In situ localization of nitric oxide synthase and direct evidence of NO production in rat retinal ganglion cells

The expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) during synaptogenesis for various phases of the pre- and postnatal developmental periods were investigated. The retinas from prenatal, lactating, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pups were immunostained for constitutive isoforms of NOS (n and eNOS) and observed with a confocal laser scanning microscope. Synthesis of NO in the RGCs was achieved by in vitro stimulation with glutamate. The intracellular NO levels were measured in real time using diaminofluorescein-2 diacetate, a fluorescence indicator of NO. Immunohistochemical analysis revealed nNOS and eNOS expressed in retinal ganglion cells during the first 2 postnatal weeks. Cultured RGCs also expressed nNOS and eNOS in vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, L-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited (P<0.001) the increase of intracellular NO 6 and 8 min after the introduction of L-arginine and glutamate to the medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glutamate in cultured RGCs.     

Role of nitric oxide in the brain during lipopolysaccharide-evoked systemic inflammation

Although the inducible isoform of nitric oxide synthase (iNOS) is a well-established source of nitric oxide (NO*) during inflammation of the central nervous system (CNS), little is known about the involvement of constitutive isoforms of NOS (cNOS) in the inflammatory process. The aim of this study was to compare the responses of the expression and activity of iNOS and the two cNOS isoforms, neuronal and endothelial (nNOS and eNOS, respectively), in the brain to systemic inflammation and their roles in the cascade of events leading to degeneration and apoptosis. A systemic inflammatory response in C57BL/6 mice was induced by intraperitoneal injection of lipopolysaccharide [LPS; 1 mg/kg body weight (b.w.)]. The relative roles of the NOS isoforms were evaluated after injection of NG-nitro-L-arginine (NNLA; 30 mg/kg b.w.), which preferentially inhibits cNOS, or 1400W (5 mg/kg b.w.), an inhibitor of iNOS. Biochemical and morphological alterations were analyzed up to 48 hr after administration of LPS. Systemic LPS administration evoked significant ultrastructural alterations in brain capillary vessels, neuropils, and intracellular organelles of neurons, astrocytes, and microglia. Apoptotic/autophagic processes occurred in many neurons of the substantia nigra (SN), which coincided with exclusive enhancement of iNOS expression and activity in this brain region. Moreover, inhibitors of both iNOS and cNOS prevented LPS-evoked release of apoptosis-inducing factor (AIF) from SN mitochondria. Collectively, the results indicate that synthesis of NO* by both the inducible and constitutive NOS isoforms contribute to the activation of apoptotic pathways in the brain during systemic inflammation.     

一氧化氮合酶活性与抑郁症的相关性

目的通过对抑郁症患者一氧化氮合酶（NOS）活性进行检测，从而研究和探讨一氧化氮合酶、一氧化氮（NO）与抑郁症之间的关系。方法采用分光光度法检测抑郁症患者治疗前后的一氧化氮合酶NOS及其亚型（结构型cNOS、诱导型iNOS）的活性，并与正常对照组比较。结果抑郁症组的NOS、cNOS活性显著低于正常对照组；治疗组的NOS、cNOS活性高于抑郁症（无显著性），但治疗后缓解组的NOS、cNOS活性均显著高于治疗前。各组iNOS的活性无显著差异。结论抑郁症病人的NOS活性下降，而且主要是结构型cNOS活性下降，经治疗缓解后有所提高。因此，NOS和NO很有可能在抑郁症的发病过程中起着重要作用。     

Lens injury stimulates axon regeneration in the mature rat optic nerve.

In mature mammals, retinal ganglion cells (RGCs) are unable to regenerate their axons after optic nerve injury, and they soon undergo apoptotic cell death. However, a small puncture wound to the lens enhances RGC survival and enables these cells to regenerate their axons into the normally inhibitory environment of the optic nerve. Even when the optic nerve is intact, lens injury stimulates macrophage infiltration into the eye, Müller cell activation, and increased GAP-43 expression in ganglion cells across the entire retina. In contrast, axotomy, either alone or combined with intraocular injections that do not infringe on the lens, causes only a minimal change in GAP-43 expression in RGCs and a minimal activation of the other cell types. Combining nerve injury with lens puncture leads to an eightfold increase in RGC survival and a 100-fold increase in the number of axons regenerating beyond the crush site. Macrophage activation appears to play a key role, because intraocular injections of Zymosan, a yeast cell wall preparation, stimulated monocytes in the absence of lens injury and induced RGCs to regenerate their axons into the distal optic nerve.     

