Viability Of Slices Research Articles

Novel histone deacetylase (HDAC) inhibitors with improved selectivity for HDAC2 and 3 protect against neural cell death

Neurodegenerative disorders, such as motor neurone disease, Alzheimer’s disease and responses to brain traumas such as stroke, involve the unwanted death of neural cells. Although the exact underlying mechanisms leading to neural cell death are not well defined, one contributory event in many situations is the over-excitation of cells caused by too much of the neurotransmitter glutamate. Drugs that inhibit enzymes called histone deacetylases (HDACs) can protect neural cells from glutamate excitotoxicity. However, current inhibitors lack specificity and although they function in vitro, they have a substantial potential for adverse side effects in vivo. HDAC2 and 3 have been implicated in neurotoxicity and here we investigated the neuroprotective potential of three novel HDAC inhibitors that show selectivity for these. The ability of these HDAC inhibitors to protect against glutamate excitotoxicity was tested using cultured organotypic cerebral slices from 7-day-old (P7) Wistar rats. Glutamate excitotoxicity was induced by 200 µM of the glutamate transporter blocker, DL-threo-β-benzyloxyaspartate (DL-TBOA). This was applied alone and alongside 1 µM of the novel HDAC2 and 3 selective inhibitors AH51, AH61 and AH62. Neural cell viability in slices was quantified from assays using the fluorescent stains, 4′,6-diamidino-2-phenylindole and ethidium homodimer-1. The induction of glutamate excitotoxicity by DL-TBOA resulted in 41.3 ± 6.1% (n = 7, P < 0.01) loss in cell viability as judged by ethidium homodimer-1 staining. All three novel HDAC inhibitors significantly prevented neural cell death in response to DL-TBOA (P < 0.01), with cell viabilities of 107.5 ± 6.01% (n = 4), 97.1 ± 16.5% (n = 3) and 106.7 ± 6.45% (n = 4) for AH51, AH61 and AH62, respectively. This study has shown that inhibitors selective for HDAC2 and 3 can protect neural cells from death and thus have potential as therapeutic agents against neurotoxicity.

A novel method for the rapid determination of beta-amyloid toxicity on acute hippocampal slices using MTT and LDH assays

It is difficult task to measure precisely the toxic effect of beta-amyloid (Aβ 1-42) peptides and also the protective effect of novel drug candidates against Aβ-peptides. The widely used MTT-assay in cell lines or primary cell cultures could be insensitive against Aβ-peptides. We describe here an easy and relevant method for testing Aβ 1-42 toxicity on acute hippocampal slices derived from rat. Brain slice viability in different conditions was measured using MTT and LDH assays. The concomitant use of these two assays can give detailed and relevant results on the toxic effect of Aβ 1-42 in oxygen-glucose deprived (OGD) acute brain slice model. Both assays are capable of quantifying tissue viability by measuring optical density (OD). We found that simultaneous application of OGD and Aβ 1-42 treatment induced a more intensive decrease in hippocampal slice viability than their separate effects. The use of MTT and LDH assay for quantifying brain slice viability proved to be an easy ex vivo method for investigating Aβ toxicity. Testing brain slices is more relevant in Alzheimer's Disease research than using in vitro cell cultures, due to maintenance of the three dimensional cellular network, the cell variability and intact cell connections.

Mechanical characteristics of native tendon slices for tissue engineering scaffold

The purpose of this study was to characterize the mechanical behavior of tendon slices with different thicknesses. Tendon slices of 100, 200, 300, 400, and 500 μm thickness were mechanically tested. The 300 μm slices were further tested for strength and modulus after 21,000-cycle fatigue testing under different applied strain levels (0, 1, 3, 5, 8, 10, and 12%). The tendon slice structure, morphology, and viability of bone marrow stromal cells (BMSCs) seeded onto the slices were also examined with histology, scanning electron microscopy, and vital cell labeling, respectively. Tendon slices 300 μm or more in thickness had similar ultimate tensile strength and Young's modulus to the intact tendon bundle. A strain of 5% or less did not cause any structural damage, nor did it change the mechanical properties of a 300 μm-thick tendon slice after 21,000-cycle fatigue testing. BMSCs were viable between and on the tendon slices after 2 weeks in tissue culture. This study demonstrated that, if tendon slices are used as a scaffold for tendon tissue engineering, slices 300 μm or more in thickness would be preferable from a mechanical strength point of view. If mechanical stimulation is performed for seeded-cell preparations, 5% strain or less would be appropriate.

