Developmental and regional differences in nitric oxide synthase activity and blood flow in the sheep brain.

Nitric oxide synthase (NOS) participates in the regulation of cerebral blood flow and neurotransmitter release and as a second messenger of glutamatergic and cholinergic systems. Developmental differences in NOS activity have been described in the rat, but not in a species with longer gestation and a larger, lobulated brain at birth. We assayed NOS activity by conversion of [14C]L-arginine to [14C]L-citrulline in 50-mg tissue samples from eight brain regions in sheep at 70, 92, 110, and 135 days gestation (term = 145 days); newborns (< 7 days); and adults to test the hypothesis that NOS activity in the brain is developmentally regulated from midgestation through adulthood and matures along the neuroaxis in parallel with the known development of cerebral blood flow and neuronal activity. Three patterns of maturation of NOS activity were evident: increasing to or exceeding adult levels before 70 days gestation in the thalamus, cerebellum, and medulla; increasing to adult levels between 70 and 92 days in the hippocampus; and increasing to adult levels after 92 days in the cortex and caudate. Additionally, there were regional differences in cortical NOS activity: at 70 and 92 days of gestation, frontal cortex NOS activity was greater than parietal or occipital activity, and at 135 days gestation and in the newborn and adult, cortical and caudate activity exceeded that in most of the more caudal regions. The up to fourfold increase in regional cortical NOS activity between 92 and 135 days gestation was associated with twofold increases in cerebral blood flow and oxygen consumption during this period. Inhibition of NOS activity with administration of 60 mg/kg of NG-nitro-L-arginine methylester (L-NAME) resulted in 27% and 25% reductions in cerebral blood flow at 93 and 133 days gestation. While the associated increases in NOS activity with increases in CBF and CMRO2 do not appear causative, at various points in gestation the development of NOS activity may participate in the development of mature patterns of cerebral blood flow regulation in parallel with development of synaptic and electrical activity.

Morphine-induced spinal cholinergic activation: in vivo imaging with positron emission tomography.

Positron emission tomography (PET) imaging of spinal cord in monkeys with a cholinergic tracer demonstrates increased spinal cholinergic activity in response to an analgesic dose of morphine, and this PET result correlates with measurement of acetylcholine spillover into spinal cord extracellular space induced by morphine, as measured by microdialysis. Previous studies in rats, mice, and sheep demonstrate activation of spinal cholinergic neurons by systemic opioid administration, and participation of this cholinergic activity in opioid-induced analgesia. Testing the relevance of this observation in humans has been limited to measurement of acetylcholine spillover into lumbar cerebrospinal fluid. The purpose of this study was to apply a recently developed method to image spinal cholinergic terminals non-invasively via PET and to test the hypothesis that the tracer utilized would reflect changes in local cholinergic activity. Following Animal Care and Use Committee approval, seven adult male rhesus monkeys were anesthetized on three separate occasions. On two of the occasions PET scans were performed using [(18)F] (+)-4-fluorobenzyltrozamicol ([(18)F]FBT), which selectively binds to the vesicular acetylcholine (ACh) transporter in the presynaptic cholinergic terminals. PET scans were preceded by injection of either saline or an analgesic dose of IV morphine (10 mg/kg). On the third occasion, microdialysis catheters were inserted in the spinal cord dorsal horn and acetylcholine concentrations in dialysates determined before and after IV morphine injection. Morphine increased cholinergic activity in the spinal cord, as determined by blood flow corrected distribution volume of [(18)F]FBT in the cervical cord compared to the cerebellum. Morphine also increased acetylcholine concentrations in microdialysates from the cervical cord dorsal horn. The one animal which did not show increased spinal cholinergic activity by PET from this dose of morphine also did not show increased acetylcholine from this morphine dose in the microdialysis experiment. These data confirm the ability to use PET to image spinal cholinergic terminals in the monkey spinal cord and suggest that acute changes in cholinergic activity can be imaged with this non-invasive technique. Following preclinical screening, PET scanning with [(18)F]FBT may be useful to investigate mechanisms of analgesic action in normal humans and in those with pain.

Hospital-based group practice and comprehensive care for children of indigent families.

The replacement of a community hospital's pediatric outpatient clinics with a physicians' group practice is described, and the effects of this development on the receipt of services by children of indigent families are analyzed. An expanded patient population, frequent telephone utilization, decreased emergency room use, and satisfactory parental perceptions are found. A comparative measure of effectiveness is obtained from a record review of the preventive care received by infants delivered by the hospital's staff obstetricians. The latter data show the group practice to be more successful than the clinics in initiating and maintaining well-child care in its facility, and in achieving the timed completion of immunizations and screening procedures. The experience indicates an acceptance by low-income families of an outpatient service that emphasizes the physician-patient relationship with limited use of outreach services and nonphysician providers. It also inidicates that such a system of care can be provided to a heterogeneous patient population within the same facility.

