Adult hippocampal neurogenesis in the context of lipopolysaccharide-induced neuroinflammation: A molecular, cellular and behavioral review.

The continuous generation of new neurons occurs in at least two well-defined niches in the adult rodent brain. One of these areas is the subgranular zone of the dentate gyrus (DG) in the hippocampus. While the DG is associated with contextual and spatial learning and memory, hippocampal neurogenesis is necessary for pattern separation. Hippocampal neurogenesis begins with the activation of neural stem cells and culminates with the maturation and functional integration of a portion of the newly generated glutamatergic neurons into the hippocampal circuits. The neurogenic process is continuously modulated by intrinsic factors, one of which is neuroinflammation. The administration of lipopolysaccharide (LPS) has been widely used as a model of neuroinflammation and has yielded a body of evidence for unveiling the detrimental impact of inflammation upon the neurogenic process. This work aims to provide a comprehensive overview of the current knowledge on the effects of the systemic and central administration of LPS upon the different stages of neurogenesis and discuss their effects at the molecular, cellular, and behavioral levels.

PI3K Signaling in Neurons: A Central Node for the Control of Multiple Functions.

Phosphoinositide 3-kinase (PI3K) signaling contributes to a variety of processes, mediating many aspects of cellular function, including nutrient uptake, anabolic reactions, cell growth, proliferation, and survival. Less is known regarding its critical role in neuronal physiology, neuronal metabolism, tissue homeostasis, and the control of gene expression in the central nervous system in healthy and diseased states. The aim of the present work is to review cumulative evidence regarding the participation of PI3K pathways in neuronal function, focusing on their role in neuronal metabolism and transcriptional regulation of genes involved in neuronal maintenance and plasticity or on the expression of pathological hallmarks associated with neurodegeneration.

EFAD transgenic mice as a human APOE relevant preclinical model of Alzheimer's disease.

Identified in 1993, APOE4 is the greatest genetic risk factor for sporadic Alzheimer's disease (AD), increasing risk up to 15-fold compared with APOE3, with APOE2 decreasing AD risk. However, the functional effects of APOE4 on AD pathology remain unclear and, in some cases, controversial. In vivo progress to understand how the human (h)-APOE genotypes affect AD pathology has been limited by the lack of a tractable familial AD-transgenic (FAD-Tg) mouse model expressing h-APOE rather than mouse (m)-APOE. The disparity between m- and h-apoE is relevant for virtually every AD-relevant pathway, including amyloid-ß (Aß) deposition and clearance, neuroinflammation, tau pathology, neural plasticity and cerebrovascular deficits. EFAD mice were designed as a temporally useful preclinical FAD-Tg-mouse model expressing the h-APOE genotypes for identifying mechanisms underlying APOE-modulated symptoms of AD pathology. From their first description in 2012, EFAD mice have enabled critical basic and therapeutic research. Here we review insights gleaned from the EFAD mice and summarize future directions.

Cholesterol-induced astrocyte activation is associated with increased amyloid precursor protein expression and processing.

Cholesterol is essential for maintaining lipid raft integrity and has been regarded as a crucial regulatory factor for amyloidogenesis in Alzheimer's disease (AD). The vast majority of studies on amyloid precursor protein (APP) metabolism and amyloid ß-protein (Aß) production have focused on neurons. The role of astrocytes remains largely unexplored, despite the presence of activated astrocytes in the brains of most patients with AD and in transgenic models of the disease. The role of cholesterol in Aß production has been thoroughly studied in neurons and attributed to the participation of lipid rafts in APP metabolism. Thus, in this study, we analyzed the effect of cholesterol loading in astrocytes and analyzed the expression and processing of APP. We found that cholesterol exposure induced astrocyte activation, increased APP content, and enhanced the interaction of APP with BACE-1. These effects were associated with an enrichment of ganglioside GM1-cholesterol patches in the astrocyte membrane and with increased ROS production. GLIA 2015;63:2010-2022.

Differential changes in Wnt7 and Dkk1 levels in astrocytes exposed to glutamate or TNFα.

