A Biosafety Study of Human Umbilical Cord Blood Mononuclear Cells Transduced with Adenoviral Vector Carrying Human Vascular Endothelial Growth Factor cDNA In Vitro.

The biosafety of gene therapy remains a crucial issue for both the direct and cell-mediated delivery of recombinant cDNA encoding biologically active molecules for the pathogenetic correction of congenital or acquired disorders. The diversity of vector systems and cell carriers for the delivery of therapeutic genes revealed the difficulty of developing and implementing a safe and effective drug containing artificial genetic material for the treatment of human diseases in practical medicine. Therefore, in this study we assessed changes in the transcriptome and secretome of umbilical cord blood mononuclear cells (UCB-MCs) genetically modified using adenoviral vector (Ad5) carrying cDNA encoding human vascular endothelial growth factor (VEGF165) or reporter green fluorescent protein (GFP). A preliminary analysis of UCB-MCs transduced with Ad5-VEGF165 and Ad5-GFP with MOI of 10 showed efficient transgene expression in gene-modified UCB-MCs at mRNA and protein levels. The whole transcriptome sequencing of native UCB-MCs, UCB-MC+Ad5-VEGF165, and UCB-MC+Ad5-GFP demonstrated individual sample variability rather than the effect of Ad5 or the expression of recombinant vegf165 on UCB-MC transcriptomes. A multiplex secretome analysis indicated that neither the transduction of UCB-MCs with Ad5-GFP nor with Ad5-VEGF165 affects the secretion of the studied cytokines, chemokines, and growth factors by gene-modified cells. Here, we show that UCB-MCs transduced with Ad5 carrying cDNA encoding human VEGF165 efficiently express transgenes and preserve transcriptome and secretome patterns. This data demonstrates the biosafety of using UCB-MCs as cell carriers of therapeutic genes.

Induction of Angiogenesis by Genetically Modified Human Umbilical Cord Blood Mononuclear Cells.

Stimulating the process of angiogenesis in treating ischemia-related diseases is an urgent task for modern medicine, which can be achieved through the use of different cell types. Umbilical cord blood (UCB) continues to be one of the attractive cell sources for transplantation. The goal of this study was to investigate the role and therapeutic potential of gene-engineered umbilical cord blood mononuclear cells (UCB-MC) as a forward-looking strategy for the activation of angiogenesis. Adenovirus constructs Ad-VEGF, Ad-FGF2, Ad-SDF1α, and Ad-EGFP were synthesized and used for cell modification. UCB-MCs were isolated from UCB and transduced with adenoviral vectors. As part of our in vitro experiments, we evaluated the efficiency of transfection, the expression of recombinant genes, and the secretome profile. Later, we applied an in vivo Matrigel plug assay to assess engineered UCB-MC's angiogenic potential. We conclude that hUCB-MCs can be efficiently modified simultaneously with several adenoviral vectors. Modified UCB-MCs overexpress recombinant genes and proteins. Genetic modification of cells with recombinant adenoviruses does not affect the profile of secreted pro- and anti-inflammatory cytokines, chemokines, and growth factors, except for an increase in the synthesis of recombinant proteins. hUCB-MCs genetically modified with therapeutic genes induced the formation of new vessels. An increase in the expression of endothelial cells marker (CD31) was revealed, which correlated with the data of visual examination and histological analysis. The present study demonstrates that gene-engineered UCB-MC can be used to stimulate angiogenesis and possibly treat cardiovascular disease and diabetic cardiomyopathy.

Influence of Recombinant Codon-Optimized Plasmid DNA Encoding VEGF and FGF2 on Co-Induction of Angiogenesis.

