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The Effects of Lipopolysaccharide on the Disrupted Blood-Brain Barrier in a Rat Model of Preeclampsia
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The Effects of Lipopolysaccharide on the Disrupted Blood-Brain Barrier in a Rat Model of Preeclampsia 1XD XMutlu Kucuk, PhD, D2X X * D3X XCanan Ugur Yilmaz, PhD, D4X X *,† D5X XNurcan Orhan, PhD, D6X X ‡ D7X XBulent Ahishali, MD, PhD,D8X X§ D9X XNadir Arican, MD, D10X X PhD,|| D1X XImdat Elmas, D12XMD, X PhD,|| € rses, MD, PhD,D14X X{ and D15X XMehmet Kaya, D16XPhD D13X XCandan Gu X # Background: Preeclampsia is a disorder characterized by high blood pressure and often proteinuria during pregnancy. It is known that a subseptic dose of bacterial lipopolysaccharide (LPS) induces production of proinflammatory cytokines, and possibly incDreasing 17X X the risk for developing preeclampsia. We investigated the effects of LPS on the blood-brain barrier (BBB) integrity in pregnant rats with N (omega)-nitro-L-arginine methyl ester (L-NAME) induced preeclampsia. Methods: Starting from the 10th day of gestation, pregnant rats were given L-NAME for 10 days to produce hypertension and proteinuria. Animals were then treated with a single injection of LPS on the 19th day of pregnancy. Arterial blood pressure and proteinuria were measured on the day of the experiment, which was 24 hours after the LPS injection. The BBB integrity was assessed by using Evans blue (EB) and horseradish peroxidase (HRP) tracers. Results: Proteinuria was observed in varying degrees, and the arterial blood pressure increased in L-NAME-treated pregnant rats (P < .01). The overall brain EB content did not increase in these preeclamptic rats when compared to pregnant animals, and LPS treatment also did not change EB content. Ultrastructurally, frequent vesicles containing HRP reaction products were observed in the capillary endothelial cells in the cerebral cortex and hippocampus of pregnant rats treated with L-NAME (P < .01). However, LPS did not change the amounts of HRP that mainly accumulated in brain capillary endothelial cells of these animals. Conclusion: Our results suggest that, in this experimental setting, LPS does not change the severity of BBB disruption observed in preeclamptic animals. Key Words: Blood-brain barrier—preeclampsia—lipopolysaccharide—horseradish peroxidase—electron microscopy © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.
From the *Department of Laboratory Animals Science, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; †Department of Pharmaceutical Bioscience, Biomedical Centrum, Uppsala University, Uppsala, Sweden; ‡Department of Neuroscience, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; §Department of Histology and Embryology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; ||Department of Forensic Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; {Department of Neurology, Ko¸c University School of Medicine, Istanbul, Turkey; and #Department of Physiology, Ko¸c University School of Medicine, Istanbul, Turkey. Received May 7, 2018; revision received July 23, 2018; accepted August 1, 2018. Conflicts of interest: The authors declare that they have no conflicts of interest. The authors have read the Journal's position on issues involved in ethical publications and affirm that this study is consistent with those guidelines. The results presented in this manuscript have not been published previously in whole or part, except in abstract format. Financial Disclosure: This study was supported by the Research Fund of Istanbul University (24210/2009). Address correspondence to Canan Ugur Yilmaz, PhD, Department of Pharmaceutical Bioscience, Biomedical Centrum, Uppsala University, Husargatan 3, Uppsala Box 591, Uppsala 75429, Sweden. E-mailes: [email protected], [email protected] 1052-3057/$ - see front matter © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2018.08.003
Journal of Stroke and Cerebrovascular Diseases, Vol. &&, No. && (&&), 2018: pp 18
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Introduction Preeclampsia is a disorder characterized by hypertension and often proteinuria in varied degrees in pregnancy. Approximately, 5%-7% of all pregnancies are complicated by preeclampsia with profound implications for both mother and fetus.1,2 Also, it is believed that the placenta plays a central role in the pathophysiology of preeclampsia. Most of the circulatory factors, mainly cytokines, and oxidative products are associated with impaired endothelial cell function in the onset of preeclampsia.3,4 In this context, generalized maternal endothelial dysfunction caused mainly by circulatory factors contributes significantly to the pathogenesis of preeclampsia.4,5 The inflammatory agents induced by an endotoxin such as lipopolysaccharide (LPS) play a role in the conversion of preeclampsia into eclampsia in in vitro and in vivo animal models of preeclampsia.6-8 The blood-brain barrier (BBB), composed of the endothelial cells of brain microvessels, is the structural and functional interface between the blood and brain parenchyma.9,10 As a highly selective permeable barrier, BBB inhibits the free diffusion of circulating molecules from the blood into the brain. The accumulated data suggest that there are 2 transport routes including transcellular (across endothelial cells) and paracellular (between endothelial cells) in the brain microvasculature. However, the transcellular and paracellular routes dynamically regulate each other; caveolae-mediated transcytosis regulates the transendothelial permeability, explicitly affecting the tight junctional structures of the paracellular pathway.11,12 Proper functioning of the BBB thus has a critical role in protecting the maternal brain from circulating factors under normal conditions. The inhibition of nitric oxide synthesis using N(omega)nitro-L-arginine methyl ester (L-NAME) in animal models has been associated with pathological changes similar to those observed in women with preeclampsia.13,14 Increased endothelial cell permeability or a “capillary leakage” syndrome has been put forward as an underlying pathophysiological event in preeclampsia.5,15 Here, important and currently unanswered questions concerning the time course of BBB disruption severity, anatomical sites of increased permeability, and size of molecules that can penetrate the BBB during LPS were raised using tracer molecules with different molecular weights in preeclamptic rat models. Therefore, the present study aimed to investigate the effect of the systemic inflammation induced by LPS on BBB permeability in experimental preeclampsia induced by L-NAME in pregnant rats.
