Effects of intermittent theta-burst transcranial magnetic stimulation on cognition and hippocampal volumes in bipolar depression.

INTRODUCTION: Repetitive transcranial magnetic stimulation (TMS) is increasingly used to treat neurocognitive symptoms in mood disorders. Intermittent theta burst stimulation (iTBS) is a brief version of TMS that may preferentially target cognitive functions. This study evaluated whether iTBS leads to cognitive improvements and associated increased hippocampal volumes in bipolar depression. METHODS: In a two-site double-blind randomised sham controlled trial (NCT02749006), 16 patients received active iTBS to the Left Dorsolateral Prefrontal Cortex (DLPF) and 15 patients received sham stimulation across four weeks. A composite neuropsychological score and declarative memory scores served as the cognitive outcomes. Hippocampal volumes were derived from T1 weighted MRI scans using the longitudinal ComBat method to harmonise data across sites. RESULTS: No significant improvements were observed in any cognitive variables in the active relative to the sham group; however, there was a trend for increased left hippocampal volume in the former. Left hippocampal volume increases were associated with improvements in nonverbal memory in the active group. CONCLUSIONS: Although cognitive improvements were not associated with iTBS, the finding that hippocampal volume increases were associated with memory improvement suggests there may be some level of prefrontal-temporal neuroplasticity that could support cognitive change in future studies of iTBS in bipolar disorder.

Lurasidone versus treatment as usual for cognitive impairment in euthymic patients with bipolar I disorder: a randomised, open-label, pilot study.

BACKGROUND: Cognitive impairment is present in euthymic patients with bipolar disorder and correlates with impairments in everyday functioning. We aimed to examine the efficacy of lurasidone adjunctive therapy compared with treatment as usual (TAU) in improving cognition. METHODS: For this randomised, open-label, pilot study, we recruited patients aged 19-65 years with euthymic bipolar I disorder from the Mood Disorder Centre in UBC Hospital (Vancouver, Canada). We included patients who were taking lithium, or valproate, or an atypical antipsychotic, or a combination of these for mood stabilisation and who showed reduced cognitive functioning (SD≤ -0·25 relative to demographics-corrected norms) on either the Trail Making Test-B or the California Verbal Learning Test-II. Patients were randomly assigned using a randomised block design with a block size of four to TAU or lurasidone adjunctive therapy (20-80 mg/day) for 6 weeks. A research coordinator masked to group allocation administered the International Society for Bipolar Disorders Battery for Assessment of Neurocognition (ISBD-BANC) at baseline and at endpoint. The primary outcome was change in global cognition score, which consisted of the mean demographics-corrected t-score value of the several ISBD-BANC measures, analysed in all patients who completed both tests. This trial is registered on ClinicalTrials.gov, number NCT02147379. FINDINGS: Between July 2, 2014, and Oct 19, 2015, 34 patients were randomly allocated to lurasidone adjunctive therapy (17 patients) or TAU (17 patients). Two patients from each group did not complete the study. The mean lurasidone dose was 48·24 (SD 15·90) mg/day. Lurasidone adjunctive therapy was more effective than TAU in improving the primary efficacy measure of ISBD-BANC global cognition score (mean difference 2·92 [95% CI 0·27-5·57]; time × treatment interaction F=5·09; p=0·032). The between-group effect size (0·82) on baseline versus study-end difference scores in the ISBD global cognition score was of moderate to large magnitude. The magnitude of benefit with lurasidone adjunctive therapy in improving global cognition (effect size 0·46) was greater compared with the improvement observed in the TAU group (0·04). Adverse events were reported by nine (60%) patients in the luradisone group and two (13%) in the TAU group. INTERPRETATION: Our results provide some preliminary evidence for the efficacy of lurasidone in improving cognition in euthymic patients with bipolar I disorder. The strengths of this study were the characterisation of the sample and use of tests sensitive to cognitive impairment in bipolar disorder. Its limitations were the sample size and inability to completely control for other medication use. Larger double-blind trials are warranted to investigate this further. FUNDING: Sumitomo Dainippon Pharma.

