Skin biopsy with investigation of small-diameter nerve fibers in human epidermis and dermis has been proven to be a useful method for confirming small-fiber neuropathy. In medical practice, small-fiber neuropathy is increasingly recognized as a leading cause of neuropathic pain. It is a prevalent complaint in medical offices, brought by patients often as a “painful burning sensation”. The prevalence of neuropathic pain is high in small-fiber neuropathies of different etiologies, especially in the elderly; 7% of population in this age group present peripheral neuropathy. Pain and paresthesia are symptoms which might cause disability and impair quality of life of patients. The early detection of small-fiber neuropathy can contribute to reducing unhealthy lifestyles, associated to higher incidence of the disease. By definition, a pure small-fiber neuropathy cannot be diagnosed by standardized nerve conduction studies, since these only reflect the function of larger, myelinated nerve fibers. A quantitative sensitivity testing may show impairment suggestive of small-fiber neuropathy, but this method is heavily dependent on patient cooperation, and does not differentiate lesions involving peripheral and central sensory pathways. Skin biopsy to investigate small-diameter nerve fibers was widely used during the last two decades in the diagnostic evaluation and follow-up of patients with peripheral neuropathy.By immunostaining for the protein gene product 9.5 (PGP 9.5), a pan-axonal neuronal marker, nerve bundles are observed as they form horizontal subepidermal neural plexuses in the papillary dermis and traverse vertically through the epidermal basement membrane, ascending between the epithelial cells as intraepidermal nerve fibers. They end superficially as free nerve endings .The intraepidermal nerve fibers are unmyelinated, as they lose their Schwann cell sheathing them at the dermal-epidermal junction. No sutures are usually needed, and minor complications  are very rare. In most cases, healing occurs within 7-10 days. The biopsy contains epidermis and dermis, including sweat glands.Biopsy is most commonly performed using a 3-to- 5mm disposable circular punch under sterile technique, and after local anesthesia with lidocaine. and later developed and standardized at the University of Minnesota, and at the Johns Hopkins University.The technique was first described by researchers at the Karolinska Institute, in Sweden, A second sample from a proximal site is chosen to detect the length-dependent cutaneous nerve fiber loss, a typical feature of axonal polyneuropathy.In most peripheral neuropathy studies, skin biopsies are obtained from the distal part of the leg  and, in some cases, also from the upper lateral aspect of the thigh (20cm below the anterior iliac spine). in 2010, established guidelines for the quantification of intraepidermal nerve fibers and recommendations for normative values. Quality control measures are recommended at all stages of processing to ensure those who will read the skin biopsy results have the best conditions to do it. Since laboratory techniques are generally not automated, artifacts and errors can occur in the processes of cutting, staining, or interpreting and counting. Intra- and interobserver measurements and inter-laboratory quality control are recommended periodically.A joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society, among others. In some cases, cutaneous vasculitis may be demonstrated.-14 Non-length-dependent values of nerve fiber density may also indicate the occurrence of sensory ganglionopathy (paraneoplastic or associated with Sjögren syndrome). In -48A skin biopsy to quantify intraepidermal nerve fibers may be considered for diagnosis in patients with symptoms suggestive of small-fiber neuropathy, and in many patients with neuropathic pain. Considering the degeneration of small nerve fibers cannot be detected by routine electrophysiological tests, this diagnosis is often difficult to establish in clinical practice. A skin biopsy does not reveal, in most cases, the etiology of the small-fiber neuropathy, but this diagnosis often promotes an earlier investigation of its causes, such as diabetes or intolerance to glucose, Intraepidermal nerve fiber densities were reduced in type 2 diabetic patients when compared to age-matched controls, and a correlation has also been shown with increased heat perception thresholds, and reduced sural sensory nerve amplitudes. An inverse association between intraepidermal nerve fiber density and severity of neuropathy has also been demonstrated.,18A number of studies have demonstrated small-diameter nerve fibers impairment in skin biopsy specimens from both diabetic patients and experimental diabetic animals. Prospective studies showed diabetes induced early degeneration of skin nerve fibers correlated with the duration of the disease.,20There is strong evidence of reduced intraepidermal nerve fiber density in patients with glucose intolerance. Intraepidermal nerve fiber density is also reduced in patients with glucose intolerance neuropathy with normal nerve conduction recordings. Therefore, quantification of intraepidermal nerve fibers could be used both for early detection of diabetic neuropathy or to assess its progression in trials and in clinical practice.,52There is an indication that lifestyle interventions can improve nerve fiber density, but this needs to be confirmed in a larger sample.,53Patients with a clinical diagnosis of idiopathic small-fiber neuropathy often present with painful burning feet. On clinical examination, decreased sensitivity to pain stimuli can be found, whereas proprioception, strength, tendon reflexes and nerve conduction are normal. The skin biopsy usually shows decreased distal intraepidermal nerve fiber density with normal proximal nerve fiber densities, indicating that this condition is typically length dependent. A skin biopsy with determination of intraepidermal nerve fiber density was also considered more sensitive than the quantitative sudomotor axonal reflex test, or the quantitative sensitivity test in the diagnosis of small fiber neuropathy.,26Repeated skin biopsies from patients with idiopathic small-fiber sensory neuropathy, with duration of 12 to 28 months, have shown a decrease in the intraepidermal nerve fiber density in the legs and, therefore, a progressive nerve fiber degeneration in this condition.-56 In patients with ganglionopathy, skin biopsies show nerve fiber loss in all sites without the proximal-distal gradient found in distal sensory polyneuropathy.Patients with sensory ganglion degeneration typically present with sensory ataxia symptoms and loss of conscious proprioception. The distribution of the sensory dysfunction is usually asymmetric and generalized.,58 Two different subtypes have been defined: one primarily associated with the HIV infection , and the other associated with the drug-specific neurotoxicity of the antiretroviral drugs . Intraepidermal nerve fiber density was reduced in a length-dependent manner in HIV-infected patients with neuropathic signs on physical examination, independently of neuropathic symptoms. Skin biopsies have become part of the diagnostic investigation of patients infected intravenously and was also applied in clinical trials on nerve growth factors.,60HIV infection is a known cause of painful neuropathy with predominant involvement of small-diameter nerve fibers.,61,42 familial dysautonomia, and congenital insensitivity to pain. In these conditions, the depletion of intraepidermal nerve fibers has been demonstrated to be more or less extensive. Similarly, in patients with Friedreich’s ataxia, which initially causes degeneration of the dorsal root ganglia and large-caliber myelinated fibers, skin biopsy revealed involvement of small-diameter nerve fibers in the pathological process.Skin biopsy has been studied in patients with hereditary neuropathies known to have small-diameter nerve fiber involvement, such as Fabry disease,,37 as previously demonstrated in sural nerve biopsies. Shortening of internodal length was recently described in dermal myelinated fibers of Charcot-Marie-Tooth 1A patients.Charcot-Marie-Tooth neuropathy is caused by mutations in myelin proteins, but skin biopsy with evaluation of dermal myelinated nerve fibers showed similar abnormalities in the molecular architecture of the axolemma,-65Recent studies have shown small-diameter nerve fibers involvement in biopsy specimens, with decreased intraepidermal nerve fiber densities in systemic lupus erythematosus (SLE). such as immunoglobulin deposition on the nerve surface and activation of the dermal vascular endothelium, leading to a decrease in the fiber density of the intraepidermal unmyelinated nerves.,65Pathogenesis of peripheral nervous system involvement in SLE is poorly understood, and different mechanisms are implicated in length-dependent small-fiber neuropathy, This adds further support to the concept that a pure neuropathy of small diameter nerve fibers can occur in SLE.A current investigation involving pain symptoms and decreased sensation of warmth in the feet of SLE patients has shown that more than a third of patients require a skin biopsy to diagnose small-fiber neuropathy, since conventional neurological examinations for neuropathy have not been shown to be sufficiently effective.,67 and it has been suggested that quantifying intraepidermal nerve fibers in skin lesions may serve as an additional tool, along with skin smear examination and nerve conduction studies, to increase diagnostic efficacy in the detection of neuropathy in leprosy control programs. Small-fiber neuropathy has been described in patients with hypothyroidism and has also been demonstrated by skin biopsy. In a recently published case report, clinical and neuropathological recovery occurred after replacement therapy.Skin biopsy has also been used to demonstrate small-diameter nerve fiber degeneration in a number of conditions other than those mentioned above. In leprosy, cutaneous nerve fibers are typically affected, Intraepidermal nerve fiber loss has also been reported in patients with Ross syndrome , erythromelalgia, type 1 complex regional pain syndrome,,74 and late-onset restless legs syndrome.,76 In restless legs syndrome it has been suggested that hyperactivity of spinal structures in this condition may be triggered by nociceptive deafferentation in a subgroup of patients with painful polyneuropathy. Skin biopsies have also been used in Parkinson’s disease to demonstrate degeneration of autonomic nerves in blood vessels, sweat glands, and erector pili muscles. A neuropathy with predominant small-fiber involvement may be a potential complication of celiac disease. Patients improved with a gluten-free diet.Chemotherapeutic agents produce acute neurotoxicity, chronic neuropathy, and painful neuropathy with skin denervation.Unexpectedly, in patients with motor neuron disease, it has also been shown that amyotrophic lateral sclerosis (ALS) reduces intraepidermal nerve fiber densities, indicating the presence of a distal and sensory axonopathy in ALS.Skin biopsy for the investigation of intraepidermal nerve fibers is a technique that has made it possible, in a relatively simple way, to visualize small-diameter unmyelinated fibers and, therefore, to evaluate small-fiber neuropathy in a more objective way than the quantitative sensory testing and thermal thresholds.diabetes mellitus and glucose intolerance, in connective tissue diseases, paraneoplastic diseases, thyroid disorders, HIV, leprosy, celiac disease, restless legs syndrome, exposure to neurotoxic drugs, hereditary diseases, amyotrophic lateral sclerosis, among other underlying causes of small-fiber neuropathies. Skin biopsy has its role in differentiating between length-dependent and non-length-dependent small-fiber neuropathies, which helps in the differential diagnosis of peripheral neuropathies, narrowing the etiological possibilities, therefore avoiding unnecessary investigation. The causes of ganglionopathy have a smaller differential range  than distal axonopathy, so the pattern aids in a narrower etiologic diagnosis, without, however, determining the causes of the ganglionopathy. The etiological investigation of small-fiber neuropathies with targeted complementary exams is important to allow the identification of treatable diseases. Skin biopsies can also be used to identify small fiber involvement in mixed neuropathies and for follow-up studies.The test contributes to earlier detection of neuropathy in diseases with dysfunctional glucose metabolism, such as


