Jiri Ruzicka

Alzheimer’s Disease: Mechanism and Approach to Cell Therapy

Alzheimer’s disease (AD) is the most common form of dementia. The risk of AD increases with age. Although two of the main pathological features of AD, amyloid plaques and neurofibrillary tangles, were already recognized by Alois Alzheimer at the beginning of the 20th century, the pathogenesis of the disease remains unsettled. Therapeutic approaches targeting plaques or tangles have not yet resulted in satisfactory improvements in AD treatment. This may, in part, be due to early-onset and late-onset AD pathogenesis being underpinned by different mechanisms. Most animal models of AD are generated from gene mutations involved in early onset familial AD, accounting for only 1% of all cases, which may consequently complicate our understanding of AD mechanisms. In this article, the authors discuss the pathogenesis of AD according to the two main neuropathologies, including senescence-related mechanisms and possible treatments using stem cells, namely mesenchymal and neural stem cells.

Hypothermic anticoagulation: testing individual responses to graded severe hypothermia with thromboelastography.

Selective incircuit blood cooling could be an effective anticoagulation strategy during hemodialysis. However, it is currently unknown what blood temperature would ensure sufficient anticoagulation. Similarly, no information exists about potential interindividual variability in response to graded hypothermia. Therefore, the aim of this study was to analyze effects of profound hypothermia on human coagulation. Furthermore, a mathematical relationship between blood temperatures and coagulation was sought to predict individual responses to blood cooling. It was designed as a laboratory study. Thromboelastography (TEG) measurements were taken at a temperature range of 38–12°C. To enable measurements below 20°C, the TEG device was placed into an air conditioned chamber allowing for setting of the temperatures over a wide range. The data were analyzed by regression analysis for pooled and individual measurements. Decreasing temperatures always led to a progressive reduction in blood coagulation by delaying the initiation of thrombus formation, as well as by decreasing the speed of its creation and growth. However, the response to cooling was not uniform and the interindividual variability exists. The relationship between blood temperature and coagulation is not linear but exponential (parameters R and K) and sigmoid (parameter &agr;-angle). The lower the blood temperature, the more significant effect on blood coagulation decline. To predict an individual response of the coagulation system over a wide range of temperatures, a mathematical modeling can be used.

The Anti-Inflammatory Compound Curcumin Enhances Locomotor and Sensory Recovery after Spinal Cord Injury in Rats by Immunomodulation

Well known for its anti-oxidative and anti-inflammation properties, curcumin is a polyphenol found in the rhizome of Curcuma longa. In this study, we evaluated the effects of curcumin on behavioral recovery, glial scar formation, tissue preservation, axonal sprouting, and inflammation after spinal cord injury (SCI) in male Wistar rats. The rats were randomized into two groups following a balloon compression injury at the level of T9–T10 of the spinal cord, namely vehicle- or curcumin-treated. Curcumin was applied locally on the surface of the injured spinal cord immediately following injury and then given intraperitoneally daily; the control rats were treated with vehicle in the same manner. Curcumin treatment improved behavioral recovery within the first week following SCI as evidenced by improved Basso, Beattie, and Bresnahan (BBB) test and plantar scores, representing locomotor and sensory performance, respectively. Furthermore, curcumin treatment decreased glial scar formation by decreasing the levels of MIP1α, IL-2, and RANTES production and by decreasing NF-κB activity. These results, therefore, demonstrate that curcumin has a profound anti-inflammatory therapeutic potential in the treatment of spinal cord injury, especially when given immediately after the injury.

Mesenchymal Stem Cells Preserve Working Memory in the 3xTg-AD Mouse Model of Alzheimer’s Disease

The transplantation of stem cells may have a therapeutic effect on the pathogenesis and progression of neurodegenerative disorders. In the present study, we transplanted human mesenchymal stem cells (MSCs) into the lateral ventricle of a triple transgenic mouse model of Alzheimer´s disease (3xTg-AD) at the age of eight months. We evaluated spatial reference and working memory after MSC treatment and the possible underlying mechanisms, such as the influence of transplanted MSCs on neurogenesis in the subventricular zone (SVZ) and the expression levels of a 56 kDa oligomer of amyloid β (Aβ*56), glutamine synthetase (GS) and glutamate transporters (Glutamate aspartate transporter (GLAST) and Glutamate transporter-1 (GLT-1)) in the entorhinal and prefrontal cortices and the hippocampus. At 14 months of age we observed the preservation of working memory in MSC-treated 3xTg-AD mice, suggesting that such preservation might be due to the protective effect of MSCs on GS levels and the considerable downregulation of Aβ*56 levels in the entorhinal cortex. These changes were observed six months after transplantation, accompanied by clusters of proliferating cells in the SVZ. Since the grafted cells did not survive for the whole experimental period, it is likely that the observed effects could have been transiently more pronounced at earlier time points than at six months after cell application.

