The opioid receptor antagonist, naloxone, protects spinal motor neurons in a murine model of alphavirus encephalomyelitis

Abstract

Spread of neuroadapted Sindbis virus (NSV) to motor neurons (MN) of the spinal cord (SC) causes severe hind limb weakness in C57BL/6 mice and models the paralysis that can accompany alphavirus and flavivirus encephalomyelitis in humans. The fate of spinal MN dictates the severity of NSV-induced paralysis, and recent data suggest that MN damage can occur indirectly via the actions of activated microglial cells. Because the opioid receptor antagonist, naloxone (NAL), blocks microglial-mediated neurodegeneration in other models, we examined its effects during NSV infection. Drug treatment prevented paralysis and enhanced the survival of MN without altering NSV tropism, replication, or clearance from SC tissue. Further studies showed that NAL most effectively inhibited paralysis in a 72-h window after NSV challenge, suggesting that the drug inhibits an early event in SC pathogenesis. Histochemical studies demonstrated that NAL blocked early microglial activation in SC tissue sections, and protein assays showed that the early induction of pathogenic IL-1 beta was blunted in SC homogenates. Finally, loss of glutamate transporter-1 (GLT-1) expression in SC, an astrocyte glutamate reuptake protein responsible for lowering toxic extracellular levels of glutamate and preventing MN damage, was reversed by NAL treatment. This GLT-1 loss proved to be highly IL-1 beta-dependent. Taken together, these data suggest that NAL is neuroprotective in the SC by inhibiting microglial activation that, in turn, maintains normal astrocyte glutamate homeostasis. We propose that drugs targeting such microglial responses may have therapeutic benefit in humans with related viral infections.

Figure 1

NAL treatment attenuates hind limb paralysis in NSV-infected mice. Hind limb paralysis was assessed in 5-6 week-old mice (n=20 per group) treated with different daily doses of NAL (20 mg/kg, 50 mg/kg, or 100 mg/kg) compared to a saline vehicle control for 7 days following NSV challenge. Each NAL-treated group showed a statistically significant difference in the onset of more severe paralysis compared to the control group (*p < 0.02).

Figure 2

NAL treatment (50 mg/kg/day) increases MN survival in NSV-infected mice without altering virus tropism, replication, or clearance from the SC. (A) The loss of MN axons in lumbar ventral nerve roots among NAL-treated animals (broken line) was significantly reduced compared to untreated controls (solid line) at two time points examined (*p < 0.05, **p < 0.01). (B, C) Immunostaining for viral antigens showed selective staining of motor neurons in both saline-treated (B) and NAL-treated animals (C) in lumbar SC sections. Magnification 400x. (D) Replication and clearance of NSV from the lumbar SC of NAL-treated animals (broken line) was unchanged compared to saline-treated controls (solid line). Each point represents the mean ± SEM of the log₁₀ PFU per gram of tissue from 3 animals. Viral growth curves were not statistically different from each other at any time point.

Figure 3

NAL treatment (50 mg/kg/day) alters an early event in NSV pathogenesis in the SC. Hind limb paralysis in 5-6 week-old mice (n=20 per group) was assessed with NAL treatment starting at 24 hours intervals after viral challenge and continued until day 7 post-infection. (A-C) Three dosing regimens (A, 24 hour delay; B, 48 hour delay; C, 72 hour delay) resulted in a statistically significant delay in the onset of more severe paralysis (*p < 0.02). (D) Delay beyond 72 hours post-infection did not alter the onset or severity of paralysis compared to untreated controls.

Figure 4

NAL treatment (50 mg/kg/day) does not affect immune cell infiltration into the SC of NSV-infected mice. (A) No difference in the number of CD45-positive mononuclear cells were found in the SC between NAL-treated animals (black bars) and saline-treated controls (grey bars). (B) Similarly, the numbers of CD3-positive T cells in SC tissue sections was the same between the two groups. Each bar represents the mean ± SEM of positive cells per cross section of lumbar SC from 3 animals at each time point.

Figure 5

NAL treatment (50 mg/kg/day) reduces the total number of lectin-positive microglial cells found in the lumber SC of NSV-infected mice. Labeled microglia were counted in SC sections from animals with or without NAL treatment at days 1-3 post-infection. Inset photo shows the representative morphology of labeled microglial cells being counted. Each bar represents the mean ± SEM of the number of lectin-positive microglia per cross-section of SC from 6 animals at each time point. NAL treatment significantly reduced the number of labeled microglia detected at day 2 (*p < 0.01) and day 3 (**p < 0.03) post-infection.

Figure 6

NAL treatment (50 mg/kg/day) selectively reduces the local induction of IL-1β in the SC of NSV-infected mice. ELISA was used to measure levels of two pro-inflammatory cytokines, IL-1β and TNF-α, in the lumbar SC homogenates from NSV-infected animals with or without NAL treatment. Each point represents the mean ± SEM of the concentration of IL-1β or TNF-α (pg/mL/μg of total protein) from 3 animals at each time point. NAL treatment (broken line) significantly dampened the local induction of IL-1β, but not TNF-α, compared to saline-treated animals (solid line) on day 3 post-infection (*p <0.05).

Figure 7

NAL treatment (50 mg/kg/day) influences SC expression of the astrocyte glutamate transporter GLT-1 during NSV infection. (A) GLT-1 and actin expression in lumbar SC lysates from mice with or without NAL treatment at different days post-infection (DPI). (B) Quantification of GLT-1 expression relative to actin expression in NAL-treated animals (broken line) compared to saline-treated controls (solid line) normalized to expression levels in uninfected controls. Each point represents the mean ± SEM of the relative integrated density of GLT-1 for 3 animals. (C) Quantification of GLT-1 expression relative to actin expression in lumbar SC lysates from IL-1β^−/− (broken line) and IL-1β^+/+ (solid line) animals. Each point represents the mean ± SEM of the relative integrated density of GLT-1 calculated from 3 animals at each time point and normalized to actin expression.