It remains a dogma in cognitive neuroscience to separate human attention and memory into distinct modules and processes. Here we propose that brain rhythms reflect the embedded nature of these processes in the human brain, as evident from their shared neural signatures: gamma oscillations (30–90 Hz) reflect sensory information processing and activated neural representations (memory items). The theta rhythm (3–8 Hz) is a pacemaker of explicit control processes , structuring neural information processing, bit by bit, as reflected in the theta-gamma code. By representing memory items in a sequential and time-compressed manner the theta-gamma code is hypothesized to solve key problems of neural computation: (1) attentional sampling (integrating and segregating information processing), (2) mnemonic updating (implementing Hebbian learning), and (3) predictive coding . In this framework, reduced alpha oscillations (8–14 Hz) reflect activated semantic networks, involved in both explicit and implicit mnemonic processes. Linking recent theoretical accounts and empirical insights on neural rhythms to the embedded-process model advances our understanding of the integrated nature of attention and memory – as the bedrock of human cognition. When awake, the human brain constantly samples, interprets, and elaborates novel information from the environment, based on former experience, to integrate them into a coherent representation of the world around . At the Human memory relies on associative neural networks , which aComplementing our understanding from psychology e.g.,  and the In this perspective article, we synthesize the state-of-the-art on neural dynamics in human attention and memory processing and propose how they may realize the elements proposed of the embedded process model , derivedIn the framework offered here, we sketch how neural rhythms may realize the elements of the embedded process model on the neural architecture of the human brain, providing a theoretical basis for a unified understanding of human attention and memory, in psychology and cognitive neuroscience. First, we characterize Cowan’s embedded process model and the brain regions involved in human memory. We will then highlight, how rhythmic neural dynamics may reflect the specific elements of the Cowan model, drawing from prevailing accounts on neural rhythms of human attention and memory. To exemplify our idea, we propose the concept of an attentional mnemonic updating loop, implemented by the theta-gamma neural code cf. .a–d, b, When awake, the human brain continuously samples novel information, interprets this information based on former experience, and integrates it into existing neural networks, to maintain a coherent representation of time and space . PsycholCowan’s model has received much attention, because it is compatible with numerous findings from experimental psychology, including chunking processes and working memory capacity , perceptIn the following, we first revisit critical brain structures involved in human memory. Then, based on the elements specified in the Cowan model, we elaborate how neural oscillations may link attention and memory processes to the neural architecture of the human brain.Of note, Cowan already suggested that neural rhythmic activity (40 Hz gamma oscillations) may play a critical role in implementing the elements of the model  and specPerceptual information is processed in primary sensory regions and fed forward into associative networks. This is well characterized for the visual system : Along tThe semantic information of different modalities  is represented in widely distributed associative networks in the neocortex (NC) and the medial temporal lobe MTL; . The MTLControl networks see  serve thLinking those neural structures to the elements of the Cowan model, central executive processes may be implemented in the PFC, in concert with further attention and memory control networks . These eWe will now turn to the critical question of how attentional and mnemonic processes are orchestrated temporally. Namely, how neural oscillatory dynamics may reflect the basic elements of the Cowan model and realize them on the neural architecture of the human brain.It is a key question, how distributed brain processes are integrated and bound into coherent experiences  is posited to implement a neural coding schema for the organization of multiple items, each item being activated or reactivated at distinct gamma bursts, nested in the theta cycle . This coComplementing its proposed role as an organizing scheme, theta-gamma coupling has been characterized as a mechanism for attentional selection , with crKey to our understanding of the specific neural network properties that may give rise to theta-gamma coupling dynamics , often functionally merged with the adjacent beta rhythm (14–20 Hz). Empirically, intracranial recordings in patients revealed a decrease in NC alpha, which preceded fast gamma oscillations in MTL networks during memory encoding . This co1Here, we propose that neural oscillatory processes may be key to our understanding of the embedded nature of attention and memory processes in the human brain , linkingPerceptual information is promoted along sensory networks, before it converges on sparse representations in conceptual , relatioThe role of neural synchronization processes in the gamma range has been well characterized for visual perceptual processes in mammals, from neurons that encode features of the same object in primary visual regions e.g., , to the In the wake human brain, gamma oscillations were first identified for object perception processes in the visual cortex using M/EEG , being hvia transcranial stimulation, facilitated perceptual processes in primary auditory networks  the prefrontal theta rhythm has been ascribed a key role in executive control processes cf. . In the In humans, the theta rhythm itself has been found to increase in PFC and MTL networks for memory encoding , as wellThe functional role of the theta rhythm for memory formation processes has recently been substantiated by rhythmic perceptual entrainment studies. These studies demonstrate that the perceptual entrainment of theta oscillations enhances memory within the visual domain , and acrTheta-gamma coupling in the human brain has been found during prefrontal control processes , WM mainWithin the theoretical model proposed by Distributed sensory processes need to be integrated  and, at In the human brain, gamma activity has been found to fluctuate at the theta rhythm in primary visual areas corresponding to potential target locations in the left versus right hemifield, facilitating the detection of a target . This stLinking frontal control processes to eye-movements, in macaques, the theta rhythm in the FEF has been shown to modulate the gamma activity in the lateral intraparietal area (LIP), as part of the attentional network, which enhanced perceptual sensitivity . The FEFTo maintain a coherent and up-to-date internal map of time and space, perceptual information must be integrated and updated continuously. Hebbian learning, the principle that cells that fire together, wire together  requiresRegarding the theta-gamma code in memory formation, the precisely timed neural input of gamma bursts to the down phase of the theta rhythm has been shown to facilitate LTP processes in the MTL of rodents  and thatper se  versus the desynchronization in the alpha band, the concept of attentional sampling at the theta pace corresponds closely to the idea of selective attention processes, while the alpha rhythm may reflect sustained attention processes . In CowaNoteworthy, Consistent with the idea that theta and gamma oscillatory processes act on functional networks shaped by alpha based gating mechanisms, desynchronized alpha power in parietal and frontal networks has often been found to coincide with increased theta power during memory formation , as wellSupport for the causal relevance of alpha oscillations in gating information processes in human primary sensory networks comes from two recent transcranial stimulation studies. Here, the experimental application of the alpha rhythm led to reduced gamma oscillations and impaired the detection of a visual stimulus, when applied to the visual cortex , and redRegarding the functional separation between the theta and the alpha rhythm, the encoding of semantic information has been associated with alpha suppression, but the explicit encoding of perceptual features was associated with high theta power . SimilarWhen awake, central executive processes  continuously sample information from the environment . Novel information is interpreted based on semantic knowledge (LTM items in NC networks) and integrated, to retain a coherent representation of time and space (in fast updating MTL networks). This up-to-date mental map allows for behavioral navigation in the light of current goals (monitored in PFC networks).Here we propose that neural rhythms reflect the integrated nature of attention and memory in the human brain, by implementing key elements of the embedded-process model see . In thisvia the frontal eye-field and the superior colliculi). This information is interpreted in the light of semantic associative networks (in the NC), the representation of time and space (in the MTL) and current goals (encoded in PFC and ACC networks), to evaluate and integrate perceptual information as they occur in real time, and to guide future behavior.In summary, the theta-gamma code implements an attentional mnemonic updating loop that samples, updates, and utilizes perceptual information to form predictions and guide behavior: The PFC initiates a neatly ordered processing sequence, sampling and integrating novel information, bit by bit, at a theta pace  dialog between PFC, MTL and NC networks. In contrast, the alpha rhythm reflects the activation of associative, semantic networks involved in explicit but also in implicit processes (the LTM storage). The alpha rhythm may also gate the implicit processing of environmental information that underlies automated behavior, namely information processing that is highly habitualized and does not rely on explicit mental elaboration cf. .The main purpose of our framework is a conceptual link between psychology and cognitive neuroscience, toward a mutually informed theory of human attention and memory. Focusing on oscillatory dynamics, we close the conceptual divide between psychology and brain mapping approaches by specifying how neural rhythms may implement the core elements of the embedded-process model of attention and memory on the neural architecture of the human brain .The past decades have seen essential advances in our functional understanding of neural rhythms in mnemonic processes. This includes the promotion of sensory signals in the visual system , the theIn this tenant, with our framework we hope to inspire the research and theory in psychology and cognitive neuroscience, and the integration of these perspectives . Namely, the theta-gamma code is proposed to remap real time events onto a faster neural timescale to solve key problems of neural computation: attentional sampling, mnemonic updating, and prediction.While initial evidence exists for each of these specific processes in isolation, the concept of their embedded nature requires the orchestration of these processes across human brain networks in shaping the functional architecture underlying human cognitive functioning. Yet, it is not well understood how neural rhythms are ultimately coordinated and interplay with each other across brain regions and distinct cognitive processes. For example, the theta-gamma code is ascribed the role of a PFC and MTL control mechanism , acting In fact, the attentional mnemonic updating loop characterized here, allows for specific hypotheses on how the theta-gamma coding schema may guide experience based visual exploration and the integration of novel visual information. For example, by combining the assessment of gaze behavior (using eye-tracking) and neurophysiological data (M/EEG), it would be intriguing to further scrutinize the interplay between the prefrontal theta phase, eye-movements and gamma bursts in perceptual and semantic networks. That is, how the theta rhythm in PFC and MTL networks may be linked to the guidance of (micro-)saccades and visual cortical processes and inform subsequent oculomotor processes.Another outstanding query on the coordination of oscillatory processes regards the co-occurrence and interplay between increased theta and reduced alpha power. One potential explanation may be a shift (tilt) in the operating rhythm, from alpha to theta, in brain regions involved in central executive processes cf. . This isIn our framework, we emphasize that the ultimate purpose of information processing is behavioral navigation. However, beyond the control of eye-movements, we do not specify how do attention and memory control processes may steer behavioral responses on the level of dynamic recruitment of neural processes in the motor system. There is good evidence that the beta rhythm (15–30 Hz) is implicated in the control of the motor system (for reviews, see A critical tool to test and further specify the assumptions on neural oscillations and their functional relevance are computational models, which allow to specify and test neurophysiological assumptions in detail. In the following, we will exemplify critical contributions that have been made to the conception of the theta-gamma code and highlight potential future directions.A computational model of how the theta-gamma code may support the restoration and sequence coding of several learned items in WM, has recently been developed based on hippocampal connectivity and Hebbian learning principles . This moAt the same time, computational models have provided a crucial alternative view e.g., , namely We hope that the perspective taken here may inform computational models, for example, by providing a concept of how WM and LTM processes may interact. In turn, attractor models may be crucial for our understanding how neural oscillatory dynamics characterized in our framework may arise from the self-organized nature of the networks of the human brain.Infancy and childhood are periods of intense brain development and learning, where developing humans built up basic representations of the world around them cf. . It is tThe theta rhythm is operative from very early in development, in the processing of novel and unexpected information ,2021 andThe framework introduced here may help us to further specify our assumptions about human brain development, at a verbal and preverbal age (for a developmental account of the Cowan model, see also Another intriguing question regards the physiological nature of neural representations (for recent developments in the field, see Regarding the primary role of the theta rhythm in cognitive control, there is also opposing evidence, indicating reduced theta activity during successful encoding . While tA major recent development is the targeted experimental manipulation of neural rhythms in the wake human brain , first, MK wrote the manuscript. MK and TG revised the manuscript. Both authors approved the submitted version.


