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Chemical liberated (LPS) by leaky gut may allow HIV to infect the brain, Einstein scientists find
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http://www.eurekalert.org
"These results demonstrate very dramatically that HIV infection of cells associated with the BBB, in conjunction with LPS exposure, contributes to BBB breakdown," says Dr. Goldstein. (See slides below). "So when HIV infection occurs, we seem to have a 1-2-3 combination of punches working in concert to facilitate entry of HIV-infected monocytes into the BBB-protected brain: HIV infection of monocytes increases their capacity to cross even an intact BBB; HIV infection in the gut releases LPS into the bloodstream allowing it to erode the BBB; and HIV infection of the cells of the BBB makes them more sensitive to the deleterious effects of LPS.".....These findings could lead to preventive or therapeutic strategies. To help maintain the integrity of the BBB in HIV-infected people, says Dr. Goldstein, one approach might be to monitor the LPS level in their bloodstream and then reduce elevated levels. "We may be able to use antibiotics that kill intestinal bacteria that make LPS, and drugs are already available that can bind to LPS and clear it from the bloodstream," says Dr. Goldstein. "Ideally, we would promptly start newly diagnosed HIV-infected patients on a treatment to reinforce their BBB's so that HIV can't penetrate it-and perhaps we could even strengthen the BBB's of people who've been infected for quite a while. But before we can prevent the tragedy of HIV-associated dementia, we need to better understand the mechanism by which these molecular and cellular 'punches' interact to undermine the BBB."
In up to 20 percent of people infected with HIV, the virus manages to escape from the bloodstream and cross into the brain, resulting in HIV-associated dementia and other cognitive disorders. Now, scientists at the Albert Einstein College of Medicine of Yeshiva University have found strong evidence that a component of the cell walls of intestinal bacteria-a chemical present in high levels in the blood of HIV-infected people-helps HIV to penetrate the usually-impregnable blood brain barrier (BBB). The findings, published in the August issue of the Journal of Virology, could lead to strategies for preventing HIV from entering the brain and causing serious complications.
"Previous research has suggested that it's not individual HIV viruses that get into the brain but rather HIV-infected immune cells known as monocytes," says Dr. Harris Goldstein, director of the Einstein-Montefiore Medical Center for AIDS Research and senior author of the study. "Using an animal model, we wanted to find out first of all whether being infected with HIV enables monocytes to do what they don't usually do-escape from blood vessels and enter brain tissue."
Overcoming HIV's inability to infect mice, Dr. Goldstein and his colleagues had previously created a transgenic mouse line, HIV-TG mice, equipped with all the genes needed to make HIV-and that produces HIV in those cells, including monocytes and T cells, in which the virus multiplies in people. The HIV-TG mice were then bred with another transgenic mouse line, GFP-TG mice, containing the gene that codes for green fluorescent protein (GFP). The result: a double transgenic mouse line, HIV/GFP-TG mice, whose HIV-infected monocytes carried the GFP gene. This meant that the monocytes could be detected-either by looking for glowing green cells under the microscope or by using polymerase chain reaction, a sensitive genetic assay capable of detecting the DNA of the GFP gene.
Next, the researchers isolated millions of monocytes-HIV/GFP-producing monocytes from the HIV/GFP-TG mice, and monocytes from the GFP-TG mice producing GFP alone-and injected each type of monocyte into control mice.
Four days later, the researchers examined the brains of the injected mice to see whether monocytes from the bloodstream had crossed their BBB's. While there was no sign of monocytes in the brains of any of the mice injected with uninfected GFP monocytes, ultrasensitive DNA analysis showed that HIV/GFP monocytes were present at very low levels in the brains of nearly one third of the mice injected with the HIV-producing monocytes. "These results demonstrated very clearly that being infected with HIV somehow gives monocytes the capacity to cross an intact BBB," says Dr. Goldstein. "But we also suspected that something else was making it easier for HIV-infected monocytes to breach the defenses protecting the brain from infection."
In 2006, scientists at the National Institutes of Health had reported that HIV infection breaks down barriers in the intestine that normally prevent intestinal bacteria from entering the bloodstream. The blood of HIV-infected people was found to contain markedly elevated levels of lipopolysaccharide (LPS), a component of certain bacteria that are normally confined to the intestine but leak out due to HIV infection. In addition, previous animal studies had shown that exposure to elevated LPS levels compromised the integrity of the BBB. "So we hypothesized that the combination of HIV-infected monocytes and elevated LPS levels would amplify the ability of HIV to cross the BBB and get into the brain," says Dr. Goldstein.