A comparative study of three different types of stem cells for treatment of rat spinal cord injury.

Three different sources of human stem cells—bone marrow-derived mesenchymal stem cells (BM-MSCs), neural progenitors (NPs) derived from immortalized spinal fetal cell line (SPC-01), and induced pluripotent stem cells (iPSCs)—were compared in the treatment of a balloon-induced spinal cord compression lesion in rats. One week after lesioning, the rats received either BM-MSCs (intrathecally) or NPs (SPC-01 cells or iPSC-NPs, both intraspinally), or saline. The rats were assessed for their locomotor skills (BBB, flat beam test, and rotarod). Morphometric analyses of spared white and gray matter, axonal sprouting, and glial scar formation, as well as qPCR and Luminex assay, were conducted to detect endogenous gene expression, while inflammatory cytokine levels were performed to evaluate the host tissue response to stem cell therapy. The highest locomotor recovery was observed in iPSC-NP-grafted animals, which also displayed the highest amount of preserved white and gray matter. Grafted iPSC-NPs and SPC-01 cells significantly increased the number of growth-associated protein 43 (GAP43+) axons, reduced astrogliosis, downregulated Casp3 expression, and increased IL-6 and IL-12 levels. hMSCs transiently decreased levels of inflammatory IL-2 and TNF-α. These findings correlate with the short survival of hMSCs, while NPs survived for 2 months and matured slowly into glia- and tissue-specific neuronal precursors. SPC-01 cells differentiated more in astroglial phenotypes with a dense structure of the implant, whereas iPSC-NPs displayed a more neuronal phenotype with a loose structure of the graft. We concluded that the BBB scores of iPSC-NP- and hMSC-injected rats were superior to the SPC-01-treated group. The iPSC-NP treatment of spinal cord injury (SCI) provided the highest recovery of locomotor function due to robust graft survival and its effect on tissue sparing, reduction of glial scarring, and increased axonal sprouting.

A green tea polyphenol epigallocatechin-3-gallate enhances neuroregeneration after spinal cord injury by altering levels of inflammatory cytokines

&NA; Spinal cord injury (SCI) is a debilitating condition which is characterized by an extended secondary injury due to the presence of inflammatory local milieu. Epigallocatechin gallate (EGCG) appears to possess strong neuroprotective properties. Here, we evaluated the beneficial effect of EGCG on recovery from SCI. Male Wistar rats were given either EGCG or saline directly to the injured spinal cord and thereafter a daily IP injection. Behavior recovery was monitored by BBB, plantar, rotarod and flat‐beam tests. The levels of inflammatory cytokines were determined on days 1, 3, 7, 10 and 14 after SCI. Additionally, NF‐&kgr;B pathway activity was evaluated. The results demonstrated that EGCG‐treated rats displayed a superior behavioral performance in a flat beam test, higher axonal sprouting and positive remodelation of glial scar. Cytokine analysis revealed a reduction in IL‐6, IL2, MIP1&agr; and RANTES levels on days 1 and 3, and an upregulation of IL‐4, IL‐12p70 and TNF&agr; 1 day following SCI in EGCG‐treated rats. Treatment with EGCG was effective in decreasing the nuclear translocation of subunit p65 (RelA) of the NF‐&kgr;B dimer, and therefore canonical NF‐&kgr;B pathway attenuation. A significant increase in the gene expression of growth factors (FGF2 and VEGF), was noted in the spinal cord of EGCG‐treated rats. Further, EGCG influenced expression of M1 and M2 macrophage markers. Our results have demonstrated a therapeutic value of EGCG in SCI, as observed by better behavioral performance measured by flat beam test, modulation of inflammatory cytokines and induction of higher axonal sprouting. HighlightsEGCG improved fuctional outcome, increased axonal sprouting and remodelate glial scar in rats with spinal cord injury.EGCG treatment attenuated canonical NFkB pathway.EGCG treatment increased the gene expression of growth factors (FGF2 and VEGF) in injured spinal cord.