Experimental Procedure Animal Protocol and Procedure The experimental procedures were conducted in timepregnant Sprague Dawley rats weighing 210-330 g. The
rats were obtained from Aziz Sancar Institute of Experimental Medicine, Istanbul University. The study and all associated procedures were approved by the Local Ethics Committee for Animal Experimentation of Istanbul University (45/2012). Animals were group housed in controlled conditions (temperature: 22 § 1°C; humidity: 55 § 5%) with access to food and water ad libitum. The female rats were caged for 24 hours with the male rat and mating was confirmed by the presence of a vaginal plug and spermatozoa in the vaginal smear. The day on which insemination was detected was designated as the day 0 of pregnancy. All efforts were made to minimize animal suffering and to reduce the number of animals used. Separate experimental groups consisting of 8 rats were submitted to each experimental procedure.
Induction of Preeclampsia Symptoms Beginning on the 10th day of pregnancy, gravid rats were given L-NAME (Sigma-Aldrich, St. Louis, MO), diluted in drinking water at .5 mg/mL for 10 days to produce arterial hypertension and proteinuria.
Administration of LPS Pregnant rats received intraperitoneal (i.p.) injections of saline or LPS (Escherichia coli serotype 026:B6; Sigma, St. Louis, MO) on the 19th day of gestation. To induce an inflammatory response, pregnant rats received a single low dose (.5 mg/kg) of LPS.16 Control pregnant animals received saline (1 mL/kg) injections. Pregnant rats were sacrificed 24 hours after the LPS/saline injection.
Measurements of Arterial Blood Pressure On the day of the experiment, polyethylene catheters (PE-50) were inserted into the femoral artery and vein of animals under chloral hydrate anesthesia (360 mg/kg). The former catheter was connected to a pressure transducer that interfaced with a data acquisition system (iWorx/ETH-256), and a personal computer to continuously monitor the arterial blood pressure. The latter catheter was used to infuse horseradish peroxidase (HRP; 40 kDa) and Evans blue (EB; 69 kDa) tracers to show BBB permeability changes.
Determination of Proteinuria Proteinuria was measured in a semiquantitative manner using urine reagent strips (Uristik, The Hague, The Netherlands) on fresh urine samples. This colorimetric assay, which is relatively specific for albumin, was graded from 1+ to 4+ that corresponded to the following approximate protein concentrations: 1+: 30 mg/dL, 2+: 100 mg/dL, 3+: 1000 mg/dL, and 4+: more than 1000 mg/dL.17 Urinary protein levels of more than 100 mg/dL were considered as positive evidence of proteinuria. Severe proteinuria was defined as 3+ on a
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urine dipstick, and mild proteinuria was defined as less than 3+.18,19
The Evaluation of BBB Permeability with EB On the day of the experiment (24 hours after LPS or saline injection), EB dye (EB; 2%, 4 mL/kg in saline) was injected through the PE-50 catheter inserted into the right femoral vein of animals under chloral hydrate (360 mg/ kg; i.p.) anesthesia and was allowed to circulate for 30 minutes. Then, rats were transcardially perfused with 200 mL of saline to remove intravascular EB dye. Brains were dissected into the following 4 regions: left and right cerebral cortex and left and right hippocampus. Each brain region was weighed, homogenized in phosphate-buffered saline, and mixed after the addition of trichloroacetic acid to precipitate protein. After centrifugation, the concentration of tracer in the supernatant was measured at 610 nm using a spectrophotofluorometer (Microplate Reader; DTX880 Multimode Detector, Beckman Coulter, London, United Kingdom). EB was expressed as mg/mg of brain tissue against a standard curve.
Electron Microscopic Assessment of HRP Permeability To acquire electron microscopic evidence of alterations in the BBB integrity electron microscopic assessment was used. In brief, the animals received an intravenous injection of HRP (type II, Sigma Chemical Co., St. Louis, MO; 200-mg/kg body weight in .2-mL saline) through a catheter inserted into the femoral vein and HRP was allowed to circulate for 30 minutes. Under sodium pentothal (50 mg/kg, i.p.) and chloral hydrate (100 mg/kg, i.p.) anesthesia, the animals were perfused transcardially with saline followed by fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in .1 M phosphate-buffered saline (PBS). Afterward, coronal sections of 50-mm thickness were cut using a Vibratome and incubated in a solution of .05% 3.30 -diaminobenzidine in .05 M Tris-HCl buffer containing .01% hydrogen peroxide for 30 minutes to yield HRP-reaction products. For ultrastructural observation, samples from the cerebral cortex and hippocampus were postfixed in 1% osmium tetroxide, dehydrated in ethanol, and then embedded in Epon. Ultrathin sections (60 nm) were then examined under a transmission electron microscope (JEOL, 1011, Tokyo, Japan) equipped with a CCD camera (MegaView III, Soft Imaging System, GmBH, Germany). During the electron microscopy procedures, en bloc staining of the samples with uranyl acetate and staining of the ultrathin sections on grids with uranyl acetate and lead citrate were omitted in an attempt to avoid misinterpretation of possible artifactual precipitates of the HRP reaction products. In this study, we used a semiquantitative method to evaluate the intensity of the HRP accumulation in barrier type of endothelial cells. We scored the degree of accumulation of vesicles containing the HRP reaction products in at least 10 brain capillaries
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in the cerebral cortex and hippocampus of each animal under transmission electron microscope. Assessment of the HRP accumulation intensity was performed using the semiquantitative histologic scoring (HSCORE) system P using the following equation: HSCORE = Pi(I + 1), where I represents the degree of HRP accumulation, and Pi is the percentage of brain capillaries for each degree, varying from 0% to 100% according to the method previously described.20,21 The degree of HRP accumulation was estimated using a subjective scale of 0-3 (0: no; 1: weak; 2: moderate; 3: strong HRP accumulation) by 2 observers who were blinded to the experimental groups.
Statistical Analysis All the other data were described as a mean § standard deviation. Group differences were determined by 1-way analysis of variance followed by Tukey test for comparison of measurements of EB dye content in brain tissue, and semiquantitative HSCORE of HRP accumulation in brain capillary endothelial cells. The Wilcoxon test for arterial blood pressure and Chi-square test for proteinuria were used after 1-way analysis of variance. In all cases, differences between the means were considered significant if P < .05.
