Expression Of Trophic Factors Research Articles

Enhancing Bone Formation Through bFGF-Loaded Mesenchymal Stromal Cell Spheroids During Fracture Healing in Mice.

This study aimed to evaluate the osteogenic potential of mesenchymal stromal cell (MSC) spheroids combined with the basic fibroblast growth factor (bFGF) in a mouse femur fracture model. To begin, MSC spheroids were generated, and the expression of key trophic factors (bFGF Bmp2, and Vegfa) was assessed using quantitative PCR (qPCR). A binding assay confirmed the interaction between the bFGF and the spheroids' extracellular matrix. The spheroid cultures significantly upregulated bFGF, Bmp2, and Vegfa expression compared to the monolayers (p < 0.001), and the binding assay demonstrated effective bFGF binding to the MSC spheroids. Following these in vitro assessments, the mice were divided into five groups for the in vivo study: (1) no treatment (control), (2) spheroids alone, (3) bFGF alone, (4) bFGF-loaded spheroids (bFGF-spheroids), and (5) non-viable (frozen) bFGF-loaded spheroids (bFGF-dSpheroids). Bone formation was analyzed by a micro-CT, measuring the bone volume (BV) and bone mineral content (BMC) of the mice four weeks post-fracture. A high dose of the bFGF (10 µg) significantly promoted bone formation regardless of the presence of spheroids, as evidenced by the increases in BV (bFGF, p = 0.010; bFGF-spheroids, p = 0.006; bFGF-dSpheroids, p = 0.032) and BMC (bFGF, p = 0.023; bFGF-spheroids, p = 0.004; bFGF-dSpheroids, p = 0.014), compared to the controls. In contrast, a low dose of the bFGF (1 µg) combined with the MSC spheroids significantly increased BV and BMC compared to the control (BV, p = 0.012; BMC, p = 0.015), bFGF alone (BV, p = 0.012; BMC, p = 0.008), and spheroid (BV, p < 0.001; BMC, p < 0.001) groups. A low dose of the bFGF alone did not significantly promote bone formation (p > 0.05). The non-viable (frozen) spheroids loaded with a low dose of the bFGF resulted in a higher BV and BMC compared to the spheroids alone (BV, p = 0.003; BMC, p = 0.017), though the effect was less pronounced than in the viable spheroids. These findings demonstrate the synergistic effect of the bFGF and MSC spheroids on bone regeneration. The increased expression of the BMP-2 and VEGF observed in the initial experiments, coupled with the enhanced bone formation in vivo, highlight the therapeutic potential of this combination. Future studies will aim to elucidate the underlying molecular mechanisms and assess the long-term outcomes for bone repair strategies.

Aging alters the expression of trophic factors and tight junction proteins in the mouse choroid plexus

BackgroundThe choroid plexus (CP) is an understudied tissue in the central nervous system and is primarily implicated in cerebrospinal fluid (CSF) production. CP also produces numerous neurotrophic factors (NTF) which circulate to different brain regions. Regulation of NTFs in the CP during natural aging is largely unknown. Here, we investigated the age and gender-specific transcription of NTFs along with the changes in the tight junctional proteins (TJPs) and the water channel protein Aquaporin (AQP1).MethodsMale and female mice were used for our study. Age-related transcriptional changes were analyzed using quantitative PCR at three different time points: mature adult, middle-aged, and aged. Transcriptional changes during aging were further confirmed with digital droplet PCR. Additionally, we used immunohistochemical analysis (IHC) for the evaluation of in vivo protein expression. We further investigated the cellular phenotype of these NTFS, TJP, and water channel proteins in the mouse CP by co-labeling them with the classical vascular marker, Isolectin B4, and epithelial cell marker, Plectin.ResultsAging significantly altered NTF gene expression in the CP. Brain-derived neurotrophic factor (BDNF), Midkine (MDK), VGF, Insulin-like growth factor (IGF1), IGF2, Klotho (KL), Erythropoietin (EPO), and its receptor (EPOR) were reduced in the aged CP of males and females. Vascular endothelial growth factor (VEGF) transcription was gender-specific; in males, gene expression was unchanged in the aged CP, while females showed an age-dependent reduction. Age-dependent changes in VEGF localization were evident, from vasculature to epithelial cells. IGF2 and klotho localized in the basolateral membrane of the CP and showed an age-dependent reduction in epithelial cells. Water channel protein AQP1 localized in the tip of epithelial cells and showed an age-related reduction in mRNA and protein levels. TJP’s JAM, CLAUDIN1, CLAUDIN2 and CLAUDIN5 were reduced in aged mice.ConclusionsOur study highlights transcriptional level changes in the CP during aging. The age-related transcriptional changes exhibit similarities as well as gene-specific differences in the CP of males and females. Altered transcription of the water channel protein AQP1 and TJPs could be involved in reduced CSF production during aging. Importantly, reduction in the neurotrophic factors and longevity factor Klotho can play a role in regulating brain aging.Graphical

