Central post-stroke pain is a severe persistent pain disease that affects 12% of stroke survivors (CPSP). These patients may have a cognitive impairment, depression, and sleep apnea, which leave them open to misdiagnosis and mistreatment. However, there has been little research on whether the neurohormone melatonin can effectively reduce pain in CPSP conditions. In the present study, we labeled melatonin receptors in various brain regions of rats. Later, we established a CPSP animal model by intra-thalamic collagenase lesions. After a rehabilitation period of three weeks, melatonin was administered using different doses (i.e., 30 mg/kg, 60 mg/kg, 120 mg/kg) for the following three weeks. Mechanical allodynia, thermal hyperalgesia, and cold allodynia behavioral tests were performed. Immediately after behavioral parameters were tested, animals were sacrificed, and the thalamus and cortex were isolated for biochemical (mitochondrial complexes/enzyme assays and LPO, GSH levels) and neuroinflammatory (TNF-α, IL-1β, IL-6) assessments. The results show that melatonin receptors were abundant in VPM/VPL regions. The thalamic lesion significantly induced pain behaviors in the mechanical, thermal planters, and cold allodynia tests. A significant decrease in mitochondrial chain complexes (C-I, II, III, IV) and enzymes (SOD, CAT, Gpx, SDH) was observed after the thalamic lesion. While there were significant increases in reactive oxygen species levels, including increases in LPO, the levels of reduced GSH were decreased in both the cortex and thalamus. Proinflammatory infiltration was noticed after the thalamic lesion, as there was a significant elevation in levels of TNF-α, IL-1β, and IL-6. Administration of melatonin has been shown to reverse the injury effect dose-dependently. Moreover, a significant increase in C-I, IV, SOD, CAT, and Gpx levels occurred in the CPSP group. Proinflammatory cytokines were significantly reduced by melatonin treatments. Melatonin seems to mediate its actions through MT1 receptors by preserving mitochondrial homeostasis, reducing free radical generation, enhancing mitochondrial glutathione levels, safeguarding the proton potential in the mitochondrial ETC by stimulating complex I and IV activities, and protecting the neuronal damage. In summary, exogenous melatonin can ameliorate pain behaviors in CPSP. The present findings may provide a novel neuromodulatory treatment in the clinical aspects of CPSP.
Keywords: central poststroke pain; neuromodulation; melatonin; inflammation; mitochondrial dysfunction
Central post-stroke pain (CPSP) is neuropathic pain due to associated lesions of the central somatosensory nervous system. This syndrome is characterized by pain and sensory abnormalities in the body parts corresponding to brain regions injured by the cerebrovascular lesion [[
Mitochondria, often referred to as the powerhouses of the cell, are present in nearly all types of human cells. The ability of cells to generate energy from ambient oxygen is facilitated by the role of mitochondria as energy providers and signaling intermediaries. The ETC transforms metabolic substrate electrons into molecular oxygen (O
To our knowledge, melatonin, called N-acetyl-5-methoxy tryptamine, is a neuro-hormone. SCN regulates the biosynthesis of melatonin in the pineal gland, and it contributes to synchronization in the light–dark cycle of the environment [[
Therefore, the present study addressed the following issues: first, whether melatonin could ameliorate pain behavior as a clinical intervention in the CPSP condition; second, whether the antinociception of melatonin was associated with oxidative stress responses, mitochondrial ETC enzymes and complexes function, or neuroinflammation.
The distribution of melatonin receptors was assessed in the various brain regions, including the ventral posteromedial thalamic nucleus (VPM, Figure 1A), somatosensory cortex (S1, Figure 1B), dentate gyrus (DG, Figure 1C), amygdalostriatal transition area (Astr, Figure 1D), secondary auditory cortex (AUD, Figure 1E), basolateral amygdala (BLA, Figure 1F), lateral hypothalamus (LH, Figure 1G), magnocellular region (Mc, Figure 1H), magnocellular nucleus of the lateral hypothalamus (Mclh, Figure 1I), nigrostriatal fibers (Ns, Figure 1J), posterior hypothalamic area (Ph, Figure 1K), retrosplenial agranular cortex (Rsa, Figure 1L), posterior thalamic nuclear group (Po., Figure 1M), ventromedial thalamic nucleus (VM, Figure 1N), ventromedial hypothalamic nucleus (VMH, Figure 1O), and reticular thalamic nucleus (RT, Figure 1P). The receptor-mediated actions of melatonin might contribute to functions of the central nervous system, especially in pain behaviors. A distribution of melatonin receptor MT1 was found in various brain areas (Table 1). The maximum count of receptor distribution was found in the somatosensory cortex S1, hippocampus, ventral posterior medial/lateral nucleus VPM/VPL, and auditory cortex AUD (see Figure 1 and Table 1).
