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Melatonin-induced neuroprotection after closed head injury is associated with increased brain antioxidants and attenuated late-phase activation of NF-B and AP-1¹

SARA M. BENI, RON KOHEN^*, RUSSEL J. REITER, DUN-XIAN TAN and ESTHER SHOHAMI²

Departments of Pharmacology and
^* Pharmaceutics School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel; and
Department of Cellular and Structural Biology, The University of Texas, San Antonio, Texas, USA

²Correspondence: Department of Pharmacology, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. E-mail: esty{at}cc.huji.ac.il

SPECIFIC AIMS

Traumatic brain injury (TBI) leads to massive production of reactive oxygen species (ROS), which in turn mediates further secondary cellular damage. Our aim was to assess whether the neuroprotective effect of melatonin, a main pineal product with controversial antioxidant properties, involves redox-dependent mechanisms.

PRINCIPAL FINDINGS

1. Effect of melatonin on clinical outcome and lesion size after CHI
Mice were subjected to closed head injury (CHI) and 1 h later the initial Neurological Severity Score (NSS), composed of 10 behavioral and motor tests, was assessed. Immediately thereafter, vehicle or melatonin (1, 5, or 10 mg/kg) were administered intraperitoneally and NSS was reevaluated 24 h later. The dose response displayed a bell shape, i.e., neuroprotection was achieved with 5 but not with 1 or 10 mg/kg (Fig. 1 A; P < 0.05 by ANOVA). Melatonin (5 mg/kg) facilitated clinical recovery (NSS=the difference between 1 h NSS and NSS at later time points) for at least 1 wk after injury (Fig. 1B ; P<0.05) and decreased lesion size (24 h) by twofold (6.17±1.04% vs. 11.08±1.01%; P<0.01).

View larger version (14K): [in this window] [in a new window]   Figure 1. Effect of melatonin on clinical recovery after CHI. A) Mice with similar NSS 1 h after CHI were randomly assigned to groups receiving vehicle (V) or 1, 5, or 10 mg/kg melatonin (M1, M5, and M10, respectively) and reevaluated 24 h later. The data are presented as the mean ± SE. Since only 5 mg/kg melatonin significantly lowered NSS, this dose was used for longer evaluation. *P < 0.05. B) Rate of recovery expressed as NSS (see text) was followed for 1 wk after CHI. Compared with the vehicle (V) group (empty symbols), the 5 mg/kg melatonin (M5)-treated group (filled symbols) displayed higher NSS at all time points during this period. *P < 0.05; **P < 0.01.

2. Effect of melatonin on post-CHI brain antioxidants
Tissue antioxidants and ascorbic acid were determined using cyclic voltametry and HPLC-EC, respectively. Melatonin (5 mg/kg) treatment was associated with a sustained (4 days) elevation of low molecular weight antioxidants oxidized at potentials of 765 and 1145 mV (P<0.05) and with a higher content of ascorbic acid (P<0.05) at the ipsilateral cortex. This effect was completely abolished at the higher dose of melatonin. Post-CHI brain antioxidants (4 days) were not affected by administration of the neuroprotective endocannabinoid 2-arachidonoyl glycerol (2-AG), pointing to the specificity of the melatonin-induced effect.

3. Effect of melatonin on redox-regulated transcription factors
DNA binding activity of two redox-regulated transcription factors, NF-B and AP-1, was assessed using an electromobility shift assay (EMSA). Melatonin did not alter the early-phase (24 h) CHI-induced activation of NF-B and AP-1, but blocked robust late-phase (8 days) activation of NF-B (Fig. 2 A) and decreased that of AP-1 to below basal levels (data not shown). We failed to observe significant changes in cytosolic levels of IB at these time points (Fig. 2B) .