Brain slice on a chip: opportunities and challenges of applying microfluidic technology to intact tissues

Isolated brain tissue, especially brain slices, are valuable experimental tools for studying neuronal function at the network, cellular, synaptic, and single channel levels. Neuroscientists have refined the methods for preserving brain slice viability and function and converged on principles that strongly resemble the approach taken by engineers in developing microfluidic devices. With respect to brain slices, microfluidic technology may 1) overcome the traditional limitations of conventional interface and submerged slice chambers and improve oxygen/nutrient penetration into slices, 2) provide better spatiotemporal control over solution flow/drug delivery to specific slice regions, and 3) permit successful integration with modern optical and electrophysiological techniques. In this review, we highlight the unique advantages of microfluidic devices for in vitro brain slice research, describe recent advances in the integration of microfluidic devices with optical and electrophysiological instrumentation, and discuss clinical applications of microfluidic technology as applied to brain slices and other non-neuronal tissues. We hope that this review will serve as an interdisciplinary guide for both neuroscientists studying brain tissue in vitro and engineers as they further develop microfluidic chamber technology for neuroscience research.

Testing neocortical slice viability in non-perfused no-magnesium artificial cerebrospinal fluid solutions

The acute in vitro brain slice model is a widely used neurophysiological research tool. When applying this method, most researchers continuously perfuse slices with carbogenated artificial cerebrospinal fluid (ACSF) to maintain pH balance and tissue oxygen delivery. Common wisdom suggests that static recordings are incompatible with submerged bath methodology because of deficiency in tissue oxygen supply. However, to our knowledge this has not been tested. In this study, we wanted to determine whether neocortical mouse slice viability could be maintained in the medium term (up to 2h) in a shallow, submerged recording bath under non-perfused, static conditions. Seizure-like events (SLEs) were generated in the slices utilizing no-magnesium ACSF and recorded for 2h under three conditions: (1) perfused ACSF condition (n=8), where slices were perfused continuously with carbogenated no-magnesium ACSF; (2) static ACSF condition (n=12), where slices were recorded in pre-carbogenated, but non-perfused (static) no-magnesium ACSF; and (3) static HEPES ACSF condition (n=12), where slices were recorded in non-perfused (static) no-magnesium ACSF with no pre-carbogenation but buffered with HEPES. SLE activity was stable for 2h across all three conditions. There was no statistically significant difference in SLE frequency, amplitude or length between static and perfused conditions. SLE frequency and amplitude were generally lower in the static HEPES buffer condition. The data indicate that robust and stable neocortical SLE activity can be generated for at least 2h in a submersion bath without ACSF perfusion if pH is adequately controlled.

Antidepressant-like and neuroprotective effects of Aloysia gratissima: Investigation of involvement of l-arginine-nitric oxide-cyclic guanosine monophosphate pathway

Ethnopharmacological relevanceAloysia gratissima (Gill. et Hook) Tronc. (Verbenaceae) is used traditionally for the treatment of headache, bronchitis, and nervous systems disorders including depression. Aim of the studyTo investigate the antidepressant-like and neuroprotective effects of Aloysia gratissima aqueous extract (AE) and the involvement of l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Materials and methodsThe antidepressant-like effect of AE was evaluated through behavioral despair in forced swimming test (FST) and tail suspension test (TST). Swiss albino mice were treated by oral route and after 1h were analyzed the time of immobility in the FST and TST. In addition, the neuroprotective effect of AE against glutamate excitotoxicity was evaluate through cell viability of hippocampal slices, phosphorylation of Akt, and the immunocontent of inducible oxide nitric synthase (iNOS) were investigated by western blotting. ResultsThe immobility time in the FST and TST were reduced by AE (100–1000 and 10–300mg/kg, respectively). The antidepressant-like effect of AE in the TST was prevented by the pretreatment with N-methyl-d-aspartate (NMDA), l-arginine or sildenafil. The subeffective dose of AE produced a synergistic antidepressant-like effect with MK-801 (an antagonist of NMDA receptor), methylene blue, l-NNA (an inhibitor of NO synthase) or ODQ (an inhibitor of soluble guanylate cyclase). In ex vivo experiments, pretreatment with AE prevented the loss of cell viability induced by glutamate, thus affording neuroprotection. Glutamate toxicity caused a decreased Akt phosphorylation and an increased iNOS expression. ConclusionsThe present study provides convincing evidence of neuroprotection and the involvement of the l-arginine-NO-cGMP pathway in the antidepressant-like effect of AE. Therefore, AE could be of potential interest for the treatment of depressive disorders and neurological conditions associated with glutamate excitotoxicity.