SPARC expression by cerebral microvascular endothelial cells in vitro and its influence on blood-brain barrier properties.

BACKGROUND: SPARC (secreted protein acidic and rich in cysteine) is a nonstructural, cell-matrix modulating protein involved in angiogenesis and endothelial barrier function, yet its potential role in cerebrovascular development, inflammation, and repair in the central nervous system (CNS) remains undetermined. METHODS: This study examines SPARC expression in cultured human cerebral microvascular endothelial cells (hCMEC/D3)-an in vitro model of the blood-brain barrier (BBB)-as they transition between proliferative and barrier phenotypes and encounter pro-inflammatory stimuli. SPARC protein levels were quantified by Western blotting and immunocytochemistry and messenger RNA (mRNA) by RT-PCR. RESULTS: Constitutive SPARC expression by proliferating hCMEC/D3s is reduced as cells mature and establish a confluent monolayer. SPARC expression positively correlated with the proliferation marker Ki-67 suggesting a role for SPARC in cerebrovascular development. The pro-inflammatory molecules tumor necrosis factor-α (TNF-α) and endotoxin lipopolysaccharide (LPS) increased SPARC expression in cerebral endothelia. Interferon gamma (IFN-γ) abrogated SPARC induction observed with TNF-α alone. Barrier function assays show recombinant human (rh)-SPARC increased paracellular permeability and decreased transendothelial electrical resistance (TEER). This was paralleled by reduced zonula occludens-1 (ZO-1) and occludin expression in hCMEC/D3s exposed to rh-SPARC (1-10 µg/ml) compared with cells in media containing a physiological dose of SPARC. CONCLUSIONS: Together, these findings define a role for SPARC in influencing cerebral microvascular properties and function during development and inflammation at the BBB such that it may mediate processes of CNS inflammation and repair.

Somatostatin preserved blood brain barrier against cytokine induced alterations: possible role in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory neurological disorder associated with demyelination, impaired blood brain barrier (BBB), axonal damage and neuronal loss. In the present study, we measured somatostatin (SST) and tumor necrosis factor-α (TNF-α) like immunoreactivity in CSF samples from MS and non-MS patients. We also examined the role of SST in cytokines and lipopolysaccharide (LPS)-induced damage to the BBB using human brain endothelial cells in culture. Most of the cerebrospinal fluid (CSF) samples studied from definite MS patients exhibited lower somatostatin (SST)-like immunoreactivity and higher expression of TNF-α in comparison to non-MS patients. Treatment of cells with cytokines and LPS blocked SST secretion and decreased SST expression. Human brain endothelial cells expressed all five somatostatin receptors (SSTRs) with increased expression of SSTR2 and 4 upon treatment with cytokines and LPS. Cytokines and LPS-induced disruption of the tight junction proteins Zonula occludens (ZO-1) organization was restored in presence of SST, SSTR2 or SSTR4 selective agonists. Furthermore, inflammation induced changes in extracellular signal-regulated kinases (ERK1/2 and ERK5) signaling and altered expression of endothelial and inducible nitric oxide synthase are modulated in presence of SST. These data indicate that decreased levels of SST contribute to failure of the BBB in MS.

Blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE.

Multiple sclerosis (MS) is a demyelinating disease characterized by the breakdown of the blood-brain barrier (BBB), and accumulation of inflammatory infiltrates in the central nervous system. Tight junctions are specialized cell-cell adhesion structures and critical components of the BBB that have previously been shown to be abnormally distributed in MS tissue. To evaluate whether experimental autoimmune encephalomyelitis (EAE) provides a suitable model for this aspect of MS disease, we examined the expression and distribution of ZO-1 over the course of disease in EAE. We observed a dramatic relocalization of ZO-1 which precedes overt clinical disease and correlates with the sites of inflammatory cell accumulation. Treatment of in vitro cultures of murine brain endothelial cells with components of EAE induction provided similar findings, with relocalization of ZO-1 and increased permeability of endothelial monolayers. BBB disruption in the EAE model appears to parallel disease progression in MS, with direct effects on the cerebrovascular endothelium, making it an ideal tool for future evaluation of tight junction breakdown and repair in MS-like pathology.

