Very few objective scar evaluations have been conducted with the burn survivor population, which limits our knowledge of the clinical recovery profile of hypertrophic scars (HSc) and donor site scars (D), thus having an impact on rehabilitation intervention and treatment prioritization. The purpose of this study was to prospectively quantify the thickness, pliability, erythema and pigmentation of post-burn HSc, donor sites  and normal skin in different anatomical locations between 2 and 7 months post-burn using objective instrumentation. A secondary objective was to compare this data with anatomic-specific normative data using the same objective instrumentation.Skin characteristics of HSc, D and N in 44 burn survivors were measured at 2, 3, 4, 5, 6 and 7 months post-burn using validated instrumentation: high-frequency ultrasound for thickness, Cutometer® to measure pliability and Mexameter® to measure erythema and pigmentation. Up to five sites were assessed on the same participant if their scar was located on the upper extremity (UE), lower extremity (LE) and trunk. A mixed model two-way analysis of variance was used to investigate the differences in means between sites at each time point and between time points at each site.The results revealed that HSc sites were thicker than D and N at all time points and UE and trunk HSc were thicker than LE HSc at 7 months post-burn, pliability of trunk HSc did not improve over time, and UE HSc was more erythematous at 7 months compared to other anatomical sites whereas D erythema decreases from 2 to 7 months.Scar management treatments should prioritize the UE and trunk sites which developed HSc during the first two months post-burn and continues to vary significantly from normal scar and normal skin at 7 months. Furthermore, these results provide preliminary evidence that the recovery profile of HSc varies at different anatomical sites and that thickness is the characteristic that distinguishes HSc from normal scar and normal skin.


Meta-analyses of randomized controlled trials (RCTs) have estimated a 13% reduction of cancer mortality by vitamin D supplementation among older adults. We evaluated if and to what extent similar effects might be expected from vitamin D fortification of foods. We reviewed the literature on RCTs assessing the impact of vitamin D supplementation on cancer mortality, on increases of vitamin D levels by either supplementation or food fortification, and on costs of supplementation or fortification. Then, we derived expected effects on total cancer mortality and related costs and savings from potential implementation of vitamin D food fortification in Germany and compared the results to those for supplementation. In RCTs with vitamin D supplementation in average doses of 820–2000 IU per day, serum concentrations of 25-hydroxy-vitamin D increased by 15–30 nmol/L, respectively. Studies on food fortification found increases by 10–42 nmol/L, thus largely in the range of increases previously demonstrated by supplementation. Fortification is estimated to be considerably less expensive than supplementation. It might be similarly effective as supplementation in reducing cancer mortality and might even achieve such reduction at substantially larger net savings. Although vitamin D overdoses are unlikely in food fortification programs, implementation should be accompanied by a study monitoring the frequency of potentially occurring adverse effects by overdoses, such as hypercalcemia. Future studies on effectiveness of vitamin D supplementation and fortification are warranted. Recent meta-analyses of randomized clinical trials have consistently suggested a reduction of cancer mortality by approximately 13% through vitamin D supplementation . We prevIn order to address these questions, we reviewed the literature on increases of vitamin D levels by vitamin supplementation, paying particular attention to such increases in the RCTs demonstrating a reduction in cancer mortality, the literature on increases in vitamin D levels by vitamin D food fortification, and the literature on costs of vitamin D fortification. Potential adverse effects of fortification by vitamin D overdoses will also be discussed. Finally, we used the results to compare those costs to potential savings from reduced cancer mortality, taking cancer mortality data from Germany as an example.We searched PubMed from inception to February 2021 for studies on the effect of vitamin D supplementation and cancer mortality as well as on the effect of vitamin D supplementation and of vitamin D food fortification (with at least 4 weeks of intervention) on serum 25-hydroxy-vitamin D (25(OH)D) levels, the most commonly employed measure of vitamin D status. The search terms are listed in the Supplement. Non-English-language articles, comments, correspondences, and studies not reporting sufficient details on the key parameters of interest  were disregarded. We used data from identified recent meta-analyses where possible.Three meta-analyses of RCTs on vitamin D supplementation and cancer mortality were published in 2019 , all of Regarding the intervention effects of supplementation on serum levels of 25(OH)D, we used data from a meta-analysis of randomized trials on vitamin D (D2 or D3) supplementation in mainly Caucasian subjects over >50 years by Autier et al. , identifFor the effects of vitamin D fortified food on serum 25(OH)D levels, we selected and extracted data from published studies identified by the literature search with at least 4 weeks of intervention .For estimated costs of food fortification in Germany, we used data from Sandmann et al. . They asWe focused on savings from prevented cancer deaths and disregarded the positive impact of vitamin D on various other health outcomes, such as osteoporosis (fracture costs). In addition to preventable cancer deaths, we calculated life-years saved as preventable years of life lost (preventable YLL) and monetary savings from prevented cancer deaths as described previously elsewhere . In brieIn the two RCTs reporting on the effects of supplementation on serum levels, increases ranged from 14.6 to 30 nmol/L. Elevations of serum 25(OH)D levels with vitamin D supplementation have been consistently found in numerous further studies. In a meta-analysis among 6207 participants , median We extracted information on the effects of intake of fortified foods on serum vitamin D levels from 10 articles ,33,34,35Data on uptake of vitamin D from fortified foods and expected effects on vitamin D inadequacy were extracted from seven studies ,38,39,40Cancer care-related savings in the most conservative scenario would amount to more than one billion € per year if fortification reduces cancer mortality similarly as supplementation with 400 IU/day. Given the comparably low costs of fortification, this would imply net savings of almost 1 billion € per year . AssuminHigher levels of vitamin D have consistently shown to be associated with lower cancer mortality , and recFortification resulted in serum increases of approximately 20 nmol/L in most studies and even up to 48 nmol/L in one study. This is comparable to the effect of daily intake of approximately 400 (up to ~800) IU vitamin D by supplements, as demonstrated in a range of intervention studies . As indiObservational studies from Canada  and FinlDifferences were also apparent in serum increases between studies focusing on supplementation and serum levels compared to the supplementation trials focusing on cancer mortality outcomes. The former found expected serum increases by 40 and 100 nmol/L for supplementation of 800 and 2000 IU/day, respectively. The latter found serum increases of only 15 and 30 nmol/L for such doses. Potential explanations might include unobserved confounders, such as differences in baseline levels, variations in trial durations (accumulation of vitamin D), differences in compliance, and differences in the extent of “contamination” of the control groups.Despite such uncertainties, our calculations suggest that food fortification with vitamin D would likely have a high impact in reducing cancer mortality. Recent meta-analyses of randomized clinical trials demonstrated cancer mortality reductions with a daily intake of vitamin D (400 to 2000 IU/day) in the range of 11% to 17% (13% on average) ,11,13. TComparing costs with the assumed benefits in terms of reduced cancer mortality alone, vitamin D fortification is expected to be highly cost saving, even much more so than population-wide or targeted daily supplementation. According to the current analysis, costs of food fortification would be approximately 95% lower compared to previously estimated costs of supplementation of the population aged 50+ 3). Even when entirely disregarding savings from reduced cancer mortality, fortification would still be expected to be highly cost-effective, with costs of only approximately 50 € per life-year saved. Of note, expected savings are entirely attributable to reductions in cancer mortality and not in cancer incidence, which is not affected by vitamin D. End-stage treatment costs among patients dying from cancer are, however, more than four-fold higher than among deaths from other causes , implyin. Even whOn the other hand, when implementing a wide-spread vitamin D fortification program, one needs to be aware of the possibility that small parts of the population may consume too much vitamin D, potentially leading to hypercalcemia and -uria by overdose . A meta-To our knowledge, no previous study comprehensively assessed the potential of vitamin D fortification of foods with respect to cancer mortality. When evaluating health impacts of vitamin D fortification of foods, other potential health effects of vitamin D also need to be considered : apart fStrengths of this study include the comprehensive literature search to assess the effect of vitamin D supplementation on serum levels and cancer mortality, on the effect of fortification of foods on serum levels, and on costs of fortification. Given the RCT-based evidence of supplementation on cancer mortality and the strong evidence on effects of fortification on serum levels being comparable to effects of supplementation, it appears plausible to assume that food fortification with vitamin D might have effects on cancer mortality comparable to supplementation.Limitations include the lack of direct evidence on the effect of food fortification on cancer mortality, heterogeneity of studies on the effects of supplementation ; uncertainty of end-of-life cancer care costs; heterogeneity of cancer-care costs regarding cancer type, stage, etc.; and lack of RCTs conducted directly in the German population. However, the prevalence of vitamin D deficiency in Germany is higher than in the U.S. ,26,56, fFuture well-designed studies of vitamin D fortification effects and of supplementation and cancer mortality are needed to assess the impact of supplementation and food fortification on serum levels and prevalence of deficiency in greater detail. Ideally, further RCTs of vitamin D supplementation and cancer-related mortality, paying particular attention to those cancers which contribute most to cancer mortality, should be undertaken, in particular in countries with widespread vitamin D insufficiency and deficiency, such as Germany. To study effects of fortification, a natural experiment design is conceivable in which serum levels of a country before and after introduction of fortification are compared along with a neighboring country with no change in policy as the comparator group. Additionally, the optimal amount of vitamin D added to foods needs to be carefully assessed in future studies. This includes not only the absolute amount of vitamin D added per 100 g but also the choice of foods, ideally considering both dietary habits of the population and potential losses of vitamin D during processing at a manufacturing or consumer level . Furthermore, future studies should aim for more comprehensive modeling of expected health outcomes and related costs beyond cancer.In summary, this study suggests that vitamin D food fortification would be expected to elevate vitamin D serum levels to an extent that would be comparable to daily supplementation with 400–1000 IU, which has been demonstrated to lower cancer mortality by 11–15%. Savings from cancer prevented end-of-life cancer care costs alone would outweigh fortification costs more than 50-fold. Food fortification with vitamin D could thus be a particularly effective and economic approach to lower the increasing burden of cancer encountered by many countries , and its


