Bryan G. Helwig

Matrix cells from Wharton's jelly form neurons and glia.

We have identified an easily attainable source of primitive, potentially multipotent stem cells from Whartons jelly, the matrix of umbilical cord. Whartons jelly cells have been propagated in culture for more than 80 population doublings. Several markers for stem cells, including c‐kit (CD117), and telomerase activity are expressed in these cells. Treatment with basic fibroblast growth factor overnight and low‐serum media plus butylated hydroxyanisole and dimethylsulfoxide induced Whartons jelly cells to express a neural phenotype. Within several hours of this treatment, Whartons jelly cells developed rounded cell bodies with multiple neurite‐like extensions, similar to the morphology of neural stem cells. Neuron‐specific enolase (NSE), a neural stem cell marker, was expressed in these cells, as shown by immunocytochemistry. Immunoblot analysis showed similar levels of NSE expression in both untreated and induced Whartons jelly cells. After 3 days, the induced Whartons jelly cells resembled bipolar or multipolar neurons, with processes that formed networks reminiscent of primary cultures of neurons. The neuron‐like cells in these cultures stained positively for several neuronal proteins, including neuron‐specific class III β‐tubulin, neurofilament M, an axonal growth‐cone‐associated protein, and tyrosine hydroxylase. Immunoblot analysis showed increasing levels of protein markers for mature neurons over time postinduction. Markers for oligodendrocytes and astrocytes were also detected in Whartons jelly cells. These exciting findings show that cells from the matrix of umbilical cord have properties of stem cells and may, thus, be a rich source of primitive cells. This study shows their capacity to differentiate into a neural phenotype in vitro.

Heat Stroke: Role of the Systemic Inflammatory Response

Heat stroke is a life-threatening illness that is characterized clinically by central nervous system dysfunction, including delirium, seizures, or coma and severe hyperthermia. Rapid cooling and support of multi-organ function are the most effective clinical treatments, but many patients experience permanent neurological impairments or death despite these efforts. The highest incidence of heat stroke deaths occurs in very young or elderly individuals during summer heat waves, with ∼ 200 deaths per year in the United States. Young, fit individuals may experience exertional heat stroke while performing strenuous physical activity in temperate or hot climates. Factors that predispose to heat stroke collapse include pre-existing illness, cardiovascular disease, drug use, and poor fitness level. For decades the magnitude of the hyperthermic response in heat stroke patients was considered the primary determinant of morbidity and mortality. However, recent clinical and experimental evidence suggests a complex interplay between heat cytotoxicity, coagulation, and the systemic inflammatory response syndrome (SIRS) that ensues following damage to the gut and other organs. Cytokines are immune modulators that have been implicated as adverse mediators of the SIRS, but recent data suggest a protective role for these proteins in the resolution of inflammation. Multi-organ system failure is the ultimate cause of mortality, and recent experimental data indicate that current clinical markers of heat stroke recovery may not adequately reflect heat stroke recovery in all cases. Currently heat stroke is a more preventable than treatable condition, and novel therapeutics are required to improve patient outcome.

Thermoregulatory, behavioral, and metabolic responses to heatstroke in a conscious mouse model

