Drug-treated Animals Research Articles

The Effect of Histone Deacetylase Inhibitors Panobinostat or Entinostat on Motor Recovery in Mice After Ischemic Stroke.

Using rigorous and clinically relevant experimental design and analysis standards, in this study, we investigated the potential of histone deacetylase (HDAC) inhibitors panobinostat and entinostat to enhance recovery of motor function after photothrombotic stroke in male mice. Panobinostat, a pan-HDAC inhibitor, is a FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor in late stage of clinical investigation. The drugs were administered every other day (panobinostat-3 or 10mg/kg; entinostat-1.7 or 5mg/kg) starting from day 5 to 15 after stroke. To imitate the current standard of care in stroke survivors, i.e., physical rehabilitation, the animals run on wheels (2h daily) from post-stroke day 9 to 41. The predetermined primary end point was motor recovery measured in two tasks of spontaneous motor behaviors in grid-walking and cylinder tests. In addition, we evaluated the running distance and speed throughout the study, and the number of parvalbumin-positive neurons in medial agranular cortex (AGm) and infarct volumes at the end of the study. Both sensorimotor tests revealed that combination of physical exercise with either drug did not substantially affect motor recovery in mice after stroke. This was accompanied by negligible changes of parvalbumin-positive neurons recorded in AGm and comparable infarct volumes among experimental groups, while dose-dependent increase in acetylated histone 3 was observed in peri-infarct cortex of drug-treated animals. Our observations suggest that add-on panobinostat or entinostat therapy coupled with limited physical rehabilitation is unlikely to offer therapeutic modality for stroke survivors who have motor dysfunction.

The first visualization of chemotherapy-induced tumor apoptosis via magnetic particle imaging in a mouse model

Imaging technologies that allow non-radiative visualization and quantification of apoptosis have a great potential for assessing therapy response, early diagnosis, and disease monitoring. Magnetic particle imaging (MPI), the direct imaging of magnetic nanoparticles as positive contrast agent and sole signal source, enables high image contrast (no tissue background signal), potential high sensitivity, and quantifiable signal intensity. These properties confer a great potential for application to tumor apoptosis monitoring. In this study, a simple and robust method was used to conjugate Alexa Fluor 647-AnnexinV (AF647-Anx), which can avidly bind to apoptotic cells, to superparamagnetic iron oxide (SPIO) nanoparticles, termed AF647-Anx-SPIO, which serves as an MPI-detectable tracer. Based on this apoptosis-specific tracer, MPI can accurately and unambiguously detect and quantify apoptotic tumor cells. AF647-Anx-SPIO showed relatively high affinity for apoptotic cells, and differences in binding between treated (apoptotic rate 67.21% ± 1.36%) and untreated (apoptotic rate 10.12 ± 0.11%) cells could be detected by MPI in vitro (P < 0.05). Moreover, the imaging signal was almost proportional to the number of apoptotic cells determined using an MPI scanner (R2 = 0.99). There was a greater accumulation of AF647-Anx-SPIO in tumors of drug-treated animals than in tumors of untreated animals (P < 0.05), and the difference could be detected by MPI ex vivo, while for in vivo imaging, no MPI imaging signal was detected in either group. Overall, this preliminary study demonstrates that MPI could be a potential imaging modality for tumor apoptosis imaging.

Abstract TP114: Histone Deacetylase Inhibitors Panobinostat and Entinostat for Motor Recovery After Ischemic Stroke in Mice