Enkephalin, an endogenous opioid peptide, is highly expressed in the reward pathway and may modulate neurotransmission to regulate reward-related behaviors, such as drug-taking and drug-seeking behaviors. Drugs of abuse also directly increase enkephalin in this pathway, yet it is unknown whether or not changes in the enkephalinergic system after drug administration mediate any specific behaviors. The use of animal models of substance use disorders (SUDs) concurrently with pharmacological, genetic, and molecular tools has allowed researchers to directly investigate the role of enkephalin in promoting these behaviors. In this review, we explore neurochemical mechanisms by which enkephalin levels and enkephalin-mediated signaling are altered by drug administration and interrogate the contribution of enkephalin systems to SUDs. Studies manipulating the receptors that enkephalin targets  implicate the endogenous opioid peptide in drug-induced neuroadaptations and reward-related behaviors; however, further studies will need to confirm the role of enkephalin directly. Overall, these findings suggest that the enkephalinergic system is involved in multiple aspects of SUDs, such as the primary reinforcing properties of drugs, conditioned reinforcing effects, and sensitization. The idea of dopaminergic-opioidergic interactions in these behaviors remains relatively novel and warrants further research. Continuing work to elucidate the role of enkephalin in mediating neurotransmission in reward circuitry driving behaviors related to SUDs remains crucial. Substance use disorders  are characterized by an inability to control drug use, continuing drug use despite adverse consequences, and relapse even after long periods of abstinence. Multiple risk factors contribute to vulnerability for developing of a SUD, such as genetic and environmental factors  of dopamine neurons in the ventral tegmental area (VTA) projecting to the NAc and/or by inhibiting the reuptake of dopamine in the NAc and is thought to be the critical mechanism underlying the primary reinforcing effects of drugs of abuse. Drug-paired cues, one important factor contributing to relapse, can also lead to increased dopamine in the NAc, which further supports other frameworks explaining the role of dopamine in various aspects and stages of SUDs, such as the opponent process and incentive salience theories  after administration  imaging in human subjects have suggested a potential role of endogenous opioids in the effects of drugs of abuse. For example, oral administration of amphetamine in male subjects reduced binding of . Enkephalins bind with high affinity to DOR and MOR  . These receptors are coupled to the Gi/o proteins, leading to inhibition of cAMP, inhibition of Ca release . Each reditions] , but morditions] . For exaditions] . TherefoSome primary regions of enkephalin release occur within the reward pathway, specifically in the NAc, VTA, and pallidum [comprised of the ventral pallidum (VP) and globus pallidus (GP)]. Interestingly, it is unclear where enkephalin in the NAc comes from, with some studies suggesting that it comes from projection neurons  leads to disinhibition of VTA dopamine neurons projecting to the NAc  of the endogenous opioid system in mediating SUDs.There are different methods and techniques to evaluate enkephalinergic involvement in reward-related pathways and behaviors. Methods for measuring enkephalin release are limited  and, while technically challenging, can distinguish between met- and leu-enkephalin . Antibodies used in these assays that bind to enkephalin peptide have limitations in selectivity. RIAs with tissue samples also cannot discriminate between intracellular expression and extracellular release of enkephalin. Other, more direct, approaches include collecting dialysate samples kephalin . Methodskephalin  and/or cPENK gene   of the via the activation of opioid receptors. Importantly, β-endorphin has been shown to be a substrate of NEP and APN, but is also degraded by other enzymes  nor enkephalin release. Similarly, using pharmacological methods to activate or inhibit DOR and/or MOR implicate opioid receptor signaling and requires highly selective ligands. While it is presumed that enkephalin is the endogenous ligand acting on those receptor systems, it is often not directly tested. Since all endogenous opioid peptides bind, to some degree, to all opioid receptors, peptides other than met- or leu-enkephalin may be responsible for the effects measured. Overall, while a wealth of literature has supported the notion that enkephalin modulates reward-related neurobiology and behavior, there is much still to be elucidated.This review primarily focuses on studies investigating PENK or enkephalin peptides, as they are more closely related to the functional role of enkephalins in reward-related behaviors. Pharmacological studies investigating the effects of DOR and/or MOR activation are not the focus of this review and are thoroughly covered elsewhere  by activating MORs in the VTA/substantia nigra. MORs do not seem to be located presynaptically on dopamine terminals in the NAc  of endogenous enkephalins in regulating dopamine neurotransmission. MOR agonists increase dopamine in the dorsal and ventral striatum  and thus driving reward-related behaviors. Acute and chronic administration of drugs of abuse have been shown to alter levels of enkephalin in reward brain regions, albeit with some inconsistent results across studies.It has been shown that amphetamine administration increases enkephalins in multiple brain regions. Amphetamine increased met-enkephalin release in NAc and PFC , 2009, aPsychostimulant administration may also alter the expression of endogenous opioid peptide mRNA, which may influence enkephalin levels and release, but there are mixed results of psychostimulant-induced changes in expression of PENK mRNA throughout reward circuitry. These may be due to differences in psychostimulant dose, time of mRNA measurement, and acute versus chronic administration. For example, psychomotor stimulants either increased, did not change, or decreased PENK mRNA in the striatum , decreasAlthough enkephalins are an endogenous ligand for MORs, few studies have investigated the effects of exogenous MOR activation on enkephalin levels. Acute morphine  and heroThere are few studies assessing the administration of exogenous opioids on PENK mRNA levels. Acute morphine did not alter PENK mRNA in NAc nor striatum  and repeTogether, these findings suggest that, while acute and chronic administration of MOR agonists may increase enkephalin release and peptide levels, chronic opioid administration mainly leads to a reduction in PENK mRNA expression, potentially compensating for the replacement of endogenous opioid peptides by exogenous opioid receptor ligands.The effects of ethanol on enkephalin levels and PENK mRNA are highly varied across studies. Acute ethanol has been shown to increase met-enkephalin in the NAc shell and striatum, decrease enkephalin in striatum, hypothalamus, and midbrain and not alter enkephalin in VTA, amygdala, hypothalamus, midbrain, brainstem, and hippocampus . It has Ethanol exposure also produces varied changes in PENK mRNA levels in various brain regions. Acute ethanol treatment and voluntary consumption increased PENK mRNA in the paraventricular nucleus of thalamus, caudate putamen, amygdala, PFC, and NAc core and shell  and decr11C] carfentanil binding in certain brain regions, such as prefrontal cortices and ventral striatum  are present in many of the same brain regions as opioid receptors , indicatThe effects of cannabinoids on enkephalins may be greater in non-reward brain regions. Subchronic THC increased PENK mRNA levels in rat hypothalamus, PAG, and mammillary nucleus , with noOther conditions have also been shown to change levels of enkephalin peptides. Consumption of palatable food leads to a surge of met- and leu-enkephalin in the anteromedial portion of the dorsal neostriatum, analyzed by LCMS . OptogenThe role of the endogenous enkephalinergic system has been evaluated in reward related behaviors as well as other potentially related (and co-morbid) behaviors and physiological functions, such as stress resiliency, pain, and emotion .For example, increasing enkephalin levels with thiorphan in the VTA  or mimicvia opioid receptor activation, contributes to the rewarding effects of various drugs of abuse. This is likely due to multiple indirect mechanisms culminating in disinhibition of dopamine, either via disinhibiting glutamate efferents in NAc or inhibiting GABAergic interneurons in the VTA.The rewarding and reinforcing effects of various drugs of abuse are also altered by attenuating endogenous enkephalin signaling with the administration of opioid receptor antagonists or by genetic deletion of PENK. Administration of opioid receptor antagonists, which presumably block the effects of endogenous enkephalins or other opioid peptides, attenuated or blunted cocaine-induced CPP , heroin Interestingly, in animals trained to discriminate morphine, systemic administration of an enkephalinase inhibitor, RB 120, did not generalize to the discriminative stimulus effects of morphine and, conversely, morphine did not generalize to the discriminative stimulus effects of RB 120. Together, these data suggest that, even though enkephalins may have some rewarding properties, endogenous enkephalin and the MOR agonists may produce different subjective effects . TherefoThe endogenous enkephalinergic system may also be involved in other aspects related to the development and maintenance of SUDs. For example, MOR activation in the NAc and VP enhances hedonic impact or “liking,” a distinct but related function to drug “wanting”  will provide a better understanding of the mechanisms involved in SUDs. In order to accomplish this, we must also have an improved appreciation of the sites of enkephalin synthesis, the sources of enkephalins, and the regulation of enkephalin catabolism. Finally, manipulating enkephalin directly and with brain region or cell type specificity will be crucial to measure enkephalinergic influence on reward-related behaviors.The studies described in this review used a multitude of techniques to probe the role of enkephalin, and each technique has limitations that can influence interpretations of results. Limitations of enkephalin measurement techniques  are due,in vivo samples (Technological advancements to improve detection and quantification of endogenous opioid peptides and their regulation by enkephalinases will help our understanding of the role of enkephalins in circuitry and reward-related behaviors. Tools for measuring extracellular enkephalin specifically, such as liquid chromatography coupled with mass spectrometry analysis of  samples  and volt samples  can be f samples . Similar samples . The abi samples , caged-o samples .The studies discussed in this review provide strong evidence that the endogenous enkephalinergic system plays an important role in modulating reward circuitry and driving maladaptive behaviors to SUDs. In order to further understand the underlying mechanisms of SUDs, more research should probe the direct involvement of enkephalins and other opioid peptides in the formation, persistence, and relapse to SUDs. Furthermore, the endogenous enkephalinergic system may also be a potential target for novel therapeutics to prevent and treat SUDs and relapse.LR and EJ contributed to the writing and revisions of this review. Both authors contributed to the article and approved the submitted version.


