A Potent Antioxidant Endogenous Neurohormone Melatonin, Rescued MCAO by Attenuating Oxidative Stress-Associated Neuroinflammation

Abstract

Ischemic stroke is an acute neurological syndrome either due to permanent or temporary obstruction of blood. Such obstruction immediately triggers abrupt pathological cascading processes, which collectively lead to neuronal cell death. Oxidative stress and neuroinflammation in ischemic stroke are critical regulating events that ultimately lead to neuronal death. Complicated interplay exists between the two processes which occur through several stages. Most often, oxidative stress precedes the inflammatory mechanisms and includes several interconnected cascades that underlie the ischemic stroke pathology. In continuation of the previously published data, here, we further ruled out the protective role of melatonin in focal cerebral ischemic injury model. Administration of 5 mg/kg dose of melatonin 30 min prior to ischemia reduced brain infarction associated with sequentially rescued neuronal apoptosis. Furthermore, melatonin attenuated neuroinflammatory markers and reactive oxygen species (ROS), induced by ischemic stroke, via halting the key players of mitogen stress family (p38/JNK). Besides, melatonin modulated the endogenously produced antioxidant enzyme, thioredoxin (Trx) pathway. These broader therapeutic efficacies of melatonin suggest that melatonin could be further investigated for its diverse therapeutic actions with multiple targets in recovering, preventing and halting the detrimental outcomes of MCAO, such as elevated oxidative stress, neuroinflammation, and neurodegeneration.

Keywords: antioxidant; ischemic stroke; melatonin; middle cerebral artery occlusion; neuroinflammation.

Figure 1

After 1 week of acclimatization, randomly selected male Sprague-Dawley were divided into the following groups: (1) Vehicle-treated sham rats; (2) MCAO rats; (3) Pre-melatonin administration to rats undergoing MCAO; (4) Pre-melatonin administration to sham rats. Melatonin or vehicle was administered as a single dose (5 mg/kg), all rats brain was isolated for further biochemical and immunohistochemical analyses.

Figure 2

(A) TTC staining was done to demarcate between ischemic and non-ischemic areas and to evaluate the neuroprotective effects of melatonin (n=5/group). The regions of interest (ROIs) selected were indicated by 1 and 4, respectively showing the frontal cortex and striatum. The parietal cortex is shown by 2, and the piriform cortex by 3. TTC staining was analyzed by one ANOVA followed by a non-parametric test (as Kruskal-Wallis test). Significance ^∗∗∗p < 0.001 showing significant difference relative to vehicle operated sham group, and ^#p < 0.05 showing significant difference relative to MCAO group (B) Representative images of H&E staining showing the degree of neutrophil infiltration in cortical and striatal tissue after ischemic insult, scale bar = 50 μm, magnification 40× and (n = 5/group). These tissue slides were processed from the stained TTC coronal sections after fixation in 4% paraformaldehyde. Infiltrated oligodendrocytes (O), neutrophils (N), and neutrophils in blood (N.b) are shown. Necrotic cells (Ne) with scalloped appearance are shown. Data were analyzed by two ways ANOVA followed by post-hoc Bonferroni Multiple Comparison test using graph-pad prism-5 software (C) Representative images of FJB staining, scale bar = 30 μm. These tissue slides were processed from the stained TTC coronal sections after fixation in 4% paraformaldehyde, and the analyzed area is frontal cortex and striatum.). Symbol ∗∗∗ showing significant difference relative to vehicle operated sham group and the value is p < 0.001 or p < 0.01, respectively, while # or ## showing significant difference values of p < 0.05 or p < 0.01, respectively, relative to MCAO group. TTC, 2,3,5-Triphenyltetrazolium chloride; FJB, Fluoro-Jade B; H&E, hemotoxylin and eosin.

Figure 3

(A) The immunoblot results of p-P38, total P-38, p-JNK, and total JNK from the ipsilateral cortex and striatum following MCAO. The immunoblot bands were quantified using ImageJ software, and the statistical differences are indicated in the corresponding graphs. The data are expressed as the mean ± SEM for n = 6 rats/group, and the number of experiments = 3. β-Actin was used as a control. Data were analyzed by two-way ANOVA followed by post-hoc Bonferroni Multiple Comparison test using graph-pad prism-5 software. The brain tissue for Western blot was collected after 24 h of vehicle or melatonin treatment and stored at −80°C until used. Symbol ∗∗∗ or ∗∗ showing significant difference relative to vehicle operated sham group and their values are p < 0.001 or p < 0.01 respectively, while # showing significant difference values of p < 0.05 relative to MCAO group. (B) The presented images indicated Immunoreactivity of p-JNK in the cortical and striatum tissue of rat brain. The p-JNK exhibits cytoplasmic localization. The data are expressed as the mean ± SEM for n = 5 rats/group, and the number of experiments = 3. Scale bar = 50 μm, magnification 40×. The immunohistochemistry slides were processed from the stained TTC coronal sections after fixation in 4% paraformaldehyde. From the thick coronal TTC sections, paraffin blocks were made, and later 4-μm-thin coronal sections were prepared by a rotary microtome. The symbol ∗∗∗ showing significant difference relative to the vehicle operated sham group and the value is p < 0.00, while # showing significant difference value of p<0.05 relative to the MCAO group. p-JNK, phospho c-Jun N-terminal kinase; JNK, c-Jun N-terminal kinase; p-P38, phospho-P38.

