Efficacy of the New Hemostatic Composite Based on Sodium Gel Carboxymethylcellulose and Aminocaproic Acid: Experimental Application after Partial Splenectomy in Rats

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Introduction. One of the most frequent complications of abdominal injuries is damage and rupture of the spleen, which in 5-41% of cases leads to death [9]. The leading strategies for surgical interventions on the spleen are to achieve effective hemostasis in the shortest possible time and to follow organ-preserving tactics in order to avoid inflammatory and infectious complications associated with the loss of the immunological function of the spleen [1, 11, 13]. An important role in the treatment of spleen injuries is in using the newest hemostatic agents. The increasing recognition of local hemostatic drugs is connected with such advantages like easiness of use, high efficacy and a minimum amount of side effects [12]. The mechanism of action of the currently used local hemostatic agents is simulation and stimulation of the stages of natural hemostasis or acceleration of the formation of a fibrin clot, escaping these stages [6]. Most often, local hemostatic drugs are a complex of sorbent (adjuvant) and thrombin component. For example, as a sorbent can be used gelatin, which swells upon contact with blood or tissue fluids, slowing their flow. Collagen matrices precipitate platelets on their surface and form blood clots. A similar mechanism of action is relevant for hemostatic agents based on oxidized cellulose. Thrombin components that directly trigger the coagulation cascade include thrombin, fibrinogen, and some coagulation factors (for example, XIII) [2]. A universal remedy for stopping parenchymal bleeding does not currently exist. Almost all of the proposed drugs, produced in the form of sponges, glue, varnish or gel, have their own drawbacks. The most frequent and dangerous drawback is the adhesion process [2].

            In this regard, the aim of this study was to develop a new local hemostatic agent with anti-adhesion activity, to study its hemostatic effectiveness and influence on morphological changes in the spleen with a focus on the analysis of lymphoid structures and the activity of T-helpers, prevailing in the T-dependent zones of white pulp and most responsive to splenectomy [1]. The sodium carboxymethyl cellulose gel (Na-CMC) was chosen as a matrix base for the new hemostatic agent; Na-CMC can be considered as an analogue of hemostatic materials based on oxidized cellulose. Moreover, a 4% concentrated sodium carboxymethyl cellulose gel, known as «Mesogel», has an anti-adhesion effect [8]. We experimentally proved the expediency of using Na-CMC in the form of a 6% concentration and the advantage of using it in the form of a gel. To enhance hemostatic properties 5% aminocaproic acid (AA) was added to the 6% Na-CMC.

            Material and research methods. As experimental animals male Wistar rats weighing 185-250 grams contained in standard vivarium conditions were used. All experiments were carried out according to the "Rules for the Use of Experimental Animals" (Appendix to the Order of the USSR Ministry of Health dated 08.08.77 No. 755) and to the directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes. The studies were carried out in three groups: 1) a control group without the use of hemostatic agents, 2) with using a standard Na-CMC 4% anti-adhesion gel «Mesogel» and 3) an experimental group using 6% sodium carboxymethyl cellulose gel with 5% aminocaproic acid (manufacturing company “Linteks”, St. Petersburg).

            To evaluate the hemostatic effect in the acute in vivo experiment in 60 animals (20 in the each group) were used special methods to determinate the stop of bleeding and the amount of blood loss. Midline laparotomy was performed under masked isoflurane anesthesia in rats. Then, the anterior edge of the spleen was resected in a standard way, using a stencil in the form of a triangle, the length of the ribs of it was 1 cm, and the depth of the wound was 3 mm. This type of the surgical wound contributed to massive bleeding. A gauze napkin with a known mass was placed under the designated wound area delimited from the peritoneum serous fluid with a plastic film. 1 ml of gel preparations was applied to the bleeding area of the wound. After modeling the wound, the stopwatch was turned on and stopped as soon as the absence of bleeding was noticed. The amount of blood loss was determined as the difference in the weight of the gauze napkin before and after the experiment. The significance of differences in the compared values was determined by calculating the average value (M ± σ) and using Student's criterion.

            In a chronic experiment in 27 rats, modeling of the spleen wound and application of hemostatic agents was performed as described above. 3 animals from each group (9 animals in every group) on the 3rd, 7th and 14th day were removed from the experiment by

poisoning with carbon dioxide into the incubator. The severity of the adhesive process was evaluated using the method of semantic differential [3]. Lower values of the semantic differential indicated a minimal severity of the adhesive process in the abdominal cavity.

