Dantrolene Significantly Improves Cerebral Blood Flow in a Rat Model of Hemorrhagic Vasospasms.

INTRODUCTION: A cerebral vasospasm (CVSP) is a potent vasoconstriction of the cerebral vasculature and the primary cause of morbidity and mortality following a subarachnoid hemorrhage. The middle cerebral artery (MCA) is commonly affected by CVSPs. Concomitant administration of dantrolene and nimodipine synergistically reduces vasospasms in aortic rings from Sprague Dawley rats. To determine if the effects observed in the systemic vasculature extend to the cerebral circulation, we investigated the effect of intravenous administration of dantrolene (2.5 mg/kg) and nimodipine (1 mg/kg and 2 mg/kg) on MCA blood flow velocity (BFV) 7 days after the induction of CVSPs. METHODS: Vasospasms were induced by bathing the left common carotid artery with autologous whole blood. Age-matched sham rats were used as controls. BFV, mean arterial pressure (MAP), and heart rate (HR) were measured with a PeriFlux 5000 Laser Doppler System, and a CODA non-invasive blood pressure system, before and after administering the drugs. Morphometric evaluations were also performed to assess vascular alterations. RESULTS: BFV was reduced by 37% with dantrolene alone (n = 6, p ≤ 0.05) and by 27% with 2 mg/kg nimodipine (n = 6, p < 0.05), while it was not affected by 1 mg/kg nimodipine. The combination of 1 mg/kg nimodipine with dantrolene, however, decreased BFV by 35% (from 435.70 ± 21.53 to 284.30 ± 23.13 perfusion units, n = 7, p ≤ 0.05). A similar reduction (31%) was obtained with dantrolene and 2 mg/kg nimodipine (from 536.00 ± 32.61 to 367.80 ± 40.93 perfusion units, n = 6, p ≤ 0.05). Neither MAP nor HR was affected by dantrolene or nimodipine alone. The combination of dantrolene with 2 mg/kg nimodipine, however, decreased MAP and increased HR. Furthermore, 7 days after the induction of vasospasms, lumen area of the left common carotid artery decreased, whereas media thickness and the wall-to-lumen ratio increased when compared to contralateral controls. The latter finding suggests that vascular remodeling was present at this stage. CONCLUSION: Altogether, our results indicate that 2.5 mg/kg dantrolene significantly reduces BFV in the MCA without altering systemic hemodynamic parameters to a similar extent than the highest dose of nimodipine or the combination of dantrolene and the lowest dose of nimodipine. Therefore, dantrolene may provide a promising alternative to lower the risk, or partially revert, CVSP.

Memantine has a nicotinic neuroprotective pathway in acute hippocampal slices after an NMDA insult.

Memantine is a non-competitive antagonist with a moderate affinity to the N-methyl-d-Aspartate (NMDA) receptor. The present study assessed memantine's neuroprotective activity using electrophysiology of ex-vivo hippocampal slices. Interestingly, a nicotinic component was necessary for memantine's neuroprotection (NP). Memantine demonstrated a bell-shaped dose-response curve of NP against NMDA. Memantine was neuroprotective at concentrations below 3 µM, but the NP declined at higher concentrations (>3 µM) when memantine inhibits the NMDA receptor. Additional evidence that memantine NP is mediated by an alternate mechanism independent of the inhibition of the NMDA receptor is supported by its ability to protect neurons when applied before or after the NMDA insult and in the presence of D(-)-2-Amino-5-phosphonopentanoic acid (APV), the standard NMDA receptor inhibitor. We found several similarities between the memantine NP mechanism and the neuroprotective nicotinic drug, the 4R cembranoid. Memantine's NP requires the release of acetylcholine, the activation of α4ß2, and is independent of MEK/MAPK signaling. Both 4R and memantine require the activation of PI3K/AKT for NP against NMDA-mediated excitotoxicity, although at different concentrations. In conclusion, our studies show memantine is neuroprotective through a nicotinic pathway, similar to the nicotinic drug 4R. This information leads to a better understanding of memantine's mechanisms of action and explains its dose-dependent effectiveness in Alzheimer's and other neurological disorders.

In Vivo Evaluation of the Acute Systemic Toxicity of (1S,2E,4R,6R,7E,11E)-Cembratriene-4,6-diol (4R) in Sprague Dawley Rats.

The tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (4R) interacts with nicotinic acetylcholine receptors, which results in neuroprotection against organophosphate toxicity, brain ischemia, and Parkinson's disease. The present study is a continuation of our previous research in which we applied a single dose of 4R 1 h before or 24 h after exposure to diisopropylfluorophosphate (DFP) (analog of the nerve agent sarin). The 4R dose robustly decreased neuroinflammation and neuronal death at both timepoints. Here, we investigated the toxicity of a single dose of 4R in male and female Sprague Dawley (SD) rats after a subcutaneous (s.c.) injection of 6, 24, or 98 mg/kg. Body weight was not affected by 4R during the 7-day observation period. No histopathologic changes in the organs were attributed to 4R. Minor hematological and blood composition variations were detected on Day 3 in the mid- and the high-dose males, but these were resolved by Day 8. At the area of the s.c. injection site, alopecia and dry skin were detected in both the 4R-treated males and females and in the female controls.

Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC).

The folate receptor alpha (FR), which is overexpressed in solid tumors including NSCLC, can be utilized for active tumor targeting to afford more effective cancer therapies. In this context, cytochrome c (Cyt c) has drawn attention to cancer research because it is non-toxic, yet, when delivered to the cytoplasm of cancer cells, can kill them by inducing apoptosis. Cyt c nanoparticles (NPs, 169 ± 9 nm) were obtained by solvent precipitation with acetonitrile, and stabilized by reversible homo-bifunctional crosslinking to accomplish a Cyt-c-based drug delivery system that combines stimulus-responsive release and active targeting. Cyt c was released under intracellular redox conditions, due to an S-S bond in the NPs linker, while NPs remained intact without any release under extracellular conditions. The NP surface was decorated with a hydrophilic folic acid-polyethylene glycol (FA-PEG) polymer for active targeting. The FA-decorated NPs specifically recognized and killed cancer cells (IC50 = 47.46 µg/mL) that overexpressed FR, but showed no toxicity against FR-negative cells. Confocal microscopy confirmed the preferential uptake and apoptosis induction of our NPs by FR-positive cancer cells. In vivo experiments using a Lewis lung carcinoma (LLC) mouse model showed visible NP accumulation within the tumor and inhibited the growth of LLC tumors.

Key genes and drug delivery systems to improve the efficiency of chemotherapy.

Cancer cells can develop resistance to anticancer drugs, thereby becoming tolerant to treatment through different mechanisms. The biological mechanisms leading to the generation of anticancer treatment resistance include alterations in transmembrane proteins, DNA damage and repair mechanisms, alterations in target molecules, and genetic responses, among others. The most common anti-cancer drugs reported to develop resistance to cancer cells include cisplatin, doxorubicin, paclitaxel, and fluorouracil. These anticancer drugs have different mechanisms of action, and specific cancer types can be affected by different genes. The development of drug resistance is a cellular response which uses differential gene expression, to enable adaptation and survival of the cell to diverse threatening environmental agents. In this review, we briefly look at the key regulatory genes, their expression, as well as the responses and regulation of cancer cells when exposed to anticancer drugs, along with the incorporation of alternative nanocarriers as treatments to overcome anticancer drug resistance.

Optimization and Characterization of Protein Nanoparticles for the Targeted and Smart Delivery of Cytochrome c to Non-Small Cell Lung Carcinoma.

The delivery of Cytochrome c (Cyt c) to the cytosol stimulates apoptosis in cells where its release from mitochondria and apoptotic induction is inhibited. We developed a drug delivery system consisting of Cyt c nanoparticles decorated with folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH) to deliver Cyt c into cancer cells and tested their targeting in the Lewis Lung Carcinoma (LLC) mouse model. Cyt c-PLGA-PEG-FA nanoparticles (NPs) of 253 ± 55 and 354 ± 11 nm were obtained by Cyt c nanoprecipitation, followed by surface decoration with the co-polymer SH-PLGA-PEG-FA. The internalization of Cyt c-PLGA-PEG-FA nanoparticles (NPs) in LLC cells was confirmed by confocal microscopy. NP caspase activation was more efficient than the NP-free formulation. Caspase activity assays showed NPs retained 88-96% Cyt c activity. The NP formulations were more effective in decreasing LLC cell viability than NP-free formulation, with IC50 49.2 to 70.1 µg/mL versus 129.5 µg/mL, respectively. Our NP system proved to be thrice as selective towards cancerous than normal cells. In vivo studies using near infrared-tagged nanoparticles show accumulation in mouse LLC tumor 5 min post-injection. In conclusion, our NP delivery system for Cyt c shows superiority over the NP-free formulation and reaches a folic acid-overexpressing tumor in an immune-competent animal model.

Accumulation of Amyloid Beta (Aß) Peptide on Blood Vessel Walls in the Damaged Brain after Transient Middle Cerebral Artery Occlusion.

It is well known that amyloid beta (Aß) peptides are generated in blood vessels, released into the brain during thrombosis, and temporarily accumulate in this organ after injury. Here we demonstrate that 24 h after transient middle cerebral artery occlusion (tMCAO), one of the standard models of focal ischemic stroke, Aß peptide accumulates in the brain, concentrating on the blood vessel walls. Because Aß oligomers are known to induce significant damage to brain cells, they act as an additional damaging factor during ischemic stroke. Considering that they have been shown to form ion channels in cells, affecting osmotic balance, we used an Aß peptide channel blocker, tromethamine (2-amino-2-(hydroxymethyl) propane-1,3-diol), to prevent this additional injury. Tromethamine injected 0.1 g/100 g body weight intraperitoneally at 5 min before tMCAO decreased water content in the damaged hemisphere, as measured by dry brain weight. Congo red staining, which binds only to Aß oligomer plaques (amyloid), showed that there was no significant presence of plaques. Therefore, we suggest that Aß peptide oligomers are responsible for some of the brain damage during stroke and that blockage of the ion channels that they form could be beneficial in treating this complex neurological syndrome.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model.