Directing Rat Hair Follicle Stem Cells Toward Neuronal Lineage With Enhanced Trophic Factor Expression.

Hair follicle stem cells (HFSCs) are promising candidates for cell-based therapies in neurodegenerative diseases because of their ability to differentiate into neural lineages and exert paracrine effects in damaged tissues. However, their clinical application faces challenges, particularly in efficiently guiding them toward neural lineages. This study explores using chick embryo extract (CEE) to enhance HFSCs' secretory capacity and neuronal differentiation. HFSCs from rat whisker pads were cultured in growth medium supplemented with either 20% FBS or a combination of 10% FBS and 10% CEE, transitioning to 20% FBS after the first subculture. We conducted gene expression profiling of lineage commitment markers and neurotrophic factors in both experimental groups, alongside morphological assessments and protein expression analyses. CEE supplementation during migration increased neuronal differentiation, evidenced by more cells with neurites and higher MAP2 expression at both the gene and protein levels. CEE also inhibited the expression of PDGFR-α, indicating a suppression of differentiation toward Schwann cells. Furthermore, we observed increased levels of trophic factors such as BDNF and VEGF at passage 3 induced by CEE supplementation. Enhancing the neuronal lineage commitment of hair follicle stem cells (HFSCs) and boosting the expression of trophic and angiogenic factors through short-term CEE preconditioning during their migratory stage presents a compelling approach. This strategy holds great promise in enhancing the effectiveness of stem cell-based therapies for neurological disorders.

Glioprotective Effects of Sulforaphane in Hypothalamus: Focus on Aging Brain.

Sulforaphane is a natural compound with neuroprotective activity, but its effects on hypothalamus remain unknown. In line with this, astrocytes are critical cells to maintain brain homeostasis, and hypothalamic astrocytes are fundamental for sensing and responding to environmental changes involved in a variety of homeostatic functions. Changes in brain functionality, particularly associated with hypothalamic astrocytes, can contribute to age-related neurochemical alterations and, consequently, neurodegenerative diseases. Thus, here, we investigated the glioprotective effects of sulforaphane on hypothalamic astrocyte cultures and hypothalamic cell suspension obtained from aged Wistar rats (24months old). Sulforaphane showed anti-inflammatory and antioxidant properties, as well as modulated the mRNA expression of astroglial markers, such as aldehyde dehydrogenase 1 family member L1, aquaporin 4, and vascular endothelial growth factor. In addition, it increased the expression and extracellular levels of trophic factors, such as glia-derived neurotrophic factor and nerve growth factor, as well as the release of brain-derived neurotrophic factor and the mRNA of TrkA, which is a receptor associated with trophic factors. Sulforaphane also modulated the expression of classical pathways associated with glioprotection, including nuclear factor erythroid-derived 2-like 2, heme oxygenase-1, nuclear factor kappa B p65 subunit, and AMP-activated protein kinase. Finally, a cell suspension with neurons and glial cells was used to confirm the predominant effect of sulforaphane in glial cells. In summary, this study indicated the anti-aging and glioprotective activities of sulforaphane in aged astrocytes.

Dimethyl Fumarate Preconditioning can Reinforce the Therapeutic Potential of Bone Marrow Mesenchymal Stem Cells through Trophic Factor Profile Enhancement.

