Adriana Miclescu

Nitric oxide and pain: ‘Something old, something new’

Challenges have emerged following the revival of nitric oxide (NO) from ‘something old’, a simple gas derived from nitrogen and oxygen with a role in the early stages of evolution, into ‘something new’, an endogenously formed biological mediator regulating a wide variety of physiological functions. Although pain is a common sensation, it encompasses multiple neurobiologic components, of which NO is only one. In pain research, the study of NO is complicated by convoluted problems related mostly to the effects of NO, which are pro‐ or anti‐nociceptive depending on the circumstances. This dual function reflects the multi‐faceted roles of the NO molecule described in physiology. This review covers current information about NO and its implications in pain mechanisms. In addition, it follows the pain pathways, demonstrating the role of NO in peripheral nociceptive transmission as well in central sensitization. This knowledge may provide the scientific basis for developing new drugs that are indicated for different types of pain, drugs that may be related to the chemical links of NO. A comprehensive approach to understanding the effects of NO will help clinicians identify novel agents that combine the pharmacological profile of native drugs with a controllable manner of NO release. Inhibitors of NO synthesis may have analgesic effects and would be of interest for treating inflammatory and neuropathic pain. Unfortunately, only a few of these compounds have reached the stage of clinical pain trials.

Methylene blue added to a hypertonic-hyperoncotic solution increases short-term survival in experimental cardiac arrest.

Objective:Methylene blue (MB), a free-radical scavenger inhibiting the production and actions of nitric oxide, may counteract excessive vasodilatation induced by nitric oxide during cardiac arrest. Effects of MB in cardiac arrest and cardiopulmonary resuscitation were investigated. Design:Randomized, prospective, laboratory animal study. Setting:University animal research laboratory. Subjects:A total of 63 piglets of both sexes. Interventions:A pig model of extended cardiac arrest (12 mins of untreated cardiac arrest and 8 mins of cardiopulmonary resuscitation) was employed to assess the addition or no addition of MB to a hypertonic saline–dextran solution. These two groups (MB and hypertonic saline–dextran group [MB group] and hypertonic saline-dextran–only group) of 21 animals were each compared with a group receiving isotonic saline (n = 21). Measurements and Main Results:Although the groups were similar in baseline values, 4-hr survival in the MB group was increased (p = .02) in comparison with the isotonic saline group. Hemodynamic variables were somewhat improved at 15 mins after restoration of spontaneous circulation in the MB group compared with the other two groups. The jugular bulb levels of 8-isoprostane-prostaglandin F2&agr; and 15-keto-dihydro-prostaglandin F2&agr; (indicators of peroxidation and inflammation) were significantly decreased in the MB group compared with the isotonic saline group. Significant differences were recorded between the three groups in levels of protein S-100&bgr; (indicator of neurologic injury), with lower levels in the MB group compared with the isotonic saline and hypertonic saline-dextran–only groups. Troponin I and myocardial muscle creatine kinase isoenzyme arterial concentrations (indicators of myocardial damage) were also significantly lower in the MB group. Conclusions:MB co-administered with a hypertonic–hyperoncotic solution increased 4-hr survival vs. saline in an experimental porcine model of cardiac arrest and reduced oxidative, inflammatory, myocardial, and neurologic injury.

Neuro- and cardioprotective effects of blockade of nitric oxide action by administration of methylene blue.

Methylene blue (MB), generic name methylthioninium (C16H18ClN3 S · 3H2O), is a blue dye synthesized in 1876 by Heinrich Caro for use as a textile dye and used in the laboratory and clinically since the 1890s, with well‐known toxicity and pharmacokinetics. It has experimentally proven neuroprotective and cardioprotective effects in a porcine model of global ischemia–reperfusion in experimental cardiac arrest. This effect has been attributed to MBs blocking effect on nitric oxide synthase and guanylyl cyclase, the latter blocking the synthesis of the second messenger of nitric oxide. The physiological effects during reperfusion include stabilization of the systemic circulation without significantly increased total peripheral resistance, moderately increased cerebral cortical blood flow, a decrease of lipid peroxidation and inflammation, and less anoxic tissue injury in the brain and the heart. The last two effects are recorded as less increase in plasma concentrations of astroglial protein S‐100β, as well as troponin I and creatine kinase isoenzyme MB, respectively.

