Search

Following acute spinal cord injury (SCI), excessive recruitment of neutrophils can result in inflammation, neural tissue loss and exacerbation of neurological outcomes. Ibrutinib is a bruton's tyrosine kinase inhibitor in innate immune cells such as the neutrophils that diminishes their activation and influx to the site of injury. The present study evaluated the efficacy of ibrutinib administration in the acute phase of SCI on neural tissue preservation and locomotor recovery. Ibrutinib was delivered intravenously at 3.125 mg/kg either immediately, 12 hours after, or both immediately and 12 hours after SCI induction in adult male C57BL/6 mice. Neutrophil influx into the lesion area was evaluated 24 hours following SCI using light microscopy and immunohistochemistry methods. Animals' body weight changes were recorded, and their functional motor recovery was assessed based on the Basso mouse scale during 28 days after treatment. Finally, spinal cord lesion volume was estimated by an unbiased stereological method. While animals' weight in the control group started to increase one week after injury, it stayed unchanged in treatment groups. However, the double injection of ibrutinib led to a significantly lower body weight compared to the control group at 4 weeks post-injury. Mean neutrophil counts per visual field and the lesion volume were significantly decreased in all ibrutinib-treated groups. In addition, ibrutinib significantly improved locomotor functional recovery in all treated groups, especially in immediate and double-injection groups. Neural tissue protection and locomotor functional recovery suggest ibrutinib treatment as a potent immunotherapeutic intervention for traumatic SCI that warrants clinical testing.

Targeted sequencing is an increasingly sought technology. Available methods, however, are often costly and yield high proportions of off-target reads. Here, we present FENGC, a scalable, multiplexed method in which target sequences are assembled into 5′ flaps for precise excision by flap endonuclease. Recovery of length-matched sequences, amplification with universal primers, and exonucleolytic removal of non-targeted genomic regions mitigate amplification biases and consistently yield ≥ 80% on-target sequencing. Furthermore, optimized sequential reagent addition and purifications minimize sample loss and facilitate rapid processing of sub-microgram quantities of DNA for detection of genetic variants and DNA methylation. Treatment of cultured human glioblastoma cells and primary murine monocytes with GC methyltransferase followed by FENGC and high-coverage enzymatic methyl sequencing provides single-molecule, long-read detection of differential endogenous CG methylation, dynamic nucleosome repositioning, and transcription factor binding. FENGC provides a versatile and cost-effective platform for targeted sequence enrichment for analysis of genetic and/or epigenetic heterogeneity.

The orexin 2 receptor plays a central role in maintaining sleep and wakefulness. Recently, it has been shown that sleep and wakefulness orchestrate the proliferation and differentiation of oligodendrocytes. Here, we explored the role of a selective orexin 2 receptor antagonist (JNJ-10397049) in proliferation and differentiation of neural progenitor cells (NPCs). We evaluated the proliferation potential of NPCs after exposure to different concentrations of JNJ-10397049 by using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide and neurosphere assays. Moreover, the expression of differentiation markers was assessed by immunocytochemistry and real-time polymerase chain reaction. JNJ-10397049 significantly increased the proliferation of NPCs at lower concentrations. In addition, orexin 2 receptor antagonist facilitated progression of differentiation of NPCs towards oligodendroglial lineage by considerable expression of Olig2 and 2',3'-cyclic-nucleotide 3'-phosphodiesterase as well as decreased expression of nestin marker. The results open a new avenue for future investigations in which the production of more oligodendrocytes from NPCs is needed.

Neural precursor cells (NPCs) are a renewable cell source that can proliferate and expand for long periods of time and give rise to the main neural cell types of the central nervous system (CNS). Establishing simple and reproducible growth culture conditions is of great importance to study the biology of NPCs and to understand the molecular basis of their behavior in healthy and diseased conditions.Here, we describe a simple free-floating , serum-free culture condition, known as the neurosphere assay, which is the most commonly used method for the isolation and expansion of NPCs harvested from the adult and fetal CNS. This culture system will result in large numbers of undifferentiated NPC progenies that represents a useful cell source for many in vitro and in vivo applications.

The renewable source of neural stem cells (NSCs) with multi-lineage differentiation capability toward neurons, astrocytes, and oligodendrocytes represents an ideal supply for cell therapy of central nervous system (CNS) diseases. In spite of this, the clinical use of NSCs is hampered by heterogeneity, poor neuronal cell yield, predominant astrocytic differentiation of NSC progeny, and possible uncontrolled proliferation and tumor formation upon transplantation. The ability to generate highly enriched and defined neural cell populations from the renewable source of NSCs might overcome many of these impediments and pave the way toward their successful clinical applications.Here, we describe a simple method for NSC differentiation and subsequent purification of neuronal progenitor cells, taking advantage of size and granularity differences between neuronal cells and other NSC progeny. This highly enriched neuronal cell population provides an invaluable source of cells for both in vitro and in vivo studies.

