Adult Mouse Brain Tissue Research Articles

CircHTT(2,3,4,5,6) — co-evolving with the HTT CAG-repeat tract — modulates Huntington's disease phenotypes

Circular RNA (circRNA) molecules have critical functions during brain development and in brain related disorders. Here, we identified and validated a circRNA, circHTT(2,3,4,5,6), stemming from the Huntington's disease (HD) gene locus that is most abundant in the CNS. We uncovered its evolutionary conservation in diverse mammalian species, and a correlation between circHTT(2,3,4,5,6) levels and the length of the CAG-repeat tract in exon-1 of HTT in human and mouse HD model systems. The mouse orthologue, circHtt(2,3,4,5,6), is expressed during embryogenesis, increases during nervous system development, and is aberrantly up-regulated in presence of the expanded CAG-tract. While an IRES-like motif was predicted in circHtt(2,3,4,5,6), the circRNA does not appear to be translated in adult mouse brain tissue. Nonetheless, a modest, but consistent fraction of circHtt(2,3,4,5,6) associates with the 40S ribosomal subunit, suggesting a possible role in the regulation of protein translation. Finally, circHtt(2,3,4,5,6) over-expression experiments in HD-relevant STHdh striatal cells revealed its ability to modulate CAG-expansion driven cellular defects in cell-to-substrate adhesion, thus uncovering an unconventional modifier of HD pathology.

Majority of alpha2,6-sialylated glycans in the adult mouse brain exist in O-glycans: SALSA-MS analysis for knockout mice of alpha2,6-sialyltransferase genes.

Sialic acids are unique sugars with negative charge and exert various biological functions such as regulation of immune systems, maintenance of nerve tissues and expression of malignant properties of cancers. Alpha 2,6 sialylated N-glycans, one of representative sialylation forms, are synthesized by St6gal1 or St6gal2 gene products in humans and mice. Previously, it has been reported that St6gal1 gene is ubiquitously expressed in almost all tissues. On the other hand, St6gal2 gene is expressed mainly in the embryonic and perinatal stages of brain tissues. However, roles of St6gal2 gene have not been clarified. Expression profiles of N-glycans with terminal α2,6 sialic acid generated by St6gal gene products in the brain have never been directly studied. Using conventional lectin blotting and novel sialic acid linkage-specific alkylamidationmass spectrometry method (SALSA-MS), we investigated the function and expression of St6gal genes and profiles of their products in the adult mouse brain by establishing KO mice lacking St6gal1 gene, St6gal2 gene, or both of them (double knockout). Consequently, α2,6-sialylated N-glycans were scarcely detected in adult mouse brain tissues, and a majority of α2,6-sialylated glycans found in the mouse brain were O-linked glycans. The majority of these α2,6-sialylated O-glycans were shown to be disialyl-T antigen and sialyl-(6)T antigen by mass spectrometry analysis. Moreover, it was revealed that a few α2,6-sialylated N-glycans were produced by the action of St6gal1 gene, despite both St6gal1 and St6gal2 genes being expressed in the adult mouse brain. In the future, where and how sialylated O-linked glycoproteins function in the brain tissue remains to be clarified.

腺相关病毒介导FMRP在成年Fmr1 KO小鼠脑组织中获得性表达

腺相关病毒介导FMRP在成年Fmr1 KO小鼠脑组织中获得性表达

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20bp) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos, and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system

