(2R,6R)-Hydroxynorketamine Treatment of Rats Exposed to Repetitive Low-Level Blast Injury

Abstract

Many military veterans who experienced blast-related traumatic brain injuries (TBIs) in the conflicts in Iraq and Afghanistan suffer from chronic cognitive and mental health problems, including post-traumatic stress disorder (PTSD). Male rats subjected to repetitive low-level blast exposure develop chronic cognitive and PTSD-related traits that develop in a delayed manner. Ketamine has received attention as a treatment for refractory depression and PTSD. (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a ketamine metabolite that exerts rapid antidepressant actions. (2R,6R)-HNK has become of clinical interest because of its favorable side-effect profile, low abuse potential, and oral route of administration. We treated three cohorts of blast-exposed rats with (2R,6R)-HNK, beginning 7-11 months after blast exposure, a time when the behavioral phenotype is established. Each cohort consisted of groups (n = 10-13/group) as follows: 1) Sham-exposed treated with saline, 2) blast-exposed treated with saline, and 3) blast-exposed treated with a single dose of 20 mg/kg of (2R,6R)-HNK. (2R,6R)-HNK rescued blast-induced deficits in novel object recognition (NOR) and anxiety-related features in the elevated zero maze (EZM) in all three cohorts. Exaggerated acoustic startle was reversed in cohort 1, but not in cohort 3. (2R,6R)-HNK effects were still present in the EZM 12 days after administration in cohort 1 and 27 days after administration in NOR testing of cohorts 2 and 3. (2R,6R)-HNK may be beneficial for the neurobehavioral syndromes that follow blast exposure in military veterans. Additional studies will be needed to determine whether higher doses or more extended treatment regimens may be more effective.

Keywords: (2R,6R)-hydroxynorketamine; blast; post-traumatic stress disorder; rat; traumatic brain injury.

FIG. 1.

(2R,6R)-HNK treatment of blast-exposed rats. (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; LTM, long-term memory; LTPM, long-term permanent memory; NOR, novel object recognition; SPI, startle pre-pulse inhibition; STM, short-term memory; VA, Department of Veterans Affairs .

FIG. 2.

Elevated zero maze (EZM) testing of cohort 1. Blast-exposed rats were treated with saline (n = 13) or (2R,6R)-HNK (n = 14) and sham-exposed were treated with saline (n = 12), as indicated in Figure 1. Rats were tested in the EZM. Shown is mean speed (A; one-way ANOVA: F_{2, 34} = 3.952, p = 0.0286), time spent in motion (B; F_{2, 34} = 4.269, p = 0.0222), distance moved (C; F_{2, 34} = 3.981, p = 0.0280), latency to enter an open arm (D; F_2,32 = 4.129, p = 0.0254), open arm entries (E; F_{2, 36} = 0.9055, p = 0.4134), and time spent in the open arms (F; F_{2, 34} = 3.489, p = 0.0430). Error bars indicate the standard error of the mean. Asterisks indicate differences between groups after a significant (p < 0.05) one-way ANOVA (*p < 0.05, Fisher's LSD). (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; LSD, least significant difference.

FIG. 3.

Novel object recognition (NOR) testing of blast-exposed rats from cohort 1 treated with (2R,6R)-HNK. Sham- and blast-exposed rats were treated with saline (n = 12 sham, n = 13 blast) or blast were treated with (2R,6R)-HNK (n = 14). Rats were tested in NOR after treatment as indicated in Figure 1. Panel (A) shows time spent exploring the objects (OB1 and OB2) during the training session. Panel (B) shows exploration of the previously presented familiar object (FO) compared to the novel object (NO) when presented 1 h (STM) after training. Asterisks indicate values significantly different (*p < 0.05, ***p < 0.001, unpaired t-tests). Panel (C) shows a discrimination index, which did not significantly differ between the groups. Panels (D) and (E) show total time spent exploring the objects during the training (one-way ANOVA: F_2,35 = 9.885, p = 0.0004) and STM (F_{2, 34} = 1.506, p = 0.2361) testing (*p < 0.05, ****p < 0.0001, Fisher's LSD). (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; LSD, least significant difference; STM, short-term memory.

FIG. 4.