Thiosemicarbazone derivate protects from AAPH and Cu2+-induced LDL oxidation

AimsSeveral lines of evidence support the hypotheses that the oxidation of low density lipoprotein (LDL) may play a crucial role in the initiation and progression of atherosclerosis. Oxidative stress is one of the causes of the overproduction of reactive species that increase the formation of oxidized LDL. Thiosemicarbazones are compounds used in anticancer, antiviral and antifungal therapy; however, its redox activity has been controversial. Thus, we tested, in vitro, a possible antioxidant activity of a thiosemicarbazone derivate, the isatin-3-N4-benzilthiosemicarbazone (IBTC). Main methodsWe measured the conjugated diene formation in serum and LDL as well as the loss of tryptophan fluorescence in LDL induced by two oxidant agents, 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH) and Cu2+. Thiobarbituric acid reactive substances (TBARS) formation in LDL and in different rat tissues was also assessed. The toxicity of IBTC was measured using aortic slices viability assay. Key findingsOur results show that IBTC significantly reduced the AAPH and Cu2+-induced formation of conjugated dienes, increased in a dose-dependent manner the lag phase and the t1/2 of tryptophan fluorescence, and reduced the TBARS formation in LDL, plasma and rat tissues, showing no toxicity to aortic slices. SignificanceThese results indicate that IBTC is a good antioxidant and a promising antiatherogenic agent for further studies in vivo.

Repair pathways evident in human liver organ slices

The extension of human liver slice culture viability for several days broadens the potential of this ex vivo model for characterizing pathways of organ injury and repair, and allows for the multiple dosing of compounds. Extended viability is demonstrated by continued synthesis of GSH and ATP, and maintenance of intracellular K+ levels. Gene expression profiling revealed the activation of regeneration pathways via increased expression of collagens (I, IV, and V), laminins, ninjurin, growth factors (EGF, epiregulin, and TGF-β1), matrix metalloproteinase-7, and insulin like growth factor 5. Collagen IV protein levels began to increase by day 4 of culture. Some markers of hepatic stellate cells, detected by RT-PCR, were up-regulated (HSP47, αSMA, pro-collagen 1a1, PDGF receptor, thrombospondin-2) with time in culture, while other markers exhibited no change or were down-regulated (αB-crystallin, synaptophysin), suggesting that the induction of regenerative pathways may in part be the role of the stellate cells as well as resident fibroblasts. Complimentary to the gene expression was evidence of regeneration in the human liver slices, as evaluated by histopathology. Improvements in organ acquisition, organ slice preparation and culture methods demonstrates that organ slice viability, integrity and morphology can be extended reproducibly for several days in culture which allows for the investigation of injury and repair processes.

NMR-based metabolomics of mammalian cell and tissue cultures

NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

Evaluation of vitro hepatotoxicity of monocrotaline by precision-cut liver slice technique

To modify the empirical method of precision-cut liver slice technique, and study the hepatotoxicity of monocrotaline by this technique. Liver slices were prepared by the domestic shaking slicer. The technique of precision-cut liver slice was established by detecting MTT reduction used as the slice viability under different culture medium, thickness of slices, pH and culture temperature. After monocrotaline and liver slices co-culture for 6, 24 h, the slice viability, enzyme activity of GPT, GOT, LDH, GGT and protein concentration were detected by MTT reduction, enzyme kinetics method and BCA protein assay method, respectively. When the thickness of slices was 200 microm and pH of medium was 6.8, culture temperature was 37 degrees C, BPM culture medium, the viability of slices could maintain on a steady level. LDH leakage was significantly increased and protein content was obviously decreased after monocrotaline co-culture for 24 h with final concentration 0.02, 0.1 and 0.5 g x L(-1). No statistically significant difference between control group and monocrotaline 3 dose groups was observed in the slice viability and the content of GPT, GOT, LDH, GGT and protein after monocrotaline co-culture for 6 h. The slice viability could retain 24 h in modified BPM medium surroundings; monocrotaline displayed liver toxicity in some degree after co-culture for 24 hours in 0.02, 0.1 and 0.5 g x L(-1) concentration.

