Comparison between stem cell therapy and stem cell derived exosomes on induced multiple sclerosis in dogs.

BACKGROUND: Multiple sclerosis (MS) is a chronic condition that primarily manifests as demyelination of neuronal axons in the central nervous system, due to the loss or attack of oligodendroglia cells that form myelin. Stem cell therapy has shown promising results for the treatment of MS due to its capability to halt the immune attack, stop apoptosis and axonal degeneration, and differentiate into oligodendrocytes. Stem cell-derived Exosomes (Exosomes) have shown great capabilities for neuronal diseases as they have growth factors, complex sets of miRNA, enzymes, proteins, major peptides, lipids, and macromolecules with anti-inflammatory, angiogenesis, and neurogenesis activities. METHODS: This study aimed to compare the healing properties of stem cells, against Exosomes for the treatment of an experimentally induced MS dog model. Dog models of MS received either a single treatment of stem cells or a single treatment of Exosomes intrathecally and the treatment process was evaluated clinically, radiologically, histopathologically, and electron microscopy and cerebrospinal fluid analysis. RESULTS: showed marked amelioration of the clinical signs in both treated groups compared to the control one, magnetic resonance scans showed the resolution of the hyperintense lesions at the end of the study period, the histopathology and electron microscopy showed marked healing properties and remyelination in treated groups with superiority of the stem cells compared to Exosomes. CONCLUSIONS: Although stem cell results were superior to Exosomes therapy; Exosomes have proven to be effective and safe important actors in myelin regeneration, and their use in diseases like MS helps to stimulate remyelination.

A novel surgical technique for treatment of cervical vertebral stenotic myelopathy (wobbler syndrome) in a filly.

Cervical vertebral stenotic myelopathy (CVSM), also known as equine wobbler syndrome or cervical ataxia, is a devastating neurological syndrome resulting from compression of the spinal cord at the cervical region. This report describes a novel surgical technique for treatment of 16-month-old Arabian filly with CVSM. The filly showed grade 4 ataxia, hypermetria, weakness of the hind limbs, stumbling during walking, and abnormal gait. Case history, clinical signs and myelography revealed spinal cord compression between the C3 and C4 and C4-C5. The filly underwent a novel surgical interference for decompression and stabilization of the point of stenosis using specially designed titanium plate and intervertebral spacer. Evidence of arthrodesis with absence of complications was confirmed by periodic radiography over eight months of postoperative care. The new technique applied in this cervical surgery was efficient for the decompression and stabilization of the vertebrae, allowing arthrodesis development and remission of the clinical signs. The obtained results encourage further assessment of this novel procedure in horses clinically affected by CVSM.

Repair of experimentally induced femoral chondral defect in a rabbit model using Lyophilized growth promoting factor extracted from horse blood platelets (L-GFequina).

Lyophilized equine platelet derived growth factors (LGF) is a novel advanced platelet rich protein growth factor. It has been successfully applied in various fields of regenerative medicine to treat a variety of inflammatory and degenerative musculoskeletal conditions. Our study aimed to evaluate the efficacy of intraarticularly injected LGF for the remedy of articular cartilage injury, commonly characterized by progressive pain and loss of joint function in osteoarthritic rabbits. Full-thickness cylindrical cartilage defects were generated in both femoral condylar articular surfaces in twenty rabbits. The left joint of all animals was injected with the adjuvant as a self-control negative, while the right joint was injected by LGF. Four- and eight-weeks post-surgery, the femoral condyles were harvested, and assessed grossly, microscopically and immunohistochemically. Cytokines (TNF-α, IL-1ß, PDGF and TGF-ß1) contents of the chondral defects were quantified by ELISA as well as the gene expression of Col I and Col II via RT-qPCR. The LGF treated defects showed significant higher ICRS (International cartilage repair society) healing scores of cartilaginous regeneration with a significant higher histological healing score on using O'Driscoll histological scoring system. Additionally, LGF significantly lowered the levels of the pro-inflammatory cytokines TNF-α and IL-1ß. It also significantly increased the anabolic and angiogenic growth factors (PDGF and TGF-ß1), and significantly elevated the expression of chondrogenic-related marker genes; Col I and Col II. The current study reveals that LGF improves chondral healing and thus it can be a superior nominee as an adjunctive therapy to positively influence regeneration of chondral defects in osteoarthritic patients.

