Effects of botulinum toxin A on endometriosis­associated pain and its related mechanism.

Endometriosis (EMS) is a common disease in women aged 25­45 years, and pain is the main clinical symptom. The primary clinical treatment is surgical excision and drug therapy targeting the ectopic lesions, but these have not been very effective. Botulinum neurotoxin serotype A (BTX­A) has been reported to be useful in the treatment of pain in a variety of diseases. Based on this, the aim of the present study was to explore the therapeutic effect and mechanism of BTX­A on EMS. A model of nerve injury induced by oxygen glucose deprivation (OGD) was constructed in PC12 cells and EMS mice. Model cells and mice were treated with different concentrations of BTX­A to observe the changes in pain behavior, to detect cell viability and the secretion of norepinephrine (NE) and methionine enkephalin (M­EK) in cells and the spinal cord, and to evaluate the expression of apoptosis­related molecules in spinal cord nerves. The results revealed that BTX­A significantly reduced the amount of writhing in model mice, enhanced the activity of PC12 OGD cells, increased the secretion of NE and M­EK in model cells and the spinal cord of mice, and decreased the apoptosis of neural cells in the spinal cord of the model mice. Therefore, it was hypothesized that BTX­A may alleviate the pain induced by EMS by increasing the secretion of analgesic substances and promoting the repair of nerve injury. The present study provided a theoretical basis for the treatment of pain induced by EMS.

Preparation of a pipette tip-based molecularly imprinted solid-phase microextraction monolith by epitope approach and its application for determination of enkephalins in human cerebrospinal fluid.

In this study, a novel molecularly imprinted polymer (MIP) monolith for highly selective extraction of enkephalins was synthesized and prepared in a micropipette tip using epitope imprinting technique. The synthesized MIPs were characterized by scanning electron microscope (SEM) and infrared spectroscopy. A molecularly imprinted solid-phase microextraction (MISPME) method was developed for extraction of enkephalins in aqueous solutions. The parameters affecting MISPME were optimized. The results indicated that this MIP monolith exhibited specific recognition capability, high enrichment efficiency and excellent reusability for enkephalins. MALDI-TOF MS analysis demonstrated that this MIP monolith can act as a useful tool for highly selective purification and enrichment of enkephalin, a kind of low abundance protein, from high-abundance proteins in human cerebrospinal fluids (CSF). Employed this MIP monolith as solid-phase microextraction column, quantitative assay of enkephalins in human CSF was developed by HPLC-ultraviolet (UV) detection in this work. The detection limits were 0.05-0.08nM. This MISPME/HPLC-UV method was used to quantify Met-enkephalin and Leu-enkephalin levels in the CSF of patients with cancer pain.

Degradation kinetics of methionine5-enkephalin by cerebrospinal fluid: in vitro studies.

Changes in the levels or biochemistry of cerebrospinal fluid (CSF) neuropeptides with opioid-like properties have been suggested to reflect alterations in specific biological processes. We have determined various kinetic parameters for methionine-enkephalin (MET) degradation by CSF samples from nonneurological patients. Study subjects included 9 males (51-67 years of age) and 5 females (47-61 years of age). Aliquots, removed from an incubation vessel containing buffer, CSF, and peptide [tyr-3',5'-H(N)MET], were analyzed for tyrosine and other degradation products. Essentially all of the labeled tyrosine from the added MET was recovered as free amino acid after 60 minutes of incubation (1:2 ratio, vol:vol; optimum pH 7.4; and temperature 37°C); other possible peptide metabolites (>3%) were not detected. Irrespective of age or gender, the peptide's degradation half-life and initial velocity values were in a limited range; t1/2 26.2 ± 5.5 and 20.8-33.8 minutes, and Iv 0.03 ±0.01 and 0.02-0.03 pg of peptide per milligram protein per minute. Km and Vmax values were 0.19 ± 0.02 and 0.17-0.21 mM, and 9.8 ± 2.2 and 7.6-12.0 µmol·L·min, respectively. Neither CSF sample storage time (up to a year) nor repeated freezing and thawing (up to 3 times over a year) altered the kinetics or products of this reaction. These preliminary findings might serve as reference values when conducting similar studies using CSF from patients diagnosed with specific neurological conditions; significant alterations in MET degradation profile in such a population could provide valuable biological markers for diagnostic and treatment purposes.

