Highlights • GH is expressed mainly as 15 kDa isoform in the green iguana neuroretina. • GH and GHR co-localize in retinal neural layers. • Kainic acid induces excitotoxic damage in the neuroretina. ...• Kainic acid administration increases the retinal GH and IGF-1 content. • GH stimulates retinal IGF-1 concentration and protects cells from KA damage.
To investigate whether and how leukemia inhibitory factor (Lif) is involved in mediating the neuroprotective effects of Norrin on retinal ganglion cells (RGC) following excitotoxic damage. Norrin is ...a secreted protein that protects RGC from
methyl-d-aspartate (NMDA)-mediated excitotoxic damage, which is accompanied by increased expression of protective factors such as Lif, Edn2 and Fgf2.
Lif-deficient mice were injected with NMDA in one eye and NMDA plus Norrin into the other eye. RGC damage was investigated and quantified by TUNEL labeling 24 h after injection. Retinal mRNA expression was analyzed by quantitative real-time polymerase chain reaction following retinal treatment.
After intravitreal injection of NMDA and Norrin in wild-type mice approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to NMDA-treated littermates, an effect which was lost in Lif-deficient mice. The mRNA expression for Gfap, a marker for Müller cell gliosis, as well as Edn2 and Fgf2 was induced in wild-type mice following NMDA/Norrin treatment but substantially blocked in Lif-deficient mice.
Norrin mediates its protective properties on RGC via Lif, which is required to enhance Müller cell gliosis and to induce protective factors such as Edn2 or Fgf2.
Oxidative/nitrosative stress is involved in NMDA receptor-mediated excitotoxic brain damage produced by the glutamate analog quinolinic acid. The purpose of this work was to study a possible role of ...peroxynitrite, a reactive oxygen/nitrogen species, in the course of excitotoxic events evoked by quinolinic acid in the brain. The effects of Fe(TPPS) (5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinate iron (III)), an iron porphyrinate and putative peroxynitrite decomposition catalyst, were tested on lipid peroxidation and mitochondrial function in brain synaptic vesicles exposed to quinolinic acid, as well as on peroxynitrite formation, nitric oxide synthase and superoxide dismutase activities, lipid peroxidation, caspase-3-like activation, DNA fragmentation, and GABA levels in striatal tissue from rats lesioned by quinolinic acid. Circling behavior was also evaluated. Increasing concentrations of Fe(TPPS) reduced lipid peroxidation and mitochondrial dysfunction induced by quinolinic acid (100μM) in synaptic vesicles in a concentration-dependent manner (10–800μM). In addition, Fe(TPPS) (10mg/kg, i.p.) administered 2 h before the striatal lesions, prevented the formation of peroxynitrite, the increased nitric oxide synthase activity, the decreased superoxide dismutase activity and the increased lipid peroxidation induced by quinolinic acid (240nmol/μl) 120 min after the toxin infusion. Enhanced caspase-3-like activity and DNA fragmentation were also reduced by the porphyrinate 24 h after the injection of the excitotoxin. Circling behavior from quinolinic acid-treated rats was abolished by Fe(TPPS) six days after quinolinic acid injection, while the striatal levels of GABA, measured one day later, were partially recovered. The protective effects that Fe(TPPS) exerted on quinolinic acid-induced lipid peroxidation and mitochondrial dysfunction in synaptic vesicles suggest a primary action of the porphyrinate as an antioxidant molecule.
In vivo findings suggest that the early production of peroxynitrite, altogether with the enhanced risk of superoxide anion (O
2
·
−) and nitric oxide formation (its precursors) induced by quinolinic acid in the striatum, are attenuated by Fe(TPPS) through a recovery in the basal activities of nitric oxide synthase and superoxide dismutase. The porphyrinate-mediated reduction in DNA fragmentation simultaneous to the decrease in caspase-3-like activation from quinolinic acid-lesioned rats suggests a prevention in the risk of peroxynitrite-mediated apoptotic events during the course of excitotoxic damage in the striatum. In summary, the protective effects that Fe(TPPS) exhibited both under
in vitro and
in vivo conditions support an active role of peroxynitrite and its precursors in the pattern of brain damage elicited by excitotoxic events in the experimental model of Huntington’s disease. The neuroprotective mechanisms of Fe(TPPS) are discussed.
