Amelioration of hippocampal dysfunction by adipose tissue-targeted stem cell transplantation in a mouse model of type 2 diabetes.

There is growing evidence that type 2 diabetes or insulin resistance is linked to cognitive impairment. We recently confirmed altered lipid composition, down-regulation of insulin receptor expression and impaired basal synaptic transmission in the hippocampus of our transgenic murine model of adipocyte insulin resistance (AtENPP1-Tg). Here we evaluated whether the correction of adipose tissue dysfunction [via the subcutaneous transplantation of mesenchymal stem cells (MSC)] can improve the hippocampal synaptic transmission in AtENPP1-Tg mice versus their wildtype littermates. Animals were simply randomized to receive MSC, then weighed weekly for 12 weeks. At euthanasia, we assessed leptin in the collected serum and hippocampal synaptic high-frequency stimulation long-term potentiation (HFS-LTP) using brain slices. MSC transplantation normalized AtENPP1-Tg body and epididymal fat weights and was associated with increased leptin levels, a sign of adipocyte maturation. More importantly, transplantation restored the deficiency observed in AtENPP1-Tg HFS-LTP, the cellular readout of memory. Our results further corroborate the role of adipocyte maturation arrest in adipose tissue and highlight a role for the adipose tissue in modulating hippocampal cellular mechanisms. Further studies are warranted to explore the mechanisms for the MSC-induced improvement of hippocampal HFS-LTP.

Adult enteric nervous system in health is maintained by a dynamic balance between neuronal apoptosis and neurogenesis.

According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.

Substance P is essential for maintaining gut muscle contractility: a novel role for coneurotransmission revealed by botulinum toxin.

Substance P (SP) is commonly coexpressed with ACh in enteric motor neurons, and, according to the classical paradigm, both these neurotransmitters excite smooth muscle via parallel pathways. We hypothesized that, in addition, SP was responsible for maintaining the muscular responsiveness to ACh. We tested this hypothesis by using botulinum toxin (BoNT/A), a known blocker of vesicular release of neurotransmitters including ACh and neuropeptides. BoNT/A was injected into rat pyloric sphincter in different doses; as control we used boiled BoNT/A. At the desired time point, pylorus was dissected out and pyloric contractility was measured ex vivo in an organ bath and by measuring phosphorylation of myosin light chain 20 (MLC20). BoNT/A (10 IU) significantly reduced the response of pyloric muscle to exogenous ACh, an effect that was accompanied by reduced MLC20 phosphorylation in the muscle. Both effects were reversed by exogenous SP. CP-96345, a NK1 receptor antagonist, blocked the ability of exogenous SP to reverse the cholinergic hyporesponsiveness as well as the reduction in MLC20 phosphorylation induced by BoNT/A. In conclusion, we have identified a novel role for SP as a coneurotransmitter that appears to be important for the maintenance of muscular responsiveness to the principal excitatory neurotransmitter, ACh. These results also provide new insight into the effects of botulinum toxin on the enteric nervous system and gastrointestinal smooth muscle.

Divergent fate and origin of neurosphere-like bodies from different layers of the gut.

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.

Evaluation of ES-derived neural progenitors as a potential source for cell replacement therapy in the gut.

BACKGROUND: Stem cell-based therapy has recently been explored for the treatment of disorders of the enteric nervous system (ENS). Pluripotent embryonic stem (ES) cells represent an attractive cell source; however, little or no information is currently available on how ES cells will respond to the gut environment. In this study, we investigated the ability of ES cells to respond to environmental cues derived from the ENS and related tissues, both in vitro and in vivo. METHODS: Neurospheres were generated from mouse ES cells (ES-NS) and co-cultured with organotypic preparations of gut tissue consisting of the longitudinal muscle layers with the adherent myenteric plexus (LM-MP). RESULTS: LM-MP co-culture led to a significant increase in the expression of pan-neuronal markers (ßIII-tubulin, PGP 9.5) as well as more specialized markers (peripherin, nNOS) in ES-NS, both at the transcriptional and protein level. The increased expression was not associated with increased proliferation, thus confirming a true neurogenic effect. LM-MP preparations exerted also a myogenic effect on ES-NS, although to a lesser extent. After transplantation in vivo into the mouse pylorus, grafted ES-NS failed to acquire a distinct phenotype al least 1 week following transplantation. CONCLUSIONS: This is the first study reporting that the gut explants can induce neuronal differentiation of ES cells in vitro and induce the expression of nNOS, a key molecule in gastrointestinal motility regulation. The inability of ES-NS to adopt a neuronal phenotype after transplantation in the gastrointestinal tract is suggestive of the presence of local inhibitory influences that prevent ES-NS differentiation in vivo.

