Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization

Substance P release from nociceptive primary afferents activates post-synaptic neurokinin-1 (NK-1) receptors causing subsequent NK-1 receptor internalization. Fluorescent immunohistochemistry is typically used to quantify NK-1 receptor internalization, an indirect measure of substance P (SP) release. However, this technique entails several limitations that restrict its application. Using simple subcellular fractionation and immunoblotting methods, we demonstrate that intrathecal SP invokes a rapid and dose-dependent increase in dorsal horn cytoplasmic NK-1 receptors. We also show that hind paw compression and noxious thermal stimulation increase cytoplasmic NK-1 receptor, when compared to sham stimulations. Fluorescent immunohistochemistry confirmed that increases in cytoplasmic NK-1 corresponded with increased NK-1 receptor internalization. Herein, we report that low-speed centrifugation and Western immunoblotting provide NK-1 internalization results consistent with those obtained by more traditional methods. These data support previous findings demonstrating a role for spinal NK-1 receptors in nociceptive processing.     

Patients' perception of asthma severity

OBJECTIVES: To identify variables patients use to determine the severity of their asthma, the perceived severity (PS), using a fuzzy decision-making analysis (FDMA). To compare these variables with those involved in the assessment of asthma severity according to the global initiative for asthma (GINA) guidelines, the objective severity (OS). PATIENTS: Outpatients (51 men, 62 women), aged (m+/-SD) 42.9+/-16.3 years with (% patients) mild intermittent (6.2), mild persistent (15.9), moderate (65.5) and severe (12.4) asthma. DESIGN: Cross sectional, observational study. METHODS: Both OS (rated by doctors) and PS (rated by patients) were rated as mild intermittent, mild persistent, moderate, or severe. Variables involved in OS assessment, variables self-assessed by patients (dyspnea, perceived treatment efficacy, asthma-related quality of life questionnaire [AQLQ]), patients' sociodemographic characteristics, and asthma characteristics, were evaluated with questionnaires. These variables were pooled, and considered as potential variables patients might use to determine their PS. They were tested against the PS measurement using FDMA. This identified variables patients actually used to determine PS. RESULTS: On the day of consultation, 68.1% of patients classed their asthma as mild intermittent or mild persistent, 23.9% as moderate persistent, and 8.0% as severe persistent. There was a significant discrepancy (p<0.01) between PS and OS with a clear patient tendency to underestimate asthma severity as compared to OS. Patients determined PS level according to variables assessing their asthma perception, i.e., AQLQ measures and dyspnea, but not variables involved in OS assessment, such as symptom frequency or knowledge of their peak flow rates. Duration of asthma and treatment characteristics were also involved. CONCLUSION: FDMA identified variables patients used to determine PS. It highlighted a discrepancy between patients' and doctors' perceptions of asthma severity, suggesting that assessment of asthma severity should consider both patients' and doctors' perceptions of the disease and includes an AQLQ measure.     

Cytoplasmic nucleolus-like inclusions (nematosomes) in spinal ganglia neurons of dog

Intracytoplasmic nucleolus-like inclusions or nematosomes in spinal ganglia neurons of dog were studied by electron microscopy. In large light neurons several types of nematosomes can be identified. All types of the inclusions are non-membrane bound and composed of filamentous or granular aggregates. Nematosomes are mostly localized in the peripheral zone of cytoplasm. The mechanism of formation and functional significance of nematosomes is not fully clarified. The presence of ribosomes and cisterns of endoplasmic reticulum in their vicinity suggests some relation to proteosynthesis; they probably represent sites of protein storage.     

Post-malaria neurological syndrome: clinical and laboratory findings in one patient

Abstract Post-malaria neurological syndrome (PMNS) is a rare complication of malaria. It follows recovery from an episode of Plasmodium falciparum malaria and is characterised by symptoms and signs of encephalopathy. Patients usually improve without any specific treatment. The pathogenesis is unknown, but it is probably immunologically mediated. The objective of this case study is to describe the first Italian patient with PMNS. A 60-year-old Italian man developed acute P. falciparum malaria after a stay in French Guinea. Twenty days after recovering from malaria, he became confused, developed generalised weakness, limb tremors, shivering and dizziness. These symptoms continued for three days, then resolved spontaneously. Neuroimaging was normal. Cerebrospinal fluid analysis revealed breakdown of the blood/brain barrier, without oligoclonal bands and normal IgG index. Our patient presented a mild diffuse encephalopathy suggestive of a generic activation of the immune system without any specific reaction against antigens within the CNS.     

Ultrastructural and quantitative investigations of the spinal ganglia neurons after ligation of the abdominal aorta

The structural changes in spinal ganglia neurons of dogs subjected to repeated 40-minute ligation of the abdominal aorta with 1-6 day survival were studied by means of electron microscopy and morphometry. The endoplasmic reticulum shows marked changes with a decrease of bound ribosomes as well as a reduction of the membranous component, especially in the peripheral zone of cytoplasm. There were frequently noticed local deposits of filamentous material. Qualitative and quantitative changes had the same time course, culminating on the second to third day of survival. Later [6 days] some signs of neuronal recovery can be observed.     

