Возможности флуоресцентной визуализации в оценке реваскуляризации гетеротопически трансплантированного сегмента трахеи приматов

Objective: to assess the potentials of using indocyanine green fluorescence angiography in evaluating revascularization of tissue-engineered construct that was obtained from the decellularized biological matrix of primate trachea, including using mesenchymal stem cells, after heterotopic tracheal allotransplantation. Material and methods. Tracheas were obtained from two male hamadryas baboons. After decellularization, 4 cm segments of tracheas were implanted under the lateral part of the latissimus dorsi in two healthy primates, one after recellularization with mesenchymal stem cells (animal 1), and the second without recellularization (animal 2). Immunosuppressive therapy was not performed. Blood flow in the transplanted segment of the trachea was evaluated 60 days after transplantation by surgical isolation of the flap of the latissimus dorsi with the transplanted segment of the trachea, while maintaining blood flow through the thoracodorsal artery. Indocyanine green near-infrared fluorescence angiography was visualized using a FLUM-808 multispectral fluorescence organoscope. Results. Sixty days after implantation, the tracheal cartilaginous framework macroscopically appeared to be intact in both animals, tightly integrated into the muscle tissue. The framework retained its natural color. After intravenous injection of indocyanine green, the tracheal vessels were visualized in both animals. Intercartilaginous vessels and portions of the cartilaginous semi-rings devoid of vessels were clearly distinguished. The entire implanted segment was almost uniformly vascularized. No local disruptions in blood supply were observed. The fluorescence brightness of the tracheal vessels was 193 ± 17 cu and 198 ± 10 cu in animals 1 and 2, respectively. The average muscle brightness in the implantation zone was 159 ± 9 cu and 116 ± 8 cu in animals 1 and 2, respectively. Conclusion. Indocyanine green fluorescence angiography is characterized by high-contrast images and high sensitivity. This facilitates vascular patency visualization and allows to assess the degree of neoangiogenesis after experimental transplantation of the tracheal segment, at different stages of experiment, without euthanizing the animal.Цель. Определение возможности использования индоцианиновой флуоресцентной ангиографии для оценки реваскуляризации тканеинженерной конструкции, полученной на основе децеллюляризированного биологического матрикса трахеи приматов, в том числе с использованием мезенхимных стволовых клеток, после ее гетеротопической аллотрансплантации. Материал и методы. Донорами трахеи послужили два самца павиана гамадрила. После децеллюляризации участки донорских трахей, по 4 см каждый, имплантированы под боковой участок широчайшей мышцы спины двум здоровым приматам, одному после рецеллюляризации мезенхимными стволовыми клетками (животное 1), второму – без проведения рецеллюляризации (животное 2). Иммуносупрессивную терапию не проводили. Наличие кровотока в трансплантированном сегменте трахеи оценивали через 60 суток после трансплантации путем хирургического выделения лоскута широчайшей мышцы спины с трансплантированным сегментом трахеи с сохранением кровотока по торакодорзальной артерии. Визуализацию инфракрасной индоцианиновой флуоресцентной ангиографии проводили с помощью мультиспектрального флуоресцентного органоскопа «FLUM-808». Результаты. Через 60 суток после имплантации хрящевой каркас трахеи макроскопически представлялся сохранным у обоих животных, плотно интегрированным в мышечную ткань, естественного цвета. После внутривенного введения индоцианина зеленого у обоих животных удалось визуализировать сосуды трахеи, четко различались межхрящевые сосуды и участки хрящевых полуколец, лишенные сосудов, весь имплантированный сегмент практически равномерно васкуляризирован, локальных нарушений кровоснабжения не отмечалось. Яркость флуоресценции сосудов трахеи у животного 1 составила 193 ± 17 у. е., у животного 2 – 198 ± 10 у. е., в то время как средняя яркость мышцы в зоне имплантации у животного 1 – 159 ± 9 у. е., а у животного 2 – 116 ± 8 у. е. Заключение. Индоцианиновая флуоресцентная ангиография характеризуется высокой контрастностью получаемых изображений, высокой чувствительностью, что может позволить визуализировать проходимость сосудистой сети и оценить степень неоангиогенеза после экспериментальной трансплантации сегмента трахеи на разных этапах эксперимента без эвтаназии животного

