pkh-26 and Disease-Models--Animal

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (hMSCs) have great promise as biologics to treat neurological and neurodegenerative conditions due to their robust antiinflammatory and neuroprotective properties. Besides, intranasal (IN) administration of EVs has caught much attention because the procedure is noninvasive, amenable for repetitive dispensation, and leads to a quick penetration of EVs into multiple regions of the forebrain. Nonetheless, it is unknown whether brain injury-induced signals are essential for the entry of IN-administered EVs into different brain regions. Therefore, in this study, we investigated the distribution of IN-administered hMSC-derived EVs into neurons and microglia in the intact and status epilepticus (SE) injured rat forebrain. Ten billion EVs labeled with PKH26 were dispensed unilaterally into the left nostril of naïve rats, and rats that experienced two hours of kainate-induced SE. Six hours later, PKH26 + EVs were quantified from multiple forebrain regions using serial brain sections processed for different neural cell markers and confocal microscopy. Remarkably, EVs were seen bilaterally in virtually all regions of intact and SE-injured forebrain. The percentage of neurons incorporating EVs were comparable for most forebrain regions. However, in animals that underwent SE, a higher percentage of neurons incorporated EVs in the hippocampal CA1 subfield and the entorhinal cortex, the regions that typically display neurodegeneration after SE. In contrast, the incorporation of EVs by microglia was highly comparable in every region of the forebrain measured. Thus, unilateral IN administration of EVs is efficient for delivering EVs bilaterally into neurons and microglia in multiple regions in the intact or injured forebrain. Furthermore, incorporation of EVs by neurons is higher in areas of brain injury, implying that injury-related signals likely play a role in targeting of EVs into neurons, which may be beneficial for EV therapy in various neurodegenerative conditions including traumatic brain injury, stroke, multiple sclerosis, and Alzheimer's disease.

Topics: Administration, Intranasal; Animals; Cells, Cultured; Disease Models, Animal; Extracellular Vesicles; Humans; Male; Mesenchymal Stem Cells; Organic Chemicals; Prosencephalon; Rats; Status Epilepticus; Treatment Outcome

Effective therapy of Acute Lung Injury (ALI) is still a major scientific and clinical problem. To define novel therapeutic strategies for sequelae of blunt chest trauma (TxT) like ALI/Acute Respiratory Distress Syndrome, we have investigated the immunomodulatory and regenerative effects of a single dose of ex vivo expanded human or rat mesenchymal stromal cells (hMSCs/rMSCs) with or without priming, immediately after the induction of TxT in Wistar rats.. We analyzed the histological score of lung injury, the cell count of the broncho alveolar lavage fluid (BAL), the change in local and systemic cytokine level and the recovery of the administered cells 24 h and 5 days post trauma.. The treatment with hMSCs reduced the injury score 24 h after trauma by at least 50% compared with TxT rats without MSCs. In general, TxT rats treated with hMSCs exhibited a lower level of pro-inflammatory cytokines (interleukin [IL]-1B, IL-6) and chemokines (C-X-C motif chemokine ligand 1 [CXCL1], C-C motif chemokine ligand 2 [CCL2]), but a higher tumor necrosis factor alpha induced protein 6 (TNFAIP6) level in the BAL compared with TxT rats after 24 h. Five days after trauma, cytokine levels and the distribution of inflammatory cells were similar to sham rats. In contrast, the treatment with rMSCs did not reveal such therapeutic effects on the injury score and cytokine levels, except for TNFAIP6 level.. TxT represents a suitable model to study effects of MSCs as an acute treatment strategy after trauma. However, the source of MSCs has to be carefully considered in the design of future studies.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Count; Cell Shape; Cytokines; Disease Models, Animal; Humans; Lung; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Organic Chemicals; Rats; Rats, Wistar; Thoracic Injuries; Transplantation, Heterologous; Transplantation, Homologous; Wounds, Nonpenetrating

Spinal cord injury (SCI) is a debilitating condition characterized by a complex of neurological dysfunctions ranging from loss of sensation to partial or complete limb paralysis. Recently, we reported that intravenous administration of neural precursors physiologically releasing erythropoietin (namely Er-NPCs) enhances functional recovery in animals following contusive spinal cord injury through the counteraction of secondary degeneration. Er-NPCs reached and accumulated at the lesion edges, where they survived throughout the prolonged period of observation and differentiated mostly into cholinergic neuron-like cells.. The aim of this study was to investigate the potential reparative and regenerative properties of Er-NPCs in a mouse experimental model of traumatic spinal cord injury.. We report that Er-NPCs favoured the preservation of axonal myelin and strongly promoted the regrowth across the lesion site of monoaminergic and chatecolaminergic fibers that reached the distal portions of the injured cord. The use of an anterograde tracer transported by the regenerating axons allowed us to assess the extent of such a process. We show that axonal fluoro-ruby labelling was practically absent in saline-treated mice, while it resulted very significant in Er-NPCs transplanted animals.. Our study shows that Er-NPCs promoted recovery of function after spinal cord injury, and that this is accompanied by preservation of myelination and strong re-innervation of the distal cord. Thus, regenerated axons may have contributed to the enhanced recovery of function after SCI.

