The use of injectable hydrogel scaffolds for encapsulating stem cells has been studied as an effective approach for cell delivery for regenerative medicine. The objective of this study is to examine the effect of hydrogel stiffness on the morphology of encapsulated mesenchymal stem cells (MSCs). Mouse MSCs were encapsulated within gelatin microspheres using a water-in-oil phase bulk emulsification method and the extent of gel stiffness was controlled by changing crosslinking time (5, 30 and 60 minutes) of gelatin matrix. The change in MSC morphology and spreading within three-dimensional microgel space was quantified by measuring cell circularity and spreading. Results shows that MSCs encapsulated within low stiffness gels exhibited high circularity compared with those within high stiffness gels.

Two dimensional (2D) tissue samples have been successfully grown using usual cell culture techniques. However, in order for cell cultures to be representative of three dimensional (3D) tissues it is necessary that they are grown in a spatial environment. Elastomers can be used for this purpose. Elastomers are a form of polymer that have elastic properties. There are multiple properties of elastomers that can be adjusted in order to provide a suitable environment for growing different cell types. The elastomer used in this project is liquid crystalline based. Its main chain is constructed of ε-caprolactam and side chains of cholesterol which gives the elastomer biocompatible properties. The use of liquid crystalline elastomers (LCE) provides a material that is anisotropic which is important for neuronal cell growth. This LCE is porous in its structure which creates a 3D matrix for cell growth making it a good candidate for supporting spatial growth of cellular structures. Elastomer pore size was measured using scanning electron microscopy (SEM) and adjusted in order to better support the growth of neuroblastoma cells. In this project human neuroblastoma cells (SHSY-5Y) were seeded within the LCE. Cells were allowed to grow for an extended period of time and imaged using confocal microscopy to better understand how the cells are growing within the LCE. The purpose of this project is to create the optimal conditions for the neuroblastoma cells by manipulating the LCE properties to control and enhance neuroblastoma cell growth.

We have previously shown that acylethanolamide oleoylethanolamide (OEA) and its metabolically stable analog AM3102 induce apoptosis in OV2008 ovarian adenocarcinoma cells independent of PPAR-α signaling pathway. This cytotoxicity is reversed in the presence of α-tocopherol, indicative of reactive oxygen species involvement in cell death. We have also shown that palmityl trifluoromethyl ketone (PTK), independent of its inhibitory effect on phospholipase A2, enhances the toxicity of OEA (ASCB 2012, Abstract #888). The enzyme neutral cholesterol ester hydrolase 1 (NCEH1) has also been shown to be a target of trifluoromethylketones (Nat Biotechnol 21:687, 2003). Using Chlorpyrifos-Oxon (CPO), a potent inhibitor of the enzyme NCEH1, we explored the relationship between OEA and NCEH1 in the OV2008 cell line. Cytotoxicity was observed in response to OEA (IC50: 14-21 µM) and CPO (IC50: 48-50 µM). When the compounds were administered together, cytotoxicity was enhanced (OEA IC50: 8-9 µM; CPO IC50: 15-19 µM) with evidence of weak synergism. However, Western blot analysis of OEA and CPO treated cells indicated no change in NCEH1 expression. Live cell labeling with dihydroethidium showed an increase superoxide by about 10% in the presence of OEA. CPO itself did not increase ROS in cells. Live cell fluorescence microscopy using MitoSOX Red also revealed an increase in superoxide in the mitochondria in the presence of OEA. The results of our study suggest that one mechanism by which OEA induces cytotoxicity in OV2008 cells is via superoxide generation. It is possible that OEA-mediated increase in ROS makes the OV2008 cells further susceptible to CPO.

Obesity is a growing concern for the nation. Increased interest has focused on the gastrointestinal tract (GI) properties that may impact nutrient absorption. To compare potential differences in GI morphology, we examined the histological characteristics of the digestive tracts of lean and obese rats. High-capacity runners (HCR) are lean, whereas low-capacity runners (LCR) are obese, yet HCR eat more than LCR. Intestines were collected from 12 HCR and 12 LCR adult male rats. After fixation with 10% formalin, a sample from the jejunum was paraffin embedded and sectioned cross-sectionally on a microtome, followed by hematoxylin and eosin (H&E) staining. Using microscopy, we were able to quantify aspects of jejunum such as villi length, diameter, and surface area, and number of crypts per unit area. Preliminary findings showed thinner mucosal layers in the LCR than in the HCR. Longer villi were seen in the HCR than LCR jejunum; however, the number of villi was greater in the LCR. Ongoing analyses will calculate the surface area of villi. The total number of villi and surface area could differentially impact nutrient absorption.

Multiple sclerosis (MS) is characterized by the demyelination of the central nervous system, which causes progressive neurological disability. Mitochondrial defects in the MS brain, including decreased levels of the neuronal mitochondrial metabolite N-acetylaspartate (NAA), have previously been identified in our lab. We suspect that demyelination in MS is due in part to depletion of NAA through two possible mechanisms. In one mechanism, oligodendrocytes break down NAA into acetate and aspartate which can enter the tricarboxylic acid (TCA) cycle altering oligodendrocyte metabolism. Changes in levels of TCA intermediates have been shown to regulate the activity of histone H3 demethylase enzymes. Preliminary data suggests that neuronal release of NAA enhances myelination by oligodendrocytes through altering trimethylation of histone H3 on lysine 4 (H3K4me3), which regulates genes associated with myelin lipid synthesis. In mechanism two, additional acetyl-CoA resulting from the catabolism of NAA may serve as a substrate for the synthesis of myelin lipids. Here, we will investigate how defects in myelin can be caused by changes in NAA by studying primary cultures of oligodendrocytes and mice that are deficient for NAA (NAT8L-KO). TCA cycle intermediates will be quantitated by mass spectrometry. Western blotting will be used to determine H3K4me3 levels. Quantitative RT-PCR and immunohistochemistry will be used to measure changes in oligodendrocyte differentiation markers. Mass spectrometry, thin layer chromatography, and magnetic resonance imaging (MRI) will be used to measure changes in myelin lipid levels in vivo in the NAA deficient NAT8L-KO mice.