Activation of phospholipase A2 is involved in indomethacin-induced damage in Caco-2 cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs), widely used in clinical practice, cause adverse effects in the gastrointestinal tract. These effects have been attributed to mechanisms such as drug-induced cyclooxygenase inhibition, oxidative stress, mitochondrial dysfunction and changes in cell membrane lipids. Our previous study showed that indomethacin (an NSAID commonly used in toxicity studies) caused activation of cytosolic phospholipase A(2) (cPLA(2)) in the rat small intestine. We hypothesized that activation of cPLA(2) is an important event in the pathogenesis of indomethacin-induced damage in enterocytes. To test this, we incubated enterocyte-like Caco-2 cells with indomethacin, with and without pretreatment with methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA(2). Cells treated with indomethacin showed decreased viability and evidence of oxidative stress and morphological cell damage. Phospholipids were degraded in these cells, with increases in the levels of lysophospholipids and arachidonic acid. There was no evidence of apoptosis in the cells in response to the drug. Pretreatment of the cells with MAFP attenuated the drug-induced effects seen. This shows that activation of phospholipase A(2) appears to be an important event in the pathogenesis of indomethacin-induced damage in Caco-2 cells. To our knowledge, this is the first report that implicates the involvement of this enzyme in NSAID-induced enteropathy.

Curcumin attenuates indomethacin-induced oxidative stress and mitochondrial dysfunction.

Oxidative stress and mitochondrial dysfunction have been implicated in the pathogenesis of indomethacin-induced enteropathy. We evaluated the potential of curcumin, a known cytoprotectant, as an agent to protect against such effects. Rats were pretreated with curcumin (40 mg/kg by intra-peritoneal injection) before administration of indomethacin (20 mg/kg by gavage). One hour later, the small intestine was isolated and used for assessment of parameters of oxidative stress. Mitochondria, brush border membranes (BBM) and surfactant-like particles (SLP) were also isolated from the tissue. Mitochondria were used for assessment of functional integrity, estimation of products of lipid peroxidation and lipid content. BBM were used for estimation of products of lipid peroxidation and lipid content, while the SLP were used for measurement of lipid content. The results showed that oxidative stress and mitochondrial dysfunction occurred in the small intestine of indomethacin-treated rats. Pre-treatment with curcumin was found to ameliorate these drug-induced changes. Significant changes were seen in some of the lipids in the mitochondria, BBM and SLP in response to indomethacin. However, curcumin did not have any significant effect on these drug-induced changes. We conclude that curcumin, by attenuating oxidative stress and mitochondrial dysfunction, holds promise as an agent that can potentially reduce NSAID-induced adverse effects in the small intestine.

Curcumin reduces indomethacin-induced damage in the rat small intestine.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in clinical medicine. Their utility is, however, often limited by the adverse effects they produce in the gastrointestinal tract. Oxidative stress has been shown to occur in the small intestine in response to the oral administration of indomethacin, an NSAID commonly used in toxicity studies. In view of this, the effect of curcumin, an agent with anti-oxidant properties, was evaluated on indomethacin-induced small intestinal damage in a rat model. Rats were pretreated with various doses of curcumin (20 mg kg(-1), 40 mg kg(-1) and 80 mg kg(-1)) before administering indomethacin at 20 mg kg(-1). Various parameters of oxidative stress and the extent of small intestinal damage produced by indomethacin, with and without pretreatment with curcumin, were measured. Macroscopic ulceration was found to occur in the small intestine in response to indomethacin. The viability of enterocytes from indomethacin-treated animals was significantly lower than those from control animals. Drug-induced oxidative stress was also evident as seen by increases in the levels of malondialdehyde and protein carbonyl and in activities of pro-oxidant enzymes such as myeloperoxidase and xanthine oxidase in indomethacin-treated rats. Concomitant decreases were seen in the activities of the antioxidant enzymes catalase and glutathione peroxidase in these animals. Pretreatment with curcumin was found to ameliorate these drug-induced changes. Thus, curcumin appears to hold promise as an agent that can potentially reduce NSAID-induced small intestinal damage.

