Temporal changes in free iron levels after brain ischemia Relevance to the timing of iron chelation therapy in stroke

Whereas iron chelators have been proposed as therapeutic agents in stroke, changes in free iron levels have never been explored after focal brain ischemia. Therefore, free and total iron levels in cortical tissue and free iron levels in plasma were measured before and after (1, 4 and 24h) photothrombotic occlusion of cortical vessels in rats. Brain ferritin expression and localization were also investigated before and after (24, 72 and 192 h) occlusion. The results showed that free iron remained below detectable levels in plasma and that the lesion exhibited high levels of free and total iron. As compared to contralateral values, free iron levels in ischemic core and penumbra increased (+50%) at 1h and returned to control values at 4h post-occlusion. In contrast, the increase in total iron levels (+20-30%) was long-lasting, but confined to the ischemic core. A time-dependent increase in the expression of both chains of ferritin was detected in regions that previously exhibited free iron accumulation. Finally, ischemic damage was reduced by the liposoluble iron chelator 2,2'-dipyridyl (20 mg/kg, i.p.) when injected 15 min or 1 h post-occlusion, yet not later (4 h). In conclusion, our results show that focal brain ischemia results in an early and transient elevation in free iron levels in the ischemic tissue and suggest that free iron excess does not originate in blood. They also highlight the importance of starting iron chelation therapy as soon as possible after stroke.     

Relevance of Post-Stroke Circulating BDNF Levels as a Prognostic Biomarker of Stroke Outcome. Impact of rt-PA Treatment

The recombinant form of tissue plasminogen activator (rt-PA) is the only curative treatment for ischemic stroke. Recently, t-PA has been linked to the metabolism of brain-derived neurotrophic factor (BDNF), a major neurotrophin involved in post-stroke neuroplasticity. Thus, the objective of our study was to investigate the impact of rt-PA treatment on post-stroke circulating BDNF levels in humans and in animals. Serum BDNF levels and t-PA/plasmin activity were measured at hospital admission and at up to 90 days in stroke patients receiving (n = 24) or not (n = 14) rt-PA perfusion. We investigated the relationships between serum BDNF with concurrent t-PA/plasmin activity, neurological outcomes and cardiovascular scores at admission. In parallel, serum BDNF levels and t-PA/plasmin activity were assessed before and after (1, 4 and 24h) the induction of ischemic stroke in rats. Our study revealed higher serum BDNF levels and better neurological outcome in rt-PA-treated than non-treated patients. However, serum BDNF levels did not predict stroke outcome when the whole cohort of stroke patients was analyzed. By contrast, serum BDNF levels when measured at admission and at day 90 correlated with cardiovascular scores, and those at day 1 correlated with serum t-PA/plasmin activity in the whole cohort of patients whereas no association could be found in the rt-PA-treated group. In rats devoid of cardiovascular risk, no difference in post-stroke serum BDNF levels was detected between rt-PA- and vehicle-treated animals and no correlation was found between serum BDNF levels and t-PA/plasmin activity. Overall, the data suggest that serum BDNF levels may not be useful as a prognostic biomarker of stroke outcome and that endothelial dysfunction could be a confounding factor when serum BDNF levels after stroke are used to reflect of brain BDNF levels.     

Time-dependent contribution of non neuronal cells to BDNF production after ischemic stroke in rats

Although brain-derived neurotrophic factor (BDNF) plays a central role in recovery after cerebral ischemia, little is known about cells involved in BDNF production after stroke. The present study testes the hypothesis that neurons are not the unique source of neosynthesized BDNF after stroke and that non neuronal-BDNF producing cells differ according to the delay after stroke induction. For this purpose, cellular localization of BDNF and BDNF content of each hemisphere were analysed in parallel before and after (4h, 24h and 8d) ischemic stroke in rats. Stroke of different severities was induced by embolization of the brain with variable number of calibrated microspheres allowing us to explore the association between BDNF production and neuronal death severity. The main results are that (a) unilateral stroke increased BDNF production in both hemispheres with a more intense and long-lasting effect in the lesioned hemisphere, (b) BDNF levels either of the lesioned or unlesioned hemispheres were not inversely correlated to neuronal death severity whatever the delay after stroke onset, (c) in the unlesioned hemisphere, stroke resulted in increased BDNF staining in neurons and ependymal cells (at 4h and 24h), (d) in the lesioned hemisphere, beside neurons and ependymal cells, microglial cells (at 24h), endothelial cells of cerebral arterioles (at 4h and 24h) and astrocytes (at 8d) exhibited a robust BDNF staining as well. Taken together, overall data suggest that non neuronal cells are able to produce substantial amount of BDNF after ischemic stroke and that more attention should be given to these cells in the design of strategies aimed at improving stroke recovery through BDNF-related mechanisms.     

