Pyrethroid Insecticides Directly Activate Microglia Through Interaction With Voltage-Gated Sodium Channels

Abstract

Microglia are considered to be the resident immune cells of the central nervous system and contribute significantly to ongoing neuroinflammation in a variety of neurodegenerative diseases. Recently, we and others identified that voltage-gated sodium channels (VGSC) are present on microglia cells and contribute to excessive accumulation of intracellular Na⁺and release of major pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). Based on this finding and the fact that pyrethroid pesticides act on VGSC, we hypothesized that exposure of microglia to the pyrethroid pesticides, permethrin and deltamethrin, would activate microglia and increase the release of TNF-α. BV2 cells or primary microglia were treated with 0–5 µM deltamethrin or permethrin in the presence or absence of tetrodotoxin (TTX), a VGSC blocker for 24–48 h. Both pyrethroids caused a rapid Na⁺influx and increased accumulation of intracellular sodium [(Na⁺)i] in the microglia in a dose- and time-dependent manner, which was significantly reduced by TTX. Furthermore, deltamethrin and permethrin increased the release of TNF-α in a dose- and time-dependent manner, which was significantly reduced by pre-treatment of cells with TTX. These results demonstrate that pyrethroid pesticides may directly activate microglial cells through their interaction with microglial VGSC. Because neuroinflammation plays a key role in many neurodegenerative diseases, these data provide an additional mechanism by which exposure to pyrethroid insecticides may contribute to neurodegeneration.

Uncontrolled inflammation in the brain is thought to play a pivotal role in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Microglia are resident immune cells in the brain, beneficial for neuroprotection and repair of neurons in the central nervous system but can become mediators of neuronal death when over-activated in response to neuronal injury or toxic stimuli (Aloisi, 2001; Gotoh et al., 2011; Su et al., 2009). Long-lasting uncontrolled microglial activation can lead to chronic neuroinflammation resulting in neuronal death and subsequently may lead to neurodegeneration (Stoll et al., 2002). Tumor necrosis factor alpha (TNF-α) is a potent pro-inflammatory cytokine that plays a pivotal role in initiating and sustaining the inflammatory process. Elevated levels of TNF-α have been found in many neurological disorders including Alzheimer’s disease (Alvarez et al., 2007) and Parkinson’s disease (Mogi et al., 1994).

A number of signal transduction pathways are involved in regulation of microglial activation and cytokine secretion (Black and Waxman, 2012). Recently, attention has turned to the fact that microglia express several ion channels, including Na⁺channels, which may participate in this process (Black et al., 2009; Pappalardo et al., 2016; Stevens et al., 2013). Voltage-gated Na⁺channels (VGSC) are transmembrane protein complexes composed of a large α-subunit and one or more small auxiliary β-subunits that modify the properties of channel-gating kinetics, expression, and cellular communication. Several recent studies have shown that VGSC in microglia regulate a variety of cellular functions such as morphological transformation, migration, proliferation, and phagocytosis in response to inflammatory stimuli with lipopolysaccharide (LPS) (Black et al., 2009; Stevens et al., 2013). We recently reported that Na⁺influx through VGSC initiates activation of microglia and subsequently triggers an inflammatory pathway by accumulation of intracellular sodium [(Na⁺)_i] after LPS exposure (Hossain et al., 2013a; Jung et al., 2013).

Pyrethroid insecticides cross the blood-brain (Singh et al., 2012a) and induce neurotoxicity by prolonging the opening of VGSC (Narahashi, 1996). Due to their potency, pyrethroids are widely used to control insect-pests in agriculture, veterinary medicine, and domestic settings in the world (Morgan, 2012). Recently, attention has focused on the potential human health risks associated with pyrethroid exposure as use of these pesticides has significantly increased (DeMicco et al., 2010; Domingues et al., 2016; Viel et al., 2015). There is also rising evidence that long-term/low-dose pyrethroid exposure may have significant neurotoxic effects (Baltazar et al., 2014). Importantly, nigrostriatal dopaminergic neurodegeneration has been reported in adult rats following 12 weeks of cypermethrin exposure (Singh et al., 2012a, b). Furthermore, exposure to the type II pyrethroid deltamethrin has been identified to cause apoptotic cell both in vitro (Hossain and Richardson, 2011) and in vivo (Hossain et al., 2015).