The neurotoxic effects of excitatory amino acids (EAAs) are suggested to be connected with the chronic loss of neuronal cells, thereby being responsible for the age-related neurodegenerative ...diseases. Therefore, it seems conceivable that the excitatory amino acid transporters may contribute to the protection of neuronal cells against the excitotoxic damage by facilitating the removal of EAAs from the brain tissue. On the other hand, previous studies have suggested that glial cell differentiation may be involved in the protection and recovery of neural function probably through the elevation of BDNF gene expression in the brain. Based on these findings, histone deacetylase (HDAC) inhibitors are assumed to induce glutamate transporter-1 (GLT-1) gene expression probably through the promotion of glial cell differentiation. Then, we examined the effects of HDAC inhibitors on GLT-1 mRNA levels in rat C6 glioma cells and found that trichostatin A can induce GLT-1 gene transcription following steroid 5α-reductase and GFAP gene expression. Therefore, it seems conceivable that glial cell differentiation may play a potential role in the removal of EAAs probably through the expression of GLT-1, thereby being involved in the protection of neuronal cells against the chronic excitotoxic insults in the brain.
The possible neuroprotective effects of two GABAergic drugs, tiagabine (TGB) and vigabatrin (VGB), against N-methyl-D-aspartate (NMDA)-induced excitotoxicity have been investigated in the isolated ...chick embryo retina model. Retina segments were incubated either with NMDA alone (100 microM) or with NMDA and TGB or VGB (10-1,000 microM, added 5 min before NMDA). Retina damage was assessed after 24 h by measuring lactate dehydrogenase (LDH) activity present in the medium and by histological analysis. Both drugs reduced LDH release in a dose-dependent manner with comparable mean maximal values of 56.6-63.7% achieved at concentration of 1 mM. Histological analysis of retina slices was in line with the biochemical assays and showed partial preservation of drug exposed retina structure with reduced edema especially in the inner plexiform layer. The present data provide pharmacological evidence that both TGB and VGB reduce the severity of NMDA-induced excitotoxic damage. Although an increase in GABAergic transmission might play a role, this in itself is insufficient to explain the neuroprotective effect of the two drugs and the exact mechanism remains to be elucidated.
Unilateral epidural applications of nickel solution to motor cortex were followed in about 1 h by contralateral forelimb myoclonus. In rats which displayed frequent myoclonal jerking during the ...45-min 2-deoxyglucose (2-DG) uptake and clearing period, autoradiographic analysis showed that glucose utilization at the nickel implant site was greater in the supragranular and infragranular layers than in the granular layer (in normal cortex, activity is greatest in the granular layer), and was also greater in the substantia nigra and other subcortical centers. The same cortical and most of the subcortical changes in 2-DG uptake were also observed when metabolic activity was assessed 1 h after myoclonus had stopped, indicating that it may not have been the seizure activity itself that had altered metabolic activity, but some process engendered by the seizures - possibly a tissue response to excitotoxic damage. In fact, rats which displayed infrequent myoclonus showed negligible increases in cortical and subcortical uptake. These results do not support an earlier claim that increased glucose consumption is the metabolic signature of the interictal activity produced by seizure-inducing metals. Indeed, the findings raise the possibility that tissue damage is responsible for interictal hypermetabolism when it is observed in animal models of epilepsy.
Unilateral epidural applications of nickel solution to motor cortex were followed in about 1 h by contralateral forelimb myoclonus. In rats which displayed frequent myoclonal jerking during the ...45-min 2-deoxyglucose (2-DG) uptake and clearing period, autoradiographic analysis showed that glucose utilization at the nickel implant site was greater in the supragranular and infragranular layers than in the granular layer (in normal cortex, activity is greatest in the granular layer), and was also greater in the substantia nigra and other subcortical centers. The same cortical and most of the subcortical changes in 2-DG uptake were also observed when metabolic activity was assessed 1 h after myoclonus had stopped, indicating that it may not have been the seizure activity itself that had altered metabolic activity, but some process engendered by the seizures — possibly a tissue response to excitotoxic damage. In fact, rats which displayed infrequent myoclonus showed negligible increases in cortical and subcortical uptake. These results do not support an earlier claim that increased glucose consumption is the metabolic signature of the interictal activity produced by seizure-inducing metals. Indeed, the findings raise the possibility that tissue damage is responsible for interictal hypermetabolism when it is observed in animal models of epilepsy.
