Absence of IFNγ expression induces neuronal degeneration in the spinal cord of adult mice

Background  

Interferon gamma (IFNγ) is a pro-inflammatory cytokine, which may be up-regulated after trauma to the peripheral or central nervous system. Such changes include reactive gliosis and synaptic plasticity that are considered important responses to the proper regenerative response after injury. Also, IFNγ is involved in the upregulation of the major histocompatibility complex class I (MHC class I), which has recently been shown to play an important role in the synaptic plasticity process following axotomy. There is also evidence that IFNγ may interfere in the differentiation and survival of neuronal cells. However, little is known about the effects of IFNγ absence on spinal cord neurons after injury.     

Is NMDA receptor activation essential for the production of locomotor-like activity in the neonatal rat spinal cord?

Previous work has established that in vitro bath application of N-methyl-D-aspartic acid (NMDA) promotes locomotor activity in a variety of vertebrate preparations including the neonatal rat spinal cord. In addition, NMDA receptor activation gives rise to active membrane properties that are postulated to contribute to the generation or stabilization of locomotor rhythm. However, earlier studies yielded conflicting evidence as to whether NMDA receptors are essential in this role. Therefore in this study, we examined the effect of NMDA receptor blockade, using D-2-amino-5-phosphono-valeric acid (AP5), on locomotor-like activity in the in vitro neonatal rat spinal cord. Locomotor-like activity was induced using 5-hydroxytryptamine (5-HT), acetylcholine, combined 5-HT and NMDA receptor activation, increased K(+) concentration, or electrical stimulation of the brain stem and monitored using suction electrode recordings of left and right lumbar ventral root discharge. We also studied the effect on locomotor capacity of selectively suppressing NMDA receptor-mediated active membrane properties; this was achieved by removing Mg(2+) ions from the bath, which in turn abolishes voltage-sensitive blockade of the NMDA receptor channel. The results show that, although NMDA receptor activation may seem essential for locomotor network operation under some experimental conditions, locomotor-like rhythms can nevertheless be generated in the presence of AP5 if spinal cord circuitry is exposed to appropriate levels of non-NMDA receptor-dependent excitation. Therefore neither NMDA receptor-mediated nonlinear membrane properties nor NMDA receptor activation in general is universally essential for locomotor network activation in the in vitro neonatal rat spinal cord.     

Developmental patterns of Ki-67, Oct-4 and α-tubulin proteins expression in the human spinal cord

The aim of this study was to analyze immunohistochemically the relationships between factors involved in processes of cell proliferation (Ki-67), differentiation (Oct-4) and primary cilia formation (α-tubulin) in the two parts of the developing human spinal cord (SC) of different origin in 11 human concepti (developmental weeks 5–10). Proliferation was highest in weeks 7–8 in the dorsal ventricular zones of the cranial (85.5%) and caudal (12.1%) SC. In the ventricular (VZ), intermediate (IZ) and marginal zones (MZ) of the cranial SC, α-tubulin and Oct-4 were moderately to strongly expressed. During weeks 5–6, moderate expression of α-tubulin and Oct-4 characterized the ventral part, with mild expression in the dorsal part of the caudal SC. In weeks 7–8, their expression increased in the VZ and IZ, and decreased in the MZ. In both parts of the SC Ki-67 and α-tubulin co-localized in the VZ. Oct-4 and Ki-67 co-localized only in the ependymal cells. In the cranial SC α-tubulin and Oct-4 co-localized (VZ and IZ), while the MZ expressed only α-tubulin. In the caudal SC, α-tubulin and Oct-4 co-localized in the VZ, while in the IZ some cells were only α-tubulin-positive. We suggest the importance of temporal–spatial expression of Ki-67 for the thickening of the cranial SC lateral wall. While in the cranial part of the SC, proliferation followed a ventral–dorsal direction, the caudal SC had a more irregular pattern. α-Tubulin was associated with cilia formation (ependymal cells) and axonic elongation of neuroblasts (MZ). Primary cilia signaling are important in control of SC proliferation and differentiation. Oct-4 expression in the SC coincided with presence of dividing neuroepithelial cells in the VZ and neuroblasts in the IZ, and could control the level of SC differentiation.     

