Elevated blood levels of inflammatory monocytes (CD14+ CD16+ ) in patients with complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a chronic pain disorder. Although its pathophysiology is not completely understood, neurogenic inflammation is thought to play a significant role. Microglia and astrocytes are activated following tissue injury or inflammation and have been reported to be both necessary and sufficient for enhanced nociception. Blood-borne monocytes/macrophages can infiltrate the central nervous system (CNS) and differentiate into microglia resulting in hypersensitivity and chronic pain. The primary aim of this study was to evaluate the proportion of the proinflammatory CD14(+) CD16(+) monocytes as well as plasma cytokine levels in blood from CRPS patients compared to age- and gender-matched healthy control individuals. Forty-six subjects (25 CRPS, 21 controls) were recruited for this study. The percentage of monocytes, T, B or natural killer (NK) cells did not differ between CRPS and controls. However, the percentage of the CD14(+) CD16(+) monocyte/macrophage subgroup was elevated significantly (P<0·01) in CRPS compared to controls. Individuals with high percentage of CD14(+) CD16(+) demonstrated significantly lower (P<0·05) plasma levels on the anti-inflammatory cytokine interleukin (IL)-10. Our data cannot determine whether CD14(+) CD16(+) monocytes became elevated prior to or after developing CRPS. In either case, the elevation of blood proinflammatoty monocytes prior to the initiating event may predispose individuals for developing the syndrome whereas the elevation of blood proinflammatory monocytes following the development of CRPS may be relevant for its maintenance. Further evaluation of the role the immune system plays in the pathogenesis of CRPS may aid in elucidating disease mechanisms as well as the development of novel therapies for its treatment.

Neuropathic central pain: epidemiology, etiology, and treatment options.

BACKGROUND: Nociceptive pain is a major problem in clinical neurology. Peripheral nerve injury may change the physiology of the dorsal horn so that pain becomes progressively centralized. OBJECTIVE: To review mechanisms underlying the plasticity of dorsal root ganglia and dorsal horn neurons that lead to central pain from a peripheral nerve injury. RESULTS: Evidence is reviewed that points to molecular changes in nociceptive terminals, ectopic firing of afferent pain fibers at the level of the dorsal root ganglia, and physiologic changes of the N-methyl-D-aspartate receptor that cause chronic nociceptive pain. CONCLUSIONS: Central sensitization is the physiologic manifestation of many severe peripherally induced pain states. It is maintained by nociceptive input and a physiologic change in the N-methyl-D-aspartate receptor. It consists of: (1) hypersensitivity at the site of injury; (2) mechanoallodynia; (3) thermal hyperalgesia; (4) hyperpathia; (5) extraterritoriality in the case of complex regional pain syndrome/reflex sympathetic dystrophy; and (6) associated neurogenic inflammation, autonomic dysregulation, and motor phenomena.

Quantitative sensory studies in complex regional pain syndrome type 1/RSD.

OBJECTIVE: Patients with complex regional pain syndrome type I (CRPSD1) may have thermal allodynia after application of a non-noxious thermal stimulus to the affected limb. We measured the warm, cold, heat-evoked pain threshold and the cold-evoked pain threshold in the affected area of 16 control patients and patients with complex regional pain syndrome type 1/RSD to test the hypothesis that allodynia results from an abnormality in sensory physiology. SETTING: A contact thermode was used to apply a constant 1 degrees C/second increasing (warm and heat-evoked pain) or decreasing (cold and cold-evoked pain) thermal stimulus until the patient pressed the response button to show that a temperature change was felt by the patient. Student t test was used to compare thresholds in patients and control patients. RESULTS: The cold-evoked pain threshold in patients with CRPSD1/RSD (p <0.001) was significantly decreased when compared with the thresholds in control patients (i.e., a smaller decrease in temperature was necessary to elicit cold-pain in patients with CRPSD1/RSD than in control patients). The heat-evoked pain threshold in patients with CRPS1/RSD was (p <0.05) decreased significantly when compared with thresholds in control patients. The warm- and cold-detection thresholds in patients with CRPS1/RSD were similar to the thresholds in control patients. CONCLUSIONS: This study suggests that thermal allodynia in patients with CRPS1/RSD results from decreased cold-evoked and heat-evoked pain thresholds. The thermal pain thresholds are reset (decreased) so that non-noxious thermal stimuli are perceived to be pain (allodynia).

