Leaky Barrier Research Articles

Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography.

We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data collected over an 18-year period indicated a leaky pre-mating barrier between ribosomal species (including Groups I and III). Whether the observed selectivity inhibits hybridization in nature is dependent on its mechanism. If the point of selectivity is the induction of gametogenesis, dissimilar ribotypes could interbreed freely, promoting displacement in cases where hybridization is lethal. If instead, selectivity occurs during the adhesion of gamete pairs, it could enable stable coexistence of A. tamarense species. In either case, hybrid inviability may impose a significant obstacle to range expansion. The nested PCR assay developed here is a valuable tool for investigation of interspecies hybridization and its consequences for the global biogeography of these important organisms.

Groundwater flow through anisotropic fault zones in multiaquifer systems

Vertical faults through the shallow crust are commonly believed to act as either barriers to horizontal groundwater flow normal to the fault, conduits to horizontal flow tangential to the fault, or a combination of both. In addition, enhanced vertical permeability has been identified as a common feature. We investigate the effects of vertical anisotropy of a fault zone on the distribution of hydraulic head within the fault, using an analytic solution. We conclude that anisotropy ratios greater than 100 result in nearly hydrostatic conditions within the fault zone, despite the existence of significant vertical flow rates. Under these conditions, the Dupuit approximation is adequate for predicting the flow from one side of the fault to the other. We then present explicit analytical solutions to problems of steady groundwater flow in a multiaquifer system cut by a single vertical fault. The fault is linear and of negligible width, is infinite in length, and acts as a conduit for vertical fluid flow. The fault may act as a leaky barrier to horizontal flow normal to the fault, as a conduit to horizontal flow tangential to the fault, or a combination of both. Examples are presented that highlight the effects of enhanced vertical permeability of a fault on aquifer interaction in a multiaquifer system. Particle tracking is used to investigate the effects of the fault on pathlines.

The emerging role of the gut in chronic heart failure

Chronic heart failure is a multisystem disease with increased sympathetic tone, an anabolic/catabolic dysbalance, and chronic inflammation. Recent studies suggest an altered morphology, permeability, and absorption of the digestive tract in chronic heart failure. Due to nonocclusive mesenterial ischaemia and disturbed intestinal microcirculation, bacterial endotoxin is thought to enter the bloodstream through the hypoperfused, oedematous gut wall, thereby triggering an inflammatory response. Circulating cytokines act as cardiosuppressors. Their plasma levels predict increased mortality in chronic heart failure. The present article focuses on specific alterations of the gastric, small intestinal, and large intestinal region in chronic heart failure. It describes the leaky intestinal barrier with an augmented bacterial biofilm that may contribute to chronic inflammation and malnutrition. Furthermore, we review methods for bowel perfusion measurement and potential therapeutic approaches. It remains unclear whether increased adherent bacteria in patients with chronic heart failure are a primary or secondary event and whether they contribute to systemic inflammation. Both lack of mucosal integrity with consecutive local and systemic inflammation and dysfunction of transport proteins may worsen the clinical symptoms of chronic heart failure. Therefore, future studies need to address the pathophysiology of the intestinal barrier whose reactivity seems to be crucial for heart function.

Interferon-γ and tumor necrosis factor-α disrupt epithelial barrier function by altering lipid composition in membrane microdomains of tight junction

Tight junctions (TJs) are specialized membrane microdomains of plasma membrane and play an important role in barrier function. IFN-γ and TNF-α have been implicated in intestinal barrier dysfunction. In the present study, we analyzed the effect of IFN-γ and TNF-α on epithelial barrier function and determined the contribution of apoptosis to this process using T84 cells, a model intestinal epithelial cell line. We found that TNF-α and IFN-γ synergistically affected the epithelial barrier and disrupted the structure of TJs. We demonstrated for the first time that treatment with TNF-α and IFN-γ changed lipid composition and fatty acyl substitutions of phospholipids in membrane microdomains of TJs. Alterations of lipid environment affected TJs barrier function and partly removed flotillin-1 and displaced occludin from membrane microdomains of TJs to detergent-soluble fractions. The distribution of claudin isoforms was unaffected by TNF-α and IFN-γ treatment. These findings indicated the interaction between inflammatory cytokines and alterations of lipid composition in membrane microdomains of TJs in the inflammatory processes. The apoptosis inhibitor did not prevent cytokine-induced decrease in TER and increase in permeability to FITC-dextran. Our results suggest that the cytokines directly influence TJ function and modulate both the membrane microdomain localization of TJ proteins and lipid composition of TJs. The effects of proinflammatory cytokines on TJ structure and function provide a mechanism in the pathophysiology of Crohn’s disease (CD). Understanding the intracellular mechanisms involved could be important in devising future therapeutic strategies to induce retightening of the leaky TJ barrier.

