Increased cerebrospinal fluid adenosine 5'-triphosphate in patients with amyotrophic lateral sclerosis.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) has been suggested to cause neuroinflammation and motor neuron degeneration by activating microglia and astrocytes in amyotrophic lateral sclerosis (ALS). Since we have developed a highly sensitive ATP assay system, we examined cerebrospinal fluid (CSF) ATP levels in patients with ALS whether it can be a useful biomarker in ALS. METHODS: Forty-eight CSF samples from 44 patients with ALS were assayed for ATP with a newly established, highly sensitive assay system using luciferase luminous reaction. CSF samples from patients with idiopathic normal pressure hydrocephalus (iNPH) were assayed as a control. Patients were divided into two groups depending on their disease severity, as evaluated using the Medical Research Council (MRC) sum score. Correlations between the CSF ATP levels and other factors, including clinical data and serum creatinine levels, were evaluated. RESULTS: CSF ATP levels were significantly higher in patients with ALS than in the iNPH (716 ± 411 vs. 3635 ± 5465 pmol/L, p < 0.01). CSF ATP levels were significantly higher in the more severe group than in the iNPH group (6860 ± 8312 vs. 716 ± 411 pmol/L, p < 0.05) and mild group (6860 ± 8312 vs. 2676 ± 3959 pmol/L, p < 0.05) respectively. ALS functional rating scale-revised (ALSFRS-R) (37.9 ± 5.7 vs. 42.4 ± 2.8, p < 0.01) and serum creatinine levels (0.51 ± 0.13 vs. 0.68 ± 0.23 mg/dL, p < 0.05) were significantly lower in the severe group than in the mild group respectively. A negative correlation of CSF ATP levels with MRC sum score was demonstrated in the correlation analysis adjusted for age and sex (r = -0.3, p = 0.08). CONCLUSIONS: Extracellular ATP is particularly increased in the CSF of patients with advanced ALS. CSF ATP levels may be a useful biomarker for evaluating disease severity in patients with ALS.

Evans Blue Reduces Neuropathic Pain Behavior by Inhibiting Spinal ATP Release.

Upon peripheral nerve injury, vesicular ATP is released from damaged primary afferent neurons. This extracellular ATP subsequently activates purinergic receptors of the spinal cord, which play a critical role in neuropathic pain. As an inhibitor of the vesicular nucleotide transporter (VNUT), Evans blue (EB) inhibits the vesicular storage and release of ATP in neurons. Thus, we tested whether EB could attenuate neuropathic pain behavior induced by spinal nerve ligation (SNL) in rats by targeting VNUT. An intrathecal injection of EB efficiently attenuated mechanical allodynia for five days in a dose-dependent manner and enhanced locomotive activity in an SNL rat model. Immunohistochemical analysis showed that EB was found in VNUT immunoreactivity on neurons in the dorsal root ganglion and the spinal dorsal horn. The level of ATP in cerebrospinal fluid in rats with SNL-induced neuropathic pain decreased upon administration of EB. Interestingly, EB blocked ATP release from neurons, but not glial cells in vitro. Eventually, the loss of ATP decreased microglial activity in the ipsilateral dorsal horn of the spinal cord, followed by a reduction in reactive oxygen species and proinflammatory mediators, such as interleukin (IL)-1ß and IL-6. Finally, a similar analgesic effect of EB was demonstrated in rats with monoiodoacetate-induced osteoarthritis (OA) pain. Taken together, these data demonstrate that EB prevents ATP release in the spinal dorsal horn and reduces the ATP/purinergic receptor-induced activation of spinal microglia followed by a decline in algogenic substances, thereby relieving neuropathic pain in rats with SNL.

Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.

