[Metabolic disorders of chronically critically ill patients caused by consequences of traumatic brain injury].

The problem of chronic critical illness therapy is relevant all over the world. Revealing the metabolic function in patients in chronic critical condition is an important link in the development of adequate treatment and rehabilitation tactics. The aim - identification of metabolic features in chronic critical patients after brain injury in the first 3 days from the moment of admission to the rehabilitation center. Material and methods. Single-center observational study included a group of 25 patients with chronic critical illness, aged 38.7±14.0 years with body mass index 20.8± 4.3 kg/m2 (min 14.5; max 29.7), who were on independent breathing through a tracheostomy tube, and who have pronounced neurological disorders in the form of depression of minimally conscious state, FOUR scale from 12 to 16 points, as well as bedsores 1-2 stage and polysegmental pneumonia. The patients underwent indirect calorimetry and analysis of biochemical parameters of protein, carbohydrate, fat and mineral metabolism, as well as the level of nitrogen excretion with urine. Results and discussion. The data obtained indicate that chronic critical ill patients with the consequences of traumatic brain injury had pronounced metabolic disorders, mainly of a protein component. Total protein level decreased up to 61.0±9.4 g/l (min 39.1; max 83.1), albumin up to 30.2±6.0 g/l (min 17.4; max 37.8), prealbumin up to 0.13± 0.06 g/l (min 0.04; max 0.23) and transferrin up to 147.7±37.7 mg/dl (min 84.0; max 209.0). The patients' requirement in protein was 106.4±38.5 g/day (min 57.1; max 160.5) or 1.55±0.46 g/kg/day (min 0.75; max 2.22). The level of resting energy expenditure measured by indirect calorimetry was 1549.1±421.8 kcal/day (min 673.0; max 2430.0) or in terms of body weight 24.8±7.6 kcal/kg/day (min 12.4; max 45.8). Conclusion. The data obtained indicate a continuing catabolic phase in patients more than 30 days after the primary injury, which led to their chronic critical condition.

Experimental protocol for development of adjuvant-free murine chronic model of allergic asthma.

BACKGROUND: Mouse models of allergic asthma play a crucial role in exploring of asthma pathogenesis and testing of novel anti-inflammatory drugs. Widely used acute asthma models usually developed with adjuvant (aluminum hydroxide (alum)) do not reproduce one of the main asthma feature - airway remodeling while chronic asthma model mimic the pathophysiology of human disease. Moreover, the use of alum causes distress in experimental animals and impedes the test of adjuvant-containing drugs. In this study, we aimed to develop a chronic adjuvant-free asthma model with pronounced asthmatic phenotype. METHODS: Female BALB/c mice were divided into 3 groups. The first group was sensitized with intraperitoneal injections of ovalbumin (OVA) emulsified in aluminum hydroxide on days 0, 14, 28 followed by two stages of intranasally challenge with OVA on days 41-43 and 62-64. The second group was subcutaneously sensitized with the same dose of OVA without adjuvant and challenged on the same days. The third group (negative control) included mice which did not received any kind of treatment (i.e. sensitization and challenge). Serum levels of OVA-specific IgE, IgG2a and IgG1 antibodies were detected by ELISA. Airway hyper-responsiveness was measured by non-invasive plethysmography on days 44 and 65. Bronchoalveolar lavage fluids (BALF) sampled in all groups on days 45 and 66 were analyzed by light microscopy. The left lung was removed for histological analysis. The IL-4 and IFNγ mRNA expression in BALF cells was evaluated by RT-PCR. RESULTS: The OVA-specific IgE antibody response was two-fold increased in mice from adjuvant-free group compared to the adjuvant group that reflects reorientation of immune response towards Th2 phenotype. At the same time, the level of OVA-specific IgG1 and IgG2a antibodies was increased in the adjuvant group. Airway hyperresponsiveness to methacholine in mice of both experimental groups was two-fold higher than in control. Analysis of cell composition in BAL has shown a significant increase in eosinophil count in both experimental groups that indicate the development of allergic inflammation. Lung histology revealed airway remodeling in both experimental groups including goblet cell hyperplasia/metaplasia, thickening of airway walls, collagen deposition in the wall of distal airways. Additionally, the tendency to develop hypertrophy of bronchial smooth muscle layer was observed. Study of gene expression in BAL cells revealed the increase of IL-4 level in both adjuvant and adjuvant-free groups while IFNγ expression in both experimental groups was similar to control group. CONCLUSION: We have developed a chronic adjuvant-free mouse asthma model which possesses all necessary features of the disease including airway remodeling and is more suitable for pre-clinical evaluation of novel therapeutic approaches including adjuvant-containing drugs.