Wnt signaling plays an important role in adult brain function, and its dysregulation has been implicated in the loss of neuronal homeostasis. Despite the existence of many studies on the participation of the Wnt pathway in adult neurons, its regulation in astrocytes has been scarcely explored. Several reports point to the presence of Wnt ligands in astrocytes and their possible impact on neuronal plasticity or neuronal death. We aimed to analyze the effect of the neurotransmitter glutamate and the inflammatory cytokine TNFα on the mRNA and protein levels of the canonical Wnt agonist Wnt7a and the antagonist Dkk1 in cultured astrocytes. Primary astrocyte cultures from rat cerebral cortices were exposed to glutamate or TNFα. Wnt7a and Dkk1 expression was analyzed by RT-qPCR and its protein abundance and distribution was assessed by immunofluorescence. We found high basal expression and protein levels of Wnt7a and Dkk1 in unstimulated astrocytes and overproduction of Dkk1 mRNA induced by the two stimuli. These results reveal the astrocytic source of the canonical Wnt ligands Wnt7a and Dkk1, whose levels are differentially regulated by glutamate and TNFα. Astrocytes are a significant source of Wnt ligands, the production of which can be differentially regulated under excitatory or proinflammatory conditions, thereby impacting neuronal function.

Role of wnt signaling in the control of adult hippocampal functioning in health and disease: therapeutic implications.

It is well recognized the role of the Wnt pathway in many developmental processes such as neuronal maturation, migration, neuronal connectivity and synaptic formation. Growing evidence is also demonstrating its function in the mature brain where is associated with modulation of axonal remodeling, dendrite outgrowth, synaptic activity, neurogenesis and behavioral plasticity. Proteins involved in Wnt signaling have been found expressed in the adult hippocampus suggesting that Wnt pathway plays a role in the hippocampal function through life. Indeed, Wnt ligands act locally to regulate neurogenesis, neuronal cell shape and pre- and postsynaptic assembly, events that are thought to underlie changes in synaptic function associated with long-term potentiation and with cognitive tasks such as learning and memory. Recent data have demonstrated the increased expression of the Wnt antagonist Dickkopf-1 (DKK1) in brains of Alzheimer´s disease (AD) patients suggesting that dysfunction of Wnt signaling could also contribute to AD pathology. We review here evidence of Wnt-associated molecules expression linked to physiological and pathological hippocampal functioning in the adult brain. The basic aspects of Wnt related mechanisms underlying hippocampal plasticity as well as evidence of how hippocampal dysfunction may rely on Wnt dysregulation is analyzed. This information would provide some clues about the possible therapeutic targets for developing treatments for neurodegenerative diseases associated with aberrant brain plasticity.

KIR-HLA receptor-ligand mismatch associated with a graft-versus-tumor effect in haploidentical stem cell transplantation for pediatric metastatic solid tumors.

Killer immunoglobulin-like receptors (KIRs) on natural killer cells (NKs) recognize groups of human leukocyte antigen (HLA) class I alleles. Cells without an inhibitory HLA ligand may trigger NK activation. Reduced risk of relapse has been reported in malignant hematologic diseases after haploidentical transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient. We performed haploidentical transplant in three children with refractory solid tumors. Our results showed that beneficial antitumor effects could be observed in the presence of inhibitory KIR-HLA mismatch. These preliminary results suggest a possible association between disease control and NK cell alloreactivity.

The detrimental effects of lipopolysaccharide-induced neuroinflammation on adult hippocampal neurogenesis depend on the duration of the pro-inflammatory response.

Adult hippocampal neurogenesis is a finely tuned process regulated by extrinsic factors. Neuroinflammation is a hallmark of several pathological conditions underlying dysregulation of neurogenesis. In animal models, lipopolysaccharide (LPS)-induced neuroinflammation leads to a neurogenic decrease mainly associated to the early inflammatory response. However, it is not well understood how the neuroinflammatory response progresses over time and if neurogenesis continues to be diminished during the late neuroinflammatory response. Moreover, it is unknown if repeated intermittent administration of LPS along time induces a greater reduction in neurogenesis. We administered one single intraperitoneal injection of LPS or saline or four repeated injections (one per week) of LPS or saline to young-adult mice. A cohort of new cells was labeled with three 5-bromo-2-deoxyuridine injections (one per day) 4 days after the last LPS injection. We evaluated systemic and neuroinflammation-associated parameters and compared the effects of the late neuroinflammatory response on neurogenesis induced by each protocol. Our results show that 1) a single LPS injection leads to a late pro-inflammatory response characterized by microglial activation, moderate astrocytic reaction and increased interleukin-6 levels. This response correlates in time with decreased neurogenesis and 2) a repeated intermittent injection of LPS does not elicit a late pro-inflammatory response although activated microglia persists. The latter profile is not accompanied by a continued long-term hippocampal neurogenic decrease. Hereby, we provide evidence that the neuroinflammatory response is a dynamic process that progresses in a milieu-dependent manner and does not necessarily lead to a neurogenic decrease, highlighting the complex interaction between the immune system and neurogenesis.

