Acid Sphingomyelinase Inhibitor, Imipramine, Reduces Hippocampal Neuronal Death after Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) broadly degrades the normal function of the brain after a bump, blow, or jolt to the head. TBI leads to the aggravation of pre-existing brain dysfunction and promotes neurotoxic cascades that involve processes such as oxidative stress, loss of dendritic arborization, and zinc accumulation. Acid sphingomyelinase (ASMase) is an enzyme that hydrolyzes sphingomyelin to ceramide in cells. Under normal conditions, ceramide plays an important role in various physiological functions, such as differentiation and apoptosis. However, under pathological conditions, excessive ceramide production is toxic and activates the neuronal-death pathway. Therefore, we hypothesized that the inhibition of ASMase activity by imipramine would reduce ceramide formation and thus prevent TBI-induced neuronal death. To test our hypothesis, an ASMase inhibitor, imipramine (10 mg/kg, i.p.), was administrated to rats immediately after TBI. Based on the results of this study, we confirmed that imipramine significantly reduced ceramide formation, dendritic loss, oxidative stress, and neuronal death in the TBI-imipramine group compared with the TBI-vehicle group. Additionally, we validated that imipramine prevented TBI-induced cognitive dysfunction and the modified neurological severity score. Consequently, we suggest that ASMase inhibition may be a promising therapeutic strategy to reduce hippocampal neuronal death after TBI.

Keywords: acid sphingomyelinase; ceramide; imipramine; neuronal death; traumatic brain injury.

Figure 1

Imipramine treatment reduced ASMase and ceramide activity after TBI. (A) Experimental procedures are demonstrated by timeline. Imipramine was injected immediately after TBI impact. Next, ASMase and ceramide analyses were performed 3 h after TBI. Histological analysis was performed 24 h after TBI. (B,C) Quantification of ceramide (C18 and C24:1) activity. (D,E) Quantification of ASMase and NSMase. Data are mean ± SEM; n = 3 from each sham group, n = 7 from TBI-vehicle group, and n = 8 from TBI-imipramine group. (F,H) Fluorescent images show effect of imipramine on ASMase activity. ASMase (red) intensity shown for hippocampal CA1 and DG in sham-operated, TBI-vehicle, and TBI-imipramine groups. Scale bar = 10 and 100 µm, respectively. (G,I) Quantification of ASMase fluorescence intensity on hippocampal CA1 and DG. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. * p < 0.05 vs. the vehicle-treated TBI group. (J,L) Fluorescent images show effect of imipramine on ceramide activity. Ceramide (red) intensity shown on hippocampal CA1 and DG in sham-operated, TBI-vehicle, and TBI-imipramine treated groups. Scale bar = 10 and 100 µm, respectively. (K,M) Quantification of ceramide fluorescence intensity on hippocampal CA1 and DG. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. * p < 0.05 vs. vehicle-treated TBI group.

Figure 2

Imipramine treatment reduces hippocampal neuron death 24 h after TBI. (A,B) Representative fluorescence images showing degenerating neurons stained with Fluoro-Jade B (FJB) in hippocampal CA1 regions after TBI. Scale bar = 10 µm. (C,D) Representative fluorescence images showing degenerating neurons stained with FJB in hippocampal DG (granular cell layer and hilus) regions after TBI. Scale bar = 50 µm. (E,F) Quantification of number of degenerating neurons in TBI-vehicle and TBI-imipramine groups. Data are mean ± SEM; n = 6 from each TBI group. * p < 0.05 vs. vehicle-treated TBI group.

Figure 3

Imipramine treatment decreased lipid peroxidation and microtubule damage 24 h after TBI. (A) Immunofluorescence images showing lipid peroxidation marker 4-HN-stained (red) hippocampal CA1 and DG. Scale bar = 100 µm. (B,C) Quantification of 4-HNE fluorescence-positive intensity in hippocampal CA1 and DG. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. * p < 0.05 vs. vehicle-treated TBI group. (D) Representative fluorescence images showing microtubule marker MAP-2-stained (green) hippocampal CA1 and DG with nuclei (blue). Scale bar = 100 µm. (E,F) Quantification of MAP-2 intensity in hippocampal CA1 and DG. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. * p < 0.05 vs. vehicle-treated TBI group.

Figure 4

Imipramine treatment reduced glial activation 24 h after TBI. (A) Representative images showing astrocytes and microglia stained with GFAP (red) in the hippocampal CA1 and DG. Scale bar = 100 µm. (B,C) Quantification of astrocyte activation in hippocampal CA1 and DG. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. * p < 0.05 vs. vehicle-treated TBI group. (D) Immunofluorescence images showing Iba-1 (green) in hippocampal CA1 and DG. Scale bar = 100 µm. (E,F) Bar graph represents activation intensity of microglia in hippocampal CA1 and DG. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. * p < 0.05 vs. vehicle-treated TBI group.

Figure 5

Imipramine restored TBI-induced delayed neuronal loss, neurological deficits, and memory dysfunction. (A) Experimental procedures are demonstrated by timeline. We performed the mNSS test every day for 7 days and then histological analysis. We performed the Morris water maze (MWM) test for five consecutive days from day 8 to 12 after TBI. (B) mNSS determined in TBI-operated groups on days 1–7 after TBI. Score of up to 18 means that all tasks failed; score of 0 means that all tasks succeeded. (C) delta-mNSS determined in TBI-operated groups. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. (D) We performed the Morris water maze (MWM) test on days 8–12 after TBI. We recorded the platform arrival time for 5 consecutive days. (E) Distance taken to arrive at platform for same schedule on 5 consecutive days of MWM. (F) MWM tracking record of sham-operated and TBI-operated groups at start and termination of MWM. Data are mean ± SEM; n = 5 from each sham group; n = 10 from each TBI group. (G) Representative images showing live neurons detected by NeuN in hippocampal CA1 and DG regions 1 week after TBI in sham-operated groups. Scale bar = 100 µm. (H,I) Quantification of number of live neurons in hippocampal CA1 and DG 1 week after TBI. Data are mean ± SEM; n = 4 from each sham group; n = 6 from each TBI group. * p < 0.05 vs. vehicle-treated TBI group.

Figure 6

Hypothetical connections between imipramine and TBI-induced neuronal death. (A) TBI-induced neuronal death mechanism; (1) under TBI condition, excessive zinc relation and translocation occur; (2) excessive zinc induces overactivation of ASMase; (3) abnormal ASMase activation increases excessive ceramide production; (4) excessive ceramide increases ROS production; (5) increased ceramide induces glial activation. Finally, neuronal death abnormally occurs in hippocampus. (B) Imipramine treatment reduces neuronal death by inhibiting ASMase in lysosomes. Imipramine decreases ASMase activation and ceramide formation, after which ROS and glial cell activation are also reduced. Therefore, neuronal death decreases in hippocampus after TBI.

Figure 7

Experimental procedures are demonstrated by timeline. Imipramine was injected immediately after TBI impact. ASMase and ceramide analyses were performed at 3 h after TBI. Histological analyses (ASMase, ceramide, FJB, 4HNE, MAP2, Iba1, and GFAP) were performed at 24 h after TBI. The mNSS test was performed every day for 7 days. Histological analysis for detecting live neurons (NeuN) was performed at 7 days after TBI. The Morris water maze (MWM) test was performed for five consecutive days from day 8 to day 12 after TBI.