The peripheral nervous system comprises an infinity of neural networks that act in the communication between the central nervous system and the most diverse tissues of the body. Along with the extension of the primary sensory neurons (axons and cell bodies), a population of resident macrophages has been described. These newly called sensory neuron–associated macrophages (sNAMs) seem to play an essential role in physiological and pathophysiological processes, including infection, autoimmunity, nerve degeneration/regeneration, and chronic neuropathic pain. After different types of peripheral nerve injury, there is an increase in the number and activation of sNAMs in the sciatic nerve and sensory ganglia. The activation of sNAMs and their participation in neuropathic pain development depends on the stimulation of pattern recognition receptors such as Toll-like receptors and Nod-like receptors, chemokines/cytokines, and microRNAs. On activation, sNAMs trigger the production of critical inflammatory mediators such as proinflammatory cytokines  and reactive oxygen species that can act in the amplification of primary sensory neurons sensitization. On the other hand, there is evidence that sNAMs can produce antinociceptive mediators  that counteract neuropathic pain development. This review will present the cellular and molecular mechanisms behind the participation of sNAMs in peripheral nerve injury–induced neuropathic pain development. Understanding how sNAMs are activated and responding to nerve injury can help set novel targets for the control of neuropathic pain. Sensory neuron–associated macrophages (sNAMs) are involved in the pathophysiology of neuropathic pain through the production of proinflammatory and pronociceptive mediators.Sensory neuron–associated macrophages are activated in the sensory ganglia after peripheral nerve injury mainly by PRRs and chemokines.Sensory neuron–associated macrophages also produce anti-inflammatory mediators that counteract neuropathic pain development such as IL-10.Understanding the interactions between injured sensory neurons and sNAMs can provide novel targets for neuropathic pain control.202 In other words, pain is a personal complex experience that includes the conscious perception of a stimulus capable of generating tissue damage and may further depend on cognitive and emotional components.224 Whereas acute pain has a protective function to avoid potential damage, chronic pain can be maladaptive and pathological, and it represents one of the most prevalent and disabling health conditions in modern society.202 Neuropathic pain is a type of chronic pain characterized by injury or disease that directly affects the somatosensory nervous system, including peripheral fibers and central neurons.202 Epidemiological studies estimate that this pathology affects an average of 7% to 10% of the general population, being one of the most prevalent health problem.190 The mechanisms involved in the development and maintenance of neuropathic pain were initially characterized as a neuronal dysfunction. Indeed, after nerve injury, a series of modifications occur across the pain pathway that includes alterations in ion channel expression and function, upregulation of neurotransmitters, and their receptors, which leads to a state of neuronal hyperexcitability.205According to the current IASP definition, pain can be defined as “an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage.”240 These cells may interact with neuronal cell bodies and their fibers distributed throughout the peripheral and central nervous system (CNS) in both pathological or homeostatic conditions. In the case of neuropathic pain, when nerve integrity is affected, immune/glial cells, which may be resident or recruited to the injured tissue, and distally to the sensory ganglia and spinal cord, are activated and release inflammatory mediators that strongly modify neuronal function, culminating in alterations of painful perception.236 Among the immune/glial cells, macrophages emerge as one of the most important cell subpopulations involved in neuroimmune interactions associated with neuropathic pain.225 This review discusses the current evidence regarding the cellular and molecular interactions between primary sensory neurons and resident macrophages associated with these peripheral neurons, known as sensory neuron–associated macrophages (sNAMs), that might play a crucial role in the development of neuropathic pain. The role of infiltrating monocytes in the site of nerve injury is also discussed. Finally, we pointed out additional mechanisms by which peripheral macrophages may also counteract neuropathic pain development.Currently, there is a growing body of evidence indicating that the cause of neuropathic pain is not restricted to changes in neuronal activity but may involve a network of interaction among neurons, glial, and immune cells.240 Although some characteristics and functions are shared among different macrophage populations, such as homeostasis maintenance and tissue protection, these cells exhibit high functional plasticity and thus have several specialized functions in each different niche/tissue.166Tissue-resident macrophage populations are present in a variety of organs across the body.181 and ontogenies of these cell populations.56 It was known that monocytes newly released from bone marrow colonize various tissues, and once mature, they may become resident macrophages with specific features. It is currently accepted that most cells in the hematopoietic compartment are regularly renewed from adult hematopoietic stem cells (HSCs); however, recent findings demonstrate that resident macrophages can self-maintain independently of HSCs because they may have an embryonic origin. In this scenario, it is known that, at least in mice, tissue macrophages are derived from 3 different developmental sources.210 Macrophages firstly appear in the yolk sac (YS) during initial fetal development without monocytic intermediates and then colonize various embryonic tissues.179 In the embryonic period 8.5 (E8.5), macrophage precursors from the YS and HSCs migrate to the fetal liver and give rise to the first monocyte cells in E12.5.142 After birth, HSC in the bone marrow produces Ly6C+ monocytes, which can migrate to different tissues and differentiate into macrophages.220Historically, distinct subpopulations of macrophages have been defined according to the anatomical location and surface markers; however, this definition has been recently expanded to subset-specific gene expression signatures63 proposed that macrophages of the liver, lung, and epidermis are originated from YS-derived erythro-myeloid progenitors. The CNS also has resident macrophages with specific characteristics, including self-sustainability and proliferation. Besides microglia, meningeal, perivascular, and choroid plexus macrophages are considered CNS interface cells that appear to be derived from the YS, demonstrating that different populations of CNS macrophages share similar ontogeny.62Based on this, some groups have performed extensive characterizations of resident macrophages in the most diverse tissues, based not only on the anatomical location and profile of phenotypic markers but also on the transcriptional and ontogeny profile. In this sense, Gomez Perdiguero et al.113 These macrophages are distributed in the large peripheral nervous system interaction network and comprise one of the most important populations of myeloid cells associated with peripheral nervous tissue. For instance, in the rat, sciatic nerve macrophages constitute 1% to 4% of the total cell population.162 Conceptually, the term NAMs defines the subset of resident tissue macrophages that are closely associated with peripheral nerves in the most diverse tissues113 and can be characterized by the type of tissue and nerve in which they reside, origin, and self-renewal characteristic. The identification of macrophages in peripheral nerves occurred many years ago. In a pioneering study by Arvidson10 when examining the sciatic nerve of animals after the systemic injection of horseradish peroxidase, an enzymatic tracer that is widely distributed in most tissues, he observed through electron microscopy, cells with similar ultrastructural characteristics macrophages and located close to the epineurial and endoneurial. Later, Gehrmann et al.55 were able to demonstrate the presence of macrophages in the sciatic nerve and the dorsal root ganglions (DRGs), where the cellular bodies of sensory neurons are located. They confirmed the presence of macrophages in the DRGs by evaluating the expression of classic cell markers, such as CR3 and MHC-II, by immunohistochemistry reaction. Despite these data, only recently, sNAMs broad characterization was performed. Importantly, it was found that sNAMs from different neuronal compartments  are mostly self-maintained in adult mice.219 Contrary, ontology analysis of sNAMs of the sciatic nerves revealed they are predominantly from late embryonic precursors that are slowly replaced by bone marrow–derived monocytes.231 Therefore, further studies are important to finally define the origin of distinct sNAMs from different neural niches. Transcriptome analysis also revealed that sNAMs share some characteristics with activated microglia. However, sNAMs-specific genes were also identified, including genes related to angiogenesis, collagen fibril organization, and peripheral nerve structural organization and axon guidance.219 This specific transcriptional profile of sNAMs is in line with their possible role in axon sprouting after peripheral nerve injury.112 Besides that, the participation of sNAMs in the pathophysiology of neuropathic pain has been extensively studied, and these studies will be discussed below.In addition to the macrophages residing in the CNS, peripheral nerves also contain resident macrophages.221 Models of peripheral nerve injury are widely used to mimic neuropathic pain and most of the common clinical characteristics of this pathology. The development of neuropathic pain models has been fundamental for characterizing pathophysiological mechanisms and has shed new light on the preclinical evaluation of potential therapeutic interventions.119Neuropathic pain, the focus of this review, can occur because of several stressors, such as viral infections, diabetic neuropathy, mechanical trauma, neurotoxic chemicals, spinal cord injury, stroke, and multiple sclerosis.43 It is currently well accepted that the interactions of immune and glial cells within the peripheral nervous system and CNS regulate neuronal excitability and sensitize the pain pathway.155 When nerve integrity is disrupted, neuroimmune interactions occur early in the local of injury and register the initial trigger for neuropathic pain development. Resident cells, including Schwann cells and sNAMs, are responsible for the production of earlier inflammatory mediators that mediate the recruitment of immune cells to the injured nerve.47 After a peripheral nerve trauma, initial recruitment of neutrophils occurs, followed by the infiltration of inflammatory CCR2+ monocytes, which might be important to amplify the immune response.93 Studies show that systemic treatment with chemotherapy drugs, which are well known as neurotoxic, promoted an increase in the number of CX3CR1+ and CCR2+ macrophages/monocytes in the peripheral nerves.146 Several studies suggested that these locally activated macrophages are directly associated with a significant increase in the levels of inflammatory mediators, which sensitize primary afferent neurons and contribute to the development of neuropathic pain.236 For instance, Cx3cr1-deficient or Ccr2-deficient showed delayed development of mechanical hypersensitivity caused by the treatment with chemotherapy drug vincristine.146 Although resident and infiltrated macrophages/monocytes at the site of nerve injury are considered essential for the development of neuropathic pain, most of the studies that claimed this possibility lack specific tools targeting only these cells to confirm this hypothesis. Given these methodological limitations, many efforts have been made to develop specific tools to precisely manipulate peripheral (resident and infiltrating) vs central 45.The injury of primary afferent neurons conducts these cells to a hyperexcitability state. Thus, the action potentials generated at their endings are easily carried forward to the second-order neurons in the dorsal horn of the spinal cord and later to supraspinal brain regions, where the painful sensation is processed and interpreted.24 In these mice, Fas ligand administration drives the death of CSF1R+ cells. Unlike CSFR1 selective antagonists, this drug fails to cross the blood–brain barrier, ensuring higher peripheral macrophage specificity. By taking advantage of MAFIA mice, Shepherd et al.183 showed alleviation of mechanical pain hypersensitivity caused by peripheral nerve injury. The authors implicated the reduction in infiltrated monocytes as responsible for the MAFIA mouse pain phenotype.183 On the other hand, more recently, it was shown that specific depletion of