To assess pain sensitivity, pain behavioral tests were conducted on sham control, lesion control, lesion + MLT (30 mg/kg), lesion + MLT (60 mg/kg), and lesion + MLT (120 mg/kg) groups. A Kruskal–Wallis analysis indicated that there were significant differences among all groups for 7D (Kruskal–Wallis test = 20.79, p < 0.05) and 14D (Kruskal–Wallis test = 20.08, p < 0.05). The mechanical hyperalgesia threshold in the Von Frey test was significantly decreased due to the lesion of the VPM/VPL, suggesting that it caused the CPSP pain behavior. However, 120 mg/kg of melatonin administration (but not the other doses of melatonin) significantly increased the mechanical hyperalgesia threshold, indicating that 120 mg/kg of melatonin administration reduced pain perception compared with the lesion control group for 7D (p < 0.05) and 14D (p < 0.05, Figure 2A). The results suggest that the higher dose of melatonin was more effective in reducing mechanical pain perception.
Concerning the cold allodynia test, a Kruskal–Wallis test indicated that there was a significant difference in the factor of the group (Kruskal–Wallis test = 31.13, p < 0.05). However, the post hoc with the Dunn test indicated that there was no significant difference in the CPSP compared with the sham control group, possibly because of the high variance in the cold allodynia scores (p > 0.05; Figure 2B); this indicates that measurement of the cold allodynia did not show pain behaviors in the animal model of CPSP.
Furthermore, the plantar test showed significant differences occurring in groups 7D (Kruskal–Wallis test = 19.16, p < 0.05) and 14D (Kruskal–Wallis test = 20.10, p < 0.05), indicating that the rats in the lesion group had a shorter paw withdrawal time for 7D and 14D compared with the sham control group (p < 0.05). Moreover, the lesion + MLT (60 mg/kg) and lesion + MLT (120 mg/kg) groups had a significantly increased paw withdrawal time compared with the lesion group for 7D and 14D (p < 0.05). Thus, the 60 mg/kg and 120 mg/kg melatonin administrations (but not the 30 mg/kg melatonin group) effectively reduced thermal hyperalgesia after 7D and 14D melatonin administrations (Figure 2C).
The oxidative stress parameters and mitochondrial antioxidant levels were assessed to verify the dose-dependent effect of melatonin (i.e., 30, 60, and 120 mg/kg) on oxidative stress in the 7 + 14 days (after 3 weeks of melatonin administration) following injury and rehabilitation.
The four mitochondrial chain complex levels were evaluated in both the thalamus and cortex tissues.
The levels of complex-i were found to be decreased in lesioned animals in both thalamic and cortical tissues. However, there was no effect of 30 mg/kg and 60 mg/kg melatonin treatments in cortical tissues. Only the highest dose of 120 mg/kg was significantly effective, while all three doses of melatonin significantly enhanced levels of complex-i in the thalamus (Kruskal–Wallis test = 22.39, p < 0.05) (Figure 3A).
Furthermore, there were no significant differences in levels of complex-ii in the thalamus among all groups (Kruskal–Wallis test = 8.91, p > 0.05), indicating that the complex-ii levels were not involved in the thalamus for the CPSP animals (Figure 3B). In the cortex, a significant difference occurred in the group in the levels of complex-ii (Kruskal–Wallis test = 17.80, p < 0.05). Post hoc Dunn tests showed that the complex-ii levels were significantly decreased in the CPSP control group compared with the sham control (p < 0.05). Compared with the CPSP control group, CPSP + MLT (60 mg/kg) and CPSP + MLT (120 mg/kg) were significantly decreased in the complex-ii levels in the cortex (Figure 3B).
The levels of complex-iii were robustly decreased in both the thalamus (Kruskal–Wallis test = 14.20, p < 0.05) and the cortex (Kruskal–Wallis test = 11.91, p < 0.05); however, melatonin treatments did not affect complex-iii levels in the thalamus and cortex (p > 0.05; Figure 3C).
