Melatonin as a potential counter-effect of hyperalgesia induced by neonatal morphine exposure.

Morphine administration in the neonatal period can induce long-term effects in pain circuitry leading to hyperalgesia induced by the opioid in adult life. This study explored a new pharmacological approach for reversing this effect of morphine. We focused on melatonin owing its well-known antinociceptive and anti-inflammatory effects, and its ability to interact with the opioid system. We used the formalin test to assess the medium and long-term effects of melatonin administration on hyperalgesia induced by morphine in early life. Newborn rats were divided into two groups: the control group, which received saline, and the morphine group, which received morphine (5µg subcutaneously [s.c.]) in the mid-scapular area, once daily for 7days, from P8 (postnatal day 8) until P14. At postnatal days 30 (P30) and 60 (P60), both groups were divided in two subgroups, which received melatonin or melatonin vehicle 30min before the formalin test. The nociceptive responses were assessed by analyzing the total time spent biting, flicking, and licking the formalin-injected hind paw; these responses were recorded during the first 5min (neurogenic/acute phase) and from 15 to 30min (inflammatory/tonic phase). Initially, animals in the morphine/vehicle group showed increased nociceptive behavior in phase II (inflammatory) of the formalin test at P30, and in the neurogenic and inflammatory phases at P60. These increased nociceptive responses were fully reversed by melatonin administration at either age. These findings show that melatonin administration is a potential means for countering hyperalgesia induced by neonatal morphine exposure in young and adult rats.

Transcranial direct current stimulation (tDCS) reverts behavioral alterations and brainstem BDNF level increase induced by neuropathic pain model: Long-lasting effect.

INTRODUCTION: Neuropathic pain (NP) is a chronic pain modality that usually results of damage in the somatosensory system. NP often shows insufficient response to classic analgesics and remains a challenge to medical treatment. The transcranial direct current stimulation (tDCS) is a non-invasive technique, which induces neuroplastic changes in central nervous system of animals and humans. The brain derived neurotrophic factor plays an important role in synaptic plasticity process. Behavior changes such as decreased locomotor and exploratory activities and anxiety disorders are common comorbidities associated with NP. OBJECTIVE: Evaluate the effect of tDCS treatment on locomotor and exploratory activities, and anxiety-like behavior, and peripheral and central BDNF levels in rats submitted to neuropathic pain model. METHODS: Rats were randomly divided: Ss, SsS, SsT, NP, NpS, and NpT. The neuropathic pain model was induced by partial sciatic nerve compression at 14 days after surgery; the tDCS treatment was initiated. The animals of treated groups were subjected to a 20 minute session of tDCS, for eight days. The Open Field and Elevated Pluz Maze tests were applied 24 h (phase I) and 7 days (phase II) after the end of tDCS treatment. The serum, spinal cord, brainstem and cerebral cortex BDNF levels were determined 48 h (phase I) and 8 days (phase II) after tDCS treatment by ELISA. RESULTS: The chronic constriction injury (CCI) induces decrease in locomotor and exploratory activities, increases in the behavior-like anxiety, and increases in the brainstem BDNF levels, the last, in phase II (one-way ANOVA/SNK, P<0.05 for all). The tDCS treatment already reverted all these effects induced by CCI (one-way ANOVA/SNK, P<0.05 for all). Furthermore, the tDCS treatment decreased serum and cerebral cortex BDNF levels and it increased these levels in the spinal cord in phase II (one-way ANOVA/SNK, P<0.05). CONCLUSION: tDCS reverts behavioral alterations associated to neuropathic pain, indicating possible analgesic and anxiolytic tDCS effects. tDCS treatment induces changes in the BDNF levels in different regions of the central nervous system (CNS), and this effect can be attributed to different cellular signaling activations.

Exogenously induced brain activation regulates neuronal activity by top-down modulation: conceptualized model for electrical brain stimulation.

