Effects of peroxynitrite on myelin basic protein expression and traffic in cultured embryonic chick brain neurons

Demyelination associated with diseases such as multiple sclerosis may be the result of oxidative damage to the myelin sheath surrounding neurons.  One putative mechanism for this damage is peroxynitrite-induced death or dysfunction of oligodendrocytes through an unknown mechanism.  In this study, cultured cerebral and cerebellar neurons from 14 day-old chick embryos were subjected to peroxynitrite to determine if this stress had a direct effect on the expression of myelin basic protein (MBP) in the central nervous system.  Immunostaining was used to visualize the MBP expression in cells treated with 0 (control), 0.1 mM, and 1 mM peroxynitrite for 24 hours.  MBP staining was visualized using fluorescence microscopy, quantified and measured to determine the amount of MBP expressed in individual cells. Results showed that peroxynitrite affected the cells from the cerebrum and cerebellum differently,  The cerebral cells treated with 1 mM of peroxynitrite up-regulated MBP expression and increased trafficking while the cells treated with 0.1 mM only increased protein trafficking.  The treated cells from the cerebellum expressed less protein and the protein trafficking with the cells appeared to be interrupted.  These results suggest a differential effect of oxidative stress on neurons from different regions of the chick brain.  Thus, the effects of oxidative stress vary in the different regions of the brain; these differences may account for the etiology of diseases such as multiple sclerosis, and has implications for its treatment and prevention.

A model of indirect nerve damage: implications for trigeminal nerve degradation associated with exposure to stressors associated with oral trauma and disease

Instances of dental pain are very prevalent and have a wide array of causes, ranging from bacterial infections to sequelae of surgical procedures.  Such damage to tissue results in pain.  Much remains unknown about the mechanisms of damage that cause pain, particularly in the nervous system.  Therefore, the purpose of this study is to develop an in vitro model that can be used to understand mechanisms of nerve damage.  Oxidative stress was selected as a method of damage to cultured chick brain neurons because oxidative stress is known to cause cellular degradation, and it is the causative agent of many diseases.  Cultured cells were analyzed using immunocytochemistry techniques after exposed to either low or high treatments of oxidative stress the hope of understanding mechanisms behind the damage.  Neurofilament, cadherin, and SNAP25 expression was measured for the treatments.  The amount of protein, staining density, percent cell stained, and total area of the cell stained were measured for each treatment group and for each protein, and these values were normalized to the controls.  The results from these experiments showed that oxidative stress causes changes in cellular morphology and widespread cellular degradation.  The results also suggest that other cells (i.e. glial cells) are responsible for coping with various stressors, such as oxidative stress.

Investigating the Long-Term Cellular Effects of Hyperglycemia on PKC Expression in Developing Chick Embryos

This research project examines the cellular effects of hyperglycemia in chick astrocytes and endothelial cells. Diabetes is a debilitating condition that affects not only maternal health but potentially fetal development and well-being as well. Among the many cellular effects of diabetes is the dysfunctional expression and/or regulation of cellular signaling molecules such as protein kinase C (PKC). This research project therefore tests this notion by artificially inducing hyperglycemia in chick embryos, and determining the effects on PKC expression in embryonic astrocytes and endothelial cells. Hyperglycemia appeared to induce acute, but not chronic, effects in astrocytes but not in endothelial cells. Results from this study will hopefully enable a better understanding of the mechanisms underlying various conditions experienced by diabetics that are caused primarily by hyperglycemia. This may have implications regarding the cellular effects of hyperglycemia on the progeny of diabetic mothers.

