Through War and Peace, These Doves Rock
By Daniel Erenstein, Neurobiology, Physiology & Behavior ‘21
“The diversity of the breeds is something astonishing,” Charles Darwin wrote in “On the Origin of Species.” He was not referring to his famous Galápagos finches. Instead, Darwin opened his foundational work by commenting on various breeds of the domestic pigeon, all descended from a common ancestor: Columba livia. Widely known as the rock dove, this species has adapted to urban environments throughout human history. Over time, we have kept pigeons for fairs, racing, message carrying in wartime, and even scientific research.
Since joining the B3 Lab at UC Davis in 2020, I have contributed to research on this model organism. The B3 name, short for Birds, Brains, and Banter, represents the lab’s main goals: to study rock doves and how stress affects their reproductive behaviors, and to advance culturally relevant science communication research and training. In April, I presented a project on how single parenting affects the amygdala, often considered the brain’s “emotional center,” at the UC Davis Undergraduate Research, Scholarship, and Creative Activities Conference. This research helps us to understand the impacts of single parenting in humans, and it could lead to insights that mitigate stresses felt by single parents and their children.
These photographs were captured in the B3 aviary via iPhone 7 camera during 2020 and 2021.
Lazarus Dies, Lazarus Lives Again
By Jesse Kireyev, History ‘21
Each of these photos captures a landscape in slow degradation. Berryessa, for all the wintergreen beauty that it holds, has experienced horrifying fires numerous times over the past few years. The natural bridge that dominates the landscape of its namesake park in Santa Cruz now remains alone, at risk of collapsing like its sibling did, forever leaving the shoreline empty of its beauty. This risk only grows as sea levels rise and as human interaction puts it at greater risk. The salt flats of the Dead Sea used to be covered in water — now nature struggles to fill the few remaining pools as the sea rapidly shrinks. Captured in these three horizons are the struggles of nature to sustain itself despite the present beauty. For all the tranquility of the Ansel Adams-esque lines jutting forth from the foreground, a great and slow war is playing itself out in the back, often hidden to the gazing eye of the unaware viewer. The horizons both serve as a reminder of the danger that lurks in our future, as well as the distant (and perhaps unreachable) hope of resurrection in the face of annihilation.
1. Berryessa Foothills, Solano, California.
Storm clouds move over the fields and lush wetlands, both morphing into the mountains hugging Lake Berryessa. Just a few months prior, the mountains had been scorched by the dizzying flames of the LNU Lightning Complex Fire, a fire whose smoke blotted out the sun for weeks in two of the largest metropolitan areas in America. The ebb-and-flow of the surroundings give us a stark reminder of just how fast a place can be destroyed and can flourish once again from the ashes. Canon EOS 5D Mark III. April, 2021.
2. West Cliff, Natural Bridges State Park, Santa Cruz, California.
Pelicans and seagulls huddle together as they hunt for fish and fight the buffeting winds. The remainders of the natural bridges, which once dominated the state beach, still serve as a helpful vantage point for the seabirds. Locals hope that this vantage point can survive, even as climate change puts the bridge at greater risk every year. Canon EOS 630, Kodak Tri-X 400TX 35mm film. June, 2017.
3. Dead Sea Salt Flats, Masada, Israel.
The salt flats are all that is left of the once sea-filled expanse below Masada. A combination of climate change and human changes to the environment are driving the evaporation of the Dead Sea, which at current rates is expected to be gone in the next three decades. Sony a700. December, 2018.
Review of recent progress in development of genetically encoded calcium indicators in imaging neural activity
By Lia Freed-Doerr, Cognitive Science, Neurobiology, Physiology & Behavior ‘22
Author’s Note: In fall quarter, I got into contact with the Tian lab in the Department of Biochemistry and Molecular Medicine in order to learn more about optogenetic techniques and the difficulties of in vivo sensing of neural dynamics and, with the mentorship of a postdoctoral researcher, I have learned more about different high-resolution sensors (or indicators) and expanded my interests to genetically encoded sensors of cellular dynamics. As I began learning about various types of imaging sensors, calcium ion (Ca2+) indicators, in particular, stuck out to me due to their variety and depth of development. As I am unable to take part in in-person projects due to COVID restrictions, to ensure my understanding of the topics I was reading about, I began to write this review.
Abstract
Methods of performing neuroscience research have progressed remarkably in recent years, providing answers to many different types of questions. Genetically encoded indicators (sensors) are of particular interest for use in answering questions about neural circuits, cell specific populations, and single cell dynamics. These indicators modulate their fluorescence in different cellular environments and allow for optical observations. Of the various cellular activities that can be measured by genetically encoded indicators, the dynamics of calcium ions (Ca2+) are of interest due to their fundamental importance in neuronal signaling. In this review, we introduce the basic underlying features of genetically encoded calcium indicators (GECIs) including characteristics of fluorescence, an overview of GECI engineering, and a brief discussion of some common variants of GECIs and their uses.
Introduction
Modern neuroscientists have found many ways to analyze the information-carrying neuronal circuits and dynamics within the brain. The continued development of genetically encoded optical indicators, specifically Ca2+ indicators, is particularly promising for analyses of single neurons or neural circuits. Optical fluorescence imaging allows large populations of neurons to be examined simultaneously and avoids major damage to the cells of interest [1]. In particular, measuring the dynamics of Ca2+ can be useful in inferring spiking activity in neurons, as Ca2+ is involved in neuronal action potentials. In this review, we will introduce the basic workings of genetically encoded sensors beginning with a ground-up introduction of fluorescence measurements and the process of engineering genetically encoded sensors. Several Ca2+ indicators will be briefly discussed in order to examine recent progress and how this can impact future studies of the brain.
Mechanics of Fluorescence Indicators
Fluorescence imaging is a valuable tool for visualizing populations of cells. It is relatively non-invasive; but, in order to use optical tools to study the cortex, some surgical procedures must still be performed. A cranial window might be installed in the animal to shine light through; alternatively, an endoscope or fiber optic cable could be installed at the desired depth within brain tissue [2].
Fluorescent proteins (FPs) internally form a barrel-like structure containing the chromophore (also known as the fluorophore), which is a trio of amino acids responsible for the protein’s fluorescence. The chromophore is autocatalytically formed as a post-translational modification, requiring just atmospheric oxygen. Genetically encoded indicators rely on a change in the chromophore environment within the labeling FP. Fluorescence is observed when light of an appropriate wavelength excites the chromophore’s electrons, which then results in the emission of a lower energy photon as the excited electrons return to a lower energy state. FPs are often connected to sensing domains, which induce the change in the chromophore environment after detecting the event of interest (e.g., Ca2+ binding for GECIs). Any number of cellular activities may induce a conformational change such as changes in pH or the binding of a ligand. Sensors can have one or two FPs with partially overlapping fluorescence spectra. Single FP-based sensors are generally preferred as indicators; the green fluorescent protein (GFP), cloned from the jellyfish Aequorea Victoria, is the most commonly used FP for single FP sensors [3]. In systems with two FPs, a Förster or fluorescence resonance energy transfer (FRET) occurs. FRET involves an energy transfer from the higher energy (more blue-shifted) donor FP to the lower energy (more red-shifted) acceptor FP. Genetically manipulating FP systems by circularly permuting FPs (fusing the original termini of the FP and introducing a new opening closer to the chromophore) can improve their performance in sensors by making the chromophore more accessible to the outside environment and, thus, more susceptible to environmental changes [4]. FPs like GFPs are also typically oligomeric in their natural environment (i.e. multiple copies stick together); but, in order to help prevent breakdown and allow for better combination with sensing domains in indicators, FPs must also be mutated to become monomeric [5].
Figure 1: A Jablonski diagram that visualizes an electron’s excitation to a higher energy level by absorption of a photon and subsequent fluorescence emission with energy decay.
To image fluorescent systems, we can use fluorescence microscopy with one or multiple photons (Fig. 2) [2]. In one-photon systems, the fluorophore absorbs energy from a light source and is excited by a single photon. Some energy is lost non-radiatively (without light) resulting in the emittance of lower energy, visible photons from the fluorophore. One-photon systems are relatively inexpensive and fast but can only penetrate tissue to a shallow depth. In contrast, multi-photon microscopy shows more promise for in vivo imaging because of its reduced out of focus emission, light scattering, and phototoxicity. The combination of the energy of multiple photons is required for excitation in such systems.
Figure 2: A diagram outlining the setup for a standard fluorescence microscopy experiment.
Genetic encoding of sensors
To introduce indicator genes into a system, methods like in utero electroporation or viral vectors can be used [6, 7]. DNA promoters or localization sequences can be used to target specific subtypes of neurons in organisms to produce transgenic animals. Transgenic animal genomes that have been modified by artificial bacterial chromosomes, CRISPR, or effector nucleases, and are particularly useful when longitudinal and intensive sampling is required. Genetic changes can be maintained throughout an animal’s lifespan and lines of transgenic animals can be bred for further testing [2]. A recombinase system administered via viral vector, like the popular Cre/loxP system, can be used to achieve high specificity [6]. In the Cre/loxP system, the loxP sequences are placed at specific target sites of genomic DNA. The Cre-recombinase protein can then target loxP sequences to modify the genetic sequence. Two mouse lines, one carrying the gene of interest flanked by loxP sequences, and the other line expressing Cre-recombinase, can be bred to produce mice expressing the gene of interest. The Cre Driver mouse line expressing Cre-recombinase can be designed to only express the gene under certain conditions. To apply Cre/loxP to genetically encoded indicator systems, a viral vector injects the indicator genes into the brain cells of a Cre Driver mouse. The indicator is only expressed where the Cre-recombinase is active. Expression would continue through one animal’s lifetime; to create a line of mice that express the desired indicator, other methods must be used [6]. Through recombinase methods, the development of transgenic animal lines is an area of active improvement.
There are several advantages to genetically encoding indicators over other methods of imaging. There are a wide variety of neuronal events that can be observed by constructing indicators from proteins that respond to cellular events, including changes in neurotransmitter concentrations, transmembrane voltage, Ca2+ dynamics, and pH [1]. Genetic encoding also allows for selective sampling of cells based on genotype. Selective sampling is not possible with chemical dyes, nor is the viewing of the evolution of neuronal dynamics during learning or development processes [8, 1]. Similar to chemical dyes, genetically encoded indicators allow for the imaging of brain activity in neurons in vitro and in animals [9]. Neurons have the machinery implanted within them to automatically report cellular dynamics of interest.
There are several different broad classes of genetically encoded indicators that are based on the dynamics of the action potential [7]. Genetically encoded voltage indicators (GEVIs) operate based on the membrane depolarization that occurs during action potentials. Other indicators, like pH and neurotransmitter sensors, detect vesicular release. Genetically encoded pH sensors (GEPIs) react to the decrease in acidity as vesicles fuse with the membrane, and genetically encoded transmitter indicators (GETIs) are used to visualize the release of neurotransmitters into the synapse [1]. Genetically encoded calcium indicators (GECIs) operate based on the rise in cytosolic Ca2+ during an action potential; however, they do not directly measure spiking activity. When an action potential occurs, Ca2+ floods into the cell. Ca2+ influx is important because calcium ions are crucial for the release of neurotransmitters from vesicles, which then go on to produce signals in other neurons. More mild calcium ion dynamics are always present in neurons, even in a resting state. Among these various classes, GECIs have been perhaps some of the most developed of these indicators and, thus, some of the most promising.
Engineering genetically-encoded calcium indicators
Performance Criteria
As GECIs are engineered, many performance criteria must be considered. Tradeoffs often occur between the various important qualities of an indicator’s performance [10]. As we optimize the indicator to produce a desired result in one criterion, another criterion often decreases in quality. Thus, development of sensors optimized for specific applications is continuous. Some of these criteria are affinity, sensitivity, kinetics, localization, and photophysical characteristics.
Affinity, represented as the dissociation constant Kd, describes what percentage of the indicator is unbound given a particular concentration of ligand.
Specificity refers to the indicator system’s ability to respond only to the target of interest, as opposed to perhaps similar molecules.
Sensitivity is usually represented by ΔF/F0, the fractional fluorescence change, which is the fluorescence signal change over a change in concentration of the target molecule. It can also be represented by signal-to-noise ratio (SNR), the relative difference between the signal of interest and background noise.
Kinetics is the rate of change in fluorescence intensity of the indicator in response to the change in ligand concentration. There tends to be a tradeoff between affinity and kinetics [8].
Photophysical qualities like brightness, photostability, and photoswitching behaviors are also important considerations. In general, brighter or more intensely emitting indicators are desired. Photostability is inversely proportional to the rate of photobleaching (the damaging of the FP so that it becomes unable to fluoresce). Additionally, some indicators have broader ranges of excitation than others, or may change their intensity or sensitivity in different light conditions, which would limit usage.
GECIs are some of the most widely used genetically encoded indicators in vivo because of their relatively high SNR and improved properties like brightness, photostability, and dynamic range [2]. However, there are still numerous obstacles to be faced in designing GECIS, and only certain variants have faced success in vivo.