In this study, we investigated the potential of two histone deacetylase (HDAC) inhibitors, panobinostat and entinostat, to enhance recovery of motor function after ischemic stroke in CD-1 male mice. Panobinostat, which is a pan-HDAC inhibitor, is an FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor and is widely expected to get approval for clinical use in near future. Stroke was induced by photothrombosis and the drugs were administered intraperitoneally every other day (panobinostat at 10 mg/kg or 30 mg/kg; entinostat at 5 mg/kg or 15 mg/kg) starting from day 5 to day 15 after stroke. The control group received only vehicle following the same regimen. In addition, all drug or vehicle-treated mice exercised for 2 hours (voluntary wheel running) starting from day 9 to day 41 after stroke. Additional control groups included sham-operated animals, and mice which had stroke but were not drug/vehicle-treated or allowed to run. Motor function of the mice was evaluated by blinded investigators using gridwalk and cylinder tests before and after stroke. Acetylation of histone 3 in the peri-infarct region of the brain samples was measured by immunoblotting. Our results indicate that lower dose of both panobinostat and entinostat marginally improved motor function in mice by day 42 after stroke, although it did not reach statistical significance. Notably, this improvement trend was lost with the higher dose of both drugs which also showed some toxicity. No statistical difference was observed in running distance of mice among experimental groups. Likewise, we did not observe statistically significant difference in stroke volumes among the experimental groups. Immunoblotting experiments indicated that both panobinostat and entinostat dose-dependently increased the level of acetylated histone in the cortical, peri-infarct region of drug-treated animals compared to the non-treated groups. In summary, our results indicate that both panobinostat and entinostat do not facilitate improvement of motor function after stroke at the tested doses. However, it is likely that lower doses of these drugs may enhance recovery of motor function after stroke.

Identifying drug targets in tissues and whole blood with thermal-shift profiling.

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.

Pharmacodynamic interaction of Tinospora cordifolia Willd. With Ocimum sanctum Linn. in isoproterenol-induced cardiac toxicity

Background:Cardiovascular diseases are the leading causes of deaths despite several advancements in the current medical interventions. Among them, myocardial infarction (MI) is the most alarming disease as about 17.1 million peoples die every year due to MI.Aim:The present study was designed to investigate the potential cardioprotective effect of combination of standardized extracts of Tinospora cordifolia (SETC) (250 mg/kg and 500 mg/kg) and Ocimum sanctum (SEOS) (50 mg/kg) in isoproterenol (ISO)-induced MI.Materials and methods:MI was induced in rats by subcutaneous injection of ISO for 2 consecutive days at an interval of 24 h. Rats were pretreated with test drugs for the period of 21 days, and ISO was administered on the 20th and 21st days. At the end of experiment, i.e., on 22nd-day electrocardiograph, a hemodynamic, biochemical, and histopathological study of heart tissues was evaluated from control and experimental groups and statistically analyzed by one-way analysis of variance followed by Tukey's test.Results:ISO-administered rats showed significant changes in electrocardiograph, mean arterial blood pressure, heart rate, biochemical markers, antioxidant parameters, and histopathology of heart. The activities of cardiac biomarkers were reduced in serum, and there was an increase in antioxidants in heart tissue of test drug-treated animals. Similarly, electrocardiograph, mean arterial blood pressure, and heart rate were restored to normalcy in all test and standard drug-treated animals.Conclusion:The SETC 500 mg/kg in combination with SEOS 50 mg/kg was found to be effective in prevention of myocardial injury induced by ISO.

TMIC-48. MACROPHAGE DEPLETION COMBINED WITH RADIATION IN A PRECLINICAL MEDULLOBLASTOMA MODEL

Abstract BACKGROUND Radiation induces a measurable immune response, yet this anti-tumor immunity is often suboptimal at eliminating residual brain tumor cells. Medulloblastomas, along with other pediatric brain tumors, tend to recur in the primary resection bed following irradiation. Infiltrating macrophage within the tumor (TAM) are radioresistant immune cells that may limit the effectiveness of radiotherapy and promote tumor regrowth through their immunosuppressive and pro-tumorigenic functions. Therefore, we explored whether CSF-1 receptor (CSF1R) inhibition, and essentially TAM depletion, could augment radiation and/or reduce local recurrence. METHODS Selected CSF1R inhibitors were tested on both human and mouse macrophage in vitro. In vivo studies were performed on a patient-derived orthotopic xenograft (PDOX) model of Group 3 medulloblastoma, Med411-FH. Macrophage depletion was achieved using the anti-murine CSF1R antibody, AFS98, injected intra-peritoneally 10 days prior to, concurrently, and after radiation. RESULTS CSF1R antibodies and small molecule tyrosine kinase inhibitors were effective at stanching macrophage survival in culture. In a PDOX medulloblastoma model, both classes of CSF1R inhibitors were effective at reducing macrophage in the brain after approximately 10 days of administration, with maximal effect of 90% depletion achieved after 21 days. In a combinatorial study of AFS98 and irradiation, TAM depletion conferred a very modest effect on tumor growth (p=0.003) and had no effect on overall survival when combined with radiation (p=0.86). Interestingly, we did observe a trend toward reduction in metastatic spinal disease following radiation in the AFS98-treated cohort. CONCLUSION CSF1R antibody administration effectively eliminated macrophage and microglia in the brain and spine, suggesting adequate blood-brain barrier penetration in our xenograft model. While CSF1R inhibition combined with radiation was ineffective at prolonging survival, further studies are needed to investigate a modest effect on brain tumor growth and an observed (yet not statistically significant) difference in metastatic spinal recurrence in drug-treated animals.