Numerous studies have confirmed the therapeutic efficacy of bone marrow-derived mesenchymal stem cells (BM-MSCs) in addressing neurologic disorders. To date, several preconditioning strategies have been designed to improve the therapeutic potential of these stem cells. This study was designed to evaluate the preconditioning effect of dimethyl fumarate (DMF) on the expression of main trophic factors in human BM-MSCs. Initially, the identity of stem cells was confirmed through the evaluation of surface markers and their capacity for osteogenic and adipogenic differentiation using flow cytometry and differentiation assay, respectively. Subsequently, stem cells were subjected to different concentrations of DMF for 72 hours and their viability was defined by MTT assay. Following 72-hour preconditioning period with 10 µM DMF, gene expression was assessed by quantitative RT-PCR. Our findings demonstrated that the isolated stem cells expressed cardinal MSC surface markers and exhibited osteogenic and adipogenic differentiation potential. MTT results confirmed that 10 µM DMF was an optimal dose for maintaining cell viability. Preconditioning of stem cells with DMF significantly upregulated the expression of BDNF, NGF, and NT-3. Despite a slight increase in transcript level of GDNF and VEGF after DMF preconditioning, this difference was not statistically significant. Our findings suggest that DMF preconditioning can enhance the expression of major neurotrophic factors in human BM-MSCs. Given the curative potential of both BM-MSCs and DMF in various neurological disease models and preconditioning outcomes, their combined use may synergistically enhance their neuroprotective properties.

MiRNA-21-5p is an important contributor to the promotion of injured peripheral nerve regeneration using hypoxia-pretreated bone marrow-derived neural crest cells.

JOURNAL/nrgr/04.03/01300535-202501000-00035/figure1/v/2024-05-14T021156Z/r/image-tiff Our previous study found that rat bone marrow-derived neural crest cells (acting as Schwann cell progenitors) have the potential to promote long-distance nerve repair. Cell-based therapy can enhance peripheral nerve repair and regeneration through paracrine bioactive factors and intercellular communication. Nevertheless, the complex contributions of various types of soluble cytokines and extracellular vesicle cargos to the secretome remain unclear. To investigate the role of the secretome and extracellular vesicles in repairing damaged peripheral nerves, we collected conditioned culture medium from hypoxia-pretreated neural crest cells, and found that it significantly promoted the repair of sensory neurons damaged by oxygen-glucose deprivation. The mRNA expression of trophic factors was highly expressed in hypoxia-pretreated neural crest cells. We performed RNA sequencing and bioinformatics analysis and found that miR-21-5p was enriched in hypoxia-pretreated extracellular vesicles of neural crest cells. Subsequently, to further clarify the role of hypoxia-pretreated neural crest cell extracellular vesicles rich in miR-21-5p in axonal growth and regeneration of sensory neurons, we used a microfluidic axonal dissociation model of sensory neurons in vitro, and found that hypoxia-pretreated neural crest cell extracellular vesicles promoted axonal growth and regeneration of sensory neurons, which was greatly dependent on loaded miR-21-5p. Finally, we constructed a miR-21-5p-loaded neural conduit to repair the sciatic nerve defect in rats and found that the motor and sensory functions of injured rat hind limb, as well as muscle tissue morphology of the hind limbs, were obviously restored. These findings suggest that hypoxia-pretreated neural crest extracellular vesicles are natural nanoparticles rich in miRNA-21-5p. miRNA-21-5p is one of the main contributors to promoting nerve regeneration by the neural crest cell secretome. This helps to explain the mechanism of action of the secretome and extracellular vesicles of neural crest cells in repairing damaged peripheral nerves, and also promotes the application of miR-21-5p in tissue engineering regeneration medicine.

Early-phase rotator training impairs tissue repair and functional recovery after spinal cord injury

Spinal cord injury (SCI) is a devastating disorder that often results in severe sensorimotor function impairment with limited recovery of function. In recent years, rehabilitation training for spinal cord injury has gradually emerged, and some of them play an important role in the repair of spinal cord injury However, the optimal training regimen for SCI remains to be determined. In this study, we explore the effects of rotarod training (began at 7 days post-injury) on the recovery of motor function after SCI, as well as its possible repair mechanism from the aspects of function and histopathological changes, the behaviors of specific trophic factors and cytokines, and the expression profile of specific genes. Multiple functional assessments showed that rotarod training initiated at 7 days post-injury is unsuitable for promoting neuro-electrophysiological improvement and trunk stability, but impaired functional coordination and motor recovery. In addition, rotarod training has negative effects on spinal cord repair after SCI, which is manifested as an increase of lesion area, a decrease in neuronal viability, a deterioration in immuno-microenvironment and remyelination, a significant reduction in the expression of trophic factors and an increase in the expression of pro-inflammatory factors. RNA sequencing suggested that the genes associated with angiogenesis and synaptogenesis were significantly downregulated and the PI3K-AKT pathway was inhibited, which was detrimental to spinal cord repair and impeded nerve regeneration. These results indicate that immediate rotarod training after SCI is currently unsuitable for rehabilitation in mice.

Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

Lack of Sod1 induces disruption of innervation accompanied by Schwann cell proliferation in skeletal muscle

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reply to: Glioblastoma, IDH-Wildtype, CNS WHO Grade 4, Associated with Deep Brain Stimulation in a Patient with Essential Tremor: Report of a Case with Molecular Characterization and Review of the Literature.

We read with interest the letter from Burns et al1 regarding a case of glioblastoma associated with deep brain stimulation (DBS) in a patient with essential tremor. Their case adds another example of a high-grade brain tumor in close proximity to DBS electrodes. The nature of the association between DBS and malignancy remains unknown. The generally accepted incidence of high-grade glioma (HGG) is ~3.56/100,0002 in the United States. With more than 280,000 patients implanted with DBS worldwide,3 we could expect perhaps 10 DBS patients per year to be affected. To date, seven cases have been catalogued in the literature from 2010 to 2022,1 however, there may be more unreported cases. It remains unclear whether the rate of HGG is higher in patients with DBS. Thus far very few cases have been reported. A caveat is that most DBS are implanted for Parkinson's disease (PD),3 and if factors such as longer implantation and stimulation times were to increase the risk of HGG, then perhaps it takes many years longer than most PD patients live post-DBS to see malignancy develop. Hence, the observed incidence might not represent true risk in relation to DBS. The risk in subpopulations of DBS patients with longer duration implantation for dystonia, epilepsy, or neuropsychiatric indications may be higher. In our case, we highlight HGG in longer-term exposure of more than 20-years of DBS in a DYT-1 dystonia patient implanted at a younger age.4 Furthermore, the tumor centered in the cerebral peduncle, which is an uncommon location for spontaneous HGG. Although any associations remain theoretical, DBS is known to increase the expression of trophic factors and drive neurogenesis.5 With this in mind, DBS driving neoplasia would not be entirely surprising. Further studies will be needed to assess for a causal relationship such as by cataloguing tumor genotypes to understand if certain profiles are present in DBS-associated malignancy, as done by Burns et al.1 Additionally, understanding tumor location relative to the volume of tissue activated and not just the electrode location would be important in future studies. Even if DBS is implicated in increasing neoplasia, the risk is likely low given the low incidence seen to date. If the benefit of DBS therapy such as in refractory dystonia or tremor outweighs the low risks, then many clinicians and patients would likely still elect for DBS except in known cases of higher risk for brain malignancy, similar to other treatments like radiation therapy with risk of secondary malignancy decades later. In conclusion, the case presented by Burns et al1 is another important addition to the literature highlighting HGG in the context of DBS, and further highlights the need for more documentation of such cases and future studies investigating any association of DBS and chronic stimulation with brain malignancy. (1) Research Project: A. Conception, B. Organization, C. Execution; (2) Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the First Draft, B. Review and Critique. A.Balanchandar: 1C, 2A, 2B, 3A. A.Boutet: 1C, 2A, 2B, 3A, 3B. A.V.: 1C, 2C, 3B. J.G.: 1C, 2B. I.Y.M.C.: 1A, 2C, 3B. D.M.: 1A, 2C, 3B. R.P.M.: 1A, 2C, 3B. A.F.: 1A, 2C, 3B. S.K.K.: 1A, 1B, 2C, 3B. A.M.L.: 1A, 1B, 2C, 3B. Ethical Compliance Statement: We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. The authors confirm that the approval of an institutional review board was not required for this work. Informed consent was obtained for this case report. Funding Sources and Conflicts of Interest: No specific funding was received for this work. The authors declare that there are no conflicts of interest relevant to this work. Financial Disclosures for the Previous 12 Months: A.Balachandar, A.Boutet, A.V., J.G., I.Y.M.C., D.M., and R.P.M. report no funding sources in the past 12 months. A.M.L. and A.F. received honoraria and/or research support from Abbott, Insightec, Boston Scientific, and Medtronic. S.K.K. received grants/grants pending from the Canadian Institutes of Health Research, and received payment for lectures including service on speaker's bureaus from Medtronic.