Accumulating evidence suggests that activation of spinal microglia contributes to the development of inflammatory and neuropathic pain. However, the role of spinal microglia in the maintenance of chronic pain remains controversial. Bone cancer pain shares features of inflammatory and neuropathic pain, but the temporal activation of microglia and astrocytes in this model is not well defined. Here, we report an unconventional role of spinal microglia in the maintenance of advanced-phase bone cancer pain in a female rat model. Bone cancer elicited delayed and persistent microglial activation in the spinal dorsal horn on days 14 and 21, but not on day 7. In contrast, bone cancer induced rapid and persistent astrocytic activation on days 7-21. Spinal inhibition of microglia by minocycline at 14 d effectively reduced bone cancer-induced allodynia and hyperalgesia. However, pretreatment of minocycline in the first week did not affect the development of cancer pain. Bone cancer increased ATP levels in CSF, and upregulated P2X7 receptor, phosphorylated p38, and IL-18 in spinal microglia. Spinal inhibition of P2X7/p-38/IL-18 pathway reduced advanced-phase bone cancer pain and suppressed hyperactivity of spinal wide dynamic range (WDR) neurons. IL-18 induced allodynia and hyperalgesia after intrathecal injection, elicited mechanical hyperactivity of WDR neurons in vivo, and increased the frequency of mEPSCs in spinal lamina IIo nociceptive synapses in spinal cord slices. Together, our findings demonstrate a novel role of microglia in maintaining advanced phase cancer pain in females via producing the proinflammatory cytokine IL-18 to enhance synaptic transmission of spinal cord nociceptive neurons.

Extracellular ATP enhances radiation-induced brain injury through microglial activation and paracrine signaling via P2X7 receptor.

Activation of purinergic receptors by extracellular ATP (eATP) released from injured cells has been implicated in the pathogenesis of many neuronal disorders. The P2X7 receptor (P2X7R), an ion-selective purinergic receptor, is associated with microglial activation and paracrine signaling. However, whether ATP and P2X7R are involved in radiation-induced brain injury (RBI) remains to be determined. Here, we found that the eATP level was elevated in the cerebrospinal fluid (CSF) of RBI patients and was associated with the clinical severity of the disorder. In our experimental model, radiation treatment increased the level of eATP in the supernatant of primary cultures of neurons and glial cells and in the CSF of irradiated mice. In addition, ATP administration activated microglia, induced the release of the inflammatory mediators such as cyclooxygenase-2, tumor necrosis factor α and interleukin 6, and promoted neuronal apoptosis. Furthermore, blockade of ATP-P2X7R interaction using P2X7 antagonist Brilliant Blue G or P2X7 knockdown suppressed radiation-induced microglial activation and proliferation in the hippocampus, and restored the spatial memory of irradiated mice. Finally, we found that the PI3K/AKT and nuclear factor κB mediated pathways were downstream of ATP-P2X7R signaling in RBI. Taken together, our results unveiled the critical role of ATP-P2X7R in brain damage in RBI, suggesting that inhibition of ATP-P2X7R axis might be a potential strategy for the treatment of patients with RBI.

Cerebrospinal fluid ATP as a potential biomarker in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS).

Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is caused by defective oxidative phosphorylation in the cerebral parenchyma, cerebral blood vessels, and leptomeningeal tissue. Although increased blood and cerebrospinal fluid (CSF) lactate level has been used as a diagnostic biomarker in patients with MELAS, no biomarkers reflecting disease activity exist. Since we have developed a highly sensitive ATP assay system using luciferase luminous reaction, we examined CSF ATP in patients with MELAS and found that it negatively correlates with disease activity and that it reflects the efficacy of the treatment. CSF ATP might be a novel disease monitoring marker for MELAS.

ATP Measurement in Cerebrospinal Fluid Using a Microplate Reader.

Imbalance in extracellular ATP levels in brain tissue has been suggested as a triggering factor for several neurological disorders. Here, we describe the most sensitive and reliable technique for monitoring the ATP levels in mice cerebrospinal samples collected by cisterna magna puncture technique and quantified using a microplate reader.

Electrochemical dual-aptamer biosensors based on nanostructured multielectrode arrays for the detection of neuronal biomarkers.