Review article: duodenal bicarbonate – mucosal protection, luminal chemosensing and acid–base balance

The duodenum serves as a buffer zone between the stomach and the jejunum. Over a length of only 25 cm, large volumes of strong acid secreted by the stomach must be converted to the neutral-alkaline chyme of the hindgut lumen, generating large volumes of CO(2). The duodenal mucosa consists of epithelial cells connected by low-resistance tight junctions, forming a leaky epithelial barrier. Despite this permeability, the epithelial cells, under intense stress from luminal mineral acid and highly elevated Pco(2), maintain normal functioning. Bicarbonate ion uniquely protects the duodenal epithelial cells from acid-related injury. The specific protective mechanisms likely involve luminal bicarbonate secretion, intracellular pH buffering and interstitial buffering. Furthermore, the duodenum plays an active role in foregut acid-base homeostasis, absorbing large amounts of H(+) and CO(2). We have studied mucosal protection and acid-base balance using live-animal fluorescence ratio microimaging and by performing H(+) and CO(2) balance studies on duodenal perfusates. On the basis of these data, we have formulated novel hypotheses with regard to mucosal protection.

Structure of the Subtropical Front in the Tasman Sea

The structure of the Subtropical Front (STF) in the southwest Pacific is established via a practical separation observed between the thermohaline properties of the subtropical and subantarctic waters (SAWs) converging at the STF. Previous examinations have been unable to separate waters in and about the STF zone, and could not identify the actual frontal interface of the STF, leaving its structure and properties largely unresolved. Consequently, other fronts in the area have been confused with the STF or misidentified. Away from the land boundaries of Australia and New Zealand, the STF appears as a well-defined zonal trending band, in which subtropical and SAWs over-ride, intrude, and interleave. The zone is bounded by quasi-parallel fronts, each associated with near definitive surface salinity values. The shallower northern front marks the equatorwards limits of surface SAW in the water column, and the deeper southern front represents the polewards limits of subsurface subtropical water (Tasman Sea Central Water). Empirical prescriptions used to approximately locate the STF over the last 70 years are explained and reconciled on comparison with the double frontal structure. Antarctic Intermediate Water (AAIW) at the STF also exhibits a double frontal structure, inside of which is maintained a pool of AAIW with distinctive properties. The AAIW frontal system forms a leaky barrier to northward movement of Southern Ocean AAIW in the eastern Tasman Sea.

Inflammatory Bowel Disease and the Apical Junctional Complex

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the underlying interstitium. This intestinal barrier is primarily regulated by the apical junctional complex (AJC) consisting of tight junctions (TJs) and adherens junctions (AJs) and is compromised in a number of intestinal diseases, including inflammatory bowel disease (IBD). In vitro studies have demonstrated that proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), that are increased in the intestinal mucosa of patients with IBD can induce a leaky mucosal barrier. There is a growing evidence that the increased permeability and altered AJC structure observed in IBD are mediated by internalization of junctional proteins. This review summarizes barrier defects observed in IBD and addresses mechanisms by which proinflammatory cytokines, such as IFN-gamma and TNF-alpha, modulate AJC structure and epithelial barrier function.

Review article: duodenal bicarbonate – mucosal protection, luminal chemosensing and acid–base balance

SummaryThe duodenum serves as a buffer zone between the stomach and the jejunum. Over a length of only 25 cm, large volumes of strong acid secreted by the stomach must be converted to the neutral–alkaline chyme of the hindgut lumen, generating large volumes of CO2.The duodenal mucosa consists of epithelial cells connected by low‐resistance tight junctions, forming a leaky epithelial barrier. Despite this permeability, the epithelial cells, under intense stress from luminal mineral acid and highly elevated Pco2, maintain normal functioning.Bicarbonate ion uniquely protects the duodenal epithelial cells from acid‐related injury. The specific protective mechanisms likely involve luminal bicarbonate secretion, intracellular pH buffering and interstitial buffering. Furthermore, the duodenum plays an active role in foregut acid–base homeostasis, absorbing large amounts of H+ and CO2.We have studied mucosal protection and acid–base balance using live‐animal fluorescence ratio microimaging and by performing H+ and CO2 balance studies on duodenal perfusates. On the basis of these data, we have formulated novel hypotheses with regard to mucosal protection.