Emerging evidence indicates that neuroinflammation play a crucial role in the pathogenesis of neurodegenerative diseases. Yet, few data related to the effects of pyrethroid pesticides on neuroinflammation exist in the literature. Most recently, one study showed that exposure to a mixture of permethrin, pyridostigmine bromide, and DEET causes microglial activation in rat hippocampus (Parihar et al., 2013) and another study showed that IL-1α and IL-1β increase in mouse brain following exposure to a high dose of cypermethrin (Maurya et al., 2012). However, the mechanism(s) responsible for this observation is not clear.

Here, we report that exposure to the pyrethroid insecticides permethrin and deltamethrin causes a rapid Na⁺ influx into microglia cells and their continued exposure for 24–48 h leads to increased accumulation of intracellular sodium [(Na⁺)_i]_. Our data further demonstrate that excessive accumulation of [(Na⁺)_i] results in microglial cell activation and release of TNF-α, which can be reduced by inhibition of VGSC with tetrodotoxin (TTX). Taken in concert, these data demonstrate that pyrethroid pesticides may cause neuroinflammation through direct activation of microglia through their interaction with microglial VGSC.

MATERIALS AND METHODS

Cell culture

Immortalized mouse (C57Bl/6) microglial cells, BV2 (a kind gift from Dr. Bin Liu, University of Florida), were cultured in minimum essential medium (MEM, Cat #10-010-CV, Mediatech Inc., Manassas, Virginia) containing 10% fetal bovine serum (Cat #35-010-CV, Mediatech Inc.), 2 mM l-glutamine (Cat #35-010-CV, Mediatech Inc.), 1 mM sodium pyruvate (Cat #25-000-CI, Mediatech Inc.), 1 mM non-essential amino acids (Cat #25-025-CI, Mediatech Inc.), and 50 IU penicillin, and 50 µg/mL streptomycin (Penicillin–Streptomycin Solution, Cat #30-001-CI, Mediatech Inc.) and maintained in a temperature (37 °C) and CO₂ (5%) controlled humidified atmosphere as described previously (Gibson et al., 2012; Hossain et al., 2013a). Stock solutions (10 mM) of permethrin and deltamethrin were each prepared in absolute ethanol and stored at 4°C for up to 1 month. Dilutions were made in MEM medium and added to the cells in the presence or absence of the Na⁺channel antagonist, tetrodotoxin (TTX, cat #T-500; Alomone Labs, Jerusalem, Israel). A 1 mM stock solution was prepared in deionized water and aliquots (30 µL/tube) stored at −20°C for up to 1 year. The dose for TTX (1 µM) was selected to block TTX-sensitive sodium channels (Roy and Narahashi, 1992) and was the basis of our previous work (Hossain and Richardson, 2011; Hossain et al., 2013a).

Primary microglia

Primary microglia were isolated from 1- to 2-day-old C57BL/6 pups as previously described (Hossain et al., 2013a). In brief, meninges were removed from brain and then collected in ice cold DMEM-F12 (GIBCO Cat #11320, Life Technologies Inc., Grand Island, New York) complete medium and dissociated in warm DMEM-F12 complete after enzymatic digestion in 0.25% trypsin EDTA (Cat #25-053-CI) in a 37 °C water bath for 30 min. Dissociated cells were passed through a nylon mesh sterile cell strainer to remove debris. The cells were then suspended in DMEM-F12 complete medium and seeded in T-75 tissue culture flasks. Following 14 days culturing, microglia were separated from mixed glial cells using EasySep mouse CD11b positive selection kit (STEMCELL Technologies Inc., Vancouver, Canada) in accordance with the manufacturer’s instructions and isolated microglia were then plated for experiments.