Multielectrode arrays (MEAs) have been increasingly used for the development of biosensors due to their capability to record signals from multiple channels, fast mass transfer rates, and high spatial resolution. Alzheimer's disease (AD) is often associated with mitochondrial dysfunction, which is closely related to reduced levels of adenosine triphosphate (ATP). Therefore, simultaneous detection of ATP together with amyloid-ß oligomers (AßO), a reliable biomarker for AD, can potentially advance the early detection of Alzheimer's disease. In this work, a dual-aptamer modified MEA chip was developed that consists of microelectrodes modified with electrodeposited 3D nanostructures (3D-GMEs). Electrodeposition methods, deposition potential, and deposition time were systematically altered and the active surface areas as well as the electrode morphologies were characterized by cyclic voltammetry and scanning electron microscopy. The nanostructured microelectrodes were sequentially modified with AßO and ATP specific aptamer receptors. To achieve the modification of different aptamer receptors at different 3D-GMEs of the same MEA chip, electrochemical cleaning was applied to individual 3D-GMEs. Ferrocene labels were attached to the aptamer receptors to enable amperometric signaling after target-aptamer binding. The developed aptasensor showed a linear detection range from 1 pM to 200 nM for the detection of AßO and from 0.01 nM to 1000 nM for the detection of ATP. Finally, ATP and AßO were detected simultaneously in the same analyte solution by the same sensor chip, which could support the early detection of AD, provide comprehensive information about the health status of the patient, and be helpful for pathological studies of neurodegenerative diseases.

A ratiometric fluorescent DNA nanoprobe for cerebral adenosine triphosphate assay.

A silver nanocluster-based ratiometric fluorescent nanosensor was developed for the determination of ATP in the cerebrospinal fluid of a mouse brain. Using this useful tool with good stability and high selectivity as well as a wide linear detection range, it was found that the ATP concentration in a mouse brain with Alzheimer's disease was 2300-fold higher than that in a normal one.

Modification of purinergic signaling in the hippocampus of streptozotocin-induced diabetic rats.

Diabetic encephalopathy is a recognized complication of untreated diabetes resulting in a progressive cognitive impairment accompanied by modification of hippocampal function. The purinergic system is a promising novel target to control diabetic encephalopathy since it might simultaneously control hippocampal synaptic plasticity and glucose handling. We now tested whether streptozotocin-induced diabetes led to a modification of extracellular ATP homeostasis and density of membrane ATP (P2) receptors in the hippocampus, a brain structure involved in learning and memory. The extracellular levels of ATP, evaluated in the cerebrospinal fluid, were reduced by 60.4+/-17.0% in diabetic rats. Likewise, the evoked release of ATP as well as its extracellular catabolism was also decreased in hippocampal nerve terminals of diabetic rats by 52.8+/-10.9% and 38.7+/-6.5%, respectively. Western blot analysis showed that the density of several P2 receptors (P2X(3,5,7) and P2Y(2,6,11)) was decreased in hippocampal nerve terminals. This indicates that the synaptic ATP signaling is globally depressed in diabetic rats, which may contribute for diabetes-associated decrease of synaptic plasticity. In contrast, the density of P2 receptors (P2X(1,2,5,6,7) and P2Y(6) but not P2Y(2)) increased in whole hippocampal membranes, suggesting an adaptation of non-synaptic P2 receptors to sense decreased levels of extracellular ATP in diabetic rats, which might be aimed at preserving the non-synaptic purinergic signaling.

Ulinastatin attenuates neuropathic pain via the ATP/P2Y2 receptor pathway in rat models.

Ulinastatin, a serine protease inhibitor, which has anti-inflammatory properties and neuroprotective effects, is used to treat acute inflammatory disorders. Recent evidence indicates that administration of ulinastatin alleviates pain in rat model of neuropathic pain (NPP). However, its effect on NPP and the underlying mechanism requires further study. In this study, we evaluated the role of intrathecal administration of ulinastatin in rats with sciatic nerve ligation and observed the effect of ulinastatin on the ATP/P2Y2 receptor pathway. We performed mechanical and thermal sensitivity measurements, immunohistochemistry and double-label immunofluoresence studies to evaluate P2Y2 receptor and adenosine 5'-monophosphate-activated protein kinase (AMPK) expression in the dorsal horn of the lumbar enlargement region of the spinal cord, and a luciferase assay for the detection of ATP levels in the cerebrospinal fluid. The results showed that ulinastatin prevented the development of mechanical allodynia and thermal hypersensitivity in the rat sciatic nerve ligation model. Ulinastatin reduced the level of extracellular ATP, down-regulated P2Y2 receptor and AMPK expression in the spinal dorsal horn of the chronic constrictive injury model. We found that increased expression of P2Y2 receptor in microglia was likely involved in the activation of microglia after nerve injury, and ulinastatin inhibited the abnormal microglia activation in the dorsal horn after nerve injury. These findings demonstrated that ulinastatin might be a potential and effective drug for the treatment of NPP via the suppression of the ATP/P2Y2 receptor pathway.