Topics: Animals; Choline O-Acetyltransferase; Dextrans; Disease Models, Animal; Erythropoietin; Fluorescent Dyes; GAP-43 Protein; Locomotion; Male; Mice; Microtubule-Associated Proteins; Myelin Sheath; Nerve Regeneration; Organic Chemicals; Recovery of Function; Rhodamines; Serotonin; Spinal Cord Injuries; Stem Cell Transplantation; Tubulin; Tyrosine 3-Monooxygenase

Alzheimer's disease (AD) is characterized by extracellular beta-amyloid plaques and intracellular tau tangles. AD-related pathology is often accompanied by vascular changes. The predominant vascular lesions in AD are cerebral amyloid angiopathy (CAA) and arteriosclerosis. Platelets circulate along the vessel wall responding immediately to vascular injury. The aim of the present study was to explore the presence and migration of platelets (thrombocytes) to sites of small vascular bleedings and/or to beta-amyloid plaques in the brain. We infused fluorescently labeled red PKH26 mouse platelets into transgenic Alzheimer mice overexpressing APP with Swedish/Dutch/Iowa mutations (APP_SDI) and explored if platelets migrate into the brain. Further we studied whether platelets accumulate in the vicinity of β-amyloid plaques. Our animal data shows that infused platelets are found in the liver and partly in the lung, while in the brain platelets were visible to a minor degree. In mice, we did not observe a significant association of platelets with beta-amyloid plaques or vessels. In the brain of Alzheimer postmortem patients platelets could be detected by immunohistochemistry for CD41 and CD62P, but the majority was found in vessels with or without beta-amyloid load, and only a few single platelets migrated deeper into the brain. Our findings suggest that platelets do not migrate into the brains of Alzheimer disease but are concentrated in brain vessels.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Blood Platelets; Brain; Cerebral Amyloid Angiopathy; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Female; Flow Cytometry; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Organic Chemicals; P-Selectin; Platelet Membrane Glycoprotein IIb

Fetal kidney cells may contain multiple populations of kidney stem cells and thus appear to be a suitable cellular therapy for the treatment of acute renal failure (ARF) but their biological characteristics and therapeutic potential have not been adequately explored. We have culture expanded fetal kidney cells derived from rat fetal kidneys, characterized them and evaluated their therapeutic effect in an ischemia reperfusion (IR) induced rat model of ARF. The fetal kidney cells grew in culture as adherent spindle shaped/polygonal cells and expressed CD29, CD44, CD73, CD90, CD105, CD24 and CD133 markers. Administration of PKH26 labeled fetal kidney cells in ARF rats resulted in a significant decrease in the levels of blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin and decreased tubular necrosis in the kidney tissues (p<0.05 for all). The injected fetal kidney cells were observed to engraft around injured tubular cells, and there was increased proliferation and decreased apoptosis of tubular cells in the kidneys (p<0.05 for both). In addition, the kidney tissues of ARF rats treated with fetal kidney cells had a higher gene expression of renotropic growth factors (VEGF-A, IGF-1, BMP-7 and bFGF) and anti-inflammatory cytokine (IL10); up regulation of anti-oxidative markers (HO-1 and NQO-1); and a lower Bax/Bcl2 ratio as compared to saline treated rats (p<0.05 for all). Our data shows that culture expanded fetal kidney cells express mesenchymal and renal progenitor markers, and ameliorate ischemic ARF predominantly by their anti-apoptotic, anti-inflammatory and anti-oxidative effects.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cell Culture Techniques; Cell Separation; Cells, Cultured; Culture Media, Conditioned; Disease Models, Animal; Fetus; Fluorescent Dyes; Growth Substances; Immunophenotyping; Inflammation Mediators; Kidney; Organic Chemicals; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Stem Cell Transplantation

During respiratory inflammation, it is generally assumed that dendritic cells differentiating from the bone marrow are immunogenic rather than immunoregulatory. Using chimeric mice, the outcomes of airways inflammation on bone marrow progenitor cells were studied.. Immune responses were analyzed in chimeric mice engrafted for >16 weeks with bone marrow cells from mice with experimental allergic airways disease (EAAD).. Responses to sensitization and challenge with the allergen causing inflammation in the bone marrow-donor mice were significantly reduced in the chimeric mice engrafted with bone marrow cells from mice with EAAD (EAAD-chimeric). Responses to intranasal LPS and topical fluorescein isothiocyanate (non-specific challenges) were significantly attenuated. Fewer activated dendritic cells from the airways and skin of the EAAD-chimeric mice could be tracked to the draining lymph nodes, and may contribute to the significantly reduced antigen/chemical-induced hypertrophy in the draining nodes, and the reduced immune responses to sensitizing allergens. Dendritic cells differentiating in vitro from the bone marrow of >16 weeks reconstituted EAAD-chimeric mice retained an ability to poorly prime immune responses when transferred into naïve mice.. Dendritic cells developing from bone marrow progenitors during airways inflammation are altered such that daughter cells have reduced antigen priming capabilities.

