Assays of 125I-Bolton-Hunter Substance P Binding Sites, Functional Sites of Tachykinin NK1 Receptor : Quantitative Receptor Autoradiographic Method

125I -Bolton-Hunter labeled tachykinins, substance P, neurokinin A, and eledoisin have been used as radioligands to differentiate multiple tachykinin receptors 11),12),30). 125I-Bolton- Hunter substance P seems to be a radioligand predominantly recognizing NK1 subtype of tachykinin receptors. As described here, using the quantitative receptor autoradiographic method, the binding sites specifically labeled with the radioligand can be vigorously analyzed. In addition to substance P, neurokinin A and neurokinin B known to be endogenously, new tachykinins, neuropeptide K31) and neuropeptide R32) have been isolated from the porcine brain and rabbit small intestine, respectively. Further characteristics of tachykinin receptors would be clarified with the method described

Blood Pressure-Independent Factors Determine the Susceptibility to Delayed Neuronal Death in the Stroke-Prone Spontaneously Hypertensive Rats.

The stroke-prone spontaneously hypertensive rat (SHRSP) is vulnerable to delayed neuronal death (DND) in the CA1 subfield of the hippocampus after the transient forebrain ischemia by the occlusion of the bilateral carotid arteries. The present study was designed to show that the genetic factors independent of high blood pressure contributed to the high incidence of DND in SHRSP. Male rats of the four strains, SHRSP/Izm, SHRSP/Ngsk, SHR/Izm and a congenic strain for the blood pressure quantitative trait locus on chromosome 1 [SHRSP.WKY-(D1Wox29-D1Arb21)/Izm]were used in the experiments. At 13 weeks of age, the bilateral carotid arteries of rats were occluded for 10 min under anesthesia with their body temperature kept at 37 degrees C. Seven days after the transient ischemia, the loss of the pyramidal cells in the CA1 was evaluated histologically. In some experiments, the blood flow was monitored with a laser Doppler flowmeter during the transient ischemia. The blood pressure in SHRSP/Izm was significantly greater than that in the other three strains. The incidence of DND, however, was not significantly different among SHRSP/Izm, SHRSP/Ngsk and the congenic strain (82, 74 and 65%, respectively), while SHR/Izm showed a significantly lower incidence (20%). Neither a significant correlation between the incidence of DND and the blood flow reduction during the occlusion, nor a significant inter-strain difference in the blood flow reduction was observed. The genetic factors independent of high blood pressure may contribute to the greater susceptibility to DND in SHRSP

Silver Nanoparticles Induce Tight Junction Disruption and Astrocyte Neurotoxicity in a Rat Blood-Brain Barrier Primary Triple Coculture Model

BACKGROUND: Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. METHOD: To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. RESULTS: A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values \u3e200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. CONCLUSION: These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes

Glutathione Related Enzyme Activities in Spontaneous Hypertensive Rat Heart

It has been reported that oxygen radicals are involved in the development of tissue injury in hypertension. To prevent o xidative stress, there are antioxidant systems inside the cells such as superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reducatase (GR), catalase (CAT) and glutathione S-transferase (GST). In this study changes in these antioxidant activities were estimated in the outer wall of the left ventricles from spontaneously hypertensive rats (SHR), stroke prone SHR (SHRSP) and normal Wister Kyoto rats (WKY). The activities of manganese-superoxide dismutase (Mn-SOD), which localizes in mitochondria and GST were lower in the left ventricles of SHR and SHRSP compared to those in WKY. Slight decrease in the GPX activity was observed in the left ventricles from SHR and SHRSP. On the other hand, the activity of GR and catalase was not different in them. The effect of Nicardipine, a calcium channel blocker, on these antioxidant activities was also esimated. Treatment of these rats with nicardipine (150 mg/kg/day) for 4 weeks improved blood pressure, from 176ﾂｱ10 mmHg to 140ﾂｱ8 mmHg in SHR (n = 5), from 201ﾂｱ11 mmHg to 167ﾂｱ5 in SHRSP (n = 5), respectively, and restored the activities of Mn-SOD, GST and GPX. Collectively, these results suggest that oxidative stress in hypertensive rat heart causes supression of antioxidant activities, which may contribute to myocardical injury, and nicardipine plays a cardioprotective role to reduce the oxidative stress in hypertensive heart

Receptor Autoradiographic Analysis of Muscarinic Receptors in the Rat Atrioventricular Node

We carried out investigations on muscarinic acetylcholine receptors (m-AChR) in the rat heart, including the atrioventricular (AV) node. The related tissue sections were incubated with 3H-quinuclidinyl benzilate (3H-QNB), then autoradiography and an image analysis coupled with computer-assisted microdensitometry were done. A single type of specific and high affinity binding sites of 3H-QNB was found to be highly concentrated in the AV node, the maximum binding capacity (Bmax) being 1.4 pmol/mg protein and with a dissociation constant (Kd) of 0.5 nM. The density and affinity of the binding to the AV node were the highest, when compared with findings in the atrium (interatrial septum) and ventricle (interventricular septum). The binding was competitively displaced by AF-DX 116, a selective antagonist for the M2 AChR subtype, with a high affinity, whereas pirenzepine, an antagonist for the M1 AChR subtype was much less potent in displacing the binding. Therefore, vagal-cholinergic stimulation presumably plays a significant role in functions of the rat AV node, probably by interacting with the specific, high affinity M2 AChR subtype

A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes.

Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs

MAP Kinase Pathways in Brain Endothelial Cells and Crosstalk with Pericytes and Astrocytes Mediate Contrast-Induced Blood–Brain Barrier Disruption

Neurointervention with contrast media (CM) has rapidly increased, but the impact of CM extravasation and the related side effects remain controversial. This study investigated the effect of CM on blood–brain barrier (BBB) integrity. We established in vitro BBB models using primary cultures of rat BBB-related cells. To assess the effects of CM on BBB functions, we evaluated transendothelial electrical resistance, permeability, and tight junction (TJ) protein expression using immunohistochemistry (IHC) and Western blotting. To investigate the mechanism of iopamidol-induced barrier dysfunction, the role of mitogen-activated protein (MAP) kinases in brain endothelial cells was examined. We assessed the effect of conditioned medium derived from astrocytes and pericytes under iopamidol treatment. Short-term iopamidol exposure on the luminal side induced transient, while on the abluminal side caused persistent BBB dysfunction. IHC and immunoblotting revealed CM decreased the expression of TJ proteins. Iopamidol-induced barrier dysfunction was improved via the regulation of MAP kinase pathways. Conditioned medium from CM-exposed pericytes or astrocytes lacks the ability to enhance barrier function. CM may cause BBB dysfunction. MAP kinase pathways in brain endothelial cells and the interactions of astrocytes and pericytes mediate iopamidol-induced barrier dysfunction. CM extravasation may have negative effects on clinical outcomes in patients