macrophages/monocytes at the site of nerve injury did not affect the development of neuropathic pain, excluding any participation of macrophages/monocytes in the local of nerve injury for the development of neuropathic pain.235 Thus, although the systemic depletion of peripheral macrophages/monocytes reduces neuropathic pain development,193 it is likely that these cells could be acting in tissues different from the local nerve injury.One of those promising examples is a recently described mouse strain in which the suicidal gene Fas is under the control of the colony-stimulating factor 1 receptor (CSF1R) promoter, called macrophage-induced fas-apoptosis (MAFIA).238 Generally, the number of macrophages peaks form 5 to 10 days after sciatic nerve injury, retracting afterward.200 In chemotherapy-induced peripheral neuropathy, an accumulation of macrophages in the DRGs was also observed by some groups, whereas others did not observe any change.238 Although the reasons for this discrepancy are not immediately apparent, it could be related to differences in the doses of the chemotherapy drug used, schedules of treatment, and evaluated time points. There is another debate regarding whether the accumulation of macrophages in the sensory ganglia after peripheral nerve injury is due to the infiltration of blood monocytes or the local proliferation of sNAMs. Thus, further studies will also be necessary to clarify this point.Besides the peripheral nerves resident sNAMs, as we mentioned above, there are also resident sNAMs in the sensory ganglia (DRGs and TGs). The injury of peripheral nerves promotes several changes at the level of sensory ganglia, including a neuroinflammatory process characterized by activation/proliferation of glial cells , and sNAMs. Early studies using different sciatic nerve trauma models described an increase in the number of macrophages/monocytes around the cell body of sensory neurons in the sensory ganglia in a time-dependent manner.117 A combination of genetic and pharmacological tools for conditional depletion of peripheral sNAMs/monocytes and microglia also prevented the development of pain hypersensitivity in a mouse model of spinal nerve transection.165 Targeting peripheral macrophage and microglia with CSFR1 inhibitor, a receptor involved in survival, proliferation, and differentiation of macrophages in different tissues, also reduced neuropathic pain caused by peripheral nerve injury.121 In addition, the clodronate-induced killing of sensory ganglia macrophages reduced neuropathic pain development caused by peripheral nerve injury (trauma) and chemotherapy (paclitaxel).238 Noteworthy, none of these treatments are selective for sNAMs in the sensory ganglia and also target infiltrating monocytes and/or microglia. Based on that, the same study that ruled out the contribution of nerve injury–infiltrating macrophages for the development of neuropathic pain provided evidence that sNAMs in the sensory ganglia play a critical role in this condition.235 However, we could not discard those peripheral monocytes could be acting in additional sites than the local of nerve injury. For instance, we recently found that after peripheral nerve injury, CCR2+ monocytes become adhered to the endothelial cells of the spinal cord microcirculation, and these cells could also have a role in central mechanisms of neuropathic painTo dissect the participation of sNAMs in the sensory ganglia for the development of neuropathic pain, some strategies were applied. The intrathecal administration of minocycline reduced the number of sNAMs in the DRG after peripheral nerve injury, which was accompanied by the downregulation of inflammatory mediators reflecting on the reduction of mechanical pain hypersensitivity.As we mentioned above, after peripheral nerve injury, the activation/accumulation of sNAMs in the sensory ganglia (DRGs) seems to play an essential role in the development of neuropathic pain. Although it is not totally clear how the peripheral nerve injury leads to the distal activation/accumulation of sNAMs in the sensory ganglia, some possible mechanisms have been proposed.233 The large family of TLRs plays a critical role in immune responses by the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs),2 such as heat shock proteins, necrotic cells, and extracellular matrix components.239 Continuous activation or dysregulation of TLRs signaling may contribute to chronic disease states and have been involved in the pathogenesis of neuroinflammation, including in neuropathic pain development.222 In this sense, several studies have indicated that the activation/proliferation of microglia in the spinal cord, after peripheral nerve injury, that accounts for neuropathic pain development might depend on TLRs stimulation.132 For instance, Shi et al. demonstrated that spinal cord microglia activation after peripheral nerve injury depends on an unidentified endogenous ligand of TLR2 derived from damaged peripheral nerves.184 More recently, it was found that after peripheral nerve injury, GT1b ganglioside is axonally transported from the cell body of sensory neurons into the spinal cord and mediates neuropathic pain development through the activation of TLR2.126Like classical immune cells, macrophages can express different innate immunity receptors, such as Toll-like receptors (TLRs) and nucleotide-binding cytoplasmic oligomerization (NLRs) receptors.108 This effect seems to be related to a decrease in the production of CCL2 in the sensory ganglia, which is a crucial chemokine in macrophages activation/infiltration.108 Although a direct TLR2 activation of sNAMs in the sensory ganglia may likely occur after peripheral nerve injury, we could not discard an indirect activation because TLR2 seems to be also expressed on different cells  of the sensory ganglia.108 Furthermore, there is evidence that TLR2 deficiency also reduced macrophages' infiltration at the nerve injury site,184 which could also indirectly affect neuroinflammation in the DRGs.Regarding the role of TLRs in sNAMs activation, TLR2 null mice also showed a reduction in the activation/accumulation of sNAMs in the sensory ganglia.132 Earlier studies have shown that TLR4 deficient mice are protected from peripheral nerve injury–induced neuropathic pain.206 This effect was attributed to reducing microglia activation in the spinal cord.197 However, no one has evaluated the impact of TLR4 deficiency in sNAMs activation in the sensory ganglia in models of traumatic peripheral nerve injury. There is also evidence that TLR4 mediates chemotherapy-induced peripheral neuropathic pain .230 In paclitaxel-induced neuropathic pain, the blockage of TLR4 reduced the accumulation of macrophages in sensory ganglia.237Another pattern recognition receptor (PRR) which has been described as important for neuropathic pain development is TLR4.237 Activation of SGCs by paclitaxel in a TLR4-dependent manner and the consequent production of proinflammatory cytokines have also been suggested as a possible mechanism involved in neuropathic pain development.226 Although the contribution of TLR4 in sensory ganglia sNAMs for paclitaxel-induced neuropathic pain has been not investigated, it could also be an alternative. In this context, it was recently found that DRGs sNAMs-expressing TLR4 mediates the development of oxaliplatin-induced neuropathic pain.180 A recent study also revealed a role for TLR9 signaling in the pathophysiology of paclitaxel-induced neuropathic pain.137 In fact, Luo and collaborators demonstrated that paclitaxel-induced neuropathic pain was impaired in TLR9 KO mice and by the intrathecal administration of a TLR9 selective antagonist. Pain hypersensitivity was also mimicked by intraplantar and intrathecal injection of a TLR9 agonist (ODN 1826). Notably, TLR9 was found in DRG sNAMs and seemed to involve the induction of proinflammatory factors, such as cytokines.Nevertheless, this was assumed as a direct effect of paclitaxel on the activation of TLR4 expressed in sensory neurons, which in turn increased the production of the macrophages chemotactic factor, CCL2.37 An important example of a receptor in this family is the nucleotide-binding oligomerization domain 2 (NOD2). Some studies indicate that microglial cells express NOD2,194 suggesting a possible role of this receptor as an innate immune sensor in the CNS. It is well established that NOD2 and TLRs act in macrophages' activation, leading to positive pressure in the proinflammatory pathways.72 We recently demonstrated that after peripheral nerve injury, the NOD2 expression is upregulated in sNAMs of the sensory ganglia.175 Using genetic inhibition of NOD2, we showed that NOD2 signaling is involved in sensory ganglia sNAMs activation/accumulation and mediates neuropathic pain development. On stimulation, NOD2 directly recruits the receptor-interacting serine/threonine-protein kinase 2, which is important for nuclear transcription factor kappa B activation and the transcription of proinflammatory genes.175 In this context, pharmacological inhibition of receptor-interacting serine/threonine-protein kinase 2 activity with a selective inhibitor (WEHI-345) also reduced the development of neuropathic pain.175 Altogether these studies provide consistent evidence that the manipulation of PRRs  or their downstream signaling in sNAMs of the sensory ganglia could be explored as targets to prevent the development of peripheral neuropathic pain.Together with TLRs, cytoplasmic nucleotide-binding oligomerization domain-like receptors (NLRs) are the most important receptors responsible for the recognition of PAMPs or DAMPs.208 These DAMPs have been shown to induce further activation of numerous cell types, including glial cells and innate immune cells, which have a well-established role in the process of neuropathic pain.178 We recently demonstrated that neutrophil-derived S100a9, an endogenous stimulator of TLR4 signaling, plays an essential role in a model of herpetic neuralgia in a mechanism dependent on activation of TLR4 in sNAMs.188 Another possibility in the activation of sNAMs PRRs after nerve injury would be by PAMPs derived from microbiota. In fact, a broader role for the microbiota as a significant modulator of systemic immunity has been proposed.173 Microbial products derived from the microbiota can be excreted or translocated across the gut mucosa into the systemic circulation during infection or inflammation.115 These processes are involved in the development of several diseases, such as autoimmune diseases, Parkinson's disease, spinal cord injury, and neuropsychiatric disorders.140 For instance, bacterial microbiota–derived peptidoglycan and methylene diphosphonate are presented in rheumatoid arthritis patients' synovial tissue, contributing to the pathogenesis through NOD2 signaling activation.143 In addition, peptidoglycan-containing immune cells were detected in the CNS of multiple sclerosis patients or animals but not in healthy controls.215 Our group has shown that germ-free mice are resistant to inflammatory pain4. We also found that peripheral nerve injury can promote a systemic increase of an undetermined stimulant of NOD2 signaling.188 Thus, it is possible that after peripheral nerve injury gut microbiota–derived PAMPs  may translocate from the luminal side of the gut into the blood to distal sites , activates PRRs signaling in sNAMs, and consequently contribute to the development of neuropathic pain. This hypothesis is supported by our unpublished data in which we found that there is impairment in the intestinal barrier permeability after spared nerve injury in mice. Furthermore, in a model of chemotherapy-induced neuropathic pain, there is an increase in the concentration of microbiota-derived lipopolysaccharides in the DRGs, which triggers a TLR4 dependent activation of sNAMs.180 Nevertheless, further studies would be important to identify the exact origin of PAMPs or DAMPs that mediate sNAMs activation in the sensory ganglia and contribute to neuropathic pain development.The involvement of PRRs in the activation of sensory ganglia sNAMs that account for neuropathic pain development raised the question of how these cells recognize or respond to peripheral nerve injury, which is assumed to be a sterile condition. Previous studies have suggested that damaged peripheral sensory neurons release DAMPs, such as fibronectin, high mobility group box-1, and heat shock proteins, which in turn can activate some TLRs.Besides the role of PRRs in the activation/accumulation of sNAMs in the sensory ganglia after peripheral nerve injury, emerging studies were designed to find additional mechanisms explaining how distal damage to primary sensory neurons could activate sensory neurons sNAMs and consequently to the development and maintenance of neuropathic pain. Among these possible mechanisms, the most characterized are those dependent