Role of purinergic signaling in experimental pneumococcal meningitis.

Excessive neutrophilic inflammation contributes to brain pathology and adverse outcome in pneumococcal meningitis (PM). Recently, we identified the NLRP3 inflammasome/interleukin (IL)-1ß pathway as a key driver of inflammation in PM. A critical membrane receptor for NLRP3 inflammasome activation is the ATP-activated P2 purinoceptor (P2R) P2X7. Thus, we hypothesized involvement of ATP and P2Rs in PM. The functional role of ATP was investigated in a mouse meningitis model using P2R antagonists. Brain expression of P2Rs was assessed by RT-PCR. ATP levels were determined in murine CSF and cell culture experiments. Treatment with the P2R antagonists suramin or brilliant blue G did not have any impact on disease course. This lack of effect might be attributed to meningitis-associated down-regulation of brain P2R expression and/or a drop of cerebrospinal fluid (CSF) ATP, as demonstrated by RT-PCR and ATP analyses. Supplemental cell culture experiments suggest that the reduction in CSF ATP is, at least partly, due to ATP hydrolysis by ectonucleotidases of neutrophils and macrophages. In conclusion, this study suggests that ATP-P2R signaling is only of minor or even no significance in PM. This may be explained by down-regulation of P2R expression and decreased CSF ATP levels.

Phosphodiesterase and adenyl-cyclase activities in the cerebral cotex of the aging rat.

In an effort to determine the factors responsible for the four-fold decrease in cyclic-AMP content of the rat cerebral cortex, observed to occur between the ages of 3 and 6 months, studies were performed on adenyl-cyclase and phosphodiesterase, the cyclic-AMP synthesizing and hydrolyzing enzymes. The activities and kinetic characteristics were determined for both enzymes as obtained from the cerebral cortex of rats ranging in age from one to 24 months. No age dependence was observed either in adenyl-cyclase activity, assayed with or without fluoride ion, or in phosphodiesterase activity. It was concluded that age related changes in factors other than the direc levels of these enzymes underlie the age related decline in cortical cyclic-AMP levels.

Role of adenosine 5'-triphosphate in vasospasm after subarachnoid hemorrhage: human investigations.

OBJECTIVE: Adenosine 5'-triphosphate (ATP) is a vasoactive compound found in high concentrations inside erythrocytes. This compound may contribute to vasospasm after subarachnoid hemorrhage (SAH). We assessed the hypothesis that ATP contributes to vasospasm in humans. METHODS: ATP and hemoglobin concentrations were measured in cerebrospinal fluid (CSF) from humans with SAH and in blood incubated in vitro. The vasoactivity of the human CSF samples and of fractionated (fractions with molecular weight greater than or less than 10 kDa) and unfractionated blood incubated in vitro was assessed by application of samples to canine basilar artery segments under isometric tension. RESULTS: ATP in human CSF declined within 72 hours of SAH to concentrations too low to contract cerebral arteries. Vasoactivity of human CSF correlated with the concentration of hemoglobin. The vasoactivity of incubated erythrocyte hemolysates remained high despite a decline in ATP concentrations. Fractionation of incubated erythrocyte hemolysates showed that for incubation periods up to 7 days, all vasoactivity was in a fraction of molecular weight greater than 10 kDa. CONCLUSION: ATP is unlikely to contribute to vasospasm because the concentrations in CSF after SAH in humans are not high enough to cause vasospasm after 72 hours. The vasoactivity of erythrocyte hemolysate is not related to the ATP or ferrous hemoglobin content but may be related to the total hemoglobin content. Therefore, ATP is unlikely to be a major cause of clinically significant delayed vasospasm.

Suramin-induced reversal of chronic cerebral vasospasm in experimental subarachnoid hemorrhage.

