Involvement of extracellular signal regulated kinases in traumatic brain injury-induced depression in rodents

Abstract

Traumatic brain injury (TBI) is the most common cause of death and acquired disability among children and young adults in the developed countries. In clinical studies, the incidence of depression is high after TBI, and the mechanisms behind TBI-induced depression remain unclear. In the present study, we subjected rats to a moderate fluid percussion into the closed cranial cavity to induce TBI. After 3 days of recovery, injured rats were given a forced swim test (FST) and novelty-suppressed feeding tests. We found that TBI rats exhibited increased duration of immobility and longer latency to begin chewing food in a new environment compared with sham-operated rats. Western blot analysis showed that TBI led to a decrease in the phosphorylated levels of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK). Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), significantly reduced the duration of immobility when administered once per day for 14 days. Consistent with behavioral tests, fluoxetine treatment reversed TBI-induced decrease in p-ERK1/2 and p-p38 MAPK levels. Pre-treatment with a selective tryptophan hydroxylase inhibitor para-chlorophenylalanine (PCPA) blocked the antidepressant effect of fluoxetine. PCPA also prevented the effect of fluoxetine on ERK1/2 phosphorylation without affecting p38 MAPK phosphorylation. Pre-treatment with ERK inhibitor SL327 but not p38 MAPK inhibitor SB203580 prevented the antidepressant effect of fluoxetine. These results suggest that ERK1/2 plays a critical role in TBI-induced depression.

FIG. 1.

Traumatic brain injury (TBI) induces cortical brain lesions. (A) Time line of the experiments. (B,C) At day 1, rats were subjected to a lateral fluid percussion injury (B) or were sham-operated (C). The levels of injury were evaluated by staining brain sections with triphenyltetrazolium chloride (TTC) at day 4. NSF, novelty suppressed feeding. Color image is available online at www.liebertpub.com/neu

FIG. 2.

Traumatic brain injury (TBI) induces depression-like behaviors in rats. (A) In the forced swim test, TBI rats (n=10) exhibited significantly longer immobility time compared with sham-operated (n=9) and naïve (n=9) rats. *p<0.05 vs. sham-operated. (B) In the novelty suppressed feeding test, the latency to begin chewing food was not different between sham-operated (n=5) and TBI (n=5) rats in the home cage. In a new environment, however, TBI rats exhibited significantly longer latency to begin chewing food compared with sham-operated rats. *p<0.05 vs. sham-operated. (C, D) In social interaction test, the duration and frequency were less in the TBI (n=4) rats than in the sham-operated (n=5) rats. *p<0.05 vs. sham-operated.

FIG. 3.

Traumatic brain injury (TBI) decreases phosphorylated levels of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) but not jun-amino-terminal kinase (JNK) in the hippocampus. Rats were subjected to fluid percussion injury on day 1, sacrificed on day 4. (A) Hippocampus and basolateral amygdala homogenates were immunoblotted with antibodies that specifically recognize dually phosphorylated ERK1/2 (both p44 and p42 isoforms), p38 MAPK, and JNK (both p54 and p46 isoforms), as well as antibodies against total ERK1/2, pMAPK, and JNK. (B) TBI led to a significant decrease in the phosphorylated levels of ERK1/2 and 38 MAPK but not JNK in the hippocampus. (C) TBI did not influence the phosphorylated levels of ERK1/2, p38 MAPK, and JNK in the amygdala.

FIG. 4.

Fluoxetine rescues traumatic brain injury (TBI)-induced depression-like behaviors. On day 1, rats were subjected to fluid percussion injury, and fluoxetine (2.5 mg/kg) or the vehicle was administered intraperitoneally to the rats from day 4 to day 17. Behavioral tests were performed on day 18. (A) In the forced swim test, TBI rats exhibited longer immobility time than those of sham-operated rats. Administration of fluoxetine had no effect on the sham-operated rats but significantly reduced immobility time in the TBI rats. *p<0.05 vs. sham-operated, #p<0.05 vs. saline. (B) In the novelty suppressed feeding test, TBI rats exhibited significantly longer latency to begin chewing food compared with sham-operated rats in a new environment. Although fluoxetine-treated TBI rats showed a trend for decreased latency to begin chewing food compared with saline-treated rats, the difference did not reach statistical significance (p>0.05). *p<0.05 vs. sham-operated. (C) In the sucrose preference test, TBI rats treated with saline exhibited lower sucrose preference than those treated with fluoxetine. *p<0.05 vs. saline. (D, E) In the social interaction test, the duration (D) and frequency (E) were restored by fluoxetine in the TBI rats. **p<0.01 vs. saline.

FIG. 5.

Fluoxetine (Flu) reverses traumatic brain injury (TBI)-induced decrease in phosphorylated levels of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in the hippocampus. On day 1, rats were subjected to fluid percussion injury and fluoxetine (2.5 mg/kg), or the vehicle was administered intraperitoneally to the rats from day 4 to day 17. On day 18, hippocampus homogenates of the ipsilateral site were immunoblotted with antibodies that specifically recognize dually phosphorylated ERK1/2 and p38 MAPK, as well as antibodies against total ERK1/2 and pMAPK. Fluoxetine treatment reversed TBI-induced decrease in the phosphorylated levels of ERK1/2 (A) and p38 MAPK (B) in the hippocampus. ###p<0.001 vs. naïve; *p<0.05, **p<0.01 vs. saline (Sal).

FIG. 6.

Block of the effects of fluoxetine on traumatic brain injury (TBI)-induced depression-like behavior and decrease in phosphorylated levels of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in the hippocampus. On day 1, rats were subjected to fluid percussion injury and fluoxetine or vehicle was administered (2.5 mg/kg, intraperitoneally [IP]) to the rats from day 4 to day 17. Para-chlorophenylalanine (PCPA) (250 mg/kg, IP) was administered 1 h before fluoxetine from day 14 to day 17. (A) The forced swim test was performed on day 18. TBI rats treated with fluoxetine exhibited shorter immobility time than those treated with saline. Pre-treatment with PCPA significantly reversed the effect of fluoxetine on the immobility time. *p<0.05 vs. saline, #p<0.05 vs. fluoxetine. (B) On day 18, Western blotting analysis showed that pre-treatment with PCPA blocked the effect of fluoxetine on the ERK1/2 phosphorylation. ###p<0.001 vs. naïve; *p<0.05, p<0.001 vs. TBI+fluoxetine. (C) PCPA had no effect on the phosphorylated level of p38MAPK. ###p<0.001 vs. naïve.

FIG. 7.

Effects of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) inhibitors on the antidepressant effect of fluoxetine. ERK1/2 inhibitor SL327 (30 mg/kg, n=7) (A) or p38 MAPK inhibitor SB203580 (0.1 mg/kg, n=3 or 2 mg/kg, n=3) (B) was administered intraperitoneally on day 17 and day 18 and the forced swim test was performed on day 18 (30 min after SL327 or SB203580 injection). SL327 completely blocked the effect of fluoxetine whereas SB203580 was ineffective. #p<0.05 vs. saline; ***p<0.001 vs. fluoxetine+dimethyl sulfoxide (DMSO).

FIG. 8.

Fluoxetine does not affect traumatic brain injury (TBI)-induced brain damage. Rats were subjected to fluid percussion injury, and fluoxetine (2.5 mg/kg) or vehicle was administered intraperitoneally to the rats from day 4 to day 17. The levels of injury were evaluated by staining brain sections with triphenyltetrazolium chloride at day 18. Color image is available online at www.liebertpub.com/neu