on chemokines (CCL2/CCR2 and CX3CL1/CX3CR1 pathways), cytokines (CSF1/CSFR1 axis), and microRNAs.171 These molecules act on receptors coupled to G proteins found in different populations of circulating and resident cells. Two important chemokine axis seem to regulate sNAMs activities: (1) the CX3CL1, also known as Fractalkine, and its receptor CX3CR134; (2) CCL2, also known as MCP-1, and its receptor CCR2 CX3CR1 is a classical marker of resident macrophages, including sNAMs, especially those originated from earlier precursors in the YS.113 CX3CR1-expressing sNAMs are in close contact with the cell body of sensory neurons in the sensory ganglia, which constitutively express the membrane-bound CX3CL1.213 The stimulation of the CX3CL1/CX3CR1 pathway in the dorsal horn of the spinal cord is a well-known mechanism involved in peripheral nerve injury–induced microglial activation/proliferation and neuropathic pain development.237 Despite all the studies that indicated that the CX3CL1/CX3CR1 pathway in microglia plays a crucial role in neuropathic pain development,124 none of these studies ruled out the possible role of this signaling in CX3CR1-expressing sNAMs of the sensory ganglia. In this context, after sciatic nerve injury or chemotherapy drug treatment, positive regulation of the CX3CL1/CX3CR1 axis in the sensory ganglia occurs.237 Furthermore, after peripheral nerve injury, membrane-bound CX3CL1 is reduced in sensory neurons' cell bodies, suggesting its release and action.101 In fact, neutralization of CX3CL1 in the sensory ganglia reduced chemotherapy-induced neuropathic pain218, which was associated with a reduction in the accumulation of sNAMs in the DRGs.81 In addition, in vincristine-induced pain, another model of CIPN, macrophages, also accumulate in the sciatic nerve and promote pain hypersensitivity in a CX3CR1-dependent manner.161 Therefore, the development of specific tools or approaches to investigate the particular contribution of the CX3CL1/CX3CR1 pathway in the spinal cord microglia or sNAMs in the periphery  for the development of neuropathic pain are necessary.Among the central communication systems of sNAMs and their microenvironments are the chemokine/chemokine receptors interaction. Chemokines are a vast group of peptides that act primarily to attract leukocytes to a given environment after infection or tissue damage.204 This chemokine recruits monocytes/macrophages by activating its highly affinity CCR2 receptor.185 This axis seems to play an essential role in the neuroinflammation process, including those associated with neuropathic pain development.237 In fact, mice lacking CCL2 or CCR2 are resistant to the development of neuropathic pain caused by peripheral nerve injury. Furthermore, pharmacological inhibition of CCL2 and CCR2 with neutralizing antibody or antagonist, respectively, also attenuates mechanical allodynia induced by peripheral nerve injury.227 Neutralization of the CCL2/CCR2 axis also protected from chemotherapy-induced neuropathic pain.83 These studies strongly support the role of the CCL2/CCR2 axis in the development of some types of neuropathic pain. However, the mechanisms by which the CCL2/CCR2 axis mediates neuropathic pain development are not totally clear, but they might be multiples.227 For instance, genetic or pharmacological inhibition of the CCL2/CCR2 pathway reduced monocytes accumulation in the sciatic nerve after traumatic nerve injury,186 suggesting a peripheral effect. On the other hand, recent data did not show any change in the accumulation of sNAMs in the sensory ganglia after peripheral nerve injury,235 indicating that the CCL2/CCR2 axis participates in the development of neuropathic pain would be preferentially at the local of the nerve injury. Supporting this hypothesis, perineural injection of CCL2 promotes pain hypersensitivity dependent on monocytes' recruitment.40 Some studies suggest a possible role for the CCL2/CCR2 pathway in the spinal cord in the pathophysiology of neuropathic pain.198 For instance, they demonstrated an increase in the expression of CCL2 by injured sensory neurons, which might be transported and released into the spinal cord, promoting the activation of CCR2-expressing glial cells.209 However, it is striking that in CCR2-RFP mouse, a mouse strain in which CCR2-expressing cells also express red fluorescent protein, no significant detection of CCR2+ cells was observed in the spinal cord either in naive condition or after peripheral nerve injury.71 There is also evidence suggesting CCL2 directly enhances primary sensory neurons excitability.223 One significant problem to address the exact role of the CCL2/CCR2 axis in the development of neuropathic pain is the lack of specific tools, especially specific antibodies, to stain CCL2 and CCR2.The well-characterized chemokine that brings blood monocytes into inflamed tissues is CCL2.232 Noteworthy, double-blind clinical trials failed to demonstrate the efficacy of a selective and safe CCR2 antagonist in diabetes and posttraumatic neuropathic pain.98 Therefore, further preclinical studies and clinical trials are necessary to further confirm the importance of the CCL2/CCR2 axis for neuropathic pain development and also the possible mechanisms underlying.199Furthermore, Ccr2 null mice have a defect to mobilize monocytes from the bone marrow; thus, even in naive conditions, these animals already have fewer monocytes in the bloodstream.235 The specific knockdown of CSF1 in sensory neurons reduced macrophages activation/accumulation in DRGs after peripheral nerve injury and neuropathic pain.235 These results indicated that the production/release of CSF1 by injured sensory neurons plays a crucial role in the direct sNAMs interaction that accounts for neuropathic pain development.Besides chemokines, cytokines' role in the activation/accumulation of sNAMs in the sensory ganglia after peripheral nerve injury was recently analyzed.189 For instance, it was found that activated or injured primary sensory neurons can release miR-21-5p as cargo in extracellular vesicles .188 These sensory neuron–derived exosomes and their miR-21-5p cargo mediate sNAMs activation/accumulation in DRGs. In fact, after release, miR-21-5p in extracellular vesicles is readily captured by sNAMs in the DRG that, in turn, induces the polarization of sNAMs into a pronociceptive and proinflammatory phenotype.Studies report that after peripheral nerve injury, there is a robust dysregulation in the expression of noncoding RNAs, including microRNAs in sensory neurons.Aside from these studies that provide several possible mechanisms involved in activation/accumulation of sNAMs in the sensory ganglia after peripheral nerve injury and, consequently, in developing neuropathic pain, further studies will be necessary to understand these crosstalks completely. For example, studies that conditionally knockdown molecular pathways or receptors in sNAMs would be required to further understand these interaction mechanisms.234Based on the evidence we have described above, it is becoming clear that peripheral macrophages  participate in the pathophysiological process involved in the genesis of neuropathic pain of different subtypes. Of interest to the community is how peripheral macrophages reciprocally influence sensory neurons excitability after nerve injury that may contribute to these pathological states. Two main effector mechanisms have been attributed to peripheral macrophages at the local of nerve injury and sensory ganglia in the induction of neuropathic pain: (1) production of proinflammatory/nociceptive cytokines and (2) production of reactive oxygen species that in turn trigger TRPA1 stimulation.127 Besides promoting leukocytes recruitment, which amplify the inflammatory/immune process in the local nerve injury, these cytokines/chemokines may also directly enhance the excitability of primary sensory neurons.223 Among cytokines produced/released by macrophages in the local of nerve injury that may affect directly and/or indirectly the excitability of primary nociceptive neurons, tumor necrosis factor (TNF), IL-1β, and IL-6 are well characterized.223 Notably, the expression of proinflammatory cytokines in injured human nerve biopsies has been reported, and this response correlates with the degree of neuropathic pain.129Resident sNAMs together with Schwann cells are the main source of the initial cytokines/chemokines cascade responsible for the recruitment of additional leukocytes, such as neutrophils, monocytes, and lymphocytes that infiltrate the local of nerve injury.235 We have shown that after spared nerve injury, the activation of NOD2 signaling in sNAMs mediates neuropathic pain development in a mechanism dependent on the production of TNF and IL-1β.175 More recently, the CSF1/CSF1R signaling-dependent activation of sNAMs also triggers neuropathic pain through the production of IL1b.235 Finally, it was suggested that sNAMs-derived IL-1β stimulates brain-derived neurotrophic factor by primary sensory neurons as a possible mechanism involved in the development of neuropathic pain.235 Nevertheless, it is striking that sensory neurons specific knockdown of brain-derived neurotrophic factor did not affect neuropathic pain development.42 Thus, the role of sNAMs-derived IL-1β in the sensory ganglia for the development of neuropathic pain is still under debate. In this context, several studies have indicated that primary sensory neurons may express receptors for proinflammatory cytokines/chemokines, including for those peripheral macrophage–derived cytokines .138 Based on that, several studies have analysed the possible effects of these cytokines on the excitability of primary sensory neurons.159 For example, both TNF and IL-1β are able to enhance the excitability of cultured primary sensory neurons in vitro. Nevertheless these results would be analysed with caution because normally cultures of primary sensory neurons also contain other cell subtypes such as SGCs, and these cells may also express receptors for these cytokines,187 hindering the interpretation of the data. One possibility to confirm the specific role of cytokines/cytokines receptor signaling directly on sensory neurons is the development of conditional animals that lack the expression of these cytokines receptors only in pain fibers. In this context, the specific knockout of gp130, a subunit of IL-6 receptor in primary nociceptive neurons, did not affect the development of neuropathic pain, suggesting no role for a direct action of IL-6 on sensory neurons in neuropathic pain8. Furthermore, the deletion of Il1r1 exclusively in the population of TRPV1+ nociceptors prevented the development of pathological pain in models of arthritis and multiple sclerosis.138 The future use of these Il1r1 conditional mice and the generation of TNF receptors conditional knockout mice in primary nociceptive neurons would be necessary to explore and confirm this possibility in models of neuropathic pain after peripheral nerve injury.The activation phenotype of sNAMs in the sensory ganglia after peripheral nerve injury has been also associated with the production of proinflammatory cytokines.203 In fact, the depletion of these cells by clodronate treatment was able to attenuate the levels of hydrogen peroxide in the injured tissue, as well as nociceptive behavior.95 It also showed that monocyte-derived ROS signals through TRPA1 receptors triggering peripheral sensibilization.203 Whereas monocytes recruitment to the site of nerve injury that increase ROS production is dependent on CCL2/CCR2 signaling, there is evidence that macrophages/monocytes activation is dependent on ATR2 signaling183 The sciatic nerve accumulated macrophages/monocytes also promote ROS production in CX3CR1-dependent manner and mediates vincristine-induced neuropathic pain.161 There are several intracellular process and pathways that generate ROS, including mitochondria, xanthine oxidase, cytochrome P450 complexes, lipoxygenases, uncoupled endothelial nitric oxide synthase, and nicotinamide adenine dinucleotide phosphate oxidases. Nox-derived ROS has been implicated in the pathophysiology of neuropathic pain.107 Notably, sNAMs of the sensory ganglia express Nox2 and increase the production of ROS after peripheral nerve injury.96 Altogether these studies indicate that peripheral macrophage–derived ROS, including Nox2 dependent, might be an interesting target for neuropathic pain control. Based on this hypothesis, pioglitazone, a PPARγ agonist, reduces cisplatin-induced neuropathic pain by reducing ROS production in the sensory ganglia.102Another possible effector mechanism by which peripheral macrophages and sNAMs contribute for neuropathic pain development is through the production of ROS. For example, ROS produced by recruited monocytes into the peripheral injured nerves mediates neuropathic pain development.229 In this