The Effects of Lipopolysaccharide on the Disrupted Blood-Brain Barrier in a Rat Model of Preeclampsia.

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 increasing 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.

Changes in electroencephalographic characteristics and blood-brain barrier permeability in WAG/Rij rats with cortical dysplasia.

PURPOSE: This study investigated the effects of cortical dysplasia (CD) on electrophysiology and blood-brain barrier (BBB) permeability in WAG/Rij rats with genetic absence epilepsy. METHODS: Pregnant WAG/Rij rats were exposed to 145cGy of gamma-irradiation on embryonic day 17 to induce CD. An electroencephalogram was recorded from cortices subdurally in the offspring of the pregnant animals. Horseradish peroxidase (HRP) was used as determinant of BBB permeability. RESULTS: A massive tissue loss in the cerebral cortex was seen in WAG/Rij rats with CD (p<0.05). There was a significant decrease in the number and duration of spike-and-wave discharges (SWDs) and an increase in the frequency of SWDs in the WAG/Rij rats with CD when compared with the properties of SWDs in intact WAG/Rij rats (p<0.01). Ultrastructurally, the accumulation of HRP reaction products in the cerebral cortex and thalamus of WAG/Rij rats was significantly higher than that of control values (p<0.01). The accumulation of HRP reaction products in the cerebral cortex and thalamus regions of WAG/Rij rats with CD increased and was higher than that of the control and WAG/Rij animals (p<0.01). CONCLUSION: In our study, we showed that number and duration of SWDs decreased and SWD frequency increased in WAG/Rij rats with CD, suggesting a shift in seizure pattern. The association of these alterations with significant loss of cortical thickness and increased BBB permeability to HRP tracer may represent a causal relation of the EEG abnormalities with cerebral structural changes in these animals.

Hyperbaric oxygen therapy for five days increases blood-brain barrier permeability.

This study aimed to explore the effects of hyperbaric oxygen (HBO2) on blood-brain barrier (BBB) integrity in rats, when administered for one (at 2.5 ATA, 3 HBO2 sessions a day) and five days (at 2.5 ATA, 3 HBO2 sessions a day for the first two days, and twice a day for the last three days). Horseradish peroxidase (HRP) was used to evaluate the BBB permeability. Superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) levels were measured in the cerebral cortex and hippocampus regions. Frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in the cerebral cortex and hippocampus of rats subjected to HBO2. The accumulation of HRP reaction products in these brain regions was significantly higher than that of control animals (P ⟨ 0.01). In animals that received HBO2, MDA levels (P ⟨ 0.01 for five days) and GSH (p ⟨ 0.05 for one day, and P ⟨ 0.01 for five days) were decreased in the cerebral cortex, whereas SOD activities slightly increased in this region. In animals that received HBO2 significant decreases in MDA (P ⟨ 0.05 for one day; P ⟨ 0.01 for five days) and GSH (P ⟨ 0.05 for five days) levels were observed in the hippocampus region, but SOD activities decreased in this region. We showed that HBO2 administered with the doses described above impaired BBB integrity in otherwise healthy rats. Therefore, we suggest that the results of this study should be taken into consideration when patients are exposed to HBO2 with the same doses.

A Case Study of Intractable Diarrhea Due to Neonatal Microvillous Inclusion Disease.

BACKGROUND: Microvillous inclusion disease (MVID) is one of the most severe congenital diarrhea disorders, caused by a genetic defect in enterocyte differentiation and polarization. CASE REPORT: We describe a neonate who presented with severe weight loss, hypernatremic dehydration and metabolic acidosis due to intractable diarrhea due to MVID, confirmed by electron microscopy. CONCLUSION: MVID can present with severe weight loss, hypernatremic dehydration and metabolic acidosis that is life threatening. The diagnosis is made by typical findings on light microscopy and electron microscope of small bowel biopsies. The only therapeutic options at this time are total parenteral nutrition and bowel rest and intestinal transplantation.

Intravenous immunoglobulins prevent the breakdown of the blood-brain barrier in experimentally induced sepsis.