Topics: Administration, Intranasal; Adoptive Transfer; Animals; Azacitidine; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; Cell Count; Decitabine; Dendritic Cells; Disease Models, Animal; DNA Modification Methylases; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Immunoglobulin E; Immunoglobulin G; Inflammation; Lipopolysaccharides; Lymph Nodes; Mice, Inbred C57BL; Organic Chemicals; Ovalbumin; Radiation Chimera; Respiratory Hypersensitivity; Skin

When introduced into the infarcted heart, bone marrow‑derived mesenchymal stem cells (MSCs) prevent the heart from deleterious remodeling and improve its recovery. The aim of the present study was to investigate the effects of Ginkgo biloba extract (EGb) 761 on the infarcted myocardium microenvironment following MSC transplantation. The established rat myocardial infarction (MI) model, with implanted PKH‑26 marked MSCs (1x105 cells), were randomly divided into two groups: The control group (injected with normal saline) and the EGb 761 treatment group (injected with 100 mg/kg/day EGb 761). The following indices for cardiac function, including the extent of inflammation, oxidative stress, MSC apoptosis and MSC differentiation were measured 1, 2 and 7 days after treatment. The anti‑inflammatory effect of EGb 761 was observed by histological examination. Compared with the respective control group, the malondialdehyde content significantly decreased and the superoxide dismutase, catalase and glutathione peroxidase activity significantly increased in the EGb761‑treated groups. In addition, the apoptotic index gradually decreased (P<0.05) with the extension of MI time in the EGb761-treated groups compared to the respective control groups, suggesting that EGb761 exhbits anti-oxidative effects. In addition, the level of the Fas protein was positively correlated with the implanted MSC apoptotic ratio. Following 7 days of MSC transplantation with EGb 761 treatment, the expression of cTnI in PKH26‑labeled MSCs was observed in the transplanted myocardium. Cardiac function, including the ejection fraction, left ventricular end‑systolic pressure and dp/dtmax significantly increased, and the left ventricular end diastolic diameters, left ventricular end‑diastolic volumes and left ventricular end‑diastolic pressure significantly decreased (P<0.05, vs. the control group). The results demonstrated that EGb 761 is important in improving cardiac function and the infarcted myocardium microenvironment. The present study indicated that the protective effects of EGb 761 on the infarcted myocardium may be mediated by improving the viability and the differentiation of the implanted MSCs into cardiomyocytes.

Topics: Animals; Apoptosis; Biomarkers; Blotting, Western; Cardiotonic Agents; Cell Differentiation; Cell Survival; Disease Models, Animal; Electrocardiography; fas Receptor; Ginkgo biloba; Inflammation; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myocardial Infarction; Myocardium; Organic Chemicals; Oxidation-Reduction; Oxidative Stress; Plant Extracts; Rats; Rats, Sprague-Dawley; Ultrasonography

In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model.. Osteoarthritis was induced at the right knee of sheep by complete resection of the anterior cruciate ligament and medial meniscus following a 3-weeks exercise regimen. Stem cells from experimental sheep were culture expanded and induced to chondrogenic lineage. Test sheep received a single dose of 2 × 10(7) autologous PKH26-labelled, chondrogenically induced ADSCs or BMSCs as 5 mls injection, while controls received 5 mls culture medium.. The proliferation rate of ADSCs 34.4 ± 1.6 hr was significantly higher than that of the BMSCs 48.8 ± 5.3 hr (P = 0.008). Chondrogenic induced BMSCs had significantly higher expressions of chondrogenic specific genes (Collagen II, SOX9 and Aggrecan) compared to chondrogenic ADSCs (P = 0.031, 0.010 and 0.013). Grossly, the treated knee joints showed regenerated de novo cartilages within 6 weeks post-treatment. On the International Cartilage Repair Society grade scores, chondrogenically induced ADSCs and BMSCs groups had significantly lower scores than controls (P = 0.0001 and 0.0001). Fluorescence of the tracking dye (PKH26) in the injected cells showed that they had populated the damaged area of cartilage. Histological staining revealed loosely packed matrixes of de novo cartilages and immunostaining demonstrated the presence of cartilage specific proteins, Collagen II and SOX9.. Autologous chondrogenically induced ADSCs and BMSCs could be promising cell sources for cartilage regeneration in osteoarthritis.

