System-Wide Immunohistochemical Analysis of Protein Co-Localization

Background: The analysis of co-localized protein expression in a tissue section is often conducted with immunofluorescence histochemical staining which is typically visualized in localized regions. On the other hand, chromogenic immunohistochemical staining, in general, is not suitable for the detection of protein co-localization. Here, we developed a new protocol, based on chromogenic immunohistochemical stain, for system-wide detection of protein co-localization and differential expression. Methodology/Principal Findings: In combination with a removable chromogenic stain, an efficient antibody stripping method was developed to enable sequential immunostaining with different primary antibodies regardless of antibody’s host species. Sections were scanned after each staining, and the images were superimposed together for the detection of protein co-localization and differential expression. As a proof of principle, differential expression and co-localization of glutamic acid decarboxylase67 (GAD67) and parvalbumin proteins was examined in mouse cortex. Conclusions/Significance: All parvalbumin-containing neurons express GAD67 protein, and GAD67-positive neurons that do not express parvalbumin were readily visualized from thousands of other neurons across mouse cortex. The method provided a global view of protein co-localization as well as differential expression across an entire tissue section. Repeate

The neuroprotective potentially of agomelatine - animal model study

BackgroundMajor depressive disorder associated the increase of cortisol level and high level of cortisolemia is correlated with the decrease of neuroprotection. Hippocampus low volume is a marker for depressive disorder. The cognitive impairment in depression is linked with hippocampus - enthorinal structures-frontal cortex disconectivity. Melatoninic agonists can increase the neuroprotection and improve depressive mood.MethodsWe formed 2 study lots each constitued of 5 male adults rats (200–250 g), held through the study duration in temperature, humidity, food and ambient stress less conditions, compared to a control lot. The studied substances were administrated intraperitoneal, daily, for 10 days, saline solution equivalent to dexametasone (0.20 mg/kg/day) (N1); 14 days we administered 10 mg/kg/daily of agomelatine through a stomach tube and the following 10 days of dexametasone and agomelatine in the same dosage (N2). The N1 lot was sacrificed in the day 11, and the N2 lot was sacrificed in the day 25.The sample brain was histopathologically processed: formalin (10%) and ethyl alcohol (96%) fixation and paraffine embeded. Microtome slices were stained in hematoxyline-eosine, trichromicGS, PAS-hematoxyline, toluidine blue and methylen blue for Nissle corpuscles. The obtained slices were studied with optical microscopy, the target of our study was hippocampus and frontal cortex.ResultsThe hippocampus and frontal cortex presents important histopathological abnormalities (neuronal death, vacuolisation, axonal fragmentation) for N1 after dexametasone. These changes are decreased in N2.ConclusionsAgomelatine on the animal model actioned with a neuroprotective effect in the hippocampus and frontal cortex front of cortisolic aggresion (marker for depression).</jats:sec

Modified variant of the Rieske iron-sulfur protein in the hippocampus of kindled rats and human epileptic patients

Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague-Dawley rat is associated with a critical developmental interval of 25 ± 1 days; the identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relationship to normal and pathological brain function. Now we report the identification, by proteomics, of a modified variant of the Rieske iron-sulfur protein, a component of the mitochondrial cytochrome bc1 complex, whose isoelectric point is shifted toward more alkaline values in the hippocampus of kindled rats. By immunohistochemistry the Rieske protein is well-expressed in the hippocampus except in the CA1 subfield, a region of selective vulnerability to seizures in humans and animal models. We also noted an asymmetric, selective expression of the Rieske protein in the subgranular neurons of the dorsal dentate gyrus, a region implicated in neurogenesis. Abnormal changes in Rieske protein immunoreactivity also were found in sections obtained from human epileptic patients. These results suggest that the Rieske protein may play a role in the response of neurons to seizure activity and could give important new insights into the molecular pathogenesis of epilepsy.Griffith Health, School of MedicineNo Full Tex