Effect of Cryopreserved Cord Blood Serum on Systemic Inflammatory Responses in Experimental Cold Wound

The effect of cryopreserved cord blood serum on systemic inflammatory responses following skin cryodestruction was studied. The investigation comprised the assessment of inflammation response indices: erythrocyte sedimentation rate, leukogram, amount of C-reactive protein and TBA reactive substances in blood serum after the treatment with the human cryopreserved cord blood serum of animals with cold wounds. It has been shown that cryodamage of rat skin led to an inflammation response accompanied with a two-fold erythrocyte sedimentation rate, 1.5 elevation of C-reactive protein to observation days 7 and 14, changes in leukogram; 1.4 rise in the level of peroxidation processes to day 7, 2.5 times to day 9, 1.9 times to day 14 and 1.5 times to day 21. Application of cryopreserved cord blood serum resulted in a reduced intensity of inflammation response to skin cryodestruction in experimental animals, that was confirmed by a 1.5 time decrease in the level of C-reactive protein to days 7 and 9, and its normalization to day 14; 2.0 times diminishing of erythrocyte sedimentation rate to day 7 and its normalization to day 14; normalization of leukogram to day 7; 1.4 times reduction of TBA reactive product level to day 7 and its normalization to day 9.

Cold injuries in humans and animals refer to severe pathological states.They are manifested in development of local vascular and tissue changes and accompanied with a systemic inflammatory response, the intensity of which depends on severity of tissue damage [9,13].According to the WHO data there is recent trend of increasing the number of people suffering of cold trauma observed in the regions with a temperate climate.The treatment of these patients is to date a complicated and diverse problem in terms of medicine and every day life, since the frostbites lead to a disability, require a long term rehabilitation and are often the cause of lifelong disability.The treatment of these patients is based either on local or/and systemic application of the drugs, the choice of those depends on the severity of tissue damage [9,13].Local treatment (antiseptics, proteolytic enzymes, ointments, laser, UV rays) contributes to the regeneration of tissues and a general one (immune and biostimulants, vitamins, pyrimidine derivatives, antioxidants) increases the nonspecific resistance of the body.However, this type of therapy has a number of disadvantages, e. g. the risk of development of allergic reactions, the need of constant monitoring of coagulogram indices, risk of disordered homeostasis, required prevention of microbial contamination [1,3,9,13].One of the ways of solving these problems is the use of cryopreserved cord blood serum (CCBS), which was reported as possessing the potential to stimulate cell proliferation and differentiation [6,11].In particular, the application of CCBS in treatment of diabetic foot syndrome can reduce the dose of insulin expenditure, and contributes to the correction of severe biochemical and immunological disorders.The effectiveness of CCBS in treatment of acute pancreatitis was associated with stimulation of reparative processes in pancreatic tissues [6,11].
The analysis of the indices of systemic inflammatory reaction in blood is of diagnostic and prognostic significance, and the success of performed therapy can be likely judged by their dynamics.For example, the severity of the body's inflammatory response to the onset and development of various pathologies is assessed using one of the standard laboratory methods, the erythrocyte sedimentation rate (ESR) [5,9,13].The change in parameters of the leukogram (LG) and the level of lipid peroxidation (LPO) in the blood is the standard criterion, characterising the activty of inflammatory response.Monitoring the level of Creactive protein (CRP) in serum is an important screening test in the diagnosis of organic lesions of organs and tissues, as well as in determining the activity of the inflammatory process.Thus, the monitoring of laboratory indices can be used to estimate the inflammation activity and grounds for the combined therapy.

