Topiramate reduces excitotoxic and ischemic injury in the rat retina

The effects of topiramate, a drug used clinically as an anti-epileptic, were investigated in excitotoxin-induced neurotoxicity models involving two different retinal primary cultures and in a rat model of retinal ischemic injury. For the in vitro studies, we used retinal-neuron cultures from rat embryos and purified retinal ganglion cells (RGCs) from newborn rats. In the retinal-neuron cultures, neurotoxicity was induced by a 10-min exposure to 1 mM glutamate or (+/-)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). In RGCs, neurotoxicity was induced by incubation for 3 days in a culture medium containing 25 microM glutamate. For the in vivo study, retinal ischemia was induced by elevating intraocular pressure to 130 mmHg for 45 min, and topiramate was administered intraperitoneally before and after the ischemia. Retinal damage was evaluated by measuring the number of cells in the ganglion cell layer (GCL) and the thickness of the inner plexiform layer (IPL), and by examining the a- and b-waves of the electroretinogram (ERG). Topiramate (> or =1 microM) markedly reduced the neuronal cell death induced by each of the excitotoxins in rat retinal-neuron cultures and in RGCs. Ischemia caused a decrease in GCL cells and in IPL thickness, and a diminution of the ERG waves. Histopathologic and functional analyses indicated that systemic treatment with topiramate prevented ischemia-induced damage in a dose-dependent manner. In conclusion, topiramate was protective against excitotoxic and ischemic retinal-neuron damage in vitro and in vivo, respectively. Therefore, it may be useful for treatment of the retina-related diseases such as central retinal artery occlusion, diabetic retinopathy, and glaucoma.     

Nicotinamide reduces hypoxic ischemic brain injury in the newborn rat

Nicotinamide reduces ischemic brain injury in adult rats. Can similar brain protection be seen in newborn animals? Seven-day-old rat pups had the right carotid artery permanently ligated followed by 2.5 h of 8% oxygen. Nicotinamide 250 or 500 mg/kg was administered i.p. 5 min after reoxygenation, with a second dose given at 6 h after the first. Brain damage was evaluated by weight deficit of the right hemisphere at 22 days following hypoxia. Nicotinamide 500 mg/kg reduced brain weight loss from 24.6 +/- 3.6% in vehicle pups (n = 28) to 11.9 +/- 2.6% in the treated pups (n = 29, P < 0.01), but treatment with 250 mg/kg did not affect brain weight. Nicotinamide 500 mg/kg also improved behavior in rotarod performance. Levels of 8-isoprostaglandin F2alpha measured in the cortex by enzyme immune assay 16 h after reoxygenation was 115 +/- 7 pg/g in the shams (n = 6), 175 +/- 17 pg/g in the 500 mg/kg nicotinamide treated (n = 7), and 320 +/- 79 pg/g in the vehicle treated pups (n = 7, P < 0.05 versus sham, P < 0.05 versus nicotinamide). Nicotinamide reduced the increase in caspase-3 activity caused by hypoxic ischemia (P < 0.01). Nicotinamide reduces brain injury in the neonatal rat, possibly by reducing oxidative stress and caspase-3 activity.     

Functional organization of rat olfactory bulb analysed by the 2-deoxyglucose method.

The spatial patterns of activity elicited in the rat olfactory bulb under different odor conditions have been analysed using the 2-deoxyglucose (2DG) technique. Rats were injected with 14C-2DG, exposed to controlled environments of amyl acetate, camphor, cage air, dimethyl disulfide, and pure air and autoradiographs prepared by the method of Sokoloff. Amyl acetate was associated with regions of glomerular layer densities in the anterolateral and mid- to posteromedial parts of the bulbar circumference, as previously reported. The extents of the densities increased with increasing concentration. Camphor odor was associated with regions of increased density in the anterodorsal and mid- to posteromedial parts of the bulb. Exposure to cage air produced scattered densities in the posteromedial and posterolateral bulb. Exposure to dimethyl disulfide gave variable results. Pure air was associated with a minimal number of small dense foci. The results with amyl acetate, camphor and cage air suggest that patterns for different odors are distinguishably different but overlapping. The regions of activity are greatest in extent and density with the highest odor concentrations. These define the regions within which more restricted and isolated foci appear at lower concentrations. The results thus provide evidence for the specific role of spatial factors in the neural processing of odor quality and odor concentration.     