The role of plasma membrane calcium channels in odorant-elicited calcium signaling of cultured human olfactory cells

Calcium, a ubiquitous intracellular messenger, drives depolarization reactions and generates action potentials in olfactory receptor neurons (ORNs) in a manner similar to those in other neurons. ORNs respond to odorants with changes in intracellular calcium concentration ([Ca2+]i). Odorant-elicited calcium fluxes in human olfactory cultured cells (HOCCs) display similar characteristics to those found in mature human olfactory neurons, so HOCCs will be tested in this experiment. Characterization of the mechanism behind calcium effluxes is important, since healthy and diseased human ORNs employ odorant-elicited [Ca2+]i effluxes. The plasma membrane calcium ATP-ase (PMCA) is a protein in the plasma membrane of ORNs known to decrease [Ca2+]i following odorant stimulation. The understanding of the mechanism behind [Ca2+]i decreases, particularly PMCA function, may further elucidate studies on physiology of olfaction, neurogenesis, and neurodevelopmental diseases. Cell responses to odorants were recorded with live cell imaging techniques and the PMCA expression was studied with immunocytochemistry. Sodium orthovanadate reversibly inhibited the PMCA in order to abolish odorant-elicited [Ca2+]i fluxes in cells. The same cells responded to two different odorants, suggesting the possibility of dual second messenger systems in HOCCs. This study also suggests the differential effect of the odorants on the PMCA; odorant solutions, which trigger specific second messengers, cAMP and IP3, affect PMCA function.

Investigating the Effects of Embryonic Sensory Experience on Fetal Development

The objective was to examine the timecourse of chick olfactory system development using anatomical and behavioral techniques.  Chick eggs take 21 days to develop and hatch.  The olfactory system begins to develop at embryonic day eight (E8) and is functional at hatching, but the timecourse of this development is poorly understood.  This timeline of development can be examined both at the cellular level by, observing protein expression patterns in the chick brain, and at the behavioral level, by studying chick odor imprinting.  Two different odorants, amyl acetate and phenylethyl alcohol were added at varying timepoints to developing chicks in ovo.  The expression pattern of the immediate early gene cFos, resulting from the odorant’s stimulation, was visualized to determine if the neural circuitry in the chick brain is developed in the embryo.  This development was also measured behaviorally: when exposed to odors in ovo, newly-hatched chicks will potentially be attracted to these familiar odors.  Odor imprinting in newborn chicks exposed to the two test odorants was studied using a Y-maze to determine if a preference for familiar odorants exists.  The results suggest that amyl acetate results in maximal cFos expression beginning around E16 and increasing until hatching, but that phenylethyl alcohol exposure causes only baseline activity, similar to chicks exposed to no odor. This neuronal activity is not necessarily indicative of imprinting, as conflicting and inconsistent behavioral results illustrate.

Hypoxic and hyperoxic stress-induced hypertrophy in cultured chick fetal cardiac myocytes

The adult heart responds to increased contraction demands by hypertrophy, or enlargement, of cardiac myocytes. Hypertrophy can occur in response to both physiological factors (generally resulting in hyperoxic conditions and cause adaptation), or pathological factors (typically resulting in hypoxic conditions which cause heart failure). The difference in the outcomes produced by pathologically- versus physiologically- induced hypertrophy suggests that the cellular signaling pathways or conditions of myocytes may be different at the cellular level. The structural and functional changes in myocytes resulting from hyperoxia (simulated using hydrogen peroxide) and hypoxia (using oxygen deprivation) were tested on fetal chick cardiac myocytes grown in vitro. Isolated myocytes grown for 2 days were exposed to stressors, then grown for an additional 1 to 6 days. Structural changes were measured using immunostaining for sarcomeric actin or MyoD (a transcription factor that regulates myosin production). Functional changes were assessed using immunostaining for calcium-calmodulin kinase (CaMK) or by measuring intracellular calcium fluxes using live cell fluorescence imaging. Hypo- or hyperoxic stress resulted in an up-regulation of actin and MyoD expression, indicating hypertrophy. However, these structural rearrangements occurring are not in themselves detrimental. Similarly, voltage-gated channels and the regulation of myosin activation by CaMK are unchanged by hyper- or hypoxic conditions. The changes in transcription regulation causing such structural changes may affect components of ligand-activated signaling pathways, and subsequent stimulations of myoctes may result in a depletion of calcium stores in the sarcoplasmic reticulum. Although this study does not indicate whether the observed changes to signaling cascades or calcium stores are beneficial or detrimental, it does suggest that changes in signaling pathways, rather than structural organization, may cause the fatal outcomes associated with pathologically-induced hypertrophy.