Physiological and exogenous factors are able to adjust sensory processing by modulating activity at different levels of the nervous system hierarchy. Accordingly, transcranial direct current stimulation (tDCS) may use top-down mechanisms to control the access for incoming information along the neuroaxis. To test the hypothesis that brain activation induced by tCDS is able to initiate top-down modulation and that chronic stress disrupts this effect, 60-day-old male Wistar rats (n = 78) were divided into control; control + tDCS; control + sham-tDCS; stress; stress + tDCS; and stress + sham-tDCS. Chronic stress was induced using a restraint stress model for 11 weeks, and then, the treatment was applied over 8 days. BDNF levels were used to assess neuronal activity at spinal cord, brainstem, and hippocampus. Mechanical pain threshold was assessed by von Frey test immediately and 24 h after the last tDCS-intervention. tDCS was able to decrease BDNF levels in the structures involved in the descending systems (spinal cord and brainstem) only in unstressed animals. The treatment was able to reverse the stress-induced allodynia and to increase the pain threshold in unstressed animals. Furthermore, there was an inverse relation between pain sensitivity and spinal cord BDNF levels. Accordingly, we propose the addition of descending systems in the current brain electrical modulation model.

Melatonin analgesia is associated with improvement of the descending endogenous pain-modulating system in fibromyalgia: a phase II, randomized, double-dummy, controlled trial.

BACKGROUND: Central disinhibition is a mechanism involved in the physiopathology of fibromyalgia. Melatonin can improve sleep quality, pain and pain threshold. We hypothesized that treatment with melatonin alone or in combination with amitriptyline would be superior to amitriptyline alone in modifying the endogenous pain-modulating system (PMS) as quantified by conditional pain modulation (CPM), and this change in CPM could be associated with serum brain-derived neurotrophic factor (BDNF). We also tested whether melatonin improves the clinical symptoms of pain, pain threshold and sleep quality. METHODS: Sixty-three females, aged 18 to 65, were randomized to receive bedtime amitriptyline (25 mg) (n = 21), melatonin (10 mg) (n = 21) or melatonin (10 mg) + amitriptyline (25 mg) (n = 21) for a period of six weeks. The descending PMS was assessed with the CPM-TASK. It was assessed the pain score on the Visual Analog Scale (VAS 0-100 mm), the score on Fibromyalgia Impact Questionnaire (FIQ), heat pain threshold (HPT), sleep quality and BDNF serum. Delta values (post- minus pre-treatment) were used to compare the treatment effect. The outcomes variables were collected before, one and six weeks after initiating treatment. RESULTS: Melatonin alone or in combination with amitriptyline reduced significantly pain on the VAS compared with amitriptyline alone (P < 0.01). The delta values on the VAS scores were-12.85 (19.93),-17.37 (18.69) and-20.93 (12.23) in the amitriptyline, melatonin and melatonin+amitriptyline groups, respectively. Melatonin alone and in combination increased the inhibitory PMS as assessed by the Numerical Pain Scale [NPS(0-10)] reduction during the CPM-TASK:-2.4 (2.04) melatonin + amitriptyline,-2.65 (1.68) melatonin, and-1.04 (2.06) amitriptyline, (P < 0.05). Melatonin + amitriptyline treated displayed better results than melatonin and amitriptyline alone in terms of FIQ and PPT improvement (P < 0.05, fort both). CONCLUSION: Melatonin increased the inhibitory endogenous pain-modulating system as assessed by the reduction on NPS(0-10) during the CPM-TASK. Melatonin alone or associated with amitriptyline was better than amitriptyline alone in improving pain on the VAS, whereas its association with amitriptyline produced only marginal additional clinical effects on FIQ and PPT. TRIAL REGISTRATION: Current controlled trail is registered at clinical trials.gov upon under number NCT02041455. Registered January 16, 2014.

Melatonin treatment entrains the rest-activity circadian rhythm in rats with chronic inflammation.

