Cerebrospinal fluid purinomics as a biomarker approach to predict outcome after severe traumatic brain injury.

Severe traumatic brain injury (TBI) is associated with high rates of mortality and long-term disability linked to neurochemical abnormalities. Although purine derivatives play important roles in TBI pathogenesis in preclinical models, little is known about potential changes in purine levels and their implications in human TBI. We assessed cerebrospinal fluid (CSF) levels of purines in severe TBI patients as potential biomarkers that predict mortality and long-term dysfunction. This was a cross-sectional study performed in 17 severe TBI patients (Glasgow Coma Scale <8) and 51 controls. Two to 4 h after admission to ICU, patients were submitted to ventricular drainage and CSF collection for quantification of adenine and guanine purine derivatives by HPLC. TBI patients' survival was followed up to 3 days from admission. A neurofunctional assessment was performed through the modified Rankin Scale (mRS) 2 years after ICU admission. Purine levels were compared between control and TBI patients, and between surviving and non-surviving patients. Relative to controls, TBI patients presented increased CSF levels of GDP, guanosine, adenosine, inosine, hypoxanthine, and xanthine. Further, GTP, GDP, IMP, and xanthine levels were different between surviving and non-surviving patients. Among the purines, guanosine was associated with improved mRS (p = 0.042; r = -0.506). Remarkably, GTP displayed predictive value (AUC = 0.841, p = 0.024) for discriminating survival versus non-survival patients up to 3 days from admission. These results support TBI-specific purine signatures, suggesting GTP as a promising biomarker of mortality and guanosine as an indicator of long-term functional disability.

Mechanisms involved in the antinociception induced by spinal administration of inosine or guanine in mice.

It is well known that adenine-based purines exert multiple effects on pain transmission. Recently, we have demonstrated that guanine-based purines may produce some antinociceptive effects against chemical and thermal pain in mice. The present study was designed to investigate the antinociceptive effects of intrathecal (i.t.) administration of inosine or guanine in mice. Additionally, investigation into the mechanisms of action of these purines, their general toxicity and measurements of CSF purine levels were performed. Animals received an i.t. injection of vehicle (30mN NaOH), inosine or guanine (up to 600nmol) and submitted to several pain models and behavioural paradigms. Guanine and inosine produced dose-dependent antinociceptive effects in the tail-flick, hot-plate, intraplantar (i.pl.) glutamate, i.pl. capsaicin and acetic acid pain models. Additionally, i.t. inosine inhibited the biting behaviour induced by spinal injection of capsaicin and i.t. guanine reduced the biting behaviour induced by spinal injection of glutamate or AMPA. Intrathecal administration of inosine (200nmol) induced an approximately 115-fold increase on CSF inosine levels. This study provides new evidence on the mechanism of action of extracellular guanine and inosine presenting antinociceptive effects following spinal administration. These effects seem to be related, at least partially, to the modulation of A1 adenosine receptors.

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats.

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats.

Effects of intermittent fasting on age-related changes on Na,K-ATPase activity and oxidative status induced by lipopolysaccharide in rat hippocampus.

Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, and lipopolysaccharide (LPS) signaling is linked to glutamate-nitric oxide-Na,K-ATPase isoforms pathway in central nervous system (CNS) and also causes neuroinflammation. Intermittent fasting (IF) induces adaptive responses in the brain that can suppress inflammation, but the age-related effect of IF on LPS modulatory influence on nitric oxide-Na,K-ATPase isoforms is unknown. This work compared the effects of LPS on the activity of α1,α2,3 Na,K-ATPase, nitric oxide synthase gene expression and/or activity, cyclic guanosine monophosphate, 3-nitrotyrosine-containing proteins, and levels of thiobarbituric acid-reactive substances in CNS of young and older rats submitted to the IF protocol for 30 days. LPS induced an age-related effect in neuronal nitric oxide synthase activity, cyclic guanosine monophosphate, and levels of thiobarbituric acid-reactive substances in rat hippocampus that was linked to changes in α2,3-Na,K-ATPase activity, 3-nitrotyrosine proteins, and inducible nitric oxide synthase gene expression. IF induced adaptative cellular stress-response signaling pathways reverting LPS effects in rat hippocampus of young and older rats. The results suggest that IF in both ages would reduce the risk for deficits on brain function and neurodegenerative disorders linked to inflammatory response in the CNS.

The hydrolysis of striatal adenine- and guanine-based purines in a 6-hydroxydopamine rat model of Parkinson's disease.

