Effect of Collagen and GelMA on Preservation of the Costal Chondrocytes' Phenotype in a Scaffold in vivo.

The aim of the study was to compare type I collagen-based and methacryloyl gelatin-based (GelMA) hydrogels by their ability to form hyaline cartilage in animals after subcutaneous implantation of scaffolds. Materials and Methods: Chondrocytes were isolated from the costal cartilage of newborn rats using 0.15% collagenase solution in DMEM. The cells was characterized by glycosaminoglycan staining with alcian blue. Chondrocyte scaffolds were obtained from 4% type I porcine atelocollagen and 10% GelMA by micromolding and then implanted subcutaneously into the withers of two groups of Wistar rats. Histological and immunohistochemical studies were performed on days 12 and 26 after implantation. Tissue samples were stained with hematoxylin and eosin, alcian blue; type I and type II collagens were identified by the corresponding antibodies. Results: The implanted scaffolds induced a moderate inflammatory response in both groups when implanted in animals. By day 26 after implantation, both collagen and GelMA had almost completely resorbed. Cartilage tissue formation was observed in both animal groups. The newly formed tissue was stained intensively with alcian blue, and the cells were positive for both types of collagen. Cartilage tissue was formed among muscle fibers. Conclusion: The ability of collagen type I and GelMA hydrogels to form hyaline cartilage in animals after subcutaneous implantation of scaffolds was studied. Both collagen and GelMA contributed to formation of hyaline-like cartilage tissue type in animals, but the chondrocyte phenotype is characterized as mixed. Additional detailed studies of possible mechanisms of chondrogenesis under the influence of each of the hydrogels are needed.

Decellularized Extracellular Matrix for Tissue Engineering (Review).

In recent years, decellularized tissues have evolved into a new, full-fledged platform for the creation of tissue-engineered constructions. Extracellular matrix (ECM) of each tissue provides a unique tissue-specific microenvironment for resident cells with the structure and biochemical signaling required for their functioning. The decellularized ECM (dECM) has been established to influence cell differentiation. The review provides recent data on the composition and functions of the ECM, methods for obtaining decellularized tissues, and their application in tissue engineering depending on their physical form (scaffold, powder, or hydrogel). The effect of the matrix source, decellularization and sterilization techniques on dECM composition has been considered. Regulatory mechanisms of cell differentiation by the extracellular matrix are discussed. Differences in the protein composition of the native and decellularized materials are presented. Application of dECM in the bioink composition for regeneration of various tissues using bioprinting technologies is also considered. It has been concluded that successful application of dECM in tissue engineering and regenerative medicine requires a permanent and biologically suitable dECM source, optimized tissue decellularization protocols, improved mechanical properties of dECM-derived bioinks, and prevention of immunological reaction of the organism.

The Technique of Thyroid Cartilage Scaffold Support Formation for Extrusion-Based Bioprinting.

During biofabrication, a tissue scaffold may require temporary support. The aim of this study was to develop an approach of human thyroid cartilage scaffold temporal support formation. The scaffold 3D-model was based on DICOM images. XY plane projections were used to form scaffold supporting part. To verify the technique, collagen hydrogel was chosen as the main scaffold component. Gelatin was applied for the supporting part. To test the applicability of the approach, a model of thyroid cartilage scaffold with the support was printed. The scaffold corresponded to a given model, although some discrepancy in geometry was observed during verification by computed tomography.

In Vivo Effects of Human Bone Marrow Mesenchymal Stromal Cells on the Development of Experimental B16 Melanoma in Mice.

Experiments on F1(CBA×C57BL/6) mice with experimental metastatic melanoma B16 F10 showed that single intravenous injection of xenogeneic bone marrow mesenchymal stromal cells (BM-MSC) in a dose of 106 cells/mouse significantly increased 100-day survival rate of tumor-bearing animals. In contrast, administration of BM-MSC in a dose of 2×106 cells/ mouse reduced survival rates in comparison with the biocontrol (injection of B16 cells alone, 5×105 cells/mouse). This phenomenon can be related to in vivo participation of BM-MSC in reprogramming of resident tissue macrophages, including tumor microenvironment, towards pro- (M1) or anti-inflammatory (M2) phenotype. This is indirectly confirmed by the data on switching from activation to inhibition of ROS-producing activity of blood mononuclears and peritoneal macrophages in tumor-bearing mice in the test of luminol-dependent zymosaninduced chemiluminescence.

