Neurotensin enhances endogenous extracellular glutamate levels in primary cultures of rat cortical neurons: involvement of neurotensin receptor in NMDA induced excitotoxicity

Primary cultures of cortical neurons were employed to investigate the modulatory effects of neurotensin on glutamate excitotoxicity and the possible neuroprotective actions of the neurotensin receptor antagonist SR48692. NT(1-13) and its biologically active fragment NT(8-13) at 10 nM (30 min) increased endogenous glutamate levels. The inactive fragment NT(1-7) (10-100 nM; 30 min) was ineffective. SR48692, applied 20 min before NT and maintained in contact with cells during NT exposure as well as a low calcium medium (from the onset of the experiment) prevented the NT(1-13)-induced increase in extracellular glutamate levels. The addition of NMDA (0.01-10 micro M; 10 min) to the medium concentration-dependently increased extracellular glutamate levels. When 0.1 nM NT(1-13) was added in combination with 0.01 micro M NMDA, in concentrations by themselves ineffective, a significant increase in glutamate levels was observed. SR48692 at 100 nM counteracted the increase in glutamate levels induced by 0.1 nM NT(1-13) plus 0.01 micro M NMDA. The inhibitor of the protein kinase C (PKC) calphostin C (0.1 micro M; 10 min before NT) prevented the increase in glutamate levels induced by the combined treatments. The morphological analysis indicated that 10 nM NT(1-13) enhanced the glutamate (10 min)-induced apoptosis. The peptide was added 30 min prior to glutamate and maintained in contact with cells during the glutamate exposure. The presence of 100 nM SR48692 (20 min before NT) antagonized this effect of NT(1-13). These findings support the view of a pathophysiological role for NT in the cerebral cortex. Thus, under pathological conditions NT by enhancing glutamate outflow and by amplifying the NMDA-mediated glutamate signaling may be involved in increasing the degeneration of cortical neurons.     

Sevoflurane protects rat mixed cerebrocortical neuronal-glial cell cultures against transient oxygen-glucose deprivation: involvement of glutamate uptake and reactive oxygen species

BACKGROUND: The purpose of this study was to clarify the role of glutamate and reactive oxygen species in sevoflurane-mediated neuroprotection on an in vitro model of ischemia-reoxygenation. METHODS: Mature mixed cerebrocortical neuronal-glial cell cultures, treated or not with increasing concentrations of sevoflurane, were exposed to 90 min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber followed by reoxygenation. Cell death was quantified by lactate dehydrogenase release into the media and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium by mitochondrial succinate dehydrogenase. Extracellular concentrations of glutamate and glutamate uptake were assessed at the end of the ischemic injury by high-performance liquid chromatography and incorporation of L-[H]glutamate into cells, respectively. Free radical generation in cells was assessed 6 h after OGD during the reoxygenation period using 2',7'-dichlorofluorescin diacetate, which reacts with intracellular radicals to be converted to its fluorescent product, 2',7'-dichlorofluorescin, in cell cytosol. RESULTS: Twenty-four hours after OGD, sevoflurane, in a concentration-dependent manner, significantly reduced lactate dehydrogenase release and increased cell viability. At the end of OGD, sevoflurane was able to reduce the OGD-induced decrease in glutamate uptake. This effect was impaired in the presence of threo-3-methyl glutamate, a specific inhibitor of the glial transporter GLT1. Sevoflurane counteracted the increase in extracellular level of glutamate during OGD and the generation of reactive oxygen species during reoxygenation. CONCLUSION: Sevoflurane had a neuroprotective effect in this in vitro model of ischemia-reoxygenation. This beneficial effect may be explained, at least in part, by sevoflurane-induced antiexcitotoxic properties during OGD, probably depending on GLT1, and by sevoflurane-induced decrease of reactive oxygen species generation during reoxygenation.     

