Aleš Homola

Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury.

Stem cell transplants into spinal cord lesions may help to improve regeneration and spinal cord function. Clinical studies are necessary for transferring preclinical findings from animal experiments to humans. We investigated the transplantation of unmanipulated autologous bone marrow in patients with transversal spinal cord injury (SCI) with respect to safety, therapeutic time window, implantation strategy, method of administration, and functional improvement. We report data from 20 patients with complete SCI who received transplants 10 to 467 days postinjury. The follow-up examinations were done at 3, 6, and 12 months after implantation by two independent neurologists using standard neurological classification of SCI, including the ASIA protocol, the Frankel score, the recording of motor and somatosensory evoked potentials, and MRI evaluation of lesion size. We compared intra-arterial (via catheterization of a. vertebralis) versus intravenous administration of all mononuclear cells in groups of acute (10–30 days post-SCI, n = 7) and chronic patients (2–17 months postinjury, n = 13). Improvement in motor and/or sensory functions was observed within 3 months in 5 of 6 patients with intra-arterial application, in 5 of 7 acute, and in 1 of 13 chronic patients. Our case study shows that the implantation of autologous bone marrow cells appears to be safe, as there have been no complications following implantation to date (11 patients followed up for more than 2 years), but longer follow-ups are required to determine that implantation is definitively safe. Also, we cannot yet confirm that the observed beneficial effects were due to the cell therapy. However, the outcomes following transplantation in acute patients, and in one chronic patient who was in stable condition for several months prior to cell implantation, are promising. It is evident that transplantation within a therapeutic window of 3–4 weeks following injury will play an important role in any type of stem cell SCI treatment. Trials involving a larger population of patients and different cell types are needed before further conclusions can be drawn.

Diffusion parameters of the extracellular space in human gliomas

Tumor cell migration through the extracellular space (ECS) might be affected by its pore size and extracellular matrix molecule content. ECS volume fraction α (α = ECS volume/total tissue volume), tortuosity λ (λ2 = free/apparent diffusion coefficient) and nonspecific uptake k′ were studied by the real‐time tetramethylammonium method in acute slices of human tissue. The diffusion parameters in temporal cortical tissue resected during surgical treatment of temporal lobe epilepsy (control) were compared with those in brain tumors. Subsequently, tumor slices were histopathologically classified according to the grading system of the World Health Organization (WHO), and proliferative activity was assessed. The average values of α, λ, and k′ in control cortex were 0.24, 1.55, and 3.66 × 10−3s−1, respectively. Values of α, λ, and k′ in oligodendrogliomas did not significantly differ from controls. In pilocytic astrogliomas (WHO grade I) as well as in ependymomas (WHO grade II), α was significantly higher, while λ and k′ were unchanged. Higher values of α as well as λ were found in low‐grade diffuse astrocytomas (WHO grade II). In cellular regions of high‐grade astrocytomas (WHO grade III and IV), α and λ were further increased, and k′ was significantly larger than in controls. Classic medulloblastomas (WHO grade IV) had an increased α, but not λ or k′, while in the desmoplastic type α and k′ remained unchanged, but λ was greatly increased. Tumor malignancy grade strongly corresponds to an increase in ECS volume, which is accompanied by a change in ECS structure manifested by an increase in diffusion barriers for small molecules. GLIA 42:77–88, 2003.

Extracellular matrix glycoproteins and diffusion barriers in human astrocytic tumours