OBJECT: The naphthylsulfonate derivative suramin is an inhibitor of growth factor receptors (receptor tyrosine kinases) and G protein-coupled P2Y receptors. Both types of these receptors are suspected of being involved in cerebral vasospasm after subarachnoid hemorrhage (SAH). In the current study, the authors examined the therapeutic effects of suramin and a selective P2X-receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in the reversal of vasospasm in an established canine double-hemorrhage model. METHODS: Twenty-four dogs underwent double blood injection into the cisterna magna, with injections given on Days 0 and 2. The dogs were divided randomly into three groups (six animals in each group) to be treated from Days 2 through 6 with the vehicle dimethyl sulfoxide, suramin, or PPADS. An additional group of six dogs received double blood injection without any treatment and served as an SAH control group. The animals were killed on Day 7. Angiography was performed on Day 0 before blood injection and again on Day 7 before the animals were killed. After the death of the animals, the basilar arteries (BAs) were collected for morphological studies and determination of tyrosine kinase expression, and the bloody cerebrospinal fluid (CSF) produced by the hemorrhages was collected for measurement of oxyhemoglobin and adenosine triphosphate (ATP). In the SAH control group, the mean diameter of the BAs on Day 7 was 46.23 +/- 6.32% of the value on Day 0 (which served as a reference of 100%). In the DMSO-treated group, the mean residual diameter of the BA was 47.77 +/- 0.8% on Day 7 compared with the value on Day 0. Suramin, but not PPADS, increased the residual diameter to 74.02 +/- 4.24% on Day 7. On Day 7 the level of ATP in the CSF was decreased and the level of oxyhemoglobin was increased, compared with values measured on Day 0. Suramin, but not PPADS, reduced tyrosine phosphorylation in the spastic BAs. CONCLUSIONS: By reducing tyrosine kinase activity, suramin may be useful in the treatment of cerebral vasospasm.

Adenosine 5'-triphosphate (ATP) concentrations in the endolymph and perilymph of the guinea-pig cochlea.

The concentration of adenosine 5'-triphosphate (ATP) in endolymph (EL), perilymph (PL) and cerebrospinal fluid (CSF), collected from anesthetized guinea pigs was determined using the luciferase-luciferin reaction. The cochlea was exposed by a ventrolateral approach and the bone overlying scala media of the third turn (EL) or scala vestibuli of the first turn (PL) was shaved to a thin layer and a small fenestrum made. For EL sampling, a double-barrelled pipette was inserted through the spiral ligament-stria vascularis complex. One barrel was filled with 150 mM KCl to record the endocochlear potential (EP) and upon the appearance of the positive EP, 0.12-1.22 microliter of fluid was aspirated into the other barrel by gentle negative pressure. For PL sampling, a single-barrelled pipette was advanced into scala vestibuli and 0.3-1.6 microliter of fluid was collected by capillarity. CSF (0.36-1.75 microliter) was obtained from the cisterna magna. The cochleae were removed and processed for light microscopy to determine the extent of tissue damage from the sampling procedure. ATP concentrations (mean +/- SEM, nM) for EL, PL and CSF were 12.95 +/- 2.4 (n = 10), 10.5 +/- 3.9 (n = 11) and 16.1 +/- 5.4 (n = 11) respectively. Differences in ATP concentrations among fluids were not statistically significant. To test the effect of hypoxia on ATP levels, a group of guinea pigs was subjected to a 90 s period of respiratory anoxia prior to sampling of EL, PL or CSF. ATP concentrations were 14.4 +/- 3.5 (n = 11), 20.7 +/- 4.1 (n = 10) and 13.5 +/- 4.6 (n = 4) for EL, PL and CSF, respectively; only PL ATP concentrations were statistically different (P = 0.018, Wilcoxon rank sum test) to basal conditions. This is the first study which demonstrates the presence of free ATP in cochlear fluids. The results indicate that ATP is present in cochlear fluids at concentrations close to those known to cause hair cell depolarization in vitro.

Haemoglobin and ATP levels in CSF from a dog model of vasospasm.

