Cerebral organoid

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Cerebral_organoid 

A flask containing human cerebral organoids

A neural, or brain organoid, describes an artificially grown, in vitro, tissue resembling parts of the human brain. Neural organoids are created by culturing pluripotent stem cells into a three-dimensional culture that can be maintained for years. The brain is an extremely complex system of heterogeneous tissues and consists of a diverse array of neurons and glial cells. This complexity has made studying the brain and understanding how it works a difficult task in neuroscience, especially when it comes to neurodevelopmental and neurodegenerative diseases. The purpose of creating an in vitro neurological model is to study these diseases in a more defined setting. This 3D model is free of many potential in vivo limitations. The varying physiology between human and other mammalian models limits the scope of animal studies in neurological disorders. Neural organoids contain several types of nerve cells and have anatomical features that recapitulate regions of the nervous system. Some neural organoids are most similar to neurons of the cortex. In some cases, the retina, spinal cord, thalamus and hippocampus. Other neural organoids are unguided and contain a diversity of neural and non-neural cells. Stem cells have the potential to grow into many different types of tissues, and their fate is dependent on many factors. Below is an image showing some of the chemical factors that can lead stem cells to differentiate into various neural tissues; a more in-depth table of generating specific organoid identity has been published. Similar techniques are used on stem cells used to grow cerebral organoids.

Instructive growth factors regulating fate decisions in embryonic NCSCs

Model development

Using human pluripotent stem cells to create in vitro neural organoids allows researchers to analyze current developmental mechanisms for human neural tissue as well as study the roots of human neurological diseases. Neural organoids are an investigative tool used to understand how disease pathology works. These organoids can be used in experiments that current in vitro methods are too simplistic for, while also being more applicable to humans than rodent or other mammalian models might be. Historically, major breakthroughs in how the brain works have resulted from studying injury or disorder in human brain function. An in vitro human brain model permits the next wave in our understanding of the human nervous system.

Culturing methods

This flow chart outlines the basic steps to create a cerebral organoid. The process takes a span of months and the size of the organoid is limited to the availability of nutrients.

An embryoid body cultivated from pluripotent stem cells is used to make an organoid. Embryoid bodies are composed of three layers: endoderm, mesoderm and ectoderm, which has the potential to be differentiated into different types of tissue.

A cerebral organoid can be formed by inducing ectoderm cells to differentiate into a cerebral organoids. The general procedure can be broken down into 5 steps. First human pluripotent stem cells are cultured. They are then cultivated into an embryoid body. Next the cell culture is induced to form a neuroectoderm. The neuroectoderm is then grown in a matrigel droplet. The matrigel provides nutrients and the neuroectoderm starts to proliferate and grow. Replication of specific brain regions in cerebral organoid counterparts is achieved by the addition of extracellular signals to the organoid environment during different stages of development; these signals were found to create change in cell differentiation patterns, thus leading to recapitulation of the desired brain region. SMAD inhibition may be used in usual cerebral organoid culturing processes to generate microglia in cerebral organoids. The lack of vasculature limits the size the organoid can grow. This has been the major limitation in organoid development. The use of a spinning bioreactor may improve the availability of nutrients to cells inside the organoid to improve organoid development. Spinning bioreactors have been used increasingly in cell culture and tissue growth applications. The reactor is able to deliver faster cell doubling times, increased cell expansion and increased extra-cellular matrix components when compared to statically cultured cells.

Components

Differentiation

It has been shown that cerebral organoids grown using the spinning bioreactor 3D culture method differentiate into various neural tissue types, such as the optic cup, hippocampus, ventral parts of the teleencephelon and dorsal cortex. Furthermore, it was shown that human brain organoids could intrinsically develop integrated light-sensitive optic cups.

The neural stem/progenitor cells are unique because they are able to self-renew and are multipotent. This means they can generate neurons and glial cells which are the two main components of neural systems. The fate of these cells is controlled by several factors that affect the differentiation process. The spatial location and temporal attributes of neural progenitor cells can influence if the cells form neurons or glial cells. Further differentiation is then controlled by extracellular conditions and cell signaling. The exact conditions and stimuli necessary to differentiate neural progenitor cells into specific neural tissues such as hippocampal tissue, optic nerve, cerebral cortex, etc. are unknown. It is believed that cerebral organoids can be used to study the developmental mechanisms of these processes.

Gene expression

To test if the neural progenitor cells and stem cells are differentiating into specific neural tissues, several gene markers can be tested. Two markers that are present during pluripotent stages are OCT4 and NANOG. These two markers are diminished during the course of development for the organoid. Neural identity markers that note successful neural induction, SOX1 and PAX6, are upregulated during organoid development. These changes in expression support the case for self-guided differentiation of cerebral organoids. Markers for forebrain and hindbrain can also be tested. Forebrain markers FOXG1 and SIX3 are highly expressed throughout organoid development. However, hindbrain markers EGR2 and ISL1 show early presence but a decrease in the later stages. This imbalance towards forebrain development is similar to the developmental expansion of forebrain tissue in human brain development. To test if organoids develop even further into regional specification, gene markers for cerebral cortex and occipital lobe have been tested. Many regions that have forebrain marker FOXG1, labeling them as regions with cerebral cortical morphology, were also positive for marker EMX1 which indicates dorsal cortical identity. These specific regions can be even further specified by markers AUTS2, TSHZ2, and LMO4 with the first representing cerebral cortex and the two after representing the occipital lobe. Genetic markers for the hippocampus, ventral forebrain, and choroid plexus are also present in cerebral organoids, however, the overall structures of these regions have not yet been formed.

Organization

Cerebral organoids also possess functional cerebral cortical neurons. These neurons must form on the radially organized cortical plate. The marker TBR1 is present in the preplate, the precursor to the cortical plate, and is present, along with MAP2, a neuronal marker, in 30-day-old cerebral organoids. These markers are indicative of a basal neural layer similar to a preplate. These cells are also apically adjacent to a neutral zone and are reelin+ positive, which indicates the presence of Cajal-Retzius cells. The Cajal-Retzius cells are important to the generation of cortical plate architecture. The cortical plate is usually generated inside-out such that later-born neurons migrate to the top superficial layers. This organization is also present in cerebral organoids based on genetic marker testing. Neurons that are early born have marker CTIP2 and are located adjacent to the TBR1 exhibiting preplate cells. Late-born neurons with markers SATB2 and BRN2 are located in a superficial layer, further away from the preplate than the early born neurons suggesting cortical plate layer formation. Additionally, after 75 days of formation, cerebral organoids show a rudimentary marginal zone, a cell-poor region. The formation of layered cortical plate is very basic in cerebral organoids and suggests the organoid lacks the cues and factors to induce formation of layer II-VI organization. The cerebral organoid neurons can, however, form axons as shown by GFP staining. GFP labeled axons have been shown to have complex branching and growth cone formation. Additionally, calcium dye imaging has shown cerebral organoids to have Ca²⁺ oscillations and spontaneous calcium surges in individual cells. The calcium signaling can be enhanced through glutamate and inhibited through tetrodotoxin.

