Methodological advice for the young at heart investigator: Triangulation to build better foundations.

In medicine and science, one is typically taught the main theories in a discipline or field along with standard models before receiving more instructions on how to apply certain methods. The aim of this work is not to address one method, but rather methodology, the study and evaluation of methods, by taking a philosophy of science detour. In this, a critique of biomedicine will be used as a starting point to address some positions regarding reductionism, specifying notions such as systems and mechanisms, as well as regarding the mind-body problem discussing psychosomatic medicine and psychoneuroimmunology. Some recommendations to make science more pluralistic, robust and translationally-relevant will then be made as a way to foster constructive debates on reductionism and the mind-body problem and, in turn, favor more interdisciplinary research.

Causal histories of psychological factors and cancer: from psychosomatic medicine to neuroimmunomodulation.

BACKGROUND: Establishing causal relationships is essential in biology and medicine. However, various notions of causality have been operationalized at different times in various fields of the life and health sciences. While this is expected from a history or sociology of science point of view, as different accounts may correspond to what is valued in terms of establishing causal relationship at different times as well as in different fields of biology and medicine, this may come as a surprise for a present-day actor in those fields. If, over time, causal accounts have not been fully dismissed, then they are likely to invite to some form of, potentially salutary, explanatory pluralism. SUMMARY: In the decades following WWII, psychosomatic medicine could propose that psychological factors cause somatic diseases. But today, most of medicine has to meet the standard of a randomized clinical trial before any causal relationship can be proposed. Instead, in biology, mechanisms seem to be the most-valued causal discourse to explain how phenomena of interest are brought about. Here, the focus will be on how psychoneuroimmunology, an interdisciplinary research field addressing interactions between the nervous system and immune system, and between behavior and health, has considered causal relationships between psychological factors and cancer. KEY MESSAGES: When it comes to causal explanations of links between psychological factors and cancer, psychoneuroimmunology is invited to consider the question of the directionality of these links as well as what and how factors causally contribute to cancer.

Expanding the notion of mechanism to further understanding of biopsychosocial disorders? Depression and medically-unexplained pain as cases in point.

Evidence-Based Medicine has little consideration for mechanisms and philosophers of science and medicine have recently made pleas to increase the place of mechanisms in the medical evidence hierarchy. However, in this debate the notions of mechanisms seem to be limited to 'mechanistic processes' and 'complex-systems mechanisms,' understood as 'componential causal systems'. I believe that this will not do full justice to how mechanisms are used in biological, psychological and social sciences and, consequently, in a more biopsychosocial approach to medicine. Here, I propose, following (Kuorikoski, 2009), to pay more attention to 'abstract forms of interaction' mechanisms. The present work scrutinized review articles on depression and medically unexplained pain, which are considered to be of multifactorial pathogenesis, for their use of mechanisms. In review articles on these disorders there seemed to be a range of uses between more 'abstract forms of interaction' and 'componential causal system' mechanisms. I therefore propose to expand the notions of mechanisms considered in medicine to include that of more 'abstract forms of interaction' to better explain and manage biopsychosocial disorders.

Do astrocytes act as immune cells after pediatric TBI?

Astrocytes are in contact with the vasculature, neurons, oligodendrocytes and microglia, forming a local network with various functions critical for brain homeostasis. One of the primary responders to brain injury are astrocytes as they detect neuronal and vascular damage, change their phenotype with morphological, proteomic and transcriptomic transformations for an adaptive response. The role of astrocytic responses in brain dysfunction is not fully elucidated in adult, and even less described in the developing brain. Children are vulnerable to traumatic brain injury (TBI), which represents a leading cause of death and disability in the pediatric population. Pediatric brain trauma, even with mild severity, can lead to long-term health complications, such as cognitive impairments, emotional disorders and social dysfunction later in life. To date, the underlying pathophysiology is still not fully understood. In this review, we focus on the astrocytic response in pediatric TBI and propose a potential immune role of the astrocyte in response to trauma. We discuss the contribution of astrocytes in the local inflammatory cascades and secretion of various immunomodulatory factors involved in the recruitment of local microglial cells and peripheral immune cells through cerebral blood vessels. Taken together, we propose that early changes in the astrocytic phenotype can alter normal development of the brain, with long-term consequences on neurological outcomes, as described in preclinical models and patients.

Early life cancer and chemotherapy lead to cognitive deficits related to alterations in microglial-associated gene expression in prefrontal cortex.