Renal damage in experimentally-induced cirrhosis in rats: Role of oxygen free radicals.

Cirrhosis with ascites is associated with impaired renal function accompanied by sodium and water retention. Although it has been suggested that mediators such as nitric oxide play a role in the development of renal failure in this situation, other mechanisms underlying the process are not well understood. This study examined the role of oxidative stress in mediating renal damage during the development of cirrhosis in order to understand mechanisms involved in the process. It was shown that carbon tetrachloride- or thioacetamide-induced cirrhosis in rats results in oxidative stress in the kidney as seen by increased lipid peroxidation and protein oxidation, accompanied by altered antioxidant status. Cirrhosis was also found to affect renal mitochondrial function, as assessed by measurement of the respiratory control ratio, the swelling of mitochondria, and calcium flux across mitochondrial membranes. Increased lipid peroxidation and changes in lipid composition were evident in the renal brush border membranes, with compromised transport of 14C glucose across these membranes. In conclusion, renal alterations produced as a result of cirrhosis in the rat are possibly mediated by oxidative stress.

Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models.

BACKGROUND/AIMS: Oxidative stress has been implicated in liver cirrhosis. Carbon tetrachloride and thioacetamide are the most widely used models to develop cirrhosis in rats and the present study compares oxidative stress in the liver induced by these compounds at different stages of cirrhosis development. METHODS: Twice-weekly intragastric or intraperitoneal administration of carbon tetrachloride or thioacetamide, respectively, produced liver cirrhosis after 3 months. Histology, serum markers and hepatic hydroxy proline content confirmed the cirrhosis. RESULTS: An increase in oxidative stress parameters was seen in mitochondria, peroxisomes and microsomes from the liver after carbon tetrachloride or thioacetamide treatment. Oxidative stress was more severe in carbon tetrachloride treated animals than thioacetamide. Mild oxidative stress was evident at 1 and 2 months of treatment and a significant increase was seen by 3 months of treatment with either compound. By this time, frank liver cirrhosis was also observed. CONCLUSIONS: These results suggest that evidence of oxygen free radicals is also found early in the development of fibrosis and cirrhosis in both models.

Intestinal mucosal alterations in rats with carbon tetrachloride-induced cirrhosis: changes in glycosylation and luminal bacteria.

Spontaneous bacterial peritonitis is a major cause of mortality after liver cirrhosis. Altered permeability of the mucosa and deficiencies in host immune defenses through bacterial translocation from the intestine due to intestinal bacterial overgrowth have been implicated in the development of this complication. Molecular mechanisms underlying the process are not well known. In order to understand mechanisms involved in translocation of bacteria, this study explored the role of oxidative stress in mediating changes in intestinal mucosal glycosylation and luminal bacterial content during cirrhosis. CCl4-induced cirrhosis in rats led to prolonged oxidative stress in the intestine, accompanied by increased sugar content of both intestinal brush border and surfactant layers. This was accompanied by changes in bacterial flora in the gut, which showed increased hydrophobicity and adherence to the mucosa. Inhibition of xanthine oxidase using sodium tungstate or antioxidant supplementation using vitamin E reversed the oxidative stress, changes in brush border membrane sugar content, and bacterial adherence. In conclusion, oxidative stress in the intestine during cirrhosis alters mucosal glycosylation, accompanied by an increased hydrophobicity of luminal bacteria, enabling increased bacterial adherence onto epithelial cells. This might facilitate translocation across the mucosa, resulting in complications such as spontaneous bacterial peritonitis.

Alterations in the intestinal glycocalyx and bacterial flora in response to oral indomethacin.

