Motor Recovery after Transplantation of Bone Marrow Mesenchymal Stem Cells in Rat Models of Spinal Cord Injury.

BACKGROUND: Neuronal tissue has a limited potential to self-renew or get repaired after damage. Cell therapies using stem cells are promising approaches for the treatment of central nervous system (CNS) injuries. However, the clinical use of embryonic stem cells is limited by ethical concerns and other scientific consequences. Bone marrow mesenchymal stromal cells (BM-MSC) could represent an alternative source of stem cells for replacement therapy. Indeed, many studies have demonstrated that MSCs can give rise to neuronal cells as well as many tissue-specific cell phenotypes. PURPOSE: Motor recovery by transplantation of bone marrow MSCs in rat models of spinal cord injury (SCI). METHODS: Bone marrow was collected from the femur of albino Wistar rats. MSCs were separated using the Ficoll-Paque density gradient method and cultured in Dulbecco's Modified Eagle Medium supplemented with 20% fetal bovine serum. Cultured MSC was characterized by immunohistochemistry and flow cytometry and neuronal-induced cells were further characterized for neural markers. Cultured MSCs were transplanted into the experimentally injured spinal cord of Wistar rats. Control (injured, but without cell transplantation) and transplanted rats were followed up to 8 weeks, analyzed using the Basso, Beattie, Bresnahan (BBB) scale and electromyography (EMG) for behavioral and physiological status of the injured spinal cord. Finally, the tissue was evaluated histologically. RESULTS: Rat MSCs expressed positivity for a panel of MSC markers CD29, CD54, CD90, CD73, and CD105, and negativity for hematopoietic markers CD34, CD14, and CD45. In vitro neuronal transdifferentiated MSCs express positivity for ß III tubulin, MAP2, NF, NeuN, Nav1.1, oligodendrocyte (O4), and negativity for glial fibrillary acid protein. All the treated groups show promising hind-limb motor recovery BBB score, except the control group. There was increased EMG amplitude in treated groups as compared to the control group. Green fluorescent protein (GFP)-labeled MSC survived and differentiated into neurons in the injured spinal cord, which is responsible for functional recovery. CONCLUSION: Our results demonstrate that BM-MSC has the potential to repair the injured cord in rat models of SCI. Thus, BM-MSC appears to be a promising candidate for cell-based therapy in CNS injury.

Globose basal cells for spinal cord regeneration.

Spinal cord injury (SCI) is a devastating condition with loss of motor and sensory functions below the injury level. Cell based therapies are experimented in pre-clinical studies around the world. Neural stem cells are located intra-cranially in subventricular zone and hippocampus which are highly invasive sources. The olfactory epithelium is a neurogenic tissue where neurogenesis takes place throughout the adult life by a population of stem/progenitor cells. Easily accessible olfactory neuroepithelial stem/progenitor cells are an attractive cell source for transplantation in SCI. Globose basal cells (GBCs) were isolated from rat olfactory epithelium, characterized by flow cytometry and immunohistochemically. These cells were further studied for neurosphere formation and neuronal induction. T10 laminectomy was done to create drop-weight SCI in rats. On the 9th day following SCI, 5 × 105 cells were transplanted into injured rat spinal cord. The outcome of transplantation was assessed by the Basso, Beattie and Bresnahan (BBB) locomotor rating scale, motor evoked potential and histological observation. GBCs expressed neural stem cell markers nestin, SOX2, NCAM and also mesenchymal stem cell markers (CD29, CD54, CD90, CD73, CD105). These cells formed neurosphere, a culture characteristics of NSCs and on induction, differentiated cells expressed neuronal markers ßIII tubulin, microtubule-associated protein 2, neuronal nuclei, and neurofilament. GBCs transplanted rats exhibited hindlimb motor recovery as confirmed by BBB score and gastrocnemius muscle electromyography amplitude was increased compared to controls. Green fluorescent protein labelled GBCs survived around the injury epicenter and differentiated into ßIII tubulin-immunoreactive neuron-like cells. GBCs could be an alternative to NSCs from an accessible source for autologous neurotransplantation after SCI without ethical issues.

Immunohistological and electrophysiological characterization of Globose basal stem cells.

OBJECTIVES: In the past few decades, variety of foetal, embryonic and adult stem and progenitor cells have been tried with conflicting outcome for cell therapy of central nervous system injury and diseases. Cellular characteristics and functional plasticity of Globose basal stem cells (GBCs) residing in the olfactory epithelium of rat olfactory mucosa have not been studied in the past by the neuroscientists due to unavailability of specific markers for GBCs. In the present research, we standardized some techniques to isolate GBCs from rat olfactory epithelium in pure form using a highly selective GBC-III antibody passaged through fluorescence activated cell sorter (FACS). We also characterized these cells immunohistologically using various pluripotent stem cell markers. This work also throws some light on ionic channels present on these stem cells which are responsible for their neuron induction potential. MATERIALS AND METHODS: Globose basal stem cells were isolated from rat olfactory epithelium using GBC-III antibody and were characterized as multipotent stem cells using various neural progenitor markers. Ionic channels on GBCs were studied with voltage clamping. RESULTS: GBCs could be isolated in pure (99% purity) form and were found to be stained positive for all neural progenitor cell markers. Voltage gated Na(+) channels were completely absent, which proves the unexcitable nature of GBCs. Leaky K(+) channels were found to be present on the GBC which was of no significance. CONCLUSION: This research work can be helpful in understanding the nature of these stem cells and utilising them in future as potent candidates for neuro-regenerative therapies.

