Repeated mild injury causes cumulative damage to hippocampal cells

Abstract

An interesting hypothesis in the study of neurotrauma is that repeated traumatic brain injury may result in cumulative damage to cells of the brain. However, post-injury sequelae are difficult to address at the cellular level in vivo. Therefore, it is necessary to complement these studies with experiments conducted in vitro. In this report, the effects of single and repeated traumatic injury in vitro were investigated in cultured mouse hippocampal cells using a well characterized model of stretch-induced injury. Cell damage was assessed by the level of propidium iodide (PrI) uptake and retention of fluorescein diacetate (FDA). Uninjured control wells displayed minimal PrI uptake and high levels of FDA retention. Mild, moderate and severe levels of stretch caused increasing amounts of PrI uptake, respectively, when measured at 15 min and 24 h post-injury, indicating increased cellular damage with increasing amounts of stretch. For repeated injury studies, cultures received a second injury 1 h after the initial insult. Repeated mild injury caused a slight increase in PrI uptake compared with single injury at 15 min and 24 h post-injury, which was evident primarily in glial cells. However, the neurites of neurones in cultures that received repeated insults showed signs of damage that were not evident after a single mild injury. The release of neurone-specific enolase (NSE) and S-100beta protein, two common clinical markers of CNS damage, was also measured following the repeated injuries paradigm. When measured at 6 h post-injury, both NSE and S-100beta were found to be elevated after repeated mild injuries when compared with the single injury group. These results suggest that cells of the hippocampus may be susceptible to cumulative damage following repeated mild traumatic insults. Both glial cells and neurones appear to exhibit increased signs of damage after repetitive injury. To our knowledge, this study represents the first report on the effects of repeated mechanical insults on specific cells of the brain using an in vitro model system. The biochemical pathways of cellular degradation following repeated mild injuries may differ considerably from those that are activated by a single mild insult. Therefore, we hope to use this model in order to investigate secondary pathways of cellular damage after repeated mild traumatic injury, and as a rapid and economical means of screening possibilities for treatment strategies, including pharmaceutical intervention.

Causal Role of Apoptosis-Inducing Factor for Neuronal Cell Death Following Traumatic Brain Injury

“…30 Briefly, dissociated cortices or hippocampi were diluted in serum-containing media [Basal Medium Eagles (Invitrogen, Carlsbad, CA) containing 10% horse serum (GIBCO), 10 g/ml gentamicin (Sigma, St. Louis, MO), 0.5% glucose (Sigma), 1 mmol/L sodium pyruvate (Invitrogen), and 1% N2 supplements (Invitrogen)] to a concentration of 500,000 cells per ml; cells were then plated in 1 ml aliquots onto collagen-coated six-well FlexPlates (FlexCell, Hillsborough, NC) coated overnight with poly-L-ornithine (500 g/ml; Sigma). All cultures were maintained in a humidified incubator (5% CO 2 , 37°C).…”

“…30,31 Briefly, cultures were fixed with 4% paraformaldehyde (all steps conducted at room temperature), permeabilized with 0.2% Triton X-100, and then incubated with primary antibodies [anti-AIF (1:200; Santa Cruz Biotechnology, Santa Cruz, CA); anti-microtubule associated protein 2 (MAP-2; 1:500; Sigma); anti-glial fibrillary acidic protein (1:300; Dakopatts, Glostrup, Denmark)]; polyclonal anti-cytochrome oxidase-antibody (CytOx: 1:200; Cell Signaling, Danvers, MA) for 1 hour, followed by incubation with secondary antibodies (Alexa 488 donkey-anti-goat, Invitrogen, 1:300; Alexa-594-conjugated goat-antimouse, 1:300, both Molecular Probes, Eugene, OR; Cy3 goat-anti-rabbit, 1:200, Jackson ImmunoResearch Laboratories, West Grove, PA; Texas red-conjugated goat anti-rabbit, 1:200, Vector Laboratories, Burlingame, CA) for 1 hour. Cultures were dehydrated with ethanol and mounted with 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining (Vector Laboratories, Burlingame, CA).…”

“…30,32,33 In brief, the Silastic membrane of the FlexPlate well was rapidly and transiently deformed by a 50-ms pulse of compressed nitrogen, which deformed the Silastic membrane and adherent cells to varying degrees controlled by pulse pressure. The extent of cell injury-produced by deforming the Silastic membrane on which the cells are grown-was dependent on the degree of deformation, or stretch.…”

“…The extent of cell injury-produced by deforming the Silastic membrane on which the cells are grown-was dependent on the degree of deformation, or stretch. Previous work conducted using rat cortical neurons and glia 32,34,35 and mouse hippocampal neurons 30 have characterized three levels of cell stretch (5.5 mm, 6.5 mm, and 7.5 mm deformations), defined as mild, moderate, and severe injury, which result in membrane deformation and biaxial strain or stretch of 31%, 38%, and 54%, respectively. This range of cell stretch has been shown to be relevant to what would occur in humans after rotational acceleration/ deceleration injury.…”

Neuroprotective Effects of Selective N-Type VGCC Blockade on Stretch-Injury-Induced Calcium Dynamics in Cortical Neurons

“…Multiple pathways are likely involved in triggering elevated [Ca 2þ ] i following injury, including depolarizationinitiated opening of voltage-gated calcium channels (VGCCs), influx through the membrane pores that form after injury (Slemmer et al, 2002), activation of glutamate receptormediated calcium influx pathways (Ahmed et al, 2002;Weber, 2004;Weber et al, 1999), and activation of storeoperated=second-messenger-operated calcium channels that respond to depleted intracellular calcium stores . Injured neurons also experience decreased cellular ATP from ischemia, as well as mitochondrial damage due to calcium accumulation (Ahmed et al, 2000;Schinder et al, 1996;Verweij et al, 2000;Young, 1992), leading to compromised sodium-potassium ATPase activity with resultant cell depolarization (Tavalin et al, 1995(Tavalin et al, , 1997.…”