Neural stem cells (NSCs) transplantation enhances plasticity and restores functions in neurological diseases. Therapeutic benefits of NSCs are due to their ability to replace the lost neurons and glial cells and also secreting a wide array of free and membrane-bound bioactive molecules that can reduce the hostility of diseased microenvironment, resolve inflammation, and rescue damaged neural cells. Membrane-encircled spherical nanostructures that are collectively known as extracellular vesicles (EVs) contain mRNA, miRNA, lipids, and specific proteins that affect different biological processes in cells located nearby or at far distances. Using EVs as an alternative non-cell-based therapy has gained huge attention, and developing methods for large-scale production of EVs is of great clinical importance. Here, we describe an efficient method to yield significant quantity of EVs from human NSCs that are expanded under free floating neurosphere assay culture system. Using the neurosphere assay in bioreactors under GMP-compliant conditions can result in scalable NSC-EVs required for human trials.

Neural precursor cells (NPCs) are a renewable cell source that can proliferate and expand for long periods of time and give rise to the main neural cell types of the central nervous system (CNS). Establishing simple and reproducible growth culture conditions is of great importance to study the biology of NPCs and to understand the molecular basis of their behavior in healthy and diseased conditions.Here, we describe a simple free-floating , serum-free culture condition, known as the neurosphere assay, which is the most commonly used method for the isolation and expansion of NPCs harvested from the adult and fetal CNS. This culture system will result in large numbers of undifferentiated NPC progenies that represents a useful cell source for many in vitro and in vivo applications.

Neural stem cells (NSCs) transplantation enhances plasticity and restores functions in neurological diseases. Therapeutic benefits of NSCs are due to their ability to replace the lost neurons and glial cells and also secreting a wide array of free and membrane-bound bioactive molecules that can reduce the hostility of diseased microenvironment, resolve inflammation, and rescue damaged neural cells. Membrane-encircled spherical nanostructures that are collectively known as extracellular vesicles (EVs) contain mRNA, miRNA, lipids, and specific proteins that affect different biological processes in cells located nearby or at far distances. Using EVs as an alternative non-cell-based therapy has gained huge attention, and developing methods for large-scale production of EVs is of great clinical importance. Here, we describe an efficient method to yield significant quantity of EVs from human NSCs that are expanded under free floating neurosphere assay culture system. Using the neurosphere assay in bioreactors under GMP-compliant conditions can result in scalable NSC-EVs required for human trials.

Spinal cord injury (SCI) is a devastating clinical problem that can lead to permanent motor dysfunction. Fingolimod (FTY720) is a sphingosine structural analogue, and recently, its therapeutic benefits in SCI have been reported. The present study aimed to evaluate the therapeutic efficacy of fingolimod-incorporated poly lactic-co-glycolic acid (PLGA) nanoparticles (nanofingolimod) delivered locally together with neural stem/progenitor cells (NS/PCs) transplantation in a mouse model of contusive acute SCI. Fingolimod was encapsulated in PLGA nanoparticles by the emulsion-evaporation method. Mouse NS/PCs were harvested and cultured from embryonic Day 14 (E14) ganglionic eminences. Induction of SCI was followed by the intrathecal delivery of nanofingolimod with and without intralesional transplantation of PuraMatrix-encapsulated NS/PCs. Functional recovery, injury size and the fate of the transplanted cells were evaluated after 28 days. The nanofingolimod particles represented spherical morphology. The entrapment efficiency determined by UV-visible spectroscopy was approximately 90%, and the drug content of fingolimod loaded nanoparticles was 13%. About 68% of encapsulated fingolimod was slowly released within 10 days. Local delivery of nanofingolimod in combination with NS/PCs transplantation led to a stronger improvement in neurological functions and minimized tissue damage. Furthermore, co-administration of nanofingolimod and NS/PCs not only increased the survival of transplanted cells but also promoted their fate towards more oligodendrocytic phenotype. Our data suggest that local release of nanofingolimod in combination with three-dimensional (3D) transplantation of NS/PCs in the acute phase of SCI could be a promising approach to restore the damaged tissues and improve neurological functions.

Background: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system characterized by infiltration of inflammatory leukocytes to the CNS followed by oligodendrocyte cell death, myelin sheath destruction, and axonal injury. A logical incidence occurring after demyelination is remyelination. G-protein coupled receptors (GPCRs) activate internal signal transduction cascades through binding to different ligands. This family of receptors are targeted by more than 40% of currently marketed drugs. GPCRs can be successfully targeted for induction of remyelination. GPCRs highly enriched in oligodendrocyte progenitor cells compared to oligodendrocytes are proposed to hamper oligodendrocyte differentiation and therefore their inhibition might induce remyelination. This study aimed to investigate the expression of GPCRs in silico and in vitro. Methods: We performed gene expression analysis using DAVID and Panther websites on a RNA-seq dataset (GSE52564 accession number). Primary embryonic neural stem/progenitor cell isolation and culture were performed and subsequently NSPCs were characterized by Immunocytochemistry with Anti-Nestin antibody. Expression of GPR37L1, EDNRB, PDGFRα, CNPase and GFAP were assessed using real-time PCR. All the experiments were conducted at Shiraz University of Medical Sciences (SUMS), Shiraz, Iran, in the year 2018. Results: The 14 most highly expressed GPCRs in oligodendrocyte progenitor cells (OPCs) compared to Oligodendrocytes were presented in our study. Conclusion: The investigation of the most highly expressed GPCRs in OPCs compared to oligodendrocyte in silico and in vitro presents the significant role of GPCRs in remyelination induction. Among the 14 GPCRs mentioned in this study, GPR37L1 is a potential remyelinating drug target and is suggested for further studies.