INTERVENTIONS: The effects of immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M on blood-brain barrier integrity and survival rates in septic rats were comparatively investigated. MEASUREMENTS: Sepsis was induced by cecal ligation and perforation in Sprague-Dawley rats. The animals were divided into the following groups: Sham, cecal ligation and perforation, cecal ligation and perforation plus immunoglobulin G (250 mg/kg, intravenous), and cecal ligation and perforation plus immunoglobulins enriched with immunoglobulin A and immunoglobulin M (250 mg/kg, intravenous). Immunoglobulins were administered 5 mins before cecal ligation and perforation and the animals were observed for behavioral changes for 24 hrs following cecal ligation and perforation. Blood-brain barrier permeability was functionally and structurally evaluated by determining the extravasation of Evans Blue and horseradish peroxidase tracers, respectively. Immunohistochemistry and Western blotting for occludin were performed. MAIN RESULTS: The high mortality rate (34%) noted in the septic rats was decreased to 15% and 3% by immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M, respectively (p < .01). Both immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M alleviated the symptoms of sickness behavior in the septic rats, with the animals becoming healthy and active. Increased extravasation of Evans Blue into the brain tissue of the septic rats was markedly decreased with the administration of both immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M (p < .01). Occludin expression remained essentially unchanged in all groups, including the cecal ligation and perforation group. In the cecal ligation and perforation group, increased luminal and abluminal vesicles containing electron-dense horseradish peroxidase-reaction product were noted in the cytoplasm of endothelial cells located in the hippocampus and the cerebral cortex. Tight junction was ultrastructurally intact, suggesting that the transcellular pathway is responsible for the blood-brain barrier breakdown in sepsis. Following immunoglobulin G or immunoglobulins enriched with immunoglobulin A and immunoglobulin M treatment, no ultrastructural evidence of leaky capillaries in the brain was observed in the septic rats, indicating the blockade of the transcellular pathway by immunoglobulins administration. CONCLUSIONS: Our study suggests that immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M improve the integrity of the blood-brain barrier and inhibits cecal ligation and perforation-induced symptoms of sickness behavior in rats.

Targeting the blood-brain barrier disruption in hypertension by ALK5/TGF-Β type I receptor inhibitor SB-431542 and dynamin inhibitor dynasore.

INTRODUCTION: In this study, we aimed to target two molecules, transforming growth factor-beta (TGF-ß) and dynamin to explore their roles in blood-brain barrier (BBB) disruption in hypertension. METHODS: For this purpose, angiotensin (ANG) II-induced hypertensive mice were treated with SB-431542, an inhibitor of the ALK5/TGF-ß type I receptor, and dynasore, an inhibitor of dynamin. Albumin-Alexa fluor 594 was used to assess BBB permeability. The alterations in the expression of claudin-5, caveolin (Cav)-1, glucose transporter (Glut)-1, and SMAD4 in the cerebral cortex and the hippocampus were evaluated by quantification of immunofluorescence staining intensity. RESULTS: ANG II infusion increased BBB permeability to albumin-Alexa fluor 594 which was reduced by SB-431542 (P < 0.01), but not by dynasore. In hypertensive animals treated with dynasore, claudin-5 immunofluorescence intensity increased in the cerebral cortex and hippocampus while it decreased in the cerebral cortex of SB-431542 treated hypertensive mice (P < 0.01). Both dynasore and SB-431542 prevented the increased Cav-1 immunofluorescence intensity in the cerebral cortex and hippocampus of hypertensive animals (P < 0.01). SB-431542 and dynasore decreased Glut-1 immunofluorescence intensity in the cerebral cortex and hippocampus of mice receiving ANG II (P < 0.01). SB-431542 increased SMAD4 immunofluorescence intensity in the cerebral cortex of hypertensive animals, while in the hippocampus a significant decrease was noted by both SB-431542 and dynasore (P < 0.01). CONCLUSION: Our data suggest that inhibition of the TGFß type I receptor prevents BBB disruption under hypertensive conditions. These results emphasize the therapeutic potential of targeting TGFß signaling as a novel treatment modality to protect the brain of hypertensive patients.

Blood-brain barrier targeted delivery of lacosamide-conjugated gold nanoparticles: Improving outcomes in absence seizures.

