Morphological Signatures of Neurogenesis and Neuronal Migration in Hypothalamic Vasopressinergic Magnocellular Nuclei of the Adult Rat.

The arginine vasopressin (AVP)-magnocellular neurosecretory system (AVPMNS) in the hypothalamus plays a critical role in homeostatic regulation as well as in allostatic motivational behaviors. However, it remains unclear whether adult neurogenesis exists in the AVPMNS. By using immunoreaction against AVP, neurophysin II, glial fibrillar acidic protein (GFAP), cell division marker (Ki67), migrating neuroblast markers (doublecortin, DCX), microglial marker (Ionized calcium binding adaptor molecule 1, Iba1), and 5'-bromo-2'-deoxyuridine (BrdU), we report morphological evidence that low-rate neurogenesis and migration occur in adult AVPMNS in the rat hypothalamus. Tangential AVP/GFAP migration routes and AVP/DCX neuronal chains as well as ascending AVP axonal scaffolds were observed. Chronic water deprivation significantly increased the BrdU+ nuclei within both the supraaoptic (SON) and paraventricular (PVN) nuclei. These findings raise new questions about AVPMNS's potential hormonal role for brain physiological adaptation across the lifespan, with possible involvement in coping with homeostatic adversities.

Longitudinal tracking of hemocyte populations in vivo indicates lineage relationships and supports neural progenitor identity in adult neurogenesis.

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types - hyaline (HC), semigranular (SGC) and granular (GC) cells - with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii.

Current progress and challenges in the development of brain tissue models: How to grow up the changeable brain in vitro?

In vitro modeling of brain tissue is a promising but not yet resolved problem in modern neurobiology and neuropharmacology. Complexity of the brain structure and diversity of cell-to-cell communication in (patho)physiological conditions make this task almost unachievable. However, establishment of novel in vitro brain models would ultimately lead to better understanding of development-associated or experience-driven brain plasticity, designing efficient approaches to restore aberrant brain functioning. The main goal of this review is to summarize the available data on methodological approaches that are currently in use, and to identify the most prospective trends in development of neurovascular unit, blood-brain barrier, blood-cerebrospinal fluid barrier, and neurogenic niche in vitro models. The manuscript focuses on the regulation of adult neurogenesis, cerebral microcirculation and fluids dynamics that should be reproduced in the in vitro 4D models to mimic brain development and its alterations in brain pathology. We discuss approaches that are critical for studying brain plasticity, deciphering the individual person-specific trajectory of brain development and aging, and testing new drug candidates in the in vitro models.

History and Recent Progress in Carotid Body Studies.

This chapter describes the history of the carotid body (CB) and the subsequent research on its structure and function. The chronological development of ideas about its anatomical structure as a ganglion, the first descriptions of its glandular nature as a ball of highly vascular tissue (glomus), the discovery of its neural crest origin and relevant embryological views as a true paraganglion toward a more conclusive understanding of its sensory nature as a chemoreceptor for chemical changes in blood have been consistently demonstrated. The knowledge of the CB neurochemistry, physiology and pathophysiology has progressed immensely in the past century and a large and compelling body of evidence for the presence of a neurogenic niche in the CB has accumulated over the last two decades, thus underlying its function and possibility for the development of cell replacement therapies.

The Carotid Body: A Tiny Structure with Many Roles.

Over the last century, the structure of the mammalian carotid body (CB) has repeatedly been studied, and our present understanding of its normal morphology is comprehensive. It has been demonstrated that the CB has an intricate internal structure and a remarkable ability to release a wide variety of neurotransmitters and neuromodulators in response to different chemical stimuli. The advances in modern cellular/molecular biological methods and newly developed single-cell electrophysiological techniques have provided an additional insight into the precise working mechanisms and roles of the CB in health and disease. Emerging experimental evidence has also shown that the CB exhibits an extraordinary structural and functional plasticity as a consequence of various environmental stimuli. Lately, the CB has attracted much clinical interest because its dysfunction relates to a number of cardiovascular and respiratory disorders. Expanding knowledge about the pathophysiological mechanisms that alter the CB cell function would certainly help to facilitate the translational research. Recent progress in cell fate experiments has further revealed that the CB is a neurogenic center with a functionally active germinal niche. This may lead to the development of promising new candidate therapies to combat these diseases and improve the quality of human life. Thus, the CB has entered the twenty-first century with its actual designation.