Engineering GECIs
Genetically encoded indicators generally are composed of an analyte-binding (sensing domain) and a fluorescent protein (reporting domain), though there are additional peptide complexes that assist in changing the conformation of the system [2]. Upon the occurrence of a sensing event, the sensing domain undergoes a conformational change which, in turn, induces a conformational change in the FP, resulting in fluorescent activity. Engineers of GECIs use two different strategies for constructing reporting domains: FRET-based indicators and single FP-based indicators [7]. When Ca2+ binds to FRET-based indicators, the spatial relationship between the donor and acceptor FPs changes so that there is a transfer of energy from the donor FP to the acceptor FP [2]. One family of indicators, Cameleon, has had some success. In this family, the sensing and peptide complex is located between two FPs with overlapping spectra. FRET-based indicators’ SNR tends to be lower, meaning it is harder to isolate the activity of a neuron from background noise. Because of these drawbacks, we mostly examine the engineering of the more commonly used single FP-based GECIs.
There are two popular designs among developers of single FP GECIs [8]. One is based on one of the earliest lines of calcium indicators, GCaMP. GCaMP consists of a circularly permuted green fluorescent protein (cpGFP) inserted between the Ca2+-binding protein, calmodulin (CaM), and another peptide called RS20, which binds CaM upon Ca2+ binding. When CaM binds Ca2+, a conformational change is induced in the cpGFP and the sensor fluoresces [10, 11]. Another recent design, the NTnC family of indicators, inserts a calcium-binding domain into a split FP [8]. Unlike GCaMP-type indicators, NTnC indicators display an inverted fluorescence response upon calcium binding (i.e., fluorescence decreases upon Ca2+ binding). They are less optimized than GCaMP variants, but it is hypothesized that their lesser Ca2+ binding capacity would interfere less with normal calcium dynamics.
Figure 3: A basic representation of the GCaMP structure.
There have been efforts to expand the color variants of GECIs. In particular, there has been much effort to develop red-fluorescing GECIs because longer red wavelengths reduce phototoxicity and have better tissue penetration [12]. However, there have been many obstacles to producing red-fluorescing GECIs. Unlike GFP, inserting calcium binding domains into red fluorescent proteins (RFPs) disrupts folding and chromophore maturation [8]. A more popular design choice is to replace the GFP in a GCaMP-style indicator with an RFP and optimize the sensor for a new FP [1].
GECIs are improved iteratively through directed evolution and linker optimization between the cpFP and the sensing domains. Site-directed mutagenesis can be used to mutate specific locations to produce novel variants. In the development of one variant of GCaMP, mutations were specifically introduced in the calcium-binding domain-cpGFP linker in a GCaMP5 scaffold to increase sensitivity [11]. Using directed evolution, mutations are randomly introduced. Then, upon testing for desired effects, the variants that produced the best results may be preserved and propagated. This process may repeat many times, producing increasingly successful indicators as the best-performing mutations survive across generations.
Challenges
Genetically encoding indicators, as a rule, comes with challenges. If we choose to use viral infection as our genetic encoding scheme, consideration must be taken to the many viral serotypes, which have varying levels of efficiency and can be toxic. Furthermore, in utero electroporation can be unpredictable, and transgenic animals may not express indicators at sufficiently high levels to be useful [1].
Sensors may affect the natural dynamics of their measured systems, affecting accuracy of results. GECIs, particularly GCaMP-based designs, may interfere with regular Ca2+ dynamics and gene expression [8]. This interference is likely due to interactions between the calcium-binding sensing domain with native proteins and the lack of availability of calcium once bound to the indicators. There have been efforts to improve and modify the calcium-binding domain so that it can bind fewer Ca2+ or otherwise improve affinity so that the indicator operates at lower concentrations of calcium.
There is also difficulty in using these indicators in vivo [2]. Especially in the mammalian brain, the SNR is highly decreased due to the amount of background noise. This reduced SNR is putting aside the level of breakdown that naturally happens in vivo vs. conditioned, cultured environments. Although many indicators have improved structural integrity in vivo, there are many that still cannot be used in living organisms.
Progress in GFP-based GECIs
There has been much development in the GCaMP series as variants are continuously improved by site-directed mutagenesis and computational design efforts [2]. The jGCaMP7 series, built from the GCaMP-6 series, provides a good example of optimization of indicators for different purposes: jGCaMP7f is optimized for fast kinetics, jGCaMP7s is optimized for high sensitivity (though it has slower kinetics), and jGCaMP7b is optimized to have a brighter baseline fluorescence [11]. All of these indicators are based on the same base scaffold but differ drastically in performance because of just a few mutations in the CaM-binding peptide, the GFP, the CaM domain, or the linkers between domains.
Progress in RFP-based GECIs
RFP-based GECIs have important advantages over GFP-based ones. Beyond the importance of color variety in tracking distinct populations of cells at once, red GECIs are also promising for reducing phototoxicity and allowing deeper imaging [12]. There are many promising RFP-based GECIs being developed, though they are generally dimmer than GFP-based indicators and may display photobleaching behaviors under blue light [1]. In particular, there are R-GECO1 variants like jRGECO1a, the RCaMP series, and, perhaps most promising, K-GECO1 [12]. There are three widely used RFPs from which red GECIs are developed; each red indicator family was generated from different RFPs. K-GECO1 has shown particular promise as it works at a distinct spectral range, allowing researchers to simultaneously work with other indicators in multicolor imaging experiments, and it also shows minimal fluorescent noise [9].
Designs of red GECIs often expand on the GCaMP design–for example, K-GECO1 follows a similar design of sandwiching the circularly permuted FP between the Ca2+-sensing domain, CaM, and a CaM-binding peptide [12]. Switching the GFP in GCaMP with an RFP comes with engineering challenges of linker optimization and preventing the breakdown of the sensor. The increased penetrative depth of red GECIs has been used to image subcortical areas like the hippocampus or medial prefrontal cortex relatively noninvasively, demonstrating the applicability of GECIs in neuroscience research [13].
There are other FP-based GECIs in development, but of particular interest is the development of near-infrared GECIs, whose spectral distinction from other indicators would help prevent photoswitching when used with optogenetic tools [8, 14].
Uses and Applications
The applications of GECIs are varied and powerful. The use of genetically encoded indicators allows for the analysis of cells of a specific type or subpopulation as they select for specific genetic qualities. The first transgenic mouse line expressing GCaMP2 in the cerebellar cortex was generated in 2007 and has allowed for characterization of certain synapses [6]. GECIs have been used to provide single-cell resolution to the decades-long study of various topographic maps in the brain and to track the communication of neural circuits [2]. In rats, GECIs have been used to monitor neural population behavior during motor learning tasks and observe the response of cells to sensory deprivation in the primary visual cortex after retinal lesion. They allow examination of ensemble and single cell-scale neural events at more and more temporally precise levels. Broadly, and perhaps more importantly, they are often used in conjunction with optogenetic and other experimental methods that allow for the inference of causation. In using these indicators, the stimulation techniques used in optogenetic experiments can also involve precise tracking of calcium or other dynamics in cells of interest [8]. These experimental approaches have caused excitement as they allow for the examination of behaviors of cells or whole organisms upon physical stimulation of even just single cells. The continued expansion of these approaches is promising.
Conclusion
Many researchers are devoted to developing new and distinct calcium indicators based on existing indicator series. With more GECIs than ever available to neuroscientists, there is some challenge in choosing which is best suited to the exploration of a particular question. With the continuing development of mouse lines and methods of genetically encoding more potent indicators with high temporal resolution, GECIs will continue to be an increasingly important tool within the neuroscientist’s toolkit that allows for population or single-cell imaging with greater resolution than ever before.
References:
- Lin M, Schnitzer M. 2016. Genetically encoded indicators of neuronal activity. Nature Neuroscience 19(9):1142-1153.
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COVID-19 Cover Art Gallery
This year, for the first time, The Aggie Transcript accepted submissions for our journal’s cover from the wider undergraduate community at UC Davis. To celebrate the release of our fifth annual print edition, we present three of the submissions that we received in this art gallery. The winning submission appears first, followed by our honorable mentions. We sincerely thank the authors and artists who submitted to our journal this year for sharing their work with us.
Unprecedented: The Science of COVID-19
By Mario Rodriguez, Wildlife, Fish & Conservation Biology, Design ‘22
This work was created with the intention of highlighting those in the medical profession and the timeline of the COVID-19 pandemic, from bottom to top: the death of loved ones, the medication and hospitalization of patients, and then the development of vaccines to combat the virus. The digital artwork was created on an iPad in the digital painting app Procreate.
Working Together to Catch Covid
By Daria Beniakoff, Biochemistry & Molecular Biology ‘21
Last year, the COVID-19 pandemic affected everyone. The hands of so many people from every direction were put to work figuring out what the virus was and how to mitigate and fight its effects, from the doctors treating patients to the scientists trying to develop treatments and vaccines to the everyday people who had to work around the circumstances. I wanted this digital medium piece to reflect the collaborative effort to contain and stop the pandemic.
Light of Hope
By Bianca Law, Design ‘23
COVID-19 survivors can retrain their smell to enjoy food and wine again
By Daniel Erenstein, Neurobiology, Physiology & Behavior ‘21
Author’s Note: Last spring, I enrolled in the inaugural offering of the University Writing Program’s wine writing course. Our instructor, Dr. Alison Bright, encouraged us to report on topics of personal interest through our news stories on the wine industry, viticulture, enology, and more. In this article, which was prepared for an audience of general science enthusiasts, I examine how biologists are making sense of a puzzling COVID-19 symptom — anosmia, or loss of smell — and what COVID-19 patients with this condition can do to overcome it. Eighteen months into this pandemic, scientists continue to study cases of COVID-19-related anosmia with dreams of a treatment on the horizon. I hope that readers feel inspired by this article to follow this in-progress scientific story. I extend my appreciation to Dr. Bright, who throughout the quarter shared approaches to rhetorical awareness that elevated my grasp of effective writing.
Image caption: Anton Ego, the “Grim Eater” from PIXAR’s Ratatouille, is reminded of his childhood by Remy’s rendition of ratatouille, a Provençal dish of stewed vegetables.
With a single bite of Remy’s latest culinary creation, the eyes of Anton Ego, a notoriously harsh food critic, dilate, and Ratatouille’s viewers are transported back in time with Monsieur Ego. The meal — a simple yet elegant serving of ratatouille, accompanied by a glass of 1947 Château Cheval Blanc — has triggered a flashback to one singular moment, a home-cooked meal during his childhood. The universal charm of this enduring scene resonates; in Ego’s eyes, many recognize how our senses of smell and taste can impact a culinary experience.
Imagine how a real-life version of this scene might change for the millions of COVID-19 patients who have lost their sense of smell [1]. Anosmia, the phenomenon of smell loss, has become one of the more perplexing COVID-19 symptoms since first observed in patients during the earliest months of the pandemic [2].
What happens when we lose our sense of smell? During the pandemic, scientists have studied smell loss, which affects more than 85 percent of COVID-19 patients according to research published this year in the Journal of Internal Medicine [3]. In fact, anosmia is so common in COVID-19 patients that physicians were offered guidance for testing olfactory function as an indicator of infection last year [4].
To simplify studies of these complicated senses, taste and smell are often examined independently of one another, even though these senses are usually experienced simultaneously.
“Smell is just — it’s so crucial to taste, which means it’s really crucial to everything that I do,” said Tejal Rao, a New York Times food critic, in a March episode of The Daily [5]. “And it’s really difficult to cook without a sense of smell if you’re not used to it. You don’t know what’s going on. It’s almost like wearing a blindfold.”
Rao, who lost her sense of smell in mid-January after contracting COVID-19, began to search for answers to this mystery from scientists. Rao’s journey started with TikTok “miracle cures” and other aromatherapies — unfortunately, they were too good to be true — but she eventually discovered the work of Dr. Pamela Dalton, a scientist at the Monell Chemical Senses Center in Philadelphia [6]. At the center, Dalton studies the emotions that are triggered by our sense of smell [7].
During simple colds or viral infections, smell is normally affected when the molecules in food and other aromas are physically blocked off from chemoreceptors in our nose by congestion. Scientists have also cited Alzheimer’s and Parkinson’s diseases, head trauma, and chemotherapy as triggers for anosmia [8]. But a separate phenomenon was occurring in the case of COVID-19.
“COVID is different in that way, because most people who lost their sense of smell did so without having any nasal congestion whatsoever,” Dalton told Rao during an interview.
One study published in October of last year by Dr. Nicolas Meunier, a French neuroscientist, aimed to investigate how the SARS-CoV-2 virus, which causes COVID-19, may disrupt sustentacular cells [9]. These structural cells express the ACE2 receptor, which the virus hijacks to gain entry into our cells, at higher levels [10]. Sustentacular cells support the specialized neurons that transmit signals from the nose to the brain.
When Meunier and his team at Paris-Saclay University in France infected hamsters with the virus, tiny hair-like projections known as cilia on the surfaces of olfactory neurons began to peel back from sustentacular cells. This disruption is a possible explanation for the difficulties with smell that COVID-19 patients experience.