OBJECTIVE: Most currently available antiepileptics are not fully effective in the prevention of seizures in absence epilepsy owing to the presence of blood-brain barrier (BBB). We aimed to test whether binding an antiepileptic drug, lacosamide (LCM), to glucose-coated gold nanoparticles (GNPs) enables efficient brain drug delivery to suppress the epileptic activity in WAG/Rij rats with absence epilepsy. METHODS: In these animals, intracranial-EEG recording, behavioral test, in vivo imaging of LCM and LCM-GNP conjugate distribution in the brain, inductively coupled plasma mass spectrometry analysis, immunofluorescence staining of glucose transporter (Glut)- 1, glial fibrillary acidic protein (GFAP), and p-glycoprotein (P-gp) and electron microscopy were performed. RESULTS: Lacosamide-GNP conjugates decreased the amplitude and frequency of spike-wave-like discharges (SWDs) and alleviated the anxiety-like behavior as assessed by EEG and elevated plus-maze test, respectively (p < 0.01). The in vivo imaging system results showed higher levels of fluorescein dye tagged to LCM-GNP in the brain during the 5-day injection period (p < 0.01). Immunofluorescence staining displayed decreased P-gp, Glut-1, and GFAP expression by LCM-GNP conjugate treatment predominantly in the cerebral cortex suggesting a potential functionality of this brain region in the modulation of neuronal activity in our experimental setting (p < 0.01). SIGNIFICANCE: We suggest that the conjugation of LCM to GNPs may provide a novel approach for efficient brain drug delivery in light of the effectiveness of our strategy not only in suppressing the seizure activity but also in decreasing the need to use high dosages of the antiepileptics to reduce the frequently encountered side effects in drug-resistant epilepsy.

Basic physiology of the blood-brain barrier in health and disease: a brief overview.

The blood-brain barrier (BBB), a dynamic interface between blood and brain constituted mainly by endothelial cells of brain microvessels, robustly restricts the entry of potentially harmful blood-sourced substances and cells into the brain, however, many therapeutically active agents concurrently cannot gain access into the brain at effective doses in the presence of an intact barrier. On the other hand, breakdown of BBB integrity may involve in the pathogenesis of various neurodegenerative diseases. Besides, certain diseases/disorders such as Alzheimer's disease, hypertension, and epilepsy are associated with varying degrees of BBB disruption. In this review, we aim to highlight the current knowledge on the cellular and molecular composition of the BBB with special emphasis on the major transport pathways across the barrier type endothelial cells. We further provide a discussion on the innovative brain drug delivery strategies in which the obstacle formed by BBB interferes with effective pharmacological treatment of neurodegenerative diseases/disorders.

Evaluation of Blood-Brain Barrier Integrity Using Vascular Permeability Markers: Evans Blue, Sodium Fluorescein, Albumin-Alexa Fluor Conjugates, and Horseradish Peroxidase.

The blood-brain barrier (BBB) constituted by endothelial cells of brain microvessels is a dynamic interface, which controls and regulates the transport of various substances including peptides, proteins, ions, vitamins, hormones, and immune cells from the circulation into the brain parenchyma. Certain diseases/disorders such as Alzheimer's disease, sepsis, and hypertension can lead to varying degrees of BBB disruption. Moreover, impairment of BBB integrity has been implicated in the pathogenesis of various neurodegenerative diseases like epilepsy. In attempts to explore the wide spectrum of pathophysiologic mechanisms of these diseases/disorders, a variety of experimental insults targeted to the BBB integrity in vitro in cell culture models and in vivo in laboratory animals have been shown to alter BBB permeability causing enhanced transport of certain tracers such as sodium fluorescein, cadaverine-Alexa fluor, horseradish peroxidase, FITC-dextran, albumin-Alexa fluor conjugates, and Evans blue dye across the barrier. The permeability changes in barrier-type endothelial cells can be assessed by intravascular infusion of exogenous tracers and subsequent detection of the extravasated tracer in the brain tissue, which enable functional and structural analysis of BBB integrity. In this chapter, we aimed to highlight the current knowledge on the use of four most commonly performed tracers, namely, Evans blue, sodium fluorescein, albumin-Alexa fluor conjugates, and horseradish peroxidase. The experimental methodologies that we use in our laboratory for the detection of these tracers by macroscopy, spectrophotometry, spectrophotofluorometry, confocal laser scanning microscopy, and electron microscopy are also discussed. Tracing studies at the morphological level are mainly aimed at the identification of the tracers both in the barrier-related cells and brain parenchyma. In addition, BBB permeability to the tracers can be quantified using spectrophotometric and spectrophotofluorometric assays and image analysis by confocal laser scanning microscopy and electron microscopy. The results of our studies conducted under various experimental settings using the mentioned tracers indicate that barrier-type endothelial cells in brain microvessels orchestrate the paracellular and/or transcellular trafficking of substances across BBB. These efforts may not only contribute to designing approaches for the management of diseases/disorders associated with BBB breakdown but may also provide new insights for developing novel brain drug delivery strategies.