History of anatomy is as long as the history of medicine itself. Development of this basic science was not possible without the dedicative effort of those physicians and scholars who were committed to discover the mysteries of human anatomy. In this regard, Iranian scholars played an important role in the development of the anatomical sciences despite the religious limitations in their societies. Mansur ibn Ilyas Shirazi is an Iranian physician of fourteenth century who wrote the first color illustrated anatomical book, Mansur's Anatomy. A considerable portion of the book has been dedicated to the central and peripheral nervous system so that he could be considered as one of the pioneers of neuroanatomy.

Background The changes induced in host immunity and the tumor microenvironment by chemotherapy have been shown to impact immunotherapy response in both a positive and a negative fashion. Temozolomide is the most common chemotherapy used to treat glioblastoma (GBM) and has been shown to have variable effects on immune response to immunotherapy. Therefore, we aimed to determine the immune modulatory effects of temozolomide that would impact response to immune checkpoint inhibition in the treatment of experimental GBM. Methods Immune function and antitumor efficacy of immune checkpoint inhibition were tested after treatment with metronomic dose (MD) temozolomide (25 mg/kg × 10 days) or standard dose (SD) temozolomide (50 mg/kg × 5 days) in the GL261 and KR158 murine glioma models. Results SD temozolomide treatment resulted in an upregulation of markers of T-cell exhaustion such as LAG-3 and TIM-3 in lymphocytes which was not seen with MD temozolomide. When temozolomide treatment was combined with programmed cell death 1 (PD-1) antibody therapy, the MD temozolomide/PD-1 antibody group demonstrated a decrease in exhaustion markers in tumor infiltrating lymphocytes that was not observed in the SD temozolomide/PD-1 antibody group. Also, the survival advantage of PD-1 antibody therapy in a murine syngeneic intracranial glioma model was abrogated by adding SD temozolomide to treatment. However, when MD temozolomide was added to PD-1 inhibition, it preserved the survival benefit that was seen by PD-1 antibody therapy alone. Conclusion The peripheral and intratumoral immune microenvironments are distinctively affected by dose modulation of temozolomide.

During neural development, embryonic neural stem cells (eNSCs) differentiate toward glial, oligodendrocytic, and neuronal cells. Dysregulation of polyunsaturated fatty acids (PUFAs) induce a wide range of neurological and developmental disorders. In this study, we investigated the effect of various concentrations and ratios of linoleic acid (LA) and alpha linolenic acid (ALA), which belong respectively to omega-6 and omega-3 PUFAs, on the proliferation and differentiation of eNSCs.Results showed that low (25 and 50μM) or high (100 and 200μM) concentrations of ALA, but not LA, and the ratio of 1:3 of LA/ALA significantly increased neurospheres size, frequency and cell numbers, in comparison to controls. Moreover, low or high concentrations of ALA, but not LA, and different ratios of LA/ALA resulted in a significant increase in mRNA expression levels of Notch1, Hes1 and Ki-67, and the differentiation of eNSCs toward astrocytes (GFAP) and oligodendrocytes (MBP), but not neurons (β-III Tubulin), with the highest increase being for LA/ALA ratio of 1:3, in comparison to controls. These results demonstrate the importance of higher concentrations of ALA in enhancing proliferation and differentiation of eNSCs, which could be used in diet to help preventing neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.

Dendritic cell (DC) vaccine efficacy is directly related to the efficiency of DC migration to the lymph node after delivery to the patient. In this research we discovered increasing cell migration by utilizing sarcosine improved anti-tumor efficacy. We hypothesized that sarcosine induced cell migration was due to chemokine or cytokine signaling. METHODS To generate DC vaccines, DCs were harvested from bone marrow of wild type C57BL/6 mice and electroporated with OVA-mRNA. Human DCs were isolated from PBMCs. DCs were treated with sarcosine at 20mM. OT-I T cells were isolated from transgenic mice and injected intravenously into B16F10-OVA tumor bearing mice. Following T cell transfer, DC vaccines were injected intradermal. In vitro migration was analyzed via transwell migration assay. In vivo migration was evaluated by flow-cytometry and immunofluorescence microscopy. Gene expression in RNA was investigated in DCs via RT-PCR and Nanostring. RESULTS Sarcosine significantly increases human and murine DC migration in vitro. In vivo murine model, sarcosine-loaded DCs had significantly increased migration to both the lymph nodes and spleen after intradermal delivery. B16F10-OVA tumor bearing mice were treated with the sarcosine-loaded DC vaccines resulted in a significant survival advantage over control and naïve DC vaccines. Gene expression in RNA was investigated in DCs. CXCR2,CXCL3 and CXCL1 were found to be upregulated in sarcosine-loaded DCs. Further metabolic analysis demonstrated the upregulation of cyclooxygenase-1 and Pik3cg. In vitro DC migration in presence of CXCR2 neutralizing antibody showed sarcosine induced migration was abrogated by adding the CXCR2 neutralizing antibody in both human and murine DCs. Animals that treated with sarcosine-loaded DC showed significantly better tumor control compares to the animals receiving anti-CXCR2 antibody one hour before DC injection. CONCLUSION Sarcosine increases the migration of murine and human DCs via the CXC chemokine pathway. This platform can be utilized to improve existing DC vaccine strategies.

