This review also provides an overview of the importance of establishing an osteogenic microniche using appropriately customized scaffolds and delineates some of the key challenges that still need to be overcome for adult stem cell skeletal regenerative therapy to become a clinical reality.Improvements in medical care, nutrition and social care are resulting in a commendable change in world population demographics with an ever increasing skew towards an aging population. As the proportion of the world’s population that is considered elderly increases, so does the incidence of osteodegenerative disease and the resultant burden on healthcare. The increasing demand coupled with the limitations of contemporary approaches, have provided the impetus to develop novel tissue regeneration therapies. The use of stem cells, with their potential for self-renewal and differentiation, is one potential solution. Adipose-derived stem cells (ASCs), which are relatively easy to harvest and readily available have emerged as an ideal candidate. In this review, we explore the potential for ASCs to provide tangible therapies for craniofacial and long bone skeletal defects, outline key signaling pathways that direct these cells and describe how the developmental signaling program may provide clues on how to guide these cells  Improvements in medical care and nutrition have resulted in a commendable change in the population demographics of the world and especially of developed countries. For example, over the last 50 years, the number of Americans over 65 years of age has increased from 12 to 37 million and is growing at an average rate of 2% year on year [In this review we explore the potential role of adult stem cells and in particular adipose-derived stem cells, a heterogeneous group of adult stem cells found in adipose tissue, that offer a suitable cell source for bone tissue regeneration in both the craniofacial skeleton and long bones. Their relative ease of harvest and abundance render this cell population as an ideal candidate for this purpose. Furthermore, this adult stem cell population is not limited by the ethical concerns that restrict the use of embryonic stem cells. Firstly, we outline how the development of the calvarium and craniofacial skeleton differs from that of the long bones. Specifically, we outline how differences in the required developmental program, in terms of signaling, may add certain nuances to the way we direct our chosen stem cell population towards bone regeneration in different anatomical locations. Attention will be given to the signaling networks that govern ASC fate and their potential to undergo osteogenic differentiation in the context of skeletal repair. We also describe how this cell population has been used in animal models of calvarial and long bone repair and the impact that this work has had or may yet have on clinical practice.The vertebrate skeleton is the product of cells from three distinct lineages. The craniofacial skeleton is formed principally from cranial neural crest cells, the axial skeleton from paraxial mesoderm and the appendicular skeleton from the lateral plate mesoderm . Subsequin situ during development and how this may influence the way adult mesenchymal stem cells may be directed in vivo for the purpose of long bone and calvarial bone tissue engineering. For more detailed accounts please refer to reviews by Rice [While a comprehensive account of the processes of endochondral and intramembranous ossification is beyond the scope of this review, the processes are outlined here in order to derive insights into how osteogenesis from mesenchymal cells proceeds  by Rice  and also by Rice .Most of the mammalian skeleton including the long bones and the axial skeleton consists of bones formed from cartilaginous templates in the process of endochondral ossification. Over the last decade studies on transgenic mice have provided insights into the molecules, that govern the key steps in this process such as mesenchymal condensation, chondrocyte differentiation, chondrocyte maturation and hypertrophy, growth plate development and osteoblastogenesis. They include the secreted proteins such as Indian Hedgehog (IHH), parathyroid hormone-related peptide (PTHrP), bone morphogenetic proteins (BMPs), wingless related integration site proteins (Wnts), fibroblast growth factors (FGFs), their receptors and several key transcriptions factors such as SRY (Sex-determining region Y) box 9 (SOX9), Runt-related transcription factor 2 (RUNX2) and Osterix (OSX).Hox genes, BMPs, and FGFs [Sox9 gene result in campomelic dysplasia, while haploinsuffuciency of SOX9 leads to chondrodysplasia in mice [Endochondral ossification begins with the condensation of mesenchymal cells, which is a poorly understood process during which mesenchymal cells aggregate and form clusters. Cell adhesion molecules such as neural-cell adhesion molecule (N-CAM) and neural cadherin (CDH2) have been implicated  as have and FGFs ,9,10. Afand FGFs . Severaland FGFs . BMP sigand FGFs . Wnt proand FGFs . Similarand FGFs . At a trand FGFs . Heteroz in mice ,13. SOX9 in mice . SOX9 al in mice .Chondrocyte proliferation is followed by maturation, chondrocyte hypertrophy and vascular invasion that brings in osteoprogenitors that differentiate into osteoblasts and develop primary ossification centers . VasculaThe majority of the bones of the face and calvaria develop though intramembranous ossification. In this process the bones develop directly from mesenchymal cells that condense and proliferate before differentiating into osteoblasts.Runx2 in mice results in complete absence of osteoblasts while haploinsufficiency of RUNX2 in humans cause cleidocranial dysplasia [Osteoblastogenesis, osteoblast differentiation during development, is controlled by a complex network at both the transcriptional level and also by extracellular signaling pathways. A key player in transcriptional control of osteoblast differentiation, Runx2, also known as Cbfa1, is essential for osteoblast differentiation . Homozygysplasia ,19. RUNXysplasia ,21. Seveysplasia .Osx in mice in the presence of normal Runx2 expression results in a lack of osteoblasts in mouse embryos [Osx expression is absent in −/−Runx2 mice [OSX is another transcription factor that is crucial for osteoblast differentiation. Deletion of  embryos . In addi−/− mice . Collect−/− mice . The tra−/− mice .Several extracellular signaling pathways exert their influence on osteoblast differentiation in both endochondral and intramembranous ossification through the key transcription factors described above. These extracellular signaling pathways are outlined here.−/−Ihh mice lack osteoblasts within the endochondral skeletal tissue but not in intramembranous bone [Runx2 and therefore fail to undergo osteoblast differentiation [Indian Hedgehog (IHH) signaling plays a critical role in endochondral development. Intriguingly, ous bone . Withoutntiation . This sintiation .Canonical Wnt signaling and more recently the non-canonical Wnt pathways have been shown to play important roles during skeletal development. Wnt ligands bind to frizzled receptors at the cell surface and LRP5 and LRP6 co-receptors. Signal transduction then proceeds with the stabilization of cytoplasmic β-catenin that enables it to translocate to the nucleus and form a transcription complex with TCF/LEF proteins , whereasNotch signaling suppresses osteoblast differentiation. Inhibition of the Notch pathway in the embryonic limb leads to increased bone mass and a reciprocal decrease in bone marrow mesenchymal progenitors which suggests that it plays a role in suppressing osteoblast differentiation and maintaining a larger progenitor pool . Notch iBmp3-null mice [BMPs are members of the transforming growth factor-β (TGF-β) superfamily and activate the signal transduction cascade via its receptor types I and II, receptor tyrosine kinases, which upon activation phosphorylate transcription factors 1, 5 and 8 . Geneticull mice . It is tull mice .et al. demonstrated that −/−fgf18 embryos had defects in osteoblast maturation despite normal Runx2 expression. In contrast FGFR1 receptor activity was shown to have a tendency to promote osteoblast differentiation at an early stage but inhibit mineralization capabilities in mature osteoblasts [FGF signaling has a complex and stage dependent effect on osteogenesis in endochondral and intramembranous bone development. FGF 2, 9, 18 and their corresponding receptors FGFR1-3 have all been implicated as playing important roles during skeletal development of the long bones and calvariae ,39,40,41eoblasts . Furthereoblasts ,44,45. Ieoblasts ,47. CleaGiven the complexity of the signaling network and developmental program which is required for osteogenesis in both the endochondral and intramembranous systems it should come as no surprise that the same signaling pathways play a role in the direction of putative adult stem cells to an osteoblastic lineage for the purposes of tissue engineering.et al. showed using a similar protocol on human fat that the SVF of human lipoaspirates contained cells of multilineage potential that were referred to as processed lipoaspirate cells [in vitro and in vivo [The term “stem cell” refers to a myriad of different cell types that share two key characteristics: self-renewal and the potential for differentiation into different cell types. Adult stem cells are not limited to the same extent by low availability and ethical concerns as are embryonic stem cells. They are, therefore, an appealing source for tissue engineering. Of the numerous kinds of adult stem cells that have been described in the literature two types are of particular relevance to bone tissue engineering: Bone marrow derived MSC (BMSCs) and ASCs. Bone marrow was the first source that was reported to contain MSCs ,49. Howete cells ,52. Thes in vivo ,54,55.