Several methods for the stimulation of skin wound repair have been proposed over the last few decades. The most promising among them are gene and stem cell therapy. Our present experiments combined several approaches via the application of human umbilical cord blood mononuclear cells (hUCB-MC) that were transfected with pBud-VEGF165-FGF2 plasmid (gene-cell therapy) and direct gene therapy using pBud-VEGF165-FGF2 plasmid to enhance healing of full thickness skin wounds in rats. The dual expression cassette plasmid pBud-VEGF165-FGF2 encodes both VEGF and FGF2 therapeutic genes, expressing pro-angiogenic growth factors. Our results showed that, with two weeks post-transplantation, some transplanted cells still retained expression of the stem cell and hematopoietic markers C-kit and CD34. Other transplanted cells were found among keratinocytes, hair follicle cells, endothelial cells, and in the derma. PCNA expression studies revealed that transplantation of transfected cells terminated proliferative processes in regenerating wounds earlier than transplantation of untransfected cells. In the direct gene therapy group, four days post-operatively, the processes of flap revascularization, while using Easy LDI Microcirculation Camera, was higher than in control wounded skin. We concluded that hUCB-MC can be used for the treatment of skin wounds and transfection these cells with VEGF and FGF2 genes enhances their regenerative abilities. We also concluded that the application of pBud-VEGF165-FGF2 plasmids is efficient for the direct gene therapy of skin wounds by stimulation of wound revascularization.

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury.

We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases, neurotrauma, and stroke. In this study, using a mini pig model of spinal cord injury, we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy. Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector (Ad5/35F) that carried an enhanced green fluorescent protein (EGFP) reporter gene in the plastic blood bag. The day after blood donation, the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate. A week after gene-modified leucoconcentrate therapy, fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury. In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed. In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes. Thus, the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions. This paper presents a proof-of-concept of simple, safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 5) on May 27, 2014.

Triple-Gene Therapy for Stroke: A Proof-of-Concept in Vivo Study in Rats.

Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC) transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury. This proof-of-principle project was aimed at evaluating the beneficial effects of the same triple-gene approach in stroke. Rats subjected to distal occlusion of the middle cerebral artery were treated intrathecally with a combination of these genes either directly or using our cell-based (UCB-MC) approach. Various techniques and markers were employed to evaluate brain injury and subsequent recovery after treatment. Brain repair was most prominent when therapeutic genes were delivered via adenoviral vector- or UCB-MC-mediated approach. Remodeling of brain cortex in the stroke area was confirmed by reduction of infarct volume and attenuated neural cell death, depletion of astrocytes and microglial cells, and increase in the number of oligodendroglial cells and synaptic proteins expression. These results imply that intrathecal injection of genetically engineered UCB-MC over-expressing therapeutic molecules (VEGF, GDNF, and NCAM) following cerebral blood vessel occlusion might represent a novel avenue for future research into treating stroke.

Spinal Cord Molecular and Cellular Changes Induced by Adenoviral Vector- and Cell-Mediated Triple Gene Therapy after Severe Contusion.

The gene therapy has been successful in treatment of spinal cord injury (SCI) in several animal models, although it still remains unavailable for clinical practice. Surprisingly, regardless the fact that multiple reports showed motor recovery with gene therapy, little is known about molecular and cellular changes in the post-traumatic spinal cord following viral vector- or cell-mediated gene therapy. In this study we evaluated the therapeutic efficacy and changes in spinal cord after treatment with the genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and neuronal cell adhesion molecule (NCAM) applied using both approaches. Therapeutic genes were used for viral vector- and cell-mediated gene therapy in two combinations: (1) VEGF+GDNF+NCAM and (2) VEGF+ANG+NCAM. For direct gene therapy adenoviral vectors based on serotype 5 (Ad5) were injected intrathecally and for cell-mediated gene delivery human umbilical cord blood mononuclear cells (UCB-MC) were simultaneously transduced with three Ad5 vectors and injected intrathecally 4 h after the SCI. The efficacy of both treatments was confirmed by improvement in behavioral (BBB) test. Molecular and cellular changes following post-traumatic recovery were evaluated with immunofluorescent staining using antibodies against the functional markers of motorneurons (Hsp27, synaptophysin, PSD95), astrocytes (GFAP, vimentin), oligodendrocytes (Olig2, NG2, Cx47) and microglial cells (Iba1). Our results suggest that both approaches with intrathecal delivery of therapeutic genes may support functional recovery of post-traumatic spinal cord via lowering the stress (down regulation of Hsp25) and enhancing the synaptic plasticity (up regulation of PSD95 and synaptophysin), supporting oligodendrocyte proliferation (up regulation of NG2) and myelination (up regulation of Olig2 and Cx47), modulating astrogliosis by reducing number of astrocytes (down regulation of GFAP and vimetin) and microglial cells (down regulation of Iba1).