RNA isolation and cDNA synthesis

Total cellular RNA was isolated using a Qiagen RNeasy mini kit (Valencia, California) and concentration was determined on a NanoDrop 2000 Spectrophotometer (Thermo Scientific), as described previously (Fortin et al., 2013). One microgram of total RNA was reverse transcribed using Superscript II kit (Invitrogen, San Diego, California) for cDNA synthesis according to the manufacturer's protocol.

Quantification of PCR products

Following PCR amplification, 16 μL of PCR product was mixed with 4 µL ethidium bromide (4:1) and then 15 μL was loaded per lane on a 1% agarose gel. After 30 min of electrophoresis, the gel was washed with distilled water and bands were then detected by UV detection using an alpha Innotech Fluorchem (San Leantro, California) imaging system and stored as a digital image. Band densitometric analysis was performed using alpha view software (San Jose, California). Actin was used as a house keeping gene to normalize the VGSC isoforms. Primers for sodium channel isoforms were designed using the Primer Blast program (NCBI) and are listed in Table 1.

TABLE 1.

Primer Sequences for qPCR Designed with Primer Blast (NCBI)

Mouse Primers	Forward	Reverse
Nav 1.1 (Scn1a)	5′-ATGTGGAGTACACCTTCACAGGAA-3′	5′-AACTCCGTCACATATGCGAATG-3′
Nav 1.2 (Scn2a)	5′-AGGTGCAATCCCATCCATAATG-3′	5′-GTTGACCACGCTCACATCAAAC-3′
Nav 1.3 (Scn3a)	5′-TGAATGTGCTACTGGTGTGCC-3′	5′-GCCTGACAGTCGCTGAAATTG-3′
Nav 1.4 (Scn4a)	5′-GTCCCCCAGCTCTAGCCAG-3′	5′-CCGGTTCCCCTTTTCAGG-3′
Nav 1.5 (Scn5a)	5′-ACCTGGACCCTTTCTATAGTACCCA-3′	5′-AGCGAATGTACCAAAATCTTCACA-3′
Nav 1.6 (Scn8a)	5′-CAAAGGGATCCGCACCCT-3′	5′-GTCATCAATGCCGGCCTC-3′
Nav 1.7 (Scn9a)	5′-GTCAGAGGAAAGCATCCGAAAG-3′	5′-GAGACTTGTTCTGCTGCTGCG-3′
Nav 1.8 (Scn10a)	5′-TGGCCCTCTTAGAATCCCAA-3′	5′-CTTCAGAGAATCCAACTCCCCA-3′
Nav 1.9 (Scn11a)	5′-ACGTGCTCTTCCACAAACTG-3′	5′-AGGACCACAGTCAGGTTTCC-3′
Nav 2.1 (Scn7a)	5′-CAAGGATTGTGTCTGCCACATAAA-3′	5′-GGCCTGGCCTGCAACC-3′
β actin	5′-AGAGGGAAATCGTGCGTGAC-3′	5′-AACCGCTCGTTGCAATAGT-3′

Cytotoxicity assay

Cell viability was assessed with AlamarBlue cell viability reagent (Invitrogen, Grand Island, New York). Cells (3 × 10⁴ cells/well) were seeded in 96 well plates and treated with permethrin or deltamethrin (0, 0.5, 1, 5, 10, 25, 50, and 100 µM) for 24 and 48 h. At the end of treatment, the cells were incubated in 10 µL AlamarBlue containing 100 µL MEM medium for 3 h at 37 °C. The formation of resorufin was measured at excitation 570 nm and emission 585 nm using a Spectramax microplate reader (Molecular Devices, Sunnyvale, California).

Cell morphology

Briefly, following 24 h exposure to 5 µM deltamethrin or permethrin, both BV2 cells and primary microglia were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Cells were also treated with LPS (10 ng/mL) as a positive control (Hossain et al., 2013a). Following fixation, the chamber was removed, cells were covered with coverslips and sealed with prolong gold without DAPI. Changes in cell morphology were examined visually by phase contrast using an inverted microscope (Zeiss Axio Observer, Thornwood, New York).

