Viability Of Slices Research Articles

Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response

Background/Objective: The conventional models currently used to evaluate various anti-tumor therapeutic agents are not sufficient for representing human pancreatic ductal adenocarcinoma (PDA), which has a unique tumor microenvironment. We aimed to produce an organotypic slice culture model from human PDA that resembles the in vivo situation and to evaluate the responses of PDA slices to established cytotoxic drugs. MethodsPDA tissues were obtained from 10 patients who underwent pancreatic resection. The tissues were sliced by a vibratome, and the tumor slices were then cultured. The viability of tumor slices during slice culture was evaluated using H&E and immunohistochemical staining, and stromal cells were demonstrated. The effects of cytotoxic drugs on PDA cell lines and slices were analyzed. ResultsTumor slices maintained their surface areas and tissue viability for at least five days during culture. Preserved proliferation and apoptosis in tumor slices were observed by the expression of Ki-67 and cleaved caspase-3. Stromal cells including macrophages (CD68+ and CD163+), T cells (CD3+, CD8+, and FOXP3+), and myeloid cells (CD11b+) were present throughout the culture period. Staurosporine, gemcitabine, and cisplatin treatment of PDA cell lines and tumor slices exerted proportional cytotoxic effects in terms of MTT viability, tumor cell number, and Ki-67 and cleaved caspase-3 expression. ConclusionsOrganotypic human PDA slice cultures preserved their viability and tumor microenvironment for at least five days during slice culture. PDA slice culture appears to be a feasible preclinical test model to assess the response to anti-tumor agents.

Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen

Monoterpenes R-pulegone (PUL) and R-menthofuran (MF), abundant in the Lamiaceae family, are frequently used in herb and food products. Although their hepatotoxicity was shown in rodent species, information about their effects in human liver has been limited. The aim of our study was to test the effects of PUL, MF and acetaminophen (APAP, as a reference compound) on cell viability and microRNA (miRNA) expression in human precision-cut liver slices. Slices from five patients were used to follow up on the inter-individual variability. PUL was toxic in all liver samples (the half-maximal effective concentration was 4.0 µg/mg of tissue), while MF and surprisingly APAP only in two and three liver samples, respectively. PUL also changed miRNA expression more significantly than MF and APAP. The most pronounced effect was a marked decrease of miR-155-5p expression caused by PUL even in non-toxic concentrations in all five liver samples. Our results showed that PUL is much more toxic than MF and APAP in human liver and that miR-155-5p could be a good marker of PUL early hepatotoxicity. Marked inter-individual variabilities in all our results demonstrate the high probability of significant differences in the hepatotoxicity of tested compounds among people.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized <em>N</em>-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Short-term hypoxia and vasa recta function in kidney slices.

Descending vasa recta (DVR) supply the inner part of outer renal medulla an area at risk for hypoxic damages. We hypothesize increased vasoreactivity after hypoxia/re-oxygenation (H/R) in DVR, which might contribute to the reduced medullary perfusion after an ischemic event. Live kidney slices (200μm) from SD rats were used for functional experiments. TUNEL assay and H&E staining were used to estimate slice viability. Kidney slices were treated with carbogen or hypoxia (1% O2) for 60 or 90 min and vasoreactivity to Ang II (10-7 M) was recorded by DIC microscopy after re-oxygenation with carbogen. Expression of NOS and NADPH enzymes mRNA were determined in iron-perfusion isolated VR. Percentage of apoptotic cells increased in control and H/R after 90 min in the medulla. Ang II- induced constriction of DVR was reduced after 90 min in control (compared to 60 min), but not after H/R. NOS enzymes mRNA expression levels decreased over 90 min hypoxia. Increased reactivity of DVR to Ang II after H/R compared to control (90 min) suggest a role of DVR in renal ischemia/reperfusion injury.

Preparation of viable adult ventricular myocardial slices from large and small mammals.