Figure 4

The immunoblot results of Nrf2, HO-1, and Trx in the cortical and striatum tissue of rat ipsilateral brain. The immunoblot bands were quantified using ImageJ software, and the statistical differences are indicated in the relative graphs. The data are expressed as the mean ± SEM for n = 6 rats/group, and the number of experiments = 3. Data were analyzed by two-way ANOVA followed by post-hoc Bonferroni Multiple Comparison test using graph-pad prism-5 software β-Actin was taken as a loading control. Significance = ^∗∗∗p<0.00, ^∗∗ or ^##p<0.01, and ^# or ^∗p<0.05. The brain tissue for Western blot was collected after 24 h of vehicle or melatonin treatment and stored at −80°C until used, Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; Trx, thioredoxin.

Figure 5

(A) Immunoreactivity of GFAP-positive cells in different groups are shown with magnification 40× and scale bar = 30 μm. The data are expressed as the mean ± SEM for n = 5 rats/group, and the number of experiment s= 3. The immunohistochemistry slides were processed from the stained TTC coronal sections after fixation in 4% paraformaldehyde. From the thick coronal TTC sections, paraffin blocks were made, and later 4-μm-thin coronal sections were prepared by a rotary microtome. The symbol ∗∗∗ showing significant difference relative to the vehicle operated sham group and the value is p<0.00, while # or ## showing significant difference value of p < 0.05 and p < 0.01, respectively, relative to MCAO group. (B) Effect of melatonin on inflammatory cytokines. Western blot results of TNF-α and IL-1β from the ipsilateral brain were analyzed by ImageJ. The statistical differences are indicated in the relative graphs. The data are expressed as the mean ± SEM for n = 6 rats/group, and the number of experiments= 3. β-Actin was used as a control. Data were analyzed by two-way ANOVA followed by post-hoc Bonferroni Multiple Comparison test using graph-pad prism-5 software. The brain tissue for western blot was collected after 24 h of vehicle or melatonin treatment and stored at −80°C until used. The symbol ^∗∗∗ or ^∗∗ showing significant difference relative to the vehicle operated sham group and their values are p < 0.00 or p < 0.01, respectively, while # or ## showing significant difference value of p<0.05 and p<0.01 respectively relative to MCAO group. Symbol ^∗∗ showing significant difference relative to vehicle operated sham group and the value is p < 0.01. (C) Immunohistochemistry results for TNF-α and IL-1β are shown; scale bar = 50 µm; magnification, 40×. Tissue sections show correspondingly elevated expression of TNF-α and IL-1β after 24 h of permanent ischemia and both proteins show cytoplasmic localization. Significance = ^∗∗∗p<0.001 and ^#p<0.05. All the morphological data are expressed as the mean ± SEM for n = 5 rats/group. The immunohistochemistry slides were processed from the stained TTC coronal sections after fixation in 4% paraformaldehyde. The TTC sections were subjected to embedding and paraffin blocks were made and later 4-μm coronal sections were made by a rotary microtome. GFAP, glial fibrillary acidic protein; TNF-α, tumor necrosis factor; IL-1β, interleukin.

Figure 6

Melatonin downregulated the NF-κB signaling pathway. (A) The immunoblot results of p-NF-κB, COX2, and iNOS in the cortical and striatum tissue of rat ipsilateral brain. The immunoblot bands were quantified using ImageJ software, and the statistical differences are indicated in the relative graphs. The data are expressed as the mean ± SEM for n=6 rats/group, and the number of experiments = 3. β-Actin was used as a control. Data were analyzed by two-way ANOVA followed by post hoc Bonferroni Multiple Comparison test using graph-pad prism-5 software. The brain tissue for western blot was collected after 24 h of vehicle or melatonin treatment and stored at −80°C until used. The symbol ∗∗∗ showing significant difference relative to the vehicle operated sham group and the value is p < 0.001, while # or ## showing significant difference value of p < 0.05 and p < 0.01 respectively relative to MCAO group. (B) Immunofluorescence reactivity of COX2 and iNOS; scale bar = 30 and 50 µm; magnification, 40×. The COX2 and iNOS-positive cells were visualized by TRITC and FITC respectively and showed cytoplasmic localization. The data are expressed as the mean ± SEM for n = 5 rats/group and the number of experiments performed=3. Symbol ** or * showing significant difference relative to vehicle operated sham group and the values are respectively p < 0.01 and p < 0.05. (C) The presented images indicated Immunoreactivity of p-NF-κB in the cortical and striatum tissue of rat brain. The p-NF-κB exhibits nuclear localization, and the number of experiments= 3.; scale bar = 20. Significance = ***p < 0.001 ^##p < 0.01. NF-κb, nuclear factor kappa light chain enhancer of activated B cells; COX2, cyclooxygenase; iNOS, inducible nitric oxide or nitric oxide synthase. Symbol ^∗∗ or * showing significant difference relative to vehicle operated sham group and the values are respectively p < 0.01 and p < 0.05.

Figure 7

Effect of melatonin (5 mg/kg) on the p-NF-κB/Trx pathways (A, B) p-NFκB and Trx were colocalized by double immunofluorescence. Scale bar = 20 μm and (n=5/group). p-NFκB showed higher expression level accompanied with Trx down expression in the MCAO group. p-NF-κB and Trx were visualized by FITC and TRITC, respectively. Significance = * and ** shows significant difference relative to sham operated vehicle group and its value are p < 0.05, and p < 0.01. ^## shows significant difference relative to MCAO operated group and its value is p < 0.01. Significance = ^# or *p < 0.05; and ** or ^##p < 0.01. FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine.

Figure 8

A graphical representation of the underlying neuroprotective mechanisms of melatonin against the MCAO-induced oxidative stress-mediated neuroinflammation and neurodegeneration.