Interactions with environment

In DishBrain, grown human brain cells were integrated into digital systems to play a simulated Pong via electrophysiological stimulation and recording. The cells "showed significantly improved performance in Pong" when embodied in a virtual game-world. In the 2020s, significant changes in how these electrophysiological systems are made and interact with brain organoids could lead to better stimulation and recording data across the organoind in 3D.

Interactions with surrounding tissues

It is not fully understood how individual localized tissues formed by stem cells are able to coordinate with surrounding tissues to develop into a whole organ. It has been shown however that most tissue differentiation requires interactions with surrounding tissues and depends on diffusible induction factors to either inhibit or encourage various differentiation and physical localization. Cerebral organoid differentiation is somewhat localized. The previously mentioned markers for forebrain and hindbrain are physically localized, appearing in clusters. This suggests that local stimuli are released once one or more cells differentiate into a specific type as opposed to a random pathway throughout the tissue. The markers for subspecification of cortical lobes, prefrontal cortex and occipital lobe, are also physically localized. However, the hippocampus and ventral forebrain cells are not physically localized and are randomly located through the cerebral organoid. Cerebral organoids lack blood vessels and are limited in size by nutrient uptake in the innermost cells. Spinning bioreactors and advanced 3D scaffolding techniques are able to increase organoid size, though the integration of in vitro nutrient delivery systems is likely to spark the next major leap in cerebral organoid development.

Assays

Cerebral organoids have the potential to function as a model with which disease and gene expression might be studied. However, diagnostic tools are needed to evaluate cerebral organoid tissue and create organoids modeling the disease or state of development in question. Transcriptome analysis has been used as an assay to examine the pathology of cerebral organoids derived from individual patients. Additionally, TUNEL assays have been used in studies as an evaluative marker of apoptosis in cerebral organoids. Other assays used to analyze cerebral organoids include the following:

Genetic modifications

Cerebral organoids can be used to study gene expression via genetic modifications. The degree to which these genetic modifications are present in the entire organoid depends on what stage of development the cerebral organoid is in when these genetic modifications are made; the earlier these modifications are made, such as when the cerebral organoid is in the single cell stage, the more likely these modifications will affect a greater portion of the cells in the cerebral organoid. The degree to which these genetic modifications are present within the cerebral organoid also depends on the process by which these genetic modifications are made. If the genetic information is administered into one cerebral organoid cell's genome via machinery, then the genetic modification will remain present in cells resulting from replication. Crispr/Cas 9 is a method by which this long-lasting genetic modification can be made. A system involving use of transposons has also been suggested as a means to generate long-lasting genetic modifications; however, the extent to which transposons might interact with a cell genome might differs on a cell to cell basis, which would create variable expressivity between cerebral organoid cells. If, however, the genetic modification is made via “genetic cargo” insertion (such as through Adeno-associated virus/ electroporation methods) then it has been found that the genetic modification becomes less present with each round of cell division in cerebral organoids.

Computational methods

Use of computational methods have been called for as a means to help improve the cerebral organoid cultivation process; development of computational methods has also been called for in order to provide necessary detailed renderings of different components of the cerebral organoid (such as cell connectivity) that current methods are unable to provide. Programming designed to model detailed cerebral organoid morphology does not yet exist.

Applications

See also: Aging brain, Neurobiological effects of physical exercise, Wetware computer, Metabolome, and Neurogenomics

There are many potential applications for cerebral organoid use, such as cell fate potential, cell replacement therapy, and cell-type specific genome assays. Cerebral organoids also provide a unique insight into the timing of development of neural tissues and can be used as a tool to study the differences across species. Further potential applications for cerebral organoids include:

Tissue morphogenesis

Tissue morphogenesis with respect to cerebral organoids covers how neural organs form in vertebrates. Cerebral organoids can serve as in vitro tools to study the formation, modulate it, and further understand the mechanisms controlling it.

Migration assays

Cerebral organoids can help to study cell migration. Neural glial cells cover a wide variety of neural cells, some of which move around the neurons. The factors that govern their movements, as well as neurons in general, can be studied using cerebral organoids.

Clonal lineage tracing

Clonal lineage tracing is part of fate mapping, where the lineage of differentiated tissues is traced to the pluripotent progenitors. The local stimuli released and the mechanism of differentiation can be studied using cerebral organoids as a model. Genetic modifications in cerebral organoids could serve as a means to accomplish lineage tracing.

Transplantation

Cerebral organoids can be used to grow specific brain regions and transplant them into regions of neurodegeneration as a therapeutic treatment. They can fuse with host vasculature and be immunologically silent. In some cases, the genomes of these cerebral organoids would first have to be edited. Recent studies have been able to achieve successful transplantation and integration of cerebral organoids into mouse brains; development of cell differentiation and vascularity was also observed after transplantation. Cerebral organoids might serve as the basis for transplantation and rebuilding in the human brain due to the similarity in structure.

Drug testing

Cerebral organoids can be used as simple models of complex brain tissues to study the effects of drugs and to screen them for initial safety and efficacy. Testing new drugs for neurological diseases could also result from this method of applying drug high-throughput screening methods to cerebral organoids. After 2015, significant effort has gone into fabricating microscale devices to generate reproducible cerebral organoids at high-throughput.

Developmental biology

Organoids can be used for the study of brain development, for example identifying and investigating genetic switches that have a significant impact on it. This can be used for the prevention and treatment of specific diseases (see below) but also for other purposes such as insights into the genetic factors of recent brain evolution (or the origin of humans and evolved difference to other apes), human enhancement and improving intelligence, identifying detrimental exposome impacts (and protection thereof), or improving brain health spans.

Disease study

Organoids can be used to study the crucial early stages of brain development, test drugs and, because they can be made from living cells, study individual patients. Additionally, the development of vascularized cerebral organoids could be used for investigating stroke therapy in the future.