Regulation of the intestinal epithelial barrier by the apical junctional complex

Epithelial intercellular junctions are important components of the epithelial barrier and are compromised in disorders such as Crohn's disease. We will highlight recent progress in understanding the role of an intercellular junction referred to as the apical junctional complex in regulating small intestinal epithelial permeability in health and disease. Recent studies have implicated aberrant regulation of the AJC as an underlying factor contributing to a leaky epithelial barrier in Crohn's patients. Consequences of increased epithelial permeability include exposure of intestinal tissue to luminal antigens/pathogens which in turn influence disease activity. Furthermore, proinflammatory cytokines released into the milieu of the epithelium in patients with Crohn's disease influence apical junctional complex and epithelial barrier function. Such cytokines induce disassembly of the apical junctional complex by promoting differential endocytosis of component proteins. Additionally, apical junctional complex proteins are targeted by pathogens that use the epithelium as a portal of entry to establish disease in the host. The epithelial apical junctional complex is important in determining epithelial barrier properties. Recent studies have highlighted contribution of proinflammatory cytokines and endocytosis of apical junctional complex proteins to the epithelial barrier defect. Continued advances in understanding of this field will yield new therapeutic targets for intestinal disorders.

Duodenal Carbonic Anhydrase: Mucosal Protection, Luminal Chemosensing, and Gastric Acid Disposal

The duodenum serves as a buffer zone between the stomach and jejunum. Over a length of only 25 cm, large volumes of strong acid secreted by the stomach must be converted to the neutral-alkaline chyme of the hindgut lumen, generating large volumes of CO2, which the duodenum then absorbs. The duodenal mucosa consists of epithelial cells connected by low-resistance tight junctions, forming a leaky epithelial barrier. Despite this high permeability, the epithelial cells, under intense stress from luminal mineral acid and highly elevated P(CO2), maintain normal functioning. Furthermore, the duodenum plays an active role in foregut acid-base homeostasis, absorbing large amounts of H+ and CO2 that are recycled by the gastric parietal cells. Prompted by the high expression of cytosolic and membrane carbonic anhydrase (CAs) in duodenal epithelial cells, and the intriguing observation that CA activity appears to augment cellular acid stress, we formulated a novel hypothesis regarding the role of CA in duodenal acid absorption, epithelial protection, and chemosensing. In this review, we will describe how luminal CO2/H+ traverses the duodenal epithelial cell brush border membrane, acidifies the cytoplasm, and is sensed in the subepithelium.

Gastrointestinal: Mycobacterium avium paratuberculosis and Crohn's disease

Uncovering the cause of inflammatory bowel disease appears to have become the holy grail of gastroenterology. For Crohn’s disease, the most popular hypothesis is that the disease results from loss of immunological tolerance to bacteria or other microorganisms that are normally present in the bowel lumen. However, several other hypotheses exist including a role for infectious agents such as atypical mycobacteria, Chlamydia species, Listeria monocytogenes, cellwall deficient Pseudomonas species, Mycoplasma species and a number of viruses including measles virus. Interest in atypical mycobacteria was recently rekindled by observations reported by Dr S Naser and others in an article published in Lancet in 2004. Using a novel method, the group was able to culture Mycobacterium avium subspecies paratuberculosis (MAP) from the peripheral blood in 50% of patients with active Crohn’s disease. This has raised the possibility that MAP may be the cause of Crohn’s disease in humans in a similar way to Johne’s disease in livestock. Recently, our group has been able to replicate the above findings in a number of patients with active Crohn’s disease. We used Ziehl-Neelsen staining to show what we believe are spheroplastic phase forms of MAP (purple spots) within the cytoplasm of a macrophage (Fig. 1) and persisting in modified TB broth after 4 months of culture (Fig. 2). To confirm the identify of these acid-fast organisms, we performed a duplex polymerase chain reaction designed to detect the unique insertion (L1 and L9) sites of the MAP-specific insertion element IS900. The amplified L9 site was sequenced and then compared to a reference DNA of MAP. Alignment of our sequence against the reference sequence showed a perfect match, thereby confirming that the Mycobacterium found in our Crohn’s disease patients was indeed MAP. These observations support the association between MAP and Crohn’s disease. However, further research is required to determine whether MAP is involved in the pathogenesis of Crohn’s disease or whether the detection of MAP in peripheral blood simply reflects translocation of organisms through a leaky epithelial barrier.

