Single administration of tripeptide alpha-MSH(11-13) attenuates brain damage by reduced inflammation and apoptosis after experimental traumatic brain injury in mice

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of alpha-MSH, the C-terminal tripeptide alpha-MSH(11-13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of alpha-MSH(11-13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg alpha-MSH(11-13) or 0.9% NaCl) showed a considerable smaller trauma in alpha-MSH(11-13) injected mice. The expression of the inflammatory markers TNF-alpha and IL-1beta as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single alpha-MSH(11-13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury

Single administration of tripeptide α-MSH(11-13) attenuates brain damage by reduced inflammation and apoptosis after experimental traumatic brain injury in mice.

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11-13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11-13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11-13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11-13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11-13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury

Effect of α-MSH(11–13) on secondary brain injury.

<p>(<b>A</b>) Neurological function was evaluated by neurological severity score (NSS; 0 point = no impairment; 15 points = maximal impairment; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071056#pone-0071056-t002" target="_blank"><b>Table 2</b></a>). NSS of each mouse is the difference of NSS post- to pre-CCI. 23 h after modest brain trauma all mice presented a moderate impaired neurological function with no difference between treatment groups (α-MSH(11–13) vs. vehicle [points]: 2±1.12 vs. 3.6±2.34). (<b>B</b>) Contusion volume increased over time between 15 min (primary lesion) and 24 h after experimental brain trauma (CCI). Tripeptide α-MSH(11–13) administrated mice developed an obvious smaller lesion volume compared to vehicle analyzed in cresyl violet stained brain slices (α-MSH(11–13) vs. vehicle [mm³]: 21±3.39 vs. 27.67±4.81; p = 0.016). The mRNA expression of inflammatory marker genes was determined in contused brain tissue by real-time RT-PCR. (<b>C</b>) TNF-α and (<b>D</b>) IL-1β were upregulated after trauma. TNF-α [% sham] mRNA expression showed a 4-fold increase from primary lesion to vehicle group (p = 0.001) and a 3.5-fold increase in tripeptide-injected group (p = 0.002). The mRNA expression of IL-1β was about 6-fold increased in vehicle (p = 0.001) and 4-fold increased in α-MSH(11–13) group (p = 0.002) at 24 h after injury. Both inflammatory marker genes showed no relevant different expression between treatment groups (p = n.s.). Data were analyzed using exact Wilcoxon-Mann-Whitney test and adjusted for multiple comparisons using Bonferroni-Holm. As this is an explorative study p-values are given for descriptive reasons only. All bar charts show mean ± S.D.</p

Mean percentage (%) of branched cells to total cells of ipsilateral and contralateral side after Iba-1 staining.

<p>Mean percentage (%) of branched cells to total cells of ipsilateral and contralateral side after Iba-1 staining.</p

Criteria of the Neurological Severity Score (NSS).

<p>Criteria of the Neurological Severity Score (NSS).</p

Time response of POMC, MC1R and MC4R mRNA expression.

<p>The mRNA expression of POMC, MC1R and MC4R was measured in contused brain tissue by real-time RT-PCR (n = 10 per group). POMC and MC4R were only slightly changed at any time after trauma (p = n.s.). MC1R increased over time and was elevated 3-fold at 12 h (p<0.001), 2.5-fold at 24 h (p<0.001) and 1.8-fold at 48 h (p = 0.001) after brain trauma compared to naive brain tissue. Similar increases were measured at 12 h (2.8-fold; p = 0.001), 24 h (2.4-fold; p<0.001) and 48 h (2-fold; p = 0.003) compared to 15 min. Data were analyzed using exact Wilcoxon-Mann-Whitney test and adjusted for multiple comparisons using Bonferroni-Holm. As this is an explorative study p-values are given for descriptive reasons only. All bar charts show mean ± S.D.</p

Effect of α-MSH(11-13) on microglial activity.

<p>As a marker for cerebral inflammation microglial cells were quantified by staining for Iba-1. (<b>A+B</b>) Total amount of positive cells was counted in an ipsilateral and contralateral predefined window with no relevant difference between groups (ipsilateral: α-MSH(11-13) 25.44±4.85 cells/mm³ vs. vehicle 27.9±6.28 cells/mm³; contralateral: α-MSH(11-13) 25.44±4.26 cells/mm³ vs. vehicle 25.5±4.88 cells/mm³; each p = n.s.). (<b>C</b>) In series Iba-1 positive cells were categorized in cells with 0 up to 6 branches. (<b>C</b>) The ratio of branched cells to total cell number in contralateral brain tissue is nearly adapted between treatment groups. (<b>E</b>) Counting of branches of ipsilateral tissue categorized into 0–1, 2, and >2 branches and summarized as mean (number of branched cells/total cell number [%]) illustrated a less activated status of microglia after α-MSH(11-13) administration compared to vehicle. Numeric data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071056#pone-0071056-t003" target="_blank"><b>Table 3</b></a>. Considering cells with one or no branch as activated, there was a lesser degree of activation in the α-MSH(11-13) treated group indicating a reduced inflammatory state (α-MSH(11-13) 7.78±8.76% vs. vehicle 17.87±9.65%, p = 0.033). Relating to cells with a maximum of two branches as activated cells, the difference was even more evident (α-MSH(11-13) 21.92±13.05% vs. vehicle 47.65±14.1%, p = 0.002; numeric data presented as mean ± S.D. are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071056#pone-0071056-t004" target="_blank"><b>Table 4</b></a>). As this is an explorative study p-values are given for descriptive reasons only.</p

Primer and Probes with optimized temperature conditions for real-time PCR.

<p>Forward, sense primer; Reverse, antisense primer; Cy5, Cyanine 5; Phos, Phosphate; FL, fluorescein; A, annealing time; E, extension time.</p

Effect of α-MSH(11-13) on apoptosis.

<p>(<b>A</b>) To determine the influence of α-MSH(11-13) on apoptosis, analysis of apoptotic cells was performed using immunofluorescent double-staining of cleaved caspase-3 (green) and neuronal nuclei (red). White arrows point to apoptotic cells. (<b>B</b>) In the quantitative analysis, α-MSH(11–13) administered group showed obvious less apoptotic cells compared to vehicle (each n = 9; one slice of vehicle was excluded due to damage; 26±10.34 vs. 54.78±19.42; p = 0.002). Data were analyzed using exact Wilcoxon-Mann-Whitney test and adjusted for multiple comparisons using Bonferroni-Holm. As this is an explorative study p-values are given for descriptive reasons only. All bar charts show mean ± S.D.</p