Kindling causes changes in the composition of the astrocytic cytoskeleton.

Changes in the astrocytic cytoskeleton were examined in amygdala kindled rats using immunocytochemical techniques. One week following kindling, there was a dramatic increase in immunoreactivity to glial fibrillary acidic protein and vimentin in astrocytes throughout amygdala, pyriform cortex and hippocampus bilaterally. Since these changes occurred in anatomical sites involved in the propagation of kindled seizures, the observed cytoskeletal reorganization in astrocytes may signify important functional alterations in the kindled brain.

Changes in inhibitory processes in the hippocampus following recurrent seizures induced by systemic administration of kainic acid.

Rats were chronically prepared with stimulation electrodes in the angular bundle and recording electrodes in the dentate gyrus under electrophysiological guidance. Following testing of dentate gyrus field potentials, the animals were given a single injection of kainic acid which caused repeated seizures and led to status epilepticus. The seizures were stopped by administration of a barbiturate anesthetic after 60 min. Changes in inhibition during seizure development were monitored by administering pulse pairs at regular intervals. The results revealed a progressive kainic acid-induced loss in inhibition that preceded the occurrence of seizures. This breakdown of inhibition was transient, and generally disappeared within 24 h. Over subsequent testing, recurrent inhibition, as measured by the double pulse test, increased beyond baseline levels. This increase persisted for at least one month and was restricted to the early phase of inhibition with a conditioning/test pulse interval of less than 50 ms. A later phase of inhibition, measured at interpulse intervals between 200 and 300 ms, showed a transient decrease which lasted about a week. These results contrast with previous reports of a long-term period of hyperexcitability following recurrent seizures. Procedural differences which might account for such discrepancies are discussed.

Astrocytes in kindling: relevance to epileptogenesis.

Astrogliosis is a prominent feature of epileptic foci, and may play a causal role in the development of seizures and the persistance of seizure disorders. We have studied morphological changes in astrocytes with respect to the evolution of seizures using the kindling model of epilepsy. Kindling-induced seizures result in a prominent hypertrophy of astrocytes that is accompanied by a reorganization of astrocytic cytoskeleton. The change in the morphology of astrocytes appears to be seizure-intensity dependent, occurs early in the kindling process, and persists for weeks following the last seizure. In addition to hypertrophy, we have observed an increase in proliferation of astrocytes in hippocampus, amygdala and piriform cortex, but no change in the expression of connexin-43 following kindling. Significantly, induction of a localized astrocyte hypertrophy prior to initiation of kindling does not result in seizures and does not facilitate kindling. Altogether these data suggest that 'gliosis' is an adaptive response to seizures.

Human adipose stem cells maintain proliferative, synthetic and multipotential properties when suspension cultured as self-assembling spheroids.

Adipose-derived stromal/stem cells (ASCs) have been gaining recognition as an extremely versatile cell source for tissue engineering. The usefulness of ASCs in biofabrication is further enhanced by our demonstration of the unique properties of these cells when they are cultured as three-dimensional cellular aggregates or spheroids. As described herein, three-dimensional formulations, or self-assembling ASC spheroids develop their own extracellular matrix that serves to increase the robustness of the cells to mechanical stresses. The composition of the extracellular matrix can be altered based on the external environment of the spheroids and these constructs can be grown in a reproducible manner and to a consistent size. The spheroid formulation helps preserve the viability and developmental plasticity of ASCs even under defined, serum-free media conditions. For the first time, we show that multiple generations of adherent ASCs produced from these spheroids retain their ability to differentiate into multiple cell/tissue types. These demonstrated properties support the idea that culture-expanded ASCs are an excellent candidate cellular material for 'organ printing'-the approach of developing complex tissue structures from a standardized cell 'ink' or cell formulation.

Low serum and serum-free culture of multipotential human adipose stem cells.

BACKGROUND: Adipose tissue provides an easily accessible and abundant source of putative stem cells for translational clinical research. Currently prevalent culture techniques include the use of FBS, a highly variable and undefined component, which brings with it the potential for adverse patient reactions. In an effort to eliminate the use of animal products in human adipose stem cell (ASC) cultures, we have developed two new culture methods, a very low human serum expansion medium and a completely serum-free medium. METHODS: Through serial testing, a highly enriched medium formulation was developed for use with and without the addition of 0.5% human serum, an amount easily obtainable from autologous blood draws. RESULTS: Very low-serum culture yielded population-doubling times averaging 1.86 days in early passage, while the serum-free formulation was associated with less robust cell growth, with doubling times averaging 5.79 days. ASC in both conditions maintained its ability to differentiate into adipo-, chondro- and osteogenic lineages in vitro, despite lower expression of CD34 in early passage. Expression of ALDH, HLA, CD133, CD184, and CD31 was comparable with that seen in cells cultured in 10% FBS. DISCUSSION: These newly developed culture conditions provide a unique environment within which to study ASCs without the interference of animal serum, and allow for the rapid expansion of autologous ASCs in culture in an animal product-free environment for use in human clinical trials.

Selective ablation of astrocytes by intracerebral injections of alpha-aminoadipate.