Abstract 16: Retinoid X Receptors (RXR) Play Essential Roles In Improving Post-ischemic Stroke Recovery In Aged Brain By Restoring Age-associated Dysfunctions Of Microglia/macrophages

After stroke, microglia and blood macrophages (MΦ) clear dead cells and cellular debris in the infarcted brain through phagocytosis. However, the phagocytic capability of MΦ declines with age. Age-related dysfunctions in MΦ also include reduced secretion of trophic factors, resulting in poor recovery after stroke. Retinoid-X-receptor (RXR) is a pleiotropic transcription factor. Our earlier studies suggest that RXR enhances MΦ phagocytic functions and improves post-stroke recovery in young mice. To assess MΦ dysfunctions with age, microglia were FACS-sorted from the brains of young adults (2-4 mo old) and aged (18-20 mo old) mice for gene expression profiling with qPCR. RXR agonist bexarotene (BEX; 5 mg/kg) or vehicle were IP injected daily for 3d prior to harvest (n=3/group). We found that compared to young adults, aged microglia have reduced expression of trophic factors ( Vegf ) and scavenger receptors ( Cd206 and Cd36 ) and that BEX treatment restored this deficit. Also, in agreement with gene expression profiles, using erythrocytes as targets for phagocytosis, we showed that BEX enhanced the phagocytic ability of MΦ in aged brain. This suggests that RXR activations restore the age-associated dysfunctions in MΦ.To further investigate the role of MΦ RXR activations in recovery after stroke, aged (18-20 months old) myeloid-specific RXRα knockout mice (Mac-RXRα-KO) and littermate control (RXRα-LoxP) of both sexes were subject to 60 min MCA occlusion. To activate RXR, BEX (5 mg/kg) or vehicle control was IP injected 24h after stroke onset and then daily for 7d (n=22/group). Sensorimotor functions were assessed using foot-fault and corner turn tests (d3, 7, 14, 28). The cognitive functions were evaluated via novel object recognition test (d30). We found that BEX significantly improved sensorimotor and cognitive functions in RXRα-LoxP mice. The beneficial effect of BEX was diminished in Mac-RXRα-KO mice, suggesting RXR signaling in MΦ plays essential role in recovery after stroke. Brain histology and serological cytokine profiles will also be discussed. In conclusion, our data show that activating RXR restores age-related dysfunctions in MΦ. More importantly, RXR in MΦ may be a potential target to improve post-stroke recovery in aged brain.

Effect of spheroid size on gene expression profiles of a mouse mesenchymal stem cell line in spheroid culture.

Mesenchymal stem cell (MSC)-based therapies offer potential for bone repair. MSC spheroid cultures may harbor enhanced therapeutic potential over MSC monolayers through increased secretion of trophic factors. However, the impact of spheroid size on trophic factor expression is unclear. We investigated the effect of spheroid size on trophic factor-related gene expression. KUM10, a murine MSC line was used. RNA-seq was used to screen the transcriptional profiles of MSC monolayer and spheroid cultures. Differentially expressed genes identified in RNA-seq were evaluated by q-PCR in cultures of 5 × 104 (S group), 5 × 105 (M group), 5 × 106 (L group) cells/well. Comparison of expression levels between KUM10 monolayer and spheroid cultures identified 2140 differentially expressed genes, of which 1047 were upregulated and 1093 were downregulated in KUM10 spheroids. Among these, 12 upregulated genes (Bmp2, Fgf9, Fgf18, Ngf, Pdgfa, Pdgfb, Tgfb1, Vegfa, Vegfc, Wnt4, Wnt5a, Wnt10a) were associated with secretory growth factors. Of these, expression of Fgf9, Fgf18, Vegfa and Vegfc was elevated in the L group, and Pdgfb and Tgfb1 was elevated in the S group. Spheroid size may impact trophic factor expression. Our results will be useful for future studies assessing the utility of MSC spheroids for treating bone injury.

Stress-Induced Changes in Trophic Factor Expression in the Rodent Urinary Bladder: Possible Links With Angiogenesis.