Topics: Adipose Tissue; Adult Stem Cells; Animals; Arthroscopy; Bone Marrow Cells; Cartilage; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Separation; Cell Survival; Cell Tracking; Cell- and Tissue-Based Therapy; Chondrocytes; Chondrogenesis; Disease Models, Animal; Fluorescent Dyes; Male; Organic Chemicals; Osteoarthritis; Regeneration; Sheep

The current study is based on the hypothesis of mesenchymal stromal cells (MSCs) contributing to soft-tissue calcification and ectopic osteogenesis in chronic kidney disease (CKD). Rat MSCs were transplanted intraperitoneally in an established three-dimensional collagen-based model in healthy control animals and two rat models of CKD and vascular calcification: (1) 5/6 nephrectomy + high phosphorus diet; and (2) adenine nephropathy. As internal controls, collagen gels without MSCs were transplanted in the same animals. After 4 and 8 weeks, MSCs were still detectable and proliferating in the collagen gels (fluorescence-activated cell sorting [FACS] analysis and confocal microscopy after fluorescence labeling of the cells). Aortas and MSC-containing collagen gels in CKD animals showed distinct similarities in calcification (micro-computed tomography [µCT], energy-dispersive X-ray [EDX] analysis, calcium content), induction of osteogenic markers, (ie, bone morphogenic protein 2 [BMP-2], Runt related transcription factor 2 [Runx2], alkaline phosphatase [ALP]), upregulation of the osteocytic marker sclerostin and extracellular matrix remodeling with increased expression of osteopontin, collagen I/III/IV, fibronectin, and laminin. Calcification, osteogenesis, and matrix remodeling were never observed in healthy control animals and non-MSC-containing collagen gels in all groups. Paul Karl Horan 26 (PKH-26)-labeled, 3G5-positive MSCs expressed Runx2 and sclerostin in CKD animals whereas PKH-26-negative migrated cells did not express osteogenic markers. In conclusion, heterotopically implanted MSCs undergo osteogenic differentiation in rat models of CKD-induced vascular calcification, supporting our hypothesis of MSCs as possible players in heterotopic calcification processes of CKD patients.

Topics: Adenine; Animals; Aorta; Biomarkers; Bone Morphogenetic Proteins; Calcification, Physiologic; Cell Differentiation; Cell Movement; Collagen; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Extracellular Matrix; Gels; Gene Expression Regulation; Genetic Markers; Kidney Function Tests; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Nephrectomy; Organic Chemicals; Osteogenesis; Pericytes; Rats; Renal Insufficiency, Chronic; Transplantation, Heterotopic; Up-Regulation

To investigate the migration of fluorescent dye PKH26-labeled BM-MSC in the Alzheimer's model rats.. Normal human bone marrow extracted for isolation of BM-MSC was cultured in vitro. The 5th passaged BM-MSC was labeled with PKH26, and observed under a fluorescence microscope for PKH26 labeling efficiency, and using flow cytometry BM-MSC surface markers was checked. The PKH26 labeled BM-MSC injected into the tail vein of the normal control group and AD animal model group, 14 days after finding the PKH26-labeled BM-MSC cells in the rat hippocampus using fluorescence microscopy. Using the Morris water maze experiment comparison of AD model and BM-MSC transplantation group of spatial learning and memory ability.. TFlow cytometry showed BM-MSC surface markers CD73 and CD105 were positive. In vitro, PKH26-labeled rate of BM-MSC was 100 %. The Morris water maze experiment comparison of BM-MSC transplantation group and AD group of animals, BM-MSC transplantation group at 13, 14 days of spatial learning and memory ability than AD animal group had significantly improved. 14 days after BM-MSCs in rat hippocampus could be found which were PKH26-positive, consistent with DAPI staining. PKH26-positive cells in animal models of AD were significantly more than those in the normal control group.. BM-MSC in AD rats not only migrates through the blood-brain barrier, but also mainly survives in the hippocampus of AD rats, and it can improve AD rat model of learning disabilities.

Topics: Alzheimer Disease; Animals; Bone Marrow Cells; Cell Movement; Cells, Cultured; Disease Models, Animal; Humans; Injections, Intravenous; Male; Mesenchymal Stem Cells; Organic Chemicals; Rats; Rats, Sprague-Dawley

Experiments have reported that granulocyte colony stimulating factor (G-CSF) can mobilize stem cells. However, few studies have examined the effect of G-CSF on bone marrow mononuclear cell (BMMC) mobilization, in particular regarding their capability to home to acutely injured liver.. The aim of this study was to evaluate the effort of G-CSF on BMMC homing to the liver following chemically-induced hepatic failure.. BMMC were isolated from mice, pre-labeled with PKH26 and infused into the mice in which hepatic injury had been induced followed by administration of G-CSF or vehicle. Livers were studied by fluorescent microscopy after transplantation of pre-labeled BMMC.. PKH26 labeled cells were found in liver tissue at 102 ± 10 cells/high power field in the BMMC+G-CSF group and 30 ± 5 cells/high power field in the BMMC group, but none in the G-CSF group and the control group (P < 0.05). In the former two groups the majority of PKH26 labeled cells colocalized with proliferative cell nuclear antigen (PCNA). The number of PCNA positive cells in the BMMC+G-CSF group was 20 ± 4 cells/high power field, while in the BMMC group it was 14 ± 2 cells/high power field, in the G-CSF group 12 ± 2 cells/high power field, and 8 ± 1 cells/high power field in the control group. Moreover, albumin expression was increased in the BMMC+G-CSF treated group (149 ± 7/high power field) relative to the BMMC group (48 ± 6/high power field), the G-CSF group (44 ± 5/high power field) and the vehicle group (30 ± 6/high power field), with the former three groups showing elevated levels as compared to vehicle control (30 ± 6) (P < 0.05).. Transplanted BMMC may home to injured liver, which appears to be enhanced by G-CSF administration.

