S-Ketamine Pretreatment Alleviates Anxiety-Like Behaviors and Mechanical Allodynia and Blocks the Pro-inflammatory Response in Striatum and Periaqueductal Gray From a Post-traumatic Stress Disorder Model

Abstract

This study aims to explore the regulatory effect of S-ketamine on the mechanical allodynia, anxiety-like behaviors and microglia activation in adult male rats exposed to an animal model of post-traumatic stress disorder (PTSD). The rat PTSD model was established by the exposure to single-prolonged stress (SPS), and 1 day later, rats were intraperitoneally injected with 5 mg/kg S-ketamine or normal saline, respectively. Paw withdrawal mechanical threshold was measured 2 days before, and 1, 3, 5, 7, 10, 14, 21 and 28 days after injection to assess mechanical allodynia in the SPS-exposed rats. For anxiety-like behaviors, the open field test and elevated plus maze test were performed at 7 and 14 days after S-ketamine treatment in the SPS-exposed rats, respectively. SPS-induced rats presented pronounced mechanical allodynia and anxiety-like behaviors, which were alleviated by S-ketamine treatment. After behavioral tests, rats were sacrificed for collecting the anterior cingulate cortex (ACC), prefrontal cortex (PFC), dorsal striatum, and periaqueductal gray (PAG). Protein levels of TNF-α, IL-1β, p-NF-κB, and NF-κB in brain regions were examined by Western blot. In addition, microglia activation in each brain region was determined by immunofluorescence staining of the microglia-specific biomarker Iba-1. Interestingly, pro-inflammatory cytokines were significantly upregulated in the dorsal striatum and PAG, rather than ACC and PFC. Activated microglia was observed in the dorsal striatum and PAG as well, and upregulated p-NF-κB was detected in the dorsal striatum. Inflammatory response, phosphorylation of NF-κB and microglia activation in certain brain regions were significantly alleviated by S-ketamine treatment. Collectively, S-ketamine is a promising drug in alleviating mechanical allodynia, anxiety-like behaviors, and pro-inflammatory responses in discrete brain regions in a model of PTSD.

Keywords: S-ketamine; microglia; post-traumatic stress disorder; pro-inflammatory cytokines; single prolonged stress.

FIGURE 1

S-ketamine alleviates chronic pain in the SPS-exposed rats. (A) Scheme diagram of behavioral tests and SPS injection in rats. Rats were randomly assigned into control + vehicle group, SPS + vehicle group, control + S-ketamine 5 mg/kg group and SPS + S-ketamine 5 mg/kg group. n = 8 per group. (B) Paw withdrawal mechanical threshold of rats in each group. ***P < 0.001, SPS + vehicle group vs. control + vehicle group; ^###P < 0.001, SPS + S-ketamine 5 mg/kg group vs. SPS + vehicle group.

FIGURE 2

S-ketamine alleviates anxiety-like behaviors in the SPS-exposed rats. Rats were randomly assigned into control + vehicle group, SPS + vehicle group, control + S-ketamine 5 mg/kg group and SPS + S-ketamine 5 mg/kg group. n = 8 per group. (A) Diagram of the open field test. (B) Movement paths in the open field. (C) Time in the center. (D) Entries in the center. (E) Distance traveled. (F) Diagram of the elevated plus maze test. (G) Movement paths in the open and closed arms. (H) Percentage of open-arm entries. (I) Percentage of time in open arms. (J) Anxiety index. *P < 0.05, **P < 0.01, control + vehicle group vs. SPS + vehicle group; ^#P < 0.05, ^##P < 0.01, SPS + S-ketamine 5 mg/kg group vs. SPS + vehicle group.

FIGURE 3

S-ketamine does not influence pro-inflammatory cytokines in the anterior cingulate cortex and prefrontal cortex. Rats were randomly assigned into control + vehicle group, SPS + vehicle group, control + S-ketamine 5 mg/kg group and SPS + S-ketamine 5 mg/kg group. n = 3 per group. (A) Protein levels of TNF-α, IL-1β, p-NF-κB, and NF-κB in the ACC. Relative intensity of TNF-α (B), IL-1β (C), p-NF-κB (D), and NF-κB (E) in the ACC. (F) Protein levels of TNF-α, IL-1β, p-NF-κB, and NF-κB in the PFC. Relative intensity of TNF-α (G), IL-1β (H), p-NF-κB (I), and NF-κB (J) in the PFC.

FIGURE 4

S-ketamine does not influence the microglial activation in the anterior cingulate cortex and prefrontal cortex. Rats were randomly assigned into control + vehicle group, SPS + vehicle group, control + S-ketamine 5 mg/kg group and SPS + S-ketamine 5 mg/kg group. n = 3 per group. (A) Immunofluorescence staining of Iba-1 in ACC and PFC. (B) The number of Iba-1-positive stained cells in the ACC. (C) Relative intensity of Iba-1-positive stained cells in the ACC. (D) The number of Iba-1-positive stained cells in the PFC. (E) Relative intensity of Iba-1-positive stained cells in the PFC.

FIGURE 5

S-ketamine alleviates microglial activation and downregulates pro-inflammatory cytokines in the dorsal striatum. Rats were randomly assigned into control + vehicle group, SPS + vehicle group, control + S-ketamine 5 mg/kg group and SPS + S-ketamine 5 mg/kg group. n = 3 per group. (A) Protein levels of TNF-α, IL-1β, p-NF-κB, and NF-κB in the dorsal striatum. Relative intensity of TNF-α (B), IL-1β (C), p-NF-κB (D), and NF-κB (E) in the dorsal striatum. (F) Immunofluorescence staining of Iba-1 in the dorsal striatum. (G) The number of Iba-1-positive stained cells in the dorsal striatum. (H) Relative intensity of Iba-1-positive stained cells in the dorsal striatum. *P < 0.05, **P < 0.01, and ***P < 0.001, control + vehicle group vs. SPS + vehicle group; ^#P < 0.05, ^###P < 0.001, SPS + S-ketamine 5 mg/kg group vs. SPS + vehicle group.

FIGURE 6

S-ketamine alleviates microglial activation and downregulates pro-inflammatory cytokines in the periaqueductal gray. Rats were randomly assigned into control + vehicle group, SPS + vehicle group, control + S-ketamine 5 mg/kg group and SPS + S-ketamine 5 mg/kg group. n = 3 per group. (A) Protein levels of TNF-α, IL-1β, p-NF-κB, and NF-κB in the PAG. Relative intensity of TNF-α (B), IL-1β (C), p-NF-κB (D), and NF-κB (E) in the PAG. (F) Immunofluorescence staining of Iba-1 in the dorsal striatum. (G) The number of Iba-1-positive stained cells in the PAG. (H) Relative intensity of Iba-1-positive stained cells in the PAG. *P < 0.05, **P < 0.01, and ***P < 0.001, control + vehicle group vs. SPS + vehicle group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001, SPS + S-ketamine 5 mg/kg group vs. SPS + vehicle group.