Locomotion-inducing sites in the vicinity of the pedunculopontine nucleus

The mesencephalic locomotor region (MLR) was identified physiologically by inducing controlled locomotion on a treadmill in the precollicular rat following application of low amplitude current pulses to areas of the pontomesencephalic tegmentum. The same brains were processed using either of two techniques known to label neurons of the pedunculopontine nucleus (PPN)-choline acetyltransferase (ChAT) immunocytochemistry or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Histological reconstruction of locomotion-inducing sites were localized within or adjacent to ChAT or NADPH-diaphorase labeled cell groups. Three dimensional reconstructions of the PPN were used to visualize the colocalization of low threshold locomotion-inducing stimulation sites within PPN neuronal aggregates. These findings lend further support to the suggestion that the PPN is part of the MLR. A theoretical framework is proposed to account for results derived from various lines of research on this area.     

3H]AMPA binding to glutamate receptor subpopulations in rat brain

The glutamate analog (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), displaced 11% of the binding of L-[3H]glutamate to rat brain membranes, amounting to 22% of the specific binding displaceable by excess non-radioactive glutamate. AMPA-sensitive L-[3H]glutamate binding was additive with that displaced by kainic acid (1 microM) plus N-methyl-D-aspartate (10 microM) when low concentrations of non-radioactive AMPA (1 microM) were employed to determine non-specific background, but partially overlapped when higher concentration of AMPA (100 microM) were used. [3H]AMPA binding was 21% specific (displaceable by non-radioactive 0.1 mM AMPA) in sodium-, calcium- and chloride-free buffer, but increased to over 30% in the presence of 0.1 M chloride. AMPA-sensitive glutamate binding and AMPA binding were both stimulated dramatically by thiocyanate and by several other anions. [3H]AMPA binding activity was resistant to freezing and thawing, optimal at 0-4 degrees C, and detectable at slightly reduced levels by filtration assays and in tissue section autoradiography. AMPA showed a heterogeneous affinity in displacement of L-[3H]glutamate, and [3H]AMPA binding showed heterogeneity with respect to AMPA, quisqualate, and glutamic acid diethyl ester. Scatchard plots gave a best fit for two sites with Kd values of 28 and 500 nM and Bmax values of 200 and 1800 fmol/mg protein, respectively. [3H]AMPA was inhibited by quisqualate (IC50 = 60 nM), L-glutamate (2 microM), (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridine-7-carboxylic acid (7-HPCA, 5 microM), kainic acid (20 microM) and glutamic acid diethyl ester (21 microM) but insensitive to L-aspartate, ibotenic acid, N-methyl-D-aspartate, (RS)-2-amino-phosphonobutyric acid and (RS)-2-amino-phosphonovaleric acid. This is consistent with labeling of a quisqualate-specific subpopulation of glutamate receptors. The high affinity (28 nM) and intermediate affinity (0.5 microM) AMPA sites had similar pharmacological specificity and brain regional distribution as determined by autoradiography. The latter revealed high densities of [3H]AMPA binding in the superficial layers of the cerebral cortex; stratum pyramidale, stratum radiatum, and stratum oriens of the hippocampus; and stratum moleculare of the dentate gyrus. Within the cerebellum, higher densities of binding were observed in the molecular layer than in the granule cell layer. In many regions, [3H]AMPA binding had a similar distribution to that of L-[3H]glutamate binding displaced by AMPA (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

CXC chemokine expression and synthesis in skeletal muscle during ischemia/reperfusion