Results Mean arterial blood pressure (MABP) values in experimental groups are presented in Table 1. On day 20 of pregnancy, MABP levels significantly increased in L-NAME-treated animals compared with pregnant animals, and pregnant animals receiving LPS only (P < .01; Table 1). Treatment with LPS did not significantly alter the MABP in pregnant and L-NAME-treated pregnant rats (Table 1). As shown in Table 2, both normal pregnant and pregnant animals receiving LPS did not show a higher degree of proteinuria in the urine. A significant correlation was found between the level of proteinuria at baseline and the percentage proteinuria enhancement at day 20 in L-NAME-treated animals with or without LPS injection (P < .01; Table 2). Table 1. Mean arterial blood pressure values of rats in the experimental groups Groups
n
Mean arterial blood pressure (mmHg)
Pregnancy Pregnancy + LPS Pregnancy + L-NAME Pregnancy + L-NAME + LPS
12 12 12 12
97 § 2.6 89 § 3.6 138 § 3.3* 140 § 7.0*
Abbreviations: L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide. Data are means § SD; n, number of animals. *P < .01 versus the values from pregnancy and pregnancy plus LPS groups.
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Table 2. The degree of proteinuria in the experimental groups Urine dipstick values (mg/dL) Groups
30 (+)
100 (++)
1000 (+++)
>1000 (++++)
Pregnancy Pregnancy + LPS Pregnancy + L-NAME Pregnancy + L-NAME + LPS
10 (83%) 9 (75%) 0 (0%) 1 (8%)
2 (17%) 3 (25%) 4 (33%) 4 (33%)
8 (67%)* 7 (59%)*
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Abbreviations: L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide. The values are expressed as the number of animals (percentage of the total). The percentage of proteinuria are described as (++), (+++), and (++++) urine dipstick values. The percentage of proteinuria in L-NAME-treated groups was significantly higher than the pregnancy and pregnancy plus LPS groups. *P < .01.
The EB dye content in the right and left cerebral cortex, and the right and left hippocampus of pregnant animals treated with L-NAME, with or without LPS injection was not higher than that observed in both pregnant without LPS and pregnant receiving LPS groups (Fig 1). Therefore, there were no significant differences in the content of EB dye among groups regardless of receiving L-NAME and/ or LPS in all brain regions examined. No sign of HRP extravasation was observed in macroscopic observation of the Vibratome sections of brains of normal pregnant rats, and pregnant rats receiving LPS only (Fig 2). However, when compared to pregnancy group, extravasation of HRP tracer was noted in pregnant treated with L-NAME, and pregnant treated with LNAME plus LPS groups (Fig 2). The HRP extravasation (brown color) was limited to midbrain and cerebral cortex regions by L-NAME treatment (Fig 2). A widespread pattern of HRP extravasation was observed in cortical and
subcortical regions of the L-NAME-treated pregnant rats exposed to the LPS challenge (Fig 2). Ultrastructurally, no HRP reaction products were observed within the cytoplasm of endothelial cells of brain capillaries in the cerebral cortex (Fig 3) and hippocampus (Fig 4) regions of both pregnant rats and pregnant rats receiving LPS only, confirming our macroscopic observations. Yet, the brain capillaries in the cerebral cortex (Fig 3) and hippocampus (Fig 4) in L-NAME-treated pregnant rats receiving LPS exhibited frequent caveolar vesicles that contained electron-dense HRP reaction products in the cytoplasm of endothelial cells. To eliminate any possibilities of leaky HRP action, tight junctions (TJs) between adjacent endothelial cells were also checked ultrastructurally in both groups, and no sign of the passage of HRP reaction products through TJs was noted, as seen in the hippocampus region (Fig 4). Endocytotic vesicles filled with HRP reaction products of different
Figure 1. Evans blue (EB) dye content in the brain regions of animals. Data are shown as means § SD. Eight animals were used in each group. Note that the EB content in brain regions did not significantly vary among the 4 groups. Abbreviation: SD, standard deviation.
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Figure 2. Photomicrographs showing the pattern of HRP extravasation in Vibratome sections of brains of rats in experimental groups. Note that in L-NAMEtreated pregnant, and in L-NAME-treated pregnant animals receiving LPS, the macroscopic evidence of HRP extravasation is observed in brown color. Abbreviations: HRP, horseradish peroxidase; L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide. (Color version of figure is available online.)
dimensions were observed in the cytoplasm of endothelial cells in L-NAME-treated pregnant animals and L-NAMEtreated pregnant animals receiving LPS (Figs 3 and 4). In L-NAME-treated pregnant rats and L-NAME-treated pregnant animals receiving LPS, the accumulation of HRP containing caveolar vesicles in the capillary endothelial cells of the cerebral cortex and hippocampus was higher compared to animals in the pregnancy group, with HSCORE significantly higher than those of the
normal pregnant and pregnant receiving LPS only (Fig 5; P < .01).
Discussion In the current study, we investigated the effect of LPS on the BBB integrity in L-NAME-treated pregnant rats. The results of the current study showed that LPS challenge did not lead to a greater extent of increased BBB
Figure 3. Electron micrographs showing capillaries from the cerebral cortex region of rats in experimental groups following HRP injection. No vesicles containing HRP reaction products are observed in the cytoplasm of endothelial cells of capillaries in cerebral cortex rats in pregnant and pregnant plus LPS groups. Capillaries endothelial cells of cerebral cortex region of brains in pregnancy plus L-NAME, and pregnancy plus L-NAME plus LPS groups display abundant cytoplasmic vesicles containing HRP reaction products (arrows). Abbreviations: HRP, horseradish peroxidase; L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide.