Gut-derived factors promote neurogenesis of CNS-neural stem cells and nudge their differentiation to an enteric-like neuronal phenotype.

Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker ß-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.

Non-Invasive Transcranial Nano-Pulsed Laser Therapy Ameliorates Cognitive Function and Prevents Aberrant Migration of Neural Progenitor Cells in the Hippocampus of Rats Subjected to Traumatic Brain Injury.

Traumatic brain injury (TBI) can lead to chronic diseases, including neurodegenerative disorders and epilepsy. The hippocampus, one of the most affected brain region after TBI, plays a critical role in learning and memory and is one of the only two regions in the brain in which new neurons are generated throughout life from neural stem cells (NSC) in the dentate gyrus (DG). These cells migrate into the granular layer where they integrate into the hippocampus circuitry. While increased proliferation of NSC in the hippocampus is known to occur shortly after injury, reduced neuronal maturation and aberrant migration of progenitor cells in the hilus contribute to cognitive and neurological dysfunctions, including epilepsy. Here, we tested the ability of a novel, proprietary non-invasive nano-pulsed laser therapy (NPLT), that combines near-infrared laser light (808 nm) and laser-generated, low-energy optoacoustic waves, to mitigate TBI-driven impairments in neurogenesis and cognitive function in the rat fluid percussion injury model. We show that injured rats treated with NPLT performed significantly better in a hippocampus-dependent cognitive test than did sham rats. In the DG, NPLT significantly decreased TBI-dependent impaired maturation and aberrant migration of neural progenitors, while preventing TBI-induced upregulation of specific microRNAs (miRNAs) in NSC. NPLT did not significantly reduce TBI-induced microglia activation in the hippocampus. Our data strongly suggest that NPLT has the potential to be an effective therapeutic tool for the treatment of TBI-induced cognitive dysfunction and dysregulation of neurogenesis, and point to modulation of miRNAs as a possible mechanism mediating its neuroprotective effects.

Endoscopic injection of skeletal muscle-derived cells augments gut smooth muscle sphincter function: implications for a novel therapeutic approach.

