Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Brazilian psychiatric brain bank: a new contribution tool to network studies

There is an urgent need for expanding the number of brain banks serving psychiatric research. We describe here the Psychiatric Disorders arm of the Brain Bank of the Brazilian Aging Brain Study Group (Psy-BBBABSG), which is focused in bipolar disorder (BD) and obsessive compulsive disorder (OCD). Our protocol was designed to minimize limitations faced by previous initiatives, and to enable design-based neurostereological analyses. The Psy-BBBABSG first milestone is the collection of 10 brains each of BD and OCD patients, and matched controls. The brains are sourced from a population-based autopsy service. The clinical and psychiatric assessments were done by an expert team including psychiatrists, through an informant. One hemisphere was perfused-fixed to render an optimal fixation for conducting neurostereological studies. The other hemisphere was comprehensively dissected and frozen for molecular studies. In 20 months, we collected 36 brains. A final report was completed for 14 cases: 3 BDs, 4 major depressive disorders, 1 substance use disorder, 1 mood disorder NOS, 3 obsessive compulsive spectrum symptoms, 1 OCD and 1 schizophrenia. The majority were male (64%), and the average age at death was 67.2 +/- 9.0 years. The average postmortem interval was 16 h. Three matched controls were collected. The pilot stage confirmed that the protocols are well fitted to reach our goals. Our unique autopsy source makes possible to collect a fairly number of high quality cases in a short time. Such a collection offers an additional to the international research community to advance the understanding on neuropsychiatric diseases.

Vascular dementia: Different forms of vessel disorders contribute to the development of dementia in the elderly brain

The diagnosis of vascular dementia (VaD) describes a group of various vessel disorders with different types of vascular lesions that finally contribute to the development of dementia. Most common forms of VaD in the elderly brain are subcortical vascular encephalopathy, strategic infarct dementia, and the multi infarct encephalopathy. Hereditary forms of VaD are rare. Most common is the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Sporadic forms of VaD are caused by degenerative vessel disorders such as atherosclerosis, small vessel disease (SVD) including small vessel arteriosclerosis, arteriolosclerosis, and lipohyalinosis, and cerebral amyloid angiopathy (CAA). Less frequently inflammatory vessel disorders and tumor-associated vessel lesions (e. g. angiocentric T-cell or angiotropic large cell lymphoma) can cause symptoms of dementia. Here, we review and discuss the impact of vessel disorders to distinct vascular brain tissue lesions and to the development of dementia in elderly individuals. The impact of coexisting neurodegenerative pathology in the elderly brain to VaD as well as the correlation between SVD and CAA expansion in the brain parenchyma with that of Alzheimer's disease (AD)-related pathology is highlighted. We conclude that "pure" VaD is rare and most frequently caused by infarctions. However, there is a significant contribution of vascular lesions and vessel pathology to the development of dementia that may go beyond tissue damage due to vascular lesions. Insufficient blood blow and alterations of the perivascular drainage mechanisms of the brain may also lead to a reduced protein clearance from extracellular space and subsequent increase of proteins in the brain parenchyma, such as the amyloid beta-protein, and foster, thereby, the development of AD-related neurodegeneration. As such, it seems to be important for clinical practice to consider treatment of potentially coexisting AD pathology in cognitively impaired patients with vascular lesions. (C) 2012 Elsevier Inc. All rights reserved.

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

Background: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. Results: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. Conclusions: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.

Coupling Between GABA(A)-R Ligand-Binding Activity and Membrane Organization in beta-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments

Correlations between GABA(A) receptor (GABA(A)-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with beta-cyclodextrin (beta-CD). The mere pre-incubation (PI) at 37A degrees C accompanying the beta-CD treatment was an underlying source of perturbations increasing [H-3]-FNZ maximal binding (70%) and K (d) (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4A degrees C) with A (DPH,heating) < A (DPH,cooling). Compared with PI samples, the beta-CD treatment reduced B (max) by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A (TMA-DPH). PI, but not beta-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with beta-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, beta-CD is not completely eliminated from the system through centrifugation washings. It was concluded that beta-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual beta-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA(A)-R density, relative to untreated samples.