Electron microscopic findings in non-alcoholic fatty liver disease: is there a difference between hepatosteatosis and steatohepatitis?

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease has long been accepted as benign; however, recent evidence suggests that the disease may progress to cirrhosis and hepatocellular carcinoma, although the natural course of the disease is still unclear. This study was designed to comparatively evaluate electron microscopic features of non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). METHODS: Quantitative and semi-quantitative ultrastructural evaluations were performed on liver biopsies from 23 patients, 10 with NAFL and 13 with NASH. RESULTS: No statistically significant difference was noted between NAFL and NASH patients in ultrastructural features of hepatocytes including megamitochondria, intramitochondrial crystalline inclusions, mitochondrial matrix granules, foamy cytoplasmic appearance, electron-lucent and glycogen-containing nuclear regions, lipofuscin granules, or an increased frequency of vesicles containing electron-dense material in peribiliary Golgi zone; however, the mitochondrial diameter was significantly higher in the NASH patients. Intercellular distance and microvilli between hepatocytes, collagen and electron-dense material accumulation in the space of Disse, electron-dense material accumulation and microvillus density in bile canaliculi did not differ significantly between the groups. CONCLUSIONS: Our data show that, although NAFL and NASH can be distinguished by their distinct light microscopic features, ultrastructural characteristics are similar, which suggests that NAFL may also have the potential to progress to fibrosis and cirrhosis like NASH.

The effects of magnesium sulfate on cyclophosphamide-induced ovarian damage: Folliculogenesis.

Cyclophosphamide (CYP) is one of the alkylating chemotherapeutic agents and its adverse effects on folliculogenesis in the ovary are well-known due to the previous scientific research on this topic. Magnesium has various effects in organisms, including catalytic functions on the activation and inhibition of many enzymes, and regulatory functions on cell proliferation, cell cycle, and differentiation. In this study, the effects of magnesium sulfate (MgSO4) on CYP induced ovarian damage were investigated. Immature Wistar-Albino female rats of 28-days were treated with pregnant mare serum gonadotrophin (PMSG) to develop the first generation of preovulatory follicles. Rats of the experimental groups were then treated with either CYP (100 mg/kg, i.p) and MgSO4 (270 mg/kg loading dose; 27 mg/kg maintenance doseX12, i.p) solely or in combination. Following in-vivo 5-bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. In the ovaries, added to the evaluation of general morphology and follicle count; BrdU and TUNEL-labeling, cleaved caspase-3 and p27 (cyclin-dependent kinase inhibitor) staining was also performed immunohistochemically and an ultrastructural evaluation was performed by transmission electron microscopy (TEM). The number of primordial follicles were decreased and multilaminar primary and atretic follicles were increased in CYP group. After MgSO4 treatment, while primordial follicle pool were elevated, the number of atretic follicles were decreased. Additionally, decreased BrdU-labeling, increased cleaved caspase 3 immunoreactivity and increased TUNEL labeling were observed in CYP group. In CYP treated animals, observations showed that while MgSO4 administration caused no alterations in BrdU proliferation index and caspase-3 immunoreactivity, it significantly reduced the TUNEL labeling. It was also observed that, while p27 immunoreactivity significantly increased in the nuclei of granulosa and theca cells in the CYP group; MgSO4 treatment significantly reduced these immunoreactivities. The ultrastructural observations showed frequent apoptotic profiles in granulosa and theca cells in both early and advanced stages of follicles in the CYP group and the MgSO4 treatment before the CYP application led to ultrastructural alleviation of the apoptotic process. In conclusion, our data suggest that MgSO4 may provide an option of pharmacologic treatment for fertility preservation owing to the beneficial effects of on chemotherapy-induced accelerated follicular apoptotic process, and the protection of the primordial follicle pool.