Measurement of Na⁺ influx and intracellular Na⁺ [(Na⁺)_i]

Na⁺influx and [(Na⁺)_i] concentration were measured as previously described (Hossain and Richardson, 2011). Briefly, after 12 h in culture, microglia cells were rinsed once with PBS and then incubated with 10 µM CoroNa Green-AM (Invitrogen, Grand Island, New York) in oxygenated Krebs–Ringer-HEPES buffer (KRHB) for 1 h at 37 °C. Cells were rinsed twice with KRHB and then test compounds were added after obtaining a stable baseline. Na⁺influx was determined by measuring fluorescence intensity every 30 s for 30 min with excitation 492 nm and emission 516 nm. Accumulation of [(Na⁺)_i] in BV2 cells was measured 24 and 48 h after permethrin or deltamethrin exposure with CoroNa green indicator as described above.

Quantification of TNF-α

The levels of TNF-α were measured in cell culture medium as previously described (Hossain et al., 2013a). Briefly, following 24 and 48 h pyrethroid exposure, cultured media were collected and levels of TNF-α were quantified by using a mouse enzyme-linked immunosorbent assay (ELISA) kit in accordance with the manufacturer’s instructions (Invitrogen, California).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.01 software (GraphPad Software, Inc., La Jolla, California). Data are presented as mean ± SEM from at least 3 individual experiments run in duplicate or triplicate. All analyses were performed on raw data using analysis of variance (ANOVA) or Student’s t-test where appropriate. For ANOVA, Bonferroni’s or Tukey’s post-hoc multiple comparison tests were performed where appropriate. The P value < .05 was considered statistically significant.

RESULTS

Permethrin and Deltamethrin Are Cytotoxic to Microglial Cells at High Concentrations

To determine the time-course and dose-response of permethrin and deltamethrin cytotoxicity, microglial cells were exposed to various concentrations (0–100 μM) of permethrin or deltamethrin for 24–48 h. Both deltamethrin and permethrin at concentrations of 25 µM and above significantly decreased cell viability in a concentration- and time-dependent manner (Figure 1A and B). Thus, the subsequent experiments were carried out with lower doses (500 nM–5 μM) of permethrin and deltamethrin which did not cause overt toxicity on BV2 cells and primary microglia (Figure 1A and B).

FIG. 1.

Cytotoxic effect of pyrethroids on BV2 cells and primary microglia cells. Cells were treated with the type I pyrethroid permethrin (A) and the type II pyrethroid deltamethrin (B) at the indicated concentrations for 24–48 h. Cell viability was determined using the alamarBlue assay and expressed as percentages of viability. Data are expressed as the mean ± SEM of 3 independent experiment run in duplicate. * Represents significantly different (P < .05) from control.

Morphological Activation of Microglia Following Exposure to Pyrethroid Insecticide

To determine whether pyrethroid insecticides directly activate microglia, we examined their morphology 24 h after 5 µM of permethrin or deltamethrin exposure. LPS (10 ng/mL) treated cells were used as a positive control (Hossain et al., 2013a). As shown in Figure 2, both permethrin- and deltamethrin-stimulated microglia cells exhibited morphological transformation compared with non-treated control cells. Control cells were uniformly round with occasional thin cytoplasmic projections. Upon pyrethroid exposure, cells became much larger, exhibiting thick cytoplasmic projections, which indicate the activation of microglia (Gibson et al., 2012; Kettenmann et al., 2011).

FIG. 2.

Activation of microglia following exposure to pyrethroid insecticides. Microglia were treated with LPS (10 ng/ml), permethrin (5 µM), or deltamethrin (5 µM) for 24 h. LPS was used as a positive control for microglial activation. Phase contrast images of BV2 cells (upper panel) and primary microglia (lower panel) in culture. Microglia with ramified morphology in a resting state (A) and transformation to phagocytic morphology when stimulated with LPS (B), permethrin (C), or deltamethrin (D). Scale bar = 20 µM.