− CD235a− CD31− CD34+. Furthermore negativity for CD106 and positivity for CD36 distinguishes them from BM-MSCs [in vitro culture [et al. used single cell-transcriptional analysis to associate low CD105 expression with increased osteogenic capacity and a combination of microfluidic analysis and FACS to identify CD90 a marker that was associated with increased osteogenic potential [et al. prospectively purified human perivascular stem cells (PSCs) from adipose tissue using FACs analysisand found a statistically significant improvement in their in vitro and in vivo osteogenic capacity relative to traditionally derived SVF [ASCs and BMSCs share many common characteristics such as multilineage differentiation capability, morphology, telomerase activity and gene expression . They al BM-MSCs . Establi culture . Recentlotential . Selectiotential . More reived SVF . This stived SVF .As described earlier many signaling pathways converge on key transcription factors to regulate and orchestrate bone development. The same pathways such as BMP, FGF, TGF-β, Notch, Wnt/β-Catenin and Hedgehog have been shown to take part in the differentiation of mesenchymal cells, such as ASCs, into osteoblasts . Deliverin vitro and in vivo [FGFs play vital and complex roles at all stages of both endochondral and intramembranous bone formation and, therefore, unsurprisingly have a biological function on ASCs in the context of osteogenic differentiation  in vivo ,70.in vivo, FGF2 has also been shown to inhibit their terminal osteogenic differentiation by antagonizing the retinoic-acid mediated up-regulation of BMPR-1B [versus proliferation, depending on stage and context [et al. provided an insight into such a tunable model, demonstrating, using a system that enabled chemical control of FGF2 secretion, that this ligand would, in the correct context, promote bone regeneration in vivo [FGF2, for example, is a well-known regulator of both osteoblastic and chondrogenic cells and acts as an autocrine/paracrine factor for bone cells. FGF2 serves as a good example of the complex ways in which FGFs can regulate bone formation . Althoug BMPR-1B ,71,72. T BMPR-1B . Other p context ,73. Givein vivo into both osteoblasts and endothelial cells [et al. also demonstrated in a calvarial defect model that VEGFA had a more potent effect in promoting ASCs mediated calvarial regeneration than both BMP2 and FGF2 [Adequate vascularization is also a pivotal part in bone tissue regeneration. Increased angiogenesis therefore benefits bone repair . It has al cells ,76,77. Band FGF2 . VEGFA pand FGF2 .The skeletal regenerative capacity of ASCs have been tested in several animal models including those for long bone defects and calvarial defects, allowing possible mimicking of the clinical condition as closely as possible.et al. for example used ASCs on an osteoinductive scaffold and showed that ASCs, which were genetically modified to overexpress BMP2, were capable of healing a critical sized unicortical femoral defect [et al. did not find any advantage of adding ASCs onto scaffold treated with rhBMP2 in a segmental femoral defect model [et al. demonstrated that ASCs overexpressing BMP6 were capable of healing rat vertebral bone defects [Peterson l defect . The saml defect . Furtherct model . De Giro defects ,82.et al. revealed that a 4 mm mouse circular parietal bone defect model represents a reliable and reproducible “critically sized” defect that fails to show more than 5% healing on average over an 8 week period in the absence of any treatment modality [co-glycolic acid) (PLGA) were able to achieve 80% healing [A landmark study by Cowan modality . The aut healing .in vivo differentiation of ASCs has been raised by studies that demonstrate the ability of HA/β-TCP, apatite coated PLGA scaffold, wet-spun starch-polycaprolactone scaffold, or coral scaffold to heal critical sized calvarial defects without the need for pre differentiation. Such an advance would be of tremendous clinical importance as it would obviate the need for pretreatment of ASCs and allow reconstruction in one step without tissue leaving the operating room [Intriguingly the prospect of ing room ,75,83.d,l-lactic-coglycolic acid) (PLGA) scaffolds have been widely used, as they are biodegradable, porous, permissive for cell attachment and differentiation. The addition of a layer of hydroxyapatite (HA) can increase the bioactivity of such scaffolds and have been used successfully, as described earlier, in the treatment of critical sized calvarial defects in mice [nHA)(BCP/PCL-nHA) have been used to drive osteogenic differentiation of ASCs through the ERK signaling pathways and others have used degradable matrices to deliver BMP4 plasmid DNA for in vivo genetic manipulation of cells for osteogenic differentiation [et al. recently demonstrated that the provision of a suitable “osteogenic microniche”, using a hydroxyapatite-coated, BMP2 protein releasing poly-l-lactic acid (PLLA) scaffold, could direct in vivo differentiation of both embryonic and induced pluripotent stem cells towards an osteogenic lineage without the formation of a teratoma in the context of skeletal regeneration in a murine calvarial defect model [It is well understood that stem cells reside in tightly controlled niches that can influence their behavior . Scaffol in mice ,84. Polyntiation ,86. Thisct model .Runx2 resulting in intramembranous ossification. A PLGA/PLLA scaffold alone, on the other hand, led to differentiation via endochondral ossification in mesenchymal cells derived from human embryonic stem cells. These data illustrate that appropriately devised scaffold-mediated microenvironments can manipulate the behavior of stem cells [Interestingly, it was demonstrated that the incorporation of HA onto a PLGA/PLLA scaffold stabilized β-Catenin and upregulated em cells .Aging has a large effect on the ability of skeletal tissue to maintain normal homeostasis and to regenerate following injury. Therefore, the implications of aging on bone physiology and also the effects of aging on resident stem cells or of any putative autologous cell population used for regenerative purposes following injury, must be considered. The progenitor pool within the bone marrow, commonly referred to as BM-MSCs, has been shown to reduce in number and to demonstrate a functional decline with age . Like otThe stem cell pool is not immune to the effects of aging. As it will mainly be an aged population that will benefit from regenerative bone strategies, it is important to understand how aging affects the stem cells planned for autologous therapeutic use. ASCs, for example, have been shown to be susceptible to the effects of aging. They show a reduced ability to proliferate, a reduced production of angiogenic and prosurvival cytokines, and a higher susceptibility to the effects of oxidative stress . Therefoin vivo models to the clinical arena represents a major step. The manipulation of ASCs and human adipose-derived stromal vascular fraction (SVF) cells for therapeutic modalities must be done according to good manufacturing practices and the regulations of the FDA and/or European Medicines Agency [ex vivo expansion of cells for two or three weeks rendering them susceptible to the possibility of genomic instability in culture [Transitioning from preclinical research in terms of s Agency . If the  culture . Attempt culture ,100.et al. used ASCs on a macroporous sheet together with milled autologous iliac crest bone graft to treat a large posttraumatic calvarial defect in a 7-year-old girl [et al. in which hASCs were used with β-TCP and BMP2 for the treatment of a large maxillary defect [Despite these restrictions some examples exist for the use of ASCs based cell therapies for bone reconstruction. Lendeckel old girl . Theslefold girl . Other ny defect .Despite the limited success of clinical applications for ASCs based bone reconstructions, many challenges still remain. For example, a clearer definition of the immunogenicity of ASCs and the elucidation of novel and more rigorous ways of screening population of ASCs, in terms of safety and suitability for their intended purpose, is needed . Good exin vitro and in vivo using calvarial and long bone animal models of skeletal defects. They therefore hold great promise as a source for bone tissue engineering and have rightly grabbed the attention of the scientific community. Despite the wealth of literature on this topic, the field of adult stem cells is still in its infancy. Evidence from the clinical application of these potential therapies is even more rudimentary. Therefore, much work still needs to be done before the true potential of ASCs for bone tissue reconstruction can be fully realized. Areas that deserve further attention include establishing a more comprehensive understanding of the signaling network that reliably leads to robust osteogenic differentiation and identification of a definitive cell surface marker profile. By understanding the cell surface markers that select for highly osteogenic subpopulations and developing techniques to harvest, screen and graft ASCs in one sitting, the prospect of a viable clinical approach for using ASCs in the treatment of skeletal defects will become more tangible. Furthermore the development of more sophisticated scaffolds that can recapitulate the stem cell niche and thereby modulate and guide ASCs for their desired therapeutic purpose will also be highly advantageous.As the change in population demographics becomes increasingly skewed towards an aging population, the need for novel therapies to reconstruct defects of skeletal tissue becomes more pressing. ASCs represent an abundant, easily accessible and multipotent cell population that has been shown repeatedly to have the potential to undergo osteogenic differentiation


There is a paucity of longitudinal data on the effect of rhinitis on asthma exacerbations during childhood. Within the context of unselected birth cohort (Manchester Asthma and Allergy Study), we investigated the association between rhinitis and asthma exacerbations from early childhood to school age.aw) using plethysmography (ages 5-11 years) and FEV1 by spirometry . Information on prescribed medication and severe asthma exacerbations (ATS definition) was extracted from participants’ medical records. The effect of rhinitis on asthma exacerbations and lung function was investigated using longitudinal analyses.Subjects were recruited prenatally and followed prospectively, attending follow-up at ages 3, 5, 8, and 11 years. A validated questionnaire was interviewer-administered to collect information on parentally-reported symptoms (n=1051). At each follow-up, current rhinitis was defined as sneezing, runny or blocked nose in the absence of cold or flu within the last 12 months, and asthma as a positive response to at least two of the following: 1) current wheeze; 2) physician-diagnosed asthma; 3) use of asthma medication. We measured specific airway resistance . However, there were no differences in the longitudinal measures of FEV1 or sRaw between asthmatic children with and without rhinitis, before or after adjustment for the use of rhinitis or asthma medication.A total of 356 children had asthma on at least one time point, of whom 198 had rhinitis. In the multivariate models adjusted for the use of intranasal and inhaled corticosteroids as well as antihistamines, asthmatic children with current rhinitis remained markedly and significantly more likely to have frequent asthma exacerbations (In the longitudinal analysis throughout childhood, children with asthma and rhinitis were also almost five times more likely to experience severe asthma exacerbations compared to children with asthma only.