The effects of CLP-induced sepsis on proliferation and apoptosis of granulosa and theca cells in rat ovary: A histochemical and ultrastructural study.

Sepsis is defined as a systemic inflammatory response to infection. This study is aimed to evaluate the effects of experimental sepsis on the proliferation and apoptosis of granulosa and theca cells in the rat ovary. 28-day-old immature Wistar-Albino female rats were treated with pregnant mare serum gonadotrophin to develop the first generation of preovulatory follicles. Sepsis was induced by cecal ligation and puncture (CLP). Following in vivo 5-Bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. Besides electron microscopic evaluation, BrdU, cleaved caspase-3, p27 immunostaining, and TUNEL labeling were performed. In CLP-operated animals, cleaved caspase-3 immunoreactivity was significantly increased in Graafian follicles. TUNEL and BrdU labeling in the ovarian follicles were not statistically different between CLP and sham-operated rats. In septic animals, p27 immunoreactivity was increased significantly in the nuclei of oocytes and decreased in the cytoplasm of granulosa and theca cells in multilaminar primary follicles compared to the sham group. In ultrastructural evaluation, increased apoptosis was observed in theca interna and granulosa cells in both the early and late stages of follicles in the CLP group. In conclusion, experimentally-induced sepsis leads to apoptosis in ovarian follicles at advanced stages of development. Our data suggest that although sepsis may not cause a potential threat to developing follicles at least in the short term, more severe damage may occur during advanced stages of follicle development.

Targeted delivery of lacosamide-conjugated gold nanoparticles into the brain in temporal lobe epilepsy in rats.

Temporal lobe epilepsy (TLE) is the most common form of epilepsy with focal seizures, and currently available drugs may fail to provide a thorough treatment of the patients. The present study demonstrates the utility of glucose-coated gold nanoparticles (GNPs) as selective carriers of an antiepileptic drug, lacosamide (LCM), in developing a strategy to cross the blood-brain barrier to overcome drug resistance. Intravenous administration of LCM-loaded GNPs to epileptic animals yielded significantly higher nanoparticle levels in the hippocampus compared to the nanoparticle administration to intact animals. The amplitude and frequency of EEG-waves in both ictal and interictal stages decreased significantly after LCM-GNP administration to animals with TLE, while a decrease in the number of seizures was also observed though statistically insignificant. In these animals, malondialdehyde was unaffected, and glutathione levels were lower in the hippocampus compared to sham. Ultrastructurally, LCM-GNPs were observed in the brain parenchyma after intravenous injection to animals with TLE. We conclude that glucose-coated GNPs can be efficient in transferring effective doses of LCM into the brain enabling elimination of the need to administer high doses of the drug, and hence, may represent a new approach in the treatment of drug-resistant TLE.

Influence of hypercholesterolemia and hypertension on the integrity of the blood-brain barrier in rats.

Hypercholesterolemia and/or hypertension impair endothelial function in peripheral vasculature; however, their impact on endothelial cells of brain microvessels is unclear. We investigated the effects of hypercholesterolemia on the integrity of the blood-brain barrier (BBB) and the activity of astrocytes during N(omega)-nitro-L-arginine methyl ester (L-NAME) hypertension followed by angiotensin (ANG) II. We found significant decreases in superoxide dismutase levels with all treatments except ANG II and L-NAME plus ANG II, and in catalase concentrations except ANG II and cholesterol plus L-NAME. Nitric oxide (NO) concentrations were significantly decreased by L-NAME but significantly increased by cholesterol. L-NAME-stimulated plasma malondialdehyde (MDA), Ox-LDL, and cholesterol levels were significantly augmented by cholesterol. Glutathione (GSH) levels significantly decreased, while MDA, TNF-alpha, and Ox-LDL levels significantly increased in cholesterol and/or L-NAME. The increase in BBB permeability by acute hypertension in hypercholesterolemic hypertensive animals was less than that observed in chronically hypertensive animals. Brain vessels of L-NAME-treated animals showed a considerable loss of immunoreactivity for tight junction proteins, occludin, and ZO-1. Immunoreactivity for occludin and ZO-1 increased in cholesterol plus L-NAME and decreased in cholesterol. Glial fibrillary acidic protein (GFAP) immunoreactivity was seen in few astrocytes in the brain sections of L-NAME-treated animals, but increased in cholesterol plus L-NAME. Positive immunoreactivity for vascular endothelial growth factor (VEGF) was observed in cholesterol and cholesterol plus L-NAME plus ANG II. We suggest that hypercholesterolemia may affect BBB integrity through increasing the expression of tight junction proteins and GFAP and leading to the production of VEGF, at least partly, via increased NO, TNF-alpha, and catalase in hypertensive conditions.