Zika Virus

Zika virus has been shown to have teratogenic effects, causing defects in fetal neurological development. Cerebral organoids have been used in studies in order to understand the process by which Zika virus affects the fetal brain and, in some cases, causes microcephaly. Cerebral organoids infected with the Zika virus have been found to be smaller in size than their uninfected counterparts, which is reflective of fetal microcephaly. Increased apoptosis was also found in cerebral organoids infected with Zika virus. Another study found that neural progenitor cell (NPC) populations were greatly reduced in these samples. The two methods by which NPC populations were reduced were increased cell death and reduced cell proliferation. TLR3 receptor upregulation was identified in these infected organoids. Inhibition of this TLR3 receptor was shown to partially halt some of the Zika induced effects. Additionally, lumen size was found to be increased in organoids infected with Zika virus. The results found from studying cerebral organoids infected with Zika virus at different stages of maturation suggest that early exposure in developing fetuses can cause greater likelihood of Zika virus-associated neurological birth defects.

Cocaine

Cocaine has also been shown to have teratogenic effects on fetal development. Cerebral organoids have been used to study which enzyme isoforms are necessary for fetal neurological defects caused by cocaine use during pregnancy. One of these enzymes was determined to be cytochrome P450 isoform CYP3A5.

Microcephaly

In one case, a cerebral organoid grown from a patient with microcephaly demonstrated related symptoms and revealed that apparently, the cause is overly rapid development, followed by slower brain growth. Microencephaly is a developmental condition in which the brain remains undersized, producing an undersized head and debilitation. Microcephaly is not suitable for mouse models, which do not replicate the condition. The primary form of the disease is thought to be caused by a homozygous mutation in the microcephalin gene. The disease is difficult to reproduce in mouse models because mice lack the developmental stages for an enlarged cerebral cortex that humans have. Naturally, a disease which affects this development would be impossible to show in a model which does not have it to begin with. To use cerebral organoids to model a human's microcephaly, one group of researchers has taken patient skin fibroblasts and reprogrammed them using four well known reprogramming factors. These include OCT4, SOX2, MYC and KLF4. The reprogrammed sample was able to be cloned into induced pluripotent stem cells. The cells were cultured into a cerebral organoid following a process described in the cerebral organoid creation section below. The organoid that resulted had decreased numbers of neural progenitor cells and smaller tissues. Additionally, the patient-derived tissues displayed fewer and less frequent neuroepithelial tissues made of progenitors, decreased radial glial stem cells, and increased neurons. These results suggest that the underlying mechanism of microcephaly is caused by cells prematurely differentiating into neurons leaving a deficit of radial glial cells.

Alzheimer's disease

Alzheimer's disease pathology has also been modeled with cerebral organoids. Affected individual's pluripotent stem cells were used to generate brain organoids and then compared to control models, synthesised from healthy individuals. It was found that in the affected models, structures similar to that of plaques caused by amyloid beta proteins and neurofibrillary tangles, that cause the disease's symptoms were observed. Previous attempts to model this so accurately have been unsuccessful, with drugs being developed on the basis of efficacy in pre-clinical murine models demonstrating no effect in human trials.

Autism spectrum disorders

Cerebral organoids can also be used to study autism spectrum disorders. In one study, cerebral organoids were cultured from cells derived from macrocephaly ASD patients. These cerebral organoids were found to reflect characteristics typical of the ASD-related macrocephaly phenotype found in the patients. By cultivating cerebral organoids from ASD patients with macrocephaly, connections could be made between certain gene mutations and phenotypic expression. Autism has also been studied through the comparison of healthy versus affected synthesised brain organoids. Observation of the two models showed the overexpression of a transcription factor FOXG1 that produced a larger amount of GABAergic inhibitory neurons in the affected models. The significance of this use of brain organoids is that it has added great support for the excitatory/inhibitory imbalance hypothesis which if proven true could help identify targets for drugs so that the condition could be treated.

The field of epigenetics and how DNA methylation might influence development of ASD has also been of interest in recent years. The traditional method of studying post-mortem neural samples from individuals with ASD poses many challenges, so cerebral organoids have been proposed as an alternate method of studying the potential effect that epigenetic mechanisms may have on the development of autism. This use of the cerebral organoid model to examine ASD and epigenetic patterns might provide insight in regards to epigenetic developmental timelines. However, it is important to note that the conditions in which cerebral organoids are cultured in might affect gene expression, and consequentially affect observations made using this model. Additionally, there is concern over the variability in cerebral organoids cultured from the same sample. Further research into the extent and accuracy by which cerebral organoids recapitulate epigenetic patterns found in primary samples is also needed.

Preterm hypoxia/ischemia

Preterm hypoxic injury remain difficult to study because of limited availability of human fetal brain tissues and inadequate animal models to study human corticogenesis. Cerebral organoid can be used to model prenatal pathophysiology and to compare the susceptibility of the different neural cell types to hypoxia during corticogenesis. Intermediate progenitors seem to be particularly affected, due to the unfolded protein response pathway. It has also been observed that hypoxia resulted in apoptosis in cerebral organoids, with outer radial glia and neuroblasts/immature neurons being particularly affected.

Glioblastomas

Traditional means of studying glioblastomas come with limitations. One example of such limitations would be the limited sample availability. Because of these challenges that come with using a more traditional approach, cerebral organoids have been used as an alternative means to model the development of brain cancer. In one study, cerebral organoids were simulated to reflect tumor-like qualities using CRISPR CAS-9. Increased cell division was observed in these genetically altered models. Cerebral organoids were also used in mice models to study tumorigenesis and invasiveness. At the same time, the growth of brain cancers is influenced by environmental factors which are not yet replicable in cerebral organoid models. Cerebral organoids have been shown to provide insight into dysregulation of genes responsible for tumor development.

Multiple Sclerosis

Multiple sclerosis is an auto-immune inflammatory disorder affecting the central nervous system. Environmental and genetic factors contribute to the development of multiple sclerosis, however the etiology of this condition is unknown. Induced pluripotent stem cells from healthy human controls, as well as from patients with multiple sclerosis were grown into cerebral organoids creating an innovative human model of this disease.

Limitations

Cerebral organoids are preferred over their 3D cell culture counterparts because they can better reflect the structure of the human brain, and because, to a certain extent, they can reflect fetal neocortex development over an extended period of time. While cerebral organoids have a lot of potential, their culturing and development comes with limitations and areas for improvement. For example, it takes several months to create one cerebral organoid, and the methods used to analyze them are also time-consuming. Additionally, cerebral organoids do not have structures typical of a human brain, such as a blood brain barrier. This limits the types of diseases that can be studied. Other limitations include:

Necrotic centers

Until recently, the central part of organoids have been found to be necrotic due to oxygen as well as nutrients being unable to reach that innermost area. This imposes limitations to cerebral organoids' physiological applicability. Because of this lack of oxygen and nutrients, neural progenitor cells are limited in their growth. However, recent findings suggest that, in the process of culturing a cerebral organoid, a necrotic center could be avoided by using fluidic devices to increase the organoid's exposure to media.

