Monika Malinowska

Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling–induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.

Cardiotoxicity of the Anticancer Therapeutic Agent Bortezomib

Recent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca(2+) fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs.

Cumulative benefits of frontal transcranial direct current stimulation on visuospatial working memory training and skill learning in rats

Transcranial direct current stimulation (tDCS) of the prefrontal cortex, which non-invasively alters cortical activity, has been established to affect executive functions in humans. We hypothesized that changes in excitability by tDCS, found to improve cognitive functions dependent on moderate prefrontal cortex activity, would operate similarly in animals as in humans. To verify this we performed experiments using a rat behavioral model of visuospatial working memory and skill learning paired with tDCS of the frontal cortex. The effect of anodal/cathodal tDCS was examined in three sessions using the allothetic place avoidance alternation task (APAAT) and later re-examined without stimulation. Stimulation had no measurable short term effect on on-going place avoidance learning. However, in the follow-up session on day 21 the rats previously treated with cathodal tDCS showed significantly more efficient place avoidance and skill retention in comparison to the controls. This demonstrates a long-term benefit of diminished excitability by frontal tDCS when paired with training on working memory and skill learning in a novel task. The presented behavioral model provides a tool to evaluate the underlying mechanisms of how tDCS modulates neural network function to support successful behavior.

Novel Higher-Order Epigenetic Regulation of the Bdnf Gene upon Seizures

Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have. In a rat model of massive neuronal activation upon kainate-induced seizures we found that elevated neuronal expression of Bdnf is associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressed Trkb remains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleus in vivo contributes to activity-dependent changes in gene expression in the brain.

Extended-focus optical coherence microscopy for high-resolution imaging of the murine brain

We propose a new method and optical instrumentation for mouse brain imaging based on extended-focus optical coherence microscopy. This in vivo imaging technique allows the evaluation of the cytoarchitecture at cellular level and the circulation system dynamics in three dimensions. This minimally invasive and non-contact approach is performed without the application of contrasting agents. The optical design achieved a resolution of 2.2 μm over a distance of 800 μm, which was sufficient to obtain a detailed three-dimensional image of a wild-type mouses brain down to the layer III of the cortex. Intrinsically contrasted microvessels and structures similar to the bodies of neurons were distinguishable.

Localization and regulation of PML bodies in the adult mouse brain

PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.

Different effects of lesions to auditory core and belt cortex on auditory recognition in dogs.

Auditory recognition memory, in contrast to memory in other modalities, is not affected by damage to the perihinal cortex, and its neural basis remains unknown. In an attempt to elucidate this problem, we investigated the role of canine auditory core and belt areas in auditory recognition. Either core or posterior belt areas were surgically removed. The core and belt regions were defined on the basis of response properties and thalamocortical connectivity established in previous studies. The animals were tested on auditory delayed matching to sample (DMS, a recognition memory task) using complex, trial-unique auditory stimuli. Both core and belt lesions impaired auditory recognition, however, the underlying deficit was different. Lesions to the core areas impaired auditory localization abilities. Lesions to the posterior belt areas did not affect this component of the recognition task, but affected auditory quality discrimination and/or recognition. The deficit following the posterior belt lesion did not increase with retention delay, suggesting that auditory belt areas do not constitute a substrate for auditory recognition memory. Their main function appears to be processing of complex sound patterns, including immediate recognition.

Cytoarchitecture and thalamic afferents of the sylvian and composite posterior gyri of the canine temporal cortex.