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Figure 4. Electron micrographs showing capillaries from the hippocampus region of rats in the experimental groups following HRP injection. No vesicles containing HRP reaction products are observed in the cytoplasm of endothelial cells of capillaries in hippocampus rats in pregnant and pregnant plus LPS groups. Capillaries endothelial cells of the hippocampus region of brains in pregnancy plus L-NAME, and pregnancy plus L-NAME plus LPS groups display abundant cytoplasmic vesicles containing HRP reaction products (arrows). High magnification of the brain capillary wall from the hippocampus of a rat in pregnancy plus L-NAME plus LPS group shows an intact TJ (arrow) along with caveolar vesicles containing HRP reaction products (arrows) in the cytoplasm of endothelial cells. Abbreviations: HRP, horseradish peroxidase; L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide.
permeability to HRP tracer into the brain parenchyma in L-NAME-treated pregnant rats. L-NAME treatment for 10 days increased the arterial blood pressure, and led to severe proteinuria in pregnant rats, indicating that L-NAME is an experimental means of inducing preeclamptic symptoms in pregnant rats. In this experimental design, we observed increased HRP extravasation in the brain parenchyma of L-NAME-treated animals, which was not further enhanced by LPS. The other method we employed to measure BBB permeability, EB dye, has a higher molecular weight and binds to albumin when it is presented to circulation. In the present study, the measurement of BBB permeability in preeclamptic rats subjected to LPS revealed no EB dye extravasation into the brain parenchyma. Our data hence shows that preeclampsia with or without LPS treatment increased BBB permeability to substances with molecular weights less than 40 kDa compared to that of higher molecular weight—EB dye (69 kDa). These data are also consistent with numerous previous findings on the low and higher molecular weight dependence of the BBB permeability.22,23
Because the levels of proinflammatory cytokines and oxidative markers in preeclamptic women and animals were already shown by numerous studies.24,25 Moreover, a recent study reported that maternal immune activation induced by LPS caused oxidative stress and inflammatory changes that could be associated with the dysfunction in the BBB and placental barrier permeability of pregnant rats and offspring.26 The plasma levels of inflammatory cytokines, including TNF-alpha and IL-6, increases at 4 hours after injection of bacterial LPS to pregnant mice on days 17 and 18.27 It is also reported that preeclampsia induced animals had higher seizure susceptibility compared to healthy pregnant rats.8 In a rat model of preeclampsia induced by LPS, it is shown that a subconvulsive dose of pentylenetetrazol induces seizure activity.7 In our previous studies, we showed that pentylenetetrazol-induced seizures lead to a severe disruption of the BBB in L-NAME-treated pregnant rats.28,29 Based on the accumulated data, we suggest that oxidative status and cytokine levels during preeclampsia might be critical in determining the BBB integrity as it is
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Figure 5. Assessment of the accumulation of HRP reaction products containing vesicles in the brain capillary endothelial cells in cerebral cortex and hippocampus regions of animals in experimental groups by semiquantitative HSCORE. Data represent mean § SD. *P < .01 versus the rest of the groups. Abbreviations: HRP, horseradish peroxidase; HSCORE, histologic scoring; SD, standard deviation.
well known that cytokines such as TNF-alpha and IL-17 disrupt the BBB integrity. In one of the recent studies, it is shown that as the water content increases in the brain, EB dye extravasation increases in the anterior cerebrum: the expression levels of the claudin-1 increases while zonula occludens-1 and occludin show no change in the posterior cerebrum in the ischemic placental rats.30 Our current data are consistent with findings that TJs close at the electron microscopy level during preeclamptic conditions with or without LPS injection. Rather than open TJs, we observed increased caveolar vesicles full of HRP reaction products in the cytoplasm of brain capillary endothelial cells of these animals, implying a role for the activation of a transcellular pathway in the increased the BBB permeability. Contrary to our results, when endothelial cells of human umbilical vein from preeclamptic women were harvested in in vitro culture, disruption of TJ proteins increase the permeability to HRP.31,32 The increase in caveolar pinocytic vesicles in the brain microvessel endothelial cells in LPS exposure to preeclamptic animals suggests that intravascular macromolecules might enter the brain through the transendothelial caveolar route. It would appear that the HRP-reaction products were internalized predominantly by caveolar vesicles of endothelial cells. The demonstration of an increased transendothelial transport corroborates the findings of our previous study which reported increased transendothelial vesicular transport of circulatory substances to the brain in
response to an L-NAME-treated rat model of preeclampsia.28 It is accepted that TJ proteins in the endothelial cells of brain microvessels are considered to control paracellular pathway of vascular permeability. In this context, we observed no open TJs of capillary endothelial cells in the cerebral cortex and hippocampus of animals in pregnancy treated with L-NAME, and pregnancy treated with LNAME plus LPS groups. We observed increased caveolar vesicles full of the HRP reaction products in the cytoplasm of brain capillary endothelial cells of animals in pregnancy treated with L-NAME, and in pregnancy treated with LNAME plus LPS groups, which imply the activation of the transcellular caveolar pathway in the increased BBB permeability.
Conclusion The current study highlights the relationship among the BBB, the inflammation, and the preeclampsia. Treatment of LPS does not change the disruption degree of BBB observed in preeclamptic conditions, and this could provide significant mechanistic insight into the cerebrovascular changes associated with proinflammatory factors during preeclampsia. Our results also suggest that enhanced caveolar pathway could be responsible for increased BBB permeability rather than paracellular as indicated by the increased number of vesicles observed in electron microscopy in this experimental setting.