Electrophysiological analysis of disturbances in reflex activity of the spinal cord in experimental botulism type a in warm — and cold-blooded animals

Summary The effect of botulism toxin on the electric acitivity of the spinal cord was studied by the electrophysiological method. The author has demonstrated on frogs and cats that in local botulism the mono-polysynaptic and electrotonic reactions are absent in the anterior roots of the spinal cord. The functional condition of motor nerves was examined. It was revealed that with development of botulism poisoning, especially in late stages, a significant prolongation of the phases of relative and absolute refractivity and decrease of lability take place.Presented by Active Member AMN SSSR V. N. Chernigovskii     

Evaluation of the activity of a novel metabotropic glutamate receptor antagonist (±)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) in the in vitro neonatal spinal cord and in an in vivo pain model

The cyclobutylglycine (±)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1α and 5α, with IC₅₀ values of 1.0±0.4 μM and 1.6±1.4 μM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent K_d values were estimated to be 0.3 μM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 μM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root–ventral root potential elicited at 8× threshold was depressed by LY393053 with IC₅₀ values of 9.0±0.7 μM and 12.7±1.7 μM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1–10 mg/kg intraperitoneally. An ED₅₀ value of 6.0 mg/kg was obtained in this test.By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.     

Edaravone effects on the expression levels of collagen type i and iv after spinal cord injury in rats北大核心

BACKGROUND: Edaravone, an effective free radical scavenger, has been reported to significantly improve the rehabilitation of limb locomotion after spinal cord injury (SCI), but the underlying mechanism remains unclear. OBJECTIVE: To explore the mechanism underlying edaravone promoting the recovery of limb locomotion in rats with SCI by observing the Basso, Beattie, Bresnahan scores and expression levels of collagen type I and IV. METHODS: Thirty-six rats were randomly allocated into three groups (n=12 per group): sham group (laminectomy plus intraperitoneal injection of normal saline), model group (SCI model by NYU impactor plus intraperitoneal injection of normal saline), and edaravone group (SCI model by NYU impactor plus intraperitoneal injection of edaravone). All rats were given the administration at the 1st day post-SCI for consecutive 7 days. The Basso, Beattie, Bresnahan scores were tested at 1, 3, 5 and 7 days post treatment. On day 7, all rats were sacrificed to remove the spinal cord, and the morphology of neurons in the spinal cord were observed by Nissl staining; the expression levels of collagen type I and IV were detected by immunohistochemistry and western blot assays. RESULTS AND CONCLUSION: Compared with the model group, the Basso, Beattie, Bresnahan scores in the edaravone group were significantly increased at day 5 post treatment (P < 0.05). Nissl staining showed a clear boundary between grey matter and white matter, and a large nucleolus in the neurocytoplasm in the sham group; there was a complete structure of neurons, slight cellular swelling and small hematoma area in the edaravone group; many and large cavitations and swollen nucleus were found in the neurons, even without nucleolus. Immunohistochemistry and western blot assay results showed that the expression levels of collagen type I and IV in the edaravone group were significantly higher than those in the model group (P < 0.05). These results indicate that edaravone can promote the recovery of limb locomotion of rats with SCI, probably via up-regulating the expression levels of collagen type I and IV. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Toll-like receptor signal transduction and the tailoring of innate immunity: a role for Mal?

Recent evidence suggests that there may be specificities in the signal transduction pathways activated by different Toll-like receptors (TLRs), with different sets of genes being induced by TLR-4 when compared with TLR-2. These differences may be because of different signalling adapters, with MyD88 being used by several TLRs, and the adapter MyD88-adapter-like (Mal) being recruited specifically by TLR-4. The set of genes being induced may be tailored for the subsequent elimination of the pathogen being recognized, as a result of differences in signal transduction pathways activated by TLRs. These findings may ultimately explain how dendritic cells control specific T-cell responses.     