Reliability in generation

The structure of cerebral organoids across different cultures has been found to be variable; a standardization procedure to ensure uniformity has yet to become common practice. Future steps in revising cerebral organoid production would include creating methods to ensure standardization of cerebral organoid generation. One such step proposed involves regulating the composition and thickness of the gel in which cerebral organoids are cultured in; this might contribute to greater reliability in cerebral organoid production. Additionally, variability in generation of cerebral organoids is introduced due to differences in stem cells used. These differences can arise from different manufacturing methods or host differences. Increased metabolic stress has also been found within organoids. This metabolic stress has been found to restrict organoid specificity. Future steps to streamline organoid culturing include analyzing more than one sample at a time.

Maturity

At the moment, the development of mature synapses in cerebral organoids is limited because of the media used. Additionally, while some electrophysiological properties have been shown to develop in cerebral organoids, cultivation of separate and distinct organoid regions has been shown to limit the maturation of these electrophysiological properties. Modeling of electrophysiological neurodevelopmental processes typical of development later in the neurodevelopmental timeline, such as synaptogenesis, is not yet suggested in cerebral organoid models. Since cerebral organoids are reflective of what happens during fetal neurodevelopment, there has been concern over how late onset diseases manifest in them. Future improvements include developing a way to recapitulate neurodegenerative diseases in cerebral organoids.

Ethics

See also: Neuroethics and Wetware computer § Ethical and philosophical implications

Sentient organoids

Ethical concerns have been raised with using cerebral organoids as a model for disease due to the potential of them experiencing sensations such as pain or having the ability to develop a consciousness. Currently it is unlikely given the simplicity of synthesised models compared to the complexity of a human brain; however, models have been shown to respond to light-based stimulation, so present models do have some scope of responding to some stimuli.

Guidelines and legislation

Steps are being taken towards resolving the grey area such as a 2018 symposium at Oxford University where experts in the field, philosophers and lawyers met to try to clear up the ethical concerns with the new technology. Similarly, projects such as Brainstorm from Case Western University aim to observe the progress of the field by monitoring labs working with brain organoids to try to begin the ‘building of a philosophical framework’ that future guidelines and legislation could be built upon.

Humanized animals

See also: Humanzee and Human intelligence § Non-human intelligence

Additionally, the "humanization" of animal models has been raised as a topic of concern in transplantation of human stem cell derived organoids into other animal models. For example, potential future concerns of this type were described when human brain tissue organoids were transplanted into baby rats, appearing to be highly functional, to mature and to integrate with the rat brain. Such models can be used to model human brain development and, as demonstrated, to investigate diseases (and their potential therapies) but could be controversial.

Risk of astronomical suffering

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Risk_of_astronomical_suffering

Scope–severity grid from Bostrom's paper "Existential Risk Prevention as Global Priority"

Risks of astronomical suffering, also called suffering risks or s-risks, are risks involving much more suffering than all that has occurred on Earth so far. They are sometimes categorized as a subclass of existential risks.

According to some scholars, s-risks warrant serious consideration as they are not extremely unlikely and can arise from unforeseen scenarios. Although they may appear speculative, factors such as technological advancement, power dynamics, and historical precedents indicate that advanced technology could inadvertently result in substantial suffering. Thus, s-risks are considered to be a morally urgent matter, despite the possibility of technological benefits.

Sources of possible s-risks include embodied artificial intelligence and superintelligence, as well as space colonization, which could potentially lead to "constant and catastrophic wars" and an immense increase in wild animal suffering by introducing wild animals, who "generally lead short, miserable lives full of sometimes the most brutal suffering", to other planets, either intentionally or inadvertently.

Types of S-risk

Artificial intelligence

Artificial intelligence is central to s-risk discussions because it may eventually enable powerful actors to control vast technological systems. In a worst-case scenario, AI could be used to create systems of perpetual suffering, such as a totalitarian regime expanding across space. Additionally, s-risks might arise incidentally, such as through AI-driven simulations of conscious beings experiencing suffering, or from economic activities that disregard the well-being of nonhuman or digital minds. Steven Umbrello, an AI ethics researcher, has warned that biological computing may make system design more prone to s-risks.

Space colonization

Space colonization could increase suffering by introducing wild animals to new environments, leading to ecological imbalances. In unfamiliar habitats, animals may struggle to survive, facing hunger, disease, and predation. These challenges, combined with unstable ecosystems, could cause population crashes or explosions, resulting in widespread suffering. Additionally, the lack of natural predators or proper biodiversity on colonized planets could worsen the situation, mirroring Earth’s ecological problems on a larger scale. This raises ethical concerns about the unintended consequences of space colonization, as it could propagate immense animal suffering in new, unstable ecosystems. Phil Torres argues that space colonization poses significant "suffering risks", where expansion into space will lead to the creation of diverse species and civilizations with conflicting interests. These differences, combined with advanced weaponry and the vast distances between civilizations, would result in catastrophic and unresolvable conflicts. Strategies like a "cosmic Leviathan" to impose order or deterrence policies are unlikely to succeed due to physical limitations in space and the destructive power of future technologies. Thus, Torres concludes that space colonization could create immense suffering and should be delayed or avoided altogether.

Magnus Vinding's "astronomical atrocity problem" questions whether vast amounts of happiness can justify extreme suffering from space colonization. He highlights moral concerns such as diminishing returns on positive goods, the potentially incomparable weight of severe suffering, and the priority of preventing misery. He argues that if colonization is inevitable, it should be led by agents deeply committed to minimizing harm.

Genetic engineering

David Pearce has argued that genetic engineering is a potential s-risk. Pearce argues that while technological mastery over the pleasure-pain axis and solving the hard problem of consciousness could lead to the potential eradication of suffering, it could also potentially increase the level of contrast in the hedonic range that sentient beings could experience. He argues that these technologies might make it feasible to create "hyperpain" or "dolorium" that experience levels of suffering beyond the human range.

Excessive criminal punishment

S-risk scenarios may arise from excessive criminal punishment, with precedents in both historical and in modern penal systems. These risks escalate in situations such as warfare or terrorism, especially when advanced technology is involved, as conflicts can amplify destructive tendencies like sadism, tribalism, and retributivism. War often intensifies these dynamics, with the possibility of catastrophic threats being used to force concessions. Agential s-risks are further aggravated by malevolent traits in powerful individuals, such as narcissism or psychopathy. This is exemplified by totalitarian dictators like Hitler and Stalin, whose actions in the 20th century inflicted widespread suffering.