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autoimmune MRL-lpr/lpr mice. Immunology 2000;100: 110-118. Furukawa S, Sameshima H, Ikenoue T. Intrapartum late deceleration develops more frequently in pre-eclamptic women with severe proteinuria. J Obst Gynaecol Res 2006;32:68-73. Kelley MA. Triage and management of the pregnant hypertensive patient. J Nurse Midwifery 1999;44:558-571. Hoffman WH, Stamatovic SM, Andjelkovic AV. Inflammatory mediators and blood brain barrier disruption in fatal brain edema of diabetic ketoacidosis. Brain Res 2009;1254:138-148. Sahin D, Yilmaz CU, Orhan N. Changes in electroencephalographic characteristics and blood-brain barrier permeability in WAG/Rij rats with cortical dysplasia. Epilepsy Behav 2017;7:70-76. Hoffmann A, Bredno J, Wendland M, et al. High and low molecular weight fluorescein isothiocyanate (FITC)-dextrans to assess blood-brain barrier disruption: technical considerations. Transl Stroke Res 2011;2:106-111. Kozler P, Pokorny J. Altered blood-brain barrier permeability and its effect on the distribution of Evans blue and sodium fluorescein in the rat brain applied by intracarotid injection. Physiol Res 2003;52:607-614. Amaral TAS, Ognibene DT, Carvalho LCRM, et al. Differential responses of mesenteric arterial bed to vasoactive substances in L-NAME-induced preeclampsia: role of oxidative stress and endothelial dysfunction. Clin Exp Hypertens 2018;40:126-135. Cotechini T, Komisarenko M, Sperou A, et al. Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth restriction and features of preeclampsia. J Exp Med 2014;211:165-179. Sim~ oes LR, Sangiogo G, Tashiro MH, et al. Maternal immune activation induced by lipopolysaccharide triggers immune response in pregnant mother and fetus, and induces behavioral impairment in adult rats. J Psychiatr Res 2018;100:71-83. Vizi ES, Szelenyi J, Selmeczy Z, et al. Enhanced tumor necrosis factor-alpha-specific and decreased interleukin10-specific immune responses to LPS during the third trimester of pregnancy in mice. J Endocrinol 2001;171:355-361. Kucuk M, Kaya M, Orhan N, et al. The effect of pentylenetetrazole-induced seizures on blood-brain barrier integrity in a rat model of preeclampsia. Turk J Vet Anim Sci 2012;36:311-318. Orhan N, Yilmaz CU, Ekizoglu O, et al. The effects of superoxide dismutase mimetic MnTMPyP on the altered blood-brain barrier integrity in experimental preeclampsia with or without seizures in rats. Brain Res 2014;1563: 91-102. Warrington J, Fan F, Murphy S, et al. Placental ischemia impairs cerebral blood flow autoregulation and increases blood-brain barrier permeability in pregnant rats. Physiol Rep 2015;2:e12134. Wang YP, Gu Y, Granger DN, et al. Endothelial junctional protein redistribution and increased monolayer permeability in human umbilical vein endothelial cells isolated during preeclampsia. Am J Obstet Gynecol 2002;186:214-220. Zhang Y, Gu Y, Li H, et al. Increased endothelial monolayer permeability is induced by serum from women with preeclampsia but not by serum from women with normal pregnancy or that are not pregnant. Hypertens Pregnancy 2003;22:99-108.
The Effects of Lipopolysaccharide on the Disrupted Blood-Brain Barrier in a Rat Model of Preeclampsia 1XD XMutlu Kucuk, PhD, D2X X * D3X XCanan Ugur Yilmaz, PhD, D4X X *,† D5X XNurcan Orhan, PhD, D6X X ‡ D7X XBulent Ahishali, MD, PhD,D8X X§ D9X XNadir Arican, MD, D10X X PhD,|| D1X XImdat Elmas, D12XMD, X PhD,|| € rses, MD, PhD,D14X X{ and D15X XMehmet Kaya, D16XPhD D13X XCandan Gu X # Background: Preeclampsia is a disorder characterized by high blood pressure and often proteinuria during pregnancy. It is known that a subseptic dose of bacterial lipopolysaccharide (LPS) induces production of proinflammatory cytokines, and possibly incDreasing 17X X the risk for developing preeclampsia. We investigated the effects of LPS on the blood-brain barrier (BBB) integrity in pregnant rats with N (omega)-nitro-L-arginine methyl ester (L-NAME) induced preeclampsia. Methods: Starting from the 10th day of gestation, pregnant rats were given L-NAME for 10 days to produce hypertension and proteinuria. Animals were then treated with a single injection of LPS on the 19th day of pregnancy. Arterial blood pressure and proteinuria were measured on the day of the experiment, which was 24 hours after the LPS injection. The BBB integrity was assessed by using Evans blue (EB) and horseradish peroxidase (HRP) tracers. Results: Proteinuria was observed in varying degrees, and the arterial blood pressure increased in L-NAME-treated pregnant rats (P < .01). The overall brain EB content did not increase in these preeclamptic rats when compared to pregnant animals, and LPS treatment also did not change EB content. Ultrastructurally, frequent vesicles containing HRP reaction products were observed in the capillary endothelial cells in the cerebral cortex and hippocampus of pregnant rats treated with L-NAME (P < .01). However, LPS did not change the amounts of HRP that mainly accumulated in brain capillary endothelial cells of these animals. Conclusion: Our results suggest that, in this experimental setting, LPS does not change the severity of BBB disruption observed in preeclamptic animals. Key Words: Blood-brain barrier—preeclampsia—lipopolysaccharide—horseradish peroxidase—electron microscopy © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.
From the *Department of Laboratory Animals Science, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; †Department of Pharmaceutical Bioscience, Biomedical Centrum, Uppsala University, Uppsala, Sweden; ‡Department of Neuroscience, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; §Department of Histology and Embryology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; ||Department of Forensic Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; {Department of Neurology, Ko¸c University School of Medicine, Istanbul, Turkey; and #Department of Physiology, Ko¸c University School of Medicine, Istanbul, Turkey. Received May 7, 2018; revision received July 23, 2018; accepted August 1, 2018. Conflicts of interest: The authors declare that they have no conflicts of interest. The authors have read the Journal's position on issues involved in ethical publications and affirm that this study is consistent with those guidelines. The results presented in this manuscript have not been published previously in whole or part, except in abstract format. Financial Disclosure: This study was supported by the Research Fund of Istanbul University (24210/2009). Address correspondence to Canan Ugur Yilmaz, PhD, Department of Pharmaceutical Bioscience, Biomedical Centrum, Uppsala University, Husargatan 3, Uppsala Box 591, Uppsala 75429, Sweden. E-mailes: [email protected], [email protected] 1052-3057/$ - see front matter © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2018.08.003
Journal of Stroke and Cerebrovascular Diseases, Vol. &&, No. && (&&), 2018: pp 18
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Introduction Preeclampsia is a disorder characterized by hypertension and often proteinuria in varied degrees in pregnancy. Approximately, 5%-7% of all pregnancies are complicated by preeclampsia with profound implications for both mother and fetus.1,2 Also, it is believed that the placenta plays a central role in the pathophysiology of preeclampsia. Most of the circulatory factors, mainly cytokines, and oxidative products are associated with impaired endothelial cell function in the onset of preeclampsia.3,4 In this context, generalized maternal endothelial dysfunction caused mainly by circulatory factors contributes significantly to the pathogenesis of preeclampsia.4,5 The inflammatory agents induced by an endotoxin such as lipopolysaccharide (LPS) play a role in the conversion of preeclampsia into eclampsia in in vitro and in vivo animal models of preeclampsia.6-8 The blood-brain barrier (BBB), composed of the endothelial cells of brain microvessels, is the structural and functional interface between the blood and brain parenchyma.9,10 As a highly selective permeable barrier, BBB inhibits the free diffusion of circulating molecules from the blood into the brain. The accumulated data suggest that there are 2 transport routes including transcellular (across endothelial cells) and paracellular (between endothelial cells) in the brain microvasculature. However, the transcellular and paracellular routes dynamically regulate each other; caveolae-mediated transcytosis regulates the transendothelial permeability, explicitly affecting the tight junctional structures of the paracellular pathway.11,12 Proper functioning of the BBB thus has a critical role in protecting the maternal brain from circulating factors under normal conditions. The inhibition of nitric oxide synthesis using N(omega)nitro-L-arginine methyl ester (L-NAME) in animal models has been associated with pathological changes similar to those observed in women with preeclampsia.13,14 Increased endothelial cell permeability or a “capillary leakage” syndrome has been put forward as an underlying pathophysiological event in preeclampsia.5,15 Here, important and currently unanswered questions concerning the time course of BBB disruption severity, anatomical sites of increased permeability, and size of molecules that can penetrate the BBB during LPS were raised using tracer molecules with different molecular weights in preeclamptic rat models. Therefore, the present study aimed to investigate the effect of the systemic inflammation induced by LPS on BBB permeability in experimental preeclampsia induced by L-NAME in pregnant rats.