Effects of propofol on extracellular acidification rates in primary cortical cell cultures: application of silicon microphysiometry to anaesthesia

Propofol depresses both cerebral oxygen consumption and glucose utilization. We tested the hypothesis that these well described effects on brain metabolism are manifest by a reduction in neuronal acid production in vitro. The rate of extracellular acidification in primary cell cultures of rat cortical neurones was measured using a novel instrument (silicon microphysiometer) after stimulation with propofol 0.3, 3 and 30 micrograms ml-1. Intralipid 10% served as a control. Propofol 3 micrograms ml-1 caused a mean decrease of 1.51 (SEM 0.71)% in baseline acidification rate, which was significantly greater than that produced by 0.3 microgram ml-1 or Intralipid alone (P < 0.05). The reduction after stimulation with propofol 30 micrograms ml-1 was 4.68 (0.35)% of baseline rates and this in turn was significantly greater than that elicited by propofol 3 or 0.3 microgram ml-1, or Intralipid (P < 0.001). We have confirmed the depressant effect of propofol on cerebral metabolism and established that propofol inhibits neuronal acid excretion in vitro.      

Stretch injury causes calpain and caspase-3 activation and necrotic and apoptotic cell death in septo-hippocampal cell cultures

Traumatic brain injury (TBI) results in numerous central and systemic responses that complicate interpretation of the effects of the primary mechanical trauma. For this reason, several in vitro models of mechanical cell injury have recently been developed that allow more precise control over intra- and extracellular environments than is possible in vivo. Although we recently reported that calpain and caspase-3 proteases are activated after TBI in rats, the role of calpain and/or caspase-3 has not been examined in any in vitro model of mechanical cell injury. In this investigation, varying magnitudes of rapid mechanical cell stretch were used to examine processing of the cytoskeletal protein alpha-spectrin (280 kDa) to a signature 145-kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3 in septo-hippocampal cell cultures. Additionally, effects of stretch injury on cell viability and morphology were assayed. One hour after injury, maximal release of cytosolic lactate dehydrogenase and nuclear propidium iodide uptake were associated with peak accumulations of the calpain-specific 145-kDa fragment to alpha-spectrin at each injury level. The acute period of calpain activation (1-6 h) was associated with subpopulations of nuclear morphological alterations that appeared necrotic (hyperchromatism) or apoptotic (condensed, shrunken nuclei). In contrast, caspase-3 processing of alpha-spectrin to the apoptotic-linked 120-kDa fragment was only detected 24 h after moderate, but not mild or severe injury. The period of caspase-3 activation was predominantly associated with nuclear shrinkage, fragmentation, and apoptotic body formation characteristic of apoptosis. Results of this study indicate that rapid mechanical stretch injury to septo-hippocampal cell cultures replicates several important biochemical and morphological alterations commonly observed in vivo brain injury, although important differences were also noted.     

Effects of LY379268, a selective group II metabotropic glutamate receptor agonist on EEG activity,cortical perfusion,tissue damage,and cortical glutamate,glucose, and lactate levels in brain-injured rats

Activating presynaptic group II metabotropic glutamate (mGlu II) receptors reduces synaptic glutamate release. Attenuating glutamatergic transmission without blocking ionotropic glutamate receptors, thus avoiding unfavorable psychomimetic side effects, makes mGlu II receptor agonists a promising target in treating brain-injured patients. Neuroprotective effects of LY379268 were investigated in rats following controlled cortical impact injury (CCI). At 30 min after CCI, rats received a single intraperitoneal injection of LY379268 (10 mg/kg/body weight) or NaCl. Changes in EEG activity and pericontusional cortical perfusion were determined before trauma, at 4, 24, and 48 h, and 7 days after CCI. Brain edema and contusion volume were determined at 24 h and 7 days after CCI, respectively. Before brain removal pericontusional cortical glutamate, glucose, and lactate were measured via microdialysis. During the early period following CCI, EEG activity and cortical perfusion were significantly reduced in rats receiving LY379268. At 7 days, cortical perfusion was significantly increased in rats treated with LY379268, while EEG activity was depressed as in control rats. While brain edema remained unchanged at 24 h, cortical contusion was significantly decreased by 56% at 7 days after CCI. Cortical glutamate, glucose, and lactate were not influenced. Significant reductions in EEG activity and contusion volume by LY379268 do not appear mediated by attenuated excitotoxicity and energetic impairment. Overall, an additional decrease in cortical perfusion seems to interfere with the anti-edematous potential of LY379268 during the early period following CCI, while an increase in perfusion in LY379268-treated rats at 7 days might contribute to tissue protection.     

Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: role of glutamate and its transporters

BACKGROUND: During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death. The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures. METHODS: Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation. Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801). Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-[3H]glutamate into cells, respectively. RESULTS: At clinically relevant concentrations (0.05-10 microm), propofol offered protection equivalent to that of MK-801. It significantly reduced lactate dehydrogenase release and increased the reduction of MTT. At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake. The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol. CONCLUSION: Propofol showed a neuroprotective effect in this in vitro model of OGD, which was apparently mediated by a GLT1-independent restoration of the glutamate uptake impaired during the injury.     

Influence of norepinephrine and dopamine on cortical perfusion,EEG activity,extracellular glutamate,and brain edema in rats after controlled cortical impact injury

Following traumatic brain injury, catecholamines given to ameliorate cerebral perfusion may induce brain damage via cerebral arteriolar constriction and increased neuronal excitation. In the present study the acute effects of norepinephrine and dopamine on pericontusional cortical perfusion (rCBF), electroencephalographic (EEG) activity, extracellular glutamate, and brain edema were investigated in rats following controlled cortical impact injury (CCI). rCBF, cerebral perfusion pressure (CPP), EEG activity, and glutamate were determined before, during, and after infusing norepinephrine or dopamine, increasing MABP to 120 mm Hg for 90 min at 4 h after CCI. Control rats received physiological saline. At 8 h after CCI, hemispheric swelling and water content were determined gravimetrically. Following CCI, rCBF was significantly decreased. In parallel to elevating MABP and CPP, rCBF was significantly increased by norepinephrine and dopamine, being mostly pronounced with norepinephrine (+44% vs. +29%). In controls, rCBF remained diminished (-45%). EEG activity was significantly increased by norepinephrine and dopamine, while pericontusional glutamate was only elevated by norepinephrine (28 +/- 6 vs. 8 +/- 4 microM). Brain edema was not increased compared to control rats. Despite significantly increasing MABP and CPP to the same extent, norepinephrine and dopamine seem to differentially influence pericontusional cortical perfusion and glutamatergic transmission. In addition to the pressure-passive increase in CPP local cerebral effects seem to account for the sustained norepinephrine-induced increase in pericontusional cortical perfusion. The significantly elevated pericontusional glutamate concentrations in conjunction with the increased EEG activity suggest a sustained metabolically driven increase in cortical perfusion during norepinephrine infusion.     

Secretion of matrix metalloproteinase-2 and -9 after mechanical trauma injury in rat cortical cultures and involvement of MAP kinase

Matrix metalloproteinases (MMP) are involved in the pathophysiology of brain injury. We recently showed that knockout mice deficient in MMP-9 expression were protected against traumatic brain injury. However, the cellular sources of MMP activity after trauma remain to be fully defined. In this study, we investigated the hypothesis that resident brain cells secrete MMP after mechanical trauma injury in vitro, and mitogen-activated protein (MAP) kinase signal transduction pathways are involved in this response. Rat primary cortical neurons, astrocytes, and co-cultures were subjected to needle scratch mechanical injury, and levels of MMP-2 and MMP-9 in conditioned media were assayed by zymography. MMP-2 and MMP-9 were increased in cortical astrocytes and co-cultures, whereas only MMP-2 was increased in neurons. Western blots showed that phosphorylated extracellular signal regulated kinase (ERK1/2) and p38 were rapidly upregulated in co-cultures after mechanical injury. No change in phosphorylated c-jun N-terminal kinase (JNK) was observed. In-gel kinase assays confirmed this lack of response in the JNK pathway. Treatment with either 10 microM of U0126 (a MAP kinase/ERK1/2 kinase inhibitor) or 10 microM of SB203580 (a p38 inhibitor) had no detectable effect on MMP-2 and MMP-9 levels after mechanical injury. However, combination treatment with both inhibitors significantly reduced secretion of MMP-9. Herein, we demonstrate that (1) resident brain cells secrete MMP after mechanical injury, (2) astrocytes are the main source of MMP-9 activity, and (3) ERK and p38 MAP kinases are upregulated after mechanical injury, and mediate the secretion of MMP-9.     