Spinal Cord Injury (SCI) is a devastating disease with poor clinical outcomes. Animal models provide great opportunities to expand our horizons in identifying SCI pathophysiological mechanisms and introducing effective treatment strategies. The present study introduces a new murine contusion model.A simple, cheap, and reproducible novel instrument was designed, which consisted of a body part, an immobilization piece, and a bar-shaped weight. The injury was inflicted to the spinal cord using an 8-g weight for 5, 10, or 15 minutes after laminectomy at the T9 level in male C57BL/6 mice. Motor function, cavity formation, cell injury, and macrophage infiltration were evaluated 28 days after injury.The newly designed instrument minimized adverse spinal movement during injury induction. Moreover, no additional devices, such as a stereotaxic apparatus, were required to stabilize the animals during the surgical procedure. Locomotor activity was deteriorated after injury. Furthermore, tissue damage and cell injury were exacerbated by increasing the duration of weight exertion. In addition, macrophage infiltration around the injured tissue was observed 28 days after injury.This novel apparatus could induce a controllable SCI with a clear cavity formation in mice. No accessory elements are needed, which can be used in future SCI studies.A simple and precise method has been introduced for creating Spinal Cord Injury (SCI) in mice by a novel device.The device consists of a body part, an immobilization piece, and a bar-shaped weight.Assessment of locomotor activity, tissue damage, and macrophage infiltration confirmed the capability of the new SCI method.Reduction of adverse spinal movements and working without any accessory elements are the key points of this new animal model of SCI.Spinal Cord Injury (SCI) is a medical problem that can cause the permanent motor and sensory dysfunction. Traffic accidents, falls, and violence are the most frequent causes of SCI, often affecting young people. Patients and even their families may encounter other problems, including reducing life quality, psychological burden, and enormous medical costs. Despite scientific and technological advances, no effective treatment has been found for SCI. Therefore, animal models help study damage mechanisms and evaluate novel treatment strategies. All SCI research centers require an economical and reproducible device without using complex surgical procedures by experienced surgeons to minimize variations in damage to the spinal cord. In this study, a simple, cheap, and reproducible novel instrument for SCI induction is introduced. The instrument consists of various parts, including a body part, an immobilization piece, and a bar-shaped weight. An 8-g weight was used for 5, 10, or 15 minutes to inflict injury to the spinal cord. Behavioral and tissue studies indicated that SCI could be induced in rodents in different severity without other elements. This instrument can be used in future investigations for SCI studies, including tissue engineering, stem cell therapy, and drugs delivery to access effective treatment.

Background Embryonic neural stem cells (eNSCs) are immature precursors of the central nervous system (CNS), with self-renewal and multipotential differentiation capacities. These are regulated by endogenous and exogenous factors such as alpha-linolenic acid (ALA), a plant-based essential omega-3 polyunsaturated fatty acid. Methods In this study, we investigated the effects of various concentrations of Alyssum homolocarpum seed oil (AHSO), containing natural ALA, stearic acid (SA), myristic acid (MA), and β-sitosterol, on proliferation and differentiation of eNSCs, in comparison to controls and to synthetic pure ALA. Results Treatment with natural AHSO (25 to 75 μM), similar to synthetic ALA, caused a significant ~ 2-fold increase in eNCSs viability, in comparison to controls. To confirm this proliferative activity, treatment of NSCs with 50 or 75 μM AHSO resulted in a significant increase in mRNA levels of notch1, hes-1 and Ki-67and NICD protein expression, in comparison to controls. Moreover, AHSO administration significantly increased the differentiation of eNSCs toward astrocytes (GFAP+) and oligodendrocytes (MBP+) in a dose dependent manner and was more potent than ALA, at similar concentrations, in comparison to controls. Indeed, only high concentrations of 100 μM AHSO, but not ALA, caused a significant increase in the frequency of neurons (β-III Tubulin+). Conclusion Our data demonstrated that AHSO, a rich source of ALA containing also other beneficial fatty acids, increased the proliferation and stimulated the differentiation of eNSCs. We suggest that AHSO’s effects are caused by β-sitosterol, SA and MA, present within this oil. AHSO could be used in diet to prevent neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.

INTRODUCTION Extracellular vesicles (EVs) have been harvested from many plant sources, some of which have anti-cancer effects and some could be used as therapeutic nanodelivery vectors. Hemp plant is a natural source of cannabinoids, of which delta 9-tetrahydroxicannabinol (THC) and cannabidiol (CBD) have proven anti-cancer proprieties. HYPOTHESIS We hypothesized that hemp EVs are enriched in cannabinoids and their application will reduce glioblastoma (GBM) tumor progression. APPROACH EVs were isolated from the hemp plant using ultracentrifugation. Nanotracking analysis, electron microscopy and liquid chromatography tandem mass spectrometry (LC-MS/MS) were utilized to characterize EVs. GBM cell lines were cultured in the neuropshere assay to evaluate hemp EVs anti-glioma effects. Fluorescent-labelled EVs were used to evaluate their brain tissue distribution in orthotopic patient-derived GBM xenografts. RESULTS Hemp EVs have a median diameter of 112.6nm with a typical lipid-bilayer structure. LC-MS/MS have shown that while cannabidiolic, cannabigerolic, and tetrahydroxicannabinolic acids represent 69.1 ± 2.1%, 19.1 ± 1.6%, 6.5 ± 0.54% of the total cannabinoids in hemp EVs, CBD and THC only make 4.75 ± 0.26%, and 0.5 ± 0.3%. Hemp EVs are potent anti-glioma agents with a 7-day LD-50 of 1.04µM and 2.4µM [based on EVs total cannabinoid content] for KR-158 and L0 GBM lines, respectively. Compared to the vehicle, overnight incubation of L0 cells with 1µM hemp EVs significantly reduced GBM cell migration (630.3 ± 61.43 vs 143.7 ± 8.7). Intranasal administration of hemp EVs led to a widespread distribution in tumor bearing brain including GBM tumor core. CONCLUSION Based on these results, hemp EVs with enriched cannabinoid content exert antiglioma effect in-vitro and when delivered intranasally, are widely distributed throughout the brain and within the tumor of PDX animals. Further experiments are ongoing to address the impact of nasally-delivered hemp EVs on tumor progression and compare to the application of purified acidic cannabinoids.