CNTF promotes the regrowth of retinal ganglion cell axons into murine peripheral nerve grafts

Autologous peripheral nerves were transplanted onto transected optic nerves of adult mice. We examined whether intraocular CNTF injections increased retinal ganglion cell (RGC) axon regeneration, and what types of RGCs regrew axons into grafts. After temporal CNTF eye injections there were more fluorogold-labelled regenerating RGCs (mean +/- s.e.m. 342+/-113.1; n=6) than in sham eye-injected mice (133+/-27.6; n=8). Greater numbers of regenerating RGCs (1198+/-367.6; n=6) were seen in mice receiving both nasal and temporal CNTF injections. The range of soma areas in regenerate and normal retinas was similar but the average size of regenerating RGCs was greater (212 microm2 vs 111 microm2). Most regenerating RGCs had large dendritic fields. The data suggest a heterogeneous response to axotomy in adult mice, large RGCs preferentially regrowing axons into PN grafts.     

Enhanced survival and regeneration of axotomized retinal neurons by repeated delivery of cell-permeable C3-like Rho antagonists

Inactivation of Rho GTPase with a single intraocular injection of Rho antagonists stimulates survival and regeneration of retinal ganglion cells (RGCs) after optic nerve injury. However, this effect is short-lived. Here we tested the impact of multiple injections of C3-like Rho antagonists on RGC viability and axon regeneration after optic nerve lesion. Our data show that both neuronal survival and axon regeneration were enhanced with repeated delivery of cell-permeable C3. We found an approximately 1.5-fold increase in RCG survival when additional Rho antagonist injections were performed after the first week from the time of lesion. In contrast, increased regeneration required early inactivation of Rho and injections performed in the second week did not further enhance regenerative outcome. These results reveal differences in the length of the therapeutic windows through which Rho inactivation acts on RGC survival or regeneration after axotomy.     

Contribution of caspase-8 to apoptosis of axotomized rat retinal ganglion cells in vivo

We have investigated the role of caspase-8 and its mode of activation during apoptosis of adult rat retinal ganglion cells (RGCs) in vivo. Retinal pro-caspase-8 expression was almost completely restricted to RGCs. Although caspase-8 is known to be involved in death-receptor-dependent apoptosis, measurable caspase-8 activity or even RGC death could be induced by neither tumor necrosis factor-alpha nor Fas ligand injections into unlesioned eyes. However, substantial caspase-8 activation could be detected after optic nerve transection as shown by a fluorogenic activity assay and Western blot analysis. Intravitreal injection of caspase-8 inhibitors significantly attenuated degeneration of RGCs and reduced the number of RGCs showing caspase-3 activation. A late peak of caspase-8 activity and additive protective effects of caspase-8 and -9 inhibition on axotomized RGCs place caspase-8 in our model rather late in the apoptosis cascade, possibly after the onset of mitochondrial dysfunction.     

Protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo

Following intraorbital transection of the optic nerve (ON) in rats, more than 80% of the retinal ganglion cell (RGC) population die by apoptosis within 14 days. Repeated intraocular injection of brain-derived neurotrophic factor (BDNF) has been efficient in enhancing RGC survival following ON axotomy. The present study was designed to define a potential survival-promoting effect of adenovirally administered BDNF on axotomized RGCs. A single injection of an adenoviral vector expressing the human BDNF gene from a CMV promoter/enhancer (Ad-BDNF) enhanced RGC survival 14 days after axotomy by 40.3%. Moreover, a combinatory treatment regimen consisting of intraocular Ad-BDNF administration and systemic application of the free radical scavenger, N-tert-butyl-(2-sulphophenyl)-nitrone (S-PBN), enhanced RGC survival by 63.0%. Our data demonstrate that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced RGC death. Further, as shown for S-PBN, therapeutic regimens that combine local virus-mediated gene delivery with systemic administration of protective compounds, may offer promising strategies for future treatment also in human neurodegenerative conditions.     

Regulation of caspase activation in axotomized retinal ganglion cells

Transection of the optic nerve initiates massive death of retinal ganglion cells (RGCs). Interestingly, despite the severity of the injury, RGC loss was not observed until several days after axotomy. The mechanisms responsible for this initial lack of RGC death remained unknown. In the current study, immunohistochemical analysis revealed that caspases-3 and -9 activation in the RGCs were not detected until day 3 post-axotomy, coinciding with the onset of axotomy-induced RGC loss. Interestingly, elevated Akt phosphorylation was observed in axotomized retinas during the absence of caspase activation. Inhibiting the increase in Akt phosphorylation by intravitreal injection of wortmannin and LY294002, inhibitors of PI3K, resulted in premature nuclear fragmentation, caspases-3 and -9 activation in the ganglion cell layer. Our findings thus indicate that the PI3K/Akt pathway may serve as an endogenous regulator of caspase activation in axotomized RGCs, thereby, contributing to the late onset of RGC death following axotomy.     

