Prenatal stress heightened adult chronic pain

This 2019 McGill rodent study found:

Prenatal stress exacerbates pain after injury. Analysis of mRNA expression of genes related to epigenetic regulation and stress responses in the frontal cortex and hippocampus, brain structures implicated in chronic pain, showed distinct sex and region-specific patterns of dysregulation.

In general, mRNA expression was most frequently altered in the male hippocampus and effects of prenatal stress were more prevalent than effects of nerve injury. Recent studies investigating chronic pain-related pathology in the hippocampus in humans and in rodent models demonstrate functional abnormalities in the hippocampus, changes in associated behavior, and decreases in adult hippocampal neurogenesis.

The change in expression of epigenetic- and stress-related genes is not a consequence of nerve injury but rather precedes nerve injury, consistent with the hypothesis that it might play a causal role in modulating the phenotypic response to nerve injury. These findings demonstrate the impact of prenatal stress on behavioral sensitivity to a painful injury.

Decreased frontal mRNA expression of BDNF and BDNF IV in male offspring following neuropathic pain or prenatal stress respectively. Relative mRNA expression of other stress-related genes (GR17, FKBP5) and epigenetic-related genes (DNMTs, TETs, HDACs, MBDs, MeCP2) in male offspring.

A drastic decrease in expression of HDAC1 was observed in all groups compared to sham-control animals. CCI: chronic constriction injury.”


The study’s design was similar to the PRS (prenatal restraint stress) model, except that the PRS procedure covered gestational days 11 to 21 (birth):

“Prenatal stress was induced on Embryonic days 13 to 17 by restraining the pregnant dams in transparent cylinder with 5 mm water, under bright light exposure, 3 times per day for 45 min.”

None of the French, Italian, and Swiss PRS studies were cited.

The limitation section included:

  1. “Although our study shows significant changes in expression of epigenetic enzymes, it didn’t examine the impact of these changes on genes that are epigenetically regulated by this machinery or their involvement in intensifying pain responses.
  2. The current study is limited by the focus on changes in gene expression which do not necessarily correlate with changes in protein expression.
  3. Another limitation of this study is the inability to distinguish the direct effects of stress in utero vs. changes in the dam’s maternal behavior due to stress during pregnancy; cross-fostering studies are needed to address this issue.
  4. Functional experiments that involve up and down regulation of epigenetic enzymes in specific brain regions are required to establish a causal role for these processes in chronic pain.”

What do you think about possible human applicability of this study’s “effects of prenatal stress were more prevalent than effects of nerve injury” finding?

What professional would recognize that if a person’s mother was stressed while pregnant, a painful injury could cause less prevalent biological and behavioral effects than their prenatal experiences?

https://www.sciencedirect.com/science/article/pii/S0166432819315219 “Prenatal maternal stress is associated with increased sensitivity to neuropathic pain and sex-specific changes in supraspinal mRNA expression of epigenetic- and stress-related genes in adulthood” (not freely available)

An epigenetic clock review by committee

This 2019 worldwide review of epigenetic clocks was a semi-anonymous mishmash of opinions, facts, hypotheses, unwarranted extrapolations, and beliefs. The diversity of viewpoints among the 21 coauthors wasn’t evident.

1. Citations of the coauthors’ works seemed excessive, and they apologized for omissions. However, Challenge 5 was titled “Single-cell analysis of aging changes and disease” and Table 1 “Major biological and analytic issues with epigenetic DNA methylation clocks” had single-cell analysis as the Proposed solution to five of the Significant issues. Yet studies such as High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations were unmentioned.

2. Some coauthors semi-anonymously expressed faith that using current flawed methodologies in the future – only more thoroughly, with newer equipment, etc. – would yield better results. If the 21 coauthors were asked their viewpoints of Proposed solutions to the top three Significant issues of epigenetic clocks, what would they emphasize when quoted?

3. Techniques were praised:

“Given the precision with which DNA methylation clock age can be estimated and evolving measures of biological, phenotype-, and disease-related age (e.g., PhenoAge, GrimAge)..”