Expression of Sodium Channel Isoform mRNA in BV2 Cells and Primary Microglia

Because Na⁺influx through VGSC initiates activation of microglia (Hossain et al., 2013a), we characterized the presence of sodium channel isoforms in BV2 cells and primary microglia by mRNA expression with standard PCR. We found that BV2 cells express a number of sodium channel isoforms including Na_v 1.2, 1.3, 1.4, 1.6, 1.8, 1.9, and 2.1 (Figure 3A) whereas primary microglia express channel isoforms Na_v 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 1.8, 1.9, and 2.1 (Figure 3B). Primary microglia expressed higher levels of Na_v 1.1. 1.2, 1.3, 1.6, 1.9, and 2.1 as compared with BV2 cells.

FIG. 3.

The presence of sodium channels isoforms in BV2 cells (A) and primary microglia (B). Sodium channel isoform presence was characterized by mRNA expression using standard PCR and agarose gel electrophoresis. Actin expression was determined to normalize the sodium channel isoform in each sample. Bar graphs demonstrate relative densities from representative blots. The values represent mean ± SEM from 3 individual experiments.

Pyrethroid Exposure Causes a Rapid Na⁺ Influx and Accumulation of [(Na⁺)_i] in Microglia Cells

Based on the presence of VGSC on microglia cells (Figure 3) and the fact that pyrethroid pesticides delay the inactivation of VGSC, we measured Na⁺influx and [(Na⁺)_i] in BV2 and primary microglia cells after exposure to pyrethroid insecticides. Cells were treated with 5 µM permethrin or deltamethrin and Na⁺influx was measured every 30 s for 30 min with CoroNa Green-AM. Both pyrethroid insecticides caused a rapid and sustained increase in Na⁺influx, which was completely abolished by 1 µM TTX (Figure 4). Permethrin increased the Na⁺influx by about 28% into BV2 cells (Figure 4A) and about 29% into primary microglia (Figure 4B). Deltamethrin increased influx about 43% into BV2 cells (Figure 4A) and about 28% into primary microglia (Figure 4B).

FIG. 4.

Pyrethroid insecticides permethrin and deltamethrin cause rapid Na⁺ influx in microglia that is inhibited by sodium channel antagonist tetrodotoxin (TTX). Representative traces of Na⁺influx in BV2 cells (A) and primary microglia (B). Data represent mean ± SEM of 3–5 individual experiments, each performed in triplicate and expressed as percentage of control.

[(Na⁺)_i] was measured 24 and 48 h after permethrin or deltamethrin exposure with or without inhibition of VGSC to determine their effects on long-term accumulation of Na⁺. Both insecticides caused a dose- and time-dependent accumulation of [(Na⁺)_i] in BV2 (Figure 5) and primary microglia cells (Figure 6). Permethrin increased [(Na⁺)_i] 124-143% in BV2 cells and 124–128% in primary microglia cells at 24 h (Figure 5A and C) after exposure with 1 and 5 µM permethrin, respectively. [(Na⁺)_i] increased to 131–157% in BV2 cells and to 138–205% in primary microglia cells at 48 h (Figure 5B and D) after exposure with 1 and 5 µM permethrin, respectively. Deltamethrin increased (Na⁺)_i 135–153% in BV2 cells and 133–152% in primary microglia cells at 24 h (Figure 6A and C) and was 166–195% in BV2 cells and159–219% in primary microglia at 48 h (Figure 6B and D) after exposure to 1 and 5 µM deltamethrin, respectively. Inhibition of VGSC with TTX significantly reduced intracellular Na⁺accumulation after 24 and 48 h of permethrin or deltamethrin treatment.

FIG. 5.

