Intracranial self-stimulation reverses impaired spatial learning and regulates serum microRNA levels in a streptozotocin-induced rat model of Alzheimer disease.

BACKGROUND: The assessment of deep brain stimulation (DBS) as a therapeutic alternative for treating Alzheimer disease (AD) is ongoing. We aimed to determine the effects of intracranial self-stimulation at the medial forebrain bundle (MFB-ICSS) on spatial memory, neurodegeneration, and serum expression of microRNAs (miRNAs) in a rat model of sporadic AD created by injection of streptozotocin. We hypothesized that MFB-ICSS would reverse the behavioural effects of streptozotocin and modulate hippocampal neuronal density and serum levels of the miRNAs. METHODS: We performed Morris water maze and light-dark transition tests. Levels of various proteins, specifically amyloid-ß precurser protein (APP), phosphorylated tau protein (pTAU), and sirtuin 1 (SIRT1), and neurodegeneration were analyzed by Western blot and Nissl staining, respectively. Serum miRNA expression was measured by reverse transcription polymerase chain reaction. RESULTS: Male rats that received streptozotocin had increased hippocampal levels of pTAU S202/T205, APP, and SIRT1 proteins; increased neurodegeneration in the CA1, dentate gyrus (DG), and dorsal tenia tecta; and worse performance in the Morris water maze task. No differences were observed in miRNAs, except for miR-181c and miR-let-7b. After MFB-ICSS, neuronal density in the CA1 and DG regions and levels of miR-181c in streptozotocin-treated and control rats were similar. Rats that received streptozotocin and underwent MFB-ICSS also showed lower levels of miR-let-7b and better spatial learning than rats that received streptozotocin without MFB-ICSS. LIMITATIONS: The reversal by MFB-ICSS of deficits induced by streptozotocin was fairly modest. CONCLUSION: Spatial memory performance, hippocampal neurodegeneration, and serum levels of miR-let-7b and miR-181c were affected by MFB-ICSS under AD-like conditions. Our results validate the MFB as a potential target for DBS and lend support to the use of specific miRNAs as promising biomarkers of the effectiveness of DBS in combatting AD-associated cognitive deficits.

Traveling waves in a model for cortical spreading depolarization with slow-fast dynamics.

Cortical spreading depression and spreading depolarization (CSD) are waves of neuronal depolarization that spread across the cortex, leading to a temporary saturation of brain activity. They are associated with various brain disorders such as migraine and ischemia. We consider a reduced version of a biophysical model of a neuron-astrocyte network for the initiation and propagation of CSD waves [Huguet et al., Biophys. J. 111(2), 452-462, 2016], consisting of reaction-diffusion equations. The reduced model considers only the dynamics of the neuronal and astrocytic membrane potentials and the extracellular potassium concentration, capturing the instigation process implicated in such waves. We present a computational and mathematical framework based on the parameterization method and singular perturbation theory to provide semi-analytical results on the existence of a wave solution and to compute it jointly with its velocity of propagation. The traveling wave solution can be seen as a heteroclinic connection of an associated system of ordinary differential equations with a slow-fast dynamics. The presence of distinct time scales within the system introduces numerical instabilities, which we successfully address through the identification of significant invariant manifolds and the implementation of the parameterization method. Our results provide a methodology that allows to identify efficiently and accurately the mechanisms responsible for the initiation of these waves and the wave propagation velocity.

Hippocampal neurogenesis and Arc expression are enhanced in high-fat fed prepubertal female pigs by a diet including omega-3 fatty acids and Bifidobacterium breve CECT8242.