Acoustic startle and sensory gating testing of cohort 1. Shown are background readings (Pre) (A; one way ANOVA: F_{2, 32} = 0.9456, p = 0.3990), acoustic startle response (Pulse) (B; F_{2, 31} = 6.164, p = 0.0056), startle after the pre-pulse (Prepulse) (C; F_{2, 32} = 1.338, p = 0.2767), and the response when the pre-pulse was subtracted from the pulse (Pulse-Prepulse) (D; F_{2, 32} = 6.779, p = 0.0035). Percent pre-pulse inhibition (%PPI) is shown in panel (E; F_{2, 33} = 1.464, p = 0.2459). Asterisks indicate differences between groups after a significant (p < 0.05) one-way ANOVA (*p < 0.05, **p < 0.01, Fisher's LSD). (2R,6R)-HNK,(2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; LSD, least significant difference.

FIG. 5.

Elevated zero maze (EZM) testing of cohort 1 at 12 days after (2R,6R)-HNK treatment. Rats in cohort 1 were retested in an EZM at 12 days after (2R,6R)-HNK administration. Shown is mean speed (A; F_{2, 30} = 3.312, p = 0.0502), time spent in motion (B; F_{2, 32} = 2.011, p = 0.1504), distance moved (C; F_{2, 29} = 4.359, p = 0.0221), latency to enter an open arm (D; F_{2, 26} = 5.909, p = 0.0077), open arm entries (E; F_{2, 28} = 2.943, p = 0.0692), and time spent in the open arms (F; F_{2, 29} = 1.223, p = 0.3091). Asterisks indicate differences between groups after a significant (p < 0.05) one-way ANOVA (*p < 0.05, **p < 0.01). (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; LSD, least significant difference.

FIG. 6.

Elevated zero maze (EZM) testing of cohort 2. Blast-exposed rats were treated with saline (n = 12) or (2R,6R)-HNK (n = 13) and sham-exposed were treated with saline (n = 13) as indicated in Figure 1. Shown is mean speed (A; one-way ANOVA: F_{2, 33} = 2.551, p = 0.0933), distance moved (B; F_{2, 34} = 1.277, p = 0.2920), latency to enter an open arm (C; F_2,33 = 4.103, p = 0.0256), open arm entries (D; F_{2, 34} = 0.5392, p = 0.5881), and time spent in the open arms (E; F_{2, 29} = 2.943, p = 0.0686). Asterisks indicate differences between groups after a significant (p < 0.05) one-way ANOVA (*p < 0.05, Fisher's LSD). (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; LSD, least significant difference.

FIG. 7.

NOR testing of cohort 2. Sham- and blast-exposed rats were treated with saline (n = 13 sham, n = 12 blast) or blast were treated with (2R,6R)-HNK (n = 13). Shown is time spent exploring the objects (OB1 and OB2) during the training session (A) or the FO and NO during STM (B) and LTM (C) testing. Asterisks indicate values significantly different (**p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t-tests). Panels (D) and (E) show a discrimination index calculated for STM (D; one-way ANOVA: F_{2, 33} = 7.446, p = 0.0021) or LTM (E; F_{2, 33} = 7.127, p = 0.0027) testing. Panels (F) to (H) show total time spent exploring the objects during training (F; F_{2, 35} = 1.494, p = 0.2384), STM (G; F_{2, 34} = 4.609, p = 0.0169), and LTM (H; F_{2, 33} = 1.124, p = 0.3371) sessions. Asterisks in panels (D) to (H) (*p < 0.05, **p < 0.01) represent Fisher's LSD performed after a significant ANOVA. (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; FO, familiar object; LSD, least significant difference; LTM, long-term memory; NO, novel object; NOR, novel object recognition; STM, short-term memory.

FIG. 8.

NOR testing of cohort 2 at 27 days after (2R,6R)-HNK administration. A subset of the rats tested in Figure 7 (n = 7 sham + veh; 8 blast + veh and 9 blast + HNK) were retested 27 days after (2R,6R)-HNK administration. The NO used in Figure 7 was replaced with an NO different from those used in the previous STM and LTM testing. The FO and NO were retained in similar locations as in Figure 7. Panel (A) shows time spent exploring the FO and NO. Asterisks indicate values significantly different (*p < 0.05, **p < 0.01, unpaired t-tests). Panel (B) shows a discrimination index (one-way ANOVA: F_{2, 21} = 6.760, p = 0.0056), and panel (C) shows total time exploring the objects (F_{2, 21} = 3.505, p = 0.0486). Asterisks in panels (B) and (C) (*p < 0.05, **p < 0.01) represent Fisher's LSD performed after a significant ANOVA. (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; FO, familiar object; LSD, least significant difference; LTM, long-term memory; NO, novel object; NOR, novel object recognition; STM, short-term memory.