context, increasing evidence suggests that peripheral macrophages also play an important role in the resolution of chronic pain.39 The identification of these regulatory mechanisms in peripheral macrophages that counteract neuropathic pain would also reveal novel targets for its treatment. For instance, we recently found that at the level of sensory ganglia there is an increase in the production of IL-27 which plays a regulatory role in the development of neuropathic pain.52 We also showed that Il-27 counteracts neuropathic pain by acting on its receptor expressed by sNAMs that in turn stimulate the production of the antinociceptive cytokine IL-10.52Concomitantly to the production of pronociceptive molecules by immune and glial cells across the pain pathway  after peripheral nerve injury, there is also evidence suggesting the production of anti-inflammatory/antinociceptive molecules.212 For example, mice deficient in CB2 receptor showed enhanced pain hypersensitivity in models of neuropathic pain.167 The mechanisms underlying the exacerbation of neuropathic pain in CB2 receptor null mice was recently investigated.150 Notably, specific deletion of CB2 receptors in myeloid cells, especially in peripheral monocytes and sNAMs of the sensory ganglia, but not in neurons, also enhance neuropathic pain to the same level of whole-body deletion.150 These results indicate that CB2 receptor signaling in peripheral macrophages limits the development of peripheral nerve injury–induced neuropathic pain. The mechanisms by which CB2R signaling modulates peripheral macrophages is not totally clear but seems to involve an increase in leptin signaling.157 It could be also due to a reduction in the production of other pronociceptive mediators derived from peripheral macrophages. In fact, activation of CB2 receptors in macrophages reduced the production of proinflammatory cytokines (TNF and IL-1β) and ROS.135 Thus, the development of CB2R agonists acting specifically in the periphery would be an interesting approach to target macrophages and to inhibit neuropathic pain development.Endogenous cannabinoids produced in the periphery and the CNS are important components of endogenous analgesia.In summary, this review pointed out the crucial participation of peripheral macrophages, especially sNAMs located in the sensory ganglia, for the development of neuropathic pain. It also described the cellular and molecular mechanisms involved in peripheral macrophages  activation/accumulation and effector functions after peripheral nerve injury that account for neuropathic pain development Fig. . In concThe authors have no conflicts of interest to declare.T.M. Cunha receives funding from the São Paulo Research Foundation (FAPESP) under grant agreements 2013/08216-2 (Center for Research in Inflammatory Disease) and 2017/50419-9. C.E.A. Silva and R.M. Guimarães have a PhD fellowship from FAPESP (2020/05446-0 and 2019/13829-0).


Hepatocellular carcinoma is a leading cause of cancer-related death worldwide. Major risk factors in liver cancer development include chronic hepatitis B or C virus, autoimmune hepatitis, diabetes mellitus, alcohol abuse, and several metabolic diseases, among others. Standard therapy shows low efficacy, and there is an urgent need for novel therapies. Recent data permit to propose that proteins that control mitochondrial morphology through changes in mitochondrial fusion or mitochondrial fission, confer susceptibility or resistance to the development of liver cancer in mouse models. Here, we review the data that suggest mitochondrial dynamics to be involved in the development of liver tumors.Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer. Due to its rising incidence and limited therapeutic options, HCC has become a leading cause of cancer-related death worldwide, accounting for 85% of all deaths due to primary liver cancers. Standard therapy for advanced-stage HCC is based on anti-angiogenic drugs such as sorafenib and, more recently, lenvatinib and regorafenib as a second line of treatment. The identification of novel therapeutic strategies is urgently required. Mitochondrial dynamics describes a group of processes that includes the movement of mitochondria along the cytoskeleton, the regulation of mitochondrial morphology and distribution, and connectivity mediated by tethering and fusion/fission events. In recent years, mitochondrial dynamic processes have emerged as key processes in the maintenance of liver mitochondrial homeostasis. In addition, some data are accumulating on the role played by mitochondrial dynamics during cancer development, and specifically on how such dynamics act directly on tumor cells or indirectly on cells responsible for tumor aggression and defense. Here, we review the data that suggest mitochondrial dynamics to be involved in the development of liver tumors. Liver cancer is the fourth leading cause of cancer mortality worldwide, with more than 840,000 new cases and 780,000 deaths currently recorded globally per year . HepatocMore recently, immune checkpoint inhibitors have been introduced into HCC clinical practice with high expectations. Since the approval of nivolumab by the FDA in 2017 for the treatment of HCC, combination therapy with anti-angiogenic drugs and immunosuppression checkpoint inhibitors has been tested in clinical studies . In lighHCC development and progression are influenced by genetic, epigenetic, and environmental factors. HCC risk factors include chronic hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection, autoimmune hepatitis, diabetes mellitus, alcohol abuse, and several metabolic diseases ,10. SpecThere are two major pathways for the development of HCC. In most cases, HCC develops in patients with chronic liver disease and cirrhosis . A seconTERT (telomerase reverse transcriptase) promoter (accounting for 60% of cases), TP53 (tumor protein 53) (30% of cases), WNT (Wingless-type MMTV integration site) signaling genes CTNNB1 (Catenin Beta 1) (30% of cases) and AXIN1 (10% of cases), and chromatin remodeling genes ARID1A (AT-Rich Interaction Domain 1A) (10% of cases) and ARID2 (AT-Rich Interaction Domain 2) (5% of cases) . M. M30]. MIn addition to its role as a mitochondrial fusion protein, MFN2 also serves as an endoplasmic reticulum (ER) protein in the connection of ER membranes to mitochondria  33], an, an33], Mitochondrial fission is catalyzed by DRP1 (Dynamin-related protein 1), MID49, MID51, FIS1 (Fission 1 homolog protein), and MFF  A. DRP1 hThe physiological relevance of mitochondrial dynamics has been studied in mammalian tissues upon genetic manipulation of specific genes. Ablation of mitochondrial fusion and fission proteins in mice is embryonically lethal ,46,47,48Loss of hepatic OPA1 also alters the morphology of mitochondria . SimultaThe expression of the mitochondrial fusion protein MFN2 is subject to regulation in the liver via hormonal and metabolic factors. Glucocorticoids repress MFN2 expression in hepatoma cells and in mouse liver . The proThere are some data on the role of mitochondrial dynamics in tumor cells and in connection with cells responsible for tumor aggression and/or defense ,58. TumoThere are some data indicating the presence of alterations in mitochondrial fusion proteins in HCC. Some results have revealed a lower average mitochondrial length in HCC tissues compared to that in adjacent nontumor tissues . These oHCC tumors are frequently characterized by loss of heterozygosity in the MFN2 gene . In some2+ ions in the ER, and high concentrations of reactive oxygen species (ROS) and mitochondrial Ca2+ [It has also been reported that MFN2 overexpression in HCC cells reduces cell proliferation and induces spontaneous apoptosis . This apial Ca2+ . In addiial Ca2+ . In turnial Ca2+ . MFN2 seial Ca2+ . NeverthConsistent with the concept that MFN2 plays an important role in the biology of the liver, hepatic MFN2 depletion promotes the generation of liver tumors in mice both with aging and in response to carcinogens . It has also been proposed that mitochondrial impairment contributes to the pathogenesis of chronic liver diseases. Endogenous MFN2 expression has been reported to decrease in patients with extrahepatic cholestasis characterized by elevated levels of toxic bile acids , and alsOPA1 is a major mitochondrial fusion protein, and it also regulates crista formation. Some data indicate that HCC tumors do not show changes in OPA1 protein or mRNA expression compared to adjacent tissue ,77. It iInterestingly, some of the cell death effects of the multikinase inhibitor sorafenib are dependent on the dysregulation of OPA1 . SorafenIn all, available data suggest the existence of alterations in the expression of MFN1 and MFN2 in HCC cancer cells, and that these alterations could be instrumental in the biological properties of those cells. In contrast, the expression of OPA1 is maintained at a high level in HCC cells, which may be key in prevention of apoptosis. Further studies are required to thoroughly analyze the roles of these proteins in the development and progression of HCC. These data also raise the possibility of using the mitochondrial fusion proteins as drug targets for the treatment of HCC.Many studies have reported that increased mitochondrial fission is possibly proto-tumorigenic and is associated with oncogene expression, metabolism, cellular behavior, and responses to stress ,82. It hThe enhanced expression of DRP1 in HCC cells is of potential biological relevance. It has been reported that dexamethasone upregulates the expression DRP1 in hepatoma FaO cells. Moreover, the effects of dexamethasone on mitochondrial respiration and on gluconeogenic activity depend on normal DRP1 activity. In this regard, inactivation of DRP1 activity via expression of a dominant-negative form of DRP1 impairs the effects of dexamethasone on oxygen consumption and on mitochondrion-dependent glucose production from lactate/pyruvate . Importantly, overexpression of DRP1 in HCC cells has been linked to enhanced tumor growth under in vivo conditions, and, vice versa, DRP1 deficiency caused a reduced tumor growth ,84. DRP1Interestingly enough, it has been reported that the expression of DRP1 is strongly increased in distant metastases of HCC compared to in primary HCC . In addiIt is likely that a number of proteins modulate DRP1 in tumor cells. The role of the large tumor suppressor 2 (LATS2), a mediator of the cell apoptotic response pathway, was proposed in a previous study . LATS2 oIn all, the available information seems to indicate that increased mitochondrial fission plays a critical role in regulating HCC cell survival, providing strong evidence for this process as a drug target in the treatment of HCC.In recent years, there have been interesting advances in the study of mitochondrial dynamics in connection with cancer. Specifically, HCC, the most common type of liver cancer, has been clearly linked to a dysregulation of mitochondrial dynamics. The majority of the available data support the proposal that mitochondrial dynamic processes are influenced in HCC tumors, in that mitochondrial fusion is reduced whereas mitochondrial fission is more active than in nontumor tissue. This proposal requires thorough demonstration as well as resolution of the mechanisms that operate in HCC tumors.Available data also suggest that mitochondrial fragmentation of HCC tumors plays a key role in cell proliferation and migration as documented in vitro and in liver tumor growth and metastasis studied using nude mice. Based on these observations, it seems relevant to explore whether mitochondrial dynamics could present pharmacological targets for the treatment of HCC, and perhaps other types of cancer. A fundamental question raised by the available evidence is whether the alterations reported in mitochondrial dynamic proteins in HCC tumors are also present in the initial phases of tumor promotion and growth. In regard to this connection, it will be key to analyze mitochondrial dynamics in HCC progenitors, and to analyze whether enhanced mitochondrial fission or reduced mitochondrial fusion indeed facilitate the tumor initiation, growth, or metastasis of liver cancer cells. Clarification of some of the above questions will be relevant not just from the point of view of the fundamental understanding of mitochondrial biology, but also from the translational perspective, and they may permit the identification of novel therapeutic strategies for HCC.Currently available data suggest that mitochondrial dynamics plays a relevant role during cancer development, and more specifically in liver cancer. Further studies on the mechanisms by which specific proteins involved in mitochondrial dynamics participate in tumor growth are of great importance. This will lead to the identification of relevant pathophysiological mechanisms and to the discovery of novel drug targets.