[Effects of vascular peptide bioregulator on cerebral microcirculation of old hypertensive rats].

Using a TV device to study brain microcirculation, we found that after a course of vascular peptide bioregulator the density of microvascular network of pia matter of old hypertensive rats (12 months) sensomotor cortex increased about 1,7 times compared to intact old rates SHR. This perfusion in the tissue of the cerebral cortex and the degree of blood oxygen saturation in the microvasculature of this tissue region raised.

Effect of Transplantation of Mesenchymal Stem Cells on the Density of Pial Microvascular Network in Spontaneously Hypertensive Rats of Different Age.

Using a TV device for studying microcirculation (×40), we analyzed the density of the whole microvascular network and the density of arterioles in the pia mater of the sensorimotor cortex in SHR rats of different ages (3-4 and 12 months) after intracerebral transplantation of human mesenchymal stem cells. We found that the density of pial microvascular network in SHR rats receiving transplantation of human mesenchymal stem cells increased to a level observed in young Wistar-Kyoto rats.

Effects of Vascular Peptide Bioregulator on the Density of Microvascular Network in the Brain Cortex of Aged Rats.

Using a TV unit for microcirculation studies, we studied the density of the entire microvascular network and its arterial compartment in the pia mater of the sensorimotor cortex in intact young and aged rats (2-3 and 22-24 months) and aged rats after a course of vascular peptide bioregulator Slavinorm. The experiments showed that the density of microvascular network in intact animals decreased by 1.6 times with aging. Treatment with the peptide bioregulator 2.5-fold increased the density of the microvascular network in aged rats in comparison with intact animals of the same age, the density of the arterial compartment of the microvascular network in the pia mater increased by 2.8 times.

Influence of High Blood Pressure on Microcirculation in Cerebral Cortex of Young Rats.

We studied the density and structure of the microvascular network of the pia mater, the blood flow rate and oxygen saturation in the sensorimotor cortex of young spontaneously hypertensive rats (SHR). The density of the microvascular network in hypertensive animals was by ~1.4 times lower than in normotensive Wistar-Kyoto rats (control) and arteriolar bed density was lower by ~1.9 times. The blood flow rate in tissue and oxygen saturation in the sensorimotor cortex in SHR rats were significantly lower than in control animals.

[Systemic errors in the estimation of systemic arterial pressure via transfer functions after experimental changes of the circulating blood volume].

The results of the experimental studies reveal the decrease of vascular rigidity with the increase of systemic error of the arterial pressure values estimated via implication of transfer functions following haemorrhage modeling in rats. Compensation of the circulating blood volume by means of dextran infusion resulted in restoration of both vascular rigidity and systemic error of estimated arterial pressure. The results can be put into medical practice as an approach for testing of non-invasive methods in cardiovascular research.

[The effect of vascular peptide bioregulator on the microcirculation in the brain cortex of old rats].

Using a TV device to study brain microcirculation, we found that after a course of vascular peptide bioregulator the density of microvascular network of pia matter of old rats (22-24 months) sensomotor cortex increased about 2,5-2,8 times compared to control old rates; and noradrenaline-induced constriction reactions and acetylcholine-induced dilative reactions of the pial arterioles increased significantly. This perfusion in the tissue of the cerebral cortex is not increased, but the degree of blood oxygen saturation in the microvasculature of this tissue region raised.

Age-related changes of microcirculation in pia mater of rats' sensorimotor cortex.

