periorbital-placement-laser-doppler-probe-for-cerebral-blood-flow

Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion ...

University of Arizona

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.

Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression.

Previous studies by our group have shown that peripheral inflammatory insult, using the lambda-carrageenan inflammatory pain (CIP) model, induced alterations in the molecular and functional properties of the blood-brain barrier (BBB). The question remained whether these changes were mediated via an inflammatory and/or neuronal mechanism. In this study, we investigated the involvement of neuronal input from pain activity on alterations in BBB integrity by peripheral inhibition of nociceptive input. A perineural injection of 0.75% bupivacaine into the right hind leg prior to CIP was used for peripheral nerve block. Upon nerve block, there was a significant decrease in thermal allodynia induced by CIP, but no effect on edema formation 1 h post-CIP. BBB permeability was increased 1 h post-CIP treatment as determined by in situ brain perfusion of [(14)C] sucrose; bupivacaine nerve block of CIP caused an attenuation of [(14)C] sucrose permeability, back to saline control levels. Paralleling the changes in [(14)C] sucrose permeability, we also report increased expression of three tight junction (TJ) proteins, zonula occluden-1 (ZO-1), occludin and claudin-5 with CIP. Upon bupivacaine nerve block, changes in expression were prevented. These data show that the lambda-carrageenan-induced changes in [(14)C] sucrose permeability and protein expression of ZO-1, occludin and claudin-5 are prevented with inhibition of nociceptive input. Therefore, we suggest that nociceptive signaling is in part responsible for the alteration in BBB integrity under CIP.

Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier.

Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood-brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O(2), 60 min), Hx (6% O(2), 60 min), or hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.

Sharon Hom

College of Nursing

University of Arizona

Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models

Sharon Hom

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University of Arizona

Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models

College of Nursing