Effect of combined intrathecal/intravenous injection of bone marrow derived stromal cells in platelet-rich plasma on spinal cord injury in companion animals.

Background: Companion animals are prone to spinal cord injuries commonly associated with severe locomotor and sensory complications, which can escalate to a state of irreversible paralysis. Stem cell therapies propose a hope for treating spinal cord injuries via differentiation into neurons and associated glial cells, halting the immune attacks, inhibiting apoptosis and necrosis, and secretion of neurotrophic factors that stimulate the regeneration process. Aim: The study aims to evaluate the use of autologous bone marrow derived stromal cells in platelet-rich plasma carrier for selected clinical cases having chronic spinal cord injuries in dogs and cats via a one-time combined intrathecal/intravenous injection. Methods: Cells were injected in five dogs and three cats suffering from disc protrusion leading to spinal cord injury and in thosewho did not respond to conventional treatment during a clinical trial. Results: Results indicated that the transplanted cells led to the restoration of the weight bearing locomotor function and spinal reflexes in a period less than 90 days with physical rehabilitation. The treatment showed minor changes in the magnetic resonance images of extruded discs. Conclusion: This study concluded that the combined intrathecal/intravenous injection of bone marrow stromal cells is a safe and promising procedure for treating chronic spinal cord injuries in companion animals.

Congenital Osteoma of the Frontal Bone in an Arabian Filly.

Congenital frontal osteoma has not been previously described in horses. This report records-for the first time-a congenital osteoma of the frontal bone in a 4-month-old Arabian filly. The filly had a frontal hard mass that was present at birth and then showed a slow and continuous growth. This mass appeared as a solitary, painless, oval dense tumor of compact bone, about 2 cm in diameter and 3 cm in length. The tumor was asymptomatic, and the skin over the mass was normal. Radiography revealed a well-defined oval, radio-dense mass projecting from the surface of the right frontal bone with no local invasion. The tumor had a broad-based attachment to frontal bone with normal frontal sinus. The mass caused disfigurement; therefore, it was removed at the owner's request. The mass was diagnosed histopathologically as osteoma. The surgical excision of the osteoma was successful without any complications, and the filly adapted remarkably well after surgery. No recurrence was reported 20 months after the surgery. In conclusion, osteoma should be listed during the differential diagnosis of the congenital craniofacial masses in horses. Early diagnosis of the frontal osteoma guarantees a successful surgical treatment and consequently prevents the future complications.

Ultrasonography and Surgical Treatment of an Unusual Case of Urethral Calculus in an Arabian Horse.

This case report records an obstructive urolithiasis due to a large calcium carbonate urethral stone in an 11-year-old Arabian stallion. The stallion had colicky pain, anuria, and reduction in food and water intakes. Palpation of the penis revealed rhythmic contractions of the urethra, a hard mass in the penile urethra at the level of the ischial arch, and a dilated urethra proximal to the mass. Rectal examination revealed a distended and turgid urinary bladder. Passing a urethral catheter revealed a complete urethral obstruction at the level of the ischial arch. Ultrasonography revealed a calculus that appeared as an irregular, hyperechoic arch-like thick line with acoustic shadowing. Subischial urethrotomy was conducted under epidural anesthesia. Uneventful recovery was seen with no recurrence or complications for 12 months of available follow-up. In conclusion, clinical, rectal, and ultrasound examinations are valuable for definite diagnosis of urethral calculi in horses and the subischial urethrotomy appears to be successful in correcting this condition in horses.