Modulation of 4-aminopyridine-induced neuronal activity and local pO(2)in rat hippocampal slices by changing the flow rate of the superfusion medium.

The brain slice preparation is the most frequently used tool for testing of pharmacological agents on the neuronal excitability. However in the absence of blood circulation in vitro, the tissue oxygenation strongly depends on the experimental conditions. It is well established that both hypoxia as well as hyperoxia can modulate the neuronal network activity. Thereby changes in tissue oxygen level during experiment may affect the final result. In the present study we investigated the effect of oxygenation on seizure susceptibility in the hippocampal slice preparation using 4-aminopyridine (4-AP) model of ictogenesis in inmature rats. We found that changing the medium perfusion rate in the range of 1-5 ml/min greatly affects the tissue oxygenation, amplitude and frequency of 4-AP-induced synchronous neuronal activity. The decrease in the flow rate as well as substitution of the oxygen in the extracellular medium with nitrogen causes a strong reduction of 4-AP-induced synchronous neuronal discharges. Our results demonstrate a significant linear correlation between the power of 4-AP-induced neuronal activity and the oxygen level in slice tissue. Also we demonstrated that the presence of medium flow is a necessary condition to support the constant level of the slice oxygenation. These data suggest that the oxygen supply of the brain slice strongly depends on experimental protocol and could modulate in vitro neuronal network excitability which should be taken into consideration when planning epilepsy-related studies.

Surface charge impact in nonsynaptic model of epilepsy in rat hippocampus.

Decreasing of surface charge screening near voltage-gated ion channels via reduction of extracellular cation divalent ions provide potent mechanism of altering cellular excitability and seizure threshold. Spontaneous field potentials were recorded from horizontal brain slices of young Wistar rats (postnatal day 10-12). Extracellular registrations wereobtained from CA1 and CA3 area of hippocampus. For induction of nonsynaptic epileptiform activity slices were perfused with artificial cerebrospinal fluid with omitted Ca2+and Mg2+ ions. Effect of different Mg2+ concentration (1, 2, and 3mmol/l) on initial stage of nonsynaptic epileptiform discharges was studied. Our results suggest that the change in Mg2+ concentration dramatically affects the probability of induction of low-Ca2+seizure-like activity (SLA), providing evidence that Mg2+ can alter cerebral excitability by affecting the surface charge and supporting the idea that surface charge could be a pharmacological target for anti-epileptic treatment.

Thrombin modulates persistent sodium current in CA1 pyramidal neurons of young and adult rat hippocampus.

Serine protease thrombin, a key factor of blood coagulation, participates in many neuronal processes important for normal brain functioning and during pathological conditions involving abnormal neuronal synchronization, neurodegeneration and inflammation. Our previous study on CA3 pyramidal neurons showed that application ofthrombin through the activation of specific protease-activated receptor 1 (PAR1) produces a significant hyperpolarizing shift of the activation of the TTX-sensitive persistent voltage-gated Na+ current (I(Nap)) thereby affecting membrane potential and seizure threshold at the network level. It was shown that PAR1 is also expressed in CA1 area of hippocampus and can be implicated in neuronal damage in this area after status epilepticus. The aim of the present study was to evaluate the effect of thrombin on I(NaP) in CA1 pyramidal neurons from adult and young rats. Using whole cell patch-clamp technique we demonstrate that thrombin application results in the hyperpolarization shift of I(NaP) activation as well as increase in the I(NaP) amplitude in both age groups. We have found that I(NaP) in pyramidal neurons of hippocampal CA 1 region is more vulnerable to the thrombin action than I(NaP) in pyramidal neurons of hippocampal CA3 region. We have also found that the immature hippocampus is more sensitive to thrombin action which emphasizes the contribution of thrombin-dependent pathway to the regulation of neuronal activity in immature brain.

Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation.

The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in efficiency of therapeutic strategies against post-ischemic pathology.

[EFFECT OF NEONATAL SEIZURES ON THE SYNAPTIC PLASTICITY OF RAT SOMATOSENSORY CORTEX].

Using an experimental model of neonatal recurrent seizures we investigated the influence of epileptic seizures in the various forms of synaptic plasticity in neurons of the somatosensory cortex. We found that early seizures do not affect the post-tetanic potentiation of the amplitude of the postsynaptic potentials and the depression of postsynaptic potentials during high-frequency stimulation. However they result in the chronic increase of the long-term potentiation of synaptic transmission. These changes of synaptic plasticity may affect the processing of the sensory information in patients with a history of recurrent seizures during early development.

A comparative study of the biological effectiveness of 14-MeV neutron pulse and continuous radiation using mouse melanoma B-16 cells.

The study was carried out using compact neutron generators with a sealed tube operating in pulsed (neutron generator ING-031) and continuous (NG-14) modes. Neutron radiation was formed due to reaction T(d,n)(4)He. The average flow of 14-MeV neutrons was 6.6×10(9) ns(-1) for ING-031 and 1.2-1.6×10(10) n s(-1) for NG-14. Duration of an impulse was ∼1 ms and pulse frequency of 50 Hz. The gamma rays of (60)Со source with an average energy of 1.25 MeV were standard radiation. Biological efficacy was estimated using the clonogenic activity of mice melanoma B-16 cells. Comparison of biological effects of neutron irradiation in pulse and continuous modes showed no significant difference between them. RBE values of pulse (ING-031) and continuous (NG-14) neutron radiation were equal-in the range of 2.4-2.6. According to the clonogenic activity of melanoma B-16 cells no dose rate effect was observed within the studied range of neutrons doses and dose rates.

Anaesthetic and postanaesthetic effect of isoflurane on the multiple-unit activity of the immature rat hippocampus.

We investigated anesthetic and postanaesthetic effect of isoflurane on the multi-unit activity (MUA) in the CA3 region of immature rat hippocampus. MUA amplitude did not significantly change during application of isoflurane. On the other hand MUA frequency significantly decreased during the anesthesia. After isoflurane discontinuation two phases of MUA frequency recovery were observed: initial rapid increase followed by a slower recovery to the control level. Comparison of recovering period of the receptor mediated systems and spontaneous field activity from isoflurane anesthesia is discussed.

The effect of neuraminidase blocker on gabazine-induced seizures in rat hippocampus.

Concentration of neuraminidase (NEU), an enzyme which cleaves negatively charged sialic acids from carbohydrate moieties of the cellular membrane, could vary depending on physiological conditions. Multiple evidences suggest that fluctuations of NEU extracellular concentrations can influence neuronal activity. In the present study we examined the effect of down regulation of endogenous NEU activity on seizure-like activity (SLA) induced by gabazine (specific blocker of inhibitory synaptic transmission) in the hippocampal CA1 pyramidal region of cultured slices. We show that in slices pretreated with the blocker of endogenous NEU, N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA), duration of synchronous oscillations induced by gabazine was considerably increased comparatively to control untreated slices. This study adds further information that changes in the level of NEU activity is an important factor, which can affect neuronal network excitability.

Mechanism of antiseizure effect of isoflurane in the immature rat hippocampus.

The volatile anesthetic isoflurane is often used in children in the management of refractory status epilepticus. However the mechanism of anticonvulsant action of isoflurane during early brain development is not clear. In this study we explore the role of excitatory and inhibitory conductances in antiseizure effect of isoflurane using combination of whole-cell patch-clamp and extracellular field potential recording techniques on two models of epilepsy in a hippocampal slice preparation from immature rat. Our data demonstrated that decreasing of excitatory synaptic transmission does not account for antiseizure effect of this volatile anesthetic agent. Isoflurane decreases the synchronization of neuronal activity mainly through the enhancing of GABAergic inhibition by influencing both phasic and tonic chloride conductances.