The extracellular matrix (ECM) and changes in the size and geometry of the extracellular space (ECS) in tumour tissue are thought to be of critical importance in influencing the migratory abilities of tumour cells as well as the delivery of therapeutic agents into the tumour. In 21 astrocytic neoplasms, the ECM composition was investigated in situ by the immunohistochemical detection of ECM glycoproteins (tenascin, laminin, vitronectin, fibronectin, collagen types I–VI). To explain the changes in ECS size and to detect barriers to diffusion in the tumour tissue, the ECM composition, the cellularity, the density of glial fibrillary acidic protein (GFAP)‐positive tumour cell processes and the proliferative activity of the tumours were compared with the size and geometry of the ECS. The ECS volume fraction and the complex of hindrances to diffusion in the ECS (i.e. the tortuosity) were revealed by the real‐time iontophoretic tetramethylammonium method. Increased proliferative activity of the tumours correlated with increased ECS volume fraction and tortuosity. The tortuosity of the tumour tissue was not significantly influenced by tumour cell density. Higher tortuosity was found in low‐grade astrocytomas associated with the presence of a dense net of GFAP‐positive fibrillary processes of the tumour cells. The increase in tortuosity in high‐grade tumours correlated with an increased accumulation of ECM molecules, particularly of tenascin. We conclude that the increased malignancy of astrocytic tumours correlates with increases in both ECS volume and ECM deposition.

Transplantation of Mesenchymal Stromal Cells in Patients with Amyotrophic Lateral Sclerosis: Results of Phase I/IIa Clinical Trial:

Amyotrophic lateral sclerosis (ALS) is a progressive untreatable neurodegenerative disorder, leading to the death of the cortical and spinal motoneurons (MNs). Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) may represent a new approach to slowing down the progression of ALS by providing neurotrophic support to host MNs and by having an anti-inflammatory effect. We have designed a prospective, nonrandomized, open-label clinical trial (phase I/IIa, EudraCT No. 2011-000362-35) to assess the safety and efficacy of autologous multipotent BM-MSCs in ALS treatment. Autologous BM-MSCs were isolated and expanded under GMP conditions. Patients received 15 ± 4.5 × 106 of BM-MSCs via lumbar puncture into the cerebrospinal fluid. Patients were monitored for 6 months before treatment and then for an 18-month follow-up period. Potential adverse reactions were assessed, and the clinical outcome was evaluated by the ALS functional rating scale (ALSFRS), forced vital capacity (FVC), and weakness scales (WSs) to assess muscle strength on the lower and upper extremities. In total, 26 patients were enrolled in the study and were assessed for safety; 23 patients were suitable for efficacy evaluation. After intrathecal BM-MSC application, about 30% of the patients experienced a mild to moderate headache, resembling the headaches after a standard lumbar puncture. No suspected serious adverse reactions (SUSAR) were observed. We found a reduction in ALSFRS decline at 3 months after application (p < 0.02) that, in some cases, persisted for 6 months (p < 0.05). In about 80% of the patients, FVC values remained stable or above 70% for a time period of 9 months. Values of WS were stable in 75% of patients at 3 months after application. Our results demonstrate that the intrathecal application of BM-MSCs in ALS patients is a safe procedure and that it can slow down progression of the disease.

The extracellular matrix and diffusion barriers in focal cortical dysplasias

Focal cortical dysplasias (FCDs) of the brain are recognized as a frequent cause of intractable epilepsy. To contribute to the current understanding of the mechanisms of epileptogenesis in FCD, our study provides evidence that not only cellular alterations and synaptic transmission, but also changed diffusion properties of the extracellular space (ECS), induced by modified extracellular matrix (ECM) composition and astrogliosis, might be involved in the generation or spread of seizures in FCD. The composition of the ECM in FCD and non‐malformed cortex (in 163 samples from 62 patients) was analyzed immunohistochemically and correlated with the corresponding ECS diffusion parameter values determined with the real‐time iontophoretic method in freshly resected cortex (i.e. the ECS volume fraction and the geometrical factor tortuosity, describing the hindrances to diffusion in the ECS). The ECS in FCD was shown to differ from that in non‐malformed cortex, mainly by the increased accumulation of certain ECM molecules (tenascin R, tenascin C, and versican) or by their reduced expression (brevican), and by the presence of an increased number of astrocytic processes. The consequent increase of ECS diffusion barriers observed in both FCD type I and II (and, at the same time, the enlargement of the ECS volume in FCD type II) may alter the diffusion of neuroactive substances through the ECS, which mediates one of the important modes of intercellular communication in the brain – extrasynaptic volume transmission. Thus, the changed ECM composition and altered ECS diffusion properties might represent additional factors contributing to epileptogenicity in FCD.

