Neurophotonic methods in approach to in vivo animal epileptic models: Advantages and limitations.

Neurophotonic technology is a rapidly growing group of techniques that are based on the interactions of light with natural or genetically modified cells of the neural system. New optical technologies make it possible to considerably extend the tools of neurophysiological research, from the visualization of functional activity changes to control of brain tissue excitability. This opens new perspectives for studying the mechanisms underlying the development of human neurological diseases. Epilepsy is one of the most common brain disorders; it is characterized by recurrent seizures and affects >1% of the world's population. However, how seizures occur, spread, and terminate in a healthy brain is still unclear. Therefore, it is extremely important to develop appropriate models to accurately explore the causal relationship of epileptic activity. The use of neurophotonic technologies in epilepsy research falls into two broad categories: the visualization of neural epileptic activity, and the direct optical influence on neurons to induce or suppress epileptic activity. An optogenetic variant of the classical kindling model of epileptic seizures, in which activatable cells are genetically defined, is called optokindling. Research is also underway concerning the application of neurophotonic techniques for suppressing epileptic activity, aiming to bring these methods into clinical practice. This review aims to systematize and describe new approaches that use combinations of different neurophotonic methods to work with in vivo models of epilepsy. These approaches overcome many of the shortcomings associated with classical animal models of epilepsy and thus increase the effectiveness of developing new diagnostic methods and antiepileptic therapy.

An Integrative Approach to the Current Treatment of HIV-Associated Neurocognitive Disorders and the Implementation of Leukemia Inhibitor Factor as a Mediator of Neurocognitive Preservation.

HIV-associated neurocognitive disorders (HANDs) continue to impact patients despite antiretroviral therapy. A combination of antiretroviral therapies can diminish the HIV viral load to near undetectable levels, but fails to preserve neurocognitive integrity. The cytokine leukemia inhibitory factor (LIF) has shown neuroprotective properties that could mitigate neurodegeneration in HANDs. The LIF promotes neurogenesis, neural cell differentiation, and survival. Combination antiretroviral therapy reduces severe forms of HANDs, but neurocognitive impairment persists; additionally, some antiretrovirals have additional adverse neurotoxic effects. The LIF counteracts neurotoxic viral proteins and limits neural cell damage in models of neuroinflammation. Adding the LIF as an adjuvant therapy to enhance neuroprotection merits further research for managing HANDs. The successful implementation of the LIF to current therapies would contribute to achieving a better quality of life for the affected population.

Pyk2/FAK Signaling Is Upregulated in Recurrent Glioblastoma Tumors in a C57BL/6/GL261 Glioma Implantation Model.

The majority of glioblastomas (GBMs) recur shortly after tumor resection and recurrent tumors differ significantly from newly diagnosed GBMs, phenotypically and genetically. In this study, using a Gl261-C57Bl/6 mouse glioma implantation model, we identified significant upregulation of proline-rich tyrosine kinase Pyk2 and focal adhesion kinase (FAK) phosphorylation levels-pPyk2 (579/580) and pFAK (925)-without significant modifications in total Pyk2 and FAK protein expression in tumors regrown after surgical resection, compared with primary implanted tumors. Previously, we demonstrated that Pyk2 and FAK are involved in the regulation of tumor cell invasion and proliferation and are associated with reduced overall survival. We hypothesized that the use of inhibitors of Pyk2/FAK in the postsurgical period may reduce the growth of recurrent tumors. Using Western blot analysis and confocal immunofluorescence approaches, we demonstrated upregulation of Cyclin D1 and the Ki67 proliferation index in tumors regrown after resection, compared with primary implanted tumors. Treatment with Pyk2/FAK inhibitor PF-562271, administered through oral gavage at 50 mg/kg daily for two weeks beginning 2 days before tumor resection, reversed Pyk2/FAK signaling upregulation in recurrent tumors, reduced tumor volume, and increased animal survival. In conclusion, the use of Pyk2/FAK inhibitors can contribute to a delay in GBM tumor regrowth after surgical resection.