Selective impairment of slow axonal transport after optic nerve injury in adult rats.

To investigate cellular responses of injured mammalian CNS neurons, we examined the slow transport of cytoskeletal proteins in rat retinal ganglion cell (RGC) axons within the ocular stump of optic nerves that were crushed intracranially. RGC proteins were labeled by an intravitreal injection of 35S-methionine, and optic nerves were examined by SDS PAGE at different times after injury. In one group of rats, the RGC proteins were labeled 1 week after crushing. From 14 to 67 d after axotomy, the labeling of tubulin and neurofilaments was reduced in relation to other labeled proteins and to the labeling of tubulin and neurofilaments in the intact optic nerve of controls. To determine whether this reduction in labeling was due to an alteration in axonal transport after axotomy, we prelabeled RGC proteins 1 week before crushing. In such experiments, the rate of slow axonal transport of tubulin and neurofilaments decreased approximately 10-fold from 6 to 60 d after injury. Our results cannot be due only to the retrograde degeneration of RGCs and injured axons caused by axotomy in the optic nerve, because fast axonal protein transport and the fluorescent labeling of many axons were preserved in the ocular stumps of these optic nerves. This selective failure of the slow axonal transport of tubulin and neurofilaments may affect the renewal of the cytoskeleton and contribute to the gradual degeneration of RGCs that is observed after axotomy. The alterations in slow transport we document here differ from the enhanced rates we previously reported when injured RGC axons regenerated along peripheral nerve segments grafted to the ocular stump of transected optic nerves (McKerracher et al., 1990).     

Neurotrophin receptor TrkB activation is not required for the postnatal survival of retinal ganglion cells in vivo.

During early postnatal development, apoptosis of retinal ganglion cells (RGCs) is regulated by target contact with the optic tectum. The neurotrophins BDNF and NT-4, but not NGF, prevent the apoptosis of retinal ganglion cells that is otherwise observed after target ablation or axotomy. Thus receptors activated by BDNF and NT-4 are candidates to mediate the early postnatal survival of RGCs. BDNF and NT-4, but not NGF, bind to all isoforms of the receptor TrkB, whether or not they contain a tyrosine kinase domain. To examine the roles of TrkB receptor isoforms in early postnatal survival, we compared RGC numbers in wild-type mice to those in a mutant lacking all isoforms of TrkB. Surprisingly, no reduction in RGCs was observed in the mutant at postnatal day 16, the latest age at which these animals are consistently viable, so TrkB signaling is not essential for target-dependent survival of these cells. In wild-type mice, RGCs also are lost gradually during adulthood, possibly due to oxidative stress. To determine whether TrkB signaling regulates this phase of RGC degeneration, RGC numbers were examined in a viable mutant of TrkB that expresses only about 25% the normal level of TrkB receptor kinase. Compared to controls, approximately 20% of the RGC were lost in mutant 3-month-old-animals. Thus, TrkB signaling is not required for survival of RGCs during the period of target-dependent survival, but does appear to reduce degeneration of RGCs in adult animals.     

Basic fibroblast growth factor applied to the optic nerve after injury increases long-term cell survival in the frog retina

The neuroprotective effects of basic fibroblast growth factor (bFGF) on the long-term survival of axotomized retinal ganglion cells (RGCs) were studied in the frog Rana pipiens. Cell loss was quantified in different regions of the ganglion cell layer using Nissl staining and tetramethylrhodamine dextran amine backfilling. All regions of the retina showed a significant decrease (32-66%) in RGC numbers between 4 and 16 weeks after axotomy. Some cells showed morphological and biochemical signs of apoptosis. A single application of bFGF to the optic nerve stump at the time of axotomy protected many of the cells 6 weeks after the injury, but this effect was lost by 12 weeks. A second application of bFGF, 6 weeks after the injury, rescued many RGCs at 12 weeks. In contrast, single or double injections of bFGF into the eyeball had no effect on RGC survival. Axotomized RGCs were significantly enlarged and elongated after axotomy, and these morphological changes were increased by bFGF treatment. In the normal retina and optic nerve, immunocytochemical staining showed bFGF-like immunoreactivity (-LI) in the pigment epithelial layer, in the outer segments of photoreceptors, and in occasional RGCs. Strong bFGF-LI was present in Müller cells and in optic nerve astrocytes and oligodendrocytes. FGF receptor-LI was present in photoreceptors, outer plexiform layer, retinal ganglion cell axons, and Müller cells. FGF receptor-LI was also observed in optic nerve glia.     