Nonsteroidal anti-inflammatory drugs (NSAIDs), used extensively in clinical medicine, tend to cause adverse effects in the gastrointestinal tract. Earlier work has shown that oral administration of indomethacin produced oxidative damage in the small intestine and attenuation of the glycocalyx layer of the mucosa. The present study assessed, in greater detail, the alterations produced in the glycocalyx of rat small intestinal mucosa in response to indomethacin, with specific reference to surfactant-like particles (SLP) and brush border membranes (BBM). Changes in gut flora in response to the drug were also studied, as it has been shown that luminal bacteria play a role in the pathogenesis of NSAID-induced intestinal damage. The levels of sugars such as sialic acid, fucose, hexose and hexosamine were increased in SLP and decreased in the BBM following indomethacin treatment, with the effects being maximal 24h after the administration of the drug. The composition of lipids in the SLP was also found to be altered. There was a significant increase in the number of bacteria in the luminal contents of the small intestine and caecum in these animals, as compared with controls. The number of bacteria adherent to the intestinal mucosa was also significantly higher in the drug-treated group. In vitro studies revealed that there was an increased tendency for bacteria to adhere to SLP isolated from indomethacin-treated rats. These results suggest that alterations in glycosylation of SLP and BBM in response to indomethacin, along with qualitative and quantitative changes in the luminal bacterial flora, may facilitate translocation of bacteria into the mucosa. These changes may contribute to the enteropathy observed as a result of NSAID treatment.

Oral glutamine attenuates indomethacin-induced small intestinal damage.

The use of NSAIDs (non-steroidal anti-inflammatory drugs), although of great therapeutic value clinically, is limited by their tendency to cause mucosal damage in the gastrointestinal tract. In the small intestine, the effects these drugs have been shown to produce include inhibition of cyclo-oxygenase, mitochondrial dysfunction and free radical-induced oxidative changes, all of which contribute to the mucosal damage seen. Glutamine is a fuel preferentially used by enterocytes and is known to contribute to maintaining the integrity of these cells. In the present study, we investigated the effect of glutamine on indomethacin-induced changes in the small intestinal mucosa. Rats were given 2% glutamine or glutamic acid or isonitrogenous amino acids, glycine or alanine, in the diet for 7 days. Indomethacin was then administered orally at a dose of 40 mg/kg of body weight. After 1 h, the small intestine was removed and used for the measurement of parameters of oxidative stress and mitochondrial and BBM (brush border membrane) function. Evidence of oxidative stress was found in the mucosa of the small intestine of drug-treated rats, as indicated by significantly increased activity of xanthine oxidase (P < 0.001) and myeloperoxidase (P < 0.001), with corresponding decreases in the levels of several free radical scavenging enzymes and alpha-tocopherol (P < 0.001 in all cases). Levels of products of peroxidation were also significantly elevated (P < 0.001 for all the parameters measured). In addition, oxidative stress was evident in isolated intestinal mitochondria and BBMs (P < 0.001 for all the parameters measured), with associated alterations in function of these organelles (P < 0.001 for all the parameters measured). Supplementation of the diet with glutamine or glutamic acid prior to treatment with indomethacin produced significant amelioration in all the effects produced by the drug in the small intestine (P < 0.001 for all the parameters measured). Glycine and alanine were found to be much less effective in these respects.

Indomethacin-induced renal damage: role of oxygen free radicals.

Nonsteroidal anti-inflammatory drugs are used extensively in clinical medicine. In spite of their therapeutic utility, however, they are known to cause significant gastrointestinal and renal toxicities, circumstances that limit their use. The side effects produced in these organs have been attributed mainly to the inhibitory effect of these drugs on the activity of cyclooxygenase, a key enzyme in prostaglandin synthesis. In addition to this, in the small intestine it is known that reactive oxygen species also contribute to the enteropathy seen in response to these drugs. In the kidney, however, there is little information whether other mechanisms contribute to the renal toxicity. This study was designed to look at the possible biochemical mechanisms involved in indomethacin-induced renal damage. Rats fasted overnight were dosed with indomethacin (20 mg/kg) by gavage and sacrificed 24 hr later. Histology of the kidney showed abnormalities in the mitochondria in the proximal tubules. Evidence of oxidative stress was found in the kidney associated with mitochondrial dysfunction and neutrophil infiltration. The lipid composition in the mitochondria was also altered. Such effects were abolished by the prior administration of arginine, a donor of nitric oxide. This study, thus, suggests that one of the mechanisms by which nonsteroidal anti-inflammatory drugs induce renal damage is through oxygen free radicals possibly generated by activated neutrophils and mitochondrial dysfunction.