Cytogenetic instability of dental pulp stem cell lines

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

Impaired blood-brain/spinal cord barrier in ALS patients

Vascular pathology, including blood-brain/spinal cord barrier (BBB/BSCB) alterations, has recently been recognized as a key factor possibly aggravating motor neuron damage, identifying a neurovascular disease signature for ALS. However, BBB/BSCB competence in sporadic ALS (SALS) is still undetermined. In this study, BBB/BSCB integrity in postmortem gray and white matter of medulla and spinal cord tissue from SALS patients and controls was investigated. Major findings include (1) endothelial cell damage and pericyte degeneration, (2) severe intra- and extracellular edema, (3) reduced CD31 and CD105 expressions in endothelium, (4) significant accumulation of perivascular collagen IV, and fibrin deposits (5) significantly increased microvascular density in lumbar spinal cord, (6) IgG microvascular leakage, (7) reduced tight junction and adhesion protein expressions. Microvascular barrier abnormalities determined in gray and white matter of the medulla, cervical, and lumbar spinal cord of SALS patients are novel findings. Pervasive barrier damage discovered in ALS may have implications for disease pathogenesis and progression, as well as for uncovering novel therapeutic targets. (C) 2012 Elsevier B.V. All rights reserved.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 mu M Ca2+, C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. (C) 2012 Elsevier Inc. All rights reserved.

Characterization of Ectonucleotidases in Human Medulloblastoma Cell Lines: ecto-5 ' NT/CD73 in Metastasis as Potential Prognostic Factor

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Brain Pathways Involved in the Modulatory Effects of Noradrenaline in Lateral Septal Area on Cardiovascular Responses

We have previously reported that stimulation of alpha-1 adrenoceptors by noradrenaline (NA) injected into the lateral septal area (LSA) of anaesthetized rats causes pressor and bradycardic responses that are mediated by acute vasopressin release into the circulation through activation of the paraventricular nucleus (PVN). Although the PVN is the final structure of this pathway, the LSA has no direct connections with the PVN, suggesting that other structures may connect these areas. To address this issue, the present study employed c-Fos immunohistochemistry to investigate changes caused by NA microinjection into the LSA in neuronal activation in brain structures related to systemic vasopressin release. NA microinjected in the LSA caused pressor and bradycardic responses, which were blocked by intraseptal administration of alpha-1 adrenoceptor antagonist (WB4101, 10 nmol/200 nL) or systemic V-1 receptor antagonist (dTyr(CH2)5(Me)AVP, 50 mu g/kg). NA also increased c-Fos immunoreactivity in the prelimbic cortex (PL), infralimbic cortex (IL), dorsomedial periaqueductal gray (dmPAG), bed nucleus of the stria terminalis (BNST), PVN, and medial amygdala (MeA). No differences in the diagonal band of Broca, cingulate cortex, and dorsolateral periaqueductal gray (dlPAG) were found. Systemic administration of the vasopressin receptor antagonist dTyr AVP (CH2)5(Me) did not change the increase in c-Fos expression induced by intra-septal NA. This latter effect, however, was prevented by local injection of the alpha-1 adrenoceptor antagonist WB4101. These results suggest that areas such as the PL, IL, dmPAG, BNST, MeA, and PVN could be part of a circuit responsible for vasopressin release after activation of alpha-1 adrenoceptors in the LSA.

Neuronal NOS Inhibitor and Conventional Antidepressant Drugs Attenuate Stress-induced Fos Expression in Overlapping Brain Regions