This Special Issue “Immunohistochemical and Physiological Research on Farm Animals” is dedicated to the application of immunohistochemical and physiological studies carried out on farm animals, including traditional  and emerging farm species .Farm species play very important economic and sociocultural roles, such as food supply, source of income, asset saving, source of employment, soil fertility, livelihood, transport, agricultural traction, agricultural diversification and sustainable agricultural production.For this reason, it is essential to study the anatomy and the physiology of these species in more detail, in order to improve the knowledge about their morpho-physiological features and to guarantee them a high level of welfare and, in turn, better conditions for farmers, including farm income.In the last few decades, there has been an exponential increase in publications on immunohistochemistry (IHC) and immunocytochemistry techniques, reflecting the current position that IHC holds in most pathological laboratories.This Special Issue attempts to demonstrate the relevance of immunohistochemical analysis and its relationship with functions in farm animals. However, taking into account the fact that different methods  are necessary to draw more reliable conclusions, the use of these methods is also considered.In addition to base research contributions, we asked authors for research with potential applied-purpose studies whose results could be useful in improving farm management practices and the responsible use of natural resources, enhancing the nutraceutical properties of animal-derived products and promoting the circular economy and, in turn, the sustainability of livestock.A total of 20 papers have been contributed for this Special Issue by 118 authors from four countries, comprising 18 research articles and 2 communications. The work of Premi and colleagues  investigBubalus bubalis). PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer (chorion/trophectoderm) of the placenta in eutherian species. Through a radioimmunoassay applied on blood samples, PAGs were recognized as the best marker for predicting embryonic mortality between 25 and 40 days of gestation in buffalo.Barile and coworkers  identifyCapra hircus) kids. Through single and double immunostaining for the first time, the NPY distribution in the abomasum, duodenum, and pancreas and the co-localization with serotonin (5-HT) were documented. Upon RLE feed supplementation, NPY immunoreactive cells increased significantly in abomasal epithelium and pancreatic islets. NPY is known to regulate gastric acid secretion while on the endocrine pancreas to suppress insulin via autocrine and/or paracrine mechanisms, and to stimulate glucagon secretion. These observations represent a baseline for future studies on the interaction between neuropeptides and polyphenols used as feed additive instead of antibiotics.De Felice and colleagues  evaluateWang and coworkers  examine Ovis aries). Collagen fibrils in their native conformation with endogen fibril-associated glycosaminoglycans can be extracted from sea urchins, obtaining a biomaterial resembling the in vivo structural microenvironment. Clinical, histopathological, immunohistochemical, and molecular experiments assess CBSS’ effects on the wound healing process. The authors demonstrate the efficacy of this pioneering skin substitute in an in vivo model. The skin substitute supported and stimulated wound healing throughout the whole process; it controlled inflammation, promoted the deposition and maturation of granulation tissue, enhanced re-epithelialization, and induced the formation of skin appendages. This application explores the possibility of deriving high-value and innovative products for veterinary innovative applications from waste materials.Melotti and coworkers  describeDe Carolis and colleagues  study ovBarbato and coworkers  evaluateBos grunniens) during different stages. ERKs are an important subfamily of mitogen-activated protein kinases (MAPKs), which regulate various cellular activities and physiological processes. For example, ERK1/2 has a pivotal role in the ovulatory process or in the mechanism of regression and functional maintenance of corpus luteum. In female yak, the expression of ERK1 and ERK2 proteins and mRNA was most pronounced in the ovary in the luteal phase and gestation period, the oviduct in the luteal phase, and the uterus in the gestation period. The histological appearance and physiological process of the main reproductive organs also varied with the different reproductive stages. These results imply that ERK1/2 plays an important role in the regulation of reproductive functions in different physiological situations.Fan and colleagues , with a Sus scrofa domesticus). Samples were analyzed using conventional histochemistry to identify the glycohistochemical profile and using immunohistochemistry to localize aquaporin 5, apelin and apelin receptor. This study demonstrates that different feed physical forms are capable of inducing morphological and functional modifications of the MG. The intense chewing activity related to the highest feed compaction and hardness promotes an increase in pig MG secretion, and saliva becomes more fluid and richer in acid glycoconjugates in order to better lubricate the bolus and protect the mouth mucosae. It has been hypothesized that the apelinergic system is likely involved in the above modifications, enhancing both the fluidity and the quantity of serous saliva.Dall’Aglio and colleagues  test thePalus and coworkers  demonstrToschi and colleagues  immunohiTian and coworkers  determinE. coli-induced inflammatory state of the uterus on the neurochemical characteristics of the gilt (crossbred Large White × Landrace pigs) caudal mesenteric ganglion (CaMG) uterus-supplying neurons. After intrauterine bacterial injection, the population of uterine neurons presenting positive staining for dopamine-β-hydroxylase  and negative staining for galanin, as well as the population of uterine neurons presenting negative staining for dopamine-β-hydroxylase but positive staining for neuropeptide Y, were decreased. Uterine inflammation causes changes in the spatial and neurochemical organization patterns of the CaMG neurons innervating the uterus.Jana and Calka  analyze Equus caballus) in a squamous cell carcinoma (SCC) using immunohistochemistry for the first time, thus illustrating an example of tumor cell adaptation during the metastatic process.Armando and coworkers  examine Oryctolagus cuniculus). TEO levels up to 180 mg/kg can play a major role in improving the productive performance, semen quality, testosterone levels, and the kidney and liver functions, analyzed through blood biochemical assay. Abdel-Wareth and Metwally  evaluateOchotona princeps), by means of sequence analysis and structure stimulation. Preziuso proved that the simulated complexes ACE2-SARS-CoV-2-RBD showed a high affinity of ACE2 of lagomorphs to the viral spike protein, suggesting that lagomorphs could be susceptible to SARS-CoV-2.Preziuso , in her Meleagris gallopavo domesticus). Considering the significance of the domestic turkey as a meat-producing animal and the regulation of MLT of feed intake, plasma analysis showed that MLT was secreted in a daily rhythm with a very high amplitude, but also responds quickly and precisely to changes in light conditions.Prusik and Lewczuk  typify tDicentrarchus labrax) in terms of growing performance, oxidative stress, and contaminant markers. The results of this study, which can be considered a pilot, point out a positive trend in the growing performance of both groups but a greater productivity of conventional fish feed compared to the organic ones on one hand, and significant differences among groups in terms of the oxidative stress and contaminant markers on the other. With regard to teleost fish, Carminato and coworkers  investigOncorhynchus mykiss) throughout the first year of development. The analysis was performed at typical time points of in vivo feeding trials  in order to establish accurate reference values, especially across the productive cycle of animals raised in standardized conditions. Verdile and colleagues  perform Danio rerio) model. The authors reveal that the effects of 3,5-T2 on fish intestines and brains can deviate from those shown in obese mammals; through the expression of different inflammatory markers, they determined that 3,5-T2 sustained or increased inflammation in the intestine when administered with the obesity-inducing diet.Imperatore and colleagues  analyze


