Biomedical subjects

Alexander K Murashov

Publications and source records attributed to Alexander K Murashov.

13 recordsLinked to original sources

PubMed2007

RNAi pathway is functional in peripheral nerve axons.

Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase-components of the RNA-induced silencing complex (RISC). Application of short-interfering RNAs against neuronal beta-tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal beta-tubulin III mRNA and corresponding protein, as well as a significant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is functional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.

Animals↗

PubMed2006

Administration of raloxifene reduces sensorimotor and working memory deficits following traumatic brain injury.

Hormonal differences between males and females have surfaced as a crucial component in the search for effective treatments after experimental models of traumatic brain injury (TBI). Recent findings have shown that selective estrogen receptor modulators (SERMs) may have therapeutic benefit. The present study examined the effects of raloxifene, a SERM, on functional recovery after bilateral cortical contusion injury (bCCI) or sham procedure. Male rats received injections of raloxifene (3.0mg/kg, i.p.) or vehicle (1.0 ml/kg, i.p.) 15 min, 24, 48, 72, and 96 h after bCCI or sham procedure. Rats were tested on both sensorimotor (bilateral tactile removal and locomotor placing tests) and cognitive tests (reference and working memory in the Morris water maze). Raloxifene-treated animals showed a significant reduction in the initial magnitude of the deficit and facilitated the rate of recovery for the bilateral tactile removal test, compared to vehicle-treated animals. The raloxifene-treated animals also showed a significant improvement in the acquisition of working memory compared to vehicle-treated animals. However, raloxifene did not significantly improve the acquisition of reference memory or locomotor placing ability. Raloxifene treatment also did not result in a significant reduction in the size of the lesion cavity. Thus, the task-dependent improvements seen following raloxifene treatment do not appear to be the result of cortical neuroprotection. However, these results suggest that raloxifene improves functional outcome following bCCI and may present an interesting avenue for future research.

Analysis of Variance↗

PubMed2005

Parallel development of cardiomyocytes and neurons in embryonic stem cell culture.

Recent studies suggest that there are strong parallels between development and patterning of the vertebrate vascular system and the nervous system. While previous observations reported generation of vascular and neuronal progenitors from embryonic stem (ES) cells, the question of parallel development of vascular and neuronal cells in the same culture has not yet been investigated. Mouse D3 ES cells were cultured for 4 days in differentiation medium IMDM with 15% FBS in 100 mm non-adhesive Petri dishes to allow cells to aggregate and form embryoid bodies. At day 5, fibronectin or all-trans retinoic acid with fibronectin was added to the culture. On day 9, the embryoid bodies were seeded on poly-L-ornithine/fibronectin-coated plates. After plating, half of the plates were treated with laminin for 3 days and maintained for 1 week in Neurobasal media with B27. Here we show that ES cells differentiate into interconnected rhythmically contracting aggregates of functional cardiomyocytes and neurons. Double immunofluorescence with anti-phospholamban, anti-SERCA2 antibodies to detect cardiomyocytes and with anti-MAP2 antibodies to detect neurons revealed the cell aggregates consisting entirely of cardiomyocytes with neuronal cells located on the periphery or covering the aggregate's surface. The observed concurrent development of cardiomyocytes and neurons suggests bidirectional communication between both cell types. We propose that crosstalk between cardiovascular and neuronal progenitors is an important mechanism for the development of both systems.

Animals↗

PubMed2004

Directed differentiation of embryonic stem cells into dorsal interneurons.

During neural development caudalization and dorsoventral patterning of the neural tube is directed by several inductive factors including retinoic acid, sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnt signaling. The purpose of the current study was to investigate whether dorsal interneurons specific for the spinal cord can be generated from mouse embryonic stem (ES) cells using known inductive signals. Here we show that specific combination of developmental signaling molecules including all trans-retinoic acid, Shh, bone morphogenetic protein 2 (BMP2), and Wnt3A can direct differentiation of ES cells into dorsal interneurons possessing appropriate neuronal markers, synaptic proteins and functional neurotransmitter machineries. We introduce a concept that Wnt3A morphogenic action relies on crosstalk with both Shh and BMP2 signaling pathways.

Animals↗

PubMed2004

Induction of VEGF and its Flt-1 receptor after sciatic nerve crush injury.

Expression of vascular endothelial growth factor and its receptors Flt-1 and Flk-1 was studied in lumbar spinal cord after sciatic nerve crush injury. Immunohistochemical staining revealed strikingly different distribution of VEGF, Flt-1, and Flk-1 in lumbar motor neurons. VEGF was observed both in the nuclei and perikarya, while Flk-1 had cytoplasmic and Flt-1 perinuclear localization. Real-time RT-PCR showed a significant increase in the expression of VEGF and Flt-1 on the injured side of the lumbar spinal cord. The increased level of VEGF was also detected by immunoblot. Here we show that lumbar motor neurons increase the expression of VEGF and Flt-1 in response to injury. We propose that VEGF/Flt-1 signaling may be involved in regeneration of the spinal motor neurons.