Evidence supporting clinical recommendations or approval for less invasive surfactant administration (LISA) has primarily examined heterogeneous or small-volume  animal-derived surfactant regimens. To address the evidence gap for larger-volume  animal-derived surfactants, the aim of this review was to evaluate and summarize LISA literature for widely used larger-volume beractant. Surfactant treatment and the LISA technique were initially summarized. The available literature on beractant with LISA was thoroughly assessed and reviewed, including a recent systematic analysis, studies from regions where access or preferences may influence reliance on larger-volume surfactants, and investigations of short- and long-term outcomes. The available literature indicated improved short-term outcomes, including less need for mechanical ventilation, death, or bronchopulmonary dysplasia, and no negative long-term developmental outcomes when beractant was administered via LISA compared with older, more invasive techniques. The rates of short-term outcomes were similar to those previously observed in examinations of LISA with small-volume surfactants, including in populations reflecting very preterm infants. As uptake of LISA is expected to increase, future research directions for larger-volume surfactants include cost-effectiveness evaluations and robust examinations of repeat dosing and surfactant reflux to further inform clinical practice. This review provides a detailed assessment of the literature describing surfactant and LISA, with a focus on studies of beractant. Collectively, the available evidence supports the use of beractant with LISA based both on short-term and long-term outcomes relative to more invasive techniques and comparability of outcomes with small-volume surfactants and may be valuable in guiding clinical decision-making. Respiratory distress syndrome (RDS) commonly affects premature infants born with surfactant deficiency resulting from inadequate lung development  and may affect up to 80% of extremely preterm infants ≤28 wGA) ,2. Early8 wGA ,2.Consequently, surfactant administration techniques have evolved to avoid prolonged exposure to mechanical ventilation and reduce the risk of adverse outcomes such as BPD ,5. The iInstead of an endotracheal tube, the LISA technique primarily uses a thin and soft catheter inserted into the trachea, permitting spontaneous breathing while receiving noninvasive respiratory support and avoiding the use of invasive and potentially harmful ventilation methods ,10. AccoThe European Consensus Guidelines recommend treating infants with RDS early with an animal-derived surfactant and identify LISA as the optimal method of surfactant administration, provided that clinicians are experienced with the technique . These rThe aim of the current literature review is to address this evidence gap by aggregating and evaluating available clinical evidence from published studies of beractant with LISA to determine whether the benefits of LISA are observed with larger volume surfactants. Here, we review beractant’s clinical characteristics and its outcomes (not specific to LISA), in the context of another commonly used animal-derived surfactant. We also summarize available clinical evidence supporting the use of LISA over more invasive techniques and address the significant knowledge gap specific to LISA with larger volume surfactants by presenting a meta-analysis of key clinical outcomes for beractant administration with LISA (versus more invasive techniques) and reviewing other available literature describing LISA outcomes with larger volume surfactants. Lastly, we review additional evidence gaps and considerations related to LISA with larger volume surfactants and address likely future developments in the field. The authors hope that their systematic assessment of available evidence will help to inform clinical practice and policy decisions regarding surfactant administration in neonates with RDS.Beractant treatment for RDSBeractant is a natural bovine-lung extract with a phospholipid concentration of 25 mg/mL  and is aBeractant and another commonly used surfactant, poractant alfa, differ in phospholipid concentration and consequently dosing characteristics. Poractant alfa is a porcine lung extract with a phospholipid concentration of 74-76 mg/mL administered in a dose of 100 mg/kg (in a volume of 1.25 mL/kg) or in a dose of 200 mg/kg (in a volume of 2.5 mL/kg) , which mPrevious systematic evaluations of randomized controlled trials and real-world evidence have shown comparable outcomes for beractant and other commonly used animal-derived surfactants ,32-34, aLISA: Past clinical evidence and current research gapThe LISA technique for surfactant administration employs a small catheter placed in the trachea under direct laryngoscopy, eliminating the need for an endotracheal tube and intubation, which are inherent in the INSURE or standard-of-care techniques. This guideline-recommended technique minimizes the need for mechanical ventilation, which is required during intubation, and can cause lung injury and increase the risk of BPD, with potentially lasting consequences ,5,6.Systematic literature reviews and meta-analyses comparing the LISA technique with mechanical ventilation, INSURE, or nasal CPAP alone have shown that LISA is associated with significantly improved outcomes for BPD or death and a reduced need for mechanical ventilation within 72 hours of birth -20,36,37A major gap in the body of evidence regarding the LISA method was the inclusion of multiple different surfactants that varied in sources and dose volumes ,45, thusBeractant and LISA: an evidence updateSystematic Evaluation of Mechanical Ventilation, BPD, and DeathA comprehensive, systematic evaluation of beractant outcomes with LISA was recently presented at the Pediatric Academic Societies (PAS) 2021 meeting . This reAmong the seven studies included in the analysis Table , two werOverall, none of the four outcomes examined favored INSURE or standard of care compared to LISA in this systematic evaluation, and the results of fixed effects and random effect modeling were comparable for the four extracted outcomes . The resAdditional Studies of Mechanical Ventilation, BPD, and DeathIn addition to the studies included in the above systematic evaluation , severalLong-Term Developmental OutcomesOne study  examined long-term developmental outcomes in infants treated with beractant and LISA. This single-center retrospective chart review of 60 preterm infants (<32 wGA) on CPAP during the first three days of life observed no significant differences in the rates of acute respiratory failure admissions, hearing loss defects, and neurological disorders between the infants treated with beractant and LISA (n = 30) versus INSURE (n = 30) during a long-term follow-up of up to 24 months of postmenstrual age . Taken tEvidence gaps and future directionsA cost-effectiveness evaluation of preterm infants with RDS observed a shorter length of NICU stay and lower total newborn intensive care unit (NICU) costs for beractant compared to other commonly used animal-derived surfactants at 209 US hospitals  . HoweverAdditional studies are also needed to more robustly examine repeat dosing with LISA, which typically involves tracheal visualization to guide the surfactant administration and may consequently impact the clinical outcomes, especially when a repeat surfactant administration is needed. In a historical cohort study of 324 infants, patients receiving surfactant with LISA required more doses of surfactant but ultimately needed less mechanical ventilation than those receiving surfactant with intubation and had other improved outcomes . With a Surfactant reflux is another outcome warranting more robust research since some groups have observed increased instances of reflux or bradycardia with LISA compared to INSURE when examining various types of surfactants ,19,36. OFinally, as the LISA technique has become more widely used in clinical practice ,12,45, tToday, there is good evidence supporting the recommendation of the LISA technique as an alternative to standard or INSURE methods of surfactant administration. As most of the recent studies analyzing the potential benefits of the LISA have been done with small-volume surfactants , some doubts could exist when LISA is performed with larger surfactant volumes. Therefore, the main objective of this review was to summarize the evidence for the effectiveness and safety of LISA administration with a larger-volume surfactant , compared to its administration with INSURE or standard-of-care techniques. When considered collectively, the available evidence summarized in this review supports the use of LISA with the larger-volume beractant.The first systematic evaluation of studies investigating the key clinical outcomes following beractant administration using the LISA technique clearly demonstrated, despite the differences in source studies, better outcomes with beractant administered via LISA vs INSURE or standard of care. Further evidence from additional studies conducted in various Asian countries further confirms the results of the systematic evaluation. The results of this review extend from previous reports and support the clinical use of beractant administered with LISA by demonstrating that this approach results in fewer adverse outcomes for preterm infants with RDS when compared with INSURE or standard of care. Importantly, the bovine-derived surfactant is included in the World Health Organization Model Lists of Essential Medicines for neonates. By focusing on this frequently used surfactant therapy, this review provides a summary of administration methods without potential confounding factors introduced by multiple surfactants from different animal sources and different doses of phospholipids. While the LISA method may eventually become replaced by surfactant nebulization or other noninvasive techniques, it is being increasingly adopted in current practice, in accordance with European treatment guidelines, with training resulting in good proficiency and comfort level among trained practitioners. We hope that the findings of this review will help guide clinical management decisions and improve patient outcomes in preterm infants with RDS and that they will be particularly informative to practitioners in regions where beractant and other larger-volume surfactants are predominantly used and access to other surfactants may be limited.