The diffusion parameters of the extracellular space are altered in focal cortical dysplasias.

Most hypotheses concerning the mechanisms underlying seizure activity in focal cortical dysplasia (FCD) are based on alterations in synaptic transmission and glial dysfunction. However, neurons may also communicate by extrasynaptic transmission, which was recently found to affect epileptiform activity under experimental conditions and which is mediated by the diffusion of neuroactive substances in the extracellular space (ECS). The ECS diffusion parameters were therefore determined using the real-time iontophoretic method in human neocortical tissue samples obtained from surgically treated epileptic patients. The obtained values of the extracellular space volume fraction and tortuosity were then correlated with the histologicaly assessed type of cortical malformation (FCD type I or II). While the extracellular volume remained unchanged (FCD I) or larger (FCD II) than in normal/control tissue, tortuosity was significantly increased in both types of dysplasia, indicating the presence of additional diffusion barriers and compromised diffusion, which might be another factor contributing to the epileptogenicity of FCD.

Extracellular diffusion parameters in the rat somatosensory cortex during recovery from transient global ischemia/hypoxia

Changes in the extracellular space diffusion parameters during ischemia are well known, but information about changes during the postischemic period is lacking. Extracellular volume fraction (α) and tortuosity (Λ) were determined in the rat somatosensory cortex using the real-time iontophoretic method; diffusion-weighted magnetic resonance imaging was used to determine the apparent diffusion coefficient of water. Transient ischemia was induced by bilateral common carotid artery clamping for 10 or 15 mins and concomitant ventilation with 6% O2 in N2. In both ischemia groups, a negative DC shift accompanied by increased potassium levels occurred after 1 to 2 mins of ischemia and recovered to preischemic values within 3 to 5 mins of reperfusion. During ischemia of 10 mins duration, α typically decreased to 0.07 ± 0.01, whereas Λ increased to 1.80 ± 0.02. In this group, normal values of α = 0.20 ± 0.01 and Λ = 1.55 ± 0.01 were registered within 5 to 10 mins of reperfusion. After 15 mins of ischemia, α increased within 40 to 50 mins of reperfusion to 0.29 ± 0.03 and remained at this level. Tortuosity (Λ) increased to 1.81 ± 0.02 during ischemia, recovered within 5 to 10 mins of reperfusion, and was increased to 1.62 ± 0.01 at the end of the experiment. The observed changes can affect the diffusion of ions, neurotransmitters, metabolic substances, and drugs in the nervous system.

Interstitial lactate, lactate/pyruvate and glucose in rat muscle before, during and in the recovery from global hypoxia

BackgroundHypoxia results in an imbalance between oxygen supply and oxygen consumption. This study utilized microdialysis to monitor changes in the energy-related metabolites lactate, pyruvate and glucose in rat muscle before, during and after 30 minutes of transient global hypoxia. Hypoxia was induced in anaesthetised rats by reducing inspired oxygen to 6% O2 in nitrogen.ResultsBasal values for lactate, the lactate/pyruvate ratio and glucose were 0.72 ± 0.04 mmol/l, 10.03 ± 1.16 and 3.55 ± 0.19 mmol/l (n = 10), respectively. Significant increases in lactate and the lactate/pyruvate ratio were found in the muscle after the induction of hypoxia. Maximum values of 2.26 ± 0.37 mmol/l for lactate were reached during early reperfusion, while the lactate/pyruvate ratio reached maximum values of 35.84 ± 7.81 at the end of hypoxia. Following recovery to ventilation with air, extracellular lactate levels and the lactate/pyruvate ratio returned to control levels within 30-40 minutes. Extracellular glucose levels showed no significant difference between hypoxia and control experiments.ConclusionsIn our study, the complete post-hypoxic recovery of metabolite levels suggests that metabolic enzymes of the skeletal muscle and their related cellular components may be able to tolerate severe hypoxic periods without prolonged damage. The consumption of glucose in the muscle in relation to its delivery seems to be unaffected.