This protocol describes the preparation of highly viable adult ventricular myocardial slices from the hearts of small and large mammals, including rodents, pigs, dogs and humans. Adult ventricular myocardial slices are 100- to 400-μm-thick slices of living myocardium that retain the native multicellularity, architecture and physiology of the heart. This protocol provides a list of the equipment and reagents required alongside a detailed description of the methodology for heart explantation, tissue preparation, slicing with a vibratome and handling of myocardial slices. Supplementary videos are included to visually demonstrate these steps. A number of critical steps are addressed that must be followed in order to prepare highly viable myocardial slices. These include identification of myocardial fiber direction and fiber alignment within the tissue block, careful temperature control, use of an excitation-contraction uncoupler, optimal vibratome settings and correct handling of myocardial slices. Many aspects of cardiac structure and function can be studied using myocardial slices in vitro. Typical results obtained with hearts from a small mammal (rat) and a large mammal (human) with heart failure are shown, demonstrating myocardial slice viability, maximum contractility, Ca2+ handling and structure. This protocol can be completed in ∼4 h.

Hypoxia-reoxygenation enhances murine afferent arteriolar vasoconstriction by angiotensin II.

We tested the hypothesis that hypoxia-reoxygenation (H/R) augments vasoreactivity to angiotensin II (ANG II). In particular, we compared an in situ live kidney slice model with isolated afferent arterioles (C57Bl6 mice) to assess the impact of tubules on microvessel response. Hematoxylin and eosin staining was used to estimate slice viability. Arterioles in the slices were located by differential interference contrast microscopy, and responses to vasoactive substances were assessed. Cytosolic calcium transients and NADPH oxidase (NOX) mRNA expression were studied in isolated afferent arterioles. SOD activity was measured in live slices. Both experimental models were subjected to control and H/R treatment (60 min). Slices were further analyzed after 30-, 60-, and 90-min hypoxia followed by 10- or 20-min reoxygenation (H/R). H/R resulted in enhanced necrotic tissue damage compared with control conditions. To characterize the slice model, we applied ANG II (10-7 M), norepinephrine (NE; 10-5 M), endothelin-1 (ET-1; 10-7 M), and ATP (10-4 M), reducing the initial diameter to 44.5 ± 2.8, 50.0 ± 2.2, 45.3 ± 2.6, and 74.1 ± 1.8%, respectively. H/R significantly increased the ANG II response compared with control in live slices and in isolated afferent arterioles, although calcium transients remained similar. TEMPOL incubation prevented the H/R effect on ANG II responses. H/R significantly increased NOX2 mRNA expression in isolated arterioles. SOD activity was significantly decreased after H/R. Enhanced arteriolar responses after H/R occurred independently from the surrounding tissue, indicating no influence of tubules on vascular function in this model. The mechanism of increased ANG II response after H/R might be increased oxidative stress and increased calcium sensitivity of the contractile apparatus.

SiRNA-Mediated RNA Interference in Precision-Cut Tissue Slices Prepared from Mouse Lung and Kidney

Small interfering RNA (siRNA)-mediated RNAi interference (RNAi) is a powerful post-transcriptional gene silencing mechanism which can be used to study the function of genes in vitro (cell cultures) and in vivo (animal models). However, there is a translational gap between these models. Hence, there is a need for novel experimental models that combine the advantages of in vitro and in vivo models (e.g., simplicity, flexibility, throughput, and representability) to study the effects of siRNA. This need may be addressed by precision-cut tissue slices (PCTS), which represent an ex vivo model that mimics the structural and functional characteristics of a whole organ. The goal of this study was to investigate whether self-deliverable siRNA (Accell siRNA) can be used in precision-cut lung slices (PCLuS) and precision-cut kidney slices (PCKS) to achieve RNAi ex vivo. PCLuS and PCKS were prepared from mouse tissue, and they were subsequently incubated up to 48 h with no siRNA (untransfected), non-targeting Accell siRNA, or Gapdh-targeting Accell siRNA. Significant Gapdh mRNA silencing was achieved (PCLuS ~ 55%; PCKS ~ 40%) without compromising the viability and morphology of slices. Fluorescence microscopy confirmed that Accell siRNA diffused into PCLuS and PCKS. Spontaneous inflammation upon incubation was observed in PCLuS and PCKS as shown by a higher mRNA expression of pro-inflammatory cytokines Il1b, Il6, and Tnfa, although Accell siRNA appeared to diminish this response in PCLuS after 24 h. In conclusion, this ex vivo transfection model can be used to evaluate the effects of siRNA in relevant biological environments.