If it is true that damage to sustentacular cells causes anosmia, loss of smell is not an irreversible brain condition. In this case, the poor connection between incoming odors and brain networks that typically process these stimuli is at fault, not direct damage to the brain itself. The sudden onset of smell loss in COVID-19 patients supports this thinking.
“It was just like a light bulb got turned off or a switch got flicked to off,” Dalton said. “And one moment they could smell. And the next moment, nothing smelled.”
But because olfactory support cells regularly regenerate, this loss of smell is only temporary, which allows for retraining of our senses. Two months of smell training, also known as olfactory training, allowed Rao to regain her sense of smell.
Olfactory training gradually exposes patients to particularly strong smells. Spices such as cinnamon or cumin, for example, were perfect for Rao’s first smell training session [5], and AbScent, a British charity offering support to patients with anosmia, sells kits with rose, lemon, and eucalyptus [8]. Scientists have found that recurring exposure to these strong scents gives the brain time to recalibrate its networks, a feature known as neuroplasticity [11].
But “you don’t just go from hurt to healed overnight,” Rao said. “It’s more like adjusting and learning how to live in a new space. That’s really just the beginning.”
Our chemical senses have the power to satisfy, to inspire, even to cause our memory to reveal itself, as 20th-century French author Marcel Proust observed in his seven-volume novel À la recherche du temps perdu, or In Search of Lost Time. Researchers have even speculated that our sense of smell can facilitate learning in other sensory domains, including vision [12].
While scientists further investigate how coronavirus causes loss of smell, olfactory training can provide an avenue in the meantime for COVID-19 patients to recover this crucial sense. Indeed, many patients are “in search of lost time,” and smell training can help them to once again experience food and wine in its sensory entirety.
References:
- Allen J, Almukhtar S, Aufrichtig A, Barnard A, Bloch M, Cahalan S, Cai W, Calderone J, Collins K, Conlen M, et al. 2021. Coronavirus in the U.S.: Latest Map and Case Count. New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/interactive/2021/us/covid-cases.html.
- Symptoms of COVID-19. 2021. Atlanta (GA): Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases, Division of Viral Diseases; [accessed 28 July 2021]. https://www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/symptoms.html.
- Lechien JR, Chiesa-Estomba CM, Beckers E, Mustin V, Ducarme M, Journe F, Marchant A, Jouffe L, Barillari MR, Cammaroto G, et al. 2021. Prevalence and 6-month recovery of olfactory dysfunction: a multicentre study of 1363 COVID-19 patients. J Intern Med. 290(2):451–461. https://doi.org/10.1111/joim.13209.
- Whitcroft KL, Hummel T. 2020. Olfactory Dysfunction in COVID-19: Diagnosis and Management. JAMA. 323(24):2512–2514. https://doi.org/10.1001/jama.2020.8391.
- Antolini T, Dorr W, Powell D, Schreppel C. 2021. A Food Critic Loses Her Sense of Smell. New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/2021/03/23/podcasts/the-daily/coronavirus-smell-food.html.
- Rao T. 2021. Will Fish Sauce and Charred Oranges Return the World Covid Took From Me? New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/2021/03/02/dining/covid-loss-of-smell.html.
- What COVID 19 is teaching us about the importance of smell, with Pamela Dalton, PhD. 17 Mar 2021, 33:31 minutes. American Psychological Association; [accessed 28 July 2021]. https://youtu.be/0pG_U13XDog.
- Schoch D. 2021. Distorted, Bizarre Food Smells Haunt Covid Survivors. New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/2021/06/15/health/covid-smells-food.html
- Bryche B, St Albin A, Murri S, Lacôte S, Pulido C, Ar Gouilh M, Lesellier S, Servat A, Wasniewski M, Picard-Meyer E, et al. 2020. Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters. Brain Behav Immun. 89(2):579–586. https://doi.org/10.1016/j.bbi.2020.06.032.
- Brann DH, Tsukahara T, Weinreb C, Lipovsek M, Van den Berge K, Gong B, Chance R, Macaulay IC, Chou HJ, Fletcher RB, et al. 2020. Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia. Sci Adv. 6(31): eabc5801.
- Kollndorfer K, Kowalczyk K, Hoche E, Mueller CA, Pollak M, Trattnig S, Schöpf V. 2014. Recovery of Olfactory Function Induces Neuroplasticity Effects in Patients with Smell Loss. Neural Plast. 1–7. https://doi.org/10.1155/2014/140419.
- Olofsson JK, Ekström I, Lindström J, Syrjänen E, Stigsdotter-Neely A, Nyberg L, Jonsson S, Larsson M. 2020. Smell-Based Memory Training: Evidence of Olfactory Learning and Transfer to the Visual Domain. Chem Senses. 45(7):593–600. https://doi.org/10.1093/chemse/bjaa049.
Surviving COVID-19: Variables of Immune Response
By La Rissa Vasquez, Neurobiology, Physiology & Behavior ‘23
Author’s Note: In this paper, I analyze autopsy reports conducted on deceased COVID-19 patients and supply a breakdown of the body’s immune response. The purpose of this paper is to provide a more generalized synopsis of how the body is affected by the virus from the onset of infection to the escalating factors that contribute to cause of death. COVID-19 and SARS-CoV-2 are referenced countless times throughout this paper, but they should not be used interchangeably. The name of the pathogenic virus is “Severe Acute Respiratory Syndrome Coronavirus 2” (SARS-CoV-2), and the name of the illness is called COVID-19 and is the common usage in forms of discussion. This paper only scratches the surface of the virus’s complexity and its effects upon the body and societies around the world.
Introduction
On December 31, 2019, the first case of the novel coronavirus was reported in Wuhan, China [1]. The first case of the virus reported in the United States was on January 22, 2020 [2]. Within 22 days, the Coronavirus had traveled across the Pacific to wreak havoc upon countries woefully unprepared. Within a year, COVID-19 has managed to bring some of the most powerful countries in the world to heel. Economies and healthcare systems across the world continue to be devastated by an adversary only 60 to 140 nanometers in diameter [3]. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) formally identified the virus as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). On March 11, 2020, the World Health Organization classified COVID-19 as a worldwide pandemic and global health crisis [4]. As of May 2021, the CDC has confirmed that the U.S. has over 32 million cases. Healthcare systems across the nation and around the world are overwhelmed by the number of infected patients. Many of them perish due to either a lack of resources or accurate and efficient testing.
SARS-CoV-2 Viral Pathogenesis
Humans have two levels of immunity. Innate immunity is the body’s first line of contact and defense against invading pathogens. Adaptive immunity learns and remembers how to effectively target and eliminate these pathogens.
Innate Immunity
Our innate immune system is composed of barrier tissues and cells specialized for defense against pathogens [5]. Barrier tissues are the first line of defense, and inside barrier tissues reside sentinel cells, which are capable of consistently recognizing repeated exposure to pathogen associated molecular patterns (PAMPs). The sentinel cells release proinflammatory mediators like cytokines, chemokines, or histamines and circulate within the blood vessels inviting more immune cells from the surrounding tissue into the bloodstream [5]. Cells such as neutrophils or monocytes differentiate into macrophages and migrate from the bloodstream and phagocytose (eat) the pathogens. Neutrophils will undergo programmed cell death, referred to as apoptosis. Macrophages will continue to phagocytose the rest of the pathogens and restore homeostasis by consuming the dead neutrophils [5].
Infection occurs when these viral pathogens in respiratory droplets from a sneeze or a cough enter a person’s mouth, nose, or eyes and attach to the ACE-2 receptors in the nose, throat, and especially the lungs. Like any virus, SARS-CoV-2 cannot replicate on its own and instead hijacks the body’s own cellular machinery. The virus inserts its own genetic information into the host cell to produce more copies of itself until the cell bursts and dies, spreading more of the virus around the body to infect more cells [6]. Infection of the host cell consists of the following five steps: attachment, penetration, biosynthesis, maturation, and release. Once a virus binds to host receptors (attachment), it enters host cells via endocytosis or membrane fusion (penetration). Once the viral contents are released inside the host cells, viral RNA are transported by protein molecules in the host cell’s cytoplasm and travel into the nucleus for replication via the nuclear pore complex (NPC). Viral mRNA then makes viral proteins (biosynthesis). Lastly, novel viral particles are made (maturation) and released [7]. This innate immune response is not as effective against SARS-CoV-2 due to the strength of the various proteins displayed in Figure 1, an ultrastructural morphology rendering, provided by the Centers for Disease Control and Prevention (CDC) Image Library on February 10 [8].
Figure 1
The SARS-CoV-2 virus contains “M (membrane), S (spike), E (envelope), and N (nucleocapsid)” proteins, which envelop the virion and act as a defensive shield [9]. The S or Spike viral surface protein, which consists of two subunits, S1 and S2, binds to the angiotensin converting enzyme 2 (ACE2) receptors of the host cells [7]. The primary role of ACE2 is the breakdown of the angiotensin II (ANG II) protein into molecules that neutralize its harmful effects. ANG II is responsible for increased inflammation and death of alveolar cells in the lungs, which reduces oxygen uptake. When the S (spike) protein of SARS-CoV-2 binds to the ACE2 receptors, they inhibit ACE2 from doing its job of regulating ANG II, allowing ANG II to freely damage tissue in the lungs. These ACE2 receptors are naturally present on the surface of the lung’s epithelial cells and other organs throughout the body, but the virus’ S protein uses these receptors to penetrate the cell membrane and replicate inside host cells. The N (nucleocapsid) protein is another viral surface protein of SARS-CoV-2, which inhibits interferons (IFN1 and IFN-β) responsible for cytokine production [10]. But if the signals for regulating proinflammatory response are disrupted by the pathogen’s surface proteins, the innate immune response becomes hyperactive and self-destructive. A malfunctioning innate immune response also compromises an adequate adaptive immune response [9].
Adaptive Immunity
Adaptive immunity consists of B-cell and T-cell responses. B-cells produce antibodies to trigger an immune response, while T-cells actively target and eliminate infected cells.
B-Cell Response
The innate immune response is not particularly equipped to combat pathogens that are especially complex and vicious because the innate immune response is non-specific and will attack anything it identifies as an invader. The adaptive immune response can target pathogens more precisely and powerfully by using proteins called antibodies, which are produced by B-cell lymphocytes that bind to antigens on the surface of pathogens [5]. Adaptive immunity can more efficiently handle foreign pathogens, like a virus, because antibodies can see through the debris of proteins and dead cells left by the cytokine storm. Antibodies uniquely bind to antigens, acting as a beacon for the adaptive immune response to converge on the invading pathogen [5]. More importantly, adaptive immunity has memory and learns how to become more effective by retaining its response to pathogens so that it can be even quicker at eliminating them after repeated exposure [5]. Widespread pandemics like COVID-19 occur because of a lack of protective antibodies in populations that have never been exposed to or vaccinated against the specificity of SARS-CoV-2 [5]. Figure 2 depicts the four ways in which antibodies attack pathogens: neutralization, complement fixation, opsonization, and antibody dependent cellular cytotoxicity.
Figure 2
Figure 2 – “Immunopathogenesis of Coronavirus Disease 2019 (COVID-19)” [3].
Neutralization is the process by which antibodies immediately bind to the surface antigens of a pathogen and block their S protein from attaching to the receptors of healthy cells, thereby neutralizing the virus’ ability to attach and insert its genetic information. Complement fixation occurs when antibodies are responsible for inviting complement proteins to bind to the antigens of the pathogen. This process coats the pathogen in attack proteins that can either initiate the complement cascade leading to cell lysis, the breakdown of the cell, or it can induce the third stage, opsonization. During opsonization, proteins called opsonins bind to the invading pathogen, acting as markers for phagocytotic cells like macrophages to identify and consume the pathogen. Lastly, antibody dependent cellular cytotoxicity (ADCC) is the process by which antibodies recognize the antigen of a pathogen and signal for natural-killer cells (NK cells) to release cytotoxic molecules which kill off the virally infected cell [5].
T-Cell Response
T-cell lymphocytes are produced by the bone marrow and mature in the thymus. They form the basis of cellular immunity because they directly attack foreign pathogens. Consequently, they are more effective than innate immune or B-cell responses at targeting intracellular pathogens like viruses [5]. Antibodies can get distracted by viral particles and proteins, so they rely on the blind T-cell lymphocytes to ignore the surrounding virus particles and eliminate the infected host cell at the source. As naive T-cells circulate the lymph nodes and spleen, they express T-cell receptors (TCR) that recognize cell surface peptides (antigens) attached to major histocompatibility complex (MHC) molecules on the surface of a specific pathogen. These surface MHC proteins tell the T-cells where to attack [5]. The dendritic cells work to activate the adaptive immune response by ingesting viral proteins and turning them into cell surface peptides that bind to MHC molecules, forming peptide-MHC complexes. The TCR of naive T-cells recognize the peptide-MHC complexes and activate the T-cell. For T-cells to become active, they also need to bind to proteins from the dendritic cell via co-simulation. They then undergo clonal expansion and differentiate into effector T-cells [5]. Effector T-cells are also referred to as cytotoxic T lymphocytes (CTLs). They travel through the body to hunt down peptide-MHC presenting pathogens and kill the infected cells by releasing cytotoxic molecules [5].
