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.

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.

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.

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.

Evaluation of fatal diving accidents in Turkey

In any kind of diving there is a risk of accidents, as the move from the topside environment to underwater can affect a diver's physiological and psychological condition. It is important to investigate dive accidents to clarify the causative factors and determine preventive measures. In this study, autopsy files of fatal dive accident cases were reviewed to evaluate demographic data, type of diving, purpose of dive, seasonal distribution, autopsy findings, and causes of death. We reviewed 56 fatal dive accident files from autopsy units in cities where dive activities are concentrated and from the archive of the Turkish Underwater Federation. Four cases were excluded from the study since we were unable to obtain autopsy reports. Of 52 cases there were 20 scuba divers, two surface-supplied divers and 30 breath-hold divers. The majority of cases involved males (94%). The average age of 50 cases was 38.6; age estimation for two cases could not be determined due to advanced putrefaction. Of these fatal dive accidents 75% took place over a period of six months between May and October. Drowning was recorded as the primary cause of death in these cases. X-ray imaging was used in four (8%) cases. A special autopsy technique was used for nine (17%) cases, to detect possible pulmonary barotrauma and arterial gas embolism. The forensic specialist who is planning to conduct the autopsy for a dive fatality should have knowledge and experience about dive physics and physiology as well as physiopathology of dysbaric injuries.

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.

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.

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.

Neuroprotective effects of intravenous immunoglobulin are mediated through inhibition of complement activation and apoptosis in a rat model of sepsis.

BACKGROUND: Intravenous (IV) immunoglobulin (Ig) treatment is known to alleviate behavioral deficits and increase survival in the experimentally induced model of sepsis. To delineate the mechanisms by which IVIg treatment prevents neuronal dysfunction, an array of immunological and apoptosis markers was investigated. METHODS: Sepsis was induced by cecal ligation perforation (CLP) in rats. The animals were divided into five groups: sham, control, CLP + saline, CLP + immunoglobulin G (IgG) (250 mg/kg, iv), and CLP + immunoglobulins enriched with immunoglobulin M (IgGAM) (250 mg/kg, iv). Blood and brain samples were taken in two sets of experiments to see the early (24 h) and late (10 days) effects of treatment. Total complement activity, complement 3 (C3), and soluble complement C5b-9 levels were measured in the sera of rats using ELISA-based methods. Cerebral complement, complement receptor, NF-κB, Bax, and Bcl-2 expressions were analyzed by western blot and/or RT-PCR methods. Immune cell infiltration and gliosis were examined by immunohistochemistry using CD3, CD4, CD8, CD11b, CD19, and glial fibrillary acidic protein antibodies. Apoptotic neuronal death was investigated by TUNEL staining. RESULTS: IVIgG and IgGAM administration significantly reduced systemic complement activity and cerebral C5a and C5a receptor expression. Likewise, both treatment methods reduced proapoptotic NF-κB and Bax expressions in the brain. IVIgG and IgGAM treatment induced considerable amelioration in glial cell proliferation and neuronal apoptosis which were increased in non-treated septic rats. CONCLUSIONS: We suggest that IVIgG and IgGAM administration ameliorates neuronal dysfunction and behavioral deficits by reducing apoptotic cell death and glial cell proliferation. In both treatment methods, these beneficial effects might be mediated through reduction of anaphylatoxic C5a activity and subsequent inhibition of inflammation and apoptosis pathways.

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.

Effects of beta-hydroxybutyrate on brain vascular permeability in rats with traumatic brain injury.

This study investigates the effect of beta-hydroxybutyrate (BHB) on blood-brain barrier (BBB) integrity during traumatic brain injury (TBI) in rats. Evans blue (EB) and horseradish peroxidase (HRP) were used as determinants of BBB permeability. Glutathione (GSH) and malondialdehyde (MDA) levels were estimated in the right (injury side) cerebral cortex of animals. The gene expression levels for occludin, glucose transporter (Glut)-1, aquaporin4 (AQP4) and nuclear factor-kappaB (NF-κB) were performed, and Glut-1 and NF-κB activities were analyzed. BHB treatment decreased GSH and MDA levels in intact animals and in those exposed to TBI (P<0.05). Glut-1 protein levels decreased in sham, BHB and TBI plus BHB groups (P<0.05). NF-κB protein levels increased in animals treated with BHB and/or exposed to TBI (P<0.05). The expression levels of occludin and AQP4 did not significantly change among experimental groups. Glut-1 expression levels increased in BHB treated and untreated animals exposed to TBI (P<0.05). While NF-κB expression levels increased in animals in TBI (P<0.01), a decrease was noticed in these animals upon BHB treatment (P<0.01). In animals exposed to TBI, EB extravasation was observed in the ipsilateral cortex regardless of BHB treatment. Ultrastructurally, BHB attenuated but did not prevent the presence of HRP in brain capillary endothelial cells of animals with TBI; moreover, the drug also led to the observation of the tracer when used in intact rats (P<0.01). Altogether, these results showed that BHB not only failed to provide overall protective effects on BBB in TBI but also led to BBB disruption in healthy animals.