Neural stem cells characterization in the vagal complex of adult ovine brain: A combined neurosphere assay/RTqPCR approach.

Neural stem cells are the effectors of adult neurogenesis, which occurs in discrete restricted areas of adult mammalian brain. In ovine species, like in rodents, in vivo incorporation of labeled DNA precursor led to characterize neurogenic proliferation in the subventricular zone and progeny migration and differentiation into the olfactory bulb. The present study addresses directly the existence of neural stem cells in the neurogenic niche of the vagal centre (area postrema) by in vitro neurosphere assay and RT-qPCR of specific markers on ex-vivo adult tissue explants, comparatively with the canonical neurogenic niche: the subventricular zone (SVZ) of the forebrain. Explants defined from the neuroanatomical patterns of in vivo BrdU incorporation yielded expandable and self-renewing spheres from both SVZ and AP. Within SVZ though, the density of sphere-forming cells was higher in ventral SVZ (SVZ-V) than in its latero-dorsal (SVZ-D) and lateral (SVZ-L) regions, which differs from the distributions of neural stem cells in mouse and swine brains. Consistently, RT-qPCR of the biomarker of neural stem cells, Sox2, yields highest expression in SVZ-V ahead of SVZ-D, SVZ-L and AP. These results are discussed with regard to previously published dynamics of adult ovine neurogenesis in vivo, and in light of corresponding features in other mammalian species. This confirms existence of neurogenetic plasticity in the vagal complex of adult mammals.

From Youthful Vigor to Aging Decline: Unravelling the Intrinsic and Extrinsic Determinants of Hippocampal Neural Stem Cell Aging.

Since Joseph Altman published his pioneering work demonstrating neurogenesis in the hippocampus of adult rats, the number of publications in this field increased exponentially. Today, we know that the adult hippocampus harbors a pool of adult neural stem cells (NSCs) that are the source of life-long neurogenesis and plasticity. The functions of these NSCs are regulated by extrinsic cues arising from neighboring cells and the systemic environment. However, this tight regulation is subject to imbalance with age, resulting in a decline in adult NSCs and neurogenesis, which contributes to the progressive deterioration of hippocampus-related cognitive functions. Despite extensive investigation, the mechanisms underlying this age-related decline in neurogenesis are only incompletely understood, but appear to include an increase in NSC quiescence, changes in differentiation patterns, and NSC exhaustion. In this review, we summarize recent work that has improved our knowledge of hippocampal NSC aging, focusing on NSC-intrinsic mechanisms as well as cellular and molecular changes in the niche and systemic environment that might be involved in the age-related decline in NSC functions. Additionally, we identify future directions that may advance our understanding of NSC aging and the concomitant loss of hippocampal neurogenesis and plasticity.

Therapeutic strategies to recover ependymal barrier after inflammatory damage: relevance for recovering neurogenesis during development.

The epithelium covering the surfaces of the cerebral ventricular system is known as the ependyma, and is essential for maintaining the physical and functional integrity of the central nervous system. Additionally, the ependyma plays an essential role in neurogenesis, neuroinflammatory modulation and neurodegenerative diseases. Ependyma barrier is severely affected by perinatal hemorrhages and infections that cross the blood brain barrier. The recovery and regeneration of ependyma after damage are key to stabilizing neuroinflammatory and neurodegenerative processes that are critical during early postnatal ages. Unfortunately, there are no effective therapies to regenerate this tissue in human patients. Here, the roles of the ependymal barrier in the context of neurogenesis and homeostasis are reviewed, and future research lines for development of actual therapeutic strategies are discussed.

