- … 703; List Biological Laboratories, Campbell, CA, USA), mouse anti-glial fibrillary acidic protein (GFAP, cat … 104; List Biological Laboratories) was injected into the superior colliculus (6.0 mm posterior and 2.0 mm lateral to the bregma, and 4-5 mm deep from the cortical …
Erythropoietin-producing hepatocyte receptor B (EphB)/ephrinB reverse signaling has been revealed to be activated in chronic ocular hypertension (COH) by increasing the apoptosis of retinal ganglion cells (RGCs). However, the exact mechanism is not well understood. The present study investigated the involvement of Ca2+ channels in the apoptosis of RGCs induced by EphB/ephrinB reverse signaling in a rat CHO model, which was established by cauterizing 3 out of the 4 episcleral veins. The expression levels of four voltage‑gated Ca2+ channel subunits (Cav3.1‑3.3 and Cav1.2) were detected using immunofluorescence and western blot analysis. TUNEL staining was performed to assess RGC apoptosis following an injection with the T type Ca2+ channel blocker. Ca2+ channels, mainly the T type, were upregulated in COH rat retinas when compared with the sham group (P<0.01). Additionally, the Cav3.2 subunit of T type calcium channels was predominantly expressed in Müller cells and RGCs, such as ephrinB2. Furthermore, an intravitreal injection of the Ca2+ channel blocker Mibefradil (3 µM) reduced EphB2‑fragment crystallizable region‑induced RGC apoptosis in normal rats. Thus, the results suggest that Ca2+ channels in a COH model may be a pathway involved in ephrinB/EphB signaling‑induced RGC apoptosis.
Multizonal Cerebellar Influence Over Sensorimotor Areas of the Rat Cerebral Cortex.
Aoki, S;Coulon, P;Ruigrok, TJH;
Cerebral Cortex (New York, N.Y. : 1991)December 29, 2017
Product: Cholera Toxin B Subunit (Choleragenoid)
from Vibrio cholerae in Low Salt
- … Under visual guid- ance, viral solution containing the French subtype of CVS-11 strain of RABV (titrating 4 × 107 plaque-forming units/ml) in con- junction with cholera toxin β-subunit (CTb, low salt; List Biological Laboratories, 1% w/v in 0.2 M phosphate buffer (PB) …
The cerebral cortex requires cerebellar input for optimizing sensorimotor processing. However, how the sensorimotor cortex uses cerebellar information is far from understood. One critical and unanswered question is how cerebellar functional entities (zones or modules) are connected to distinct parts of the sensorimotor cortices. Here, we utilized retrograde transneuronal infection of rabies virus (RABV) to study the organization of connections from the cerebellar cortex to M1, M2, and S1 of the rat cerebral cortex. RABV was co-injected with cholera toxin β-subunit (CTb) into each of these cortical regions and a survival time of 66-70 h allowed for third-order retrograde RABV infection of Purkinje cells. CTb served to identify the injection site. RABV+ Purkinje cells throughout cerebellar zones were identified by reference to the cerebellar zebrin pattern. All injections, including those into S1, resulted in multiple, zonally arranged, strips of RABV+ Purkinje cells. M1 injections were characterized by input from Purkinje cells in the vermal X-zone, medial paravermis (C1- and Cx-zones), and lateral hemisphere (D2-zone); M2 receives input from D2- and C3-zones; connections to S1 originate from X-, Cx-, C3-, and D2-zones. We hypothesize that individual domains of the sensorimotor cortex require information from a specific combination of cerebellar modules.