Children that survive leukemia are at an increased risk for cognitive difficulties. A better understanding of the neurobiological changes in response to early life chemotherapy will help develop therapeutic strategies to improve quality of life for leukemia survivors. To that end, we used a translationally-relevant mouse model consisting of leukemic cell line (L1210) injection into postnatal day (P)19 mice followed by methotrexate, vincristine, and leucovorin chemotherapy. Beginning one week after the end of chemotherapy, social behavior, recognition memory and executive function (using the 5 choice serial reaction time task (5CSRTT)) were tested in male and female mice. Prefrontal cortex (PFC) and hippocampus (HPC) were collected at the conclusion of behavioral assays for gene expression analysis. Mice exposed to early life cancer + chemotherapy were slower to progress through increasingly difficult stages of the 5CSRTT and showed an increase in premature errors, indicating impulsive action. A cluster of microglial-related genes in the PFC were found to be associated with performance in the 5CSRTT and acquisition of the operant response, and long-term changes in gene expression were evident in both PFC and HPC. This work identifies gene expression changes in PFC and HPC that may underlie cognitive deficits in survivors of early life exposure to cancer + chemotherapy.

Translationally relevant mouse model of early life cancer and chemotherapy exposure results in brain and small intestine cytokine responses: A potential link to cognitive deficits.

Survivors of acute lymphoblastic leukemia (ALL), the most common childhood cancer, are at increased risk for long-term cognitive problems, including executive function deficits. The chemotherapeutic agent methotrexate (MTX) is used to treat most ALL patients and is closely associated with cognitive deficits. To address how early life cancer chemotherapy leads to cognitive deficits, we developed a translationally relevant mouse model of leukemia survival that exposed mice to leukemic cells and chemotherapeutic drugs (vincristine and MTX, with leucovorin rescue) in early life. Male and female mice were tested several weeks later using novel object recognition (recognition memory) and 5-choice serial reaction time task (executive function). Gene expression of proinflammatory, white matter and synapse-associated molecules was assessed in the prefrontal cortex and small intestine both acutely after chemotherapy and chronically after cognitive testing. Early life cancer-chemotherapy exposure resulted in recognition memory and executive function deficits in adult male mice. Prefrontal cortex expression of the chemokine Ccl2 was increased acutely, while small intestine expression of the proinflammatory cytokine tumor necrosis factor-alpha was elevated both acutely (both sexes) and chronically (males only). Inflammation in the small intestine was correlated with prefrontal cortical proinflammatory and synaptic gene expression changes, as well as to executive function deficits. Collectively, these data indicate that the current protocol results in a robust mouse model in which to study cognitive deficits in leukemia survivors, and suggest that small intestine inflammation may represent a novel contributor to adverse CNS consequences of early life chemotherapy.

Experimental sepsis-associated encephalopathy is accompanied by altered cerebral blood perfusion and water diffusion and related to changes in cyclooxygenase-2 expression and glial cell morphology but not to blood-brain barrier breakdown.

Sepsis-associated encephalopathy (SAE) refers to brain dysfunction, including delirium, occurs during severe infection and is associated with development of post-traumatic stress disorder. SAE has been proposed to be related to reduced cerebral blood flow (CBF), blood-brain barrier breakdown (BBB), white matter edema and disruption and glia cell activation, but their exact relationships remain to be determined. In the present work, we set out to study CBF using Arterial Spin Labeling (ASL) and grey and white matter structure with T2- and diffusion magnetic resonance imaging (dMRI) in rats with cecal ligation and puncture (CLP)-induced encephalopathy. Using immunohistochemistry, the distribution of the vasoactive prostaglandin-synthesizing enzyme cyclooxygenase-2 (COX-2), perivascular immunoglobulins G (IgG), aquaporin-4 (AQP4) and the morphology of glial cell were subsequently assessed in brains of the same animals. CLP induced deficits in the righting reflex and resulted in higher T2-weighted contrast intensities in the cortex, striatum and at the base of the brain, decreased blood perfusion distribution to the cortex and increased water diffusion parallel to the fibers of the corpus callosum compared to sham surgery. In addition, CLP reduced staining for microglia- and astrocytic-specific proteins in the corpus callosum, decreased neuronal COX-2 and AQP4 expression in the cortex while inducing perivascular COX-2 expression, but did not induce widespread perivascular IgG diffusion. In conclusion, our findings indicate that experimental SAE can occur in the absence of BBB breakdown and is accompanied by increased water diffusion anisotropy and altered glia cell morphology in brain white matter.