Individuals in the early phases of psychotic illness have disturbed metacognitive capacity, which has been linked to a number of poor outcomes. Little is known, however, about the neural systems associated with metacognition in this population. The purpose of this study was to elucidate the neuroanatomical correlates of metacognition. We anticipated that higher levels of metacognition may be dependent upon gray matter density (GMD) of regions within the prefrontal cortex. Examining whole-brain structure in 25 individuals with early phase psychosis, we found positive correlations between increased medial prefrontal cortex and ventral striatum GMD and higher metacognition. These findings represent an important step in understanding the path through which the biological correlates of psychotic illness may culminate into poor metacognition and, ultimately, disrupted functioning. Such a path will serve to validate and promote metacognition as a viable treatment target in early phase psychosis. Schizophrenia is a chronic and disabling psychiatric disorder , affectiIndividuals with prolonged psychosis have compromised metacognitive capacity which has been linked to symptoms ,10,11,12Advances in technology over the last several decades have enabled increased ability to understand psychosis, moving across levels of analysis from basic science and the study of molecules to investigations of higher order disturbances in human consciousness. The purpose of this study was to elucidate the neural substrates underlying metacognitive capacity in EPP. One possible route to studying neural correlates of metacognition is to employ magnetic resonance imaging (MRI) techniques. Neuroimaging data allow for the examination of psychosis as a brain disorder with altered structure and function ,26. We pThe socio-demographic characteristics and average scores for measures of symptoms and metacognition are reported in Voxel based morphometry (VBM) analyses indicated that metacognition was positively correlated to gray matter density (GMD) in several locations , with hiBy including the Positive and Negative Syndrome Scale (PANSS) negative symptom cluster as a regressor, we demonstrated that the relationships of mPFC and VS with metacognition were not better accounted for by the association between total metacognition and negative symptoms . SignifiTaken together, the above findings support our hypothesis that metacognition as measured by the Metacognitive Assessment Scale Abbreviated (MAS-A) is subserved by specific neuroanatomical structures. Specifically, even after controlling for negative symptoms , higher The implication of mPFC in synthetic metacognition is consistent with a number of studies that suggest mPFC may play an important role in metacognitive processes . These rThe second finding, that total metacognition was associated with GMD of the ventral striatum (VS), was not an a priori prediction but nonetheless may have important implications. The VS is thought to consist primarily of the nucleus accumbens and olfactoray tubercle , with thAs mentioned above, individuals in the early phase of psychosis appear to experience metacognitive deficits ,19 that Early scholars described psychosis as an interruption in goal directed behavior secondary to a complex, biologically based decomposition and unbinding of associations that collapsed higher order understanding of self and others . HoweverA number of caveats should be considered when interpreting the findings of this study. First, the relatively modest sample used here included primarily young, African American males within 5 years of initial diagnosis of psychotic illness. It is therefore possible that a sample with more females or individuals of varied ethnic backgrounds, or perhaps even those experiencing their very first acute episode of illness, may have different results. Because there was no comparison group in this study, it is also difficult to say exactly how the observed metacognitive deficits are related to structural abnormalities. It is possible that the observed link between the study variables was due to some other unforeseen population-specific factor. The current study did account for factors such as antipsychotic drug exposure or current symptomatology and other potential confounds related to volumetric measurements such as VBM  are difficult to control. In terms of VBM, blurring is 3 dimensional and therefore does not respect boundaries along tissue classes, leading to increased probability of either diluting existing signal or misinterpreting boundary shift as signal. Additionally, only a single imaging analysis technique was employed in the present study. Other techniques, such as Diffusion Tensor Imaging (DTI) analysis may yield insight into to how white matter integrity affects metacognitive capacity.In addition to these limitations, a number of questions remain. Medial PFC and ventral striatum GMD is linked to metacognitive capacity, but exactly how does this occur? Do such neurobiological alterations mediate the impact of metacognitive deficits on other aspects of the illness and, ultimately, behavior? Do the structures that subserve metacognition also play a role in other important psychological phenomenon, such as poor awareness of illness that are thought to be linked to metacognitive processing? Answers to these and other questions seem essential in order to develop fully elucidate neural correlates and treatments that adequately intervene at the level most proximate to function.Twenty-five individuals with EPP were recruited through the Indiana University Psychotic Disorders Program, within the Indiana University School of Medicine. All study procedures were completed via two concurrent studies both approved by the Indiana University Institutional Review Board, protocol numbers 1112007648  and 1011002975 . After receiving an explanation of study procedures, subjects gave their written informed consent prior to enrollment. At a baseline visit, demographic information and medical history were recorded. Patients were ages 18–35, within the first five years of psychotic illness onset, including diagnoses of schizophrenia, schizophreniform disorder, schizoaffective disorder, or Psychotic disorder not otherwise specified, as confirmed by a trained psychiatrist or psychologist via clinical interview and/or administration of the Structured Clinical Interview for Diagnosis (SCID) for the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) . All patMetacognitive capacity was assessed in a two-step process. First a narrative about self and illness was elicited using the Indiana Psychiatric Illness Interview (IPII) . The IPISymptoms were assessed via the Positive and Negative Syndrome Scale (PANSS), which is a 30-item scale designed to be completed by a trained rater at the conclusion of an interview and chart review . PreviouAt the imaging visit, patients underwent a magnetic resonance imaging (MRI) scan on a 3T Tim Trio scanner  with a 32-channel phased array head coil. Brain structure was characterized with a high-resolution T1-weighted whole-brain magnetization prepared rapid gradient echo (MPRAGE) scan with the following parameters: 160 3D sagittal slices, echo time/repeat time/inversion time = 2.91/2300/900 ms; slice resolution = 100%; Echo spacing = 7.7 ms, flip angle = 9°, field of view = 240 × 256 mm, voxel size = 1 × 1 × 1.2 mm.Gray matter density was quantified using unmodulated, normalized data using VBM, the VBM8 toolbox  within tp < 0.005, with a cluster-level family-wise error (FWE) correction of p < 0.05 to correct for multiple comparisons. Data from significant clusters were extracted using the MarsBaR (toolbox (http://marsbar.sourceforge.net) within SPM8) for visualization and post-hoc analyses with the Statistical Package for the Social Sciences (SPSS) 22 .To quantify the relationship of gray matter density to metacognitive capacity, we conducted a multiple regression analysis, with age, sex (as a dummy variable), and total intracranial volume as regressors along with MAS-A total scores. Given that metacognition is associated with negative symptoms in schizophrenia , we condThis is the first study to examine the neural correlates of metacognition, as measured by the MAS-A, in EPP. The present study demonstrated that increased GMD of the mPFC and VS is associated with higher levels of metacognition. The correlations among these areas and metacognitive processing fit the existing literature on the functions currently believed to be mediated by both areas. Such associations represent an important step in understanding the neurobiological underpinnings of disrupted metacognition in EPP, which ultimately culminates into poor outcomes. It also promotes and validates metacognition as an important treatment target in early intervention for psychosis and provides neurobiological marker(s) to track changes due to psychotherapeutic interventions  and may lead to more accurate predictions of real-life/clinical outcomes.


In the mammalian central nervous system, excitatory glutamatergic synapses harness neurotransmission that is mediated by ion flow through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). AMPARs, which are enriched in the postsynaptic membrane on dendritic spines, are highly dynamic, and shuttle in and out of synapses in an activity-dependent manner. Changes in their number, subunit composition, phosphorylation state, and accessory proteins can all regulate AMPARs and thus modify synaptic strength and support cellular forms of learning. Furthermore, dysregulation of AMPAR plasticity has been implicated in various pathological states and has important consequences for mental health. Here we focus on the mechanisms that control AMPAR plasticity, drawing particularly from the extensive studies on hippocampal synapses, and highlight recent advances in the field along with considerations for future directions. In a simplified model, at synapses, PSD proteins trap and anchor surface AMPARs in response to increases in neuronal or synaptic signaling and release the receptors when activity levels are low. How different forms of activity-dependent synaptic plasticity affect the distribution and composition of synaptic nanodomains is an extremely exciting and promising topic for future research. The postsynaptic nanodomain might be equally matched by the heterogeneous presynaptic organization, for example, representing hotspots of synaptic vesicle priming and fusion.LTP of synaptic strength can be induced by a variety of electrical, pharmacological and behavioral paradigms. Classical LTP, as originally described by Bliss and Lømo  can be s+-influx through AMPARs, depolarization of the postsynaptic compartment, and activation of NMDARs to permit Ca2+-influx; this sets off a cascade of phosphorylation events to potentiate synaptic transmission. The primary change following tetanic stimulation is the gross increase in AMPAR number at the synapse, but hidden within this is a series of subtle temporal and subunit-specific effects. The primary signaling effector (and the most studied molecule) is CaMKII in the postsynaptic neuron. This kinase is transiently activated following LTP induction  plays a key role in LTD. AKAP150 can interact with calcineurin and drives NMDAR-dependent removal of AMPARs from the synapse . GluA2 is endocytosed upon phosphorylation at S880 by interacting with PICK1, which also involves PICK1-mediated inhibition of actin polymerization via the Arp2/3 complex -3,5-dihydroxyphenylglycine, DHPG) induces a rapid removal of synaptic AMPARs. Whilst not covered here, we direct the reader to several excellent review articles on the subject  and compare it to some pre-set value, and then be able to adjust their synaptic protein complement to offset changes in external activity. In most mammals large changes in network activity happen on a diurnal basis with the onset of sleep  decreased the mEPSC amplitude, with the overall effect of maintaining the firing rate of the neuron despite the activity manipulation. Neurons thus adjust their synaptic AMPAR number in a manner that opposes the external changes in activity. Moreover, this is cell-wide and multiplicative such that the differences in individual synaptic weights are conserved. Consequently, this phenomenon—the activity-dependent bidirectional change in mEPSC amplitude—has been termed “synaptic scaling” as all of the individual postsynaptic strengths across the entire neuron are apparently scaled up or down by a uniform amount. Crucially, such a scaling process retains the information encoded in the relative original strengths of the connections, and thus a strong synapse will still be stronger than its weak neighbor after scaling . Further studies have indicated that GluA1 and GluA2 increase in a coordinated fashion during scaling up, and AMPA/NMDA ratios are also conserved , but unlike LTP/LTD, synaptic scaling appears to rely strongly