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What Elements of the Inflammatory System Are Necessary for Epileptogenesis In Vitro?(1,2).

Park KI, Dzhala V, Saponjian Y, Staley KJ - eNeuro (2015)

Bottom Line: Organotypic hippocampal brain slices can be maintained in culture independently of the systemic inflammatory system, and the rapid course of epileptogenesis in these cultures supports the idea that inflammation is not necessary for epilepsy.However, this preparation still retains key cellular inflammatory mediators.These data support the idea that although the inflammatory system, neurons, and glia share key intercellular signaling molecules, neither systemic nor CNS-specific cellular elements of the immune and inflammatory systems are necessary components of epileptogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Massachusetts General Hospital , Boston, Massachusetts 02129 ; Seoul Paik Hospital, Inje University , Seoul 100-032, South Korea.

ABSTRACT
Epileptogenesis in vivo can be altered by manipulation of molecules such as cytokines and complement that subserve intercellular signaling in both the inflammatory and central nervous systems. Because of the dual roles of these signaling molecules, it has been difficult to precisely define the role of systemic inflammation in epileptogenesis. Organotypic hippocampal brain slices can be maintained in culture independently of the systemic inflammatory system, and the rapid course of epileptogenesis in these cultures supports the idea that inflammation is not necessary for epilepsy. However, this preparation still retains key cellular inflammatory mediators. Here, we found that rodent hippocampal organotypic slice cultures depleted of T lymphocytes and microglia developed epileptic activity at essentially the same rate and to similar degrees of severity as matched control slice cultures. These data support the idea that although the inflammatory system, neurons, and glia share key intercellular signaling molecules, neither systemic nor CNS-specific cellular elements of the immune and inflammatory systems are necessary components of epileptogenesis.

No MeSH data available.


Related in: MedlinePlus

Long-term assays of epileptogenesis in microglia-depleted versus control hippocampal slice cultures. A, Examples of slice culture brightfield micrographs at DIV0 prior to clodronate treatment of upper slice. B, Brightfield micrographs of the same slice cultures on DIV6 at the conclusion of clodronate treatment to the upper slice and empty liposome treatment of the lower slice. No deleterious effects of clodronate are evident at this magnification. C, Cumulative group mean lactate production, assayed in the spent culture media at 3-4 daintervals during twice weekly media changes. N = 3 slices each group; all slices from the same animal. D, Cumulative group mean LDH release, assayed in the spent culture media. Same groups slices and media as for lactate assays in panel C. All values are expressed as mean ± SEM. Scale bar, 250 μm.
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Figure 6: Long-term assays of epileptogenesis in microglia-depleted versus control hippocampal slice cultures. A, Examples of slice culture brightfield micrographs at DIV0 prior to clodronate treatment of upper slice. B, Brightfield micrographs of the same slice cultures on DIV6 at the conclusion of clodronate treatment to the upper slice and empty liposome treatment of the lower slice. No deleterious effects of clodronate are evident at this magnification. C, Cumulative group mean lactate production, assayed in the spent culture media at 3-4 daintervals during twice weekly media changes. N = 3 slices each group; all slices from the same animal. D, Cumulative group mean LDH release, assayed in the spent culture media. Same groups slices and media as for lactate assays in panel C. All values are expressed as mean ± SEM. Scale bar, 250 μm.

Mentions: As epileptogenesis progresses in organotypic slices, more SLAs are generated (Dyhrfjeld-Johnsen et al., 2010), and just as in human epilepsy (Lazeyras et al., 2000; Canas et al., 2010), the increased epileptiform activity is associated with increases in local lactate (Berdichevsky et al., 2013). Ictal excitotoxic neuronal injury releases the cytoplasmic LDH to the culture media (Berdichevsky et al., 2012, 2013). To be certain that we had not missed a critical compensation that normalized epileptogenesis in the slices depleted of microglia, we used the lactate and LDH assays to follow the detailed time course of epileptogenesis in vitro. Mouse hippocampal slices were exposed to clodronate (0.2 mg/ml) at DIV0-6 (Fig. 6A,B), and lactate and LDH levels were assayed at each subsequent media change in three depleted versus three control slices from the same animal. Cumulative lactate production was slightly reduced in microglial-deficient slices (Fig. 6C), but this reduction was in line with the 10% reduction in total cell number due to microglial depletion (Benarroch, 2013), did not reach statistical significancew at any time point, and was much less than the lactate reduction during anticonvulsant treatment (Berdichevsky et al., 2012). Cumulative LDH release was increased early in the clodronate-treated slices (Fig. 6D), consistent with microglial cell death, but this difference was not sustained statisticallyx.