The intestinal tract is an essential digestive organ of the human body, and damage to the intestinal barrier will lead to various diseases. Functional oligosaccharides are carbohydrates with a low degree of polymerization and exhibit beneficial effects on human intestinal health. Laboratory experiments and clinical studies indicate that functional oligosaccharides repair the damaged intestinal tract and maintain intestinal homeostasis by regulating intestinal barrier function, immune response, and intestinal microbial composition. Functional oligosaccharides treat intestinal disease such as inflammatory bowel disease (IBD) and colorectal cancer (CRC) and have excellent prospects for therapeutic application. Here, we present an overview of the recent research into the effects of functional oligosaccharides on intestinal health. With the continuous improvement of people’s quality of life in recent years, the diet structure has changed from simple to complex. Today, the global nutrition situation is complicated. On the one hand, hunger and malnutrition are the dominant concerns in low- and middle-income countries. On the other hand, millions of people are at increased risk of developing diet-related chronic diseases, for example, intestinal disease, heart disease, and diabetes. Taking inflammatory bowel disease (IBD) as an example, the incidence of IBD has increased year by year worldwide over the past decade ,2, with The intestine is a vital digestive organ responsible for the digestion and absorption of nutrients, and the intestinal barrier prevents pathogenic bacteria, toxins, and other harmful substances from entering the intestine’s circulatory system ,9,10. ThCurrent treatment strategies for intestinal diseases include micro-ecological regulation therapy , surgicaClinicalTrials.gov, accessed on 1 June 2022) related to assessing beneficial effects of functional oligosaccharides on human health, including 28 IBS-related studies and 33 IBD-related studies  , FOS, anAkkermansia muciniphila by affecting specific populations in microbial groups; reduced the adhesion, invasion, and colonization of intestinal pathogens represented by Escherichia coli; and inhibited the occurrence and development of enteritis, thereby maintaining intestinal health [Functional oligosaccharides can be selectively fermented into short-chain fatty acids (SCFA) in the gut  to maintl health ,35.Bifidobacterium than sucrose FOS. The molecular mechanism of the metabolism of FOS, GOS, and milk-derived oligosaccharides by probiotics has also been studied, and the unique intake mechanism for functional oligosaccharides plays an active role. A brief summary of the coincidence relationship between common functional oligosaccharides and probiotics is shown in There is a correspondence between functional oligosaccharides and probiotics. Functional oligosaccharides exhibit a complex degree of polymerization and glycosidic bonding ,37, and Based on previous and our own research on functional oligosaccharides affecting proliferation and colonization of probiotics and considering the specificity and complexity of the interaction between intestinal flora and functional oligosaccharides, it is critical to further study the effects of functional oligosaccharides with different structures on the changes in intestinal metabolites, bacterial gene expression, and potential molecular mechanisms in maintaining intestinal barrier function.Functional oligosaccharide plays a positive role in the intestinal immune barrier. Indirectly, functional oligosaccharides can be fermented by probiotic to produce SCFA, which regulate the activity of T cells, B cells, and dendritic cells ,64. For The human gastrointestinal tract has no relevant enzyme system to hydrolyze functional oligosaccharides ,91. HoweAkkermansia in a dose- and time-dependent manner in mice, with 300 mg/kg of berberine showing a two-fold higher proliferation rate than 200 mg/kg. The investigators also demonstrated that this proliferation works by promoting the secretion of mucins, especially mucin-2 [Functional oligosaccharides can also affect the production of mucin and antimicrobial peptides by host cells ,99. For  mucin-2 . The strThe colon environment, including imbalanced intestinal microflora and mutations in the Wnt signaling pathway are the leading causes of CRC ,103. TheThe clinical data also show that functional oligosaccharides have a positive effect on the immunological indexes of colon cancer and microbial flora abundance . HoweverIBD is a chronic nonspecific gastrointestinal inflammatory disease that destroys the intestinal mucosal structure and floral balance, leading to abnormal systemic biochemical indexes . The etiLactobacilus and Bifidobacterium, thereby repairing intestinal mucosal damage during RT and preventing the occurrence and development of IBD [The DSS-induced mouse colitis model is one of the widely recognized models for studying the pathogenesis of IBD and evaluating potential therapeutic methods . Growingt of IBD . Our pret of IBD , alleviat of IBD .Future studies need to understand how functional oligosaccharides regulate the disease-related signaling pathways, drive different cellular processes and regulate intestinal functions, and conduct the mechanism of functional oligosaccharides as a drug adjuvant or substitute in the treatment of IBD.IBS is a chronic disease affected by stress and eating habits. It is characterized by abdominal pain, mucosal and immune functions, and changes in the intestinal microbial structure. Dietary patterns, the intestinal microbial structure, inflammatory response, and other factors can aggravate the symptoms of irritable bowel syndrome. Dietary interventions are recommended to control the disease due to the efficacy and tolerance of common drug treatments.Bifidobacterium species [Bifidobacterium, which is not conducive to the thorough treatment of IBS patients. In view of the pathogenic factors and pathogenesis of IBS and the complexity and diversity of individual microbial communities, we need to consider these two interventions for further research and consider individualized diagnoses according to clinical symptoms.Evidence shows that the ecological imbalance of intestinal and mucosal colon microflora in IBS is usually characterized by the reduction of the  species ,126,127. species ,129. Som species . In cont species . In factGlycans generally have complex monosaccharide composition, glycosidic bond type and degree of polymerization, and their structural complexity is much higher than that of proteins and nucleic acids. In the past decade, glycoscience, with the support of governments, has made a lot of progress, and it has revealed the role of glycans in inflammatory responses and immune system regulation, cardiovascular diseases, intestinal diseases, and cancers. A variety of functional oligosaccharides have shown kinds of activities in intestinal barrier protection and repair and have demonstrated the great promise of glycans in intestinal disease treatment. However, the structure-activity relationship and molecular mechanism have not been fully elucidated. Glycan-based products used in related research are often a mixture of glycans with slightly different structural characters and subject to variations in different preparation methods and raw material sources. Recent studies have shown that small changes in the structure of glycans have significant effects on their activities, so the accurate analysis and preparation of glycan products is the key to clarify their structure-activity relationships and develop functional glycan products, for example, structural analysis and pharmacokinetic study of glycans by liquid chromatography-tandem mass spectrometry (LC-MS/MS) . On the


Alterations in sleep and circadian rhythms are prevalent features in several neurodegenerative diseases. Manifestations in this regard are diverse, varying according to the type of neurodegenerative disease, the stage in which the patient is in the course of neurodegeneration, and the pharmacological treatment, among others. Patients with Alzheimer's disease (AD) report difficulty in falling asleep, frequent night-time awakenings, and excessive daytime sleepiness (EDS), in concordance with alterations in the sleep architecture and sleep efficiency often observed through objective evaluations , non-rapid eye movement sleep parasomnias, and narcolepsy. The review by Chapelle et al. gave additional insight into the relationship between epilepsy and dreaming. The authors reported the available evidence demonstrating that the content of dreams is altered among epilepsy patients, mainly related to familiar settings surrounded by a negative context in detriment of success and sexuality-related settings. Additionally, the frequency of dream recall is affected in those patients.Rabinowitz et al. provided an interesting report, revealing that more stable circadian rest-activity rhythm was related to slower cognitive decline among cognitively healthy adults. The authors also demonstrated that such relationship was pronounced among women, people of color, aged individuals, and apolipoprotein E e4 allele carriers. Similarly, Liu et al. discussed the circadian regulation of several factors that could increase the risk of stroke. The authors comprehensively addressed the variation in stroke severity and its adverse outcomes according to the time of day in which it occurs and the discrepancy in the pharmacological treatment efficacy depending on the time of day in which it is administered.Sleep and circadian alterations often precede the diagnosis of neurodegenerative diseases. Insomnia, sleep fragmentation, and SDB are all independently associated with an increased risk of developing several neurodegenerative diseases  could be associated with cognitive decline. These authors demonstrated that moderate to severe OSA patients (apnea-hypopnea index ≥15) exhibited disturbed functional connectivity from the dorsal anterior insula, an important area involved in cognitive processing, to several areas relevant to cognition. The report presented by Kazukauskiene et al. demonstrated that an interaction between OSA and high levels of N-terminal pro-B-type natriuretic peptide in males with coronary artery disease increases the risk of cognitive functioning problems. Furthermore, Needham et al. proposed a dichotomous role for brain-type fatty acid binding protein (FABP7) in the sleep-mediated pathogenesis of AD. The intracellular lipid chaperone FABP7 regulates fatty acid uptake and transport, being implicated in sleep regulation and