The composite posterior and sylvian gyri of the canine temporal cortex show cytoarchitectonic features of poorly differentiated isocortex. Quantitative evaluation of connections examined with retrogradely transported fluorescent tracers indicated that both gyri received strong thalamic projections from the medial geniculate body (MG) and the lateromedial-suprageniculate (LM-Sg) complex, and a weaker projection from the posterior (Po) nuclei. On the basis of the connectivity patterns and cytoarchitectonic features we distinguished the anterior (CPa) and posterior (CPp) areas in posterior composite gyrus and the anterior (SA), dorsal (SD) and posterior (SP) sylvian areas. Afferents from individual thalamic nuclei were focused in distinct areas, forming dominant projections, and diminished gradually in the adjacent areas as non-dominant projections. The most prominent MG projection arose from the dorsal caudal (MGdc) nucleus. Its ventral subdivision sent a dominant projection into the SP and CPa, whereas the dorsal MGdc subdivision was connected with the SA, SD and CPp areas. The most substantial connections from the LM-Sg complex were directed to areas SA, SD and CPp, with weak connections to areas CPa and SP. A gradient of density of LM-Sg afferents was distributed in the opposite direction to that sent from the MGdc. The origin of the CPa and SP afferents in the ventral MGdc, like connections reaching the posterior ectosylvian cortex, suggest that these areas are related to processing of auditory information. In contrast, areas SA and CPp, receive dominant projections from the polymodal LM-Sg, and therefore may constitute successive steps in a hierarchy of cortical areas.

Vascular Cognitive Impairment Linked to Brain Endothelium Inflammation in Early Stages of Heart Failure in Mice

Background Although advanced heart failure (HF) is a clinically documented risk factor for vascular cognitive impairment, the occurrence and pathomechanisms of vascular cognitive impairment in early stages of HF are equivocal. Here, we characterize vascular cognitive impairment in the early stages of HF development and assess whether cerebral hypoperfusion or prothrombotic conditions are involved. Methods and Results Tgαq*44 mice with slowly developing isolated HF triggered by cardiomyocyte‐specific overexpression of G‐αq*44 protein were studied before the end‐stage HF, at the ages of 3, 6, and 10 months: before left ventricle dysfunction; at the stage of early left ventricle diastolic dysfunction (with preserved ejection fraction); and left ventricle diastolic/systolic dysfunction, respectively. In 6‐ to 10‐month‐old but not in 3‐month‐old Tgαq*44 mice, behavioral and cognitive impairment was identified with compromised blood‐brain barrier permeability, most significantly in brain cortex, that was associated with myelin sheet loss and changes in astrocytes and microglia. Brain endothelial cells displayed increased E‐selectin immunoreactivity, which was accompanied by increased amyloid‐β1‐42 accumulation in piriform cortex and increased cortical oxidative stress (8‐OHdG immunoreactivity). Resting cerebral blood flow measured by magnetic resonance imaging in vivo was preserved, but ex vivo NO‐dependent cortical arteriole flow regulation was impaired. Platelet hyperreactivity was present in 3‐ to 10‐month‐old Tgαq*44 mice, but it was not associated with increased platelet‐dependent thrombogenicity. Conclusions We report for the first time that vascular cognitive impairment is already present in the early stage of HF development, even before left ventricle systolic dysfunction. The underlying pathomechanism, independent of brain hypoperfusion, involves preceding platelet hyperreactivity and brain endothelium inflammatory activation.

Assessment of the influence of viscoelasticity of cornea in animal ex vivo model using air-puff optical coherence tomography and corneal hysteresis

Application of the air‐puff swept source optical coherence tomography (SS‐OCT) instrument to determine the influence of viscoelasticity on the relation between overall the air‐puff force and corneal apex displacement of porcine corneas ex vivo is demonstrated. Simultaneous recording of time‐evolution of the tissue displacement and air pulse stimulus allows obtaining valuable information related in part to the mechanical properties of the cornea. A novel approach based on quantitative analysis of the corneal hysteresis of OCT data is presented. The corneal response to the air pulse is assessed for different well‐controlled intraocular pressure (IOP) levels and for the progression of cross‐linking‐induced stiffness of the cornea. Micrometer resolution, fast acquisition and noncontact character of the air‐puff SS‐OCT measurements have potential to improve the in vivo assessment of mechanical properties of the human corneas.