Topics: Albumins; Animals; Biopsy; Bone Marrow Cells; Bone Marrow Transplantation; Carbon Tetrachloride; Cell Movement; Cells; Disease Models, Animal; Flow Cytometry; Fluorescent Dyes; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Immunohistochemistry; Liver; Liver Failure, Acute; Male; Mice; Mice, Inbred BALB C; Organic Chemicals

Neural and mesenchymal stem cells have been proposed as alternative sources of cells for transplantation into the brain in neurodegenerative disorders. However, the endogenous factors controlling their engraftment within the injured parenchyma remain ill-defined. Here, we demonstrate significant engraftment of undifferentiated exogenous mesenchymal or neural stem cells throughout the lesioned area in a rat model for Huntington's disease, as late as 8 weeks post-transplantation. We show that stem cell factor (SCF), strongly up-regulated within host cells in the damaged striatum, is able to activate the SCF receptor c-kit and its signaling pathway and to promote the migration and proliferation of mesenchymal and neural stem cells in vitro. Furthermore, c-kit receptor blockade alters neural stem cell distribution within the lesioned striatum. Host SCF expression is observed in atypical cells expressing glial fibrillary acidic protein and doublecortin in the lesioned striatum and in migrating doublecortin-positive progenitors. Taken together, these data demonstrate that SCF produced in situ in the lesioned striatum is an important factor in promoting the engraftment of stem cells within the lesioned brain.

Topics: Analysis of Variance; Animals; Cell Count; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Corpus Striatum; Disease Models, Animal; Doublecortin Protein; Embryo, Mammalian; Huntington Disease; Male; Nerve Tissue Proteins; Neurons; Organic Chemicals; Proto-Oncogene Proteins c-kit; Rats; Rats, Wistar; Stem Cell Factor; Stem Cell Transplantation; Stem Cells; Time Factors

This study presents a novel biodegradable alginate delivery system for antibiotics and bone cells to treat infected bone defects. About 2 x 10(7) New Zealand rabbit mesenchymal stem cells (MSCs) and 5 mL vancomycin solution (50 mg/mL) were added to 5 mL of 2.5% (w/v) sodium alginate solution to form biodegradable antibiotic and MSCs alginate beads 3 mm in diameter. The alginate beads were then cultured in an osteogenic medium for 14 days. The profiles of antibiotics released from the alginate beads were evaluated using the method of high performance liquid chromatography (HPLC). The expression of osteogenic genes, including Cbfa1 and osteopontin, in the alginate beads was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The alkaline phosphatase activity, calcium level, and mineral deposition of the cultured cells within the alginate beads were assessed. Analytical results demonstrated that the concentrations of vancomycin eluted from the alginate beads were, for 14 days, well above the minimal inhibitory concentration of Staphylococcus aureus. Osteopontin and mRNA of Cbfa1 were detected and increased alkaline phosphatase activity and calcium levels were noted, as was a substantial mineral deposition in cultured MSCs. The PKH 26-labeled MSCs and vancomycin alginate beads were implanted in rabbit bony cavities for in vivo analysis. Implanted PKH 26-labeled MSCs were identified in the newly formed bony trabeculae in all specimens at 2 and 4 months after implantation and there was abundant mineral deposition. The results of in vitro study demonstrated sustained elution of vancomycin from the alginate carrier for 14 days and good osteogenic differentiation of cultured MSCs in the alginate carrier matrix. The results of in vivo study demonstrated the implanted MSCs participating in new bone formation. Based on experimental evidence, development of a biodegradable alginate carrier system for antibiotics and bone cells is possible, providing a potential treatment procedure for infected bone defects.