Myeloid cell plasticity in the evolution of central nervous system autoimmunity
- … Mice were injected intraperitoneally with 300 ng pertussis toxin (List Biological) on days 0 and 2. For adoptive transfer, mice were immunized as described, without pertussis toxin, and the draining lymph nodes (inguinal, brachial, and …
Myeloid cells, including macrophages and dendritic cells, are a prominent component of central nervous system (CNS) infiltrates during multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). Although myeloid cells are generally thought to be pro-inflammatory, alternatively-polarized subsets can serve non-inflammatory and/or reparative functions. Here we investigate the heterogeneity and biological properties of myeloid cells during central nervous system autoimmunity. Myeloid cell phenotypes in chronic active MS lesions were analyzed by immunohistochemistry. In addition, immune cells were isolated from the CNS during exacerbations and remissions of EAE and characterized by flow cytometric, genetic and functional assays. Myeloid cells expressing iNOS, indicative of a pro-inflammatory phenotype, were detected in the actively demyelinating rim of chronic active MS lesions, whereas macrophages expressing mannose receptor (CD206), a marker of alternatively-polarized human myeloid cells, were enriched in the quiescent lesion core. During EAE, CNS-infiltrating myeloid cells, as well as microglia, shifted from expression of pro- to non-inflammatory markers immediately prior to clinical remissions. Murine CNS myeloid cells expressing the alternative lineage marker arginase-1 (Arg1) were partially derived from iNOS+ precursors and were deficient in activating encephalitogenic T cells compared with their Arg1- counterparts. These observations demonstrate the heterogeneity of CNS myeloid cells, their evolution during the course of autoimmune demyelinating disease, and their plasticity on the single cell level. Future therapeutic strategies for disease modification in individuals with MS may be focused on accelerating the transition of CNS myeloid cells from a pro- to a non-inflammatory phenotype. This article is protected by copyright. All rights reserved.
Hydrogen postconditioning promotes survival of rat retinal ganglion cells against ischemia/reperfusion injury through the PI3K/Akt pathway
- … Statistical significance was set at P < .05. 3. Results. 3.1. RGC density after retinal IRI. We applied the anterograde tracer, FITC-conjugated cholera toxin B (CTB-FITC; List Biological, Campbell, CA) to count the number of surviving RGCs at one week after retinal IRI …
Retinal ischemia/reperfusion injury (IRI) plays a crucial role in the pathophysiology of various ocular diseases. Our previous study have shown that postconditioning with inhaled hydrogen (H2) (HPC) can protect retinal ganglion cells (RGCs) in a rat model of retinal IRI. Our further study aims to investigate potential mechanisms underlying HPC-induced protection. Retinal IRI was performed on the right eyes of rats and was followed by inhalation of 67% H2 mixed with 33% oxygen immediately after ischemia for 1 h daily for one week. RGC density was counted using haematoxylin and eosin (HE) staining, retrograde labelling with cholera toxin beta (CTB) and TUNEL staining, respectively. Visual function was assessed using flash visual evoked potentials (FVEP) and pupillary light reflex (PLR). The phosphorylated Akt was analysed by RT-PCR and western blot. The results showed that administration of HPC significantly inhibited the apoptosis of RGCs and protected the visual function. Simultaneously, HPC treatment markedly increased the phosphorylations of Akt. Blockade of PI3K activity by inhibitors (LY294002) dramatically abolished its anti-apoptotic effect and lowered both visual function and Akt phosphorylation levels. Taken together, our results demonstrate that HPC appears to confer neuroprotection against retinal IRI via the PI3K/Akt pathway.
Renitence vacuoles facilitate protection against phagolysosomal damage in activated macrophages
- … used for assays of macropinocytosis was purchased from R&D Systems (Minneapolis, MN). LPS from Salmonella typhimuirum (no. 225) was purchased from List Biological Laboratories (Campbell, CA). EIPA was purchased from Tocris (Minneapolis, MN). 35 mm dishes …
As professional phagocytes, macrophages are susceptible to endolysosomal membrane damage inflicted by the pathogens and noxious particles they ingest. Whether macrophages have mechanisms for limiting such damage is not well understood. Previously we reported a phenomenon, termed inducible renitence, in which LPS activation of macrophages protected their endolysosomes against damage initiated by the phagocytosis of silica beads. To gain mechanistic insight into the process, we analyzed the kinetics of renitence and morphological features of LPS-activated versus resting macrophages following silica bead-mediated injury. We discovered novel vacuolar structures that form in LPS-activated but not resting macrophages following silica bead phagocytosis. Because of their correlation with renitence and damage-resistant nature, we termed these structures renitence vacuoles. Renitence vacuoles formed coincident with silica bead uptake in a process associated with membrane ruffling and macropinocytosis. However, unlike normal macropinosomes, which shrink within 20 minutes of formation, renitence vacuoles persisted around bead-containing phagosomes. Renitence vacuoles fused with lysosomes, whereas associated phagosomes typically did not. These findings are consistent with a model in which renitence vacuoles, as persistent macropinosomes, prevent fusion between damaged phagosomes and intact lysosomes and thereby preserve endolysosomal integrity.