     For histological ìnvestigation, fragments of the spleen of experimental animals, including the area of the wound and applicated hemostatic agents, were embedded with paraffin according to standard methods. Sections 5 μm thick were stained with hematoxylin and eosin for morphometric and Van Gieson analyzes to evaluate the severity of fibrotic changes. Using the microscope with 40x magnification the density of the arrangement of lymphoid nodules was calculated. The area of lymphoid nodules and the sizes of germinal centers were identified using the ImageJ computer program at a 40x magnification. Using colorimetric histograms, the blood supply to the spleen was indirectly evaluated. Monoclonal antibodies Anti-CD4 (M3350) (Glostrup, Denmark) were used for immunohistochemical staining of CD4 + T-lymphocytes. Diaminobenzidine was used as a chromogen. Following staining with hematoxylin was performed. The results of this study were evaluated semi-quantitatively in arbitration units on a 5-point scale, taking into account the staining intensity and the number of positively stained cells. The significance of differences in the compared values was evaluated by calculating the average value (M ± σ) and using the calculation of the confidence interval (for data with a normal distribution of the attributes) and the Mann-Whitney criterion (U). Differences were considered statistically significant if p <0.05.

Research results and discussion.     In the acute experiment time of stop of the bleeding was: in the control group – 323.67 ± 1.27 s.; group 4% Na-CMC «Mesogel» - 233 ± 1.52 s.; the study group 6% Na-CMC+AA 5% - 100.4 ± 1.39 s. In a comparative aspect, it was proved that the new composite has expressed hemostatic properties, significantly reducing the time of bleeding by 69% (222.27 s) (p<0.01) comparing to the control group and «Mesogel» by 42% (132.76 s) (p<0.05). Moreover, the new composite significantly reduces blood loss during spleen injury by 69% (285.6 mg) (p<0.01), which is significantly higher than the hygroscopic properties of «Mesogel» (while using «Mesogel» it was possible to reduce the blood loss only by 21% (65.8 mg) (p<0.05)).  The initial data corresponds to the blood loss: control group – 411.6 ± 2.15 mg; 4% Na-CMC 345.8 ± 2.13 mg; 6% Na-CMC + AA5% -126 ± 1.62 mg.

            In a chronic experiment, all animals survived. All animals from the control group were presented with adhesive process. On the 3rd day, the coefficient of severity of the adhesive process was 1.856 ± 0.23 points; the adhesion process was presented mostly with omental adhesions and was located in the area of manipulation - the mesogastric region.

            On day 7, planar and omental adhesions dominated, sometimes the deformation of the edges of the spleen or narrowing the diameter of the intestine were presented; single traction adhesions were found. The coefficient of the adhesive process severity was 2.344 ± 0.11 points. On day 14, the adhesion process occupied 80% of the abdominal cavity. Tractional, cord-like adhesions dominated, planar and omental adhesions were less common, significant deformation of internal organs was noted. The coefficient of severity of the adhesive process was 4.143 ± 0.24 points.

            During the autopsy of the animals from the group were the new composite of 6% Na-CMC+5% AA and «Mesogel» was used, adhesion manifestations on the 3rd day were absent. On the 7th day after the application of the new composite 6%Na-CMC+5%AA, the coefficient of the adhesive process severity was 0.481±0.277 points, after using 4% Na-CMC («Mesogel») – 0.326±0.163 points, without significant differences between these groups, but significantly different comparing to the control group (p<0.05). Only single omental adhesions connected to the postoperative edge of the spleen were found. On the 14th day, the severity of the adhesion process after applying the new composite of was 6%Na-CMC+5%AA 0,486 ± 0,27 points, after the application of «Mesogel» – 0.641 ± 0.32 points. The kind of adhesions was similar to 7 days of the experiment. There were no significant differences between these groups, which indicates that 6%Na-CMC+5% AA, like «Mesogel», shows the presence of effective anti-adhesion activity (p <0.05 relative to the control).

Histological investigation.

    On the 3rd day after partial splenectomy, the white pulp on the histological sections is represented by a small number (7-9) of large lymphoid nodules with unsharply defined borders. On the 7th day, the number of lymphoid nodules increases in 2-3 times (p <0.05) and remains elevated by the 14th day of the experiment (p <0.01). The area of lymphoid follicles from the 3rd to the 14th day does not significantly change (Fig. 1). 

 

      

 

        The amount of CD4 + T-lymphocytes increases from 3 to 7 days and remains elevated by 14 days (Fig. 2).

 

In general, changes in the white pulp indicate the activation of the immune function of the spleen on the 7th day of the experiment, which coincides with the dynamic of the inflammatory process and the formation of adhesions. On the 14th day, based on the volume of lymphoid tissue and the number of CD4 + T-lymphocytes, the activity of white pulp remains increased comparing to the groups where hemostatic and anti-adhesion preparations were used, which indicates a greater intensity of inflammatory processes associated with massive adhesions process.In the parenchyma of the spleen fibrotic changes were absent. However, the development of an adhesive process involving the nearly located pancreas was noted in all animals of this experimental subgroup.