Microglia are resident immune cells of the central nervous system (CNS). The exact role of microglia in CNS disorders is not clear due to lack of tools to discriminate between microglia and infiltrating myeloid cells. Here, we present a novel reporter mouse model targeting a microglia-specific marker, TMEM119, for studying microglia in health and disease. By placing a reporter cassette (GSG-3xFlag-P2A-tdTomato) between the coding sequence of exon 2 and 3′UTR of the Tmem119 gene using CRISPR/Cas9 technology, we generated a Tmem119-tdTomato knock-in mouse strain. Gene expression assay showed no difference of endogenous Tmem119 in the CNS of Tmem119tdTomato/+ relative to wild-type mice. The cells expressing tdTomato were recognized by immunofluorescence staining using commercially available anti-TMEM119 antibodies. Additionally, immunofluorescence and flow cytometry techniques revealed that tdTomato+ cells are detected throughout the CNS, but not in peripheral tissues of Tmem119tdTomato/+ mice. Aging does not influence TMEM119 expression as tdTomato+ cells were detectable in the CNS of older mice (300 and 540 days old). Further immunofluorescence characterization shows that tdTomato+ cells colocalize with Iba1+ cells in the brain, but not with neurons, astrocytes or oligodendrocytes. Moreover, flow cytometry analysis of brain tissues of adult mice demonstrates that the majority of microglia CD45loCD11b+ cells (96.3%) are tdTomato-positive; and a minority of infiltrating CD45hiCD11b+ myeloid cells (5.3%) are also tdTomato-positive, which we further characterized and found that tdTomato expression is in part of choroid plexus macrophages but not in meningeal and perivascular macrophages. Functionally, using an acute injury model, we measured time-lapse activation of tdTomato-labeled microglia by transcranial two-photon microscopy in live Tmem119tdTomato/+ mice. Taken together, the Tmem119-tdTomato reporter mouse model is a valuable tool to specifically study the role of microglia in health and disease.

Gene expression profile of Sox2 and Nestin both were regulated by histone deacetylase in C17.2 neural stem cells

Histone deacetylation is a key modulator involved in proliferation, apoptosis and mRNA transcription . However, the effects of histone deacetylation in C17.2 neural stem cells (NSCs) are still not fully understood. In this study, C17.2 NSCs were used to evaluate the effects of nicotinamide and trichostatin A (TSA) on the transcription of several important genes. Here, we confirmed that the mRNA expression of sox1, sox2, p53 and bax in E14.5 NSCs were significantly higher than in C17.2 NSCs. There was no difference between E14.5 and C17.2 NSCs in the transcription of nestin. The transcription levels of p53 and nestin were significantly higher in C17.2 NSCs than in cerebral cortex, hippocampus, cerebellar cortex and olfactory bulb. The expression levels of sox1, sox2 and bax were upregulated in olfactory bulb as compared to the other three brain tissues. To further assess the effects of histone deacetylation in C17.2 NSCs, we used two histone deacetylase (HDAC) inhibitors, nicotinamide and TSA, to explore the differential changes in the expression of genes. The results indicated that the mRNA transcription of sox2, p53, nestin and bax was generally downregulated after exposure to nicotinamide and TSA, but the difference was statistically significant only for sox2 and nestin. In addition, there was no transcription of sox1. Thus our studies demonstration that C17.2 NSCs have different mRNA expression profiles compared to E14.5 NSCs and adult mouse brain tissues. Furthermore, HDAC inhibitors nicotinamide and TSA regulate gene expression through different histone acetylation.

Isolation of Neural Stem Cells from Whole Brain Tissues of Adult Mice.

A population of neural stem cells exists in the adult mammalian central nervous system. Purification and characterization of neurospheres provide valuable tools to study the regulation and differentiation of neural stem cells both in vitro and in vivo. Successful stimulation and production of neurospheres can ultimately be used for therapeutic purposes. The currently available methods are limited by their poor yield and the large number of animals required to compensate for that. Here, we describe a procedure to purify neurospheres from adult mouse whole brain. We provide detailed steps on how to propagate, passage, and maintain the adult neurospheres, and how to differentiate the pure neurospheres into the lineage of interest. Using this method, neurospheres can be easily derived from adult mouse whole brain. The derived adult neurospheres maintain their homogenous undifferentiated status while retaining their differentiation potential. This new protocol facilitates adult neurospheres isolation, purification, maintenance, and differentiation. © 2019 by John Wiley & Sons, Inc.