Effects of intravenous immunoglobulin therapy on behavior deficits and functions in sepsis model.

BACKGROUND: We aim to demonstrate behavioral alterations in a sepsis model using intravenous (IV) immunoglobulin G (IgG) and immunoglobulins enriched with IgA and IgM (IgGAM). METHODS: We divided 48 Wistar albino rats into five groups: control group, sham-operated group (only antibiotic treatment), cecal ligation and puncture (CLP) group (CLP plus antibiotic treatment), IgG group (250 mg/kg IV IgG) and IgGAM group (250 mg/kg IV IgGAM). Intravenous immunoglobulins were given 5 min after the CLP procedure. Experimental animals put into three behavioral tasks 10, 30 and 60 days after the surgery; to evaluate the locomotor activity, an open field test was performed, elevated plus maze test was used to measure anxiety levels, and depressive state was assessed by forced swimming test. The effects of therapy which were acquired from the results of these tests were used to estimate the behavioral changes after CLP. RESULTS: The mortality rate of 50% in the septic rats decreased to 30 and 20% with the administration of IgG and IgGAM, respectively. Significant changes on locomotor activity and depressive-like behavior were reported in the sepsis group; on the other hand, the treatment with immunoglobulins reduced the symptoms. Treatment with immunoglobulins attenuated the sepsis-related anxiogenic-like responses. Behavioral alterations returned to normal on day 60 in all groups. CONCLUSIONS: Sepsis caused deterioration on behavioral parameters. Immunoglobulin treatments alleviated the symptoms of functional disturbances and caused early reversal of behavioral deficits in septic animals.

The effects of superoxide dismutase mimetic MnTMPyP on the altered blood-brain barrier integrity in experimental preeclampsia with or without seizures in rats.

We investigated the effects of a cell-permeable superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) on blood-brain barrier (BBB) integrity following pentylenetetrazole (PTZ)-induced seizures in experimental preeclampsia symptoms induced by N(omega)-nitro-l-arginine methyl ester (l-NAME) in pregnant rats. To show the functional and morphological alterations in BBB integrity, quantitative analysis of sodium fluorescein (NaFlu) extravasation, immunohistochemistry and electron microscopic assessment of horseradish peroxidase (HRP) permeability were performed. Varying degrees of proteinuria were seen and arterial blood pressure increased in l-NAME-treated pregnant rats (p<0.01). MnTMPyP pretreatment and convulsive PTZ challenge significantly decreased the immunoreactivity of occludin in hippocampal capillaries in l-NAME-treated pregnant rats (p<0.01). BBB permeability to NaFlu significantly increased in pregnant rats treated with l-NAME plus PTZ (p<0.01), but MnTMPyP pretreatment did not significantly decrease NaFlu penetration into the brain parenchyma in these animals. 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 and l-NAME plus PTZ with the abundance being more in the latter group. MnTMPyP pretreatment caused a marked reduction in the frequency of HRP reaction product containing vesicles in both experimental settings. In conclusion, the results of the present study provide evidence that MnTMPyP plays an important role in limiting the enhanced vesicle-mediated transcellular transport in BBB endothelium in a rat model of preeclampsia and the differences in the way of transports of NaFlu and HRP might be responsible for the different effects of MnTMPyP on the BBB permeability to these two tracers.

The effects of atorvastatin on oxidative stress in L-NAME-treated rats.