Topics: Alginates; Alkaline Phosphatase; Animals; Anti-Bacterial Agents; Bone and Bones; Calcium; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Drug Carriers; Drug Delivery Systems; Drug Implants; Mesenchymal Stem Cell Transplantation; Organic Chemicals; Osteomyelitis; Osteopontin; Rabbits; Vancomycin

The present study analyzed whether grafts of the mesencephalic progenitor cell line CSM14.1 into the neonatal rat caudate putamen (CPu) differentiate into neurons and whether this is accompanied by a functional improvement in 6-hydroxydopamine (6-OHDA)-lesioned animals. As in previous studies, a neuronal differentiation of CSM14.1 cells transplanted into the CPu of adult animals could not be observed, so we here used neonatal rats, because graft location and host age seemingly are crucial parameters for neural transplant differentiation and integration. Rats bilaterally lesioned at postnatal day 1 by intraventricular 6-OHDA-injections 2 days later received 100,000 CSM14.1 cells prelabelled with the fluorescent dye PKH26 into the right CPu. Five weeks after grafting, the cylinder test was performed, and the data compared with data from age-matched intact controls and bilaterally lesioned-only animals. Brain slices immunostained for tyrosine hydroxylase (TH) were quantified by optical densitometry. We observed a significant preference of left forelimb use exclusively in transplanted animals. In these rats, TH-containing perikarya were found in the grafted CPu, presumedly leading to the significant increase of TH-immunoreactive fibers in this region. Moreover, confocal laser microscopy revealed a differentiation of transplanted PKH26-labelled CSM14.1 cells into neuronal nuclei antigen or TH-immunoreactive cells. Thus, CSM14.1 cells differentiate into TH-containing neurons, which most probably contribute to the preferred forelimb use, indicating a functional integration of CSM14.1 cells into the host basal ganglia loops during early postnatal development. These findings that are in contrast to observations in adult rats suggest instructive cues for neuronal differentiation and integration given by the neonatal microenvironment.

Topics: Animals; Animals, Newborn; Behavior, Animal; Cell Differentiation; Cell Line; Cell Movement; Disease Models, Animal; Male; Mesencephalon; Neostriatum; Organic Chemicals; Oxidopamine; Parkinsonian Disorders; Psychomotor Performance; Rats; Rats, Wistar; Stem Cell Transplantation; Stem Cells; Tyrosine 3-Monooxygenase

Over the years, researchers have carried out experiments with Mycobacterium leprae obtained from either human multibacillary lesions, or infected armadillo tissues, or infected footpad tissues of conventional mice as well as athymic nu/nu mice. In general, these sources of leprosy bacilli are satisfactory for most biochemical and mouse footpad studies, but less than satisfactory for studies in cell biology and immunology where contaminating host tissues pose a serious problem. We examined the utility of a procedure for eliminating mouse footpad tissue from M. leprae suspension using sodium hydroxide solution and its subsequent effect on the viability of the organism by determining the rate of palmitic acid oxidation, bacterial membrane integrity, and growth in the mouse footpad. We found that treating M. leprae suspension, obtained from infected nu/nu mouse footpad, with 0.1N NaOH for 3 min was sufficient to remove the majority of mouse tissue without adversely affecting the viability of the organism. This is a simple and rapid method to get suspensions of nu/nu footpad-derived viable M. leprae essentially free of host tissues, which can be a research reagent for studying the host-pathogen relationship in leprosy. We also report here a method for labeling M. leprae with the fluorescent dye PKH26, without compromising on the viability of the organism. This method may be useful in intracellular trafficking studies of M. leprae or in other cell biology studies that require tracking of the bacteria using fluorescent tag. We observed the staining to be stable in vitro over considerable lengths of time and did not affect the viability of the bacteria.

Topics: Animals; Cell Membrane; Disease Models, Animal; Fluorescent Dyes; Foot; Leprosy; Macrophages, Peritoneal; Mice; Mice, Nude; Microscopy, Electron, Scanning; Mycobacterium leprae; Organic Chemicals; Oxidation-Reduction; Palmitic Acid; Sodium Hydroxide; Staining and Labeling

Autologous chondrocyte implantation (ACI) is an established technique to repair joint surface defects. Although there is some indirect evidence that the expanded chondrocytes are required to achieve proper healing, the role they play in the repair process is not clear yet. To monitor the persistence and the phenotype of the injected chondrocytes in the repair tissue (RT) we have optimized a fluorescent labeling protocol for articular chondrocytes, which allows cell tracking in vivo for up to 14 weeks, using the fluorescent dye PKH26. We have combined in vivo cell tracking, with the immune-detection of collagen type II protein in a goat model of ACI. Our data indicate that the implanted cells can persist for at least 14 weeks in the defects, can participate in the integration with the surrounding tissues, and become structural part of the RT, rich in collagen type II and sulfated proteoglycans. Albeit with a small number of samples, our data provide proof of principal that the implanted chondrocytes can contribute to structural cartilage repair in a goat model of ACI.