Arc protein, a remnant of ancient retrovirus, forms virus-like particles, which are abundantly generated by neurons during epileptic seizures, and affects epileptic susceptibility in rodent models.

A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or Arc protein) of retroviral ancestry resides in the genome of all tetrapods for millions of years and is expressed endogenously in neurons. It is a well-known protein, very important for synaptic plasticity and memory consolidation. Activity-dependent Arc expression concentrated in glutamatergic synapses affects the long-time synaptic strength of those excitatory synapses. Because it modulates excitatory-inhibitory balance in a neuronal network, the Arc gene itself was found to be related to the pathogenesis of epilepsy. General Arc knockout rodent models develop a susceptibility to epileptic seizures. Because of activity dependence, synaptic Arc protein synthesis also is affected by seizures. Interestingly, it was found that Arc protein in synapses of active neurons self-assemble in capsids of retrovirus-like particles, which can transfer genetic information between neurons, at least across neuronal synaptic boutons. Released Arc particles can be accumulated in astrocytes after seizures. It is still not known how capsid assembling and transmission timescale is affected by seizures. This scientific field is relatively novel and is experiencing swift transformation as it grapples with difficult concepts in light of evolving experimental findings. We summarize the emergent literature on the subject and also discuss the specific rodent models for studying Arc effects in epilepsy. We summarized both to clarify the possible role of Arc-related pseudo-viral particles in epileptic disorders, which may be helpful to researchers interested in this growing area of investigation.

The Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer.

BACKGROUND: The significant challenge in treating triple-negative breast cancer (TNBC) lies in its high rate of distant metastasis. To address this, inhibiting metastasis formation in TNBC is vital. Rac is a key player in cancer metastasis. Previously, we developed Ehop-016, a Rac inhibitor that successfully reduced tumor growth and metastasis in mice. In this study, we assessed the effectiveness of HV-107, a derivative of Ehop-016, in inhibiting TNBC metastasis at lower doses. METHODS: Rho GTPases activity assays were performed with the use of GST-PAK beads and Rac, Rho, and Cdc42 GLISA. Cell viability was assessed through trypan blue exclusion and MTT assays. Cell cycle analysis was conducted using flow cytometry. To evaluate invading capabilities, transwell assays and invadopodia formation assays were performed. Metastasis formation studies were conducted using a breast cancer xenograft mouse model. RESULTS: HV-107 inhibited Rac activity by 50% in MDA-MB-231 and MDA-MB-468 cells at concentrations of 250-2000 nM, leading to a 90% decrease in invasion and invadopodia activity. Concentrations of 500 nM and above caused dose-dependent reductions in cell viability, resulting in up to 20% cell death after 72 h. Concentrations exceeding 1000 nM upregulated PAK1, PAK2, FAK, Pyk2, Cdc42, and Rho signallings, while Pyk2 was downregulated at 100-500 nM. Through in vitro experiments, optimal concentrations of HV-107 ranging from 250 to 500 nM were identified, effectively inhibiting Rac activity and invasion while minimizing off-target effects. In a breast cancer xenograft model, administration of 5 mg/kg HV-107 (administered intraperitoneally, 5 days a week) reduced Rac activity by 20% in tumors and decreased metastasis by 50% in the lungs and liver. No observed toxicity was noted at the tested doses. CONCLUSION: The findings indicate that HV-107 exhibits promising potential as a therapeutic medication utilizing Rac inhibition mechanisms to address metastasis formation in TNBC.

Adjuvant effect of dendritic cells activator Imiquimod in genetic immunization with HIV-1 p55 Gag.