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo.

Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.

RNA interference and amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neuro-degenerative disorder characterized by progressive loss of motor neurons. The etiology and molecular pathogenesis of cell death in most sub-types of the disease are largely unknown. The best documented cause of moto-neuron degeneration is the mutation in the superoxide dismutase-1 (SOD1) gene, which occurs in 10% of the familial forms of ALS. Discovery of RNA interference (RNAi), which plays an important role in the regulation of gene expression, has proven to be a powerful tool to study the pathogenesis and to develop innovative treatment options for hereditary diseases, including familial variants of ALS. This review summarizes current research advances in RNAi in relation to ALS.

Genetically modified human umbilical cord blood cells expressing vascular endothelial growth factor and fibroblast growth factor 2 differentiate into glial cells after transplantation into amyotrophic lateral sclerosis transgenic mice.

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF(165)) and human fibroblast growth factor 2 (FGF(2)) (pBud-VEGF-FGF(2)). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models ((G)93(A) mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal ß(3)-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF(2). Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific ß(3)-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF(2), hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF(2) on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.

Potential role of dental stem cells in the cellular therapy of cerebral ischemia.

Stem cell based therapies for cerebral ischemia (CI) utilize different cell sources including embryonic stem cells (ESCs), neural stem cells (NSCs), umbilical cord blood cells (UCBCs), mesenchymal stem cells (MSCs), and some immortalized cell lines. To date, experimental studies showed that all of these cell sources have been successful to some extent in attenuating the ischemic damage and improving functional recovery after brain injury. Bone marrow derived MSCs seem to be the most widely used and well characterized cell source, which can be also employed for autologous transplantation. Currently, there are two main theories behind the therapeutic effect of stem cell transplantation for treating CIs. The first concept is cell replacement theory in which transplanted stem cells differentiate into progenitor and specialized somatic cells to supersede dying cells. The other hypothesis is based on immuno-modulatory, neuro-protective and neuro-trophic abilities of stem cells which help reducing stroke size and increasing the recovery of behavioral functions. Recent studies focusing on alternative stem cell sources have revealed that dental stem cells (DSCs), including dental pulp stem cells (DPSCs) and dental follicle cells (DFCs) possess properties of MSCs and NSCs. They differentiate into neural linage cells and some other cell types such as osteocytes, adipocytes, chondrocytes, muscle cells and hepatocytes. This review is intended to examine stem cell therapy approaches for CI and emphasize potential use of DSCs as an alternative cell source for the treatment of brain ischemia.

Retrogradely transported siRNA silences human mutant SOD1 in spinal cord motor neurons.

The transgenic mouse model of familial amyotrophic lateral sclerosis (ALS) expressing human mutant ((G)93(A)) copper/zinc superoxide dismutase (SOD(1)) is an attractive model for studying the therapeutic effects of RNA interference (RNAi) because of the specific silencing of the mutant gene expression. We studied small interfering RNA (siRNA)-mediated down-regulation of human mutant (G)93(A) SOD(1) gene in lumbar spinal cord of ALS mice. siRNA was applied onto the proximal nerve stump of severed sciatic nerves. One day after surgery the lumbar spinal cords were processed for RT-PCR examination. Treatment with specific siRNA resulted in 48% decrease in human SOD(1) mRNA levels in lumbar spinal cord, but had no effect on the abundance of mouse ChAT and SNAP(25) mRNAs which were used as randomly selected internal controls, the mark of a specific silencing of SOD(1). Our findings demonstrate for the first time that siRNA, targeting mutant human SOD(1) mRNA, is taken up by the sciatic nerve, retrogradely transported to the perikarya of motor neurons, and inhibits mutant SOD(1) mRNA in (G)93(A) transgenic ALS mice.