Changes in (Na⁺)_i in microglia following exposure to pyrethroid insecticide permethrin. Increase (Na⁺)_i in BV2 cells (A–B) and primary microglia (C–D). Inhibition of VGSC with TTX (1 µM) prevented permethrin-induced excessive accumulation of (Na⁺)_i. Data represent mean ± SEM of 3–5 individual experiments, each performed in triplicate and expressed as percentage of control. * Represents significantly different from control (p < 0.05) and # represents significantly different from permethrin (p < 0.05).

FIG. 6.

Changes in (Na⁺)_i in microglia following exposure to deltamethrin. Increases (Na⁺)_i in BV2 cells (A–B) and primary microglia (C–D). Inhibition of VGSC with TTX (1µM) prevented excessive accumulation of (Na⁺)_i caused by deltamethrin. Data represent mean ± SEM of 3–5 individual experiments, each performed in triplicate and expressed as percentage of control. * Represents significantly different from control (p < 0.05) and # represents significantly different from deltamethrin (p < 0.05).

Inhibition of VGSC Reduces TNF-α Release Following Pyrethroid Exposure

TNF-α is a potent pro-inflammatory cytokine that plays a crucial role in initiating and sustaining the inflammatory process, and persistent activation of microglia has been found to increase the release of TNF-α (Lull and Block, 2010). Thus, release of TNF-α was measured 24 and 48 h after permethrin or deltamethrin exposure with or without inhibition of VGSC. Pyrethroid stimulated-BV2 and -primary microglia released TNF-α in dose- and time-dependent manner (Figs. 7 and 8). Permethrin increased the release of TNF-α from BV2 cells about 34-fold at 24 h (Figure 7A) and about 100-fold at 48 h (Figure 7B), whereas release was increased by 100-fold at 24 h (Figure 7C) and 325-fold at 48 h (Figure 7D) in primary microglia. In contrast to permethrin, deltamethrin increased the release of TNF-α from BV2 cells about 75-fold at 24 h (Figure 8A) and about 110-fold at 48 h (Figure 8B), whereas release was increased by 135-fold at 24 h (Figure 8C) and 300-fold at 48 h (Figure 8D) in primary microglia. Effects of both pyrethroids on the release of TNF-α were significantly reduced by TTX.

FIG. 7.

TTX reduces TNF-α secretion in response to permethrin. Pre-treatment of cells with the VGSC antagonist TTX (1µ M) prevented permethrin-induced TNF-α secretion from BV2 cells (A–B) and primary microglia (C–D). Data represent mean ± SEM of 3–5 individual experiments, each performed in triplicate and expressed as ng/ml. *Indicates significantly different from control (p < .05). #Indicate significant differences between permethrin-treated cells and permethrin plus TTX-treated cells (p < .05).

FIG. 8.

TTX reduces TNF-α secretion in response to deltamethrin. Pre-treatment with the VGSC antagonist TTX (1µM) prevented deltamethrin-induced TNF-α secretion from BV2 cells (A–B) and primary microglia (C–D). Data represent mean ± SEM of 3–5 individual experiments, each performed in triplicate and expressed as ng/ml. *Indicates significantly different from control (p < .05) and #Indicate significant differences between deltamethrin-treated cells and deltamethrin plus TTX-treated cells (p < .05).

DISCUSSION

Microglia are beneficial for neuroprotection and repair of neurons in the central nervous system (Gotoh et al., 2011), but can become mediators of neuronal injury when over-activated in response to neuronal insult (Lee et al., 2010). Persistent activation of microglia leads to chronic neuroinflammation by increasing the release of toxic reactive oxygen species (ROS) or inflammatory cytokines such as TNF-α (Lull and Block, 2010). Here, we demonstrate that exposure to low levels of the pyrethroid insecticides permethrin and deltamethrin causes a rapid and sustained Na⁺ influx into microglial cells through VGSC, leading to excessive accumulation of intracellular Na⁺, which subsequently results in microglial activation and increased release of pro-inflammatory cytokine TNF-α.

