Immunoexpression of proliferating cell nuclear antigen ( PCNA ) in spleen of splenectomized rats with preservation of inferior pole , submitted to hyperbaric oxygenation

IFellow PhD degree, Postgraduate Program in Surgical and Ophthalmological Applied Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte-MG, Brazil. Intellectual and scientific content of the study, acquisition and interpretation of data, manuscript preparation. IIGraduate student, Pharmacy, College of Health Sciences, EMESCAM, Vitoria-ES, Brazil. Acquisition and interpretation of data, manuscript preparation. IIIGraduate student, Pharmacy, College of Health Sciences, EMESCAM, Vitoria-ES, Brazil. Interpretation of data. IVGraduate student, Pharmacy, College of Health Sciences, EMESCAM, Vitoria-ES, Brazil. Acquisition and interpretation of data, manuscript preparation. VPhD, Associate Professor, College of Health Sciences, EMESCAM, Vitoria-ES, Brazil. Analysis and interpretation of data. VIMD, Pathologist, Santa Casa de Misericórdia de Vitória Hospital, Brazil. Analysis and interpretation of data. VIIAssociate Professor, Department of Morphology, College of Health Sciences, EMESCAM, Vitoria-ES, Brazil. Intellectual and scientific content of the study, manuscript preparation, critical revision. VIIIPhD, Chairman, Full Professor, Department of Surgery, College of Health Sciences, EMESCAM, Vitoria-ES, Brazil. Conception, design, intellectual and scientific content of the study; statistical analysis; manuscript writing; critical revision. IXPhD, Chairman, Full Professor, Department of Surgery, Medical School, UFMG, Belo Horizonte-MG, Brazil. Manuscript writing, critical revision.


Introduction
For a long time the spleen was considered a non-essential organ to life, and its removal suggested that it caused no serious damage to the patient 1 .In the last decades, however, it was confirmed that splenectomy in children and adults results in high mortality in the postoperative period caused by fulminating sepsis 2 .Infectious complications were also observed in experimental animals 3 .For these reasons, conservative spleen surgeries were again taken into account.Subtotal splenectomy with inferior pole preservation (ESTPI) in which blood irrigation is maintained by the vessels of the gastrosplenic ligament is one of the most recently described.
The investigation of the inferior pole of the spleen of dogs and rats [4][5][6] submitted to ESPTI showed alterations in the viability of the remaining tissue, in the immediate postoperative period.Rats submitted to this procedure, followed by hyperbaric oxygenation (HBO) at 100% pure oxygen, in conventional microscopy, showed larger lymphatic follicles, increase of the amount of cells and vessels and more lymphocytic cell proliferation in the inferior pole of the spleen 7,8 .Considering these findings, we proposed to evaluate the immunohistochemistry expression (IHC) of the proliferating cell nuclear antigen (PCNA) which main function is to increase the activity of delta DNA polymerase during DNA replication, event that precedes cell division process 9 .In this context, PCNA investigation might contribute to a better comprehension of the cellular processes that occur during the growth and proliferation of the inferior pole of the spleen after HBO.

Study design
Twenty male Wistar rats submitted to ESTPI were used in this study.The animals were randomly divided in two groups according to whether they were submitted or not to HBO and were later subdivided in four groups according to the day of specimen collection of the inferior pole of the spleen for IHC analysis 8 (Table 1).

Surgical procedure and hyperbaric oxygenation
The animals were anesthetized with 75mg/Kg ketamine chloridrate (Vetaset ® , Fort Lodge -Iowa, USA) associated with 5mg/Kg of xylazine chloridrate (Kensol ® König -Avellaneda, Argentina) via intraperitoneal injection.Next, the rats were submitted to laparotomy for the realization of ESTPI which remained irrigated by the vessels of the gastrosplenic ligament 4 .
The spleen superior portion was removed according to Paulo et al. 10 .After the end of the anesthetic effect, the animals in group B were submitted to HBO in an appropriate chamber, according to what was established in the protocol 10 : gradual compression up to 2, 5 atmospheres, being 1 atm at sea level and 1.5 atm registered at the chamber manometer, maintained at this pressure for 90 minutes, followed by gradual decompression of the chamber for 25 minutes.This procedure was carried out twice a day for three days and once a day for seven days.

