Christopher Kirk Thompson

Neurological symptoms involving the central nervous system (CNS) and peripheral nervous system (PNS) are common complications of acute COVID-19 as well as post-COVID conditions. Most research into these neurological sequalae focuses on the CNS, disregarding the PNS. Guinea pigs were previously shown to be useful models of disease during the SARS-CoV-1 epidemic. However, their suitability for studying SARS-CoV-2 has not been experimentally demonstrated. To assess the suitability of guinea pigs as models for SARS-CoV-2 infection and the impact of SARS-CoV-2 infection on the PNS, and to determine routes of CNS invasion through the PNS, we intranasally infected wild-type Dunkin-Hartley guinea pigs with ancestral SARS-CoV-2 USA-WA1/2020. We assessed PNS sensory neurons (trigeminal ganglia, dorsal root ganglia), autonomic neurons (superior cervical ganglia), brain regions (olfactory bulb, brainstem, cerebellum, cortex, hippocampus), lungs, and blood for viral RNA (RT-qPCR), protein (immunostaining), and infectious virus (plaque assay) at three- and six-days post infection. We show that guinea pigs, which have previously been used as a model of SARS-CoV-1 pulmonary disease, are not susceptible to intranasal infection with ancestral SARS-CoV-2, and are not useful models in assessing neurological impacts of infection with SARS-CoV-2 isolates from the early pandemic.

Mitochondrial health is critical for normal brain development, and mitochondrial toxicants impair energy production and induce mitophagy, resulting in network fission. This can substantially impact cellular and molecular pathways essential for brain development. Recent studies have shown that triclosan, a once widely used antibacterial and antifungal agent, acts as a mitochondrial uncoupler. However, the impact of triclosan on mitochondrial function in the developing brain has yet to be thoroughly investigated. We performed a series of experiments designed to assess the effects of triclosan on mitochondrial form and function in the developing Xenopus laevis tadpole brain. We injected tadpole brains with TMRM, a fluorescent reporter of mitochondrial membrane potential. After two hours, we imaged the brain using high-resolution confocal microscopy to obtain a baseline level of TMRM fluorescence, then immediately treated tadpoles with either triclosan (10uM, 5uM, 1uM, 0.5uM 0.1uM), FCCP (0.5uM), or control, and then continued imaging the brain once every five min for 30 min. We found that 10uM triclosan significantly decreased TMRM fluorescence in mitochondria in the end feet of radial glial cells in ∼ 15-20 min; lower concentrations operated over a longer time course. We also observed that mitochondrial networking was disrupted as they underwent fission. We also measured metabolic function in vivo in whole tadpoles using an XF Seahorse Flux Analyzer, acutely exposing tadpoles to either triclosan (30uM, 10uM, 1uM) or control. Triclosan immediately increased oxygen consumption rates dose-dependently, suggesting mitochondrial uncoupling. These results show that triclosan has the capacity to impair mitochondrial form and function in developing neural tissue.

PFOS is a banned stain repellent that is a type of PFAS, a category of chemicals commonly referred to as “forever chemicals.” These substances are known for their bioaccumulative nature and their resistance to degradation in the environment. Emerging data indicates that PFOS may disrupt thyroid hormone (TH) signaling, which plays a pivotal role in healthy vertebrate development, particularly neural development. Our study investigates whether PFOS interferes with TH-dependent developmental changes, with focus on the developing brain, in Xenopus laevis larvae. To assess this, we exposed five-day old larvae to varying concentrations of PFOS (25 μg/L, 2.5 μg/L, and 250 ng/L). Additionally, some larvae were exposed to either 15 μg/L or 1.5 μg/L of thyroxine to evaluate PFOS’s potential interference with TH-dependent developmental mechanisms. After four days, we euthanized larvae and examined body length and hind limb size, dissected out brains and performed immunostaining for markers of neuronal proliferation and apoptosis. We found that PFOS significantly impeded overall body growth in a dose-dependent manner, irrespective of TH status. Moreover, PFOS impaired TH-stimulated hind limb development. In the brain, PFOS had no effect on proliferation but hindered TH-stimulated growth of the optic tectum. Last, PFOS increased apoptosis in the optic tectum, indicative of cytotoxicity. These findings suggest that PFOS may have the capacity to disrupt TH-dependent mechanisms involved in brain development. The observed pattern of change also suggests that its impact is cytotoxic and may not stem solely from a canonical endocrine disruption mechanism.