on signaling linked to GluA2 (see below).Investigations of homeostatic plasticity have often relied on simple, neuronal culture preparations. Experimentally, activity manipulation is achieved in a variety of ways: global pharmacological blockade of synaptic AMPARs and NMDARs heavily suppresses network activity, as does the addition of TTX that prevents action potentials, whereas GABAn Figure . In a fin Figure . Both TT+ channel, which when overexpressed, reduces AP firing, see Burrone et al., 2+-flux is supported by the finding that blocking all Ca2+ channels with NiCl2, or L-type Ca2+ channels with nifedipine, have the same outcome.Studies have also used local perfusion of drugs to selectively perturb synapses. Interestingly, global action potential firing and local spontaneous mEPSC events appear to play different but overlapping roles in regulating AMPARs. Sutton et al.  has demoin vivo  blocks this form of plasticity both in cultures and As of now, multiple proteins have been implicated in synaptic scaling, including Arc  underscores the subtleties still being elucidated (Khodosevich et al., Whereas AMPAR auxiliary proteins undeniably expand the variety of AMPAR function, in what way do AMPARs with distinct subunit composition contribute to their functional diversity? As discussed above, GluA2-lacking CP-AMPARs participate in different forms of plasticity. Intriguingly, however, a recent report has raised questions about the subunit-specific requirements for recruiting AMPARs to synapses during LTP (Granger et al., in vivo synapses in their native milieu, especially in behaving animals, will likely uncover new aspects of AMPAR plasticity that may have been lost in in vitro preparations.Live super-resolution light microscopy has only just begun to reveal the intricacies of molecular movement at the synapse. Whilst not quite reaching electron microscopy levels of resolution, the advantage of being able to image live tissues at resolutions well below 100 nm makes the technique highly attractive for studying the behavior of synaptic proteins in response to activity. Where exactly do AMPARs undergo exo-endocytic recycling and is this dependent on the subunit composition? Are the nanodomains described for AMPARs and PSD components mirrored by the organization of presynaptic structures? At the active zone, precisely where does presynaptic vesicle fusion take place and in what manner does the released glutamate affect the diffusion properties of synaptic and extra-synaptic AMPARs? Furthermore, targeting super-resolution imaging to In another direction, different disease states are now being unraveled, with the causative genes and protein products being identified in humans and reassembled in animal models. Many pathological states of the brain feature deficits in synaptic transmission at their core, which have been termed “synaptopathies”, and the aging global population has created a serious social and medical issue that neuroscientists must play their part in solving.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, we provided the first experimental evidence that the pyramidal neurons in the PrL received a direct inhibitory input mediated by bipolar NPY+-GABAergic projection neurons in the IL. Deletion of the anxiety-related neuroligin 2 gene caused a decrease of this direct synaptic inhibition that originated from the IL. Thus, our data suggested that activation of the IL might not only directly activate the corresponding downstream anxiolytic pathway, but also suppress the PrL-related anxiogenic pathway and thus could differentially bias the regulation of fear expression and extinction.Anxiety disorders are thought to reflect deficits in the regulation of fear memories. While the amygdala has long been considered a site of storage of fear memories, newer findings suggest that the prefrontal cortex (PFC) is essential in the regulation of amygdala-dependent memories and fear expression. Here, activation of the prelimbic cortex (PrL) enhances the expression of fear, while an elevated activity in the infralimbic cortex (IL) enhances fear extinction. Despite the presence of these facts, we still know very little about the synaptic interconnectivity within the PFC. The aim of the present study was to investigate the inhibitory circuits between prelimbic and IL using morphological and electrophysiological methods. Our immunohistochemical analysis revealed that the distribution of PV Mental disorders, such as anxiety disorders including panic disorder and post-traumatic stress disorder, are thought to reflect deficits in regulation of emotional memories.9 that is critical for the expression of conditioned fear.11 On the other hand, the infralimbic cortex (IL) in the ventral part of the PFC contributes the majority of PFC inputs to the central nucleus of the amygdala13 that plays a key role in the expression of fear extinction.16 Thus, published data make it very clear that the PFC is not functionally monolithic, but that there exists a dorsal–ventral functional dichotomy, such that the activation of the PrL drives and enhances the expression of fear, while an elevated activity in the IL suppresses and terminates these behaviors after extinction.17 Successful extinction requires the activation of an intact IL, which suppresses conditioned increases in amygdala activity, and subsequently reduces fear responses. Consequently, failure to retrieve extinction, as may occur in diseases like panic disorder and post-traumatic stress disorder, is thought to reflect a lack of IL-mediated suppression of amygdala activity, leading to persistent fear responses.17 Together, these data strongly suggest that the dichotomic circuit between IL and PrL represents a common node in the central regulation circuits that bi-directionally modulates the fear expression.17Within the PFC, the dorsally located prelimbic cortex (PrL) projects primarily to the basal amygdala nucleus21 In particular, the calcium-binding protein parvalbumin (PV) is a crucial marker in defining the most predominant interneuron subtype within the cerebral cortex,22 which comprises ~40% of the total GABAergic cortical interneuron population.23 Neuropeptide Y (NPY) has been shown to be important in the modulation of anxiety.25 NPY-expressing-neurons are less abundant, but widely distributed throughout the depth of the cortex and are more frequent in layers II–III and VI.26 Despite the existence of many data about the GABAergic interneurons in the cerebral cortex, there are few detailed studies examining the GABAergic inhibitory neurons in the PFC.28In the cerebral cortex, the diversity of GABAergic interneurons is manifested by their different morphological, electrophysiological and neurochemical features. So far, over 20 different subtypes of GABAergic interneurons have been classified based on the specific proteins they express.29 They are differentially localized with respect to the postsynaptic specializations of excitatory and inhibitory synapses.32 One member of the neuroligin family, neuroligin 2 (Nlgn2) is preferentially localized in inhibitory synapses,31 and determines and fine-tunes the function of central inhibitory synapses.37 Our previous data demonstrated that deletion of the corresponding gene Nlgn2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.34 Furthermore, Nlgn2-deficient mice display a decrease in pain sensitivity and a slight decrease in motor co-ordination, and, most importantly, a marked increase in anxiety-like behavior.38Neuroligins are proteins belonging to a family of postsynaptic cell adhesion molecules that are expressed ubiquitously in the brain.40 Despite the presence of an increasing amount of experimental evidences that emphasizes the importance of the dichotomy of the PFC and its connections with the amygdala for regulating fear behavior,6 we still know very little about the fear-related synaptic circuit within the PFC.17 How circuits are organized in agranular cortices like the PFC, which lacks a granular L4 layer, and how GABAergic neurons that play a vital role in neural circuitry are distributed in this area, is even less known.43 Therefore, detailed knowledge about the involving GABAergic neuronal circuit between IL and PrL could become a key link in our understanding of physiology and pathophysiology in the central regulation of fear and anxiety behaviors.So far cortical circuit organization has been studied predominantly in sensory cortices such as the visual and somatosensory cortices.6 By convention, most cortical GABAergic neurons are referred to as GABAergic interneurons, as they typically project a highly ramified axon to neurons in their close vicinity. On the other hand, it has been shown that a small population of these GABAergic neurons can also give rise to long-range cortico-cortical projections. To distinguish them from GABAergic interneurons, this class of GABAergic neurons is referred to as GABAergic projection neurons, although their functional relevance in the cortical network is still at issue.44 These GABAergic projection neurons have been suggested to account for only 0.5% of the whole population of GABAergic neurons and part of them have been assumed to be NPY+-GABAergic neurons.44 In the present study, we hypothesized that pyramidal neurons of IL might activate NPY+-GABAergic neurons and directly inhibit the pyramidal neurons in the ipsilateral PrL. In this way, IL would not only lead to an activation of the downstream central nucleus of the amygdala,13 but also lead to inhibition of the ipsilateral PrL, resulting in reduced activation of the basal amygdala nucleus and finally an anxiolytic effect.15 In addition, we investigated whether deletion of Nlgn2 would influence inhibitory transmission from IL to PrL, and thus may be causally involved in increased anxiety-like behaviors that have been reported in these mice.38To the best of our knowledge, there are so far no published data demonstrating a direct inhibitory synaptic connection between IL and PrL.Detailed information about immunohistochemistry and electrophysiology is provided in 45 NPY–eGFP mice46 and Nlgn2-KO mice34 (10–16 weeks old) were used.All experiments were performed in accordance with the European Communities Council Directive (86/EEC), and were approved by the State Office for Nature, Environment and Consumer Protection of North Rhine-Westphalia, Germany (LANUV NRW). For immunohistochemistry and electrophysiology, adult male wild-type C57BL/6 mice, transgenic PV–eGFP mice,Coronal sections of 25 μm in thickness were cut using cryostat  from the PFC of the brains (from 1.98 to 1.54 mm anterior to Bregma) for immunohistochemistry and immunofluorescent procedures. For detection of PV and NPY, standard immunohistochemistry staining procedures were performed . Distributions of positively stained cells were analyzed using ImageJ software  in the anterior cingulated cortex (ACC), PrL, IL and motor cortex 2 (M2). For each region, mean numbers of cells as well as cell density (cell × mm−2) were calculated across all the layers in above regions. For better comparison, the mean numbers of cells have also been calculated as cell × mm−3 that were presented in +-GABAergic neurons, the layer specific distribution of different subtypes was calculated as percentage of total numbers .For quantification, the brain areas and the layer borders were defined according to the mouse brain atlas51 Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded at a holding potential of −70 mV in the presence of the 10 μM AMPA-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 50 μM NMDA-receptor antagonist 2-amino-5-phosphono-valeric acid (AP5) and 2 μM glycine receptor antagonist strychnine. For assessing neuronal firing properties, square current pulses  were injected from a holding level corresponding to −70 mV holding potential every 5 s. For each neuron, several parameters were estimated .All recordings were performed in neurons of PFC  as described before  short process multipolar53  near53 (54  and (iii (54 (55 . Short m+ neurons were fast-spiking (average frequency ~60 Hz) with mild frequency adaptation within 500 ms (f1/flast appeared to be significantly lower than that of neurogliaform cells in PrL and IL (P=0.03), while short multipolar cells in M2 had higher adaptation ratio similar to that of neurogliaform cells , which then developed into a train of spikes of increasing frequency up to a certain maximum  was placed directly on the soma of a small unidentified neuron in layers V–VI of ipsilateral IL  elicited a single action potential . The miniature IPSCs in PrL were significantly attenuated in Nlgn2-KO mice . In addition, iontophoretically evoked bicuculline-sensitive IPSCs from layer V of IL to pyramidal neurons in layer II of ipsilateral PrL were also significantly reduced in Nlgn2-KO mice as compared with their wild-type littermates . Thus these data indicate that deletion of Nlgn2-gene causes a significant attenuation of overall inhibitory inputs to pyramidal neurons in PrL as well as the direct inhibition from IL to PrL.