INTRODUCTION: Immune cell migration is critical for cellular immunotherapy efficacy in the treatment of glioblastoma (GBM). Sarcosine is a non-toxic metabolite that is associated with a migratory phenotype in prostate cancer cells. We utilized sarcosine to increase migration of dendritic cells (DC) and T cells to increase efficacy of cellular immunotherapy. HYPOTHESIS: Sarcosine increases cellular migration and will improve tumor outcomes when combined with cellular immunotherapy platforms in the treatment of GBM. METHODS: DC and T cell migration was evaluated in vitro using transwell plates and in vivo using flowcytometry and immunofluorescence microscopy. The impact of sarcosine loaded cells on tumor outcomes was measured by testing a DC vaccine platform in the treatment of murine tumor models. Genomic expression of cytokines on sarcosine treated cells was tested using RT-PCR. RESULTS: Cells were efficiently loaded with sarcosine by simply adding sarcosine to the culture media (20mM). DCs and T cells loaded with sarcosine demonstrated increased migration in vitro (p

Background: Oligodendrocytes are myelin-producing cells, which develop from oligodendrocyte precursor cells (OPCs) and may be damaged in multiple sclerosis (MS) and traumatic brain injuries. Promotion of neural stem cell (NSC) differentiation into oligodendrocytes in vivo is a viable strategy to replace the lost myelinating cells and restore motor functions in such debilitating diseases. Crocus sativus L. or saffron has been used as a spice since ancient times. Saffron and its major active component, crocin, are well-known for their medicinal and neuroprotective activities. The current study aimed at assessing the effect of saffron extract and crocin on the differentiation of NSCs into OPCs. Methods: To isolate NSCs, 14-day embryonic rats cortices were cultured, using the neurosphere assay. NSCs were cultured in a complete NSC medium under 4 different treatment conditions: 1, Negative control group, 10 ng/mL of basic fibroblast growth factor (bFGF); 2, Positive control group, 10 ng/mL of bFGF plus 30 ng/mL of platelet-derived growth factor-AA (PDGF-AA); 3, Crocin group, crocin extract plus 10 ng/mL of bFGF; and 4, Saffron group, saffron extract plus 10 ng/mL of bFGF in 2 or 5 days. The level of Olig2 protein, an early OPC marker, was evaluated by flow cytometry 2 days after the treatment. The level of sox10 mRNA expression, involving in OPC maturation processes, was assessed in the study groups via quantitative real-time polymerase chain reaction (PCR), 5 days after the treatment. All the experiments were done in triplicate. The data were analyzed using one-way ANOVA with Graph Pad Prism version 6.01; P < 0.05 was considered as the level of significance. Results: The present data demonstrated that, similar to the positive control group, the rate of olig2+ cells in the crocin and saffron extract groups did not exceed 80% indicating that these components could significantly increase olig2 expression in NSC differentiation compared with that of the negative controls (P < 0.05). Moreover, the mean expression level of sox10 gene in the positive control, saffron and crocin groups was 1 ± 0.012, 0.72 ± 0.13, and 0.835 ± 0.015, respectively, which significantly higher than that of the negative control group (P < 0.05). Conclusions: It can be concluded that crocin and saffron are the proper alternatives for PDGF-AA in the enhancement of NSC differentiation into oligodendrocytes.

Tumor resistance to conventional therapies is a major challenge toward the eradication of cancer, a life-threatening disease. This resistance mainly results from tumor heterogeneity and more specifically from the existence of "stem-like" cells that remain in a quiescent state for long periods of time and thus escape commonly used anti-cancer drugs resulting in treatment failure. Therefore, targeting this subpopulation would present a viable strategy to overcome tumor burden. This daunting task requires a deep and thorough understanding of the biology of the quiescent stem-cell population, their interaction with tumor microenvironments, and mechanisms used to sustain themselves despite aggressive therapies. In this chapter, we describe detailed technical procedures for the isolation of quiescent or infrequently dividing stem-like cells in cultured glioblastoma tumor cells using carboxy fluorescein succinimidyl ester (CFSE) staining and flow cytometric analysis. Quiescent glioblastoma cells with stem-like features are characterized and subsequently isolated based on their ability to retain the CFSE labeling.