Intracerebral microdialysis study of glutamate reuptake in awake, behaving rats.

The central nervous system has high-affinity uptake systems for the clearance of amino acid transmitters. These systems are found in both neurons and astrocytes. Previous studies have shown that the uptake of amino acid transmitters by astrocytes in culture can be modulated by adrenergic agents. The objectives of this study were to develop a methodology that evaluates the brain's reuptake capacity for glutamate in awake, behaving animals and to determine whether glutamate reuptake is under alpha-adrenergic regulation in the intact central nervous system. Male Sprague-Dawley rats weighing 250-450 g were used in this study. The extraction fraction of L-[3H]glutamate with [14C]mannitol as a reference was measured. The cortical extraction fraction of L-[3H]glutamate corrected for [14C]mannitol (EL-glu) reaches steady state rapidly and is both stable and repeatable. EL-glu is a measure of L-glutamate reuptake and not metabolism. EL-glu is decreased in a dose-dependent manner by the addition of the glutamate reuptake blocker D,L-threo-beta-hydroxyaspartic acid or unlabeled L- glutamate. In addition, EL-glu is increased in a dose-dependent manner by the alpha1-adrenergic agonist phenylephrine, and this increase is blocked by the alpha-adrenergic antagonist phentolamine.

Effect of plasma levels of large neutral amino acids and degree of parkinsonism on the blood-to-brain transport of levodopa in naive and MPTP parkinsonian monkeys.

We investigated the transport of levodopa from blood to brain in control and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian monkeys by using intracerebral microdialysis. The degree of parkinsonism was established by the monkey's clinical score and its postmortem striatal dopamine level. We administered levodopa plus carbidopa either orally or by intravenous injection. We estimated the blood-to-brain transport of levodopa as the ratio of its concentration in the brain's extracellular fluid to that in arterial plasma. This ratio was reduced in the MPTP parkinsonian monkeys, and we found an inverse relationship between the monkeys' ability to transport levodopa from blood to brain and their degree of parkinsonism. The oral administration of a high-protein meal or the intravenous infusion of large neutral amino acids before the administration of the levodopa plus carbidopa reduced the transport of levodopa into the brain, as measured by microdialysate collected from the striatum. We found that in two animals an intraperitoneal injection of the beta-adrenergic agonist isoproterenol increased the blood-to-brain transport of levodopa. Pharmacologic manipulation of the transport of levodopa from blood to brain may offer a new strategy for the treatment of Parkinson's disease.

Beta 2-adrenergic agonist as adjunct therapy to levodopa in Parkinson's disease.

We studied the effect of the beta 2-adrenergic agonist albuterol on Parkinson's disease (PD) patients receiving chronic levodopa treatment. The albuterol-treated patients demonstrated reduced parkinsonian symptoms and an increased ability to tap their index finger between two points 20 cm apart, and were able to perform a "walk test" in 70% of their control time. Three patients currently on chronic albuterol therapy still show amelioration of their parkinsonian symptoms, and two have reduced their daily levodopa dose. This study suggests that beta 2-adrenergic agonists as adjunct therapy to levodopa may be beneficial in PD.

Changes in brain dopamine receptors in MPTP parkinsonian monkeys following L-dopa treatment.