Neutrophil elastase inhibition reduces cerebral ischemic damage in the middle cerebral artery occlusion

It has been reported that activated neutrophils are involved in the development of cerebral damage induced by ischemia. Activated neutrophils release a lot of mediators including toxic oxygen metabolites, elastase and cytokines which damage brain tissue. Therefore, we investigated roles of neutrophil elastase in the development of cerebral damage using an elastase inhibitor, ONO-5046. The rat middle cerebral artery (MCA) was occluded by a thrombus induced by photochemical reaction between green light and the photosensitizer dye, Rose Bengal. Photochemical reaction causes endothelial injury followed by formation of a platelet and fibrin-rich thrombus at the site of the irradiation. Photochemical reaction is routinely used in our laboratory to produce arterial occlusion in experimental animals. Twenty-four hours after the MCA occlusion, the size of cerebral damage was measured by histochemical technique. Water content in the brain was measured and neuronal deficits were examined 24 h after the MCA occlusion. ONO-5046 was administered at various doses as continuous infusion for 24 h, starting just after the MCA occlusion or from 3 h after. ONO-5046 at doses of 10 and 30 mg/kg/h significantly (p<0.05 and p<0.01, respectively) reduced the size of cerebral damage and water content (p<0.05, p<0.01, respectively) in different eight rats. Further, ONO-5046 at a dose of 30 mg/kg/h significantly (p=0.01) improved neuronal deficits. ONO-5046 which was administered starting from 3 h after the MCA occlusion, also reduced the size of cerebral damage. Neutropenia by anti-neutrophil antibody injection significantly (p<0. 01) reduced the size of cerebral damage. Elastase released from activated neutrophils may play a key role in the development of cerebral damage.     

Ascending reticular activating system in the rat: A 2-deoxyglucose study

The autoradiographic [14C]2-deoxyglucose (2-DG) method was used to map ascending pathways which are influenced by arousing electrical stimulation of the midbrain reticular formation (RET) in alert rats. The major finding was that RET stimulation produces selective patterns of metabolic activation and suppression in discrete brain regions. The regions activated were limited to specific intralaminar, medial and anterior thalamic nuclei, and to the entire auditory system. Conversely, a large suppression of 2-DG uptake was observed in most of the cerebral cortex, limbic and extrapyramidal structures, whereas at the same time some brain regions were left unaffected. Striking similarities were found between the functional pathways affected differentially by RET stimulation and well-defined cholinergic pathways which originate in the midbrain tegmentum. Structures which showed metabolic activation are part of the dorsal cholinergic pathway, whereas the regions suppressed are part of the ventral cholinergic pathway and its higher-order projections. The results support the conclusion that cholinergic pathways represent the thalamic and extrathalamic divisions of the reticular activating system. Our observations provide the first anatomical demonstration that RET stimulation has widespread and differential effects on cerebral metabolism. They also support the concept that arousing electrocortical desynchronization involves reticular activation of thalamocortical neurons, which in turn have widespread suppressive influences on cortical metabolism.     

Chronic ethanol treatment reduces inhibition in CA1 of the rat hippocampus

The effect of chronic ethanol exposure on inhibition in the rat hippocampal slice was investigated using paired-pulse stimulation techniques with stimulation in stratum radiatum or stratum oriens of CA1. Experimental animals were fed ethanol in a liquid diet for 20 weeks and were withdrawn for at least 8 weeks prior to electrophysiological recording. Prior ethanol treatment had no effect on basic input-output relationships for the extracellular population spike. Ethanol treatment significantly reduced the recurrent inhibition produced by antidromic stimulation in a manner dependent upon stimulus intensity. In addition, with orthodromic paired-pulse stimulation of either stratum radiatum or oriens, a trend toward an augmentation of the facilitation of population spike amplitude was observed, suggesting that feedforward inhibition may also be reduced. These results are similar to those found with treatments that reduce inhibition. Therefore, we conclude that chronic ethanol exposure produces an enduring disruption of inhibitory neuronal function in the rat hippocampus.     