Pressure-induced left ventricular hypertrophy in canine model of aortic stenosis using nylon tie

The aim of this study was to develop an experimentally-induced canine model of left ventricular hypertrophy through banding of the ascending aorta using nylon ties. Seven clinically normal dogs free of cardiovascular disease were used. Nylon tie was used in banding the mid-ascending aorta. Clinical, radiographic and echocardiographic evaluations were done at 1.5, 3 and 6 months. Dogs were euthanized at 6 months for post mortem and histopathological evaluation. Clinically, dogs did not exhibit any signs of cardiovascular disease at 1.5 or 3 months, while at 6 months two dogs (28.6 %) exhibited mild weight loss, exercise intolerance and heart murmurs. Radiographic evaluation revealed significant increase in cardiac size only at 6 months based on measurement of the cardiothoracic area evaluation. Echocardiography revealed increased left ventricular wall thickness starting from 1.5 month, although this increase was statistically significant at 3 and 6 months (p > 0.05). Left ventricular hypertrophy was confirmed by post mortem examination. Histopathological sections of left ventricle in all dogs demonstrated myocyte hypertrophy and interstitial fibrosis. This model simulates the naturally occurring ventricular hypertrophy using a rapid and economic technique. Such models are required to understand pathogenesis of heart disease and to develop effective treatment strategy.

Evaluation of treatment of experimentally induced canine model of multiple sclerosis using laser activated non-expanded adipose derived stem cells.

Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system that destroys oligodendrocytes. This work aims to evaluate the treatment of experimentally induced MS in dogs using laser activated non-expanded adipose derived stem cells. The results showed amelioration of the clinical signs over time confirmed by the resolution of the previous lesions on MRI. Positive migration of the injected cells to the site of lesion, increased remyelination detected by Myelin Basic Proteins, positive differentiation into Olig2 positive oligodendrocytes, prevented the glial scar formation and restored axonal architecture. The study concluded that treatment using laser activated stem cells holds a promising therapeutic option for treatment of MS in a canine model.

Hydrogen sulfide enhances the effectiveness of mesenchymal stem cell therapy in rats with heart failure: In vitro preconditioning versus in vivo co-delivery.

Stem cell therapy represents a promising therapeutic avenue for cardiac disorders, including heart failure. Although stem cell transplantation showed encouraging preliminary results, the outcomes of clinical studies are still unsatisfactory. This study aimed to compare the outcomes of two therapeutic approaches, in vivo co-delivery of sodium hydrogen sulfide (NaHS) concomitant with bone marrow-derived mesenchymal stem cell (BMSC) transplantation and in vitro preconditioning of BMSCs with NaHS, both of which are intended to promote the success of stem cell therapy in rats with isoprenaline-induced heart failure. Heart failure developed 4 weeks after the subcutaneous injection of isoprenaline (170 mg/kg) for 4 consecutive days. The in vivo approach involved the co-delivery of intraperitoneally administered NaHS concomitant with BMSC transplantation for a period of 14 days. The in vitro approach involved preconditioning BMSCs with NaHS for 30 min before transplantation. Compared to treatment with BMSCs alone, in vitro preconditioning of BMSCs with NaHS improved left ventricular function as measured by echocardiography and electrocardiography and enhanced stem cell homing, proliferation and differentiation as manifested by higher cardiac expression of GATA-4 and myocyte enhancer factor 2. Moreover, the measurement of cardiac transforming growth factor beta 1 levels and histopathological investigation revealed mitigated fibrosis and myocardial injury scores. Compared with BMSC therapy alone, the in vivo approach enhanced stem cell homing and differentiation, alleviated fibrosis and augmented vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression. In conclusion, NaHS can potentiate the efficiency of BMSC therapy for heart failure by in vitro preconditioning or in vivo co-delivery. The in vitro approach is superior with regard to improving cardiac function in addition to enhancing stem cell proliferation, while the in vivo approach is superior with regard to increasing cardiac VEGF and eNOS expression.

Graphene Oxide-Gold Nanosheets Containing Chitosan Scaffold Improves Ventricular Contractility and Function After Implantation into Infarcted Heart.

