We examined serial changes of diffusion- (DWI) and T2-weighted (T2WI) magnetic resonance images 30 minutes to 3 hours after intraluminal suture occlusion of the middle cerebral artery (MCA) in eight rats and after sham occlusion in four. We correlated the abnormal areas on DWI and T2WI with postmortem areas of infarction determined by 2,3,5-triphenyltetrazolium chloride (TTC), 24 hours after the operation. The 30-minute DWI in each MCA-occluded rat demonstrated increased signal intensity in the ipsilateral MCA territory, while T2WI showed no changes. At 3 hours, the ipsilateral DWI signal intensity increased further and the area of abnormality slightly increased. In some animals, the 3-hour T2WI disclosed an area of hyperintensity significantly smaller than that seen on the 30-minute DWI. TTC staining demonstrated an extensive MCA infarction in all rats with permanent MCA occlusion, confirmed by hematoxylin and eosin staining. The percent infarcted area of coronal brain sections, as determined by TTC staining, correlated significantly with areas on similar DWI sections at both 30 minutes and 3 hours. Sham-occluded control animals did not display any changes on DWI, T2WI, or TTC staining. The present study suggests that DWI is a very sensitive modality for detecting early ischemic brain injury, being highly correlated with post-mortem area of infarction, and may be useful to assess pharmacologic intervention.
The concept of repairing the brain with growth factors has been pursued for many years in a variety of neurodegenerative diseases including primarily Parkinson's disease (PD) using glial cell line-derived neurotrophic factor (GDNF). This neurotrophic factor was discovered in 1993 and shown to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. These observations led to a series of clinical trials in PD patients including using infusions or gene delivery of GDNF or the related growth factor, neurturin (NRTN). Initial studies, some of which were open label, suggested that this approach could be of value in PD when the agent was injected into the putamen rather than the cerebral ventricles. In subsequent double-blind, placebo-controlled trials, the most recent reporting in 2019, treatment with GDNF did not achieve its primary end point. As a result, there has been uncertainty as to whether GDNF (and by extrapolation, related GDNF family neurotrophic factors) has merit in the future treatment of PD. To critically appraise the existing work and its future, a special workshop was held to discuss and debate this issue. This paper is a summary of that meeting with recommendations on whether there is a future for this therapeutic approach and also what any future PD trial involving GDNF and other GDNF family neurotrophic factors should consider in its design.
Foetal bovine serum influence on in vitro extracellular vesicle analyses INTRODUCTION Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles most notably from either endosomal (i.e., exosomes) or plasma membrane origins (i.e., microvesicles/ectosomes) and released from nearly all mammalian cells (Colombo et al., 2014). An interest in EV research has increased over the past decade, in part due to their participation in complex intercellular communication (Roy et al., 2018). Though EVs are abundant in blood and other biofluids, the investigation of in vitro-derived EVs provides a critical tool for understanding various mechanisms associated with their biogenesis, molecular composition, packaging of specific payloads, and cellular trafficking. Once released, EVs traffic to target cells where they may be taken up to release their payloads via specific mechanisms, and thereby exert their physiological influence (Colombo et al., 2014; Kowal et al., 2014). Although engineered micelles and liposomes have previously been utilized as lipid nanocarriers (Fiandaca & S., 2013; Fiandaca et al., 2011) for many therapeutic applications, EVs have garnered recent interest as drug delivery vehicles (Elsharkasy et al., 2020). Currently, there exist vastly heterogeneous cell culture conditions for EV production and isolation (Consortium, 2017). Therefore, there is a current need to define more standard cell culture conditions for investigating EVs that may accelerate the translation of therapeutic clinical-grade EVs (Lener et al., 2015; Lötvall et al., 2014; Théry et al., 2018). Herein, we present a mini-review on recent investigations reporting the influence of foetal bovine serum (FBS)-supplemented media formulations on cultured cell physiology, EV production/release, and its contaminating presence of vesicular and non-vesicular particles. Additionally, we describe potential solutions and provide recommendations to aid in vitro EV investigators. CELL CULTURE CONDITIONS FOR EV INVESTIGATIONS: SERUM USAGE AND CONCERNS An international survey observed 83% of International Society for Extracellular Vesicles (ISEV) respondents utilize conditioned cell culture media as their starting material (Gardiner et al., 2016). FBS is a common additive in cell culture and 52% of ISEV respondents utilize serum-containing media for downstream EV analyses, with 59% and 57% of those respondents performing in vitro and in vivo functional studies, respectively (Gardiner et al., 2016). Serum usage, in part due to its ill-defined composition, provides a variety of contaminating particles (e.g., EVs, lipoproteins, and protein complexes, which differ in their physical properties, yet also have similar size, density, and/or RNA components) that confound these investigative results. FBS SUPPLEMENTATION AND GENERAL CONCERNS The growth factors and other constituents within FBS appear to provide a nourishing ecosystem for many cultured cells (Bettger & Mckeehan, 1986). Despite this nourishing milieu, the presence of FBS in culture has raised spec...
The complex multifactorial nature of polygenic Alzheimer's disease (AD) presents significant challenges for drug development. AD pathophysiology is progressing in a non-linear dynamic fashion across multiple systems levels - from molecules to organ systems - and through adaptation, to compensation, and decompensation to systems failure. Adaptation and compensation maintain homeostasis: a dynamic equilibrium resulting from the dynamic non-linear interaction between genome, epigenome, and environment. An individual vulnerability to stressors exists on the basis of individual triggers, drivers, and thresholds accounting for the initiation and failure of adaptive and compensatory responses. Consequently, the distinct pattern of AD pathophysiology in space and time must be investigated on the basis of the individual biological makeup. This requires the implementation of systems biology and neurophysiology to facilitate Precision Medicine (PM) and Precision Pharmacology (PP). The regulation of several processes at multiple levels of complexity from gene expression to cellular cycle to tissue repair and system-wide network activation has different time delays (temporal scale) according to the affected systems (spatial scale). The initial failure might originate and occur at every level potentially affecting the whole dynamic interrelated systems within an organism. Unraveling the spatial and temporal dynamics of non-linear pathophysiological mechanisms across the continuum of hierarchical self-organized systems levels and from systems homeostasis to systems failure is key to understand AD. Measuring and, possibly, controlling space- and time-scaled adaptive and compensatory responses occurring during AD will represent a crucial step to achieve the capacity to substantially modify the disease course and progression at the best suitable timepoints, thus counteracting disrupting critical pathophysiological inputs. This approach will provide the conceptual basis for effective disease-modifying pathway-based targeted therapies. PP is based on an exploratory and integrative strategy to complex diseases such as brain proteinopathies including AD, aimed at identifying simultaneous aberrant molecular pathways and predicting their temporal impact on the systems levels. The depiction of pathway-based molecular signatures of complex diseases contributes to the accurate and mechanistic stratification of distinct subcohorts of individuals at the earliest compensatory stage when treatment intervention may reverse, stop, or delay the disease. In addition, individualized drug selection may optimize treatment safety by decreasing risk and amplitude of side effects and adverse reactions. From a methodological point of view, comprehensive "omics"-based biomarkers will guide the exploration of spatio-temporal systems-wide morpho-functional shifts along the continuum of AD pathophysiology, from adaptation to irreversible failure. The Alzheimer Precision Medicine Initiative (APMI) and the APMI cohort program (APMI-CP) have commenced to facili...
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