Craig Cady

Estradiol regulation of astroglia and apolipoprotein E: an important role in neuronal regeneration.

The effects of ovarian hormone on neuronal growth and function are well known. However, equally important, but often neglected, are ovarian hormone effects on glia. Our in vivo and in vitro studies show that estradiol modifies both neuronal growth and glial activity and these effects are tightly linked. Estradiol stimulates neurite growth and the release of the glial apolipoprotein E (apoE) in culture studies. Estradiol-stimulated neurite growth in these cultures requires apoE. Estradiol replacement in ovariectomized mice transiently increases the expression of apoE, the low density lipoprotein receptor related protein (LRP) and synaptophysin throughout the brain. Continuous estradiol replacement over two months loses effect on apoE, LRP, and synaptophysin and suppresses reactive gliosis. Estrous cycle variation of glial activation (GFAP) and apoE are not identical. We propose that estradiol (and other ovarian hormones) functions as a zeitgeber to co-ordinate neuronal-glial interactions. Co-ordination assures temporally appropriate excitatory and inhibitory interactions between glia and neurons. With aging and the loss of ovarian cyclicity, some of this co-ordination must be diminished. These observations present significant clinical implications. Approaches to hormone therapy (HT), for diminishing the risk of chronic neurological diseases, need to consider the temporal nature of ovarian hormones in brain repair and plasticity. Moreover, approaches must consider apoE genotype. The neuroprotective effects of HT in numerous chronic age-related diseases may represent effective co-ordination of repair processes rather than direct disease-specific actions. Moreover, the role of glial-derived proteins in neuroprotection should not be ignored.

Time course of response to estradiol replacement in ovariectomized mice: brain apolipoprotein E and synaptophysin transiently increase and glial fibrillary acidic protein is suppressed.

The current study examined the effect of long-term estradiol replacement in ovariectomized mice. Estradiol-17beta (E2) pellets or vehicle pellets were implanted at the time of ovariectomy (OVX) in young adult female mice. Five mice from each group were sacrificed at 5, 14, 28 and 49 days after OVX and pellet replacement. Western blotting of homogenates from somatosensory cortex, hippocampus, olfactory bulb and cerebellum was performed to obtain concentrations of glial fibrillary acidic protein (GFAP), apolipoprotein E (apoE) and synaptophysin (SYN). At 5 days after OVX, GFAP levels were not affected by E2 replacement. In contrast to GFAP, synaptophysin and apoE concentrations were significantly elevated by 15% and 25%, respectively, in the E2-replaced group compared to the vehicle-replaced group at 5 days but by 14 days concentrations were equivalent. Late in the time course of this study, at 49 days, GFAP concentrations were higher in the E2-deprived mice but did not increase in the E2-replaced group. Immunocytochemistry for GFAP confirmed this observation. Of note was that these effects occurred in all four brain regions measured. These observations suggest that estradiol is able to suppress reactive gliosis. In addition, E2 replacement in OVX mice is associated with transiently higher levels of apoE and synaptophysin.

Single walled carbon nanotube composites for bone tissue engineering

The purpose of this study was to develop single walled carbon nanotubes (SWCNT) and poly lactic‐co‐glycolic acid (PLAGA) composites for orthopedic applications and to evaluate the interaction of human stem cells (hBMSCs) and osteoblasts (MC3T3‐E1 cells) via cell growth, proliferation, gene expression, extracellular matrix production and mineralization. PLAGA and SWCNT/PLAGA composites were fabricated with various amounts of SWCNT (5, 10, 20, 40, and 100 mg), characterized and degradation studies were performed. Cells were seeded and cell adhesion/morphology, growth/survival, proliferation and gene expression analysis were performed to evaluate biocompatibility. Imaging studies demonstrated uniform incorporation of SWCNT into the PLAGA matrix and addition of SWCNT did not affect the degradation rate. Imaging studies revealed that MC3T3‐E1 and hBMSCs cells exhibited normal, non‐stressed morphology on the composites and all were biocompatible. Composites with 10 mg SWCNT resulted in highest rate of cell proliferation (p < 0.05) among all composites. Gene expression of alkaline phosphatase, collagen I, osteocalcin, osteopontin, Runx‐2, and Bone Sialoprotein was observed on all composites. In conclusion, SWCNT/PLAGA composites imparted beneficial cellular growth capabilities and gene expression, and mineralization abilities were well established. These results demonstrate the potential of SWCNT/PLAGA composites for musculoskeletal regeneration and bone tissue engineering (BTE) and are promising for orthopedic applications.