Abnormal conduction and improper electrical impulse propagation are common in heart after myocardial infarction (MI). The scar tissue is non-conductive therefore the electrical communication between adjacent cardiomyocytes is disrupted. In the current study, we synthesized and characterized a conductive biodegradable scaffold by incorporating graphene oxide gold nanosheets (GO-Au) into a clinically approved natural polymer chitosan (CS). Inclusion of GO-Au nanosheets in CS scaffold displayed two fold increase in electrical conductivity. The scaffold exhibited excellent porous architecture with desired swelling and controlled degradation properties. It also supported cell attachment and growth with no signs of discrete cytotoxicity. In a rat model of MI, in vivo as well as in isolated heart, the scaffold after 5 weeks of implantation showed a significant improvement in QRS interval which was associated with enhanced conduction velocity and contractility in the infarct zone by increasing connexin 43 levels. These results corroborate that implantation of novel conductive polymeric scaffold in the infarcted heart improved the cardiac contractility and restored ventricular function. Therefore, our approach may be useful in planning future strategies to construct clinically relevant conductive polymer patches for cardiac patients with conduction defects.

Contribution of volume overload to progression of cardiovascular disease in a rat model of chronic kidney disease.

Volume overload is a common phenomenon in patients with chronic kidney disease that is associated with cardiovascular risk factors. However, its contribution to the development of adverse cardiovascular outcomes in those patients is not fully understood. Thus, the present work investigated the effect of salt-induced volume overload on cardiac functions and geometry in a rat model of chronic kidney disease. Thirty adult male Sprague-Dawley rats were randomly divided. One set of animals received a sham operation, while another set of animals underwent uninephrectomy. Rats were then fed either a normal-salt (0.4%) or high-salt (8.0%) diet for 6 weeks. The salt-loaded, uninephrectomized rats were treated with indapamide (3 mg·kg-1·day-1, orally) for 6 weeks. We found that uninephrectomized rats subjected to a high-salt diet (8.0%) for 6 weeks presented with hypertension, proteinuria, decreased renal Klotho expression, and deterioration in cardiac hemodynamics and histology. Echocardiography to assess cardiac function showed that ejection fraction and fractional shortening were positively correlated with relative renal Klotho expression. In conclusion, salt-induced volume overload in a rat model of chronic kidney disease has an adverse cardiovascular outcome and is associated with inflammatory activation and decrease in renal Klotho expression.

Histological Evaluation of Experimentally Induced Critical Size Defect Skin Wounds Using Exosomal Solution of Mesenchymal Stem Cells Derived Microvesicles.

BACKGROUND AND OBJECTIVES: The present study investigated whether MSCs derived microvesicles (MVs) or (Exosomes) can exert therapeutic effects on an experimental model of cutaneous injury and explored the underlying involving mechanisms. METHODS AND RESULTS: Three bilateral full thickness circular wounds were created on the back of two groups of dogs using 2-cm dermal punch. The wounds were at least 2.5 cm apart. Saline was subcutaneously injected in 4 places around each wound area in group-I (control), whereas an equal volume of exosomal solution of MSCs derived MVs was similarly injected in group-II. The findings demonstrated that MSCs derived MVs had significantly promoted cutaneous wound healing, collagen synthesis, and vascularization at wound sites. The application of the exosomal solution had not only promoted the generation of newly formed vessels, but also have accelerated their development and maturation leading to a faster healing process. CONCLUSIONS: MSC-Exosomes appeared to be a superior candidate for treating cutaneous wounds than their originator cells, and may represent a promising opportunity to develop a novel cell-free therapy approach that might overcome the obstacles and risks associated with the use of native or engineered stem cells transplantation therapy.

Neupogen and mesenchymal stem cells are the novel therapeutic agents in regeneration of induced endometrial fibrosis in experimental rats.