Materials and methods
The work was carried out on in Sphinx rats in accordance with the requirements of the Committee in Bioethics (Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv), coordinated with the Directive of the European Parliament and the Council of the European Union dated of September 22 nd , 2010.
The wounds were modeled on lateral surface of the femur, using a cryoinstrument with an actively cooled applicator (-195°C) with a diameter of 8.0 mm.The duration of cryoexposure was 60 seconds.
The animals were divided into three groups of 10 individuals each.The control group (CG) of the rats were injected with physiological saline (0.15 M sodium chloride solution, Yuriya-Pharm, Ukraine), the experimental group (EG) was treated with human CCBS (procured according to technological regulations, developed at the IPC&C of the National Academy of Sciences of Ukraine).The animals of intact group (IG) were not subjected to any influences.The administration protocol was in accordance with the instructions for the application of the medical immunobiological preparation Cryocell-Cryocord (Interdepartmental Scientific Center of Cryobiology and Cryomedicine of the NAS, AMS and Ministry of Health Care of Ukraine) containing the serum cord blood.The injections were started on the 3 rd day after cryodestruction every other day by 0.1 ml/kg of body weight intramuscularly (into a healthy paw) for 9 days.The dosage was calculated as described by Yu.R. Rybolovlev [10].
The erythrocyte sedimentation rate in the blood of rats was measured by Panchenkov's micromethod [8,12].The parameters of blood leukogram were determined by counting 500 cells (using microscope Granum R 4003, China) in the smears stained with azur-eosin, according to Romanowsky-Giemsa [3].The level of C-reactive protein in the blood serum was determined by latex agglutination using the Granum kits (Ukraine).The level of lipid peroxidation products in the blood serum of rats was evaluated by the content of the products reacting with thiobarbituric acid (TBARS) using a spectrophotometer Hitachi-U3210 (Japan) [2].

