Differential Collective Cell Migratory Behaviors Modulated by Phospholipid Nanocarriers.

Phospholipid nanocarriers have been widely explored for theranostic and nanomedicine applications. These amphiphilic nanocarriers possess outstanding cargo encapsulation efficiency, high water dispersibility, and excellent biocompatibility, which render them promising for drug delivery and bioimaging applications. While the biological applications of phospholipid nanocarriers have been well documented, the fundamental aspects of the phospholipid-cell interactions beyond cytotoxicity have been less investigated. In particular, the effect of phospholipid nanocarriers on collective cell behaviors has not been elucidated. Herein, we evaluate the interactions of phospholipid nanocarriers possessing different functional groups and sizes with normal and cancerous immortalized breast epithelial cell sheets with varying metastatic potential. Specifically, we examine the impact of nanocarrier treatments on the collective migratory dynamics of these cell sheets. We observe that phospholipid nanocarriers induce differential collective cell migratory behaviors, where the migration speed of normal and cancerous breast epithelial cell sheets is retarded and accelerated, respectively. To a certain extent, the nanocarriers are able to alter the migration trajectory of the cancerous breast epithelial cells. Furthermore, phospholipid nanocarriers could modulate the stiffness of the nuclei, cytoplasm, and cell-cell junctions of the breast epithelial cell sheets, remodel their actin filament arrangement, and regulate the expressions of the actin-related proteins. We anticipate that this work will further shed light on nanomaterial-cell interactions and provide guidelines for rational and safer designs and applications of phospholipid nanocarriers for cancer theranostics and nanomedicine.

Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke.

Stroke is the second leading cause of death in the world and a major cause of long-term disability. Recent evidence has provided insight into a newly described inflammatory mechanism that contributes to neuronal and glial cell death, and impaired neurological outcome following ischemic stroke - a form of sterile inflammation involving innate immune complexes termed inflammasomes. It has been established that inflammasome activation following ischemic stroke contributes to neuronal cell death, but little is known about inflammasome function and cell death in activated microglial cells following cerebral ischemia. Microglia are considered the resident immune cells that function as the primary immune defense in the brain. This study has comprehensively investigated the expression and activation of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in isolates of microglial cells subjected to simulated ischemic conditions and in the brain following ischemic stroke. Immunoblot analysis from culture media indicated microglial cells release inflammasome components and inflammasome activation-dependent pro-inflammatory cytokines following ischemic conditions. In addition, a functional role for NLRC4 inflammasomes was determined using siRNA knockdown of NLRC4 and pharmacological inhibitors of caspase-1 and -8 to target apoptotic and pyroptotic cell death in BV2 microglial cells under ischemic conditions. In summary, the present study provides evidence that the NLRC4 inflammasome complex mediates the inflammatory response, as well as apoptotic and pyroptotic cell death in microglial cells under in vitro and in vivo ischemic conditions.

SIRT2 Inhibition Confers Neuroprotection by Downregulation of FOXO3a and MAPK Signaling Pathways in Ischemic Stroke.

Sirtuin 2 (SIRT2) is a family member of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases which appears to have detrimental roles in an array of neurological disorders such as Parkinson's disease (PD) and Huntington's disease (HD). In light of the recently emerging roles of sirtuins in normal physiology and pathological conditions such as ischemic stroke, we investigated the role of SIRT2 in ischemic stroke-induced neuronal cell death. Primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) under in vitro ischemic conditions, and subsequently tested for the efficacy of SIRT2 inhibitors AK1 and AGK2 in attenuating apoptotic cell death caused by OGD. We have also evaluated the effect of SIRT2 inhibition in C57BL/6 mice subjected to 1 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion, which is a model for ischemic reperfusion injury in vivo. Significant reductions in apoptotic cell death were noted in neurons treated with AK1 or AGK2, as evidenced by reduced cleaved caspase-3 and other apoptotic markers such as Bim and Bad. In addition, downregulation of phosphorylated-AKT and FOXO3a proteins of the AKT/FOXO3a pathway, as well as a marked reduction of JNK activity and its downstream target c-Jun, were also observed. When tested in animals subjected to MCAO, the neuroprotective effects of AGK2 in vivo were evidenced by a substantial reduction in ipsilateral infarct area and a significant improvement in neurological outcomes. A similar reduction in the levels of pro-apoptotic proteins in the infarct tissue, as well as downregulation of AKT/FOXO3a and JNK pathway, were also noted. In summary, the current study demonstrated the neuroprotective effects of SIRT2 inhibition in ischemic stroke, and identified the downregulation of AKT/FOXO3a and MAPK pathways as intermediary mechanisms which may contribute to the reduction in apoptotic cell death by SIRT2 inhibition.

Transcriptome analysis reveals intermittent fasting-induced genetic changes in ischemic stroke.

Genetic changes due to dietary intervention in the form of either calorie restriction (CR) or intermittent fasting (IF) are not reported in detail until now. However, it is well established that both CR and IF extend the lifespan and protect against neurodegenerative diseases and stroke. The current research aims were first to describe the transcriptomic changes in brains of IF mice and, second, to determine whether IF induces extensive transcriptomic changes following ischemic stroke to protect the brain from injury. Mice were randomly assigned to ad libitum feeding (AL), 12 (IF12) or 16 (IF16) h daily fasting. Each diet group was then subjected to sham surgery or middle cerebral artery occlusion and consecutive reperfusion. Mid-coronal sections of ipsilateral cerebral tissue were harvested at the end of the 1 h ischemic period or at 3, 12, 24 or 72 h of reperfusion, and genome-wide mRNA expression was quantified by RNA sequencing. The cerebral transcriptome of mice in AL group exhibited robust, sustained up-regulation of detrimental genetic pathways under ischemic stroke, but activation of these pathways was suppressed in IF16 group. Interestingly, the cerebral transcriptome of AL mice was largely unchanged during the 1 h of ischemia, whereas mice in IF16 group exhibited extensive up-regulation of genetic pathways involved in neuroplasticity and down-regulation of protein synthesis. Our data provide a genetic molecular framework for understanding how IF protects brain cells against damage caused by ischemic stroke, and reveal cellular signaling and bioenergetic pathways to target in the development of clinical interventions.