When using a 4% of Na- Na-CMC known as «Mesogel», significant changes of the density of lymphoid follicles and their area from day 3 to day 14 of the experiment were not identified. However, it should be noted that on the 7th day the amount of lymphoid nodules was 2-3 times lower than in the control group (where hemostatic and anti-adhesions drugs weren't used). On day 3, the area of lymphoid follicles was significantly smaller than in the control (p<0.01), which indicates less active immune response of the spleen. Moreover, the use of the 4% Na - carboxymethyl cellulose gel prevented the development of adhesions and reduced the severity of the immune response from the spleen.

        In the third group of animals, where a new composite consisting of 6% sodium carboxymethyl cellulose gel and 5% aminocaproic acid was applied to the wound surface of the spleen after resection, the size and amount of lymphoid follicles significantly decreased from the 3rd to 7th day of the experiment (p<0.01). A pronounced decrease in the size of lymphoid follicles on day 7 was associated with the highest amount of CD4 + T-lymphocytes comparing with 3rd and 14th (p <0.01) days of the experiment.

         The amount of lymphoid follicles and CD4 + T-lymphocytes decreased by 14 days. The highest activity of white pulp in this group of animals was recorded on the 3rd day of the experiment. Adhesions, fibrotic changes and signs of degenerative changes in the splenic parenchyma were absent, which indirectly indicates the absence of local negative effects of the new hemostatic composite.

In modern surgical practice, a combined drug of similar action is absent. Despite the large selection of solid hemostatic forms (sponges, plates, adhesives), these products, like any foreign body, are the trigger mechanism of the adhesive process. So, for example, «Sergisel fibrillary» is a non-woven hemostatic drug, according to the mechanism of action specified in the instructions [4], it causes oxidation of body tissues, in fact, a chemical burn, and triggers a cascade of formation of connective tissue. On the other hand, modern anti-adhesion drugs used in the clinic (glycerin, dextrans, hyalouranic acid, saline, etc.) don’t help stop bleeding [10]. Thus, the new composite has pronounced advantages, demonstrating a combined hemostatic and anti-adhesion effect.

Conclusions.

Thus, based on the data obtained, the following conclusions can be made:

1. The new composite 6% Na-CMC+AA5% reliably has demonstrated hemostatic properties, significantly exceeding its prototype – «Mesogel». Reduced time of bleeding by 68% (222,27 s); blood loss values by 69% (285,6 mg) (p<0.01).
2. 6% Na-CMC + 5% AA reduces the immunological reactivity of the white pulp of the spleen, associated with the development of inflammatory processes in the postoperative period, which is one of the key points in preventing the development of adhesions, both at the area of manipulation and in the abdominal cavity as a whole.
3. The drug has a high anti-adhesive activity. The severity of the adhesion process on the 3rd day of the experiment was rated 0 points; on the 7th and 14th day it was minimal: 0.481 ± 0.27 and 0.486 ± 0.27 points, respectively (p<0.05).
4. The new composite 6% Na-CMC+AA5% can be recommended for further clinical researches, with the aim of using it in abdominal surgery during operations on the spleen, as a combined action drug.

About the authors

Darya S Soldatova

Federal state budget educational institution of higher health care of the Kursk state medical university of the ministry of health of Russia, Kursk, Russia

Author for correspondence.
Email: dascha22118@mail.ru
ORCID iD: 0000-0002-9278-2737

assistant of the department of operative surgery and topographic anatomy named after A. D. Myasnikov

Russian Federation, Kursk, Russian Federation

Aleksandr I Begin

Federal state budget educational institution of higher health care of the Kursk state medical university of the ministry of health of Russia, Kursk, Russia

Email: abezin@yandex.ru
ORCID iD: 0000-0003-3776-9449
SPIN-code: 1250-5549

M.D., Professor, head of the department of operative surgery and topographic anatomy named after A. D. Myasnikov, Kursk state medical university

Russian Federation, Kursk, Russian Federation

Tatyana A Ishunina

Federal state budget educational institution of higher health care of the Kursk state medical university of the ministry of health of Russia, Kursk, Russia

Email: ishunina@gmail.com
ORCID iD: 0000-0002-2743-7515
SPIN-code: 6334-5020

Ph.D., Associate Professor at the department of histology, embryology, cytology

Russian Federation, Kursk, Russian Federation

References

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