A preclinical study on the protective effect of melatonin against methotrexate-induced small intestinal damage: effect mediated by attenuation of nitrosative stress, protein tyrosine nitration, and PARP activation.

PURPOSE: One of the major toxic side effects of methotrexate (MTX) is enterocolitis. To date, there is no efficient standard treatment for this side effect. Nitrosative stress is reported to play a critical role in MTX-induced mucositis. The purpose of this study is to investigate whether pretreatment with melatonin, an inhibitor of nitro-oxidative stress, prevents MTX-induced mucositis in rats. METHODS: Rats were pretreated with melatonin (20 and 40 mg/kg body weight) i.p. daily 1 h before MTX (7 mg/kg body weight) administration for three consecutive days. After the final dose of MTX, the rats were killed and the small intestines were used for analysis. RESULTS: The small intestines of MTX-treated rats showed moderate to severe injury. The villi were distorted, blunted, and atrophied and focally absent in various segments of the small intestines. Crypt abscesses were also found, suggesting an inflammatory response. Pretreatment with melatonin had a dose-dependent protective effect on MTX-induced mucositis. Morphology was saved to a moderate extent with 20 mg melatonin pretreatment, and near-normal morphology was achieved with 40 mg melatonin pretreatment. Damage to the villi and crypt abscess was reduced. The villi/crypt ratio was almost restored. Melatonin pretreatment protected the small intestines from MTX-induced damage by attenuating nitrosative stress, protein tyrosine nitration and PARP expression. CONCLUSION: Because of its versatility in protecting against nitro-oxidative stress and reducing inflammation, we suggest that melatonin could be beneficial in ameliorating MTX-induced enteritis in humans.

Isolating globose Basal stem cells from albino wistar rats using a highly specific monoclonal antibody.

INTRODUCTION: Olfactory mucosa which is situated in the roof of the nasal cavity possesses an extremely peculiar and exceptional type of pluripotent stem cells called Globose Basal Cells (GBCs) which help in lifelong regeneration of the olfactory mucosa. Previous literature doesn't provide much knowledge on the cytological, histochemical and electrophysiological properties of these cells, as they have never been isolated in pure form. MATERIAL AND METHODS: Olfactory mucosa was obtained from six Albino Wistar rats by using standardized surgical and chemical separation procedures. GBCs were isolated by using different chemical, surgical and fluorescent techniques. RESULTS: In this research work, we standardized the techniques for isolating these stem cells in pure form from rat olfactory mucosa by tagging them with GBC-III antibody and separating them from other epithelial cells by using Fluorescence Activated Cell Sorter (FACS). GBC-III antibody is a mouse monoclonal IgM antibody which recognizes a 40 kDa surface antigen, which is a laminin receptor surface protein present on the GBCs. It is a highly specific marker for GBCs, unlike the earlier antibodies used, like GBC-I, which were nonspecific markers for GBCs and showed positive reactions, even with Horizontal Basal Cells (HBCs), sustentacular cells (Sus) and duct cells. This study also standardized the techniques for surgically excising the olfactory mucosa from the nasal septum and chemically separating the olfactory epithelium from the lamina propria. CONCLUSION: GBCs are an important group of cells which can be exploited in future to study and treat neuro-degenerative disorders like multiple sclerosis, brain ischaemia, etc. and spinal cord trauma, as they reside in a niche similar to the microenvironment in the central nervous system and have the similar ectodermal development as the neuronal and non-neuronal cells of the CNS. Moreover, olfactory epithelium is easily accessible for autologous transplantation of GBCs for different CNS disorders.

Propylthiouracil attenuates acetaminophen-induced renal damage in the rat.

BACKGROUND: To date, there is no specific antidote to acetaminophen poisoning. Propylthiouracil (PTU) has been shown to be protective against acetaminophen (APAP)-induced liver damage in rats; however, the nephroprotective effect of propylthiouracil has not been studied yet. METHODS: In order to verify this, rats were given different doses of PTU (100, 200 or 400 mg/kg per body weight, orally) 1 h before a nephrotoxic dose of APAP (1,000 mg/kg per body weight, intraperitoneally (i.p.)). RESULTS: Propylthiouracil pretreatment significantly reduced APAP-induced nephrotoxicity in a dose-dependant manner, as evidenced by reduction in plasma creatinine and by amelioration of renal pathology (interstitial congestion, tubular cell degeneration and necrosis). CONCLUSION: The mechanism of protection by PTU is probably not due to the sparing effect of non-protein thiol (approximately 95% of which is reduced glutathione), as similar depletion of renal glutathione was observed regardless of PTU pretreatment; other mechanisms are suggested.