In addition to generation of action potentials in neurons, VGSC mediate a number of cellular functions in non-neuronal cells, including microglia (Black et al., 2009; Eder, 2005). A number of recent studies demonstrate that microglia express VGSC and that the blockade of these channels affects multiple functions of these immune cells (Aloisi, 2001; Black and Waxman, 2012; Mantegazza et al., 2010; Waxman, 2008). Experimental studies demonstrated that the VGSC contribute to phagocytic activity of microglia when stimulated with LPS (Black et al., 2009). LPS exposure initiated a series cellular events including microglial activation that are dependent on interaction of with VGSC, resulting in elevation of [(Na⁺)_i], activation of NADPH oxidase and secretion of pro-inflammatory cytokines (Black et al., 2009; Hossain et al., 2013a; Jung et al., 2013). Here, we found that Na⁺ influx through VGSC activates microglia and subsequently induces inflammatory responses following exposure to permethrin and deltamethrin. The activation of microglia was accompanied by its phagocytic morphology (retraction of cell processes, increased size, and an amoeboid shape) with increased TNF-α release (Kim and Joh, 2006; Lull and Block, 2010). These findings are similar to that we (Hossain et al., 2013a) and others (Black et al., 2009; Jung et al., 2013; Liu et al., 2010) previously reported, that the VGSC antagonist TTX and antiepileptic drugs reduce microglial activation by LPS. Thus, we hypothesized that pyrethroid insecticides could activate microglia through direct interaction with VGSC.

Pyrethroid insecticides interact with VGSC and prolong sodium current activation, but their actions are varied based on the differences in their chemical structures and 3-dimensional configuration (Soderlund, 2012; Soderlund et al., 2002). For example, Type I pyrethroids (ie, allethrin and permethrin) lack a cyano moiety and hold the channels open for relatively short period (milliseconds) whereas Type II pyrethroids (ie, cyhalothin and deltamethrin) have an α-cyanophenoxy benzyl moiety in structure (Ecobicon, 2001; Narahashi, 1992, 1996) and hold the channels open for longer period (seconds). It has been reported that and that type I and type II pyrethroids may actually bind to separate sodium channel sites (Song et al., 1996). Although there are some similarities between type I and type II pyrethroids in their actions on sodium channels, the pattern of modification of sodium currents is different between the 2 types of pyrethroids (Motomura and Narahashi, 2001). For example, deltamethrin-modified sodium channels opened much longer than tetramethrin-modified sodium channels (Motomura and Narahashi, 2001). Furthermore, an electrophysiology study using Xenopus oocytes indicated that permethrin decaying sodium tail current recovered more rapidly than that of deltamethrin (Choi and Soderlund, 2006). Therefore, this may explain why there was more accumulation of intracellular Na⁺ in microglia following exposure to deltamethrin than permethrin.

There are several VGSC isoforms present in brain microglia (Black et al., 2009; Craner et al., 2005; Jung et al., 2013; Sontheimer et al., 1994), but evidence suggests that Na_v1.6 plays a predominant role in modulating microglial function (Black and Waxman, 2012; Hossain et al., 2013a). Electrophysiological studies demonstrated that mouse, rat, and human Na_v 1.6 produce persistent sodium current visible at the end of depolarization, which is not commonly seen with other sodium channels isoforms (Tan and Soderlund, 2010). Continued sodium currents were also found with Na_v 1.6 expressed in oocytes and HEK293 cells following exposure to pyrethroids (Tan and Soderlund, 2010). In contrast to BV2 cells, primary microglia appeared more sensitive to pyrethroid insecticides, as indicated by differences in the accumulation of intracellular Na⁺ and release of TNF-α between BV2 cells and primary microglia. This may be the result of differences in VGSC isoform expression, as we found that Na_v1.6 is more abundantly expressed in primary microglia than the BV2 cells (Hossain et al., 2013a) and Na_v1.6 is particularly sensitive to modification by pyrethroids (Tan and Soderlund, 2010). However, it should also be noted that these primary microglia are isolated from neonatal mice and developing mammals are more sensitive to pyrethroids than adults (Shafer et al., 2005; Wakeling et al., 2012). However, this is thought to be more likely the result of lower levels of pyrethroid detoxication enzymes in the young rather than the expression of juvenile-specific isoforms such as Na_v1.3 (Meacham et al., 2008).