We assessed the therapeutic effect of exogenous melatonin (MEL), dexamethasone (DEXA), and a combination of both on nociceptive response induced by chronic inflammation and on the rest-activity circadian rhythm in rats. A total of 64 animals were randomly divided into eight groups of eight rats each: one control group and seven groups with complete Freund's adjuvant-inflamed animals (CFA; injection into the footpad). One of the CFA-inflamed groups did not receive any treatment; the other six were treated with melatonin (MEL), dexamethasone (DEXA), melatonin plus dexamethasone (MELDEXA), and their respective vehicles. Fifteen days after CFA injection, animals were treated with intraperitoneal injection of MEL (50 mg/kg) or its vehicle (8% ethanol in saline), DEXA (0.25 mg/kg) or its vehicle (saline), and MEL plus DEXA or their vehicles, for 8 days. The von Frey test was performed 24 h after the last administration of each treatment regimen. Hind paw thickness was measured using a pachymeter during the treatment days. The degree of swelling and histological findings were analyzed. All treated groups significantly reduced the severity of inflammation when compared with their vehicles (repeated-measures analysis of variance [ANOVA], p < 0.05 for all analyses). Inflamed animals treated with dexamethasone alone or associated with melatonin showed marked inhibition of histological findings. On the other hand, the group treated with melatonin remained with moderate inflammation. The CFA group showed a decrease in the mean rest-activity circadian rhythm, determined by the number of touch-detections per hour during water intake in comparison with the control group; only the group treated with melatonin showed a synchronized rest-activity rhythm. At the end of treatment, a significant increase was observed in hind paw withdrawal threshold on the von Frey test in the treated groups (one-way ANOVA, p < 0.05 for all). Our findings showed that melatonin (50 mg/kg) has strong chronobiotic and antinociceptive effects, but only mild anti-inflammatory effects. This evidence supports the hypothesis that melatonin can induce phase advance and circadian rhythm synchronization in rats with chronic inflammation.

Morphine treatment in early life alters glutamate uptake in the spinal synaptosomes of adult rats.

Morphine exposure during the neonatal period can promote changes in pain signaling pathways that can be expressed as an increased nociceptive response in adult life. Glutamate is the major excitatory neurotransmitter in primary afferent terminals and plays a critical role in normal spinal excitatory synaptic transmission. Considering the importance of a better understanding of the mechanisms that underlie nociceptive changes throughout the life course, the aim of this study was investigate the effects of repeated morphine administration at postnatal days 8 (P8) to 14 (P14) on glutamate uptake in spinal synaptosomes at P30 and P60. The morphine group showed decreased [3H]-glutamate uptake as compared to control groups in both P30 and P60. These findings suggest that morphine exposure in early life leads to changes in glutamatergic signaling at least until the 60th day of age, which may lead to increased levels of glutamate in the spinal synaptic cleft and, consequently, an increased nociceptive response in adult life. Thus, this study highlights the importance of conducting research in this field to provide a comprehensive knowledge of the long-term effects of early-life morphine treatment on nociceptive pathways.

After-effects of consecutive sessions of transcranial direct current stimulation (tDCS) in a rat model of chronic inflammation.

Transcranial direct current stimulation (tDCS) induces cortical excitability changes in animals and humans that can last beyond the duration of stimulation. Preliminary evidence suggests that tDCS may have an analgesic effect; however, the timing of these effects, especially when associated with consecutive sessions of stimulation in a controlled animal experiment setting, has yet to be fully explored. To evaluate the effects of tDCS in inflammatory chronic pain origin immediately and 24 h after the last treatment session, complete Freund's adjuvant (CFA) was injected (100 µl) in the right footpad to induce inflammation. On the 15th day after CFA injection, rats were divided into two groups: tDCS (n = 9) and sham (n = 9). The tDCS was applied for 8 days. The hot plate and Von Frey tests were applied immediately and 24 h after the last tDCS session. Eight 20-min sessions of 500 µA anodal tDCS resulted in antinociceptive effects as assessed by the hot plate test immediately (P = 0.04) and 24 h after the last tDCS session (P = 0.006), for the active tDCS group only. There was increased withdrawal latency in the Von Frey test at 24 h after the last session (P = 0.01). Our findings confirm the hypothesis that tDCS induces significant, long-lasting, neuroplastic effects and expands these findings to a chronic pain model of peripheral inflammation, thus supporting the exploration of this technique in conditions associated with chronic pain and peripheral inflammation, such as osteoarthritis.