The adaptive immune response is stimulated by the recognition of pathogen-associated molecular patterns (PAMPs). Within 1-2 weeks after infection, the B-cells produce antibodies while T-cells simultaneously increase proinflammatory cytotoxic molecules in a forceful attempt to eliminate the virus [7]. The uptick in Interleukin cytokines abbreviated as IL-1, IL-6, IL-8, and so on, flood the body with proinflammatory substances, which “chronically increase the stimulation of T-cells, resulting in a cytokine storm and T-cell exhaustion” [9]. T-cell exhaustion not only means that the virus is overwhelming the body’s antibodies but also draining the strength of the T-cell’s ability to eliminate the virus at the source of infected host cells. SARS-CoV-2 is a “high-grade chronic viral infection because it decreases the responsiveness of T-cells leading to a decreased effector function and lower proliferative capacity” [9]. T-cell exhaustion is also linked to an increase in inhibitory receptors that can initiate apoptosis in T-cells. This results in the destruction of T-cells and their co-receptors, further suppressing the T-cells, as well as B-cells and NK cells, all of which are white blood cells (lymphocytes). Thus, explaining the general lymphopenia (the lack of lymphocytes) observed in severe COVID-19 cases and the increased number of cytokines [9]. Viral entry and attachment to ACE2 receptors trigger a vicious cycle of both innate and adaptive immune responses, mounting an intense attack by secreting proinflammatory substances that invite more lymphocytes to try and kill the virus. This releases more cytokines and chemokines [11]. The downregulation of the ACE2 enzyme results in a cascade of chemical reactions that lead to further inflammation and destruction of cells, weakening and damaging the body’s own immune response.
pathologies of a pandemic:
COVID-19 Autopsies
Once the SARS-CoV-2 attaches to alveolar type II cells, it propagates within the cells. Most viral particles cause apoptosis, releasing more self-replicating pulmonary toxins. Figure 3 displays normal ACE2 receptors located in the type II pneumocytes. Healthy alveoli are unobstructed to allow efficient diffusion of oxygen and carbon dioxide with red blood cells.
Figure 3
Figure 3 – “Type I pneumocytes are very thin in order to mediate gas exchange with the bloodstream (via diffusion). Type II pneumocytes secrete a pulmonary surfactant in order to reduce the surface tension within the alveoli” [12].
In contrast to Figure 3, Figure 4 shows the histopathology of alveolar damage (A) and inflammation (B) of the epithelial cells. As the epithelial cells detach from the alveolar wall the alveoli structures collapse and no longer inflate making it hard for patients with severe cases of COVID to breathe [13]. This results in diffuse alveolar damage with fibrin rich hyaline membranes and hemorrhages in the lungs [13]. The histopathology also detected multinucleated cells that lead to pulmonary fibrosis (scarring in the lungs). Infected cells are “abnormally large and often polynucleated cells showing a large cytoplasm with intense staining for the COVID-19 RNA probe” [13]. The viral Spike protein is also largely detected in the histopathology of COVID cases (C). The nuclei of Spike-positive cells appear an intense red stain and have abnormally enlarged cytoplasts (panel h) [13].
Figure 4
Figure 4 – “Histopathological evidence of alveolar damage, inflammation and SARS-CoV-2 infection in COVID-19 lungs” [13].
The cellular destruction detected in the histopathology is macroscopically reflected in the physical damage of lung tissue displayed in Figure 5.
Figure 5
Figure 5 – “Macroscopic appearance of COVID lungs” [13].
In all pathological examinations of patients that died of COVID, their lungs sustained macroscopic damage [13]. Severe cases of COVID reveal congested and firm lungs (A) with “hemorrhagic areas and loss of air spaces (a’, c’)” [13]. As the virus ravages the body, some patients rapidly deteriorate and develop severe inflammation and clotting in the lungs (B) which shows “the thrombosis of large pulmonary vessels, often with multiple thrombi and in one case determining an extensive infarction in the right lobe (Fig. 5B panels a and b)” [13]. The lung’s architecture crumbles as cells lose their integrity and continue to die, thus resulting in the development of Acute Respiratory Distress (ARDS). ARDS develops in about 5% of COVID-19 patients, and of all the infected, the mortality rate remains around 1 to 2% [14]. Autopsies are beginning to reveal that rather than a singular cause of death, many factors seem to be responsible for higher mortality rates in patients that develop critical cases of COVID-19.
The fallout from the hyperactive immune response disrupts regular oxygen diffusion from the alveoli into the capillaries and consequently to the rest of the body. This commonly leaves fluid and dead cells, resulting in pneumonia, a condition in which patients experience symptoms such as coughing, fever, and rapid or shallow breathing [14]. If oxygen levels in the blood continue to drop, patients rely on breathing assistance by a ventilator to forcefully push oxygen into damaged lungs “riddled with white opacities where black space—air—should be” [14]. The presence of opacities in the lungs indicate the development of pneumonia into ARDS, which was found in the autopsy of a 77-year-old man with a history of comorbidities, including hypertension and the removal of his spleen (splenectomy) [15]. The decedent exhibited chills and an intermittent fever but no cough for 6 days. On March 20, 2020, emergency medical services responded to a call, stating that the deceased was experiencing weakness, fever, and shortness of breath. In route to the hospital, the decedent went into cardiac arrest and died shortly after reaching the hospital [15]. A postmortem nasopharyngeal swab was administered and came back positive for SARS-CoV-2.
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Figure 6 |
Figure 7 |
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| Figure 6 – Normal chest X-Ray of healthy lungs [16]. | Figure 7 – “Lesion segmentation results of three COVID-19 cases displayed using the software post-processing platform” [17]. |
Figure 7 shows opacities in the CT “of typical COVID-19 infection cases at three different infection stages: the early stage, progressive stage, and severe stage” [17]. Figure 7 highlights these opacities in red, which appear to intensify and cover more of the lung CT as the virus increases in severity (a-c). Patient 4 (c) exhibits what medical examiners refer to as a “complete whiteout” of the lungs. Indicating reduced air flow, whereas the normal scan of healthy lungs (Figure 6) has a black background, representing the transparency of free and unrestricted airflow.
The postmortem radiography of the deceased 77-year-old man showed “Diffuse, dense bilateral airspace consolidations (complete “whiteout”)” [15]. In most cases of severe COVID-19 “the greatest severity of CT findings is visible around day 10 after the symptom onset. Acute respiratory distress syndrome is the most common indication for transferring patients with COVID-19 to the ICU” [18].
ARDS in connection to SARS-CoV-2 was first documented in Wuhan, Hubei, China in December 2019 with over 90,000 deaths associated with organ dysfunction, particularly progressive respiratory failure and the formation of blood clots resulting in the highest mortality rates [19]. Another autopsy from Hamburg, Germany conducted on the first 12 documented consecutive cases of COVID-19 related deaths revealed that there was not only profuse alveolar damage in 8 out of the 12 patients but also a high rate of clotting resulting in death. 75% of patients that died were males within an age range of 52 to 87 years and 7 out of 12 patients autopsied (58%) presented venous thromboembolism, as displayed in Figure 7. A pulmonary embolism was the direct cause of death in 4 of the deceased [20].
Figure 8
Figure 8 – “Macroscopic autopsy findings: A. Patchy aspect of the lung surface (case 1). B. Cutting surface of the lung in case 4. C. Pulmonary embolism (case 3). D. Deep venous thrombosis (case 5)” [20].
The formation of clots results in pulmonary vasoconstriction, or the constriction of arteries and halting of blood delivery to the arteries and capillaries in the lungs. Blood cannot travel to the lungs, so oxygen levels drop. As a result, a cytokine storm from our hyperactive immune system occurs, destroying the alveolus and the endothelium and causing clots to form. Smaller clots come together and form a fatal giant blood clot, or the clots can break apart and travel to other parts of the body, causing a blockage and inadequate blood supply to organs or other parts of the body [19]. If the blood supply to fingers, toes, and other extremities is cut off by a clot, it is referred to as ischemia and often results in the amputation of digits and appendages once the flesh begins to die [19].
When SARS-CoV-2 enters the alveolar cells in the lungs via the ACE2 receptors, it can directly attack organs and indirectly cause damage to other organs by triggering a hyperactive immune response (cytokine storm). When the viral particles trigger a cytokine storm, they cause further inflammation of the lungs resulting in plummeting oxygen levels and the formation of blood clots in the arteries (arterial thrombosis).
Conclusion
SARS-CoV-2 is a multi-organ scourge, but it primarily attacks the lung by first attaching its spike protein to the host cell’s ACE2 receptors. This prevents the lungs from regulating their function because it inhibits ANG II protein breakdown, causing increased alveolar damage and inflammation of the lungs. The virion proteins create proinflammatory responses in the innate immune response and compromise an effective adaptive immune response. As the virus progresses the number of neutrophils from the innate immune response increase while the number of helpful lymphocytes (T-cells and B-cells) decrease. The ACE2 receptors overstimulate the innate and adaptive immune response to produce more proinflammatory molecules to eliminate the virus, thus causing more destruction to the body and its immune response. Autopsies of COVID-19 victims show ongoing cellular death and collapse of the respiratory system caused by inflammation and alveolar damage that eventually develop into ARDS. Extreme inflammation induced by the immune response causes difficulties in breathing and clotting in the lungs. Radiography of progressive stages of COVID identify opacities in lung CTs indicating obstructed airways and alveolar deterioration. Postmortem examinations reveal gross destruction of the lung tissue, such as pulmonary artery thrombosis, vasoconstriction, lung infarction, or pulmonary embolism. Progressive organ and respiratory failure and abnormal clotting are all contributing factors to the cause of death in the most severe cases of COVID-19.
SARS-CoV-2 efficiently exploits weaknesses not only within our innate and adaptive immune systems across sex, age, race, and ethnicity, but it also exploits weaknesses within our societies. The etymological origins of Pandemic are rooted in pandēmos , which is Greek for ‘all’ (pan)+ ‘people’ (demos). When simplified, pandemic literally means “all people” but the priorities of leadership across the world reveal that not all people suffer the burden of this pandemic equally. Regarding the United States’ approach to the pandemic, this quote from the Atlantic’s article “Why Some People Get Sicker Than Others” is sufficient; “the damage of disease and a global pandemic is not a mystery. Often, it’s a matter of what societies choose to tolerate. America has empty hotels while people sleep in parking lots. Food is destroyed every day while people go hungry. Americans are forced to endure the physiological stresses of financial catastrophe while corporations are bailed out. With the coronavirus, we do not have vulnerable populations so much as we have vulnerabilities as a population. Our immune system is not strong” [21].
References:
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The Relationship Between Itch and Pain in Itch Pathways
By Nathifa Nasim, Neurobiology, Physiology & Behavior ‘22
Author’s Note: Itch is not a stranger to any of us, but growing up with eczema, I have always been hyper aware of it. As far back as I can remember, burning hot showers and painful levels of scratching temporarily alleviated the maddening sensation of itch without my understanding of how pain was linked to itch. Once I joined the Carstens Lab studying the relationship between itch and pain, these memories were rekindled, and I became interested in not only understanding itch, which we know so little of, but how these two sensations interact. This paper was also written for my UWP 104E class.
Introduction
Itch is an everyday sensation that nearly all people have experienced. Its origins lie in its role as a defense mechanism — when faced with irritant stimuli, the scratching urge produced by itch can remove potentially harmful substances [1]. However, despite its evolutionary advantages, itch is often a source of discomfort and for many can dramatically impact their quality of life. Itch not only includes acute itch such as mosquito bites, but debilitating chronic itch can stem from different diseases such as cancer, HIV/AIDS, liver/kidney failure, atopic dermatitis, and other skin disorders [2]. However, despite the widespread impacts of itch, much of its mechanisms and pathways still remain elusive.
Itch is a somatosensory sensation, relying on the nervous system for detection and perception, therefore similar to other somatosensory sensations such as heat, touch, vibration, and most importantly, pain. Pain and itch have an antagonistic relationship, meaning each sensation has an opposing effect on the other. This is evident in “painful” scratching which relieves the feeling of itch, and that morphine administration reduces pain while increasing itch [3]. The intersection between the two sensations translates to potential treatment as well: chronic itch, for example, can be treated by medications similar to chronic pain [2]. Researching the interplay between itch and pain can help illuminate the pathophysiology of itch and how it is perceived as a sensation different from pain, and consequently lead to a better understanding of treating itch.
Currently, there are numerous models and theories proposed to explain this overlap, however, there is no consensus amongst itch researchers on which model(s) may best explain the relationship between pain and itch. This review will be an overview of the various models of itch transduction and perception and how they have evolved with the accumulation of new research. It will also cover the basic mechanisms of itch at the level of the periphery and spinal cord and how it interacts with pain.