Topics: Animals; Cartilage; Chondrocytes; Disease Models, Animal; Extracellular Matrix; Female; Fluorescent Dyes; Goats; Graft Survival; Joint Diseases; Organic Chemicals; Phenotype; Surgical Flaps

Cultured annulus fibrosus cells within an atelocollagen honeycomb-shaped scaffold with a membrane seal were allografted into the lacunas of intervertebral discs of which the nucleus pulposus had been vaporized using an indocyanine green dye-enhanced laser. Regeneration of the intervertebral disc was assessed based on the viability and histologic status of the allografted annulus fibrosus cells, as well as the prevention of narrowing disc space.. To study the regeneration of intervertebral disc after laser discectomy using tissue-engineering methods.. Intervertebral disc is the most avascular tissue in the human body, and its ability to regenerate is as low as that of articular cartilage. When nucleotomy is carried out, little regeneration of the annulus fibrosus is observed; consequently, intervertebral disc degeneration is inevitable.. Annulus fibrosus cells isolated from 20 Japanese white rabbits were labeled with a PKH-26 fluorescent dye and seeded within an atelocollagen honeycomb-shaped scaffold with a membrane seal. Annulus fibrosus cells cultured in atelocollagen honeycomb-shaped scaffold with a membrane seal for 1 week were allografted into the lacunas of intervertebral discs of recipient rabbit of which the nucleus pulposus had been vaporized using an ICG dye-enhanced laser. Soft radiograph photographs of the lumbar spine of these anesthetized rabbits were taken, the disc space measured, and the lumbar spine extracted 2, 4, 8, and 12 weeks after the operation. The proliferation of allografted annulus fibrosus cells with 5-bromo-2'-deoxyuridine/PKH-26 fluorescent labels was assessed using consecutive frozen sections, and safranin-O staining carried out for histologic evaluation.. The allografted annulus fibrosus cells were viable and showed proliferation activity with a hyaline-like cartilage being produced. The narrowing of the intervertebral disc space of the cell translation group was significantly prevented, as shown, up to 12 postoperative weeks.. The annulus fibrosus cells cultured in an ACHMS-scaffold were allografted into the lacunae of nucleus pulposus (obtained using laser vaporization), as well as the hole of annulus fibrosus (obtained by laser fiber insertion) of rabbit intervertebral discs. These cells were viable and showed cell proliferation in the disc tissues of recipients.

Topics: Animals; Bromodeoxyuridine; Cell Division; Cell Survival; Cells, Cultured; Disease Models, Animal; Female; Fluorescent Dyes; Graft Survival; Intervertebral Disc; Intervertebral Disc Displacement; Laser Therapy; Organic Chemicals; Rabbits; Regeneration; Tissue Engineering; Transplantation, Homologous; Treatment Outcome

Infusing monocytes that have been stimulated to produce fibrinolytic activators and factors that regulate cell proliferation, migration and maturation, might enhance venous thrombus resolution. The aim of this study was to determine the time course of infused monocyte recruitment into venous thrombus in an appropriate model of this disease.. Thrombus was induced in the inferior vena cava of male Wistar rats using reduced flow (80-90% stenosis). The vessel wall was examined at 1hr by scanning electron microscopy. Resolving thrombi with surrounding vena cava were obtained at 1, 7, 14 and 21 days after induction (n = 8). Sections, taken at 0.5 mm intervals (10-15 sections per thrombus), were stained using haematoxylin, Martius Scarlet Blue and antibodies against monocytes, platelets and fibrin. Sections from human venous thrombi (n = 4) were similarly stained. The area occupied by monocytes (in relative pixel units, RPU) was determined using computer aided image analysis. Peripheral rat blood monocytes were extracted, fluorescently labelled and injected intravenously into 7 rats prior to thrombus induction, Vena cava with thrombus was harvested 1 h, 2, 3, 4, 7, 14 and 25 days after induction and their fluorescence measured. The fluorescent content of the caval wall and thrombus was analysed in greater detail at 2 and 25 days after thrombus induction (n = 4 at each time interval).. Experimental thrombi were structurally similar to human thrombus and resolved within 14-21 days. Scanning electron microscopy showed minimal endothelial damage at 1 h with signs of early thrombus formation (platelet, red cell leukocyte and fibrin deposition). Neutrophils were the predominant leukocyte in the thrombus at 1 day, with monocytes making up only 0.3% (0.04% sem) of the area of the thrombus. There was a steady increase in thrombus monocyte content and by 21 days the percentage area of thrombus covered by monocytes had increased by over 35 fold to 11.5% (2.3% sem) (p <0.001). Initially, monocytes appeared around the edge of the thrombus and became more evenly distributed through the thrombus as resolution progressed. Labelled monocytes could be found in the circulation up to 1 week after infusion. The fluorescent content (RPU) of the thrombus increased over 25 days (mean RPU At 2 days 0.012, sem 0.005; mean RPU at 25 days 1.062, sem 0.252, p = 0.008). The number of labelled monocytes in the vessel wall peaked at 2 days and decreased thereafter.. The structure of thrombi produced by this model was comparable to that of human venous thrombi. Endogenous and injected monocytes migrated into the thrombus during natural resolution, possibly via the vein wall. Monocyte targeting could therefore be used to develop novel treatments for venous thrombosis, with the aim of reducing post-thrombotic complications.