The important role for βVLDLs binding at the fourth cysteine of first ligand-binding domain in the low-density lipoprotein receptor

The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH₂ terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease.     

Immune modulation of ovalbumin-induced lung injury in mice using β-glucosylceramide and a potential role of the liver

CD1d-restricted natural killer T (NKT) cells are implicated in the pathogenesis of asthma. β-Glucosylceramide (GC), a naturally occurring lipid, was previously shown to alter NKT cell distribution in the liver. We hypothesized that GC can affect lung and liver NKT cell distribution and ameliorate asthma. Mice were sensitized by intra-peritoneal injection of ovalbumin (OVA) for 2 weeks followed by repeated intranasal OVA challenges to induce lung injury mimicking asthma. OVA induced asthma groups were either treated by intranasal instillation of normal saline, intranasal instillation of GC or inhaled budesonide. To investigate the role of the liver, hepatic fibrosis was induced using carbon tetrachloride prior to asthma induction. Allergen induced bronchoconstriction was measured prior to sacrifice. Isolated lymphocytes from lungs, livers and spleens were analyzed for OVA induced proliferation and flow cytometry. Liver and lung histology, serum aminotransferase and anti-OVA antibodies level were assessed. Treatment with GC significantly reduced OVA induced airway responsiveness (p < 0.001) similar to inhaled budesonide. GC significantly reduced the peri-bronchial and peri-vascular inflammatory infiltration mainly through an effect on T cells, as suggested by decreased T cell proliferation (p = 0.009). Liver CD4 and NKT cells significantly increased after GC treatment suggesting liver involvement. Inducing hepatic fibrosis blunted the propagation of asthma in spite of sufficient increase of serum anti-OVA titers. GC has an immunomodulatory effect on a murine model of experimental asthma. We also suggest that the liver acts as an immunomodulatory organ and might have a regulatory effect on pulmonary diseases.     

Is there a role for leukocyte and CRP measurements in the diagnosis of acute appendicitis in the elderly?

Objectives: The diagnosis of acute appendicitis is still difficult and the results are unsatisfactory in three particular patient groups: in children, in fertile-age women and in elderly patients. As our population ages, the challenge for expedient diagnosis and intervention in older age groups will become more and more significant. The present study aimed at clarifying the role of leukocyte count and C-reactive protein (CRP) measurements in the diagnosis of acute appendicitis in the elderly. In particular, are there patients with acute appendicitis but unelevated leukocyte count and CRP? Methods: Eighty-three consecutive elderly patients underwent appendectomy for suspected acute appendicitis. The mean leukocyte count and CRP value were calculated in patients with an uninflamed appendix (group A) and in those with acute appendicitis (group B). The percentages of patients with: (1) both values unelevated; (2) only leukocyte count elevated; (3) only CRP value elevated; (4) both values elevated were calculated within the groups A and B. Results: There was no statistically significant difference in leukocyte counts or CRP values between patients with an uninflamed appendix (group A) and those with acute appendicitis (group B). When the patients were divided into the four subgroups, the most conspicuous finding was that group B (acute appendicitis, n=73) contained no patients with both values unelevated. Conclusions: Although elevated leukocyte count and CRP value cannot effectively establish the diagnosis of acute appendicitis in the elderly, unelevated values exclude it. Accordingly, appendectomy is not recommended to be performed in an elderly patient with unelevated leukocyte count and CRP value, although clinical symptoms and signs indicate acute appendicitis.     