Bioenergetics dysfunction, mitochondrial permeability transition pore opening and lipid peroxidation induced by hydrogen sulfide as relevant pathomechanisms underlying the neurological dysfunction characteristic of ethylmalonic encephalopathy

Hydrogen sulfide (sulfide) accumulates at high levels in brain of patients with ethylmalonic encephalopathy (EE). In the present study, we evaluated whether sulfide could disturb energy and redox homeostasis, and induce mitochondrial permeability transition (mPT) pore opening in rat brain aiming to better clarify the neuropathophysiology of EE. Sulfide decreased the activities of citrate synthase and aconitase in rat cerebral cortex mitochondria, and of creatine kinase (CK) in rat cerebral cortex, striatum and hippocampus supernatants. Glutathione prevented sulfide-induced CK activity decrease in the cerebral cortex. Sulfide also diminished mitochondrial respiration in cerebral cortex homogenates, and dissipated mitochondrial membrane potential (ΔΨm) and induced swelling in the presence of calcium in brain mitochondria. Alterations in ΔΨm and swelling caused by sulfide were prevented by the combination of ADP and cyclosporine A, and by ruthenium red, indicating the involvement of mPT in these effects. Furthermore, sulfide increased the levels of malondialdehyde in cerebral cortex supernatants, which was prevented by resveratrol and attenuated by glutathione, and of thiol groups in a medium devoid of brain samples. Finally, we verified that sulfide did not alter cell viability and DCFH oxidation in cerebral cortex slices, primary cortical astrocyte cultures and SH-SY5Y cells. Our data provide evidence that bioenergetics disturbance and lipid peroxidation along with mPT pore opening are involved in the pathophysiology of brain damage observed in EE.

Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound

Precision cut liver slices (PCLiS) represent a promising tool in reflecting hepatotoxic responses. However, the culture of PCLiS varies considerably between laboratories, which can affect the performance of the liver slices and thus the experimental outcome. In this study, we describe an easily accessible culture method, which ensures optimal slice viability and functionality, in order to set the basis for reproducible and comparable PCLiS studies. The quality of the incubated rat PCLiS was assessed during a 24h culture period using ten readouts, which covered viability (lactate dehydrogenase-, aspartate transaminase- and glutamate dehydrogenase-leakage, ATP content) and functionality parameters (urea, albumin production) as well as histomorphology and other descriptive characteristics (protein content, wet weight, slice thickness). The present culture method resulted in high quality liver slices for 24h. Finally, PCLiS were exposed to increasing concentrations of acetaminophen to assess the suitability of the model for the detection of hepatotoxic responses. Six out of ten readouts revealed a toxic effect and showed an excellent mutual correlation. ATP, albumin and histomorphology measurements were identified as the most sensitive readouts. In conclusion, our results indicate that rat PCLiS are a valuable liver model for hepatotoxicity studies, particularly if they are cultured under optimal standardized conditions.

Hypoxia/re‐oxygenation increases the response of afferent arterioles to angiotensin II ‐ Investigation in mouse kidney slices