+- and NPY+-GABAergic neurons was different in PrL and IL as compared with M2 and ACC; (2) the PrL could be divided in a dorsal and a ventral part; (3) IL directly inhibited the ipsilateral pyramidal neurons of both dorsal and ventral part of PrL; (4) this direct inhibition was not mediated by PV+-, but by NPY+-GABAergic projection neurons in IL; (5) deletion of Nlgn2 caused significant attenuation of the inhibitory transmission from IL to PrL. Given the importance of the dichotomic network between IL and PrL in central control of emotion, activation of IL would suppress the activation of PrL-related pathways and thus differentially change the activation of downstream limbic areas and subsequently shape the fear expression and fear extinction both in physiology and pathophysiology.The present study provided five novel findings: (1) the distribution of PV+- and NPY+-neurons was very low in layer II and III of PrL and IL (+− and NPY+−neurons, the PFC can be divided into a 'cortical type' comprising ACC and dorsal PrL and a 'prefrontal type' comprising ventral PrL and IL.In the present study, the distribution patterns of PV and NPY were similar between ACC and M2  and 2. TL and IL . They ca43 This would be especially interesting for layers II/III, where the expression of PV+- and NPY+-neurons was very rare. Not only will it be interesting to know which subtypes of GABAergic neurons are expressed in layer II/III of PrL and IL, also the interconnectivity between these neurons and the pyramidal neurons is largely unknown. Furthermore, molecular analysis on the single cell level57 will greatly contribute to our understanding of neuronal circuits in the PFC.To comprehend the functional consequences of the above data, it would be worthwhile to investigate the distribution and the specific projections of other GABAergic neurons, such as calretinin-, somatostatin-positive GABAergic neurons, which are at present unexplored.dorsal and the 'prefrontal type' PrLventral. This classification might be premature yet in the absence of a clear functional understanding of their contribution to the neocortical network. However, it is quite likely that both parts are interconnected with different brain areas with different neuronal oscillatory features and therefore need quite different equipped GABAergic neuronal circuits.Although we do not know the functional consequences yet, we want to suggest dividing the PrL in two parts: the 'cortical type' PrL3 and has been functionally implicated in processes of emotional regulation.6 The dichotomic effects of IL and PrL on fear expression are mediated by their outputs to different targets within the amygdala.17 In addition, non-amygdala outputs of IL and PrL are also emerging as important targets for emotional regulation.59 Our present data provide the first experimental evidence that IL and PrL might reciprocally regulate the activity of each other, such that the activation of IL leads to direct inhibition of pyramidal neurons in PrL (The PFC has extensive connections with the subcortical limbic areas and thalamuss in PrL .44 Previous data revealed that a subset of GABAergic neurons also project axons to remote neocortical regions.63 In addition, it has been shown that microstimulation of the neocortex elicits monosynaptic inhibitory postsynaptic potentials in the remote ipsilateral cortex.65 It has therefore been proposed that neurons in different cortical areas may need to be connected reciprocally and symmetrically via GABAergic projection neurons for synchronization of gamma-oscillations in multiple cortical areas,66 although their functional significance has remained uncertain.44 In the present study, we showed for the first time that IL provides direct inhibitory input to ipsilateral ventral and dorsal PrL by activation of NPY+-GABAergic projection neurons in IL (44 Thus, activation of these NPY+-GABAergic projection neurons in IL will lead to direct inhibition of pyramidal neurons in ipsilateral PrL and thus attenuate the activation of its downstream targets. One would thus expect that activation of the NPY+-GABAergic projection neuron-mediated inhibition would alleviate the activation of PrL-related anxiogenic pathway, and accentuate the IL-related anxiolytic pathway.17 To comprehend the neuronal circuits between PrL and IL, it would be worthwhile to further investigate that the synaptic inhibition originates from GABAergic neurons located in PrL that reciprocally inhibit the pyramidal neurons in IL.By convention cortical glutamatergic neurons are considered the sole originators of long-range projections, while cortical GABAergic interneurons are typically described as only projecting their axons locally.ns in IL . This dain vivo,31 Nlgn2 appears to function primarily at inhibitory synapses.33 Furthermore, our previous findings demonstrated that Nlgn2 binds the scaffolding protein gephyrin and functions as a specific activator of collybistin, thus guiding the clustering of inhibitory neurotransmitter receptors. Deletion of Nlgn2 perturbs GABAergic and glycinergic synaptic transmissions and leads to a loss of postsynaptic specializations.37 As a consequence of this, Nlgn2-KO mice demonstrate heightened anxiety-related behavior on multiple measures.38 Given the broad expression of Nlgn2 at inhibitory synapses throughout the brain,31 this selective anxiety-related phenotype is somehow surprising. One possible explanation is that the loss of Nlgn2 could be partially compensated by other Nlgn isoforms in many brain regions. However, there was no compensatory increase in Nlgn1 or Nlgn3 levels in Nlgn2-KO mice.38 As Nlgn2 is differentially expressed in different brain areas,31 the functional roles of Nlgn2-related control of maturation of inhibitory synapses in PFC and limbic areas might be more predominant than in other brain areas. Indeed, our present result showed that deletion of Nlgn2-gene could not be compensated in PFC and therefore significantly diminished the overall GABAergic inhibitory inputs to PrL, especially the inhibition from IL to ipsilateral PL (38Consistent with its localization teral PL . These d67 but it must also include the neuronal circuit between IL and PrL that reciprocally regulates the activity of each other, although many details about the related neuronal circuits are still elusive. Our present results provide the first experimental evidence for the existence of such a direct inhibition from IL to ipsilateral PrL. Within this reciprocal neuronal circuit between IL and PrL, the inhibitory input from PrL to IL will predominate during fear expression/renewal, while the inhibitory input from IL to PrL predominates during fear extinction/extinction recall. In this way, fear extinction results not only from increased activity within the IL-related inhibitory circuit, but it also occurs via simultaneous decreased activity of the PrL-related excitatory circuit mediated by NPY+-GABAergic projection neurons in IL. Given the existence of the reciprocal inhibitions between IL and PrL, this simultaneous deactivation of PrL may be necessary to additionally remove the possible reversal inhibition from PrL to IL and to further facilitate the IL-related extinction learning.Taken together, we propose that the dichotomic neuronal circuit in PFC does not only contain the PrL-related excitatory circuit and the IL-related inhibitory circuit including their downstream target areas,68 A detailed knowledge of the role of neuromodulators within the neuronal circuits throughout the different layers of the PFC could lead to a deepened understanding of neuronal processing under various physiological and pathophysiological conditions.Future studies are necessary to determine the detailed synaptic interconnectivities within this reciprocal neuronal circuit between IL and PrL. Furthermore, it is important to consider the exact subtypes of GABAergic neurons that are studied. Indeed, for several neuromodulators, it is already known that they are co-localized in a subtype-specific way.


Ypt/Rab gene family), an evolutionarily conserved ubiquitously expressed subfamily of Rab GTPases, has been implicated in regulating vesicular trafficking through the recycling of endosomes. Rabs have been grouped into different subfamilies based on the distinct unambiguous sequence motifs. Three members: Rab11a, Rab11b and Rab25 make up the Rab11 GTPase subfamily. In this review article, we describe an overview over Rab11 subfamily with a brief structural aspect and its roles in implicating different disease progression.Membrane/protein trafficking in the secretory/biosynthetic and endocytic pathways is mediated by vesicles. Vesicle trafficking in eukaryotes is regulated by a class of small monomeric GTPases: the Rab protein family. Rab proteins represent the largest branch of the Ras superfamily GTPases, and have been concerned in a variety of intracellular vesicle trafficking and different intracellular signalling pathways. Rab11 (a subfamily of the  Overview over Rab family proteins and Rab11 subfamilyviz., growth, protein trafficking and different signalling pathways. So far, over 70 Rab proteins are encoded by the vertebrates; while most of them are ubiquitously expressed but some of these are tissue-specific also. A variety of Rabs are defective and abnormally expressed in different diseases including cancers  monomeric GTP-binding molecules (GTPases) forming the largest branch of the Ras superfamily, have been found to be present from yeast to humans. These proteins participate in receptor cargo collection during transport vesicle formation and enable motor proteins to interact with membranes to drive vesicle motility, mediating the complex events of precise docking and fusion of transport vesicles with their targets. In addition to the aforesaid general functions, Rabs also act as key players in performing a variety of cellular functions, -25 kDa m cancers -4. In adthaliana -7 have aYpt/Rab gene family, is associated with recycling endosomes (REs) and has been implicated in regulating vesicular trafficking through recycling of endosomal compartment (REC) and early endosomes (EEs) to the trans- Golgi network (TGN) and plasma membrane through recycling pathways  that are essential for prenyltransferase recognition It is the most common form of dementia and has been characterized by the cerebral deposition of β-amyloid (Aβ) peptides as amyloid plaques which are generated from amyloid precursor protein (APP) by β and γ secretases. APP and the secretases are membrane associated proteins -18. AggrHuntington disease (HD)Drosophila model of HD can be prevented by enhanced neuronal expression of Rab11. Inhibition of Rab11 function in fibroblasts of HD patients has been observed to perturb vesicle formation from recycling endosomes. Rab11 is involved in synaptic dysfunction prior to the onset of HD symptoms, with the aim of finding a possible early intervention to disease progression. Rab11 ameliorates synaptic dysfunction due to expression of mutant huntingtin (the causative protein in HD) by normalizing synaptic vesicle size, which consequently ameliorates locomotor deficits in Drosophila larvae  exert an important role in cancers of multiple lineages, including breast, colon, lung, ovarian, renal, endometrial, prostate, bladder and carcinoid types . It has Rab11, a master regulator of intracellular membrane trafficking routes, is the most characterized molecule among all the Rab proteins. It has been implicated in the progression of a number of human diseases. It is noteworthy to mention that Rab11 has an extensive protein-protein interactions network and a variety of distinct cargo molecules. But still, several significant questions remain unclear including how Rab11 mediated pathways are regulated; what is the precise molecular mechanism of the upstream activation of Rab11 subfamily members. Indeed, understanding the precise molecular mechanism for Rab11 in membrane recruitment is a key question elucidating its functional role within the cell. A more complete picture of the molecular and cellular events controlled by Rab11 proteins will uncover additional important physiological processes of the Rab11 subfamily members and will prompt further research in molecular mechanisms for many of the diseases in which it has been implicated. Further, understanding how the various interdependent cellular processes occur is now an important goal in the field of membrane trafficking pathways which is yet to be addressed. The members of Rab11 GTPase family are involved in a variety of disease settings including cancer progression. As the actions of mechanisms of these GTPases are identified; the signalling and trafficking pathways in which they operate may present novel targets for therapeutic invention.The authors declared no conflicts of interest.