Disrupting Epileptiform Activity by Preventing Parvalbumin InterneuronDepolarization BlockJ Neurosci. 2021;41(45):9452-9465.doi:10.1523/JNEUROSCI.1002-20.2021. Epub 2021 Oct 5.Călin A, Ilie AS, Akerman CJ. Inhibitory synaptic mechanisms oppose epileptic network activity in the brain. Thebreakdown in this inhibitory restraint and propagation of seizure activity has beenlinked to the overwhelming of feedforward inhibition, which is provided in large partby parvalbumin-expressing (PV) interneurons in the cortex. The underlying cellularprocesses therefore represent potential targets for understanding and preventing thepropagation of seizure activity. Here we use an optogenetic strategy to test thehypothesis that depolarization block in PV interneurons is a significant factor duringthe loss of inhibitory restraint. Depolarization block results from the inactivationof voltage-gated sodium channels and leads to impaired action potential firing. Weused focal NMDA stimulation to elicit reproducible epileptiform discharges inhippocampal organotypic brain slices from male and female mice and combined this withtargeted recordings from defined neuronal populations. Simultaneous patch-clamprecordings from PV interneurons and pyramidal neurons revealed epileptiform activitythat was associated with an overwhelming of inhibitory synaptic mechanisms and theemergence of a partial, and then complete, depolarization block in PV interneurons. Tocounteract this depolarization block, we developed protocols for eliciting pulsedmembrane hyperpolarization via the inhibitory opsin, archaerhodopsin. This opticalapproach was effective in counteracting cumulative inactivation of voltage-gatedchannels, maintaining PV interneuron action potential firing properties during theinhibitory restraint period, and reducing the probability of initiating epileptiformactivity. These experiments support the idea that depolarization block is a point ofweakness in feedforward inhibitory synaptic mechanisms and represents a target forpreventing the initiation and spread of seizure activity. It is well known that inhibitory activity restrains overexcitation, and that failureof this protective mechanism is central to seizure initiation. There are several reasons, still debated, why inhibitory restraint can fail. Onepotential mechanism is that accumulation of chloride ions in neurons innervated by intenselyfiring interneurons shifts the reversal potential for GABA (EGABA) in adepolarizing direction, thereby attenuating the inhibitory action of GABA or even convertingit to excitation. A further consequence of intracellular chloride accumulation is that itleads to an outward flux of potassium ions carried by the potassium-chloride co-transporter.Accumulation of extracellular potassium can then lead to depolarization of excitatoryneurons. A further potential mechanism is that interneurons themselves are over-depolarized,because they receive an intense barrage of glutamatergic excitation, possibly exacerbated bythe effect of extracellular potassium accumulation. Such over-depolarization prevents interneurons from firing repetitively becausesodium channels eventually become inactivated, a phenomenon known as ‘depolarization block’,and as a consequence GABA release fails. When depolarization block occurs, further actionpotentials cannot be triggered and thus interneurons become silent, leading to an escape ofexcitatory neurons from the inhibitory restraint. This might result in the triggering of theseizure. Several studies have provided evidence, in different pre-clinical models, for eachof these mechanisms, which are not mutually exclusive. However, a direct test of causality for any of these hypotheses is not trivial.Epilepsy is characterised by seizures, and therefore a deep understanding of the mechanismsof seizure initiation and propagation is pivotal for the field. The role of inhibitoryneurons in seizure initiation is among the most intensely studied. They used an established in vitro model based on NMDA-evoked epileptiform discharges(EDs) in organotypic hippocampal cultures. This model, which preserves aspects of theanatomy of the hippocampus, allows repeated EDs to be elicited. Călin et al performed both voltage and current clamp recording in PV interneurons andpyramidal cells in the CA1 sub-field, while pressure-applying NMDA to CA3.In their recent publication, Călin and colleagues used an elegant experimental design todetermine whether depolarization block of parvalbumin-positive (PV) interneuron accompaniesseizure initiation. This is accompanied by a decrease in both the frequency and the amplitude of actionpotentials recorded from PV interneurons, consistent with impending depolarization block. To test whether the decrease in spike frequency and amplitude was indeed because ofover-depolarization, Călin et al expressed the hyperpolarizing opsin archaerhodopsin (Arch).Trains of short light pulses designed to hyperpolarize the membrane potential, and therebyrelease sodium channels from inactivation, during the pre-ED period led to an increase in spike frequency and amplitude,consistent with rescue from depolarization block. Furthermore, this manipulation wassufficient to decrease the probability of initiation of EDs without interfering with theironset delay and morphology. This observation suggests that depolarization block of PV positive interneurons inthe critical period before an ictal event is indeed a mechanism by which the inhibitoryconstraint can fail, thereby triggering seizure initiation.Firstly, they showed that, while NMDA is applied, a pre-ED period can be detected duringwhich inhibitory constraint is still active, but that this fades away before the initiationof a full-blown ED. The importance of the advance reported by Călin and colleagues is that it highlightsthe potential to tailor future treatments to counteract depolarization block in interneuronswith spatial and temporal control, for example by using closed-loop optogenetics or otheradvanced manipulations. Another potential therapeutic avenue may be to use gene or RNAtherapy to overexpress sodium channel splice variants with faster recovery from inactivation. This latter approach would have to be targeted specifically to interneurons, andwould need to be studied closely to determine whether they could interfere withphysiological interneuron activity. For example, although a SCN1A splicevariant with faster recovery from inactivation has been lost during evolution, probably toprotect from a gain of function effect, in the case of a pathological hyperactivitycondition such as epilepsy, its reintroduction in interneurons may be therapeutic.This study is arguably the most direct test of causality available, to show howdepolarization block in PV expressing interneurons can lead to seizure initiation. This hadbeen postulated and tested in previous studies, but never directly., a model of chronic epilepsy with spontaneous seizures is necessary,with all the technical difficulties of recording and manipulating membrane potentials in theintact brain. A systematic experimental design to test different hypotheses underlining thefailure of inhibitory restraint in the same in vivo model could be a significant stepforward in the field. Nevertheless, the work by Călin and colleagues opens new avenues forthe understanding of epilepsy and seizure initiation, as well as for developing newpotential therapeutic approaches to stop seizure initiation and spreading.Depolarization block of PV-positive interneurons may not be the only event occurring in thelead-up to seizure initiation, and it will be important to understand how it interacts withother mechanisms. A potential limitation of the study by Călin et al is that the data wereobtained in a model of acutely evoked seizures in vitro. To be verified invivo


Swine are popular large mammals for cardiac preclinical testing due to their similarities with humans in terms of organ size and physiology. Recent studies indicate an early neonatal regenerative capacity for swine hearts similar to small mammal laboratory models such as rodents, inspiring exciting possibilities for studying cardiac regeneration with the goal of improved clinical translation to humans. However, while swine hearts are anatomically similar to humans, fundamental differences exist in growth mechanisms, nucleation, and the maturation of pig cardiomyocytes, which could present difficulties for the translation of preclinical findings in swine to human therapeutics. In this review, we discuss the maturational dynamics of pig cardiomyocytes and their capacity for proliferative cardiac regeneration during early neonatal development to provide a perspective on swine as a preclinical model for developing cardiac gene- and cell-based regenerative therapeutics. Heart failure is a highly prevalent disease in humans with no effective long-term treatment strategy beyond organ transplantation . Loss ofSwine are widely utilized as a cardiac preclinical model, especially for testing surgical and mechanical interventions for heart disease . At a grCardiac regeneration is the formation of new muscle to replace damaged muscle, via the proliferation of cardiomyocytes with minimal fibrotic scarring. While adult human heart regeneration remains elusive, the mechanisms underlying the regenerative potential of the heart can be elucidated by studying laboratory and preclinical model systems. Nonmammalian ectothermic vertebrates, such as zebrafish, newts, and axolotls, can regenerate their hearts after injury via cardiomyocyte proliferation throughout life, but adult endothermic mammals do not have this capacity . HoweverIn humans, functional recovery following myocardial infarction soon after birth has been described anecdotally, hinting at a potential for early neonatal heart regenerative capacity ,9. LimitStudying porcine models of heart regeneration could offer insight into human postnatal cardiac regenerative potential and facilitate development of effective therapeutics for human cardiac regenerative repair. As a large mammal model, juvenile pigs  have been frequently utilized to test cardiac regenerative therapies for clinical translation. However, differences in cardiomyocyte growth characteristics in swine, such as extensive multinucleation and ongoing cardiomyocyte mitotic activity extending well into adolescence could be distinctive to pigs , which mPig hearts undergo rapid growth to nearly triple in size in the first month after birth . Pig carPig cardiomyocytes grow longitudinally relative to nucleation before transitioning to a diametric mode of hypertrophic growth at 4–6 postnatal months, when cell cycle withdrawal finally occurs. However, cytokinetic arrest occurs in the first week after birth in porcine cardiomyocytes, similar to rodents, with most of the persisting mitotic activity resulting in extensive multinucleation. Pig cardiomyocytes exhibit four or more nuclei per cardiomyocyte beyond two postnatal weeks, and about 60–90% of cardiomyocytes are multinucleated in juvenile pigs that are commonly utilized for preclinical cardiac studies . These dMammalian cardiac regeneration was first reported in mice where regenerative repair without scarring was observed when cardiac injury occurred before P7, but injury in older mice resulted in scar formation and compromised heart function . SimilarAltogether, these reports indicate that there exists a shared early neonatal window of innate cardiac