Twenty-two monkeys (Macaca fascicularis) were utilized in this study. Ten animals were rendered parkinsonian with serial injections of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Five of these parkinsonian monkeys received L-dopa/carbidopa treatment, and five animals did not. The remaining twelve animals did not receive MPTP. Eight of these animals received no L-dopa treatment, two animals were treated chronically with L-dopa/carbidopa and two animals received L-dopa/carbidopa only on the day of sacrifice. All animals were given weekly scored neurologic examinations throughout the study. Their movement was quantitated in an activity box. All animals were sacrificed by an overdose of sodium pentobarbital. The parkinsonian animals were sacrificed 107-355 days after their last MPTP injection. The brains were removed and frozen. Punch samples were taken from the caudate and putamen for tissue dopamine determination. Selected areas of the basal ganglia were cut into 20 microns sections for quantitative receptor autoradiography. The density of D1 and D2 receptors was evaluated in the basal ganglia of these animals at the level of the anterior commissure. For the D2 assay, total binding was determined using various concentrations of [3H]spiperone in buffer containing 300 nm mianserin. For the D1 assay, total binding was determined using various concentrations of [3H]SCH-23390. Tissue isotope concentration was determined from the autoradiographs. The MPTP parkinsonian monkeys showed a mean striatal dopamine depletion of 93.5% and a mean clinical score of 9.0. The untreated parkinsonian monkeys demonstrated an increase in the number of D2 sites as compared to controls. This increase was greatest in the lateral putamen.(ABSTRACT TRUNCATED AT 250 WORDS)

N-methyl-4-phenylpyridinium (MPP+) potentiates the killing of cultured hepatocytes by catecholamines.

The role of catecholamines in the toxicity of MPTP (N-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine) was explored. The killing of cultured hepatocytes by dopamine and 6-hydroxydopamine was enhanced following inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a manipulation known to sensitize such cells to an oxidative stress. The participation of activated oxygen species in the cell injury under such circumstances was shown by the ability of catalase and the ferric iron chelator deferoxamine to protect the hepatocytes. The toxicity of catecholamines was also potentiated by the mitochondrial site I (NADH dehydrogenase) inhibitor rotenone. MPP+ (N-methyl-4-phenyl-pyridinium), the putative toxic metabolite of MPTP is also a site I inhibitor. Incubation of hepatocytes with MPP+ similarly potentiated the toxicity of 6-hydroxydopamine, dopamine, and norepinephrine under conditions where MPP+ alone or catecholamines alone did not kill cells. Hepatocytes that had accumulated dopamine from the medium were killed by a subsequent exposure to MPP+ in the absence of a catecholamine in the medium. Hepatocytes that had not been pretreated with dopamine were not affected by the subsequent exposure to MPP+. These data indicated that catecholamines render hepatocytes more susceptible to the toxicity of MPP+ and suggest that the presence of catecholamines in specific neurons in the brain may be related to the selective neurotoxicity of MPTP.

Changes in brain catecholamines and dopamine uptake sites at different stages of MPTP parkinsonism in monkeys.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to produce parkinsonism in primates. We have studied the changes in brain catecholamines and the distribution of desipramine insensitive mazindol binding sites in MPTP parkinsonian primates at different levels of parkinsonism. Thirty-seven monkeys (Macaca fascicularis) were utilized in this study. Twelve naive animals received no treatment and served as controls. Twenty-five animals were rendered parkinsonian with serial injections of MPTP. All animals were given scored neurologic examinations throughout the study. Their movement was quantitated in an activity box. The animals were sacrificed 30-360 days after their last MPTP injection. The clinical exam of the MPTP parkinsonian monkeys demonstrated mildly to severely affected animals. There was an exponential decrease in brain catecholamine levels with increased clinical parkinsonism. The MPTP parkinsonian animals showed the greatest decrease (67-99.8%) in tissue dopamine levels in the caudate nucleus. The putamen followed closely in severity (48-99.8%) and the nucleus accumbens was much less affected (0-40%). The percent reduction of norepinephrine in the anterior pole of the frontal cortex (0-48%) was similar in degree to the decreased dopamine levels in the nucleus accumbens. Mazindol binding was decreased 30-98% in the caudate nucleus, 20-97% in the putamen, 0-26% in the nucleus accumbens, 80-96% in the substantia nigra pars compacta and 49-94% in the ventral tegmental area. In the striatum, the decreased mazindol binding was more pronounced laterally and posteriorly. In each animal, there was good correlation between tissue dopamine levels and the number of mazindol binding sites.