The differential response to neuronal hyperexcitation and neuroinflammation of the hippocampal neurogenic niche.

Hippocampal neurogenesis is a tightly regulated process in which neural stem cells (NSCs) get activated, enter in the cell cycle and give rise to neurons after a multistep process. Quiescent and activated NSCs, neural precursors, immature and mature neurons and newborn astrocytes coexist in the neurogenic niche in a strictly controlled environment which maintains the correct functioning of neurogenesis. NSCs are the first step in the neurogenic process and are a finite and, mostly, non-renewable resource, therefore any alteration of the intrinsic properties of NSCs will impact the total neurogenic output. Neuronal hyperexcitation is a strong activator of NSCs prompting them to divide and therefore increasing neurogenesis. However, neuronal hyperactivity is not an isolated process but often also involves excitotoxicity which is subsequently accompanied by neuroinflammation. Neuroinflammation normally reduces the activation of NSCs. It is technically difficult to isolate the effect of neuronal hyperexcitation alone, but neuroinflammation without neuronal hyperexcitation can be studied in a variety of models. In order to shed light on how the balance of neuronal hyperexcitation and neuroinflammation affect NSCs we analyzed proliferation and morphology of NSCs. We used two models of neuronal hyperactivity [an epilepsy model induced by KA, and a model of traumatic brain injury (TBI)] and different models of inflammation (LPS, Poly I:C, IFN-α and IL-6). We observed that only those models that induce neuronal hyperactivity induce NSCs activation but neuroinflammation causes the opposite effect. We also analyzed the response of other cell types in the neurogenic niche, focusing on astrocytes.

Neural Progenitor Cells and the Hypothalamus.

Neural progenitor cells (NPCs) are multipotent neural stem cells (NSCs) capable of self-renewing and differentiating into neurons, astrocytes and oligodendrocytes. In the postnatal/adult brain, NPCs are primarily located in the subventricular zone (SVZ) of the lateral ventricles (LVs) and subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). There is evidence that NPCs are also present in the postnatal/adult hypothalamus, a highly conserved brain region involved in the regulation of core homeostatic processes, such as feeding, metabolism, reproduction, neuroendocrine integration and autonomic output. In the rodent postnatal/adult hypothalamus, NPCs mainly comprise different subtypes of tanycytes lining the wall of the 3rd ventricle. In the postnatal/adult human hypothalamus, the neurogenic niche is constituted by tanycytes at the floor of the 3rd ventricle, ependymal cells and ribbon cells (showing a gap-and-ribbon organization similar to that in the SVZ), as well as suprachiasmatic cells. We speculate that in the postnatal/adult human hypothalamus, neurogenesis occurs in a highly complex, exquisitely sophisticated neurogenic niche consisting of at least four subniches; this structure has a key role in the regulation of extrahypothalamic neurogenesis, and hypothalamic and extrahypothalamic neural circuits, partly through the release of neurotransmitters, neuropeptides, extracellular vesicles (EVs) and non-coding RNAs (ncRNAs).

The vasculature of neurogenic niches: Properties and function.

In the adult rodent brain, neural stem cells (NSCs) reside in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. In these areas, NSCs and their progeny integrate intrinsic signals and extrinsic cues provided by their microenvironment that control their behavior. The vasculature in the SVZ and SGZ, and the choroid plexus (ChP) in the SVZ, have emerged as critical compartments of the neurogenic niches as they provide a rich repertoire of cues to regulate NSC quiescence, proliferation, self-renewal and differentiation. Physical contact between NSCs and blood vessels is also a feature within the niches and supports different processes such as quiescence, migration and vesicle transport. In this review, we provide a description of the brain and choroid plexus vasculature in both stem cell niches, highlighting the main properties and role of the vasculature in each niche. We also summarize the current understanding of how blood vessel- and ChP-derived signals influence the behavior of NSCs in physiological adulthood, as well as upon aging.

Rearrangement of cell types in the rat carotid body neurogenic niche induced by chronic intermittent hypoxia.