Dendritic cells (DC) are important antigen-presenting cells that have abilities to induce and maintain T-cell immunity, or attenuate it during hyperimmunization. Additional activation of DCs may be useful for vaccination purposes. Imiquimod is known to be a specific agonist of the Toll-like receptors (TLR7), which are located mainly on DCs. To study the effect of DC stimulation on the effectiveness of an HIV-1 p55 gag DNA vaccine in a mice model, we employed 25, 50, and 100 nM of Imiquimod as an adjuvant. Subsequently, Western blot analysis was used to quantify p55 protein production after the immunization. To characterize T-cells immune response, both the frequency of IFN-γ -secreting cells and IFN-γ and IL-4 production were measured, via an ELIspot assay and ELISA, respectively. Low concentrations of Imiquimod were found to effectively stimulate Gag production and the magnitude of the T-cell immune response, whereas higher concentrations reduced vaccination effects. Our results show that the adjuvant effects of Imiquimod depend on concentration. The use of Imiquimod may be helpful to study DC to T cell communication, including possible induction of immunotolerance.

The Effects of Amyloid-ß on Metabolomic Profiles of Cardiomyocytes and Coronary Endothelial Cells.

BACKGROUND: An increasing number of experimental and clinical studies show a link between Alzheimer's disease and heart diseases such as heart failure, ischemic heart disease, and atrial fibrillation. However, the mechanisms underlying the potential role of amyloid-ß (Aß) in the pathogenesis of cardiac dysfunction in Alzheimer's disease remain unknown. We have recently shown the effects of Aß1 - 40 and Aß1 - 42 on cell viability and mitochondrial function in cardiomyocytes and coronary artery endothelial cells. OBJECTIVE: In this study, we investigated the effects of Aß1 - 40 and Aß1 - 42 on the metabolism of cardiomyocytes and coronary artery endothelial cells. METHODS: Gas chromatography-mass spectrometry was used to analyze metabolomic profiles of cardiomyocytes and coronary artery endothelial cells treated with Aß1 - 40 and Aß1 - 42. In addition, we determined mitochondrial respiration and lipid peroxidation in these cells. RESULTS: We found that the metabolism of different amino acids was affected by Aß1 - 42 in each cell type, whereas the fatty acid metabolism is consistently disrupted in both types of cells. Lipid peroxidation was significantly increased, whereas mitochondrial respiration was reduced in both cell types in response to Aß1 - 42. CONCLUSION: This study revealed the disruptive effects of Aß on lipid metabolism and mitochondria function in cardiac cells.

Amount of Melanin Granules in Human Hair Defines the Absorption and Conversion to Heat of Light Energy in the Visible Spectrum.

One of the known important functions of hair is protection from extensive sunlight. This protection is accomplished in large part due to natural hair pigmentation which is known to reflect the number of melanin granules (melanosomes) in the hair shaft, and melanin variants. Melanin takes in excessive light energy and converts it to heat in a process called absorption; heat is then dissipated into the environment as infrared radiation, thereby protecting the underlying skin. We used transmission electron microscopy (TEM) to visualize the melanosome counts in samples of human hair, and used thermal microscopy to measure the temperature changes of the samples when exposed to green and blue light lasers. In our experiments green light conversion to heat was highly correlated to the number of melanosomes, whereas blue light conversion to heat was less correlated, which may be because the reddish melanosomes it contains are less effective in absorbing energy from the blue spectrum of light. Anyway, we have shown the metals accumulation in the melanin can be easily visualized with TEM. We confirmed that the amount of melanin granules in human hair defines the conversion to heat of light energy in the visible spectrum.

Multilayer subwavelength gratings or sandwiches with periodic structure shape light reflection in the tapetum lucidum of taxonomically diverse vertebrate animals.

Eye shine in the dark has attracted many researchers to the field of eye optics, but the initial studies of subwavelength arrangements in tapetum began only with the development of electronic microscopy at the end of the 20th century. As a result of a number of studies, it was shown that the reflective properties of the tapetum are due to their specialized cellular subwavelength microstructure (photonic crystals). These properties, together with the mutual orientation of the crystals, lead to a significant increase in reflection, which, in turn, enhances the sensitivity of the eye. In addition, research confirmed that optical mechanisms of reflection in the tapetum are very similar even for widely separated species. Due to progress in the field of nano-optics, researchers now have a better understanding of the main principles of this phenomenon. In this review, we summarize electron microscopic and functional studies of tapetal structures in the main vertebrate classes. This allows data on the microstructure of the tapetum to be used to improve our understanding of the visual system.