Lysophosphatidylcholine: an enigmatic lysolipid

increased endothelial permeability of pulmonary microvessels is the major contributor of acute lung injury, sepsis, and acute respiratory distress syndrome ([2][1], [7][2]). Studies have established that change in cell shape resulting in concurrent formation of gaps between endothelial cells is a

Interferon‐γ induces internalization of epithelial tight junction proteins via a macropinocytosis‐like process

Increased epithelial permeability is observed in inflammatory states. However, the mechanism by which inflammatory mediators such as IFN-gamma increase epithelial permeability is unknown. We recently observed that IFN-gamma induces disassembly of tight junctions (TJ); in this study we asked whether such TJ disassembly is mediated by endocytosis of junctional proteins. The role of three major internalization pathways in disruption of TJ in IFN-gamma-treated intestinal epithelial cells was analyzed using selective inhibitors and markers of the pathways. No role for the clathrin- and caveolar-mediated endocytosis in the IFN-gamma-induced internalization of TJ proteins was observed. However, inhibitors of macropinocytosis blocked internalization of TJ proteins and junctional proteins colocalized with macropinocytosis markers, dextran and phosphatidylinositol-3,4,5-trisphosphate. Internalized TJ proteins were identified in early and recycling endosomes but not in late endosomes/lysosomes. These results for the first time suggest that IFN-gamma produces a leaky epithelial barrier by inducing macropinoytosis of TJ proteins.

Duodenal alkaline secretion: its mechanisms and role in mucosal protection against gastric acid

<h2>Abstract</h2> Duodenal mucosa, especially its proximal portion, is exposed to intermittent pulses of gastric acid (H⁺). This review summarises the mechanisms of duodenal bicarbonate (HCO₃⁻) secretion and their role in protecting duodenal epithelium against gastric H⁺. Duodenal epithelium is a leaky barrier against gastric H⁺, which diffuses into duodenocytes, but fails to damage them due to: (a) an enhanced expression of cyclooxygenase, producing protective prostaglandins and expression of nitric oxide synthase, releasing nitric oxide, both stimulating duodenal HCO₃⁻ secretion and (b) the release of several neurotransmitters also stimulating HCO₃⁻ secretion such as vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, acetylcholine and melatonin. At the apical duodenocyte membrane, several HCO₃⁻/Cl⁻ anion exchangers operate in response to luminal H⁺ to extrude HCO₃⁻ into duodenal lumen. In baso-lateral duodenocyte membrane, both non-electrogenic and electrogenic Na⁺–HCO₃⁻ cotransporters are activated after exposure of duodenum to gastric H⁺, causing inward movement of HCO₃⁻ from extracellular fluid to duodenocytes. There are also at least three Na⁺/H⁺ exchangers, eliminating H⁺ which diffused into these cells. The <i>Helicobacter pylori</i> infection with gastric metaplasia in the duodenum and <i>bacterium</i> inoculation results in the inhibition of HCO₃⁻ secretion by its endogenous inhibitor dimethyl arginine, resulting in ulcerogenesis.

Quantitative Assessment of the Cell Penetrating Properties of RI-Tat-9: Evidence for a Cell Type-Specific Barrier at the Plasma Membrane of Epithelial Cells

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.

Investigation of the electrical properties and reliability of amorphous SiCN

Amorphous SiCN (a-SiCN) is a candidate for barrier dielectric and has lower dielectric constant ( k∼5) relative to the commonly used barrier SiN ( k∼7). In this study, we investigate the leaky behavior and barrier characteristics of a-SiCN ( k: 4∼5) doped with different nitrogen concentration. The leaky behavior of a-SiCN is Poole–Frenkel at high electric field. This is similar to SiN. Thermal stability of a-SiCN films is good enough to meet the prescription in back-end-of-line fabrication process. The bias-temperature stress (BTS) test has been conducted on a-SiCN to investigate the barrier ability against copper penetration. We find that a-SiCN could sustain the stress of electric field up to 4 mV/cm at 150 °C. In addition, a-SiCN films with higher nitrogen concentration exhibit better barrier property against copper penetration. The SIMS spectra also have been used to monitor the distributions of copper after different BTS conditions, confirming our inference on leakage mechanism.

The influence of high-temperature annealing on SiC schottky diode characteristics

The influence of high temperature (up to 800C) annealing on the current-voltage characteristics of n-type 6H-SiC Schottky diodes is presented. Our experimental results indicate that high-temperature annealing can result in the improvement of the forw ard and reverse electrical characteristics of SiC Schottky diodes by repairing any leaky low barrier secondary diode parallel to the primary diode that may be present due to the barrier inhomogeneities at the Schottky contact interface.

Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation.