The efficacy and the specificity of the putative astrotoxin, alpha-aminoadipate, were examined in this study. The integrity of astrocytes was evaluated at several time points following a single injection of alpha-aminoadipate into amygdala of adult rats using immunohistochemistry. The density and the morphological appearance of neurons and the response of microglia were also examined. The injection of alpha-aminoadipate disrupted the astrocytic network in that region. There was a profound loss of glial fibrillary acidic protein-positive and S100 beta-positive astrocytes, normally present in the region, while vimentin immunohistochemistry revealed the presence of deformed cell processes, presumably astrocytic. The presence of reactive microglia at the injection site was suggestive of an active degenerative process, while the normal neuronal density and appearance, as compared to controls, suggested that the damage was confined to astrocytes. The confirmed effectiveness and cellular specificity of alpha-aminoadipate in vivo makes it a potentially important experimental tool for attempting to decipher the functional significance of astrocytes.

Tyrosine phosphorylation of glycoproteins in the adult and developing rat brain.

The tyrosine phosphorylation of glycoproteins in the adult and developing rat brain was investigated. Immunoblotting with anti-tyr(P) antibodies identified a glycoprotein with an apparent Mr of 180,000 (GP180) as the major tyrosine-phosphorylated protein in the concanavalin A (con A)-binding fraction prepared from forebrain homogenates. This glycoprotein had the same electrophoretic mobility as the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. Tyrosine-phosphorylated GP180 was enriched 24-fold in isolated PSDs relative to homogenates. Digestion with endoglycosidase F/N-glycosidase F demonstrated that GP180 present in total homogenates and PSD-GP180 present in isolated PSDs contained similar amounts of N-linked oligosaccharide suggesting that they are the same glycoprotein. The tyrosine phosphorylation of GP180 in homogenates varied between brain regions with the highest levels occurring in cortical areas and the amygdala and low or undetectable amounts being present in hindbrain regions. Incubation of homogenates with adenosine triphosphate (ATP) resulted in the tyrosine phosphorylation of GP180 in all regions examined except the cerebellum and identified a second con A-binding glycoprotein, GP110, which was phosphorylated on tyrosine. GP180 was not phosphorylated on tyrosine following the incubation of cerebellar homogenate, synaptic membranes, or PSDs and ATP. Tyr(P)-GP180 was not detected prior to the onset of synaptogenesis, increased in parallel with the formation of synapses during the first 4 weeks of postnatal development of the frontal cortex and hippocampus, and then decreased 50-60% to adult levels. The results suggest that GP180 corresponds to the PSD glycoprotein PSD-GP180 and are consistent with a role for this glycoprotein in synaptic development and signal transduction at the synapse.

Hippocampal volume and food-storing behavior are related in parids.

The size of the hippocampus has been previously shown to reflect species differences and sex differences in reliance on spatial memory to locate ecologically important resources, such as food and mates. Black-capped chickadees (Parus atricapillus) cached more food than did either Mexican chickadees (P. sclateri) or bridled titmice (P. wollweberi) in two tests of food storing, one conducted in an aviary and another in smaller home cages. Black-capped chickadees were also found to have a larger hippocampus, relative to the size of the telencephalon, than the other two species. Differences in the frequency of food storing behavior among the three species have probably produced differences in the use of hippocampus-dependent memory and spatial information processing to recover stored food, resulting in graded selection for size of the hippocampus.

Females have a larger hippocampus than males in the brood-parasitic brown-headed cowbird.

Females of the brood-parasitic brown-headed cowbird (Molothrus ater) search for host nests in which to lay their eggs. Females normally return to lay a single egg from one to several days after first locating a potential host nest and lay up to 40 eggs in a breeding season. Male brown-headed cowbirds do not assist females in locating nests. We predicted that the spatial abilities required to locate and return accurately to host nests may have produced a sex difference in the size of the hippocampal complex in cowbirds, in favor of females. The size of the hippocampal complex, relative to size of the telencephalon, was found to be greater in female than in male cowbirds. No sex difference was found in two closely related nonparasitic icterines, the red-winged blackbird (Agelaius phoeniceus) and the common grackle (Quiscalus quiscula). Other differences among these species in parental care, migration, foraging, and diet are unlikely to have produced the sex difference attributed to search for host nests by female cowbirds. This is one of few indications, in any species, of greater specialization for spatial ability in females and confirms that use of space, rather than sex, breeding system, or foraging behavior per se, can influence the relative size of the hippocampus.

Activation of astrocytes during epileptogenesis in the absence of neuronal degeneration.

The issue of whether neuronal degeneration is a primary factor in activation of astrocytes during epileptogenesis was addressed using the kindling model of epilepsy. No degenerative changes specific to the kindling process were observed in brain sections from kindled animals, sampled from the olfactory bulbs through to cerebellum and processed with the degeneration-sensitive cupric silver stain. Also, examination of lectin-stained sections did not reveal any reactive microglia. At the same time, reactive astrocytes, as judged by an increase in glial fibrillary acidic protein immunoreactivity and a de novo vimentin immunoreactivity, were prominent in amygdala, piriform cortex, entorhinal cortex and hippocampus. These results suggest that loss of neurones is not a prerequisite for establishment of epilepsy-prone state, that seizures of short duration do not necessarily result in neuronal death, and that in kindling, astrocytes are activated by factors that are not related to neuronal degeneration, but which are likely associated with abnormal neuronal activity.