Exotic risks

According to David Pearce, there are other potential s-risks that are more exotic, such as those posed by the many-worlds interpretation of quantum mechanics.

Mitigation strategies

To mitigate s-risks, efforts focus on researching and understanding the factors that exacerbate them, particularly in emerging technologies and social structures. Targeted strategies include promoting safe AI design, ensuring cooperation among AI developers, and modeling future civilizations to anticipate risks. Broad strategies may advocate for moral norms against large-scale suffering and stable political institutions. According to Anthony DiGiovanni, prioritizing s-risk reduction is essential, as it may be more manageable than other long-term challenges, while avoiding catastrophic outcomes could be easier than achieving an entirely utopian future.

Induced amnesia

Induced amnesia has been proposed as a way to mitigate s-risks in locked-in conscious AI and certain AI-adjacent biological systems like brain organoids.

Cosmic rescue missions

David Pearce's concept of "cosmic rescue missions" proposes the idea of sending probes to alleviate potential suffering in extraterrestrial environments. These missions aim to identify and mitigate suffering among hypothetical extraterrestrial life forms, ensuring that if life exists elsewhere, it is treated ethically. However, challenges include the lack of confirmed extraterrestrial life, uncertainty about their consciousness, and public support concerns, with environmentalists advocating for non-interference and others focusing on resource extraction.

Molten-salt battery

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Molten-salt_battery 

FZSoNick 48TL200: sodium–nickel battery with welding-sealed cells and heat insulation

Molten-salt batteries are a class of battery that uses molten salts as an electrolyte and offers both a high energy density and a high power density. Traditional non-rechargeable thermal batteries can be stored in their solid state at room temperature for long periods of time before being activated by heating. Rechargeable liquid-metal batteries are used for industrial power backup, special electric vehicles and for grid energy storage, to balance out intermittent renewable power sources such as solar panels and wind turbines.

In 2023, the use of molten salts as electrolytes for high-energy rechargeable lithium metal batteries was demonstrated.

History

Thermal batteries originated during World War II when German scientist Georg Otto Erb developed the first practical cells using a salt mixture as an electrolyte. Erb developed batteries for military applications, including the V-1 flying bomb and the V-2 rocket, and artillery fuzing systems. None of these batteries entered field use during the war. Afterwards, Erb was interrogated by British intelligence. His work was reported in "The Theory and Practice of Thermal Cells". This information was subsequently passed on to the United States Ordnance Development Division of the National Bureau of Standards. When the technology reached the United States in 1946, it was immediately applied to replacing the troublesome liquid-based systems that had previously been used to power artillery proximity fuzes. They were used for ordnance applications (e.g., proximity fuzes) since WWII and later in nuclear weapons. The same technology was studied by Argonne National Laboratories and other researchers in the 1980s for use in electric vehicles.

Rechargeable configurations

Since the mid-1960s much development work has been undertaken on rechargeable batteries using sodium (Na) for the negative electrodes. Sodium is attractive because of its high reduction potential of −2.71 volts, low weight, relative abundance, and low cost. In order to construct practical batteries, the sodium must be in liquid form. The melting point of sodium is 98 °C (208 °F). This means that sodium-based batteries operate at temperatures between 245 and 350 °C (470 and 660 °F). Research has investigated metal combinations with operating temperatures at 200 °C (390 °F) and room temperature.

Sodium–sulfur

Main article: Sodium–sulfur battery

The sodium–sulfur battery (NaS battery), along with the related lithium–sulfur battery employs cheap and abundant electrode materials. It was the first alkali-metal commercial battery. It used liquid sulfur for the positive electrode and a ceramic tube of beta-alumina solid electrolyte (BASE). Insulator corrosion was a problem because they gradually became conductive, and the self-discharge rate increased.

Because of their high specific power, NaS batteries have been proposed for space applications. An NaS battery for space use was successfully tested on the Space Shuttle mission STS-87 in 1997, but the batteries have not been used operationally in space. NaS batteries have been proposed for use in the high-temperature environment of Venus.

A consortium formed by Tokyo Electric Power Co. (TEPCO) and NGK Insulators Ltd. declared their interest in researching the NaS battery in 1983, and became the primary drivers behind the development of this type ever since. TEPCO chose the NaS battery because its component elements (sodium, sulfur and ceramics) are abundant in Japan. The first large-scale field testing took place at TEPCO's Tsunashima substation between 1993 and 1996, using 3 × 2 MW, 6.6 kV battery banks. Based on the findings from this trial, improved battery modules were developed and were made commercially available in 2000. The commercial NaS battery bank offers:

• Capacity : 25–250 kWh per bank
• Efficiency of 87%
• Lifetime of 2,500 cycles at 100% depth of discharge (DOD), or 4,500 cycles at 80% DOD

Sodium–nickel chloride (Zebra) battery

Main article: ZEBRA battery

The Citroën Berlingo First Electric "Powered by Venturi" used a ZEBRA storage battery; a specially-prepared version was driven from Shanghai to Paris in 2010.

A lower-temperature variant of molten-salt batteries was the development of the ZEBRA (originally, "Zeolite Battery Research Africa"; later, the "Zero Emissions Batteries Research Activity") battery in 1985, originally developed for electric vehicle applications. The battery uses NaNiCl
₂ with Na⁺-beta-alumina ceramic electrolyte.

The NaNiCl
₂ battery operates at 245 °C (473 °F) and uses molten sodium tetrachloroaluminate (NaAlCl
₄), which has a melting point of 157 °C (315 °F), as the electrolyte. The negative electrode is molten sodium. The positive electrode is nickel in the discharged state and nickel chloride in the charged state. Because nickel and nickel chloride are nearly insoluble in neutral and basic melts, contact is allowed, providing little resistance to charge transfer. Since both NaAlCl
₄ and Na are liquid at the operating temperature, a sodium-conducting β-alumina ceramic is used to separate the liquid sodium from the molten NaAlCl
₄. The primary elements used in the manufacture of these batteries have much higher worldwide reserves and annual production than lithium.

It was invented in 1985 by the Zeolite Battery Research Africa Project (ZEBRA) group at the Council for Scientific and Industrial Research (CSIR) in Pretoria, South Africa. It can be assembled in the discharged state, using NaCl, Al, nickel and iron powder. The positive electrode is composed mostly of materials in the solid state, which reduces the likelihood of corrosion, improving safety. Its specific energy is 100 Wh/kg; specific power is 150 W/kg. The β-alumina solid ceramic is unreactive to sodium metal and sodium aluminum chloride. Lifetimes of over 2,000 cycles and twenty years have been demonstrated with full-sized batteries, and over 4,500 cycles and fifteen years with 10- and 20-cell modules. For comparison, LiFePO₄ lithium iron phosphate batteries store 90–110 Wh/kg, and the more common LiCoO₂ lithium-ion batteries store 150–200 Wh/kg. A nano lithium-titanate battery stores 72 Wh/kg and can provide power of 760 W/kg.