Non-suicidal self-injurious behavior, endogenous opioids and monoamine neurotransmitters.

BACKGROUND: Self-inflicted injury, including cutting or burning, is the most frequent reason for psychiatric visits to medical emergency departments. This behavior, particularly when there is no apparent suicidal intent, is poorly understood from both biological and clinical perspectives. OBJECTIVE: To examine the role of endogenous opioids and monoamine neurotransmitters in non-suicidal self-injury (NSSI). METHODS: We compared cerebrospinal fluid (CSF) levels of endogenous opioids, 5 hydroxyindolacetic acid (5-HIAA) and homovanillic acid (HVA) in individuals with a history of repetitive non-suicidal self-injury with a diagnostically-matched group of individuals who had never engaged in non-suicidal self-injury. History of suicidal behavior, demographic background and psychopathology was assessed. All patients were diagnosed with a Cluster B personality disorder (i.e. borderline, antisocial, narcissistic or histrionic) (N=29) and had a history of at least one suicide attempt. Fourteen participants had a history of repeated non-suicidal self-injurious behavior (NSSI) in adulthood and 15 did not (no NSSI). RESULTS: The NSSI group had significantly lower levels of CSF beta-endorphin and met-enkephalin when compared with the non-NSSI group. CSF dynorphin, HVA and 5-HIAA levels did not differ. Severity of depression, hopelessness and overall psychopathology was greater in the NSSI group. CONCLUSION: beta-endorphin and met-enkephalin, opioids acting upon receptors involved in mediating stress-induced and physical pain analgesia respectively, are implicated in NSSI. Serotonergic and dopaminergic dysfunctions do not appear to be related to NSSI. Based on our findings, we propose a model of non-suicidal self-injury. Our results suggest that drugs acting on the opioid system warrant exploration as pharmacological treatments for NSSI.

Capillary liquid chromatography and tandem mass spectrometry for the quantification of enkephalins in cerebrospinal fluid.

A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.

[Met-enkephalin in the cerebrospinal fluid as an indicator of central nervous system injury in meningitis and encephalitis]. / Met-enkefalina plynu mózgowo-rdzeniowego jako wskaznik uszkodzenia osrodkowego ukladu nerwowego w zapaleniu opon mózgowo-rdzeniowych i mózgu.

THE AIM: Assessment of met-enkephalin level in the cerebrospinal fluid (CSF) of patients with inflammatory process of the central nervous system (CNS) was performed to estimate the role of opioid system in viral and bacterial meningitis, and encephalitis. MATERIAL AND METHOD: The met-enkephalin level, protein concentration and pleocytosis were analysed in the CSF of 53 patients with viral or bacterial meningitis, encephalitis, and in the control group of patients without inflammatory disease of the CNS. RESULTS: The biggest differences have been observed between the groups of patients with bacterial meningitis and those without inflammatory disease of the CNS, but they were statistically insignificant. There was a lack of correlation between met-enkephalin level and some factors of inflammatory process in CSF, such as pleocytosis and protein concentration. We have not revealed any correlation between etiological agent of CNS infection and opioid system of the brain. CONCLUSION: Despite the fact that, we observed in the study statistically insignificant changes, we suggest to continue investigations, including additional parameters which are characteristic for the CNS diseases.

Cerebrospinal fluid levels of opioid peptides in fibromyalgia and chronic low back pain.

BACKGROUND: The mechanism(s) of nociceptive dysfunction and potential roles of opioid neurotransmitters are unresolved in the chronic pain syndromes of fibromyalgia and chronic low back pain. METHODS: History and physical examinations, tender point examinations, and questionnaires were used to identify 14 fibromyalgia, 10 chronic low back pain and 6 normal control subjects. Lumbar punctures were performed. Met-enkephalin-Arg6-Phe7 (MEAP) and nociceptin immunoreactive materials were measured in the cerebrospinal fluid by radioimmunoassays. RESULTS: Fibromyalgia (117.6 pg/ml; 85.9 to 149.4; mean, 95% C.I.; p = 0.009) and low back pain (92.3 pg/ml; 56.9 to 127.7; p = 0.049) groups had significantly higher MEAP than the normal control group (35.7 pg/ml; 15.0 to 56.5). MEAP was inversely correlated to systemic pain thresholds. Nociceptin was not different between groups. Systemic Complaints questionnaire responses were significantly ranked as fibromyalgia > back pain > normal. SF-36 domains demonstrated severe disability for the low back pain group, intermediate results in fibromyalgia, and high function in the normal group. CONCLUSIONS: Fibromyalgia was distinguished by higher cerebrospinal fluid MEAP, systemic complaints, and manual tender points; intermediate SF-36 scores; and lower pain thresholds compared to the low back pain and normal groups. MEAP and systemic pain thresholds were inversely correlated in low back pain subjects. Central nervous system opioid dysfunction may contribute to pain in fibromyalgia.