Delayed cerebral ischemia (DCI) is a feared and significant medical complication following aneurysmal subarachnoid hemorrhage (aSAH). It occurs in about 30% of patients surviving the initial hemorrhage, mostly between days 4 and 10 after aSAH. Clinical deterioration attributable to DCI is a diagnosis of exclusion and especially difficult to diagnose in patients who are comatose or sedated. The latter are typically patients with a high grade on the World Federation of Neurosurgical Societies scale (WFNS grade 4–5), who represent approximately 40–70% of the patient population with ruptured aneurysms. In this group of patients, the incidence of DCI is often underestimated and higher when compared to low WFNS grade patients. To overcome difficulties in diagnosing DCI, which is especially relevant in sedated and comatose patients, the article reports the most recent recommendation for definition of DCI and discusses their advantages and problematic issues in neurocritical care practice. Finally, appropriate neuromonitoring techniques and their clinical impact in high-grade SAH patients are summarized. Delayed cerebral ischemia (DCI) is a feared and significant medical complication following aneurysmal subarachnoid hemorrhage (aSAH). It occurs in about 30% of patients surviving the initial hemorrhage, mostly between days 4 and 10 after aSAH. The known clinical symptoms such as decrease in the level of consciousness and focal signs such as aphasia and hemiparesis may be reversible or otherwise progress to cerebral infarction resulting in an unfavorable outcome or even death . The latAngiographic vasospasm can be associated with reductions in cerebral perfusion measured with 15O-positron emission tomographic imaging . HoweverThroughout the literature, various means of defining vasospasm are used. To overcome difficulties to diagnose DCI, which is especially relevant in sedated and comatose patients, an excellent summary of possible definitions and their clinical impact, evaluated in 580 aSAH patients, was performed by Frontera and colleagues : (1) cliDelayed cerebral ischemia is defined as “the occurrence of focal neurological impairing  or a decrease of at least two points on the Glasgow Coma Scale [either on the total score or one of its individual components ]. This should last for at least 1 h, and is not apparent immediately after aneurysm occlusion and cannot be attributed to other causes by means of clinical assessments, cerebral CT or MRI scanning, and appropriate laboratory studies. Nevertheless, the diagnosis of DCI remains fairly subjective.Cerebral infarction is defined as presence of cerebral infarction on CT or MRI scan of the brain within 6 weeks after SAH, or on the latest CT or MRI scan made before death within 6 weeks, or proven at autopsy, not present on the CT or MR scan between 24 and 48 h after early aneurysm occlusion, and not attributable to other causes (such as clipping or endovascular treatment). Hypodensities on CT imaging resulting from ventricular catheter or intraparenchymal hematoma should not be regarded as cerebral infarctions from DCI.To remember, cerebral infarction correlates with the territory of angiographic vasospasm in only 25–81% of SAH patients . The terThough knowing that the simple association of radiographic evidence of vasospasm with clinical features of cerebral ischemia does not take into account the different factors that may contribute to DCI, most neurointensivist know a series of cases showing an excellent correlation between an observed clinical deterioration and angiographic narrowing, with deficits easily reversible with an increase of mean arterial blood pressure. In our view, these concern mainly low WFNS grade patients, while in high-WFNS grade patients, the large spectrum of difficult-to-diagnose DCI/infarction is common. The diagnosis of DCI is limited and the diagnosis of cerebral infarction possible only retrospectively once an infarct is found radiographically . ObviousThere are a series of worth-to-measure techniques, some of them shortly summarized in the following, aimed to detect “asymptomatic” ischemia in high-WFNS grade patients.(1)Class I; Level of Evidence B, revised recommendation from previous guidelines); (B) (new recommendation) induction of hypertension for patients with DCI unless blood pressure is elevated at baseline or cardiac status precludes it ; and (C) prophylactic hypervolemia before the development of angiographic spasm is not recommended .Intracranial pressure (ICP) and cerebral perfusion pressure (CPP): ICP and CPP monitoring, obviously important components of neurocritical care, are widely used in comatose aSAH patients. ICP is preferentially measured as external ventricular drainage (EVD), while in absence of hydrocephalus/slit ventricles, an ICP probe may be used instead, allowing continuous CPP monitoring. A high ICP (>20 mmHg) is considered as an indirect but unspecific warning sign, is associated with energy failure, high secretion of pro-inflammatory interleukins, and possibly aggravates vasospasm. Optimal CPP is targeted individually depending on several factors (ruptured aneurysm treated and presence of DCI). Since CPP might not be available in all patients, current guidelines  recommen(2)ischemia-producing cortical spreading depolarizations (CSDs) are likely to be involved in DCI development Disturbed cerebrovascular autoregulation in the first 5 days after SAH significantly increases the odds of DCI occurrence and was shown to have a better predictive value than classical TCD monitoring protocols Neurological wake-up test . Clinical deteriorations, such as occurrence of focal neurological impairing such as aphasia and hemiparesis are warning signs for potentially DCI, which are difficult to evaluate in high-grade SAH patients. The effect of interruption of sedatives and analgesics on neurologic assessment, hemodynamic changes, brain metabolism, and brain tissue oxygen have shown that (1) IS-trials were not attempted on one-third of eligible days due to safety concerns, and that (2) one-third of performed trials had to be stopped due to a critical increase in ICP and impending brain tissue hypoxia. All IS-trials were associated with cardiopulmonary stress, and detection of a new neurological deficit that led to changes in management occurred in only one trial Monitoring of cerebral oxygenation. The regionally monitoring brain tissue partial pressure of oxygen sensor and the globally measuring jugular bulb oximetry allowing further analysis of arterial-venous differences of energy-related factors are commonly used techniques for detection of cerebral hypoxia/ischemia. A decrease of brain tissue PO2 (<15−10 mmHg) and/or jugular bulb oxygenation (<70%) are warning signs and sensibilize the neurointensivist for occurring processes leading to progressive, clinically not detectable ischemia, infarction, and unfavorable outcome in this high-risk SAH population.(6)Cerebral metabolism. Lacking clinical features to diagnose a neurological deterioration in high-WFNS grade patients, cerebral microdialysis provides information on the metabolic state of the injured brain and allows for determination of a variety of markers of ischemia and immunocompetence in the cerebral extracellular fluid (ECF). This is of relevance since both the blood–brain and blood–CSF barriers may be variably disrupted following aSAH, rendering blood and CSF levels of such markers less reliable. Probes are implanted directly into the region of interest, typically after aneurysm clipping. Profound changes in extracellular glucose, lactate, glutamate, and glycerol in patients with signs of acute or delayed ischemia have been shown  on admission . Most, bIn conclusion, the use of clear definitions to classify: (1) occurring deficits such as AFND and DCI, (2) pathomechanisms such as CSD, cerebral hypoxia, disturbed cerebral autoregulation, cerebral metabolism, and inflammation alterations, and (3) resulting lesion using precise time windows and assessment techniques will allow to better share experiences on the disease and improve its understanding. Advanced neuromonitoring techniques may offer the neurointensivists complementary tools to better perceive the pathological condition and engage appropriate treatments that would otherwise lead to progressive, clinically not detectable ischemia, infarction, and unfavorable outcome in high-risk SAH population.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice. Congenital infection with human cytomegalovirus (HCMV) is the most common viral infection of the fetus and occurs in 0.5–2.0% of all live births in most regions in the world. Infection of the fetus can result in a spectrum of end-organ disease, including long term damage to the central nervous system (CNS). Although less than 10% of infected infants exhibit clinical evidence of end-organ disease, up to 10% of the total number of infected infants develop hearing loss. Mechanisms of disease leading to hearing loss are poorly understood because of the limited availability of pathological specimens and accessibility of the inner ear. Existing small animal models fail to recapitulate many features of this infection of the inner ear. In this report we describe a mouse model in which newborn animals infected peripherally with murine CMV develop hearing loss following hematogenous spread of virus to the inner ear. Hearing loss occurs in 30–50% of animals and characteristics of hearing loss in infants with congenital HCMV infection, including delayed onset of hearing loss, progressive hearing loss, and unilateral hearing loss were present in infected mice. Our findings suggest that host derived inflammatory responses and not direct virus-mediated cytopathology are responsible for hearing loss. Findings from this study provide insight into potential mechanisms of hearing loss in infants with congenital HCMV infection. Human cytomegalovirus (HCMV) is the largest human herpesviruses and an important pathogen in immunocompromised hosts ,2. Althond trimester human fetus, this model has allowed study of the effects of CNS infection on brain development, including auditory development that takes place postnatally in the mouse. In this report, we describe findings in this model that are consistent with the loss of spiral ganglion neurons and/or inflammation in the stria vascularis in infected mice as mechanisms of hearing loss. In addition, our results also suggested that virus-induced inflammation is likely a major component of mechanisms leading to hearing loss in MCMV infected mice.The mechanism(s) of disease leading to hearing loss following intrauterine HCMV infection are not understood. In addition, hearing loss can develop months after the newborn period in about 50% of infected infants