Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

Background

The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues.

Methodology/Principal Findings

Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180–250 µm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing.

Conclusions/Significance

Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins.     

The potential role of metalloproteinases in neurogenesis in the gerbil hippocampus following global forebrain ischemia

Background

Matrix metalloproteinases (MMPs) have recently been considered to be involved in the neurogenic response of adult neural stem/progenitor cells. However, there is a lack of information showing direct association between the activation of MMPs and the development of neuronal progenitor cells involving proliferation and/or further differentiation in vulnerable (Cornus Ammoni-CA1) and resistant (dentate gyrus-DG) to ischemic injury areas of the brain hippocampus.

Principal Findings

We showed that dynamics of MMPs activation in the dentate gyrus correlated closely with the rate of proliferation and differentiation of progenitor cells into mature neurons. In contrast, in the damaged CA1 pyramidal cells layer, despite the fact that some proliferating cells exhibited antigen specific characteristic of newborn neuronal cells, these did not attain maturity. This coincides with the low, near control-level, activity of MMPs. The above results are supported by our in vitro study showing that MMP inhibitors interfered with both the proliferation and differentiation of the human neural stem cell line derived from umbilical cord blood (HUCB-NSCs) toward the neuronal lineage.

Conclusion

Taken together, the spatial and temporal profiles of MMPs activity suggest that these proteinases could be an important component in neurogenesis-associated processes in post-ischemic brain hippocampus.     

Neuronal deletion of caspase 8 protects against brain injury in mouse models of controlled cortical impact and kainic acid-induced excitotoxicity

Background

Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8 ^−/−), in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system.

Methodology/Principal Findings

Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml) or TRAIL (250 ng/mL) plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI) model of traumatic brain injury (TBI) and seizure-induced brain injury caused by kainic acid (KA). Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8 ^−/− mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.

Conclusions

Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma.     

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro

Background

Riboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.

Results

Results indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.

Conclusion

Thus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.     

Brain deletion of insulin receptor substrate 2 disrupts hippocampal synaptic plasticity and metaplasticity

Objective

Diabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits and the corresponding neurophysiological structural and functional alterations are linked to both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. To elucidate the specific role of brain insulin signalling in neuronal functions that are relevant for cognitive processes we have investigated the behaviour of neurons and synaptic plasticity in the hippocampus of mice lacking the insulin receptor substrate protein 2 (IRS-2).

Research Design and Methods

To study neuronal function and synaptic plasticity in the absence of confounding factors such as hyperglycaemia, we used a mouse model with a central nervous system- (CNS)-restricted deletion of IRS-2 (NesCreIrs2KO).

Results

We report a deficit in NMDA receptor-dependent synaptic plasticity in the hippocampus of NesCreIrs2KO mice, with a concomitant loss of metaplasticity, the modulation of synaptic plasticity by the previous activity of a synapse. These plasticity changes are associated with reduced basal phosphorylation of the NMDA receptor subunit NR1 and of downstream targets of the PI3K pathway, the protein kinases Akt and GSK-3β.

Conclusions

These findings reveal molecular and cellular mechanisms that might underlie cognitive deficits linked to specific defects of neuronal insulin signalling.     

Change in Brain Magnetic Resonance Spectroscopy after Treatment during Acute HIV Infection

Objective

Single voxel proton magnetic resonance spectroscopy (MRS) can be used to monitor changes in brain inflammation and neuronal integrity associated with HIV infection and its treatments. We used MRS to measure brain changes during the first weeks following HIV infection and in response to antiretroviral therapy (ART).

Methods

Brain metabolite levels of N-acetyl aspartate (NAA), choline (tCHO), creatine (CR), myoinositol (MI), and glutamate and glutamine (GLX) were measured in acute HIV subjects (n = 31) and compared to chronic HIV+individuals (n = 26) and HIV negative control subjects (n = 10) from Bangkok, Thailand. Metabolites were measured in frontal gray matter (FGM), frontal white matter (FWM), occipital gray matter (OGM), and basal ganglia (BG). Repeat measures were obtained in 17 acute subjects 1, 3 and 6 months following initiation of ART.