Neurological symptoms associated with COVID-19, acute and long term, suggest SARS-CoV-2 affects both the peripheral and central nervous systems (PNS/CNS). Although studies have shown olfactory and hematogenous invasion into the CNS, coinciding with neuroinflammation, little attention has been paid to susceptibility of the PNS to infection or to its contribution to CNS invasion. Here we show that sensory and autonomic neurons in the PNS are susceptible to productive infection with SARS-CoV-2 and outline physiological and molecular mechanisms mediating neuroinvasion. Our infection of K18-hACE2 mice, wild-type mice, and golden Syrian hamsters, as well as primary peripheral sensory and autonomic neuronal cultures, show viral RNA, proteins, and infectious virus in PNS neurons, satellite glial cells, and functionally connected CNS tissues. Additionally, we demonstrate, in vitro, that neuropilin-1 facilitates SARS-CoV-2 neuronal entry. SARS-CoV-2 rapidly invades the PNS prior to viremia, establishes a productive infection in peripheral neurons, and results in sensory symptoms often reported by COVID-19 patients.

Lead (Pb) poisoning during early development is associated with behavioral and cognitive deficits, but the specific mechanisms by which Pb impairs brain development are not fully understood. One potential mechanism is that Pb poisoning may impair thyroid hormone (TH)- mediated changes in brain development To address this issue, we performed experiments to assess the effects of Pb poisoning on (TH) -dependent changes in cellular and molecular mechanisms in the developing Xenopus laevis tadpole brain. We treated stage 48 tadpoles to combinations of 1000 ppb Pb bath for seven days and added one of three different concentrations of thyroxine (T ₄ ) for the final two days of treatment. We found that lead exposure decreased body length, including in T ₄ -treated tadpoles. We also performed immuno-staining for proliferative marker pH3 and found that Pb disrupts T ₄ -induced increases in neuronal proliferation. Finally, we used syGlass VR data visualization software to measure volume of the forebrain, midbrain, and hindbrain in 3D and found that Pb exposure impaired T ₄ -mediated changes in brain volume. Last, we found that Pb poisoning reduced the T ₄ -mediated increase in proliferating cell nuclear antigen (PCNA), a TH-sensitive gene. These results illustrate that Pb poisoning impairs some TH-dependent changes in the developing brain.

Neurological symptoms associated with COVID-19, acute and long-term, suggest SARS-CoV-2 affects both central and peripheral nervous systems. Although studies have shown olfactory and hematogenous entry into the brain and neuroinflammation, little attention has been paid to the susceptibility of the peripheral nervous system to infection or to alternative routes of CNS invasion. We show that neurons in the central and peripheral nervous system are susceptible to productive infection with SARS-CoV-2. Infection of K18-hACE2 mice, wild-type mice, golden Syrian hamsters, and primary neuronal cultures demonstrate viral RNA, protein, and infectious virus in peripheral nervous system neurons and satellite glial cells, spinal cord, and specific brain regions. Moreover, neuropilin-1 facilitates SARS-CoV-2 neuronal infection. Our data show that SARS-CoV-2 rapidly invades and establishes a productive infection in the peripheral and central nervous system via direct invasion of neurons prior to viremia, which may underlie some cognitive and sensory symptoms associated with COVID-19.

This article describes a collaboration among a group of university faculty, undergraduate students, local governments, local residents, and U.S. Army staff to address long-standing concerns about the environmental health effects of an Army ammunition plant. The authors describe community-responsive scientific pilot studies that examined potential environmental contamination and a related undergraduate research course that documented residents’ concerns, contextualized those concerns, and developed recommendations. We make a case for the value of resource-intensive university–community partnerships that promote the production of knowledge through collaborations across disciplinary paradigms (natural/physical sciences, social sciences, health sciences, and humanities) in response to questions raised by local residents. Our experience also suggests that enacting this type of research through a university class may help promote researchers’ adoption of “epistemological pluralism”, and thereby facilitate the movement of a study from being “multidisciplinary” to “transdisciplinary”.