Endometrial fibrosis is the presence of intrauterine adhesions (IUAs) after any uterine surgery or curettage and it results in infertility and recurrent pregnancy loss. We evaluated the role of human mesenchymal stem cells (hMSCs) as a therapeutic agent of endometrial fibrosis. We also compared the effect of MSCs with the effect of estrogen and neupogen either each alone or as a combined therapy with MSCs. This experimental study was performed on 84 albino rats which were divided into seven groups (n=12 rats/group) as follows, group1: normal control rats, group 2: induced fibrosis, group 3: induced fibrosis that received oral estrogen, group 4: induced fibrosis that received hMSCs, group 5: induced fibrosis that received hMSCs and estrogen, group 6: induced fibrosis that received neupogen, and group 7: induced fibrosis that received hMSCs and neupogen. The extent of fibrosis, vascularization, and inflammation were evaluated by; qRT-PCR for interleukin 1 (IL-1), interleukin 6 (IL-6), TNF, vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß), and RUNX; ELISA for connective tissue growth factor (CTGF); Western blotting for collagen-I; immunohistochemistry examination for VEGF and RUNX-2; and histopathological assessment. In therapeutic groups either by hMSCs alone or combined with estrogen or neupogen; fibrosis and inflammation (IL-1, IL-6, TNF, TGF-ß, RUNX, CTGF, and collagen-I) were significantly decreased but vascularization (VEGF) was significantly increased (P<0.05) compared with induced fibrosis group. The most significant result was obtained in fibrosis that received combined therapy of hMSCs and neupogen (P=0.000). Stem cells and neupogen are a highly effective alternative regenerative agents in endometrial fibrosis.

Prophylactic supplementation of resveratrol is more effective than its therapeutic use against doxorubicin induced cardiotoxicity.

Resveratrol (RSV), a polyphenolic compound and naturally occurring phytoalexin, has been reported to exert cardio-protective effects in several animal studies. However, the outcome of initial clinical trials with RSV was less effective compared to pre-clinical studies. Therefore, RSV treatment protocols need to be optimized. In this study we evaluated prophylactic versus therapeutic effect of resveratrol (RSV) in mitigating doxorubicin (Dox)-induced cardiac toxicity in rats. To investigate prophylactic effects, RSV was supplemented for 2 weeks along with Dox administration. After 2 weeks, Dox treatment was stopped and RSV was continued for another 4 weeks. To study therapeutic effects, RSV treatment was initiated after 2 weeks of Dox administration and continued for 4 weeks. Both prophylactic and therapeutic use of RSV mitigated Dox induced deterioration of cardiac function as assessed by echocardiography. Also RSV treatment (prophylactic and therapeutic) prevented Dox induced myocardial damage as measured by cardiac enzymes (LDH and CK-MB) in serum. Which was associated with decrease in Dox induced myocardial apoptosis and fibrosis. Interestingly our study also reveals that prophylactic use of RSV was more effective than its therapeutic use in mitigating Dox induced apoptosis and fibrosis in the myocardium. Therefore, prophylactic use of resveratrol may be projected as a possible future adjuvant therapy to minimize cardiotoxic side effects of doxorubicin in cancer patients.

Does vitamin C have the ability to augment the therapeutic effect of bone marrow-derived mesenchymal stem cells on spinal cord injury?

Methylprednisolone (MP) is currently the only drug confirmed to exhibit a neuroprotective effect on acute spinal cord injury (SCI). Vitamin C (VC) is a natural water-soluble antioxidant that exerts neuroprotective effects through eliminating free radical damage to nerve cells. Bone marrow mesenchymal stem cells (BMMSCs), as multipotent stem cells, are promising candidates in SCI repair. To evaluate the therapeutic effects of MP, VC and BMMSCs on traumatic SCI, 80 adult male rats were randomly divided into seven groups: control, SCI (SCI induction by weight-drop method), MP (SCI induction, followed by administration of 30 mg/kg MP via the tail vein, once every other 6 hours, for five times), VC (SCI induction, followed by intraperitoneal administration of 100 mg/kg VC once a day, for 28 days), MP + VC (SCI induction, followed by administration of MP and VC as the former), BMMSCs (SCI induction, followed by injection of 3 × 106 BMMSCs at the injury site), and BMMSCs + VC (SCI induction, followed by BMMSCs injection and VC administration as the former). Locomotor recovery was assessed using the Basso Mouse Scale. Injured spinal cord tissue was evaluated using hematoxylin-eosin staining and immunohistochemical staining. Expression of transforming growth factor-beta, tumor necrosis factor-alpha, and matrix metalloproteinase-2 genes was determined using real-time quantitative PCR. BMMSCs intervention better promoted recovery of nerve function of rats with SCI, mitigated nerve cell damage, and decreased expression of transforming growth factor-beta, tumor necrosis factor-alpha, and matrix metalloproteinase-2 genes than MP and/or VC. More importantly, BMMSCs in combination with VC induced more obvious improvements. These results suggest that VC can enhance the neuroprotective effects of BMMSCs against SCI.