View larger version (21K): [in this window] [in a new window]   Figure 2. Effect of melatonin on post-CHI nuclear translocation of NF-B. A) NF-B DNA binding was determined in brain nuclear extracts using EMSA (insert). Each experiment was repeated three times and relative optical densities (CHI/Sham) were obtained using Bio-Rad Multi-Analyst. Quantitative data are expressed as mean ± SE. CHI induced a robust nuclear translocation of NF-B that was significantly inhibited by melatonin at the later (8 d) but not the earlier (24 h) post-traumatic phase. V: vehicle; M5: 5 mg/kg melatonin; C: competition with access of unlabeled NF-B oligonucleotide; S: sham; p65: Supershift assay with (+) or without (–) preincubation with anti-p65 antibody. *P < 0.05. B) Cytosolic IB was determined by Western blot using monoclonal anti-IB (insert). Quantitative data, expressed as relative optical densities, represent the mean ± SE of all experiments. Neither CHI nor melatonin treatment affected IB levels at the time points studied.

CONCLUSIONS AND SIGNIFICANCE

Melatonin has been shown to exert neuroprotection in a variety of oxidative-stress associated neuropathologies, including brain trauma and ischemia. Hence it was suggested that melatonin-induced neuroprotection is mediated via its nonreceptor activities as a free radical scavenger and an inhibitor of lipid peroxidation. However, some antioxidant activity of melatonin has been strongly argued in recent studies, and our results shed some light on this controversy. We assessed whether the neuroprotective effect of melatonin in our CHI model involved redox-dependent mechanisms.

Our results demonstrate that melatonin does indeed induce neuroprotection, but its antioxidant properties are not reflected only as a radical neutralizing agent. Rather, we suggest that neuroprotection is mediated via the potentiation of other brain antioxidants (e.g., ascorbic acid and other, yet unidentified compounds) that, by altering the cell’s redox state, attenuate the subsequent activation of NF-B and AP-1. Since melatonin-induced effects on brain antioxidants lasted much longer than its own half-life, it is unlikely that our findings reflect simply the accumulation of melatonin or its metabolite in the injured tissue. The endocannabinoid 2-AG recently shown to reduce infarct volume and enhance clinical recovery in our CHI model failed in the present study to affect post-traumatic brain antioxidants, hence confirming the specificity of the melatonin-induced effects. Notably, loss of effect of high-dose melatonin is a typical feature of antioxidants, some of which may become pro-oxidants under certain conditions, and was accompanied by loss of neuroprotection.

In agreement with previous studies, we demonstrate rapid activation of AP-1 and a longer, more progressive activation of NF-B following CHI. AP-1 is involved in the control of cell proliferation and differentiation, and NF-B regulates the expression of numerous genes encoding acute-phase proteins, cell adhesion molecules, anti- and pro-oxidant enzymes, and cytokines. Proinflammatory cytokines play crucial role in brain trauma and can act synergistically with ROS to induce cell damage. Thus, a therapeutic strategy such as the use of melatonin to block persistent activation NF-B may either stimulate beneficial mechanisms (e.g., antioxidant enzymes) or inhibit destructive responses such as inflammation. This may contribute to the "decision-making" process of transcription factors toward a "survival signal" rather than a "death signal" during the secondary phase after TBI (Fig. 3 ). Failure of melatonin to block the early activation of AP-1 and NF-B may point to distinct regulatory mechanisms for their activation in the early (as opposed to later) postinjury phases.

View larger version (38K): [in this window] [in a new window]   Figure 3. A schematic diagram showing basic cellular mechanisms induced by traumatic brain injury and their proposed modification by exogenous melatonin. CHI activates NF-B via several signaling pathways. These include activation of NMDA receptors, a downstream Ras-GTP, PKB/Akt pathway, TNF release, which activates TNF receptors, and production of ROS, which impairs the cellular redox state. Melatonin may act as a direct radical scavenger, thus reducing ROS levels at early time points (hours), or can indirectly modify endogenous anti- and/or pro-oxidant enzymes, resulting in elevated levels of low molecular weight antioxidants (LMWA) at later, postinjury phases (days). Lower oxidative burden on the cell leads to a lower degree of activation of transcription factors AP-1 and NF-B. The delicate balance between death and survival signals maintained under normal conditions is switched in favor of the death signals after trauma, and is now turned in favor of survival signals after treatment with melatonin. This is observed in vivo in a better long-lasting outcome. Events related to melatonin appear as dotted lines and half-filled arrows. Width of arrows represents magnitude of response. Empty arrows indicate pathways not investigated in the present study.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0323fje

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