Influence of aversive stimulation on haloperidol-induced catalepsy in rats.

Catalepsy - an immobile state in which individuals fail to change imposed postures - can be induced by haloperidol. In rats, the pattern of haloperidol-induced catalepsy is very similar to that observed in Parkinson's disease (PD). As some PD symptoms seem to depend on the patient's emotional state, and as anxiety disorders are common in PD, it is possible that the central mechanisms regulating emotional and cataleptic states interplay. Previously, we showed that haloperidol impaired contextual-induced alarm calls in rats, without affecting footshock-evoked calls. Here, we evaluated the influence of distinct aversive stimulations on the haloperidol-induced catalepsy. First, male Wistar rats were subjected to catalepsy tests to establish a baseline state after haloperidol or saline administration. Next, distinct cohorts were exposed to open-field; elevated plus-maze; open-arm confinement; inescapable footshocks; contextual conditioned fear; or corticosterone administration. Subsequently, catalepsy tests were performed again. Haloperidol-induced catalepsy was verified in all drug-treated animals. Exposure to open-field, elevated plus-maze, open-arm confinement, footshocks, or administration of corticosterone had no significant effect on haloperidol-induced catalepsy. Contextual conditioned fear, which is supposed to promote a more intense fear, increased catalepsy over time. Our findings suggest that only specific defensive circuitries modulate the nigrostriatal system mediating the haloperidol-induced cataleptic state.

Rapid antidepressant effects of deep brain stimulation of the pre-frontal cortex in an animal model of treatment-resistant depression.

A significant proportion of depressed patients fail to respond to treatment with antidepressant drugs. Such patients might nonetheless respond to deep brain stimulation of the prefrontal cortex. Deep brain stimulation has also been shown to normalize behaviour in the chronic mild stress (CMS) model of depression. However, these studies have involved animals that are in general treatment responsive. Thus, this is not the ideal situation in which to investigate how deep brain stimulation is effective where antidepressant drugs are not. Here, we studied the behavioural effects of deep brain stimulation in treatment-resistant animals. Wistar rats were exposed to chronic mild stress and concurrent treatment with saline or one of three antidepressant drugs, imipramine, citalopram and venlafaxine. Individuals were selected from the CMS-exposed drug-treated groups that had failed to increase their sucrose intake by week 5 of drug treatment. All animals were then implanted with deep brain stimulation electrodes in the ventro-medial prefrontal cortex, and tested for sucrose intake and in the elevated plus maze and novel object recognition test, following 2 × 2 h of deep brain stimulation. The selected drug-treated animals were found to be antidepressant-resistant in all three tests. With a single exception (sucrose intake in imipramine-treated animals), deep brain stimulation reversed the anhedonic, anxiogenic and dyscognitive effects of CMS in all four conditions, with no significant differences between saline- and drug-treated animals. These data provide a proof of principle that deep brain stimulation of the prefrontal cortex can be effective in a rat model of resistance to chronic antidepressant treatment, replicating the clinical effect of deep brain stimulation in treatment-resistant depression.

Neurotoxicity of Inhalation Anesthetics in the Neonatal Rat Brain: Effects on Behavior and Neurodegeneration in the Piriform Cortex.