The ZEBRA's liquid electrolyte freezes at 157 °C (315 °F), and the normal operating temperature range is 270–350 °C (520–660 °F). Adding iron to the cell increases its power response. ZEBRA batteries are currently manufactured by FZSoNick and used as a power backup in the telecommunication industries, Oil&Gas and Railways. It is also used in special electric vehicles used in mining. In the past it was adopted in the Modec Electric Van, the Iveco Daily 3.5-ton delivery vehicle, the prototype Smart ED, and the Th!nk City. In 2011 the US Postal Service began testing all-electric delivery vans, one powered by a ZEBRA battery.

In 2010 General Electric announced a Na-NiCl
₂ battery that it called a sodium–metal halide battery, with a 20-year lifetime. Its cathode structure consists of a conductive nickel network, molten salt electrolyte, metal current collector, carbon felt electrolyte reservoir and the active sodium–metal halide salts. In 2015, as a result of a global restructuring, the company abandoned the project. In 2017 Chinese battery maker Chilwee Group (also known as Chaowei) created a new company with General Electric (GE) to bring to market a Na-NiCl battery for industrial and energy storage applications.

When not in use, Na-NiCl
₂ batteries are typically kept molten and ready for use because if allowed to solidify they typically take twelve hours to reheat and charge. This reheating time varies depending on the battery-pack temperature, and power available for reheating. After shutdown a fully charged battery pack loses enough energy to cool and solidify in five-to-seven days depending on the amount of insulation.

Sodium metal chloride batteries are very safe; a thermal runaway can be activated only by piercing the battery and also, in this unlikely event, no fire or explosion will be generated. For this reason and also for the possibility to be installed outdoor without cooling systems, make the sodium metal chloride batteries very suitable for the industrial and commercial energy storage installations.

Sumitomo studied a battery using a salt that is molten at 61 °C (142 °F), far lower than sodium based batteries, and operational at 90 °C (194 °F). It offers energy densities as high as 290 Wh/L and 224 Wh/kg and charge/discharge rates of 1C with a lifetime of 100–1000 charge cycles. The battery employs only nonflammable materials and neither ignites on contact with air nor risks thermal runaway. This eliminates waste-heat storage or fire- and explosion-proof equipment, and allows closer cell packing. The company claimed that the battery required half the volume of lithium-ion batteries and one quarter that of sodium–sulfur batteries. The cell used a nickel cathode and a glassy carbon anode.

In 2014 researchers identified a liquid sodium–cesium alloy that operates at 50 °C (122 °F) and produced 420 milliampere-hours per gram. The new material was able to fully coat, or "wet," the electrolyte. After 100 charge/discharge cycles, a test battery maintained about 97% of its initial storage capacity. The lower operating temperature allowed the use of a less-expensive polymer external casing instead of steel, offsetting some of the increased cost of cesium.

Innovenergy in Meiringen, Switzerland has further optimised this technology with the use of domestically sourced raw materials, except for the nickel powder component. Despite the reduced capacity compared with lithium-ion batteries, the ZEBRA technology is applicable for stationary energy storage from solar power. In 2022, the company operated a 540 kWh storage facility for solar cells on the roof of a shopping center, and currently produces over a million battery units per year from sustainable, non-toxic materials (table salt).

Liquid-metal batteries

See also: Ambri (company)

Professor Donald Sadoway at the Massachusetts Institute of Technology has pioneered the research of liquid-metal rechargeable batteries, using both magnesium–antimony and more recently lead–antimony. The electrode and electrolyte layers are heated until they are liquid and self-segregate due to density and immiscibility. Such batteries may have longer lifetimes than conventional batteries, as the electrodes go through a cycle of creation and destruction during the charge–discharge cycle, which makes them immune to the degradation that afflicts conventional battery electrodes.

The technology was proposed in 2009 based on magnesium and antimony separated by a molten salt. Magnesium was chosen as the negative electrode for its low cost and low solubility in the molten-salt electrolyte. Antimony was selected as the positive electrode due to its low cost and higher anticipated discharge voltage.

In 2011, the researchers demonstrated a cell with a lithium anode and a lead–antimony cathode, which had higher ionic conductivity and lower melting points (350–430 °C). The drawback of the Li chemistry is higher cost. A Li/LiF + LiCl + LiI/Pb-Sb cell with about 0.9 V open-circuit potential operating at 450 °C had electroactive material costs of US$100/kWh and US$100/kW and a projected 25-year lifetime. Its discharge power at 1.1 A/cm² is only 44% (and 88% at 0.14 A/cm²).

Experimental data shows 69% storage efficiency, with good storage capacity (over 1000 mAh/cm²), low leakage (< 1 mA/cm²) and high maximal discharge capacity (over 200 mA/cm²). By October 2014 the MIT team achieved an operational efficiency of approximately 70% at high charge/discharge rates (275 mA/cm²), similar to that of pumped-storage hydroelectricity and higher efficiencies at lower currents. Tests showed that after 10 years of regular use, the system would retain about 85% of its initial capacity. In September 2014, a study described an arrangement using a molten alloy of lead and antimony for the positive electrode, liquid lithium for the negative electrode; and a molten mixture of lithium salts as the electrolyte.

A recent innovation is the PbBi alloy which enables lower melting point lithium-based battery. It uses a molten salt electrolyte based on LiCl-LiI and operates at 410 °C.

Ionic liquids have been shown to have prowess for use in rechargeable batteries. The electrolyte is pure molten salt with no added solvent, which is accomplished by using a salt having a room temperature liquid phase. This causes a highly viscous solution, and is typically made with structurally large salts with malleable lattice structures.

Thermal batteries (non-rechargeable)

Not to be confused with Thermal energy storage.

Technologies

Thermal batteries use an electrolyte that is solid and inactive at ambient temperatures. They can be stored indefinitely (over 50 years) yet provide full power in an instant when required. Once activated, they provide a burst of high power for a short period (a few tens of seconds to 60 minutes or more), with output ranging from watts to kilowatts. The high power is due to the high ionic conductivity of the molten salt (resulting in a low internal resistance), which is three orders of magnitude (or more) greater than that of the sulfuric acid in a lead–acid car battery.