Overview of Itch Mechanisms
Itch Activation at the Level of the Epidermis
Pruriceptors are neurons capable of detecting itch; these can be activated by either mechanical stimuli, a scratchy fabric for instance, or chemical stimuli, such as poison ivy. For simplicity, and as the chemical pathway is currently better understood, the paper will focus on chemical itch from here onwards. Similar to other somatosensory neurons, the cell bodies of the primary pruriceptive neurons reside in the dorsal root ganglion (DRG), close to the spinal cord, with axons stretching to both the periphery and the spinal cord [4, 5]. Unlike other sensory modalities, itch is specific to the outermost epidermis only, as opposed to pain, which can be felt in the muscle and bone. The pruriceptors’ branched sensory nerve endings which terminate in the epidermis are studded with membrane receptors activated by various “itchy” mediators [4]. The receptors differ in the mediators they respond to but can be broadly grouped into histamine receptors, serotonin receptors, G Protein-coupled receptors (GPCRs), toll-like receptors (TLRs), or cytokine and chemokine receptors [4,5].
Once acute itch is triggered by an irritant, keratinocytes, mast cells, and immune cells release chemical mediators which trigger vasodilation, inflammation, and the arrival of more immune cells to clear the irritant. The chemical mediators can include histamine, serotonin, proteases, cytokines, and chemokines, each of which is associated with a certain receptor [4]. The activation of itch from internal factors in disease differs from acute itch in that it is instead dependent on unknown mediators in the bloodstream from drugs or diseased organs [4]. Despite the origin of the chemical mediators, however, once released they bind to the receptors on the free nerve endings and activate them. The receptors then depend on various ion channels to depolarize the pruriceptor neuron which conveys the sensory information to the spinal cord via its axon [4, 5].
Itch Transmission to the Spinal Cord
The pruriceptive DRG neurons’ axon also ends in the spinal dorsal horn. These then synapse onto interneurons in the spinal cord which connect to projection neurons that carry the sensory information to the brain. The interneurons are important for transmission as well as modulation of itch via excitatory and inhibitory synapses [4, 5, 6]. Electrophysiological responses to itch stimuli in primates have identified the projection neurons as belonging to the spinothalamic tract, which carries axons to the thalamus. This tract also conveys pain and temperature and is consequently an area of itch interaction with other modalities [5, 7]. In addition to interneurons, descending modulation in the spinal cord can also regulate itch. After applying a cervical cold block to mice – activity of the upper cervical spinal cord level was essentially stopped – mice were unable to relieve itch and decrease neuronal firing when the lumbar spinal cord neurons below were stimulated by an itchy substance. This suggests that there is some level of descending modulation that was disrupted when the upper spinal cord was damaged [8].
Areas of Itch and Pain Interaction
Having briefly discussed the pathways for itch perception, it is important to note how often it converges with that of pain. Firstly, pruriceptors are in fact pruriceptive nociceptive neurons, meaning they are a subset of nociceptors, or pain-sensitive neurons. Although there are many non-pruriceptive nociceptors (neurons sensitive to pain but not itch), studies have pointed towards most pruriceptors being stimulated by pain as well as itch [1, 2]. One method of explaining this convergence is the expression of TRPV1 ion channels in pruriceptors. Although these are important for detecting itch, it is also expressed in nociceptors, and is stimulated by the classic pain stimulus found in peppers, capsaicin [4].
The relationship between itch and pain continues to the spinal cord. As mentioned, the spinothalamic tract (STT) is of special interest in understanding the distinction between itch and pain, as both sensations traverse the same pathway. Transecting the anterolateral funiculus where the tract ascends has eliminated sensitivity to itch, pain, and temperature, establishing the common usage of the tract by these sensations [6]. Electrophysiological recordings of primate STT neurons when given different types of sensory stimuli also revealed that two thirds of the nociceptors were sensitive to itch stimuli as well as pain, again highlighting the apparent overlap between itch and pain in the spinal cord [9].
The relationship between itch and pain is best understood as antagonistic. Recordings of STT pruriceptive neurons showed that after being stimulated by histamine (itch/pruritic stimuli) the neuronal firing decreased when the skin was scratched. However, the same neuron increased firing after scratching in response to capsaicin. Although being activated by both pain and itch stimuli, the difference in response to scratching suggests an antagonistic relationship between pruriceptive and nociceptive neurons via inhibitory interneurons [6].
The intersection between pain and itch raises the question of the brain’s perception of pain and itch as distinct in the presence of much overlap. There are numerous theories and models attempting to explain the nature of this relationship, which will be overviewed in the following sections.
Classical Models of Itch and Pain Discrimination
Intensity Model
Given observations on the overlaps between itch and pain, itch was first theorized to be a subset of pain in the intensity model. This postulates that polymodal neurons (sensitive to many modalities) differentiate between itch and pain through patterns of firing or “intensity” due to weak or strong stimulation [1, 4, 6, 10]. The model was tested by delivering electrical pulses to the skin that varied in frequency. Although the results seemingly disproved the theory as it only increased the intensity of itch felt, rather than transforming it to pain, the theory has not yet been discounted [6]. Itch stimuli has been shown to trigger lower firing rates compared to painful stimuli in both peripheral and STT neurons, suggesting that firing rates do have some role in itch perception [6, 9]. Furthermore, both itch and pain stimuli give rise to “bursting” patterns of action potentials, and the interburst interval is shorter in response to capsaicin/pain. This suggests some level of temporal coding, when information is coded based on the timing of action potentials or intervals between them. This aligns with the intensity model as a polymodal neuron could code for itch and pain depending on the rate of action potentials or their intervals [9].
The intensity model’s basic principle lies in neurons activated by both pain and itch, and seemingly aligned with the previously mentioned research identifying pruriceptors as a subset of nociceptors and activated by both pain and itch. However, the discovery of itch-specific neurons further complicated the validity of the model, lending support to the labeled line model instead.
Labeled Line or Specificity Theory
Labeled line refers to the idea that there exists a specific, separate “line” or neural pathway devoted to the sensation of itch – the opposite of the intensity model’s polymodal neurons. As early as the 1800s, researchers discovered there are specific spots on the skin which are activated by different sensory modalities: coolness, heat, pain, etc., giving rise to the labeled line theory. Recent electrophysiological studies have supported this for different sensations through establishing the presence of sensory fibers and spinal relay neurons tuned to only one sensory modality [11].
The labeled line theory’s validity for itch was confirmed by the presence of itch specific neurons. GRPR3+ neurons in the spinal cord were identified that differed from STT neurons in that they carried purely itch information [3, 12]. This was evident as when these neurons were treated with a toxin, not only was there loss of itch behavior (scratching), there was no change in pain behavior (wiping) [12]. The discovery of these itch specific neurons was emphasized by the consequent discovery of MrgprA3+ neurons in the dorsal horn which were itch specific as well; their deletion also resulted in loss of itch behavior only [7]. Furthermore, the neurons gave rise to purely itch behavior regardless of the nature of the stimulus – precisely as predicted by the labeled line [7]. These discoveries gave significant support to the labeled line theory, yet the presence of itch neurons activated by pain remained a dilemma.
Modified Models of Itch and Pain
The theories of intensity and labeled line represent the two ends of the spectrum in understanding pain signalling – the first depends on polymodal neurons, and the latter on itch specific neurons. The discovery of neurons that fall under both complicate their validity, and suggest that an accurate model should include neurons sensitive to both itch and pain while capable of differentiating between the two [1].
Spatial Contrast Model
Spatial models expand on the intensity model, and do not require itch and pain specific neurons. It proposes that itch is felt in “spatial contrast,” or when a small population of nociceptors are activated, and pain is felt when a larger population is activated due to a stronger stimulus [6,10]. In a study, it was observed that a spicule (small pointed end) of both histamine (itch stimuli) and capsaicin resulted in itch sensation, yet an injection of only capsaicin resulted in pain activation [13]. This could be explained by the spatial contrast theory in that the spicule activated a small number of nociceptors, resulting in itch, whereas the more widespread injection stimulated a larger number of nociceptors, resulting in pain.
According to the model, a small number of even non-pruriceptive nociceptors activated should result in itch, eliminating the need for a labeled line. However, there remains an obstacle in this model as well – there was no decrease of itch sensation relative to pain when the area of exposure to stimuli increased, although the model predicts this should in theory activate a greater number of receptors [6, 13].
Selectivity Theory and Population Coding
The population coding theory – also known as the selectivity theory – modifies the labeled line, proposing that although there are specific sensory labeled lines, the antagonistic interaction between them shapes perception of itch. It takes into account the overlap between nociceptors and pruriceptors as well as pain’s inhibition of itch, proposing that pruriceptors are a smaller subset of nociceptors and are linked to them by inhibitory interneurons [1,11]. Theoretically, activation of the larger nociceptive population – including pruriceptors – is felt as pain, as the activation of the pain neurons “masks” the sensation of itch. Yet if only the smaller itch specific subset is activated, this is felt as purely itch, as there is no activation, and consequently no inhibition from the nociceptive neurons [1, 11, 14].
There have been numerous studies that appear to support this hypothesis. In one, the vesicular glutamate transporter VGLUT2 was deleted from DRG nociceptive neurons, affecting their ability to signal. This resulted in spontaneous itch in mice along with a decrease in pain behavior and, importantly, itch behavior resulting from capsaicin injections [14]. These results were paralleled in another study where blocking pruriceptors had no effect on pain, yet deleting TRPV1 in a group of nociceptive neurons led to capsaicin to be perceived as itch [15]. These two studies suggest groups of nociceptors are involved in inhibiting and masking itch, as deleting their receptors results in itch signaling instead, supporting the population coding theory.
It is also necessary to identify an inhibitory neuron to explain the antagonistic relationship between the nociceptors and pruriceptors; Bhlhb5 neurons are one such interneuron. When Bhlhb5 was knocked out in mice, there was increased itch behavior that ultimately resulted in lesions from itching and licking [16]. This suggests that the interneuron, and perhaps other interneurons as well, are responsible for inhibiting and regulating itch, further bolstering the support for the population coding model.
Gate Control and Leaky Gate Model
The gate control theory hypothesizes that nociceptive transmission neurons in the spinal cord receive input from both nociceptive primary neurons, and Aβ fibers: primary neurons attuned to non-nociceptive stimuli such as touch. These Aβ fibers in turn inhibit the nociceptive neurons via interneurons, effectively creating a “gate” that can halt transmission of pain or itch [10]. The previously mentioned Bhlhb+ interneurons support this gate control model as well [10, 16].
This model was recently further refined into the “leaky gate” theory. This builds on the intensity theory and modifies the gate control theory by substituting Grp+ neurons in the role of Aβ fibers. Grp+ spinal cord neurons receive strong input from pain sensory neurons and weak input from itch specific neurons, coding for itch in an intensity dependent manner and inhibiting pain. This model is different from gate control in that it lets weak pain signals “leak” through while suppressing strong pain signals to prevent an overwhelming pain sensation. When strongly activated by pain, these interneurons inhibit pain, whereas due to weak activation from itch, it does not inhibit pain [10]. This model is able to explain the phenomenon that itch is often accompanied with a prickly, burning pain: it proposes that itch is not strong enough to inhibit pain sensation, resulting in a weak pain sensation accompanying itch [10].
Conclusion
A few of the major theories of itch perception have been discussed in an attempt to illuminate how itch is attenuated by the presence of pain in an inverse relationship. The intensity theory and the labeled line theory are both supported by the presence of polymodal neurons and itch specific neurons, respectively. However, given their opposing views, the accuracy of both theories is undermined by support for the other; this indicates the need for a model that is able to reconcile itch specificity with neurons attuned to both itch and pain.
The following models attempted to ease the apparent discord between the two previous models. The spatial model expands on the intensity model while providing a possible mechanism by which pain and itch could be felt from the same population of neurons. On the other hand, the population coding model expands on itch specific neurons of the labeled line while accommodating the inverse relationship between itch and pain. Lastly, the leaky gate model combines aspects of both intensity and selectivity theories.
These theories attempt to explain itch and pain crosstalk; the importance of understanding this relationship is seen in both acute and chronic itch pathophysiology in cases of crosstalk dysfunction. The previously discussed Bhlhb+ neurons are a prime example of the consequences of impaired itch and pain interaction [2, 16]. Research has shown that knocking out these interneurons – thereby severing the connection between itch and pain – results in chronic itch-like behavior such as lesions from scratching [16]. This suggests that chronic itch may result from uninhibited, unregulated itch when pain is no longer permitted to suppress itch [2, 16].
This example highlights the importance of the application of the interaction between pain and itch. Not only does understanding the intersection between the two sensations provide a better understanding of itch mechanisms, the very intersection itself has an important role in itch pathophysiology, of which there is much that is still unknown. With the advent of new discoveries of new aspects of the itch pathway, these current models will continue to develop.
References:
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Transgender Health: Barriers to Healthcare and Physiological Differences
By Ana Nazmi Glosson, Neurobiology, Physiology & Behavior ‘21
Author’s Note: I initially wrote this literature review for UWP 104F in Winter 2020. I chose to focus on a topic that was, and is, very dear to me. I believe that readers would benefit from an overview of transgender specific health, as it is a subsection of science that is often unknown or overlooked. I wrote this while personally researching TGD healthcare and the availability of transitional therapies, and realizing firsthand the barriers to access, and lack of available information.
