A new tool for exploring the effects of repeated low-amplitude mechanical impacts onto in vitro neuronal networks is presented. The experimental setup, HAMr, is specifically designed to allow variability in impact conditions while ensuring a highly repeatable result. HAMr’s functionality to induce inflammation related to mild traumatic brain injuries has been validated by assessing its capability to induce elevated expression of the inflammatory protein IL-1 β in in vitro neuronal cell cultures. The two main results obtained for the inflammatory response in dissociated cortical networks, presented for a range of impact force amplitude and total number of impacts, can be summarized as follows. First, the results demonstrate a strong correlation between ensuing inflammation level and numbers of impact. Second, the results indicate a possible existence of a safe threshold of number of impacts that does not initiate an inflammatory response.
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We would like to thank Anton Schuetze-Coburn, Michael Ray and Rodney Yates for help with machining and design of HAMr, the USC Viterbi Machine Shop and the WSU Spokane Microscopy Core Lab for access to their equipment. Parijat Sengupta’s work was supported by the ISP/Applied Sciences Laboratory, WSU
Appendix A: Biological Methods
Appendix A: Biological Methods
A.1 Preparation of Cortical Networks for Impact Experiments
Cortices isolated from embryonic day-18 C57BL/6 (CRL line) mice brain (BrainBits LLC, Springfield, IL) were used to make 2-dimensional neuronal networks used in this study. Under sterile conditions, cortical tissue was digested at 30 ∘C for 25 minutes in 2 mg/ml papain solution in Hibernate-E minus Ca 2+ (Brain Bits LLC, Springfield, IL), mechanically dissociated in Hibernate-E medium (Brain Bits LLS, Springfield, IL) supplemented with 0.5 mM Glutamax (Life Technologies, Carlsbad, CA), and strained with a 40 μm cell strainer (BD Biosciences, San Jose, CA) to remove debris and tissue fragments [26, 27]. Cells were spun down at 200 g for 3 minutes and the pellet was re-suspended in warm Dulbecco’s Modified Eagles Medium (DMEM) with high glucose (Cat #D-6429, Sigma, St. Louis, MO) supplemented with 10 % fetal bovine serum (FBS) and 0.5 mM Glutamax. Approximately 105 cells in 40 μl were plated on poly-D-lysine coated 35 mm diameter glass-bottomed circular imaging dishes (MatTek Corporation, Ashland, MA). Cells were incubated inside a 5 % CO 2 incubator at 37 ∘C. After 1.5 hours, 2 ml warm pre-equilibrated media was added to the cultures. After 4 hours half of the growth medium was replaced with NbActiv4 (Brain Bits LLC, Springfield, IL) medium. By day 3 in vitro (DiV-03) cells formed an intricate network with numerous connections. Cells were maintained in culture by feeding every third day by replacing half of the culture medium with warm, fresh NbActiv4 medium pre-equilibrated in the incubator. A cocktail of penicillin/streptomycin and gentamicin was used to keep cultures free from microbial contamination an bacterial infection. All experiments reported here used DIV-15 to DIV-20 cultures. Cell type analyses for these networks using immunostaining with neuronal marker NeuN (ABN78 from Millipore, Billerica, MA), a suitable Alexa-633 conjugated secondary antibody (Life Technologies, Carlsbad, CA) and cell nuclei marker Hoechst (Fig. 8) show that 44±3 % (n=6 isolations) of cells are neurons in these networks between DIV-11 and DIV-24 in culture. For the impact experiments, networks were taken out of the incubator for pressure pulse exposure and then again returned to the incubator for 4 hours. Impact-exposed networks were then fixed, immunostained for IL-1 β protein and other cellular markers, and then imaged using a fluorescence microscope.
A.2 Determination of IL-1 β Expression Levels in Neurons
IL-1 β expression levels in neurons were determined by immunostaining these networks with suitable antibodies. After impact experiment and incubation, networks were washed twice with warm phosphate buffered saline (PBS) solution, and then fixed with 3.7 % paraformaldehyde (PFA) solution in PBS for 20 minutes at room temperature. Samples were washed thrice with PBS/50 mM glycine solution to neutralize PFA, and then treated with 0.25 % Triton-X for 10 minutes to make cell membranes permeable. A blocking solution made of 3 % bovine serum albumin (BSA), 5 % goat serum and 50mM glycine in PBS was used to minimize nonspecific labeling with antibodies. Then each network was treated with 2-3 drops of Image-IT FX (Life Technologies, Carlsbad, CA) for 30 minutes at room temperature in humid environment. For immunostaining, networks were first incubated with primary antibody (IL-1 β, goat, AF-401-NA, (R&D Systems, Minneapolis, MN); Cy3 conjugated MAP-2, rabbit, AB2290C3 (Millipore, Billerica, MA); NeuN, rabbit, ABN78 (Millipore, Billerica, MA); Alexa-488 conjugated NeuN, rabbit, ABN78A4 (Millipore, Billerica, MA)) for 2 hours, washed tree times with PBS, and then treated with a suitable donkey anti-goat Alexa-633 or Alexa-488 labelled secondary antibody (Life Technologies, Carlsbad, CA) for 2 hours. All reactions were done at room temperature. Samples were washed twice and then labelled with NucBlue reagent (Life Technologies, Carlsbad, CA). Immunostained samples were stored at 4 ∘C and were imaged within three days of preparation.
For fluorescence imaging experiments a fluorescence microscope (Axio Imager M2, Zeiss) coupled to X-Cite Series 120Q (EXFO) illumination system and a digital camera (Axio Cam MRm, Zeiss) was used. For all experiments the detector gain and the channel exposure times used for recording images from immunostained samples were kept constant. For every network, 6-10 fields (1000 μ m×800 μm for fluorescence microscope) were imaged from different regions of the network. Higher fluorescence intensity was interpreted as higher concentration of IL-1 β protein.
Image processing and analyses were done using ImageJ software (National Institutes of Health, MD). For every image, neuronal cell bodies were selected (∼50 cells per image, 4-6 images per network) using Region of Interest (ROI) Manager tool, and average fluorescence intensity for each ROI was measured. Finally, data from multiple networks were combined, and the change in fluorescence intensity is presented as percent increase using the average values of non-impactor exposed samples as controls. All experiments were done using 4 independent cell isolations. Data from all preparations were combined and presented as average ± standard deviation.
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Koumlis, S., Buecker, D., Moler, G. et al. HAMr: A Mechanical Impactor for Repeated Dynamic Loading of In vitro Neuronal Networks. Exp Mech 55, 1441–1449 (2015). https://doi.org/10.1007/s11340-015-0052-y