Pneumopericardium Secondary to Pneumomediastinum in a Golden Retriever Dog.

Pneumopericardium is a rare finding that has been previously reported following spontaneous, traumatic, or iatrogenic causes. A 3-year old Golden Retriever dog was admitted with respiratory distress after falling from a height. Clinical and electrocardiographic findings were nonspecific. Thoracic radiography revealed hyperinflated lung with sharp outlining of the mediastinal structures. A well-demarcated region of radiolucent gas opacity was seen surrounding the cardiac silhouette. Echocardiography revealed intense hyper-reflective shadows all over the heart. Echocardiographic measurements were within the reference range. The dog responded well to conservative medical therapy. Pneumopericardium was reported secondary to pneumomediastinum; pneumopericardium is self-limiting unless other complications develop.

Canine neonatal transcranial ultrasonography from birth until closure of bregmatic fontanelle.

Ultrasonography is a valuable diagnostic tool that has been used for diagnosis of neonatal brain diseases. The purpose of the present study was to describe the sequential ultrasonographic appearance of the normal canine neonatal brain from birth till closure of the bregmatic fontanelle. In total, 16 clinically normal neonates of mixed breed dogs were used. The bregmatic fontanelle was used as an acoustic window to record 5 transcranial scans (3 transverse, 1 sagittal, and 1 parasagittal scans) at 3, 10, 20, and 30 days of age. The appearance, echogenicity, and developmental differentiation of the structures within the cranium were described. Good images were obtained at 10 and 20 days of age. At 30 days of age, the obtained images presented poor details, as the fontanelle was small. Data obtained from this study represent the basis of brain ultrasound in neonates until 30 days of age, which could be beneficial in diagnosing congenital brain diseases.

Homing and reparative effect of intra-articular injection of autologus mesenchymal stem cells in osteoarthritic animal model.

BACKGROUND: This work aimed to study the homing evidence and the reparative effect of mesenchymal stem cells (MSCs) in the healing process of induced osteoarthritis in experimental animal model (donkeys). METHODS: Twenty-seven donkeys were equally divided into 3 groups based on the observation period after induction of arthritis (3, 6 and 9 weeks) to achieve different degrees of osteoarthritis. Each group was subdivided into three subgroups of three animals each based on the follow-up period (1, 2 and 6 months) after treatment. The induction was done through intra-articular (IA) injection of 2 ml of Amphotericin-B in both carpal joints. MSCs were harvested in a separate procedure, labeled with green fluorescent protein (GFP) using monster GFP vector and suspended in hyaluronic acid for IA injection. Treatment approaches consisted of cell-treatment using MSCs suspended in 3 ml of hyaluronic acid (HA) for the right carpal joint; and using the same amount of (HA) but without MSCs for the left contralateral carpal joint to serve as a control. Animals were assessed clinically and radiologically before and after treatment. Synovial fluid was also evaluated. Histopathologically; articular cartilage structural changes, reduction of articular cartilage matrix staining, osteophyte formation, and subchondral bone plate thickening were graded. Data was summarized using median and percentile for scores of histopathologic grading. Comparison between groups was done using non-parametric Mann Whitney test. RESULTS: The reparative effect of MSCs was significant both clinically and radiologically in all treated groups (P < 0.05) compared to the control groups. Fluorescence microscopy of sections of the cell-treated joints of all animals indicated that the GFP-transduced injected cells have participated effectively in the reparative process of the damaged articular surface and have integrated within the existing articular cartilage. The cells were associated with the surface of the cartilage and, were also detected in the interior. CONCLUSIONS: Homing was confirmed by the incorporation of injected GFP-labeled MSCs within the repaired newly formed cartilage. Significant recovery proves that the use of IA injection of autologous MSCs is a viable and a practical option for treating different degrees of osteoarthritis.