Effect of Treatment with Cryopreserved Fetal Neuronal Cells on Prooxidant-Antioxidant Balance in Rats with Experimental Cranio-Skeletal Injury

In the response to cranio-skeletal injury, complicated by bleeding, the content of primary and secondary products of lipid peroxidation (LPO) in liver homogenate significantly increased, and achieved a maximum level to the 14th day of the experiment and was significantly higher than the control level during 25 days of observation. Within the 3rd–14th days the accumulation of LPO products occured on the background of significant decrease of superoxide dismutase (SOD) activity, without any changes in catalase activity and compensatory increase of ceruloplasmin content to the 3rd day of the experiment. From 14th to 25th day following treatment with cryopreserved fetal neuronal cells we observed less pronounced deviations of lipid peroxidation in terms of content of primary LPO products (diene conjugates), and content of secondary LPO products (TBA-active) from the 3rd to 7th day. Catalase content was reduced through the 3rd–14th days of the experiment, increased by the 25th day; from the 14th day we observed a protective effect of cryopreserved fetal neuronal cells in terms of SOD activity. There was virtually no correcting effect found in blood serum ceruloplasmin content.

Intensification of lipid peroxidation (LPO) plays an important role among the main pathogenic mechanisms of severe trauma, especially accompanied with bleeding.At early stage of the disease the activation of lipid peroxidation is of adaptive nature: the permeability of cell membranes increases moderately and transmembrane circulation of substances is thereby facilitated [9].In case of trivial trauma the antioxidant defence system compensates the excessive formation of reactive oxygen species and free radicals and supports the prooxidant-antioxidant balance.And vice versa, severe trauma causes a systemic response to inflammation, corresponding increase in the intensity of free radical oxidation, and failed antioxidant defence, that coinsides to an acute phase of inflammation [2].The systemic nature of these processes needs a new approaches to treat injuries.
To date much attention has been paid to the use of fetal neuronal cells which are referred to 'polyfunctional' modulators of supra-systemic effect [3,4].It has been established that therapeutic potential of fetal cells is caused by a wide range of produced biologically active substances possessing angiogenic, antiapoptotic, antioxidant and mitogenic effects [8].This makes them a prospective medicine product to reduce the intensity of systemic response to inflammation, including violations of free radical oxidation and antioxidant defence during severe injury.
The research aim was to study the dynamics of LPO indices and enzyme link of antioxidant defence during early and late manifestations of traumatic disease under experimental cranio-skeletal injury complicated by bleeding, and after treatment with cryopreserved fetal neuronal cells.
The suspension of cryopreserved fetal neuronal cells (cFNCs) of rat was injected intraperitoneally to the animals of the first experimental group 12 hours later the injury (5×10 6 cells per 100 g body mass [4]).A suspension of fetal neuronal cells was obtained at the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine (Kharkiv) by mechanical dissociation of brain fragments of rats embryos (11 days of gestation).Cryopreservation was performed according to the method described previously [5].The second experimental group of animals were intraperitoneally injected with an equivalent volume of physiological solution.The rats were sacrificed to the 3 rd , 7 th , 14 th and 25 th days after injury by decapitation after a previous thiopental sodium anaesthesia (60 mg/kg body mass) [15].
Lipid peroxidation was evaluated in rats by measuring the content of TBA-active products and diene conjugates in liver homogenate [11,13].Activity of enzymatic link of antioxidant defence was determined by the activity of superoxide dismutase (SOD) [1], and catalase [7] in liver homogenate, as well as by content of ceruloplasmin in blood serum [6].
Statistical analysis was done using Statistica 10.0 software.To compare the samples we applied Student's t-test and Mann-Whitney's test.
All the manipulations with laboratory animals were carried-out in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986) and Guidelines of State Pharma-cological Center of the Ministry of Health of Ukraine on Preclinical Studies of Medicine Products (Kyiv, 2001).