The carotid body (CB) is a prototypical acute oxygen (O2 )-sensing organ that mediates reflex hyperventilation and increased cardiac output in response to hypoxaemia. CB overactivation, secondary to the repeated stimulation produced by the recurrent episodes of intermittent hypoxia, is believed to contribute to the pathogenesis of sympathetic hyperactivity present in sleep apnoea patients. Although CB functional plasticity induced by chronic intermittent hypoxia (CIH) has been demonstrated, the underlying mechanisms are not fully elucidated. Here, we show that CIH induces a small increase in CB volume and rearrangement of cell types in the CB, characterized by a mobilization of immature quiescent neuroblasts, which enter a process of differentiation into mature, O2 -sensing and neuron-like, chemoreceptor glomus cells. Prospective isolation of individual cell classes has allowed us to show that maturation of CB neuroblasts is paralleled by an upregulation in the expression of specific glomus cell genes involved in acute O2 -sensing. CIH enhances mitochondrial responsiveness to hypoxia in maturing neuroblasts as well as in glomus cells. These data provide novel perspectives on the pathogenesis of CB-mediated sympathetic overflow that may lead to the development of new pharmacological strategies of potential applicability in sleep apnoea patients. KEY POINTS: Obstructive sleep apnoea is a frequent condition in the human population that predisposes to severe cardiovascular and metabolic alterations. Activation of the carotid body, the main arterial oxygen-sensing chemoreceptor, by repeated episodes of hypoxaemia induces exacerbation of the carotid body-mediated chemoreflex and contributes to sympathetic overflow characteristic of sleep apnoea patients. In rats, chronic intermittent hypoxaemia induces fast neurogenesis in the carotid body with rapid activation of neuroblasts, which enter a process of proliferation and maturation into O2 -sensing chemoreceptor glomus cells. Maturing carotid body neuroblasts and glomus cells exposed to chronic intermittent hypoxia upregulate genes involved in acute O2 sensing and enhance mitochondrial responsiveness to hypoxia. These findings provide novel perspectives on the pathogenesis of carotid body-mediated sympathetic hyperactivation. Pharmacological modulation of carotid body fast neurogenesis could help to ameliorate the deleterious effects of chronic intermittent hypoxaemia in sleep apnoea patients.

Extracellular vesicles in the glioblastoma microenvironment: A diagnostic and therapeutic perspective.

Glioblastoma (GBM), is the most malignant form of gliomas and the most common and lethal primary brain tumor in adults. Conventional cancer treatments have limited to no efficacy on GBM. GBM cells respond and adapt to the surrounding brain parenchyma known as tumor microenvironment (TME) to promote tumor preservation. Among specific TME, there are 3 of particular interest for GBM biology: the perivascular niche, the subventricular zone neurogenic niche, and the immune microenvironment. GBM cells and TME cells present a reciprocal feedback which results in tumor maintenance. One way that these cells can communicate is through extracellular vesicles. These vesicles include exosomes and microvesicles that have the ability to carry both cancerous and non-cancerous cargo, such as miRNA, RNA, proteins, lipids, and DNA. In this review we will discuss the booming topic that is extracellular vesicles, and how they have the novelty to be a diagnostic and targetable vehicle for GBM.

Promoting Endogenous Neurogenesis as a Treatment for Alzheimer's Disease.

Alzheimer's disease (AD) is the most universal neurodegenerative disorder characterized by memory loss and cognitive impairment. AD is biologically defined by production and aggregation of misfolded protein including extracellular amyloid ß (Aß) peptide and intracellular microtubule-associated protein tau tangles in neurons, leading to irreversible neuronal loss. At present, regulation of endogenous neurogenesis to supplement lost neurons has been proposed as a promising strategy for treatment of AD. However, the exact underlying mechanisms of impaired neurogenesis in AD have not been fully explained and effective treatments targeting neurogenesis for AD are limited. In this review, we mainly focus on the latest research of impaired neurogenesis in AD. Then we discuss the factors affecting stages of neurogenesis and the interplay between neural stem cells (NSCs) and neurogenic niche under AD pathological conditions. This review aims to explore potential therapeutic strategies that promote endogenous neurogenesis for AD treatments.