In histological studies using retinas, eyes are commonly fixed with aldehyde derivatives administered by immersion or perfusion. However, the histology of rat retinas chemically fixed as a whole eye ...is typically inferior to the histology of retinas that are immediately fixed after acute dissection from the rest of the eye. Chemical fixation without dissection often results in neuronal swelling resembling excitotoxic damage induced by ischemia because the retina is protected by the sclera and is thus poorly accessible to immersion or perfusion fixation techniques. In order for the acute dissection technique to work properly, it must be completed in a timely manner, which may be difficult under some circumstances. Microwave irradiation is an alternative method for fixing tissues that are inaccessable to chemicals. We examined the effectiveness of microwave irradiation of the whole eye as a substitute for acute retinal dissection. To study the feasibility of microwave methods, we compared retinal morphology using microwave irradiation to morphology using conventional immersion fixation methods.
Eyes were removed from rats, placed in a container with 2 or 20 ml artificial cerebrospinal fluid (aCSF) and irradiated with a household microwave oven. For morphological comparison, control eyes were immersed in a chemical fixative containing 1% paraformaldehyde and 1.5% glutaraldehyde. All eyes were embedded in araldite for evaluation by light microscopy.
Retinal segments acutely isolated before immersion fixation revealed intact histology whereas retinal segments exposed to 60 min of simulated ischemia showed severe neuronal degeneration. Using an immersion technique, the retinas of chemically fixed whole eyes showed neuronal swelling similar to excitotoxic ischemic damage, suggesting that conventional immersion methods provide poor whole eye fixation. The neuronal degeneration observed with conventional immersion fixation was not found in retinas of whole eyes fixed with 20 sec of microwave irradiation. During microwave irradiation the temperature in the bathing aCSF rose to 55–72°C. In some eyes, overcooking produced chromatin clumping and a small loss of contrast in staining.
Although nuclear clumping and diminished staining occasionally result from overcooking, ischemic damage is well controlled with microwave fixation of enucleated eyes. When the optimal conditions are defined, microwave fixation may be preferable for retinal histology if chemical fixation following acute dissection is not feasible.
Recent data suggest that brain damage in ischemia, hypoglycemia, and several other brain diseases is caused by excitotoxic mechanisms which are triggered by presynaptic release of glutamate and ...related excitatory amino acids, and which involve an abnormal postsynaptic influx of calcium into cells containing a high density of glutamate receptors. This contention is supported by results demonstrating reduction of infarct size in focal ischemia due to middle cerebral artery (MCA) occlusion, and amelioration of neuronal necrosis in hypoglycemic coma, by antagonist which block the NMDA type of glutamate receptor. These results underscore the pathogenetic role of calcium influx into energy-compromised cells since the NMDA receptor-linked ion channel has a high conductance to calcium. The issue has been clouded by the inability of NMDA antagonists to ameliorate brain damage due to cardiac arrest, or to forebrain ischemia in rats and gerbils. In these conditions, however, an AMPA receptor blocker (NBQX) has been found efficacious. These results demonstrate that the pathophysiology of ischemic lesions is different in the cardiac arrest type of ischemia and in lesions due to MCA occlusion, and demand an explanation of the differences in therapeutic response. Tentatively, the cardiac arrest type of ischemia is so dense that multiple calcium conductances are activated in the energy-deprived tissue, explaining why any drug which acts on only one of them (such as an NMDA antagonist) cannot prevent cellular calcium overload. Furthermore the ultimate brain damage, which is often conspicuously delayed, may be secondary to upregulation of synaptic efficacy, causing increased calcium cycling and calcium-related damage. In this situation, an AMPA receptor blocker may be efficacious because it blocks "fast" excitation and Na+ influx, an "upstream" event which causes "downstream" calcium influx via multiple pathways. In the perifocal ("penumbra") zone of a stroke lesion, the situation is different since depolarisation is initially moderate and/or intermittent. Furthermore, since ATP is still produced (albeit at a reduced rate) the problem is one of a disturbed pump/leak relationship. Then, blockade of a major calcium-carrying channel by NMDA receptor blockers, or of the trigger to depolarisation by an AMPA receptor antagonist, may improve the pump/leak relationship and carry cells in the penumbra over a critical period.
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