To test complex-iv levels in the thalamus and cortex, a Kruskal–Wallis test was used, indicating that there were significant group differences in the thalamus (Kruskal–Wallis test = 22.08, p < 0.05) and cortex (Kruskal–Wallis test = 20.61, p < 0.05). The Dunn post hoc indicated that the CPSP group had significantly decreased complex-iv levels in the thalamus (p < 0.05) and cortex (p < 0.05) compared with the sham control group. Melatonin treatments in 30~120 mg/kg doses could increase complex-iv levels in the thalamus and cortex (p < 0.05; Figure 3D).
Therefore, our results suggested that melatonin was significantly effective in rescuing levels of complex-i and complex-iv. Melatonin treatments significantly decreased the complex-ii levels in the cortex for the CPSP animal.
To assess the effect of lesion and melatonin treatments on electron transport chain enzymes, we used a Kruskal–Wallis test that indicated that there were significant group differences in the levels of glutathione peroxidase (GPx) in the thalamus (Kruskal–Wallis test = 18.14, p < 0.05) and the cortex (Kruskal–Wallis test = 17.39, p < 0.05). The Dunn post hoc indicated that the lesions reduced GPx levels in the thalamus (p < 0.05) and the cortex (p < 0.05); moreover, melatonin treatments increased GPx levels in the thalamus (p < 0.05) and the cortex, particularly under 120 mg/kg doses of melatonin (p < 0.05; Figure 4A).
Furthermore, levels of catalase were reduced significantly after CPSP in both the thalamus (Kruskal–Wallis test = 17.67, p < 0.05) and the cortex (Kruskal–Wallis test = 18.36, p < 0.05) (Figure 5B). However, treatments with 30–120 mg/kg doses of melatonin may have increased catalase levels in the thalamus (p < 0.05) and the cortex (p < 0.05; Figure 4B).
Furthermore, after CPSP, the CPSP animals demonstrated significantly decreased superoxide dismutase levels in the thalamus (Kruskal–Wallis test = 22.07, p < 0.05) and the cortex (Kruskal–Wallis test = 22.21, p < 0.05) compared with the sham control group. However, 30–120 mg/kg doses of melatonin treatments significantly increased superoxide dismutase levels in the thalamus (p < 0.05) and the cortex (p < 0.05; Figure 4C).
The hallmark of mitochondrial oxidative stress is the presence of ROS, e.g., OH– (hydroxyl radical), O
The levels of reduced glutathione after CPSP were significantly decreased in the thalamus (Kruskal–Wallis test = 21.44, p < 0.05) and cortex (Kruskal–Wallis test = 23.08, p < 0.05), suggesting the presence of oxidative stress in these brain areas. While treatment with 30 mg/kg had no effect on thalamic tissue (p > 0.05), both 60 mg/kg and 120 mg/kg treatments were associated with significant improvements in levels of reduced glutathione in the thalamus (p < 0.05) and the cortex (p < 0.05; Figure 5B).
In determining neuroinflammation in both CPSP rats and melatonin-treated rats, a one-way ANOVA was used. Neurodegeneration due to stroke leads to mitochondria-related oxidative stress, which may activate microglia, thereby triggering the infiltration of neuro-inflammatory cytokines such as TNF-α (Kruskal–Wallis test = 21.52, p < 0.05) (Figure 6A), IL-6 (Kruskal–Wallis test = 20.30, p < 0.05) (Figure 6B), and IL-1b (Kruskal–Wallis test = 24.04, p < 0.05) (Figure 6C) into the whole brain tissue. There was a significant increase in neuroinflammatory cytokines in the lesion animals in the whole brain compared with the sham control group (p < 0.05). Treatments with melatonin in 60 mg/kg and 120 mg/kg doses significantly reduced IL-1b levels, suggesting that higher doses of melatonin of 60–120 mg/kg could ameliorate neuroinflammation responses.
In previous studies, melatonin receptors have been identified using RT-PCR in various tissues in humans and rats, such as in the cerebellum, SCN, entorhinal cortex, pars tuberalis, pineal gland, neurohypophysis, hypothalamus, bone marrow, blood, and spleen [[
Collagenase microinjections can be successfully induced in CPSP, indicating decreases in the withdrawal threshold and showing mechanical hyperalgesia. The quick paw withdrawal response also decreased with cold allodynia, and there was a decrease in paw withdrawal latency in plantar tests; additionally, melatonin alleviated the pain caused by CPSP in all pain behavior tests. However, the effect of melatonin was shown to be dose-dependent, with data suggesting that higher doses of 120 mg/kg have a more significant effect than lower (30 mg/kg) and medium (60 mg/kg) doses.