Effects of isoflurane and propofol on glutamate and GABA transporters in isolated cortical nerve terminals

BACKGROUND: Depression of glutamate-mediated excitatory transmission and potentiation of gamma-aminobutyric acid (GABA)-mediated inhibitory transmission appear to be primary mechanisms by which general anesthetics produce anesthesia. Since effects on transmitter transport have been implicated in anesthetic actions, the authors examined the sensitivity of presynaptic glutamate and GABA transporters to the effects of a representative volatile (isoflurane) and a representative intravenous (propofol) anesthetic. METHODS: A dual-isotope (l-[3H]glutamate and [14C]GABA) approach allowed simultaneous comparisons of anesthetic effects on three independent assays of glutamate and GABA transporters in adult rat cerebral cortex: transmitter uptake into isolated nerve terminals (synaptosomes), transmitter binding to lysed and washed synaptosomes (synaptic membranes), and carrier-mediated release (reverse transport) of transmitter from preloaded synaptosomes using a modified superfusion system. RESULTS: Isoflurane produced small but statistically significant inhibition of l-[3H]glutamate and [14C]GABA uptake, while propofol had no effect. Inhibition of uptake by isoflurane was noncompetitive, an outcome that was mimicked by indirectly affecting transporter function through synaptosomal depolarization. Neither isoflurane nor propofol affected l-[3H]glutamate or [14C]GABA binding to synaptic membranes or Ca(2+)-independent carrier-mediated l-[3H]glutamate or [14C]GABA release (reverse transport). CONCLUSIONS: These findings suggest that isoflurane and propofol at clinical concentrations do not affect excitatory glutamatergic transmission or inhibitory GABAergic transmission directly effects on their presynaptic neuronal transporters.     

高氧液对氧糖剥夺离体神经元损伤的保护作用

目的 观察高氧液对氧糖剥夺(OGD)离体神经元损伤的保护作用。方法 新生小鼠神经元原代培养14d后．造成OGD离体神经元缺血模型，4h后复氧复糖(ROG)．模拟再灌注模型。随机分组：对照组(A组，n=6)；治疗组在ROG时，根据剂量的不同分为两组：B组(n=6)高氧液浓度为33．3％，C组(n=6)高氧液浓度为20．0％。各组分别于ROG后4、12、24h取神经元细胞记数后行四唑盐(MTT)比色试验。并取ROG 24h各组神经元细胞做台盼蓝染色，计阳性细胞数百分比。另取原代培养神经元，造成OGD离体神经元缺血模型30min后，分A组(n=10)和B组(n=10)ROG 4h后，免疫组化SABC法染色．比较Ⅰ型一氧化氮合酶(nNOS)和Ⅱ型-氧化氮合酶(iNOS)活性。结果 ROG后，B组和C组MTT各时点吸光值(OD)均高于A组，且台盼蓝染色阳性细胞数百分比B组和C组均低于A组，但B组和C组间差异不显著。免疫组化染色结果显示，B组nNOS和iNOS的表达灰度值均高于A组。结论 高氧液能减轻OGD离体神经元损伤，抑制神经元nNOS和iNOS活性是其可能机制之一。     

Pre- or postinsult administration of lidocaine or thiopental attenuates cell death in rat hippocampal slice cultures caused by oxygen-glucose deprivation

Lidocaine and thiopental improve recovery when administrated during hypoxia and ischemia; however, the effect of pre- or postinsult treatment alone is unknown. We applied either lidocaine or thiopental to hippocampal slice cultures from 20-day-old rats either before or after 10 min of oxygen-glucose deprivation (OGD). Propidium iodide (PI) fluorescence was used as an indicator of neuronal death for 7 days after OGD. OGD-induced neuronal death, in both the Cornus Ammonis 1 (CA1) and the dentate gyrus regions, peaked the first day after ischemia. Preinsult administration of either lidocaine (10, 100 microM) or thiopental (250, 600 microM) significantly reduced the damage measured on the first and second days after OGD; these drugs also significantly decreased the summed daily post-OGD PI fluorescence in both regions. Postinsult administration of lidocaine (10, 100 microM) or thiopental (250, 600 microM) significantly decreased the PI fluorescence on the first day after OGD; postinsult administration of these drugs also attenuated the summed daily post-OGD PI. These data indicate that the administration of lidocaine or thiopental either before or directly after OGD reduced neuronal damage in this in vitro model of cerebral ischemia. Postischemic administration is frequently the first opportunity for treatment. IMPLICATIONS: Lidocaine or thiopental applied either 10 min before or 10 min directly after oxygen-glucose deprivation reduced neuronal cell death in rat hippocampal slice cultures. Postinsult administration is often the first opportunity for treatment after stroke; lidocaine and thiopental reduced damage caused by oxygen-glucose deprivation, an in vitro model of stroke.     