Experimental Procedure Animal Protocol and Procedure The experimental procedures were conducted in timepregnant Sprague Dawley rats weighing 210-330 g. The
rats were obtained from Aziz Sancar Institute of Experimental Medicine, Istanbul University. The study and all associated procedures were approved by the Local Ethics Committee for Animal Experimentation of Istanbul University (45/2012). Animals were group housed in controlled conditions (temperature: 22 § 1°C; humidity: 55 § 5%) with access to food and water ad libitum. The female rats were caged for 24 hours with the male rat and mating was confirmed by the presence of a vaginal plug and spermatozoa in the vaginal smear. The day on which insemination was detected was designated as the day 0 of pregnancy. All efforts were made to minimize animal suffering and to reduce the number of animals used. Separate experimental groups consisting of 8 rats were submitted to each experimental procedure.
Induction of Preeclampsia Symptoms Beginning on the 10th day of pregnancy, gravid rats were given L-NAME (Sigma-Aldrich, St. Louis, MO), diluted in drinking water at .5 mg/mL for 10 days to produce arterial hypertension and proteinuria.
Administration of LPS Pregnant rats received intraperitoneal (i.p.) injections of saline or LPS (Escherichia coli serotype 026:B6; Sigma, St. Louis, MO) on the 19th day of gestation. To induce an inflammatory response, pregnant rats received a single low dose (.5 mg/kg) of LPS.16 Control pregnant animals received saline (1 mL/kg) injections. Pregnant rats were sacrificed 24 hours after the LPS/saline injection.
Measurements of Arterial Blood Pressure On the day of the experiment, polyethylene catheters (PE-50) were inserted into the femoral artery and vein of animals under chloral hydrate anesthesia (360 mg/kg). The former catheter was connected to a pressure transducer that interfaced with a data acquisition system (iWorx/ETH-256), and a personal computer to continuously monitor the arterial blood pressure. The latter catheter was used to infuse horseradish peroxidase (HRP; 40 kDa) and Evans blue (EB; 69 kDa) tracers to show BBB permeability changes.
Determination of Proteinuria Proteinuria was measured in a semiquantitative manner using urine reagent strips (Uristik, The Hague, The Netherlands) on fresh urine samples. This colorimetric assay, which is relatively specific for albumin, was graded from 1+ to 4+ that corresponded to the following approximate protein concentrations: 1+: 30 mg/dL, 2+: 100 mg/dL, 3+: 1000 mg/dL, and 4+: more than 1000 mg/dL.17 Urinary protein levels of more than 100 mg/dL were considered as positive evidence of proteinuria. Severe proteinuria was defined as 3+ on a
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urine dipstick, and mild proteinuria was defined as less than 3+.18,19
The Evaluation of BBB Permeability with EB On the day of the experiment (24 hours after LPS or saline injection), EB dye (EB; 2%, 4 mL/kg in saline) was injected through the PE-50 catheter inserted into the right femoral vein of animals under chloral hydrate (360 mg/ kg; i.p.) anesthesia and was allowed to circulate for 30 minutes. Then, rats were transcardially perfused with 200 mL of saline to remove intravascular EB dye. Brains were dissected into the following 4 regions: left and right cerebral cortex and left and right hippocampus. Each brain region was weighed, homogenized in phosphate-buffered saline, and mixed after the addition of trichloroacetic acid to precipitate protein. After centrifugation, the concentration of tracer in the supernatant was measured at 610 nm using a spectrophotofluorometer (Microplate Reader; DTX880 Multimode Detector, Beckman Coulter, London, United Kingdom). EB was expressed as mg/mg of brain tissue against a standard curve.
Electron Microscopic Assessment of HRP Permeability To acquire electron microscopic evidence of alterations in the BBB integrity electron microscopic assessment was used. In brief, the animals received an intravenous injection of HRP (type II, Sigma Chemical Co., St. Louis, MO; 200-mg/kg body weight in .2-mL saline) through a catheter inserted into the femoral vein and HRP was allowed to circulate for 30 minutes. Under sodium pentothal (50 mg/kg, i.p.) and chloral hydrate (100 mg/kg, i.p.) anesthesia, the animals were perfused transcardially with saline followed by fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in .1 M phosphate-buffered saline (PBS). Afterward, coronal sections of 50-mm thickness were cut using a Vibratome and incubated in a solution of .05% 3.30 -diaminobenzidine in .05 M Tris-HCl buffer containing .01% hydrogen peroxide for 30 minutes to yield HRP-reaction products. For ultrastructural observation, samples from the cerebral cortex and hippocampus were postfixed in 1% osmium tetroxide, dehydrated in ethanol, and then embedded in Epon. Ultrathin sections (60 nm) were then examined under a transmission electron microscope (JEOL, 1011, Tokyo, Japan) equipped with a CCD camera (MegaView III, Soft Imaging System, GmBH, Germany). During the electron microscopy procedures, en bloc staining of the samples with uranyl acetate and staining of the ultrathin sections on grids with uranyl acetate and lead citrate were omitted in an attempt to avoid misinterpretation of possible artifactual precipitates of the HRP reaction products. In this study, we used a semiquantitative method to evaluate the intensity of the HRP accumulation in barrier type of endothelial cells. We scored the degree of accumulation of vesicles containing the HRP reaction products in at least 10 brain capillaries
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in the cerebral cortex and hippocampus of each animal under transmission electron microscope. Assessment of the HRP accumulation intensity was performed using the semiquantitative histologic scoring (HSCORE) system P using the following equation: HSCORE = Pi(I + 1), where I represents the degree of HRP accumulation, and Pi is the percentage of brain capillaries for each degree, varying from 0% to 100% according to the method previously described.20,21 The degree of HRP accumulation was estimated using a subjective scale of 0-3 (0: no; 1: weak; 2: moderate; 3: strong HRP accumulation) by 2 observers who were blinded to the experimental groups.