There is concern that clinical use of anesthetic drugs may cause neurotoxicity in the developing brain and subsequent abnormal neurobehavior. We therefore evaluated neurotoxic effects of inhalation anesthetics in the neonatal rat brain, using in vivo histological and neurobehavioral outcomes. Wistar rats (n=79, postnatal day 15) were subjected to a clinically relevant single exposure of urethane, isoflurane, sevoflurane, or placebo, without surgery. At 48 h and 96 h, behavioral parameters were recorded and the animals were sacrificed. In cryosectioned brains, total cells and dying cells in layer II of the piriform cortex were counted using unbiased stereology. At 48 h, cell numbers in layer II of the piriform cortex of all drug-treated animals were reduced versus controls (p=0.01). The effect persisted at 96 h in isoflurane- and urethane-exposed animals. Piriform cortical layer II neurons undergoing degeneration, detected histologically by pyknotic nuclei and eosinophilic cytoplasm, were increased in the animals treated with isoflurane (1.9 ± 0.7 at 96 h) and urethane (2.4 ± 0.8 at 96 h) versus sevoflurane (0.8 ± 0.3 at 96 h) and controls (0.9 ± 0.2 at 96 h). Sevoflurane- and isoflurane-treated animals exhibited increased activity and decreased suckling compared with controls, and sevoflurane-exposed animals also displayed increased rearing behavior at both timepoints.

COMPARATIVE STUDY OF BUTANOLIC AND ETHANOLIC EXTRACTS OF TRIBULUS TERRESTRIS FRUITS IN 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN-INDUCED HEPATOTOXICITY IN RATS

Objective: To compare hepatoprotective activity of ethanol and butanol fraction of Tribulus terrestris fruits against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatic damage in albino rats.Methods: The degree of liver protection was assessed in terms of biochemical parameters, liver marker enzymes, and lipid peroxidation (LPO) in drug-treated and TCDD-intoxicated rats.Results: The results revealed that the elevated serum marker enzymes and biochemical parameters in TCDD-treated rats were significantly reduced toward normal levels in saponin rich fraction of T. terrestris drug-treated animals when comparable to ethanolic extract of T. terrestris drug-treated group. The plant extracts have got a notable inhibitory activity on the formation of LPO products in the basal as well as in the presence of inducers.Conclusion: The plant extracts hindered oxidative damage that occurred to the hepatocytes during TCDD toxicity may be responsible for the hepatoprotective activity in the present study.

Gestational Exposure to the Synthetic Cathinone Methylenedioxypyrovalerone Results in Reduced Maternal Care and Behavioral Alterations in Mouse Pups.

The member of synthetic cathinone family, methylenedioxypyrovalerone (MDPV), is a frequently used psychoactive drug of abuse. The objective of our study was to determine the effect of MDPV (administered from the 8th to the 14th day of gestation) on the behavior of neonatal and adolescent mice, as well as its effect on maternal care. We measured maternal care (pup retrieval test, nest building), locomotor activity (open field test), and motor coordination (grip strength test) of dams, whereas on pups we examined locomotor activity at postnatal day 7 and day 21 (open field test) and motor coordination on day 21 (grip strength test). On fresh-frozen brain samples of the dams we examined the expression of two important peptides implicated in the regulation of maternal behavior and lactation: tuberoinfundibular peptide 39 (TIP39) mRNA in the thalamic posterior intralaminar complex, and amylin mRNA in the medial preoptic nucleus. We detected decreased birth rate and survival of offspring, and reduced maternal care in the drug-treated animals, whereas there was no difference between the motility of treated and control mothers. Locomotor activity of the pups was increased in the MDPV treated group both at 7 and 21 days of age, while motor coordination was unaffected by MDPV treatment. TIP39 and amylin were detected in their typical location but failed to show a significant difference of expression between the drug-treated and control groups. The results suggest that chronic systemic administration of the cathinone agent MDPV to pregnant mice can reduce birth rate and maternal care, and it also enhances motility (without impairment of motor coordination) of the offspring.

Neratinib + Valproate] exposure permanently reduces ERBB1 and RAS expression in 4T1 mammary tumors and enhances M1 macrophage infiltration.