Tumor resistance to conventional therapies is a major challenge toward the eradication of cancer, a life-threatening disease. This resistance mainly results from tumor heterogeneity and more specifically from the existence of "stem-like" cells that remain in a quiescent state for long periods of time and thus escape commonly used anti-cancer drugs resulting in treatment failure. Therefore, targeting this subpopulation would present a viable strategy to overcome tumor burden. This daunting task requires a deep and thorough understanding of the biology of the quiescent stem-cell population, their interaction with tumor microenvironments, and mechanisms used to sustain themselves despite aggressive therapies. In this chapter, we describe detailed technical procedures for the isolation of quiescent or infrequently dividing stem-like cells in cultured glioblastoma tumor cells using carboxy fluorescein succinimidyl ester (CFSE) staining and flow cytometric analysis. Quiescent glioblastoma cells with stem-like features are characterized and subsequently isolated based on their ability to retain the CFSE labeling.

INTRODUCTION: While the KD holds promise as a therapeutic option for brain cancer patients, stringency of the diet impacts compliance. We have previously demonstrated that a high fat/low carbohydrate diet, similar to the Ketogenic Diet [KD], can reduce tumor progression and enhance survival in an orthotopic xenograft model. However, while this diet is less restrictive than the classic KD it still involves significant changes to a patient’s diet. Two of the primary physiological changes that occur when on the KD are a reduction in glucose and an increase in ketone bodies. These physiological changes are mimicked by providing ketone esters [KE] in the diet, and we [AMP, DPD], have recently shown that ketone esters can reduce glucose, elevate ketone bodies and enhance survival in a metastatic cancer model. HYPOTHESIS: We hypothesize that KE [1,3 butanediol acetoacetate diester] will reduce glucose, elevate ketones, reduce tumor progression and enhance survival in an orthotopic xenograft GBM model using a PDX model. APPROACH: NON/SCID animals implanted with patient-derived GBM cells were fed a standard diet [SD], or SD + KE [20%] till they reached endpoint. Body weight, plasma glucose, and ketones were measured weekly and overall survival assessed. RESULTS: While the KE is bitter and can have poor compliance, we found that supplementing with 1% Stevia increased palatability based on food consumption and body weight. Comparing to SD, KE supplemented diet reduced plasma glucose (145.5 ± 5.3 vs 121.7 ± 5.7), increased ketone bodies [ß-hydroxybutarate, 0.7 ± 0.15 vs.1.3 ± 0.1] and enhanced median survival [47 ± 6.2 vs 60.8 ± 1.9, days]. CONCLUSION: Ketone esters can be effectively delivered orally together with a standard diet, and produce similar physiological changes [reduction in glucose and elevation in ketones] and enhance survival as the more restrictive KD in NON/SCID animals.

Neural stem cell (NSC) culture is a remarkable tool to investigate the potential therapeutic benefits of drugs in neurological diseases. The purpose of this study was to determine the effect of melatonin on proliferation and differentiation of NSCs in vitro. NSCs were isolated and expanded from mouse embryonic E14 cortex, and the effect of various concentrations of melatonin (0.05, 0.1, 0.5, 1, 5 and 10 μM) on NSC proliferation was assessed by MTT and neurosphere assay. Results showed that melatonin significantly increased NSC viability and NSC proliferation in a dose-dependent manner, in comparison to controls. Similarly, neurosphere formation frequency and cell counts increased significantly with increasing melatonin concentrations and reached its peak at 0.5 μM, in comparison to controls. Moreover, NSCs treated with either low (0.05 µM) or high concentrations (5 µM) of melatonin showed that the mean percentage of glial fibrillary acidic protein (GFAP) positive cells were not significantly different in PDGF or melatonin at 5 μM, in comparison to controls. However, low melatonin concentrations (0.05 µM) showed a slight significant increase in comparison to controls and PDGF. On the other hand, both concentrations of melatonin treatment significantly increased the percentage of myelin basic protein (MBP) positive cells (oligodendrocytes), in comparison to controls and to PDGF. Our results demonstrated, for the first time, that melatonin increased oligodendrocyte differentiation from NSCs. These results suggest that melatonin might have a potential therapeutic effect for some neurological diseases that involve oligodendrocyte and myelin pathologies.

Traumatic brain injury (TBI) is one of the leading causes of death and disabilities worldwide. It affects approximately 1.5 million people each year and is associated with severe post-TBI symptoms such as sensory and motor deficits. Several neuro-therapeutic approaches ranging from cell therapy interventions such as the use of neural stem cells (NSCs) to drug-based therapies have been proposed for TBI management. Successful cell-based therapies are tightly dependent on reproducible preclinical animal models to ensure safety and optimal therapeutic benefits. In this chapter, we describe the isolation of NSCs from neonatal mouse brain using the neurosphere assay in culture. Subsequently, dissociated neurosphere-derived cells are used for transplantation into the ipsilateral cortex of a controlled cortical impact (CCI) TBI model in C57BL/6 mice. Following intra-cardiac perfusion and brain removal, the success of NSC transplantation is then evaluated using immunofluorescence in order to assess neurogenesis along with gliosis in the ipsilateral coronal brain sections. Behavioral tests including rotarod and pole climbing are conducted to evaluate the motor activity post-treatment intervention.