Interleukin-1 reduces food intake and body weight in rat by acting in the arcuate hypothalamus.

A reduction in food intake is commonly observed after bacterial infection, a phenomenon that can be reproduced by peripheral administration of Gram-negative bacterial lipopolysaccharide (LPS) or interleukin-1beta (IL-1ß), a pro-inflammatory cytokine released by LPS-activated macrophages. The arcuate nucleus of the hypothalamus (ARH) plays a major role in food intake regulation and expresses IL-1 type 1 receptor (IL-1R1) mRNA. In the present work, we tested the hypothesis that IL-1R1 expressing cells in the ARH mediate IL-1ß and/or LPS-induced hypophagia in the rat. To do so, we developed an IL-1ß-saporin conjugate, which eliminated IL-R1-expressing neurons in the hippocampus, and micro-injected it into the ARH prior to systemic IL-1ß and LPS administration. ARH IL-1ß-saporin injection resulted in loss of neuropeptide Y-containing cells and attenuated hypophagia and weight loss after intraperitoneal IL-1ß, but not LPS, administration. In conclusion, the present study shows that ARH NPY-containing neurons express functional IL-1R1s that mediate peripheral IL-1ß-, but not LPS-, induced hypophagia. Our present and previous findings indicate that the reduction of food intake after IL-1ß and LPS are mediated by different neural pathways.

Gas Diffusion in the CNS.

Gases have been long known to have essential physiological functions in the CNS such as respiration or regulation of vascular tone. Since gases have been classically considered to freely diffuse, research in gas biology has so far focused on mechanisms of gas synthesis and gas reactivity, rather than gas diffusion and transport. However, the discovery of gas pores during the last two decades and the characterization of diverse diffusion patterns through different membranes has raised the possibility that modulation of gas diffusion is also a physiologically relevant parameter. Here we review the means of gas movement into and within the brain through "free" diffusion and gas pores, notably aquaporins, discussing the role that gas diffusion may play in the modulation of gas function. We highlight how diffusion is relevant to neuronal signaling, volume transmission, and cerebrovascular control in the case of NO, one of the most extensively studied gases. We point out how facilitated transport can be especially relevant for gases with low permeability in lipid membranes like NH3 and discuss the possible implications of NH3 -permeable channels in physiology and hyperammonemic encephalopathy. We identify novel research questions about how modulation of gas diffusion could intervene in CNS pathologies. This emerging area of research can provide novel and interesting insights in the field of gas biology.

Neural pathways involved in infection-induced inflammation: recent insights and clinical implications.

Although the immune and nervous systems have long been considered independent biological systems, they turn out to mingle and interact extensively. The present review summarizes recent insights into the neural pathways activated by and involved in infection-induced inflammation and discusses potential clinical applications. The simplest activation concerns a reflex action within C-fibers leading to neurogenic inflammation. Low concentrations of pro-inflammatory cytokines or bacterial fragments may also act on these afferent nerve fibers to signal the central nervous system and bring about early fever, hyperalgesia and sickness behavior. In the brain, the preoptic area and the paraventricular hypothalamus are part of a neuronal network mediating sympathetic activation underlying fever while brainstem circuits play a role in the reduction of food intake after systemic exposure to bacterial fragments. A vagally-mediated anti-inflammatory reflex mechanism has been proposed and, in turn, questioned because the major immune organs driving inflammation, such as the spleen, are not innervated by vagal efferent fibers. On the contrary, sympathetic nerves do innervate these organs and modulate immune cell responses, production of inflammatory mediators and bacterial dissemination. Noradrenaline, which is both released by these fibers and often administered during sepsis, along with adrenaline, may exert pro-inflammatory actions through the stimulation of ß1 adrenergic receptors, as antagonists of this receptor have been shown to exert anti-inflammatory effects in experimental sepsis.

Microbiota-gut-brain research: A critical analysis.