BDNF as an effect modifier for gender effects on pain thresholds in healthy subjects.

BDNF is an important marker of neuronal plasticity. It has also been associated with pain processing. Increased BDNF levels are observed in chronic pain syndromes. In order to understand the role of BDNF associated with other factors such as gender on experimental pain we aimed to determine whether experimental heat or pressure pain threshold is correlated with brain derived neurotrophic factor (BDNF) level, gender and age. Heat pain threshold and pressure pain threshold were measured in 49 healthy volunteers (27 females). The multivariate linear regression models (on heat and pressure pain thresholds) revealed a significant effect of gender (p=0.001 for both models), serum BDNF (p<0.004 for both models) and interaction between BDNF and gender (<0.001 for both models). In fact, when adjusting for BDNF levels and age, heat and pressure pain thresholds were significantly reduced in women as compared to men (p<0.001 for both models). These effects were not observed when gender was analyzed alone. These finding suggests that experimental heat and pressure pain threshold is gender-related and BDNF dependent. In fact BDNF has a facilitatory effect on pain threshold in females but has an opposite effect in males; supporting the notion that BDNF is an effect modifier of the gender effects on pain threshold in healthy subjects.

Morphine treatment alters nucleotidase activities in rat blood serum.

Morphine has been widely used in neonatal pain management. However, this treatment may produce adaptive changes in several physiologic systems. Our laboratory has demonstrated that morphine treatment in neonate rats alters nucleoside triphosphate diphosphohydrolase (NTPDase) activity and gene expression in central nervous system structures. Considering the relationship between the opioid and purinergic systems, our aim was to verify whether treatment with morphine from postnatal days 8 (P8) through 14 (P14) at a dose of 5 µg per day alters NTPDase and 5'-nucleotidase activities in rat serum over the short, medium, and long terms. After the in vivo assay, the morphine group showed increased hydrolysis of all nucleotides at P30, and a decrease in adenosine 5'-diphosphate hydrolysis at P60. Moreover, we found that nucleotidase activities change with age; adenosine 5'-triphosphate hydrolysis activity was lower at P16, and adenosine 5'-monophosphate hydrolysis activity was higher at P60. These changes are very important because these enzymes are the main regulators of blood nucleotide levels and, consequently, nucleotide signaling. Our findings showed that in vivo morphine treatment alters nucleotide hydrolysis in rat blood serum, suggesting that purine homeostasis can be influenced by opioid treatment during the neonatal period.

Physiological level of norepinephrine increases adenine nucleotides hydrolysis in rat blood serum.