Effects of isoflurane on hippocampal seizures at immature rats in vivo.

In the present study we examined the effect of isoflurane on seizure-like activity at hippocampal CA3 pyramidal region of immature rats in vivo. We found that isoflurane in clinically relevant concentrations effectively stops hippocampal seizures. When animal was under isoflurane anesthesia seizure-like activity still can be evoked by application of proconvulstant agents, but this activity was qualitatively different from that obtained in nonanesthetized rats. This observation suggests that the mechanism by which seizures are terminated differ from the mechanism responsible for preventing of the initiation of seizures.

Possibility of multiquantal transmission at single inhibitory synapse in cultured rat hippocampal neurons.

Miniature, spontaneous and evoked inhibitory postsynaptic currents were studied using the whole-cell patch-clamp technique on synaptically connected cultured hippocampal neurons, at a holding potential of -75 mV. All experiments were done in tetrodotoxin-containing solution to exclude an action potential generation. Spontaneous miniature inhibitory postsynaptic currents were observed in Ca2+-free solution. The distribution of miniature inhibitory postsynaptic currents was skewed to larger current amplitudes and could be fitted reliably by one Gaussian with the mean at 10.0 +/- 1.2 pA (n = 7). Spontaneously occurring whole-cell spontaneous inhibitory postsynaptic currents were recorded in physiological solution (Ca2+ 2 mM). The average amplitude of spontaneously occurring currents depended on membrane potential and reversed at -18 +/- 5 mV (n = 5). The amplitude distribution of spontaneous inhibitory postsynaptic currents had one peak clearly detectable with the mean of 20.0 +/- 2.0 pA (n = 6) or 10.0 +/- 2.0 pA (n = 2). Inhibitory postsynaptic stimulus-evoked currents arose in responses to gradual activation of neurotransmitter release by direct extracellular electrical stimulation of a single presynaptic bouton by short depolarizing pulses. The current-voltage relation of the averaged amplitudes of evoked inhibitory postsynaptic currents was linear and reversed at potential predicted by the Nernst equation for corresponding intra- and extracellular Cl- concentrations. The time-course of decay of miniature, spontaneous and evoked inhibitory postsynaptic currents was fitted by a sum of two exponents and their time-constants were the same in the range of standard deviation. The stimulus-evoked inhibitory postsynaptic currents fluctuated with regard to the discrete aliquot values of their peak amplitudes in all the investigated synapses from a measurable minimum of about 8 pA to 200 pA. The evoked inhibitory postsynaptic currents were assumed as superimposition of statistically independent quantal events. Fitting amplitude histograms of evoked inhibitory postsynaptic currents with several Gaussian curves resulted in peaks that were equidistant with the mean space of 20 +/- 3 pA (n = 10), which was assumed as one quantum (quantum size) to construct the Poisson's distribution. A possibility of simultaneous multiquantal release at single inhibitory synapses of rat hippocampal neurons was discussed.

[Several principles for epidemiologic evaluation of individual regions of the Trans-Caucasian plains-piedmont natural focus of plague]. / O nekotorykh printsipakh épidemiologicheskoi otsenki otdel'nykh uchastkov Zakavkazskogo ravninno-predgornogo prirodnogo ochaga chumy.

The authors analyse many-year data concerning the epizootological study of seven landscape-geographical regions of the Eastern Transcaucasus differing by natural conditions and the enzootic intensity. Quantitative assessment of natural nidality determined by the level (norm) of many-year indices calculated for the years of 1955--1974 are presented for each region. Flea vectors were more numerous in localities of stable enzootic manifestation; also the numbers of Meriones erythrourus (carrier rodent) was more stable than in the regions of temporary occurrence of the causative agent and those free of plague. Many-year level of the flea (X. conformis) abundance index equal to one served as the threshold value determining the territory enzooticity.