Parkinson's disease (PD) is characterized by a progressive neurodegeneration in the substantia nigra and a striatal dopamine decrease. Striatal extracellular adenosine and ATP modulate the dopaminergic neurotransmission whereas guanosine has a protective role in the brain. Therefore, the regulation of their levels by enzymatic activity may be relevant to the clinical feature of PD. Here it was evaluated the extracellular nucleotide hydrolysis from striatal slices 4 weeks after a unilateral infusion with 6-OHDA into the medial forebrain bundle. This infusion increased ADP, AMP, and GTP hydrolysis by 15, 25, and 41%, respectively, and decreased GDP hydrolysis by 60%. There was no change in NTPDases1, 2, 3, 5, 6, and 5'-nucleotidase transcription. Dopamine depletion changes nucleotide hydrolysis and, therefore, alters the regulation of striatal nucleotide levels. These changes observed in 6-OHDA-lesioned animals may contribute to the symptoms observed in the model and provide evidence to indicate that extracellular purine hydrolysis is a key factor in understanding PD, giving hints for new therapies.

Effects of a highly palatable diet on lipid and glucose parameters, nitric oxide, and ectonucleotidases activity.

Obesity has reached epidemic proportions worldwide and is stimulated by the ready availability of food rich in fat and sugar (highly palatable diet). This type of diet increases the risks of obesity-associated pathologies, such as insulin resistance and cardiovascular disease. Nitric oxide, a potent endogenous vasodilator, is decreased in these pathologies, mostly as a result of insulin resistance. Ectonucleotidases are ecto and soluble enzymes that regulate the availability of the nucleotides ATP, ADP, and AMP and the nucleoside adenosine in the vascular system, thereby affecting vasoconstriction, vasodilatation, and platelet aggregation homeostasis. The aim of this study was to evaluate the effects of a highly palatable diet on serum lipid and glucose parameters, nitric oxide, and ectonucleotidase activity. Forty male Wistar rats were fed 1 of 2 diets for either 45 days or 4 months: standard chow (SC, n = 10) or a highly palatable diet enriched with sucrose (HP, n = 10). Body mass, visceral fat mass, glucose tolerance, cholesterol (total, high-density lipoprotein (HDL) and non-HDL), serum triacylglycerol, liver triacylglycerol, and free glycerol were increased in the HP group after 45 days and after 4 months, whereas insulin levels were not different between the groups at either time. Furthermore, levels of nitric oxide metabolites and ATP, ADP, and AMP hydrolysis were significantly lower in the HP group (p < 0.05) after 4 months. In conclusion, the consumption of the HP diet for 4 months induced overall corporal and metabolic changes, and decreased nitric oxide metabolites and ectonucleotidase activity, thereby promoting an appropriate environment for the development of cardiovascular diseases, without apparent changes in insulin levels.

Chronic treatment with cyclosporine affects systemic purinergic parameters, homocysteine levels and vascular disturbances in rats.

Vascular disease is a major cause of morbidity and mortality among transplanted recipients and cyclosporine (CsA) treatment has been consistently implicated in this event. In this study we assessed total blood homocysteine levels (tHcy), ecto-nucleotidase activities and adenine nucleotide/nucleoside levels searching for parameters related to the mechanisms of vascular damage induced by chronic CsA treatment in non-transplanted rats. Thirty male Wistar rats were divided in three groups: control group treated with corn oil, CsA 5mg/kg and CsA 15 mg/kg, administered by daily gastric gavage during 8 weeks. CsA 15 mg/kg treatment increased blood levels of tHcy. Both CsA treatments (5mg/kg and 15 mg/kg) decreased adenine nucleotides hydrolysis by ecto-nucleotidases in serum, which negatively correlated with tHcy levels (r: -0.74, r: -0.63 and r: -0.63, p<0.004, for ATP, ADP and AMP, respectively). CsA 15mg/kg induced a statistically significant increase in ADP and decrease in adenosine (ADO) plasma levels compared to control group. THcy levels were positively correlated with plasma ADP levels and negatively correlated with ADO levels (r: 0.84, p<0.0001 and r: -0.68, p<0.0001, respectively). Rats under CsA 15 mg/kg treatment presented cell injury and inflammatory responses in the endothelium and intima layer of the aorta artery. In conclusion, blood ecto-nucleotidases activity, tHcy, and ADP and ADO levels may be implicated in vascular injury induced by CsA treatment.

Increased plasma levels of brain derived neurotrophic factor (BDNF) in patients with fibromyalgia.

Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival and synaptic plasticity of the central and peripheral nervous system. BDNF appears to modulate nociceptive sensory inputs and pain hypersensitivity and has been studied in pathological situations, including chronic pain conditions and major depression. Increased serum BDNF levels have been recently reported in fibromyalgia (FM). In the present study, we assessed plasma BDNF levels in patients with FM and controls. Plasma BDNF was measured from 30 female patients with FM and 30 healthy age- and gender-matched volunteers using an enzyme immunoassay. FM patients showed higher levels of BDNF (FM = 167.1 +/- 171.2 pg/mL) when compared with the control group (control = 113.8 +/- 149.6 pg/mL) (P = 0.049; Mann-Whitney test). Six out of 30 controls presented superior values to the medium (15/15) of the patients with fibromyalgia (129 pg/mL) (P = 0.029, Fisher exact test). There was no correlation between plasma BDNF levels and age, disease duration, pain score, number of pain points and HAM-D score. Our results confirm previous findings of increased plasma BDNF levels in patients with FM, suggesting that BDNF may be involved in the pathophysiology of Fibromyalgia, despite high levels of depression.

Peripheral nucleotide hydrolysis in rats submitted to a model of electroconvulsive therapy.

Electroconvulsive therapy (ECT) is an efficacious and safe method for the treatment of mood disorders. Its utilization is accompanied by a myriad of biochemical and cellular changes, which are far from fully understood. The present work investigates in rat serum the effects of seizures induced by electroconvulsive shocks (ECS), an animal model of ECT, on enzymes that hydrolyze ATP, ADP and AMP to adenosine. Two different models of ECS were used, consisting in the application of one or eight ECS sessions, and respectively named acute or chronic. Serum samples were collected at several time points after the single shock in the acute and after the eighth and last shock in the chronic model. A single shock produced a sudden and short-lived inhibition of enzymatic activity (P<0.01 for ADP and AMP), whereas in the chronic model significant increases were noticed starting as early as 12 h after the last shock, remaining significantly elevated until the last measurement 7 days later for ATP and ADP. Analysis of hydrolysis was assessed at the selected time point of 7 days in cerebrospinal fluid samples, also demonstrating a significant activation in the chronic model (P<0.0001 for ATP and ADP). These results support the idea that adenosine nucleotides may be involved in the biochemical mechanisms underlying longer lasting therapeutic effects associated with ECT, and suggest that peripheral markers can possibly contribute to the evaluation of activity in the central nervous system.

Antinociceptive effects of intracerebroventricular administration of guanine-based purines in mice: evidences for the mechanism of action.

It is well known that adenine-based purines exert multiple effects on pain transmission. However, less attention has been given to the potential effects of guanine-based purines (GBPs) on pain transmission. The aim of this study was to investigate the effects of intracerebroventricular (i.c.v.) guanosine and GMP on mice pain models. Mice received an i.c.v. injection of vehicle (saline or 10 muM NaOH), guanosine (5 to 400 nmol), or GMP (240 to 960 nmol). Additional groups were also pre-treated with i.c.v. injection of the A(1)/A(2A) antagonist caffeine (15 nmol), the non-selective opioid antagonist naloxone (12.5 nmol), or the 5'-nucleotidase inhibitor AOPCP (1 nmol). Measurements of CSF purine levels and cortical glutamate uptake were performed after treatments. Guanosine and GMP produced dose-dependent antinociceptive effects. Neither caffeine nor naloxone affected guanosine antinociception. Pre-treatment with AOPCP completely prevented GMP antinociception, indicating that conversion of GMP to guanosine is required for its antinociceptive effects. Intracerebroventricular administration of guanosine and GMP induced, respectively, a 180- and 1800-fold increase on CSF guanosine levels. Guanosine was able to prevent the decrease on cortical glutamate uptake induced by intraplantar capsaicin. This study provides new evidence on the mechanism of action of GBPs, with guanosine and GMP presenting antinociceptive effects in mice. This effect seems to be independent of adenosine and opioid receptors; it is, however, at least partially associated with modulation of the glutamatergic system by guanosine.

Pentylenetetrazol kindling alters adenine and guanine nucleotide catabolism in rat hippocampal slices and cerebrospinal fluid.