Extracellular adenosine 5'-triphosphate (ATP) and its breakdown products, adenosine 5'-diphosphate (ADP) and adenosine, have significant effects on a variety of biological processes. NTPDase enzymes, responsible for adenine nucleotides hydrolysis, are considered the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATP and ADP hydrolysis in rat blood serum are higher during the dark (activity) phase compared to the light (rest) phase. In nocturnal animals (e.g., rats), important physiological changes occur during the dark phase, such as increased circulating levels of melatonin, corticosterone, and norepinephrine (NE). This study investigated the physiological effects, in vivo and in vitro, of melatonin, dexamethasone, and NE upon nucleotides hydrolysis in rat blood serum. For in vivo experiments, the animals received a single injection of saline (control), melatonin (0.05 mg/kg), dexamethasone (0.1 mg/kg), or NE (0.03 mg/kg). For in vitro experiments, melatonin (1.0 nM), dexamethasone (1.0 µM), or NE (1.0 nM) was added directly to the reaction medium with blood serum before starting the enzyme assay. The results demonstrated that ATP and ADP hydrolysis in both in vitro and in vivo experiments were significantly higher with NE treatment compared to control (in vitro: ATP = 36.63%, ADP = 22.43%, P < 0.05; in vivo: ATP = 44.1%, ADP = 37.28%, P < 0.001). No significant differences in adenine nucleotides hydrolysis were observed with melatonin and dexamethasone treatments. This study suggests a modulatory role of NE in the nucleotidases pathway, decreasing extracellular ATP and ADP, and suggests that NE might modulate its own release by increasing the activities of soluble nucleotidases.

Morphine exposure in early life increases nociceptive behavior in a rat formalin tonic pain model in adult life.

Considering the importance of a deeper understanding of the effect throughout life of opioid analgesia at birth, our objective was to determine whether morphine administration in early life, once a day for 7 days in 8-day-old rats, alters the nociceptive response over the short (P16), medium (P30), and long term (P60) and to evaluate which system is involved in the altered nociceptive response. The nociceptive responses were assessed by the formalin test, and the behavior analyzed was the total time spent in biting and flicking of the formalin-injected hindpaw, recorded during the first 5 min (phase I) and from 15-30 min (phase II). The morphine group showed no change in nociceptive response at P16, but at P30 and P60, the nociceptive response was increased in phase I, and in both phases, respectively. At P30 and P60, the animals received a non-steroidal anti-inflammatory drug (indomethacin) or NMDA receptor antagonist (ketamine) 30 min before the formalin test. The increase in the nociceptive response was completely reversed by ketamine, and partially by indomethacin. These results indicate that early morphine exposure causes an increase in the nociceptive response in adult life. It is possible that this lower nociception threshold is due to neuroadaptations in nociceptive circuits, such as the glutamatergic system. Thus, this work demonstrates the importance of evaluating clinical consequences related to early opioid administration and suggests a need for a novel design of agents that may counteract opiate-induced neuroplastic changes.

Lifetime behavioural changes after exposure to anaesthetics in infant rats.

The aim of this study was to assess the effect of acute use of general anaesthetic with or without a surgical procedure, at post-natal day 14 (P14), on behavioural responses in the short-, medium- and long-term, evaluated in open field (OF) and elevated plus-maze (EPM) tests. Fourteen-day-old male Wistar rats were divided into two experimental designs (ED): inhalation and intravenous anaesthetic, and these groups were subdivided into: 1st ED - control (C), isoflurane (ISO), isoflurane/surgery (ISO-SUR); 2nd ED - control (C), fentanyl/S(+)-ketamine (FK) and fentanyl+ketamine-s/surgery (FK-SUR). In the OF the following were found: (a) in the 1st ED: an increase in the locomotor activity in the ISO group at P14, and ISO and ISO-SUR groups at P30; the ISO-SUR group showed a reduced latency to leave the first quadrant at P30 and P60; (b) in the 2nd ED: FK and FK-SUR groups presented increased locomotor activity at P30, and the FK group showed a reduction in the number of faecal boluses. In the EPM the following were found: FK and FK-SUR groups presented an increase in the number of non-protected head-dipping (NPHD) movements and in the number of entries and time spent in open arms at P30; the FK group showed an increased number of protected head-dipping movements, NPHD and entries and time spent in the open arms at P60. The behavioural changes observed may be related to locomotor activity (1st ED) and anxiety level (2nd ED) and they may result from changes in neurotransmitters/hormones (DA, 5HT, CRH) and glutamate/NMDA receptors, respectively.

24-hour temporal pattern of NTPDase and 5'-nucleotidase enzymes in rat blood serum.