neurodegenerative diseases, but the mechanisms underlying this are unclear. According to these authors' hypothesis, depending on the availability of arachidonic acid (AA) and docosahexaenoic acid (DHA), FABP7 could bind to either AA, leading to a cascade of events promoting wakefulness aligned with AD pathogenesis, or to DHA with beneficial effects including activation of anti-inflammatory pathways and neuroprotection.Studies focusing on the mechanisms by which sleep and circadian alterations could increase the risk of neurodegeneration and vice versa are paramount to disentangling this complex relationship. Neurodegeneration inherent to each disease affects relevant nuclei and pathways associated with sleep and circadian regulation, consequently leading to or increasing the magnitude of sleep and circadian alterations  compliance in Down Syndrome patients with OSA. The findings demonstrated an appropriate tolerance to CPAP treatment among these patients, indicating the suitability of this population for future clinical trials aiming to evaluate the preventive role of sleep in the context of AD.Interventional studies aid in the unraveling of possible relationships of causality between sleep, circadian rhythms, and neurodegenerative diseases. In summary, the studies presented in this Research Topic reinforce a bidirectional relationship between sleep and circadian alterations and neurodegenerative diseases. Nevertheless, future evaluations are required to address the several gaps and limitations within the current knowledge. To accomplish this, the consideration of the following items would be of great value: (i) inclusion of appropriate assessments of sleep and circadian health using validated questionnaires and objective measurements; (ii) incorporation of circadian function markers other than the rest-activity rhythm to increase reliability of the findings and dissociate the effects related to sleep to those related to the circadian rhythms; (iii) interpretation of findings based on pharmacological treatment, mental health disorders, disorientation, decreased exposure to natural light, loss of autonomy, and dependence of caregivers schedules; (iv) incorporation of molecular markers to evaluate the impact of sleep and circadian health on the physiopathology of the disease besides the clinical manifestations; (v) performance of experimental and interventional studies to further understand the possible mechanisms and relationships of causality within this context. Accordingly, while there is sufficient evidence related to the impact of neurodegenerative diseases on sleep, the evidence in the reverse direction is mainly based on association and correlational studies. These would collectively contribute to improving the knowledge in this regard, ultimately enlightening promising strategies to prevent or delay the progression of neurodegenerative diseases.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


During the first 2 years of the COVID-19 pandemic, empirical efforts in the psychological sciences have been unequivocally focused on understanding the psychosocial impact on resilience and vulnerability. While current empirical work is guided by different existing theoretical models of resilience and vulnerability, the emerging datasets have also pointed to a necessity for an update of these models. Due to the unique features and developments specific to the current pandemic such as the occurrence of repeated collective stressors of varying durations, in the current position paper, we introduce the Wither or Thrive model of Resilience (With:Resilience). It integrates key aspects of prevailing psychological resilience frameworks within the context of the COVID-19 pandemic, and extends them by (1) moving away from single scale approaches towards a higher-order latent expression of resilience and vulnerability incorporating also non-clinical mental health markers, (2) proposing different trajectories of resilience-vulnerability emerging across repeated stressors over long periods of time, and (3) by incorporating multiple influencing factors including aspects of the socio-economic concept of social cohesion as well as separate mediating processing mechanisms. We propose that With:Resilience will enable a more nuanced approach and appropriate analytical investigation of the vast incoming data on mental health and resilience during the COVID-19 pandemic, and we suggest some concrete methodological approaches. This framework will assist in the development of actionable public health guidelines for society in the present and future pandemic contexts as well as aid policy making and the interventional sciences aimed at protecting the most vulnerable amongst us. The SARS-CoV-2 pandemic has had an immense impact on the daily lives of people across the globe. A vast empirical effort has been launched by researchers across the globe to document and understand the psychological impact, in terms of mental health and psychologically resilient functioning, resulting from the pandemic. While there exists an abundance of theoretical and empirical models of resilience and vulnerability in the field of psychology  or resilience questionnaires such as the Connor-Davidson Resilience Scale  and the mediating processing mechanisms through which they exert influence over the dynamic resilience-vulnerability process to enable coping with the stressor. This proposition of our model is in line with the important framework of dynamic resilience processes proposed by Kalisch and colleagues . Thus, iFifth, and crucially, not only do the predictors, and the regulating mechanisms through which they function, precipitate the outcome of dynamic resilience-vulnerability at any given moment and its process over time for an individual, they function differentially between persons. In line with other prevailing theoretical models of resilience, the With:Resilience framework posits that different predictors and mechanisms may become salient in different individuals within a given context and at a particular timepoint depending upon the nature of the stressor encountered. As such, this stressor- and context-dependent differential activation of predictors and mechanisms could potentially lead to varying outcomes of dynamic resilience-vulnerability for different individuals. Therefore, distinct potential trajectories of dynamic resilience-vulnerability process will likely emerge as a result.In sum, the proposed model accounts for (1) multi-method approaches to resilience and vulnerability, (2) the dynamic nature of resilience-vulnerability processes over time, (3) a variety of factors that influence resilience-vulnerability through (4) different processing mechanisms, and (5) interindividual as well as situational and context-specific differences. In the following we discuss in more detail all the different categories of influencing factors we integrate in our With:Resilience Model.A key set of predictors that influence the resilience-vulnerability outcome, and eventually the process over time, are factors that are unique to an individual, which can be characterized as relatively stable characteristics, and some of which can even be innate and inherent in nature. This category of predictors encompasses all biological, psychological and historical aspects that are specific to an individual and that impact the particular way an individual may respond to stressors. For example, a person high on neuroticism or with a history of childhood maltreatment is considered at risk of an adverse resilience-vulnerability trajectory . By doing this, it can, for example, account for different effects such as first lockdown shock effects  or lockdown fatigue effects (observed in further lockdowns extending over many months) and identify how different types of people can respond to such repeated collective stressors over time in different ways: some more resilient than others.Thirdly, the framework proposes a rather comprehensive set of factors ranging from psychological, biological, contextual and demographic individual factors as well as social intersubjective factors including social capacities and social cohesion markers. All these factors act as predictors influencing the different trajectories of resilience-vulnerability over time.Fourthly, the model proposes another class of variables as mediators between prediction factors and the classes of different trajectories of resilience-vulnerability outcomes. These processing mechanisms represent neurobiological, psychophysiological, socio-emotional as well as socio-cognitive regulating mechanisms such as epigenetic changes, autonomic regulation mechanisms, emotional regulations strategies, cognitive flexibility, appraisal, attributional and perceptual styles as well as social skills that in turn allow for social support strategies. As these intermediate processing mechanisms mediate whether specific factors will have detrimental effects on mental health or not, it is there where intervention research can most efficiently elicit adaptive changes.We further propose statistical methods of how to adequately model the complex interaction between influencing factors, processing mechanisms onto different classes of time trajectories of resilience-vulnerability outcomes. Importantly, the framework also has implications for public health policies and policymakers with respect to imposition of repeated long-term social isolation measures.In sum, the With:Resilience model provides an important theory-driven, integrative framework with a dynamic view on resilience and vulnerability that allows for appropriate modeling of complex longitudinal pandemic-related data, and helps identify which factors allow people to wither or to thrive when exposed to unprecedented, repeated and continuous stressors. As countries around the globe continue to battle with the fourth and the fifth waves of the pandemic with further lockdowns and social isolation orders, such a model will help to integrate existing datasets, help choose the adequate methodologies to model these complex data evolving over time, and assist in the development of actionable guidelines for society as well as the clinical sciences that are necessary to protect the most vulnerable amongst us.