We studied the density of the microvascular network in the pia mater, the tissue perfusion and saturation of the oxygen of sensorimotor cortex of hypertensive rats of different ages. Our investigations shown that the microvascular density in the pia mater was not only decreased, but also was increased. The perfusion of sensorimotor cortex was reduced and tissue saturation was enlarged. By the age of 12 months orienting-exploratory rats behavior became worse significantly in all major indicators of the test «open field¼.

[THE AGING OF MICROVASCULAR NETWORK FORMED IN CORTEX FOLLOWING INTRACEREBRAL TRANSPLANTATION OF MESENCHYMAL STEM CELLS].

Using a TV device to study microcirculation in brain we found that intracerebral transplantation of mesenchymal stem cells to 12-months old rats led to a significant increase (circa 1,5-fold times) of microvascular density in pia tissue and to increased constriction reactions of pia arterioles in response to noradrenalin application on a brain surface. Both microvascular density and pia arterioles reactivity was completely preserved in aging until 22-24 months.

[Age-related changes in blood microcirculation functional state of the brain cortex of rats].

Male Wistar-Kyoto rats, aged 22-24 months, were intracerebrally transplanted syngeneic mesenchymal stem cells (MSC). Cognitive functions of these animals were tested in 3 weeks. The density of microvascular bed, the tissue blood flow, saturation of mixed blood in cortex microvessels were measured under the standard conditions, under the impact of noradrenaline and in the context of global ischemia. The control groups consisted of rats aged 2-3 months and 22-24 months. MSC transplantation have a positive impact on the microcirculation in brain cortex of old animals: reduction of vascular density was prevented, level of saturation in the blood vessels was increased, which enabled animals to endure easier extreme impacts, but did not correct age-related deterioration of cognitive functions.

[Efficacy of mesenchymal stem cells intracerebral transplantation for the correction of age-related cerebral microcirculation alterations in rats].

Using a television-based vital microscopy method and immunohystochemical analysis, we have assessed the effect of intracerebral transplantation of syngeneic mesenchymal stem cells (MSC) on the brain cortex structure and the microcirculation in the pia mater of old rats. Using "open field" system, we have studied the effect of MSC transplantation on position-finding and discovery behavior of older animals. We have found that density of microvascular network of the pia mater increased ca. 1.9-fold in MSC recipients, compared to age-matched intact animals. Density of the arteriolar area of microvascular network of the pia mater increased ca. 2-fold. Reactivity of the newly formed arterioles was nearly equal to that of native microvessels. Intracerebral transplantation procedure itself was traumatic for brain cortex of rats, but it had no effect on the microcirculation in the contralateral hemisphere. Intracerebral transplantation of MSC did not improve locomotor behavi- or and emotional stage of old rats, did not increase their position-finding and discovery activity.

Effect of intracerebral transplantation of mesenchymal stem cells on reactivity of pial arterioles in old rats.

Using a TV device for studying microcirculation (×160), we analyzed the responses of arterioles in the pia mater of the sensorimotor cortex in young (2-3 months) and old (22-24 months) rats after local application of a vasoconstrictor (norepinephrine, 10(-6) M) or vasodilator (acetylcholine, 10(-6) M). The responses of the arterioles were evaluated by changes in their diameter and by the number of responding vessels in the field of view. The constrictor responses of the pial arteries to norepinephrine did not significantly differ in intact young and old rats. The number and degree of dilatory responses to acetylcholine in old rats were lower than in young animals by 14 and 30%, respectively. Intracerebral transplantation of mesenchymal stem cells to old rats had practically no effect on reactivity of pial arterioles to acetylcholine, while the number of constricted vessels in response to norepinephrine increased by ~20%.

[Mesenchymal stem sell donor age effect on the cerebral cortex microvascular net density in old age rats-recipients of transplant].

Male Wistar-Kyoto rats aged 22-24 months were intracerebrally transplanted with syngenic bone marrow mesenchymal stem cells (BM MSC) established from the donor aged 3-4 months and 20-22 months, respectively. Using a TV device to study microcirculation in vivo, we have established that transplantation of BM MSC from young donors increased a density of the microvascular network in the pia mater of the sensorimotor cortex in old rats approximately 1.9-fold, comparing to age-matched controls, while a density of the arteriolar compartment increased approximately 2.1-fold. Transplantation of BM MSC from old donors did not lead to the significant increase in the density of the microvascular network in the pia mater, while a density of the arteriolar compartment increased approximately 1.5-fold.