Hypoxia/re‐oxygenation (H/R) plays an important role in the pathogenesis of acute kidney injury (AKI). However, the influence of H/R on cortical microvessel function, especially that of afferent arterioles, is sparsely investigated. We hypothesize that H/R increases the vasoreactivity to angiotensin II (Ang II), which may promote the development of kidney injury. To test the hypothesis, we applied an innovative in situ model of live kidney slices.Experiments were performed in acute kidney slices (200 μm) of C57BL/6 mice. The TUNEL assay was used to estimate viability of slices by determining the percentage of apoptotic cells. For functional experiments, kidney slices were situated in a chamber at 37°C. Bath solution was equilibrated with carbogen. The afferent arterioles were visualized by differential interference contrast applied to an inverted microscope. To characterize arteriolar reactivity in this model of kidney slices, Ang II, norepinephrine, endothelin‐1 and ATP were administered. For H/R experiments, the slices were transferred into a hypoxia chamber (1% O2) for 30, 60 or 90 minutes and re‐oxygenated for 10 or 20 minutes.H/R did not significantly influence the apoptotic cell portion in the slice compared to control conditions. Afferent arterioles responded to the bolus application of Ang II (10−7 mol/l) with a maximum constriction, to 44.5 ± 2.8 % of the initial diameter. Norepinephrine (10−5 mol/l), endothelin‐1 (10−5 mol/l) and ATP (10−5 mol/l) reduced the diameter under control conditions to 50.0 ± 2.2%, 45.3 ± 2.6% and 74.1 ± 1.8, respectively. Inhibition of the NO‐synthesis by L‐NAME (10−4 mol/l) significantly enhanced the vasoconstriction to Ang II (32.3 ± 2.3 %). H/R increased the Ang II response of afferent arterioles compared to control conditions. Longer periods of hypoxia were associated with greater arteriolar vasoconstriction to Ang II (30/20 vs. 90/20: 39.7 ± 4.1% vs. 25.9 ± 2.0 %.). Also, increasing the duration of re‐oxygenation resulted in increased vasoconstriction (90/10 vs. 90/20: 34.7 ± 3.5% vs. 25.9 ± 2.00%).TUNEL staining and functional testing with vasoactive substances indicates the usefulness of the in situ model for the investigation of afferent arteriolar function. The increased response to Ang II in H/R suggests this effect as one component in the multifactorial genesis of AKI.Support or Funding InformationGerman Research Foundation (DFG PA 479/10‐2)

Castanea sativa Mill. Bark Extract Protects U-373 MG Cells and Rat Brain Slices Against Ischemia and Reperfusion Injury.

Ischemic brain injury is one of the most important causes of death worldwide. The use of one-drug-multi-target agents based on natural compounds is a promising therapeutic option for cerebral ischemia due to their pleiotropic properties. This study assessed the neuroprotective properties of Castanea sativa Mill. bark extract (ENC) in human astrocytoma U-373 MG cells subjected to oxygen-glucose deprivation and reperfusion and rat cortical slices subjected to ischemia-like conditions or treated with glutamate or hydrogen peroxide. Neuroprotective effects were determined by assessing cells or slices viability (MTT assay), ROS formation (DCFH-DA assay), apoptosis (sub G0/G1 peak), nuclear fragmentation and chromatin condensation (DAPI staining) as well as changes in lysosomes and mitochondria morphology (Acridine Orange and Rhodamine123 staining, respectively). ENC treatment before injury on U-373 MG cells (5-50 μg/ml) and cortical slices (50-100 μg/ml) provided neuroprotection, while lower or higher concentrations (100 μg/ml U-373 MG cells, 200 μg/ml brain slices) were ineffective. ENC addition during reperfusion or after the injury was not found to be effective. The results suggest that ENC might hold potential as preventive neuroprotective agent, and indicate the importance of further studies exploring its mechanism of action. J. Cell. Biochem. 118: 839-850, 2017. © 2016 Wiley Periodicals, Inc.

In vitro 6-hydroxydopamine-induced toxicity in striatal, cerebrocortical and hippocampal slices is attenuated by atorvastatin and MK-801

Parkinson's disease (PD) involves the loss of striatal dopaminergic neurons, although other neurotransmitters and brain areas are also involved in its pathophysiology. In rodent models to PD it has been shown statins improve cognitive and motor deficits and attenuate inflammatory responses evoked by PD-related toxins. Statins are the drugs most prescribed to hypercholesterolemia, but neuroprotective effects have also been attributed to statins treatment in humans and in animal models. This study aimed to establish an in vitro model of 6-hydroxydopamine (6-OHDA)-induced toxicity, used as an initial screening test to identify effective drugs against neural degeneration related to PD. The putative neuroprotective effect of atorvastatin against 6-OHDA-induced toxicity in rat striatal, cerebrocortical and hippocampal slices was also evaluated. 6-OHDA (100μM) decreased cellular viability in slices obtained from rat cerebral cortex, hippocampus and striatum. 6-OHDA also induced an increased reactive oxygen species (ROS) production and mitochondrial dysfunction. Co-incubation of 6-OHDA with atorvastatin (10μM) or MK-801 (50μM) an N-methyl-d-aspartate (NMDA) receptor antagonist, partially attenuated the cellular damage evoked by 6-OHDA in the three brain areas. Atorvastatin partially reduced ROS production in the hippocampus and striatum and disturbances of mitochondria membrane potential in cortex and striatum. 6-OHDA-induced toxicity in vitro displays differences among the brain structures, but it is also observed in cerebrocortical and hippocampal slices, besides striatum.