Moreover, our previous study showed reduced levels of endogenous melatonin due to CPSP [[
Tissue damage due to stroke may cause mitochondrial dysfunction, thereby inducing an oxidative stress cascade and apoptosis and leading to mitochondrial membrane permeabilization (MMP). This may give rise to matrix calcium levels causing electron transport chain (ETC) dysfunction in mitochondria and ETC failure, and lead to the generation of reactive oxygen and reactive nitrogen species by mitochondria (Figure 7).
Our results showed that melatonin treatments had a significant effect on mitochondrial function and are consistent with previous reports. However, melatonin has shown protective effects in various diseases, such as AD and PD, in which mitochondrial dysfunction is one of the causes of the condition [[
The brain is particularly susceptible to damage under oxidative stress because it is rich in phospholipids and proteins that are susceptible to oxidative damage and may not have significant quantities of antioxidant defense enzymes. Free-radical-mediated injury may play an important role in the severity of the disease. Superoxide Dismutase (SOD) down-regulation marks the amount of increased superoxide free radicals in mitochondria, while catalase (CAT) is important for the protection of cells from oxidative damage by ROS and catalyzes the breakdown of hydrogen peroxide to water and oxygen.
The hallmark of mitochondrial oxidative stress is the presence of ROS, e.g., OH– (hydroxyl radical), O
In addition, melatonin administration decreased lipid peroxidation (LPO) by interacting with lipid bilayers, stabilizing mitochondrial inner membranes, and restoring oxidative damage by augmenting GSH levels and glutathione peroxidase (GPx) activities. Hence, it may improve the ETC activity of mitochondria by safeguarding the electron flow and thereby increasing ATP production.
Oxidative stress cascades produce reactive oxygen or nitrogen species (ROS/RNS), leading to mitochondrial dysfunction. This oxidative stress cascade causes lipid peroxidation and activates p38MAPK and, thus, causes the high activity of the NFκB transcription factor and, in turn, increased proinflammatory cytokines such as TNF-α and IL-6; this cycle continues [[
However, our study has certain limitations. We did not assess the role of melatonin in opiate and dopaminergic signaling. SCN has melatonin, opioid, and dopamine receptors, which may interfere with melatonin's antinociception signaling. Thus far, melatonin has been found to have no adverse effects. Therefore, exogenous melatonin administration in chronic pain conditions may be a promising clinical intervention for drug tolerance and addiction.
Twenty-five male Sprague Dawley rats (~8 weeks of age) were purchased from BioLASCO, Yi-Lan, Taiwan. They were housed (one rat per cage) in an animal room under a 12 h light–dark cycle with air conditioning and 60% humidity and received a chow diet and water ad libitum. All experiments were performed in accordance with the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee.
The experimental procedure is shown in Figure 8A. At the beginning of the experiment, animals were allowed habituation and pre-training from days 0 to 6. On day 7, the lesion was induced. Then, there was a rehabilitation period of three weeks following the lesion. After three weeks following the rehabilitation period, melatonin was administrated. Behavior tests were conducted for the baseline and lesioned animals and for the melatonin treatment (after the first 7 days of melatonin treatment, 7D; after the next 14 days of melatonin treatment, 14D; see Figure 8A). The surgery was performed with the stereotaxic instrument; the successful induction of lesion VPA and VPL is shown in Figure 8B.
CPSP was induced by thalamic lesions, and the procedures were performed using the method described by Kuan et al. in 2015 [[
At −20 °C, frozen tissue samples were sliced into 10 μm thick cryo-sections using a cryostat microtome. The non-specific antibody binding was blocked with 3% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 0.3% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) for 30 min. The sections were incubated in the primary antibody solution (1:100 dilution, MT1, Mel 1a Receptor, MTNR1A: AMR-031, Alomone labs, Israel) for 30 min. Then, the sections were incubated in secondary antibody solution (1:1000 dilution, Alexa Fluor 488-labeled goat anti-rabbit IgG, Thermo Fisher Scientific, 29851 Willow Creek Road, Eugene, OR, USA.) for 1 h and counterstained with DAPI for 3 min. Except where otherwise noted, all dilutions and thorough washings between phases were carried out using PBS. The images of the sections were obtained using a Pannoramic 250 FLASH II slide scanner, and the total count of receptor distribution in different regions of rat brains was performed using Zen software on panoramic images.