The Anti-Epileptic Effects of Carbenoxolone In Vitro and In Vivo.

Gap junctions (GJs) are intercellular junctions that allow the direct transfer of ions and small molecules between neighboring cells, and GJs between astrocytes play an important role in the development of various pathologies of the brain, including regulation of the pathological neuronal synchronization underlying epileptic seizures. Recently, we found that a pathological change is observed in astrocytes during the ictal and interictal phases of 4-aminopyridin (4-AP)-elicited epileptic activity in vitro, which was correlated with neuronal synchronization and extracellular epileptic electrical activity. This finding raises the question: Does this signal depend on GJs between astrocytes? In this study we investigated the effect of the GJ blocker, carbenoxolone (CBX), on epileptic activity in vitro and in vivo. Based on the results obtained, we came to the conclusion that the astrocytic syncytium formed by GJ-associated astrocytes, which is responsible for the regulation of potassium, affects the formation of epileptic activity in astrocytes in vitro and epileptic seizure onset. This effect is probably an important, but not the only, mechanism by which CBX suppresses epileptic activity. It is likely that the mechanisms of selective inhibition of GJs between astrocytes will show important translational benefits in anti-epileptic therapies.

Accumulation of amyloid beta (Aß) and amyloid precursor protein (APP) in tumors formed by a mouse xenograft model of inflammatory breast cancer.

Accumulation of amyloid in breast cancer is a well-known phenomenon, but only immunoglobulin light-chain amyloidosis (AL) or transthyretin (TTR) amyloid had been detected in human breast tumor samples previously. We recently reported that another amyloidogenic peptide, amyloid beta (Aß), is present in an aggregated form in animal and human high-grade gliomas and suggested that it originates systemically from the blood, possibly generated by platelets. To study whether breast cancers are also associated with these Aß peptides and in what form, we used a nude mouse model inoculated with triple-negative inflammatory breast cancer cell (SUM-149) xenografts, which develop noticeable tumors. Immunostaining with two types of specific antibodies for Aß identified the clear presence of Aß peptides associated with (a) carcinoma cells and (b) extracellular aggregated amyloid (also revealed by Congo red and thioflavin S staining). Aß peptides, in both cells and in aggregated amyloid, were distributed in clear gradients, with maximum levels near blood vessels. We detected significant presence of amyloid precursor protein (APP) in the walls of blood vessels of tumor samples, as well as in carcinoma cells. Finally, we used ELISA to confirm the presence of elevated levels of mouse-generated Aß40 in tumors. We conclude that Aß in inflammatory breast cancer tumors, at least in a mouse model, is always present and is concentrated near blood vessels. We also discuss here the possible pathways of Aß accumulation in tumors and whether this phenomenon could represent the specific signature of high-grade cancers.

In Vitro and In Vivo Study of the Short-Term Vasomotor Response during Epileptic Seizures.

Epilepsy remains one of the most common brain disorders, and the different types of epilepsy encompass a wide variety of physiological manifestations. Clinical and preclinical findings indicate that cerebral blood flow is usually focally increased at seizure onset, shortly after the beginning of ictal events. Nevertheless, many questions remain about the relationship between vasomotor changes in the epileptic foci and the epileptic behavior of neurons and astrocytes. To study this relationship, we performed a series of in vitro and in vivo experiments using the 4-aminopyridine model of epileptic seizures. It was found that in vitro pathological synchronization of neurons and the depolarization of astrocytes is accompanied by rapid short-term vasoconstriction, while in vivo vasodilation during the seizure prevails. We suggest that vasomotor activity during epileptic seizures is a correlate of the complex, self-sustained response that includes neuronal and astrocytic oscillations, and that underlies the clinical presentation of epilepsy.