PURPOSE: Obesity during childhood has become a pandemic disease, mainly caused by a diet rich in sugars and fatty acids. Among other negative effects, these diets can induce cognitive impairment and reduce neuroplasticity. It is well known that omega-3 and probiotics have a beneficial impact on health and cognition, and we have hypothesized that a diet enriched with Bifidobacterium breve and omega-3 could potentiate neuroplasticity in prepubertal pigs on a high-fat diet. METHODS: Young female piglets were fed during 10 weeks with: standard diet (T1), high-fat (HF) diet (T2), HF diet including B. breve CECT8242 (T3) and HF diet including the probiotic and omega-3 fatty acids (T4). Using hippocampal sections, we analyzed by immunocytochemistry the levels of doublecortin (DCX) to study neurogenesis, and activity-regulated cytoskeleton-associated protein (Arc) as a synaptic plasticity related protein. RESULTS: No effect of T2 or T3 was observed, whereas T4 increased both DCX+ cells and Arc expression. Therefore, a diet enriched with supplements of B. breve and omega-3 increases neurogenesis and synaptic plasticity in prepubertal females on a HF diet from nine weeks of age to sexual maturity. Furthermore, the analysis of serum cholesterol and HDL indicate that neurogenesis was related to lipidic demand in piglets fed with control or HF diets, but the neurogenic effect induced by the T4 diet was exerted by mechanisms independent of this lipidic demand. CONCLUSION: Our results show that the T4 dietary treatment is effective in potentiating neural plasticity in the dorsal hippocampus of prepubertal females on a HF diet.

Intracranial Self-stimulation of the Medial Forebrain Bundle Ameliorates Memory Disturbances and Pathological Hallmarks in an Alzheimer's Disease Model by Intracerebral Administration of Amyloid-ß in Rats.

No curative or fully effective treatments are currently available for Alzheimer's disease (AD), the most common form of dementia. Electrical stimulation of deep brain areas has been proposed as a novel neuromodulatory therapeutic approach. Previous research from our lab demonstrates that intracranial self-stimulation (ICSS) targeting medial forebrain bundle (MFB) facilitates explicit and implicit learning and memory in rats with age or lesion-related memory impairment. At a molecular level, MFB-ICSS modulates the expression of plasticity and neuroprotection-related genes in memory-related brain areas. On this basis, we suggest that MFB could be a promising stimulation target for AD treatment. In this study, we aimed to assess the effects of MFB-ICSS on both explicit memory as well as the levels of neuropathological markers ptau and drebrin (DBN) in memory-related areas, in an AD rat model obtained by Aß icv-injection. A total of 36 male rats were trained in the Morris water maze on days 26-30 after Aß injection and tested on day 33. Results demonstrate that this Aß model displayed spatial memory impairment in the retention test, accompanied by changes in the levels of DBN and ptau in lateral entorhinal cortex and hippocampus, resembling pathological alterations in early AD. Administration of MFB-ICSS treatment consisting of 5 post-training sessions to AD rats managed to reverse the memory deficits as well as the alteration in ptau and DBN levels. Thus, this paper reports both cognitive and molecular effects of a post-training reinforcing deep brain stimulation procedure in a sporadic AD model for the first time.

Phase-locking patterns underlying effective communication in exact firing rate models of neural networks.

Macroscopic oscillations in the brain have been observed to be involved in many cognitive tasks but their role is not completely understood. One of the suggested functions of the oscillations is to dynamically modulate communication between neural circuits. The Communication Through Coherence (CTC) theory proposes that oscillations reflect rhythmic changes in excitability of the neuronal populations. Thus, populations need to be properly phase-locked so that input volleys arrive at the peaks of excitability of the receiving population to communicate effectively. Here, we present a modeling study to explore synchronization between neuronal circuits connected with unidirectional projections. We consider an Excitatory-Inhibitory (E-I) network of quadratic integrate-and-fire neurons modeling a Pyramidal-Interneuronal Network Gamma (PING) rhythm. The network receives an external periodic input from either one or two sources, simulating the inputs from other oscillating neural groups. We use recently developed mean-field models which provide an exact description of the macroscopic activity of the spiking network. This low-dimensional mean field model allows us to use tools from bifurcation theory to identify the phase-locked states between the input and the target population as a function of the amplitude, frequency and coherence of the inputs. We identify the conditions for optimal phase-locking and effective communication. We find that inputs with high coherence can entrain the network for a wider range of frequencies. Besides, faster oscillatory inputs than the intrinsic network gamma cycle show more effective communication than inputs with similar frequency. Our analysis further shows that the entrainment of the network by inputs with higher frequency is more robust to distractors, thus giving them an advantage to entrain the network and communicate effectively. Finally, we show that pulsatile inputs can switch between attended inputs in selective attention.

Cav-1 Protein Levels in Serum and Infarcted Brain Correlate with Hemorrhagic Volume in a Mouse Model of Thromboembolic Stroke, Independently of rt-PA Administration.

Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for acute ischemic stroke. However, its administration is still limited due to the associated increased risk of hemorrhagic transformation (HT). rt-PA may exacerbate blood-brain barrier (BBB) injury by several mechanisms that have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae, has been linked to the endothelial barrier function. The effects of rt-PA on Cav-1 expression remain largely unknown. Here, Cav-1 protein expression after ischemic conditions, with or without rt-PA administration, was analyzed in a murine thromboembolic middle cerebral artery occlusion (MCAO) and in brain microvascular endothelial bEnd.3 cells subjected to oxygen/glucose deprivation (OGD). Our results show that Cav-1 is overexpressed in endothelial cells of infarcted area and in bEnd.3 cell line after ischemia but there is disagreement regarding rt-PA effects on Cav-1 expression between both experimental models. Delayed rt-PA administration significantly reduced Cav-1 total levels from 24 to 72 h after reoxygenation and increased pCav-1/Cav-1 at 72 h in the bEnd.3 cells while it did not modify Cav-1 immunoreactivity in the infarcted area at 24 h post-MCAO. Importantly, tissue Cav-1 positively correlated with Cav-1 serum levels at 24 h post-MCAO and negatively correlated with the volume of hemorrhage after infarction, the latter supporting a protective role of Cav-1 in cerebral ischemia. In addition, the negative association between baseline serum Cav-1 levels and hemorrhagic volume points to a potential usefulness of baseline serum Cav-1 levels to predict hemorrhagic volume, independently of rt-PA administration.

Protocol to assess rewarding brain stimulation as a learning and memory modulating treatment: Comparison between self-administration and experimenter-administration.

Intracranial electrical self-stimulation (ICSS) is a useful procedure in animal research. This form of administration ensures that areas of the brain reward system (BRS) are being functionally activated, since the animals must perform an operant response to self-administer an electrical stimulus. Rewarding post-training ICSS of the medial forebrain bundle (MFB), an important system of the BRS, has been shown to consistently improve rats' acquisition and retention in several learning tasks. In the clinical setting, deep brain stimulation (DBS) of different targets is currently being used to palliate the memory impairment that occurs in some neurodegenerative diseases. However, the stimulation of the MFB has only been used to treat emotional alterations, not memory disorders. Since DBS stimulation treatments in humans are exclusively administered by external sources, studies comparing the efficacy of that form of application to a self-administered stimulation are key to the translationality of ICSS. This protocol compares self-administered (ICSS) and experimenter-administered (EAS) stimulation of the MFB on the spatial Morris Water Maze task (MWM). c-Fos immunohistochemistry procedure was carried out to evaluate neural activation after retention. Results show that the stimulation of the MFB improves the MWM task regardless of the form of administration, although some differences in c-Fos expression were found. Present results suggest that MFB-ICSS is a valid animal model to study the effects of MFB electrical stimulation on memory, which could guide clinical applications of DBS. The present protocol is a useful guide for establishing ICSS behavior in rats, which could be used as a learning and memory-modulating treatment.

Global phase-amplitude description of oscillatory dynamics via the parameterization method.

In this paper, we use the parameterization method to provide a complete description of the dynamics of an n-dimensional oscillator beyond the classical phase reduction. The parameterization method allows us, via efficient algorithms, to obtain a parameterization of the attracting invariant manifold of the limit cycle in terms of the phase-amplitude variables. The method has several advantages. It provides analytically a Fourier-Taylor expansion of the parameterization up to any order, as well as a simplification of the dynamics that allows for a numerical globalization of the manifolds. Thus, one can obtain the local and global isochrons and isostables, including the slow attracting manifold, up to high accuracy, which offer a geometrical portrait of the oscillatory dynamics. Furthermore, it provides straightforwardly the infinitesimal phase and amplitude response functions, that is, the extended infinitesimal phase and amplitude response curves, which monitor the phase and amplitude shifts beyond the asymptotic state. Thus, the methodology presented yields an accurate description of the phase dynamics for perturbations not restricted to the limit cycle but to its attracting invariant manifold. Finally, we explore some strategies to reduce the dimension of the dynamics, including the reduction of the dynamics to the slow stable submanifold. We illustrate our methods by applying them to different three-dimensional single neuron and neural population models in neuroscience.

Intracranial Self-Stimulation Modulates Levels of SIRT1 Protein and Neural Plasticity-Related microRNAs.

