Nurcan Orhan

Effect of the methylenetetrahydrofolate reductase gene polymorphisms on homocysteine, folate and vitamin B12 in patients with bipolar disorder and relatives

We investigated the effect of polymorphic variants of c.1298A>C (Glu429Ala) and c.677C>T (Ala222Val) in methylenetetrahydrofolate (MTHFR) gene on the total homocysteine (tHcy), folate and B12 levels in patients with bipolar disorder, first-degree relatives of patients, and controls. The c.677C>T and c.1298A>C polymorphisms in MTHFR were determined by polymerase chain reaction-restriction fragment length polymorphism in 197 bipolar patients, 278 relatives and 238 controls. tHcy and folate and vitamin B12 levels were measured by Fluorescence Polarization Immunoassay and Electrochemiluminescence, respectively. The tHcy was significantly increased in patients and relatives. In contrast, folate and B12 were significantly lower in patients and relatives. Gender was not considered as a significant determinant in the multivariate analysis. Genotypes of c.1298A>C and c.677C>T were correlated with tHcy, folate and B12. Patients and relatives carrying TT and/or AA and AC genotypes had elevated tHcy and reduced folate and B12 levels. High tHcy but low folate and vitamin B12 levels may be a risk factor for development of bipolar disorder.

Combined effects of ACE and MMP‐3 polymorphisms on migraine development

Migraine is a primary headache disorder which involves both genetic and environmental components. Since angiotensin-converting enzyme (ACE) and matrix metalloproteinase (MMP) share the same homology, we investigated whether the MMP-3 and ACE I/D gene variants are involved in migraine risk and whether the ACE variant might act in combination with the MMP-3 genetic variant in patients with migraine. This is the first study to evaluate the association between MMP-3 and ACE polymorphisms, and migraine. Genotypes were determined by polymerase chain reaction. The frequencies of 5A5A genotypes of the MMP-3 and D allele of ACE were significantly elevated, but II genotypes of the ACE and 6A allele of MMP-3 significantly decreased in all patients. The combined DD/5A5A and ID/5A5A genotypes increased the risk of migraine. Individuals who were homozygous for the deletion (D) allele showed increased ACE activity. Subjects with the 5A5A genotype and/or D allele or with the combined DD/5A5A or ID/5A5A might be more susceptible to migraine development. In contrast, subjects with the II and/or 6A6A genotypes may be protected from migraine development. The greater activity of the 5A5A and DD genotypes might result in vascular reactivity that is more pronounced in migraine. Taken together, our data suggest that numerous genes may influence ACE activity. Discovery of new genes might better clarify the pathogenesis of migraine and open an avenue to therapeutic strategies against migraine.

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. (Crit Care Med 2012; 40:–1220)

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.

Levetiracetam decreases the seizure activity and blood-brain barrier permeability in pentylenetetrazole-kindled rats with cortical dysplasia

This study investigates the effects of levetiracetam (LEV) on the functional and structural properties of blood-brain barrier (BBB) in pentylenetetrazole (PTZ)-kindled rats with cortical dysplasia (CD). Pregnant rats were exposed to 145 cGy of gamma-irradiation on embryonic day 17. In offsprings, kindling was induced by giving subconvulsive doses of PTZ three times per week for 45 days. While all kindled rats with CD died during epileptic seizures evoked by the administration of a convulsive dose of PTZ in 15 to 25 min, one week LEV (80 mg/kg) pretreatment decreased the mortality to 38% in the same setting. LEV caused a remarkable decrease (p<0.01) in extravasation of sodium fluorescein dye into the brain tissue of kindled animals with CD treated with convulsive dose of PTZ. Occludin immunoreactivity and expression remained essentially unchanged in all groups. Immunoreactivity for glial fibrillary acidic protein (GFAP) was observed to be slightly increased by acute convulsive challenge in kindled rats with CD while LEV pretreatment led to GFAP immunoreactivity comparable to that of controls. An increased c-fos immunoreactivity in kindled rats with CD exposed to convulsive PTZ challenge was also observed with LEV pretreatment. Tight junctions were ultrastructurally intact, whereas LEV decreased the increased pinocytotic activity in brain endothelium of kindled rats with CD treated with convulsive dose of PTZ. The present study showed that LEV decreased the increased BBB permeability considerably by diminishing vesicular transport in epileptic seizures induced by convulsive PTZ challenge in kindled animals with CD.

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.

Paraoxonase-1 55/192 genotypes in schizophrenic patients and their relatives in Turkish population.

Background Oxidative stress and free radical-induced toxicity have been implicated in the pathophysiology of schizophrenia. In this study, we examined paraoxonase (PON1)-55/192 polymorphisms and PON1 activity in patients with schizophrenia, first-degree relatives of schizophrenic patients, and healthy controls. Methods This study consisted of 292 healthy participants, 267 unrelated patients with schizophrenia and 311 first-degree relatives of schizophrenic patients. PON1 55 (rs 854560) and PON1 192 (rs 662) polymorphisms were performed by restriction fragment length polymorphism. Results The frequencies of the QQ and LL genotypes were significantly overpresented in controls compared with those of schizophrenic patients and their relatives. In contrast, the RR genotype was more prevalent in patients than their relatives and healthy controls. The frequencies of the LM and QR genotypes in relatives were higher than controls. Serum PON1 activities of controls were significantly higher when compared with both schizophrenic patients and their relatives. The RR and LL genotypes were associated with a significantly increased PON1 activity as compared with QR or QQ and MM or LM genotypes, respectively, in all groups. Conclusion This is the first study that shows the association between PON1-55/192 polymorphisms and schizophrenia. Our data suggest that the subjects carrying R allele or RR genotype might be susceptible to schizophrenia and subjects with QQ or LL might be protected against schizophrenia. First-degree relatives of schizophrenic patients have higher heterozygote genotypes, suggesting that this group can shift either to patient or control group depending on their allele types and environmental factors. PON1 genetic variations are also associated with PON1 activities. Reduced PON1 activity in patients and their relatives might result from the combined effects of more than one polymorphic variant in PON1 or other genes and/or increased oxidative stress, supporting the hypothesis that reactive oxygen species-mediated cellular damage might contribute to the neuropathology of schizophrenia.

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 Racines 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.

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.

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.