Alterations In Immunostaining Research Articles

Influence of Cannabinoid Receptor Deficiency on Parameters Involved in Blood Glucose Regulation in Mice.

Cannabinoids are known to influence hormone secretion of pancreatic islets via G protein-coupled cannabinoid receptor type 1 and 2 (CB1 and CB2). The present study was designed to further investigate the impact of cannabinoid receptors on the parameters involved in insulin secretion and blood glucose recognition. To this end, CB1 and CB2 receptor knockout mice (10–12 week old, both sexes) were characterised at basal state and compared to wild-type mice. The elimination of cannabinoid receptor signalling resulted in alterations of blood glucose concentrations, body weights and insulin levels. Changes were dependent on the deleted receptor type and on the sex. Analyses at mRNA and protein levels provided evidence for the impact of cannabinoid receptor deficiency on the glucose sensing apparatus in the pancreas. Both receptor knockout mouse lines showed decreased mRNA and protein amounts of glucose transporters Glut1 and Glut2, combined with alterations in immunostaining. In addition, pancreatic glucokinase expression was elevated and immunohistochemical labelling was modified in the pancreatic islets. Taken together, CB1 and CB2 signalling pathways seem to influence glucose sensing in β-cells by affecting glucose transporters and glucokinase. These alterations were more pronounced in CB2 knockout mice, resulting in higher blood glucose and lower plasma insulin levels.

Fructose-1,6-bisphosphate ameliorates lipopolysaccharide-induced dysfunction of blood–brain barrier

Fructose-1,6-bisphosphate (FBP), a glycolytic intermediate, has neuroprotective effects in various brain injury models. However, its effects on blood-brain barrier (BBB) are largely unknown. In this study, we investigated the effects of FBP on lipopolysaccharide (LPS)-induced BBB dysfunction in in vitro BBB model comprising co-culture of mouse brain endothelial cell line, bEnd.3 and mouse primary astrocyte and explored its action mechanism therein involved. LPS induced the impairment of endothelial permeability and transendothelial electrical resistance (TEER). The functional changes were confirmed by alterations in immunostaining for junctional proteins occludin, ZO-1 and VE-cadherin, such as the loss of cortical staining pattern and appearance of intercellular gaps in endothelial cells. Co-administration of FBP alleviated the deleterious effects of LPS on BBB permeability and TEER in a dose dependent manner. And also FBP inhibited the LPS-induced changes in the distribution of endothelial junctional proteins, resulting in the better preservation of monolayer integrity. FBP suppressed the production of reactive oxygen species (ROS) but did not affect cyclooxygenase-2 expression and prostaglandin E₂ production in endothelial cells stimulated with LPS. Taken together, these data suggest that FBP could ameliorate LPS-induced BBB dysfunction through the maintenance of junctional integrity, which might be mediated by downregulation of ROS production.

Exposure to Lipopolysaccharide and/or Unconjugated Bilirubin Impair the Integrity and Function of Brain Microvascular Endothelial Cells

BackgroundSepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC).Methodology/Principal FindingsMonolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB.ConclusionsLPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

GABA A receptor γ subunits in the hippocampus of the rat after perforant pathway lesion

Immunohistochemical and Western blotting techniques were employed to examine the alterations in immunostaining of the γ-amino butyric acid (GABA) receptor subunits gamma 1/3 and 2 within the hippocampus of the rat brain at 1, 3, 7, 14, and 30 days after a unilateral perforant pathway lesion. At 1, 3, and 7 days post-lesion, we observed a remarkable decrease in gamma 1/3 neuropil staining in the deafferented zone (i.e., the outer molecular layer of the dentate gyrus ipsilateral to the lesion), although at 3 and 7 days post-lesion, staining intensity was considerably recovered. At 14 days post-lesion, the gamma 1/3 immunostaining was indistinguishable from that of controls and it appeared yet more robust at 30 days post-lesion. We also observed a slight decrease in gamma 2 neuropil staining until 7 days post-lesion, and an increase in gamma 2 staining at 30 days post-lesion. Western blot analysis demonstrated data that was relatively consistent with our immunohistochemical observations, although gamma 3 was hardly detectable. Our study suggests that gamma subunits of the GABA A receptor in the dentate gyrus display a plastic response to the deafferentation of the perforant pathway.