INTRODUCTION: Sphincter function is a common problem in gastroenterology and leads to disorders such as GERD and fecal incontinence. OBJECTIVE: We hypothesized that transplantation of skeletal muscle-derived cells (MDCs) into GI sphincters may improve their function, leading to a more physiological approach to treating these disorders. DESIGN: We performed experiments to test the potential of MDCs to survive and differentiate within the GI smooth muscle in order to gain further knowledge on the biology of skeletal muscle transplantation in GI smooth muscle sphincters as well as to test the safety and feasibility of endoscopic injection of MDCs in a large animal model. SETTING: Animal laboratory. INTERVENTIONS: Adult male Sprague-Dawley rats and adult male beagle dogs were used. Rat-derived and dog-derived MDCs were prepared in vitro and labeled with DiI. DiI-labeled, rat-derived MDCs (200,000/4 muL phosphate buffered saline solution) were injected bilaterally in the pyloric wall of rats, and survival, differentiation, and in vitro contractility were assessed 1 month after transplantation. Dog-derived MDCs (4.0 x 10(6) cells) were also injected into the lower esophageal sphincter of 3 beagle dogs by using a standard variceal sclerotherapy needle after baseline esophageal manometry and pH monitoring. The dogs were treated with daily cyclosporine, and 2 weeks later esophageal manometry was repeated and the esophagus was examined histologically. Differentiation of grafted cells was assessed by immunofluorescence, using specific antibodies to markers of the smooth muscle phenotype (smooth muscle actin) and of the skeletal muscle phenotype (skeletal muscle myosin). RESULTS: In rats, grafted MDCs were visualized based on DiI fluorescence and were found to be localized within the muscle wall and in the muscularis mucosa. In vitro organ bath studies showed a significant increase in the contractile response of the pyloric sphincter to exogenous acetylcholine. In dogs, MDC injection resulted in a significant increase in baseline lower esophageal sphincter pressure. Further, in 1 dog with significant baseline acid reflux, MDC injection resulted in a reduction of acid reflux, with the fraction of time with pH <4 decreasing from 26.5% to 1.5%. Transplanted MDCs were seen adding bulk to the lower esophageal area and were well-integrated into the surrounding tissue. Immunofluorescence analysis revealed weak expression of skeletal muscle myosin in grafted MDCs and no expression of smooth muscle actin in either rats or dogs. LIMITATIONS: Animal study. CONCLUSION: MDCs can survive and integrate into GI smooth muscle and augment their contractile response. Thus, they may have potential for the treatment of a variety of conditions.

The D2/D3 agonist PD128907 (R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol) inhibits stimulated pyloric relaxation and spontaneous gastric emptying.

BACKGROUND: Enteric neuronal dopamine (DA) inhibits acetylcholine release and gastric motility; this has been thought to be mediated via neuronal dopamine-2 receptor (D2R). The aim of this study was to investigate the modulation of gastric motility by the dopamine-3 receptor (D3R). METHODS: Adult Sprague-Dawley rats were used. Pyloric relaxation in response to electrical field stimulation (EFS) was assessed in an organ bath in the presence of varying concentrations of a selective D3R agonist, PD128907. Gastric emptying was assessed by the phenol red method after rats were treated with varying doses of PD128907 or DA with and without a selective D3R antagonist, L-nafadotride. RESULTS: Immunoblotting and immunohistochemistry confirmed the presence of D3R in the myenteric neurons in the rat pylorus. D3R activation reduced EFS-induced relaxation of pyloric strips in a dose-dependent manner and significantly delayed gastric emptying compared with vehicle. The D3R antagonist partially reversed the effect of DA on gastric emptying. CONCLUSIONS: Our data suggest a novel role for D3R in regulation of gastric motility. D3R activation delays gastric emptying, an effect that may be due to impairment of pyloric relaxation. D3R antagonists therefore hold promise as useful agents for treatment of gastric motility disorders.

Blood-Brain Barrier Dysfunction After Smoke Inhalation Injury, With and Without Skin Burn.

Only a handful of published reports exist today that describe neurological complications following smoke inhalation injury. In this study, we characterize acute pathophysiological changes in the brain of sheep exposed to smoke inhalation, with- and without third-degree skin burn that models the injuries sustained by human victims of fire accidents. Blood-brain barrier integrity and hemorrhage were analyzed throughout the brain using specific histological stains: Hematoxylin & Eosin, Luxol fast blue, Periodic acid-Schiff (PAS), and Martius, Scarlet and Blue (MSB). Our data show that, following smoke inhalation injury, alone and in combination with third-degree skin burn, there was a significant increase in the number of congested and dilated blood vessels in the frontal cortex, basal ganglia, amygdala, hippocampus, pons, cerebellum, and pituitary gland as compared to sham-injured controls. Positive PAS staining confirmed damage to the basement membrane of congested and dilated blood vessels throughout the brain. Severe rupturing of blood vessels, microvascular hemorrhaging and bleeding throughout the brain was also observed in the injured groups. No significant changes in hemodynamics and PaO2 were observed. Our data demonstrate for the first time that acute smoke inhalation alone results in diffuse blood-brain barrier dysfunction and massive bleeding in the brain in the absence of hypoxia and changes in hemodynamics. These findings provide critical information and prompt further mechanistic and interventional studies necessary to develop effective and novel treatments aimed at alleviating CNS dysfunction in patients with smoke and burn injuries.