The mechanical pain behavior test was performed by the Von Frey device. The animals were put on an elevated mesh and given 30 min to explore. A specified force was applied while maintaining compression with an elastic filament. The diameters of filaments require a wide range of forces to induce buckling. The shorter the filament, the higher the force required to buckle it. It helped to record the minimal force/pressure at which the animals reacted (limb withdrawal) due to the painful stimulus. Each hind limb underwent three trials, and the average of the minimal pressure was used to determine the threshold. A 5 min break separated every trial.
The Thermal Plantar Instrument measures pain sensitivity according to Hargreave's Method (IITC Inc. Life Science, 23924 Victory Blvd Woodland Hills, CA 91367, USA). It measures the infrared heat stimulus response in rodents and functions by focusing the infrared source below the plexiglass surface (instrument) and pressing the button of the instrument. Rats would then be placed on a plexiglass surface for about 30 min. The radiant heat would be focused on the hind paw below the surface of the glass floor; paw withdrawal latency and infrared intensity are recorded automatically. Each rat was tested three times for each hind paw, with a 5 min interval between tests.
The procedure of cold allodynia was performed in accordance with the method described by Flatters and Bennett [[
After the behaviors, the animals were sacrificed. After brain extraction from the rat's skull, the brain was rinsed with cold PBS to remove excess blood. Then, the tissues were isolated on dry ice with forceps and scissors. Because the previous studies demonstrated that the thalamus and cerebral cortex contributed to chronic pain [[
Brain tissues were homogenized in 230 mM mannitol, 70 mM sucrose, 1.0 mM EDTA, and 10 mM Tris-HCl, pH 7.40, at a ratio of 10 mL of homogenization medium/g of tissue, and mitochondria were extracted from these tissues. To obtain mitochondrial preparations, the homogenate was centrifuged at 700× g for 10 min and the supernatant was centrifuged at 8000× g for 10 min to pellet the mitochondria that were then washed under the same circumstances [[
Protein content was measured with the Lowry method using a Folin phenol reagent [[
Preparing 0.1 mL of the sample or standard (10 μg, 20 μg, 40 μg, 80 μg, and 100 μg/mL), 0.1 mL of 2 N NaOH was added and hydrolyzed at 100 °C for 10 min in a heating block or boiling water bath. Hydrolysate was allowed to cool down, and 1 mL of freshly mixed complex-forming reagent (2% (w/v) Na
Malondialdehyde (MDA), an end product of lipid peroxidation, was measured quantitatively in the brain [[
The amino acids glutamine, cysteine, and glycine are combined to form the water-soluble tripeptide known as glutathione (GSH). GSH, which can reach millimolar quantities in some tissues, is the most prevalent intracellular small-molecule thiol due to the thiol group's powerful reducing agent. Glutathione S-transferases (GST) and glutathione peroxidases catalyze the detoxification of a variety of electrophilic substances and peroxides using GSH as an essential antioxidant (GPx). 5,5′-dithio-bis(2-nitrobenzoic acid), often known as DTNB and Ellman's [[
A mixture of homogenate and 4% sulphosalicylic acid was maintained at 4 °C for one hour before being centrifuged at 1200× g for 15 min at 4 °C. The supernatant was collected, and phosphate buffer (0.1 M, pH 8) and DTNB (0.4% w/v in 0.1 M phosphate buffer, pH 8) were added to it. At 412 nm, absorbance was immediately measured.
Complex—I: Also known as NADH: ubiquinone oxidoreductase, Type I NADH dehydrogenase, and mitochondrial complex I. It was assayed with the method described by King and Howard [[
Complex—II: Also known as succinate dehydrogenase (SDH); it was assayed with the modified method of King (1967) [[
Complex—III: This complex is also known as Coenzyme Q—cytochrome c reductase. MTT, 10 mg/mL, in 0.1 PBS and DMSO were used as reagents [[
Complex—IV: This complex is also known as Cytochrome c oxidase. This assay was performed using the method described by Sottocasa (1967) [[
Cyto-C was reduced by the addition of several crystals of sodium borohydride (light-sensitive) and neutralized with 100 mM HCl (pH 7). Then, 700 µL of phosphate buffer was added to the solution of 100 µL of reduced Cyto-C (50 µL) and 10 µL of the sample and the change in OD was checked at 550 for 180 min.