regenerative capacity for the first few days after birth in pigs and mice. During this time, injury to the heart results in regenerative healing and functional recovery without scar formation. However, in both model systems, this ability is lost beyond the first few days after birth. Comparing postnatal maturational and regenerative mechanisms between the model systems could facilitate an understanding of key processes that regulate mammalian cardiac regenerative potential.Some of the hallmark characteristics of terminal cardiomyocyte maturation as determined in rodents are: cardiomyocyte mitotic arrest, switch to hypertrophic mode of growth via increased cardiomyocyte cross-sectional size, sarcomeric maturation with switch to adult contractile protein isoforms, and a decline of mononucleated-diploid cardiomyocytes ,16. ThesA significant presence of mononucleated-diploid cardiomyocytes has been highlighted as imperative for cardiomyocyte proliferation and hyperplastic growth during early postnatal development in rodents. In addition, cardiac regenerative capacity in rodents is dependent on the presence of a proliferative mononucleated-diploid cardiomyocyte population . FurtherEct2  and Lmnb2 (Lamin B2) were significantly downregulated by P7 in pig hearts. Both genes are critical for successful proliferation, the depletion of which results in the onset of polyploidy in rodent cardiomyocytes [In neonatal pigs, while mitotic activity remains high at P7, cytokinesis-specific genes are downregulated at P7 compared to P0 by RNA analysis . Interesmyocytes ,20. Thusmyocytes , all of myocytes ,7, similStudying such shared processes, including via the direct comparison of existing sequencing datasets during neonatal development, could facilitate the identification of new target genes and pathways critical for cardiomyocyte proliferation and heart regeneration . UnderstVarious therapeutic avenues have been explored utilizing swine as a large mammal model for human heart regenerative repair. In preclinical studies, porcine myocardial infarct models are utilized to cause rapid cardiomyocyte death in one region of the heart. The established methods for inducing myocardial infarction in pigs are: ischemia/reperfusion by temporary coronary occlusion ,21, ischFactors involved in cardiomyocyte growth and development have been manipulated to assess preclinical therapeutic efficacy in the hearts of young swine after cardiac injury. The extracellular matrix protein agrin promotes cardiac regeneration in neonatal mice, and the potential benefit of local administration of agrin by retrograde delivery in coronary arteries after myocardial infarction (MI) was tested in three-month-old pigs ,24. A siMultiple genetic mechanisms have been demonstrated to promote adult cardiomyocyte proliferation in mice. Recent efforts have been directed towards determining if these mechanisms are effective in promoting cardiomyocyte renewal in young adult pigs after injury. The development of adeno-associated virus (AAV) serotypes, such as AAV6 and AAV9, which infect porcine cardiomyocytes, has facilitated these gene therapy approaches that potentially could be translated to human patients.MicroRNAs (miR) have been manipulated to induce cardiac regeneration in rodents , and miRSav-shRNA was injected into the border zone of infarcted three-month-old pig hearts. At three months post-injection, AAV9-mediated Sav knockdown hearts exhibited improved cardiac function, less scarring, and improved vascularity compared to vector-injected controls. In pigs analyzed at 1-month post-treatment, mitotic activity assessed by EdU incorporation and pHH3 staining in cardiomyocytes, together with sarcomere breakdown and Aurora B kinase indicative of cytokinesis, were increased, providing evidence for cardiomyocyte proliferation and renewal after injury. Together, these studies support the possibility of an AAV9-mediated gene therapy approach to promote cardiomyocyte renewal in adult pig hearts through the targeted inhibition of the Hippo pathway.Among the most effective ways to stimulate cardiomyocyte proliferation in neonatal and adult rodents is inactivation of the Hippo signaling pathway which controls organ size . The pot9) of human embryonic stem cell (hESC)-derived cardiomyocytes post-MI [Multiple stem/progenitor cell therapy approaches for cardiac regenerative repair have been tested in various mammalian model systems. The beneficial effects on cardiac recovery in most of these studies likely stem from paracrine or anti-inflammatory effects that promote cardiomyocyte survival and function after injury . In one  post-MI . Despite post-MI . Another post-MI . Success post-MI . HoweverThe regeneration of myocardium with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has long been a goal in the field. However, the immature phenotype together with limited proliferative capacity of iPSC-CMs have so far impeded their use for in vivo cardiac therapeutics . A stratde novo generation of cardiomyocytes in pigs is limited by the lack of genetic reporters and lineage-tracing tools available in mice [de novo generation of cardiomyocytes. Even when cytokinetic assays are performed, the identification of dividing cardiomyocytes with characteristic post-abscission midbodies indicative of successful cytokinesis can be challenging, especially in histological sections. An additional caveat of cardiac regenerative studies in pigs and rodents is that the robust induction of cardiomyocyte cell cycle activity can lead to cardiac dysfunction in the long term [Definitive assessment of  in mice . Thus, aong term ,38. ThusThe multinucleation and growth characteristics of cardiomyocytes are very different among mammals, including mice, pigs, and humans ,18. TypiDespite such challenges, several of the regenerative therapeutics that have been preclinically tested in swine do provide evidence of beneficial effects on cardiac performance and myocardial integrity after cardiac injury  21,24,2,36. TreaAt the organ level, human and pig cardiac physiology is sufficiently similar for the preclinical testing of mechanical or surgical innovations for treating human heart disease. Due to the great similarities in the anatomy and function of juvenile swine and human hearts, such as similar heart size, body weight, and cardiac load, swine are often utilized for preclinical tests of biomechanical devices, with great success . FurtherPigs have been popular as preclinical models for investigation of new therapies for translation to human disease. However, despite an increasing number of reports of successful preclinical testing of drugs, tissue-engineered patches, AAV-mediated gene therapies, and cell-based therapies designed to promote cardiac repair after injury, none of these approaches have been safely and efficaciously translated into the clinic for healing the human heart. In this review, we discuss the proliferative and regenerative potential of cardiomyocytes in neonatal pigs alongside preclinical therapeutic strategies for inducing cardiac regenerative repair in juvenile pig injury models. While pigs and rodents exhibit a similar early neonatal period of transient regenerative capacity, an early neonatal window of cardiac regenerative capacity in human infants has not been definitively demonstrated. However, there is potential for the discovery of novel factors for inducing cardiac regeneration in humans using the porcine model, especially when taken together with studies in rodents.de novo cardiomyocyte generation, as opposed to pleiotropic beneficial effects that result in cardiac functional recovery post-injury, is necessary to accurately interpret cardiac regenerative strategies in swine for clinical translation to human disease. Still, pigs remain an attractive model for preclinical surgical and interventional approaches due to their similarities with human hearts in terms of anatomy and physiology. With the advent of gene targeting and recent advances in xenotransplantation, the use of the pig system for studies of cardiac regeneration and repair is likely to accelerate in the years to come.A limitation of the pig model is that cardiomyocyte maturational dynamics and growth mechanisms are distinct in swine compared to other mammals studied so far. Unlike humans or rodents, mature cardiomyocytes in juvenile and adult swine are highly multinucleated, which could limit the translation of results when older swine are utilized as preclinical models for regenerative therapeutic strategies. Thus, increased rigor in identifying


Arterial stiffness (AS) is one of the earliest detectable signs of structural and functional alterations of the vessel wall and an independent predictor of cardiovascular events and death. The emerging field of metabolomics can be utilized to detect a wide spectrum of intermediates and products of metabolism in body fluids that can be involved in the pathogenesis of AS. Research over the past decade has reinforced this idea by linking AS to circulating acylcarnitines, glycerophospholipids, sphingolipids, and amino acids, among other metabolite species. Some of these metabolites influence AS through traditional cardiovascular risk factors , while others seem to act independently through both known and unknown pathophysiological mechanisms. We propose the term ‘arteriometabolomics’ to indicate the research that applies metabolomics methods to study AS. The ‘arteriometabolomics’ approach has the potential to allow more personalized cardiovascular risk stratification, disease monitoring, and treatment selection. One of its major goals is to uncover the causal metabolic pathways of AS. Such pathways could represent valuable treatment targets in vascular ageing. Blood pressure (BP), traditional blood lipid biomarkers , and glucose are widely used in everyday clinical practice for cardiovascular (CV) risk assessment. Unfortunately, the classical biomarkers can only partially explain the distribution of CV risk in the general population. Many metabolic, inflammatory, and hemodynamic drivers of the residual CV risk are thus missed, and a more tailored approach to CV risk stratification is clearly needed. Currently, doctors may be misled by normal cholesterol and glucose blood levels when assessing CV risk in patients whose CV health can also be affected by quite different metabolite species. Prehypertensive patients with increased arterial stiffness (AS) may be unfairly withheld from antihypertensive treatment, and those with excellent vascular function may be overtreated. To overcome these clinical uncertainties, more randomized controlled trials (RCTs) looking beyond traditional CV risk factors are needed.Recent large-scale RCTs have partially addressed residual inflammatory CV risk by targeting tubuline polymerization ,2 and neTo date, residual metabolic CV risk has been addressed mainly in observational and laboratory research. These studies have often implemented metabolomics, which is an emerging analytical profiling technique ,8,9. An AS describes the rigidity of the arterial wall, i.e., the capability of an artery to expand and contract in response to pressure changes. The pressure load of each heartbeat in large conduit vessels is borne mainly by two extracellular matrix proteins: elastin and collagen. An increase in the collagen/elastin ratio during the ageing process is a classical determinant of AS . Other mAS is an important determinant of target organ damage and an