[Treatment of chronic anal fissure with botulinic toxin type A]. / Tratamiento de la fisura anal crónica con toxina botulínica tipo A.

UNLABELLED: Anal fissure is a disease that generally affects to young people, but it can present at any age. PURPOSE: To show our experience in the treatment of chronic anal fissure with botulinum toxin type A. MATERIAL AND METHODS: This is a prospective, experimental and longitudinal study realized in the Coloproctology Unit in the General Hospital of Mexico City and in Medical North of Monterrey, Mexico, between June 2002 and November 2004. Direct variable was healing with 25 units of botulinum toxin. Secondary variables: age, sex, symptomatology, evolution time, localization of fissure, evolution after application of botulinum toxin, complications and time ofhealing. RESULTS: 35 (67.30%) were female and 17 (32.69%), male; age range, 21 to 64 years, with a medium of 43. At twelve months of toxin, application of 34 (65.38%) patients were asymptomatic, but in four fissure persisted. The other 18 patients were operated on due to persistence of the fissure. In a follow up of 18 months, 30 patients cured (57.69%), 4 improved (7.69%) and 18 (34.61%) were failures; one died by myocardial infarction and there were no recurrences. CONCLUSIONS: Botulinum toxin type A is a good alternative in the treatment of chronic anal fissure. Healing or improved with the application of 25 units of botulinum toxin was 65.38%.

A preclinical model for the analysis of genetically modified human skin in vivo.

Although skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer, it is a challenging site when attempting gene delivery. In addition to the transience of gene expression, important obstacles to cutaneous gene therapy have included the inability to sustain gene expression in a large proportion of keratinocytes within a given skin compartment. In this study, we have developed a novel experimental strategy that allows long-term regeneration of entirely genetically engineered human skin on the backs of NOD/SCID mice. Primary human keratinocytes were infected with a retroviral vector encoding the enhanced green fluorescent protein (EGFP) produced by transient transfection of 293T cells. EGFP expression allowed cell-sorting selection of a polyclonal population of productively transduced keratinocytes that were assembled in a live fibroblast-containing fibrin dermal matrix and orthotopically grafted onto mice. Epifluorescent illumination of the transplanted zone allowed in vivo monitoring of the genetically modified graft. EGFP-positive human skin was present on mice for 22 weeks after grafting. In addition, frozen sections prepared from the grafts displayed consistently strong EGFP-based fluorescence in all epidermal strata at every time point examined. Persistence of transgene expression was further confirmed through EGFP protein immunodetection. Purified EGFP-positive keratinocytes grafted as part of the fibrin-based artificial skin were capable of generating multilayer human epidermis on mice, with well-developed granulosum and corneum strata, and clearly defined rete ridges. Finally, the large proportion of transduced keratinocytes in our grafts allowed us to study, for the first time, the long-term in vivo clonal reconstitution pattern of the regenerated skin. Analysis of the provirus insertion sites indicates that a discrete number of epidermal stem cell clones was responsible for the maintenance of human skin regenerated in NOD/SCID recipients.

A comparison of targeting performance of oncoretroviral versus lentiviral vectors on human keratinocytes.

The epidermis, like other rapidly renewing tissues, relies on a stem cell compartment to undergo constant regeneration. In order to develop realistic and long-lasting therapeutic approaches for some skin disorders, gene transfer to these critical cells must be obtained. While efficient retroviral ex vivo targeting and transgene integration in human keratinocytes is tightly dependent on proliferation, transferring genetic information to quiescent cells in culture also presents advantages, including the possibility of targeting putative dormant epidermal stem cells. In the present study we compared the efficiency of transduction achieved with a third-generation of human immunodeficiency virus (HIV)-based lentiviral vector to that obtained with a Moloney murine leukemia oncoretroviral vector (MLV) on proliferating and quiescent human keratinocytes growing in vitro in standard Rheinwald and Green cultures as well as in confluent organotypic cultures. Each viral vector contained the enhanced green fluorescent protein (EGFP) as a reporter gene. The lentiviral vector, but not the MLV vector, led to EGFP expression both in nondividing and proliferating epidermal cell populations in vitro. This feature was clearly evident when direct targeting of human keratinocytes, forming part of the epidermal component of an organotypic skin culture, was attempted. Keratinocytes modified by both MLV and the lentiviral vector allowed long-term regeneration of genetically engineered human skin on the backs of immunodeficient nonobese diabetic/severe combined immunodeficiency disorders (NOD/SCID) mice. However, EGFP transgene expression in the context of the MLV (long-terminal repeat [LTR]-driven) or lentiviral vector (cytomegalovirus [CMV]-driven) demonstrated clear differences both in quantitative terms and in the in vivo localization pattern.