Methylene blue protects the cortical blood-brain barrier against ischemia/reperfusion-induced disruptions

Objectives:To investigate the effects of cardiac arrest and the reperfusion syndrome on blood–brain barrier permeability and evaluate whether methylene blue counteracts blood–brain barrier disruption in a pig model of controlled cardiopulmonary resuscitation. Design:Randomized, prospective, laboratory animal study. Setting:University-affiliated research laboratory. Subjects:Forty-five piglets. Interventions:Forty-five anesthetized piglets were subjected to cardiac arrest alone or 12-min cardiac arrest followed by 8 mins cardiopulmonary resuscitation. The first group (n = 16) was used to evaluate blood–brain barrier disruptions after untreated cerebral ischemia after 0, 15, or 30 mins after untreated cardiac arrest. The other two groups received either an infusion of saline (n = 10) or infusion of saline with methylene blue (n = 12) 1 min after the start of cardiopulmonary resuscitation and continued 50 mins after return of spontaneous circulation. In these groups, brains were removed for immunohistological analyses at 30, 60, and 180 mins after return of spontaneous circulation. Measurements and Main Results:An increase of injured neurons and albumin immunoreactivity was demonstrated with increasing duration of ischemia/reperfusion. Less blood–brain barrier disruption was observed in subjects receiving methylene blue as demonstrated by decreased albumin leakage (p < .01), water content (p < .05), and neuronal injury (p < .01). Methylene blue treatment reduced cerebral tissue nitrite/nitrate content (p < .05) and the number of inducible and neuronal nitric oxide synthase-activated cortical cells during administration (p < .01). Meanwhile, the number of cortical endothelial nitric oxide synthase-activated cells increased over time (p < .001). Conclusion:Cerebral tissue water content, blood–brain barrier permeability and neurologic injury were increased early in reperfusion after cardiac arrest. Methylene blue exerted neuroprotective effects against the brain damage associated with the ischemia/reperfusion injury and ameliorated the blood–brain barrier disruption by decreasing nitric oxide metabolites.

Evidence for Time-dependent Maximum Increase of Free Radical Damage and Eicosanoid Formation in the Brain as Related to Duration of Cardiac Arrest and Cardio-pulmonary Resuscitation

Recovery of neurological function in patients following cardiac arrest and cardiopulmonary resuscitation (CPR) is a complex event. Free radical induced oxidative stress is supposed to be involved in this process. We studied levels of 8-iso-PGF 2 f (indicating oxidative injury) and 15-keto-dihydro-PGF 2 f (indicating inflammatory response) in venous plasma obtained from the jugular bulb in a porcine model of experimental cardiopulmonary resuscitation (CPR) where 2, 5, 8, 10 or 12 min of ventricular fibrillation (VF) was followed by 5 or 8 min of closed-chest CPR. A significant increase of 8-iso-PGF 2 f was observed immediately following restoration of spontaneous circulation in all experiments of various duration of VF and CPR. No such increase was seen in a control group. When compared between the groups there was a duration-dependent maximum increase of 8-iso-PGF 2 f which was greatest in animals subjected to the longest period (VF12 min+CPR8 min) of no or low blood flow. In contrast, the greatest increase of 15-keto-dihydro-PGF 2 f was observed in the 13 min group (VF8 min+CPR5 min). Thus, a time-dependent cerebral oxidative injury occurs in conjunction which cardiac arrest and CPR.

Effectiveness of multidisciplinary rehabilitation treatment for patients with chronic pain in a primary health care unit

ABSTRACT Background In recent years, multidisciplinary rehabilitation (MDR) became an alternative treatment option for chronic non-cancer pain. MDR is mostly available in specialized pain units, usually at rehabilitation centers where the level of knowledge and therapeutically options to treat pain conditions are considered to be high. There is strong evidence that MDR in specialized pain units is affecting pain and improves the quality of life in a sustainable manner. There are few studies about MDR outcome in primary health care, especially in those units situated in rural areas and with a different population than that encountered in specialized hospitals. That, in spite of the fact that the prevalence of pain in the patients treated in primary care practice is about 30%. The aim of this study is to analyze the effectiveness of MDR for chronic non-cancer patients in a primary health care unit. Methods This study included a total of 51 patients with chronic pain conditions who were admitted and completed the local MDR-program at the primary health care unit in Arvika, Sweden. The major complaint categories were fibromyalgia (53%), pain from neck and shoulder (28%) or low back pain (12%). The inclusion criteria were age between 16 and 67 years and chronic non-cancer pain with at least 3 months duration. The multidisciplinary team consisted of a general practitioner, two physiotherapists, two psychologists and one occupational therapist. The 6-week treatment took place in group sessions with 6-8 members each and included cognitive-behavioral treatment, education on pain physiology, ergonomics, physical exercises and relaxation techniques. Primary outcomes included pain intensity, pain severity, anxiety and depression scores, social and physical activity, and secondary outcomes were sick leave, opioid consumption and health care utilization assessed in the beginning of the treatment and at one year follow-up. Data was taken from the Swedish Quality Register for Pain Rehabilitation (SQRP) and the patients’ medical journal. Results One year after MDR treatment, sick leave decreased from 75.6% to 61.5% (p <0.05). Utilization of health-care during one year decreased significantly from 27.4 to 20.1 contacts (p = 0.02). There were significant improvements concerning social activity (p = 0.03) and depression (p <0.05), but not in anxiety (p = 0.1) and physical activity (p = 0.08). Although not statistically significant, some numerical decrease in the mean levels of pain intensity, pain severity and opioid consumption were reported one year after MDR (p > 0.05). Conclusions The results obtained one year after rehabilitation indicated that patients with chronic noncancer pain might benefit from MDR in primary health care settings. Implications This study suggests that MDR in primary care settings as well as MDR at specialized pain units may lead to better coping in chronic non-cancer pain conditions with lower depression scores and higher social activity, leading to lower sick leave. This study demonstrated that there is a place for MDR in primary health care units with the given advantage of local intervention in rural areas allowing the patients to achieve rehabilitation in their home environment.