The Effect of Human Mesenchymal Stem Cells Derived from Wharton’s Jelly in Spinal Cord Injury Treatment Is Dose-Dependent and Can Be Facilitated by Repeated Application

Human mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs) were used for the treatment of the ischemic-compression model of spinal cord injury in rats. To assess the effectivity of the treatment, different dosages (0.5 or 1.5 million cells) and repeated applications were compared. Cells or saline were applied intrathecally by lumbar puncture for one week only, or in three consecutive weeks after injury. Rats were assessed for locomotor skills (BBB, rotarod, flat beam) for 9 weeks. Spinal cord tissue was morphometrically analyzed for axonal sprouting, sparing of gray and white matter and astrogliosis. Endogenous gene expression (Gfap, Casp3, Irf5, Cd86, Mrc1, Cd163) was studied with quantitative Real-time polymerase chain reaction (qRT PCR). Significant recovery of functional outcome was observed in all of the treated groups except for the single application of the lowest number of cells. Histochemical analyses revealed a gradually increasing effect of grafted cells, resulting in a significant increase in the number of GAP43+ fibers, a higher amount of spared gray matter and reduced astrogliosis. mRNA expression of macrophage markers and apoptosis was downregulated after the repeated application of 1.5 million cells. We conclude that the effect of hWJ-MSCs on spinal cord regeneration is dose-dependent and potentiated by repeated application.

The safety and efficacy of a new anticoagulation strategy using selective in-circuit blood cooling during haemofiltration—an experimental study

BACKGROUND Selective in-circuit blood cooling was recently shown to be an effective anticoagulation strategy during short-term haemofiltration. The aim of this study was to examine the safety of this novel method and circuit life. METHODS Fourteen pigs were randomly assigned to receive continuous haemofiltration with anticoagulation achieved either by selective cooling of an extracorporeal circuit (ECC) (COOL; n = 8) or through systemic heparinization (HEPARIN; n = 6). Before (T0) as well as 1 (TP1) and 6 h (TP6) after starting the procedure the following parameters were assessed: animal status, variables reflecting haemostasis, oxidative stress, inflammation and function of blood elements. RESULTS All animals remained haemodynamically stable with unchanged body core temperature and routine biochemistry. Regional ECC blood cooling did not alter clinically relevant markers of haemostasis, namely activated partial thromboplastin and prothrombin times, thrombin-antithrombin complexes, von Willebrand factor and plasminogen activator inhibitor-1. Platelet aggregability, serum levels of free haemoglobin, leukocyte count, oxidative burst and blastic transformation of T-lymphocytes were all found to be stable over the treatment period in both groups. ECC blood cooling affected neither plasma malondialdehyde concentrations (a surrogate marker of oxidative stress) nor plasma levels of cytokines (tumour necrosis factor-α, interleukin-6 and -10). While the patency of all circuits treated with systemic heparin was well maintained within the pre-selected period of 24 h, the median filter lifespan in the COOL group was 17 h. CONCLUSION Utilizing clinically relevant markers, selective in-circuit blood cooling was demonstrated to be a safe and feasible means of achieving regional anticoagulation in healthy pigs. The long-term safety issues warrant further evaluation.

Artificial Lung Ventilator Working in the Hyperbaric Chamber

The aim of this article is to identify and interpret the data provided by modern digitally controlled ventilators that provide the greatest help in evaluating respiratory mechanics during mechanical ventilation. In intensive care, respiratory mechanics can be assessed in dynamic conditions (no flow interruption) or static conditions (occlusion techniques) to record compliance and resistance and to monitor pressure, flow, and volume. Real-time visualization of the pressure curve is crucial for monitoring during volume-controlled ventilation, in which pressure is the dependent variable. The system for forced inspiration in hyperbaric chamber was designed, simulated and evaluated. The system is scheduled for animal experiments on anestesied pig and it is based on pressure control ventilation mode. The whole system is controlled by microcontroller which is connected to personal computer. The control system is equipped also with high resolution analog-digital converter with on chip digital lowpass filter, which is used for precise measuring of some physical quantities by means of sensors e.g. oxygen concentration in hyperbaric chamber, temperature and pressure in hyperbaric chamber.

Evaluation by volunteers of respirator characteristics in modes used in non-invasive ventilation

We studied the medical personnels power of distinction various types of respirators in CPAP and CPAP+pressure support (PS) modes.