Epidural analgesia in the management of severe vaso-occlusive sickle cell crisis.

OBJECTIVES: To determine whether continuous epidural analgesia could effectively decrease pain and thereby improve the management of severe vaso-occlusive crisis in children with sickle cell disease who were unresponsive to conventional analgesic therapy. DESIGN: Retrospective observational study. SETTING: A tertiary care hospital with a large pediatric sickle cell patient referral population. PATIENTS: The study describes nine children in 11 painful vaso-occlusive crises, unresponsive to high-dose systemic opioids, nonsteroidal anti-inflammatory drugs, and adjunctive measures, who underwent continuous epidural analgesia to control pain. OUTCOME MEASURES: Subjective pain scores, arterial oxygen saturation monitoring, and plasma lidocaine levels. METHODS: Placement of an epidural catheter for the administration of a continuous infusion of local anesthetic, alone, or in combination with fentanyl, in the management of vaso-occlusive crisis. RESULTS: An initiation of epidural analgesic therapy, 8 of 9 patients reported severe pain (8 to 10 on a scale of 0 to 10, 0 = no pain, 10 = the worst pain they ever experienced). Analgesic was immediate (pain score 0 to 2) in 8 of 9 patients, and continuously effective in 9 of 11 crises. Five patients required either the addition of fentanyl or changing the local anesthetic from lidocaine to bupivacaine to maintain analgesia for 2 to 5 days. In 7 of 9 patients, oxygen saturation dramatically increased from 87 to 95% to 99 to 100% after epidural analgesia was initiated. In all patients, plasma lidocaine levels ranged from 1.1 to 4.6 mg/L and dose-related toxicity did not occur. One patient developed hypotension secondary to high sympathetic blockade (T-4), one had an inadvertent dural puncture during insertion of the catheter, one had the epidural catheter removed for fever, and one achieved analgesia only transiently. There were no other complications, and epidural analgesia was not associated with sedation, respiratory depression, or limitation of movement. All epidural catheters were cultured on removal, and colonization did not occur. CONCLUSIONS: Epidural analgesia with local anesthetics administered alone or in combination with fentanyl effectively and safely treats the pain of sickle cell vaso-occlusive crisis unresponsive to conventional pain management and does so without causing sedation, respiratory depression, or significant limitation on ambulation. Furthermore, early treatment of painful crisis with this technique may improve oxygenation, a critical factor in the evolution of further sickling.

Reproducibility of repeated measures of cholinergic terminal density using.

UNLABELLED: [18F](+)-4-fluorobenzyltrozamicol (FBT), which selectively binds to the vesicular acetylcholine transporter in the presynaptic cholinergic neuron, has previously been shown to be a useful ligand for the study of cholinergic terminal density in the basal ganglia with PET. The goal of this study was to assess the test-retest variability of [18F]FBT and PET measurements under baseline conditions in the basal ganglia. METHODS: After approval from the Animal Care and Use Committee, 6 rhesus monkeys underwent a series of 2 [18F]FBT PET scans (time between scans, 32-301 d) under isoflurane anesthesia. Each scan was initiated on the bolus injection of the radiotracer and consisted of 26 frames acquired during 180 min. Arterial blood samples were collected over the course of each scan to determine the metabolite-corrected arterial input function. Tissue time-activity curves were obtained from the scan data by drawing regions of interest over the basal ganglia and cerebellum. The distribution volume ratio for the basal ganglia was then determined for each scan by taking the ratio of the basal ganglia (specific binding) to cerebellum (nonspecific binding) distribution volume. Distribution volumes were derived using the Logan graphic analysis technique as well as a standard 3-compartment model. Additionally, the radioactivity concentration ratio was calculated as the ratio of the average [18F]FBT concentration in the basal ganglia to that in the cerebellum during the last half of the study (85-170 min). The constant K1, determined using the standard 3-compartment model, was used as an index of blood flow changes between studies. RESULTS: For all subjects, the test-retest variability was less than 15% for the distribution volume ratio and 12% for the radioactivity concentration ratio. Good agreement was found between the distribution volume ratio calculated using the graphic technique and the standard 3-compartment model. Using K1 as an index, the variability in blood flow seen in both the basal ganglia and the cerebellum was significantly reduced in their ratio. CONCLUSION: These results show the reproducibility of [18F]FBT and PET measurements in the basal ganglia.

S-nitroso-l-cysteine releases norepinephrine in rat spinal synaptosomes.

