Acute Kahweol Treatment Attenuates Traumatic Brain Injury Neuroinflammation and Functional Deficits

Abstract

Traumatic brain injury (TBI) affects millions worldwide with devastating long-term effects on health and cognition. Emerging data suggest that targeting the immune response may offer promising strategies to alleviate TBI outcomes; kahweol, an anti-inflammatory diterpene that remains in unfiltered coffee, has been shown to be beneficial in neuronal recovery. Here, we examined whether kahweol could alleviate brain trauma-induced injury in a mouse model of TBI and its underlying mechanisms. TBI was induced by controlled cortical impact (CCI) and various doses of kahweol were intraperitoneally administered following injury. Contusion volume, brain edema, neurobehavioral deficits, and protein expression and activity were evaluated in both short-term and long-term recovery. We found that kahweol treatments significantly reduced secondary brain injury and improved neurobehavioral outcomes in TBI mice. These changes were accompanied by the attenuation of proinflammatory cytokine secretion, decreased microglia/macrophage activation, and reduction of neutrophil and leukocyte infiltration. In addition, continuous kahweol treatment further improved short-term TBI outcomes compared to single-dosage. Collectively, our data showed that kahweol protects against TBI by reducing immune responses and may serve as a potential therapeutic intervention for TBI patients.

Keywords: coffee; coffee diterpene kahweol; innate immunity; leukocyte infiltration; neuroinflammation; traumatic brain injury.

Figure 1

Kahweol treatment improved acute TBI outcomes. (A) Representative cresyl violet-stained brain sections of kahweol-treated TBI animals. Dotted area indicates contusion region that lacks staining in the ipsilateral cortex at day 3 post-injury. Scale bar = 1 mm. (B) When compared with Veh group, Kah 5- and Kah 10-treated groups significantly had smaller contusion volume. There was no significant change in brain contusion volume between Veh- and Kah 20-treated groups. n = 10 for Veh, Kah 5, and Kah 10 groups and n = 6 for Kah 20 group. *p < 0.05 versus vehicle group. (C) Brain water content was reduced by Kah 5 and Kah 10 treatment at day 3 post-injury. n = 11 for sham group, n = 10 for Veh and Kah 5 groups, and n = 12 for Kah 10 group. * p < 0.05 versus vehicle group. In addition, Kah 5 and Kah 10 significantly reduced (D) modified neurological severity score (mNSS) at day 3 post-CCI, improved (E) beam score on day 3, reduced (F) the beam walk latencies at days 2 and 3, and improved (G) the rotarod performance at days 2–3 post-injury. n = 21 for Veh and Kah 5 groups and n = 19 for Kah 10 group. * p < 0.05 versus vehicle group. (H) Representative cresyl violet-stained TBI brain section shows areas (white box) selected for Fluoro-Jade C (FJC)-positive cell counting. Scale bar = 1 mm. (I) Representative FJC-stained sections of a vehicle-treated and Kah 5-treated animal at three days post-injury. Inset shows FJC-positive cells at a higher magnification. Scale bar = 50 μm in the full image and 10 μm in the inset. (J) Quantification shows that Kah 5 significantly reduced the number of degenerating neurons at three days post-injury. n = 5 for Veh group and n = 6 for Kah 5 and Kah 10 groups. * p < 0.05 versus the Veh group. Throughout, data are the mean ± SD. Veh, vehicle; Kah 5, kahweol 5 mg/kg; Kah 10, kahweol 10 mg/kg; Kah 20, kahweol 20 mg/kg.

Figure 2

Single-dose kahweol treatment reduced long-term brain tissue loss and improved long-term neurobehavioral functions after TBI. (A) Representative cresyl violet-stained brain sections of Veh- and Kah 5-treated TBI animals at days 1, 3, 7, and 28 post-injury. Dotted area indicates the contusion region that lacks staining in the ipsilateral cortex. Scale bar = 1 mm. (B) Kah 5 treatment significantly reduced contusion volume from days 1 to 28 after TBI. n = 6 to 10 for Veh and Kah 5 groups. *p < 0.05 versus Veh group. When compared with the Veh group, Kah 5-treated animals showed significantly reduced (C) modified neurological severity score (mNSS) on days 3 and 28 post-TBI, better (D) beam score, and reduced (E) beam walk latencies from days 3 to 28 post-injury, improved (F) forelimb function from days 7 to 28, and enhanced (G) rotarod performance. n = 4 to 8 per group. * p < 0.05 versus Veh group. Throughout, data are the mean ± SD. Veh, vehicle; Kah 5, kahweol 5 mg/kg.

