UCB Student Abstracts

To improve techniques for cryopreserving tissues and organs, we compared the actions of cryoprotectants on the viability of cells (mixed cell populations kidney, spleen and lungs) from a cryopreserved Syrian Hamster body compared to the viability of the cryopreserved cultured cells from the same organs. Female hamsters (120–140 g) were anesthetized with 0.2 ml of ketamin (100mg/ml) intramuscularly, chilled and placed on a surgical platform. The spinal cord was then severed and organs harvested. Cells from the spleen, kidney, and lungs were incubated at 35°C and 5% CO₂ for 7 days in DMEM medium (with high glucose and without sodium pyruvate). Cells were then immersed in solutions of cryoprotectants (20% Glycerol, 20% DMSO, 10% Glycerol plus 10% DMSO, or 7.5% Glycerol plus 7.5% DMSO plus 0.2 M Glucose) mixed with blood plasma substitute Hextend or pure Hextend (Abbott, N. Chicago, Il). Cultures were then cooled by either 1. Fast Freezing - cells submerged directly into liquid nitrogen -196°C and remained there for 7 days, or 2. Slow Freezing - cells cooled at 1°C/min to -80°C, remained at -80°C for 1 day, then transferred into liquid nitrogen, where they remained for 6 days. Cells were then thawed and allowed to incubate at 35°C and 5% CO₂ for 14 days in DMEM medium (with high glucose and without sodium pyruvate). The viability of fibroblasts (the cell type we primarily observed) were then determined every 7 days by their ability to attach to the surface of Petri dishes and divide to confluence. Despite bacterial contamination that resulted in some cases, the dividing cells from the slow freezing method with 7.5% Glycerol plus 7.5% DMSO plus 0.2 M Glucose showed the best results. This contrasts the results from the whole body preservation experiments in which a 10% Glycerol plus 10% DMSO mixture showed the best results.

(This experiment was done as a transition to high pressure freezing that will be first done on cultured cells and then on whole hamster body. High pressure will be used instead/with cryoprotectants to minimize ice crystallization.)

Curcumin (CUR), an active compound derived from the curry spice turmeric, inhibits proliferation and maturation of neuroglia in vitro. This activity may be related to its anti-inflammatory and anti-oxidant properties. Due to its lipophilic nature and small molecular size, CUR is able to cross cell membranes, including the blood-brain barrier; thereby inhibiting transcription factors associated with the inflammatory response and also acting as a free radical scavenger. CUR has no known side effects. Using a mixed colony of neuroglial cells, namely astrocytes (AST) and oligodendrocytes (OLG), from C-6 rat glioma 2B-clone cells, we studied the effects of varying doses of CUR over two different time periods. In a past series of experiments, 4, 5, 10, 15, and 20 μM doses were applied over a 6-day period. It was found that concentrations as low as 5 μM inhibited proliferation, while 20 μM was cytotoxic. In an effort to find the minimum effective dose and effects over an increased duration of administration, doses of 3, 5, and 10 μM were applied over a period of 12 days. In both series CUR inhibited neuroglia proliferation in a dose-dependent manner. Cytotoxicity occurred at 20 μM in the 6-day trials while in the 12-day trials it occurred at 10 μM; this implies an additive effect. Interestingly, CUR has a selective effect on the different types of neuroglia. In an assay that tested for AST enzyme marker, a decrease in activity was measured. In contrast, a marker enzyme for myelogenesis by OLG had increased levels indicating a stimulatory effect by CUR. The inhibitory and stimulatory effects of CUR may prove to be useful in prevention-treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's.

Two neuroglial cell types, ependymal cells and subventricular zone (SVZ) astrocytes, have been implicated as multipotential stem cells that have the ability to divide, self-renew, and give rise to neurons, astrocytes, and oligodendrocytes. Although no other neural cells are known to function as stem cells, growth factors could be used to stimulate neuroglial cells into this progenitor state. To better understand this role of growth factors, we treated C-6 rat glioma 2B-clone cells, a mixed culture of astrocytes and oligodendrocytes, with varying concentrations of Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF). We treated the neuroglial cells with EGF concentrations of 25, 50, 100 ng/ml and FGF concentrations of 40, 80, 160 ng/ml for 14 days. Cell growth was measured by cell counts and cell differentiation was measured by the activity of the specific enzyme glutamine synthetase (GS) for astrocytes and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) for oligodendrocytes. We previously discovered that increased proliferation and decreased differentiation of the neuroglial cells were most effective with the 50 ng/ml dose for EGF and 80 ng/ml dose for FGF during our 8 day trial. In this study, we discover that once again the 50 ng/ml of EGF surpasses the other concentrations in proliferation but that this increase in cell growth reaches its optimal value by day 10, with a decline in cell proliferation seen from day 10 to 14. With FGF, the most effective dose was also 80 ng/ml, but it reached an optimal growth value by day 8 and declined thereafter. Using both GS and CNP enzyme assays, we further discovered that by day 14 enzymatic activity was decreased with all treatments compared to the control, the most effective doses being 50 ng/ml for EGF and 80 ng/ml for FGF. Despite the decline in cell growth, enzymatic activity was still repressed. These findings show that local growth factors have the ability to maintain neuroglial cells in a highly proliferative yet immature state, similar to that of stem cells.

Altered hypothalamic sensitivity to circulating hormones, constitutes the feedback mechanism used by the neuroendocrine system to modulate endocrine profiles over time. Endocrine profiles, in turn, dictate gene expression and ultimately drive changes in whole organism phenotype over the lifespan. While the causal link between endocrine profiles and phenotypes associated with development and aging has been widely recognized, acknowledging that altered hypothalamic sensitivity plays a crucial role in timing the onset of phenotypic shifts remains understated. Mapping hypothalamic sensitivity at different points in the lifespan, will establish feedback thresholds associated with youthful & aged phenotypes and determine if hypothalamic sensitivity can be used as an effective neuroendocrine biomarker. In addition, tracking the effects of caloric restriction (CR) on hypothalamic sensitivity will help determine if the youthful phenotype maintained by CR is reflected in the hypothalamus. Of the many endocrine signal receptors that may be involved in maintaining hypothalamic feedback sensitivity, our study has selected to begin mapping those for estrogen (E). Altered hypothalamic sensitivity to E is known to schedule reproductive maturation and influence reproductive senescence. Taking estrogen receptor-alpha (ER-α) immunoreactivity as an index of sensitivity to E, we counted ER-α-positive and negative cells in the pre-optic, arcuate and ventromedial hypothalamus of young (6 weeks), ad-libitum (AL) fed old (22 months), and calorie restricted (CR) old (22 months) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each sampled section of pre-optic, arcuate and ventromedial hypothalamus. Results from the pre-optic nucleus show a 38% reduction in ER-α-positive cells and a 19% reduction in total cell numbers in AL-old mice, compared to only 18% reduction in ER-α-positive cells and 13% reduction in total cell numbers in the CR-old mice. This indicates that pre-optic estrogen sensitivity declines with age and that CR counteracts this decline. Preliminary cell counts from the ventromedial and arcuate nucleus suggest similar trends.