Decellularized extracellular matrix components isolated from animal sources have shown much clinical promise in the treatment of several wound care indications including a variety of dermal ulcerations (diabetic, pressure, venous, e.g.), full-thickness trauma wounds affecting the skin and skeletal muscle, and post-operative surgical wounds. The purpose of this study was to evaluate the biocompatibility of three sources of decellularized matrix wound care products using a novel tissue engineered dermal cellular proliferation assay. Deceullarized basement matrices derived from porcine small intestinal submucosa (pSIS), porcine peritoneal membrane (pPM), and ovine forestomach (oFS), respectively, were tested for their ability to support the engraftment and proliferation of human neonatal dermal fibroblasts. Cells were seeded onto each respective membrane using a custom, non-adherent, cell culture system and cultured for 2 weeks. At 3, 5, 7, and 14 days time points cultures were assayed for proliferation and matrix integration using the PrestoBlue cell viability assay and fluorescent confocal laser scanning microscopy, respectively. Human dermal fibroblast attachment to all three materials was relatively low compared to conventional two-dimensional culture systems. However, pSIS displayed a consistently, but not statistically significant, greater level of fluorescence intensity in the cell viability assay, and qualitatively displayed a superior ability to support engraftment of dermal fibroblasts in comparison to pPM and oFS matrices. These data indicate that pSIS supports the attachment and growth of dermal cells in vitro, which may underscore the success of this material in pre-clinical and clinical wound healing studies.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by deposition of amyloid plaques in the brain. Amyloid-β (AB) fragments (1-42) are the main components of plaques and are formed by the sequential cleavage of amyloid precursor protein (APP) by β-secretase and ϒ-secretase. Another enzyme, α-secretase, cleaves within the amyloid-β sequence preventing it from aggregating. Previous studies have shown that peripheral administration of a non-aggregating form of the metabolic hormone amylin, an amyloid with similar aggregating properties, improves cognitive function in mouse models of AD and reduces amyloid-β plaque levels. However, how this hormone regulates amyloid-β levels is not known. The purpose of our research was to study the effects of amylin administration on the levels of pro-aggregation soluble amyloid-β fragments (1-42) the expression of α- and β-secretases and the C-terminal fragments that result from enzyme cleavage in the APP/PS1 mouse model of AD. Our data indicate that amylin treatment reduced formic acid-soluble Amyloid-β 1-42 fragments (aggregates). These reductions were paralleled by general upregulation of cleavage enzymes. Taken together, our data demonstrate that amylin may be a potential new therapeutic target for Alzheimer’s disease through its ability to regulate amyloid-beta processing and aggregation.

Keywords: Amylin, Alzheimer’s disease, amyloid-β, APP, α-secretase, β-secretase.

Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases today. Oxidative stress has been proposed as a key pathological feature in AD. Previous studies in our laboratory show that administration of peptide hormone amylin in mouse models of AD improves cognitive function and reduces oxidative stress damage to membranes. However, how amylin provides neuroprotection against oxidative stress damage is not known. To address this question we treated APP/PS1 mice, a mouse model of AD, with amylin for two months and also treated primary hippocampal neurons from these mice with various doses of amylin. From these treatments, we determined the levels of various oxidative enzymes and stress signaling proteins known to be altered in the AD brain and important in the regulation of oxidative stress damage. Our data shows that, in vitro, amylin is able to dose-dependently protect primary hippocampal neurons from oxidative insults (H₂O₂). Further, in vivo, a two month treatment with amylin is able to reduce AD-associated upregulation of the Heme-oxygenase-1 (HO-1), an enzyme known to be activated in response to high levels of oxidative stress. Importantly, these reductions in HO-1 paralleled upregulations of MnSOD expression, a classic endogenous antioxidant enzyme, and reductions in levels of phosphorylated JNK, known to be increased during cellular stress. Taken together our data demonstrates that amylin may be improving CNS function through its ability to reduce oxidative stress damage, both by upregulating endogenous protective systems and preventing the activation of cellular cascades.

Chronic inflammatory responses have adverse effects on the memory retaining areas of the brain, such as hippocampus and neocortex. Understanding how inflammation influences hippocampal neurobiology during aging is of significant importance to the study of normal senescence and neurodegenerative diseases, and knowledge of the various factors involved in cognitive impairment may lead to a better understanding of what causes the adverse effects to memory and what treatments could be used to fight memory loss. Microglia are an important line of defense against various brain insults, and as such are a key component of the inflammatory cascade. By examining the brains of cognitively assessed canines, we will determine the inflammatory response in the hippocampus using microglial immunohistochemistry as a marker for immune function. Immunostaining was performed on hippocampal sections of both normal and behaviorally impaired canines. Quantitative analyses will be conducted using unbiased stereological methods (Stereologer, St. Petersburg, FL). We hypothesize that we will observe elevated numbers of activated microglia in the hippocampi of cognitively impaired animals than in the hippocampi of aged, non-impaired canines, and that the observed increases will correlate with behavioral performance. Our results in the canine may provide a more relevant model of natural aging and disease processes in humans than do genetically modified mice, and help elucidate future targets in the inflammatory cascade upon which to focus treatments that may benefit patients with age-related cognitive impairment, Alzheimer’s Disease, and dementias.