Statistical Analysis All the other data were described as a mean § standard deviation. Group differences were determined by 1-way analysis of variance followed by Tukey test for comparison of measurements of EB dye content in brain tissue, and semiquantitative HSCORE of HRP accumulation in brain capillary endothelial cells. The Wilcoxon test for arterial blood pressure and Chi-square test for proteinuria were used after 1-way analysis of variance. In all cases, differences between the means were considered significant if P < .05.
Results Mean arterial blood pressure (MABP) values in experimental groups are presented in Table 1. On day 20 of pregnancy, MABP levels significantly increased in L-NAME-treated animals compared with pregnant animals, and pregnant animals receiving LPS only (P < .01; Table 1). Treatment with LPS did not significantly alter the MABP in pregnant and L-NAME-treated pregnant rats (Table 1). As shown in Table 2, both normal pregnant and pregnant animals receiving LPS did not show a higher degree of proteinuria in the urine. A significant correlation was found between the level of proteinuria at baseline and the percentage proteinuria enhancement at day 20 in L-NAME-treated animals with or without LPS injection (P < .01; Table 2). Table 1. Mean arterial blood pressure values of rats in the experimental groups Groups
n
Mean arterial blood pressure (mmHg)
Pregnancy Pregnancy + LPS Pregnancy + L-NAME Pregnancy + L-NAME + LPS
12 12 12 12
97 § 2.6 89 § 3.6 138 § 3.3* 140 § 7.0*
Abbreviations: L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide. Data are means § SD; n, number of animals. *P < .01 versus the values from pregnancy and pregnancy plus LPS groups.
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Table 2. The degree of proteinuria in the experimental groups Urine dipstick values (mg/dL) Groups
30 (+)
100 (++)
1000 (+++)
>1000 (++++)
Pregnancy Pregnancy + LPS Pregnancy + L-NAME Pregnancy + L-NAME + LPS
10 (83%) 9 (75%) 0 (0%) 1 (8%)
2 (17%) 3 (25%) 4 (33%) 4 (33%)
8 (67%)* 7 (59%)*
-
Abbreviations: L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide. The values are expressed as the number of animals (percentage of the total). The percentage of proteinuria are described as (++), (+++), and (++++) urine dipstick values. The percentage of proteinuria in L-NAME-treated groups was significantly higher than the pregnancy and pregnancy plus LPS groups. *P < .01.
The EB dye content in the right and left cerebral cortex, and the right and left hippocampus of pregnant animals treated with L-NAME, with or without LPS injection was not higher than that observed in both pregnant without LPS and pregnant receiving LPS groups (Fig 1). Therefore, there were no significant differences in the content of EB dye among groups regardless of receiving L-NAME and/ or LPS in all brain regions examined. No sign of HRP extravasation was observed in macroscopic observation of the Vibratome sections of brains of normal pregnant rats, and pregnant rats receiving LPS only (Fig 2). However, when compared to pregnancy group, extravasation of HRP tracer was noted in pregnant treated with L-NAME, and pregnant treated with LNAME plus LPS groups (Fig 2). The HRP extravasation (brown color) was limited to midbrain and cerebral cortex regions by L-NAME treatment (Fig 2). A widespread pattern of HRP extravasation was observed in cortical and
subcortical regions of the L-NAME-treated pregnant rats exposed to the LPS challenge (Fig 2). Ultrastructurally, no HRP reaction products were observed within the cytoplasm of endothelial cells of brain capillaries in the cerebral cortex (Fig 3) and hippocampus (Fig 4) regions of both pregnant rats and pregnant rats receiving LPS only, confirming our macroscopic observations. Yet, the brain capillaries in the cerebral cortex (Fig 3) and hippocampus (Fig 4) in L-NAME-treated pregnant rats receiving LPS exhibited frequent caveolar vesicles that contained electron-dense HRP reaction products in the cytoplasm of endothelial cells. To eliminate any possibilities of leaky HRP action, tight junctions (TJs) between adjacent endothelial cells were also checked ultrastructurally in both groups, and no sign of the passage of HRP reaction products through TJs was noted, as seen in the hippocampus region (Fig 4). Endocytotic vesicles filled with HRP reaction products of different
Figure 1. Evans blue (EB) dye content in the brain regions of animals. Data are shown as means § SD. Eight animals were used in each group. Note that the EB content in brain regions did not significantly vary among the 4 groups. Abbreviation: SD, standard deviation.
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Figure 2. Photomicrographs showing the pattern of HRP extravasation in Vibratome sections of brains of rats in experimental groups. Note that in L-NAMEtreated pregnant, and in L-NAME-treated pregnant animals receiving LPS, the macroscopic evidence of HRP extravasation is observed in brown color. Abbreviations: HRP, horseradish peroxidase; L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide. (Color version of figure is available online.)
dimensions were observed in the cytoplasm of endothelial cells in L-NAME-treated pregnant animals and L-NAMEtreated pregnant animals receiving LPS (Figs 3 and 4). In L-NAME-treated pregnant rats and L-NAME-treated pregnant animals receiving LPS, the accumulation of HRP containing caveolar vesicles in the capillary endothelial cells of the cerebral cortex and hippocampus was higher compared to animals in the pregnancy group, with HSCORE significantly higher than those of the
normal pregnant and pregnant receiving LPS only (Fig 5; P < .01).