OBJECTIVES: Current evidence suggests that the beneficial vascular effects of statins are not limited to the statins' lipid-lowering properties; these drugs can also improve vascular endothelial cell function. Nω-nitro-l-arginine methyl ester (L-NAME) is a potent synthetic nitric oxide inhibitor, and long-term oral L-NAME treatment is used to induce vascular lesions in experimental animal models. METHODS: We determined the effects of statins on protein carbonyl (PCO), lipid hydroperoxides (LHP), oxidized low-density lipoproteins (ox-LDL) and antioxidants such as paraoxonase 1 (PON1) and total thiols (T-SH) in long-term L-NAME-treated rats. Adult male Wistar rats were divided into three groups, namely, control, L-NAME-treated (1 mg/mL in drinking water for three weeks), and atorvastatin plus L-NAME-treated (4 mg/kg/day atorvastatin for 1 week during the third week of L-NAME treatment) groups. RESULTS: In the L-NAME group, the ox-LDL, LHP and PCO were higher and the PON1 and T-SH were lower than the concentrations observed for the controls. When compared with the L-NAME group, the L-NAME plus atorvastatin group had significantly lower ox-LDL and LHP and higher PON1 activities. Additionally, the elevated total cholesterol (TC) and low-density lipoprotein-C (LDL-C) in the L-NAME group were decreased by atorvastatin administration. TC and LDL-C were positively correlated with ox-LDL and LHP and negatively correlated with PON1 in all groups. High-density lipoprotein-C (HDL-C) was negatively correlated with ox-LDL. CONCLUSION: PON1 prevents LDL oxidation and inactivates LDL-derived oxidized phospholipids; its activity showed a pronounced decrease in the L-NAME treatment group and was increased in the atorvastatin group. Based on our findings, we concluded that the atorvastatin had HDL-related antioxidant activity as well as lipid-lowering properties.

The effects of hyperbaric air and hyperbaric oxygen on blood-brain barrier integrity in rats.

Hyperbaric oxygen (HBO) treatment yields conflicting results on blood-brain barrier (BBB) integrity under various pathological conditions and the effects of HBO on healthy brain is poorly understood. In this experimental study, the effects of HBO on BBB integrity were investigated in comparison with hyperbaric air (HBA) in intact rats. Four sessions of HBA or HBO were applied to intact rats in 24h. BBB integrity was functionally and structurally evaluated by determining extravasation of Evans blue (EB) dye and horseradish peroxidase (HRP) tracers. In immunohistochemical evaluation, relative staining intensity for occludin, a tight junction (TJ) protein, and aquaporin 4 (AQP4), a water-channel protein, was detected in the barrier type of microvessels of brain by image analysis. BBB permeability to EB dye significantly increased in animals in HBO treatment group compared to those in HBA and control groups (p<0.05). The immunoreactivity of occludin, a tight junction protein, remained essentially unaltered in capillaries of hippocampus in all groups. In animals exposed to HBO, AQP4 immunoreactivity significantly increased in parietal cortex compared to those in HBA and control groups (p<0.01). Ultrastructurally, frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in cerebral cortex and hippocampus of rats subjected to both HBA and HBO. Our results indicate that the HBO administration to intact rats increased BBB permeability to both EB and HRP while HBA increased only HRP extravasation in these animals. The results of this study suggest that HBA also impairs the BBB integrity in intact rats as well as HBO.

Vagus nerve stimulation inhibits seizure activity and protects blood-brain barrier integrity in kindled rats with cortical dysplasia.

AIMS: This study investigates the effects of vagus nerve stimulation (VNS) on seizure severity and blood-brain barrier (BBB) integrity in kindled rats with cortical dysplasia (CD). MAIN METHODS: Pregnant rats were exposed to 145 cGy of gamma-irradiation on day 17 of pregnancy. In offsprings, kindling was induced by giving subconvulsive doses of pentylenetetrazole. Left VNS was performed for 48 h at output currents of 0.5 or 1 mA. Horseradish peroxidase (HRP) was used to study the BBB permeability. Immunohistochemistry for occludin and P-glycoprotein (P-gp) was also performed. KEY FINDINGS: Kindled rats with CD exhibited seizures with mean Racine's scores of 3.57 ± 1.2 during video EEG recording. Kindled animals with CD receiving VNS at 0.5 and 1.0 mA did not exhibit either clinical or electrophysiological signs of seizure. Immunostaining for occludin, a tight junction protein, in hippocampus remained relatively intact in all groups. VNS-treated and -untreated kindled animals with CD revealed intense immunostaining for P-gp in hippocampal formation (P<0.01). Electron microscopic observations revealed frequent transport vesicles containing electron-dense HRP reaction products in the cytoplasm of brain capillary endothelial cells in both cerebral cortex and hippocampus of kindled animals with CD. Those which were exposed to 1 mA VNS were observed to have brain capillary endothelial cells largely devoid of HRP reaction products in both cerebral cortex and hippocampus. SIGNIFICANCE: The results of this study suggest that VNS therapy at 1 mA inhibits seizure activity and protects BBB integrity by limiting the enhancement of transcellular pathway in kindled animals with CD.

Topiramate reduces blood-brain barrier disruption and inhibits seizure activity in hyperthermia-induced seizures in rats with cortical dysplasia.