Deep brain stimulation (DBS) of reward system brain areas, such as the medial forebrain bundle (MFB), by means of intracranial self-stimulation (ICSS), facilitates learning and memory in rodents. MFB-ICSS has been found capable of modifying different plasticity-related proteins, but its underlying molecular mechanisms require further elucidation. MicroRNAs (miRNAs) and the longevity-associated SIRT1 protein have emerged as important regulatory molecules implicated in neural plasticity. Thus, we aimed to analyze the effects of MFB-ICSS on miRNAs expression and SIRT1 protein levels in hippocampal subfields and serum. We used OpenArray to select miRNA candidates differentially expressed in the dentate gyrus (DG) of ICSS-treated (3 sessions, 45' session/day) and sham rats. We further analyzed the expression of these miRNAs, together with candidates selected after bibliographic screening (miR-132-3p, miR-134-5p, miR-146a-5p, miR-181c-5p) in DG, CA1, and CA3, as well as in serum, by qRT-PCR. We also assessed tissue and serum SIRT1 protein levels by Western Blot and ELISA, respectively. Expression of miR-132-3p, miR-181c-5p, miR-495-3p, and SIRT1 protein was upregulated in DG of ICSS rats (P < 0.05). None of the analyzed molecules was regulated in CA3, while miR-132-3p was also increased in CA1 (P = 0.011) and serum (P = 0.048). This work shows for the first time that a DBS procedure, specifically MFB-ICSS, modulates the levels of plasticity-related miRNAs and SIRT1 in specific hippocampal subfields. The mechanistic role of these molecules could be key to the improvement of memory by MFB-ICSS. Moreover, regarding the proposed clinical applicability of DBS, serum miR-132 is suggested as a potential treatment biomarker.

Orexin-1 receptor blockade differentially affects spatial and visual discrimination memory facilitation by intracranial self-stimulation.

Intracranial self-stimulation (ICSS) of the medial forebrain bundle is an effective treatment to facilitate memory. Performance in both explicit and implicit memory tasks has been improved by ICSS, and this treatment has even been capable of recovering loss of memory function due to lesions or old age. Several neurochemical systems have been studied in regard to their role in ICSS effects on memory, however the possible involvement of the orexinergic system in this facilitation has yet to be explored. The present study aims to examine the relationship between the OX1R and the facilitative effects of ICSS on two different types of memory tasks, both carried out in the Morris Water Maze: spatial and visual discrimination. Results show that the OX1R blockade, by intraventricular administration of SB-334867, partially negates the facilitating effect of ICSS on spatial memory, whereas it hinders ICSS facilitation of the discrimination task. However, ICSS treatment was capable of compensating for the severe detrimental effects of OX1R blockade on both memory paradigms. These results suggest different levels of involvement of the orexinergic system in the facilitation of memory by ICSS, depending on the memory task.

Modeling the long term effects of thermoregulation on human sleep.

The connection between human sleep and energy exertion has long been regarded as part of the reasoning for the need to sleep. A recent theory proposes that during REM sleep, energy utilized for thermoregulation is diverted to other relevant biological processes. We present a mathematical model of human sleep/wake regulation with thermoregulatory functions to gain quantitative insight into the effects of ambient temperature on sleep quality. Our model extends previous models by incorporating equations for the metabolic processes that control thermoregulation during sleep. We present numerical simulations that provide a quantitative answer for how humans adjust by changing the normal sleep stage progression when it is challenged with ambient temperatures away from thermoneutral. We explore the dynamics for a single night and several nights. Our results indicate that including the effects of temperature is a vital component of modeling sleep.

Evaluation of long-term rt-PA effects on bEnd.3 endothelial cells under ischemic conditions; changes in ZO-1 expression and glycosylation of the bradykinin B2 receptor.