The irreversible ERBB1/2/4 inhibitor neratinib has been shown in vitro to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the [neratinib + valproate] drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to [neratinib + valproate] for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to [neratinib + valproate] grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination. In vitro exposure of 4T1 tumor cells to [neratinib + valproate] variably reduced the expression of histone deacetylases 1-11. In vivo, prior exposure of tumors to [neratinib + valproate] permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that [neratinib + valproate] evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies.

Inhibition of Vascular Endothelial Growth Factor Receptor Decreases Regenerative Angiogenesis in Axolotls.

Angiogenesis is crucial for tissue growth and repair in mammals, and is chiefly regulated by vascular endothelial growth factor (VEGF) signaling. We evaluated the effect of chemical inhibition of VEGF receptor signaling in animals with superior regenerative ability, axolotl salamanders, to determine the impact on vascularization and regenerative outgrowth. Following tail amputation, treated animals (100nM PTK787) and controls were examined microscopically and measured over the month-long period of regeneration. Treatment with VEGFR inhibitor decreased regenerative angiogenesis; drug-treated animals had lower vascular densities in the regenerating tail than untreated animals. This decrease in neovascularization, however, was not associated with a decrease in regenerative outgrowth or with morphological abnormalities in the regrown tail. Avascular but otherwise anatomically normal regenerative outgrowth over 1mm beyond the amputation plane was observed. The results suggest that in this highly regenerative species, significant early tissue regeneration is possible in the absence of a well-developed vasculature. This research sets the groundwork for establishing a system for the chemical manipulation of angiogenesis within the highly regenerative axolotl model, contributing to a better understanding of the role of the microvasculature within strongly proliferative yet well-regulated environments. Anat Rec, 300:2273-2280, 2017. © 2017 Wiley Periodicals, Inc.

The Roles of Dopamine D1 Receptor on the Social Hierarchy of Rodents and Nonhuman Primates.

Background:Although dopamine has been suggested to play a role in mediating social behaviors of individual animals, it is not clear whether such dopamine signaling contributes to attributes of social groups such as social hierarchy.Methods:In this study, the effects of the pharmacological manipulation of dopamine D1 receptor function on the social hierarchy and behavior of group-housed mice and macaques were investigated using a battery of behavioral tests.Results:D1 receptor blockade facilitated social dominance in mice at the middle, but not high or low, social rank in the groups without altering social preference among mates. In contrast, the administration of a D1 receptor antagonist in a macaque did not affect social dominance of the drug-treated animal; however, relative social dominance relationships between the drug-treated and nontreated subjects were altered indirectly through alterations of social affiliative relationships within the social group.Conclusions:These results suggest that dopamine D1 receptor signaling may be involved in social hierarchy and social relationships within a group, which may differ between rodents and primates.

Retinoic acid and sodium butyrate suppress the cardiac expression of hypertrophic markers and proinflammatory mediators in Npr1 gene-disrupted haplotype mice.

The objective of the present study was to examine the genetically determined differences in the natriuretic peptide receptor-A (NPRA) gene (Npr1) copies affecting the expression of cardiac hypertrophic markers, proinflammatory mediators, and matrix metalloproteinases (MMPs) in a gene-dose-dependent manner. We determined whether stimulation of Npr1 by all-trans retinoic acid (RA) and histone deacetylase (HDAC) inhibitor sodium butyric acid (SB) suppress the expression of cardiac disease markers. In the present study, we utilized Npr1 gene-disrupted heterozygous (Npr1(+/-), 1-copy), wild-type (Npr1(+/+), 2-copy), gene-duplicated (Npr1(++/+), 3-copy) mice, which were treated intraperitoneally with RA, SB, and a combination of RA/SB, a hybrid drug (HB) for 2 wk. Untreated 1-copy mice showed significantly increased heart weight-body weight (HW/BW) ratio, blood pressure, hypertrophic markers, including beta-myosin heavy chain (β-MHC) and proto-oncogenes (c-fos and c-jun), proinflammatory mediator nuclear factor kappa B (NF-κB), and MMPs (MMP-2, MMP-9) compared with 2-copy and 3-copy mice. The heterozygous (haplotype) 1-copy mice treated with RA, SB, or HB, exhibited significant reduction in the expression of β-MHC, c-fos, c-jun, NF-κB, MMP-2, and MMP-9. In drug-treated animals, the activity and expression levels of HDAC were significantly reduced and histone acetyltransferase activity and expression levels were increased. The drug treatments significantly increased the fractional shortening and reduced the systolic and diastolic parameters of the Npr1(+/-) mice hearts. Together, the present results demonstrate that a decreased Npr1 copy number enhanced the expression of hypertrophic markers, proinflammatory mediators, and MMPs, whereas an increased Npr1 repressed the cardiac disease markers in a gene-dose-dependent manner.