MicroRNA profiling identifies a novel compound with antidepressant properties.

Patients with traumatic brain injury (TBI) are frequently diagnosed with depression. Together, these two leading causes of death and disability significantly contribute to the global burden of healthcare costs. However, there are no drug treatments for TBI and antidepressants are considered off-label for depression in patients with TBI. In molecular profiling studies of rat hippocampus after experimental TBI, we found that TBI altered the expression of a subset of small, non-coding, microRNAs (miRNAs). One known neuroprotective compound (17ß-estradiol, E2), and two experimental neuroprotective compounds (JM6 and PMI-006), reversed the effects of TBI on miRNAs. Subsequent in silico analyses revealed that the injury-altered miRNAs were predicted to regulate genes involved in depression. Thus, we hypothesized that drug-induced miRNA profiles can be used to identify compounds with antidepressant properties. To confirm this hypothesis, we examined miRNA expression in hippocampi of injured rats treated with one of three known antidepressants (imipramine, fluoxetine and sertraline). Bioinformatic analyses revealed that TBI, potentially via its effects on multiple regulatory miRNAs, dysregulated transcriptional networks involved in neuroplasticity, neurogenesis, and circadian rhythms- networks known to adversely affect mood, cognition and memory. As did E2, JM6, and PMI-006, all three antidepressants reversed the effects of TBI on multiple injury-altered miRNAs. Furthermore, JM6 reduced TBI-induced inflammation in the hippocampus and depression-like behavior in the forced swim test; these are both properties of classic antidepressant drugs. Our results support the hypothesis that miRNA expression signatures can identify neuroprotective and antidepressant properties of novel compounds and that there is substantial overlap between neuroprotection and antidepressant properties.

Hippocampal stem cells promotes synaptic resistance to the dysfunctional impact of amyloid beta oligomers via secreted exosomes.

BACKGROUND: Adult hippocampal neurogenesis plays an important role in synaptic plasticity and cogntive function. We reported that higher numbers of neural stem cells (NSC) in the hippocampus of cognitively-intact individuals with high Alzheimer's disease (AD) pathology (plaques and tangles) is associated with decreased synaptic amyloid beta oligomers (Aßο), an event linked to onset of dementia in AD. While these findings suggest a link between NSC and synaptic resistance to Aßο, the involved mechanism remains to be determined. With this goal in mind, here we investigated the ability of exosomes secreted from hippocampal NSC to promote synaptic resilience to Aßo. METHODS: Exosomes isolated from media of hippocampus NSC (NSC-exo) or mature hippocampal neuronal (MN-exo) cultures were delivered intracerebroventricularly (ICV) to mice before assessment of Aßο-induced suppression of hippocampal long-term potentiation (LTP) and memory deficits. Aßο binding to synapses was assessed in cultured hippocampal neurons and on synaptosomes isolated from hippocampal slices from wild type mice and from an inducible mouse model of NSC ablation (Nestin-δ-HSV-TK mice) treated with exosomes. Expression of CaMKII and of AMPA and NMDA glutamate receptor subunits in synaptosomes was measured by western blot. Small RNA Deep sequencing was performed to identify microRNAs enriched in NSC-exo as compared to MN-exo. Mimics of select miRNAs were injected ICV. RESULTS: NSC-exo, but not MN-exo, abolished Aßo-induced suppression of LTP and subsequent memory deficits. Furthermore, in hippocampal slices and cultured neurons, NSC-exo significantly decreased Aßo binding to the synapse. Similarly, transgenic ablation of endogenous NSC increased synaptic Aßo binding, which was reversed by exogenous NSC-exo. Phosphorylation of synaptic CaMKII was increased by NSC-exo, while AMPA and NMDA receptors were not affected. Lastly, we identified a set of miRNAs enriched in NSC-exo that, when injected ICV, protected the synapses from Aßo-binding and Aßo-induced LTP inhibition. CONCLUSIONS: These results identify a novel mechanism linking NSC-exo and synaptic susceptibility to Aßo that may underscore cognitive resilience of certain individuals with increased neurogenesis in spite of AD neuropathology and unmask a novel target for the development of a new treatment concept for AD centered on promoting synaptic resilience to toxic amyloid proteins.

