Carvacrol, a Food-Additive, Provides Neuroprotection on Focal Cerebral Ischemia/Reperfusion Injury in Mice

Carvacrol (CAR), a naturally occurring monoterpenic phenol and food additive, has been shown to have antimicrobials, antitumor, and antidepressant-like activities. A previous study demonstrated that CAR has the ability to protect liver against ischemia/reperfusion injury in rats. In this study, we investigated the protective effects of CAR on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that CAR (50 mg/kg) significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. This neuroprotection was in a dose-dependent manner. Post-treatment with CAR still provided protection on infarct volume when it was administered intraperitoneally at 2 h after reperfusion; however, intracerebroventricular post-treatment reduced infarct volume even when the mice were treated with CAR at 6 h after reperfusion. These findings indicated that CAR has an extended therapeutic window, but delivery strategies may affect the protective effects of CAR. Further, we found that CAR significantly decreased the level of cleaved caspase-3, a marker of apoptosis, suggesting the anti-apoptotic activity of CAR. Finally, our data indicated that CAR treatment increased the level of phosphorylated Akt and the neuroprotection of CAR was reversed by a PI3K inhibitor LY-294002, demonstrating the involvement of the PI3K/Akt pathway in the anti-apoptotic mechanisms of CAR. Due to its safety and wide use in the food industry, CAR is a promising agent to be translated into clinical trials

Loss of Ahi1 impairs neurotransmitter release and causes depressive behaviors in mice.

Major depression is becoming one of the most prevalent forms of psychiatric disorders. However, the mechanisms of major depression are still not well-understood. Most antidepressants are only effective in some patients and produce some serious side effects. Animal models of depression are therefore essential to unravel the mechanisms of depression and to develop novel therapeutic strategies. Our previous studies showed that Abelson helper integration site-1 (Ahi1) deficiency causes depression-like behaviors in mice. In this study, we characterized the biochemical and behavioral changes in Ahi1 knockout (KO) mice. In Ahi1 KO mice, neurotransmitters including serotonin and dopamine were significantly decreased in different brain regions. However, glutamate and GABA levels were not affected by Ahi1 deficiency. The antidepressant imipramine attenuated depressive behaviors and partially restored brain serotonin level in Ahi1 KO mice. Our findings suggest that Ahi1 KO mice can be used for studying the mechanisms of depression and screening therapeutic targets

Fluorescent staining showed the decrease of serotonin in control and Ahi1 KO mice.

<p>The brain slices were stained with anti-serotonin antibody and DAPI. Significant decrease of fluorescent density of serotonin was observed in the hypothalamus area in Ahi1 KO mice (200X).</p

Serotonin and its metabolite 5-HIAA were decreased in different brain regions of Ahi1 KO mice.

<p>The levels of serotonin (A) and its metabolite 5-HIAA (B) were examined in amygdala (Amy), brainstem (BS), cortex (Ctx), hippocampus (Hip), and hypothalamus (Hypo) regions of control mice and AHi1 KO mice. (C) The ratios of 5-HIAA to serotonin in the five regions were calculated. *P<0.05 versus control mice. N = 6–8.</p

Impramine improved depressive behaviors and partially restored serotonin level in Ahi1 KO mice.

<p>(A) After mice were treated with antidepressants imipramine (20 mg/kg) for three weeks, the immobility time in tail suspension test (TST) and force swimming test (FST) was determined in control and Ahi1 KO mice. After treatment with imipramine (IM) or normal saline (NS), serotonin (B) and dopamine (C) levels in brains were determined. *P<0.05 versus normal saline-treated Ahi1 KO mice. N = 6–8.</p

Ahi1 KO did not affect glutamate and GABA levels in mouse brains.

<p>The levels of dopamine (A) and its metabolite DOPAC (B) and HVA (C) were determined in different brain regions of control mice and AHi1 KO mice. (D) The ratios of HVA to dopamine in different brain regions were also calculated. *P<0.05 versus control mice. N = 6–8.</p

Loss of Ahi1 affected the increase of depression-related neurotransmitters with age.

<p>The levels of serotonin (A) and dopamine (B) in the brainstem tissues were examined at the age of 4 d, 10 d, 1 m, 2 m and 4 m. The content of GABA(C) and glutamate (D) in the region of brainstem was determined at the age of 4 d, 10 d, and 1 m. *P<0.05 versus control mice. N = 6.</p

Ahi1 KO caused depressive behaviors in young mice.

<p>(A) Western blot analysis showed Ahi1 expression in the hypothalamic tissues from control mice and Ahi1 KO mice. (B) Immobility time in tail suspension test was shown in control mice and Ahi1 KO mice at the age of 1 m and 2 m. (C) Immobility time in forced swimming test was shown in control mice and Ahi1 KO mice at the age of 1 m and 2 m. (D and E) Sucrose preference test was performed and total consumed liquid (D) and the percentage of 1% sucrose (E) were calculated. *P<0.05 versus control mice. N = 6–8 mice.</p

MAO activity was increased in brains of Ahi1 KO mice.

<p>MAO activity was determined by the ELISA method. MAO activity was significantly increased in Ahi1 KO mouse. *P<0.05, versus control mice. N = 6–8.</p

The protection of CAR on infarct volume was in a dose-dependent manner.

<p>Mice were administered (i.p.) with CAR at doses of 5, 25, and 50 mg/kg at 2 h before ischemia. Cerebral infarct volume was determined by TTC staining after 75 min of ischemia and 24 h of reperfusion. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p