Recent evidence indicates that the administration of inhibitors of neuronal nitric oxide synthase (nNOS) induces antidepressant-like effects in animal models such as the forced swimming test (FST). However, the neural circuits involved in these effects are not yet known. Therefore, this study investigated the expression of Fos protein, a marker of neuronal activity, in the brain of rats submitted to FST and treated with the preferential nNOS inhibitor, 7-nitroindazole (7-NI), or with classical antidepressant drugs (Venlafaxine and Fluoxetine). Male Wistar rats were submitted to a forced swimming pretest (PT) and, immediately after, started receiving a sequence of three ip injections (0, 5, and 23 h after PT) of Fluoxetine (10 mg/kg), Venlafaxine (10 mg/kg), 7-NI (30 mg/kg) or respective vehicles. One hour after the last drug injection the animals were submitted to the test session, when immobility time was recorded. After the FST they were sacrificed and had their brains removed and processed for Fos immunohistochemistry. Independent group of non-stressed animals received the same drug treatments, or no treatment (naive). 7-NI, Venlafaxine or Fluoxetine reduced immobility time in the FST, an antidepressant-like effect. None of the treatments induce significant changes in Fos expression per se. However, swimming stress induced significant increases in Fos expression in the following brain regions: medial prefrontal cortex, nucleus accumbens, locus coeruleus, raphe nuclei, striatum, hypothalamic nucleus, periaqueductal grey, amygdala, habenula, paraventricular nucleus of hypothalamus, and bed nucleus of stria terminalis. This effect was attenuated by 7-NI, Venlafaxine or Fluoxetine. These results show that 7-NI produces similar behavioral and neuronal activation effects to those of typical antidepressants, suggesting that these drugs share common neurobiological substrates.

Expression analysis of stem cell-related genes reveal OCT4 as a predictor of poor clinical outcome in medulloblastoma

Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P <= 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients.

Paradoxical effects of brain death and associated trauma on rat mesenteric microcirculation: an intravital microscopic study

OBJECTIVE: Experimental findings support clinical evidence that brain death impairs the viability of organs for transplantation, triggering hemodynamic, hormonal, and inflammatory responses. However, several of these events could be consequences of brain death-associated trauma. This study investigated microcirculatory alterations and systemic inflammatory markers in brain-dead rats and the influence of the associated trauma. METHOD: Brain death was induced using intracranial balloon inflation; sham-operated rats were trepanned only. After 30 or 180 min, the mesenteric microcirculation was observed using intravital microscopy. The expression of P-selectin and ICAM-1 on the endothelium was evaluated using immunohistochemistry. The serum cytokine, chemokine, and corticosterone levels were quantified using enzyme-linked immunosorbent assays. White blood cell counts were also determined. RESULTS: Brain death resulted in a decrease in the mesenteric perfusion to 30%, a 2.6-fold increase in the expression of ICAM-1 and leukocyte migration at the mesentery, a 70% reduction in the serum corticosterone level and pronounced leukopenia. Similar increases in the cytokine and chemokine levels were seen in the both the experimental and control animals. CONCLUSION: The data presented in this study suggest that brain death itself induces hypoperfusion in the mesenteric microcirculation that is associated with a pronounced reduction in the endogenous corticosterone level, thereby leading to increased local inflammation and organ dysfunction. These events are paradoxically associated with induced leukopenia after brain damage.

Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

Computation of direction selectivity in retinal starburst amacrine cell dendrites – studied using electrophysiological recordings and two-photon imaging

Neuronal circuits in the retina analyze images according to qualitative aspects such as color or motion, before the information is transmitted to higher visual areas of the brain. One example, studied for over the last four decades, is the detection of motion direction in ‘direction selective’ neurons. Recently, the starburst amacrine cell, one type of retinal interneuron, has emerged as an essential player in the computation of direction selectivity. In this study the mechanisms underlying the computation of direction selective calcium signals in starburst cell dendrites were investigated using whole-cell electrical recordings and two-photon calcium imaging. Analysis of the somatic electrical responses to visual stimulation and pharmacological agents indicated that the directional signal (i) is not computed presynaptically to starburst cells or by inhibitory network interactions. It is thus computed via a cell-intrinsic mechanism, which (ii) depends upon the differential, i.e. direction selective, activation of voltage-gated channels. Optically measuring dendritic calcium signals as a function of somatic voltage suggests (iii) a difference in resting membrane potential between the starburst cell’s soma and its distal dendrites. In conclusion, it is proposed that the mechanism underlying direction selectivity in starburst cell dendrites relies on intrinsic properties of the cell, particularly on the interaction of spatio-temporally structured synaptic inputs with voltage-gated channels, and their differential activation due to a somato-dendritic difference in membrane potential.