What Elements of the Inflammatory System Are Necessary for Epileptogenesis In Vitro?(1,2).

Park KI, Dzhala V, Saponjian Y, Staley KJ - eNeuro (2015)

Long-term assays of epileptogenesis in microglia-depleted versus control hippocampal slice cultures. A, Examples of slice culture brightfield micrographs at DIV0 prior to clodronate treatment of upper slice. B, Brightfield micrographs of the same slice cultures on DIV6 at the conclusion of clodronate treatment to the upper slice and empty liposome treatment of the lower slice. No deleterious effects of clodronate are evident at this magnification. C, Cumulative group mean lactate production, assayed in the spent culture media at 3-4 daintervals during twice weekly media changes. N = 3 slices each group; all slices from the same animal. D, Cumulative group mean LDH release, assayed in the spent culture media. Same groups slices and media as for lactate assays in panel C. All values are expressed as mean ± SEM. Scale bar, 250 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4596089&req=5

Figure 6: Long-term assays of epileptogenesis in microglia-depleted versus control hippocampal slice cultures. A, Examples of slice culture brightfield micrographs at DIV0 prior to clodronate treatment of upper slice. B, Brightfield micrographs of the same slice cultures on DIV6 at the conclusion of clodronate treatment to the upper slice and empty liposome treatment of the lower slice. No deleterious effects of clodronate are evident at this magnification. C, Cumulative group mean lactate production, assayed in the spent culture media at 3-4 daintervals during twice weekly media changes. N = 3 slices each group; all slices from the same animal. D, Cumulative group mean LDH release, assayed in the spent culture media. Same groups slices and media as for lactate assays in panel C. All values are expressed as mean ± SEM. Scale bar, 250 μm.
Mentions: As epileptogenesis progresses in organotypic slices, more SLAs are generated (Dyhrfjeld-Johnsen et al., 2010), and just as in human epilepsy (Lazeyras et al., 2000; Canas et al., 2010), the increased epileptiform activity is associated with increases in local lactate (Berdichevsky et al., 2013). Ictal excitotoxic neuronal injury releases the cytoplasmic LDH to the culture media (Berdichevsky et al., 2012, 2013). To be certain that we had not missed a critical compensation that normalized epileptogenesis in the slices depleted of microglia, we used the lactate and LDH assays to follow the detailed time course of epileptogenesis in vitro. Mouse hippocampal slices were exposed to clodronate (0.2 mg/ml) at DIV0-6 (Fig. 6A,B), and lactate and LDH levels were assayed at each subsequent media change in three depleted versus three control slices from the same animal. Cumulative lactate production was slightly reduced in microglial-deficient slices (Fig. 6C), but this reduction was in line with the 10% reduction in total cell number due to microglial depletion (Benarroch, 2013), did not reach statistical significancew at any time point, and was much less than the lactate reduction during anticonvulsant treatment (Berdichevsky et al., 2012). Cumulative LDH release was increased early in the clodronate-treated slices (Fig. 6D), consistent with microglial cell death, but this difference was not sustained statisticallyx.

Bottom Line: Organotypic hippocampal brain slices can be maintained in culture independently of the systemic inflammatory system, and the rapid course of epileptogenesis in these cultures supports the idea that inflammation is not necessary for epilepsy.However, this preparation still retains key cellular inflammatory mediators.These data support the idea that although the inflammatory system, neurons, and glia share key intercellular signaling molecules, neither systemic nor CNS-specific cellular elements of the immune and inflammatory systems are necessary components of epileptogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Massachusetts General Hospital , Boston, Massachusetts 02129 ; Seoul Paik Hospital, Inje University , Seoul 100-032, South Korea.

ABSTRACT
Epileptogenesis in vivo can be altered by manipulation of molecules such as cytokines and complement that subserve intercellular signaling in both the inflammatory and central nervous systems. Because of the dual roles of these signaling molecules, it has been difficult to precisely define the role of systemic inflammation in epileptogenesis. Organotypic hippocampal brain slices can be maintained in culture independently of the systemic inflammatory system, and the rapid course of epileptogenesis in these cultures supports the idea that inflammation is not necessary for epilepsy. However, this preparation still retains key cellular inflammatory mediators. Here, we found that rodent hippocampal organotypic slice cultures depleted of T lymphocytes and microglia developed epileptic activity at essentially the same rate and to similar degrees of severity as matched control slice cultures. These data support the idea that although the inflammatory system, neurons, and glia share key intercellular signaling molecules, neither systemic nor CNS-specific cellular elements of the immune and inflammatory systems are necessary components of epileptogenesis.

No MeSH data available.


Related in: MedlinePlus