Click histochemistry for whole-mount staining of brain structures

Labeling of the replicating DNA with synthetic thymidine analogs is commonly used for marking the dividing cells. However, until now this method has only been applied to histological sections. A growing number of current approaches for three-dimensional visualization of large tissue samples requires detection of dividing cells within whole organs. Here we describe a method for labeling dividing cells with 5-ethynyl-2'-deoxyuridine (EdU) and their further detection in whole brain structures (for example, hippocampus) using the Cu (I) -catalyzed [3 + 2] cycloaddition reaction (so-called click-reaction). The presented method can be used for brain neurogenesis studies as well as for whole-mount staining of any preparations in which the terminal ethynyl group has been introduced.•New click histochemistry method based on Cu (I) -catalyzed [3 + 2] cycloaddition reaction allows whole-mount staining of brain structures and other tissues.•Our whole-mount click histochemistry method allows to visualize dividing cells in 3D and can be used in neurogenesis studies, i.e. for birthdating dividing early progenitors and further tracking of proliferation, survival, migration, differentiation, and fate of their progeny.•Our whole-mount click histochemistry staining demonstrates high staining specificity, high signal intensity, and low background levels in young and adult mouse brain tissue.

A Simple Technique for Three-Dimensional Imaging and Segmentation of Brain Vasculature U sing Fast Free-of-Acrylamide Clearing Tissue in Murine.

Objective Fast Free-of-Acrylamide Clearing Tissue (FACT) is a recently developed protocol for the whole tissue three-dimensional (3D) imaging. The FACT protocol clears lipids using sodium dodecyl sulfate (SDS) to increase the penetration of light and reflection of fluorescent signals from the depth of cleared tissue. The aim of the present study was using FACT protocol in combination with imaging of auto-fluorescency of red blood cells in vessels to image the vasculature of a translucent mouse tissues. Materials and Methods In this experimental study, brain and other tissues of adult female mice or rats were dissected out without the perfusion. Mice brains were sliced for vasculature imaging before the clearing. Brain slices and other whole tissues of rodent were cleared by the FACT protocol and their clearing times were measured. After 1 mm of the brain slice clearing, the blood vessels containing auto-fluorescent red blood cells were imaged by a z-stack motorized epifluorescent microscope. The 3D structures of the brain vessels were reconstructed by Imaris software. Results Auto-fluorescent blood vessels were 3D imaged by the FACT in mouse brain cortex. Clearing tissues of mice and rats were carried out by the FACT on the brain slices, spinal cord, heart, lung, adrenal gland, pancreas, liver, esophagus, duodenum, jejunum, ileum, skeletal muscle, bladder, ovary, and uterus. ConclusionThe FACT protocol can be used for the murine whole tissue clearing. We highlighted that the 3D imaging of cortex vasculature can be done without antibody staining of non-perfused brain tissue, rather by a simple auto- fluorescence.

Isolation and Phenotyping of Adult Mouse Microglial Cells.

Microglia are the resident macrophages of the central nervous system parenchyma and fulfill crucial roles in brain development, homeostasis, and inflammation. The isolation of a pure microglia population from brain tissue enables the examination of microglial phenotypes without the interference of other cell populations. Microglial extractions from the neonatal brain have been described in various protocols, yet the more established and complex adult mouse brain poses a greater challenge. Here we describe a refined protocol including enzymatic and mechanical dissociation of adult mouse brain tissue and removal of myelin by Percoll density gradient. Microglial cells were subsequently extracted by an immunomagnetic approach. This isolation procedure enables the use of functionally viable cells for various applications such as cell culture, flow cytometry, functional assays including bacteria- or bead-based phagocytosis, stimulation assays, and transcriptome profiling techniques such as qRT-PCR and microarray/RNA sequencing.