Recombinant tissue plasminogen activator (rt-PA) has proven effective in the treatment of acute ischemic stroke, despite the increased risk of hemorrhagic transformation (HT), its major associated complication. Although it is known that HT is related to blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. We assessed time-dependent effects of rt-PA on the bEnd.3 murine brain endothelial cell line subjected either to normoxia or to 2.5 h of oxygen and glucose deprivation (OGD), evaluating a longer period than has previously been done, beyond 6 h post-reoxygenation. Parameters of cell viability, metabolic activity, ionic and transcellular permeability, as well as levels of claudin-5, zonula occludens-1 (ZO-1) and bradykinin B2 receptor (B2R) protein expression were analyzed at 24, 48 and 72 h post-reoxygenation with or without the administration of rt-PA. rt-PA treatment increased both the ionic and transcellular permeability until 72 h and did not modify cell viability or metabolic activity or the expression of claudin-5, ZO-1 and B2R under normoxia at any analyzed time. Under OGD conditions, rt-PA exacerbated OGD effects on metabolic activity from 48 to 72 h, increased transcellular permeability from 24 to 72 h, significantly decreased ZO-1 protein levels at the plasma membrane and increased B2R glycosylation at 72 h post-reoxygenation. Our findings suggest that a long-term analysis is necessary to elucidate time-dependent molecular mechanisms associated to BBB breakdown due to rt-PA administration under ischemia. Thus, protective BBB therapies after ischemic stroke and rt-PA treatment should be explored at least until 72 h after OGD and rt-PA administration.

Rewarding deep brain stimulation at the medial forebrain bundle favours avoidance conditioned response in a remote memory test, hinders extinction and increases neurogenesis.

Intracranial Self-Stimulation (ICSS) at the medial forebrain bundle consistently facilitates learning and memory in rats when administered post-training or when administered non-concurrent to training, but its scope regarding remote memory has not yet been studied. The present work aims to test whether the combination of these two forms of ICSS administration can cause a greater persistence of the facilitating effect on remote retention and affect neurogenesis in the dentate gyrus (DG) of the hippocampus. Rats were trained in active avoidance conditioning and tested in two retention sessions (10 and 90 days) and later extinction. Subjects received an ICSS session after each of the five avoidance acquisition sessions (post-training treatment) and half of them also received ten additional ICSS sessions during the rest period between retention tests (non-concurrent treatment). All the stimulated groups showed a higher performance in acquisition and retention sessions, but only the rats receiving both ICSS treatments showed greater resistance to extinction. Remarkably, at seven months, rats receiving the non-concurrent ICSS treatment had a greater number of DCX-positive cells in the DG as well as a higher amount of new-born cells within the granular layer compared to rats that did not receive this additional ICSS treatment. Our present findings significantly extend the temporal window of the facilitating effect of ICSS on active avoidance and demonstrate a neurogenic effect of rewarding medial forebrain bundle stimulation.

The uncoupling limit of identical Hopf bifurcations with an application to perceptual bistability.

We study the dynamics arising when two identical oscillators are coupled near a Hopf bifurcation where we assume a parameter ϵ uncouples the system at [Formula: see text]. Using a normal form for [Formula: see text] identical systems undergoing Hopf bifurcation, we explore the dynamical properties. Matching the normal form coefficients to a coupled Wilson-Cowan oscillator network gives an understanding of different types of behaviour that arise in a model of perceptual bistability. Notably, we find bistability between in-phase and anti-phase solutions that demonstrates the feasibility for synchronisation to act as the mechanism by which periodic inputs can be segregated (rather than via strong inhibitory coupling, as in the existing models). Using numerical continuation we confirm our theoretical analysis for small coupling strength and explore the bifurcation diagrams for large coupling strength, where the normal form approximation breaks down.

Altered expression of dopaminergic cell fate regulating genes prior to manifestation of symptoms in a transgenic rat model of Huntington's disease.

Hyperactivity of the dopaminergic pathway is thought to contribute to clinical symptoms in the early stages of Huntington's disease (HD). It is suggested to be result of a reduced dopaminergic inhibition by degeneration of medium spiny neurons in the striatum. Previously, we have shown that the number of dopaminergic cells is increased in the dorsal raphe nucleus (DRN) of HD patients and transgenic HD (tgHD) rats during the manifestation phase of the disease; as well as in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) of tgHD rats. To address whether these changes are secondary to neurodegeneration or take place in the pre-manifest phase of the disease, we examined the expression of genes controlling neuronal cell fate and genes that define dopaminergic cell phenotype. In the SNc-VTA of tgHD rats, Msx1 was upregulated, which correlated with an altered expression of transcription factors Zbtb16 and Tcf12. Zbtb16 was upregulated in the DRN and it was the only gene that showed a correlated expression in the tgHD rats between SNc-VTA and DRN. Zbtb16 may be a candidate for regionally tuning its cell populations, resulting in the increase in dopaminergic cells observed in our previous studies. Here, we demonstrated an altered expression of genes related to dopaminergic cell fate regulation in the brainstem of 6 months-old tgHD rats. This suggests that changes in dopaminergic system in HD precede the manifestation of clinical symptoms, contradicting the theory that hyperdopaminergic status in HD is a consequence of neurodegeneration in the striatum.