ABSTRACT
Transgender and gender diverse (TGD) individuals are people whose gender identity does not match the biological sex they were assigned at birth. Transgender is an umbrella term for many gender identities, and individuals may identify as male, female, or outside the gender binary. This population faces more barriers to healthcare access than cisgender individuals, or people whose gender identity does match their sex assigned at birth. Lack of access to knowledgeable healthcare providers, as well as provider bias, creates an environment of hostility for a TGD patient. Transgender people have unique health needs that healthcare professionals must be educated on in order to properly serve this community. Emerging literature is beginning to identify health concerns among transgender people who have undergone hormone replacement therapy (HRT) that may require specialized treatment and attention. This review attempts to answer the following question: What does current research tell us about barriers and educational gaps in healthcare of transgender individuals, and what physiological differences in this population, compared to cisgender individuals, make this research important? Further studies are essential to properly providing healthcare to this population.
Key Concepts: Transgender and gender diverse, hormone replacement therapy, culturally competent healthcare.
INTRODUCTION
Historically, TGD individuals have faced many barriers to healthcare access—and many of these barriers still exist [1-9]. This paper aims to review the specifics of these barriers and educational gaps. Current research suggests that a lack of education from physicians and provider biases against transgender people are primary reasons why transgender individuals—especially TGD youth— struggle to access safe and culturally component heathcare [3,5-7]. Transgender people are less likely to seek healthcare, and if they do seek it, they are less likely to receive proper, unbiased access with educated professionals [1,3]. This review also presents literature on unique physiological differences between transgender and cisgender individuals in order to properly express why clinical research is needed to increase baseline education [10-16]. The critical health differences between the TGD population and other groups means a team of doctors and specialists—primary care physician, gender specialist, surgeon, and endocrinologist—must collaborate to provide culturally competent care. TGD individuals may choose to medically transition and undergo gender-affirming therapies such as gender-affirming surgery (GAS) or hormone replacement therapy (HRT). Given the nature of this topic, it is important to note that much of the research in this review is from ground-breaking preliminary studies that have not yet been repeated with larger sample sizes beyond initial investigation.
DISCUSSION
Healthcare Access Among TGD Individuals
TGD individuals of all ages face challenges to healthcare on both a personal and institutional level. Increasing numbers of TGD people, including older adults, are openly living with their gender identity, meaning this is a critical area of research. TGD adults frequently struggle with insurance access; they are less likely to have insurance access compared to non-TGD LGBTQ+ individuals, and those that do have access are more likely to face healthcare discrimination [7,8]. One study found that individuals with Medicaid were more likely to be refused hormone replacement therapy, and more likely to lack a surgeon to perform gender-affirming surgery in their network, as compared to individuals with private insurance [7]. TGD adults who are part of other disadvantaged communities, such as being an ethnic minority or having lower socioeconomic status, face additional obstacles and higher levels of healthcare refusal [1,8]. Older LGBT adults are far more likely to have physical and mental health struggles than their non-LGBT counterparts, but older TDG adults are the most likely to have those struggles within the LGBT community [9,17]. Older TGD adults are more likely to live alone and have a community of “chosen family” instead of partners or children, which adds a layer of complexity to difficult end-of-life care decisions and increases senior care costs [9,17]. These circumstances show the need for thoughtful and individualized care for TDG individuals of all ages, necessitating competent and knowledgeable providers to navigate these sensitive topics.
Adolescence is a very stressful time in people’s lives, and recent literature shows that young TGD individuals are especially vulnerable [3,5,11]. Surveying the adolescent population directly allows researchers to analyze experiences and suggestions from youth to further improve healthcare. Currently, there is not much information on transgender youth, though the field of research has begun to grow rapidly in the past few years. In everyday life, TGD individuals are often misgendered or referred to as names that they do not identify with anymore. In the context of medical care, this leads to individuals being less likely to seek continuing care. Even without malicious intent, these actions may be incredibly damaging to the TGD individual. In a medical setting, misgendering patients may foster unspoken feelings of distrust and alienation between the patient and their doctors. This is critical because transgender individuals are less likely to continue seeking routine and specialized healthcare if they feel uncomfortable in the medical environment [2,3]. In order for healthcare professionals to serve this population, practices must be as friendly as possible. Requesting and consistently using the individual’s pronouns and preferred name is a critical first step [2-3,5]. Surveyed youth suggested that healthcare providers should ask all individuals these questions, instead of only those known or assumed to be LGBTQ+ [2]. This will lead to the subpopulation not being immediately singled out in a healthcare environment, as well as creating a welcoming space for patients who may not otherwise volunteer this information. Another suggestion was healthcare providers using gender-neutral decor in exam rooms [4]. In settings such as a gynecologist office, traditionally feminime or masculine imagery and furnishings can further alienate TGD individuals and reduce the likelihood of patient continuation. The language used in medical forms should be adjusted to encompass diverse gender expressions. Given the fact that many TGD individuals identify outside of the gender binary, medical records should allow patients to write in their identity rather than check one of two boxes [3]. The gender binary is essentially the rigid classification system of two genders, male or female, a system which is commonly rejected by members of the LGBTQ+ community and their allies. Since gender identity and the language that individuals use to express their personal sense of self is incredibly varied, giving patients more freedom to define and communicate their gender identity would allow them a greater sense of expression. This may also require reform of electronic healthcare systems to include this information, which is currently not common practice. In one study, the vast majority (79%) of TGD youth indicate they would appreciate the professional record of preferred name and pronouns [5].
A common method of surveying the adolescent population is in-depth interviews of a small sample size. These thorough accounts of real experiences are very useful, as researchers can gain a more holistic insight into the individual’s life and experiences. The downside of this research approach is the small sample size, which may lead to results that are not as applicable to larger audiences as would be the case with a larger sample size. In order to best reach this population, researchers target LGBTQ+ programs, but for many reasons, a large subset of the TGD population cannot safely participate in those programs, and therefore are not included in reviews such as this. Voices of closeted LGBTQ+ community members in general are rarely heard, meaning this subset of the population is almost always left out.
Research also suggests that preferences regarding the inclusion of gender identity information in medical records differ greatly if the patient is closeted or “out” [2]. There are factors that should be taken into account with medical records disclosing transgender identity. For instance, a TGD minor may privately disclose their gender identity or preferred pronouns to their healthcare provider. If this TGD youth was not “out” to their parents, and the healthcare provider made a note, their parents might find this while viewing their medical records. This could potentially be damaging or even dangerous to the patient, so healthcare providers should be careful with handling such delicate information. Additionally, TGD care—especially for patients that are in the process of transitioning—involves many aspects of healthcare; a team of culturally competent therapists, physicians, specialists, nurses, and staff must all be properly informed to contribute to a holistically supportive team.
Sexual Health Needs
Research into sexual health needs of young transgender people demonstrates TGD youth have unique sexual health needs that are not currently being met by their healthcare providers. Healthcare providers tend to be less knowledgeable about TGD-specific health issues, which differ from cisgender individuals [3,13,15]. Distinct aspects of TGD individuals include hormone replacement therapy (HRT), gender-affirming surgery (GAS), reversible puberty blockers, and same-sex STI transmission. Compared to previous generations, youth today are more likely to come out as transgender at a younger age, but many healthcare providers are not properly relaying healthcare information to their patients [3]. When providers fail to relay crucial information to their patients, it poses risk to the patients that could otherwise be avoided. For instance, a doctor who is unknowledgeable on STI transmission among two people that were assigned the same sex at birth, or even a doctor with personal prejudices against TGD patients, might not inform patients of essential sexual health information, thus putting the patients at higher risk. Sexual education information for teenagers is lacking, and this issue is amplified for TGD youth, many of whom receive absolutely no relevant information from professionals and alternatively turn to unvetted online sources. Healthcare providers need to stay up to date on the current literature for LGBTQ+ patients and have an obligation to confirm their patients receive adequate and age-appropriate information on topics of sexual health.
Transgender men or non-binary individuals who have been prescribed testosterone, a gender-affirming hormone replacement therapy, may suddenly experience an ovulatory event after a long period of time [15]. Testosterone can stop ovulation by suppressing the hypothalamic-pituitary-adrenal axis, but this research study is the first to show that after an extended period of time, such as several years, some individuals may “overcome” these suppressed hormones and suddenly ovulate [15]. This is important for healthcare professionals to be aware of because their patients may not be on contraceptives and will likely not expect this after suppressed ovulation. Unplanned pregnancy may result among patients who partake in sexual intercourse with sperm-producing individuals. Healthcare providers have an obligation to inform their patients of medical issues such as this, as pregnancy for a transitioning TGD individual can be an extremely emotionally stressful event, especially in the face of body and gender dysphoria.
An emerging branch of literature involves TGD patients and gynecological care. TGD patients are less likely to seek this type of care, and when they do, healthcare providers may have personal biases against treating transgender patients [4,6-7]. Transgender men or transmasculine individuals were found less likely to seek cervical cancer screenings, the main preventative test against cervical cancer. This is because of a variety of barriers on both a personal level and a wider institutional level. On a personal level, traumatic experiences with past healthcare, misgendering, and overall gender dysphoria contribute to transgender men not seeking cervical cancer screenings [4]. Institutionally, research suggests incompetent provider education is a primary barrier to accessing satisfactory healthcare. This leads to a reduced number of transgender men or transmasculine individuals continuing cervical cancer screening [4].. Healthcare professionals should focus on ways to retain transgender men as patients throughout their transition and changing gender identity, as well as providing culturally competent healthcare to this population.
In a study on gynecological health of transmasculine people, healthcare professionals were surveyed on their willingness to provide healthcare to TGD individuals. It was found that personal biases and attitudes against TGD individuals were the greatest barriers [6]. This contradicts other studies, which indicate healthcare providers’ lack of knowledge to be the biggest obstacle to accessing safe healthcare. Professional training should account for transphobic beliefs among healthcare professionals [6].
Much of the research on TGD populations are groundbreaking pilot studies, and conducting more large scale clinical studies and research is highly recommended for improving healthcare for transgender individuals [2,5,17]. Another recommendation is to standardize inclusive and informed education on transgender topics in medical school curricula and continuing education programs [3,5,8]. Informed and supportive healthcare professionals are absolutely vital in addressing health and continued patient retention among TGD individuals. More research must be done to determine the extent of additional training needed to properly serve this population.
HRT and Physiological Differences
Literature has begun to explore and emphasize that physiological differences exist between transgender individuals who are undergoing gender-affirming hormone replacement therapy (HRT) and cisgender individuals [11-17]. Hormone replacement therapy is suggested to be gender-affirming to a patient with gender dysphoria by helping their body match their preferred gender identity, and has been found to be correlated with better body- and self-perception, as well as lower sexual distress [13]. This is incredibly important in increasing the holistic wellness of a transgender patient. Limited available research suggests that transitioned TGD individuals are at greater risk for certain cardiovascular diseases, such as heart attacks, compared to the general population [16]. When researching the impact of HRT on adolescents, one pilot study found key body composition differences in regards to cardiovascular health, suggesting this population has unique cardiometabolic needs that differ from both cisgender males and cisgender females [11,16]. Similarly, in regards to resting state network, individuals on HRT were found to have “intermediate” levels of physiological values unique and distinct from cisgender male or female individuals [11,16]. For the purpose of this paper, we can think of resting state networks as networks and patterns of activity between spatially separated areas in the brain, which are helpful in analysing organization, when the brain is not processing a specific task.. This information is preliminary—and it is important to keep up with developing research—but it suggests the extreme importance of larger repeat studies. Questions for further research include long-term effects of HRT on adolescents. Additionally, research should be conducted on the distinct physiological values of individuals on HRT. In particular, do these values (the intermediate state) change the longer the individual is on HRT? If a patient were to stop HRT, would this “intermediate” state revert to values similar to their gender assigned at birth?
Another question to consider would be whether or not this intermediate state is reversible if the patient were to stop HRT for a period of time. However, such a question would bring up many ethical concerns for the psychological well-being of the study participants, as well as physical concerns of abruptly stopping medical therapy. One longitudinal pilot study found that transgender individuals on HRT had altered resting state functional connectivity in emotional, cognition, and sensorimotor ways after undergoing gender-affirming surgery [15]. These studies suggest that the brains of TGD individuals have the ability to form altered synaptic connections in a way that is different from cisgender people. Much more research is required in order to pinpoint any major connections and the implications of treating this population. These medical differences could be very important in areas such as proper drug dosage. Healthcare professionals must recognize these differences, and continue to push for more research to ensure transgender patients receive the competent care they need. Much of this research contributes to some sense of a gender binary, given that this “intermediate” state is defined as being between “the two” genders; furthermore, a TGD individual may not aspire to follow a binary gender, and providers should be thoughtful and individualized in the language they use with patients. The majority of these studies were composed of very few individuals. These results suggest that healthcare professionals must stay informed with research findings in order to keep their patients updated.