In this article of EBioMedicine, An Cheng and colleagues show that blocking the function of fatty acid-binding proteins (FABPs) pharmacologically, alleviates the severity of the inflammatory demyelinating neurological condition in the experimental autoimmune encephalomyelitis (EAE) mouse model MS is a multifactorial autoimmune and neurodegenerative condition. The presence of proinflammation brought about by reactive T cells, microglia and astrocytes, oxidative stress, and mitochondrial dysfunction that perpetuate degeneration of neurons and glial cells, including oligodendrocytes, are common hallmarks of early MS. Hence, therapeutic targets that suppress proinflammatory activities by the immune cells and astrocytes and promote the remyelination capability of oligodendrocytes are priorities in MS research.Fatty acids are known to be involved in the inflammatory response, oxidative stress, and mitochondrial dysfunction . Moreover, myelin (a lipid-rich membrane structure) production is dependent on fatty acid synthesis made by oligodendrocytes FABPs are lipid chaperones that regulate of fatty acid metabolism with differential subtype expressions that confer tissue and cell specificity. For example, the brain FABP (FABP7) is expressed by astrocytic cells while epidermal FABP (FABP5) is expressed in immune cells such as microglia. There is early, but limited, clinical evidence showing that FABP is involved in MS. A recent paper showed that increased FABP4 is associated with higher disability in MS patients Several studies targeting FABP to limit proinflammation and injuries to the central nervous system have demonstrated overlapping pathological hallmarks across neurodegenerative diseases including MS. For example, mice lacking FABP4 and 5 have reduced EAE severity associated with impaired proinflammatory activities Cheng and colleagues show that MF6, a novel ligand which binds FAB5 and 7, reduces EAE severity and attenuates proinflammatory activities by limiting reactive astrocytes consistent with previous findings Several questions remain. It is tempting, at first, to think that inhibiting lipid metabolism may be key to limiting MS progression or at least in the EAE model. However not all fatty acids are increased in MS as summarized in a recent systematic review Although MF6 has been shown to be well-tolerated in an animal study, other factors need to be considered. A recent epigenome-wide study showed that narcolepsy, a sleep disorder, and MS had overlapping DNA methylation in FABP7 suggesting that its downregulation (or inhibition) may impact sleep physiology The feasibility of modulating complex fatty acid metabolic processes as a multitarget treatment of neuro-inflammatory and -degenerative conditions such as MS requires precise mechanistic control including disease timing, cell specificity, and host fatty acid metabolome profiles to achieve a holistic precision medicine approach. The findings presented in this study make even more of a case for targeting multiple cell types responsible for proinflammation, oxidative stress, mitochondrial dysfunction, and myelination. This signals an exciting time in the next generation of treatments targeting FABP in MS and possibly in other neurodegenerative diseases that share some overlapping pathologies.CKL wrote and reviewed the commentary and approved the final version.The author declares no conflict of interest.


Prospective epidemiological studies concur in an association between habitual coffee consumption and a lower risk of type 2 diabetes. Several aspects of these studies support a cause–effect relationship. There is a dependency on daily coffee dose. Study outcomes are similar in different regions of the world, show no differences between sexes, between obese versus lean, young versus old, smokers versus nonsmokers, regardless of the number of confounders adjusted for. Randomized controlled intervention trials did not find a consistent impact of drinking coffee on acute metabolic control, except for effects of caffeine. Therefore, lowering of diabetes risk by coffee consumption does not involve an acute effect on the post-meal course of blood glucose, insulin or insulin resistance. Several studies in animals and humans find that the ingestion of coffee phytochemicals induces an adaptive cellular response characterized by upregulation and de novo synthesis of enzymes involved in cell defense and repair. A key regulator is the nuclear factor erythroid 2-related factor 2 (Nrf2) in association with the aryl hydrocarbon receptor, AMP-activated kinase and sirtuins. One major site of coffee actions appears to be the liver, causing improved fat oxidation and lower risk of steatosis. Another major effect of coffee intake is preservation of functional beta cell mass via enhanced mitochondrial function, lower endoplasmic reticulum stress and prevention or clearance of aggregates of misfolded proinsulin or amylin. Long-term preservation of proper liver and beta cell function may account for the association of habitual coffee drinking with a lower risk of type 2 diabetes, rather than acute improvement of metabolic control. The perception of coffee has experienced a remarkable transition from a stimulant drink which may stress your cardiovascular system to a beverage that is good for your health. The latter view is derived from a large number of prospective cohort studies which observed an association of socioeconomic or lifestyle factors including habitual coffee consumption and clinical outcomes including type 2 diabetes mellitus, non-alcoholic fatty liver disease, liver cancer, gout, kidney stones and Parkinson’s disease .In this paper, we discuss the association of habitual coffee consumption and a lower risk of type 2 diabetes. In addition to epidemiological studies, data are available from a considerable number of randomized short-term intervention trials with metabolic endpoints, and from Mendelian randomization studies. Finally, we consider that coffee constituents appear to exert similar molecular effects at the cellular level as reported for phytochemicals of other dietary plants. From these data, a picture emerges how coffee consumption promotes resistance to the development of type 2 diabetes.This paper is a narrative review and commentary based on a survey of all papers listed by PubMed for the search items coffee, caffeine or chlorogenic acid, in combination with the items diabetes, glucose tolerance or insulin resistance, respectively. Other papers were retrieved from the reference list of reviews published on coffee, caffeine or chlorogenic acid versus metabolic endpoints. To the best of our knowledge, we included and dis-cussed all published human trials of coffee consumption versus metabolic endpoints. For the discussion of a possible molecular mechanism, we conducted an additional search in PubMed with the items coffee/caffeine/chlorogenic acid in combination with the items Nrf2/anti-oxidative/anti-inflammatory, respectively.Although epidemiological studies cannot prove a causal relationship all recent meta-analyses considered it probable that coffee consumption lowers the risk of type 2 diabetes ,2,3,4. UObservational studies suffer from different characteristics of subgroups drinking none, little or much coffee. Consumption of none or little coffee may be due to intestinal intolerance, religious reasons or simply dislike or prejudice. It is virtually impossible to correct for this type of confounding. The number of confounders considered in the various studies is limited (listed in ). For inNonetheless, it seems improbable that residual confounding accounts for the association of coffee consumption with a lower risk of type 2 diabetes, for the following reasons. (i) Results of prospective cohort studies are surprisingly similar in different regions of the world  although lifestyle and cultural background differ. (ii) There is no significant difference in outcome for men and women. (iii) Sub-analyses for study participants with obesity (body mass index > 25), with age above 50 years or for non-smokers also observed an inverse association of coffee consumption and risk of type 2 diabetes ,7. (iv) Another epidemiological approach is to correlate changes in coffee consumption pattern with diabetes outcome. Three large prospective studies in the USA of 16–20 years duration and documentation of dietary habits every four years were analyzed for changes in coffee consumption . An incrTaken together, epidemiological studies concur on an inverse association of habitual coffee consumption with risk of type 2 diabetes. The association with diabetes risk is as robust as that of other lifestyle factors not tested in randomized long-term trials such as physical activity, sitting time, sleep duration, smoking or exposure to traffic noise and fine dust .Genome-wide association studies have identified several genetic variants seen more often in coffee drinkers than non-drinkers ,13,14,15Several Mendelian randomization studies have searched for a higher diabetes risk in carriers of gene variants promoting coffee consumption, but results remained inconclusive or did not show a causal link with incident type 2 diabetes or other health outcomes ,16,17. OAs described above, changes in coffee consumption in individuals followed in prospective studies was accompanied with an inverse change of diabetes risk while this was not the case for tea . In thisWe conclude that there is no major influence of individual genetic characteristics associated with coffee consumption regarding the risk of type 2 diabetes. The responsible molecular mechanism involved seems directly linked to coffee consumption and not dependent on genetic variants promoting coffee/caffeine consumption.It has been difficult to identify acute metabolic effects of coffee consumption in randomized controlled intervention trials except for effects of caffeine. The major pharmacological activity of caffeine is inhibition of adenosine receptors and modulation of the purinergic system because of the molecular similarity to adenosine ,21. As aThe acute metabolic effects of caffeinated coffee or caffeine did not persist after longer periods of coffee consumption. A 24-week trial with 126 overweight Asian participants reported no significant change of insulin sensitivity after daily consumption of 4 cups of caffeinated coffee compared to a coffee-like placebo drink. There was also no difference in fasting glucose . A trialTaken together, habitual consumption of caffeinated coffee does not impact metabolic control of glucose and insulin levels but may have a mild effect in favor of caffeine-mediated body fat/visceral fat loss. In the long term, these changes may be sufficient for lowering the risk of type 2 diabetes, because lifestyle intervention trials have shown that modest lowering of body weight by 5%–7% is already associated with a decreased diabetes rate . HoweverIt, therefore, may be concluded that the presence of caffeine in coffee and the accompanying modest decrease in body fat is not essential for its apparent diabetes-protective effect although there may be a minor contribution. Other constituents of coffee must be viewed as candidates for lowering the risk of type 2 diabetes.Decaffeinated coffee contains only very small amounts of caffeine  so that Three trials did not report on a control group with water or placebo fluid consumption instead of coffee, and therefore, do not provide sound evidence ,39,40. Tn = 14) there was no difference to the water control (n = 15) with respect to the glucose and insulin response to an oral glucose load, or with regard to insulin resistance, in healthy, middle-aged and overweight persons [n = 17) versus water (n = 13) [After 8 weeks of daily drinking decaffeinated coffee ( persons . Another(n = 13) .The trials usually included 10–17 participants, and these were young, metabolically healthy and lean persons (overweight middle-aged participants in only one study). Several endpoints were