Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.
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Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition
PLoS ONE
pone.0172677.g002:Essential components of B27 supplement.(A) Low magnification confocal images of Nissl staining in cultures at 8 DIV, conditions are indicated on the left side of images, scale bars, 500 μm. (B) Corresponding neuron counts in CA3c, CA3b and CA1. Statistical differences are indicated for comparisons between B27 group versus other groups. Error bars indicate SD. Statistical significance is indicated as **, representing p < 0.01. (C) Confocal images of Nissl staining in CA3c, CA3b and CA1, scale bars, 50 μm. n = 3 cultures, each condition.
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We hypothesized that only BSA and a subset of components of B27 with concentrations indicated in Table 2 (these components make up the widely used N2 supplement) are essential to maintain viable slice cultures. The following compositions were tested: BSA only; BSA and insulin (BSA + ins); BSA, insulin and selenium (BSA + ins + Se); BSA + insulin + Se + transferrin + putrescine + progesterone; and B27. The concentration of each component is listed in Table 2. Supplements were added to Neurobasal-A medium, which was further supplemented with GlutaMAX and gentamycin as described in Methods. Confocal images taken on 8 DIV showed that cultures kept in medium supplemented with BSA only or with BSA + insulin had missing or fragmented pyramidal layers, indicating poor neuronal survival (Fig 2A). Slices maintained in media with 3 other supplements showed intact pyramidal layers and maintained hippocampal morphology. To further compare these 3 supplements, we quantified the neuron numbers in CA3c, CA3b and CA1. Compared with B27 group, BSA+ins+Se+transferrin+putrescine+progesterone group had a significantly lower number of neurons in CA3b (ANOVA with post hoc Holm-Sidak analysis in this and subsequent statistical tests, p = 0.007, n = 3 cultures, each condition) (Fig 2B). BSA + insulin + selenium group showed similar neuron numbers to B27 group in CA3c, CA3b and CA1 (p = 0.222, 0.410, 0.398, respectively, n = 3 cultures, each condition). We conclude that BSA, insulin and selenium were essential and sufficient supplements to Neurobasal-A medium for neuronal survival in organotypic hippocampal cultures until 8 DIV.
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