Results

After adjustment for age we identified elevated BG tCHO/CR in acute HIV cases at baseline (median 14 days after HIV infection) compared to control (p = 0.0014), as well as chronic subjects (p = 0.0023). A similar tCHO/CR elevation was noted in OGM; no other metabolite abnormalities were seen between acute and control subjects. Mixed longitudinal models revealed resolution of BG tCHO/CR elevation after ART (p = 0.022) with tCHO/CR similar to control subjects at 6 months.

Interpretation

We detected cellular inflammation in the absence of measurable neuronal injury within the first month of HIV infection, and normalization of this inflammation following acutely administered ART. Our findings suggest that early ART may be neuroprotective in HIV infection by mitigating processes leading to CNS injury.     

HIV DNA Reservoir Increases Risk for Cognitive Disorders in cART-Na?ve Patients

Objectives

Cognitive impairment remains frequent in HIV, despite combination antiretroviral therapy (cART). Leading theories implicate peripheral monocyte HIV DNA reservoirs as a mechanism for spread of the virus to the brain. These reservoirs remain present despite cART. The objective of this study was to determine if the level of HIV DNA in CD14⁺ enriched monocytes predicted cognitive impairment and brain injury.

Methods

We enrolled 61 cART-naïve HIV-infected Thais in a prospective study and measured HIV DNA in CD14⁺ enriched monocyte samples in a blinded fashion. We determined HAND diagnoses by consensus panel and all participants underwent magnetic resonance spectroscopy (MRS) to measure markers of brain injury. Immune activation was measured via cytokines in cerebrospinal fluid (CSF).

Results

The mean (SD) age was 35 (6.9) years, CD4 T-lymphocyte count was 236 (139) and log₁₀ plasma HIV RNA was 4.8 (0.73). Twenty-eight of 61 met HAND criteria. The log₁₀ CD14⁺ HIV DNA was associated with HAND in unadjusted and adjusted models (p = 0.001). There was a 14.5 increased odds ratio for HAND per 1 log-value of HIV DNA (10-fold increase in copy number). Plasma CD14⁺ HIV DNA was associated with plasma and CSF neopterin (p = 0.023) and with MRS markers of neuronal injury (lower N-acetyl aspartate) and glial dysfunction (higher myoinositol) in multiple brain regions.

Interpretation

Reservoir burden of HIV DNA in monocyte-enriched (CD14⁺) peripheral blood cells increases risk for HAND in treatment-naïve HIV+ subjects and is directly associated with CSF immune activation and both brain injury and glial dysfunction by MRS.     

Cloned Myogenic Cells Can Transdifferentiate In Vivo into Neuron-Like Cells

Background

The question of whether intact somatic cells committed to a specific differentiation fate, can be reprogrammed in vivo by exposing them to a different host microenvironment is a matter of controversy. Many reports on transdifferentiation could be explained by fusion with host cells or reflect intrinsic heterogeneity of the donor cell population.

Methodology/Principal Findings

We have tested the capacity of cloned populations of mouse and human muscle progenitor cells, committed to the myogenic pathway, to transdifferentiate to neurons, following their inoculation into the developing brain of newborn mice. Both cell types migrated into various brain regions, and a fraction of them gained a neuronal morphology and expressed neuronal or glial markers. Likewise, inoculated cloned human myogenic cells expressed a human specific neurofilament protein. Brain injected donor cells that expressed a YFP transgene controlled by a neuronal specific promoter, were isolated by FACS. The isolated cells had a wild-type diploid DNA content.

Conclusions

These and other results indicate a genuine transdifferentiation phenomenon induced by the host brain microenvironment and not by fusion with host cells. The results may potentially be relevant to the prospect of autologous cell therapy approach for CNS diseases.     

Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain

Background

Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain.

Methodology/Principal Findings

We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.

Conclusions

MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.     

Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

Background

Traumatic brain injury (TBI) induces a complex sequence of apopototic cascades that contribute to secondary tissue damage. The aim of this study was to investigate the effects of salidroside, a phenolic glycoside with potent anti-apoptotic properties, on behavioral and histological outcomes, brain edema, and apoptosis following experimental TBI and the possible involvement of the phosphoinositide 3-kinase/protein kinase B (PI3K)/Akt signaling pathway.

Methodology/Principal Findings

Mice subjected to controlled cortical impact injury received intraperitoneal salidroside (20, or 50 mg/kg) or vehicle injection 10 min after injury. Behavioral studies, histology analysis and brain water content assessment were performed. Levels of PI3K/Akt signaling-related molecules, apoptosis-related proteins, cytochrome C (CytoC), and Smac/DIABLO were also analyzed. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, was administered to examine the mechanism of protection. The protective effect of salidroside was also investigated in primary cultured neurons subjected to stretch injury. Treatment with 20 mg/kg salidroside_significantly improved functional recovery and reduced brain tissue damage up to post-injury day 28. Salidroside_also significantly reduced neuronal death, apoptosis, and brain edema at day 1. These changes were associated with significant decreases in cleaved caspase-3, CytoC, and Smac/DIABLO at days 1 and 3. Salidroside increased phosphorylation of Akt on Ser473 and the mitochondrial Bcl-2/Bax ratio at day 1, and enhanced phosphorylation of Akt on Thr308 at day 3. This beneficial effect was abolished by pre-injection of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, delayed administration of salidroside at 3 or 6 h post-injury reduced neuronal damage at day 1. Salidroside treatment also decreased neuronal vulnerability to stretch-induced injury in vitro.

Conclusions/Significance

Post-injury salidroside improved long-term behavioral and histological outcomes and reduced brain edema and apoptosis following TBI, at least partially via the PI3K/Akt signaling pathway.     

Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research

Background

Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert.

Methodology/Principal Findings

Here, we show that thy1-driven expression of yellow fluorescent protein (YFP) in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury.

Conclusions/Significance

We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.     

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice

Background

Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.

Methods

In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.

Results

Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.

Conclusion

In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.     

Protective effect of a sesamin derivative, 3-bis (3-methoxybenzyl) butane-1, 4-diol on ischemic and hypoxic neuronal injury

Background

Stroke is one of the leading causes of neuronal death. Sesamin is known for neuroprotection by its antioxidant and anti-inflammatory properties but it lacks blood–brain barrier (BBB) activity. A panel of sesamin derivatives was screened and 3-bis (3-methoxybenzyl) butane-1,4-diol (BBD) was selected for high BBB activity and tested for its neuroprotective effect.

Methods

The focal cerebral ischemia of Sprague–Dawley rats and hypoxia models of murine BV-2 microglia or PC12 cells under oxygen/glucose deprivation were used for in vivo and in vitro test, respectively. Lipid peroxidation and superoxide dismutase (SOD) activity from the ischemic brain were tested and reactive oxygen species (ROS), cytokine production, prostaglandin (PGE₂) and related signaling pathways from hypoxic cells were examined by ELISA or Western blot assay, respectively.

Results

BBD showed a protective effect when given 90 min after the focal cerebral ischemia. It also reduced lipid peroxidation and preserved SOD activity from the ischemic brain. The mechanism of BBD was further confirmed by attenuating ROS, cytokine production, and PGE₂ release from hypoxic BV-2 or PC12 cells. BBD significantly reduced hypoxia-induced c-Jun N-terminal kinases (JNK) and modulated AKT-1 and caspase-3 (survival and apoptotic pathways) in BV-2 cells, and inhibited hypoxia-induced JNK and cyclooxygenase-2 activation in PC12 cells.

Conclusions

The neuroprotective effect of BBD on ischemia/hypoxia models was involved with antioxidant and anti-inflammatory effects. The result would help the development of new CNS drug for protection of ischemia/hypoxia injury.     