Retinoic acid negatively regulates β4 integrin expression and suppresses the malignant phenotype in a Lewis lung carcinoma cell line

Retinoic acid (RA) is a potent inhibitor of the malignant phenotype and of tumour cell growth. We observed thatin vitro RA treatment of a highly metastatic lung carcinoma cell line (C87) induced a marked reduction in the amount of the4 integrin subunit. The downregulation of this adhesion molecule was assessed by immunofluorescence, immunoprecipitation, and northern analysis. In order to investigate the effects of RA on the malignant phenotype in C87 cells we performed morphological and functional analysis after RA treatment. We found that RA was able to produce marked changes in C87 cell shape, increasing the number of flat cells (90% of the total cell population), and significantly inhibiting the malignant and invasive phenotype of C87 cells. RA treatment suppressed their clonogenic potential in soft agar (control, 20±5; RA, 0), and strongly reduced their chemotactic and chemoinvasive capacity (chemotaxis: control, 231±5; RA, 28±0; chemoinvasion: control, 132±11; RA=2±1). FACS analysis and cell count, however, indicated that RA reduced the growth of C87 cells only partially. After 72 h of treatment we observed only a 10% reduction in the S phase fraction of the cell population. Finally, the reduced lung colony-forming ability, observed after i.v. injection of RA-treated cells (lung foci/animal: RA-treated cells, 1±0.1; untreated, 8.5±0.8), further supports the conclusion that in this murine lung carcinoma cell line a marked reduction in the expression of the 4 integrin subunit is associated with a marked inhibition of the malignant phenotype.     

Optimization of the method for α-l-fucosidase, β-d-galactosidase and β-d-glucuronidase determination in serum from hemolyzed blood

Purpose

Adaptation of the colorimetric method for the determination of β-d-galactosidase, β-d-glucuronidase and α-l-fucosidase activities in serums from hemolyzed blood, the material currently being discarded.

Evaluation of a commercial microarray as a confirmation test for the presence of extended-spectrum β-lactamases in isolates from the routine clinical setting

Since the diagnostic characteristics of the Check-KPC ESBL microarray as a confirmation test on isolates obtained in a routine clinical setting have not been determined, we evaluated the microarray in a random selection of 346 clinical isolates with a positive ESBL screen test (MIC >1 mg/L for cefotaxime or ceftazidime or an ESBL alarm from the Phoenix or Vitek-2 expert system) collected from 31 clinical microbiology laboratories in the Netherlands in 2009. Using sequencing as the reference method the sensitivity of the microarray was 97% (237/245), the specificity 98% (97/99), the positive predictive value 99% (237/239) and the negative predictive value 92% (97/105).     

Dynamic alterations of gene expression of nicotinic acetylcholine receptor α7, α4 and β2 subunits in an acute MPTP-lesioned mouse model

Epidemiologic studies show that the prevalence of Parkinson's disease (PD) is lower in smokers than in nonsmokers. Nicotine, a potent agonist of nicotinic acetylcholine receptors (nAChRs), excites midbrain dopaminergic neurons and this may contribute to the anti-parkinsonian effects. However, the alterations in gene expression of nAChR subunits using an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse PD model remain unclear. In the present study, we profile the time course of nAChR α7, α4 and β2 subunit expression levels using a comparative RT-PCR approach after acute MPTP injection. The results fall into four categories. (1) MPTP treatment transiently increased nAChR α7 (after last injection of MPTP 3 and 24 h), α4 and β2 (24 h) mRNA expression in the substantia nigra (SN) and striatum. (2) Compared to cortical and hippocampal tissues, this transient increase of nAChR subunit expression specifically occurred in the SN and striatum. (3) In the acute MPTP model, time-courses of altered expression for nAChR α7, α4 and β2 subunits closely mirrored the deficits observed in animal motor activity. (4) Stereological data showed that after administration of MPTP for 24 h, there was a robust astrogliosis in the SN associated with significant dopaminergic neurodegeneration. These changes followed or paralleled MPTP-induced elevation in the levels of α7, α4 and β2 mRNAs. Collectively, our results demonstrate that nAChRs are important targets in the MPTP neurotoxic process. These data suggest that therapeutic strategies targeted toward nAChR α7, α4 and β2 subunits may have potential for developing new treatments for PD.