Возможности клеточной терапии в торакальной хирургии

Introduction. Bronchial fistula often occurs after lung surgery. This is an extremely severe complication with high mortality. Despite the variety methods for the treatment of bronchial fistula, the results are not always effective. In our paper, we presented a method of preventing the development of bronchial fistula based on the cell therapy. The objective of the study was to evaluate the effects of the cell therapy on the regeneration of damaged bronchial tissue in an experiment on non-human primates. Methods and materials. The study was performed on 6 non-human primates. Laboratory animals were divided into 2 groups: experimental and control. The left pneumonectomy with the formation of a stump of the main bronchus was done in each primate. In the experimental group, a regenerative product was introduced into the stump; in the control group, 0.9 % NaCl solution was used. The regenerative product consisted of autologous platelet-rich plasma activated by thermal lysis and the suspension of the xenogeneic fibroblast’s culture. The primates were observed for 7, 14 and 21 days, the peripheral blood was taken to study the dynamics of biologically active substances. A histological examination of the autopsy material was performed at the end of the follow-up time. Results. On the early postoperative period, a typical inflammatory reaction was observed and resulted by the intraoperative tissue damage. Then the morphological changes were different in both group. The inflammatory were mild, the restoration of the natural function of the mucous membrane and the formation of scar tissue were earlier in the experimental group than in the control group. An analysis of the dynamics of plasma mediators in the postoperative period revealed an earlier dominance of regenerative processes in the experimental group. Conclusion. The results of the study showed that the cell therapy stimulates the tissue regeneration, thereby preventing the failure of the bronchial stump suture.Введение. Бронхиальные свищи чаще всего возникают после оперативного вмешательства на легких и являются крайне тяжелым осложнением с высокой смертностью. Способы лечения бронхиальных свищей, несмотря на их разнообразие, не всегда являются эффективными. В работе представлен способ профилактики развития бронхиальных свищей, основанный на клеточной терапии. Цель – оценить влияние клеточной терапии на регенерацию поврежденной ткани бронха в эксперименте на нечеловекообразных приматах. Методы и материалы. Исследование было выполнено на 6 особях нечеловекообразных приматов. Лабораторные животные были разделены на 2 группы: опытная и контрольная. Каждому примату была выполнена левая пневмонэктомия с формированием культи главного бронха. В опытной группе в зону культи был введен регенеративный продукт, в контрольной группе – 0,9 % раствор NaCl. Регенеративный продукт состоял из аутологичной плазмы, обогащенной тромбоцитами, активированной температурным лизисом, и культуры ксеногенных фибробластов. Период наблюдения за приматами составил 7, 14 и 21 суток, в течение которого забирали периферическую кровь для изучения динамики биологически активных веществ. По окончании периода наблюдения было проведено гистологическое исследование аутопсийного материала. Результаты. В первые сутки в обеих группах наблюдалась типичная воспалительная реакция, связанная с интраоперационным повреждением тканей, но в дальнейшем морфологические изменения в обеих группах были различными. В опытной группе воспалительные явления были слабо выражены, восстановление непрерывности слизистой оболочки и формирование рубцовой ткани начинало происходить раньше, чем в группе контроля. Анализ динамики плазменных медиаторов в послеоперационном периоде выявил более раннее доминирование репаративных процессов в опытной группе. Заключение. Результаты исследования показали, что клеточная терапия способствует ускорению процессов регенерации тканей, тем самым предотвращая несостоятельность шва культи бронха

Age-Related Changes in the Clustering of Blood Populations in Cynomolgus Monkeys Depend on Sex and Immune Status

Non-anthropoid primates cynomolgus monkeys (Macaca fascicularis), also known as crab-eating macaques, are increasingly used in biomedical and preclinical studies due to their evolutionary proximity to humans, sharing similar diets, infectious and senile diseases. Age-related changes and sexual dimorphism of the immune system of C. monkeys have not been sufficiently characterized in literature, though age and sex differences affect the course of diseases and sensitivity to medications. Aging in C. monkeys is accompanied by an increase in CD3+CD4+CD8+ (DP-T) cells, plasma B-cells, and a decrease in platelets. Erythromyeloid bias has also been noticed in older animals. There was an increase in eosinophils, haematocrit (HCT) and haemoglobin concentration (HGB). Senile decline in the function of the immune system had sex differences. An increase in the number of monocytes, cytotoxic lymphocytes (CTL) and a decrease in the T-helper population were more pronounced in older females. A significant reduction in the number of B-cells and activated T-cells was detected in males only. A moderate correlation with the regression model of aging was established for DP-T, HCT and HGB. The reduction in the B cells count in males and the increase in CTL level in females are moderately correlated with age. Other blood cell populations did not show significant correlations in the regression models due to their high sample variability. The novel cell population CD3-CD20loCD16/CD56+, presumably NK-cells subset, was revealed. This cell population demonstrated an increase trend with age in both sexes. Population-statistical age norms for different sexes for young and very old macaques were established. The blood population clusters associated with sex and immune status in older animals were also identified

Fluorescence imaging in evaluating the revascularization of heterotopically transplanted primate trachea segment

Objective: to assess the potentials of using indocyanine green fluorescence angiography in evaluating revascularization of tissue-engineered construct that was obtained from the decellularized biological matrix of primate trachea, including using mesenchymal stem cells, after heterotopic tracheal allotransplantation. Material and methods. Tracheas were obtained from two male hamadryas baboons. After decellularization, 4 cm segments of tracheas were implanted under the lateral part of the latissimus dorsi in two healthy primates, one after recellularization with mesenchymal stem cells (animal 1), and the second without recellularization (animal 2). Immunosuppressive therapy was not performed. Blood flow in the transplanted segment of the trachea was evaluated 60 days after transplantation by surgical isolation of the flap of the latissimus dorsi with the transplanted segment of the trachea, while maintaining blood flow through the thoracodorsal artery. Indocyanine green near-infrared fluorescence angiography was visualized using a FLUM-808 multispectral fluorescence organoscope. Results. Sixty days after implantation, the tracheal cartilaginous framework macroscopically appeared to be intact in both animals, tightly integrated into the muscle tissue. The framework retained its natural color. After intravenous injection of indocyanine green, the tracheal vessels were visualized in both animals. Intercartilaginous vessels and portions of the cartilaginous semi-rings devoid of vessels were clearly distinguished. The entire implanted segment was almost uniformly vascularized. No local disruptions in blood supply were observed. The fluorescence brightness of the tracheal vessels was 193 ± 17 cu and 198 ± 10 cu in animals 1 and 2, respectively. The average muscle brightness in the implantation zone was 159 ± 9 cu and 116 ± 8 cu in animals 1 and 2, respectively. Conclusion. Indocyanine green fluorescence angiography is characterized by high-contrast images and high sensitivity. This facilitates vascular patency visualization and allows to assess the degree of neoangiogenesis after experimental transplantation of the tracheal segment, at different stages of experiment, without euthanizing the animal