One design uses a fuze strip (containing barium chromate and powdered zirconium metal in a ceramic paper) along the edge of the heat pellets to initiate the electrochemical reaction. The fuze strip is typically fired by an electrical igniter or squib which is activated with an electric current.

Another design uses a central hole in the middle of the battery stack, into which the high-energy electrical igniter fires a mixture of hot gases and incandescent particles. This allows much shorter activation times (tens of milliseconds) vs. hundreds of milliseconds for the edge-strip design. Battery activation can be accomplished by a percussion primer, similar to a shotgun shell. The heat source should be gasless. The standard heat source typically consists of mixtures of iron powder and potassium perchlorate in weight ratios of 88/12, 86/14, or 84/16. The higher the potassium perchlorate level, the higher the heat output (nominally 200, 259, and 297 cal/g respectively). This property of unactivated storage has the double benefit of avoiding deterioration of the active materials during storage and eliminating capacity loss due to self-discharge until the battery is activated.

In the 1980s lithium-alloy anodes replaced calcium or magnesium anodes, with cathodes of calcium chromate, vanadium or tungsten oxides. Lithium–silicon alloys are favored over the earlier lithium–aluminium alloys. The corresponding cathode for use with the lithium-alloy anodes is mainly iron disulfide (pyrite) replaced by cobalt disulfide for high-power applications. The electrolyte is normally a eutectic mixture of lithium chloride and potassium chloride.

More recently, other lower-melting, eutectic electrolytes based on lithium bromide, potassium bromide, and lithium chloride or lithium fluoride have also been used to provide longer operational lifetimes; they are also better conductors. The so-called "all-lithium" electrolyte based on lithium chloride, lithium bromide, and lithium fluoride (no potassium salts) is also used for high-power applications, because of its high ionic conductivity. A radioisotope thermal generator, such as in the form of pellets of ⁹⁰SrTiO₄, can be used for long-term delivery of heat for the battery after activation, keeping it in a molten state.

Uses

Thermal batteries are used almost exclusively for military applications, notably for nuclear weapons and guided missiles. They are the primary power source for many missiles such as the AIM-9 Sidewinder, AIM-54 Phoenix, MIM-104 Patriot, BGM-71 TOW, BGM-109 Tomahawk and others. In these batteries the electrolyte is immobilized when molten by a special grade of magnesium oxide that holds it in place by capillary action. This powdered mixture is pressed into pellets to form a separator between the anode and cathode of each cell in the battery stack. As long as the electrolyte (salt) is solid, the battery is inert and remains inactive. Each cell also contains a pyrotechnic heat source, which is used to heat the cell to the typical operating temperature of 400–550 °C.

Electron counting

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/Electron_counting

In chemistry, electron counting is a formalism for assigning a number of valence electrons to individual atoms in a molecule. It is used for classifying compounds and for explaining or predicting their electronic structure and bonding. Many rules in chemistry rely on electron-counting:

• Octet rule is used with Lewis structures for main group elements, especially the lighter ones such as carbon, nitrogen, and oxygen,
• 18-electron rule in inorganic chemistry and organometallic chemistry of transition metals,
• Hückel's rule for the π-electrons of aromatic compounds,
• Polyhedral skeletal electron pair theory for polyhedral cluster compounds, including transition metals and main group elements and mixtures thereof, such as boranes.

Atoms are called "electron-deficient" when they have too few electrons as compared to their respective rules, or "hypervalent" when they have too many electrons. Since these compounds tend to be more reactive than compounds that obey their rule, electron counting is an important tool for identifying the reactivity of molecules. While the counting formalism considers each atom separately, these individual atoms (with their hypothetical assigned charge) do not generally exist as free species.

Counting rules

Two methods of electron counting are "neutral counting" and "ionic counting". Both approaches give the same result (and can therefore be used to verify one's calculation).

• The neutral counting approach assumes the molecule or fragment being studied consists of purely covalent bonds. It was popularized by Malcolm Green along with the L and X ligand notation. It is usually considered easier especially for low-valent transition metals.

• The "ionic counting" approach assumes purely ionic bonds between atoms.

It is important, though, to be aware that most chemical species exist between the purely covalent and ionic extremes.

Neutral counting

• Neutral counting assumes each bond is equally split between two atoms.
• This method begins with locating the central atom on the periodic table and determining the number of its valence electrons. One counts valence electrons for main group elements differently from transition metals, which use d electron count.

E.g. in period 2: B, C, N, O, and F have 3, 4, 5, 6, and 7 valence electrons, respectively.

E.g. in period 4: K, Ca, Sc, Ti, V, Cr, Fe, Ni have 1, 2, 3, 4, 5, 6, 8, 10 valence electrons respectively.

• One is added for every halide or other anionic ligand which binds to the central atom through a sigma bond.
• Two is added for every lone pair bonding to the metal (e.g. each Lewis base binds with a lone pair). Unsaturated hydrocarbons such as alkenes and alkynes are considered Lewis bases. Similarly Lewis and Bronsted acids (protons) contribute nothing.
• One is added for each homoelement bond.
• One is added for each negative charge, and one is subtracted for each positive charge.

Ionic counting

• Ionic counting assumes unequal sharing of electrons in the bond. The more electronegative atom in the bond gains electron lost from the less electronegative atom.
• This method begins by calculating the number of electrons of the element, assuming an oxidation state.

E.g. for a Fe²⁺ has 6 electrons

S²⁻ has 8 electrons

• Two is added for every halide or other anionic ligand which binds to the metal through a sigma bond.
• Two is added for every lone pair bonding to the metal (e.g. each phosphine ligand can bind with a lone pair). Similarly Lewis and Bronsted acids (protons) contribute nothing.
• For unsaturated ligands such as alkenes, one electron is added for each carbon atom binding to the metal.

Electrons donated by common fragments

Ligand	Electrons contributed (neutral counting)	Electrons contributed (ionic counting)	Ionic equivalent
X	1	2	X−; X = F, Cl, Br, I
H	1	2	H−
H	1	0	H+
O	2	4	O2−
N	3	6	N3−
CO	2	2	CO
NR3	2	2	NR3; R = H, alkyl, aryl
CR2	2	4	CR2−2
Ethylene	2	2	C2H4
cyclopentadienyl	5	6	C5H−5
benzene	6	6	C6H6

"Special cases"

The numbers of electrons "donated" by some ligands depends on the geometry of the metal-ligand ensemble. An example of this complication is the M–NO entity. When this grouping is linear, the NO ligand is considered to be a three-electron ligand. When the M–NO subunit is strongly bent at N, the NO is treated as a pseudohalide and is thus a one electron (in the neutral counting approach). The situation is not very different from the η³ versus the η¹ allyl. Another unusual ligand from the electron counting perspective is sulfur dioxide.

