Experimental autoimmune encephalopathy (EAE)-induced hippocampal neuroinflammation and memory deficits are prevented with the non-opioid TLR2/TLR4 antagonist (+)-naltrexone

Abstract

Multiple sclerosis (MS) is associated with burdensome memory impairments and preclinical literature suggests that these impairments are linked to neuroinflammation. Previously, we have shown that toll-like receptor 4 (TLR4) antagonists, such as (+)-naltrexone [(+)-NTX], block neuropathic pain and associated spinal inflammation in rats. Here we extend these findings to first demonstrate that (+)-NTX blocks TLR2 in addition to TLR4. Additionally, we examined in two rat strains whether (+)-NTX could attenuate learning and memory disturbances and associated neuroinflammation using a low-dose experimental autoimmune encephalomyelitis (EAE) model of MS. EAE is the most commonly used experimental model for the human inflammatory demyelinating disease, MS. This low-dose model avoided motor impairments that would confound learning and memory measurements. Fourteen days later, daily subcutaneous (+)-NTX or saline injections began and continued throughout the study. Contextual and auditory-fear conditioning were conducted at day 21 to assess hippocampal and amygdalar function. With this low-dose model, EAE impaired long-term, but not short-term, contextual fear memory; both long-term and short-term auditory-cued fear memory were spared. This was associated with increased mRNA for hippocampal interleukin-1β (IL-1β), TLR2, TLR4, NLRP3, and IL-17 and elevated expression of the microglial marker Iba1 in CA1 and DG regions of the hippocampus, confirming the neuroinflammation observed in higher-dose EAE models. Importantly, (+)-NTX completely prevented the EAE-induced memory impairments and robustly attenuated the associated proinflammatory effects. These findings suggest that (+)-NTX may exert therapeutic effects on memory function by dampening the neuroinflammatory response in the hippocampus through blockade of TLR2/TLR4. This study suggests that TLR2 and TLR4 antagonists may be effective at treating MS-related memory deficits.

Keywords: Auditory fear conditioning; Contextual fear conditioning; Experimental autoimmune encephalomyelitis; Hippocampus; Multiple sclerosis; Rats; Toll-like receptors.

Copyright © 2020 Elsevier B.V. All rights reserved.

Figures

Figure 1.

Schematic of timeline and experimental design. Four cohorts of rats were used for this study. Cohorts 1-3 were Dark Agouti (DA) rats, whereas cohort 4 was Sprague-Dawley (SD) rats. Motor scores were assessed daily from day −1 to day 26 in all cohorts. On day 0, all rats were injected with either MOG or vehicle. Fourteen days later, half of each group received 3 daily injections of either (+)-NTX or saline until the end of the study. On day 21, cohorts 1, 2, and 4 were conditioned in a contextual fear conditioning paradigm. Cohorts 1 and 4 were tested for long-term memory four days later (day 25), whereas cohort 2 was tested for short-term memory 1-2 hrs later (day 21). The third cohort of rats was used to measure serum anti-MOG antibody levels and mRNA expression of various inflammatory markers in the brain that may have been modulated by EAE and/or (+)-NTX, but not by the behavioral experience. This cohort was euthanized on day 26. Lastly, hippocampi from the fourth cohort (also euthanized on day 26) were assessed via immunohistochemistry.

Figure 2.

Reduction of TLR nitric oxide release by (+)-NTX. Percent nitrite in supernatant (a stable indicator of nitric oxide release) from BV-2 cells treated with agonists of TLR4, TLR1/2; TLR2/6, or TLR7/8, and the indicated concentrations of (+)-NTX. Data are presented as mean ± SEM and analyzed using a two-way ANOVA, n=4 wells per group. Experiments were replicated 5 times. * p

Figure 3.

Long-term memory in Dark Agouti (A-C) and Sprague Dawley (D-F) control or rats with EAE that were treated with (+)-NTX or saline. (A,D) Percentage of time spent freezing in the conditioning context (hippocampal-dependent memory), or (B,E) in a novel context in the presence of the tone (amygdala-dependent memory) 4 days after conditioning. (C,F) Percentage of time spent exploring the context during conditioning prior to shock. Data are presented as mean ± SEM and analyzed using a two-way ANOVA. DA rat: n=8 vehicle-saline and vehicle-(+)-NTX; n=6 MOG-saline and MOG-(+)-NTX. SD rat: n=8 per group. * p

Figure 4.

Short-term memory in control or rats with EAE that were treated with (+)-NTX or saline. (A) Percentage of time spent freezing in the conditioning context (hippocampal-dependent memory), or (B) in a novel context in the presence of the tone (amygdala-dependent memory) 1-2 hours after conditioning. (C) Percentage of time spent exploring the context during conditioning prior to shock. Data are presented as mean ± SEM and analyzed using a two-way ANOVA, n=8 vehicle-saline, vehicle-(+)-NTX, and MOG-saline; n=6 MOG-(+)-NTX group.

Figure 5.

Assessment of IL-1β mRNA in (A) hippocampus and (B) amygdala of naïve rats or rats with EAE that were treated with (+)-NTX or saline. Data are presented as mean ± SEM and analyzed using a one-way ANOVA. Hippocampus: n=8 naïve; n=4 MOG-saline; n=7 MOG-(+)-NTX. Amygdala: n=8 naïve, n=7 MOG-saline; n=8 MOG-(+)-NTX. * p

Figure 6.

Microglial (Iba1) and astrocyte (GFAP) cell immunoreactivity of hippocampal CA1 and DG of control rats or rats with EAE that were treated with (+)-NTX or saline. Data are presented as mean ± SEM and analyzed using a two-way ANOVA, n=8 for all groups except n=7 for vehicle-(+)-NTX group in DG tissue. * p

Figure 7.

Gene expression of the pattern recognition receptors (A) TLR2 and (B) TLR4, (C) the inflammasome NLRP3, and (D) the proinflammatory cytokine IL-17 in the hippocampus of naïve rats or rats with EAE that were treated with (+)-NTX or saline. Data are presented as mean ± SEM and analyzed using a one-way ANOVA, n=8 naïve; n=4 MOG-saline; n=7 MOG-(+)-NTX. * p

Figure 8.

Increased serum anti-MOG IgG antibody concentration in rats with EAE that were treated with (+)-NTX or saline (mg/ml). Data are presented as mean ± SEM and analyzed using a one-way ANOVA, n=6 naive; n=5 MOG-saline; n=4 MOG-(+)-NTX. * p

Figure 9.

Assessment of (+)-NTX administration on motor disturbances in Dark Agouti (DA) and Sprague-Dawley (SD) rats with EAE. Motor scores throughout (A) Cohort 1 (DA rat), (B) Cohort 2 (DA rat), (C) Cohort 3 (DA rat), and (D) Cohort 4 (SD rat). Data are presented as mean ± SEM and analyzed using a repeated measures two-way ANOVA, n=8 for all groups except MOG-(+)-saline (n=7) and MOG-(+)-NTX (n=6) groups in Cohort 1 and MOG-saline (n=7) group in Cohort 3.