Publisher Correction: Evidence linking microRNA suppression of essential prosurvival genes with hippocampal cell death after traumatic brain injury.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Nano-Pulsed Laser Therapy Is Neuroprotective in a Rat Model of Blast-Induced Neurotrauma.

We have developed a novel, non-invasive nano-pulsed laser therapy (NPLT) system that combines the benefits of near-infrared laser light (808 nm) and ultrasound (optoacoustic) waves, which are generated with each short laser pulse within the tissue. We tested NPLT in a rat model of blast-induced neurotrauma (BINT) to determine whether transcranial application of NPLT provides neuroprotective effects. The laser pulses were applied on the intact rat head 1 h after injury using a specially developed fiber-optic system. Vestibulomotor function was assessed on post-injury days (PIDs) 1-3 on the beam balance and beam walking tasks. Cognitive function was assessed on PIDs 6-10 using a working memory Morris water maze (MWM) test. BDNF and caspase-3 messenger RNA (mRNA) expression was measured by quantitative real-time PCR (qRT-PCR) in laser-captured cortical neurons. Microglia activation and neuronal injury were assessed in brain sections by immunofluorescence using specific antibodies against CD68 and active caspase-3, respectively. In the vestibulomotor and cognitive (MWM) tests, NPLT-treated animals performed significantly better than the untreated blast group and similarly to sham animals. NPLT upregulated mRNA encoding BDNF and downregulated the pro-apoptotic protein caspase-3 in cortical neurons. Immunofluorescence demonstrated that NPLT inhibited microglia activation and reduced the number of cortical neurons expressing activated caspase-3. NPLT also increased expression of BDNF in the hippocampus and the number of proliferating progenitor cells in the dentate gyrus. Our data demonstrate a neuroprotective effect of NPLT and prompt further studies aimed to develop NPLT as a therapeutic intervention after traumatic brain injury (TBI).

Evidence linking microRNA suppression of essential prosurvival genes with hippocampal cell death after traumatic brain injury.

The underlying molecular mechanisms of how dysregulated microRNAs (miRNAs) cause neurodegeneration after traumatic brain injury (TBI) remain elusive. Here we analyzed the biological roles of approximately 600 genes - we previously found these dysregulated in dying and surviving rat hippocampal neurons - that are targeted by ten TBI-altered miRNAs. Bioinformatic analysis suggests that neurodegeneration results from a global miRNA-mediated suppression of genes essential for maintaining proteostasis; many are hub genes - involved in RNA processing, cytoskeletal metabolism, intracellular trafficking, cell cycle progression, repair/maintenance, bioenergetics and cell-cell signaling - whose disrupted expression is linked to human disease. Notably, dysregulation of these essential genes would significantly impair synaptic function and functional brain connectivity. In surviving neurons, upregulated miRNA target genes are co-regulated members of prosurvival pathways associated with cellular regeneration, neural plasticity, and development. This study captures the diversity of miRNA-regulated genes that may be essential for cell repair and survival responses after TBI.

Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

Preserved neurogenesis in non-demented individuals with AD neuropathology.