To test this hypothesis, his team injected control mice with very low doses of LPS that were comparable to the levels in the bloodstream of HIV-infected individuals and would only minimally weaken their BBB's. Three hours later, half the mice were intravenously injected with HIV-and-GFP-producing monocytes, while the remaining mice were intravenously injected with GFP-producing monocytes that were otherwise normal.
Four days later, monocytes could not be detected in the brains of any of the 15 mice that were pre-treated with LPS and then injected with normal monocytes producing GFP alone. By contrast, monocytes were readily detected in the brains of about 25% of mice pre-treated with LPS and then injected with HIV-and-GFP-producing monocytes.
"Clearly, HIV-infected monocytes uniquely benefit from the LPS that is present in high amounts in the blood of HIV-infected people," says Dr. Goldstein. "So when HIV-infected monocytes are 'knocking on the door' of the BBB and starting to crack it open, the LPS facilitates their entry by making the BBB more permeable, apparently by weakening blood vessel structure."
If HIV-infected monocytes and LPS in the bloodstream can be considered a one-two punch for entry into the brain, a third punch-simply having a systemic HIV infection-also seems to help soften up the BBB. In making this discovery, Dr. Goldstein used his HIV-TG mouse strain, in which HIV is known to replicate inside brain cells associated with the BBB. These HIV-TG mice, along with control mice, were injected with LPS and, three hours later, intravenously injected with HIV-and-GFP-producing monocytes from the HIV/GFP-TG mouse strain.
Four days later, HIV-producing monocytes could be detected in the brains of about 25 percent of the control mice, as in the preceding experiment. By contrast, more than twice as many (70 percent) of the brains of HIV-TG mice that support systemic HIV infection contained HIV-producing monocytes. Even more impressive: When present, HIV-producing monocytes were three times more numerous in the brains of HIV-TG mice than in the brains of control mice.
"These results demonstrate very dramatically that HIV infection of cells associated with the BBB, in conjunction with LPS exposure, contributes to BBB breakdown," says Dr. Goldstein. (See slides below). "So when HIV infection occurs, we seem to have a 1-2-3 combination of punches working in concert to facilitate entry of HIV-infected monocytes into the BBB-protected brain: HIV infection of monocytes increases their capacity to cross even an intact BBB; HIV infection in the gut releases LPS into the bloodstream allowing it to erode the BBB; and HIV infection of the cells of the BBB makes them more sensitive to the deleterious effects of LPS."
These findings could lead to preventive or therapeutic strategies. To help maintain the integrity of the BBB in HIV-infected people, says Dr. Goldstein, one approach might be to monitor the LPS level in their bloodstream and then reduce elevated levels. "We may be able to use antibiotics that kill intestinal bacteria that make LPS, and drugs are already available that can bind to LPS and clear it from the bloodstream," says Dr. Goldstein. "Ideally, we would promptly start newly diagnosed HIV-infected patients on a treatment to reinforce their BBB's so that HIV can't penetrate it-and perhaps we could even strengthen the BBB's of people who've been infected for quite a while. But before we can prevent the tragedy of HIV-associated dementia, we need to better understand the mechanism by which these molecular and cellular 'punches' interact to undermine the BBB."
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Other Einstein researchers involved in the study were Drs. Hongwei Wang and Jinglin Sun.