independent CV risk factor. There is strong evidence that aortic stiffness, measured as carotid–femoral pulse wave velocity (cf-PWV), independently predicts CV risk ,27 and aAnother reflector of arterial function is the arterial pressure waveform  Figure . It is aThe methodologies used to determine AS fall under five categories: (1) devices that use a probe or tonometer to record the pulse wave with a transducer, (2) devices using cuffs placed around the limbs or the neck, which record the arrival of the pulse wave oscillometrically, (3) ultrasonography approaches, (4) magnetic-resonance-imaging-based approaches, and (5) invasive measurements with pressure catheters. These methods differ in their applicability, strengths, and limitations, which have been addressed in detail in previous reviews ,36.To date, AS assessment has primarily been used for research purposes. Although the independent prognostic value of cf-PWV is now overwhelming, it still remains to be proven whether its measurement truly facilitates clinical decisions. More large-scale trials comparing clinical approaches with and without cf-PWV monitoring are needed. Development of pharmacological agents that directly target AS would also help to pave its way into clinical practice. Current therapeutic options that ameliorate AS include antihypertensives  inhibitors , calciumMetabolomics is a field of science that combines high-throughput analytical techniques with bioinformatics and focuses on chemical processes involving LMWMs (<1500 Da), including amino acids (AAs), peptides, lipids, carbohydrates, and nucleic and fatty acids (FAs). It reflects changes in the genome, transcriptome, and proteome and thus represents the endpoint of the ‘omics’ cascade ,46. The Two distinct approaches, targeted and untargeted metabolomics, can be followed to analyze a set of LMWMs in biofluids or tissues. The aim of the targeted approach is to quantify only a preselected set of known LMWMs based on internal or external standards . This apTo carry out a high-quality metabolomics study, the following steps are usually followed: experimental design, sample collection and extraction, data acquisition, and data analysis and interpretation. Accordingly, (1) a targeted or nontargeted animal or human study is designed; (2) samples of blood, urine, tissue extract, or some other type of biological specimen are collected; (3) mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, or infrared spectroscopy are employed; and (4) uni-, bi-, or multivariate statistical methods, or a combination of these are used. The two most common analytical platforms for data acquisition and processing in metabolomics are MS and NMR spectroscopy . The NMRBecause of the inherent sensitivity to even subtle changes in the actual phenotype, metabolomics is steadily gaining popularity in different areas of medical research. Altered lipid metabolism in CV disease (CVD), in particular, has been the focus of a number of recent studies ,56. Meta‘Arteriometabolomics’ involves identification and quantification of thousands of LMWMs, metabolic pathways, and their interactions with arterial structural and functional parameters. Current CV risk assessment relies on traditional risk markers , but analytical metabolomics methods and AS assessment offer an opportunity to further interrogate the human metabolome and vascular system . The linL-carnitine is an endogenous vitamin-like molecule involved in lipid metabolism, synthesized in vivo ) and supplemented by diet  . When caSubclinical vascular effects of carnitine deficiency and supplementation have been previously studied. Carnitine attenuates free FA-induced ED and modulates platelet activation by limiting the production of endogenous reactive oxygen species (ROS) . StudiesP-cresyl sulfate [Interestingly, L-carnitine supplementation may increase the blood levels of a gut microbe-dependent metabolite trimethylamine N-oxide (TMAO) . Dietary sulfate , phenyla sulfate , and equ sulfate ), as wel sulfate ,86,87 haSome recent metabolomics studies link AS to higher circulating levels of ACs. Accumulation of ACs has been associated with cellular stress , inflammPhospholipids are amphiphilic molecules that serve mainly as structural components in cellular membranes, lipoproteins, and natural surfactants, among others . They coThe most abundant phospholipids in cellular membranes and lipoproteins are phosphatidylcholines (PCs) . If a PCTo date, PCs and lysoPCs are probably the most thoroughly studied phospholipids in regard to AS. In an observational study of patients with symptomatic atherosclerosis, we found that lower serum levels of PC-acyl-acyl C32:2 and several lysoPCs  were related to increased AS, increased resting heart rate, or ED . KurotanPolonis et al. identified four down-regulated lysoPCs  that were independently associated with high PWV values in patients with hypertension . PositivSphingolipids and their metabolites are involved in various biological functions and are particularly abundant in tissues of the central nervous system . Like glPrevious studies suggest that sphingolipids may be important mediators of vascular ageing. The sphingolipid precursor Ser was associated with PWV in a large-scale study of female twins by Menni et al. . Ceramid−/− mice, and this was accompanied by complete reversal of aortic intima-media thickening and PWV [Glycosphingolipids are formed when sugars  are sequentially added via specific glycosyltransferases (glucosylceramide synthase (GCS), lactosylceramide synthase (LCS)) to ceramide. Like ceramides, this group of lipids can also be causally involved in AS. In a study by Chatterjee et al., inhibition of GCS and LCS drastically and dose-dependently ameliorated atherosclerosis in ApoE and PWV . Subsequ and PWV . Circula and PWV .SM production from ceramide and PC is catalyzed by SM synthase (SMS). SM concentration is increased in the human atheromatous aorta and coronary vessels ,137. In Acylglycerols are esters formed by esterification of glycerol and one to three fatty acids, yielding monoacylglycerol (MAG), diacylglycerol (DAG), or triacyglycerol (TAG). TAGs are the major source of circulating FAs and energy . A positCEs are formed when cholesterol is esterified with long-chain FAs, a reaction catalyzed by acyl-CoA cholesterol acyltransferase (ACAT-1 and ACAT-2) in the cell  and LCATAAs are organic compounds that contain a basic amino group, a carboxylic acid functional group, and a side chain that determines the characteristics specific to each AA . They arA metabolomics study in symptomatic peripheral arterial disease patients conducted by our group showed associations between PWV and aromatic AAs , tyrosine (Tyr)) . This fiSome derivatives of the aromatic AA tryptophan (Trp) and His have also been shown to correlate with AS. Higher circulating levels of indoxyl sulfate, an uremic toxin that is produced by the metabolism of dietary Trp, showed positive associations with aortic stiffness in patients with CAD  and typeAnother potential target to attenuate vascular ageing is the arginine (Arg) metabolic pathway. Being the substrate for nitric oxide synthase (NOS), Arg has important nitric oxide (NO) dependent vasodilatory and antithrombotic effects but also several NO independent effects that help to maintain and improve vascular health . For theHomocysteine (Hcy) is an intermediate product in the biosynthesis of the AAs Met and cysteine and a key determinant of the methylation cycle. Higher circulating Hcy levels are associated with AS ,181, altTaurine (Tau) is a biogenic amine with antioxidant and vasodilatory effects . SupplemGlucose (Glc) is a monosaccharide that is vital for fueling both aerobic and anaerobic cellular respiration. Dietary Glc comes in different forms such as mono- , di- , or polysaccharides . Excess Glc in the organism is stored as glycogen or converted to fat. These sources help to meet the energy needs during exercise, between meals, and while sleeping. In starvation, Glc can also be derived from the process of gluconeogenesis.A rapid elevation in blood Glc levels after a meal is an independent risk factor for CVD and a greater risk factor than fasting glucose . PreviouUric acid (UA) is a purine derivative and a well established CVD risk marker ,209. HowPolyphenols are naturally occurring compounds found largely in fruits, vegetables, cereals, and beverages . NumerouUrinary caffeine, paraxanthine, and theophylline excretions were associated with decreased PWV in a study by Ponte et al., suggesting a protective effect of caffeine intake beyond its BP-lowering effect . The acuUrine isocitrate (a citric acid cycle intermediate), hydroxymethylglutarate (a precursor to cholesterol and coenzyme Q10 synthesis), and formiminoglutamate (an intermediate in the catabolism of L-His to L-glutamic acid) were independently associated with AS in Korean adults . The resIt is clear from the literature review given above that ‘arteriometabolomics’ is a powerful research tool that allows simultaneous exploration of metabolic and hemodynamic alterations. Combining the increasingly growing knowledge from metabolome analysis with the constantly evolving understanding of AS could lead to the development and validation of novel biomarker panels with a predictive power beyond conventional circulating CVD markers and peripheral BP measurement. In clinical practice, a more detailed overview of subclinical metabolic and hemodynamic  derangements could be most helpful for low to medium CV risk patients in whom traditional biomarkers/parameters give borderline or equivocal results. It could also help monitor disease  progression and treatment efficacy.Metabolic pathways that prove to be causally associated with AS could emerge as novel therapeutic targets for vascular ageing. Destiffening medicines capable of ameliorating the intrinsic elastic properties of the arterial wall, regardless of BP and cardiac output, would further help protect against CV complications . Recently, the concept of supernormal vascular ageing (SUPERNOVA) was proposed . AccordiA thorough understanding of pathophysiology is a prerequisite for the development of novel therapies for any disease. Metabolomics studies in humans have so far largely focused on systemic shifts in metabolism. Significantly less is known about the local features of metabolism in different organs  and regions of the body . The research approach based on arteriovenous gradients (AVGs) assessment isolates local changes in the levels of LMWMs of interest from changes in the systemic circulation and is therefore a more direct reflection of the (patho)physiology of the study region. In such an approach, for any given LMWM, venous/arterial ratio <1 is consistent with net uptake by the organ or region of the body, whereas the ratio >1 suggests net release. AVG assessment has been previously used to study local metabolism in the kidneys of patients with chronic kidney disease ,230 and There is already a significant amount of scientific evidence to support the link between AS and metabolism. Some LMWMs seem to influence the arterial wall through traditional CV risk factors, while others may act independently through both known and unknown pathophysiological mechanisms. The integration of metabolomics methods into arterial wall research broadens our understanding of the pathophysiology of AS and may lead to the discovery of more accurate CV biomarkers and novel destiffening therapies.