analyzed at different points of time, usually without correction for the high number of statistical tests performed. Taken together, the trials do not provide evidence for an acute effect on metabolic control after consumption decaffeinated coffee. Long-term trials would be required to analyze for a delayed impact of metabolic control and the prevention of metabolic deterioration or with diabetes as clinical endpoint. Such trials currently do not appear feasible.A summary of conclusions from observational and randomized controlled studies of coffee consumption is given in Box 1Prospective cohort studies find a lower risk of type 2 diabetes associated with habitual coffee consumption.The association with diabetes risk is dose dependent, seen world-wide and in both sexes, and is also found for decaffeinated coffee.The association with diabetes risk is seen regardless of the number of potential confounders adjusted for.Changes of coffee consumption over time are accompanied by a change of diabetes risk.Mendelian randomization studies do not provide consistent results on the association of diabetes risk with a genetic background favoring caffeine/coffee consumption. However, the genetic effect size on caffeine/coffee intake is modest.Randomized controlled trials do not observe a consistent acute impact of coffee consumption on metabolic control except for some beneficial effects of caffeine on appetite and body fat mass. Long term trials with diabetes as endpoint are not feasible.Because randomized controlled trials of short duration failed to uncover a consistent caffeine-independent modulatory effect of coffee consumption on systemic insulin or glucose levels after a meal or glucose challenge, other physiological responses appear to be relevant. These include delayed impact on metabolic control and prevention of metabolic deterioration.One approach to identify relevant physiological responses to coffee is to screen for an impact on a wide variety of circulating components of metabolism, other than insulin and glucose. A recent analysis of the Nurses’ Health Study II identified three cholesteryl esters associated with coffee consumption and lower diabetes risk. Conversely, five diacylglycerols and seven triacylglycerols showed negative associations with coffee-related diabetes risk factors . A lipidMany phenolic phytochemicals exhibit concentration-dependent toxic properties which include DNA damage, mutagenesis, carcinogenesis and cell death. Organs affected include the liver, intestine and kidney. Tumor cells appear to be more susceptible than normal cells ,59,60,61The studies have been performed with high doses of phenolic compounds from foods like coffee, tea or other edible plants. Despite these potentially toxic properties, vegetarian food is well tolerated by the human organism. The reason is the subtoxic dose of phytochemicals taken up with our daily diet. Postprandial blood concentrations of major dietary phenolics may reach a few µmol/l but usually are lower ,63. CytoCoffee appears to be a safe beverage in that regard except for high intakes of caffeine, especially during pregnancy . RandomiAfter consumption of one cup of coffee, peak blood concentrations of chlorogenic acid metabolites were about 1 µmol/l, whereas pyridine derivatives trigonelline and 1-methylpyridinium reached peak concentrations of 6 and 1 µmol/l. Peak concentrations are below the micromolar range for all other phytochemicals in coffee, except for caffeine . Even atMost of these interactions are weak at micromolar or submicromolar concentrations but randomized controlled trials of coffee consumption in humans report one major cellular response, the increased expression of proteins involved in the defense against free radicals, xenobiotics or UV irradiation, improved cell regeneration, DNA repair and cell survival, as well as dampening of pro-inflammatory activities . These rAs described in detail previously , Nrf2 is2O2 may be either generated directly by phenolics extra- or intracellularly, in the presence of Cu(II) or Fe(III) ions [There is continuous de novo synthesis of Nrf2, most of which is captured by cytosolic Keap1 and channeled to proteasomal degradation. Keap1 is highly sensitive to an electrophile attack by phytochemicals because of its 17 cysteine residues and readily loses its ability to recycle Nrf2 and prevent its translocation to the nucleus where it targets the DNA sequences described above. Phytochemicals may also indirectly activate the Nrf2/Keap1 system by increasing the intracellular levels of oxygen radical species or nitric oxide all of which target cysteine residues of Keap 1. Free oxygen radical species including HII) ions ,60,93,94II) ions ,99,100.Electrophilic phytochemicals including those of coffee may also target the aryl hydrocarbon receptor ,74. This+ which promotes mitochondrial function, autophagy and a gene expression pattern supporting cell survival during various forms of stress including those mediated by phytochemicals [While the Nrf2/Keap1 and AHR systems respond to increased levels of electrophiles or free radicals, another approach of sensing danger in cells is monitoring the energy level. The response to phytochemical stress is expected to affect cellular energy reserve. A low energy state is characterized by low ATP/AMP and ADP/ATP ratios as well as low glucose levels. This condition leads to the activation of AMP-activated protein kinase (AMPK). This group of enzymes increase energy production and decreases energy uses by phosphorylation and promoting acetylation of many metabolic enzymes, histones and transcription factors. There is also activation of histone deacetylases of the sirtuin family by provision of NADhemicals ,105,106.hemicals ,107.The possible health effect of coffee is supported by findings that several different components of coffee are able to activate the Nrf2/Keap1 or AHR systems in isolated cells, animals and humans. Chlorogenic acid and its degradation product caffeic acid are strong activators of Nrf2 activity ,109,110 Taken together, there is evidence that consumption of coffee activates a cell protective, Nrf2 and AHR dependent cell response in a systemic manner  which inThe upregulation of cell protective regulatory circuits in response to the ingestion of coffee appears to occur in many organs of the body, as judged from analyses of liver, hepatocytes, stomach, blood lymphocytes, endothelial cells, muscle cells and the small intestine ,124,125.Coffee and liver: In vivo imaging using a luciferase-reporter gene system in rodents showed that the liver is the main organ responding to coffee ingestion with the expression of Nrf2-regulated genes . FeedingIn accordance with the animal experiments, prospective studies of coffee consumption versus incident NAFLD or liver cirrhosis in humans reported an inverse relationship ,133. TheCoffee and beta cells: Loss of functional beta cell mass in relation to insulin requirements is the critical process causing type 2 diabetes . The lowProspective studies indicate that increased insulin secretion and hyperinsulinemia precedes type 2 diabetes in most people ,136. TheNrf2 gene substantially reduced the defense response of beta cells towards oxidative and nitrosative damage, disturbed mitochondrial function and diminished glucose-stimulated beta cell proliferation [keap 1 gene led to activation of Nrf2 which largely prevented diabetes development and beta cell loss in the spontaneously diabetic db/db mice, and also prevented diabetes development in a high calorie-diet model [Upregulation of Nrf2 activity in beta cells by phytochemicals counteracts the damaging effects of glucolipotoxicity (in vitro) or a high fat diet (in vivo) ,145,146.feration ,149. Conet model . Preservet model .Coffee phytochemicals such as 5-O-caffeoylquinic acid, pyrocatechol and melanoidins as well as polyphenols from other plants can preserve cell function in periods of metabolic stress also by interfering with the aggregation of misfolded proteins into amyloids ,153,154.Taken together, coffee phytochemicals can sustain beta cell function and survival . There iBox 2Prospective epidemiological studies find a robust association of habitual coffee consumption (caffeinated or decaffeinated) with a lower risk of type 2 diabetes.Results of Mendelian randomization studies remain inconclusive, possibly because of a small effect size.Randomized controlled trials do not show a consistent effect of coffee intake on acute metabolic control, except for some effects of caffeine.Metabolomic analyses also do not provide a clear picture how coffee might modulate metabolic control.Phytochemicals of coffee or other dietary plants are known to induce an adaptive cell response characterized by activation of Nrf2, AHR, AMPK and sirtuins.Most coffee phytochemicals and metabolites accumulate in the liver. The resulting Nrf2-dependent toxic stress response improves mitochondrial function, lipid oxidation and reduces the risk of steatosis.Data on modulation of the gut microbiota are scarce, but there seems to be an improved intestinal barrier function which will contribute to the prevention of steatosis.Coffee phytochemicals support the preservation of pancreatic beta cell function via Nrf2-mediated resistance to cell damage during periods of high insulin secretion. In addition, coffee constituents directly interact with misfolded peptides and prevent the formation of cell-toxic amyloids.Long-term effects of habitual coffee consumption appears to maintain proper function of liver and beta cells rather than improve acute metabolic control.A summary of conclusions is given in Although epidemiological studies consistently find an association of habitual coffee consumption formal proof of a cause-effect relationship is lacking. However, several aspects of observational prospective studies strongly argue against a major influence of residual confounding and support an antidiabetic effect of coffee. Results of prospective observational studies are surprisingly similar for different regions of the world, including coffee-dose dependency, no difference between male versus female, obese versus lean, younger versus older study participants, regardless of the number of confounders adjusted for. Analyses for habitual consumption of decaffeinated coffee yielded similar results as for caffeinated coffee ,6,7. ChaShort-term randomized controlled trials comparing consumption of coffee versus water or a placebo drink did not provide clues about the possible mechanism of diabetes prevention. Aside from acute consequences of caffeine intake there were no consistent effects on diabetes-relevant metabolic parameters such as the insulin and glucose response to a glucose load or to a meal. Insulin sensitivity was not modulated (see detailed description above). We conclude that a diabetes-preventive effect of coffee does not bear on acute metabolic responses but must exhibit a delayed impact on metabolic control and prevent metabolic deterioration.One consistent biochemical response to the consumption of coffee phytochemicals is an improved antioxidative defense in animals and humans, such as elevated levels of glutathione, catalase and superoxide dismutase ,156,157.The liver is the primary organ for the accumulation of coffee phytochemicals and metabolites . The ensBesides the liver, the other prominent target for a long-term diabetes-preventive effect of coffee consumption is probably the pancreatic beta cell. In the prediabetic period, beta cells are exposed to metabolic stress associated with obesity and insulin resistance. Glucolipotoxicity involves deficient mitochondrial function and endoplasmic reticulum stress which leads to beta cell dysfunction, dedifferentiation or death ,135,136.Taken together, habitual coffee consumption may lower the risk of type 2 diabetes by preventing the deterioration of liver and beta cell function during chronic metabolic stress preceding the manifestation of overt diabetes.