Human Immunodeficiency Virus Type 1 Infection Increases the In Vivo Capacity of Peripheral Monocytes To Cross the Blood-Brain Barrier into the Brain and the In Vivo Sensitivity of the Blood-Brain Barrier to Disruption by Lipopolysaccharide
Journal of Virology, August 2008, p. 7591-7600, Vol. 82, No. 15
Hongwei Wang,1 Jinglin Sun,1 and Harris Goldstein1,2*
Departments of Microbiology & Immunology,1 Pediatrics, Albert Einstein College of Medicine, Bronx, New York 104612
"investigation of the mechanism by which HIV-1-infected monocytes migrate across the BBB from the peripheral circulation into the brain has been limited by the absence of an in vivo model of HIV-1 infection......Although there are differences between the biological behaviors of murine and human mononuclear phagocytes such as microglia and macrophages, mouse models provide informative in vivo systems for studying BBB function and the deleterious effect of HIV-1 on the CNS. Knockout mouse models.....In the current study, we demonstrated that monocytes from JR-CSF mice displayed an increased capacity to cross both the intact and compromised BBB (blood-brain-barrier) into the brain parenchyma and that the BBB of JR-CSF mice is more susceptible to disruption by inflammatory signals, such as LPS, than that of control wild-type (WT) mice......The capacity of HIV-1-infected monocytes to enter the brain was increased when the integrity of the BBB was partially compromised by the injection of LPS, particularly in the brains of JR-CSF mice, where the BBB is composed of cells carrying the HIV-1 provirus. The BBB in the JR-CSF mice was more sensitive to disruption by LPS than the BBB of control mice, indicating that HIV-1 infection may increase the sensitivity of the components that form the BBB to becoming compromised by systemic LPS. It is possible that increased susceptibility of the BBB in the JR-CSF mice that support HIV-1 replication to LPS treatment may be related to the capacity of HIV-1-infected monocytes to alter the brain microvascular proteome....The results of this study demonstrate that carriage of the HIV-1 provirus by monocytes increased their ability to enter the brain, particularly after partial compromise of the BBB in response to LPS treatment, and indicate the applicability of these mice as an in vivo system to study the mechanism by which HIV-1-infected monocytes migrate into the brain and how these migrated cells subsequently cause CNS damage....This in vivo mouse model would also permit examination of the capacities of various therapies to protect the BBB from compromise by determining the effects of candidate treatments on preventing or decreasing the migration of HIV-1-infected monocytes across the BBB. These future studies should contribute to our understanding of the role of the immune system and other in vivo factors in controlling traffic of HIV-1-infected monocytes across the BBB and their role in compromising subsequent BBB function.
ABSTRACT
Human immunodeficiency virus type 1 (HIV-1), introduced into the brain by HIV-1-infected monocytes which migrate across the blood-brain barrier (BBB), infects resident macrophages and microglia and initiates a process that causes HIV-1-associated neurocognitive disorders. The mechanism by which HIV-1 infection circumvents the BBB-restricted passage of systemic leukocytes into the brain and disrupts the integrity of the BBB is not known. Circulating lipopolysaccharide (LPS), which can compromise the integrity of the BBB, is significantly increased in HIV-1-infected individuals. We hypothesized that HIV-1 infection increases monocyte capacity to migrate across the BBB, which is further facilitated by a compromise of BBB integrity mediated by the increased systemic LPS levels present in HIV-1-infected individuals. To investigate this possibility, we examined the in vivo BBB migration of monocytes derived from our novel mouse model, JR-CSF/EYFP mice, which are transgenic for both a long terminal repeat-regulated full-length infectious HIV-1 provirus and ROSA-26-regulated enhanced yellow fluorescent protein. We demonstrated that JR-CSF/EYFP mouse monocytes displayed an increased capacity to enter the brain by crossing either an intact BBB or a BBB whose integrity was partially compromised by systemic LPS. We also demonstrated that the JR-CSF mouse BBB was more susceptible to disruption by systemic LPS than the control wild-type mouse BBB. These results demonstrated that HIV-1 infection increased the ability of monocytes to enter the brain and increased the sensitivity of the BBB to disruption by systemic LPS, which is elevated in HIV-1-infected individuals. These mice represent a new in vivo system for studying the mechanism by which HIV-1-infected monocytes migrate into the brain.