BACKGROUND: The chemokines keratinocyte-Derived Cytokine (KC) and macrophage inflammatory protein (MIP)-2, murine equivalents of human interleukin 8, have been implicated in remote injury after acute hind limb ischemia/reperfusion (I/R). These studies were designed to determine whether the cytokines responsible for remote tissue injury are also synthesized and accumulate in the ischemic or reperfused hind limb. METHODS: B6, 129SF2/J mice were subjected to either 3 hours of unilateral hind limb ischemia alone (IA) or 3 hours of ischemia followed by 4 or 24 hours of reperfusion (I/R). After IA or I/R, experimental and control (nonischemic) contralateral hind limbs were harvested for analysis of protein content, messenger RNA (mRNA), tissue edema, and viability. RESULTS: IA did not increase KC or MIP-2 mRNA or protein levels. In contrast, I/R resulted in a 15- and 10-fold increase in KC mRNA after 4 and 24 hours of reperfusion, respectively. KC protein levels were increased 10-fold after 4 hours of reperfusion and 30-fold after 24 hours (vs IA or sham; P < .001). MIP-2 mRNA transiently increased 42-fold after 4 hours of reperfusion but decreased to basal levels after 24 hours of reperfusion. Despite the relative increase in MIP-2 mRNA by 4 hours of reperfusion, significantly increased (8- to 10 fold) MIP-2 protein levels were not detected until 24 hours of reperfusion only in the reperfused limbs. Tissue edema was increased significantly (P < .01) compared with sham after just 4 hours of reperfusion and remained increased at 24 hours. Tissue viability decreased 52% after 4 hours of reperfusion and did not change significantly by 24 hours. CONCLUSIONS: Skeletal muscle is a site of significant ongoing chemokine synthesis during reperfusion. The persistent increase in muscle chemokine levels at 24 hours of reperfusion was not associated with increased edema or injury. The role of these chemokines during reperfusion may be further investigated by local or oral administration of chemokines or chemokine receptor antagonists. CLINICAL RELEVANCE: I/R injury remains an important clinical problem across a variety of surgical specialties. In the critical care arena, serum levels of proinflammatory cytokines have been useful in predicting the mortality associated with acute respiratory distress syndrome and sepsis. In this article, the data presented indicate that murine skeletal muscle produces potent proinflammatory neutrophil and macrophage chemokines during reperfusion, but not during ischemia. These findings suggest that measurement of tissue and/or serum levels of chemokines during reperfusion may be an important adjunct to predicting tissue injury along with ongoing inflammation during the clinical course of reperfusion injury. Within the vascular system, severe inflammatory responses are usually associated with thrombotic events. New techniques to noninvasively image thrombin activation (by using magnetic resonance imaging) in reperfused limbs may coincide with the pattern of murine skeletal muscle chemokine expression in humans. The data suggest that reperfusion is when chemokine mRNA and protein synthesis increase. Within the time periods studied in these experiments, the chemokine component of the inflammatory response remained in the reperfused, rather than the systemic nonreperfused, tissue. This observation may underestimate the degree of the systemic response to ischemia because the single mouse hind limb represents only 7% of the mouse total body area, whereas the human limb represents nearly 18% of the adult body area. Despite this shortcoming, these data provide potential temporal and quantitative information regarding the location and magnitude of chemokine synthesis in skeletal muscle during reperfusion.     

Genetic and Environmental Factors in Parkinson's Disease Converge on Immune Function and Inflammation

Idiopathic Parkinson's disease (iPD) is a movement disorder characterized by the degeneration of dopaminergic neurons and aggregation of the protein α-synuclein. Patients with iPD vary in age of symptom onset, rate of progression, severity of motor and non-motor symptoms, and extent of central and peripheral inflammation. Genetic and environmental factors are believed to act synergistically in iPD pathogenesis. We propose that environmental factors (pesticides and infections) increase the risk for iPD via the immune system and that the role of PD risk genes in immune cells is worthy of investigation. This review highlights the major PD-relevant genes expressed in immune cells and key environmental factors that activate immune cells and, alone or in combination with other factors, may contribute to iPD pathogenesis. By reviewing these interactions, we seek to enable the future development of immunomodulatory approaches to prevent or delay onset of iPD. © 2020 International Parkinson and Movement Disorder Society     

Natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents

This is the first report of natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents. We examined the association of demographic characteristics of this cohort with three outcomes: CD16+ cell absolute count, lytic units per peripheral blood mononuclear cell (PBMC), and lytic units per natural killer (NK) cell. We also examined the association of CD4, CD38, and antiretroviral therapy (ART) use with these outcomes in the subset of HIV-infected adolescents. Adolescents participating in an on-going longitudinal study (the REACH study) were sampled for CD16+ cell count and NK function. This cross-sectional analysis was performed on 412 subjects with NK cell data available. HIV-positive males had higher numbers of CD3-/CD16+/CD56+ NK cells than HIV-positive females. However, for the HIV-negative subjects, we did not observe a gender-related effect for absolute NK cell numbers. Gender, however, was a significant covariate for the analysis, using lytic units per PBMC as the unit of measurement, with males showing higher values than females. Age was not a predictive covariate for any of the three assessments of NK cell number and function examined. Our observations concerning the HIV-positive individuals indicate that reduced CD4+ T cell counts were associated with decreased circulating CD3-/CD16+/CD56+ NK cells. We also observed an association between elevation of CD8+/CD38+/DR+ lymphocytes and lower NK lytic units per PBMC. The results of our multivariate models indicate that there is a reduced number of NK cells and reduced lytic units per PBMC in patients receiving single or multidrug antiretroviral therapy. There are changes in circulating NK cell number and function in HIV-infected adolescents, in comparison with high-risk HIV-negative adolescents. The data suggest that these changes may occur early in the course of HIV disease but that quantitative changes continue to occur with advancing depletion of the CD4+ T cell pool.     

Transmyocardial coil implants: a novel approach to transmyocardial revascularization

BACKGROUND: Transmyocardial laser revascularization (TMLR) has potential benefit for patients with end-stage coronary artery disease and intractable angina not amenable to conventional revascularization techniques. Neovascularization has been proposed to occur around the laser channels. Our aim was to determine the feasibility of a novel nonlaser myocardial revascularization technique and its effect on angiogenesis in a nonischemic porcine model. METHODS: In the first phase, six transmyocardial stainless steel coil implants (TMI) were deployed to the lateral wall of the left ventricle in each of 6 pigs. The animals were sacrificed at 8 and 12 weeks, with a single animal dying prematurely at 4 weeks, and the myocardium was assessed for new vessel growth. In the second phase, 8 implants were deployed in each of 12 pigs with regular fluoroscopic follow-up until sacrifice at 2 weeks to assess implant stability. RESULTS: The deployment procedure was safe and feasible with no complications evident. A significant increase in new vessels at implant sites with 5.43 +/- 3.67, 4.97 +/- 2.44, and 3.57 +/- 2.29 seen per high power field at 12, 8, and 4 weeks, respectively, compared to 1.00 +/- 1.06 (p < 0.0001) in control myocardium. There was no evidence of implant migration in Phase 2. CONCLUSIONS: TMIs can feasibly be deployed in the nonischemic pig model with a high success rate. The presence of angiogenesis at the implant site and the maintenance of this reaction for 3 months implies that TMI may offer an alternative to TMLR while providing a platform for delivery of angiogenic factors.     

GABA(A) receptor changes in delta subunit-deficient mice: altered expression of alpha4 and gamma2 subunits in the forebrain

The delta subunit is a novel subunit of the pentameric gamma-aminobutyric acid (GABA)(A) receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the delta subunit have been produced by gene-targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABA(A) receptors were limited to deletion of the delta subunit or whether alterations in other GABA(A) receptor subunits were also present in the delta subunit knockout (delta-/-) mice. Immunohistochemical studies of wild-type mice confirmed the restricted distribution of the delta subunit in the forebrain. Regions with moderate to high levels of delta subunit expression included thalamic relay nuclei, caudate-putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no delta subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in delta-/- and wild-type mice demonstrated substantial changes in the alpha4 and gamma2 subunits of the GABA(A) receptor in the delta-/- mice. gamma2 Subunit expression was increased, whereas alpha4 subunit expression was decreased in delta-/- mice. Importantly, alterations of both the alpha4 and the gamma2 subunits were confined primarily to brain regions that normally expressed the delta subunit. This suggests that the additional subunit changes are directly linked to loss of the delta subunit and could reflect local changes in subunit composition and function of GABA(A) receptors in delta-/- mice.