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Highlights • SARS-CoV-2 may infect immature olfactory sense neurons. • Olfactory sense neurons and the blood brain barrier may allow infection of the brain. • The distribution of ACE2, or novel cellular entry proteins, will limit viral spread. • Animal models may be of limited value in studying COVID-19 and the brain. • Post-mortem histological approaches may be insufficient to study COVID-19 in the CNS., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China in December 2019. On February 11, the World Health Organization (WHO) announced the name for the new illness caused by SARS-CoV-2: COVID-19. By March 11, the outbreak of COVID-19 was declared a pandemic by the WHO. This virus has extensively altered daily life for many across the globe, while claiming hundreds of thousands of lives. While fundamentally a respiratory illness, many infected individuals experience symptoms that involve the central nervous system (CNS). It is likely that many of these symptoms are the result of the virus residing outside of the CNS. However, the current evidence does indicate that the SARS-CoV-2 virus can use olfactory neurons to travel from the periphery into the CNS, and that the virus may also enter the brain through the blood-brain barrier (BBB). We discuss how the virus may use established infection mechanisms (ACE2, TMPRSS2, and Cathepsin L), as well mechanisms still under consideration (NRP1 and BASIGIN) to infect and spread throughout the CNS. Confirming the impact of the virus on the CNS will be crucial in dealing with the long-term consequences of the epidemic.

Alzheimer's disease (AD) pathology is characterized by amyloid plaques containing amyloid beta (Aβ) peptides, neurofibrillary tangles containing hyperphosphorylated tau protein, and neuronal loss. In addition, Aβ deposition in brain microvessels, known as cerebral amyloid angiopathy (CAA), increases blood-brain barrier (BBB) permeability and induces vascular dysfunction which aggravates AD pathology. The aim of the present study was to characterize neurovascular dysfunction in the Tg-SwDI mouse model of AD. Isolated brain capillaries from wild type (WT) and Tg-SwDI mice were used to evaluate the expression of monomeric and aggregated forms of Aβ, P-glycoprotein (P-gp), the receptor for advance glycation end-products (RAGE) and the tight junction (TJs) proteins occludin and claudin-5. Cultured brain endothelial cells were used to analyze barrier function via fluorescein flux. Isolated capillaries from Tg-SwDI mice contained increased levels of aggregated and oligomeric Aβ compared to WT animals. Isolated capillaries from Tg-SwDI had decreased levels of P-gp, which transports Aβ from brain to blood, and increased levels of RAGE, which transports Aβ from blood to brain. In addition, the TJ protein occludin was decreased in Tg-SwDI mice relative to WT mice, which correlated with an increase in BBB permeability in cultured brain endothelial cells. These findings demonstrated that Tg-SwDI mice exhibit Aβ aggregation that is due, in part, to impaired Aβ clearance driven by both a decrease in P-gp and increase in RAGE protein levels in brain capillaries. Aβ aggregation promotes a decrease in the expression of the TJ protein occludin, and as consequence an increase in BBB permeability.

The amyloid β-peptide (Aβ) is transported across the blood-brain barrier (BBB) by binding with the receptor for advanced glycation end products (RAGE). Previously, we demonstrated that the Aβ fraction 25–35 (Aβ25–35) increases RAGE expression in the rat hippocampus, likely contributing to its neurotoxic effects. However, it is still debated if the interaction of Aβ with RAGE compromises the BBB function in Alzheimer’ disease (AD). Here, we evaluated the effects of Aβ25–35 in an established in vitro model of the BBB. Rat brain microvascular endothelial cells (rBMVECs) were treated with 20 μM active Aβ25–35 or the inactive Aβ35–25 (control), for 24 h. Exposure to Aβ25–35 significantly decreased cell viability, increased cellular necrosis, and increased the production of reactive oxygen species (ROS), which triggered a decrease in the enzyme glutathione peroxidase when compared to the control condition. Aβ25–35 also increased BBB permeability by altering the expression of tight junction proteins (decreasing zonula occludens-1 and increasing occludin). Aβ25–35 induced monolayer disruption and cellular disarrangement of the BBB, with RAGE being highly expressed in the zones of disarrangement. Together, these data suggest that Aβ25–35-induces toxicity by compromising the functionality and integrity of the BBB in vitro.

Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aβ) plaques in the brain. Unfortunately, its use is underappreciated due to its low resolution and brightness as stated in previous studies using bright field microscopy. Here, we modified a previous method to localize both plaques and tangles in brains from humans and a transgenic rodent model of AD for fluorescence microscopic visualization. The plaque staining affinities displayed by CR were compared with fibrillar pattern labeling seen with Thioflavin S. This study summarizes the optimization of protocols in which various parameters have been finetuned. To determine the target CR potentially binds, we have performed double labeling with different antibodies against Aβ as well as phosphorylated Tau. The plaque staining affinities exhibited by CR are compared with those associated with the diffuse pattern of labeling seen with antibodies directed against different epitopes of Aβ. Neither CP13, TNT2 or TOC1 binds all the neurofibrillary tangles as revealed by CR labeling in the human brain. Additionally, we also evaluated double labeling with AT8, AT180, and PHF1. Interestingly, PHF-1 shows 40% colocalization and AT8 shows 15% colocalization with NFT. Thus, CR is a much better marker to detect AD pathologies in human and rodent brains with higher fluorescence intensity relative to other conventional fluorescence markers.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, whose hallmark is the loss of dopamine terminals in the substantia nigra pars compacta (SNpc). PD is usually diagnosed after the appearance of motor symptoms, when about 70% of neurons in the SNpc have already been lost. Because of that, it is important to search for new methods that aid in the early diagnosis of PD. In recent years, microRNAs (miRs) have emerged as potential biomarkers for a variety of diseases and hold the potential to be used to aid in the diagnosis of PD. Therefore, the aim of this study was to characterize if specific miRs are differentially expressed in serum in a mouse model of PD. To induce PD-like damage, mice were subcutaneously injected with 25 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) by administering 10 doses over a period of 5 weeks, with 3.5 days between doses. Expression of 71 different microRNAs was quantified in serum separated from blood collected at day 35, using next-generation sequencing. Histological analysis and quantification of neurotransmitters were performed to confirm dopaminergic neurodegeneration. Chronic MPTP treatment induced loss of dopaminergic terminals in the SNpc and caudate putamen, confirmed by a decrease in the number of tyrosine hydroxylase and dopamine transporter positive cells. In addition, MPTP decreased the concentration of dopamine and its metabolites in the SNpc, simulating the damage observed in PD. From the 71 miRs analyzed, only 4 were differentially expressed after MPTP treatment. Serum levels of miR19b, miR124, miR126a and miR133b were significantly decreased in MPTP-treated mice compared to control. These data suggest that specific miRs are downregulated in a pre-clinical model of PD and hold the potential to be used as biomarkers to aid in the diagnosis of this disease. Further experiments need to be conducted to validate the use of these miRs as biomarkers of PD in additional pre-clinical models as well as in samples from patients diagnosed with PD.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

Bath salts, or synthetic cathinones, have cocaine-like or amphetamine-like properties and induce psychoactive effects via their capacity to modulate serotonin (5-HT) and dopamine (DA). Structurally distinct synthetic cathinones are continuously being generated to skirt existing drug laws. One example of these modified compounds is cathinone phthalimide (CP), which has already appeared on the global market. The lack of toxicological studies on the effects of CP on monoaminergic systems led to the development of the present study in order to generate an acute toxicity profile for CP, and to clarify whether it primarily affects both dopamine and serotonin, like the synthetic cathinones mephedrone and methylone, or primarily affects dopamine, like 3, 4-methylenedioxypyrovalerone (MDPV). For the first time, the toxicity profile of CP (10µM - 1000µM) is reported. In pheochromocytoma cells, exposure to CP induced cell death, and altered mitochondrial function, as well as intracellular DA and 5-HT levels; at the same time, reduced glutathione (GSH) levels remained unaffected. This seems to indicate that CP functions like mephedrone or methylone. The role of CP metabolites, the effect of CP induced hyperthermia on neurotoxicity, and its ability to traverse the blood-brain barrier warrant further consideration.