Emerging Roles of Sirtuins in Ischemic Stroke.

Ischemic stroke is one of the leading causes of death worldwide. It is characterized by a sudden disruption of blood flow to the brain causing cell death and damage, which will lead to neurological impairments. In the current state, only one drug is approved to be used in clinical setting and new therapies that confer ischemic neuroprotection are desperately needed. Several targets and pathways have been indicated to be neuroprotective in ischemic stroke, among which the sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases has emerged as important modulators of several processes in the normal physiology and pathological conditions such as stroke. Recent studies have identified some members of the sirtuin family are able to ameliorate the devastating consequences of ischemic stroke by conferring neuroprotection by means of reducing neuronal cell death, oxidative stress, and neuroinflammation whereas some sirtuins are found to be detrimental in the pathophysiology of ischemic stroke. This review summarizes implications of sirtuins in ischemic stroke and the experimental evidences that demonstrate the potential of sirtuin modulators as neuroprotective therapy for ischemic stroke.

Disease-modifying effect of intravenous immunoglobulin in an experimental model of epilepsy.

Novel therapies that prevent or modify the development of epilepsy following an initiating brain insult could significantly reduce the burden of this disease. In light of evidence that immune mechanisms play an important role in generating and maintaining the epileptic condition, we evaluated the effect of a well-established immunomodulatory treatment, intravenous immunoglobulin (IVIg), on the development of epilepsy in an experimental model of epileptogenesis. In separate experiments, IVIg was administered either before (pre-treatment) or after (post-treatment) the onset of pilocarpine status epilepticus (SE). Our results show that both pre- and post-treatment with IVIg attenuated acute inflammation in the SE model. Specifically, IVIg reduced local activation of glial cells, complement system activation, and blood-brain barrier damage (BBB), which are all thought to play important roles in the development of epilepsy. Importantly, post-treatment with IVIg was also found to reduce the frequency and duration of subsequent spontaneous recurrent seizures as detected by chronic video-electroencephalographic (video-EEG) recordings. This finding supports a novel application for IVIg, specifically its repurposing as a disease-modifying therapy in epilepsy.

Visualization of mouse barrel cortex using ex-vivo track density imaging.

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo T1- and T2-weighted imaging and gradient echo T1/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3mm)(3) block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system.

MRI changes and complement activation correlate with epileptogenicity in a mouse model of temporal lobe epilepsy.

The complex pathogenesis of temporal lobe epilepsy includes neuronal and glial pathology, synaptic reorganization, and an immune response. However, the spatio-temporal pattern of structural changes in the brain that provide a substrate for seizure generation and modulate the seizure phenotype is yet to be completely elucidated. We used quantitative magnetic resonance imaging (MRI) to study structural changes triggered by status epilepticus (SE) and their association with epileptogenesis and with activation of complement component 3 (C3). SE was induced by injection of pilocarpine in CD1 mice. Quantitative diffusion-weighted imaging and T2 relaxometry was performed using a 16.4-Tesla MRI scanner at 3 h and 1, 2, 7, 14, 28, 35, and 49 days post-SE. Following longitudinal MRI examinations, spontaneous recurrent seizures and interictal spikes were quantified using continuous video-EEG monitoring. Immunohistochemical analysis of C3 expression was performed at 48 h, 7 days, and 4 months post-SE. MRI changes were dynamic, reflecting different outcomes in relation to the development of epilepsy. Apparent diffusion coefficient changes in the hippocampus at 7 days post-SE correlated with the severity of the evolving epilepsy. C3 activation was found in all stages of epileptogenesis within the areas with significant MRI changes and correlated with the severity of epileptic condition.

Inhibition of histone deacetylase in utero causes sociability deficits in postnatal mice.

Exposure to sodium valproate (VPA) in utero increases the risk of language impairment and a diagnosis of autism spectrum disorder (ASD). Mice exposed to VPA while in utero have also shown postnatal social deficits. Inhibition of histone deacetylase (HDAC) is one of VPA's many biological effects. The main objective of this study was to test the hypothesis that HDAC inhibition causes these behavioral outcomes following prenatal VPA exposure in mice. We exposed embryonic mice to VPA, the HDAC inhibitor trichostatin A (TSA), or vehicle controls. TSA (1mg/kg) inhibited HDAC in embryonic tissue at a level comparable to 600 mg/kg VPA, resulting in significant increases in histone H3 and H4 acetylation, and histone H3 lysine 4 tri-methylation. Postnatally, decreases in ultrasonic vocalization, olfactory motivation and sociability were observed in TSA and VPA-exposed pups. Treated mice exhibited elevated digging and grooming suggestive of mild restrictive and repetitive behaviors. Olfactory social preference, social novelty and habituation were normal. Together, these data indicate that embryonic HDAC inhibition alone can cause abnormal social behaviors in mice. This result serves as a molecular understanding of infant outcomes following mild VPA exposure in utero.