Distinct characteristics of microglia from neurogenic and non-neurogenic regions of the human brain in patients with Mesial Temporal Lobe Epilepsy.

The study of microglia isolated from adult human brain tissue provides unique insight into the physiology of these brain immune cells and their role in adult human brain disorders. Reports of microglia in post-mortem adult human brain tissue show regional differences in microglial populations, however, these differences have not been fully explored in living microglia. In this study biopsy tissue was obtained from epileptic patients undergoing surgery and consisted of both cortical areas and neurogenic ventricular and hippocampal (Hp) areas. Microglia were concurrently isolated from both regions and compared by immunochemistry. Our initial observation was that a greater number of microglia resulted from isolation and culture of ventricular/Hp tissue than cortical tissue. This was found to be due to a greater proliferative capacity of microglia from ventricular/Hp regions compared to the cortex. Additionally, ventricular/Hp microglia had a greater proliferative response to the microglial mitogen Macrophage Colony-Stimulating Factor (M-CSF). This enhanced response was found to be associated with higher M-CSF receptor expression and higher expression of proteins involved in M-CSF signalling DAP12 and C/EBPß. Microglia from the ventricular/Hp region also displayed higher expression of the receptor for Insulin-like Growth Factor-1, a molecule with some functional similarity to M-CSF. Compared to microglia isolated from the cortex, ventricular/Hp microglia showed increased HLA-DP, DQ, DR antigen presentation protein expression and a rounded morphology. These findings show that microglia from adult human brain neurogenic regions are more proliferative than cortical microglia and have a distinct protein expression profile. The data present a case for differential microglial phenotype and function in different regions of the adult human brain and suggest that microglia in adult neurogenic regions are "primed" to an activated state by their unique tissue environment.

The Neurotoxic Effect of Ochratoxin-A on the Hippocampal Neurogenic Niche of Adult Mouse Brain.

Ochratoxin A (OTA) is a common secondary metabolite of Aspergillus ochraceus, A. carbonarius, and Penicillium verrucosum. This mycotoxin is largely present as a contaminant in several cereal crops and human foodstuffs, including grapes, corn, nuts, and figs, among others. Preclinical studies have reported the involvement of OTA in metabolic, physiologic, and immunologic disturbances as well as in carcinogenesis. More recently, it has also been suggested that OTA may impair hippocampal neurogenesis in vivo and that this might be associated with learning and memory deficits. Furthermore, aside from its widely proven toxicity in tissues other than the brain, there is reason to believe that OTA contributes to neurodegenerative disorders. Thus, in this present in vivo study, we investigated this possibility by intraperitoneally (i.p.) administering 3.5 mg OTA/kg body weight to adult male mice to assess whether chronic exposure to this mycotoxin negatively affects cell viability in the dentate gyrus of the hippocampus. Immunohistochemistry assays showed that doses of 3.5 mg/kg caused a significant and dose-dependent reduction in repetitive cell division and branching (from 12% to 62%). Moreover, the number of countable astrocytes (p < 0.001), young neurons (p < 0.001), and mature neurons (p < 0.001) negatively correlated with the number of i.p. OTA injections administered (one, two, three, or six repeated doses). Our results show that OTA induced adverse effects in the hippocampus cells of adult mice brain tissue when administered in cumulative doses.

The endogenous progenitor response following traumatic brain injury: a target for cell therapy paradigms.