The glutamate antagonist MK-801 reduces focal ischemic brain damage in the rat

Excessive activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor has been implicated in the sequence of neurochemical events that results in irreversible neuronal damage in cerebral ischemia. The effects of the NMDA antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) upon the amount of ischemic brain damage has been assessed quantitatively in the lightly anesthetized rat. Focal cerebral ischemia was produced by the permanent occlusion of one middle cerebral artery (MCA), and the animals were killed 3 hours after the arterial occlusion. MK-801 (0.5 mg/kg) was administered intravenously either 30 minutes prior to MCA occlusion or 30 minutes after the induction of ischemia. Pretreatment with MK-801 reduced the volume of ischemic damage both in the cerebral cortex (by 38% compared with untreated rats with MCA occlusion; p less than 0.01) and in the caudate nucleus (by 18% compared with controls; p less than 0.05). Treatment with MK-801, initiated 30 minutes after MCA occlusion, reduced the volume of ischemic damage in the cerebral cortex (by 52% compared with controls; p less than 0.01). The volume of ischemic damage in the caudate nucleus was minimally influenced by MK-801 treatment initiated after MCA occlusion. The antiischemic effects of MK-801 were readily demonstrable despite the hypotension that MK-801 induced in rats anesthetized with halothane (0.5%), nitrous oxide (70%), and oxygen (30%). The potency of MK-801 in reducing ischemic brain damage, even when administered after the induction of ischemia, highlights the potential use of NMDA receptor antagonists for the treatment of focal cerebral ischemia in humans.     

Effects of substantia nigra lesions on forebrain 2-deoxyglucose retention in the rat

A method was developed for the measurement of regional 2-deoxyglucose (2-DG) retention in rat brain by injecting tracer quantities of tritiated 2-DG intravenously, dissecting out individual brain regions, making extracts of the tissue, and counting aliquots of the extracts. This technique permits the separation of unreacted 2-DG from 2-deoxyglucose-6-phosphate (2-DGP) by ion exchange chromatography as well as the performance of other biochemical measurements on the extracts.Using this method, the effect of unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra on 2-DG retention and 2-DGP formation by the striattum and the cerebral cortex was investigated. Animals were studied both 3 days and 2–4 weeks after lesioning. The location and efficacy of the lesions were verified histologically, behaviorally (by observing rotational behavior), and biochemically (by assay of striatal dopamine concentration or tyrosine hydroxylase activity). The lesions induced a mean asymmetry of less than 10% in 2-DG retention and in 2-DGP formation in striatum and cerebral cortex. This result was verified by [¹⁴C]2-DG autoradiography. Systemic administration of amphetamine (5 mg/kg) or apomorphine HBr (1.5 mg/kg) elicited rotational behavior, but did not induce a marked asymmetry of 2-DG retention in the regions studied. It is concluded that unilateral lesions of the nigrostriatal dopaminergic pathway have, at most, a modest effect on 2-DG retention by forebrain structures. We also conclude that vehicle injections may produce morphological and chemical evidence of brain injury, including small but reproducible changes in deoxyglucose retention.     

Learning-related activation in the auditory system of the rat produced by long-term habituation: a 2-deoxyglucose study

Autoradiography with [14C]2-deoxyglucose (2-DG) was used to examine the functional activity of the rat auditory system during long- and short-term habituation of the acoustic startle reflex. The data showed that presentation of the acoustic stimulus to long-term habituated rats resulted in a learning-related metabolic enhancement that was significantly greater than the response evoked by the same acoustic stimulus in the inexperienced rats. This enhancement was localized to brainstem and midbrain auditory nuclei and no significant changes occurred at thalamocortical levels of the auditory pathway. The largest difference in 2-DG uptake between long- and short-term habituated rats was in the lateral superior olivary nucleus (LSO). The LSO activation suggests that olivocochlear efferents may operate in a central feedback control of peripheral auditory input during long-term habituation. Findings of enhanced metabolism from the cochlear nuclei to the central nucleus of the inferior colliculus indicated that active processes of neuronal plasticity take place in the lower auditory system during long-term habituation. The results provide the first demonstration of how a nonassociative learning experience such as long-term habituation modifies the metabolic activity of the auditory system. The findings support the conclusion that auditory responses of behaving animals to acoustic stimuli are dependent not only on the physical parameters of a stimulus, but also on its learned behavioral significance.     

Cilostazol reduces ischemic brain damage partly by inducing metallothionein-1 and -2

The neuroprotective effect of cilostazol, an antiplatelet drug, was examined after 24 h permanent middle cerebral artery (MCA) occlusion in mice, and explored the possible underlying mechanism by examining metallothionein (MT)-1 and -2 induction in vivo. Cilostazol (30 mg/kg) was intraperitoneally administered at 12 h before, 1 h before, and just after MCA occlusion. Mice were euthanized at 24 h after the occlusion, and the neuronal damage was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cilostazol significantly reduced the infarct area and volume, especially in the cortex. Real-time RT-PCR revealed increased mRNA expressions for MT-1 and -2 in the cortex of normal brains at 6 h after cilostazol treatment without MCA occlusion. MT-1 and -2 immunoreactivity was also increased in the cortex of such mice, and this immunoreactivity was observed in the ischemic hemisphere at 24 h after MCA occlusion (without cilostazol treatment). The strongest MT-1 and -2 immunoreactivity was detected in MCA-occlused mice treated with cilostazol [in the peri-infarct zone of the cortex (penumbral zone)]. These findings indicate that cilostazol has neuroprotective effects in vivo against permanent focal cerebral ischemia, especially in the penumbral zone in the cortex, and that MT-1 and -2 may be partly responsible for these neuroprotective effects.     