Apolipoprotein E may be a critical factor in hormone therapy neuroprotection.

In this review we examine the evidence for ovarian hormone neuroprotection in chronic neurological diseases, including stroke. We propose that neuroprotection may involve the ability of estrogens to modulate apolipoprotein E (apoE) and its receptor, the low density lipoprotein receptor related protein (LRP). Results from numerous studies have demonstrated that (1) nerve regeneration is severely delayed in apoE-gene knockout (KO) mice as compared to wild-type (WT) littermates; (2) 17beta estradiol replacement in ovariectomized mice resulted in a significant increase in levels of apoE and LRP, in the olfactory bulb (OB) and other brain areas; (3) estradiol treatment increased both apoE and neurite outgrowth in cortical and olfactory neuronal cultures; and (4) estradiol treatment had no effect on neurite outgrowth in cultures deprived of apoE or in the presence of apoE4. In essence these studies suggest that apoE is a critical intermediary for the beneficial effects of 17beta estradiol on nerve repair, which can lead to functional reorganization (plasticity). Future studies of HT should evaluate the effects of apoE genotype and production estradiol on neuroprotection.

Estradiol replacement increases the low-density lipoprotein receptor related protein (LRP) in the mouse brain

Numerous epidemiology studies have shown protective effects of hormone therapy (HT) on chronic neurological diseases. We have proposed that some of the neuroprotective effects of estrogen are mediated by apolipoprotein E (apoE). Polymorphisms of receptors for apoE modify the risk for dementia. To our knowledge, no reports exist showing CNS effects of estrogen replacement on members of the low-density lipoprotein receptor family. The current study focused on the effect of estradiol-17beta (E2) replacement on protein expression of two members of the receptor family, the low-density lipoprotein receptor (LDL-r) and low-density lipoprotein receptor related protein (LRP) in ovariectomized mice. Five days of E2 replacement significantly increased LRP expression in the hippocampus, olfactory bulb and neocortex but not in cerebellum. In contrast, E2 treatment decreased LDL-r protein expression in olfactory bulb. HT modification of both apoE and LRP could have wide-spread effects on cellular function given LRPs manifold signaling functions.

Three Brief Epileptic Seizures Reduce Inhibitory Synaptic Currents, GABAA Currents, and GABAA-Receptor Subunits

Summary:  Purpose: Cellular mechanisms activated during seizures may exacerbate epilepsy. γ‐Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in brain, and we hypothesized that brief epileptic seizures may reduce GABA function.

Neocortical and Hippocampal Glial Fibrillary Acidic Protein Immunoreactivity Shows Region-Specific Variation during the Mouse Estrous Cycle

Ovarian hormones modulate both neuronal and glial activation during the estrous cycle. These effects are particularly well characterized in the hypothalamus. Ovarian hormones also affect brain regions not directly related to reproductive function. In this study we used glial fibrillary acidic protein (GFAP) immunocytochemistry to quantify astroglial cells and process density in both the neocortex and hippocampus during the estrous cycle. Our data show that the density of GFAP immunoreactive processes in the hippocampus peaks on proestrus although cell density does not change. In contrast, both GFAP immunoreactive cell and process densities are elevated on diestrus and proestrus in the supragranular layer of the somatosensory cortex and reach a nadir on estrus and metestrus. This activation pattern is not apparent in the motor or cingulate cortex. Neocortical GFAP immunoreactivity appears to follow the distribution of estrogen receptor-α-like immunoreactivity. Our data show that ovarian hormones have regionally specific effects on glial activation within the neocortex. Characterizing glial activation by ovarian hormones is important since astroglia are the source of numerous trophic factors and play an important, although often unrecognized, role in neuronal metabolism and function.

In vitro evaluation of three-dimensional single-walled carbon nanotube composites for bone tissue engineering.