Although there is ample evidence that central nervous system progenitor pools respond to traumatic brain injury, the reported effects are variable and likely contribute to both recovery as well as pathophysiology. Through a better understanding of the diverse progenitor populations in the adult brain and their niche-specific reactions to traumatic insult, treatments can be tailored to enhance the benefits and dampen the deleterious effects of this response. This review provides an overview of endogenous precursors, the associated effects on cognitive recovery, and the potential of exogenous cell therapeutics to modulate these endogenous repair mechanisms. Beyond the hippocampal dentate gyrus and subventricular zone of the lateral ventricles, more recently identified sites of adult neurogenesis, the meninges, as well as circumventricular organs, are also discussed as targets for endogenous repair. Importantly, this review highlights that progenitor proliferation alone is no longer a meaningful outcome and studies must strive to better characterize precursor spatial localization, transcriptional profile, morphology, and functional synaptic integration. With improved insight and a more targeted approach, the stimulation of endogenous neurogenesis remains a promising strategy for recovery following traumatic brain injury.

Wnt Signaling in the Adult Hippocampal Neurogenic Niche.

The subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) is a neurogenic niche of the adult brain that contains neural stem cells (NSCs) able to generate excitatory glutamatergic granule neurons, which integrate into the DG circuit and contribute to hippocampal plasticity, learning, and memory. Thus, endogenous NSCs could be harnessed for therapeutic purposes. In this context, it is critical to characterize the molecular mechanisms controlling the generation and functional integration of adult-born neurons. Adult hippocampal neurogenesis is tightly controlled by both cell-autonomous mechanisms and the interaction with the complex niche microenvironment, which harbors the NSCs and provides the signals to support their maintenance, activation, and differentiation. Among niche-derived factors, Wnt ligands play diverse roles. Wnts are secreted glycoproteins that bind to Frizzled receptors and co-receptors to trigger the Wnt signaling pathway. Here, we summarize the current knowledge about the roles of Wnts in the regulation of adult hippocampal neurogenesis. We discuss the possible contribution of the different niche cells to the regulation of local Wnt signaling activity, and how Wnts derived from different cell types could induce differential effects. Finally, we discuss how the effects of Wnt signaling on hippocampal network activity might contribute to neurogenesis regulation. Although the evidence supports relevant roles for Wnt signaling in adult hippocampal neurogenesis, defining the cellular source and the mechanisms controlling secretion and diffusion of Wnts will be crucial to further understand Wnt signaling regulation of adult NSCs, and eventually, to propose this pathway as a therapeutic target to promote neurogenesis.

Assessing the Role of Ependymal and Vascular Cells as Sources of Extracellular Cues Regulating the Mouse Ventricular-Subventricular Zone Neurogenic Niche.

Neurogenesis persists in selected regions of the adult mouse brain; among them, the ventricular-subventricular zone (V-SVZ) of the lateral ventricles represents a major experimental paradigm due to its conspicuous neurogenic output. Postnatal V-SVZ neurogenesis is maintained by a resident population of neural stem cells (NSCs). Although V-SVZ NSCs are largely quiescent, they can be activated to enter the cell cycle, self-renew and generate progeny that gives rise to olfactory bulb interneurons. These adult-born neurons integrate into existing circuits to modify cognitive functions in response to external stimuli, but cells shed by V-SVZ NSCs can also reach injured brain regions, suggesting a latent regenerative potential. The V-SVZ is endowed with a specialized microenvironment, which is essential to maintain the proliferative and neurogenic potential of NSCs, and to preserve the NSC pool from exhaustion by finely tuning their quiescent and active states. Intercellular communication is paramount to the stem cell niche properties of the V-SVZ, and several extracellular signals acting in the niche milieu have been identified. An important part of these signals comes from non-neural cell types, such as local vascular cells, ependymal and glial cells. Understanding the crosstalk between NSCs and other niche components may aid therapeutic approaches for neuropathological conditions, since neurodevelopmental disorders, age-related cognitive decline and neurodegenerative diseases have been associated with dysfunctional neurogenic niches. Here, we review recent advances in the study of the complex interactions between V-SVZ NSCs and their cellular niche. We focus on the extracellular cues produced by ependymal and vascular cells that regulate NSC behavior in the mouse postnatal V-SVZ, and discuss the potential implication of these molecular signals in pathological conditions.