Figure 3

Kahweol treatment attenuated proinflammatory cytokines/chemokines secretion levels after TBI. (A) Representative chemiluminescence images of proteome profiler array from Veh- and Kah 5-treated TBI animals at 1-day post-injury. The red box indicates selected candidate proteins that were reduced by Kah 5 treatment. A1.2, A23.24, and F1.2 were the loading controls in the panels. n = 2 per group; each independent experiment includes three individual animals. Quantification analysis showed that increased levels of (B) IL-1β, (C) MIP-1α, (D) MIP-2, and (E) TIMP-1 in the vehicle group were markedly reduced in the Kah 5-treated day 1 TBI animals. n = 3 for sham group and n = 6 for Veh and Kah 5 groups. * p < 0.05 versus Veh group. ^# p < 0.05 versus Contra group. Data are the mean ± SD. Veh, vehicle; Kah 5, kahweol 5 mg/kg; Contra, contralateral; Ipsi, ipsilateral.

Figure 4

Kahweol treatment reduced neuroinflammation after TBI. (A) Representative immunofluorescence of microglia/macrophage marker Iba1 (red) and nuclear marker DAPI (blue) in the Veh- and Kah 5-treated animals at three days following TBI. The inset represents higher magnification of the Iba1-positive cells. Scale bar = 50 μm in the full image and 10 μm in the inset. (B) Representative cresyl violet-stained TBI brain section showed areas (white box) selected for double-positive cell counting. Scale bar = 1 mm. (C) Quantification of Iba1-positive cells showed that increased levels of inflammatory microglia/macrophage in the Veh-treated group (compared versus sham-injured group) were attenuated in Kah 5-treated animals 3 days post-injury. n = 3 per group. * p < 0.05 versus Veh group. (D) Representative immunostaining of neutrophils marker Ly6G (green) and nuclear marker DAPI (blue) in the Veh- and Kah 5-treated animals at three days following TBI. The inset image shows the Ly6G-positive cells at a higher magnification. Scale bar = 50 μm in the full image and 10 μm in the inset. (E) Increased levels of neutrophils infiltration in the vehicle-treated group were reduced in the Kah 5-treated group. n = 3 mice for the sham-groups, n = 4 for the Veh group, and n= 5 for the Kah 5 group. * p < 0.05 versus the Veh group. Throughout, data are mean ± SD. Veh, vehicle; Kah 5, kahweol 5 mg/kg; Iba-1, ionized calcium-binding adapter molecule 1; Ly6G, Lymphocyte antigen 6 complex locus G6D.

Figure 5

Multiple-dose kahweol treatment reduced brain injury in acute phase following TBI, but did not affect long-term neurobehavioral functions. (A) Representative IHC staining of leukocyte marker CD45 in the sham-injured, Veh-treated, and Kah 5- and Kah cont.-treated TBI animals. Scale bar = 50 μm. (B) Quantification of CD45-positive cells showed that increased levels of leukocyte infiltration in the Veh-treated group (compared versus sham-injured group) were markedly reduced in Kah 5-treated animals at three days post-injury. Multiple-dose Kah treatment further attenuated leukocyte infiltration compared to single-dose treatment in acute phase following injury. n = 3 per group. * p < 0.05 versus the Veh group. ^# p < 0.05 versus Kah 5 group. (C) Representative cresyl violet-stained brain sections of Veh-, Kah 5-, and Kah cont.-treated animals at day 3 following TBI. Dotted area indicates the contusion region in the ipsilateral cortex. Scale bar = 1 mm. (D) Continuous treatment of Kah further reduced contusion volume in the Kah cont. group compared to the Kah 5 group at day 3 post-injury. n = 6 for Veh group and n = 5 for Kah 5 and Kah cont. groups. * p < 0.05 versus the Veh group. ^# p < 0.05 versus Kah 5 group. Kah cont. treatment reduced (E) modified neurological severity score (mNSS) at day 7 post-injury, improved the (F) forelimb functions at days 3 and 7, reduced the (G) beam walk latencies at days 3 and 7, and improved the (H) rotarod performance at days 1, 3, and 7 compared to vehicle post-injury. However, there were no significant differences in neurobehavioral outcomes between the Kah 5- and Kah cont.-treated groups. n = 6 to 12 per group. * p < 0.05 versus the Veh group. Throughout, the data are mean ± SD. Veh, vehicle; Kah 5, kahweol 5 mg/kg; Kah cont., multiple-dose kahweol.