The purpose of this study was to develop three-dimensional single-walled carbon nanotube composites (SWCNT/PLAGA) using 10-mg single-walled carbon nanotubes (SWCNT) for bone regeneration and to determine the mechanical strength of the composites, and to evaluate the interaction of MC3T3-E1 cells via cell adhesion, growth, survival, proliferation, and gene expression. PLAGA (polylactic-co-glycolic acid) and SWCNT/PLAGA microspheres and composites were fabricated, characterized, and mechanical testing was performed. MC3T3-E1 cells were seeded and cell adhesion/morphology, growth/survival, proliferation, and gene expression analysis were performed to evaluate biocompatibility. Imaging studies demonstrated microspheres with uniform shape and smooth surfaces, and uniform incorporation of SWCNT into PLAGA matrix. The microspheres bonded in a random packing manner while maintaining spacing, thus resembling trabeculae of cancellous bone. Addition of SWCNT led to greater compressive modulus and ultimate compressive strength. Imaging studies revealed that MC3T3-E1 cells adhered, grew/survived, and exhibited normal, nonstressed morphology on the composites. SWCNT/PLAGA composites exhibited higher cell proliferation rate and gene expression compared with PLAGA. These results demonstrate the potential of SWCNT/PLAGA composites for musculoskeletal regeneration, for bone tissue engineering, and are promising for orthopedic applications as they possess the combined effect of increased mechanical strength, cell proliferation, and gene expression.

Biocompatibility of human Whartons Jelly Mesenchymal Stem Cells on poly-caprolactone and collagen based nanofiber mats

Nanofiber scaffolds were fabricated to analyze the proliferation of human Whartons Jelly Mesenchymal Stem Cells (hWJMSCs) for skin tissue engineering applications. Poly-caprolactone (PCL) and PCL mixed with collagen scaffolds were fabricated using electrospinning. ImageJ analysis was carried out on SEM images to characterize the structural and morphological properties of the nanofiber scaffolds. Tensile testing was also performed to quantify the mechanical properties of the scaffolds. The scaffolds were then seeded with hWJMSCs to study cell proliferation over five days in order to determine the feasibility for tissue regeneration. The average fiber diameters for the PCL scaffold and for the PCL/collagen scaffold were 0.542 μm and 0.633 μm, respectively. The Youngs Moduli of the PCL and PCL/collagen scaffolds were 0.00370 Pa and 0.00683 Pa. respectively. After analyzing the data, it can be concluded that the PCL/collagen scaffold is better suited for regeneration of damaged or diseased nervous tissue.

Effect of Valproic Acid on Cell Proliferation of Wharton’s Jelly MSC in PCL Nanofiber Scaffolds

The field of tissue engineering and regenerative medicine is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. The process involves seeding cells onto biocompatible scaffolds that temporarily act as a supporting structure for cells to attach and grow. Scaffolds for tissue regeneration must present a viable microenvironment for the living cells to adhere, proliferate, and exhibit the necessary tissue function. Electrospinning is an emerging area where polymeric fibers can be fabricated in the micro-nano scale. The flexibility of this process allows for including a wide array of synthetic and natural biocompatible polymers in the scaffold composition, inclusion of bioactive molecules (e.g. DNA, proteins) for enhancing therapeutic applications, and ability to control material and mechanical properties via the electrospinning process — all advantageous parameters that contribute to the promise of utilizing electrospun scaffolds in tissue repair. Biocompatible materials, such as polycaprolactone (PCL), have been used extensively to fabricate scaffolds using electrospinning technique, to study cell compatibility and to evaluate cell functionality for nerve tissue engineering applications. The objective of this study is to quantify the effects of the addition of valproic acid to PCL nanofiber scaffolds created through the electrospinning process with regards to cell proliferation. Valproic acid is a commonly used therapeutic drug for the treatment of epilepsy and bipolar disorder. To determine the effects of the presence of valproic acid (VA), Wharton’s jelly mesenchymal stem cells (MSC) are seeded to the two scaffolds. Wharton’s jelly MSC are multipotent adult stem cells present in the umbilical cord and drawn from their matrix [1,2,3]. These stem cells have renowned ability for use in cell therapy and organ regeneration. This study tests the hypothesis that the presence of valproic acid in PCL nanofiber scaffolds will enhance cell proliferation. Structural and morphological characterization of the scaffolds is also carried out. Fiber diameter and tensile properties of the scaffolds with and without valproic acid are also observed. Such studies will enable us to understand the effects of drugs such as valproic acid on stem cells and will aid in designing scaffolds for applications in nerve regeneration.© 2013 ASME