For tumors to initiate and sustain rapid growth, they need to alter their metabolic reprograming to support this growth. While many oncogenes upregulate metabolic pathways in cancer, here, we focus on how mutant p53 interacts with the mevalonate pathway (MVA) and functions as a continual on switch, causing tumor cells to grow uncontrollably. The relationship of mutant p53 and the MVA pathway in promoting tumorigenesis and the pleiotropic effects within the tumor microenvironment as a result of MVA pathway upregulation are discussed in this review. Many individuals are prescribed a statin drug to decrease their circulating cholesterol levels by blocking the MVA pathway. Several studies have suggested the potential of repurposing statins to treat various cancers, since tumor cells upregulate and rely substantially on the MVA pathway. We evaluate previous studies on statins and cancer incidence and patient mortality, as well as future research to optimize the use of statins as a cancer therapy.Tumor cells have the ability to co-opt multiple metabolic pathways, enhance glucose uptake and utilize aerobic glycolysis to promote tumorigenesis, which are characteristics constituting an emerging hallmark of cancer. Mutated tumor suppressor and proto-oncogenes are frequently responsible for enhanced metabolic pathway signaling. The link between mutant p53 and the mevalonate (MVA) pathway has been implicated in the advancement of various malignancies, with tumor cells relying heavily on increased MVA signaling to fuel their rapid growth, metastatic spread and development of therapy resistance. Statin drugs inhibit HMG-CoA reductase, the pathway’s rate-limiting enzyme, and as such, have long been studied as a potential anti-cancer therapy. However, whether statins provide additional anti-cancer properties is worthy of debate. Here, we examine retrospective, prospective and pre-clinical studies involving the use of statins in various cancer types, as well as potential issues with statins’ lack of efficacy observed in clinical trials and future considerations for upcoming clinical trials. Normal, non-cancerous cells rely primarily on mitochondrial oxidative phosphorylation to maintain metabolic homeostasis. Under oxygenated, aerobic conditions, cells metabolize glucose through oxidative phosphorylation . For thiDuring anaerobic conditions, there is increased demand for rapid ATP production in order to fuel cells with ample energy due to a lack of oxygen . AlthougThe reprograming of energy metabolism by tumor cells is an established hallmark of cancer . AlteratWhile aerobic glycolysis is primarily favored, tumor cells expand their energy production by involving protein biosynthesis and fatty acid synthesis as well. The mTOR pathway is known for regulating cell growth through facilitating biosynthesis of proteins, lipids and nucleic acids . More spTumorigenic processes, including cell proliferation, migration and angiogenesis, require significant energy, so cancer cells adopt fundamental changes in energy metabolism, nutrient uptake and substrate utilization. Most cancers are characterized by the acquisition of profound mutations in tumor suppressor genes  ,25,26 orMutant p53 has been shown to interact with nuclear SREBP-2 and enhance transcription of genes of the MVA pathway  33]. Ex. Ex33]. The MVA pathway is a complex signaling pathway that is involved in the production of cholesterol, vitamin D, lipoproteins, dolichol, ubiquinone and isoprenoids , and theFurther, the condensation of two farnesyl-PP molecules produces squalene, resulting in the formation of sterols, which are then converted to cholesterol. Rapidly dividing cancer cells require elevated levels of cholesterol to support membrane biogenesis, lipid raft formation within the cell membrane and a number of cancer cell functions, including proliferation, migration and invasion ,42.In the non-cholesterol branch of the MVA pathway, farnesyl-PP and geranylgeranyl-PP are both essential for protein prenylation, which is required for the membrane localization, anchoring and activity of several signaling proteins . IncludeThe Hippo pathway is a potent tumor suppressor pathway that includes the oncogenes yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). When the Hippo pathway is activated, YAP and TAZ are phosphorylated, which inhibits their activity. YAP and TAZ are activated by the MVA pathway , and thiEpithelial mesenchymal transition (EMT) is the process whereby cells lose E-cadherin expression and differentiate into mesenchymal cells with stem-like features and enhanced migratory potential . The indCSCs exhibit indefinite potential for self-renewal, which makes them key drivers of tumor initiation and metastases. These cells are thought to arise through subpopulations of stem cells, which are maintained by a mutated stem cell niche affording aberrant proliferation . The preAnother pivotal cell type in the process of metastasis are cancer-associated fibroblasts (CAFs). In normal development, fibroblasts are key producers of connective tissue and participate in wound healing by producing cytokines and chemokines, including TGF-β. In the context of the tumor microenvironment, cues such as DNA damage, physiological stressors and inflammatory signals arising from tumors, instruct the activation of CAFs. CAFs contribute to the production of matrix proteases, allowing for remodeling of the tumor matrix that creates paths for tumor cell invasion. CAF-induced matrix production also leads to advantageous consequences for tumors, including pro-survival and pro-proliferative signaling, collapsed blood vessels leading to hypoxia, reduced immune cell motility and metabolic dysregulation . The adaWhile hyperactivity of the MVA pathway leads to malignant transformation, it is important to note that ablation of MVA signaling may impair the function and survival of cells that may have a role in cancer immune surveillance. Although resting T cells rely on oxidative phosphorylation, activated T cells shift to aerobic glycolysis, fueling MVA metabolism . Since cOverexpression of SREBP-1 and SREBP-2 is a major contributor to therapy resistance in cancer . LipogenIn many cancers, the MVA pathway is activated to increase cell growth, survival and therapy resistance. MVA pathway activity was shown to be increased in lapatinib- and trastuzumab-resistant HER2+ breast cancer cells . TreatmeThe MVA pathway is also important for the prenylation of proteins. The MVA enzyme, geranylgeranyl transferase II, and its corresponding substrate, Rab11b, promote overexpression of Arf6 . This ulAs previously mentioned, YAP from the Hippo pathway is activated in response to MVA pathway upregulation. Both YAP and TAZ overexpression are contributors to drug resistance in cancer . OverexpTherapy resistance is heavily influenced by the MVA pathway and its downstream constituents. Targeting this pathway therapeutically may battle drug resistance and treat cancer patients more effectively.Statins, bisphosphonates, geranylgeranyl transferase inhibitors, farnesyltransferase inhibitors and squalene synthase inhibitors are all classes of drugs that target the MVA pathway. Statins inhibit the pathway early on at the rate-limiting enzyme, HMG-CoA reductase, and are effective in lowering the circulating cholesterol levels in the blood and protecting against heart attacks and strokes . BisphosStatin drugs are one of the most prescribed classes of drugs and are predominantly prescribed as a cholesterol-lowering medication to protect against the risk of heart attack and stroke . StatinsLovastatin became the first FDA-approved statin in 1987 , and theWith respect to their solubility, statins are sub-divided into lipophilic and hydrophilic groups , with liStatins inhibit the MVA pathway and therefore endogenous cholesterol production, and they are currently approved as single-agent or combination therapies to reduce plasma cholesterol and reduce coronary heart disease . ThroughMany researchers have indulged in pre-clinical research to better understand the mechanism through which statins have shown effectiveness in a number of cancers.Atorvastatin has been shown to inhibit cell adhesion, proliferation and invasion and induced apoptosis, G1 cell-cycle arrest and autophagy in ovarian and breast cancer cell lines ,112. MecThe use of fluvastatin in combination with many current anti-cancer therapies demonstrated promising results. Fluvastatin and cisplatin resulted in robust inhibition of proliferation, G2/M cell arrest, impaired expression of Ras GTPase proteins and increased early apoptosis in ovarian cancer cells . FluvastLovastatin treatment in lung cancer cells has demonstrated increased apoptosis and inhibited G1 phase cell progression by increasing the cell-cycle checkpoint inhibitors p21 and p27 and decreasing cyclin D1 expression . In an oIn liver cancer cells, pitavastatin inhibited growth and colony formation, induced activated caspase 3 and 9 apoptosis and induced G1 phase cell-cycle arrest . AdditioPravastatin treatment on hepatocarcinoma significantly decreased cell proliferation compared to sorafenib only or a combination therapy both in vitro and in vivo . PravastRosuvastatin treatment on prostate cancer cells inhibited cell proliferation and spheroid formation and inhibited EMT through increased E-cadherin expression and decreased vimentin and Zeb-1 expression . RosuvasSimvastatin treatment in breast cancer cells increased the expression of miR-140-5p, a known tumor suppressor gene in many cancers, through activation of the transcription factor NRF1, resulting in overall anti-proliferative and pro-apoptotic effects . CervicaIn vivo, varying statins  were capable of