No abstract

The recent public awareness of the incidence and possible long-term consequences of traumatic brain injury only heightens the need to develop effective approaches for treating this neurological disease. In this report, we identify a new therapeutic target for traumatic brain injury by studying the role of astrocytes, rather than neurons, after neurotrauma. We use in vivo multiphoton imaging and show that mechanical forces during trauma trigger intercellular calcium waves throughout the astrocytes, and these waves are mediated by purinergic signalling. Subsequent in vitro screening shows that astrocyte signalling through the 'mechanical penumbra' affects the activity of neural circuits distant from the injury epicentre, and a reduction in the intercellular calcium waves within astrocytes restores neural activity after injury. In turn, the targeting of different purinergic receptor populations leads to a reduction in hippocampal cell death in mechanically injured organotypic slice cultures. Finally, the most promising therapeutic candidate from our in vitro screen (MRS 2179, a P2Y1 receptor antagonist) also improves histological and cognitive outcomes in a preclinical model of traumatic brain injury. This work shows the potential of studying astrocyte signalling after trauma to yield new and effective therapeutic targets for treating traumatic brain injury.

Cell adhesion and morphology are affected by the mechanical properties of the extracellular matrix. Using polyacrylamide gels as cell substrates, the cellular response to substrate compliance was investigated in pure neuronal, pure astroglial, or mixed co-cultures. Substrates used spanned a large range of stiffnesses including that of brain tissue. In both pure and mixed cultures, immature (vimentin+) astroglia adhered best to stiffest gels. Mature (GFAP+) astrocyte adhesion peaked on intermediate stiffness, while pure GFAP+ astroglial adhesion displayed no intermediate preference and increased with stiffness. Neurite length was constant with stiffness; however, primary dendrite number was lowest on intermediate gels. Pure neuronal cultures were more adherent to hard gels, while mixed cultures had no stiffness preference. Furthermore, we investigated the role of stiffness in the modulation of the neurotoxic effect of glutamate. Exposure to two glutamate concentrations (500 and 1000 M) of cultured spinal cord neurons induced cell death. The damage elicited by 500 m glutamate to neurons in a mixed culture of spinal cord cells is most severe on soft 300 Pa gels. The neurotoxic effect of glutamate on neurons cultured on hard gels where astrocytes are present was strongly attenuated compared with that observed on soft gels, where there is a relatively low number of astrocytes. Our data suggest that mechanical stiffness of the substrate affects the response of both neurons and astroglia, and this response is varied by interaction between the two cell types.

Neuronal morphology plays a significant role in determining how neurons function and communicate [1][2][3] . Specifically, it affects the ability of neurons to receive inputs from other cells 2 and contributes to the propagation of action potentials 4,5 . The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network 6,7 . Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease 8 , schizophrenia 9,10 , and mental retardation 11 . The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations 12 .The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1. Protocol Before You Begin: 1) E18 rat dissection:Standard dissection methods of E18 hippocampal neurons have previously been described 13 . In order to use the Bonfire program to analyze the morphological characteristics of the neurites, 8 bit .tif images of individual neurons must be obtained. This can be accomplished in a number of ways depending on the e...