An In Vitro Trauma Model to Study Rodent and Human Astrocyte Reactivity

Abstract

Protocols are presented describing a unique in vitro injury model and how to culture and mature mouse, rat, and human astrocytes for its use. This injury model produces widespread injury and astrocyte reactivity that enable quantitative measurements of morphological, biochemical, and functional changes in rodent and human reactive astrocytes. To investigate structural and molecular mechanisms of reactivity in vitro, cultured astrocytes need to be purified and then in vitro "matured" to reach a highly differentiated state. This is achieved by culturing astrocytes on deformable collagen-coated membranes in the presence of adult-derived horse serum (HS), followed by its stepwise withdrawal. These in vitro matured, process-bearing, quiescent astrocytes are then subjected to mechanical stretch injury by an abrupt pressure pulse from a pressure control device that briefly deforms the culture well bottom. This inflicts a measured reproducible, widespread strain that induces reactivity and injury in rodent and human astrocytes. Cross-species comparisons are possible because mouse, rat, and human astrocytes are grown using essentially the same in vitro treatment regimen. Human astrocytes from fetal cerebral cortex are compared to those derived from cortical biopsies of epilepsy patients (ages 1-12 years old), with regard to growth, purity, and differentiation. This opens a unique opportunity for future studies on glial biology, maturation, and pathology of human astrocytes. Prototypical astrocyte proteins including GFAP, S100, aquaporin4, glutamate transporters, and tenascin are expressed in mouse, rat, and human in vitro matured astrocyte. Upon pressure-stretching, rodent and human astrocytes undergo dynamic morphological, gene expression, and protein changes that are characteristic for trauma-induced reactive astrogliosis.

The density difference between tissue and neural probes is a key factor for glial scarring

“…Hence, given that the probes used here are very similar apart from with respect to density, we propose that the mechanical forces between probes and neural tissues that arise because of differences in inertial forces every time the animal accelerates or because of difference in gravitational pull on the implants trigger astrocyte reactivity and the development of a glial scar. This is in line with recent findings that the mechanical stimulation of astrocytes renders them reactive, with high expression levels of GFAP29.…”

Impact of Simulated Microgravity on Oligodendrocyte Development: Implications for Central Nervous System Repair

“…Neural progenitors were purified from a cell suspension of human fetal cortex at 15–17 weeks of gestation using a percoll gradient [7]. The volume of the filtered cell suspension in growth medium (DMEM/F12, 10% FBS, 1∶1000 gentamicin) was determined and a final 30% percoll solution was made by mixing one part of a HBSS-buffered percoll solution to two parts of cell suspension.…”

Stretch in Brain Microvascular Endothelial Cells (cEND) as an <em>In Vitro</em> Traumatic Brain Injury Model of the Blood Brain Barrier

“…For instance, a method for studying the effect of mechanical injury to cells has been established for astrocytes, neurons, glial cells and aortic endothelial cells 7,8,9 . The in vitro trauma model established for the study of rodent and human astrocyte reactivity 10 employed a pressure control device identical to what we use for our model. The same method was applied to induce injury through stretch in mouse brain microvessel endothelial cells (bEnd3) 11 and cortical neurons 12,13 as well as, cerebral endothelial cells from newborn piglets 14 .The device deforms the bottom of the culture well thereby producing mechanical stretch injury 10 .…”

“…The in vitro trauma model established for the study of rodent and human astrocyte reactivity 10 employed a pressure control device identical to what we use for our model. The same method was applied to induce injury through stretch in mouse brain microvessel endothelial cells (bEnd3) 11 and cortical neurons 12,13 as well as, cerebral endothelial cells from newborn piglets 14 .The device deforms the bottom of the culture well thereby producing mechanical stretch injury 10 . It inflicts injury upon cultured cells by the application of air pressure above the cells.…”