Effects of methyl-beta-cyclodextrin on blood-brain barrier permeability in angiotensin II-induced hypertensive rats.

The blood-brain barrier (BBB) permeability primarily increases in cerebral venules during acute hypertension. Methyl-ß-cyclodextrin (MßCD), a cholesterol-depleting agent, decreases the expression of caveolins disrupting caveolar structures. We aimed to determine the effects of MßCD on the BBB permeability of angiotensin (ANG) II-induced hypertensive rats. Three minutes after MßCD administration (5 mg/kg), acute hypertension was induced by ANG II (60 µg/kg). Evans blue (EB) and horseradish peroxidase (HRP) tracers were used to assess BBB permeability. Immunohistochemistry for caveolin (Cav)-1 and tight junction protein claudin-5 was performed. EB dye content significantly increased in both cerebral cortices and left hippocampus in MßCD (P < 0.05), right cerebral cortex and both hippocampi in ANG II (P < 0.05; P < 0.01), and both cerebral cortices and hippocampi in MßCD plus ANG II groups compared with controls (P < 0.05; P < 0.01). A significant decrease in claudin-5 immunostaining intensity was observed in animals treated with MßCD compared with controls (P < 0.05). Cav-1 immunostaining intensity increased in ANG II group (P < 0.05). Ultrastructurally, HRP reaction products were observed in endothelial cells of the microvessels in the hippocampus region in MßCD group while the tracer was mainly localized in astrocytes and neurons in ANG II, and MßCD plus ANG II groups. The endothelial cells of the venules in the cerebral cortex of the animals in the latter experimental groups also showed an abundance of caveolar vesicles devoid of HRP reaction products. Our results revealed that MßCD did not provide overall protective effects on BBB integrity in acute hypertension and even led to BBB disruption in normotensive animals.

Morphological and functional changes of blood-brain barrier in kindled rats with cortical dysplasia.

Cortical dysplasia (CD) is one of the major causes contributing to epileptogenesis associated with blood-brain-barrier (BBB) disturbances. The current study investigated the functional and ultrastructural changes of BBB in pentylenetetrazole (PTZ)-kindled rats with CD. Pregnant rats on E17 were exposed to 145 cGy of gamma-irradiation and offspring were used for experiments. The rats were given PTZ three times per week to induce kindling. The permeability of BBB was determined by using sodium fluorescein (NaFlu). Immunohistochemistry for occludin, GFAP and c-fos, western-blot analysis for occludin and electron microscopy for the ultrastructural alterations in BBB were performed. The brain level of NaFlu did not increase in rats with CD and/or kindling. Following administration of a convulsive dose of PTZ, a significant increase in BBB permeability was observed in kindled rats with CD. Occludin immunoreactivity and expression remained essentially unchanged in all groups. Slightly enhanced immunoreactivity for GFAP was observed in all groups except control. c-fos immunoreactivity in brain sections of kindled rats with CD displayed a striking increase by convulsive PTZ challenge. Tight junctions were ultrastructurally intact, whereas markedly increased number of pinocytotic vesicles was noted in brain endothelium of kindled rats with CD by convulsive dose of PTZ. The present study showed that epileptic seizures induced by convulsive PTZ challenge during kindling-mediated epileptogenesis in the presence of CD changed both functional and ultrastructural properties of the BBB and considerably enhanced transendothelial vesicular transport, while paracellular pathway was apparently not involved in this setting.

Clinicopathological features of ocular cystinosis.

A 31-year-old woman who presented with photophobia was found to have bilateral corneal and conjunctival crystal deposition. Ocular cystinosis was diagnosed upon observation of typical crystals and lack of systemic involvement. In vivo confocal microscopy confirmed crystal deposition of the corneas and conjunctivae bilaterally. Optical coherence tomography showed stromal hyper-reflectivity due to crystals within the corneal stroma. Transmission electron microscopy of the conjunctiva demonstrated pathognomonic intralysosomal cystine crystals inside fibroblasts and macrophages. Clinicopathological characteristics of ocular cystinosis are well described by this exceptional case.