The anterogradely transported BDNF promotes retinal axon remodeling during eye specific segregation within the LGN

Neurotrophins have been implicated in regulating many aspects of neuronal development and plasticity, including dendritic and axonal elaboration, by acting primarily as target derived trophic factors. Recently, we have shown that brain-derived neurotrophic factor (BDNF) is produced by retinal ganglion cells (RGCs) and travels in an anterograde direction along the optic nerve in neonatal rats. Here, we have assessed whether the anterogradely transported BDNF plays a role in shaping the retinogeniculate connectivity during development. We used intraocular injections of antisense oligonucleotides to suppress selectively retinal synthesis and anterograde transport of BDNF in rat pups. We found that in the absence of endogenous BDNF, RGC axons retract from their target in the dorsal lateral geniculate nucleus (dLGN). The blockade of BDNF action at the retinal level with the tyrosine kinase inhibitor, K252a, failed to produce this effect, suggesting an anterograde action of the endogenous BDNF. Moreover, the effects of BDNF removal on RGC fibers were evident only during a narrow temporal window coincident with the critical period for the retinothalamic refinement, indicating a role for BDNF on growth and elaboration of RGC axons rather than on their maintenance. Altogether these results propose a novel role for BDNF in the elaboration of retinogeniculate axons.     

Involvement of nitric oxide pathway in the acute anticonvulsant effect of melatonin in mice

Melatonin, the major hormone produced by the pineal gland, is shown to have anticonvulsant effects. Nitric oxide (NO) is a known mediator in seizure susceptibility modulation. In the present study, the involvement of NO pathway in the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced clonic seizures was investigated in mice. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) significantly increased the clonic seizure threshold induced by intravenous administration of PTZ. This effect was observed as soon as 1 min after injection and lasted for 30 min with a peak effect at 3 min after melatonin administration. Combination of per se non-effective doses of melatonin (10 and 20 mg/kg) and nitric oxide synthase (NOS) substrate L-arginine (30, 60 mg/kg) showed a significant anticonvulsant activity. This effect was reversed by NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg), implying an NO-dependent mechanism for melatonin effect. Pretreatment with L-NAME (30 mg/kg) and N(G)-nitro-L-arginine (L-NNA, 10 mg/kg) inhibited the anticonvulsant property of melatonin (40 and 80 mg/kg) and melatonin 40 mg/kg, respectively. Specific inducible NOS (iNOS) inhibitor aminoguanidine (100 and 300 mg/kg) did not affect the anticonvulsant effect of melatonin, excluding the role of iNOS in this phenomenon, while pretreatment of with 7-NI (50 mg/kg), a preferential neuronal NOS inhibitor, reversed this effect. The present data show an anticonvulsant effect for melatonin in i.v. PTZ seizure paradigm, which may be mediated via NO/L-arginine pathway by constitutively expressed NOS.     

In vivo imaging of murine retinal ganglion cells

Current methods for in vivo retinal ganglion cells (RGCs) imaging involve either retrograde or intravitreal injection of chemical or biological tracers, which are invasive and may require repeated injection for serial long-term assessment. We have developed a confocal scanning laser ophthalmoscope technique (blue-light CSLO or bCSLO) to image retinal ganglion cells (RGCs) in mice expressing cyan fluorescent protein under the control of a Thy-1 promoter. Fluorescent spots corresponding to CFP-expressing retinal ganglion cells were discernable with the bCSLO. 96.1+/-2.6% of CFP expressing cells also were retrograde labeled with DiI indicating the bCSLO imaged fluorescent spots are RGCs. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator to reflect the integrity of RGCs, and provides a non-invasive method for longitudinal study of the mechanism of RGC degeneration and the effect of neuroprotective agents.     

Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons

In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.     