Dopamine receptor changes in untreated and (+)-PHNO-treated MPTP parkinsonian primates.

Fifteen monkeys (Macaca fascicularis) were utilized in this study. Seven naive animals received no treatment and served as controls. Eight animals were rendered parkinsonian with serial injections of MPTP. Three of the parkinsonian monkeys were treated with (+)-4-propyl-9-hydroxynaphthoxasine [(+)-PHNO], a selective dopamine D2 agonist. (+)-PHNO (2-5 micrograms/kg/h) was administered continuously using subcutaneous osmotic pumps. All animals were given weekly scored neurologic examinations throughout the study. Their movement was quantitated in an activity box. The animals were sacrificed 30-120 days after their last MPTP injection by an overdose of sodium pentobarbital. The brains were removed, frozen and cut into 20-microns sections. The density of D1 and D2 receptors was studied in the basal ganglia of these animals at the level of the anterior commissure. For the D2 assay, total binding was determined using various concentrations of [3H]spiperone in buffer containing 300 nm mianserine. For the D1 assay, total binding was determined using various concentrations of [3H]SCH-23390. Tissue isotope concentration was determined from the autoradiographs. The parkinsonian animals demonstrated 90-97% dopamine depletion in the striatum. There was a 75-90% decrease in free movement in the untreated parkinsonian monkeys and their composite clinical score was 8.9 on a scale of 0-16 (zero being normal). Control monkey scores averaged 0.6. The untreated parkinsonian monkeys demonstrated an increase in the number of D2 sites as compared to controls. This increase was greatest at the lateral putamen. The (+)-PHNO-treated monkeys demonstrated increased activity, a neurologic score of 3.4, and a 40-70% decreased in D2 sites in both caudate and putamen. There was no change in the number of D1 binding sites in both the untreated and the (+)-PHNO-treated parkinsonian monkeys as compared to controls.

Comparison of the effects of intracerebrally administered MPP+ (1-methyl-4-phenylpyridinium) in three species: microdialysis of dopamine and metabolites in mouse, rat and monkey striatum.

Intracerebral microdialysis in 3 awake species allowed the measurement of the basal output of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole-acetic acid (5-HIAA) from rat and mouse striatum and monkey caudate in vivo. The DOPAC/HVA ratios in dialysates from mouse and rat striatum were about 1 and 2 respectively, but only 0.09 in monkey caudate dialysates. The extracellular levels of the metabolites correlated well with reported tissue levels, while extracellular DA levels were 3 orders of magnitude lower than tissue concentrations. The effects of the intracerebrally administered dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) were essentially similar in the 3 species. In all cases an immediate, massive release of DA was accompanied by a pronounced decrease in the output of the metabolites. Basal DA release was no longer detectable 5-12 h after MPP+ administration and a second MPP+ perfusion failed to increase the release of DA.

Cerebral metabolism of parkinsonian primates 21 days after MPTP.

This study evaluates the changes in the local cerebral metabolic rate for glucose (LCMRg) in primates exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The LCMRg was evaluated 21 days following the last dose of MPTP. At this time, all MPTP-injected animals demonstrated parkinsonism and striatal dopamine was reduced to less than 3% of control values. The structures whose LCMRg was most affected were the motor cortex, the intermediate zone of the putamen, the external segment of the globus pallidus, the medial part of the ventrolateral nucleus of the thalamus (VLm), visual cortex, locus ceruleus, and the dorsolateral segment of the substantia nigra pars compacta. The structure whose increase in LCMRg correlated most closely to the clinical severity of parkinsonism was the external segment of the globus pallidus.

Gyroxin, a toxin from the venom of Crotalus durissus terrificus, is a thrombin-like enzyme.