Quasiperiodic perturbations of heteroclinic attractor networks.

We consider heteroclinic attractor networks motivated by models of competition between neural populations during binocular rivalry. We show that gamma distributions of dominance times observed experimentally in binocular rivalry and other forms of bistable perception, commonly explained by means of noise in the models, can be achieved with quasiperiodic perturbations. For this purpose, we present a methodology based on the separatrix map to model the dynamics close to heteroclinic networks with quasiperiodic perturbations. Our methodology unifies two different approaches, one based on Melnikov integrals and the other one based on variational equations. We apply it to two models: first, to the Duffing equation, which comes from the perturbation of a Hamiltonian system and, second, to a heteroclinic attractor network for binocular rivalry, for which we develop a suitable method based on Melnikov integrals for non-Hamiltonian systems. In both models, the perturbed system shows chaotic behavior, while dominance times achieve good agreement with gamma distributions. Moreover, the separatrix map provides a new (discrete) model for bistable perception which, in addition, replaces the numerical integration of time-continuous models and, consequently, reduces the computational cost and avoids numerical instabilities.

Arc protein expression after unilateral intracranial self-stimulation of the medial forebrain bundle is upregulated in specific nuclei of memory-related areas.

BACKGROUND: Intracranial Self-Stimulation (ICSS) of the medial forebrain bundle (MFB) is a deep brain stimulation procedure, which has a powerful enhancement effect on explicit and implicit memory. However, the downstream synaptic plasticity events of MFB-ICSS in memory related areas have not been described thoroughly. This study complements previous work studying the effect of MFB-ICSS on the expression of the activity-regulated cytoskeleton-associated (Arc) protein, which has been widely established as a synaptic plasticity marker. We provide new integrated measurements from memory related regions and take possible regional hemispheric differences into consideration. RESULTS: Arc protein expression levels were analyzed 4.5 h after MFB-ICSS by immunohistochemistry in the hippocampus, habenula, and memory related amygdalar and thalamic nuclei, in both the ipsilateral and contralateral hemispheres to the stimulating electrode location. MFB-ICSS was performed using the same paradigm which has previously been shown to facilitate memory. Our findings illustrate that MFB-ICSS upregulates the expression of Arc protein in the oriens and radiatum layers of ipsilateral CA1 and contralateral CA3 hippocampal regions; the hilus bilaterally, the lateral amygdala and dorsolateral thalamic areas as well as the central medial thalamic nucleus. In contrast, the central amygdala, mediodorsal and paraventricular thalamic nuclei, and the habenular complex did not show changes in Arc expression after MFB-ICSS. CONCLUSIONS: Our results expand our knowledge of which specific memory related areas MFB-ICSS activates and, motivates the definition of three functionally separate groups according to their Arc-related synaptic plasticity response: (1) the hippocampus and dorsolateral thalamic area, (2) the central medial thalamic area and (3) the lateral amygdala.

Increased training compensates for OX1R blockage-impairment of spatial memory and c-Fos expression in different cortical and subcortical areas.

It has been suggested that the orexin system modulates learning and memory-related processes. However, the possible influence that training could have on the effect of the blockade of orexin-A selective receptor (OX1R) on a spatial memory task has not been explored. Therefore, the present study attempts to compare the effects of OX1R antagonist SB-334867 infusion on spatial memory in two different conditions in the Morris Water Maze (MWM). This experiment evaluated the animals' performance in weak training (2 trials per session) vs strong training (6 trials per session) protocols in a spatial version of the MWM. We found that in the 2-trial condition the post-training SB-334867 infusion had a negative effect on consolidation as well as on the retention and reversal learning of the task 72 h later. This effect was not apparent in the 6-trial condition. In addition, while the strong training groups showed a general increase in c-Fos expression in several brain areas of the hippocampal-thalamic-cortical circuit, SB-334867 administration had the opposite effect in areas that have been previously reported to have a high density of OX1R. Specifically, the SB-infused group in the 2-trial condition showed a decrease in c-Fos immunoreactivity in the dentate gyrus, granular retrosplenial and prelimbic cortices, and centrolateral thalamic nucleus. This was not observed for subjects in the 6-trial condition. The activation of these areas could constitute a neuroanatomical substrate involved in the compensatory mechanisms of training upon SB-334867 impairing effects on a MWM spatial task.