Modulation of methylmercury uptake by methionine: Prevention of mitochondrial dysfunction in rat liver slices by a mimicry mechanism

Methylmercury (MeHg) is an ubiquitous environmental pollutant which is transported into the mammalian cells when present as the methylmercury-cysteine conjugate (MeHg–Cys). With special emphasis on hepatic cells, due to their particular propensity to accumulate an appreciable amount of Hg after exposure to MeHg, this study was performed to evaluate the effects of methionine (Met) on Hg uptake, reactive species (RS) formation, oxygen consumption and mitochondrial function/cellular viability in both liver slices and mitochondria isolated from these slices, after exposure to MeHg or the MeHg–Cys complex. The liver slices were pre-treated with Met (250μM) 15min before being exposed to MeHg (25μM) or MeHg–Cys (25μM each) for 30min at 37°C. The treatment with MeHg caused a significant increase in the Hg concentration in both liver slices and mitochondria isolated from liver slices. Moreover, the Hg uptake was higher in the group exposed to the MeHg–Cys complex. In the DCF (dichlorofluorescein) assay, the exposure to MeHg and MeHg–Cys produced a significant increase in DFC reactive species (DFC-RS) formation only in the mitochondria isolated from liver slices. As observed with Hg uptake, DFC-RS levels were significantly higher in the mitochondria treated with the MeHg–Cys complex compared to MeHg alone. MeHg exposure also caused a marked decrease in the oxygen consumption of liver slices when compared to the control group, and this effect was more pronounced in the liver slices treated with the MeHg–Cys complex. Similarly, the loss of mitochondrial activity/cell viability was greater in liver slices exposed to the MeHg–Cys complex when compared to slices treated only with MeHg. In all studied parameters, Met pre-treatment was effective in preventing the MeHg- and/or MeHg–Cys-induced toxicity in both liver slices and mitochondria. Part of the protection afforded by Met against MeHg may be related to a direct interaction with MeHg or to the competition of Met with the complex formed between MeHg and endogenous cysteine. In summary, our results show that Met pre-treatment produces pronounced protection against the toxic effects induced by MeHg and/or the MeHg–Cys complex on mitochondrial function and cell viability. Consequently, this amino acid offers considerable promise as a potential agent for treating acute MeHg exposure.

Hydrogel embedding of precision‐cut liver slices in a microfluidic device improves drug metabolic activity

A microfluidic-based biochip made of poly-(dimethylsiloxane) was recently reported for the first time by us for the incubation of precision-cut liver slices (PCLS). In this system, PCLS are continuously exposed to flow, to keep the incubation environment stable over time. Slice behavior in the biochip was compared with that of slices incubated in well plates, and verified for 24 h. The goal of the present study was to extend this incubation time. The viability and metabolic activity of precision-cut rat liver slices cultured in our novel microflow system was examined for 72 h. Slices were incubated for 1, 24, 48, and 72 h, and tested for viability (enzyme leakage (lactate dehydrogenase)) and metabolic activity (7-hydroxycoumarin (phase II) and 7-ethoxycoumarin (phase I and II)). Results show that liver slices retained a higher viability in the biochip when embedded in a hydrogel (Matrigel) over 72 h. This embedding prevented the slices from attaching to the upper polycarbonate surface in the microchamber, which occurred during prolonged (>24 h) incubation in the absence of hydrogel. Phase II metabolism was completely retained in hydrogel-embedded slices when medium supplemented with dexamethasone, insulin, and calf serum was used. However, phase I metabolism was significantly decreased with respect to the initial values in gel-embedded slices with medium supplements. Slices were still able to produce phase I metabolites after 72 h, but at only about ∼10% of the initial value. The same decrease in metabolic rate was observed in slices incubated in well plates, indicating that this decrease is due to the slices and medium rather than the incubation system. In conclusion, the biochip model was significantly improved by embedding slices in Matrigel and using proper medium supplements. This is important for in vitro testing of drug metabolism, drug-drug interactions, and (chronic) toxicity.

Viability Assessment of Primo-node Slices From Organ Surface Primo-vascular Tissues in Rats

Abstract The primo-vascular system is a novel thread-like structure which is recently rediscovered, but its cellular properties are largely unknown. In this study, a slice preparation for primo-nodes was developed to facilitate study of the cellular properties of primo-node cells in vitro. Slices (4–8 slices; 200 μm thick) were sectioned from single primo-nodes collected from the abdominal organ surface of rats and incubated in oxygenated Krebs solution at 25°C or 31°C for up to 7 hours. Trypan blue staining and whole-cell patch-clamp recordings were performed to estimate the viability of cells in the slices. Viability was largely maintained during the first 3 hours, but subsequently decreased (from 80% to 21%, p < 0.001). In addition, the viability of slices incubated at 31°C was higher than those incubated at 25°C (80% vs. 47%, p < 0.001). In whole-cell patch-clamp experiments, high resistance seals readily formed and primo-node cells showed a mean resting membrane potential (-38 mV) comparable to that recorded with sharp electrodes and outwardly-rectifying current-voltage relationships. The results show that the primo-node slices developed in this study maintained viability for up to 4 hours in vitro.

Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes

Adult hippocampal slice cultures were used in the modeling of apoptotic aspects of neurodegeneration. Slice viability was determined by the use of trypan blue (TB) staining, and apoptosis was assessed by caspase-3 immunohistochemistry. A large number of pyramidal cells showed signs of degeneration 30 min after sectioning (58.4% of the total number of pyramidal cells), as they exhibited TB uptake, and about 71.6% of these neurons became stained by the third day in culture, when patches in the stratum oriens also demonstrated distinct TB staining. By the sixth day of culturing, almost all cells in the pyramidal cell layer became TB positive (88.4%). The caspase-3 immunoreactivity displayed a different pattern, as the most intense immunoreactivity, detected mainly in the pyramidal cells, peaked 6 h after culturing, and then decreased steadily. The present data show that in adult hippocampal slices a large number of pyramidal cells initiate apoptotic processes as a result of irreparable damage sustained during slice preparation and culture maintenance, and support the notion that apoptosis is an integral part of the neurodegenerative processes not only in vivo but also in vitro. Elucidation of mechanisms for the apoptotic processes in adult hippocampal slice cultures could lead to the development of new therapeutic strategies; moreover, the utilization of adult hippocampal slice cultures could be a viable alternative technique to in vivo experiments in studying the mechanisms responsible for neurodegeneration.

Stability of brain neocortical slice seizure-like activity during low-magnesium exposure: Measurement and effect of artificial cerebrospinal fluid temperature

Low-magnesium perfusion is an established method for inducing seizure-like activity in neocortical slices and is widely utilized as a model of human epilepsy. However, many contentious issues remain around optimizing in vitro cortical slice experimental protocols. In this study we investigated the stability of low-magnesium seizure-like event (SLE) activity at different perfusion temperatures; with an aim towards determining whether time-dependent effects in this preparation could be attributed to a reduction in tissue viability. The results showed that SLE characteristics changed significantly and consistently over a 3h recording period, independent of artificial cerebrospinal fluid temperature; there was a median increase in event frequency of 61% (p<0.001) following 2h of recording which remained elevated after 3h (52%, p<0.001). Event amplitude was significantly reduced after 3h (38% reduction, p<0.001). Similar effects were recorded whether perfusion temperature was maintained at 26°C or 36°C. No evidence was found to implicate a reduction in slice viability in these time-dependent changes in SLE behaviour. The results indicate that low-magnesium-induced SLE activity is not stable for long periods and the likelihood of time-related changes in slice performance must be considered when planning and interpreting experiments utilizing the low-magnesium seizure model.

Guanosine-5′-monophosphate induces cell death in rat hippocampal slices via ionotropic glutamate receptors activation and glutamate uptake inhibition

Guanine derivatives modulate the glutamatergic system through displacement of binding of glutamate to its receptors acting as antagonist of glutamate receptors in moderate to high micromolar concentrations. Guanosine-5′-monophosphate (GMP) is shown to be neuroprotective against glutamate- or oxygen/glucose deprivation-induced neurotoxicity and also against NMDA-induced apoptosis in hippocampal slices. However, in this study we are showing that high extracellular GMP concentrations (5 mM) reduced cell viability in hippocampal brain slices. The toxic effect of GMP was not blocked by dipyridamole, a nucleoside transport inhibitor, nor mimicked by guanosine, suggesting an extracellular mode of action to GMP which does not involve its hydrolysis to guanosine. GMP-dependent cell damage was not blocked by P1 purinergic receptor antagonists, neither altered by adenosine A 1 or A 2A receptor agonists. The blockage of the ionotropic glutamate receptors AMPA or NMDA, but not KA or metabotropic glutamate receptors, reversed the toxicity induced by GMP. GMP (5 mM) induced a decrease in glutamate uptake into hippocampal slices, which was reversed by dl-TBOA. Therefore, GMP-induced hippocampal cell damage involves activation of ionotropic glutamate receptors and inhibition of glutamate transporters activity.