The typical core temperature (T(c)) profile displayed during heatstroke (HS) recovery consists of initial hypothermia followed by delayed hyperthermia. Anecdotal observations led to the conclusion that these T(c) responses represent thermoregulatory dysfunction as a result of brain damage. We hypothesized that these T(c) responses are mediated by a change in the temperature setpoint. T(c) (+/- 0.1 degrees C; radiotelemetry) of male C57BL/6J mice was monitored while they were housed in a temperature gradient with ambient temperature (T(a)) range of 20-39 degrees C to monitor behaviorally selected T(a) (T(s)) or an indirect calorimeter (T(a) = 25 degrees C) to monitor metabolism (V(O(2))) and calculate respiratory exchange ratio (RER). Responses to mild and severe HS (thermal area 249.6 +/- 18.9 vs. 299.4 +/- 19.3 degrees C.min, respectively) were examined through 48 h of recovery. An initial hypothermia following mild HS was associated with warm T(s) (approximately 32 degrees C), approximately 35% V(O(2)) decrease, and RER approximately 0.71 that indicated reliance on fatty acid oxidation. After 24 h, mild HS mice developed hyperthermia associated with warm T(s) (approximately 32 degrees C), approximately 20% V(O(2)) increase, and RER approximately 0.85. Severe HS mice appeared poikilothermic-like in the temperature gradient with T(c) similar to T(s) (approximately 20 degrees C), and these mice failed to recover from hypothermia and develop delayed hyperthermia. Cellular damage (hematoxylin and eosin staining) was undetectable in the hypothalamus or other brain regions in severe HS mice. Overall, decreases and increases in T(c) were associated with behavioral and autonomic thermoeffectors that suggest HS elicits anapyrexia and fever, respectively. Taken together, T(c) responses of mild and severe HS mice suggest a need for reinterpretation of the mechanisms of thermoregulatory control during recovery.

Tissue and circulating expression of IL-1 family members following heat stroke

Interleukin-1 (IL-1) is thought to have a significant role in the pathophysiology of heat stroke (HS), although little is known regarding the actions or expression patterns of the IL-1 family. This study tested the hypotheses that following HS IL-1 family gene expression is dynamic, while loss of IL-1 signaling enhances recovery. IL-1 family expression was determined in plasma, spleen, and liver from C57BL/6J mice (n=24 control, n=20 HS) at maximum core temperature (Tc,Max), hypothermia, and 24 h post-HS (24 h). Soluble IL-1 receptor subtype I (sIL-1RI) protein expression peaked at 24 h (14,659.01±2,016.28 pg/ml, P<0.05), while sIL-1RII peaked at hypothermia (19,099.30±1,177.07 pg/ml). IL-1α gene expression in the spleen (ninefold) and liver (fourfold) along with IL-1RI (threefold spleen and fivefold liver) were maximal at hypothermia. Spleen IL-1β gene expression peaked at Tc,Max (fourfold) but at hypothermia (fourfold) in liver. Gene expression of the IL-1 family member IL-18 peaked (2.5-fold) at Tc,Max but was similar at all other time points. Subsequent studies revealed that despite accruing a greater heating area (298±16 vs. 247±13°C·min, P<0.05), IL-1RI knockout (KO) mice (n=14) showed an attenuated hypothermia depth (28.5±0.2 vs. 27.3±0.5°C, P<0.05) and duration (675±82 vs. 1,283±390 min, P<0.05) with a higher 24 h Tc (36.9 vs. 34.1°C, P<0.05) compared with C57BL/6J mice (n=8). The current results demonstrate that following HS IL-1 family gene expression is altered and IL-1RI KO mice display Tc responses consistent with a more rapid recovery.

Central nervous system administration of interleukin-6 produces splenic sympathoexcitation.

Interleukin-6 (IL-6) is a multifunctional cytokine that has been shown to play a pivotal role in centrally-mediated physiological responses including activation of the hypothalamic-pituitary-adrenal axis. Cerebral spinal fluid (CSF) concentrations of IL-6 are elevated in multiple pathophysiological conditions including Alzheimers disease, autoimmune disease, and meningitis. Despite this, the effect of IL-6 on central regulation of sympathetic nerve discharge (SND) remains unknown which limits understanding of sympathetic-immune interactions in health and disease. In the present study we determined the effect of intracerebroventricular (i.c.v, lateral ventricle) administration of IL-6 on splenic SND in urethane-chloralose-anesthetized rats. A second goal was to determine if icv injected IL-6 enters the brain parenchyma and acts as a volume transmission signal to access areas of the brain involved in regulation of sympathetic nerve outflow. i.c.v administration of IL-6 (10 ng, 100 ng, and 400 ng) significantly and progressively increased splenic SND from control levels in baroreceptor-denervated Sprague-Dawley rats. Administration of 100-ng and 400-ng IL-6 resulted in significantly higher SND responses when compared to those elicited with a 10-ng dose. Sixty minutes following icv administration, fluorescently labeled IL-6 was not distributed throughout the parenchyma of the brain but was localized to the periventricular areas of the ventricular system. Brain sections counter-stained for the IL-6 receptor (IL-6R) revealed that IL-6 and the IL-6R were co-localized in periventricular areas adjoining the third ventricle. These results demonstrate that icv IL-6 administration increases splenic SND, an effect likely achieved via signaling mechanisms originating in the periventricular cells.