Examples

• H₂O

For a water molecule (H₂O), using both neutral counting and ionic counting result in a total of 8 electrons.

This figure of the water molecule shows how the electrons are distributed with the covalent counting method. The red ones are the oxygen electrons, and the blue ones are electrons from the hydrogen atoms.

Neutral counting

Atom	Electrons contributed	Electron count
H.	1 electron x 2	2 electrons
O	6 electrons	6 electrons
		Total = 8 electrons

The neutral counting method assumes each OH bond is split equally (each atom gets one electron from the bond). Thus both hydrogen atoms have an electron count of one. The oxygen atom has 6 valence electrons. The total electron count is 8, which agrees with the octet rule.

This figure of the water molecule shows how the electrons are distributed with the ionic counting method. The red ones are the oxygen electrons, and the blue ones are electrons from hydrogen. All electrons in the OH bonds belong to the more electronegative oxygen.

Ionic counting

Atom	Electrons contributed	Electron count
H+	none	0 electron
O2-	8 electrons	8 electrons
		Total = 8 electrons

With the ionic counting method, the more electronegative oxygen will gain electrons donated by the two hydrogen atoms in the two OH bonds to become O^2-. It now has 8 total valence electrons, which obeys the octet rule.

• CH₄, for the central C

neutral counting: C contributes 4 electrons, each H radical contributes one each: 4 + 4 × 1 = 8 valence electrons

ionic counting: C⁴⁻ contributes 8 electrons, each proton contributes 0 each: 8 + 4 × 0 = 8 electrons.

Similar for H:

neutral counting: H contributes 1 electron, the C contributes 1 electron (the other 3 electrons of C are for the other 3 hydrogens in the molecule): 1 + 1 × 1 = 2 valence electrons.

ionic counting: H contributes 0 electrons (H⁺), C⁴⁻ contributes 2 electrons (per H), 0 + 1 × 2 = 2 valence electrons

conclusion: Methane follows the octet-rule for carbon, and the duet rule for hydrogen, and hence is expected to be a stable molecule (as we see from daily life)

• H₂S, for the central S

neutral counting: S contributes 6 electrons, each hydrogen radical contributes one each: 6 + 2 × 1 = 8 valence electrons

ionic counting: S²⁻ contributes 8 electrons, each proton contributes 0: 8 + 2 × 0 = 8 valence electrons

conclusion: with an octet electron count (on sulfur), we can anticipate that H₂S would be pseudo-tetrahedral if one considers the two lone pairs.

• SCl₂, for the central S

neutral counting: S contributes 6 electrons, each chlorine radical contributes one each: 6 + 2 × 1 = 8 valence electrons

ionic counting: S²⁺ contributes 4 electrons, each chloride anion contributes 2: 4 + 2 × 2 = 8 valence electrons

conclusion: see discussion for H₂S above. Both SCl₂ and H₂S follow the octet rule - the behavior of these molecules is however quite different.

• SF₆, for the central S

neutral counting: S contributes 6 electrons, each fluorine radical contributes one each: 6 + 6 × 1 = 12 valence electrons

ionic counting: S⁶⁺ contributes 0 electrons, each fluoride anion contributes 2: 0 + 6 × 2 = 12 valence electrons

conclusion: ionic counting indicates a molecule lacking lone pairs of electrons, therefore its structure will be octahedral, as predicted by VSEPR. One might conclude that this molecule would be highly reactive - but the opposite is true: SF₆ is inert, and it is widely used in industry because of this property.

• RuCl₂(bpy)₂

The geometry of cis-Dichlorobis(bipyridine)ruthenium(II).

RuCl₂(bpy)₂ is an octahedral metal complex with two bidentate 2,2′-Bipyridine (bpy) ligands and two chloride ligands.

Neutral counting

Metal/ligand	Electrons contributed	Electron count
Ru(0)	d8 (8 d electrons)	8 electrons
bpy	4 electrons x 2	8 electrons
Cl .	1 electron x 2	2 electrons
		Total = 18 electrons

In the neutral counting method, the Ruthenium of the complex is treated as Ru(0). It has 8 d electrons to contribute to the electron count. The two bpy ligands are L-type ligand neutral ligands, thus contributing two electrons each. The two chloride ligands halides and thus 1 electron donors, donating 1 electron each to the electron count. The total electron count of RuCl₂(bpy)₂ is 18.

Ionic counting

metal/ligand	electrons contributed	number of electrons
Ru(II)	d6 (6 d electrons)	6 electrons
bpy	4 electrons x 2	8 electrons
Cl−	2 electrons x 2	4 electrons
		Total = 18 electrons

In the ionic counting method, the Ruthenium of the complex is treated as Ru(II). It has 6 d electrons to contribute to the electron count. The two bpy ligands are L-type ligand neutral ligands, thus contributing two electrons each. The two chloride ligands are anionic ligands, thus donating 2 electrons each to the electron count. The total electron count of RuCl₂(bpy)₂ is 18, agreeing with the result of neural counting.

• TiCl₄, for the central Ti

neutral counting: Ti contributes 4 electrons, each chlorine radical contributes one each: 4 + 4 × 1 = 8 valence electrons

ionic counting: Ti⁴⁺ contributes 0 electrons, each chloride anion contributes two each: 0 + 4 × 2 = 8 valence electrons

conclusion: Having only 8e (vs. 18 possible), we can anticipate that TiCl₄ will be a good Lewis acid. Indeed, it reacts (in some cases violently) with water, alcohols, ethers, amines.

• Fe(CO)₅

neutral counting: Fe contributes 8 electrons, each CO contributes 2 each: 8 + 2 × 5 = 18 valence electrons

ionic counting: Fe(0) contributes 8 electrons, each CO contributes 2 each: 8 + 2 × 5 = 18 valence electrons

conclusions: this is a special case, where ionic counting is the same as neutral counting, all fragments being neutral. Since this is an 18-electron complex, it is expected to be isolable compound.

• Ferrocene, (C₅H₅)₂Fe, for the central Fe:

neutral counting: Fe contributes 8 electrons, the 2 cyclopentadienyl-rings contribute 5 each: 8 + 2 × 5 = 18 electrons

ionic counting: Fe²⁺ contributes 6 electrons, the two aromatic cyclopentadienyl rings contribute 6 each: 6 + 2 × 6 = 18 valence electrons on iron.

conclusion: Ferrocene is expected to be an isolable compound.