obstructive hydrocephalus, however, was not evident in spite of his severe disc edema and visual loss. After partial removal of the right acoustic schwannoma, symptoms of intracranial hypertension, such as vomiting and headache, developed and MRI demonstrated evidence of obstructive hydrocephalus. Placement of a ventricular-peritoneal shunt relieved the symptoms of intracranial hypertension, but visual acuity in his left eye was reduced to hand motion due to secondary optic atrophy. In patients with similar symptoms it is suggested that, in addition to tumor removal, early treatment to decrease intracranial pressure should be considered when visual function is progressively impaired by the symptoms of prolonged papilledema.