The antinociceptive effect of Delta9-tetrahydrocannabinol in the arthritic rat.

Our study addressed the hypothesis that spinal release of endogenous opioids underlies Delta9-tetrahydrocannabinol (Delta9-THC)-induced antinociception in Freund's adjuvant-induced arthritic and nonarthritic rats. The paw-pressure test was used to assess the antinociceptive effects of Delta9-THC versus those of morphine, and opioid and cannabinoid receptor-selective antagonists were used to characterize the involved receptors. Cerebrospinal fluid was collected after Delta9-THC injection (i.p.) for the measurement of endogenous opioid peptides. Our results indicate that morphine or Delta9-THC is equally potent and efficacious in both nonarthritic and arthritic rats. Delta9-THC-induced antinociception is attenuated by the kappa opioid receptor antagonist, nor-binaltorphimine, in arthritic rats only. Delta9-THC induces increased immunoreactive dynorphin A (idyn A) levels in nonarthritic rats while decreasing idyn A in arthritic rats. We hypothesize that the elevated idyn A level in arthritic rats contributes to hyperalgesia by interaction with N-methyl-D-aspartate receptors, and that Delta9-THC induces antinociception by decreasing idyn A release.

Human chromaffin cell graft into the CSF for cancer pain management: a prospective phase II clinical study.

A number of pre-clinical studies have demonstrated the value of adrenal medullary allografts in the management of chronic pain. The present longitudinal survey studied 15 patients transplanted for intractable cancer pain after failure of systemic opioids due to the persistence of undesirable side-effects. Before inclusion, all the patients had their pain controlled by daily intrathecal (I-Th) morphine administration. The main evaluation criteria of analgesic activity of the chromaffin cell allograft was the complementary requirement of analgesics and in particular the consumption of I-Th morphine required to maintain effective pain control. Out of the 12 patients who profited from enhanced analgesia with long-term follow-up (average 4.5 months), five no longer required the I-Th morphine (with prolonged interruption of systemic opioids as well), two durably decreased I-Th morphine intake and five were stabilized until the end of their follow-up. Durable decline and stabilization were interpreted as indicative of analgesic activity by comparison with the usual dose escalation observed during disease progression. In most cases, we noted a relationship between analgesic responses and CSF met-enkephalin levels. The results of this phase II open study demonstrate the feasibility and the safety of this approach using chromaffin cell grafts for long-term relief of intractable cancer pain. However, while analgesic efficacy was indicated by the reduction or stabilization in complementary opioid intake, these observations will need to be confirmed in a controlled trial in a larger series of patients.

Intrathecal grafting of unencapsulated adrenal medullary tissue can bring CD4 T lymphocytes into CSF: a potentially deleterious event for the graft.

Adrenal medullary tissue including chromaffin cells was grafted intrathecally in cancer patients to relieve intractable pain. The central nervous system (CNS) is considered an immune privileged site. Therefore, non-HLA-matched and unencapsulated tissue was grafted in 15 patients and 1 sham control in a series of at least 20 grafts. We observed an increase in CSF lymphocyte counts in 15/20 allografts (75%). In contrast to peripheral blood, CD4 T cells predominated in the CSF, but failed to exhibit an activated phenotype (CD25+ CD45RO+ HLA-DR+). The positive effect of graft on pain, the high met-enkephalin levels, the absence of any increase in CSF cytokine levels particularly for IFN-gamma or IL-2 (but not IL-10 and IL-6), indirectly indicated that the graft was tolerated despite the presence of CSF lymphocytes. The single treatment failure and three of four cases of partial efficacy occurred in grafts where CSF lymphocytes were present. Moreover, when assayed (n = 7), the CD4+ CSF lymphocytes still retained the capacity to exhibit ex vivo a normal or enhanced frequency of T CD4 cells producing IFN-gamma and IL-2. Taken together, our observations indicate that impairment of the local immunosuppressive balance can lead to activation of those CSF CD4 T cells and drive a rejection process. This study suggests further work on the purification and/or the immunoisolation of tissues grafted in the CNS will be necessary, particularly when the possibility of long-term and repeated grafting is considered.