Substantive evidence supports a role of chronic stress in the development, maintenance, and even enhancement of functional bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Increased urinary frequency and bladder hyperalgesia have been reported in rodents exposed to a chronic stress paradigm. Here, we utilized a water avoidance stress (WAS) model in rodents to investigate the effect of chronic stress on vascular perfusion and angiogenesis. Female Wistar-Kyoto rats were exposed to WAS for 10 consecutive days. Bladder neck tissues were analyzed by western immunoblot for vascular endothelial growth factor (VEGF) and nerve growth factor precursor (proNGF). Vascular perfusion was assessed by fluorescent microangiography followed by Hypoxyprobe testing to identify regions of tissue hypoxia. The expression of VEGF and proNGF in the bladder neck mucosa was significantly higher in the WAS rats than in the controls. There was a trend toward increased vascular perfusion, but without a statistically significant difference from the control group. The WAS rats displayed a 1.6-fold increase in perfusion. Additionally, a greater abundance of vessels was observed in the WAS rats, most notably in the microvasculature. These findings show that chronic psychological stress induces factors that can lead to increased microvasculature formation, especially around the bladder neck, the region that contains most nociceptive bladder afferents. These findings may indicate a link between angiogenesis and other inflammatory factors that contribute to structural changes and pain in IC/BPS.

Chronic Trazodone and Citalopram Treatments Increase Trophic Factor and Circadian Rhythm Gene Expression in Rat Brain Regions Relevant for Antidepressant Efficacy.

Trazodone is an efficacious atypical antidepressant acting both as an SSRI and a 5HT2A and 5HT2C antagonist. Antagonism to H1-histaminergic and alpha1-adrenergic receptors is responsible for a sleep-promoting action. We studied long-term gene expression modulations induced by chronic trazodone to investigate the molecular underpinning of trazodone efficacy. Rats received acute or chronic treatment with trazodone or citalopram. mRNA expression of growth factor and circadian rhythm genes was evaluated by qPCR in the prefrontal cortex (PFCx), hippocampus, Nucleus Accumbens (NAc), amygdala, and hypothalamus. CREB levels and phosphorylation state were evaluated using Western blotting. BDNF levels were significantly increased in PFCx and hippocampus by trazodone and in the NAc and hypothalamus by citalopram. Likewise, TrkB receptor levels augmented in the PFCx after trazodone and in the amygdala after citalopram. FGF-2 and FGFR2 levels were higher after trazodone in the PFCx. The CREB phosphorylation state was increased by chronic trazodone in the PFCx, hippocampus, and hypothalamus. Bmal1 and Per1 were increased by both antidepressants after acute and chronic treatments, while Per2 levels were specifically augmented by chronic trazodone in the PFCx and NAc, and by citalopram in the PFCx, amygdala, and NAc. These findings show that trazodone affects the expression of neurotrophic factors involved in antidepressant responses and alters circadian rhythm genes implicated in the pathophysiology of depression, thus shedding light on trazodone's molecular mechanism of action.

Deletion of Nox4 enhances remyelination following cuprizone-induced demyelination by increasing phagocytic capacity of microglia and macrophages in mice.

NOX4 is a major reactive oxygen species-producing enzyme that modulates cell stress responses. We here examined the effect of Nox4 deletion on demyelination-remyelination, the most common pathological change in the brain. We used a model of cuprizone (CPZ)-associated demyelination-remyelination in wild-type and Nox4-deficient (Nox4-/- ) mice. While the CPZ-induced demyelination in the corpus callosum after 4 weeks of CPZ intoxication was slightly less pronounced in Nox4-/- mice than that in wild-type mice, remyelination following CPZ withdrawal was significantly enhanced in Nox4-/- mice with an increased accumulation of IBA1-positive microglia/macrophages in the demyelinating corpus callosum. Consistently, locomotor function, as assessed by the beam walking test, was significantly better during the remyelination phase in Nox4-/- mice. Nox4 deletion did not affect autonomous growth of primary-culture oligodendrocyte precursor cells. Although Nox4 expression was higher in cultured macrophages than in microglia, Nox4-/- microglia and macrophages both showed enhanced phagocytic capacity of myelin debris and produced increased amounts of trophic factors upon phagocytosis. The expression of trophic factors was higher, in parallel with the accumulation of IBA1-positive cells, in the corpus callosum in Nox4-/- mice than that in wild-type mice. Nox4 deletion suppressed phagocytosis-induced increase in mitochondrial membrane potential, enhancing phagocytic capacity of macrophages. Treatment with culture medium of Nox4-/- macrophages engulfing myelin debris, but not that of Nox4-/- astrocytes, enhanced cell growth and expression of myelin-associated proteins in cultured oligodendrocyte precursor cells. Collectively, Nox4 deletion promoted remyelination after CPZ-induced demyelination by enhancing microglia/macrophage-mediated clearance of myelin debris and the production of trophic factors leading to oligodendrogenesis.