Targeting Oxidative Injury and Cytokines' Activity in the Treatment with Anti‐Tumor Necrosis Factor‐α Antibody for Complex Regional Pain Syndrome 1

Cytokines and oxygen free radicals have been implicated in the potential pathogenic development of complex regional pain syndrome (CRPS). We aimed to analyze the relationship between clinical status, circulating levels of cytokines, and markers of oxidative damage during the treatment with anti‐TNFα antibodies. The patient chosen for treatment had not had improvement through a number of conventional therapies and fulfilled the current diagnostic criteria for CRPS‐1. We investigated the clinical variables before and after systemic administration of 1.4 mg/kg anti‐TNFα antibody (infliximab), repeated after 1 month in a dose of 3 mg/kg. Blood samples were collected before and after anti‐TNFα antibodies administration, and plasma was analyzed for 8‐isoprostane‐prostaglandin F2α (8‐iso‐PGF2α, a marker of oxidative injury) and cytokines (TNF‐α, IL‐4, IL‐6, IL‐7, IL‐8, IL‐10, IL‐17A). Plasma concentrations of 8‐iso‐PGF2α were measured with radioimmunoassay (RIA), and the kinetics of cytokines were detected in plasma by antibody‐based proximity ligation (PLA). Pathologically high levels of 8‐iso‐PGF2α were found in the patient. Immediately after each administration of infliximab, the levels of 8‐iso‐PGF2α decreased. Although the patient showed an improvement of the cutaneous dystrophic symptoms and diminished pain associated with these lesions, the levels of circulating TNFα increased after the administration of anti‐TNFα antibodies. In a patient with CRPS‐1 treated with anti‐TNFα antibodies, we report increased levels of circulating TNFα and a temporary mitigation of oxidative stress as measured by plasma F2‐isoprostane. This case report provides evidence 2 supporting the indication of monitoring the oxidative stress biomarkers during treatment with anti‐TNFα antibodies in CRPS 1.

Differential analgesic effects of subanesthetic concentrations of lidocaine on spontaneous and evoked pain in human painful neuroma: A randomized, double blind study

Abstract Background Both peripheral nerve injury and neuroma pain are the result of changes in sodium channel expression. Lidocaine selectively inhibits the spontaneous ectopic activity by binding to sodium channels. Subanesthetics concentrations of lidocaine are able to produce a differential block of the ectopic discharges, but not propagation of impulses, suppressing differentially the associated neuropathic pain symptoms. The aim of this study was to investigate the differences between the analgesic effects of lidocaine 0.5% and a control group of lidocaine 0.1% on several neuroma related pain modalities. Methods Sixteen patients with neuropathic pain due to painful neuromas caused by nerve injury participated in this randomized, double-blind experiment. The patterns of sensory changes were compared before and after injection of 1ml lidocaine 0.5% and 0.1% close to the neuroma, the sessions being 1–2 weeks apart. Spontaneous and evoked pains were assessed using a visual analogue scale (VAS), quantitative and qualitative sensory testing. The primary end-point measure was defined as the change in pain score measured from baseline until 60min after injection. Assessments of spontaneous pain and evoked pain were done post injection at 15s, 30s, 1min, and at 5-min intervals for the first 30-min post injection and then every 10-min to 1 hr post injection. The assessments of pain were performed between the limbs in the following order: spontaneous pain, then assessment of dynamic mechanical allodynia and then hyperalgesia. Results Lidocaine dose-dependently reduced spontaneous and evoked pain scores by more than 80% with maximum effects between 1 and 5min for evoked pain and between 3 and 15min for spontaneous pain. While evoked pain normalized rapidly reaching about 50% of the control level 20min after the injection, spontaneous pain levels continue to be lower in comparison with baseline values for more than 60min. When comparing the time course of analgesia between spontaneous and evoked pain, lidocaine-induced a greater reduction of evoked pain, but with shorter duration than spontaneous pain. The differences between evoked pain and spontaneous pain were statistically significant in both groups (lidocaine 0.5% group; p = 0.02 and lidocaine 0.1% group; p = 0.01). Reproducibility was high for all assessed variables. Surprisingly, both lidocaine concentrations produced a sensory loss within the area with hyperalgesia and allodynia: hypoesthesia occurred earlier and lasted longer with lidocaine 0.5% (between 30s and 5min) in comparison with lidocaine 0.1% (p = 0.018). Conclusion Differential analgesic effects of subanesthetic concentrations of local lidocaineon evoked and spontaneous pain in human neuroma suggest that different mechanisms underlie these two key clinical symptoms. Spontaneous pain and evoked pain need an ongoing peripheral drive and any possible CNS amplification change is temporally closely related to this peripheral input. Implications Painful neuroma represents a clinical model of peripheral neuropathic pain that could lead to a significant step forward in the understanding of pain pathophysiology providing the opportunity to study spontaneous and evoked pain and the underlying mechanisms of neuropathic pain. The proposed model of neuropathic pain allows testing new substances by administration of analgesics directly where the pain is generated.