INTRODUCTION
Human immunodeficiency virus type 1 (HIV-1) enters the brain during the establishment and systemic dissemination of HIV-1 infection and either directly or indirectly causes a broad range of HIV-1-associated neurocognitive disorders, including asymptomatic neurocognitive impairment, HIV-1-associated mild neurocognitive disorder, and HIV-1-associated dementia (HAD) (6). HAD, a devastating neurological disease that is a frequent consequence of HIV-1 infection (42, 43), is associated with the pathological findings of HIV-1 encephalitis, characterized by multinucleated giant-cell formation, microglial nodules, astrogliosis, myelin pallor, and neocortical atrophy (48). The major route by which HIV-1 enters the brain is transmigration of HIV-1-infected monocytes across the blood-brain barrier (BBB) early in the course of infection (23, 27, 36, 44). After entry into the brain, these peripheral HIV-1-infected monocytes produce HIV-1 that subsequently infects resident brain microglia and macrophages (3, 7, 37, 38, 66). HAD is not caused by direct HIV-1 infection of neurons but rather is a consequence of the cumulative neurotoxic effects of multiple factors produced by HIV-1-infected or HIV-1-exposed cells in the brain, such as microglia and macrophages (33, 48, 67). Circulating monocytes are normally precluded from migrating into the brain by the BBB, an anatomical barrier that restricts the exchange of cells and soluble factors from the blood into the brain parenchyma (47, 57). The BBB is composed of several components that include specialized endothelial cells which form tight junctions at cellular contact points, the end feet of astrocytes that surround the blood vessels, the capillary basement membrane, and pericytes which are embedded in the basement membrane (31). The BBB prevents migration of inflammatory cells from the periphery into the brain, with the exception of a small number of leukocytes engaged in immune surveillance that can pass across the BBB without altering BBB integrity. The restricted passage of leukocytes from the systemic circulation into the brain across the BBB may be disrupted during the course of HIV-1 infection (53, 54). Compromise of the integrity of the BBB allows circulating HIV-1-infected monocytes to enter the brain, which further disrupts BBB integrity and permits the influx into the brain of more HIV-1-infected monocytes; this further disrupts BBB integrity and allows the entry of increased numbers of HIV-1-infected monocytes, which subsequently infect resident brain monocytes and microglia with HIV-1 (23, 25). The precise mechanism by which HIV-1-infected monocytes cross the intact BBB and subsequently disrupt the integrity of the BBB is not known.
The plasma of HIV-1-infected individuals contains markedly elevated levels of lipopolysaccharide (LPS) due to the increased microbial translocation across a gut mucosa barrier which is compromised by HIV-1-mediated depletion of mucosal CD4 T lymphocytes (11, 22). The mean LPS plasma level (75 pg/ml) in HIV-1 progressors is markedly higher than the plasma level of LPS (14 pg/ml) shown to activate the systemic immune system and induce inflammatory cytokine production in LPS-injected HIV-1-naive human volunteers (22) and was associated with in vivo activation of the innate and adaptive immune systems of HIV-1 progressors (11, 22). The in vivo integrity of the BBB can be compromised by exposure to elevated levels of LPS in the circulation (70). We hypothesized that the increased levels of systemic LPS in HIV-1-infected individuals disrupt BBB integrity and permit the influx of increasing numbers of HIV-1-infected monocytes, which further compromise BBB integrity, and permit the entry of increasing numbers of HIV-1-infected monocytes, which ultimately initiates the development of HAD. We also postulated that HIV-1 infection of monocytes may increase their capacity to migrate across the intact and partially compromised BBB. An in vitro model of the BBB that consists of brain microvascular endothelial cells and astrocytes cultured on opposite sides of a semipermeable coated membrane has permitted investigation of the mechanism by which HIV-1-infected monocytes cross and damage the BBB (52, 68). Although this model has been very useful for studying some aspects of BBB function, it does not fully recapitulate the in vivo function of the BBB. As an in vivo model to investigate whether HIV-1-infection alters the capacity of monocytes to cross the intact BBB and whether HIV-1 infection increases the sensitivity of the BBB to disruption by systemic LPS, we used our well-characterized system consisting of JR-CSF mice, which are transgenic for an infectious HIV-1 provirus (13, 46, 64).
Our JR-CSF transgenic mouse line circumvents the block of HIV-1 entry into mouse cells by carrying as a transgene a full-length infectious HIV-1 provirus derived from the primary R5-tropic clinical isolate HIV-1JR-CSF, and these mice display plasma viremia at levels comparable to that observed in HIV-1-infected patients (46, 64). Furthermore, infectious HIV-1 is produced by monocytes and microglia from the JR-CSF mice, and LPS-stimulated JR-CSF mouse monocytes and microglia produce higher levels of MCP-1 than monocytes and microglia from LPS-stimulated control mice (65). Because JR-CSF mouse monocytes and microglia carry an HIV-1 provirus regulated by the HIV-1 long terminal repeat, we used this model to investigate whether HIV-1 infection of monocytes increases their in vivo capacity to cross the intact and LPS-compromised BBB and migrate into the brain. In the current study, we demonstrated that monocytes from JR-CSF mice displayed an increased capacity to cross both the intact and compromised BBB into the brain parenchyma and that the BBB of JR-CSF mice is more susceptible to disruption by inflammatory signals, such as LPS, than that of control wild-type (WT) mice.