Opposite changes in dopamine metabolites and met-enkephalin levels in the ventricular CSF of patients subjected to thalamic electrical stimulation.

High-frequency electrical stimulations of thalamic nuclei are currently used for the suppression of parkinsonian or essential tremor and for the relief of some types of intractable pain in man. However, the mechanisms by which such stimulations exert their therapeutic effects are essentially unknown. Attempts were made to provide some insight into these mechanisms by measuring the levels of the dopamine metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and met-enkephalin-like immunoreactivity in ventricular cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) or multiple sclerosis (MS) after a 30-minute therapeutic electrical stimulation of the ventralis intermedius nucleus of the thalamus. In nonstimulated control patients, the levels of these compounds did not significantly differ in two CSF samples taken 30 minutes apart. In stimulated patients, a decrease in dopamine metabolite levels associated with a relative increase in met-enkephalin-like immunoreactivity were observed in the CSF sample taken after the 30-minute stimulation as compared to the sample taken immediately before the stimulation. In contrast, the levels of 5-HIAA remained unaffected by the stimulation. These data confirmed the existence of negative interactions between dopaminergic and enkephalinergic systems in man similar to those previously demonstrated in rats. In addition, they suggest that alterations in dopaminergic or enkephalinergic neurotransmission might be involved in the therapeutic action of thalamic electrical stimulation in patients with parkinsonian symptoms and other patients.

Opioid receptor ligands in human hepatic encephalopathy.

BACKGROUND/AIMS: Opioid peptides may contribute to some of the manifestations of hepatic encephalopathy. To address the role of the opioid system in the pathogenesis of hepatic encephalopathy, three representative opioid ligands were measured in plasma and cerebrospinal fluid of patients with hepatic encephalopathy. METHODS: Plasma and cerebrospinal fluid were obtained in three groups of patients: group 1: patients with hepatic encephalopathy; group 2: patients with lumbar back pain; group 3: healthy controls. Met-enkephalin, leu-enkephalin and beta-endorphin levels were measured in extracted plasma and cerebrospinal fluid samples by radioimmunoassay. RESULTS: Plasma met-enkephalin levels were 656% (p<0.05) and 301% (p<0.05) and cerebrospinal fluid met-enkephalin levels were 1481% (p<0.01) and 645% (p<0.05) higher when compared to healthy control and pain control patients, respectively. Although plasma and cerebrospinal leu-enkephalin levels were elevated in patients with hepatic encephalopathy, the increases were not statistically significant. Plasma and cerebrospinal beta-endorphin levels were similar in the three study groups. CONCLUSIONS: The results of this study support accumulating data on the role of the delta opioid receptor ligand met-enkephalin in the pathogenesis of hepatic encephalopathy, and provide a rationale for the use of opioid receptor antagonists in the treatment of hepatic encephalopathy.

Role of opioids in hypoxic pial artery dilation is stimulus duration dependent.