Evaluation of the protein biomarkers and the analgesic response to systemic methylene blue in patients with refractory neuropathic pain: a double-blind, controlled study.

Aim This study was carried out in patients with neuropathic pain in order to assess the analgesic effects and changes in protein biomarkers after the administration of methylene blue (MB), a diaminophenothiazine with antioxidant and anti-inflammatory properties, and with inhibitory effects on nitric oxide. Materials and methods Ten patients with chronic refractory neuropathic pain were randomized to receive either MB (10 mg/mL Methylthioninium chloride) 2 mg/kg (MB group) or MB 0.02 mg/kg (control group) infused over 60 minutes. Sensory function and pain (Numerical Rating Scale) were evaluated at baseline and at 60 minutes after the start of the infusion. The patients kept a pain diary during the next 24 hours and for the following 4 days. Plasma and urinary concentrations of 8-isoprostane-prostaglandin F2α (8-iso-PGF2α) and plasma protein biomarkers prior to and after the infusions were measured with radioimmunoassay and with proximity extension assay. Results A decrease of the Numerical Rating Scale at 60 minutes in comparison with baseline was observed in the MB (P=0.047) group. The decrease was significant between the MB and the control group on the day of and day after MB infusion (P=0.04 and P=0.008, respectively). There was no difference in systemic protein expressions between groups except for prolactin (PRL) (P=0.02). Three patients demonstrated diminished dynamic mechanical allodynia. Conclusion MB decreased the pain levels in patients with chronic therapy-resistant neuropathic pain on the first 2 days after administration. Known as an endocrine modulator on the anterior pituitary gland, MB infusion produced a decrease of PRL. The detailed role of PRL effects in chronic neuropathic pain remains undetermined.

Cerebral protection in experimental cardiopulmonary resuscitation (with special reference to the effects of methylene blue)

Objective: The present study was designed to study the effects of cardiac arrest and cardiopulmonary resuscitation (CPR) on blood-brain barrier (BBB) permeability and subsequent neurological injury. It also tests the cerebral effects of MB on the maintenance of BBB integrity, the production of nitric oxide (NO) and regulation of nitric oxide synthases (NOS) in cerebral cortex.Intervention: The control group (CA, n=16) underwent 12 min cardiac arrest without subsequent CPR, after which the brain of the animals was removed immediately or after 15 and 30 min. The other two groups with 12 min cardiac arrest and subsequent 8 min CPR received either an infusion of saline (CA-MB group, n=10) or an infusion of saline with MB (CA+MB, n= 12) started one minute after the start of CPR and continued 50 min after return of spontaneous circulation (ROSC). In both the latter (CA-MB and CA+MB) groups the brains were removed for histological analysis at the following time points: 30, 60, 180 min after ROSC.Main Results: In all the groups an increase of necrotic neurons and albumin immunoreactivity was demonstrated with increasing duration of ischemia and reperfusion time. The immunohistochemistry analysis indicated less blood brain barrier disruption in the animals receiving MB (CA+MB group) evidenced by decreased albumin leakage (P<0.01), water content (P=0.02), potassium (P=0.04), but also decreased neuronal injury (P<0.001) in this group in comparison with the group that was not treated with MB (CA-MB). Similarly, MB treatment reduced nitrite/nitrate ratio (P=0.02), iNOS expression (P<0.01), nNOS expression (P<0.01).Conclusion: Cerebral edema, increase BBB permeability and neurologic injury are observed early in ischemia induced by cardiac arrest. MB markedly reduced BBB disruption and subsequent neurologic injury. These cerebral cortical effects after the exposure to MB appear to be associated with a decrease of NO measured by nitrate/nitrite and different effects on NOS.