RESULTS
JR-CSF/EYFP mouse monocytes but not control mouse monocytes cross the intact BBB into the brain. We previously demonstrated that bone marrow-derived monocytes from JR-CSF mice support HIV-1 replication, produce HIV-1 that infects activated human peripheral blood mononuclear cells in vitro (13), and introduce disseminated HIV-1 infection in vivo into SCID mice implanted with pieces of human fetal thymus and liver (thy/liv-SCID-hu mice) (64). We postulated that JR-CSF mouse monocytes that carry an integrated long terminal repeat-regulated HIV-1 provirus could be used to recapitulate the behavior of HIV-1-infected monocytes and enable us to study the process of in vivo migration of HIV-1-infected monocytes across the BBB into the brain. To establish a system where these migrant cells could be detected in the brains of recipient mice by PCR and fluorescence microscopy, we crossed the JR-CSF mice with R26-EYFP mice, which carry an EYFP gene under the control of the ubiquitously expressed ROSA26 promoter, to generate JR-CSF/EYFP mice which express both transgenes. To determine whether monocytes expressing HIV-1 proteins displayed an increased capacity to cross the intact BBB, JR-CSF/EYFP or R26-EYFP mouse monocytes (5 x 106 cells) were intravenously injected into BALB/c SCID mice. Four days after injection of the monocytes, circulating leukocytes were flushed from the blood vessels of the mice by intracardiac injection of PBS, their brains were harvested, the meninges were removed, and their brain DNA was extracted. Carriage of the EYFP transgene permitted us to detect the presence of transferred JR-CSF/EYFP and R26-EYFP mouse monocytes in the brains of the recipient mice by PCR amplification with EYFP-specific primers of DNA extracted from the mouse brains. No EYFP DNA was detected in the brains of any of the injected mice after a single round of PCR. After a second round of PCR amplification, while no EYFP DNA was detected by PCR in any of the 10 BALB/c SCID mice injected with R26-EYFP mouse monocytes, EYFP DNA was detected by PCR in 3 of 10 BALB/c SCID mice injected with JR-CSF/EYFP mouse monocytes (Fig. 1). Analysis of the brain DNA with PCR amplification using exonuclease-specific primers demonstrated that comparable levels of DNA were present in the brain samples analyzed. Variability in detection of EYFP DNA in the peripheral blood likely reflects the differential rate of in vivo clearance of the injected cells. These results indicated that HIV-1-infected monocytes display an increased capacity to cross an intact BBB and enter the brain.
JR-CSF/EYFP mouse monocytes and not control R26-EYFP mouse monocytes cross the BBB of LPS-treated BALB/c mice. Treatment of mice with a low dose of LPS (3 mg/kg) partially compromises the integrity of the BBB without disrupting the capillary structure (9, 69), and this approach has been used to investigate the in vivo capacity of factors to modulate BBB permeability (1, 63). We postulated that LPS-mediated compromise of the BBB is relevant to HIV-1 infection because the BBB in HIV-1-infected individuals may be partially compromised in a similar manner by their increased levels of LPS in plasma (11). Therefore, we examined whether the JR-CSF/EYFP mouse monocytes that support HIV-1 replication displayed an increased capacity to migrate across a BBB whose integrity was partially compromised by LPS and enter the brain. Three hours after BALB/c mice were intraperitoneally injected with LPS (3 mg/kg), the mice were intravenously injected with either R26-EYFP or JR-CSF/EYFP mouse monocytes (5 x 106 cells). Four days later, the mouse brains were harvested and the DNA was extracted and analyzed with a single round of PCR with EYFP-specific primers (Fig. 2). We did not detect passage of R26-EYFP mouse monocytes into the brains of any of the 15 BALB/c mice injected with R26-EYFP mouse monocytes. In contrast, we detected migration of JR-CSF/EYFP mouse monocytes into the brains of 4 of 15 BALB/c mice injected with JR-CSF/EYFP mouse monocytes. These results indicated that monocytes carrying the JR-CSF provirus displayed an increased capacity to cross a BBB whose integrity was partially compromised by LPS treatment.