Modulation of Specific Sphingosine-1-Phosphate Receptors Augments a Repair Mediating Schwann Cell Phenotype.

Transdifferentiation of Schwann cells is essential for functional peripheral nerve regeneration after injury. By activating a repair program, Schwann cells promote functional axonal regeneration and remyelination. However, chronic denervation, aging, metabolic diseases, or chronic inflammatory processes reduce the transdifferentiation capacity and thus diminish peripheral nerve repair. It was recently described that the sphingosine-1-phosphate receptor (S1PR) agonist Fingolimod enhances the Schwann cell repair phenotype by activation of dedifferentiation markers and concomitant release of trophic factors resulting in enhanced neurite growth. Since Fingolimod targets four out of five S1PRs (S1P1, S1P3-5) possibly leading to non-specific adverse effects, identification of the main receptor(s) responsible for the observed phenotypic changes is mandatory for future specific treatment approaches. Our experiments revealed that S1P3 dominates and that along with S1P1 acts as the responsible receptor for Schwann cell transdifferentiation as revealed by the combinatory application of specific agonists and antagonists. Targeting both receptors reduced the expression of myelin-associated genes, increased PDGF-BB representing enhanced trophic factor expression likely to result from c-Jun induction. Furthermore, we demonstrated that S1P4 and S1P5 play only a minor role in the adaptation of the repair phenotype. In conclusion, modulation of S1P1 and S1P3 could be effective to enhance the Schwann cell repair phenotype and thus stimulate proper nerve repair.

Study on NGF and VEGF during the Equine Perinatal Period-Part 1: Healthy Foals Born from Normal Pregnancy and Parturition.

Simple SummaryThe end of pregnancy, the birth and the sudden need for the fetus to adapt to the extra-uterine environment make the perinatal period eventful in all species. Trophic factors, such as nerve growth factor and vascular endothelial growth factor, as well as thyroid hormones, take part in the processes associated with the final maturation of the fetus. Our aim is to evaluate the levels of trophic factors and thyroid hormones obtained at parturition from a population of healthy mares and foals and in the first 72 h of foal life, as well as the expression of trophic factors in fetal membranes. The levels of both trophic factors decreased over time in foal plasma and a positive correlation was found between their levels at each time point, but no correlation was found with the thyroid hormone levels. Vascular endothelial growth factor was expressed in all fetal membranes, while nerve growth factor and its receptors were not expressed in the amnion. The close relationship between the two trophic factors in foal plasma over time and their fine expression in placental tissues appear to be key regulators of fetal development and adaptation to extra-uterine life.The importance of trophic factors, such as nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) during the perinatal period, is now emerging. Through their functional activities of neurogenesis and angiogenesis, they play a key role in the final maturation of the nervous and vascular systems. The present study aims to: (i) evaluate the NGF and VEGF levels obtained at parturition from the mare, foal and umbilical cord vein plasma, as well as in amniotic fluid; (ii) evaluate NGF and VEGF content in the plasma of healthy foals during the first 72 h of life (T0, T24 and T72); (iii) evaluate NGF and VEGF levels at parturition in relation to the selected mares’ and foals’ clinical parameters; (iv) evaluate the relationship between the two trophic factors and the thyroid hormone levels (TT3 and TT4) in the first 72 h of life; (v) assess mRNA expression of NGF, VEGF and BDNF and their cell surface receptors in the placenta. Fourteen Standardbred healthy foals born from mares with normal pregnancies and parturitions were included in the study. The dosage of NGF and VEGF levels was performed using commercial ELISA kits, whereas NGF, VEGF and BDNF placental gene expression was performed using semi-quantitative real-time PCR. In foal plasma, both NGF and VEGF levels decreased significantly over time, from T0 to T24 (p = 0.0066 for NGF; p < 0.0001 for VEGF) and from T0 to T72 (p = 0.0179 for NGF; p = 0.0016 for VEGF). In foal serum, TT3 levels increased significantly over time from T0 to T24 (p = 0.0058) and from T0 to T72 (p = 0.0013), whereas TT4 levels decreased significantly over time from T0 to T24 (p = 0.0201) and from T0 to T72 (p < 0.0001). A positive correlation was found in the levels of NGF and VEGF in foal plasma at each time point (p = 0.0115; r = 0.2862). A positive correlation was found between NGF levels in the foal plasma at T0 and lactate (p = 0.0359; r = 0.5634) as well as between VEGF levels in the foal plasma at T0 and creatine kinase (p = 0.0459; r = 0.5407). VEGF was expressed in all fetal membranes, whereas NGF and its receptors were not expressed in the amnion. The close relationship between the two trophic factors in foal plasma over time and their fine expression in placental tissues appear to be key regulators of fetal development and adaptation to extra-uterine life.