Cell-cell interactions of the mucosal epithelia are important for the maintenance and establishment of epithelial barrier function. During events of inflammation, such cell-cell interactions are often disrupted, resulting in a leaky epithelial barrier, which in turn can lead to various inflammatory and infective dysfunctions. Human junctional adhesion molecule (huJAM), found on the mucosal epithelia and vascular endothelia of many major organ systems, is a membrane glycoprotein which resolves to a doublet band of approximately 40 and approximately 37 kDa under SDS-PAGE analysis, representing differentially glycosylated forms of the same protein. huJAM was localized to the lateral membrane of Caco-2 cells (a human colonic epithelial cell line) monolayers, in an area basolateral of the epithelial tight junctions (TJ). Through functional and biochemical assays, we show huJAM to be able to homotypically associate and to participate in TJ restitution after trypsin-EDTA disruption. Furthermore, we also observed a migration of huJAM expression toward areas of cell-cell contacts during events of cell adhesion and monolayer formation. These qualities makes huJAM a likely player in the regulation of cell-cell contacts and the subsequent formation of TJs.

Glycerol disrupts tight junction-associated actin microfilaments, occludin, and microtubules in Sertoli cells.

Intratesticular injections of glycerol have been shown to result in a marked and prolonged reduction of spermatogenesis, accompanied by increased permeability of the blood-testis barrier. Because the permeability of the blood-testis barrier is regulated by Sertoli cell tight junctions, and tight junction organization is regulated by the cytoskeleton, we undertook to examine the effects of glycerol treatment on cytoskeletal actin microfilaments and microtubules, and on the tight junction protein, occludin, in Sertoli cells. Adult rats received a single intratesticular injection of either saline (controls) or a 10% glycerol solution. At 24 hours and 7, 15, and 21 days after injection, testes were collected and prepared for routine histology, cryosectioning, or whole seminiferous tubule immunohistochemical staining; and the preparations were viewed by light and confocal microscopy. In saline-injected testes, Sertoli cells had a cytoskeletal and junctional organization that resembled that of normal testes. F-actin microfilaments, located in the basal region, were arranged in regular bundles or chords that circumscribed the perimeter of each Sertoli cell at the level of the tight junction. Occludin colocalized with tight junction-associated actin filament distribution and microtubules formed a geometric array associated with spermatogenic cells. In contrast, in glycerol-treated Sertoli cells, microfilament and microtubule organization and occludin distribution were partially or completely disrupted. From these results we conclude that glycerol treatment either directly or indirectly disrupts tight junction-associated F-actin and occludin and tubulin organization in rat Sertoli cells. Perturbation of the tight junction-associated proteins could explain the increase in permeability of the blood-testis barrier observed after glycerol treatment. Impaired spermatogenesis following glycerol treatment is likely a consequence of a leaky blood testis barrier and disrupted Sertoli cell cytoskeleton. Glycerol injections may serve as a useful tool in studying the relationship between cytoskeletal organization and the stabilization of Sertoli-Sertoli cell junctions.

Cell death in the choroid plexus following transient forebrain global ischemia in the rat.

Following a complete disruption of blood flow to the brain, cerebral ischemia, a specific neuronal population, namely the CA1 pyramidal neurons in the hippocampus, will die a delayed type of cell death. This is often referred to as "delayed neuronal death" (DND). It is not known why it takes around 48 hours for these cells to die. It is very often speculated that events, intrinsic to the CA1 neurons, regulate their demise, whereas it is less often considered that extrinsic mechanisms also could play an important role for the development of DND. We discovered that in addition to the CA1 pyramidal neurons, cells in the choroid plexus were TUNEL (terminaldeoxynucleotidyl-mediated biotin-dUTP nick-end labeling)-positive following transient forebrain global ischemia. The time course and the number of TUNEL-positive cells were determined. A dramatic increase in the number of TUNEL-positive cells in the choroid plexus was seen at 18, 24, and at 36 hours of recovery, but not at 48 hours of recovery following 15 minutes of transient forebrain global ischemia. No TUNEL-positive cells were seen at 24 hours of recovery in the CA1 region. The cell death in the choroid plexus thus preceded the occurrence of cell death in the CA1 region. Massive cell death in the choroid plexus will inevitably lead to a leaky blood-CSF barrier, which in turn will allow substances to enter the ventricular system and from there reach the brain parenchyma. We, therefore, conclude that choroid plexus cell death may adversely affect the outcome of CA1 pyramidal neurons following transient forebrain global ischemia, through, e.g., a disruption of the blood-cerebro spinal fluid barrier. Alternatively, the choroid plexus may produce factors, which can affect the outcome of neurons.