The BBB in JR-CSF mice is more sensitive to disruption by systemic LPS. We previously demonstrated that HIV-1 replication occurs in the brains of JR-CSF mice (65). We used these mice to examine whether systemic viremia and local production of HIV-1 proteins was associated with increased sensitivity of the BBB to disruption by LPS treatment, as indicated by increased migration of monocytes from the peripheral circulation into the brain. JR-CSF mice or BALB/c mice were intraperitoneally injected with LPS (3 mg/kg), and 3 h later the mice were injected with monocytes (5 x 106 cells) isolated from JR-CSF/EYFP mice. After 4 days, the injected mice were sacrificed and their peripheral blood, spleens, and brains were analyzed for the presence of transferred monocytes by EYFP-specific PCR. PCR analysis of the brains of the LPS-treated BALB/c mice injected with JR-CSF/EYFP mouse monocytes demonstrated passage of JR-CSF/EYFP mouse monocytes into the brains of 3 out of 10 injected BALB/c mice (Fig. 3A). In contrast, we detected passage of JR-CSF/EYFP mouse monocytes into the brains of 7 out of 10 injected JR-CSF mice (Fig. 3B). The relative number of JR-CSF/EYFP mouse monocytes in the brains of the injected BALB/c or JR-CSF mice was determined by comparing the intensity of the EYFP-PCR-amplified product detected in mouse brains normalized to the intensity of the exonuclease-specific PCR products of the brain DNA by densitometric analysis. Densitometric analysis of the PCR products detected in the mouse brains where JR-CSF mouse monocytes entered indicated that threefold-higher numbers of JR-CSF/EYFP mouse monocytes penetrated the BBB of JR-CSF mice (mean = 1,178) than the BBB of BALB/c mice (mean = 373).
Migration of the injected JR-CSF/EYFP monocytes across the BBB into the brain parenchyma was visualized by examining 20 coronal slices from the brains of control uninjected JR-CSF mice and the brains of LPS-treated JR-CSF mice 4 days after intravenous injection with JR-CSF/EYFP monocytes. The expression of EYFP permitted highly sensitive and specific visualization and localization of the fluorescent JR-CSF/EYFP mouse monocytes in the parenchyma of the brains. Fluorescent cells were not detected in the coronal sections from the brains of uninjected mice, ruling out the presence of cells displaying nonspecific fluorescence. In contrast, several EYFP-expressing cells were observed in the parenchyma of JR-CSF mice injected with JR-CSF/EYFP monocytes. Representative photomicrographs of sections from three JR-CSF mice evaluated by fluorescence microscopy are shown in Fig. 3C. Taken together, these results indicate that HIV-1-infected monocytes migrated across the BBB into the brain parenchyma, where they could potentially introduce HIV-1 infection and initiate neurotoxic processes.
Cerebral blood vessels in LPS-treated JR-CSF mice display increased disruption of BBB-specific cerebral vascular tight junctions. We investigated whether the functional evidence for increased compromise of BBB integrity indicated by the passage of monocytes into the brains of JR-CSF mice correlated with anatomical evidence of increased disruption of the BBB. The BBB is composed of tight junctions between epithelial cells formed by the interaction of continuous intramembranous strands of several proteins, including ZO-1 and occludin (20). Anatomical compromise of the BBB due to HIV-1 infection could be detected by visualizing breaks in the intramembranous strands by immunohistological analysis of ZO-1 and occludin in cerebral blood vessels (20). All of the cerebral blood vessels in untreated WT and JR-CSF mice displayed strong and continuous interendothelial patterns of equivalent intensity of occludin and ZO-1, indicating the presence of an intact BBB (Fig. 4). To determine if systemic treatment of mice with LPS more severely compromised the functional activity of the BBB in JR-CSF mice, we compared the effect of peripheral LPS injection on the integrity of ZO-1 and occludins in the cerebral vessels of JR-CSF mice to that for control BALB/c mice. In the brains of LPS-treated BALB/c mice, some cerebral vessels displayed intermittent breaks in occludin and ZO-1 continuity. In contrast, a markedly greater disruption of the anatomical BBB of the cerebral blood vessels in the LPS-treated JR-CSF mice was observed, as indicated by the weak and fragmented expression of occludin and ZO-1 by the majority of cortical blood vessels (Fig. 4A). Semiquantitative measurement of the extent of BBB integrity compromise in the brains of LPS-treated mice was performed as described previously (20) and demonstrated that significantly more vessels (P < 0.02) displayed BBB disruption in JR-CSF mouse brains than in BALB/c mouse brains (Fig. 4B). Taken together, these results indicated that the BBB of JR-CSF mice was more susceptible to disruption by LPS than the BBB of WT mice.
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