Exactly why these techniques have at times produced inexplicable results wasn’t examined, though. Two examples:

  • In Reversal of aging and immunosenescent trends, the Levine PhenoAge methodology estimated that the 51-65 year old subjects’ biological ages at the beginning of the study averaged 17.5 years less than their chronological age. Comparing that to the Horvath average biological age of 3.95 years less raised the question: exactly why did PhenoAge show such a large difference?
  • The paper mentioned the GrimAge methodology findings about “smoking-related changes.” But it didn’t explain why the GrimAge methylation findings most closely associated with smoking history also accurately predicted future disease risk with non-smokers.

Eluding explanations for these types of findings didn’t help build confidence in the methodologies.

4. A more readable approach to review by committee could have coauthors – in at least one section – answer discussion questions, as Reversing epigenetic T cell exhaustion did with 18 experts.

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1824-y “DNA methylation aging clocks: challenges and recommendations”

A review of fetal adverse events

This 2019 Australian review subject was fetal adversities:

“Adversity during the perinatal period is a significant risk factor for the development of neurodevelopmental disorders long after the causative event. Despite stemming from a variety of causes, perinatal compromise appears to have similar effects on the developing brain, thereby resulting in behavioural disorders of a similar nature.

These behavioural disorders occur in a sex‐dependent manner, with males affected more by externalizing behaviours such as attention deficit hyperactivity disorder (ADHD) and females by internalizing behaviours such as anxiety. The term ‘perinatal compromise’ serves as an umbrella term for intrauterine growth restriction, maternal immune activation, prenatal stress, early life stress, premature birth, placental dysfunction, and perinatal hypoxia.

The above conditions are associated with imbalanced excitatory-inhibitory pathways resulting from reduced GABAergic signalling. Methylation of the GAD1/GAD67 gene, which encodes the key glutamate‐to‐GABA synthesizing enzyme Glutamate Decarboxylase 1, resulting in increased levels of glutamate is one epigenetic mechanism that may account for a tendency towards excitation in disorders such as ADHD.

The posterior cerebellum’s role in higher executive functioning is becoming well established due to its connections with the prefrontal cortex, association cortices, and limbic system. It is now suggested that disruptions to cerebellar development, which can occur due to late gestation compromises such as preterm birth, can have a major impact on the region of the brain to which it projects.

Activation of the maternal hypothalamic-pituitary adrenal (HPA) axis and placental protection. Psychological stress is perceived by the maternal HPA axis, which stimulates cortisol release from the maternal adrenal gland.

High levels of maternal cortisol are normally prevented from reaching the fetus by the 11β-hydroxysteroid dehydrogenase 2 (HSD11B2) enzyme, which converts cortisol to the much less active cortisone. Under conditions of high maternal stress, this protective mechanism can be overwhelmed, with the gene encoding the enzyme becoming methylated, which reduces its expression allowing cortisol to cross the placenta and reach the fetus.”


The reviewers extrapolated many animal study findings to humans, although most of their own work was with guinea pigs. The “suggest” and “may” qualifiers were used often – 22 and 37 times, respectively. More frequent use of the “appears,” “hypothesize,” “propose,” and “possible” terms was justified.

As a result, many reviewed items such as the above graphic and caption should be viewed as hypothetical for humans rather than reflecting solid evidence from quality human studies.

The reviewers focused on the prenatal (before birth) period more than the perinatal (last trimester of pregnancy to one month after birth) period. There were fewer mentions of birth and early infancy adversities.

https://onlinelibrary.wiley.com/doi/abs/10.1111/jne.12814 “Perinatal compromise contributes to programming of GABAergic and Glutamatergic systems leading to long-term effects on offspring behaviour” (not freely available)

Organismal aging and cellular senescence

I’ll curate this 2019 German review through its figures:

“With the discovery of beneficial aspects of cellular senescence and evidence of senescence being not limited to replicative cellular states, a redefinition of our comprehension of aging and senescence appears scientifically overdue.

Figure 1. Current determinants and relevant open questions, marking the processes of aging and senescence as discussed in the text. Aspects represented in green are considered as broadly accepted or scientifically consolidated. Novel aspects that are yet unproven, or are under debate, are highlighted in red.

SASP = senescence-associated secretory phenotype. AASP = putative aging-associated secretory phenotype as suggested in the text.