Pseudarthrosis (PSA) is a possible complication of femoral shaft fracture treatment. It is often associated with reduced bone quality and can, therefore, adversely affect quality of life. Its treatment poses a major challenge for orthopaedic surgeons. Several authors have set forth different surgical approaches for the treatment of pseudarthrosis, such as internal fixation with plate and screws, replacement of an intramedullary nail or prosthetic replacement. In cases associated with bone loss, osteopenia, or comminution of fracture fragments, autologous or homologous bone grafts may also be used. The chronic outcomes of the surgical treatment of femoral shaft pseudarthrosis, even when consolidation is achieved, are linked to disabling sequelae of clinical-functional relevance, deserving an adequate medico-legal evaluation. The purpose of this retrospective study is to analyse a clinical case series of patients treated for atrophic femoral shaft pseudarthrosis at the IRCCS Orthopaedic Institute Galeazzi, Milan, Italy, from 2014 to 2020 and their orthopaedic-traumatological and medico-legal implications. Pseudarthrosis (PSA) has been defined in different ways in the literature. It has been described as a lack of radiographic consolidation at six months after the traumatic event and is associated with the necessity for revision surgery. According to the Food and Drug Administration (FDA), the diagnosis of PSA is made after at least nine months from the trauma with no visible signs of progressive healing for three months ,2.A higher incidence of PSA has been observed in femoral shaft fractures in recent years. However, it is an infrequent complication compared with pseudarthrosis of the humeral shaft , whose iNumerous techniques have been proposed for the treatment of femoral shaft pseudarthrosis, including electromagnetic fields , low-intUndoubtedly, the major severity of the conditions of road polytrauma patients and the increase in the average survival age have led to therapeutic problems due to the greater complexity of fractures and associated comorbidities such as obesity, diabetes, Chronic Obstructive Pulmonary Disease (COPD), correlated visceral injuries, and neurological diseases, including the pathological consequences of the recent severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) pandemic .In the 1970–1980s, femoral shaft PSA was considered an exceptional complication, while delayed union was less rare, affecting open fractures, especially with bone loss and infection. In closed fractures, treated conservatively, a muscle flap was sometimes interposed between the fragments, which constituted an insuperable barrier for the repairing tissue. Furthermore, it was believed that the non-consolidation was caused by an insufficient period of immobilisation in the plaster cast and/or by premature weight-bearing loading and walking .In the past, femur PSA was believed to be mostly related to errors in the treatment method. Recent research has shown that non-union, both atrophic and hypertrophic, is related to a certain degree of vascularisation and mesenchymal stem cells (MSCs) with osteogenic capacity.The non-consolidation of a femur fracture causes pain, especially during loading, altered and excessive motility at the fracture focus, functional limitations of the contiguous joints and muscle hypotrophy. It can therefore cause serious damage to the “functioning” capabilities of the injured lower limb, with consequent qualitative and quantitative alterations of everyday life activities. The lack of bone continuity of the femoral shaft and the consequent preternatural motility with instability to biomechanical stress prevent lower limb loading and severely reduce active and passive mobility, as well as walking. According to the disability model of the International Classification of Functioning, Disability and Health (ICF), adopted by the World Health Organization (WHO), five levels of disability involving the lower limbs have been identified: level 0 interference with the function is not demonstrable; level 1 interference is associated with vigorous exercise; level 2 analgesic claudication is presented and limits the distance travelled or requires the constant and regular use of an orthosis or a cane; level 3 presence of analgesic claudication with constant use of two sticks associated with orthoses; level 4 is impossible to walk . By adopThis study is centred on 16 patients, both male and female, with atrophic PSA after femoral shaft fracture, treated at the IRCCS Orthopaedic Institute Galeazzi, Milan, Italy, between 2014 to 2020. Patient charts, operation, and follow-up notes were retrospectively analysed.The indication for revision surgery was given in case of rupture of the fixation tools, severe osteopenia, cortical bone defect of ≥2 cm at the site of PSA or in the presence of disabling clinical symptoms.Surgical treatment of atrophic PSA of the femoral stem requires complete removal of the interposed necrotic tissue, remodelling of bone fragments and the restoration of fracture stability. Plate osteosynthesis, applied with an axial compression mechanism, is the best choice for most surgeons. The application of cortico-spongy grafts can increase the likelihood of consolidation. The graft, harvested from the patients themselves (autologous) or from a donor (homologous), enhances the mechanical stability of the synthesis to ensure total immobility of the fragments; in some cases, two plates mounted in parallel can be used for the treatment of humeral shaft PSA, although it may determine increased soft tissue dissection and a higher risk of bleeding. Open reduction and internal fixation with a long compression plate combined with homologous bone grafting is our treatment of choice, even in osteoporosis patients with reduced bone quality. However, in selected cases previously treated with an intramedullary nail, we have considered dynamisation of the nail when possible or replacement of the intramedullary nail after further reaming the femoral canal. Only in case of impossibility to perform a revision of the surgical synthesis has prosthetic replacement been considered.Healing after the second surgery was radiographically assessed through the presence of a callus in the PSA site with the restoration of bone continuity and through a physical and clinical examination to determine the solidity of consolidation.As preoperative antibiotic prophylaxis, cefazolin 2 g was administered intravenously, except for two patients who were given clindamycin 600 mg intravenously due to allergy concerns. Antibiotic prophylaxis was continued until the fifth postoperative day.Given the attention to residual haemostasis at the end of the surgical revision, we never used surgical drains. The surgical wound dressing was renewed every two days.The rehabilitation of the lower limb muscles started with isometric exercises and passive mobilisation of the affected limb the day after the surgery. Some cases may even call for pharmacological interventions (e.g. the administration of anabolic steroids) ,21.®, DJO global®, Lewisville, TX, USA) was placed after surgery in all the patients for thirty days. The 20% partial weight bearing started at four weeks after surgery. At eight weeks, in the presence of radiographic signs of healing, such as the appearance of normal osteo-reparative phenomena at the level of the pseudarthrosis focus, the patient was granted a tolerance load with two crutches.An articulated knee brace with long splints  at the time of trauma, which has been initially treated with an intramedullary nail or plate and screws. Nine cases involved the left side and seven the right side. In all cases, atrophic PSA resulted after a fracture caused by a high-energy trauma, mainly a car or motorcycle event.In our series, eight PSA occurred at the proximal third of the femoral shaft, four at the middle third and four at the distal third. Two patients were obese (body mass index ≥30), four were polytrauma patients with multiple district involvement, one patient had type 1 neurofibromatosis, four patients had multiple comorbidities , three patients were smokers, while none had alcohol abuse issues. In one case, the femoral fracture was linked to a point exposure, classified as type 1 according to the Gustilo-Anderson classification.Eight patients were treated by intramedullary nail, and eight patients by plate and screws. Two patients had undergone an intermediate surgery to dynamise the medullary nail, previously implanted, in order to address the consolidation delay.All these procedures were performed in other hospitals and came to our observation after finding the non-union of the fracture.All patients considered underwent revision surgery with new internal fixation.The time between the first osteosynthesis and the revision surgery for PSA was, on average, 454 days .Seven patients experienced the rupture of the implants previously implanted, while in the remaining nine, the implants were still intact when removed by revision surgery. All patients were treated by the same surgeon and the mean intervention time was 163 min (range 40–236).® plate, Zimmer Inc., Warsaw, IN, USA) associated with homologous bone graft, using a standard lateral surgical approach to the femoral shaft. After the removal of the previous implants, the focus of pseudarthrosis was exposed, removing the fibrous tissue between the bone fragments, and the bone stumps were reduced under fluoroscopic control with a C-arm. A bone graft splint was applied as a medial buttress, and internal fixation was performed with a plate and screw at the lateral side. The anatomical planes were then sutured before applying a sterile dressing  after further reaming of the medullary canal.Two patients were treated by revisioning the intramedullary nail using an antegrade nail with a larger diameter . In two patients, the onset of non-union in the distal third of the femoral diaphysis was associated with significant bone resorption and marked arthritic degeneration of the knee cartilage. In these cases, it was not possible to proceed with internal fixation, but it was necessary to implant a knee mega prosthesis   .In all cases, during the revision surgery, a lower level of callus formation at the PSA site was found intraoperatively, compatible with atrophic pseudarthrosis, and probably due to excessive rigidity with a lack of stimulus for the callus formation. The presence of thin shoots of loose fibrous tissue in the interfragmentary space was associated with the loss of bone substance of the fracture stumps, which also appeared sclerotic. The mean follow-up was of 15.4 months (range 6–38). The demographic characteristics of the patients considered and the different treatments applied in our study are summarised in Recovery from pseudarthrosis was reported in 13 patients (81% of cases), including patients who had had total hip arthroplasty and mega prosthesis of the knee implants.In three patients, the normal reparative healing processes did not occur in the follow-up period. These patients had multiple comorbidities, such as type 2 diabetes mellitus, hypertension, and COPD. No patient had a history of infection at the fracture site, so all the PSA were considered non-septic. There were no postoperative infections after revision surgery in any patient, although some complications did occur. In the postoperative course, four patients were transfused with concentrated red blood cell units for post-surgical anaemia. A supra-fascial fluid collection with no signs of infection was observed in one patient, while another one was left with a dystrophic scar, adhering to the underlying anatomical planes.Magnetotherapy was used by five patients to stimulate the bone healing of the PSA focus, leading to a progression of osteo-repairing phenomena.Recovery from pseudarthrosis occurred in all five patients (100%) initially treated with an intramedullary nail and subsequently treated with plate and screws and bone graft implant.Patients who achieved PSA consolidation after neo-osteosynthesis experienced mild thigh and knee pain, muscle hypotonia of varying degrees, reduced knee range of motion (ROM) with flexion block between 90–110°, and varying degrees of disability. The level of disability was grade 1 in