Microbiota-gut-brain (MGB) research is a fast-growing field of inquiry with important implications for how human brain function and behaviour are understood. Researchers manipulate gut microbes ("microbiota") to reveal connections between intestinal microbiota and normal brain functions (e.g., cognition, emotion, and memory) or pathological states (e.g., anxiety, mood disorders, and neural developmental disorders such as autism). Many claims are made about causal relationships between gut microbiota and human behaviour. By uncovering these relationships, MGB research aims to offer new explanations of mental health and potential avenues of treatment.So far, limited evaluation has been made of MGB's methods and its core experimental findings, many of which are extensively reiterated in copious reviews of the field. These factors, plus the self-help potential of MGB, have combined to encourage uncritical public uptake of MGB discoveries. Both social and professional media focus on the potential for dietary intervention in mental health, and causal relationships are assumed to be established.Our target article has two main aims. One is to examine critically the core practices and findings of experimental MGB research and to raise questions about them for brain and behavioural scientists who may not be familiar with the field. The other is to challenge the way in which MGB findings are presented. Our positive goal is to suggest how current problems and weaknesses may be addressed, in order for both scientific and public audiences to gain a clearer picture of MGB research and its strengths and limitations.

Does television reflect the evolution of scientific knowledge? The case of attention deficit hyperactivity disorder coverage on French television.

Biomedical findings mature from uncertain observations to validated facts. Although subsequent studies often refute initial appealing findings, newspapers privilege the latter and often fail to cover refutations. Thus, biomedical knowledge and media reporting may diverge with time. Here we investigated how French television reported on three scientific questions relative to attention deficit hyperactivity disorder (ADHD) from 1995 to 2010: i) is ADHD mainly genetic in origin, ii) does methylphenidate treatment decrease the risk of academic underachievement, and iii) are brain imaging techniques able to reveal ADHD in individual patients? Although scientific evidence regarding these questions has evolved during these 16 years, we observed that nine out of ten TV programs broadcast between 2007 and 2010 still expressed only opinions against the current scientific consensuses. The failure of TV programs to reflect the evolution of the scientific knowledge might be related to a biased selection of medical experts.

Blood-brain borders: a proposal to address limitations of historical blood-brain barrier terminology.

Many neuroscientists use the term Blood-Brain Barrier (BBB) to emphasize restrictiveness, often equating or reducing the notion of BBB properties to tight junction molecules physically sealing cerebral endothelial cells, rather than pointing out the complexity of this biological interface with respect to its selectivity and variety of exchange between the general blood circulation and the central nervous tissue. Several authors in the field find it unfortunate that the exquisitely dynamic interfaces between blood and brain continue to be viewed primarily as obstructive barriers to transport. Although the term blood-brain interface is an excellent descriptor that does not convey the idea of a barrier, it is important and preferable for the spreading of an idea beyond specialist communities to try to appeal to well-chosen metaphors. Recent evidence reviewed here indicates that blood-brain interfaces are more than selective semi-permeable membranes in that they display many dynamic processes and complex mechanisms for communication. They are thus more like 'geopolitical borders'. Furthermore, some authors working on blood-brain interface-relevant issues have started to use the word border, for example in border-associated macrophages. Therefore, we suggest adopting the term Blood-Brain Border to better communicate the flexibility of and movement across blood-brain interfaces.

Lipopolysaccharide-induced anhedonia is abolished in male serotonin transporter knockout rats: an intracranial self-stimulation study.

A growing body of evidence suggests that pro-inflammatory cytokines contribute to the pathogenesis of depression. Previously, it has been shown that cytokines (e.g. interferon-α therapy) induce major depression in humans. In addition, administration of the cytokine-inducer lipopolysaccharide (LPS) provokes anhedonia (i.e. the inability to experience pleasure) in rodents. Furthermore, serum pro-inflammatory cytokine levels are increased in depressed patients. Nevertheless, the etiology of cytokine-induced depression is largely unknown. Previously, it has been shown that selective serotonin re-uptake inhibitors decrease serum pro-inflammatory cytokine levels and that pro-inflammatory cytokines increase activity of the serotonin transporter (SERT). The purpose of this study was to explore the effect of partial and complete lack of the SERT in LPS-induced anhedonia assessed in the intracranial self-stimulation (ICSS) paradigm. A single intraperitoneal injection of LPS was used to induce a pro-inflammatory immune response in male serotonin transporter wild type (SERT(+/+)), heterozygous (SERT(+/-)) and knockout (SERT(-/-)) rats. Body weight and ICSS thresholds were measured daily. Although LPS reduced body weight in all genotypes, loss of body weight was less pronounced in SERT(-/-) compared to SERT(+/+) rats. Remarkably, LPS-induced anhedonia was totally abolished in SERT(-/-) rats and as expected was still present in SERT(+/+) and to a lesser extent in SERT(+/-) rats. Therefore, it is concluded that an intact SERT function is needed for pro-inflammatory cytokine-induced anhedonia and weight loss in rats.