Evaluation of common housekeeping proteins under ischemic conditions and/or rt-PA treatment in bEnd.3 cells.

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the only pharmacological approved treatment for ischemic stroke, despite its associated increasing risk of hemorrhagic transformation. Since many of rt-PA effects in blood-brain barrier (BBB) are not well characterized, the study of protein changes in BBB cells after rt-PA administration may help to understand its adverse effects. Our aim was to analyze protein levels of four commonly used housekeeping proteins: ß-Actin, α-Tubulin, GAPDH and HPRT in bEnd.3 endothelial cell line subjected to oxygen and glucose deprivation (OGD) conditions and rt-PA treatment to determine their reliability as Western blot loading controls. bEnd.3 monolayers were subjected to 2.5 h of OGD and reperfusion with/without 20 µg/ml of rt-PA. At 3, 6, 24 and 72 h post-OGD, protein levels were analyzed by Western blot using Stain-Free technology. OGD significantly decreased ß-Actin, α-Tubulin, GAPDH and HPRT protein levels at 3, 6, 24 and 72 h post-OGD without significant rt-PA treatment effects except for the GAPDH levels increase in control condition at 3 h post-OGD. The present study clearly demonstrated that ß-Actin, α-Tubulin, GAPDH and HPRT proteins are not suitable as loading controls for Western Blot analysis in bEnd.3 cells after OGD. SIGNIFICANCE: We reported altered levels of ß-Actin, α-Tubulin, GAPDH and HPRT housekeeping proteins in bEnd.3 endothelial cell line after an ischemic insult. Therefore, we demonstrated that these proteins are not suitable as loading controls for Western Blot analysis in our experimental conditions and we recommended the use of Stain-Free gels as an alternative to traditional housekeeping proteins normalization.

Low Levels of Caveolin-1 Predict Symptomatic Bleeding After Thrombolytic Therapy in Patients With Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Experimental models of cerebral ischemia demonstrate that the decrease in the caveolin-1 membrane protein results in an increase in endothelial permeability. Because this phenomenon is responsible for hemorrhagic transformation (HT) after cerebral ischemia, we aimed to determine whether caveolin-1 levels may predict bleeding after recombinant tissue-type plasminogen activator (r-tPA) administration in patients with acute stroke. METHODS: We studied 133 patients with a first hemispheric stroke treated with r-tPA within 4.5 hours of symptom onset. HT was evaluated and classified on cranial computed tomography at 24 hours and was considered as symptomatic HT (sHT) if associated with neurological deterioration. Serum caveolin-1 levels were analyzed before and at 2, 24, and 72 hours post-r-tPA administration in patients and in 40 healthy controls. RESULTS: Baseline caveolin-1 levels were higher in patients than controls (0.24 [0.17-0.40] versus 0.07 [0.0-0.20] ng/mL; P<0.000). Twenty six (19.5%) patients had HT, which was symptomatic in 7 (5.3%). Patients with parenchymal hemorrhage-2 and sHT had lower baseline caveolin-1 levels than the rest of patients (0.08 [0.04-0.19] versus 0.26 [0.14-0.40]; P=0.019 and 0.08 [0.02-0.17] versus 0.26 [0.13-0.41]; P=0.019, respectively). The levels remained stable in the first 72 hours in patients with parenchymal hemorrhage-2 and sHT, whereas in the rest of patients levels decreased in this time. Caveolin-1 levels ≤0.17 ng/mL had the highest predictive capacity of sHT (86% sensitivity, 65% specificity, 99% negative predictive value, 12% positive predictive value). After adjustment for confounders, caveolin-1 levels ≤0.17 ng/mL independently predicted sHT (odds ratio, 11.6; 95% confidence interval, 11.3-102.8; P=0.027). CONCLUSIONS: Low serum levels of caveolin-1 are an independent predictor of sHT after r-tPA administration. Because of the small sample size, further research is needed to validate these data.