Alterations in ApoE and ApoJ in Relation to Degeneration and Regeneration in a Mouse Model of Entorhinal Cortex Lesion

Apolipoproteins are primarily involved in the transport of lipid and cholesterol within the central nervous system (CNS) and are thought to play a role in synaptic remodeling, repair, and regeneration after brain injury. In the present study, alterations in apolipoproteins E (apoE) and J (apoJ) were examined in the molecular layers of the dentate gyrus after unilateral chemical lesioning of the entorhinal cortex (ECL), at days 0, 1, 3, 7, 28, and 90 days following injury. Alterations in immunostaining for these proteins were assessed in relation to accumulation of silver-labeled degeneration products and alterations in synaptophysin and GAP-43 immunoreactivity. Quantitative analysis of synaptophysin and GAP-43 immunostaining highlighted synaptic loss and fiber degeneration initially (3–7 days post-ECL), with subsequent terminal sprouting and reactive synaptogenesis occurring at longer survival periods (28–90 days post-ECL). Increased apoE and apoJ immunoreactivity was evident first within the neuropil (*P < 0.05 and **P < 0.01) followed by intense glial staining by day 7 post-ECL. By day 28 apoE and apoJ immunostaining had returned almost to baseline levels. However, at day 90 post-ECL, neuropil apoE and apoJ immunoreactivity was dramatically increased compared to contralateral levels (**P < 0.01 and ***P < 0.0001, respectively). Silver-labeled degeneration products were found to be in abundance at day 3 postlesion; however, by day 7 this was reduced leaving only a thin band of material within the MML and at day 90 post-ECL, dentate silver staining was similar to that of controls. The results indicate that apoE and apoJ are upregulated after injury and parallel clearance of cholesterol and lipid debris from the site of injury. This coordinated alteration in apolipoproteins may redistribute lipid material to sprouting fibers to promote neurite extension and may play an important role in long-term plasticity changes following injury.

Cholestasis with altered structure and function of hepatocyte tight junction and decreased expression of canalicular multispecific organic anion transporter in a rat model of colitis.

Cholestasis is frequently associated with inflammatory bowel disease. Because some cholestasis is resulted from altered hepatocyte tight junctions (TJs) or the canalicular multispecific organic anion transporter, we have investigated the following topics in a rat model of inflammatory bowel disease: (1) alterations in hepatocyte TJs and in the canalicular multispecific organic anion transporter, (2) etiologic factors for cholestasis, and (3) effects of antibiotics on cholestasis. Rats with trinitrobenzene sulfonic acid-induced colitis were studied 24 hours after treatment. Hepatocyte TJs and the canalicular multispecific organic anion transporter were evaluated by immunostaining for TJ-associated proteins, 7H6 and ZO-1, and multidrug resistance protein 2 (mrp2). To investigate etiologic factors causing cholestasis, portal endotoxin and proinflammatory cytokines were examined. The effects of polymyxin B, penicillin G, or metronidazole on immunostaining for 7H6, ZO-1, mrp2, and cholestasis were investigated. (1) Immunostaining for 7H6 and ZO-1 colocalized outlining the bile canaliculi and immunostaining for mrp2 localized on the canalicular membrane in controls. Treatment with trinitrobenzene sulfonic acid induced significant cholestasis and caused translocation of immunostaining for 7H6, but not that for ZO-1, to the cytoplasm and diminished immunostaining for mrp2 on the canaliculus membrane. (2) The levels of portal endotoxin, but not proinflammatory cytokines, was increased. (3) Polymyxin B, but not the other antibiotics, prevented alterations in immunostaining for both 7H6 and mrp2, and cholestasis. We described that both hepatocyte TJs and the canalicular multispecific organic anion transporter were altered and that gut-derived endotoxin levels in the portal blood were increased in this rat colitis model.

Lamina-Specific Synaptic Activation Causes Domain-Specific Alterations in Dendritic Immunostaining for MAP2 and CAM Kinase II

Polyribosomal complexes are selectively localized beneath postsynaptic sites on neuronal dendrites; this localization suggests that the translation of the mRNAs that are present in dendrites may be regulated by synaptic activity. The present study tests this hypothesis by evaluating whether synaptic activation alters the immunostaining pattern for two proteins whose mRNAs are present in dendrites: the dendrite-specific cytoskeletal protein MAP2 and the alpha-subunit of CAMKII. High-frequency stimulation of the perforant path projections to the dentate gyrus, which terminate in a discrete band on the dendrites of dentate granule cells, produced a two-stage alteration in immunostaining for MAP2 in the dendritic laminae. Five minutes of stimulation (30 trains) caused a decrease in MAP2 immunostaining in the lamina in which the activated synapses terminate. After more prolonged periods of stimulation (1-2 hr), there was an increase in immunostaining in the sideband laminae just proximal and distal to the activated band of synapses. The same stimulation paradigm produced a modest increase in immunostaining for alpha-CAMKII in the activated laminae, with no detectable changes in the sideband laminae. The alterations in immunostaining for MAP2 were diminished, but not eliminated, by inhibiting protein synthesis; the increases in CAMKII were not. These findings reveal that patterned synaptic activity can produce domain-specific alterations in the molecular composition of dendrites; these alterations may be caused in part by local protein synthesis and in part by other mechanisms.