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Open access

I. Šulla, V. Balik and M. Šarišský

Abstract

This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was < 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.

Open access

I. Šulla, V. Balik, D. Maženský and V. Danielisová

Abstract

It is well known that neuronal death, clinically manifested as paresis or plegia, is the end result of many pathological events affecting the central nervous system. However, several aspects of pathophysiological mechanisms involved in the development of tetra- or paraplegia caused by spinal cord traumatic or ischemic damage are only insufficiently understood and their histopathological manifestations remain poorly documented. That is why the authors decided to report on light-microscopic changes observed in 30 μm thick spinal cord sections cut from L3-S1 segments processed by the Nauta staining method in a group of 6 dogs with ischemic paraplegia induced by 30 min of a high thoracic aorta occlusion, and in a different group of 6 dogs with traumatic paraplegia induced by 5 min spinal cord compression with 200 g metallic rod. Both experimental groups (ischemic and compression) of spinal cord injuries (SCI) comprised the same number of mongrel dogs of both sexes, weighing 18-25 kg. In addition, each of the experimental groups had 3 normal dogs that served as controls. All experimental procedures were accomplished under general anaesthesia induced by pentobarbital and maintained by a mixture of halothane and oxygen. Following the 72 hour survival period, all 18 animals were euthanized by transcardial perfusion with 3,000 ml of saline and fixed by 3,000 ml of 10 % neutral formaldehyde during deep pentobarbital anaesthesia. The histopathological manifestation of neural tissue damage caused by ischemia or compression was similar. The light-microscopic images in both groups were characterised by argyrophilia and the swelling of grey matter neurons. However, in the dogs with traumatic SCIs, the changes only reached about 750 μm cranially and caudally from the necrotic epicentre. These findings indicated that the events taking part in secondary spinal cord injury mechanisms are similar in both, ischemic as well as in traumatic SCI.

Open access

I. Šulla, V. Balik, S. Horňák and V. Ledecký

Abstract

Spinal cord injuries (SCI) in dogs are not frequent, but they are serious pathological conditions accompanied with high morbidity and mortality. The pathophysiology of SCI involves a primary insult, disrupting axons, blood vessels, and cell membranes by mechanical force, or causes tissue necrosis by ischemia and reperfusion. The primary injury is followed by a cascade of secondary events, involving vascular dysfunction, edema formation, continuing ischemia, excitotoxicity, electrolyte shifts, free radical production, inflammation, and delayed apoptotic cell death. The most frequent cause of SCI in dogs is an acute intervertebral disc extrusion, exogenous trauma or ischemia. Neurological symptomatology depends on the location, size and the type of spinal cord lesions. It is characterized by transient or permanent, incomplete or complete loss of motor, sensory, autonomic, and reflex functions caudal to the site of the lesion. In a case of partial spinal cord (SC) damage, one of the typical syndromes develops (e. g. Brown-Séquard syndrome, central SC syndrome, ventral SC syndrome, dorsal SC syndrome, conus medullaris syndrome, or traumatic cauda equina syndrome). The severe transversal spinal cord lesion in the cervical region causes paresis or plegia of all four extremities (tetraparesis, tetraplegia); in thoracic or lumbosacral region, paresis or plegia of the pelvic extremities (paraparesis, paraplegia), i. e. sensory-motor deficit, urinary and foecal incontinence and sexual incompetence. The central nervous system in mammals does not regenerate, so the neurological deficit in dogs following severe SCI persists for the rest of their lives and animals display an image of permanent suffering. The research strategy presented here involved a PubMed, Medline (Ovid) and ISI Web of Science literature search from Januray 2001 to December 2017 using the term “canine spinal cord injury” in the English language; also references from selected papers were scanned and relevant articles included.

Open access

I. Šulla, V. Balik, J. Petrovičová, V. Almášiová, K. Holovská and Z. Oroszová

Abstract

Spinal cord injuries (SCI) with their tragic consequences belong to the most serious pathological conditions. That is why they have stimulated basic research workers, as well as health care practitioners, to search for an effective treatment for decades. Animal experimental models have been essential in these efforts. We have jointly decided to test and standardize one of the spinal cord injury compression models in rats. Twentythree adult female Wistar rats weighing 250-320 g were utilized. Employing general anaesthesia along with a mixture of sevoflurane with O2, 2 rats (sham controls) had their vertebral arch of either Th8 or Th9 vertebra removed (laminectomy). The other 21 experimental rats with similar laminectomies were divided into 3 subgroups (n = 7) which received compression impact forces of 30, 40 or 50 g (subgroups-1, -2, and -3, respectively) applied on their exposed spinal medulla for 15 minutes. All rats were observed for 28 days after the experimental procedure and their motor functions were assessed by the Basso, Beattie, Bresnahan (BBB) test 6 hours, 7, 21 and 28 days after the simulated SCI. All 23 rats survived the surgical procedures. The control rats were without any neurological deficits. There were, in every experimental subgroup, 1 or 2 rats with extreme BBB scores. So the rats with the maximum and minimum BBB values were excluded. Then, the results acquired in the residual 5 rats in each group were averaged and statistically analysed by the Tukey multiple comparisons test. Statistically significant intersubgroup differences were found at all survival times equal to or longer than 7 post SCI days. The goal of the SCI experiment was to generate a reproducible and reliable, submaximal spinal cord trauma model. The statistical analyses demonstrated that this objective was best achieved in the subgroup-2 with the 40 g compression.

Open access

I. Šulla, V. Balik, S. Horňák and V. Ledecký

Abstract

Severe spinal cord injuries (SCI), causing physical handicaps and accompanied by many serious complications, remains one of the most challenging problems in both, human and veterinary health care practices. The central nervous system in mammals does not regenerate, so the neurological deficits in a dog following SCI persists for the rest of its life and the affected animals display an image of permanent suffering. Diagnostics are based on: neurological examination, plain x-rays of vertebral column, x-rays of the vertebral column following intrathecal administration of a water-soluble contrast medium (myelography), x-rays of the vertebral column following epidural administration of a contrast medium (epidurography), computed tomography (CT) and/or magnetic resonance imaging (MRI). Currently, only limited therapeutic measures are available for the dogs with SCIs. They include: the administration of methylprednisolone sodium succinate (MPSS) during the acute stage; early spinal cord decompression; stabilisation of vertebral fractures or luxations; prevention and treatment of complications, and expert rehabilitation. Together with the progress in the understanding of pathophysiologic events occurring after SCI, different therapeutic strategies have been instituted, including the local delivery of MPSS, the utilisation of novel pharmacological agents, hypothermia, and stem/precursor cell transplantation have all been tested in the experimental models and preclinical trials with promising results. The aim of this review is the presentation of the generally accepted methods of diagnostics and management of dogs with SCIs, as well as to discuss new therapeutic modalities. The research strategy involved a PubMed, Medline (Ovid), Embase (Ovid) and ISI Web of Science literature search from January 2001 to December 2017 using the term “spinal cord injury”, in the English language literature; also references from selected papers were scanned and relevant articles included.