Abstract
This session was designed to provide current information on what the organizers consider to be special problems related to toxicology of the central nervous system, in particular, issues that confront pathologists dealing with issues of toxicity from pharmaceuticals, agrochemicals, and environmental contaminants. The panel of speakers included accomplished academics, researchers, and industrial pathologists with wide respect and recognition by their peers. Topics covered were the blood brain barrier (BBB), the cerebrospinal fluid (CSF) barrier, the circumventricular organs (CVOs), direct delivery of agents to the brain, the dilemma of neuronal necrosis, and finally, new approaches to the assessment of synaptic connections.
Dr. Colin Willis of Arizona State University (Tempe, AZ) provided current information on the BBB, the regulated interface that mediates selective transcellular transport of nutrients and essential components from the blood into the brain parenchyma (Willis, Leach, et al. 2004; Willis, Nolan, et al. 2004; Willis and Davis 2008). Despite the increasing body of evidence for the involvement of glia in maintaining the BBB, few studies have specifically addressed glial/endothelial/extracellular matrix interactions and the mechanisms by which glia may regulate the BBB. Imaging studies to assess BBB integrity were presented using gadolinium and fluorescent dextran tracers (4–70 kDa) as magnetic resonance imaging (MRI) probes. Parallel experiments used confocal and electron microscopy to examine the neurovascular unit. These studies, inducing astrocyte loss by treatment with the glial toxicant 3-chloropropanediol, demonstrated the critical role of astrocytes in regulating the integrity of BBB, changes in the expression of tight and adherens junction proteins in vascular endothelial cells as well as vascular permeability, and a cascade of events involving microglial activation, inflammatory response, and remodeling of the extracellular matrix. Laminin (basal lamina) and macrophages play a role in restoring astrocytic population, although it is uncertain whether the macrophages are recruited from the microglia or elsewhere. Comparisons were drawn between regions with robust BBB and the area postrema, a CVO that lacks BBB properties.
Dr. Conrad Johanson, Director of Neurosurgery Research at the Alpert Medical School at Brown University (Providence, RI), enhanced our understanding of the relationship of the CSF to the ependyma, choroid plexus, and the CVOs (Johanson et al. 2008; Smith et al. 2004). Attendees learned that the choroid plexus, an epithelial interface between blood and cerebrospinal fluid (CSF), serves as a regulatory “gateway” for solute and fluid transport into the ventricles (Wolburg and Paulus 2010). Additionally, CVOs are located at specific locations in the third ventricle (neurohypophysis, vascular organ of the lamina terminalis, subfornical organ, pineal gland, subcommissural organ) and fourth ventricle (area postrema) (Duvernoy and Risold 2007). These six small, commonly ignored structures lack a BBB but instead receive and transduce neural and hormonal signals directly from both the blood and the CSF to make homeostatic adjustments correcting body fluid imbalances. Emphasis was placed on the need for remedial pharmacologic strategies to protect the CSF and periventricular brain regions from elevated pressure, ischemia, pathogen invasion, and toxic compounds. Epithelial cells of the choroid plexus have a slow turnover and a limited regenerative capacity, which may have implications for plexus damage by chemicals. With advancing age, potentially deleterious waste products accumulate in the CSF, which may contribute to central nervous system (CNS) deterioration.
Dr. Mark Butt, founder of Tox Path Specialists, LLC (Walkersville, MD), is no stranger to commercial neuropathology issues and the Society of Toxicologic Pathology podium. He addressed issues of direct delivery of therapeutic agents to the CNS, notably biologics such as proteins and cells (Bolon et al. 2008; Hovland et al. 2007). Methods discussed included catheterization of the intrathecal or intracerebroventricular spaces; intranasal delivery; parenchymal injection/infusion; direct implantation of cells, microparticles, or polymer implants; electrical stimulation via implanted electrodes; and introduction of gene therapy vectors. Advantages and disadvantages of the various approaches as well as differentiation of possible test article/therapy changes were addressed. Mark’s presentation concluded by emphasizing the importance of examining a sufficient quantity of neural tissue, including the central, peripheral, and autonomic nervous systems, to perform the evaluation at the correct time and to use the appropriate staining methodologies. With direct drug delivery, some CNS injury is inherently unavoidable. It is, however, important to discriminate between the effects of the medical device itself and those of the compounds delivered by it.
Dr. Alok Sharma, a board-certified pathologist with Covance Laboratories (Madison, WI) and a student of mesial temporal lobe epilepsy models (Sharma et al. 2007; Sharma et al. 2008), presented information on the dilemma of selective neuronal necrosis in toxicologic studies. An important question for toxicologic pathologists engaged in safety studies is to discriminate whether seizures cause brain damage, or chemically induced brain damage causes seizures. Alok, in a well-illustrated presentation, focused on the process of seizure-related neuronal damage resulting from excitotoxicity, secondary to excessive depolarization, causing release of elevated amounts of synaptic glutamate. That process causes increased concentration of intracellular calcium, triggering various pathways to cell death by necrosis and/or apoptosis.
The concluding speaker, Dr. Lee Martin of the Department of Pathology, Johns Hopkins University School of Medicine (Baltimore, MD), discussed new ways to examine toxic effects on synapses, an area long neglected in conventional toxicologic neuropathology examinations (Lesuisse and Martin 2002; Martin et al. 2009; Portera-Cailliau et al. 1997a; Portera-Cailliau et al. 1997b). He reviewed quantitative approaches to assessment of synapses using monitoring of synaptophysin, transmitter-identified synaptic terminals, receptor subunits, and DNA damage profiling. He related the current ability to genetically express jellyfish and coral fluorescent proteins in mammalian cells, which has revolutionized experimental approaches and redefined fluorescence microscopy as applied to synaptic pathology. For example, neuromuscular junction and motor neuron toxicity can be studied using mice expressing enhanced-GFP under the control of the Hlxb9 promoter. This genetic construct drives enhanced-GFP expression specifically in motor neurons and their entire cell body and peripheral axon to the neurouscular junction. Lee discussed the importance of these mouse models and their potential applications to toxicologic pathology.