Inhibitory activity of plant stilbenoids against nitric oxide production by lipopolysaccharide-activated microglia.

Microglia-driven inflammatory processes are thought to play an important role in ageing and several neurological disorders. Since consumption of a diet rich in polyphenols has been associated with anti-inflammatory and neuroprotective effects, we studied the effects of twenty-five stilbenoids isolated from Milicia excelsa, Morus alba, Gnetum africanum, and Vitis vinifera. These compounds were tested at 5 and 10 µM on BV-2 microglial cells stimulated with bacterial lipopolysaccharide. Ten stilbenoids reduced lipopolysaccharide-induced nitric oxide production at 5 and/or 10 µM. Two tetramers, E-vitisin A and E-vitisin B, were the most effective molecules. Moreover, they attenuated the expression of the inducible NO synthase protein and gene.

Minding the gut: extending embodied cognition and perception to the gut complex.

Scientific and philosophical accounts of cognition and perception have traditionally focused on the brain and external sense organs. The extended view of embodied cognition suggests including other parts of the body in these processes. However, one organ has often been overlooked: the gut. Frequently conceptualized as merely a tube for digesting food, there is much more to the gut than meets the eye. Having its own enteric nervous system, sometimes referred to as the "second brain," the gut is also an immune organ and has a large surface area interacting with gut microbiota. The gut has been shown to play an important role in many physiological processes, and may arguably do so as well in perception and cognition. We argue that proposals of embodied perception and cognition should take into account the role of the "gut complex," which considers the enteric nervous, endocrine, immune, and microbiota systems as well as gut tissue and mucosal structures. The gut complex is an interface between bodily tissues and the "internalized external environment" of the gut lumen, involved in many aspects of organismic activity beyond food intake. We thus extend current embodiment theories and suggest a more inclusive account of how to "mind the gut" in studying cognitive processes.

The calm mouse: an animal model of stress reduction.

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.

Cytokines in the Brain and Neuroinflammation: We Didn't Starve the Fire!

In spite of the brain-protecting tissues of the skull, meninges, and blood-brain barrier, some forms of injury to or infection of the CNS can give rise to cerebral cytokine production and action and result in drastic changes in brain function and behavior. Interestingly, peripheral infection-induced systemic inflammation can also be accompanied by increased cerebral cytokine production. Furthermore, it has been recently proposed that some forms of psychological stress may have similar CNS effects. Different conditions of cerebral cytokine production and action will be reviewed here against the background of neuroinflammation. Within this context, it is important to both deepen our understanding along already taken paths as well as to explore new ways in which neural functioning can be modified by cytokines. This, in turn, should enable us to put forward different modes of cerebral cytokine production and action in relation to distinct forms of neuroinflammation.

Hormonal, hypothalamic and striatal responses to reduced body weight gain are attenuated in anorectic rats bearing small tumors.

Lack of compensatory or even reduced food intake is frequently observed in weight-losing cancer patients and contributes to increased morbidity and mortality. Our previous work has shown increased transcription factor expression in the hypothalamus and ventral striatum of anorectic rats bearing small tumors. mRNA expression of molecules known to be involved in pathways regulating appetite in these structures was therefore assessed in this study. Given that pain, pro-inflammatory cytokines and metabolic hormones can modify food intake, spinal cord cellular activation patterns and plasma concentrations of cytokines and hormones were also studied. Morris hepatoma 7777 cells injected subcutaneously in Buffalo rats provoked a 10% lower body weight and 15% reduction in food intake compared to free-feeding tumor-free animals 4 weeks later when the tumor represented 1-2% of body mass. No differences in spinal cord activation patterns or plasma concentration of pro-inflammatory cytokines were observed between groups. However, the changes in plasma ghrelin and leptin concentrations found in food-restricted weight-matched rats in comparison to ad libitum-fed animals did not occur in anorectic tumor-bearing animals. Real-time PCR showed that tumor-bearing rats did not display the increase in hypothalamic agouti-related peptide mRNA observed in food-restricted weight-matched animals. In addition, microarray analysis and real-time PCR revealed increased ventral striatal prostaglandin D synthase expression in food-restricted animals compared to anorectic tumor-bearing rats. These findings indicate that blunted hypothalamic AgRP mRNA expression, probably as a consequence of relatively high leptin and low ghrelin concentrations, and reduced ventral striatal prostaglandin D synthesis play a role in maintaining cancer-associated anorexia.