On the Role of Platelet-Generated Amyloid Beta Peptides in Certain Amyloidosis Health Complications.

As do many other immunity-related blood cells, platelets release antimicrobial peptides that kill bacteria, fungi, and even certain viruses. Here we review the literature suggesting that there is a similarity between the antimicrobials released by other blood cells and the amyloid-related Aß peptide released by platelets. Analyzing the literature, we also propose that platelet-generated Aß amyloidosis may be more common than currently recognized. This systemic Aß from a platelet source may participate in various forms of amyloidosis in pathologies ranging from brain cancer, glaucoma, skin Aß accumulation, and preeclampsia to Alzheimer's disease and late-stage Parkinson's disease. We also discuss the advantages and disadvantages of specific animal models for studying platelet-related Aß. This field is undergoing rapid change, as it evaluates competing ideas in the light of new experimental observations. We summarized both in order to clarify the role of platelet-generated Aß peptides in amyloidosis-related health disorders, which may be helpful to researchers interested in this growing area of investigation.

Electron microscopy study of the central retinal fovea in Pied flycatcher: evidence of a mechanism of light energy transmission through the retina.

We present unique ultrastructural data on avian retinal cells. Presently and earlier (Zueva et al., 2016) we explored distribution of intermediate filaments (IFs) in retinal cells of the Pied flycatcher (Ficedula hypoleuca, Passeriformes, Aves) in the central foveolar zone. This retinal zone only contains single and double cone photoreceptors. Previously we found that continuous IFs span Müller cells (MC) lengthwise from the retinal inner limiting membrane (ILM) layer up to the outer limiting membrane (OLM) layer. Here we describe long cylindrical bundles of IFs (IFBs) inside the cone inner segments (CIS) adjoining the cone plasma membrane, with these IFBs following along the cone lengthwise, and surrounding the cone at equal spacing one from the other. Double cones form a combined unit, wherein they are separated by their respective plasma membranes. Double cones thus have a common external ring of IFBs, surrounding both cone components. In the layer of cilia, the IFBs that continue into the cone outer segment (COS) follow on to the cone apical tip along the direction of incident light, with single IFs separating from the IFB, touching, and sometimes passing in-between the light-sensitive lamellae of the COS. These new data support our previous hypothesis on the quantum mechanism of light energy propagation through the vertebrate retina (Zueva et al., 2016, 2019).

Accumulation of Amyloid Beta (Aß) Peptide on Blood Vessel Walls in the Damaged Brain after Transient Middle Cerebral Artery Occlusion.

It is well known that amyloid beta (Aß) peptides are generated in blood vessels, released into the brain during thrombosis, and temporarily accumulate in this organ after injury. Here we demonstrate that 24 h after transient middle cerebral artery occlusion (tMCAO), one of the standard models of focal ischemic stroke, Aß peptide accumulates in the brain, concentrating on the blood vessel walls. Because Aß oligomers are known to induce significant damage to brain cells, they act as an additional damaging factor during ischemic stroke. Considering that they have been shown to form ion channels in cells, affecting osmotic balance, we used an Aß peptide channel blocker, tromethamine (2-amino-2-(hydroxymethyl) propane-1,3-diol), to prevent this additional injury. Tromethamine injected 0.1 g/100 g body weight intraperitoneally at 5 min before tMCAO decreased water content in the damaged hemisphere, as measured by dry brain weight. Congo red staining, which binds only to Aß oligomer plaques (amyloid), showed that there was no significant presence of plaques. Therefore, we suggest that Aß peptide oligomers are responsible for some of the brain damage during stroke and that blockage of the ion channels that they form could be beneficial in treating this complex neurological syndrome.

Quantum mechanism of light energy propagation through an avian retina.