CONCLUSION
Transgender individuals face discrimination in everyday life, as well as in the medical world. This is a large problem because transgender patients have specific healthcare needs that differ from cisgender patients and must be approached and treated differently. Many of these studies are pilot studies and were only published in the last several years. Several recent studies have attempted to classify barriers transgender individuals face, specific health differences, and what steps healthcare providers need to be taking. As research in transgender healthcare continues, it is important to note that not all transgender people can be grouped under one umbrella. Subpopulations exist within the TGD community, each with their own healthcare concerns, physiological health differences, and types of care they seek and receive. In order to better treat these populations, healthcare professionals cannot treat every transgender person with identical care. This emerging research, especially on topics of physiological differences, should not be used to discourage TGD individuals from their necessary transitional therapies. Rather, a more comprehensive understanding should help healthcare providers give their patients stronger, evidence-backed information about their medical choices. In addition, there are barriers that this discussion barely touched on, such as cost, insurance issues, and overall accessibility. Many more studies are required to identify the best ways to combat transgender barriers to healthcare access in order to address the physiological differences between TGD and cisgender individuals.
References:
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- Johnson M, Wakefield C, Garthe K. 2020. Qualitative socioecological factors of cervical cancer screening use among transgender men. Prev Med Rep [Internet]. 17:101052. doi: 10.1016/j.pmedr.2020.101052.
- Sequeira GM, Kidd K, Coulter RWS, Miller E, Garofalo R, Ray KN. 2020. Affirming Transgender Youths’ Names and Pronouns in the Electronic Medical Record. JAMA Pediatr [Internet]. 174(5):501-503. doi: 10.1001/jamapediatrics.2019.6071.
- Shires DA, Prieto L, Woodford MR, Jaffee KD, Stroumsa D. 2019. Gynecologic Health Care Providers’ Willingness to Provide Routine Care and Papanicolaou Tests for Transmasculine Individuals. J Womens Health [Internet]. 28(11):1487-1492. doi: 10.1089/jwh.2018.7384.
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Among Virions
By Jordan Chen, Biochemical Engineering ‘24
What are viruses? Miniscule packages of protein and genetic material, smaller than all but the smallest cells, relatively simple structures on the boundaries of what we consider living. Undetectable to the human eye, these invisible contagions are rarely on the minds of the average person, occupying a semantic space in public consciousness more often than they are understood for their material reality. Stories are more likely to be described as “viral” than an actual virus, yet when the COVID-19 pandemic washed over the world at the end of 2019, the public suddenly had to confront that which was seemingly abiotic, simple, and small. However, the impact of the COVID-19 pandemic exceeded that unassuming material reality. With the shuttering of the global economy, mass death, political crisis, confusion, hysteria, and science without immediate answers, it’s become clear that the sum of COVID-19’s viral components is much more than the whole.
To emphasize this idea in the piece, coronavirus virions are depicted as massive and detailed larger than earth bodies, in a vital bloody red, surrounding and overwhelming the relatively simply shaded globe. What was formerly small, simple, and nonliving, can now be dramatically understood as larger than life, having created complex predicaments, and having taken on a life of its own in its assault against the world. This digital artwork was created in Blender.
Reproductive and Developmental Health Effects of PFAS on Animal Models: A Review of Current Literature
By Anna Maddison, Environmental Toxicology ‘21, Janaé Bonnell, Environmental Toxicology ‘22, Dr. Michele La Merrill
Authors’ Note: This literature review was conducted for the Office of Environmental Health Hazard Assessment in the California Environmental Protection Agency under a contract issued to Dr. Michele La Merrill. We wanted to understand the current research on the reproductive and developmental toxicity of PFHxS, PFBS, PFHxA, PFHpA, PFNA, PFDA, and ADONA to draw conclusions and make recommendations for future policy and research. It is important to understand the health effects of substances such as PFAS chemicals that are present in our food, water, and consumer products to develop regulatory standards that protect public health.
Abstract
Per- and polyfluoroalkyl (PFAS) chemicals are used in the production of many industrial processes and consumer goods, and they have been widely detected in humans and animals. PFOS and PFOA have been comprehensively studied and are being phased out of use, but there are other understudied PFAS chemicals with effects that should be considered in regulatory affairs regarding public health and safety. In this study, we focus on the reproductive and developmental effects of seven PFAS chemicals: perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorohexane sulfonic acid (PFHxS), 4,8-dioxia-3H-perfluorononanoic acid (ADONA), perfluorobutane sulfonic acid (PFBS), perfluoroheptanoic acid (PFHpA), perfluorodecanoic acid (PFDA). This literature review presents the observed reproductive and developmental effects of these chemicals on animal models, which can be used to help establish legislative priorities and draw attention to current gaps in published literature.
Keywords: PFAS, review, animal model, PFBS, PFHxS, PFHpA, PFHxA, PFDA, PFNA, ADONA
Introduction
Poly- and perfluoroalkyl (PFAS) chemicals are widespread synthetic chemicals that are highly mobile, persistent in the environment, and are known to bioaccumulate in humans and animals[1,2]. PFAS chemicals have extensive use due to their unique anti-wetting abilities, as well as their ability to act as a surfactant, a molecule that lowers the surface tension between two liquids[3]. These properties have led to their use in oil- and water-repellent textiles, coatings, and fire-retardant products. PFAS chemicals can also be found in drinking water, production facilities and industries, and many commercial household products[1,4]. Humans are mainly exposed to PFAS through their diet but are unable to metabolize these chemicals in their bodies[5,6].
These chemicals have found use in multiple industries for over 60 years[7]. PFAS production peaked in the 1990s, with PFOS and PFOA being the most popularly used PFAS chemicals. However, after studies linked these chemicals to adverse human health effects, including damage to the immune system and liver, reproductive and developmental harm, hormone disruption, and cancer, the United States had voluntarily phased out their use[1,5,2]. The industry has since shifted to favor PFASs with shorter chains, as these were believed to be less harmful to human health[7,8]. Although no association between chain length and toxicity has been proven, it is still being explored.
To this end, perfluorohexane sulfonic acid (PFHxS), perfluorobutane sulfonic acid (PFBS), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and 4,8-dioxia-3H- perfluorononanoic acid (ADONA) have been increasingly used for industrial purposes as alternatives to PFOS and PFOA[6,8]. These PFAS chemicals have shorter carbon backbone chains, so it is believed they may have less negative health effects than PFOA and PFOS, but this has not been proven. However, their health effects are not as well-explored as those of PFOS and PFOA, thus necessitating new research into the long-term health impacts of exposure to these replacement PFAS chemicals.
Multiple epidemiological studies in humans have suggested that there is an association between PFAS exposure and adverse reproductive and developmental health impacts. After exposure during pregnancy, lower levels of thyroid hormones T3 and thyroxine have been observed in both pregnant women and fetuses, as well as adverse birth outcomes and behavioral effects on children exposed in-utero[6,9]. Some of these PFAS chemicals, such as PFNA, are being detected in both seminal plasma and breast milk of humans[10].
Individuals who are not fully developed may be more vulnerable to PFAS exposure and therefore, more at risk for any toxic effects. The purpose of this literature review is to survey reproductive and developmental effects of PFAS exposure to inform on potential literary gaps and future regulatory priorities.
Methods
Search Procedure
The research for this literature review was conducted by systematically choosing articles discovered through the search engine PubMed. The initial database of articles was created from searching the abbreviated forms of the chemical names (PFHxS, PFBS, PFHxA, PFHpA, PFNA, PFDA, ADONA) in PubMed using the filter “Other Animals” (Table 1). Only papers in English were considered for inclusion. An exception was made for ADONA due to our search yielding an excess of papers written by an author by that name. Instead the search term “ADONA NOT adona [author]” was used. Even with the reduced search, many of the results did not include the PFAS chemical ADONA, and subsequently, were not included.
Table 1: Contains a list of searches and number of results yielded for each one.
| Search Date | Search Term(s) | Number of Results |
| April 15th, 2020 | PFHxS | 124 |
| April 25th, 2020 | PFBS | 78 |
| April 25th, 2020 | PFHxA | 54 |
| April 25th, 2020 | PFHpA | 29 |
| April 25th, 2020 | PFNA | 189 |
| April 25th, 2020 | PFDA | 198 |
| April 26th, 2020 | PFHxS | 125 |
| May 12th, 2020 | PFHpA | 30 |
| May 13th, 2020 | PFDA | 199 |
| May 15th, 2020 | “ADONA NOT adona [author]” | 14 |
| May 29th, 2020 | PFDA | 200 |
Study Selection
The titles and abstracts of each article were scanned for relevance to reproductive toxicity. This includes in vitro studies on isolated cells, different toxicological effects based on sex, responses affecting reproductive organs, and developmental effects in offspring. To limit the scope, this excluded human studies, bioconcentration studies that did not specify reproductive organs or unique developmental patterns, wildlife studies, and studies focused on other organ systems not in the context of reproduction or development. Additionally, we chose to exclude studies which used a mixture of chemicals or in which the PFAS chemical was analyzed as a metabolite of a parent chemical. This was due to the fact that it is uncertain which chemical actually caused an effect. Relevant articles were examined in depth to determine significant toxicological effects of the PFAS chemicals. The number of relevant articles in comparison to the total articles which arose from our search can be seen in Table 2. Other notable responses, from papers which included reproductive effects, were denoted in our accompanying spreadsheet. Due to time constraints, the results for PFDA were summarized with less depth and were instead included as an appendix.
Table 2: Contains the number of resulting studies chosen for inclusion in this review.
| Chemical | Articles Used in Our Research |
| Perfluorononanoic Acid PFNA | 17 |
| Perfluorohexanoic Acid PFHxA | 5 |
| Perfluorohexane Sulfonic Acid PFHxS | 11 |
| 4,8-3H-Perfluorononanoic Acid ADONA | 1 |
| Perfluorobutane Sulfonic Acid PFBS | 15 |
| Perfluoroheptanoic Acid PFHpA | 2 |
| Perfluorodecanoic Acid PFDA | 16 |
Results
PFNA
Seventeen studies contained relevant reproductive and/or developmental effects of PFNA exposure. All of these studies except for one, which dosed Xenopus laevis (African clawed toad, African claw-toed frog or the platanna), examined the effects of PFNA exposure on Danio rerio (zebrafish), Rattus (rats), or Mus (mice). These studies are compiled in Supplementary Table 1 (ST1).
PFNA exposure in Danio rerio caused embryo disfigurement and altered motion patterns, as well as other reproductive and developmental effects. In recently fertilized eggs, PFNA exposure resulted in an increase in malformation rate[6], specifically, in the rate of ventricular edema[19]. Studies also showed correlation between PFNA exposure and locomotion: Danio rerio exposed as embryos travelled less distance [11][13][6], their activity level increased[24] [13], they bumped into the mirror more[13], their startle response and burst activity increased[6], the amount of time they spent in the middle of the water, and their velocity changed[13][11]. Additionally, exposure to PFNA caused reproductive effects such as an increase in the number of opaque embryos[19][, a decrease in hatching rate[19][15], a decrease in the number of eggs in females[15], and an increase in yolk sac area[11]. Observed developmental effects included a decrease in body length[13], abnormally enlarged follicles in the thyroid, and increased T3 hormone levels[18].
In Mus and Rattus, the most observed reproductive effects occurred in the testis. In Rattus, cell viability in Sertoli[12] and testicular[23] cells decreased, DNA damage in testicular cells increased[23], vacuoles formed in the Sertoli cells[12], the number of germ cells that degenerated or were TUNEL-positive increased[12][20], and the percentage of apoptotic cells in the testis increased[20]. In Mus, intratesticular and serum levels of testosterone, testicular glucose level, and testicular lactate concentration decreased[14][10]. Also in Mus, seminiferous tubules had intraepithelial vacuolation where small cavities formed in the tubules, marginal condensation of chromatin in round spermatids, and giant cells and exfoliation of germ cells in the lumen of the tubules[14]. Reproductive effects in mammals exposed to PFNA included reduced prenatal and postnatal survival[16], maternal weight decreased[16][25], and lower birth weight and higher blood pressure in pups[25]. Additionally, there was a sex-specific difference in that serum concentration of PFNA decreased more rapidly in adult females than males[17]. Finally, the developmental effects of PFNA exposure in Mus who had not experienced puberty included a decrease in weight, an increase in absolute and relative liver weight, and hepatocellular hypertrophy[14][10].
In Xenopus laevis, PFNA exposure resulted in increased embryo mortality and malformations including stunted tadpole length, multiple edemas, gut miscoiling, microcephaly in which the offspring had abnormally small heads, and skeletal kinking[22].
PFHxA
Five articles on reproductive and/or developmental effects of PFHxA, which showed statistically significant responses, were found to be relevant to our study. These studies are compiled in Supplementary Table 2 (ST2). Each of the five papers studied a different animal: Danio rerio, Daphnia magna, anuran Xenopus laevis, Mus, and Brachionus calyciflorus. In both Danio rerio and anuran Xenopus laevis embryos, a decrease in body length was observed in the animals exposed to PFHxA[26][28]. In Daphnia magna, reproductive output increased with chronic exposure to PFHxA and mobility decreased with acute exposure to PFHxA[27]. In the Mus, indirect exposure in utero to PFHxA resulted in pups taking longer to open their eyes, a decrease in their body weight, and a lower ratio of liver to body weight[29]. Xenopus also saw liver effects in the form of swollen livers in tadpoles[28. In Brachionus calyciflorus, the rate of population increase decreased, and the mictic ratio, the ratio between eggs that require fertilization and those that do not, and egg size increased[30].