seven cases and grade 2 in two cases.One patient treated with knee mega prosthesis presented decreased joint ROM, with a deficit of 15° both in flexion and in extension compared with the contralateral knee, and chronic pain, which increased with load and active mobilisation. The other patients treated with knee or hip resection prosthesis had a normal postoperative course, with normal or moderately reduced ROM, occasional pain, mild hypotonia, and 1–2 degree of disability.During the follow-up, none of the patients showed intolerance to the fixation implants or their mobilisation and in no case was the implanted material removed.Pseudarthrosis is a possible complication of femoral shaft fractures that require adequate therapeutic planning, which needs to be aimed towards two fundamental goals: mechanical stability to allow the early recovery of joint function and walking, and protection of vascularity, to minimise trophic damages, secondary to excessive cruentation at the PSA focus. Definitive surgery must ensure adequate stability of the fracture site, as well as accurate bone alignment, to allow for optimal functionality of the operated limb.The ultimate objective is to reduce the risk of permanent disability, often severe in a patient affected by PSA, especially in young patients incurring such a complication after a high-energy trauma.About possible treatments for PSA, Kouzelis et al. have demonstrated that reaming the femoral canal has an important osteo-inductive biological effect due to reaming debris, which acts as an autologous endogenous graft . AnotherInternal fixation of the plate and screw, however, may not be enough if associated with osteopenia, comminute fracture, or loss of bone stock. High complication rates have been reported in such cases, including misalignment, reduced mobility and persistent non-union . For thiIn other cases, as reported by Raju Vaishya et al., the unsuccessful internal fixation of distal femoral shaft fractures, associated with bone loss and arthritis of the knee with or without ligament instability, can be treated with a cemented mega prosthesis, especially in elderly patients .Earlier studies have presented results comparable with our own. To treat femoral shaft PSA, we often use internal fixation with plate and screws associated with the medial bone graft. This surgery allows for the removal of all the fibrotic tissue interposed. It enables the establishment of a stable, but at the same time flexible, system for adequate bone tissue formation. The use of autologous and homologous bone grafts constitutes a potentially invaluable tool; it makes it possible to rely on growth factors stimulating the formation of the reparative callus, thus obtaining a further stabilisation of the PSA focus, considering that patients with such a condition usually have poor bone quality.However, surgery must always be carefully planned based on fracture type, patient age, the site of the fracture and bone conditions in order to obtain good results both in terms of the healing process and quality of life.The examination of the proposed cases and the scientific literature allows us to exhibit some orthopaedic-traumatic and medico-legal remarks.The fracture healing process is influenced by many potentially interfering biological factors. For example, factors related to bone biology and genetic predisposition, patient-related factors such as lifestyle habits and comorbidities and some related to fracture characteristics such as topography or soft tissue injuries .Patient age is very important in determining the recovery process: in children and young adults, the periosteum is rich in osteoblasts and is well vascularised, whereas elderly patients usually exhibit a partially fibrous periosteum, associated with slower callus formation .In patients with osteoporosis, delayed expression of estrogen receptors was detected during the healing process and is associated with reduced callus formation. Nikolaou et al. reported the average consolidation time of femoral shaft fractures treated with a medullary nail in patients with osteoporosis to be three weeks longer than in healthy patients . FurtherThe activity of osteoprogenitor cells is also influenced by genetic factors. In fact, the recruitment, differentiation, and proliferation of osteogenic cells during the initial phase of fracture healing are reduced due to the low production of growth factors, such as BMPs and the qualitative and quantitative deficit of MSCs ,33. ManiIn addition, there is no discounting the role plaid by lifestyle habits such as smoking, alcohol and non-steroidal anti-inflammatory drugs (NSAIDs) abuse, stress, and poor eating habits, which constitute noteworthy risk factors for non-consolidation ,39. In aSeveral risk factors are closely linked to the type of fracture, such as the mechanism of injury as high or low energy trauma, vascular damage, and impairment of soft tissues. The number, the comminution of the fragments and the extent of the interfragmentary gap affect the healing process .In light of such findings, all factors related to the patient and the fracture’s type need to be thoroughly accounted for during the forensic evaluation of unsuccessful femoral shaft fracture treatment. Such factors often play a decisive role in the development of pseudarthrosis and can determine the negative outcomes of orthopaedic surgical treatments. At the same time, it will also be necessary to verify whether adequate biological therapies have been administered to stimulate the reparative process. Therefore, attention in the evaluation of any profiles of medical professional responsibility must be focused on the patients themselves, without being limited to an exclusive judgment on mechanical stability and correctness of internal fixation. The latter is necessary but not enough for a positive outcome of the treatment. We must also always consider the biological environment of the fracture. From 2020, moreover, the SarsCoV-2 pandemic has been disrupting the regular provision of clinical-instrumental controls, creating management difficulties not attributable to the individual surgeons, limited in part by the introduction of the applications of telemedicine which made it possible to overcome some organisational difficulties .The literature herein analysed and the results obtained from the analysis of our sample of patients underline the importance of the anatomo-functional damage caused by a femoral shaft pseudarthrosis, which can determine a 3 or 4 disability level, with severe or total functional impotence on the injured lower limb.For the treatment of femoral pseudarthrosis, an accurate assessment of the patient’s general condition, age, type of fracture and bone quality is essential to choosing the most suitable treatment to create the best possible conditions conducive to bone healing.From a medico-legal perspective, it is apparent that chronic consolidation outcomes after revision surgery of femoral shaft PSA cannot be compared with the results after a consolidated femoral fracture with mild functional repercussions. The PSA of the femur recovered after a second osteosynthesis with bone grafts is burdened by a major anatomical and functional impairment. Such impairment is caused by the double surgical access, the second one wider and more complex; by a morphologically and biomechanically less valid callus than the first intention consolidation one; by mild and unavoidable angular or rotational bone deformities; by greater residual hypotonia; by more significant repercussions in joint functions linked to the long therapeutic path and the myotendinous scars, especially of the knee.The physical damage resulting from the outcomes of femoral shaft PSA consolidated after a revision osteosynthesis treatment and the bone grafts, will be more substantial than the permanent sequelae following a femoral diaphyseal fracture consolidated ab initio. The presence of any limitations at the hip and/or knee range of motion, deformity and/or dysmetria will require an adequate medico-legal evaluation.


Background: Wandering among people with dementia (PwD) is associated with a high risk of injury and death. The stigma of dementia prevents Chinese dementia families from seeking information and support earlier, which increases the demand for long-term care facilities. Despite universal recognition of the importance of care facilities, healthcare providers in care facilities still lack the relevant nursing knowledge and skills, including non-pharmacological interventions (NPIs) that have been proven to be effective in preventing wandering. Systematic and culturally appropriate NPI programmes for healthcare providers to manage wandering among PwD in long-term care facilities are still lacking. We aimed to develop an evidence-based and culturally appropriate NPI programme for wandering in PwD to guide healthcare providers in nursing homes to prevent wandering and its adverse outcomes. Methods: The NPI programme was developed according to the framework of the Belgian Centre for Evidence-Based Medicine (CEBAM). We, (1) performed a systematic literature search to summarize the available evidence, (2) developed evidence-based recommendations for the NPI programme based on the existing evidence, and (3) carried out a validation process to revise the content of the recommendations and to determine the grades of recommendations, including group meetings with experts and a survey for end-users. Results: Based on 22 publications and validation from 7 experts and 76 end users, we developed 21 recommendations covering 4 domains: (1) caregiver education, (2) preventing excessive wandering, (3) promoting safe walking, and (4) preventing people with dementia from going missing. We created almost all recommendations of the four domains with accompanying levels of evidence and grades of recommendations. Conclusions: By combining the evidence with expert and end-user opinions, a comprehensive NPI programme was developed to support institutional healthcare providers to prevent wandering and its adverse outcomes. The benefits of this programme are currently being tested. With the incredible speed of population ageing, someone develops dementia every three seconds, and the number of people with dementia is set to increase to 152 million by the year 2050 . In 2020Wandering, a common trait found in PwD, refers to seemingly aimless or disoriented ambulation throughout a facility, often with a wide range of behaviours such as lapping, pacing, and random ambulation ,8. The cIn order to prevent these events, effective strategies should be taken to reduce wandering and ensure the safety of PwD. Despite universal recognition of the severe impact of dementia on people’s lives, Chinese people are generally unwilling to discuss dementia and mental health issues with others, and the stigma of dementia and the current one-child family structure in China might leave PwD without adequate support at home, increasing the demand for long-term care facilities ,16. HoweStudies on pharmacological and non-pharmacological interventions (NPIs) for wandering have been widely carried out. As pharmacological interventions could lead to unwanted side effects and negative consequences , personaHealthcare providers who care for PwD in nursing institutions require further training to increase their knowledge and capacities. Based on a systematic literature search, we aimed to evaluate and summarize the existing professional knowledge and the best evidence about NPIs for wandering in PwD to develop a systematic and culturally appropriate NPI programme. This optimized NPI programme will serve as a valuable tool to educate institutional healthcare providers on how to prevent and manage wandering and its adverse outcomes to improve the quality of life of PwD.We used the procedure of the Belgian Centre for Evidence-Based Medicine (CEBAM) model  to develWe assembled a working group to develop the recommendations: a neurologist nurse, a geriatric psychiatric specialist nurse, and a geriatric specialist nurse. Collectively, they have more than ten years of experience in dementia care. Based on a previous literature review and the combined experience of the working and author groups, we identified the research question: What NPIs are currently available to help healthcare providers prevent wandering in PwD? Through discussion and consensus within the author and working groups, we derived four domains: (1) caregiver education, (2) preventing excessive wandering, (3) promoting safe walking, and (4) preventing PwD from going missing.According to the conceptual framework provided by the 6S pyramid , we syst(i) To retrieve evidence-based decision information, we undertook an initial search of UpToDate and BMJ Best Practice from the inception of the project to 12 July 2021. The relevant search terms included wandering and dementia. Furthermore, we performed a systematic literature search in the Cochrane Library and the Joanna Briggs Institute (JBI) evidence-based practice to retrieve articles published between 2016 and 2021. Search terms consisted of both keywords and medical subject heading terms.