Mining, whether current or inactive, generally increases salt concentrations in catchment watersheds due to precipitation on and through exposed rock surfaces. Practices like mountaintop removal mining have exacerbated this issue, with measurements of salt concentrations in nearby catchment systems well above normal levels. Nevertheless, the impact of the ionic composition of mining effluent on aquatic animal health is not well understood. This is a particularly important issue in Appalachia because it is home to an enormous diversity of organisms, including a huge array of amphibians that live in streams that receive mining effluent from operating and abandoned mines. To investigate this issue, we examined the effects of reconstituted mining effluent on the development of wild-caught wood frog (Lithobates sylvaticus) tadpoles. We collected day-old fertilized eggs from a creek near Blacksburg, VA in early March, 2018 and raised them to hatch. Tadpoles were then assigned to either sulfate or chloride-based reconstituted mining effluent diluted to six different conductivities (100 μS/cm - 2,400 μS/cm). After 7 or 14 days of treatment, tadpoles were euthanized and fixed in paraformaldehyde. We imaged the heads and bodies of tadpoles for morphometric analysis before dissecting out brains and immunostaining them for phospho-histone H3, which labels dividing progenitor cells in the brain. We found that sulfate-based reconstituted mining effluent significantly lowered progenitor cell division at 1200 μS/cm at Day 7 and at 600 μS/cm at Day 14 relative to control. Chloride-based reconstituted mining effluent was less impactful, with no significant differences observed at Day 7 and significantly lowered progenitor cell division at 2400 μS/cm at Day 14. In addition, both treatments slowed growth of some head morphological features, including head size and interocular distance. Chloride treatment slowed growth of body length at Day 14 at 600 μS/cm, whereas sulfate-based reconstituted mining effluent had no effect on body length. These data show that sulfate-based mining effluent has a substantial impact on aspects of neural development, whereas chloride-based reconstituted mining effluent had less effect. In contrast, chloride-based reconstituted mining effluent had a much greater impact than sulfate on body morphology and growth. These experiments demonstrate that the chemical composition of salts in mining effluent can have divergent effects on the development of amphibians.

Melanophores are pigmented cells that change the distribution of melanosomes, enabling animals to appear lighter or darker for camouflage, thermoregulation, and UV‐ protection. A complex series of hormonal and neural mechanisms regulates melanophore pigment distribution, making these dynamic cells a valuable tool to screen toxicants as they rapidly respond to changes in the environment. We found that maltol, a naturally occurring flavor enhancer and fragrance agent, induces melanophore pigment aggregation in a dose‐dependent manner in Xenopus laevis tadpoles. To determine if maltol affects camouflage adaptation, we placed tadpoles into maltol baths situated over either white or black background. Maltol induced pigment aggregation in a similar dose‐dependent pattern regardless of background color. We also tested how maltol treatment compares to melatonin treatment and found that the degree of pigment aggregation induced by maltol is similar to treatment with melatonin, but maltol induces over a much longer time course. Last, maltol had no effect on mRNA expression in the brain of genes that regulate camouflage‐related pigment aggregation. Our results suggest that maltol does not exert its effects via the camouflage adaptation mechanism nor via melatonin‐related mechanisms. These results are the first to identify a putative toxicological effect of maltol exposure in vivo and rules out several mechanisms by which maltol may exert its effects on pigment aggregation.

Toxcast/Tox21 is a massive federally run research effort dedicated to better understanding the potential toxicity of thousands of compounds in a high throughput manner. Among this list of compounds is equilin, an estrogen-like compound that was flagged as a potential thyroid hormone agonist. Here we examine if equilin acts like a thyroid hormone agonist on cellular and molecular mechanisms of brain development in Xenopus laevis tadpoles. To examine the effect of equilin, tadpoles were divided into eight groups and received 4 days of exposure. The experimental groups were as follows: 1 μL, 10 μL, and 100 μL of equilin, 1 μL, 10μM, and 100 μM of 17-β estradiol as an estrogen control, 15 μg/mL thyroxine (T₄) as a thyroid hormone control, and a no-exposure control. After 4 days of treatment, animals were treated with CldU to label dividing cells for 2hr and then euthanized in MS-222. After fixation, body length was measured and the brains dissected out. IHC was performed on brains for CldU to label proliferating neural progenitor cells. Brains were then whole-mounted and analyzed using confocal microscopy. We found that equilin did not increase the number of dividing progenitor cells in a T₄-like manner. Instead, equilin decreased proliferation in a dose-dependent manner, as did estradiol. The same paradigm was performed separately staining for caspase-3 and h2ax, finding that equilin increased cell death in contrast to CNTL and T₄. In another experiment, RNA was extracted from tadpole brains in each group and qPCR was performed to assess change in expression of thyroid hormone-sensitive genes, Equilin did not affect gene expression in a thyroid hormone-like manner. Our data indicate that equilin does not act as a thyroid hormone agonist in the Xenopus laevis nervous system but instead acts similarly to estradiol. Our data strongly suggest that equilin is not a TH disruptor, contrary to the findings of the ToxCast/Tox21 dataset.