We investigated the effects of topiramate (TPM), a novel broad spectrum anticonvulsant, on seizure severity, survival rate and blood-brain barrier (BBB) integrity during hyperthermic seizures in rats with cortical dysplasia (CD). Offsprings of irradiated mothers were used in this study. To show the functional and morphological alterations in BBB integrity, quantitative analysis of Evans blue (EB) extravasation, immunohistochemistry and electron microscopic assessment of horseradish peroxidase (HRP) permeability were performed. Rats with CD exposed to hyperthermia exhibited seizures with mean Racine's scores of 3.92 ± 1.2. Among the rats with CD pretreated with TPM, 21 of 24 rats showed no sign of seizure activity upon exposure to hyperthermia (p<0.01). The immunoreactivity of occludin, a tight junction protein, remained essentially unaltered in capillaries of hippocampus in all groups. In animals with CD exposed to hyperthermia, the significantly increased p-glycoprotein immunoreactivity in hippocampus (p<0.01) was slightly decreased by TPM pretreatment. Hyperthermic seizures increased BBB permeability to EB in animals with CD, but TPM pretreatment decreased the penetration of the tracer into the brain in these animals (p<0.01). Ultrastructurally frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in cerebral cortex and hippocampus of rats with CD subjected to hyperthermia-induced seizures, and TPM pretreatment prevented the development of HRP reaction products in these animals. The results of this study suggest that TPM inhibits seizure activity and maintains BBB integrity in the course of febrile seizures in the setting of CD.

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.

The effects of hyperbaric oxygen therapy on blood-brain barrier permeability in septic rats.

The mechanisms underlying the changes in blood-brain barrier (BBB) integrity in septic encephalopathy are poorly understood. The present study was designed to examine whether hyperbaric oxygen therapy (HBOT) influences the response of BBB to sepsis induced by cecal ligation and puncture (CLP) in rats. Cerebral cortical and hippocampal tissue levels of tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA) and glutathione (GSH) levels were measured. BBB permeability was functionally and structurally evaluated by determining extravasation of Evans blue (EB) and horseradish peroxidase (HRP) tracers, respectively. Immunohistochemistry and western blotting for occludin were performed. HBOT did not alter TNF-α levels in CLP operated rats while a significant decrease was noted when the therapy was subjected to intact rats. MDA levels in animals subjected to CLP plus HBOT were significantly decreased. In septic rats, the decreased GSH levels were significantly increased by HBOT. While HBOT attenuated the increased BBB permeability to EB in rats subjected to CLP (P<0.01), no macroscopic alteration was observed in the enhanced HRP extravasation. An increase in HRP extravasation was also observed by HBOT in intact animals. Occludin immunoreactivity and expression remained essentially unchanged in the brain capillaries of animals in all groups. Ultrastructurally, frequent vesicles containing HRP reaction products were observed in brain capillary endothelial cells of animals treated with CLP and/or HBOT. In conclusion, our results revealed that HBOT did not provide overall protective effects on the BBB integrity in septic conditions and even led to BBB disruption in intact animals.

Effects of levetiracetam on blood-brain barrier disturbances following hyperthermia-induced seizures in rats with cortical dysplasia.

AIMS: The mechanisms underlying the changes in blood-brain barrier (BBB) integrity and the generation of seizures in childhood associated with preexisting brain lesions like cortical dysplasia (CD) are poorly understood. We investigated the effects of levetiracetam (LEV) on BBB integrity and the survival during hyperthermic seizures in rats with CD. MAIN METHODS: Pregnant rats were exposed to 145 cGy of gamma-irradiation on embryonic day 17. On postnatal day 28, hyperthermia-induced seizures were evoked in offspring with CD. To show the functional and morphological alterations in BBB integrity, quantitative analysis of sodium fluorescein (NaFlu) extravasation, immunohistochemistry and electron microscopy were performed. KEY FINDINGS: Seizure scores and mortality rates were decreased by LEV during hyperthermia-induced seizures in rats with CD (P<0.01). Increased NaFlu extravasation into brain by hyperthermia-induced seizures in animals with CD was decreased by LEV (P<0.01). While glial fibrillary acidic protein (GFAP) immunoreactivity slightly increased in brain sections of animals with CD during hyperthermia-induced seizures, LEV led to GFAP immunoreactivity comparable to that of controls. Decreased occludin immunoreactivity and expression in CD plus hyperthermia-induced seizures was increased by LEV. Opening of tight junctions and abundance of pinocytotic vesicles representing ultrastructural evidences of BBB impairment and severe perivascular edema were observed in animals with CD exposed to hyperthermia-induced seizures and LEV treatment led to the attenuation of these findings. SIGNIFICANCE: These results indicate that LEV may present a novel approach for the protection of the BBB besides its antiepileptic impact on hyperthermic seizures in the setting of CD.