脂多糖预处理改善脂多糖诱导的中脑脑片神经元损伤

To identify the protective effect of lipopolysaccharide (LPS) preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations (0, 1, 3, 6 or 10 ng/mL) of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was determined by measurement of the activity of lactic acid dehydrogenase (LDH). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons and CD11b/c equivalent-immunoreactive (OX-42-IR) microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-alpha (TNF-alpha) levels in the culture media were detected by enzyme-linked immunosorbent assays (ELISA). In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191+/-12 in the control slices to 46+/-4, and the LDH activity elevated obviously (P < 0.01), along with remarkably increased number of OX-42-IR cells and production of TNF-alpha (P < 0.01). Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss (the number of TH-IR neurons increased to 126+/-12 and 180+/-13, respectively) and markedly reduced LDH levels (P < 0.05), accompanied by significant decreases of OX-42-IR microglia activation and TNF-alpha production (P < 0.05). Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-alpha production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson's disease.

Profile of glutamate uptake and cellular viability in hippocampal slices exposed to oxygen and glucose deprivation: Developmental aspects and protection by guanosine

Stroke syndromes are a major cause of disability in middle and later life resulting in severe neuronal degeneration and loss of brain functions. In situations with energy failure, glutamate transport is impaired and high levels of this amino acid accumulate on the synaptic cleft. Our group has showed that guanosine exerts neuroprotection against neurotoxicity situations. The aim of this work is draw a post-ischemic profile of glutamate uptake and cell damage using an oxygen and glucose deprivation model (OGD) in hippocampal slices from young (P10) and adult (P60) rats, analyzing guanosine effect. OGD decreases glutamate uptake in both ages and recovery times, although decrease in cell viability was only observed 1 and 3 h after OGD in young and adult animals, respectively. Guanosine partially protected cell damage from 1 h in P10 and at 3 h in P60 rats and avoided glutamate uptake decrease from P10 rats at 3 h. The impairment of glutamate transporters since immediately after the insult observed here is probably due to an energetic failure; loss of cell viability was only observed from 1 h after OGD. The mechanism by which guanosine acts in the ‘ischemic’ model used here is still unknown, but evidence leads to its antiapoptotic effect.

Glutamate-induced Toxicity in Hippocampal Slices Involves Apoptotic Features and p38MAPK Signaling

Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apoptosis in vivo and in cell cultures. However, glutamate-induced apoptosis and the signaling pathways related to glutamate-induced cell death in acute hippocampal slices remain elusive. Hippocampal slices exposed to 1 or 10 mM glutamate for 1 h and evaluated after 6 h, showed reduced cell viability, without altering membrane permeability. This action of glutamate was accompanied by cytochrome c release, caspase-3 activation and DNA fragmentation. Glutamate at low concentration (10 microM) induced caspase-3 activation and DNA fragmentation, but it did not cause cytochrome c release and, it did not alter the viability of slices. Glutamate-induced impairment of hippocampal cell viability was completely blocked by MK-801 (non-competitive antagonist of NMDA receptors) and GAMS (antagonist of KA/AMPA glutamate receptors). Regarding intracellular signaling pathways, glutamate-induced cell death was not altered by a MEK1 inhibitor, PD98059. However, the p38 MAPK inhibitor, SB203580, prevented glutamate-induced cell damage. In the present study we have shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38 MAPK pathway.

Multilayer PDMS microfluidic chamber for controlling brain slice microenvironment

A novel three-layer microfluidic polydimethylsiloxane (PDMS) device was constructed with two fluid chambers that holds a brain slice in place with microposts while maintaining laminar perfusate flow above and below the slice. Our fabrication technique permits rapid production of PDMS layers that can be applied to brain slices of different shapes and sizes. In this study, the device was designed to fit the shape and thickness (530-700 microm) of a medullary brain slice taken from P0-P4 neonatal rats. Medullary slices in this chamber spontaneously produced rhythmic, respiratory-related motor output for up to 3 h, thereby demonstrating that brain slice viability was maintained for prolonged periods. This design is unique in that it achieves independent control of fluids through multiple channels in two separate fluid chambers. The laminar flow exhibited by the microfluidic chamber allows controlled solutions to target specific areas of the brain slice based on the input flow rates. To demonstrate this capability, a stream of Na(+)-free solution was focused on one half of a medullary slice to abolish spontaneous neural activity in only that half of the brain slice, while the other half remained active. We also demonstrated that flow of different solutions can be focused over the midline of the brain slice. The multilayer brain slice chamber design can integrate several traditional types of electrophysiology tools that are commonly used to measure neurophysiological properties of brain slices. Thus, this new microfluidic chamber is advantageous for experiments that involve controlled drug or solution delivery at high spatiotemporal resolution.