Because methionine enkephalin contributes to and dynorphin opposes dilation during a 10-min hypoxic exposure, opioids modulate pial artery dilation to this stimulus. However, such modulation may be dependent on the duration of hypoxia. The present study was designed to characterize the modulation of hypoxic pial dilation by opioids as a function of stimulus duration in newborn pigs equipped with a closed cranial window. Hypoxic dilation was decremented in both moderate and severe groups (PO2 approximately 35 and 25 mmHg, respectively) during 20-min and 40-min exposure periods compared with the response during 5 or 10 min of stimulation (24 +/- 1, 25 +/- 1, 18 +/- 1, and 14 +/- 1% for 5, 10, 20, and 40 min of moderate hypoxia; means +/- SE). Moderate and severe hypoxia had no effect on cerebral spinal fluid (CSF) methionine enkephalin or dynorphin concentration during a 5-min exposure period. During a 10-min exposure, however, both opioids were increased in CSF. During 20- and 40-min exposure periods, CSF dynorphin continued to increase, whereas methionine enkephalin steadily decreased (962 +/- 18, 952 +/- 21, 2,821 +/- 15, 2,000 +/- 81, and 1,726 +/- 58 pg/ml methionine enkephalin for control, 5, 10, 20, and 40 min of moderate hypoxia, respectively). The mu-opioid (methionine enkephalin) antagonist beta-funaltrexamine had no influence on dilation during the 5-min exposure, decremented the 10- and 20-min exposures, but had no effect on 40-min exposure hypoxic dilation. Whereas the kappa-opioid (dynorphin) antagonist norbinaltorphimine similarly had no effect on a 5-min exposure dilation, it, in contrast, potentiated 10-, 20-, and 40-min exposure hypoxic dilations (23 +/- 1 vs. 23 +/- 1, 24 +/- 1 vs. 32 +/- 1, 16 +/- 1 vs. 24 +/- 2, and 13 +/- 1 vs. 23 +/- 3% for 5, 10, 20, and 40-min hypoxic dilation before and after norbinaltorphimine). These data show that opioids do not modulate hypoxic pial dilation during short but do so during longer exposure periods. Moreover, hypoxic pial dilation is diminished during longer exposure periods. Decremented hypoxic pial dilation during longer exposure periods results, at least in part, from decreased release of methionine enkephalin and accentuated release of dynorphin. These data suggest that the relative role of opioids in hypoxic pial dilation changes with the stimulus duration.

One-year chromaffin cell allograft survival in cancer patients with chronic pain: morphological and functional evidence.

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.

Increased cerebrospinal fluid concentration of aspartate but decreased concentration of nitric oxide breakdown products in women experiencing visceral pain during active labour.

The aim of the present study was to investigate some putative neurotransmitters involved in nociception and pain in parturients during active labour experiencing intense visceral pain. The concentration of the excitatory amino acid aspartate was significantly increased, and there was a tendency for an increase in glutamate, in lumbar cerebrospinal fluid (CSF) of parturients in active vaginal labour compared with control patients without pain subjected to elective caesarean section. The CSF concentration of the nitric oxide breakdown product nitrate was significantly decreased in parturients compared with control patients and healthy volunteers. No significant differences in the concentrations of substance P, substance P-endopeptidase or met-enkephalin were detected between parturients and controls. Our data suggest a paradoxical negative relationship between CSF concentrations of excitatory amino acids and nitric oxide in labour pain. The mechanisms behind this finding is unclear at present.

Role of PACAP in the relationship between cAMP and opioids in hypoxia-induced pial artery vasodilation.

The opioids methionine enkephalin and leucine enkephalin contribute to hypoxic pial artery dilation in the newborn pig, and adenosine 3',5'-cyclic monophosphate (cAMP) analogs have been shown to elevate cerebrospinal fluid (CSF) opioid concentration. The present study was designed to investigate the contribution of cAMP to hypoxic dilation and to determine whether an endogenous activator of adenylate cyclase, pituitary adenylate cyclase-activating peptide (PACAP), could modulate the cAMP-induced release of opioids to contribute to hypoxic pial dilation in piglets equipped with closed cranial windows. An alpha level of P < 0.05 was considered significant in all statistical tests. Moderate and severe hypoxia (PO2 approximately 35 and 25 mmHg, respectively) induced pial artery dilation that was attenuated by the Rp diastereomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate (Rp-8-BrcAMPS), a cAMP antagonist (24 +/- 1 and 36 +/- 2% vs. 21 +/- 1 and 30 +/- 1% for moderate hypoxia and 34 +/- 1 and 46 +/- 2% vs. 24 +/- 1 and 32 +/- 1% for severe hypoxia before and after Rp-8-BrcAMPS, respectively). These responses were associated with an increased CSF cAMP (1,046 +/- 25, 1,366 +/- 28, and 1,735 +/- 47 fmol/ml for control, moderate, and severe hypoxia, respectively). Hypoxic pial dilation was also accompanied by an increase in CSF methionine enkephalin (1,101 +/- 62, 3,283 +/- 119, and 3,835 +/- 129 pg/ml for control, moderate, and severe hypoxia, respectively). Hypoxic dilation additionally increased CSF PACAP (1,727 +/- 86, 2,268 +/- 157, and 7,980 +/- 238 pg/ml for control, moderate, and severe hypoxia, respectively). PACAP (10(-8) and 10(-6) M) elicited pial dilation that was associated with increased CSF cAMP and blunted by Rp-8-BrcAMPS. PACAP-induced dilation was also accompanied by increases in the opioid methionine enkephalin (1,059 +/- 23, 1,483 +/- 34, and 2,108 +/- 77 pg/ml for control and 10(-8) and 10(-6) M PACAP, respectively). These data show that cAMP contributes to hypoxic pial artery dilation. Hypoxia increases CSF PACAP, whereas PACAP elevates CSF opioid concentration. These data, therefore, suggest that PACAP modulates cAMP-induced opioid release, thereby contributing to hypoxic pial dilation.