H3K9me3 Inhibition Improves Memory, Promotes Spine Formation, and Increases BDNF Levels in the Aged Hippocampus.

Cognitive decline is a debilitating condition associated with not only neurodegenerative diseases but also aging in general. However, effective treatments have been slow to emerge so far. In this study, we demonstrate that epigenetic regulation of key synaptic proteins may be an underlying, yet reversible, cause of this decline. Our findings suggest that histone 3 trimethylation is a probable target for pharmacological intervention that can counteract cognitive decline in the aging brain. Finally, we provide support to the hypothesis that, by manipulating the enzyme that regulates H3K9me3 (using a newly developed specific inhibitor of SUV39H1), it is possible to alter the chromatin state of subjects and restore memory and synaptic function in the aging brain.

Effect of Eisenia foetida Extract against Cisplatin-Induced Kidney Injury in Rats

ABSTRACTKidney injury is a deleterious side effect accompanied by therapeutic uses of cisplatin as an antineoplastic agent. However, no therapeutic option is available against this complication. This study was designed to evaluate the protective role of a glycoprotein extract obtained from Eisenia foetida against cisplatin-induced nephrotoxicity. Rats were treated with cisplatin (7.5 mg/kg, intraperitoneally, i.p.) and Eisenia foetida extract (300 and 500 mg/kg, i.p. and/or oral). Serum creatinine (Cr) and blood urea nitrogen (BUN) were significantly elevated in cisplatin-treated rats. A significant amount of lipid peroxidation was detected in drug-treated animals. Furthermore, kidney histopathological findings revealed acute tubular necrosis and hyaline cast formation caused by cisplatin. Eisenia foetida extract administration (300 and 500 mg/kg, i.p.) significantly reduced serum BUN and creatinine and lipid peroxidation in kidney tissue. Moreover, cisplatin-induced histopathological lesions were alleviated by Eisenia foetida extract. This investigation concluded that Eisenia foetida extract ameliorated cisplatin-induced nephrotoxicity. This protection might be mediated by preventing cisplatin-induced oxidative stress.

Astrocyte Cultures Mimicking Brain Astrocytes in Gene Expression, Signaling, Metabolism and K+ Uptake and Showing Astrocytic Gene Expression Overlooked by Immunohistochemistry and In Situ Hybridization.

Based on differences in gene expression between cultured astrocytes and freshly isolated brain astrocytes it has been claimed that cultured astrocytes poorly reflect the characteristics of their in vivo counterparts. This paper shows that this is not the case with the cultures of mouse astrocytes we have used since 1978. The culture is prepared following guidelines provided by Drs. Monique Sensenbrenner and John Booher, with the difference that dibutyryl cyclic AMP is added to the culture medium from the beginning of the third week. This addition has only minor effects on glucose and glutamate metabolism, but it is crucial for effects by elevated K+ concentrations and for Ca2+ homeostasis, important aspects of astrocyte function. Work by Liang Peng and her colleagues has shown identity between not only gene expression but also drug-induced gene upregulations and editings in astrocytes cultured by this method and astrocytes freshly isolated from brains of drug-treated animals. Dr. Norenberg's laboratory has demonstrated identical upregulation of the cotransporter NKCC1 in ammonia-exposed astrocytes and rats with liver failure. Similarity between cultured and freshly isolated astrocytes has also been shown in metabolism, K+ uptake and several aspects of signaling. However, others have shown that the gene for the glutamate transporter GLT1 is not expressed, and rat cultures show some abnormalities in K+ effects. Nevertheless, the overall reliability of the cultured cells is important because immunohistochemistry and in situ hybridization poorly demonstrate many astrocytic genes, e.g., those of nucleoside transporters, and even microarray analysis of isolated cells can be misleading.