FIG. 9.

Elevated zero maze (EZM) testing of cohort 3. Blast-exposed rats were treated with saline (n = 12) or (2R,6R)-HNK (n = 13) and sham-exposed were treated with saline (n = 14) as indicated in Figure 1. Shown is mean speed (A; one-way ANOVA: F_{2, 32} = 1.075, p = 0.3532), time in motion (B; F_{2, 32} = 4.044, p = 0.0272), distance moved (C; F_{2, 31} = 1.564, p = 0.2252), latency to enter an open arm (D; F_{2, 27} = 3.600, p = 0.0398), open arm entries (E; F_{2, 34} = 0.5129, p = 0.6033), and time spent in the open arms (F; F_{2, 27} = 3.802, p = 0.0351). Asterisks indicate differences between groups after a significant (p < 0.05) one-way ANOVA (*p < 0.05, Fisher's LSD). (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; LSD, least significant difference.

FIG. 10.

NOR testing of cohort 3. Sham- and blast-exposed rats were treated with saline (n = 12 sham, n = 13 blast) or blast were treated with (2R,6R)-HNK (n = 14). Shown is time spent exploring the objects (OB1 and OB2) during the training session (A) or the FO and NO during STM (B) and LTM (C) testing. Asterisks indicate values significantly different (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-tests). Panels (D) and (E) show a discrimination index calculated for STM (D; one-way ANOVA: F_{2, 34} = 16.40, p < 0.0001) and LTM (E; F_{2, 34} = 11.27, p = 0.0002) testing. Panels (F) to (H) show total time spent exploring the objects during training (F; F_{2, 36} = 2.189, p = 0.1267), STM (G; F_{2, 36} = 7.363, p = 0.0021), and LTM (H; F_{2, 36} = 2.286, p = 0.1162) sessions. Asterisks in panels (D) to (H) (**p < 0.01, ***p < 0.01, ****p < 0.0001) represent Fisher's LSD performed after a significant ANOVA. (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; FO, familiar object; LSD, least significant difference; LTM, long-term memory; NO, novel object; NOR, novel object recognition; STM, short-term memory.

FIG. 11.

Acoustic startle and sensory gating testing of cohort 3. Sham- and blast-exposed rats were treated with saline (n = 12 sham, n = 13 blast), or blast were treated with (2R,6R)-HNK (n = 14). Shown are background readings (Pre) (A; one-way ANOVA: F_{2, 36} = 0.1612, p = 0.8517), acoustic startle response (Pulse) (B; F_{2, 32} = 3.313, p = 0.0492), startle after the pre-pulse (Prepulse) (C; F_{2, 35} = 1.121, p = 0.3373), and response when the pre-pulse was subtracted from the pulse (Pulse-Prepulse) (D; F_{2, 33} = 3.446, p = 0.0437). Percent pre-pulse inhibition (%PPI) is shown in panel (E) (F_{2, 33} = 2.323, p = 0.1138). Asterisks indicate differences between groups after a significant (p < 0.05) one-way ANOVA (*p < 0.05, Fisher's LSD). (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; LSD, least significant difference.

FIG. 12.

NOR testing of cohort 3 at 27 days after (2R,6R)-HNK administration. A subset of the rats tested in Figure 11 (n = 7 sham + veh; 8 blast + veh and 9 blast + HNK) were retested 27 days after (2R,6R)-HNK administration. The NO used in Figure 11 was replaced with an NO different from those used in the previous STM and LTM testing. The FO and NO were retained in similar locations. Panel (A) shows time spent exploring the FO and NO. Asterisks indicate values significantly different (*p < 0.05, **p < 0.01, unpaired t-tests). Panel (B) shows a discrimination index (one-way ANOVA: F_{2, 21} = 6.706, p = 0.0056), and panel (C) shows total spent time exploring the objects (F_{2, 21} = 2.013, p = 0.1575). Asterisks in panel (B) (*p < 0.05, **p < 0.01) represent Fisher's LSD performed after a significant ANOVA. (2R,6R)-HNK, (2R,6R)-hydroxynorketamine; ANOVA, analysis of variance; FO, familiar object; LSD, least significant difference; LTM, long-term memory; NO, novel object; NOR, novel object recognition; STM, short-term memory.