Role of nitric oxide, cyclic nucleotides, and the activation of ATP-sensitive K+ channels in the contribution of adenosine to hypoxia-induced pial artery dilation.

Previously, it had been observed that nitric oxide (NO) contributes to hypoxia-induced pial artery dilation in the newborn pig. Additionally, it was also noted that activation of ATP-sensitive K+ channels (KATP) contribute to cGMP-mediated as well as to hypoxia-induced pial dilation. Although somewhat controversial, adenosine is also thought to contribute to hypoxic cerebrovasodilation. The present study was designed to investigate the role of NO, cyclic nucleotides, and activation of KATP channels in the elicitation of adenosine's vascular response and relate these mechanisms to the contribution of adenosine to hypoxia-induced pial artery dilation. The closed cranial window technique was used to measure pial diameter in newborn pigs. Hypoxia-induced artery dilation was attenuated during moderate (PaO2 approximately 35 mm Hg) and severe hypoxia (PaO2 approximately 25 mm Hg) by the adenosine receptor antagonist 8-phenyltheophylline (8-PT) (10(-5) M) (26 +/- 2 vs. 19 +/- 2 and 34 +/- 2 vs. 22 +/- 2% for moderate and severe hypoxia in the absence vs. presence of 8-PT, respectively). This concentration of 8-PT blocked pial dilation in response to adenosine (8 +/- 2, 16 +/- 2, and 23 +/- 2 vs. 2 +/- 2, 4 +/- 2, and 6 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after 8-PT, respectively). Similar data were also obtained using adenosine deaminase as a probe for the role of adenosine in hypoxic pial dilation. Adenosine-induced dilation was associated with increased CSF cGMP concentration (390 +/- 11 and 811 +/- 119 fmol/ml for control and 10(-4) M adenosine, respectively). The NO synthase inhibitor, L-NNA, and the cGMP antagonist, Rp 8-bromo cGMPs, blunted adenosine-induced pial dilation (8 +/- 1, 14 +/- 1, and 20 +/- 3 vs. 3 +/- 1, 5 +/- 1, and 8 +/- 3% for 10(-8), 10(-6), and 10(-4) M adenosine before and after L-NNA, respectively). Adenosine dilation was also blunted by glibenclamide, a KATP antagonist (9 +/- 2, 14 +/- 3, 21 +/- 4 vs. 4 +/- 1, 8 +/- 2, and 11 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after glibenclamide, respectively). Finally, it was also observed that adenosine-induced dilation was associated with increased CSF cAMP concentration and the cAMP antagonist, Rp 8-bromo cAMPs, blunted adenosine pial dilation. These data show that adenosine contributes to hypoxic pial dilation. These data also show that NO, cGMP, cAMP, and activation of KATP channels all contribute to adenosine induced pial dilation. Finally, these data suggest that adenosine contributes to hypoxia-induced pial artery dilation via cAMP and activation of KATP channels by NO and cGMP.

Relationship between vasopressin and opioids in hypoxia induced pial artery vasodilation.