inhibiting Ras protein translocation, with the exception of pravastatin, demonstrating anti-tumor effects of statins on pancreatic cancer xenograft mice . Using mThere is a large body of research that suggests all statins have some anti-cancer capabilities. As a result, additional research, particularly clinical trials, are necessary to further optimize the use of statins as a therapy in various cancers.With statins so widely prescribed, many researchers have retrospectively investigated whether there is a link between statin use and cancer incidence. Many other diseases act as comorbidity risk factors in the development of cancer. For instance, patients with heart failure have a higher risk of developing cancer. Heart failure patients taking a statin had a 16% lower risk of cancer incidence . This reThere is also compelling evidence to support the role of statins in reducing cancer mortality and improving patient survival. Lung cancer patients with tumors harboring a p53 mutation, which accounts for about 50% of all lung cancer cases, were responsive to statin therapy, with higher levels of cancer cell apoptosis, inhibited cell proliferation and more tightly controlled lipid raft regulation . SimilarRetrospective studies demonstrated that statin use contributed to a lower risk of mortality in breast cancer patients . More spSystematic analysis of epidemiological studies demonstrated improved overall survival in pancreatic patients using statin therapies . AdditioNationwide retrospective analysis demonstrated that statin use post-diagnosis improved survival in women with epithelial ovarian cancer . This isOverall, several studies suggest that statins may be effective when used as anti-cancer agents, which justifies additional research into their effectiveness in clinical trials for cancer patients.The repertoire of retrospective and prospective studies as well as pre-clinical in vitro and in vivo research presents a compelling tale to support the hypothesis that statins may be a useful adjunct to cancer therapy. As a result, a slew of clinical trials were launched to look into the matter further. Simvastatin combined with fluorouracil, adriamycin and cyclophosphamide therapy showed improvement in the objective response rate and pathological response in patients with locally advanced breast cancer . The useMany of these trials were poorly designed due to various reasons, including: intervening with statins in late-stage cancer, evaluating statin efficacy in a variety of cancers, dosing used being based on treating hypercholesterolemia and not taking into account the half-life and dosing schedule of the statins used . There mAs tumor heterogeneity causes significant disparities in therapeutic responses within the tumor, often leading to recurrence or resistance , it is cSimvastatin specifically demonstrated efficacy in lung cancer cells harboring p53 missense mutations through inhibition of cancer cell growth, reduced lipid rafts and induced tumor cell apoptosis . SimilarIn a similar line, the lipophilicity of the statin used may have an impact on cancer response. Lipophilic statins have a greater capability to passively diffuse through cellular membranes and can therefore penetrate both hepatocytes as well as non-hepatocytes ,163. LipThe dose, timing its implementation and duration of usage for each statin may influence its efficacy. Many studies have significantly shown a strong link between the combinational use of statins and other cancer therapies proving to be efficacious, especially in reducing the risk of cardiotoxicity ,168,169.Cancer is a whole-body disease, complicating other organ function with metastases leading to physiological imbalance. Cancer and heart disease are two leading causes of mortality, with numerous studies implicating tumor-induced changes as a major contributor to cardiac dysfunction in patients . In factIn addition, given the benefit of statins in LDL cholesterol independent diseases, it has been proposed that the effects of these agents are pleiotropic, and we are just beginning to uncover the molecular pathways that they influence. In fact, in addition to the blockbuster drug’s influence on hypercholesterolemia, statins have had clinical significance in periodontal disease and rheumatoid arthritis with evidence of decreased inflammation ,180, kidThere is compelling evidence that the use of statins as a potential cancer therapy should be investigated further in clinical trials. From a pharmaceutical perspective, de novo drug design is extremely time consuming, quite costly and is a high-risk process, with 90% of potential drug candidates failing in clinical trials ,186. On Thalidomide has already been repurposed from its original approved use as a morning sickness medication to its present use as a multiple myeloma therapy . ThalidoThe combination of a heterogeneous tumor microenvironment and dysregulated metabolic pathways mediated by mutant tumor suppressor and proto-oncogenes promotes tumor survival and therapeutic resistance. The mevalonate pathway has been shown to be elevated in a variety of malignancies, impacting many cellular processes, and ultimately promoting tumorigenesis. As a result, the mevalonate pathway could be targeted by statins to inhibit tumor growth and metastasis. Despite the fact that several studies have been conducted over the last few decades to investigate the use of statins to treat various cancers, there has been limited concrete evidence of success in clinical trials. Statins may show efficacy in specific cancer types, after tailored dosage and timing of application, and when used in specific combination treatments. All of these issues should be taken into consideration in order to optimize the use of statin drugs as a cancer therapy and improve clinical success.


While chronic disease is a risk factor for dementia  studies addressing multimorbidity and dementia risk are limited. Multimorbidity leads to steeper dementia progression  impacting cognitive dysfunction, cognitive decline and impairment . Multimorbidity has generally been examined using a dose- response disease count, something cited as impractical for clinicians due to information deficits on identifying disease specific pathways . Thus, differentiating disease clusters and dementia risk will provide insight into the pathophysiology of dementia, while informing priority targets for early intervention, potentially reducing burden and secondary care costs.The COVID-19 pandemic has resulted in delayed diagnosis and treatment for cancer patients and increases in elective surgery waiting lists. The impact on other ‘long-term’ conditions (LTCs) is unclear. We examined the effects of the pandemic on the recorded incidence of 20 LTCs to inform decisions on treatment pathways and resource allocation.We used national data defined by the NHAIS, including basic demographics for all patients aged fifty-five plus and registered with a GP in Northern Ireland. The linked data comprised: Patient Admissions and Discharges (from 2013-2021); Electronically captured prescribing data, area-type of usual residence; Multiple Deprivation Indicators; and GRO mortality data (including cause of death). We identified multimorbidity through validated algorithms for ICD-10 coding of thirty morbidities in administrative hospital diagnostics data . The same data identified dementia-free patients at baseline. Latent Class Analysis assigned patient groups by the patterning of multimorbidity states, following them through the study period, with analysis in relation to subsequent diagnoses of both clinical dementia and mortality. As age has a potential modifier effect we tested through age group stratified analyses. A series of multinomial logistic regression models further examine (for the multimorbidity sub-groups of interest) risks associated socio-demographic, socio-economic, medication use and household indicators at the individual, family and area level.Multimorbidity occurs in two thirds, approximately, of this hospital population of older adults. By age sixty-five years, most people are multimorbid. We identify subgroups of multimorbidity associated with dementia onset and mortality, combinations of diabetes, hypertension, chronic kidney disease and cancer multimorbidity are highlighted as higher-risk conditions for increased dementia incidence and mortality. Greater disease burden is associated with frequent healthcare service use, medication use and is more common in the most socioeconomically deprived, compared with the least deprived.Evidence on multimorbidity patterns and dementia is limited . Our study suggests that people with combinations of diabetes, hypertension, chronic kidney disease and cancer multimorbidity are associated with increased risks for dementia and socio demographic and economic circumstances further increase this risk. Overlapping combinations comprising chronic kidney disease + hypertension, or diabetes + hypertension, were a significant proportion of total secondary care costs for patients with multimorbidity; combinations comprising chronic heart failure + chronic kidney disease + hypertension had the highest proportion of total cost from potentially preventable emergency admissions . Our finding support the body and mind connection in dementia development. Thus, we note that cardiovascular health is associated with dementia . Often described as a heart-brain connection, cardiovascular disease leads to higher dementia risk due to a number of mechanisms including but not limited to chronic inflammation . Identifying such high-risk groups will facilitate tailored interventions for dementia prevention and the reduction of healthcare use and costs.