Treatment of BV2 cells and primary microglia with permethrin or deltamethrin for 24 h increased the secretion of TNF-α to similar extent as previously observed with 10–100 ng/mL LPS (Hossain et al., 2013a). Inhibition of VGSC by TTX completely prevented the pyrethroid-induced accumulation of intracellular sodium, but was unable to abolish the secretion of TNF-α, suggesting additional mechanisms may be involved. One possibility relates to the observation that voltage-sensitive Ca²⁺channels may mediate the actions of some pyrethroid compounds (Clark and Symington, 2012; Hossain and Richardson, 2011; Soderlund et al., 2002), particularly at higher doses. Because the effects of the highest concentration (5 µM) of permethrin and deltamethrin on TNF-α release were not completely blocked by TTX, it is possible that the remaining effect was Na⁺-independent and Ca²⁺ channels may be involved in this mechanism. Indeed, we previously found that the rise in Ca²⁺ influx into the SK-N-AS is secondary to the activation of Na⁺ channels following exposure to deltamethrin (Hossain and Richardson, 2011). Evidence from our microdialysis studies also demonstrate that the inhibition of L-type calcium channel can prevent the Na⁺ channel-independent effects of pyrethroids on release of extracellular neurotransmitters in rat (Hossain et al., 2013b; Hossain et al., 2006, 2008). Similarly, Cao et al. (2011) showed that deltamethrin potently stimulates calcium influx in cortical neurons and that secondary calcium influx may be mediated by L-type calcium channels or reversal of the Na⁺/Ca²⁺ exchanger. Furthermore, reverse operation of Na⁺/Ca²⁺ exchanger has been found to contribute to intracellular Ca²⁺ overload during anoxia, ischemia, and malignant hyperthermia (Altamirano et al., 2014; Barrientos et al., 2009). Another possibility relates to Na⁺/H⁺ exchangers (NHE) that can also regulate the cellular functions of microglia (Hossain et al., 2013a; Liu et al., 2010). We and others previously reported that the inhibition of NHE significantly reduced production of TNF-α after activation of microglia with LPS (Hossain et al., 2013a; Liu et al., 2010). Our previous study demonstrated that LPS initiates microglial activation through VGSC and thereafter, this process is maintained through NHE-1 (Hossain et al., 2013a). Finally, we cannot exclude the potential contribution of TTX-resistant VGSC present in microglia because we used a concentration of TTX (1 µM) that would not inhibit those channels. Thus, additional studies are required to determine the relevance of these channels or alternate pathways to the effect of permethrin and deltamethrin on TNF-α release.

Together, the data reported here demonstrate that pyrethroid insecticides may contribute to neuroinflammation by directly activating VGSC present in microglia leading to release of the pro-inflammatory cytokine TNF-α. Co-treatment of microglial cells with TTX resulted in reduction of intracellular Na⁺ accumulation and decreased production of TNF-α, demonstrating the requirement of VGSC. Because sustained neuroinflammation can lead to neurodegeneration (Hossain et al., 2015) and microglial activation has been observed following pyrethroid exposure (Singh et al., 2011), these data suggest that pyrethroid-induced neurodegenerations may be the result of direct interactions of pyrethroids with microglia rather than a secondary effect from interaction with VGSC on neurons. Additional in vivo experiments are required to confirm or refute this hypothesis.

FUNDING

The content of this article is soley the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors declasre that they have no competing financial interests or conflicts of interest. This research was supported in part by NIH Grants: R01ES015991, R01ES021800, P30ES005022, R21NS072097, and U01NS079249; R25ES020721 (to J.R.R.) and ASPET SURF Program (to J.L.).