Regional brain monoamine levels and utilization in middle-aged rats.

Significant changes in monoamine levels and utilization were noted in certain brain regions of middle-aged Fisher 344 rats when compared with young adult controls. In the prefrontal cortex and septum, 3,4 dihydroxyphenylglycol (MHPG) and the MHPG/norepinephrine (NE) ratio were decreased. The septum also showed increases in dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) but there was a decrease in the DOPAC/DA ratio. The striatum showed an increase in the MPHG/NE ratio and an increase in DOPAC. The hippocampus and thalamus showed an increase in 5-hydroxyindoleacetic acid (5HIAA). This demonstrates that selected neurotransmitter systems in the brain are altered at an early stage of senescence. This could lead to ensuing neurological deficits.     

Decreased brain levels of ascorbic acid in rats exposed to high pressures

Ascorbic acid was repeatedly monitored in vivo in the striatum of rats subjected to an increasing pressure (100 bar/h compression rate; 0.5 bar partial pressure of O2 He-O2 mixture, up to 120 bar (121 ATA), to which they were exposed for 1 h. Measurements were performed using differential pulse voltammetry and carbon fiber microelectrodes. High-pressure-exposed animals exhibited a dramatic decrease of striatal ascorbic acid. This decrease was detectable at pressures as low as 50 bar and significant over 70 bar (75% of the control level), and the lower level (25% of the control level) was reached shortly after the end of the compression period. This finding is discussed in relation to the physiological role of ascorbic acid in the brain, e.g., its participation in the defense mechanisms against reactive O2 intermediates and lipid peroxidations and its probable involvement in neurotransmission. Emphasis is placed on a possible increased sensitivity of nerve cell membrane phospholipids to peroxidation under stressful hyperbaric situations.     

Plasma BDNF levels vary in relation to body weight in females

Brain derived neurotrophic factor (BDNF) has been implicated in the pathophysiology of depression as well as neuropsychiatric and neurodegenerative disorders. Recent studies show a role of BDNF in energy metabolism and body weight regulation. We examined BDNF levels in plasma and cerebrospinal fluid (CSF) samples from age matched elderly depressed and control subjects. Also, the association of BDNF levels with age, gender, body weight, body mass index (BMI), and cognitive performance was evaluated. We did not find any significant differences in plasma and CSF BDNF levels between depressed and control subjects. Plasma BDNF levels were negatively correlated with age (but not with BMI and body weight), when analyses were performed including both depressed and control subjects. A significant reduction in plasma BDNF levels was observed in females as compared to male subjects, and the change in BDNF levels were significantly and positively related to body weight in females. Furthermore, significant increases in Total Recall and Delayed Recall values were found in females as compared to males. In conclusion, the lower BDNF levels observed in females suggest that changes in peripheral BDNF levels are likely secondary to an altered energy balance. However, further studies using larger sample size are warranted.     

Mesenchymal stem cell therapy of brain ischemic stroke in rats

Mesenchymal stem cell (MSCs)-based therapy is a promising attempt to improve the recovery after stroke. Our experiments were carried out on inbred Wistar-Kyoto rats. MSCs were isolated, expanded in culture, and labeled with vital fluorescent dye PKH-26. Animals were subjected to middle cerebral artery occlusion (MCAO). After three days, MCAO 5 × 10⁶ isolated MSCs were injected into the tail vein of the experimental rats. The control animal group received PBS injections (negative control). Therapy results were evaluated by the following parameters: behavioral and neurological testing, the inured brain areas, damaged brain structures, neuron state, and vessel quantity in the region close to with necrosis zone. It was shown that control animals (PBS injection) did not return to their initial behavioral and neurological state within 6 weeks, while the experimental animals (MSCs injection), within 2–3 weeks after MCAO, had parameters like intact rats. The size of the damaged region in the control group was larger than in the experimental group by a factor of approximately 1.3. The damage in MSC-treated rats was limited to the neocortex; caudate nucleus, capsula externa and piriform cortex remained uninjured. The small vessel quantity in the “border” regions was twice as high as compared to the control group and approximately equal to the number of vessels in an intact brain. For the first time, we demonstrated that the vessel quantity in the neocortex and caudate nucleus of the contralateral hemisphere after MSC transplantation was twice as high as in control rats. It is concluded that the MSC transplantation exerts a beneficial influence upon the brain tissue reparation after stroke.     