Therapeutic potential of hair follicle-derived stem cell intranasal transplantation in a rat model of ischemic stroke

BackgroundStem cell-based therapy has received considerable attention as a potential candidate in the treatment of ischemic stroke; however, employing an appropriate type of stem cells and an effective delivery route are still challenging. In the present study, we investigated the therapeutic effect of safe, noninvasive, and brain-targeted intranasal administration of hair follicle-derived stem cells (HFSCs) in a rat model of ischemic stroke.MethodsStem cells were obtained from the adult rat hair follicles. In experiment 1, stroke was induced by 30 min middle cerebral artery occlusion (MCAO) and stem cells were intranasally transplanted immediately after ischemia. In experiment 2, stroke was induced by 120 min MCAO and stem cells were administered 24 h after cerebral ischemia. In all experimental groups, neurological performance, short-term spatial working memory and infarct volume were assessed. Moreover, relative expression of major trophic factors in the striatum and cortex was evaluated by the quantitative PCR technique. The end point of experiment 1 was day 3 and the end point of experiment 2 was day 15.ResultsIn both experiments, intranasal administration of HFSCs improved functional performance and decreased infarct volume compared to the MCAO rats. Furthermore, NeuN and VEGF expression were higher in the transplanted group and stem cell therapy partially prevented BDNF and neurotrophin-3 over-expression induced by cerebral ischemia.ConclusionsThese findings highlight the curative potential of HFSCs following intranasal transplantation in a rat model of ischemic stroke.

Abstract TP235: Delayed FGF21 Administration Improves Cerebrovascular Remodeling And White Matter Repair After Focal Stroke In Diabetic Mice

Introduction: Type 2 diabetes mellitus (T2DM) is a major comorbidity exacerbating ischemic brain injury and impairing post-stroke recovery. Our previous study suggested that recombinant human fibroblast growth factor (rFGF) 21 might be a potent therapeutic targeting multiple aspects of pathophysiology in T2DM stroke. As a continuation of our previous work investigating the therapeutic effects of rFGF21 in T2DM stroke, this study aims to evaluate the potential effects of rFGF21 on cerebrovascular remodeling during a delayed phase after T2DM stroke. Methods: Permanent distal middle cerebral artery occlusion was performed in heterozygous non-diabetic db/+ and homozygous diabetic db/db mice. Daily rFGF21 administration was initiated one week after stroke induction and maintained for up to two weeks thereafter. Multiple markers associated with post-stroke recovery, including angiogenesis, oligodendrogenesis, white matter integrity and neurogenesis, were assessed up to three weeks after stroke. Results: Our results showed an impairment in post-stroke vascular remodeling under T2DM condition, reflected by the decreased expression of trophic factors in brain microvessels and impairments of angiogenesis. The defected cerebrovascular remodeling was accompanied by the decreased oligodendrogenesis and neurogenesis. However, delayed rFGF21 administration normalized post-stroke hyperglycemia and improved neurological outcomes, which may partially via the promotion of pro-angiogenic trophic factor expression in brain microvessels and cerebrovascular remodeling. The better cerebrovascular remodeling may also contribute to oligodendrogenesis, white matter integrity and neurogenesis after T2DM stroke. Conclusion: Delayed rFGF21 administration may improve neurological outcomes in T2DM stroke mice, at least in part by normalizing the metabolic abnormalities and promoting cerebrovascular remodeling and white matter repair.

Chronic trazodone treatment increases trophic factor and circadian rhythm gene expression in rat brain regions relevant for antidepressant efficacy

Chronic trazodone treatment increases trophic factor and circadian rhythm gene expression in rat brain regions relevant for antidepressant efficacy