Pentylenetetrazol (PTZ) is commonly used as a convulsant drug. The enhanced seizure susceptibility induced by kindling is probably attributable to plastic changes in the synaptic efficacy. Adenosine and guanosine act both as important neuromodulators and neuroprotectors with mostly inhibitory effects on neuronal activity. Adenosine and guanosine can be released per se or generated from released nucleotides (ATP, ADP, AMP, GTP, GDP, and GMP) that are metabolized and rapidly converted to adenosine and guanosine. The aim of this study was to evaluate nucleotide hydrolysis by ecto- and soluble nucleotidases (hippocampal slices and CSF, respectively) after PTZ-kindling (stages 3, 4, or 5 seizures) or saline treatment in rats. Additionally, the levels of purines in rat cerebrospinal fluid (CSF), as well as ecto-NTPDases (1, 2, 3, 5, 6 and 8) and ecto- 5'-nucleotidase expression were determined. Ecto-enzyme assays demonstrated that ATP, AMP, GDP, and GMP hydrolysis enhanced when compared with controls. In addition, there was an increase of ADP, GDP, and GMP hydrolysis by soluble nucleotidases in PTZ-kindling rats compared to control group. The HPLC analysis showed a marked increase in PTZ-kindled CSF concentrations of GTP, ADP, and uric acid, but GDP, AMP, and hypoxanthine concentrations were decreased. Such alterations indicate that the modulatory role of purines in CNS could be affected by PTZ-kindling. However, the physiological significance of these findings remains to be elucidated.

In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats.

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.

Ecto-hydrolysis of adenine nucleotides in rat blood platelets are altered by ovariectomy.

There is evidence that estrogen is associated with a reduction on cardiovascular disease risk through inhibition of platelet aggregation and action on vascular function. The process of haemostasis can also be affected by adenine nucleotides and adenosine. Consequently, regulation of enzymes that hydrolyze these nucleotides in the bloodstream is essential in the modulation of the processes of platelet aggregation, vasodilatation and coronary flow. Ecto-ATP diphosphohydrolase and ecto-5'-nucleotidase from platelets are enzymes related to nucleotide hydrolysis. In the present study, we examined the effect of ovariectomy (OVX) and estradiol replacement therapy (ER) on the activity of the enzymes that degrade adenine nucleotides in platelets of female rats. The OVX group significantly decrease the hydrolysis of ATP, ADP and AMP by 42, 52 and 29.3%, respectively, when compared to a control group. ER did not reverse the inhibition of nucleotide hydrolysis observed in OVX rats. Our findings indicate that hormonal deprivation affects ATP, ADP and AMP hydrolysis by platelets and consequently the level of these nucleotides and adenosine in the circulation. Since, ADP is the most important platelet agonist and recruiting agent present in the microenvironment of the thrombus, our findings was contribute to a better comprehension of the cardiovascular complications described in alterations of sexual hormonal status.

NTPDase and 5'-nucleotidase activities in synaptosomes of hippocampus and serum of rats subjected to homocysteine administration.

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.

The effect of stress upon hydrolysis adenine nucleotides in blood serum of rats.

Alterations of enzyme activities involved in adenine nucleotide hydrolysis have been reported in spinal cord and blood serum after repeated restraint stress. On the other hand, no effect was observed in the spinal cord of rats after acute stress. In the present study, we investigated the effect of acute stress on the hydrolysis of adenine nucleotides in rat blood serum. Adult male Wistar rats were submitted to 1-h restraint stress and were sacrificed at 0, 6, 24 and 48 h. Increased ATP and ADP hydrolysis were observed in the blood serum of stressed rats 24 h after stress (58% and 54%, respectively, when compared to controls). On the other hand, the AMP hydrolysis was increased after 6 h (68% when compared to controls) and at 24 h (94% when compared to controls) after stress. The results suggest that altered activity of soluble enzymes in serum may be a biochemical marker for stress situations.

Chronic stress effects on adenine nucleotide hydrolysis in the blood serum and brain structures of rats.

We have previously observed that adenosine 5'-diphosphate (ADP) hydrolysis was decreased 25% in spinal cord synaptosomes of chronically stressed male rats, while no changes were observed in ATPase activity. In the present study, we investigated the effect of chronic stress on the hydrolysis of adenine nucleotides in two cerebral structures (frontal cortex and hypothalamus) and in the blood serum of male rats. Adult male Wistar rats were submitted to 1-h restraint stress/day for 45 days (chronic) and were sacrificed 24 h after the last session of stress. Adenosine 5'-triphosphate (ATP) or ADP hydrolysis was assayed in the synaptosomal fraction obtained from the frontal cortex and hypothalamus of control and chronically stressed animals. No effects on ADP or ATP hydrolysis were observed in any of the cerebral structures analyzed after chronic stress. On the other hand, reduced ADP hydrolysis was observed in the blood serum of chronic stressed rats. It is possible that the effects observed in the blood serum may represent an adaptation to chronic stress and may reflect different functions of nucleotides and/or enzymes in these tissues. It is possible that altered levels of ADPase activity in the serum may be a biochemical marker for chronic stress situations.