Simvastatin acutely reduces ischemic brain damage in the immature rat via Akt and CREB activation

We have previously shown that simvastatin (Sim) has long-lasting neuroprotective effects in a neonatal model of hypoxia–ischemia. Herein we evaluated the neuroprotective effect of different doses and duration of Sim treatment and further addressed its mechanism of action. Neonatal rats were subjected to occlusion of the right carotid artery followed by 2.5 h hypoxia (hypoxia–ischemia, HI). Sim was given at the dose of 10 or 5 mg/kg, s.c. from postnatal day 1 (PN1) to PN7, or at 20 mg/kg from PN4 to PN7, or at 20 mg/kg in a single administration 18 h before the onset of the ischemic procedure. Low-dose treatments or a single administration of the drug were effective in reducing HI-induced brain damage and its behavioural outcomes. Sim increased both Akt and CREB phosphorylation in neuronal cells and treatment with wortmannin completely blocked neuroprotection and p-Akt. These data demonstrate that even a single prophylactic Sim administration protects from hypoxic ischemic brain damage and that neuroprotection is in part obtained by preserving Akt and stimulating CREB phosphorylation in neuronal cells. Prophylactic Sim administration set in motion biochemical events that are known to increase brain tolerance to harmful factors, suggesting that the drug may exert neuroprotection by inducing pharmacological preconditioning.     

Phosphodiesterase-4 inhibition reduces proteolysis and atrogenes expression in rat skeletal muscles

Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE-4-selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram-induced muscle cAMP increase was accompanied by a decrease in ubiquitin-proteasome system (UPS) activity and atrogin-1 mRNA, a ubiquitin-ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin-1, MuRF-1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE-4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy.     

High-resolution 2-deoxyglucose autoradiography in quick-frozen slabs of neonatal rat olfactory bulb

We have used rapid freezing and freeze-substitution fixation to permit electron microscopic study of [3H]2-deoxyglucose autoradiographs. The techniques minimize diffusion of label into processing fluids and, by inference, migration of label within tissue. Slabs of olfactory bulbs from 12-day-old rats were quick-frozen after one hour of exposure to physiological olfactory stimuli. In light microscopic autoradiographs at low magnification, the neuropil of individual olfactory glomeruli appeared uniformly labeled with different levels of labeling in different glomeruli. At higher magnification, glomerular neuropil labeling consisted of small unlabeled regions surrounded by label clusters, suggesting greater deoxyglucose uptake by olfactory nerve terminals as compared with their postsynaptic dendrites. Periglomerular neurons were labeled differentially. Some microglia and glia precursor cells were heavily labeled in all bulbar laminae. The ultrastructure of cells and neuropil in all bulbar laminae was well-preserved. Cell processes and organelles could be identified in both stained sections and unstained electron microscopic autoradiographs. These experiments demonstrate the feasibility of combining quick-freezing with freeze substitution, in order to extend the resolution of studies using diffusable tracers such as 2-deoxyglucose. The results suggest that this is a promising method for assessing several controversies concerning deoxyglucose incorporation and neuronal and glial metabolism.     

Glycolytic activity and fructose 2, 6-bisphosphate changes in rat brain during ischemia

The brain ischemia of spontaneously hypertensive rat was produced gradually by bilateral ligation of the common carotid arteries. The cortical blood flow was measured with a laser doppler flowmeter before and after ligation of the arteries. At the specified intervals, the brain was frozen in situ with liquid nitrogen. The concentration of blood glucose and glycolytic intermediate in frozen brain were measured and the relationship between glycolytic activity and the concentrations of effectors to PFK-1, such as fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, AMP, ATP, Pi and citrate, was investigated. The changes in glycolytic intermediates, pyridine and adenine nucleotides concentration showed that ischemic change occurred in the brain tissue after 30 min of bilateral ligation of the common carotid arteries, in correlation with the decrease in cortical blood flow. The rate of lactate formation increased during the 30-60 min interval and finally decreased during 60-120 min period of ischemia. This indicates that anaerobic glycolysis was accelerated during the early stages of ischemia. The most potent activator of PFK-1, fructose 2, 6-bisphoshate, increased from 5.3 or 6.7 nmol g during the initial stage of ischemia, and this change preceded the activation of glycolysis and the increase in fructose 1,6-bisphosphate concentration, a result indicated that fructose 2,6-bisphosphate does participate in the activation of glycolysis during brain ischemia in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kanic acid alters cholinergic responses in the rat retina: A 2-deoxyglucose study