The effects of thiopental and propofol on cell swelling induced by oxygen/glucose deprivation in the CA1 pyramidal cell layer of rat hippocampal slices

Cellular swelling has been implicated as an early process after cerebral ischemia. We compared the effects of two commonly used IV anesthetics, thiopental and propofol, on hippocampal CA1 pyramidal cell swelling induced by oxygen/glucose deprivation (OGD) in vitro. Experiments were performed in rat hippocampal slices. Cell swelling in the CA1 pyramidal cell layer was evaluated by determining light transmittance (LT) change through the slices and by histopathological examination. For LT experiments, OGD was induced for 10 min by superfusing slices with glucose-free artificial cerebrospinal fluid equilibrated with 95% nitrogen and 5% CO(2). Thiopental and propofol were present 10 min before and during the period of OGD. The results showed that thiopental (100 and 400 microM), but not propofol (40 and 160 microM), significantly prolonged latency to the peak of LT increase after the onset of OGD. Consistent with the LT experiments, histopathological examination revealed that thiopental, but not propofol, attenuated CA1 pyramidal cell expansion and the gap diminution between CA1 pyramidal cells induced by OGD. These results suggest that thiopental, but not propofol, reduces the neuronal cell swelling caused by OGD. Whether the reduction of cell swelling is related to reduction in cell injury caused by OGD remains to be investigated. IMPLICATIONS: We demonstrated that thiopental, but not propofol, attenuates ischemic neuronal swelling induced by oxygen/glucose deprivation in an in vitro ischemic model.     

Ceramide-induced cell death in the prostate cancer cell line LNCaP has both necrotic and apoptotic features

BACKGROUND: Prostate cancer is the second leading cause of cancer death in men. The most common treatment of prostate cancer is androgen ablation therapy which leads to regression of the tumor due to increased cell death. However, at later stages, the tumor becomes resistant to androgen ablation. Ceramide is a lipid second messenger that mediates cell death in prostate cancer cells. Previous studies suggested that ceramide may cause either apoptosis or growth arrest in the androgen-responsive prostate cancer cell line LNCaP. However, the molecular details of ceramide-induced cell death in LNCaP cells remain to be elucidated. METHODS: To investigate the mechanisms of cell death in LNCaP cells, we used various methods, including cell viability assays, fluorescence image analysis, internucleosomal DNA fragmentation analysis, Western blotting, and protein kinase assays. RESULTS: Ceramide caused LNCaP cell death without exhibiting typical signs of apoptosis, such as internucleosomal DNA fragmentation and poly(ADP)-ribose-polymerase (PARP) proteolysis. In addition, the general caspase inhibitor z-VAD-fmk did not alter ceramide-induced cell death in LNCaP cells, whereas it efficiently inhibited thapsigargin-induced apoptosis under similar conditions. However, ceramide treatment of LNCaP cells resulted in nuclear fragmentation, which is characteristic of apoptosis. Ceramide induced a strong and prolonged activation of c-Jun N-terminal Kinase (JNK) that correlated very well with the time course of cell death. Whereas the PKC inhibitor bisindolylmaleimide prevented phorbol ester-induced apoptosis in LNCaP cells, it did not affect ceramide-induced cell death. These results suggest that LNCaP cell death induced by ceramide progresses through a novel pathway that is more necrotic than apoptotic.     

Plasticity of excitation and inhibition in the receptive field of primary auditory cortical neurons after limited receptor organ damage

Permanent receptor organ damage can cause plasticity of topographic cortical maps of that receptor surface while temporary receptor organ damage, and conditions mimicking such damage, can unmask new excitatory inputs in central sensory neurons receiving input from that receptor surface. Cortical plasticity is associated with an anatomically or pharmacologically defined decrease in inhibition in cortex. It is therefore widely proposed that a reduction incentral inhibition underlies cortical neural plasticity. Here I demonstrate that small receptor organ damage results, in primary auditory cortical (A1) neurons, in loss of one component of functionally defined afferent inhibition but unmasking of another component of afferent inhibition along with new excitatory responses. Overall, there did not appear to be any change in the strength of afferent inhibition or in the strength of excitation. Thus, auditory receptor organ damage can unmask new excitatory inputs as well as inhibitory inputs from within the receptive field of the neurons.