(ii) We searched existing guidelines published from 2016 to 2021 in various databases, including the National Institute for Health and Clinical Excellence (NICE), the Registered Nurses’ Association of Ontario (RNAO), and the National Guideline Clearinghouse (NGC). The relevant search terms included wandering and dementia. Further, we included appropriate guidelines when searching other databases such as PubMed. Due to the absence of pertinent guidelines for the past six years, we extended the timeframe for searches.(iii) Since there were few intervention studies on the wandering behaviour of dementia patients, we supplemented the search of databases such as PubMed, Embase, and CINAHL. We restricted the timeframe for searches of publications from January 2016 to December 2021. We developed the search strategies according to specific database requirements and employed keywords and medical subject heading terms, which were the same throughout.(iv) By referring to the International Alzheimer’s Association member associations , we searThe participants were older adults with any form of dementia.The study was about specific NPIs for wandering in PwD.Articles meeting the following criteria were excluded:The study only included pharmacological interventions.Interventions were implemented in hospitals.Conference abstracts, protocols, introductions, or reviews other than systematic ones.Eligible articles had to meet the following criteria:Both authors, who had received a period of training beforehand, independently assessed the guidelines using the Appraisal of Guidelines and Research and Evaluation II (AGREE II) instrument . The AGRThe methodological quality of the included articles was evaluated by two authors independently using the JBI Critical Appraisal Checklist for the following study types: systematic reviews , analytiTwo authors independently used a standard data extraction form to extract the relevant data. In case of disagreements, a consensus was necessary for resolution. An overview of all included publications is provided in Data were extracted from each study and then used to inform the first draft of possible recommendations drawn up by the author group. The consensus method was used to establish the critical recommendations of the intervention programme. When there was disagreement regarding the evidence or rationale for/against a particular point, a majority opinion was required for resolution.The level of evidence was evaluated using the JBI Level of Evidence for Effectiveness . The JBIWe conducted additional validation of the recommendations. The findings were discussed within the author group, and recommendations were revised if necessary and applicable.(1) The first draft of the NPI programme was developed into a consultation questionnaire e-mailed to the experts ; informed consent was also obtained. Afterwards, group meetings were organized to assess and improve each formulated recommendation. The author group modified the recommendations according to the opinions put forward by the experts and refined the NPI programme following expert validation.(2) An online survey was set up to assess the importance and familiarity of recommendations in the first domain, and the feasibility and degree of completion of each formulated recommendation in the last three domains. The survey was then e-mailed to potential end users, including health care professionals in hospitals and institutions and family members of PwD. The respondents were asked to score recommendations on a scale of 1 to 10 for (i) importance, (ii) familiarity, (iii) feasibility, and (iv) degree of completion.(3) The final NPI programme was identified, and the grades of recommendation were assigned by the author groups and experts using the JBI Grades of Recommendation . The folBased on a broad search, we identified 230 articles, 55 of which we removed because they were duplicates. Further, we included five additional papers: three by extending the timeframe, and two from the reference lists. In a restrictive analysis of titles and abstracts, we excluded 102 articles from further analysis. Next, we subjected 78 articles to full-text analysis; 56 items did not meet the inclusion criteria. Finally, 22 articles constituted the evidence and validation base upon which we developed the recommendations. The results of the search and selection process are illustrated in In terms of the study design, we included one guideline , three sSome articles involved many aspects, and we broke them down to discuss them; therefore, the number of articles discussed in this section was greater than 22. Two studies ,55 reporOf the 22 eligible articles, we directly included 5; we evaluated 1 using the AGREE II instrument and appraised 9 studies using the JBI Critical Appraisal Checklist. Directly included articles encompassed three clinical decision-making studies, one evidence summary, and one recommended practice. We used the seven articles from the Alzheimer’s Society only as search clues and supplementary references. There is little insight into NPIs since physicians continue to prioritize pharmacological interventions. The expert selection was mainly in the nursing field through the group discussion. In total, seven experts confirmed the recommendations and helped to improve them further. End users, including health care professionals in hospitals and institutions and family members of PwD, were asked to assess the importance, familiarity, feasibility, and degree of completion of the recommendations. The importance score of the first domain of the programme and the feasibility and degree of completion score of the last three domains were all above average, so the recommendations were not deleted. The evaluation result is shown in We formulated 21 recommendations covering four domains: (1) caregiver education, (2) preventing excessive wandering, (3) promoting safe walking, and (4) preventing PwD from going missing. The recommendations of the four domains are presented in Whereas wandering within safe limits may have some benefits, such as providing physical exercise and social contact and improving appetite ,13, the Getting lost is inevitable, although extensive interventions may be put in place. Healthcare providers should be educated on the response plan in advance to search for PwD who go missing in a short time period ,28.Keep calm.Contact the local police immediately and provide a recent colour photo of the missing person, a description of his or her clothes, and details about past walking experiences, favourite places, or anywhere the person may have gone.Search the house and the surrounding buildings immediately.The initial 6 to 12 h of the search should cover an eight-mile radius around the location where the lost person was last seen, concentrating on open, populated areas, including the inside of easily accessible buildings.If the missing person has not been found, intense foot searches should focus on natural and sparsely populated areas, beginning within a two-mile radius of the last known location and extending from there, and ponds, gardens, and tree lines should be carefully searched.Search strategies should not be based on personal characteristics and experiences since PwD often exhibit unpredictable behaviour when lost.If the missing person has not been found, searches should continue through the night.If PwD travelled by bus or subway, initial search efforts should focus on locating the vehicle.When PwD become lost, the following steps should be taken ,50,52,56The cause of wandering is multifaceted as it may be an expression of unmet needs, such as a desire to relieve boredom or socialize with others , or it mAdditionally, physical exercise should not be unnecessarily limited, as it may ensure PwD get enough light during the day and improve sleep quality to reduce wandering ,45,61. RAs PwD get older, their vision, muscle strength, and coordination weaken, making them vulnerable to falls . ProvidiIn addition to environmental interventions, regular supervised exercise can reduce the desire to wander and the risk of falls and related injuries, to promote safe walking for PwD ,65,66,67For PwD with spatial disorientation, environmental cues to reduce the risk of loss and assist them in wayfinding may be effective, such as printed or graphic signage, personal items such as photographs, and posters and murals on walls ,28. WayfHigh-tech strategies should be employed to reduce attempts at exiting. For example, monitoring systems (such as warning bells and devices above doors) tracking the patient’s position, and sending signals (such as bells and buzzers) when a door was opened ,52,64,72Few systematic NPI programmes are available for healthcare providers in nursing homes to prevent and manage the wandering behaviour of PwD. In the present article, a comprehensive set of recommendations to provide healthcare providers with a NPI programme was developed by maintaining a systematic approach. Compared with previous studies on NPIs for wandering in PwD, the outstanding feature of the NPI programme developed in this work is that the four domains, including caregiver education, preventing excessive wandering, promoting safe walking, and preventing PwD from going missing, were sorted out by different purposes and priorities. Therefore, healthcare providers could make targeted choices. Most existing studies on NPIs for wandering are not comprehensive and systematic and only cover part of the contents of this NPI programme, such as the response plan on getting lost ,50, apprMoreover, the comprehensive set of recommendations for NPIs regarding wandering in PwD in this study could be better applied in practice with the participation of experts and end users. The procedure of the CEBAM attaches great importance to the validation process with emphasis on the involvement of experts to revise recommendations of the programme if applicable . For demn = 7) who replied to the survey. Second, the primary source of recommendations is the lack of high-quality studies, such as randomised controlled trials, and there are few guidelines on wandering, with only one published in recent years. In addition, the quality of some of the articles included was not assessed formally. We recommend additional training for healthcare providers before using the programme in the future, as merely providing a programme such as this will not be enough to improve practices. The final programme requires further testing in clinical practice to evaluate its feasibility and effectiveness and should be improved based on more high-quality research.Readers must be aware of several limitations of this study. The first limitation is the relatively small number of experts (As the disease progresses, the needs of PwD vary, and adults with dementia cannot receive adequate support at home, resulting in increased demand for long-term care facilities. There are few systematic and culturally appropriate NPI programmes for healthcare providers to manage the BPSD of dementia, such as wandering. This article developed a comprehensive NPI programme regarding wandering in PwD to provide culturally appropriate recommendations for healthcare providers in nursing homes. These recommendations can serve as a valuable tool to enrich their knowledge about wandering and guide them in preventing wandering and its adverse outcomes. The benefits of this programme are currently being tested.