Figure 2. Theories on the causality and purpose of aging. Graphically summarized are four contrasting concepts crystallized from current evidence addressing the inductive driving force of aging. Apart from a stochastic deleteriome, there are arguments for a pseudo-programmed, programmed or at least partially programmed nature of aging.

Figure 3. Comparative representation of the aging and senescence processes highlighting different levels of interaction and putative sites of interventions.

(1) As discussed in the text, causative mechanisms of aging are still not well understood, however, multiple factors including genetic, epigenetic and stress-related effects seem to have an orchestrated role in the progression of aging. Senescence on the other hand, is seen as a programmed response to different kinds of stressors, which proceed in defined stages. Whether, in analogy, aging also follows a defined program or sequential stages is not known.

(2) Senescence involves autocrine and paracrine factors, which are responsible for a ‘seno-infection’ or bystander effect in neighboring cells. There is currently no direct evidence for a similar factor composition propagating the aging process via a kind of ‘gero-infection’.

(3) Accumulation of senescent cells has been described as a hallmark of aging; however, whether they are a causative factor or a consequence of tissue and organismal aging is still unknown. As discussed in the text, it appears possible that aging and senescence mutually influence each other through positive feedback at this level, leading to accelerated tissue damage and aging.

(4,5) Clearance of senescent or aging cells might constitute putative targets for interventional approaches aimed to reduce or reverse the impact of aging and improve cell and tissue homeostasis by inducing a ‘rejuvenation’ process.

Figure 4. Pathological and beneficial functions of aging and senescence, according to current knowledge. In red are represented pathological consequences and in green beneficial functions of aging and senescence.

The impact of aging has mainly been described at the organismal level, since a complete cellular functional profile has not yet been established. Accordingly, whether beneficial consequences of the aging process exist at the cellular level is unclear.”


The reviewers’ position on Figure 2 was:

“In our view, recent evidence that senescence is based on an unterminated developmental growth program and the finding that the concept of post-mitotic senescence requires the activation of expansion, or ‘growth’ factors as a second hit, favor the assumption that aging underlies a grating of genetic determination similarly to what is summarized above under the pseudo-programmed causative approach.”

Their position on Figure 4’s beneficial effects of aging began with the sentence:

“If we assume that aging already starts before birth, it can be considered simply a developmental stage, required to complete the evolutionary program associated with species-intrinsic biological functions such as reproduction, survival, and selection.”

Cited studies included:

https://www.mdpi.com/2073-4409/8/11/1446 “Dissecting Aging and Senescence-Current Concepts and Open Lessons”

Epigenetic transgenerational inheritance extends to the great-great-grand offspring

This 2019 rodent study by the Washington State University labs of Dr. Michael Skinner continued to F4 generation great-great-grand offspring, and demonstrated that epigenetic inheritance mechanisms are similar to imprinted genes:

“Epigenetic transgenerational inheritance potentially impacts disease etiology, phenotypic variation, and evolution. An increasing number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease.

Imprinted genes are a special class of genes since their DNA methylation patterns are unchanged over the generation and are not affected by the methylation erasure occurring early in development. The transgenerational epigenetic alterations in the germline appear to be permanently reprogrammed like imprinted genes, and appear protected from this DNA methylation erasure and reprogramming at fertilization in the subsequent generations. Similar to imprinted genes, the epigenetic transgenerational germline epimutations appear to have a methylation erasure in the primordial germ cells involving an epigenetic molecular memory.

Comparison of the transgenerational F3 generation, with the outcross to the F4 generation through the paternal or maternal lineages, allows an assessment of parent-of-origin transmission of disease or pathology. Observations provided examples of the following:

  1. Pathology that required combined contribution of both paternal and maternal alleles to promote disease [testis and ovarian disease];
  2. Pathology that is derived from the opposite sex allele such as father to daughter [kidney disease] or mother to son [prostate disease];
  3. Pathology that is derived from either parent-of-origin alleles independently [obesity];
  4. Pathology that is transmitted within the same sex, such as maternal to daughter [mammary tumor development]; and
  5. Pathology that is observed only following a specific parent-of-origin outcross [both F4 male obesity and F4 female kidney disease in the vinclozolin lineage].”