Metastatic disease still lacks effective treatments, and remains the primary cause of cancer mortality. Therefore, there is a critical need to develop better strategies to inhibit metastatic cancer. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that is activated by a myriad of cell surface receptors to promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activity of metastatic cancer cells with an IC50 of 1 µM. EHop-016 also inhibits the activity of the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, and cell migration in metastatic cancer cells. Herein, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where EHop-016 administration at 25 mg/kg body weight (BW) significantly reduced mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by UPLC MS/MS, EHop-016 was detectable in the plasma of nude mice at 17 to 23 ng/ml levels at 12 h following intraperitoneal (i.p.) administration of 10 to 25 mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis In Vivo was confirmed by immunohistochemistry of excised tumors and by In Vitro tube formation assays of endothelial cells. Moreover, EHop-016 affected cell viability by down-regulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities in metastatic cancer cells. In conclusion, EHop-016 has potential as an anticancer compound to block cancer progression via multiple Rac-directed mechanisms.

Emerging Roles for Platelets in Inflammation and Disease.

Platelets and their interaction with cells of the immune system contribute through a variety of molecular mechanisms to support hemostasis and inflammation. These simple yet essential cells exert their effects in lymphocytes, monocytes, and neutrophils, both recruiting and modulating their function after activation. Emerging evidence is starting to define the mechanisms that allow platelets to also play pivotal roles in host defense. For example, platelet cell-surface expression of toll-like receptors allows platelets to direct neutrophil activation toward extracellular trap formation and facilitate the elimination of blood pathogens. In addition to these well-known receptors, two of the most recently discovered platelet receptors, C-type lectin receptor 2 (CLEC-2), and TREM-like transcript-1 (TLT-1), have been shown to modulate hemostatic and inflammation-related roles in platelets. This review will discuss the evolution of our understanding of platelet functions from hemostasis to inflammation, and highlight novel mechanisms that platelets use to mediate hemostasis under inflammatory pressure.

EFhd2 is a novel amyloid protein associated with pathological tau in Alzheimer's disease.

EFhd2 is a conserved calcium-binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tau(P301L) mutant. This association was validated in human tauopathies, such as Alzheimer's disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl-insoluble fractions derived from human AD brains also indicated that EFhd2 co-localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co-localizes with pathological tau proteins in AD brains, confirming the co-aggregation of EFhd2 and pathological tau. Furthermore, EFhd2's coiled-coil domain mediated its self-oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2's amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau-mediated neurodegeneration.

Functional and structural analysis of the conserved EFhd2 protein.

EFhd2 is a novel protein conserved from C. elegans to H. sapiens. This novel protein was originally identified in cells of the immune and central nervous systems. However, it is most abundant in the central nervous system, where it has been found associated with pathological forms of the microtubule-associated protein tau. The physiological or pathological roles of EFhd2 are poorly understood. In this study, a functional and structural analysis was carried to characterize the molecular requirements for EFhd2's calcium binding activity. The results showed that mutations of a conserved aspartate on either EF-hand motif disrupted the calcium binding activity, indicating that these motifs work in pair as a functional calcium binding domain. Furthermore, characterization of an identified single-nucleotide polymorphisms (SNP) that introduced a missense mutation indicates the importance of a conserved phenylalanine on EFhd2 calcium binding activity. Structural analysis revealed that EFhd2 is predominantly composed of alpha helix and random coil structures and that this novel protein is thermostable. EFhd2's thermo stability depends on its N-terminus. In the absence of the N-terminus, calcium binding restored EFhd2's thermal stability. Overall, these studies contribute to our understanding on EFhd2 functional and structural properties, and introduce it into the family of canonical EF-hand domain containing proteins.

A novel calcium-binding protein is associated with tau proteins in tauopathy.

Tauopathies are a group of neurological disorders characterized by the presence of intraneuronal hyperphosphorylated and filamentous tau. Mutations in the tau gene have been found in kindred with tauopathy. The expression of the human tau mutant in transgenic mice induced neurodegeneration, indicating that tau plays a central pathological role. However, the molecular mechanism leading to tau-mediated neurodegeneration is poorly understood. To gain insights into the role that tau plays in neurodegeneration, human tau proteins were immunoprecipitated from brain lysates of the tauopathy mouse model JNPL3, which develops neurodegeneration in age-dependent manner. In the present work, a novel EF-hand domain-containing protein was found associated with tau proteins in brain lysate of 12-month-old JNPL3 mice. The association between tau proteins and the novel identified protein appears to be induced by the neurodegeneration process as these two proteins were not found associated in young JNPL3 mice. Consistently, the novel protein co-purified with the pathological sarkosyl insoluble tau in terminally ill JNPL3 mice. Calcium-binding assays demonstrated that this protein binds calcium effectively. Finally, the association between tau and the novel calcium-binding protein is conserved in human and enriched in Alzheimer's disease brain. Taken together, the identification of a novel calcium-binding protein associated with tau protein in terminally ill tauopathy mouse model and its confirmation in human brain lysate suggests that this association may play an important physiological and/or pathological role.