Intraocular injections of the neuroexcitatory toxin, kainic acid, did not alter the output of the retinal ganglion cells, as determined by the rate of glucose use in the stratum griseum superficialis of the superior colliculus. However, significant differences were observed in cholinergic interactions of the ganglion cells after kainic acid treatment. Intraocular injection of kainic acid prevented the increase in the stratum griseum superficialis activity typically produced by systemic injection of the acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). In addition, the retinal ganglion cells were strikingly sensitive to intraocular injections of acetylcholine 1 week after exposure to kainic acid, reflected in the marked increased glucose utilization in the stratum griseum superficialis. This responsiveness to acetylcholine may be entirely due to the 80% decrease in acetylcholinesterase in the retina observed 1 week after kainic acid exposure or in part to a supersensitivity of the ganglion cells following the period of acetylcholine depletion.     

Uncrossed and crossed inhibition in the inferior colliculus of the cat: a combined 2-deoxyglucose and electrophysiological study

The cat inferior colliculus (IC) was studied with 2-deoxyglucose (2-DG). By presenting high-frequency tone bursts to one ear and white noise bursts simultaneously to the other, a band of reduced or inhibitory labeling was revealed in the central nucleus (ICC) of the IC ipsilateral to the ear receiving the tone bursts. It was concluded that this ipsilateral inhibition might be related to the organization of excitatory/inhibitory units in ICC. In the opposite ICC, narrow bands of increased labeling were seen. In some animals, the positions of single units were marked, and tone frequencies were presented under 2-DG, which were the same as these units' characteristic frequencies (CFs). The positions of the units coincided with the position of the inhibitory bands, indicating that they were functional isofrequency-inhibitory contours. Unlike higher auditory centers, the binaural inhibitory areas were in register with and not orthogonal to the excitatory isofrequency contours. The inhibitory contours were generally larger than the excitatory contours and became even larger in more caudal sections. Both the inhibitory and excitatory contours extended into dorsal cortex areas of IC. In two other cats, high-frequency tone bursts and white noise bursts were presented to the same ear, and both a band of increased and a band of reduced labelling were found in the IC contralateral to this ear. The inhibitory band was always lateral to the excitatory band and was often smaller. They did not become larger in more caudal sections. The position of a unit in one cat was marked by pontamine sky blue, and the position of the unit coincided with the position of the excitatory band. It was concluded that this lateral inhibitory band represents high-frequency inhibitory sidebands of cells with CFs lower than the stimulating tone. It is concluded that the 2-DG method might reveal hitherto unknown inhibitory systems if stimuli could be combined with diffuse stimuli that raised the general background activity of sensory systems.     

Functional activation in the auditory system of the rat produced by arousing reticular stimulation: a 2-deoxyglucose study

The 2-deoxyglucose (2-DG) autoradiographic method was used to map the activity in the auditory pathway during behaviorally arousing electrical stimulation of the mesencephalic reticular formation (RET). Uptake of 2-DG during RET stimulation was compared to the effect of a frequency-modulated tone (4-5 kHz, 60 dB SPL) and to controls without stimulation. The major finding was a specific pattern of increased metabolic activation throughout the auditory pathway evoked during RET stimulation. The observed increases in 2-DG uptake were always greater in RET-stimulated rats as compared to sound-stimulated or control rats. The dorsal cochlear nucleus (DCN) showed the largest incorporation of 2-DG among the auditory nuclei of the brainstem in RET-stimulated rats. In the central nucleus of the inferior colliculus a layered pattern made of 3 discrete bands of high 2-DG uptake was visible in RET-stimulated rats. The medial geniculate (MG) and the auditory cortex (AC) also showed highly significant increases in 2-DG uptake induced by RET stimulation. The method provided correlations between classical morphological schemes of parcellation on nuclei and functionally defined areas of increased 2-DG uptake. Our observations represent the first anatomical demonstration of the activating effects of RET stimulation in a sensory system, and they support the concepts of arousing reticular mechanisms for sensory control.