Plasma BDNF and PDGF-AA levels are associated with high TCD velocity and stroke in children with sickle cell anemia

Sickle cell anemia (SCA) associated cerebrovascular disease includes vascular remodeling, abnormal cerebral blood flow (CBF) and infarction. We studied the relationships between plasma brain derived neurotropic factor (BDNF), platelet derived growth factors (PDGF-AA and -AB/BB) and high trans-cranial Doppler (TCD) velocity, an indication of CBF velocity. Baseline plasma samples from 39 children (19 SCA with abnormal/high TCD [SATCD], 13 SCA with normal TCD [SNTCD] and 7 healthy non-SCA), were assayed for BDNF, PDGF-AA and -AB/BB plus 11 other cytokines. The sensitivity, specificity and usefulness of these biomarkers for stroke prediction was investigated. All subject groups were of similar age and gender distribution. Mean BDNF was significantly higher among SATCD than SNTCD (p=0.004) as was mean PDGF-AA (p=0.001). Similarly, mean PDGF-AA was higher among SCA subjects who developed stroke than those who did not (p=0.012). Elevated BDNF and PDGF-AA were good predictors of the presence of abnormally high CBF velocity and were both associated with severity of anemia. Elevated PDGF-AA predicted risk for stroke development. Stroke incidence and high TCD velocity were associated with elevated BDNF and PDGF-AA. These findings suggest a role for BDNF and PDGF-AA in the patho-physiological mechanism of cerebrovascular disease in SCA.     

Circulating antigens levels in different clinical forms of the Schistosoma mansoni infection

With the aim to evaluate the circulating cathodic antigen (CCA) levels in relation to the different clinical phases of Schistosoma sp. infection a sandwich ELISA using monoclonal antibody 5H11 was performed. The sera of three groups of 25 Brazilian patients with acute, intestinal and hepatosplenic forms of S. mansoni infection were tested and compared to a non-infected control group. Patients and control groups were matched for age and sex and the number of eggs per gram of feces was equally distributed among the three patient groups. Sensitivity of 100%, 72%, 52% of the assay was observed for the intestinal, hepatosplenic and acute toxemic groups respectively. The specificity was 100%. Intestinal and hepatosplenic groups presented CCA levels significantly higher in comparison to those observed for acute patients (F-ratio = 2,524; p = 0.000 and F-ratio = 6,314; p = 0.015 respectively). There was no significant difference of CCA serum levels between hepatosplenic and intestinal groups (F-ratio = 1,026; p = 0.316).     

Variations of brain histamine levels in germ-free and nephrectomized rats

The influence of nephrectomy on brain and peripheral tissue histamine and on brain norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid was studied in germ-free and conventionally housed rats. The conventional controls had higher levels of histamine in the hypothalamus than the germ-free control animals, but no differences existed for histamine in whole brain minus the hypothalamus or in peripheral tissues. Nephrectomy increased brain histamine and 5-hydroxyindoleacetic acid levels in both germ-free and conventional rats, but had no effect on norepinephrine, dopamine or serotonin. In contrast, the histamine level in the heart of the nephrectomized germ-free animals was lower than that for germ-free controls. There were no changes in the heart or liver histamine levels of the conventional nephrectomized rats.     

Robust changes in expression of brain-derived neurotrophic factor (BDNF) mRNA and protein across the brain do not translate to detectable changes in BDNF levels in CSF or plasma

Adult rats were treated acutely with peripheral kainic acid (KA), and changes in brain-derived neurotrophic factor (BDNF) mRNA and protein were tracked over time across multiple brain regions. Despite robust elevation in both mRNA and protein in multiple brain regions, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable. Primary neurons were then treated with KA. BDNF was similarly elevated within neurons, but was undetectable in neuronal media. Thus, while deficits in BDNF signaling have been implicated in a number of diseases, these data suggest that extracellular concentrations of BDNF may not be a facile biomarker for changes in neurons.     