Choline acetyltransferase and acetylcholinesterase in canine spinal ganglia: increase of choline acetyltransferase activity following sciatic nerve lesion

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in the dorsal spinal ganglia, the dorsal spinal root and the spinal cord of the normal adult dogs and following one side transection of the sciatic nerve in the intervals 5, 10, 15 and 21 days respectively. In the spinal ganglia of normal dogs very low ChAT activity was found; it was three orders lower than AChE activity. Within 5-10 days after the nerve section ChAT activity increased almost five times in the spinal ganglia while AChE activity remained without any changes. The elevation of ChAT activity correlated with that in the dorsal roots at 15th day and in the dorsal spinal cord at 21st day after the nerve section. Histochemical "direct-colouring" thiocholine method showed AChE-positive cells were distributed mainly in the peripheral area of the spinal ganglia. The spinal ganglion cells ranged from intensely AChE-positive to AChE-negative without correlation between cell size and AChE activity. The ChAT activity changes were evaluated in correlation to the cholinergic function in the spinal ganglion neurons.     

Alterations of the expression and activity of midbrain nitric oxide synthase and soluble guanylyl cyclase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice

The study was aimed at investigating the expression and the activity of neuronal nitric oxide synthase, and of soluble guanylyl cyclase and phosphodiesterase activities that regulate guanosine 3',5'-cyclic monophosphate level in the midbrain, in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections. Adult male mice of the C57/BL strain were given three i.p. injections of physiological saline or three i.p. injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine solution in physiological saline at 2 h intervals (summary 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dose: 40 mg/kg), and were killed 3, 7, or 14 days later. mRNA, protein level, and/or activities of neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase and guanosine 3',5'-cyclic monophosphate were determined. Immunohistochemistry showed about 75% decrease in the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed increased midbrain guanylyl cyclase and total nitric oxide synthase activities at 3, 7, and 14 days post-treatment. The specific neuronal nitric oxide synthase inhibitor 7-nitroindazole (10 microM) and the specific inducible nitric oxide synthase inhibitor 1400W (10 microM) inhibited the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced excess in nitric oxide synthase activity by 63-70 and 13-25%, respectively. The increases in total midbrain nitric oxide synthase activity were accompanied by elevated guanosine 3',5'-cyclic monophosphate, enhanced expression of neuronal nitric oxide synthase and of the beta1 subunit of guanylyl cyclase at both mRNA and protein levels that persisted up to the end of the observation period, and by enhanced neuronal nitric oxide synthase and guanylyl cyclase beta1 immunoreactivities in substantia nigra pars compacta 7 and 14 days after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The increases in guanylyl cyclase activity were found to occur exclusively due to increased maximal enzyme activity. No 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced change in phosphodiesterase activity has been detected in any brain region studied. 7-Nitroindazole prevented a significant increase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced midbrain guanosine 3',5'-cyclic monophosphate level and neurodegeneration of dopaminergic neurons. These results raise the possibility that the nitric oxide/guanylyl cyclase/guanosine 3',5'-cyclic monophosphate signaling pathway may play a role in maintaining dopaminergic neurons function in substantia nigra pars compacta.     

Development of GABA-sensitive spasticity and rigidity in rats after transient spinal cord ischemia: a qualitative and quantitative electrophysiological and histopathological study

Transient spinal cord ischemia may lead to a progressive degeneration of spinal interneurons and subsequently to increased hind limb motor tone. In the present work we sought to characterize the rigidity and spasticity components of this altered motor function by: i) tonic electromyographic activity measured in gastrocnemius muscle before and after ischemia, ii) measurement of muscle resistance during the period of ankle flexion and corresponding changes in electromyographic activity, iii) changes in Hoffmann reflex, and, iv) motor evoked potentials. In addition the effect of intrathecal treatment with baclofen (GABAB receptor agonist; 1 microg), nipecotic acid (GABA uptake inhibitor; 300 microg) and dorsal L2-L5 rhizotomy on spasticity and rigidity was studied. Finally, the changes in spinal choline acetyltransferase (ChAT) and vesicular glutamate transporter 2 and 1 (VGLUT2 and VGLUT1) expression were characterized using immunofluorescence and confocal microscopy. At 3-7 days after ischemia an increase in tonic electromyographic activity with a variable degree of rigidity was seen. In animals with modest rigidity a velocity-dependent increase in muscle resistance and corresponding appearance in electromyographic activity (consistent with the presence of spasticity) was measured during ankle rotation (4-612 degrees /s rotation). Measurement of the H-reflex revealed a significant increase in Hmax/Mmax ratio and a significant loss of rate-dependent inhibition. In the same animals a potent increase in motor evoked potential amplitudes was measured and this change correlated positively with the increased H-reflex responses. Spasticity and rigidity were consistently present for a minimum of 3 months after ischemia. Intrathecal treatment with baclofen (GABA B receptor agonist) and nipecotic acid (GABA uptake inhibitor) provided a significant suppression of spasticity, rigidity, H-reflex or motor evoked potentials. Dorsal L2-L5 rhizotomy significantly decreased muscle resistance but had no effect on increased amplitudes of motor evoked potentials. Confocal analysis of spinal cord sections at 8 weeks-12 months after ischemia revealed a continuing presence of ChAT positive alpha-motoneurons, Ia afferents and VGLUT2 and VGLUT1-positive terminals but a selective loss of small presumably inhibitory interneurons between laminae V-VII. These data demonstrate that brief transient spinal cord ischemia in rat leads to a consistent development of spasticity and rigidity. The lack of significant suppressive effect of dorsal L2-L5 rhizotomy on motor evoked potentials response indicates that descending motor input into alpha-motoneurons is independent on Ia afferent couplings and can independently contribute to increased alpha-motoneuronal excitability. The pharmacology of this effect emphasizes the potent role of GABAergic type B receptors in regulating both the spasticity and rigidity.