Older people represent the fastest growing group with epilepsy diagnosis. For example, cerebrovascular disease may underlie roughly 30–50% of epilepsy in older adults and seizures are also an underrecognized comorbidity of Alzheimer's disease (AD). As a result, up to 10% of nursing home residents may take antiseizure medicines (ASMs). Despite the greater incidence of epilepsy in older individuals and increased risk of comorbid seizures in people with AD, aged animals with seizures are strikingly underrepresented in epilepsy drug discovery practice. Increased integration of aged animals into preclinical epilepsy drug discovery could better inform the potential tolerability and pharmacokinetic interactions in aged individuals as the global population becomes increasingly older. Quite simply, the ASMs on the market today were brought forth based on efficacy in young adult, neurologically intact rodents; preclinical information concerning the efficacy and safety of promising ASMs is not routinely evaluated in aged animals. Integrating aged animals more often into basic epilepsy research may also uncover novel treatments for hyperexcitability. For example, cannabidiol and fenfluramine demonstrated clear efficacy in syndrome-specific pediatric models that led to a paradigm shift in the perceived value of pediatric models for ASM discovery practice; aged rodents with seizures or rodents with aging-related neuropathology represent an untapped resource that could similarly change epilepsy drug discovery. This review, therefore, summarizes how aged rodent models have thus far been used for epilepsy research, what studies have been conducted to assess ASM efficacy in aged rodent seizure and epilepsy models, and lastly to identify remaining gaps to engage aging-related neurological disease models for ASM discovery, which may simultaneously reveal novel mechanisms associated with epilepsy. Seizures and epilepsy have a high incidence worldwide. At least 10% of the population will experience seizures at some point in life , alcoholPreclinical antiseizure drug discovery is booming , 22. NumSeizure vulnerability is not uniform with advanced age , 16, theUnfortunately, most studies on epilepsy have employed younger adult animals; the study of epilepsy (and other comorbid conditions) in aged animals is rather limited , 38. ThuModeling the sequelae of epilepsy in aged animals is challenging from a variety of perspectives, including species-specific physiological changes with aging, the interaction of comorbidities observed in people with epilepsy, and the feasibility and cost limitations of inducing clinically relevant neurological disease in aged animals. These factors limit current models, especially concerning the replication of age-related conditions , 38.Most of the studies conducted in older animals have compared seizure susceptibility between sexes . Few havA agonists better than juvenile mice, mimicking patient responses (The chemoconvulsant pentylenetetrazol (PTZ), has provided insight into some of the pathways and genes involved in epilepsy in the aged and degenerating brain . In the esponses . Furtheresponses . During esponses .The relevance of tau in aging-related epilepsy has also been interrogated using the acute PTZ seizure model. Tau, a microtubule-associated protein, is a natively unfolded protein in human brains and has several biological roles, including microtubule assembly and stabilization . There aKainic acid (KA)-induced status epilepticus (SE) in mice revealed that some of the most affected areas secondary to seizure induction are the hippocampus, cortex, amygdala, and thalamus , 40. NonKindling of aged mice elicits similar biological outcomes as the chemoconvulsants mentioned above. The severity of and susceptibility to hippocampal and CA3 kindling in old C57BL/6 mice increases with advanced age , 44. MorAligning with clinical risk factors for epilepsy in old individuals , traumatThe Ras homolog enriched in brain protein  may explain some differences in epilepsy development between young and older individuals . Aged miOther studies revealed that there is a progressive age-related increase in hippocampal hyperexcitability . Variation in the levels of the proto-oncogene c-Fos, responsible, among other things, for synaptic plasticity  and the In conclusion, although few additional aged mouse studies have been reported since the last comprehensive review was published , these sDuring the last decade, studies of epilepsy and aging effects in rats have been even less frequently conducted than in mice, likely as a result of advances in genetic models of aging-related neurological disorders favoring mice, as well as the financial constraints and resource limitations associated with aged rats. Nonetheless, the most notable studies carried out in recent years are summarized in Studies in rats highlight the relevance of other pathological mechanisms that are different from those observed in mice. Pilocarpine-induced epilepsy in aged rats triggers an important switch in glial phenotype and response . FurtherClearly, there is untapped value in integrating aged models more frequently to better understand the molecular pathways of epilepsy and seizure. As we have been able to successfully uncover many novel therapeutic targets based on findings in pediatric genetic epilepsy models, it is entirely plausible that integrating aged rodents may similarly carry broad relevance to other epilepsy indications, including developmental epileptic encephalopathies (DEE).Aging, in general, is associated with cellular, molecular, anatomical, and physiological changes . There aAging shifts the responsiveness of the immune system , 78, i.eMicroglia, particularly when activated, are the primary source of inflammatory cytokines in the brain. IL-1, IL-6, and IFN-α levels are increased in the brains of old vs. younger adult mice and rats , 83. AddProteins are repaired by the chaperone system and eliminated by autophagy or ubiquitin systems , 84. TheThe aggregation of tau and β-amyloid, two proteins implicated in AD, can similarly explain the close relationship between neurodegeneration and epilepsy. Although the mechanisms by which these proteins may act on epilepsy are not fully understood, tau reduces PP2A activity, increasing GSK3β and CDK5 activity, activating PI3K/AKT pathways, and promotes NMDAR subtypes to drive glutamate release . These fAging and oxidative stress are closely related  Figure . Aging iStudies of naturally aged rodents and genetic SAM models demonstrate that there is an accumulation of oxidation products in the brain over time . OxidatiSE changes the redox potential and decreases ATP levels, reducing the production and supply of energy to the brain . MoreoveMitochondrial dysfunction is another pathological feature of both aging and epilepsy , 91 Fig, which cExcitotoxicity describes the toxic actions of excitatory neurotransmitters, mainly glutamate, where exacerbated or prolonged activation of glutamate receptors initiates a cascade of neurotoxicity that ultimately leads to loss of neuronal function and cell death , 93 Fig. Both inThe blood-brain barrier (BBB) consists of endothelial cells, tight junctions, astrocytes, pericytes, and the extracellular matrix , 96–98. Although BBB disruption is associated with other neurological disorders, increased permeability may be relevant in understanding differences in drug response between older and younger people with epilepsy. The increased permeability may contribute to better drug uptake, resulting in differential response . This efAging is accompanied by progressive atrophy of tissues and aggregation of stochastic damage to DNA, RNA, protein, and lipid macromolecules , 100. LoIn epilepsy, studies have not yet conclusively revealed the effect of an acute seizure on neuronal death and the role of seizure-induced neuronal death in acquired epileptogenesis. Isolated seizures probably do not kill neurons. However, severe, and repetitive seizures do . ANormal aging evokes molecular and cellular changes in the brain that may also delay recovery after injury . Aging dPreclinical and clinical evidence increasingly supports that serotonin (5-HT) has an important role in epilepsy and SUDEP , 110. ClEpilepsy in older people is particularly concerning when it arises due to comorbid conditions because of an elevated mortality risk. Mortality specifically from AD in people with epilepsy has increased over 216% in the last 20 years, whereas people with epilepsy alone have seen a significant (27%) decrease in mortality . WhetherAging causes substantial biological changes that play an important role in other related neurological disorders, such as epilepsy or AD. These physiological events may be relevant to understand differential sensitivity to ASMs of older adults with epilepsy compared to younger individuals, as well as elucidate the reason for the increased incidence of epilepsy in this demographic.Older adults represent the fastest-growing demographic with epilepsy , 34. WhiScn1a+/− mouse model of Dravet syndrome  or AD , 32. A hStroke and cerebrovascular disease are highly associated with epilepsy , 71, 119Epilepsy is an under-recognized comorbidity of AD . RoughlyUnfortunately, the number of preclinical studies of the effect of aging on epilepsy is limited. There are several plausible limitations. One reason is the resource limitations associated with mimicking the physiological conditions of aging itself. Second, aging naturally deteriorates biological resilience and processes. The coexistence of chronic comorbidities, such as high cholesterol or hypertension, results in a “new physiological state” that is difficult to mimic in rodents. Further, such reduced systems as rodent models may infrequently consider additive impacts such as environmental conditions to reduce variables in a reasonably powered study. Third, older adults (over 65 years of age) are a more frequently polymedicated population , 124. IdUltimately, twenty-first century epilepsy drug discovery could strategically rely on developing new animal models based on the mutations or pathophysiological events produced in both stroke and AD to better reproduce late-onset epilepsy in an efficient manner. These models would fill the above-detailed preclinical void. Interdisciplinary integration of clinical observations into preclinical research would dynamically improve translational research . Rather We propose that some of the characteristics that animal models should have to study the role of epilepsy in advanced age and vice versa are:Use a range of ages of “old” animals to understand heterogeneity in the geriatric population. This includes rodents older than 16 months.Studies should be performed with both sexes to dissect the age-related effects of gonadal hormones or other sex-specific factors on functional outcomes and susceptibility to epilepsy.Use epilepsy models that phenotypically reproduce the symptomatology observed in older people with epilepsy, including those age-associated comorbidities such as stroke and AD-associated genotypes.Increase emphasis on establishing a pharmacokinetic/pharmacodynamic relationship in aged animals to inform on the potential for adverse effects liability in clinical populations. This is particularly critical to older population, who have decreased renal and hepatic function and may be subject to polypharmacy.Study the response to repeated seizures both acutely and chronically.Include biomarkers to validate of age-related changes that are translationally feasible.Older adults are more vulnerable to seizures and epilepsy, but less resistant to ASMs than young people with epilepsy. Preclinical studies in aged animals and the effect of advanced age on epilepsy and seizures are unfortunately limited. Little work has been conducted to define ASM efficacy in preclinical models of late-onset epilepsy. This review updates the comprehensive review by Kelly  and exteAP, LL, KK, and MB-H contributed to the literature review and writing of this manuscript. All authors contributed to the article and approved the submitted version.This work was supported by ITHS KL2 (KL2TR002317) and R01AG067788 to MB-H.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.