Silver nanoparticles (AgNPs) have been widely used because of their antimicrobial properties. However, several reports suggest that AgNPs exposure promote cardiac effects that involve nitric oxide (NO) and oxidative stress (OS). Nevertheless, there are no studies related to AgNPs-induced effects in cardiac physiology. The aim of this study was to evaluate the AgNPs direct actions on coronary vascular tone and cardiac contractility using Langendorff rat heart preparation. Low concentrations of AgNPs (0.1 and 1 μg/mL) increased NO derived from inducible NO-synthase (iNOS), without modifying cardiac parameters. Meanwhile, high concentrations (10 and 100 μg/mL) promoted a sustained vasoconstriction and increased cardiac contractility related to OS, leading to rhabdomyolysis. Furthermore, AgNPs were internalized in the cardiac muscle, hindering classic actions induced by phenylephrine (Phe) and acetylcholine (ACh). These data suggest that AgNPs affect cardiac physiology in function of the concentration and in part of the NO generation, NOS expression and OS.

Traumatic brain injury (TBI) is defined as damage to the brain that consequently disrupts normal function. Neuronal death, a hallmark of TBI, has been related to the development of neurodegenerative disorders like Parkinson's disease (PD), where loss of dopaminergic neurons and dopaminergic dysfunction are observed. To date, no in vitro model exists in which the dopaminergic damage observed in TBI is replicated. In this study, we evaluated the effects of in vitro simulated TBI on human dopaminergic neurons. To simulate TBI, neurons were subjected to 0%, 5%, 10%, 15%, 25% and 50% deformation. 24 h after injury, cell viability and apoptosis were determined by lactate dehydrogenase (LDH) release and DNA fragmentation, as well as ethidium homodimer and caspase 3/7 staining. Dopamine (DA) levels were determined by ELISA. Levels of tyrosine hydroxylase (TH) and DA transporter (DAT) were determined by western blot. Only 50% stretch increased LDH release and ethidium homodimer staining, suggesting the induction of necrosis. On the contrary, 25% and 50% stretch increased DNA fragmentation while 15%, 25% and 50% increased caspase 3/7 staining, suggesting that moderate and severe TBI promote apoptosis. Levels of intracellular DA decreased in a stretch-dependent manner with 15%, 25% and 50% stretch, which were related with a decrease in TH expression. Extracellular DA levels increased only at 50%. Levels of DAT remained unchanged regardless of treatment. These data support the use of stretch as a model to simulate TBI in vitro in human dopaminergic neurons, replicating the acute effects of TBI in the dopaminergic system.

For the past five years, Dr. Daniel Acosta has served as the Deputy Director of Research at the National Center for Toxicological Research (NCTR), a principle research laboratory of the U.S. Food and Drug Administration (FDA). Over his career at NCTR, Dr. Acosta has had a major impact on developing and promoting the use of in vitro assays in regulatory toxicity and product safety assessments. As Dr. Acosta nears his retirement we have dedicated this paper to his many accomplishments at the NCTR. Described within this paper are some of the in vitro studies that have been conducted under Dr. Acosta's leadership. These studies include toxicological assessments involving developmental effects, and the development and application of in vitro reproductive, heart, liver, neurological and airway cell and tissue models.

Traumatic brain injury (TBI) occurs when external mechanical forces induce brain damage as result of impact, penetration or rapid acceleration/deceleration that causes deformation of brain tissue. Depending on its severity, TBI can be classified as mild, moderate or severe and can lead to blood-brain barrier (BBB) dysfunction. In the present study, we evaluated the effects of uniaxial high-speed stretch (HSS) at 0, 5, 10 and 15% on a pure culture of primary rat brain endothelial cells as an in vitro model of TBI to the BBB. LDH release, viability and apoptosis analysis, expression of tight junction proteins and endothelial permeability were evaluated 24 h after a single stretch episode. HSS slightly increased cell death and apoptosis at 10 and 15%, while LDH release was increased only at 15% stretch. Occludin expression was increased at 10% stretch, while claudin-5 expression was increased at 5% stretch, which also decreased the endothelial permeability. In summary, 15% HSS induced low levels of cell death, consistent with mild TBI and very low percentages of HSS (5%) enhanced the BBB properties, promoting the formation of a stronger barrier. These data support the use of 15% HSS as valuable tool in the study of mild TBI to the BBB in vitro.