Discussion In the current study, we investigated the effect of LPS on the BBB integrity in L-NAME-treated pregnant rats. The results of the current study showed that LPS challenge did not lead to a greater extent of increased BBB
Figure 3. Electron micrographs showing capillaries from the cerebral cortex region of rats in experimental groups following HRP injection. No vesicles containing HRP reaction products are observed in the cytoplasm of endothelial cells of capillaries in cerebral cortex rats in pregnant and pregnant plus LPS groups. Capillaries endothelial cells of cerebral cortex region of brains in pregnancy plus L-NAME, and pregnancy plus L-NAME plus LPS groups display abundant cytoplasmic vesicles containing HRP reaction products (arrows). Abbreviations: HRP, horseradish peroxidase; L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide.
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Figure 4. Electron micrographs showing capillaries from the hippocampus region of rats in the experimental groups following HRP injection. No vesicles containing HRP reaction products are observed in the cytoplasm of endothelial cells of capillaries in hippocampus rats in pregnant and pregnant plus LPS groups. Capillaries endothelial cells of the hippocampus region of brains in pregnancy plus L-NAME, and pregnancy plus L-NAME plus LPS groups display abundant cytoplasmic vesicles containing HRP reaction products (arrows). High magnification of the brain capillary wall from the hippocampus of a rat in pregnancy plus L-NAME plus LPS group shows an intact TJ (arrow) along with caveolar vesicles containing HRP reaction products (arrows) in the cytoplasm of endothelial cells. Abbreviations: HRP, horseradish peroxidase; L-NAME, N(omega)-nitro-L-arginine methyl ester; LPS, lipopolysaccharide.
permeability to HRP tracer into the brain parenchyma in L-NAME-treated pregnant rats. L-NAME treatment for 10 days increased the arterial blood pressure, and led to severe proteinuria in pregnant rats, indicating that L-NAME is an experimental means of inducing preeclamptic symptoms in pregnant rats. In this experimental design, we observed increased HRP extravasation in the brain parenchyma of L-NAME-treated animals, which was not further enhanced by LPS. The other method we employed to measure BBB permeability, EB dye, has a higher molecular weight and binds to albumin when it is presented to circulation. In the present study, the measurement of BBB permeability in preeclamptic rats subjected to LPS revealed no EB dye extravasation into the brain parenchyma. Our data hence shows that preeclampsia with or without LPS treatment increased BBB permeability to substances with molecular weights less than 40 kDa compared to that of higher molecular weight—EB dye (69 kDa). These data are also consistent with numerous previous findings on the low and higher molecular weight dependence of the BBB permeability.22,23
Because the levels of proinflammatory cytokines and oxidative markers in preeclamptic women and animals were already shown by numerous studies.24,25 Moreover, a recent study reported that maternal immune activation induced by LPS caused oxidative stress and inflammatory changes that could be associated with the dysfunction in the BBB and placental barrier permeability of pregnant rats and offspring.26 The plasma levels of inflammatory cytokines, including TNF-alpha and IL-6, increases at 4 hours after injection of bacterial LPS to pregnant mice on days 17 and 18.27 It is also reported that preeclampsia induced animals had higher seizure susceptibility compared to healthy pregnant rats.8 In a rat model of preeclampsia induced by LPS, it is shown that a subconvulsive dose of pentylenetetrazol induces seizure activity.7 In our previous studies, we showed that pentylenetetrazol-induced seizures lead to a severe disruption of the BBB in L-NAME-treated pregnant rats.28,29 Based on the accumulated data, we suggest that oxidative status and cytokine levels during preeclampsia might be critical in determining the BBB integrity as it is
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Figure 5. Assessment of the accumulation of HRP reaction products containing vesicles in the brain capillary endothelial cells in cerebral cortex and hippocampus regions of animals in experimental groups by semiquantitative HSCORE. Data represent mean § SD. *P < .01 versus the rest of the groups. Abbreviations: HRP, horseradish peroxidase; HSCORE, histologic scoring; SD, standard deviation.
well known that cytokines such as TNF-alpha and IL-17 disrupt the BBB integrity. In one of the recent studies, it is shown that as the water content increases in the brain, EB dye extravasation increases in the anterior cerebrum: the expression levels of the claudin-1 increases while zonula occludens-1 and occludin show no change in the posterior cerebrum in the ischemic placental rats.30 Our current data are consistent with findings that TJs close at the electron microscopy level during preeclamptic conditions with or without LPS injection. Rather than open TJs, we observed increased caveolar vesicles full of HRP reaction products in the cytoplasm of brain capillary endothelial cells of these animals, implying a role for the activation of a transcellular pathway in the increased the BBB permeability. Contrary to our results, when endothelial cells of human umbilical vein from preeclamptic women were harvested in in vitro culture, disruption of TJ proteins increase the permeability to HRP.31,32 The increase in caveolar pinocytic vesicles in the brain microvessel endothelial cells in LPS exposure to preeclamptic animals suggests that intravascular macromolecules might enter the brain through the transendothelial caveolar route. It would appear that the HRP-reaction products were internalized predominantly by caveolar vesicles of endothelial cells. The demonstration of an increased transendothelial transport corroborates the findings of our previous study which reported increased transendothelial vesicular transport of circulatory substances to the brain in
response to an L-NAME-treated rat model of preeclampsia.28 It is accepted that TJ proteins in the endothelial cells of brain microvessels are considered to control paracellular pathway of vascular permeability. In this context, we observed no open TJs of capillary endothelial cells in the cerebral cortex and hippocampus of animals in pregnancy treated with L-NAME, and pregnancy treated with LNAME plus LPS groups. We observed increased caveolar vesicles full of the HRP reaction products in the cytoplasm of brain capillary endothelial cells of animals in pregnancy treated with L-NAME, and in pregnancy treated with LNAME plus LPS groups, which imply the activation of the transcellular caveolar pathway in the increased BBB permeability.
Conclusion The current study highlights the relationship among the BBB, the inflammation, and the preeclampsia. Treatment of LPS does not change the disruption degree of BBB observed in preeclamptic conditions, and this could provide significant mechanistic insight into the cerebrovascular changes associated with proinflammatory factors during preeclampsia. Our results also suggest that enhanced caveolar pathway could be responsible for increased BBB permeability rather than paracellular as indicated by the increased number of vesicles observed in electron microscopy in this experimental setting.
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