PFHxS
Using the methods described above, fifteen articles examining the effects of PFHxS on Mus, Rattus, Gallus gallus domesticus, and Danio rerio were within the scope of our literature review. These studies are compiled in Supplementary Table 3 (ST3). In the mammals, notable, visible reproductive and developmental effects were observed. In general, male mammals dosed with PFHxS seemed to present more deviations from the control opposed to females. For example, when PFHxS was orally administered or injected via IV, male Rattus took a significantly longer time to expel it from their plasma than female Rattus[38]. Other instances of this trend were apparent when male pups displayed increased anogenital distance[35] and decreased weight gain[36] at more concentrations than females. Other effects of PFHxS in mammals included decreased activity[32] and increased liver and thyroid weight[35] in pups who were respectively dosed neonatally and exposed indirectly in utero as well as directly for two weeks. In Gallus gallus domesticus, PFHxS egg injections decreased pipping success, which is the chick’s ability to break themselves out from their shell, and decreased the mass of embryos[37]. Finally, in Danio rerio embryos an EC50 value was calculated to be 84.5μM using the concentration at which they died or suffered from an adverse effect, defined to be non-inflated swim bladder, pericardial or yolk sac edemas, or scoliosis, as a reference[34]. Altered motion patterns and body lengths were also observed[6][26].
ADONA
Only one article was found that showed reproductive effects of ADONA. This study is shown in Supplementary Table 4 (ST4). Pregnant Rattus norvegicus were exposed to ADONA via oral administration which resulted in a decrease in pup weight, maternal food consumption while pregnant, maternal weight gain, and the number of pups that survived[40]. A higher dosage was abandoned after two days due to death, significant body weight loss, reduced food consumption, decreased activity, dehydration, coldness to touch, pale extremities, rales or rattling sounds in the lungs, ungroomed coat, urine-stained fur, and ptosis or drooping eyelids in the pregnant Rattus[40].
PFBS
Seventy-nine articles resulted from a PubMed search on perfluorobutane sulfonic acid (PFBS) using the above-described methods. Of those seventy-nine articles, fifteen were deemed to be within the scope of this review. These studies are compiled in Supplementary Table 5 (ST5). These studies investigated the effects of PFBS on Mus musculus (mice), Oryzias melastigma (marine medaka), Caenorhabditis elegans, Danio rerio, Rattus norvegicus (rats), Xenopus laevis, and C. riparius (harlequin flies). A multitude of adverse reproductive effects was observed as a result of PFBS exposure. Multiple studies demonstrated that PFBS affects hormone levels in both ICR Mus and Oryzias, generally including a decrease in estrogen levels in all animals and a testosterone decrease in males[7][43]. One study noted that males were experiencing estrogenic changes and females were experiencing antiestrogenic changes as a result of PFBS exposure[43]. PFBS exposure also leads to alterations in levels of follicle-stimulating hormone and progesterone[7][43]. In several egg-producing organisms studied, it was found that overall egg production was significantly decreased[42][43] and that this effect could also occur after several generations of exposure to PFBS[48]. In both Mus and Oryzias, significant decreases in uterus and ovary size were recorded in both exposed organisms[43][9]. These species also had significant changes in the number of follicles and oocytes in various stages after exposure[7][43][9]. Lastly, organisms were observed to have decreased length after exposure to PFBS, an effect that was usually more prominent in males[3][48][49].
First-generation offspring from exposed parents were observed to have changes in hormone levels that would affect growth and development, generally including increases in luteinizing hormone[43][9] and significant alterations in T3 concentration[3][9]. Reduced estrogen levels and elevation of thyroid-stimulating hormone were also observed[9]. The length of diestrus in Mus and Rattus was also significantly increased after parental exposure to PFBS[9][8]. Significant decreases in uterus and ovary size were recorded in the offspring of exposed Mus and Oryzias[43][9]. Mus offspring were also observed to have significant changes in the number of follicles and oocytes in various stages[9]. Furthermore, organismal body weight was observed to be influenced by parental PFBS exposure, although it both increased and decreased in separate experiments[3][8][48]. A study exploring the influence of maternal and paternal dosage on Oryzias offspring observed greater negative impacts on offspring related to paternal exposure to PFBS rather than maternal exposure, such as swimming hyperactivity[44]. Offspring of exposed parents also experienced deformities, such as increased tail and craniofacial malformations in Danio rerio[49]. Lastly, it was observed that PFBS exposure delayed both the age of vaginal opening[9] and preputial separation in Mus and Rattus with parents exposed to PFBS[8].
Of the fifteen studies included, only two focused on the multigenerational effects of PFBS on organismal reproduction and development. PFBS was observed to affect T4 hormone concentration in F2 generation Oryzias after a life-cycle exposure during the F0 generation[3]. In the same species, both the weight of F2 generation eggs, as well as their lipid and protein content, were significantly increased after ancestral exposure during the F0 generation development[43].
PFHpA
A search of PubMed using the above methods yielded thirty results for the chemical PFHpA. Of these thirty results, two were found to be within the scope of this review. These studies are compiled in Supplementary Table 6 (ST6). These reports studied the effects of multiple chemicals, including PFHpA, on Danio rerio and Xenopus laevis via exposure in solution. Both studies are described here due to the limited number of papers. The lowest-observed-adverse-effect levels in the Menger[6] and Kim[28] studies were found to be 89 µM and <0.25 mM (250 μM) renewed daily, respectively. The findings in these studies suggest that PFHpA is both a possible teratogen negatively affecting the embryo or fetus and a developmental toxicant in multiple animal species[28]. A dose-response relationship was exhibited regarding PFHpA dosage and rates of both malformations and mortality in Xenopus embryos[28]. Observed malformations included reduction of body length in 24% of Xenopus tadpoles dosed with 1,000 µM of PFHpA as embryos, as well as enlarged, abnormal livers[28]. However, the observation of reduced tadpole body length was only noted at a dosage of 1,000 µM of PFHpA, which is comparable to the LC50 value in this study, found to be 942.4 µM[28]. There was also evidence of potential behavioral effects in developing organisms. For example, Danio rerio exposed to PFHpA had a significant decrease in swimming distance recorded at the highest dosage[6]. No studies listed in PFHpA searches investigated the reproductive hazard of the chemical.
PFDA
Of the two hundred articles listed for PFDA on PubMed using the above search methods, sixteen studies were considered relevant to be included within this report. These sixteen studies are summarized in Appendix 1 (A1) and a subset of them is highlighted here. Both the viability and maturation of pig oocytes was shown to be negatively impacted by exposure to PFDA[50]. The thyroid was shown to be affected in multiple studies, with thyroid hormone levels being both decreased[59][61][62] and increased[57] in different reports. PFDA also adversely affected the sexual organs of exposed organisms, including the seminal vesicle[56] and seminiferous tubules[60]. In rats, hamsters, and guinea pigs, degeneration of the seminiferous tubules was observed following PFDA exposure[60].
Outside of the 184 remaining studies not included in Appendix 1, three studies stated that PFDA may be estrogen-like in its action. However, these three papers did not contain health endpoints that were within the scope of this report, such as gene expression and carcinogenesis[64][65][66]. These papers are referenced within the sources of this report but were not included within the review of reproductive and developmental health, nor were they included within Appendix 1 (A1).
Discussion
While the toxicity of PFBS, PFHxS, PFHpA, PFHxA, PFDA, PFNA, and ADONA, which are used as substitutes for PFOS and PFOA, is still being investigated, the current body of scientific research suggests that exposure to these PFAS chemicals poses potential reproductive and developmental risk to an organism.
In several species, PFNA exposure resulted in several effects that could interfere with normal procreation and aging systems. Observed deviations from control groups included embryo malformation, offspring mortality, altered behavioral patterns, morphological abnormalities, weight changes, and different hormone levels. There was evidence to suggest that males retain more PFNA than their female counterparts, and several studies suggested that male sex organs were impacted by PFNA exposure. Relative to the other PFAS chemicals studied in this paper, PFNA had a high amount of significant papers with reproductive and developmental effects. This may suggest that it poses a higher risk than some of the other chemicals, but it should be considered in the context that PFNA also had a higher number of search results than the other chemicals and therefore, may just be more well studied. A limitation in these findings that should be considered is the apparent lack of corresponding research on female sexual organs. Also, several effects were not observed to have a dose-dependent response, while others were only seen at lower concentrations.
PFHxA only had five relevant studies and each used a different species as test subjects. Additionally, there were four studies, two used in the results of PFHxA and two not used due to a lack of significant results, which also did their experiments with other PFAS chemicals, and in comparison, individuals dosed with PFHxA had milder, if any, effects. This suggests that PFHxA may have lower reproductive and developmental toxicity than other PFAS chemicals in this paper. This could be taken into consideration when assessing the risk of exposure associated with this chemical.
PFHxS, similar to PFNA, had more toxic effects in males than females. Unlike the studies using PFNA, a majority of these did test both males and females in the same conditions. Exposed males retained a higher serum concentration of PFHxS for longer and displayed changes in anogenital distance and weight at a larger range of dosage concentrations than females.
There was one relevant paper that addressed the reproductive and developmental effects of ADONA exposure. This, coupled with the potential conflict of interest presented by the funding, makes it impossible to draw any conclusions regarding the toxicity of this chemical. Even so, effects were observed from exposure to ADONA. These included changes in weight, mortality rate, and food consumption. To further understand the reproductive and developmental toxicity of ADONA, further research is necessary.
Multiple species demonstrated adverse reproductive and developmental health outcomes after exposure to PFBS. Alterations to hormone levels and morphological abnormalities were observed following PFBS dosage. Also, PFBS was seen to potentially influence hormonal sex changes in exposed fish. Several studies emphasized the impacts PFBS exposure may have on offspring. The above changes were seen in offspring after parental exposure, as well as alterations to egg development and the size of female reproductive organs. It was also noted that paternal exposure to PFBS tends to more negatively impact the health of offspring than maternal exposure does. Compared to other chemicals included within this report, PFBS had a relatively high number of studies that met the requirements for inclusion.
Only two relevant studies were found regarding PFHpA, each using a different test species. These studies investigated the developmental effects of PFHpA on organisms exposed as embryos or tadpoles. Findings suggested that PFHpA may be teratogenic and toxic to developing organisms based on resulting morphological abnormalities and behavioral alterations in exposed organisms. However, no studies were found to investigate the reproductive impacts of PFHpA exposure, leading to a gap in the available research on this chemical.
The studies included on PFDA cover its effect on cell viability, hormone levels, and sex organs, as well the potential developmental toxicity and teratogenicity of PFDA exposure. However, within these studies, there are limitations. Multiple studies did not include information on the age at which organisms, usually Rattus, were dosed, making it more difficult to fully comprehend the effects of PFDA on stages of animal growth. Compared to several of the chemicals included in this paper, PFDA has a relatively higher number of papers that met the criteria for inclusion. Studies have been conducted on this chemical since the 1980s, far preceding work on some of the other chemicals in this report. Furthermore, while the effects of PFDA on hormones and the thyroid have been investigated in several studies, little work appears to have been done on the multigenerational and offspring effects of PFDA. Lastly, five of the sixteen studies investigated the effects of PFDA only on males, leaving further information on female-specific effects to be desired. Both multigenerational and female-specific studies could be considered potential avenues for further research.
Overall, these PFAS chemicals have demonstrated that they may have potential for reproductive and developmental health effects, and their effects on humans should be investigated to ascertain human risk.
Conclusion
The objective of the present study was to compile and summarize the toxic effects of different PFAS chemicals on the reproduction and development of animals in controlled settings. While there are variations in the species used, as well as the method in which they were exposed and the concentration tested, exposure to all seven PFAS chemicals impacted aspects of development, and six had effects on reproduction. These effects should be considered in designing additional research on the outcomes of PFAS exposure and in assessing the risk of these chemicals on living organisms.
Acknowledgements
This work was conducted under California Environmental Protection Agency, Office of Environmental Health Hazard Assessment contract 17-E0024. The authors appreciate the support of Dr. David Furlow, professor of Neurobiology, Physiology, and Behavior and University Honors Program Director at University of California, Davis, and Dr. Sarah Elmore at the Office of Environmental Health Hazard Assessment.
Abbreviations
| DPF◇ | days post-fertilization |
| E2⛊ | estrogen/estradiol |
| FSH╋ | follicle stimulating hormone |
| GD⊕ | gestation day |
| GnRH◬ | Gonadotropin-releasing hormone |
| HPF▽ | hours post-fertilization |
| KT-11⌧ | 11-keto-testosterone |
| LH⋕ | luteinizing hormone |
| P4 | progesterone |
| PFAA✿ | perfluoroalkyl acids |
| PND⋈ | postnatal day |
| PPD⤮ | postpartum day |
| T⌓ | testosterone |
| T3⍙ | 3,3’,5-triiodothyronine |
| T4⌗ | thyroxine |
| TBG‡ | thyroxine-binding globulin |
| TSHↀ | thyroid-stimulating hormone |
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