Rare individuals remain cognitively intact despite the presence of neuropathology usually associated with fully symptomatic Alzheimer's disease (AD), which we refer to as Non-Demented with Alzheimer's disease Neuropathology (NDAN). Understanding the involved mechanism(s) of their cognitive resistance may reveal novel strategies to treat AD-related dementia. In the pursuit of this goal, we determined the number of hippocampal neural stem cells (NSCs) and investigated the expression of several miRNAs in NDAN and AD subjects. Laser-capture microdissection of autopsy human hippocampus DG and qRT-PCR miRNA analyses were combined with immunofluorescence in this study. The number of SOX2(+) NSCs in the DG was significantly increased in NDAN individuals as compared to AD subjects. Further, the prevalence of SOX2(+) NSCs was found to correlate with cognitive capacity. Neurogenesis-regulating miRNAs were decreased in NDAN individuals as compared to AD patients. An increased number of NSCs and new neurons in NDAN individuals is associated with a unique expression of regulating miRNAs and strongly support a role of neurogenesis in mediating, in part, the ability of these individuals to resist the pathological burden of AD.

Development of a novel imaging system for cell therapy in the brain.

INTRODUCTION: Stem cells have been evaluated as a potential therapeutic approach for several neurological disorders of the central and peripheral nervous system as well as for traumatic brain and spinal cord injury. Currently, the lack of a reliable and safe method to accurately and non-invasively locate the site of implantation and track the migration of stem cells in vivo hampers the development of stem cell therapy and its clinical application. In this report, we present data that demonstrate the feasibility of using the human sodium iodide symporter (hNIS) as a reporter gene for tracking neural stem cells (NSCs) after transplantation in the brain by using single-photon emission tomography/computed tomography (SPECT/CT) imaging. METHODS: NSCs were isolated from the hippocampus of adult rats (Hipp-NSCs) and transduced with a lentiviral vector containing the hNIS gene. Hipp-NSCs expressing the hNIS (NIS-Hipp-NSCs) were characterized in vitro and in vivo after transplantation in the rat brain and imaged by using technetium-99m ((99m)Tc) and a small rodent SPECT/CT apparatus. Comparisons were made between Hipp-NSCs and NIS-Hipp-NSCs, and statistical analysis was performed by using two-tailed Student's t test. RESULTS: Our results show that the expression of the hNIS allows the repeated visualization of NSCs in vivo in the brain by using SPECT/CT imaging and does not affect the ability of Hipp-NSCs to generate neuronal and glial cells in vitro and in vivo. CONCLUSIONS: These data support the use of the hNIS as a reporter gene for non-invasive imaging of NSCs in the brain. The repeated, non-invasive tracking of implanted cells will accelerate the development of effective stem cell therapies for traumatic brain injury and other types of central nervous system injury.

Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.

Cognitive deficits in survivors of traumatic brain injury (TBI) are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays) to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive) or surviving (Fluoro-Jade-negative) pyramidal neurons obtained by laser capture microdissection (LCM). In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER) stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.

Molecular cloning of the rat proteinase-activated receptor 4 (PAR4).

BACKGROUND: The proteinase-activated receptor 4 (PAR4) is a G-protein-coupled receptor activated by proteases such as thrombin and trypsin. Although activation of PAR4 has been shown to modulate rat gastrointestinal motility, the rat PAR4 sequence was unknown until now. This study aimed to identify the rat PAR4 cDNA. RESULTS: The cDNA coding for the rat PAR4 homologue was cloned from the duodenum. Northern blots demonstrated a 3.0 kb transcript in the duodenum. Protein homology with mouse and human counterparts was 90% and 75% respectively. PAR4 is expressed predominantly in the esophagus, stomach, duodenum and the spleen. When expressed in COS cells, PAR4 is activated by trypsin (1 nM), thrombin (50 nM), mouse PAR4 specific peptide (500 microM) and a putative rat PAR4 specific activating peptide (100 microM), as measured by intracellular Ca2+-changes. CONCLUSIONS: We have identified and characterized cDNA encoding the rat PAR4 homologue. PAR4 is expressed predominantly in the upper gastrointestinal tract. It is activated by trypsin, thrombin and its newly identified rat PAR4 specific activating peptide.