An experimental Staphylococcus aureus meningitis model for investigating induced leptomeningeal and subpial inflammation in rats: a transmission electron microscopy study.

OBJECTIVE: To evaluate leptomeningeal and subpial inflammatory responses of experimental Staphylococcus aureus bacteriemia following intraperitoneal and intravenous applications and to compare the inflammatory reactions in different regions of central nervous system. MATERIAL AND METHODS: Forty anesthetized rats were divided into four groups equal in number. The rats in group-I were given 1 ml suspension of Staphylococcus aureus intraperitoneally. Group-II was the control group of group I; it was administrated 1 ml 0.9% NaCl in water intraperitoneally. The rats in group-III were given the same amount of bacteria intravenously. Group IV was the control group of the group-III; it was administrated 1 ml 0.9% NaCl solution intravenously. The rats were sacrificed on the 21st day. Inflammatory changes of different regions of the central nervous system were examined under transmission electron microscopy. Statistical analysis was done by using variance analysis, Bonferroni, Tamhane post hoc, Student's t and univariate tests. RESULTS: Thoracic and occipital regions were the most vulnerable zones. Increasing of collagen tissue was the most detected inflammatory change. CONCLUSION: This experimental model can be used for inducing subpial and leptomeningeal inflammations and it may be developed for investigations of pathogenesis of leptomeningitis during systemic infections.

Long-term L-NAME treatment potentiates the blood-brain barrier disruption during pentylenetetrazole-induced seizures in rats.

We investigated whether the severity of blood-brain barrier disruption caused by pentylenetetrazole-induced seizures is modified by long-term nitric oxide synthase inhibition in rats. Rats were given N-omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, in drinking water for 4 weeks, and then treated with pentylenetetrazole to induce seizures. Damage to the blood-brain barrier was investigated using Evans blue dye extravasation. Serum nitric oxide concentration was decreased in L-NAME-treated rats (P<0.01). L-NAME and/or pentylenetetrazole treatments elevated systolic blood pressure of animals (P<0.01). L-NAME caused an increase in the mortality rate after pentylenetetrazole injection leading to the death of animals at about 15 min after the onset of the seizure. Pentylenetetrazole-induced seizures in rats treated with L-NAME caused a significant increase in Evans blue dye extravasation into cerebral cortex, diencephalon and cerebellum, as compared with seizures evoked by pentylenetetrazole injection to L-NAME-untreated rats (P<0.01). Data presented here suggest that the degree of blood-brain barrier disruption induced by seizures is more pronounced in long-term nitric oxide deficiency.

Effects of lipopolysaccharide on blood-brain barrier permeability during pentylenetetrazole-induced epileptic seizures in rats.

We investigated the effects of lipopolysachharide (LPS) on functional and structural properties of the blood-brain barrier (BBB) during pentylenetetrazole (PTZ)-induced epileptic seizures in rats. Arterial blood pressure was significantly elevated during epileptic seizures irrespective of LPS pretreatment. Plasma levels of interleukin (IL)-1, interleukin (IL)-6, nitric oxide (NO) and malondialdehyde (MDA) increased while catalase concentrations decreased in animals treated with LPS, PTZ and LPS plus PTZ. The significantly increased BBB permeability to Evans blue (EB) dye in the cerebral cortex, diencephalon and cerebellum regions of rats by PTZ-induced seizures was markedly reduced upon LPS pretreatment. Immunoreactivity for tight junction proteins, zonula occludens-1 and occludin, did not change in brain vessels of animals treated with PTZ and LPS plus PTZ. Glial fibrillary acidic protein immunoreactivity was increased in LPS, but not in PTZ and LPS plus PTZ. These results indicate that LPS pretreatment reduces the passage of EB dye bound to albumin into the brain, at least partly, by increasing plasma NO and IL-6 levels during PTZ-induced epileptic seizures. We suggest that LPS may provide protective effects on the BBB integrity during epileptic seizures through transcellular pathway, since the paracellular route remained unaffected by LPS and LPS plus PTZ.