Inducible and neuronal nitric oxide synthases (NOS) have complementary roles in recovery sleep induction

Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. We have previously shown that nitric oxide (NO) generation increases in the basal forebrain (BF) during sleep deprivation (SD). Moreover, both NO synthase (NOS) inhibition and a NO scavenger prevented recovery sleep induction, while administration of a NO donor during the spontaneous sleep-wake cycle increased sleep, indicating that NO is necessary and sufficient for the induction of recovery sleep. Next we wanted to know which NOS isoform is involved in the production of recovery sleep. Using in vivo microdialysis we infused specific inhibitors of NOS into the BF of rats during SD, and found that an inhibitor of inducible NOS (iNOS), 1400W, prevented non-rapid eye movement (NREM) recovery, while an inhibitor of neuronal NOS (nNOS), L-N-propyl-arginine, decreased REM recovery but did not affect NREM recovery. Using immunoblot analysis we found that iNOS was not expressed during the spontaneous sleep-wake cycle, but was induced by prolonged wakefulness (increased by 278%). A known iNOS inducer, lipopolysaccharide, evoked an increase in sleep that closely resembled recovery sleep, and its effects were abolished by 1400W. These results suggest that the elevation of NO produced by induction of iNOS in the BF during prolonged wakefulness is a specific mechanism for producing NREM recovery sleep and that the two NOS isoforms have a complementary role in NREM and REM recovery induction.     

Neuroprotective effects of erythropoietin on glutamate and nitric oxide toxicity in primary cultured retinal ganglion cells

Erythropoietin receptor (EpoR) is expressed in the central nervous system (CNS), however, no clear consensus has been obtained whether Epo acts as a prosurvival factor in neurons. Because retinal ganglion cell (RGC) death is a common cause of reduced visual function in several ocular diseases, we explored whether Epo might potentially be beneficial in protecting RGCs from glutamate and nitric oxide (NO)-induced cytotoxicity, using isolated RGCs by a two-step panning method. Brain-derived neurotrophic factor (BDNF) was used as a positive control. EpoR mRNA was expressed in isolated RGCs, and EpoR protein was expressed on the RGCs in the normal and ischemic retinas. Epo had less potential to improve the survival of primary RGCs in serum-free medium than BDNF. In these cells, BDNF, but not Epo, downregulated the expression of Bim, a proapoptotic Bcl-2 family member that plays a key role in cytokine-mediated cell survival, suggesting a possible mechanism for this difference. When RGCs were cultured with glutamate or an NO-generating reagent, the survival of RGCs was compromised, and Bcl-2 expression was decreased in these cells. Both Epo and BDNF significantly reduced RGC death induced by glutamate and NO. In agreement with this, these factors reversed the Bcl-2 expression. These findings suggest that Epo may be a potent neuroprotective therapeutic agent for the treatment of ocular diseases that are characterized by RGC death.     

Altered Expression of nNOS/NIDD in the Retina of a Glaucoma Model of DBA/2J Mice and the Intervention by nNOS Inhibition

The NIDD gene, neuronal NOS (nNOS)-interacting DHHC domain-containing protein with dendritic mRNA, codes a protein that upregulates nNOS enzyme activity by the interaction with the postsynaptic density protein 95/discsslarge/zon occlusens-1 (PDZ) domain of nNOS. Glial cell activation, especially Müller cells, may be an important factor contributing to retinal ganglion cell (RGC) death in glaucoma. The present study was to measure nNOS and NIDD expression in DBA/2J mice, a mouse model of glaucoma, and their correlation with glaucomatous phenotypes. Slit-lamp biomicroscopy, fundus photography, intraocular pressure (IOP) measurement, histology, and optic nerve axon counts were used to examine the ocular phenotypes of DBA/2J mice. Quantitative real-time PCR(RT-PCR) and Western blot analysis were used to analyze mRNA and protein expression of nNOS and NIDD. Their spatial distribution was evaluated by immunohistochemistry. Immunofluorescence was performed to observe the colocalization of nNOS and NIDD and the association of NIDD with Müller cells. The results showed that the prevalence and severity of ocular abnormalities, IOP, optic nerve cupping, and optic nerve atrophy increased with age. The mRNA and protein expression of nNOS reached the peak at 9 months old. The protein of NIDD underwent a similar change, while the mRNA of NIDD significantly increased at 6 months old. The expression of NIDD physically coexisted with nNOS in Müller cells. Administration of NOS inhibitor N^G-Nitro-l-arginine-methyl-ester (L-NAME) by intraperitoneal injection (i.p.) prevented RGCs from apoptosis as shown in the increase of Brn-3a (RGC marker) expression, which was accompanied by decreased expression of NIDD. The spatiotemporal changes of nNOS/NIDD expression and its interference suggest that NIDD–nNOS axis may play a role in the degenerative process of RGC in glaucoma.