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO2 , field potentials and changes in extracellular potassium concentration ([K+ ]o ). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K+ ]o and pO2 changes as compared to continuous illumination.

Tabernaemontana catharinensis ethyl acetate fraction presents antinociceptive activity without causing toxicological effects in mice

Ethnopharmacological relevanceTabernaemontana catharinensis (Apocynaceae) is a medicinal plant used for the treatment of painful and inflammatory disorders. Here, we investigated the antinociceptive potential of the ethyl acetate fraction (Eta) from T. catharinensis leaves and assessed its toxic effects in mice to validate its popular use. Materials and methodsAdult male Swiss mice (30–35g) were used. The Eta antinociceptive effect (200–800mg/kg, oral route (p.o.)) was evaluated in the acetic acid, formalin, capsaicin and tail-immersion tests. Adverse effects were analyzed using rotarod and open-field tests, body temperature, biochemical analysis and gastric lesions assessment. To evaluate the acute (OECD 423) or sub-acute (OECD 407) toxicity of the Eta, it was administered orally at a single (2000mg/kg) or repeated doses (100–400mg/kg/day for 28 days), respectively. Mortality, behavioral changes, biochemical and hematological parameters were evaluated. The Eta effect on cellular viability also was evaluated. ResultsEta (200–800mg/kg) inhibited the nociception caused by acetic acid (93.9±1.5%), formalin (86.2±10.8%) or capsaicin (75.4±3.3%) without inducing gastric lesions. Moreover, Eta neither altered the body temperature, biochemical parameters, nor forced or spontaneous locomotor activity of mice. The acute administration of the Eta (2000mg/kg) promoted a decrease in blood glucose levels and alanine aminotransferase activity. In the sub-acute toxicity study, Eta increased the aspartate aminotransferase activity (400mg/kg) and platelet distribution width (200mg/kg). Furthermore, Eta did not alter the cellular viability in cortical slices. ConclusionsEta presents antinociceptive effects and mild toxicity in mice. These results support its traditional use as a potential analgesic.

Ilex paraguariensis hydroalcoholic extract exerts antidepressant-like and neuroprotective effects: involvement of the NMDA receptor and the L-arginine-NO pathway.

Ilex paraguariensis St. Hilaire (Aquifoliaceae) is a typical plant from South America. Preclinical studies have reported the effect of I. paraguariensis-based preparations on different alterations in the brain. This study aimed to examine the antidepressant-like and neuroprotective effects of I. paraguariensis hydroalcoholic extract (IpHE). The role of the N-methyl-D-aspartate receptor and the L-arginine-nitric oxide pathway in the IpHE antidepressant-like effect was also evaluated. Using the tail suspension test, we showed that IpHE (0.1-10 mg/kg, orally) exerts an antidepressant-like effect similar to that of ketamine (1 mg/kg, intraperitoneally). The antidepressant-like effect depends on the N-methyl-D-aspartate receptor and L-arginine-nitric oxide pathway modulation as we observed a combinatory effect using subeffective doses of IpHE (0.01 mg/kg, orally) and ketamine (0.1 mg/kg, intraperitoneally) or MK-801 (0.001 mg/kg, intraperitoneally). Also, pretreatment of mice with L-arginine (750 mg/kg, intraperitoneally) abolished the antidepressant-like effect of IpHE. This effect coincides with the neuroprotective effect, given that glutamate toxicity (10 mmol/l) did not decrease cell viability in hippocampal or cortical slices from IpHE-treated mice. The chromatographic profile of IpHE showed the presence of the methylxanthines caffeine and theobromine. Administration of methylxanthines (2.7 µg/kg) in mice produced an antidepressant-like effect, but not neuroprotection. We suggest that methylxanthines are at least in part responsible for the antidepressant-like effect of IpHE; further studies are necessary to determine the biological compounds responsible for the neuroprotective effect.

Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue.

Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45–150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6–8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

Passiflora actinia hydroalcoholic extract and its major constituent, isovitexin, are neuroprotective against glutamate-induced cell damage in mice hippocampal slices.