An in vivo model of wound healing in genetically modified skin-humanized mice.

Cutaneous wound-healing disorders are a major health problem that requires the development of innovative treatments. Whithin this context, the search for reliable human wound-healing models that allow us to address both mechanistic and therapeutic matters is warranted. In this study, we have developed a novel invivo wound-healing model in a genetically modified human context. Our model is based on the regeneration of human skin on the back of nude mice by transplantation of a cultured bioengineered skin equivalent previously designed in our laboratory. In this setting, human keratinocytes in the epidermal compartment were genetically modified with a retroviral vector encoding the enhanced green fluorescent protein (EGFP). After stable engraftment of the EGFP skin was achieved (9-12 wk after grafting), a small circular full thickness wound was performed on this mature human skin. A wide variety of parameters involved in wound healing were monitored, including tissue architecture, cell proliferation, epidermal differentiation, dermal remodelling, and basement membrane regeneration. Wounded gene-targeted skin-humanized mice re-capitulated native skin wound-healing features. In addition, when keratinocyte growth factor (KGF), a growth factor that has been shown to improve wound healing, was added to wounds during 3 d, the re-epithelialization was significantly accelerated. The present wound-healing model system provides a suitable in vivo tool to test gene transfer strategies for human skin repair. It also serves as a complementary platform for studies in genetically modified mice and as a model to evaluate pharmaceutical therapeutic approaches for impaired wound healing.

[Hemorrhoidal desarterialization guided by Doppler. A surgical alternative in hemorrhoidal disease management]. / Desarterialización hemorroidaria guiada por Doppler. Una alternativa quirúrgica en el manejo de la enfermedad hemorroidaria.

BACKGROUND: Hemorrhoidal disease is a common condition that includes 30% of patients seen for the first time at the Colon and Rectal Unit of the Gastroenterology Service, Hospital General in Mexico City. This study shows experience with hemorrhoidal desarterialization guided by Doppler in patients with internal hemorrhoidal disease stages II or III in the Coloproctology Units of the Hospital General in Mexico City and at the North Medical Unit in Monterrey, Nuevo Leon, Mexico. MATERIALS AND METHODS: A prospective, longitudinal, and descriptive study was performed in 56 patients with diagnosis of internal hemorrhoids stages II or III, not complicated, and treated by hemorrhoidal artery ligation guided by Doppler between July 1, 2002 and April 31, 2003. RESULTS: 49 patients were included, 32 male (66%) and 17 female (34%), aged between 21 and 75 years with mean age of 43.5 years; 40 patients had internal hemorrhoids stage II (82%) and nine, stage III (18%). With a follow-up of 4 months or longer, all patients were symptomless: bleeding and hemorrhoidal prolapse disappeared. CONCLUSIONS: Doppler-Guided hemorrhoidal desarterialization is indicated in patients with internal hemorrhoids stages II or III; it is a simple technique that requires a short learning curve, minimal anesthetic and surgical materials, and pain after the procedure is mild and short-lived in the majority of patients.

When astrocytes become harmful: functional and inflammatory responses that contribute to Alzheimer's disease.

A growing body of research suggests that astrocytes play roles as contributors to the pathophysiology of Alzheimer's disease (AD). Several lines of evidence propose that activated astrocytes produce and release proinflammatory molecules that may be critical for the generation of amyloid-ß peptide (Aß). However, accumulating evidence indicates that Aß may activate astrocytes, which leads to an increase in cytokines that has been suggested to be a causative factor in the cognitive dysfunction of AD; thus, a vicious circle may be created. Intrinsic inflammatory mechanisms may provide a regulatory system that is capable of influencing the neuronal microenvironment that affects neuronal survival. In this article, we address the evidence surrounding the interactions of dysfunctional astrocytes with neighboring neurons that may initiate a cascade of events that culminates with neuronal injury and the expression of the hallmark lesions of AD. Comprehensive knowledge of the molecular mechanisms underlying the participation of astrocytes in neurodegeneration could aid the development of therapies to restore proper astrocyte function that can be used in AD patients to prevent or alleviate the progression of the disease in a more efficient and comprehensive manner.