The estimation of catalase was performed with the method described by Luck [[
The Ellman method was used to carry out the assay [[
The enzyme superoxide dismutase was assayed using the modified method by Kono et al. [[
Rat IL-1β, IL-6, and TNF-α -immunoassay kits (Abcam) were used to perform IL-1β, IL-6, and TNF-α quantifications. The 4.5 h solid-phase ELISA used in quantizing the rat IL-1β, IL-6, and TNF-α immunoassay was created to quantify the amounts of these three substances. An enzyme-linked immunosorbent test (ELISA) uses a solid-phase sandwich and a microtiter plate reader. From the standard curves, proinflammatory cytokine concentrations were determined.
The pre-coated plate was filled with the sample (100 μL). After that, the plate was sealed and left to sit at room temperature for two hours. With wash buffer, the plate was washed four times. Each well received 100 μL of diluted detection antibody solution, and the plate was then sealed and incubated at room temperature for 2 h. The plate was then cleaned with wash buffer four times. After adding 100 μL of diluted Streptavidin-HRP solution to each well, the plate was sealed and incubated at room temperature for 30 min. The plate was cleaned with a wash buffer four more times. Each well received 100 μL of diluted TMB substrate solution, and the plate underwent a 15 min incubation period in the dark. Positive wells then took on a bluish color. The reaction was stopped by adding 100 μL of stop solution to each well. Positive wells changed from blue to yellow. After halting the experiment, the absorbance at 450 nm was detected after 15 min.
The drug used in the study was melatonin, which was obtained from Sigma-Aldrich, USA. Melatonin was intraperitoneally administered (dose-dependently 30, 60, 120 mg/kg i.p.) [[
All data were analyzed using a Kruskal–Wallis test (a nonparametric analysis) followed by a Dunn post hoc test for multiple comparisons. * p < 0.05 was considered statistically significant.
Chronic melatonin administrations have been shown to alleviate pain behaviors in animal models of CPSP in various ways, such as by preserving mitochondrial homeostasis, reducing free radical generation, enhancing mitochondrial glutathione levels, and safeguarding the proton potential in the mitochondrial ETC by stimulating complex I and IV activities and reducing CPSP-induced neuroinflammation. Therefore, we suggested that administrations of exogenous melatonin could be promising clinical interventions in CPSP patients.
Graph: Figure 1 MT1 receptor distribution using FITC (florescent green) and DAPI (blue) staining in the specific brain regions. (A) Ventral posteromedial thalamic nucleus (VPM), (B) Somatosensory cortex (S1), (C) Dentate gyrus (DG), (D) Amygdalostriatal transition area (Astr), (E) Secondary auditory cortex (AUD), (F) Basolateral amygdala (Bla), (G) Lateral hypothalamus (LH), (H) Magnocellular region (Mc), (I) Magnocellular nucleus of lateral hypothalamus (Mclh), (J) Nigrostriatal fibers (Ns), (K) Posterior hypothalamic area (Ph), (L) Retrosplenial agranular cortex (Rsa), (M) Posterior thalamic nuclear group (Po), (N) Ventromedial thalamic nucleus (VM), (O) Ventromedial hypothalamic nucleus (VMH), and (P) Reticular thalamic nucleus (RT). Scale bar is 100 μm.
Graph: Figure 2 Effects of melatonin treatments on various pain tests, including Vonfrey pain, plantar, and cold allodynia tests. (A) Effect of melatonin administration on paw withdrawal threshold (g) in rats during a Von Frey pain test on days 7 and 14 after thalamic hemorrhage (n = 5, per group). Data expressed as mean ± SD. The figure shows pain hypersensitivity in central stroke rats 7 days and 14 days post-injury, * p < 0.05. (B) Effect of melatonin administration in cold allodynia in rats on days 7 and 14 after thalamic hemorrhage (n = 6, per group). The cold allodynia scores, as accounted by paw withdrawal, were demonstrated more in CPSP rats as compared with the sham control; MLT (30 mg/kg, i.p.) gave the same response as CPSP rats (not shown in figure); MLT (60 mg/kg i.p.) showed an effective response, but some rats showed both prolonged and quick paw withdrawal responses, whereas MLT (120 mg/kg i.p.) demonstrated a much better effect on CPSP rats. The cold allodynia score was almost zero on the 14th day of melatonin administration, which suggests a healing effect of melatonin. (C) Effect of melatonin administration on paw withdrawal latency in plantar test in rats on days 7 and 14 after thalamic hemorrhage (n = 5, per group). Data expressed as mean ± SD. The figure shows pain hyperalgesia in central stroke rats 7 days and 14 days post-injury, * p < 0.05.