The study showed that epigenetically inherited legacies extend to the fifth generation. Do any of us know our ancestors’ medical histories back to our great-great-grandparents?

Will toxicologists take their jobs seriously enough to look for possible effects in at least one generation that had no direct toxicant exposure?

https://www.sciencedirect.com/science/article/pii/S0012160619303471 “Epigenetic transgenerational inheritance of parent-of-origin allelic transmission of outcross pathology and sperm epimutations”

Reversing epigenetic T cell exhaustion

This 2019 worldwide discussion among 18 experts concerned T cell exhaustion:

“‘T cell exhaustion’ is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours.

Key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations.”


There were nearly a dozen viewpoints on “What do we mean by T cell exhaustion and/or dysfunction and how would you define this state?” 🙂

Answers to the question “What are the key controversies and outstanding research questions?” included:

  • “What are the cellular signalling and transcriptional pathways that drive the conversion to an exhausted T cell phenotype, and how can the chromatin and transcriptional changes of exhaustion be reversed in individual exhausted cells?
  • Whether and how we can manipulate signalling pathways to both activate and maintain T cell responses remain open questions, as does the question of whether pharmacological manipulations can reverse the epigenetic changes associated with exhaustion versus expand less-exhausted populations.
  • We need to define better the effects of the microenvironment on the induction of T cell exhaustion, the developmental trajectories of exhaustion and the point at which and extent to which exhaustion can be reversed. Understanding the consequences of unleashing T cells from exhaustion will also be crucial to designing the most effective therapeutic interventions.
  • When and how exhausted T cell populations are formed. The original view that they are terminally differentiated descendants of formerly ‘normal’ effector T cells has been challenged.
  • Whether the predysfunctional T cells themselves, or their more differentiated (and phenotypically dysfunctional) progeny, form the ultimate effector pool for control of human tumours.
  • How do the functions and states (subpopulations) of exhausted T cells change over time? Can the epigenetic state of exhaustion be reversed to form true effector or memory T cells, and is this required for improved cancer immunotherapy?
  • There is no definitive marker for exhausted T cells, although TOX may prove to be useful. Transcriptional profiles are informative, but epigenetic changes are more specific and robust. A major clinical question is whether exhausted T cells can be, or indeed need to be, reprogrammed to achieve therapeutic benefit.”

https://www.nature.com/articles/s41577-019-0221-9 “Defining ‘T cell exhaustion'” (not freely available)

Get outside today

This 2019 Finnish review focused on vitamin D’s immune system effects:

“The epigenome of human monocytes is at multiple levels sensitive to vitamin D. These data served as the basis for the chromatin model of vitamin D signaling, which mechanistically explains the activation of a few hundred primary vitamin D target genes.

Vitamin D and its receptor are able to antagonize the pro-inflammatory actions of the transcription factors nuclear factor activated T cells (NF-AT) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) in T cells. In this way, vitamin D reduces autoimmunity, such as the onset and progression of multiple sclerosis, as well as chronic inflammation.

Population-wide recommendations do not take inter-individual variations into account, such as a different molecular response to vitamin D, which are expressed by the vitamin D response index. Instead of population-based recommendations for vitamin D3 supplementation there should be personalized recommendations in order to reach a vitamin D status that is optimized for an individual’s health protection.

Trained immunity implies that immune cells memorize challenges, to which they are exposed in their rather short lifespan, in form of changes of their epigenome leading to subtype specification. The stabilization of the epigenomes of the subtypes of monocytes, macrophages and dendritic cells by vitamin D can prevent or delay the onset of common age-related diseases.”


One of the five elements of the clinical trial Reversal of aging and immunosenescent trends was daily 3,000 IU vitamin D3 supplementation for nine months. That study’s monocyte findings included:

“Analysis of CyTOF‐defined immune cell populations revealed the most robust changes to be decreases in total and CD38‐positive monocytes and resulting increases in the lymphocyte‐to‐monocyte ratio (LMR). The changes in mean monocyte populations persisted 6 months after discontinuation of treatment, and the increase in LMR remained highly significant at 18 months as well.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753645/ “Vitamin D Signaling in the Context of Innate Immunity: Focus on Human Monocytes”