Connexins-Based Hemichannels/Channels and Their Relationship with Inflammation, Seizures and Epilepsy.

Connexins (Cxs) are a family of 21 protein isoforms, eleven of which are expressed in the central nervous system, and they are found in neurons and glia. Cxs form hemichannels (connexons) and channels (gap junctions/electric synapses) that permit functional and metabolic coupling between neurons and astrocytes. Altered Cx expression and function is involved in inflammation and neurological diseases. Cxs-based hemichannels and channels have a relevance to seizures and epilepsy in two ways: First, this pathological condition increases the opening probability of hemichannels in glial cells to enable gliotransmitter release, sustaining the inflammatory process and exacerbating seizure generation and epileptogenesis, and second, the opening of channels favors excitability and synchronization through coupled neurons. These biological events highlight the global pathological mechanism of epilepsy, and the therapeutic potential of Cxs-based hemichannels and channels. Therefore, this review describes the role of Cxs in neuroinflammation and epilepsy and examines how the blocking of channels and hemichannels may be therapeutic targets of anti-convulsive and anti-epileptic treatments.

An electrochemiluminescent method for glutamate measurement in small microdialysate samples in asphyxiated young rats.

Glutamate (Glu) quantification has been performed by a combination of intracerebral microdialysis through which the samples are obtained and analyzed by high performance liquid chromatography (HPLC); its measurement requires a large expenditure of time (15-30 min per sample) and special training. Therefore, an alternative method is presented here, based on the electrochemiluminescence produced by the use of an enzymatic reactor, containing glutamate-oxidase, mixed and incubated with microdialysate from dorsal striatum (DS) and prefrontal cortex (PFC) of young rats asphyxiated during the neonatal period, under a global asphyxia model in order to test this method. Using this approach, we found high extracellular Glu concentration in the DS of asphyxiated animals, but only during K+ stimulation, while in the PFC, only a delay in the rise of Glu after K+ stimulation was observed, without any difference in extracellular Glu content when compared with controls. This new method permitted a fast measurement of Glu in brain dialysate samples, it significantly reduces the cost of the analysis per sample, since only a single device and pump are needed without using columns and high pressure inside the system or complex hardware and software to control pumps, detector, fraction collector or any other peripheral used in HPLC.

Increase in the extracellular glutamate level during seizures and electrical stimulation determined using a high temporal resolution technique.

BACKGROUND: Glutamate has been measured using different methods to determine its role under normal and pathological conditions. Although microdialysis coupled with HPLC is the preferred method to study glutamate, this technique exhibits poor temporal resolution and is time consuming. The concentration of glutamate in dialysis samples can be measured via glutamate oxidase using the Amplex Red method. METHODS: A new device has been designed and constructed to rapidly deposit dialysis samples onto a polycarbonate plate at Cartesian coordinates (every five seconds). The samples were added to an enzymatic reaction that generates hydrogen peroxide from glutamate, which was quantified using fluorescence detection. Fluorescence emission was induced by laser excitation, stimulating each spot automatically, in addition to controlling the humidity, temperature and incubation time of the enzymatic reaction. RESULTS: The measurement of standard glutamate concentrations was linear and could be performed in dialysis samples. This approach was used to determine the effect of the convulsant drugs bicuculline and 4-aminopyridine on the extracellular glutamate concentration. Seizure activity was associated with a considerable increase in glutamate that correlated with altered EEG patterns for both drugs. CONCLUSIONS: These results indicate that this method is able to read samples with high temporal resolution, and it is easy to use compared with classical methods such as high-performance liquid chromatography, with the advantage that a large number of samples can be measured in a single experimental series. This method provides an alternative approach to determine the concentrations of neurotransmitters or other compounds that generate hydrogen peroxide as a reaction product.

A novel hydro-pneumatic fluid percussion device for inducing traumatic brain injury: assessment of sensory, motor, cognitive, molecular, and morphological outcomes in rodents.

Introduction: The fluid percussion method is widely used to induce brain injury in rodents. However, this approach has several limitations, including variability in the resulting damage, which is attributed to factors such as manual control of the mass used to generate the desired pressure. To address these issues, several modifications to the original method have been proposed. Methods: In this study, we present a novel device called the Hydro-pneumatic Fluid Percussion Device, which delivers fluid directly to a lateral region of the brain to induce injury. To validate this model, three groups of male and female rats were subjected to lateral fluid percussion using our device, and the resulting damage was evaluated using sensory, motor, and cognitive tests, measurements of serum injury biomarkers, and morphological analysis via cresyl violet staining. Results: Our results demonstrate that this new approach induced significant alterations in all parameters evaluated. Discussion: This novel device for inducing TBI may be a valuable alternative for modeling brain injury and studying its consequences.

Nano dot blot: An alternative technique for protein identification and quantification in a high throughput format.

BACKGROUND: Dot blot technique has been used in a similar way to western blotting, with the major difference being the lack of protein separation with electrophoresis. Protein samples are spotted over a membrane paper, the identification and quantification of a protein is achieved by immunodetection procedures such as colorimetry, fluorescence or chemiluminescence. This technique is widely accepted, but it uses large amounts of sample and antibodies to reveal the presence of the target protein. Significant milestones have been reached to achieve better results with the use of less sample and reagents; however, the ninety-six-well format is still in use. NEW METHOD: In this work, we propose an innovation to this technique, reducing the amount of sample and antibodies to identify a specific protein when compared to the regular dot blot method. Procedure consists of using a sample volume of approximately 200 nanoliters deposited with a multineedle device developed by our group. RESULTS: Five samples of standard protein or antigen can be spotted in a Cartesian format to identify and quantify the protein involved in physiological or pathological conditions. In addition, at least five replicates of sample or antigen are used to enable better statistics to calculate the concentration of every standard and the protein present in a sample. CONCLUSIONS: Hundreds of samples can be deposited in a few minutes and analyzed in a single experimental session. To validate this method, which we called nano dot blot, six proteins involved in the inflammation process were tested in acute and chronic rat models of seizures.

Evidence of sexual dimorphism in D1 and D2 dopaminergic receptors expression in frontal cortex and striatum of young rats.

D1 and D2 receptors are key mediators of dopaminergic signaling in the brain, and since the manifestations of pathologies related to dopamine are different in female and male patients, it is important to analyze if there are sex-related differences in dopaminergic markers. To contribute to the knowledge in this regard, the objective of this report was to characterize the particular expression level of D1 and D2 dopamine receptors in young male and female rats. Striatum (STR) and frontal cortex (CTX) were obtained from intact 30-days old animals, and the D1 and D2 expression level was analyzed by Western blot. The results show a greater expression of D1, but less of D2, in female CTX compared with males, whereas in STR, both D1 and D2 receptors shows predominance in females. These results support the evidence of dimorphic expression in dopaminergic markers, outside of the sex-related brain nuclei, and suggests an early effect of hormones in establishing long life characteristics in dopaminergic circuits.