Topics: Animals; Cell Adhesion; Cell Survival; Disease Models, Animal; Fluorescent Dyes; Histocytochemistry; Humans; Male; Microscopy, Electron, Scanning; Monocytes; Organic Chemicals; Rats; Rats, Wistar; Time Factors; Vena Cava, Inferior; Venous Thrombosis

Primary gastrointestinal cancer frequently spreads to the mesentery, omentum and other parts of the peritoneum, and these deposits are generally considered to be induced by intraperitoneal seeding from the primary lesion. However, a few peritoneal metastatic cases or cases with positive intraperitoneal lavage cytology, without serosal infiltration, have been reported. Most of peritoneal dissemination is certainly attended with serosal involvement of gastrointestinal malignancy. Nevertheless, we observe an unusual case of peritoneal dissemination without definite serosal invasion of the malignancy. And peritoneal dissemination is likely to be concomitant with lymph node metastasis in both cases with and without definite serosal invasion. In this study, we examined peritoneal cancer dissemination from the viewpoint of lymphogenous metastasis. For the model of lymphatic invasion, we established an animal experimental model of mesenteric lymph vessel obstruction. With these models, lymphangiographical studies were made on the fourth postoperative day (ten animals each) and we obtained mesenteric lymphangiograms of extensive mesenteric lymph vessels and reflux of lymph distal to the obstruction point from all ten animals. Next, in these experimental models, fluorescent-labelled tumor cells (rat hepatoma cell line, N1-S1) were infused from the mesenteric lymph node distal to the obstruction point on the fourth postoperative day (five animals each), and the migration of these tumor cells was investigated via fluorescent micrography. Subsequently, the fluorescent-labelled tumor cells were revealed in the mesenteric lymph nodes, mesenteric lymph vessels, interstitial tissues of the mesentery, submucosal lymph nodules and mucosal layer of the small intestine. Hence, lymphatic invasion and obstruction may cause extensive peritoneal dissemination via the lymphatic route.

Topics: Animals; Cell Movement; Disease Models, Animal; Fluorescent Dyes; Liver Neoplasms, Experimental; Lymph Nodes; Lymphatic Metastasis; Lymphography; Male; Microscopy, Fluorescence; Organic Chemicals; Peritoneal Neoplasms; Rats; Rats, Wistar; Risk Factors; Staining and Labeling; Tumor Cells, Cultured

To quantify S-antigen-specific (S-Ag) T cells in the retina after adoptive transfer, and to evaluate their role in the initiation and progress of destructive ocular inflammation in experimental autoimmune uveoretinitis (EAU).. Lewis rats were administered 10 x 10(6) S-Ag-specific T cells from the SP35 cell line or 10 x 10(6) concanavalin A-stimulated syngeneic spleen cell lymphoblasts labeled with lipophilic PKH26 fluorescent dye immediately before intravenous inoculation. Labeled cells in each retina were counted at various times from 4 to 120 hours after cell transfer by fluorescence microscopic analysis of each dissociated retina. Recipient eyes were examined within the same period by light and confocal microscope.. SP35 T cells showed a biphasic distribution in the retina. The first peak of 160 cells/retina was noted at 24 hours. A steady decline of labeled cells at 48 and 72 hours was followed by a rapid increase at 96 and 120 hours. Concanavalin A-stimulated, control-labeled cell populations showed an identical peak at 24 hours but a persistent decline thereafter; only two or three T cells were present in each retina at 120 hours. Concurrent inoculation of SP35 cells and nonspecific T cell blasts did not produce more SP35 cells than control cells in the retina at any time. Microscopic analysis showed mononuclear cell infiltration of the iris, ciliary body, and aqueous humor at 48 hours, which intensified rapidly and persisted through 120 hours. Retinal inflammation did not begin until 80 hours. Mononuclear cell adherence to vascular endothelium and perivascular macrophage infiltration of the innermost layers progressed to edema, and profound destructive inflammation and loss of retinal stratification were observed at 120 hours.. There is no evidence of a blood-ocular or blood-retinal barrier to activated T cell blasts. Autologous S-Ag does not provoke a more rapid entry of specific T cells at that site. The data confirm that anterior segment inflammation precedes retinal inflammation, even though S-Ag-specific T cells were present in the retina within a few hours after cell transfer. Because S-Ag is clearly present in the retina, delay in antigen presentation at that site may account for the temporal difference between retinal and anterior segment inflammation.

Topics: Adoptive Transfer; Animals; Arrestin; Autoimmune Diseases; Concanavalin A; Cytokines; Disease Models, Animal; Fluorescent Dyes; Lymphocyte Activation; Lymphocyte Count; Male; Organic Chemicals; Rats; Rats, Inbred Lew; Retina; Retinitis; T-Lymphocytes; Uveitis, Anterior; Uveitis, Posterior