We report a simple method for the isolation of gyroxin, a protein from the venom of the South American rattlesnake Crotalus durissus terrificus. The intravenous injection of gyroxin into mice produces temporary episodes characterized by opisthotonos and rotations around the long axis of the animal. We found gyroxin to be a glycoprotein with thrombin-like and esterase activities. Gyroxin loses its ability to produce the gyroxin syndrome, its thrombin-like activity and its esterase activity with heat, dithiothreitol, phenylmethylsulfonyl fluoride or diisopropylfluorophosphate. We also report that three other thrombin-like enzymes, crotalase from the eastern diamondback rattlesnake (Crotalus adamanteus), ancrod from the Malayan pit viper (Agkistrodon rhodostoma) and a thrombin-like enzyme from the Central American rattlesnake (Crotalus durissus durissus), produce the gyroxin syndrome in mice. These enzymes may work by releasing neuroactive peptides from endogenous precursors.

Flow dependent changes in the effective surface area of microdialysis probes.

The relative efficiencies of microdialysis probes were determined both in vitro and in vivo using tritiated water. Tritiated water (THO) freely distributes throughout the fluid spaces of an experimental animal and, at equilibrium, the brain extracellular concentration of THO is the same as the plasma concentration. Microdialysis probes were inserted into the right caudoputamen of anesthetized rats. The rats were injected with THO and after one hour microdialysis samples were collected at flow rates between 0.2 and 10.0 ul/min. The in vitro relative efficiency for THO was computed as the ratio of the THO concentration in the dialysate to that of the solution the probe was immersed in. The in vivo relative efficiency was computed as the ratio of the concentration of THO in the brain dialysate to that measured in the plasma of the rat. Both the in vitro and in vivo relative efficiencies for THO decrease with increasing flow rates, but they differ from each other except at very low flow rates (less than 0.25 ul/min). The in vitro relative efficiency at a given probe flow is the maximum efficiency that can be attained in vivo at that flow. The surface of effective exchange (Se) is the fraction of that maximum which is attained in vivo. This study also demonstrates how the effective surface area can be computed at any probe flow rate and how it can be used as a correction factor.

A simple method of analyzing profiles in time-difference direct optical scanning gel chromatography.

A simple method is presented for the analysis of time-difference, large-zone chromatography profiles. The method is derived from basic theory and is applicable to multicomponent as well as single-component systems. Simple computer simulations are used to demonstrate the inaccuracies of earlier, more empirical methods. This method has been tested on several proteins using an inexpensive, semi-automated, data acquisition and control system.

Characterization of a homogeneous arginyl- and lysyl-tRNA synthetase complex isolated from rat liver. Kinetic mechanism for lysyl-tRNA synthetase.

Bisubstrate kinetics and end product and dead end inhibition studies were performed on lysyl-tRNA synthetase isolated from rat liver. The kinetic patterns obtained are consistent with a sequential ordered mechanism of substrate addition, tRNA bound first, followed by lysine, and then by ATP. Pyrophosphate and AMP are released in a random fashion with aminoacylated tRNA the last product to dissociate from the enzyme. This is the first report of a kinetic mechanism for lysyl-tRNA synthetase.

A re-examination of some properties of fatty acyl-CoA micelles.

Three separate techniques have been employed to estimate the critical micelle concentration: spin labeling using 6-doxylstearoyl-CoA, gel permeation chromatography, and analytical ultracentrifugation. The first method is a labeling technique. The latter two methods utilize no potentially interfering probe and provide a value for the aggregation number for palmitoyl-CoA. All three methods provide a critical micelle concentration for palmitoyl-CoA no lower than 30 to 60 microM. The latter methods provide an aggregation number near 40 and certainly no larger than 200. These values are inconsistent with the values suggested earlier (Zahler, W. L., Barden, R. E., and Cleland, W. W. (1968) Biochim. Biophys. Acta 164, 1-11). The spin-labeled analogues, 6- and 16-doxylstearoyl-CoA, were shown not to micellize, yet these analogues were good inhibitors for citrate synthase. These observations will require the re-examination of a large body of literature in which inhibition of enzymes by fatty acyl-CoA at concentrations below 30 microM was simply ascribed to the formation of micelles.