Localization of PPAR isotypes in the adult mouse and human brain.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain.

Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue

Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV–GFP mice, aged 8–12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

Dynamic Readers for 5-(Hydroxy)Methylcytosine and Its Oxidized Derivatives

Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice

SUMMARYFetal alcohol spectrum disorders (FASDs) are characterized by life-long changes in gene expression, neurodevelopment and behavior. What mechanisms initiate and maintain these changes are not known, but current research suggests a role for alcohol-induced epigenetic changes. In this study we assessed alterations to adult mouse brain tissue by assaying DNA cytosine methylation and small noncoding RNA (ncRNA) expression, specifically the microRNA (miRNA) and small nucleolar RNA (snoRNA) subtypes. We found long-lasting alterations in DNA methylation as a result of fetal alcohol exposure, specifically in the imprinted regions of the genome harboring ncRNAs and sequences interacting with regulatory proteins. A large number of major nodes from the identified networks, such as Pten signaling, contained transcriptional repressor CTCF-binding sites in their promoters, illustrating the functional consequences of alcohol-induced changes to DNA methylation. Next, we assessed ncRNA expression using two independent array platforms and quantitative PCR. The results identified 34 genes that are targeted by the deregulated miRNAs. Of these, four (Pten, Nmnat1, Slitrk2 and Otx2) were viewed as being crucial in the context of FASDs given their roles in the brain. Furthermore, ∼20% of the altered ncRNAs mapped to three imprinted regions (Snrpn-Ube3a, Dlk1-Dio3 and Sfmbt2) that showed differential methylation and have been previously implicated in neurodevelopmental disorders. The findings of this study help to expand on the mechanisms behind the long-lasting changes in the brain transcriptome of FASD individuals. The observed changes could contribute to the initiation and maintenance of the long-lasting effect of alcohol.

Characterization of the Early CNS Stress Biomarkers and Profiles Associated with Neuropsychiatric Diseases

Neuropsychiatric disorders (including dementia) have high personal, family, and social costs. Although many neuropsychiatric disorders share common patterns of symptoms and treatments, there are no validated biomarkers that define the underlying molecular mechanisms in the central nervous system (CNS). We hypothesize that there are early and common molecular changes in the CNS that will serve as sensitive indicators of CNS molecular stress and that will be predictive of neuropathological changes resulted in increasing the risk for neuropsychiatric diseases. Using the rodent model, we showed that systemic exposure to three diverse CNS stressors with different mechanisms of action (ketamine, low-dose and high-dose ionizing radiation, interferon-α) induced the expression of troponin T1 (Tnnt 1) within hours in adult mouse brain tissue. Tnnt 1 expression was induced in neuronal (not glial) cells, the hippocampal zone of neurogenesis, cerebral cortex, amygdale, and choroid plexus, which are important CNS locations in behavior and mental health. We also identified nine neural signaling pathways that showed a high degree of concordance in their transcriptional response in mouse brain tissue for hours after low-dose irradiation, in the aging human brain (unirradiated), and in brain tissue from patients with Alzheimer’s disease. Our studies provide new molecular information on shared mechanisms and expression profiles of diverse neuropsychiatric disorders. This knowledge will be fundamental for developing molecular signatures of early CNS stress biomarker for early diagnosis and treatment of neuropsychiatric diseases.

Sublimation of New Matrix Candidates for High Spatial Resolution Imaging Mass Spectrometry of Lipids: Enhanced Information in Both Positive and Negative Polarities after 1,5-Diaminonapthalene Deposition

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

Enhanced Sensitivity for High Spatial Resolution Lipid Analysis by Negative Ion Mode Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry

We have achieved enhanced lipid imaging to a ~10 μm spatial resolution using negative ion mode matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry, sublimation of 2,5-dihydroxybenzoic acid as the MALDI matrix, and a sample preparation protocol that uses aqueous washes. We report on the effect of treating tissue sections by washing with volatile buffers at different pHs prior to negative ion mode lipid imaging. The results show that washing with ammonium formate, pH 6.4, or ammonium acetate, pH 6.7, significantly increases signal intensity and number of analytes recorded from adult mouse brain tissue sections. Major lipid species measured were glycerophosphoinositols, glycerophosphates, glycerolphosphoglycerols, glycerophosphoethanolamines, glycerophospho-serines, sulfatides, and gangliosides. Ion images from adult mouse brain sections that compare washed and unwashed sections are presented and show up to 5-fold increases in ion intensity for washed tissue. The sample preparation protocol has been found to be applicable across numerous organ types and significantly expands the number of lipid species detectable by imaging mass spectrometry at high spatial resolution.

Characterization of GD3 ganglioside as a novel biomarker of mouse neural stem cells

Neural stem cells (NSCs) are undifferentiated neural cells characterized by their high proliferative potential and the capacity for self-renewal with retention of multipotency. Over the past two decades, there has been a huge effort to identify NSCs morphologically, genetically, and molecular biologically. It is still controversial, however, what bona fide NSCs are. To define and characterize NSCs more systematically, it is crucial to explore novel cell-surface marker molecules of NSCs. In this study, we focused on GD3, a b-series ganglioside that is enriched in the immature brain and the subventricular zone (SVZ) of the postnatal and adult brain, and evaluated the usefulness of GD3 as a cell-surface biomarker for identifying NSCs. We demonstrated that GD3 was expressed in more than 80% of NSCs prepared from embryonic, postnatal, and adult mouse brain tissue by the neurosphere culture method. The percentage of GD3-expressing NSCs in neurospheres was nearly the same as it was in neurospheres derived from embryonic, postnatal, and adult brains but decreased drastically to about 40% after differentiation. GD3(+) cells isolated from embryonic mouse striata, postnatal, and adult mouse SVZs by fluorescence-activated cell sorting with an R24 anti-GD3 monoclonal antibody efficiently generated neurospheres compared with GD3(-) cells. These cells possessed multipotency to differentiate into neurons, astrocytes, and oligodendrocytes. These data indicate that GD3 is a unique and powerful cell-surface biomarker to identify and isolate NSCs.

Mapping and quantifying mammalian transcriptomes by RNA-Seq

We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41-52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3' untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 x 10(5) distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.

Repeated Restraint and Nerve Growth Factor Administration in Male and Female Mice: Effect on Sympathetic and Cardiovascular Mediators of the Stress Response

Chronic stress and increased sympathetic nerve activity have been associated with cardiovascular disorders such as hypertension, myocardial infarction and stroke. The aim of this study was to investigate the role of nerve growth factor (NGF) on the expression of tyrosine hydroxylase (TH), vascular-endothelial growth factor (VEGF) and leptin receptor (OB-R) in brain, adrenal and cardiovascular tissues of adult male and female mice following a chronic stress procedure. It was found that daily restraint for 10 consecutive days alters TH levels in hypothalamic and brainstem areas related to sympathetic activation, in both male and female mice. Chronic stress procedure also modifies heart and aorta VEGF levels in male mice, and adrenal glands TH in female mice. The NGF administration in stressed mice reverted the stress-induced up-regulation of TH levels in male and female mice hypothalamic nuclei and in male locus coeruleus. Administration of NGF in stressed animals also down-regulated OB-R levels in the hypothalamus of both male and female mice and in the female aorta. Our findings indicate that repeated restraint in mice has an effect on TH and VEGF protein content at different brain and peripheral sites involved in the sympathetic and cardio-vascular response to stressful stimuli. NGF administration is able to counteract some of these stress-induced changes. Since NGF is known to be up-regulated during stress, a possible functional significance of our observations is that the circulating NGF released during and following stress may serve to prevent possible deficits and/or damage linked to stress-induced sympathetic and cardiovascular activation.