and be progressive in other infants, suggesting that ongoing virus replication could contribute to hearing loss ,12. Stud1–104 copies of MCMV DNA per mg of cochlear tissue could be detected in 100%, 77%, and 50% of cochlea at PNd14, 4 mo, and 12 mo, respectively  and mock infected controls using 2 modalities, (1) Distortion Product Otoacoustic Emission (DPOAE), and (2) Auditory Brainstem Evoked Response (ABR). DPOAE was used to measure otoacoustic emissions from the inner ear in MCMV and mock-infected mice with DPOAE thresholds defined as the lowest stimulus intensity at which an otoacoustic emission was detectable. DPOAE thresholds were measured at 4 frequencies  and although thresholds were high for both infected and mock-infected mice, we observed an increased DPOAE threshold at only a single frequency (8 kHz) in MCMV infected mice .Auditory brain stem response (ABR) testing served as our standard measure of auditory function. ABR sound pressure levels (SPL) thresholds were assigned as the lowest intensity stimulus at which measurable ABR waveforms were detected as depicted in recordings from a mock and MCMV infected mouse . In thisIn a significant number of infants congenitally infected with HCMV hearing loss can be unilateral, thus overall hearing function can be estimated by determining auditory function for the best and worst ear ,12. FurtAn important clinical feature of hearing loss associated with congenital HCMV infection is delayed onset hearing loss and/or progressive hearing deficits in a significant number of infants ,12. SeriABR thesholds were noted in about 50% of Smith MCMV-infected mice with documented hearing loss . SignifiTo investigate the influence of virus inoculum on progressive hearing loss, hearing was analyzed as a function of time post infection in mice given 50 and 200PFU of BAC cloned MCMV. There was an increase in ABR thresholds between PNd42 and 78 in mice given both 50PFU and 200PFU as compared to mock infected mice . ImportaThe findings that, (i) virus persisted in the inner ear for over four months , (ii) anTo determine if further histologic analysis of the inner ear in mice with increased ABR thresholds could point to mechanisms of disease in the auditory system, cochlear tissue from a group of mock and MCMV infected animals with and without hearing loss were analyzed. No obvious differences in cochlear histology were found between mock infected, infected mice with normal hearing, and infected mice with abnormal hearing . BecauseEven though the development of hearing loss in MCMV infected mice was related to the magnitude of the intial viral inoculum, the lack of a direct relationship between cochlear viral DNA and increased ABR thresholds raised the possibility that MCMV-induced inflammation and not direct viral cytopathic effects, could be responsible for hearing loss in MCMV infected mice. This mechanism was also suggested by both limited cytopathology in the cochlea  and the Although the association of hearing loss with congenital HCMV infection has been recognized for over 4 decades, the pathogenesis of this sequelae of congenital HCMV infection remains poorly understood. Early studies of temporal bones from a small number of infants and older children with congenital HCMV infections described viral antigens and viral particles in the inner ear, inflammatory cellular infiltrates in the inner ear, and degeneration, calcifications, and fibrosis of structures in the inner ear in older children . More reDeficits in hearing in MCMV infected mice were documented primarily by findings of increased ABR thresholds. Although our findings in DPOAE testing suggested normal hair cell function in infected animals, elevated stimulus thresholds in our DPOAE protocol could have obscured abnormal test results. However, the lack of cytopathology in the Organ of Corti, increased ABR thresholds, and the loss of spiral ganglion neurons suggested that hearing loss in this model was consistent with lesions in the auditory system other than hair cell loss. In contrast to our results, two recent studies utilizing intracranial (IC) inoculation of newborn mice with MCMV to produce hearing loss documented hair cell loss ,19. ThesOther small animal models of CMV associated hearing loss have been reported, including congenital infections in guinea pigs (gpCMV). Although this model includes hematogenous spread of virus to the inner ear, it is also associated with modest and often inconsistent inner ear infection, hearing loss in animals without inner ear infection, and confounding issues associated with the severity of maternal infection required to reproducibly achieve intrauterine transmission . This laLarger initial inoculums of MCMV resulted in increased amounts of virus in the inner ear and an increase in the incidence of hearing loss suggesting a direct relationship between virus replication in the cochlea and hearing loss; however, by PNd 42 there was no correlation between virus in the cochlea and hearing loss suggesting that persistent virus replication in the cochlea was not directly related to hearing loss. Thus, it could be argued that only the quantity of virus present in the cochlea early in infection was related to the development of hearing loss. Possible explanations that could account for these findings include the increased likelihood of infection of specific cell types in the inner ear following infection with larger inoculums or, alternatively, inoculum dependent induction of cochlear inflammation early in infection that was not reflected in levels of virus replication at later time points. Thus, larger virus inoculums resulted in increased virus replication and as we observed, a corresponding increase in expression of some proinflammatory cytokines and chemokines early in infection . These eObservations in other small animal models of hearing loss as well as findings in human temporal bones are consistent with a mechanism(s) of virus-induced inflammation; however, specific lesions and the pathways leading to hearing deficits have not been well described. Virus infected cells and mononuclear cells in the spiral ganglion are common to rodent models as well as tissue from infected fetuses, findings suggesting that disruption of spiral ganglion function could contribute to hearing deficits in MCMV infected mice. The loss of spiral ganglion neurons following MCMV infection in our model are similar to findings reported in a more limited analysis of a murine model of CMV associated hearing loss . Thus, lA decrease in the number of spiral ganglion neurons provided a potential mechanism of hearing loss in infected animals secondary to loss of afferent input into cochlear nuclei during this critical time of auditory development ,36. StudThe finding of virus infected cells and CD3+ mononuclear infiltrates in the stria vascularis suggested additional mechanisms of hearing loss in MCMV infected mice. This region of the inner ear is critical for maintaining the unique electrolyte composition of the extracellular fluid within the Organ of Corti that contributes to electrical potential of the endocochlear fluid, the endocochlear potential ,47. HairIn summary, we have described a model of hearing loss in mice inoculated early in the postnatal period with MCMV that closely recapitulates key aspects of hearing loss associated with congenital HCMV infection. Our studies have documented the dependence of viral replication in the cochlea on the initial viral inoculum size and in turn, the level of expression of proinflammatory molecules in the cochlea as well subsequent increases in the ABR thresholds on the level of virus replication during this early time in auditory development. Although virus infected cells could be detected in various regions of the inner ear, significant cytopathology or tissue damage was absent arguing that the mechanism(s) of hearing loss following this infection could have a significant inflammatory component.The University of Alabama-Birmingham Institutional Animal Care and Use Committee (IACUC) guidelines were followed for all animal experiments and breeding. These guidelines are in strict compliance with the NIH (OLAW Assurance Number—A32555-01) guidelines. Animals were euthanized with carbon dioxide under regulated flow. Following carbon dioxide, cervical dislocation was implemented to ensure animals were completely euthanized prior to carrying out experimental protocols, according to the UAB Animal Resource Program (ARP) guidelines. An approved protocol (APN09024) was obtained from IACUC (UAB) for all experiments involving animals and animal tissues carried out at UAB. All procedures involving animals and their care conducted at the Faculty of Medicine, University of Rijeka were approved by the Veterinary Department of the Ministry of Agriculture, Croatia —Assurance no. A5905-01 (effective until 10/31/2019) and were conducted in compliance with institutional guidelines, as well as with national  and international Directive 2010/63/EU of The European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, Guide for the Care and Use of Laboratory Animals, National Institutes of Health, The National Academies Press, Washington, DC) laws and policies.Pathogen-free BALB/c mice were obtained from a colony propagated from animals purchased from Charles River Laboratories. All breeding and experiments were done in accordance with the guidelines of the University of Alabama at Birmingham Institutional Animal Care and Use Committee and with the University of Rijeka-Croatia animal use policies. Auditory testing including ABR and DPOAE (see below) measurements were performed in anesthetized animals (ketamine (75 mg/kg) and medetomidine (1 mg/kg) delivered i.p.). Animals were maintained at 37°C during all testing and monitored for movement.Virus stocks ) were propagated in mouse embryonic fibroblasts. In some experiments, a virus that has been determined by sequence analysis and characterization in-vivo to be wild type Smith virus was recovered from an infectious BAC clone of MCMV kindly provided by Dr. Ulrich Koszinowski (University of Munich) . Virus rDPOAEs were measured using Tucker Davis Technologies  System III equipment and software using published protocols . StimuliABRs were also measured with the TDT System III system, using tone pips at 4, 8, 16, and 32 kHz (90 dB to 20 dB in -10 dB steps), as well as broadband click stimuli (90 dB to 20 dB in -5 dB steps). Signal was detected using 3 stainless steel needle electrodes , amplified, band-pass filtered , and averaged over 750 sweeps. The ABR threshold was defined as the lowest intensity signal at which an ABR waveform was detectable. We adapted protocols from several sources including material provided during a training course in audiometric