To investigate whether Passiflora actinia hydroalcoholic extract and its major constituent, isovitexin, protect mice hippocampal brain slices from glutamate-induced neurotoxicity. Neuroprotective effect of the extract against glutamate-induced excitotoxicity (10 mm) was evaluated through cell viability of hippocampal slices. The extract or its flavonoids were directly applied to hippocampal slices and then subjected to glutamate-induced toxicity. Alternatively, hippocampal slices from extract-treated mice were also subjected to the same toxicity protocol. Mice supplementation with the extract protected hippocampal slices from in-vitro neurotoxicity. When directly applied to hippocampal slices, the extract showed a higher neuroprotective potential than a commercial dry extract of Passiflora incarnata, which was related to P. actinia extract which had higher isovitexin and total flavonoid content expressed as isovitexin. Isovitexin, but not apigenin, induced a similar neuroprotective response when applied alone, at a concentration equivalent to that found in the extract. This study highlights new neuropharmacological activity of the Passiflora genus, suggesting that it can act as modulator of the glutamatergic system. The search for improved pharmacotherapies with novel mechanisms of action has been shown of great importance for the treatment of resistant neurological and psychiatric disorders.

Creatine affords protection against glutamate-induced nitrosative and oxidative stress

Creatine has been reported to exert beneficial effects in several neurodegenerative diseases in which glutamatergic excitotoxicity and oxidative stress play an etiological role. The purpose of this study was to investigate the protective effects of creatine, as compared to the N-Methyl-d-Aspartate (NMDA) receptor antagonist dizocilpine (MK-801), against glutamate or hydrogen peroxide (H2O2)-induced injury in human neuroblastoma SH-SY5Y cells. Exposure of cells to glutamate (60–80 mM) or H2O2 (200–300 μM) for 24 h decreased cellular viability and increased dichlorofluorescein (DCF) fluorescence (indicative of increased reactive oxygen species, ROS) and nitric oxide (NO) production (assessed by mono-nitrogen oxides, NOx, levels). Creatine (1–10 mM) or MK-801 (0.1–10 μM) reduced glutamate- and H2O2-induced toxicity. The protective effect of creatine against glutamate-induced toxicity involves its antioxidant effect, since creatine, similar to MK-801, prevented the increase on DCF fluorescence induced by glutamate or H2O2. Furthermore, creatine or MK-801 blocked glutamate- and H2O2-induced increases in NOx levels. In another set of experiments, the repeated, but not acute, administration of creatine (300 mg/kg, po) in mice prevented the decreases on cellular viability and mitochondrial membrane potential (assessed by tetramethylrhodamine ethyl ester, TMRE, probe) of hippocampal slices incubated with glutamate (10 mM). Creatine concentration-dependent decreased the amount of nitrite formed in the reaction of oxygen with NO produced from sodium nitroprusside solution, suggesting that its protective effect against glutamate or H2O2-induced toxicity might be due to its scavenger activity. Overall, the results suggest that creatine may be useful as adjuvant therapy for neurodegenerative disease treatments.

Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation: Effects of culture medium

Precision-cut liver slices (PCLS) are an ex vivo model for metabolism and toxicity studies. However, data on the maintenance of the morphological integrity of the various cell types in the slices during prolonged incubation are lacking. Therefore, our aims were to characterize morphological and functional changes in rat PCLS during five days of incubation in a rich medium, RegeneMed®, and a standard medium, Williams' Medium E. Although cells of all types in the slices remain viable, profound changes in morphology were observed, which were more prominent in RegeneMed®. Slices underwent notable fibrosis, bile duct proliferation and fat deposition. Slice thickness increased, resulting in necrotic areas, while slice diameter decreased, possibly indicating cell migration. An increased proliferation of parenchymal and non-parenchymal cells (NPCs) was observed. Glycogen, albumin and Cyp3a1 were maintained albeit to a different level in two media. In conclusion, both hepatocytes and NPCs remain viable and functional, enabling five-day toxicity studies. Tissue remodeling and formation of a new capsule-like cell lining around the slices are evident after 3–4days. The differences in effects between media emphasize the importance of media selection and of the recognition of morphological changes in PCLS, when interpreting results from toxicological or pharmacological studies.