A simple method of rat renal brush border membrane preparation using polyethylene glycol precipitation.

A simple method for preparation of brush border membranes (BBM) from rat kidney using polyethylene glycol (PEG) precipitation has been described. This method avoids the use of cations for the preparation, which might alter membrane lipid composition. These preparations were assessed for enrichment of marker enzymes, contamination by subcellular structures, lipid composition and transport function. An enrichment of 11.8910-fold of alkaline phosphatase, 13.9500-fold of amino peptidase and 13.6500-fold of gamma-glutamyl transpeptidase and an approximate yield of 60% were seen in the final membrane preparation as compared to the homogenate. There was very little contamination of basolateral membranes, peroxisomes, microsomes or lysosomes in the final membrane preparation. Analysis of sugars indicated high content of fucose and sailic acid as compared to hexoses. Isolated membranes appeared as vesicles as seen by electron microscopy. Lipid analysis indicated the presence of various neutral and phospholipids with a high content of sphingomyelin along with a cholesterol/phospholipid ratio of 0.4850. The isolated membrane vesicles were able to transport glucose. This study has shown a simple method of renal brush border membrane preparation, which is comparatively pure and functionally active.

Indomethacin-induced free radical-mediated changes in the intestinal brush border membranes.

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause small intestinal damage but the pathogenesis of this toxicity is not well established. Our earlier work has shown that villus enterocytes are most susceptible to the effects of indomethacin, a commonly used NSAID. This study looked at the acute effect of indomethacin on brush border membranes (BBM), which are present mainly in the villus cells and are in immediate contact with the contents of the small intestinal lumen. Evidence of oxidative stress was found in the mucosa of the small intestine of rats dosed with indomethacin, as indicated by increased activity of xanthine oxidase with corresponding decrease in the levels of several free radical scavenging enzymes. These changes were associated with an increase in peroxidation parameters in the BBM and a fall in the level of alpha-tocopherol. These BBM also exhibited impairment in glucose transport. Significant changes were seen in the lipid composition of these membranes, with upregulation of an 85kDa isoform of phospholipase A(2). Pretreatment of animals with allopurinol, arginine or zinc protected against these effects of indomethacin. Thus this study suggests that in an acute model of indomethacin dosing there is impairment in structure and function of the BBM in enterocytes, with the effects possibly mediated by free radicals and phospholipases.

Indomethacin-induced mitochondrial dysfunction and oxidative stress in villus enterocytes.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause small intestinal damage but the pathogenesis of this toxicity is not well established. Intestinal epithelial cells are thought to be affected by these drugs in the course of their absorption. These cells are of different types, viz. villus, middle and crypt cells. There is little information on which of these cells, if any, are particularly vulnerable to the effects of NSAIDs. This paper aimed to study the effects of indomethacin, an NSAID commonly used in toxicity studies, on different populations of enterocytes. Effects of the drug were assessed in terms of oxidative damage, mitotic activity, mitochondrial function and lipid composition in enterocytes isolated from the small intestine of rats that had been orally administered indomethacin. In addition, the effects of arginine and zinc in protecting against such changes were assessed. Cell viability, tetrazolium dye (MTT) reduction and oxygen uptake were significantly reduced in villus tip cells from rats dosed with the drug. Thymidine uptake was higher in the crypt cell fraction from these rats. Similarly, products of lipid peroxidation were elevated in the villus tip cells with a corresponding decrease in the level of the anti-oxidant, alpha-tocopherol. In isolated mitochondrial preparations from various enterocyte fractions, significant functional impairment and altered lipid composition were seen mainly in mitochondria from villus cells. Arginine and zinc pre-treatment were found to protect against these effects. These results suggest for the first time that the villus tip cells are more vulnerable to the damaging effects of indomethacin and that oxidative stress is possibly involved in this damage.