Animals↗

PubMed2004

17beta-Estradiol enhances neuronal differentiation of mouse embryonic stem cells.

Existing protocols show a variety in the percentage of neurons that can be generated from mouse embryonic stem (ES) cells. In the current study, we compared effects of various differentiating conditions, including gelatin and poly-l-ornithine/fibronectin coatings, and NGF and 17beta-estradiol treatments on the total yield of neurons, as well as, neurite growth and branching. Here, we show that combination of fibronectin coating with 17beta-estradiol increased number of generated neurons over 50%. Poly-l-ornithine/fibronectin increased the percent of neurons in all cultures, suggesting its direct influence on neurogenesis. Addition of 17beta-estradiol reduced mean neurite length in culture, but significantly increased branching. Our results indicate a substrate-dependent regulation of estrogen-induced ES cells differentiation into neuronal cells.

Animals↗

PubMed2004

Estrogen increases retrograde labeling of motoneurons: evidence of a nongenomic mechanism.

Estrogen has a variety of neurotrophic effects mediated via different signaling cascades, including ERK and phosphatidylinositol 3-kinase (PI3K) pathways. In this study, we investigated effects of estrogen and inhibitors for ERK and PI3K applied directly onto the cut sciatic nerve on retrograde labeling of lumbar motoneurons. A mix of retrograde tracer (Fluorogold) and 17beta-estradiol, in combination with an antagonist for estrogen receptors ICI 182,780, an inhibitor of ERK1/2 pathway (U0126), an inhibitor of PI3K (LY-294002), or a protein synthesis inhibitor (cycloheximide), was applied to the proximal stump of the transected sciatic nerve for 24 h. Coapplication of Fluorogold with 17beta-estradiol produced a significant increase in the number of retrograde-labeled lumbar motoneurons, compared with Fluorogold alone. Estrogen potentiation of retrograde labeling was inhibited by application of ICI 182,780, U0126, LY-294002, and cycloheximide. Immunohistochemical analysis of the sciatic nerve, 24 h following crush injury, revealed accumulation of phospho-ERK in regenerating nerve fibers. The data suggest a role for estrogen, ERK, PI3K, and protein synthesis in the uptake and retrograde transport of Fluorogold. We propose that estrogen action in peripheral nerve fibers is mediated via the ERK and PI3K signaling pathways and is reliant on local protein synthesis.

Animals↗

PubMed2004

Transplantation of neuronal and glial precursors dramatically improves sensorimotor function but not cognitive function in the traumatically injured brain.

Embryonic stem (ES) cells have been investigated in various animal models of neurodegenerative disease; however, few studies have examined the ability of ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to examine the ability of pre-differentiated murine ES cells (neuronal and glial precursors) to improve functional outcome. Rats were prepared with a unilateral controlled cortical impact injury or sham and then transplanted 7 days later with 100K ES cells (WW6G) (~30% neurons) or media. Two days following transplantation rats were tested on a battery of behavioral tests. It was found that transplantation of ES cells improved behavioral outcome by reducing the initial magnitude of the deficit on the bilateral tactile removal and locomotor placing tests. ES cells also induced almost complete recovery on the vibrissae --> forelimb placing test, whereas, media-transplanted rats failed to show recovery. Acquisition of a reference memory task in the Morris water maze was not improved by transplantation of ES cells. Histological analysis revealed a large number of surviving ES cells in the lesion cavity and showed migration of ES cells into subcortical structures. It was found that transplantation of ES cells prevented the occurrence of multiple small necrotic cavities that were seen in the cortex adjacent to the lesion cavity in media transplanted rats. Additionally, ES cells transplants also significantly reduced lesion size. Results of this study suggest that ES cells that have been pre-differentiated into neuronal precursors prior to transplantation have therapeutic potential.

Animals↗

PubMed2003

Differential expression of endothelin receptors in regenerating spinal motor neurons in mice.

On day 4 after sciatic nerve crush injury, expression and localization of endothelin receptors ET(A) and ET(B) in the lumbar spinal cord were examined. Immunohistochemical staining with antibodies to ET(A) and ET(B) receptors showed cytoplasmic distribution of ET(A) receptors in motor neurons, whereas ET(B) receptors were localized in the perinuclear region. On the injured side of the lumbar spinal cord, when compared to contralateral, results demonstrated an up-regulation of ET(B) and a down-regulation of ET(A) receptors expression at the level of both mRNA and protein. These results suggest that ET(B) receptors may play a role in the regeneration of axotomized motor neurons.