The blood–brain barrier (BBB) is a physical frontier that separates the brain tissue from all the circulating substances in the bloodstream to keep the chemical microenvironment for the proper functioning of the brain. The BBB is formed by neurons, astrocytes, pericytes, and endothelial cells from the brain microvessels; the interaction and communication between these cell types give the BBB its low permeability. During development, the migration and phenotype acquisition of the endothelial cells is driven by factors produced by astrocytes and pericytes, triggering complex signaling pathways that lead to the developing of the barrier properties on the endothelial cells. In this chapter, we will review the events that lead to the formation of the BBB, how all the different cell types interact and communicate and how the BBB is affected during different brain pathologies.

The blood-brain barrier (BBB) is essential to maintain the proper microenvironment for brain function. Although formed by different cell types, the endothelial cells (ECs) of the brain microvessels provide the BBB with its selective permeability. To study the BBB in vitro, EC lines as well as primary isolated ECs have been used. In this chapter, we will provide a detailed protocol on how to isolate and culture primary brain microvascular endothelial cells from different species for use as in vitro models of the BBB. When performed properly, this protocol will allow one to obtain a pure culture of brain microvascular endothelial cells with which to analyze the effects of therapeutic and toxic agents on BBB functions.

Traumatic brain injury (TBI) is one of the major causes of disability in the USA. It occurs when external mechanical forces induce brain damage that causes deformation of brain tissue. TBI is also associated with alterations of the blood-brain barrier (BBB). Using primary rat brain microvascular endothelial cells as an in vitro BBB model, the effects of biaxial stretch were characterized at 5, 10, 15, 25, and 50% deformation using a commercially available system. The results were compared to the effects of mild and moderate TBI in vivo, induced by the weight-drop method in mice. In vitro, live/dead cells, lactate dehydrogenase (LDH) release, caspase 3/7 staining, and tight junction (TJ) protein expression were evaluated 24 h after a single stretch episode. In vivo, Evans blue extravasation, serum levels of S100β, and TJ protein expression were evaluated. Stretch induced a deformation-dependent increase in LDH release, cell death, and activation of caspase 3/7, suggesting the induction of apoptosis. Interestingly, low magnitudes of deformation increased the expression of TJ proteins, likely in an attempt to compensate for stretch damage. High magnitudes of deformation decreased the expression of TJ proteins, suggesting that the damage was too severe to counteract. In vivo, mild TBI did not affect BBB permeability or the expression of TJ proteins. However, moderate TBI significantly increased BBB permeability and decreased the expression of these proteins, similar to the results obtained with a high magnitude deformation. These data support the use biaxial stretch as valuable tool in the study of TBI in vitro.

Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules.

With the advent of nanotechnology, the use and applications of silver nanoparticles (AgNPs) have increased, both in consumer products as well as in medical devices. However, little is known about the effects of these nanoparticles on human health, more specific in the cardiovascular system, since this system represents an important route of action in terms of distribution, bioaccumulation and bioavailability of the different circulating substances in the bloodstream. A collection of studies have addressed the effects and applications of different kinds of AgNPs (shaped, sized, coated and functionalized) in several components of the cardiovascular system, such as endothelial cells, isolated vessels and organs as well as integrative animal models, trying to identify the underlying mechanisms involved in their actions, to understand their implication in the field of biomedicine. The purpose of the present review is to summarize the most relevant studies to date of AgNPs effects in the cardiovascular system and provide a broader picture of the potential toxic effects and exposure risks, which in turn will allow pointing out the directions of further research as well as new applications of these versatile nanomaterials.