A novel xenogeneic co-culture system to examine neuronal differentiation capability of various adult human stem cells

Background

Targeted differentiation of stem cells is mainly achieved by the sequential administration of defined growth factors and cytokines, although these approaches are quite artificial, cost-intensive and time-consuming. We now present a simple xenogeneic rat brain co-culture system which supports neuronal differentiation of adult human stem cells under more in vivo-like conditions.

Methods and Findings

This system was applied to well-characterized stem cell populations isolated from human skin, parotid gland and pancreas. In addition to general multi-lineage differentiation potential, these cells tend to differentiate spontaneously into neuronal cell types in vitro and are thus ideal candidates for the introduced co-culture system. Consequently, after two days of co-culture up to 12% of the cells showed neuronal morphology and expressed corresponding markers on the mRNA and protein level. Additionally, growth factors with the ability to induce neuronal differentiation in stem cells could be found in the media supernatants of the co-cultures.

Conclusions

The co-culture system described here is suitable for testing neuronal differentiation capability of numerous types of stem cells. Especially in the case of human cells, it may be of clinical relevance for future cell-based therapeutic applications.     

Minocycline inhibition of monocyte activation correlates with neuronal protection in SIV neuroAIDS

Background

Minocycline is a tetracycline antibiotic that has been proposed as a potential conjunctive therapy for HIV-1 associated cognitive disorders. Precise mechanism(s) of minocycline''s functions are not well defined.

Methods

Fourteen rhesus macaques were SIV infected and neuronal metabolites measured by proton magnetic resonance spectroscopy (¹H MRS). Seven received minocycline (4 mg/kg) daily starting at day 28 post-infection (pi). Monocyte expansion and activation were assessed by flow cytometry, cell traffic to lymph nodes, CD16 regulation, viral replication, and cytokine production were studied.

Results

Minocycline treatment decreased plasma virus and pro-inflammatory CD14+CD16+ and CD14^loCD16+ monocytes, and reduced their expression of CD11b, CD163, CD64, CCR2 and HLA-DR. There was reduced recruitment of monocyte/macrophages and productively infected cells in axillary lymph nodes. There was an inverse correlation between brain NAA/Cr (neuronal injury) and circulating CD14+CD16+ and CD14^loCD16+ monocytes. Minocycline treatment in vitro reduced SIV replication CD16 expression on activated CD14+CD16+ monocytes, and IL-6 production by monocytes following LPS stimulation.

Conclusion

Neuroprotective effects of minocycline are due in part to reduction of activated monocytes, monocyte traffic. Mechanisms for these effects include CD16 regulation, reduced viral replication, and inhibited immune activation.     

Spontaneous Recovery of the Injured Higher Olfactory Center in the Terrestrial Slug Limax

Background

Of all organs and tissues in adult mammals, the brain shows the most limited regeneration and recovery after injury. This is one reason why treating neurological damage such as ischemic injury after stroke presents such a challenge. Here we report a novel mode of regeneration which the slug''s cognitive center, the procerebrum, shows after surgical lesioning in the adult. It is well known that the land slug Limax possesses the capacity to demonstrate conditioned food aversion. This learning ability critically depends on the procerebrum, which is the higher olfactory center in the brain of the terrestrial mollusk.

Principal Findings

In the present study, after a 1-month recovery period post-surgical lesioning of the procerebrum we investigated whether the brain of the slug shows recovery from damage. We found that learning ability, local field potential oscillation, and the number of cells in the procerebrum (PC) all recovered spontaneously within 1 month of bilateral lesioning of the PC. Moreover, neurogenesis was enhanced in the lesioned PC. However, memory acquired before the surgery could not be retrieved 1 month after surgery although the procerebrum had recovered from injury by this time, consistent with the notion that the procerebrum is the storage site of odor-aversion memory, or deeply involved in the memory recall process.

Significance

Our findings are the first to demonstrate that a brain region responsible for the associative memory of an adult organism can spontaneously reconstitute itself, and can recover its function following injury.