Although nitric oxide (NO) participates in development of hypersensitivity states in the spinal cord thought to underlie chronic pain, it also participates in analgesia produced by various drugs. In rats with a hypersensitivity state following peripheral nerve injury, spinal administration of an NO donor or l-cysteine alone produced no effect, whereas their combination, which yields s-nitroso-l-cysteine (SNC) powerfully reduced hypersensitivity. In the current study, we examined the ability of SNC to stimulate release of a known spinal analgesic neurotransmitter, norepinephrine (NE), as a possible mechanism of analgesic action of NO in the spinal cord. SNC (but not the NO donor alone or decomposed SNC) produced a concentration-dependent release of NE from rat spinal cord synaptosomes. The d-isomer of SNC was less potent than the l-isomer, and the effect of SNC was partially blocked by l-, but not d-leucine, implicating an interaction with the l-amino acid transporter. SNC-induced NE release was partially Na(+) dependent, but largely Ca(2+) independent. NE uptake inhibitors partially antagonized the effect of SNC, but guanylate cyclase inhibitors were without effect. These data are therefore consistent with NO stimulating NE release in the spinal cord via reaction with thiol containing compounds, such as cysteine, entry into NE terminals via active transport, and production of both exocytotic and carrier mediated release.

Nitric oxide synthase inhibition does not impair visual or spatial discrimination learning.

Nitric oxide (NO) is a candidate retrograde messenger involved in synaptic plasticity, and is linked to the cholinergic system in the brain. We examined the role of NO in the acquisition of visual and spatial discriminations by daily administration of either saline or 1-nitroarginine methyl ester (L-NAME), an NO synthase inhibitor. Brains were assayed for NO synthase activity and two presynaptic cholinergic markers: hemicholinium-3 (HC-3) binding, which determines the number of sodium-dependent high-affinity choline uptake sites, and activity of choline acetyltransferase (ChAT), which is the synthetic enzyme for acetylcholine. In both behavioral tasks, the acquisition rate was not different between groups. L-NAME reduced NO synthase activity by 85% in all brain areas assayed and HC-3 binding by 38% in hippocampus and 48% in posterior cortex. ChAT activity was not different between groups in any region assayed. These data suggest that NO does not play a role in visual or spatial discrimination learning. However, NO synthase inhibition may play a role in the regulation of cholinergic activity.

Atypical beta-adrenoceptor in bovine adrenal medulla.

Bovine adrenal medullary membranes were incubated with [125I]cyanopindolol to assess beta-adrenoceptor binding. Binding was saturable and specific; a single low affinity site (Kd = 750 pM) was identified. [125I]Cyanopindolol binding was displaced by micromolar concentrations of classic beta-adrenoceptor antagonists and by sodium-4-[-2-[2-hydroxy-2-(-3-chloro-phenyl) ethylamino] propyl] phenoxyacetate. These data are similar to reported binding of beta 3-adrenoceptors and may explain beta-adrenoceptor agonist modulation of chromaffin cell degranulation in this catecholamine rich environment.

Neuropathic pain in rats is associated with altered nitric oxide synthase activity in neural tissue.

Peripheral nerve injury may lead to a chronic neuropathic pain state that results from an increase in excitability of central neurons. This central sensitization is mediated via an N-methyl-D-aspartic acid (NMDA) receptor and may involve the production of nitric oxide (NO). As NO is suggested to play a role in nociceptive transmission following nerve injury, we examined for altered NO synthase activity at multiple levels of peripheral and spinal neural tissue in a rat model of neuropathic pain. Peripheral neuropathy was induced in rats (N = 12) by ligation of the left L5 and L6 nerve roots. Six other rats had sham surgery. An ipsilateral decrease in paw withdrawal threshold to mechanical stimuli confirmed the presence of a neuropathic pain state. Samples of the lumbar and thoracic spinal cords, L4, L5, and L6 dorsal root ganglia (DRGs), and the sciatic nerves were obtained from the lesioned and contralateral sides at 2 and 4 weeks after neuropathic surgery (N = 6 per group). In the lumbar spinal cord, a bilateral decrease in nitric oxide synthase (NOS) activity was observed 2 and 4 weeks after neuropathic surgery. NOS activity was increased in the ipsilateral L5 and 6 DRGs 2 weeks following neuropathic surgery. An increase in NOS activity in the DRG may be an early mechanism for inducing more central changes. The bilaterally decreased NOS activity in the lumbar spinal cord may be secondary to a negative feedback mechanism resulting from increased NO production in the spinal dorsal root ganglia. Multiple alterations in expression of NOS activity that occur in both peripheral and central processing may play a role in the pain behavior resulting from peripheral nerve injury. (Preliminary results of these studies have been presented in abstract form at the annual meetings of the Society for Neuroscience, 1994, and the American Society of Anesthesiologists, 1994).