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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

Microglial depletion from slices of nude mouse. A, Comparison of the density of Iba1-positive cells in area CA1 in control slices from different species (n = 5-6 per group) versus the percentage of slices that displayed seizure activity (n = 5-8 per group; different slices used for Iba-1 staining and recording). Across species, there was no significant correlation between microglial density and fraction of epileptic slices (R = −0.10, p = 0.94). B, Examples of hippocampal cultures from nude mice at each time point. Liposomal (Lipo) clodronate (top, 0.02 mg/ml) or liposome-control (bottom) was applied from DIV0 to 6. C, D, Double immunostaining was performed with NeuN and Iba-1 antibodies. Quantification at CA1 reveals that liposomal clodronate did not affect the neuronal populations of nude mouse (p = 0.33), whereas it depleted all microglia (n = 5 per group). E, The proportion of epileptic slices and total duration of seizure-like activities in microglia-depleted slices did not differ significantly from control slices, whereas the frequency of seizure-like activity was somewhat lower in microglia-depleted slices (n = 8-10 per group). All values are expressed as mean ± SEM. *p < 0.05; N.S, not significant. Scale bar, 100 μm.
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Figure 5: Microglial depletion from slices of nude mouse. A, Comparison of the density of Iba1-positive cells in area CA1 in control slices from different species (n = 5-6 per group) versus the percentage of slices that displayed seizure activity (n = 5-8 per group; different slices used for Iba-1 staining and recording). Across species, there was no significant correlation between microglial density and fraction of epileptic slices (R = −0.10, p = 0.94). B, Examples of hippocampal cultures from nude mice at each time point. Liposomal (Lipo) clodronate (top, 0.02 mg/ml) or liposome-control (bottom) was applied from DIV0 to 6. C, D, Double immunostaining was performed with NeuN and Iba-1 antibodies. Quantification at CA1 reveals that liposomal clodronate did not affect the neuronal populations of nude mouse (p = 0.33), whereas it depleted all microglia (n = 5 per group). E, The proportion of epileptic slices and total duration of seizure-like activities in microglia-depleted slices did not differ significantly from control slices, whereas the frequency of seizure-like activity was somewhat lower in microglia-depleted slices (n = 8-10 per group). All values are expressed as mean ± SEM. *p < 0.05; N.S, not significant. Scale bar, 100 μm.

Mentions: T lymphocytes are a part of the adaptive immune system that play an important role in activating the innate immune, i.e. inflammatory system, of the brain (Ransohoff and Brown, 2012) and have been hypothesized to contribute to epileptogenesis (Fabene et al. 2008; Marchi et al., 2011). To investigate the role of this cellular component of the immune system on epilepsy more precisely, we compared wild-type organotypic slices to organotypic slices depleted of either T lymphocytes or both microglia and T lymphocytes by treating slices prepared from nude mice with clodronate (0.02 mg/ml). The numbers of microglia were counted in area CA1 in the three different preparations (slice cultures from wild-type (WT), nude (T lymphocyte-deficient) mice, and slices from nude mice treated with clodronate, n = 5-6 each group). Nude mice showed fewer microglia per field (p = 0.003r vs rat, p = 0.01s vs WT mouse; Fig. 5A). However, there was no correlation between the density of microglia and the incidence of epileptic slices across species (Fig. 5A). In mice, a lower concentration (0.02 mg/ml) of clodronate eliminated microglia as effectively as the higher concentration used in rat slices (0.2 mg/ml), so we used the lower concentration in the following experiments. Clodronate at 0.02 mg/ml did not alter neuronal populations (Fig. 5C,D). Epileptogenesis proceeded at a similar rate in clodronate-treated nude mouse slices as in control slices. Recordings at DIV12 demonstrated SLAs in 40.0% of double-deficient slices (n = 10) versus 62.5% in control slices prepared from nude mice (n = 8) (p = 0.36t). Analysis of seizure parameters (Fig. 5E) revealed that the total duration of SLAs in clodronate-treated nude mouse slices was not different from that of control slices (20.0 ± 12.0 s vs 33.1 ± 18.4 s; p = 0.54u). SLAs were rarely observed in both groups. However, the frequency of SLAs in the double-deficient preparations was decreased compared to controls (0.5 ± 0.2 SLAs/h vs 1.6 ± 0.5 SLAs/h; p = 0.051v). Thus, we conclude that epileptogenesis is not dependent on T lymphocytes and/or microglia in this model.