Results and discussion
The analysis of the obtained results showed that bleeding-complicated cranio-skeletal injury resulted in a significant intensification of lipid peroxidation processes in the experimental rats.The content of TBA-active products in liver homogenate of animals not treated with cFNCs in all the terms of post-traumatic period was significantly higher than in the control and achieved a maximum to the 14 th day of observation.After treatment with cFNCs the studied parameter was also significantly higher than the control level in all periods of observation with a maximum to the 14 th day of observation (Table 1).Analysis of indices dynamics Примітка: значущі відмінності по відношенню до контрольної групи: * -р ≤ 0,01; # -р ≤ 0,001.
Note: the differences are statistically significant if compared with the control: * -p ≤ 0.01; #p ≤ 0.001.
Отже, через 14 діб вміст ТБК-активних продуктів ПОЛ у тварин обох дослідних груп був най-in both experimental groups showed that there was a statistically significant increase in the content of TBAactive products of LPO to the 14 th day, and then a significant reduction to the 25 th day if compared to the previous observation term (the 14 th day).It was established that to the 3 rd and 7 th day of post-traumatic period and conducted cell therapy the content of LPO TBAactive products in liver homo-genate was signifi-cantly lower if compared with the group of animals not treated with cFNCs.However, to the 14 th and 25 th day of the experiment there were no significant differences between the experimental groups in terms of this index.
Thus, after 14 days the content of TBA-active products of LPO in animals of both experimental groups was the highest.Application of cFNCs had protective effect on the intensity of formation of LPO secondary products to the 3 rd and 7 th day of post-traumatic period.
control, but in case of conducted cell therapy it was significantly decreased by the 14 th and 25 th day if compared with the animals, not treated with cFNCs.The maximum content of diene conjugates in animals of both groups was observed by the 7 th day.Analysis of dynamics of studied index changes revealed a significant increase to the 7 th day of the experiment in liver homogenate of animals of both experimental groups.To the 14 th day the reduction of content of diene conjugates was observed in the animals, not treated with cFNCs, moreover to the 25 th day it was significantly lower if compared with the 7 th day.In the group of animals which underwent cell therapy, this index was significantly lower to the 14 th and 25 th days if compared with the 7 th day of the study (Table 1).
Thus, the use of cFNCs therapy resulted in less accumulation of LPO primary products to the 14 th and 25 th days following cranio-skeletal injury complicated by bleeding.
After 3, 7, 14 and 25 days post cranio-skeletal injury complicated by bleeding, the indices of enzymatic link of antioxidant protection were changed as follows.
In the group of animals not treated with cFNCs the SOD activity in liver homogenate was lower than in the control groups during the whole observation period (Table 2).By the 3 rd , 7 th and 14 th days of experiment this index did not differ significantly from the previous observation periods, and to the 25 th day increased significantly.By the 3 rd and 7 th days after cFNCs application the SOD activity was lower if compared with the control, by the 14 th and 25 th days it increased, achieving the control level and was significantly higher if compared with the indices assessed by the 3 rd and 7 th days.It has been found that introduction of cFNCs resulted in an increase of SOD activity in liver homogenate if compared to the first group of animals to the 14 th and 25 th days of the experiment.Thus, treatment with cryopreserved fetal neuronal cells contributed to SOD activity recovery.
Catalase activity in liver homogenate (Table 2) in the animals not treated with cFNCs did not differ significantly from the index in the control animals to the 3 rd -14 th observation days, and to the 25 th day it was lower.Application of cFNCs resulted in insignificant decrease of catalase activity in liver homogenate of the animals to the 3 rd and 7 th days of posttraumatic period, and to the 14 th day it was significantly lower than the control.After 25 days this index did not significantly differ from the control.Comparing the catalase activity in the liver homogenate of animals of the experimental groups showed that to the 7 th day after administration of cFNCs it was lower than the index in animal with spontaneous course, and to the 25 th day it was higher.Примітка: значущі відмінності по відношенню до контрольної групи: 1 -р ≤ 0,05; 2 -р ≤ 0,01; 3 -р ≤ 0,001; 4 -р ≤ 0,1.
The content of ceruloplasmin in blood serum of animals of the group not treated with cFNCs significantly increased 3 days later the injury if compared with the control (Table 2).To the 7 th , 14 th and 25 th days of the experiment it was reduced and did not differ from the control.In the group treated with cFNCs the content of ceruloplasmin was significantly increased to the 3 rd day post treatment if compared with the control group, to the 7 th and 14 th days it returned to the control level, and increased again after 25 days.
Thus, the enzymatic link of antioxidant protection of animal organism with cranio-skeletal injury complicated by bleeding, was changed in all the studied periods.Within 14 days of the experiment the level of SOD in liver homogenate was decreased, then increased after 25 days, but did not achieved the control level.Catalase activity of liver homogenate was changed to the 25 th observation day and was lower than the control.Content of ceruloplasmin in blood serum increased   2. Накопичення продуктів ПОЛ у гомогенаті печінки групи тварин з мимовільним перебігом відбувалося на фоні істотного зниження активності СОД, відсутності відхилень активності каталази через 3-14 діб експерименту та збільшення вмісту церулоплазміну на 3-ю добу.

Conclusions
1.In response to cranio-skeletal injury complicated by bleeding the content of primary and secondary products of LPO in liver homogenate of animals maximally increased to the 14 th day of the experiment and was significantly higher than the control values within 25 days.
2. The accumulation of lipid peroxidation products was observed in the liver homogenate of group of animals with spontaneous course, which was accompanied with a significant reduction in the activity of SOD, not changed catalase activity through 3-14 days of the experiment and increase of ceruloplasmin content to the 3 rd day.
3. The application of cryopreserved fetal neuronal cells resulted to the less changes (if compared with non-treated animals) of lipid peroxidation indices of liver homogenate: diene conjugates (from the 14 th to 25 th days) and LPO TBA-active products (from the 3 rd to 7 th days).In these experimental conditions there was revealed a decrease of catalase content 3-14 days later and its recovery after 25 days, protective effect on SOD activity from the 14 th day and no corrective effect on the content of ceruloplasmin in blood serum.