Circulating immune complexes in multiple sclerosis. Relation to clinical and biological parameters

Circulating immune complexes were looked for on 38 clinically definite MS (multiple sclerosis) patients compared to 35 other neurological patients and 26 healthy subjects. 29% of the MS sera and 8.6% of the other neurological sera were positive, whereas none of the control sera were positive. These differences are significant. Clinical status was analysed as regards the age at onset, the duration and course of the disease, the disability of the patients and the treatment they received. CSF (cerebrospinal fluid) parameters studied were pleocytosis, concentration of total protein, electrophoresis. The results suggest that IC (immune complex) are more frequent during the first 10 years of the MS disease, in patients with blood-brain barrier damage and in patients without oligoclonal IgG.     

Transcranial low-level laser therapy in an in vivo model of stroke: Relevance to the brain infarct,microglia activation and neuroinflammation

Stroke is the main cause of death and functional disability. The available therapy affects only 5% of patients, and new therapeutic approaches have been constantly tested. Transcranial photobiomodulation (PBM) is promising for its neuroprotective effect on brain injuries. Thus, the present study investigated the PBM effects in an in vivo model of ischemic stroke induced by photothrombosis (PT). Five different groups of Wistar rats were submitted or not to a daily dose of fish oil or/and laser sessions for 2 months. The ischemia volume was evaluated by stereology; GFAP, Iba and NeuN by immunohistochemistry; TNF-α, IL-1β, IL-6, IL-10 and TGF-β by ELISA assay. PBM influenced both the lesion volume and the GFAP. Furthermore, PBM and Ω-3 or both reduced Iba RNAm. PBM reduced TNF-α, IL-1β, IL-6, brain damage, neuroinflammation and microglial activation, and it increased astroglial activity in peri-lesioned region after stroke.     

脑源性神经营养因子在大鼠后足切割疼痛中的作用

目的:观察脑源性神经营养因子(BDNF)对大鼠后足切割疼痛的影响。方法:采用纵行切割大鼠后足作为疼痛模型,运用免疫组织化学与免疫荧光双标记方法,观察大鼠后足切割后不同时间点(1-72hr)BDNF在相应节段背根神经节与脊髓内表达的变化。腹腔或鞘内注射BDNF抗体中和内源性BDNF后,以Von Frey尼龙纤维刺激后足行机械痛敏评价。结果:大鼠后足切割后1-24hr内,BDNF在切割侧L42-L5脊髓后角表达明增加,BDNF主要位于后角神经元内与神经末梢,星形胶质细胞与小胶质细胞内未见明显表达;在L42-L5背根神经节,BNDF免疫阳性细胞百分比在切割后1-24hr内也明显增加,增加的主要为大直经神经元;鞘内给予BDNF抗体可明显增加大鼠后足切割后的缩足阈值,而腹腔给予BDNF抗体对大鼠的缩足阈值影响较小。结论:BDNF参与了大鼠后足切割后机械痛敏的过程。     

A novel herbal treatment reduces depressive-like behaviors and increases BDNF levels in the brain of stressed mice

Aims

Depression is a chronic, recurring and potentially life-threatening illness. Current treatments for depression are characterized by a low success rate and associated with a wide variety of side effects. The aim of the present study was to evaluate the behavioral and biological anti-depressant effects of a novel herbal treatment (NHT), as well as to assess its potential side effects, in comparison to treatment with the selective serotonin reuptake inhibitor escitalopram.

Main methods

Depressive-like behavior was evaluated using the forced swim test (FST) and the tail suspension test (TST). Sexual behavior was evaluated following treatment by measuring latency before first mount and number of total mounts. Brain derived neurotrophic factor (BDNF) levels were evaluated using enzyme-linked immunosorbent assay. Serotonin transporter (SERT) levels in the pre-frontal cortex (PFC) and hypothalamus were evaluated using high affinity binding assay.

Key findings

(1) The NHT reduced depressive-like behavior in the FST and TST; (2) BDNF levels in the PFC of mice treated both with the NHT and escitalopram were increased; (3) SERT levels in the hypothalamus were significantly higher in the NHT group, in comparison to escitalopram and the control groups, and significantly lower in the PFC of the NHT group in comparison to the escitalopram group; and (4) the NHT led to less sexual dysfunction, compared to treatment with escitalopram.

Significance

Our NHT has the potential of being highly efficacious in treating depression in humans, while causing minimal to no influence on sexual function.