Circadian rhythms represent an important mechanism to prepare the organism for environmental variations. ATP, ADP, AMP, and adenosine can act as extracellular messengers in a range of biological processes and are metabolized by a number of enzymes, including NTPDases and 5'-nucleotidase. In the present study the authors report that ATPase and ADPase activities present 24-h temporal variations that peak during dark (activity) span. These findings suggest that this enzymatic temporal pattern in blood serum might be important for the normal physiology and function of the organism through the maintenance of extracellular nucleotides at physiological levels.

Neonatal morphine exposure alters E-NTPDase activity and gene expression pattern in spinal cord and cerebral cortex of rats.

The neonate opioid system has been frequently investigated, and studies have shown that exposure to drugs in early life can have implications for nervous system development. It has been proposed that adenosine is involved in opioid antinociception, and ATP is involved in central and peripheral mechanisms of nociception. Extracellular nucleotides can be hydrolyzed by E-NTPDases and ecto-5'nucleotidase, which present the functions of removing ATP and generating adenosine. In this study, we evaluated ATP, ADP, and AMP hydrolysis in synaptosomes from spinal cord and cerebral cortex of rats at postnatal day 16 after repeated morphine exposure in early life (postnatal day 8 to 14). Additionally, we evaluated E-NTPDase (1, 2 and 3) and ecto-5'nucleotidase gene expression by semi-quantitative RT-PCR analysis. We observed an increase in ATP hydrolysis in the cerebral cortex, and a decrease in ADP hydrolysis in spinal cord. Expression levels of E-NTPDase 1 decreased in cerebral cortex and increased in spinal cord. Our findings highlight the importance of the purinergic system in young rats submitted to repeated morphine exposure by showing that in the neonatal period such exposure is capable of affecting the control system for nucleotide levels, which can promote changes in modulation or transmission of painful stimuli.

A neuroprotective exercise protocol reduces the adenine nucleotide hydrolysis in hippocampal synaptosomes and serum of rats.

Regular and moderate exercise has been considered as an interesting neuroprotective strategy. However, the molecular mechanisms by which physical exercise alters brain function are unclear. Purinergic signaling seems to modulate the pathophysiology of ischemic neuronal damage, since it has been described a neuroprotective activity of adenosine and a dual role of ATP. In the present study, we investigated the effect of daily moderate intensity exercise on ectonucleotidase activities in synaptosomes from hippocampus and the soluble nucleotidases from blood serum of rats. Adult male Wistar rats were assigned to non-exercised (sedentary) group and exercised during 20-min sessions on different programs. The effects of physical activity on hydrolysis of ATP, ADP and AMP were assayed in the synaptosomal fraction obtained from the hippocampus and serum approximately 16 h after the last training session. Our data demonstrated that a neuroprotective exercise protocol, daily 20 min of training in treadmill during 2 weeks, diminished significantly the ADP hydrolysis and there is a trend to reduce the ATP hydrolysis in both hippocampal synaptosomes and blood serum of rats. We suggest that the neuroprotective exercise protocol may modulate nucleotidase activities.

Long-term effect of morphine administration in young rats on the analgesic opioid response in adult life.

Neonates, infants and children are often exposed to pain from invasive procedures during intensive care and during the post-operative period. Opioid anesthesia and post-operative opioid analgesia have been used in infants and result in clinical benefits. The objectives of this study were to verify the effect of repeated 5 microg morphine administration (subcutaneous), once a day for 7 days in 8-day-old rats, at P8 until P14. To verify the long-term effect of morphine, the animals were submitted to a second exposure of 5mg/kg (intraperitoneal) of morphine at P80 until P86. Animals that received morphine for 7 days, at P14 did not develop tolerance, however at P80, rats demonstrated greater morphine analgesia. At P86, after 7 days of morphine administration, animals showed classical tolerance. These findings may have important implications for the human neonate, suggesting a possible explanation for the differences in the requirements of morphine observed in the youngest patients.