Human umbilical cord blood cells transfected with VEGF and L(1)CAM do not differentiate into neurons but transform into vascular endothelial cells and secrete neuro-trophic factors to support neuro-genesis-a novel approach in stem cell therapy.

Genetically modified mono-nuclear cell fraction from human umbilical cord blood (HUCB) expressing human vascular endothelial growth factor (VEGF) and mouse neural L(1) cell adhesion molecule (L(1)CAM) were used for gene-stem cell therapy of transgenic (G)93(A) mice adopted as an animal amyotrophic lateral sclerosis (ALS) model. We generated non-viral plasmid constructs, expressing human VEGF(165) (pcDNA-VEGF) and mouse neural L(1) cell adhesion molecule (pcDNA-mL(1)CAM). Mono-nuclear fraction of HUCB cells were transiently transfected by electro-poration with a mixture of expression plasmids (pcDNA-VEGF+pcDNA-mL(1)CAM). Sixteen transgenic female and male mice were randomly assigned to three groups: (1) transplantation of genetically modified HUCB cells expressing L(1) and VEGF (n=6), (2) transplantation of un-transfected HUCB cells (n=5), and (3) control group (n=5). In first two experimental groups 1x10(6) cells were injected retro-orbitally in pre-symptomatic 22-25-week-old (G)93(A) mice. Our results demonstrate that HUCB cells successfully grafted into nervous tissue of ALS mice and survived for over 3 months. Therefore, genetically modified HUCB cells migrate in the spinal cord parenchyma, proliferate, but instead of transforming into nerve cells, they differentiate into endothelial cells forming new blood vessels. We propose that: (A) expression of mouse neural L(1)CAM is responsible for increased homing and subsequent proliferation of transplanted cells at the site of neuro-degeneration, (B) expression of human VEGF directs HUCB cell differentiation into endothelial cells, and (C) neuro-protective effect may stem from the delivery of various neuro-trophic factors from newly formed blood vessels.

Modulation of neurotransmitter release by carbon monoxide at the frog neuro-muscular junction.

Carbon monoxide (CO) is an endogenous gaseous messenger, which regulates numerous physiological functions in a wide variety of tissues. Using extracellular microelectrode recording from frog neuro-muscular preparation the mechanisms of exogenous and endogenous CO action on evoked quantal acetyl-choline (Ach) release were studied. It was shown that CO application increases Ach-release in dose-dependent manner without changes in pre-synaptic Na+ and K+ currents. The effect of exogenous CO on Ach-release was decreased by prior application of guanylate cyclase inhibitor ODQ and prevented by application of a cyclic guanylate monophosphate (cGMP) analog 8Br-cGMP. Pre-treatment of the preparation with adenylate cyclase inhibitor MDL-12330A has completely abolished the effect of CO, whereas elevation of intracellular level of cyclic adenosine monophosphate (cAMP) mimicked and eliminated CO action. Application of cGMP-activated phosphodiesterase-2 inhibitor EHNA did not prevent CO action, whereas inhibition of cGMP-inhibited phosphodiesterase-3 by quazinone has partially blocked the effect of CO. Utilizing immuno-histochemical methods CO-producing enzyme heme-oxygenase-2 (HO-2) was shown to be expressed in skeletal muscle fibers, mostly in sub-sarcolemmal region, karyolemma and sarcoplasmic reticulum. Zn-protoporphirin-IX, the selective HO-2 blocker, has depressed Ach-release, suggesting the tonic activating effect of endogenous CO on pre-synaptic function. These results suggest that facilitatory effect of CO on Ach-release is mediated by elevation of intracellular cAMP level due to activation of adenylate cyclase and decrease of cAMP breakdown. As such, endogenous skeletal muscle-derived CO mediates tonic retrograde up-regulation of neuro-transmitter release at the frog neuro-muscular junction.

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.

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.

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.

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.

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.

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.