Despite all attempts the problem of regeneration in damaged central nervous system (CNS) has remained challenging due to its cellular complexity and highly organized and sophisticated connections. In this regard, stem cell therapy might serve as a viable therapeutic approach aiming either to support the damaged tissue and hence to reduce the subsequent neurological dysfunctions and impairments or to replace the lost cells and re-establish damaged circuitries. Adult neural stem/progenitor cells (NS/PCs) are one of the outstanding cell sources that can be isolated from the subventricular zone (SVZ) of the lateral ventricles. These cells can differentiate into neurons, astrocytes, and oligodendrocytes. Implanting autologous NS/PCs will greatly benefit the patients by avoiding immune rejection after implantation, better survival, and integration with the host tissue. Developing safe and efficient methods in small animal models will provide us with the opportunity to optimize procedures required to achieve successful human autologous NS/PC transplantation in near future. In this chapter, a highly controlled and safe biopsy method for harvesting stem cell containing tissue from the SVZ of adult rat brain is introduced. Then, isolation and expansion of NS/PCs from harvested specimen as well as the techniques to verify proliferation and differentiation capacity of the resulting NS/PCs are discussed. Finally, a method for assessing the biopsy lesion volume in the brain is described. This safe biopsy method in rat provides a unique tool to study autologous NS/PC transplantation in different CNS injury models.

Many neurological injuries are likely too extensive for the limited repair capacity of endogenous neural stem cells (NSCs). An alternative is to isolate NSCs from a donor and expand them in vitro as transplantation material. Numerous groups have already transplanted neural stem and precursor cells. A caveat to this approach is the undefined phenotypic distribution of the donor cells, which has three principle drawbacks: (1) Stem-like cells retain the capacity to proliferate in vivo. (2) There is little control over the cells’ terminal differentiation, e.g., a graft intended to replace neurons might choose a predominantly glial fate. (3) There is limited ability of researchers to alter the combination of cell types in pursuit of a precise treatment. We demonstrate a procedure for differentiating human neural precursor cells (hNPCs) in vitro, followed by isolation of the neuronal progeny. We transplanted undifferentiated hNPCs or a defined concentration of hNPC-derived neurons into mice, then compared these two groups with regard to their survival, proliferation and phenotypic fate. We present evidence suggesting that in vitro-differentiated-and-purified neurons survive as well in vivo as their undifferentiated progenitors and undergo less proliferation and less astrocytic differentiation. We also describe techniques for optimizing low-temperature cell preservation and portability.

Stroke is one of the leading causes of morbidity and mortality in developed countries and an immense amount of medical care resources are devoted to combat the poststroke debilitating consequences. The key to develop effective and clinically applicable treatment methodologies is a better understanding of the pathophysiology of the disease, including the root causes and targets for pharmacology. Developing these foundations requires the use of standard animal models that mimic the physicochemical process of the diseases that can reliably replicate results in order to test and fine-tune therapeutic modalities. Middle cerebral artery occlusion (MCAO), endothelin-1-induced ischemic stroke, photothrombosis, devascularization, embolization, and spontaneous infarction using hemorrhage are some examples of different animal models. Reliability of MCAO has been proved and due to the ability to induce reperfusion similar to tissue plasminogen activator (tPA) therapy, this model is widely used in preclinical studies. Here, we describe a detailed methodology on how to develop MCAO stroke in rodents using intra-arterial insertion of filament to occlude the middle cerebral artery. This approach allows for the study of a wide array of basic pathophysiology mechanisms, regenerative medicine and rehabilitation therapy.

The co-occurrence of several anomalies in the hepatobiliary system is uncommon. In the present study, hepatic lobe anomalies occurring in combination with hepatic artery and biliary variations were observed in an adult male cadaver. There are no previous reports in the literature on the coexistence of such anatomical variations. Preoperative diagnosis of such coexisting anomalies is very difficult. Hence, a thorough knowledge of these variations will enable surgeons to select the most appropriate hepatobiliary surgical procedure and postoperative management.

There exists substantial evidence that a peridural membrane (PM) is present in the spinal canal of humans and, like the pleura and peritoneum, has one or more physiologic functions. Innervation of the PM suggests that it may become a source of pain if injured. Although debated, the physiology of this structure has important implications with respect to neuraxial distribution of drugs and for back and radiating pain. This review, separated into embryological, anatomic, and physiologic discussions, provides an in-depth summary of the observations of this connective tissue. The discrepancies between accounts are highlighted within each section. Focused research to clearly elucidate the true nature of the PM, especially as related to neuraxial distribution of drugs and back and radiating pain, is warranted.

Individual tumour cells display diverse functional behaviours in terms of proliferation rate, cell-cell interactions, metastatic potential and sensitivity to therapy. Moreover, sequencing studies have demonstrated surprising levels of genetic diversity between individual patient tumours of the same type. Tumour heterogeneity presents a significant therapeutic challenge as diverse cell types within a tumour can respond differently to therapies, and inter-patient heterogeneity may prevent the development of general treatments for cancer. One strategy that may help overcome tumour heterogeneity is the identification of tumour sub-populations that drive specific disease pathologies for the development of therapies targeting these clinically relevant sub-populations. Here, we have identified a dye-retaining brain tumour population that displays all the hallmarks of a tumour-initiating sub-population. Using a limiting dilution transplantation assay in immunocompromised mice, label-retaining brain tumour cells display elevated tumour-initiation properties relative to the bulk population. Importantly, tumours generated from these label-retaining cells exhibit all the pathological features of the primary disease. Together, these findings confirm dye-retaining brain tumour cells exhibit tumour-initiation ability and are therefore viable targets for the development of therapeutics targeting this sub-population.