Patterns of Gene Expression Associated with Recovery and Injury in Heat-stressed Rats

BackgroundThe in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model.ResultsWe heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response.ConclusionsPathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.

The impact of acute-stressor exposure on splenic innate immunity: A gene expression analysis

Exposure to intense, acute-stressors modulates immune function. We have previously reported, for example, that exposure to a single session of inescapable tailshock suppresses acquired and potentiates innate immune responses mediated by the spleen. The mechanisms for these changes remain unknown, however, they likely involve stress-induced modulation of cytokines. Cytokines operate in coordinated networks that include other immunoregulatory factors. Broad-scoped analyses are required to gain an understanding of the net-impact of stress on these immunoregulatory factors and the immune system. The goal of this study, therefore, is to examine the impact of acute-stressor exposure on network-wide changes in splenic immunoregulatory factor expression. One hundred and sixty-one genes linked to innate immune responses were quantified in the spleen following exposure to tailshock using an RT-PCR based gene array. Expression changes in 17 of the measured genes were confirmed using individual RT-PCR reactions. Further assessment of the expression changes using Exploratory Gene Association Networks (EGAN) identified important ontologies, processes and pathways that are indicative of a broader impact of stress on the immune system. Interestingly, EGAN identified several linkages between immunoregulatory factors that may be important in explaining previous results concerning the functional consequences of stress on splenic immunity. Additional processes, some of which are novel to this study, were also uncovered that may be important in directing future studies examining the impact of stress on the immune system. In this way, these analyses provide a better understanding of how acute stressor exposure modulates splenic immunity and may function as predictive tool for future related studies.

A physiological systems approach to modeling and resetting of mouse thermoregulation under heat stress

Heat stroke (HS) is a serious civilian and military health issue. Due to the limited amount of experimental data available in humans, this study was conducted on a mouse mathematical model fitted on experimental data collected from mice under HS conditions, with the assumption there is good agreement among mammals. Core temperature (T(c)) recovery responses in a mouse model consist of hypothermia and delayed fever during 24 h of recovery that represent potential biomarkers of HS severity. The objective of this study was to develop a simulation model of mouse T(c) responses and identify optimal treatment windows for HS recovery using a three-dimensional predictive heat transfer simulation model. Several bioenergetic simulation variables, including nonlinear metabolic heat production (W/m³), temperature-dependent convective heat transfer through blood mass perfusion (W/m³), and activity-related changes in circadian T(c) were used for model simulation. The simulation results predicted the experimental data with few disparities. Using this simulation model, we tested a series of ambient temperature treatment strategies to minimize hypothermia and delayed fever to accelerate HS recovery. Using a genetic algorithm, we identified eight time segments (ambient temperature = 27, 30, 31, 29, 28, 28, 27, 26°C) of 110 min total duration that optimized HS recovery in our model simulation.

45 Central activation of sympathetic neural circuits alters splenic cytokine gene expression

#44 Hyperbaric oxygen therapy ameliorates stress-impaired healing P. Gajendrareddy , G. Ilangovan , P. Kuppusamy , C.K. Sen , M.P. Horan , P.T. Marucha a a Department of Periodontics, University of Illinois, Chicago, USA b Department of Oral Biology, Center for Biomed, EPR Spectroscopy and Imaging, USA c Department of Internal Medicine, Center for Biomed, EPR Spectroscopy and Imaging, USA d Departments of Surgery and Molecular and Cellular Biochemistry, The Ohio State University, Columbus, USA