Results and discussion
The results of measuring the ESR in animals at different observation terms are shown in Fig. 1.To day 7 of the experiment, the ESR values in all the group ssignificantly exceeded the indices in the IG, and this can be considered as a manifestation of acute phase proteins during the inflammatory response to cryodamage of integumentary tissues.In the blood of the CG animals, the value of ESR was (12.9 ± 0.9) mm/hr, which was 4.7 times higher than that of the IG.After the CCBS administration, the coefficient of ESR decreased twice if compared to that of the CG animals.To day 14 of the experiment, this index was significantly decreased in the blood of animals in both CG and EG.On the 14 th day of observation it was (7.6 ± 0.8) mm/hr in CG, and it was 2.8 times higher comparing to the IG animals.Introduction of the CCBS contributed to the normalization of the studied index, which was 2.2 times less than in the CG animals.To day 21 of the experiment, the values of ESR in the CG and EG animals were back to the norm.Thus, the therapeutic efficiency of the CCBS administration was confirmed by a decrease in the ESR value in the blood of rats to day 7 and its normalization on the 14 th day of observation.
Результаты исследований уровня СРБ в сыворотке крови показали, что на 7-е сутки наблюдения  The results of studying the blood LG values of experimental animals are presented in the Table .To day 7 of the experiment, the total number of neutrophils in blood of the animals was 1.5 times higher than in the IG.The rise in the numbers of both segmented (in 1.5 times) and band neutrophils (in 1.6 times).On the background of an increased number of neutrophils a relative lymphopenia was noticed, i. e. the number of lymphocytes was 1.2 times less than in IG.In the EG animals the studied index was normal.It was found that in the rats of CG and EG to day 14 the blood LG corresponded to the values of IG.Thus to day 14 of experiment we observed normalization of the percentage of leukocytes even with no treatment, that confirmed a proceeding active reparation.Thus, after the administration of the CCBS to the rats with cold wounds, the blood LG values corresponded to the norm already to day 7 of the experiment.
The results of studies of the CRP level in blood serum showed that to day 7 of observation it was (15.2 ± 1.8) mg/l in the animals of CG, and this was 2.5 times higher than in the IG (Fig. 2).Introduction of the CCBS contributed to a 1.5 times decrease in the level of CRP to days 7 and 9 of observation.To day 14 its content in the blood serum of the CG animals did not significantly differ from the indices to day 9. On the 14 th day after the administration of the CCBS, the normalization of this index was found, as evidenced by the absence of statistically significant differences in comparison with the IG.To day 21 of observation, the level of CRP in the animals of all groups was consistent with the results of the IG.Thus, a positive effect of the CCBS was manifested in a statistically significant decrease in the level of CRP to day 9 of observation with its coming back to the norm to the 14 th day.
After modeling the cold wounds to days 7, 9 and 14 of the experiment in the animals of the CG we found a rise in the ESR, CRP and LPO indices and after the administration of the CCBS the severity of systemic manifestations of inflammation in response to cryodestruction of the skin was decreased.That testified to the normalization of the level of CRP and the values of ESR in the blood to day 14 of the experiment, as well as in the normalization of blood LG values to day 7 and the TBARS level to day 9.
It is known that normal values of ESR testify to the absence of any inflammation.Thus the restoration of previously decreased ESR may demonstrate the effectiveness of the therapy, although it could not be a specific indicator for any particular disease [7,12].We have found that administration of the CCBS was followed by a double decrease of the ESR index already to day 7 of the experiment.Obviously, this facilitated the elimination of acute-phase and fibron-like proteins from the erythrocyte membrane.These proteins are weakening a membrane electrostatic charge.One of the main representatives of acute-phase proteins, determining the activity of inflammatory process, is CRP, which activity lies in detection of potentially toxic substances, and their removal from blood [5,7,12].Therapeutic effectiveness of CCBS can be manifested at any level of regulation (molecular, cellular, systemic) in the form of suppression of inflammatory processes in tissues and enhancement of the reparation.For example, the action of CCBS can be mediated by the interaction of the serum components with mediators of inflammation, that contributes to an inhibition of the synthesis of acute-phase proteins and partial restoration of native pentameric conformational form of the protein molecules [3,11].In addition, the effect of the CCBS on inflammatory processes in cold wounds is manifested in a decrease of the proinflammatory cytokines level, reduced hypercoagulation accompanied by concomitant activation of fibrinolysis, and stimulation of inductive and productive phases of an immune response.A high correlation of the CRP level with the disease severity can be considered a more reliable marker of inflammation if compared with the parameters of ESR and the чества лимфоцитов диагностической ценности не представляет.
У экспериментальных животных после применения КСКК снижалась интенсивность воспали-leukogram [7].Various populations of leukocytes are involved into patho-and sanogenesis under several pathological conditions, as well as in the onset and development of an inflammatory reaction.Since neutrophilic leukocytes are the main cells that provide antimicrobial protection, an increase in their total number, in particular that of band neutrophils, indicates an active inflammation in infected wounds [7].The found lymphopenia has a relative character and does not have diagnostic value without determining an absolute number of lymphocytes.
The concentration of TBARS in blood serum reflects the activity of LPO processes in an organism, which are accompanied with cell membranes damage, and, consequently, the disorder of active and passive ion transport, the complications in proton transfer through the mitochondrial respiratory chain and, finally, the development of hypoxic changes in tissues [1].Leukocytes, especially neutrophils, being a rich source of various active species of oxygen (superoxide anion, hydroxyl radical, singlet oxygen, hydrogen peroxide), also contribute to an increase in the activity of peroxide processes in blood serum [7].The emergence and development of inflammation, including the one accompanying cold trauma, results in an active prooxidant milieu and, accordingly, an impaired balance between the production of free radicals and the mechanisms of antioxidant control.This can lead to depolarization of cell membranes, hypertrophy, apoptosis, tissue necrosis [1,4].Oxidation of lipids is accompanied with apperance of chemoattractants enhancing the migration of phagocytes into the inflammation focus [3,6].Ultimately, the damaging effect of LPO products leads to cell destruction, a reduction in the repair capabilities of tissues.
Thus positive effect of CCBS can be caused by biologically active substances, being its components and having a versatile activity.The revealed therapeutic effect can be stipulated by the effect of CCBS on the processes of cell metabolism (stimulation of oxygen supply, activation of oxidative phosphorylation and transport of glucose into cells, increased energy production, inhibition of LPO processes), direct antioxidant reactivity of CCBS, the capability to stabilize biomacromolecules, determining the activity of regulatory systems [3].

Conclusions
Cryoinjury of rats' skin resulted in an inflammatory reaction to days 7 and 14, and it was accompanied with an increased ESR (two times), CRP (in 1.5 times), a change in the leukogram, an increase in the level of peroxidation processes to day 7 (in 1.4 times), to day 9 (in 2.5 times), on the 14 th day (in 1.9 times), and on 21 st days (in 1.5 times).
The intensity of inflammatory response to cryodestruction of the skin of experimental animals decreased, and this was manifested in 1.5 times reduction of CRP content to days 7 and 9, and its normalization to the 14 th day; 2.0 times decrease in ESR to day 7 and its normalization to the 14 th day; normalization of the blood count to day 7; the level of TBARS was 1.4 times reduced to day 7 with its normalization to the 9 th day.
We express our gratitude to the Head of the Department of Cryobiology of Reproductive Systems of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of UkraineProf.Olga S. Prokopyuk, MD for advisory assistance.