Based on the clonal evolution model and the assumption that the vast majority of tumor cells are able to propagate and drive tumor growth, the goal of cancer treatment has traditionally been to kill all cancerous cells. This theory has been challenged recently by the cancer stem cell (CSC) hypothesis, that a rare population of tumor cells, with stem cell characteristics, is responsible for tumor growth, resistance, and recurrence. Evidence for putative CSCs has been described in blood, breast, lung, prostate, colon, liver, pancreas, and brain. This new hypothesis would propose that indiscriminate killing of cancer cells would not be as effective as selective targeting of the cells that are driving long-term growth (ie, the CSCs) and that treatment failure is often the result of CSCs escaping traditional therapies. The CSC hypothesis has gained a great deal of attention because of the identification of a new target that may be responsible for poor outcomes of many aggressive cancers, including malignant glioma. As attractive as this hypothesis sounds, especially when applied to tumors that respond poorly to current treatments, we will argue in this article that the proposal of a stemlike cell that initiates and drives solid tissue cancer growth and is responsible for therapeutic failure is far from proven. We will present the point of view that for most advanced solid tissue cancers such as glioblastoma multiforme, targeting a putative rare CSC population will have little effect on patient outcomes. This review will cover problems with the CSC hypothesis, including applicability of the hierarchical model, inconsistencies with xenotransplantation data, and nonspecificity of CSC markers.

Despite all attempts the problem of regeneration in damaged central nervous system (CNS) has remained challenging due to its cellular complexity and highly organized and sophisticated connections. In this regard, stem cell therapy might serve as a viable therapeutic approach aiming either to support the damaged tissue and hence to reduce the subsequent neurological dysfunctions and impairments or to replace the lost cells and re-establish damaged circuitries. Adult neural stem/progenitor cells (NS/PCs) are one of the outstanding cell sources that can be isolated from the subventricular zone (SVZ) of the lateral ventricles. These cells can differentiate into neurons, astrocytes, and oligodendrocytes. Implanting autologous NS/PCs will greatly benefit the patients by avoiding immune rejection after implantation, better survival, and integration with the host tissue. Developing safe and efficient methods in small animal models will provide us with the opportunity to optimize procedures required to achieve successful human autologous NS/PC transplantation in near future. In this chapter, a highly controlled and safe biopsy method for harvesting stem cell containing tissue from the SVZ of adult rat brain is introduced. Then, isolation and expansion of NS/PCs from harvested specimen as well as the techniques to verify proliferation and differentiation capacity of the resulting NS/PCs are discussed. Finally, a method for assessing the biopsy lesion volume in the brain is described. This safe biopsy method in rat provides a unique tool to study autologous NS/PC transplantation in different CNS injury models.

Cultivation of neural stem/progenitor cells (NS/PCs) in PuraMatrix (PM) hydrogel is an option for stem cell transplantation. The efficacy of a novel method for placing adult rat NS/PCs in PM (injection method) was compared to encapsulation and surface plating approaches. In addition, the efficacy of injection method for transplantation of autologous NS/PCs was studied in a rat model of brain injury. NS/PCs were obtained from the subventricular zone (SVZ) and cultivated without (control) or with scaffold (three-dimensional cultures; 3D). The effect of different approaches on survival, proliferation, and differentiation of NS/PCs were investigated. In in vivo study, brain injury was induced 45 days after NS/PCs were harvested from the SVZ and phosphate buffered saline, PM, NS/PCs, or PM+NS/PCs were injected into the brain lesion. There was an increase in cell viability and proliferation after injection and surface plating of NS/PCs compared to encapsulation and neural differentiation markers were expressed seven days after culturing the cells. Using injection method, transplantation of NS/PCs cultured in PM resulted in significant reduction of lesion volume, improvement of neurological deficits, and enhancement of surviving cells. In addition, the transplanted cells could differentiate in to neurons, astrocytes, or oligodendrocytes. Our results indicate that the injection and surface plating methods enhanced cell survival and proliferation of NS/PCs and suggest the injection method as a promising approach for transplantation of NS/PCs in brain injury.

Patients with psychiatric disorders exhibit several neurobehavioral and neuropsychological alterations compared to healthy controls. However, signature endpoints of these behavioral manifestations have not yet been translated into clinical tests for diagnosis and follow-up measures. Recently, neuroproteomic approaches have been utilized to identify unique signature markers indicative of these disorders. Development of reliable biomarkers has the potential to revolutionize the diagnosis, classification, and monitoring of clinical responses in psychiatric diseases. However, the lack of biological gold standards, the evolving nosology of psychiatric disorders, and the complexity of the nervous system are among the major challenges that have hindered efforts to develop reliable biomarkers in the field of neuropsychiatry and drug abuse. While biomarkers currently have a limited role in the area of neuropsychiatry, several promising biomarkers have been proposed in conditions such as dementia, schizophrenia, depression, suicide, and addiction. One of the primary objectives of this review is to discuss the role of proteomics in the development of biomarkers specific to neuropsychiatry. We discuss and evaluate currently available biomarkers as well as those that are under research for clinical use in the future.