It has been observed that a vasopressin receptor antagonist attenuates hypoxic hyperemia in fetal sheep, whereas methionine enkephalin (Met) and leucine enkephalin (Leu) contribute to hypoxia-induced pial artery dilation in newborn pigs. This study was designed to investigate the relationship between vasopressin and opioids in hypoxia-induced pial artery dilation in the newborn pig by use of the closed cranial window technique. Hypoxia-induced pial artery dilation was attenuated during moderate [arterial Po2 (PaO2) approximately 35 mmHg] and severe hypoxia (PaO2 approximately 25 mmHg) by the vasopressin receptor antagonist, [beta-mercapto-beta beta-cyclopentamethylenepropionyl, 2-O-Me-Tyr2, Arg8]vasopressin (MeAVP, 5 micrograms/kg i.v.; 29 +/- 1 vs. 14 +/- 2 and 37 +/- 2 vs. 18 +/- 2% for moderate and severe hypoxia in absence vs. presence of MeAVP, respectively, n = 7). Hypoxia-induced dilation was accompanied by increased cerebrospinal fluid (CSF) vasopressin concentration (26 +/- 1 vs. 67 +/- 4 and 26 +/- 1 vs. 99 +/- 4 pg/ml for control vs. moderate and control vs. severe hypoxia, n = 5). Vasopressin increased CSF Met (895 +/- 28, 1,147 +/- 63, 1,327 +/- 48, and 1,600 +/- 75 pg/ml for control and 40, 400, and 4,000 pg/ml vasopressin, respectively, n = 7). CSF Leu concentration was similarly increased by vasopressin. Furthermore, MeAVP attenuated the release of Met during moderate hypoxia (910 +/- 38 and 2,682 +/- 49 vs. 911 +/- 38 and 2,110 +/- 84 pg/ml for control and moderate hypoxia in absence and presence of MeAVP, respectively, n = 5). MeAVP had similar effects on hypoxia-induced Leu release. These data show that vasopressin contributes to hypoxia-induced pial artery dilation and that vasopressin increases CSF Met and Leu concentrations. These data also suggest that elevated CSF vasopressin concentrations that occur during hypoxemia result in opioid release, which subsequently contributes to hypoxic pial artery dilation.

Relationship between opioids and prostaglandins in hypoxia-induced vasodilation of pial arteries in the newborn pig.

Previously, it has been observed that methionine enkephalin and leucine enkephalin contribute to hypoxia-induced pial artery dilation in the newborn pig. It has also been observed that the cyclooxygenase inhibitor indomethacin attenuates hypoxic hyperemia in piglets. The present study was designed to determine the relationship between opioids and prostaglandins in hypoxia-induced pial artery dilation. Newborn pigs equipped with closed cranial windows were used to measure pial artery diameter and collect cortical periarachnoid cerebrospinal fluid (CSF) for assay of opioids and prostaglandins. Hypoxia-induced artery vasodilation was mildly attenuated during moderate hypoxia (PaCO2 approximately 35 mm Hg), while this response was blunted during severe hypoxia (PaO2 approximately 25 mm Hg) by indomethacin, 5 mg/kg iv (23% +/- 1 % vs 18% +/- 1% and 33% +/- 2% vs 21% +/- 2% for moderate and severe hypoxia in the absence and presence of indomethacin, respectively). Hypoxic dilation was accompanied by increased CSF prostaglandin E2 (PGE2) concentration (1260 +/- 37 vs 1734 +/- 67 and 1256 +/- 33 vs 2859 +/- 189 pg/ml for moderate and severe hypoxia, respectively). Similar changes in CSF 6 keto PGF1alpha concentration during hypoxia were also observed. Topical PGE2 (10,100 ng/ml) increased CSF methionine enkephalin (874 +/- 35, 1290 +/- 44, and 1791 +/- 143 pg/ml for control, 10 and 100 ng/ml PGE2 respectively). Similar increases in CSF methionine enkephalin concentration were observed for topical PGI2. Additionally, these two prostaglandins also increased CSF leucine enkephalin concentration. Furthermore, while indomethacin had no effect on the release of CSF methionine enkephalin during moderate hypoxia, it attenuated the release of this opioid during severe hypoxia (786 +/- 27 and 2633 +/- 74 vs 781 +/- 51 and 2467 +/- 52; 926 +/- 15 and 3489 +/- 156 vs 898 +/- 11 and 2314 +/- 124 pg/ml for control and moderate/severe hypoxia before and after indomethacin, respectively). Similar effects of indomethacin on hypoxic release of leucine enkephalin were also observed. These data indicate that prostaglandins contribute to hypoxic pial dilation. Additionally, these data show that prostaglandins release the opioids methionine enkephalin and leucine enkephalin. Finally, these data suggest that elevated prostaglandin concentrations during severe hypoxia release opioids which in turn contribute to hypoxic pial dilation.