Haemoglobin and adenosine 5'-triposphate (ATP) released from lysed erythrocytes have been postulated to be responsible for delayed cerebral vasospasm after subarachnoid haemorrhage (SAH). However, the concentrations of haemoglobin and ATP in cerebrospinal fluid (CSF) in patients or in an animal model of vasospasm have not been reported. In this study, 12 mongrel dogs underwent a double blood injection via the cisterna magna on day 0 and 2, after an initial collection of CSF. On day 3, 5 or 7, the dogs were sacrificed after a second collection of CSF. An angiogram was recorded on day 0 and on the day of sacrifice. Results showed that the diameter of the dog's basilar artery was reduced 20% on day 3 (P > 0.05), 35% on day 5 (P < 0.05) and 45% on day 7 (P < 0.05). The concentrations of OxyHb, deOxyHb and MetHb in CSF were increased (P < 0.05), and all peaked on day 3. OxyHb and MetHb remained significantly higher than control (day 0) from day 3 to day 7, while deOxyHb remained at a high level on day 5 but returned to normal on day 7. In contrast, ATP was decreased (P < 0.05) on days 5 and 7 after SAH compared with day 0. The results indicate that haemoglobin might be involved in the development of cerebral vasospasm. The possible role of ATP in vasospasm remains unclear.

[The acid-base balance of cerebrospinal fluid]. / Równowaga kwasowo-zasadowa plynu mózgowo-rdzeniowego.

On the basis of literature the mechanisms of acid-base balance in cerebrospinal fluid (CSF) in physiological and pathological conditions were discussed. The author focused on acid-base balance in CSF in the course of meningitis and meningoencephalitis and the influence of its disorders in the pathogenesis of those illnesses of central nervous system.

Technique for collection of cerebrospinal fluid from the cisterna magna in rat.

Cerebrospinal fluid (CSF) is in direct contact with the extracellular space in the central nervous system (CNS), and biological changes in the brain can be reflected in CSF. In the present article, a procedure for collection of CSF in rats is described. The technique quickly and reliably yields large quantities of CSF (50-150 microl) in rats. More importantly, blood contamination of the CSF is avoided. Furthermore, detections of ATP and interleukin (IL)-1 beta in the CSF have been carried out. ATP concentration in the CSF samples was between 8.3 and 15.8 nM, with an average of 10.5+/-0.83 nM (mean+/-SEM). The concentrations of IL-1beta were below the detection limit in the CSF in the laminectomy control rats, but it increased to 0.26+/-0.07 ng/ml at 1h after spinal cord injury. This technique offers an alternative method to surgical cannulation for the collection of CSF in rats.

Effects of surgery and asphyxia on levels of nucleosides, purine bases, and lactate in cerebrospinal fluid of fetal lambs.

During severe oxygen shortage, the fetal brain resorts to anaerobic metabolism and ATP becomes catabolized. High levels of nucleosides, hypoxanthine, and xanthine (ATP catabolites) in cerebrospinal fluid (CSF) may therefore be associated with increased neonatal neurologic morbidity. In 22 fetal lambs (3 to 5 d after surgery, gestational age 123.5 +/- 3.5 d), arterial oxygen content was progressively reduced to 35% of the baseline value with a balloon occluder around the maternal common internal iliac artery. This resulted in a 1-h period of asphyxia, leading to a pH of 7.02 +/- 0.03 and a base excess of -17.0 +/- 1.0 mM. Mortality was 50%. CSF was sampled from the spinal cistern and analyzed using HPLC. During reoxygenation, hypoxanthine and xanthine may serve as substrate for xanthine oxidase with concomitant production of oxygen-derived free radicals, which may aggravate cerebral damage. The main difference between surviving and nonsurviving animals was the speed of increment of ATP catabolites in CSF: in the surviving group levels increased steadily, recovery values being significantly elevated compared with asphyxia values, whereas in the nonsurviving group the rise was rapid and levels during asphyxia did not differ significantly from levels during recovery. We conclude that 1) catheterization of the spinal cistern leads to increased levels of CSF hypoxanthine, xanthine, and inosine, and 2) during fetal asphyxia, levels of these ATP catabolites and lactate in CSF increase. 3) Maximum levels are reached during the recovery period and are similar for surviving and nonsurviving animals, but during asphyxia CSF levels of hypoxanthine and lactate were higher in the nonsurviving fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)