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

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

Microglial depletion from slices of nude mouse. A, Comparison of the density of Iba1-positive cells in area CA1 in control slices from different species (n = 5-6 per group) versus the percentage of slices that displayed seizure activity (n = 5-8 per group; different slices used for Iba-1 staining and recording). Across species, there was no significant correlation between microglial density and fraction of epileptic slices (R = −0.10, p = 0.94). B, Examples of hippocampal cultures from nude mice at each time point. Liposomal (Lipo) clodronate (top, 0.02 mg/ml) or liposome-control (bottom) was applied from DIV0 to 6. C, D, Double immunostaining was performed with NeuN and Iba-1 antibodies. Quantification at CA1 reveals that liposomal clodronate did not affect the neuronal populations of nude mouse (p = 0.33), whereas it depleted all microglia (n = 5 per group). E, The proportion of epileptic slices and total duration of seizure-like activities in microglia-depleted slices did not differ significantly from control slices, whereas the frequency of seizure-like activity was somewhat lower in microglia-depleted slices (n = 8-10 per group). All values are expressed as mean ± SEM. *p < 0.05; N.S, not significant. Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Microglial depletion from slices of nude mouse. A, Comparison of the density of Iba1-positive cells in area CA1 in control slices from different species (n = 5-6 per group) versus the percentage of slices that displayed seizure activity (n = 5-8 per group; different slices used for Iba-1 staining and recording). Across species, there was no significant correlation between microglial density and fraction of epileptic slices (R = −0.10, p = 0.94). B, Examples of hippocampal cultures from nude mice at each time point. Liposomal (Lipo) clodronate (top, 0.02 mg/ml) or liposome-control (bottom) was applied from DIV0 to 6. C, D, Double immunostaining was performed with NeuN and Iba-1 antibodies. Quantification at CA1 reveals that liposomal clodronate did not affect the neuronal populations of nude mouse (p = 0.33), whereas it depleted all microglia (n = 5 per group). E, The proportion of epileptic slices and total duration of seizure-like activities in microglia-depleted slices did not differ significantly from control slices, whereas the frequency of seizure-like activity was somewhat lower in microglia-depleted slices (n = 8-10 per group). All values are expressed as mean ± SEM. *p < 0.05; N.S, not significant. Scale bar, 100 μm.
Mentions: T lymphocytes are a part of the adaptive immune system that play an important role in activating the innate immune, i.e. inflammatory system, of the brain (Ransohoff and Brown, 2012) and have been hypothesized to contribute to epileptogenesis (Fabene et al. 2008; Marchi et al., 2011). To investigate the role of this cellular component of the immune system on epilepsy more precisely, we compared wild-type organotypic slices to organotypic slices depleted of either T lymphocytes or both microglia and T lymphocytes by treating slices prepared from nude mice with clodronate (0.02 mg/ml). The numbers of microglia were counted in area CA1 in the three different preparations (slice cultures from wild-type (WT), nude (T lymphocyte-deficient) mice, and slices from nude mice treated with clodronate, n = 5-6 each group). Nude mice showed fewer microglia per field (p = 0.003r vs rat, p = 0.01s vs WT mouse; Fig. 5A). However, there was no correlation between the density of microglia and the incidence of epileptic slices across species (Fig. 5A). In mice, a lower concentration (0.02 mg/ml) of clodronate eliminated microglia as effectively as the higher concentration used in rat slices (0.2 mg/ml), so we used the lower concentration in the following experiments. Clodronate at 0.02 mg/ml did not alter neuronal populations (Fig. 5C,D). Epileptogenesis proceeded at a similar rate in clodronate-treated nude mouse slices as in control slices. Recordings at DIV12 demonstrated SLAs in 40.0% of double-deficient slices (n = 10) versus 62.5% in control slices prepared from nude mice (n = 8) (p = 0.36t). Analysis of seizure parameters (Fig. 5E) revealed that the total duration of SLAs in clodronate-treated nude mouse slices was not different from that of control slices (20.0 ± 12.0 s vs 33.1 ± 18.4 s; p = 0.54u). SLAs were rarely observed in both groups. However, the frequency of SLAs in the double-deficient preparations was decreased compared to controls (0.5 ± 0.2 SLAs/h vs 1.6 ± 0.5 SLAs/h; p = 0.051v). Thus, we conclude that epileptogenesis is not dependent on T lymphocytes and/or microglia in this model.

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