Taking into account the ultrastructure of the Pied Flycatcher foveal retina reported earlier and the earlier reported properties of Müller cell (MC) intermediate filaments (IFs) isolated from vertebrate retina, we proposed a quantum mechanism (QM) of light energy transfer from the inner limiting membrane level to visual pigments in the photoreceptor cells. This mechanism involves electronic excitation energy transfer in a donor-acceptor system, with the IFs excited by photons acting as energy donors, and visual pigments in the photoreceptor cells acting as energy acceptors. It was shown earlier that IFs with diameter 10 nm and length 117 µm isolated from vertebrate eye retina demonstrate properties of light energy guide, where exciton propagates along such IFs from MC endfeet area to photoreceptor cell area. The energy is mostly transferred via the contact exchange quantum mechanism. Our estimates demonstrate that energy transfer efficiencies in such systems may exceed 80-90%. Thus, the presently developed quantum mechanism of light energy transfer in the inverted retina complements the generally accepted classic optical mechanism and the mechanism whereby Müller cells transmit light like optical fibers. The proposed QM of light energy transfer in the inverted retina explains the high image contrast achieved in photopic conditions by an avian eye, being probably also active in other vertebrates.

Tissue Transparency In Vivo.

In vivo tissue transparency in the visible light spectrum is beneficial for many research applications that use optical methods, whether it involves in vivo optical imaging of cells or their activity, or optical intervention to affect cells or their activity deep inside tissues, such as brain tissue. The classical view is that a tissue is transparent if it neither absorbs nor scatters light, and thus absorption and scattering are the key elements to be controlled to reach the necessary transparency. This review focuses on the latest genetic and chemical approaches for the decoloration of tissue pigments to reduce visible light absorption and the methods to reduce scattering in live tissues. We also discuss the possible molecules involved in transparency.

Accumulation of Innate Amyloid Beta Peptide in Glioblastoma Tumors.

Immunostaining with specific antibodies has shown that innate amyloid beta (Aß) is accumulated naturally in glioma tumors and nearby blood vessels in a mouse model of glioma. In immunofluorescence images, Aß peptide coincides with glioma cells, and enzyme-linked immunosorbent assay (ELISA) have shown that Aß peptide is enriched in the membrane protein fraction of tumor cells. ELISAs have also confirmed that the Aß(1-40) peptide is enriched in glioma tumor areas relative to healthy brain areas. Thioflavin staining revealed that at least some amyloid is present in glioma tumors in aggregated forms. We may suggest that the presence of aggregated amyloid in glioma tumors together with the presence of Aß immunofluorescence coinciding with glioma cells and the nearby vasculature imply that the source of Aß peptides in glioma can be systemic Aß from blood vessels, but this question remains unresolved and needs additional studies.

Platelet-generated amyloid beta peptides in Alzheimer's disease and glaucoma.

Amyloid beta (Aß) peptides have been implicated in both Alzheimer's disease (AD) and glaucoma and have been shown to be the key etiological factor in these dangerous health complications. On the other hand, it is well known that Aß peptide can be generated from its precursor protein and massively released from the blood to nearby tissue upon the activation of platelets due to their involvement in innate immunity and inflammation processes. Here we review evidence about the development of AD and glaucoma neuronal damage showing their dependence on platelet count and activation. The correlation between the effect on platelet count and the effectiveness of anti-AD and anti-glaucoma therapies suggest that platelets may be an important player in these diseases.

Localization of αA-Crystallin in Rat Retinal Müller Glial Cells and Photoreceptors.

Transparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level of light scattering in their cytoplasm. It has been shown that both cell types contain (1) beaded intermediate filaments as well as (2) α-crystallin globulins. It is known that genetic and chemical alterations to these specialized proteins induce cytoplasmic opaqueness and visual complications. Crystallins were described previously in the retinal Müller cells of frogs. In the present work, using immunocytochemistry, fluorescence confocal imaging, and immuno-electron microscopy, we found that αA-crystallins are present in the cytoplasm of retinal Müller cells and in the photoreceptors of rats. Given that Müller glial cells were recently described as "living light guides" as were photoreceptors previously, we suggest that αA-crystallins, as in other highly transparent cells, allow Müller cells and photoreceptors to minimize intraretinal scattering during retinal light transmission.