testing at Jackson Labs  and published protocols .th section). Immunohistochemical (IHC) staining for the IE-1 protein (pp89) encoded by MCMV IE-1 was performed as described previously [Animals were exhaustively perfused initially with phosphate buffered saline  and then with 4% paraformaldehyde (PFA) as described . Cochleaeviously . Anti-CDeviously .Nucleic acids were isolated from cochleae using several protocols depending on downstream applications. In the initial experiments, nucleic acids were isolated using a QIAGEN M48 robot and MagAttract RNA Tissue Mini M48 kit as per the manufacturer’s instructions . In latter experiments, RNA and DNA nucleic acids were isolated using SQ Total RNA Kit and HP Total RNA Isolation Kit  with minor modifications. Quantitative real-time PCR was performed by amplification of a fragment of immediate-early 1 (IE1) exon 4 using the primers forward, 5'-GGC TCC ATG ATC CAC CCT GTT A-3' and reverse, 5'-GCC TTC ATC TGC TGC CAT ACT-3'. The probe (5'-AGC CTT TCC TGG ATG CCA GGT CTC A-3') was labeled with reporter dye FAM and quencher dye TAMRA. To quantify viral genome copy number, serial dilutions (log10) of a plasmid with the cloned IE1 exon 4 PCR product was used to create a standard curve. This assay has a sensitivity of between 50–100 copies of MCMV DNA. For the detection of inflammatory network genes, the Mouse Antiviral Response PCR Array  and for proinflammatory chemokines and cytokines (PAMM-150Z) were used according to manufacturer's protocol. Quantitative real-time PCR was conducted using TaqMan Gene Expression master mix (Qiagen). Samples were run in triplicate using a two-step amplification protocol on the Applied Biosystems 7500 Real-Time PCR System  of life and 50ng reverse transcribed and assayed using PCR arrays as described in Materials and Methods. Number of animals are listed in parentheses and PCR results compared to RNA prepared from cochlea of mock infected animals . Results are expressed as fold increase over signal from cochlea obtained in mock infected animals with statistical measures of differences shown as (TIF)Click here for additional data file.S1 FigNewborn mice infected with 200 PFU of tissue culture derived Smith strain MCMV, or mock infected, were sacrificed on postnatal day (PNd) 11 and after perfusion with PBS and fixation, the inner ears were removed, decalcified and embedded in paraffin as described in Materials and Methods. After sectioning, de-paraffinization, sections were stained with hematoxylin and eosin (H&E) and photographed (x40). Panel C and F represent digitally enlarged images of Panel B and E respectively.(TIF)Click here for additional data file.S2 FigAfter extensive perfusion, temporal bones from mice infected with 200PFU Smith MCMV were harvested on PNd7, fixed and embedded in paraffin as described in Materials and Methods. Sections were cut and stained with H&E . Boxed areas demonstrated large cell with characteristics of MCMV infected cell. Other sections were reacted with anti-MCMV IE-1 antibodies and developed with HRP anti-mouse IgG followed with diaminobenzidine to produce brown color in infected cells . Scale bars shown in lower right corner.(TIF)Click here for additional data file.S3 FigCochlea from mock infected and MCMV infected mice were harvested on PNd7, decalcified, fixed and embedded in paraffin as described in Materials and Methods. MCMV infected cells were stained with anti-IE-1 antibodies and then developed with HRP anti-mouse IgG and diaminobenzidine (brown). This section reveals infected cell in marginal layer of stria vascularis. Panel A at 20x  and panel B at 100x . Panel C is digitally enlarged image of panel B.(TIF)Click here for additional data file.S4 Fig○; n = 25) and MCMV infected  mice. Mice were tested at PNd42 as described in Materials and Methods. (*) indicates significant difference in response at 8 kHz.DPOAE revealed a significant differences at only one frequency between mock infected ((TIF)Click here for additional data file.S5 FigAfter extensive perfusion, cochlea from mice between PNd42-60 were harvested, decalcified, fixed and embedded in paraffin as described in Materials and Methods. Following sectioning and staining with H&E, individual sections were viewed by blinded observer with experience in cochlear histology. Sections from 10 different cochlea were submitted for review and four representative sections are presented this figure. Samples 127 and 133 were from mock infected animals and samples 147 and 211 were from infected animals. The ABR thresholds for these ears are displayed at the bottom of each panel.(TIF)Click here for additional data file.S6 FigAfter extensive perfusion, cochlea from mock infected and MCMV infected mice were harvested on PNd7, decalcified, fixed and embedded in paraffin as described in Materials and Methods. Following sectioning, tissue was reacted with anti-Tuji 1 followed by HRP conjugated anti-mouse IgG and developed with diaminobenzidine(brown). Note the decreased density of Tuji 1+ cells in section from infected mouse .(TIF)Click here for additional data file.


Rheumatoid arthritis (RA) is considered to be, in many respects, an archetypal autoimmune disease that causes activation of pro-inflammatory pathways resulting in joint and systemic inflammation. RA remains a major clinical problem with the development of several new therapies targeted at cytokine inhibition in recent years. In RA, biologic therapies targeted at inhibition of tumor necrosis factor alpha (TNFα) have been shown to reduce joint inflammation, limit erosive change, reduce disability and improve quality of life. The cytokine TNFα has a central role in systemic RA inflammation and has also been shown to have pro-inflammatory effects in the brain. Emerging data suggests there is an important bidirectional communication between the brain and immune system in inflammatory conditions like RA. Recent work has shown how TNF inhibitor therapy in people with RA is protective for Alzheimer's disease. Functional MRI studies to measure brain activation in people with RA to stimulus by finger joint compression, have also shown that those who responded to TNF inhibition showed a significantly greater activation volume in thalamic, limbic, and associative areas of the brain than non-responders. Infections are the main risk of therapies with biologic drugs and infections have been shown to be related to disease flares in RA. Recent basic science data has also emerged suggesting that bacterial components including lipopolysaccharide induce pain by directly activating sensory neurons that modulate inflammation, a previously unsuspected role for the nervous system in host-pathogen interactions. In this review, we discuss the current evidence for neuro-inflammation as an important factor that impacts on disease persistence and pain in RA. Rheumatic diseases include some of the most common chronic disorders worldwide. Of these, rheumatoid arthritis (RA) is considered to be, in many respects, an archetypal autoimmune disease  enzymes (mainly COX-2) and generation of prostanoids. Under usual physiological conditions, these inflammatory mediators are absent in the brain or found in low levels. After brain injury or insult, there can be induction of pro-inflammatory mediators in astrocytes, microglia, neurons and endothelial cells which results in the development of neuro-inflammatory conditions. Recently, the COX-2 inhibitor celecoxib has been shown to reduce functional connectivity measured by functional MRI in an animal model of osteoarthritis  sensory neurons and demonstrated an induction of calcium flux and induced action potential firing. S. aureus was found to directly activate nociceptors in the Nav1.8-lineage , DMARDs or biological agents for RA and spondyloarthritis with respiratory tract infections being the most common  infections leading to activation of macrophages, cell recruitment, granuloma formation and maintenance  developed opportunistic infections  including oesophageal candidiasis, aspergillosis, candida sepsis, coccidiomycosis, cytomegalovirus, herpes zoster, and nocardiasis  may be compromised in RA with macrophages being recruited from the systemic circulation via intercellular adhesion molecule (ICAM) expression on the endothelium  in placebo-controlled trials found that serious infections requiring anti-microbial therapy or hospitalization occurred in 126 patients in the treatment arm and 26 patients on placebo   was used by Leombruno and colleagues to investigate the safety of TNF inhibitor therapy in RA , the median time from commencement of treatment to time of first opportunistic infection is 16.2 months (6.0–26.0)  was used to assess the neurological effects of TNF inhibitor therapy in 10 patients with active RA  provides a variety of techniques for assessing neurological involvement of disease processes by detecting changes in brain morphometry [T1 weighted imaging (T1w)] and tissue microstructure , brain metabolism by magnetic resonance spectroscopy (MRS) and actual brain functionality by fMRI as described above. However, there are far fewer reports of neuroimaging abnormalities for RA compared to other related autoimmune diseases. In Sjögren's syndrome significant loss of brain tissue and presence of white matter lesions are observed . This reflects the lower incidence of neurological symptoms and the more subtle effects of cerebrovascular involvement that have been so far detected.The concept of neuroinflammation as a significant component of disease pathophysiology in RA has only recently been recognized. Animal models have recently demonstrated that infectious agents including LPS can directly activate the immune system stimulate pain responses. Coupled with the strong link between RA and incidence of infection, it may be time to reconsider the balance between maintaining disease remission using disease-modifying immune-modulatory drugs and preventing disease flares induced by infection. However, the link between infection and immune-modulatory therapies remains controversial and comparison of studies is made difficult by differing biologic therapies used, different diseases treated and different, concurrent medications regimes. Further, high-powered studies are required to establish the period of highest risk and susceptibility to infections and the overall risk of infections for patients taking biological treatments for RA.Future work is needed to understand how the RA disease process impacts on pain activation in RA, whether sensitisation is a general phenomenon in RA or found in specific subsets of patients and what specific infectious triggers contribute to an autoimmune pro-inflammatory network that leads to chronic pain and inflammation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.