Animals↗

PubMed2003

Effect of 17 beta-estradiol on gene expression in lumbar spinal cord following sciatic nerve crush injury in ovariectomized mice.

Previously, we observed that estrogen treatment enhances regeneration of the sciatic nerve after crush injury [Brain Res. 943 (2002) 283]. In this research, we studied expression of estrogen receptors and effects of estrogen on gene expression in the lumbar spinal cord, following sciatic nerve crush injury. Using the Atlas Mouse 1.2 Array, changes in the expression of 267 of 1176 genes were registered 4 days after nerve injury. Those genes that exhibited a change in signal intensity ratios of 2-fold or greater were selected as up-regulated (42) or down-regulated (21). In estrogen treated mice, we have observed up-regulation of the genes known to control apoptosis, cell proliferation, and growth, which might account for the positive effects of estrogen on the regeneration of motor neurons. Immunohistochemical staining revealed estrogen receptor-alpha and estrogen receptor-beta localized in the nucleus and cytoplasm of lumbar motor neurons, and in the regenerating neurites of the sciatic nerve. Expression of estrogen receptor-alpha and estrogen receptor-beta mRNA in lumbar spinal cord was shown by traditional RT-PCR. Using real-time quantitative RT-PCR, we demonstrated increased expression of estrogen receptors-alpha and -beta mRNA on the injured side of the lumbar spinal cord. Western blot analysis showed the accumulation of ERs in regenerating sciatic nerve, and revealed a 40% increase of activated ERK1/2 in estrogen treated mice, compared to placebo. Our findings indicate that: (i). axotomized motor neurons increase expression of estrogen receptors-alpha and -beta mRNA, (ii). estrogen mediates the expression of genes which accelerate the growth and maturation of axons, and (iii). estrogen receptors are transported from the perikaryon into regenerating neurites, and estrogen promotes regeneration locally through the non-genomic ERK-activated signaling pathway.

Animals↗

PubMed2003

Development of the mouse vestibular system in the absence of gravity perception.

The tilted mutant mouse, which lacks otoconia in the inner ear, was used to study development of the mouse vestibular system in the absence of gravity perception. Otoconia are dense particles composed of proteins and calcium carbonate crystals suspended in the gelatinous macular membrane. They enhance, and are largely responsible for, sensitivity to gravity. Morphometric analysis of the vestibular ganglion showed that the mutant developed more slowly than the normal controls, both in rate of development and cell number, particularly during the first week of post-natal development. The mutant ganglia also exhibited a reduction of cells during the first 6 days of post-natal development.

Animals↗

PubMed2002

17Beta-estradiol stimulates regeneration of sciatic nerve in female mice.

In ovariectomized mice with and without estrogen replacement, regeneration of the sciatic nerve after crush injury was studied. Functional recovery, quantified with sciatic functional index was significantly accelerated in estrogen-treated mice throughout the regeneration. On semi-thin sections of sciatic nerves in estrogen-treated mice we registered a greater total number of regenerating nerve fibers at the first week, and a higher mean axonal area at the third week of regeneration. Our results demonstrated that estrogen treatment enhances regeneration of the sciatic nerve.

Animals↗

PubMedDate not supplied

Predifferentiated embryonic stem cells prevent chronic pain behaviors and restore sensory function following spinal cord injury in mice.

Embryonic stem (ES) cells have been investigated in repair of the CNS following neuronal injury and disease; however, the efficacy of these cells in treatment of postinjury pain is far from clear. In this study, we evaluated the therapeutic potential of predifferentiated mouse ES cells to restore sensory deficits following spinal cord injury (SCI) in mice. The pain model used unilateral intraspinal injection of quisqualic acid (QUIS) into the dorsal horn between vertebral levels T13 and L1. Seven days later, 60,000 predifferentiated ES cells or media were transplanted into the site of the lesion. Histological analysis at 7, 14, and 60 days post-transplantation revealed that animals receiving ES cell transplants suffered significantly less tissue damage than animals receiving media alone. Transplanted cells provided immediate effects on both spontaneous and evoked pain behaviors. Treatment with ES cells resulted in 0% (n = 28) excessive grooming behavior versus 60% (18 of 30) in media-treated animals. In the acetone test (to assess thermal allodynia), mice recovered to preinjury levels by 12 days after ES cell transplant, whereas control animals injected with media after SCI did not show any improvement up to 60 days. Similarly, the von Frey test (to assess mechanical allodynia) and the formalin test (to assess nociceptive hyperalgesia) showed that transplantation of predifferentiated ES cells significantly reduced these pain behaviors following injury. Here we show that predifferentiated ES cells act in a neuroprotective manner and provide antinociceptive and therapeutic effects following excitotoxic SCI.

Animals↗