Graph: Figure 3 Effect of CPSP and melatonin treatment on mitochondria chain complexes, including (A) mitochondrial complex-i (NADH: ubiquinone oxidoreductase; n = 5, per group), (B) complex-ii (n = 4, per group), (C) complex-iii (n = 5, per group), and (D) complex-iv (n = 5, per group) levels in the thalamus and cortex after two weeks of melatonin administration. Data expressed as mean ± SD; * p < 0.005 as compared with CPSP.
Graph: ijms-24-05413-g003b.tif
Graph: Figure 4 Effect of CPSP and melatonin on ETC chain enzymes A, including (A) gluthathione peroxidase (n = 4, per group), (B) catalase (n = 4, per group), and (C) superoxidase dismutase (n = 5, per group) levels in the thalamus and the cortex after two weeks of melatonin administration. Data expressed as mean ± SD; * p < 0.005 as compared with CPSP.
Graph: ijms-24-05413-g004b.tif
Graph: Figure 5 Effect of lesions and melatonin treatments on oxidative stress, including (A) lipid peroxidation (n = 5, per group) and (B) reduced glutathione (n = 5, per group) levels in the thalamus and the cortex after two weeks of melatonin administration. Data expressed as mean ± SD; * p < 0.005 as compared with CPSP.
Graph: Figure 6 Effect of lesions and melatonin administration on neuroinflammation, including (A) TNF-a (n = 5, per group), (B) IL-6 (n = 6, per group), and (C) IL-1b (n = 6, per group) levels in the whole brain after two weeks of melatonin administration. Data expressed as mean ± SD; * p < 0.005 as compared with CPSP.
Graph: Figure 7 The schematic representation showing an ETC chain reaction in mitochondria in normal physiological conditions, effect of lesion, and effect of exogenous melatonin treatment on ETC. In normal conditions, the electron from the transport chain progresses to form water, but due to the lesion, the oxygen free radical produces nitric oxide radicals, giving rise to RNS. Mitochondrial superoxide dismutase (SOD) neutralizes the highly reactive superoxide radical O2–, known as reactive oxygen species (ROS). Under CPSP conditions due to the lesion, ROS and RNS levels increase and melatonin helps to reduce ROS and RNS.
Graph: Figure 8 (A) Flowchart for experimental design. (B) Microinjection of collagenases to induce lesions of the VPL/VPM in the animal model of central post-stroke pain.
Table 1 Melatonin receptor distribution in regions of the brain.
Region of Interest Annotation Area Relative Mask Area Total Count Receptor Density S1 7.67 mm2 0.12% 3170 413.1 mm2 VM 0.47 mm2 0.1% 211 449.45 mm2 RT 0.34 mm2 0.23% 265 768.52 mm2 Hippocampus 5.50 mm2 1.14% 4648 844.86 mm2 MEPD/MEPV 0.42 mm2 0.2% 415 996.89 mm2 VPM/VPL 2.26 mm2 0.88% 3274 1450.59 mm2 LH (lateral hypothalamus) 0.68 mm2 0.44% 592 867.58 mm2 Vmhyp nu (ventromedial hypothalamus) 2.12 mm2 0.09% 579 273.11 mm2 AUD 4.11 mm2 0.33% 3428 833.96 mm2 PH (posterior hypothalamus) 0.26 mm2 0.27% 163 635.45 mm2 BLA/BMP 0.73 mm2 0.08% 223 303.86 mm2 S2 1.01 mm2 0.23% 751 744.1 mm2
T.K.: Methodology, Validation, Investigation, Project Administration; A.C.-W.H.: Formal Analysis, Writing—Review and Editing, Supervision; B.-C.S.: Writing—Review and Editing, Supervision, Funding Acquisition. All authors have read and agreed to the published version of the manuscript.
The study received approval from the Institutional Animal Care and Use Committee (IACUC) of Academia Sinica.
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.
The authors declare that they have no competing interest.
We would like to thank the Institute of Biomedical Sciences of Academia Sinica in Taiwan.
By Tavleen Kaur; Andre Chih-Wei Huang and Bai-Chuang Shyu
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