[Detection of Staphylococcus aureus toxins using immuno-PCR].

A highly sensitive test system, based on the method of immuno-PCR, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was obtaining supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were used as DNA tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not less than 10 pg/mL.

Effect of intracerebral transplantation of mesenchymal stem cells on pial microcirculation in rats.

We studied the effect of intracerebral transplantation of bone marrow mesenchymal stem cells on microcirculation (density of microvascular network and reactivity of arterioles) in the pia mater of 2-3-month-old rats. It was found that after transplantation of mesenchymal stem cells, the density of pial microcirculatory network in the contralateral hemisphere significantly increased (by 1.7 times; p<0.05) in comparison with both intact animals and controls. The number of arterioles in the studied area increased most markedly (by ≈2.5 times; p<0.05) in comparison with other groups. Intracerebral transplantation of mesenchymal stem cells or conditioned culture medium (α-MEM) had no effect on reactivity of pial arterioles.

Effect of transplantation of mesenchymal stem cells on the density of pial microvascular network in rats of different age.

Using a TV device for studying microcirculation (×40), we studied the density of the whole microvascular network and arteriolar its compartment in the pia mater of the sensorimotor cortex in rats of different age (2-3, 12, and 24 months) after intracerebral transplantation of mesenchymal stem cells or nutrient medium (control). The density of the microvascular network in the pia mater remained practically unchanged until 1 year, but then decreased by 1.8 times with adding (up to 2 years). MSC transplantation 1.5-1.8-fold increased the density of the pial microvessels in animals of all age groups in comparison with intact and control rats; the density of the arteriolar compartment increased by 2.1-2.4 times. Intracerebral injection of MSC to 1-year-old animals prevented pathological decrease in the density of microvascular network during the next year of life.

[Age-related changes of microcirculation in pia mater of rats' sensorimotor cortex].

We studied the density of whole microvascular network and separately the density of arterioles in the pia mater of sensorimotor cortex of rats of different ages. Also pial arteriolar reactivity on exposure to norepinephrine or acetylcholine chloride was evaluated. The microvascular density and the arteriolar reactivity in the pia mater were not significantly changed before the age of 12 months. In the age of 22-24 months the density of whole microvascular network decreased 1,7 times at the mean and the density of arterioles decreased 1,2 times. There were no significant changes of pia arterioles constriction during the animals life but dilatation was noticeably worse in senility. Orientation and exploratory behavior got worse to the age of 22-24 months: the number of behavioral acts in the "open field" test decreased 1,5-2,3 times in comparison with young animals.

[The effect of intracerebral transplantation of mesenchymal stem cells on orientation and exploratory behavior of aged animals].

Male Wistar-Kyoto rats (12 and 22-24 months old) were intracerebrally transplantated with syngeneic mesenchymal stem cells (MSC). Three weeks or one year later, orientation and exploratory behavior of the same animals (not 2 years old) was assessed using an «open field¼ test. Older rats (22-24 months old) were inhibited in the basic behavioral acts in comparison to younger animals (2-3 months old). Transplantation of MSC did not cause improvement of an orientation and exploratory behavior of aged rats. Morphological and immunohistochemical analysis suggests intracerebral MSC transplantation led to the injury of the cortex of the ipsilateral hemisphere.

[The effect of intracerebral mesenchymal stem cells transplantation on the density of microvascular network of the pial matter of the rat brain cortex].

Using a TV installation for studying the microcirculation (with 30-160-fold magnification), the density of microvascular network in the pia matter of the rat brain sensomotor cortex was determined after intracerebral transplantation of mesenchymal stem cells (MSC) or (as control) of the MSC cultivation nutrition medium, or of saline. The results have shown that intracerebral transplantation does not change density of microvascular network in the pia mater of the ipsilateral hemisphere. Transplantation of the MSC led to a 1.8-fold increase of density of the pia matter of the contralateral hemisphere as compared with control animals; the number of arterioles in the same zone was 2.5-fold higher than in intact rats.