Assessment of the anticonvulsant potency of various benzylamide derivatives in the mouse maximal electroshock-induced seizure threshold model.

The aim of this study was to assess the anticonvulsant potency of 6 various benzylamide derivatives [i.e., nicotinic acid benzylamide (Nic-BZA), picolinic acid 2-fluoro-benzylamide (2F-Pic-BZA), picolinic acid benzylamide (Pic-BZA), (RS)-methyl-alanine-benzylamide (Me-Ala-BZA), isonicotinic acid benzylamide (Iso-Nic-BZA), and (R)-N-methyl-proline-benzylamide (Me-Pro-BZA)] in the threshold for maximal electroshock (MEST)-induced seizures in mice. Electroconvulsions (seizure activity) were produced in mice by means of a current (sine-wave, 50Hz, 500V, strength from 4 to 18mA, ear-clip electrodes, 0.2-s stimulus duration, tonic hindlimb extension taken as the endpoint). Nic-BZA, 2F-Pic-BZA, Pic-BZA, Me-Ala-BZA, Iso-Nic-BZA, and Me-Pro-BZA administered systemically (ip) in a dose-dependent manner increase the threshold for maximal electroconvulsions in mice. Linear regression analysis of Nic-BZA, 2F-Pic-BZA, Pic-BZA, MeAla-BZA, IsoNic-BZA, and Me-Pro-BZA doses and their corresponding threshold increases allowed determining threshold increasing doses by 20% (TID20 values) that elevate the threshold in drug-treated animals over the threshold in control animals. The experimentally derived TID20 values in the MEST test for Nic-BZA, 2F-Pic-BZA, Pic-BZA, Me-Ala-BZA, Iso-Nic-BZA, and Me-Pro-BZA were 7.45mg/kg, 7.72mg/kg, 8.74mg/kg, 15.11mg/kg, 21.95mg/kg and 28.06mg/kg, respectively. The studied benzylamide derivatives can be arranged with respect to their anticonvulsant potency in the MEST test as follows: Nic-BZA>2F-Pic-BZA>Pic-BZA>Me-Ala-BZA>Iso-Nic-BZA>Me-Pro-BZA.

Cannabidiol, a Cannabis sativa constituent, inhibits cocaine-induced seizures in mice: Possible role of the mTOR pathway and reduction in glutamate release

Cannabidiol (CBD), a major non-psychotomimetic constituent of Cannabis sativa, has therapeutic potential for certain psychiatric and neurological disorders. Studies in laboratory animals and limited human trials indicate that CBD has anticonvulsant and neuroprotective properties. Its effects against cocaine neurotoxicity, however, have remained unclear. Thus, the present study tested the hypothesis that CBD protects against cocaine-induced seizures and investigated the underlying mechanisms. CBD (30mg/kg) pre-treatment increased the latency and reduced the duration of cocaine (75mg/kg)-induced seizures in mice. The CB1 receptor antagonist, AM251 (1 and 3mg/kg), and the CB2 receptor antagonist, AM630 (2 and 4mg/kg), failed to reverse this protective effect, suggesting that alternative mechanisms are involved. Synaptosome studies with the hippocampus of drug-treated animals revealed that cocaine increases glutamate release, whereas CBD induces the opposite effect. Finally, the protective effect of this cannabinoid against cocaine-induced seizure was reversed by rapamycin (1 and 5mg/kg), an inhibitor of the mammalian target of rapamycin (mTOR) intracellular pathway. In conclusion, CBD protects against seizures in a model of cocaine intoxication. These effects possibly occur through activation of mTOR with subsequent reduction in glutamate release. CBD should be further investigated as a strategy for alleviating psychostimulant toxicity.