The mammalian ovary has two main functions—producing mature oocytes for fertilization and secreting hormones for maintaining the ovarian endocrine functions. Both functions are vital for female reproduction. Primordial follicles are composed of flattened pre-granulosa cells and a primary oocyte, and activation of primordial follicles is the first step in follicular development and is the key factor in determining the reproductive capacity of females. The recent identification of the phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling pathway as the key controller for follicular activation has made the study of primordial follicle activation a hot research topic in the field of reproduction. This review systematically summarizes the roles of the PI3K/PTEN signaling pathway in primordial follicle activation and discusses how the pathway interacts with various other molecular networks to control follicular activation. Studies on the activation of primordial follicles have led to the development of methods for the in vitro activation of primordial follicles as a treatment for infertility in women with premature ovarian insufficiency or poor ovarian response, and these are also discussed along with some practical applications of our current knowledge of follicular activation. The mammalian ovary is a key component of the female reproductive system and contains follicles at various developmental stages . In addiFollicles are the functional units of ovaries, and in most mammalian species primordial follicles are formed before or shortly after birth . A largeOnce the primordial follicle pool is formed, the primordial follicles have three possible developmental fates—to maintain a quiescent state as primordial follicles, to be activated and enter the growth phase, or to undergo death . The balThe activation of primordial follicles and subsequent follicular development is an irreversible process . The chaOnly a small proportion of primordial follicles are recruited into the growing follicle pool ,23, and According to their structural characteristics and substrate specificity, PI3Ks are divided into three classes  . Among tPten gene results in over-activation of the entire pool of primordial follicles, and this is followed by the premature depletion of all the primordial follicles, thus revealing the important roles of PI3K and PTEN in follicular activation [As a negative regulator of PI3K function, PTEN reverts PIP3 back to PIP2 and thus suppresses PI3K signaling ,41. For tivation ,26  in pre-granulosa cells can inactivate mTORC1 signaling, and this suppresses follicular activation and prevents the awakening of dormant oocytes [Rptor in oocytes does not affect follicular development or female reproductive capacity [Another study from Liu’s group showed that conditional knockout of  oocytes ,58. Howecapacity . These r−/−1 mouse ovaries [−/−Tsc1 mice with Y719F/KitY719FKit mice to further determine how the KITL–KIT and the intraoocyte PI3K signaling pathways function to awaken dormant oocytes. Phosphorylation of KIT Y719 is responsible for activating the downstream PI3K signaling, and Y719F/KitY719FKit mice carry a point mutation that prevents this phosphorylation [–/–Tsc1, Y719F/KitY719FKit mice prevented the KIT-mediated activation of PI3K in oocytes. Thus, binding of KITL to Kit receptor results in the phosphorylation of KIT Y719, and this further activates PI3K signaling in mouse oocytes. The activated PI3K phosphorylates Akt, and this leads to the shuttling of Foxo3 from the nucleus to the cytoplasm resulting in the activation of primordial follicles [The first stage in primordial follicle activation is the differentiation and proliferation of pre-granulosa cells, which is followed by oocyte growth in the ovary ,61,62. Z ovaries . They thrylation . The Y71ollicles   stimulates the activation of primordial follicles in both mouse ovaries and human cortical pieces using a new in vivo system  with dissolved EGF in liquid Matrigel, suggesting the role of EGF in follicle development . Furthernull mouse model showed that a lack of Amh does not affect fertility in female mice [null female mice contain fewer primordial follicles and more growing follicles than those of wild-type mice [AMH is a dimeric glycoprotein belonging to the transforming growth factor-β superfamily of growth and differentiation factors  and was ale mice , but priype mice . Similarype mice . Taken tAMH is widely applied in the clinic for predicting the ovarian response to controlled ovarian stimulation treatment, because AMH is mainly produced in the growing follicles, which are capable of responding to exogenous gonadotrophins ,95. For The IGF system, which is composed of insulin and insulin-like growth factors 1 (IGF-1) and 2 (IGF-2), participates in cell growth, proliferation, and survival . IGFs, pTGFβ1 is a member of the TGFβ superfamily of proteins, which function in the regulation of cell growth and differentiation ,104. RecLKB1 are associated with Peutz–Jeghers syndrome (PJS) in humans [Lkb1 in mouse oocytes leads to excessive activation of the primordial follicle pool resulting in POI in adulthood [Lkb1 conditional knockout mice, indicating that elevated mTOR signaling is responsible for the excessive follicle activation and growth [LKB1 is a serine/threonine kinase that plays roles in regulating cellular energy homeostasis, the establishment of cell polarity, vascular development, and tumor suppression . LKB1 ren humans , and condulthood . Rapamycd growth . Togethep27-deficient mice have shown that follicle assembly is accelerated and that the primordial follicle pool is prematurely activated, leading to POI in early adulthood [p27 is a regulatory protein that controls cell cycle progression by binding to cycle-dependent kinase complexes ,110. It dulthood . OverallRecent studies of semaphorins have focused on their roles in cancer, the immune system, the vascular system, and organogenesis, and Sema6c has been found to play a role in the neonatal mouse ovary ,117,118.+-dependent deacetylases that have recently emerged as key sensors in metabolism, cancer development, and aging [The sirtuins are a family of NADnd aging ,121. As nd aging . The expnd aging , and thend aging . Based oExosomes derived from mesenchymal stem cells (MSC-exos) have great therapeutic potential due to their ethical acceptance, abundant tissue sources, low immunogenicity, and rapid renewal properties . Like thWith regards to female fertility, an increasing number of studies have shown that MSCs can improve ovarian function in POI or natural-aging animal models ,131,132.In recent years, more and more women in many societies are delaying marriage and postponing motherhood, and this is increasing the number of women seeking in vitro fertilization (IVF) in order to have their own children. However, IVF cannot help women with POR or POI because they only have undeveloped follicles in their ovaries, and these do not respond to hormones. In recent years, primordial follicle in vitro activation (IVA) has provided a new potential treatment for infertile women with POR and POI. IVA is a new in vitro assisted reproductive technology that treats infertility in POR patients by activating the primordial/small follicles in vitro . Based o3 cubes followed by treatment with Akt stimulators for two days. The cubes were then autografted beneath the serosa of Fallopian tubes for development into follicles. After antral follicles had appeared, the patients were injected with FSH. With the emergence of preovulatory follicles, hCG was injected for the final maturation of the oocytes. These oocytes were aspirated and used in IVF to form embryos  showed follicular development, and oocytes were successfully obtained from the growing follicles of six patients. Following IVF, embryo transfers were carried out in four patients and three pregnancies were identified that resulted in one miscarriage and two successful deliveries . In 2016In recent years, drug-free IVA, i.e., only fragmentation of the ovarian cortex, has appeared as a simplified IVA procedure for POR and POI patients and has been reported to allow retrieval of more oocytes and to achieve higher pregnancy rates. Drug-free IVA showed that disrupting the Hippo signaling pathway through ovarian cortical fragmentation could effectively promote follicle growth and increase the clinical pregnancy rate. Using this simplified drug-free IVA procedure, two case reports showed successful pregnancies in patients with POI in 2018 and 2019 ,140. AnoResearch into mTORC1 signaling in the activation of primordial follicles has shown that the efficiency of primordial follicle activation can be improved by the combination of mTOR stimulators and PI3K activators . This suAlthough IVA has been used in the treatment of POI patients, there is still much to do to improve this approach. Due to the complex clinical manifestations, complex etiology, and heterogeneity of POI, the application of this technology needs to consider individual differences among patients. The use of drugs for activating primordial follicles might result in negative feedback from downstream effectors. For example, inhibition of PTEN will not always leads to a constant over-activation of the PI3K pathway, but rather the opposite, which might lead to unsatisfactory clinical outcomes and low success rates of IVA.In this review, we highlight the roles of the PI3K/PTEN signaling pathway in primordial follicle activation along with other pathways and factors involved in the regulation of dormancy and the activation of primordial follicles . Primord