Specimen collection from the inferior pole of the spleen and euthanasia
The twenty rats were submitted to a new laparotomy on the 15 th and 45 th postoperative day, according to the subgroup, for the collection of specimen from the spleen inferior pole, followed by euthanasia (Table 1).The splenic fragments were fixed on neutral-buffered formalin 10% (pH 7.0) and sent to the laboratory of anatomical pathology.
Immunoexpression of proliferating cell nuclear antigen (PCNA) in spleen of splenectomized rats with preservation of inferior pole, submitted to hyperbaric oxygenation Acta Cirúrgica Brasileira -Vol.28 (10)  A slide with a spleen section was used as negative control (Figure 1A) without using the specific antibody and, as positive control (Figure 1B) with human amygdala fragments.The positive nuclei presented brownish or yellowish coloration and were considered positive, no matter the staining intensity.Quantitative analysis of PCNA expression was done in optical microscope.The pathologist chose the most representative areas with x100 augmentation (immersion).The level of positivity was determined by the formula described below 9 : Data was represented by median.The difference between the groups was evaluated with the non-parametric Mann Whitney test and p<0.05 was considered significant.

Results
PCNA was immunopositive in all subgroups with a varying positivity level between 53.0% and 84.0%.Among the subgroups which were not submitted to hyperbaric oxygenation, there was no difference in the positivity level.However, there was a difference of positivity level among the groups of animals submitted or not to hyperbaric oxygenation.In the groups which were not treated, there was no difference in positivity level.B15 subgroup (Figure 2A) presented a positivity level higher than that of A15 (p<0.01 (Figure 2B) and the same difference was observed between the groups A45 and B45 (p<0.01).45 days of postoperative resulted in higher level of positivity than that of 15 days, only in animals submitted to hyperbaric oxygenation (p<0.01)(Table 2).

Discussion
Many experimental studies have been conducted in order to better understand the biological properties of hyperbaric oxygenation.Among the most used markers to access cell proliferation PCNA stands out, and its immune expression has been investigated in many studies.In this study, a significant difference was noted in PCNA immunoexpression in the group treated with HBO.
HBO is used in the treatment of many inflammatory diseases and ischemic conditions such as wounds, carbon monoxide intoxication and acute necrotic infection 12 .The increase of oxygen dissolution in the blood causes oxygenation in hypoxic areas 12 .HBO increases free radical generation that oxidizes membrane proteins and lipids and inhibits bacterial metabolic functions.Hyperoxia in normal tissues causes fast significant vasoconstriction which is compensated by the increase of oxygen transportation in the plasma and the micro-vascular blood flow in the ischemic tissue is effectively improved 16 .In conventional histologic analysis, Costa-Val et al. 17 did not find spleen and liver cellular proliferation in rats submitted to HBO at 2, 5 atm for 90 minutes in 20 consecutive days, but found a significant reduction of extra medullar hepatic eritropoyesis 17 .
There are no reports in the literature on the evaluation of spleen cellular proliferation after HBO using PCNA.In order to understand the effects of this therapy on spleen regeneration references on the regeneration of other organs were considered, especially those on liver regeneration.
Although there was a decrease in renal tissue necrosis, HBO did not increase PCNA immunoexpression, due probably to insufficient HBO time at 2, 5 atm (2 days for 90 minutes) 11 .
Many authors [12][13][14] have studied the effect of HBO in liver regeneration in rats after 70% hepatectomy using PCNA to evaluate proliferative activity.There was an increase in cellular proliferation with a positivity index varying from 4% to 69% in treated groups and of 1, 4% to 50% in non-treated groups.Pressure in these groups varied from 2.0 to 2.8 atm, being 2.5 atm the most used.
The great variety of protocols described may confirm differences in positivity index found in many studies.These protocols include continuous treatment for many days; however, there are reports in which HBO effects are observed in a few hours after the beginning of treatment 18 .This is the reason why most recent studies investigate HBO effects between 24-48 hours of postoperative period.It is possible that studies on gene expression will help to understand the effects of early HBO.

Conclusion
The quantitative analysis of proliferating cell nuclear antigen positive suggests that hyperbaric oxygenation increases cellular proliferation, contributing to splenic regeneration.
Committee in Animal Experimentation (n° 004/08-CETEA) of the Federal University of Minas Gerais, Brazil.The study was conducted at the Laboratory of IHC of Research Center EMESCAM in collaboration with the Laboratory of Cellular and Molecular Biology of Human Cancer of the Federal University of Espirito Santo, Brazil (UFES).
2013 -693 Immunohistochemistry analysisThe specimens collected from the inferior pole of the spleen were kept in formalin for 24-48 hours and processed in paraffin for IHC analysis.Sections were cut at 3mm thickness and placed on pre-treated glass slides (AutoFrost ® -CancerDiagnostic -United States of America).Immunostaining was done by anti-PCNA primary antibody (Mouse monoclonal [PC-10] to PCNA, BSA and Azide free [ab80576] -Abcam ® ).For the IHC procedure, glass slides were: deparaffinized with xylol at room temperature twice at 10 minutes each; hydrated in 100% ethanol, 95% ethanol, 80% ethanol and distilled water; antigenic recuperation: heating the slides in 10mmol/L citrate buffer pH 6.0 at 98 0 C for 30 minutes; maintained at room temperature for thirty minutes and washed in phosphate-buffered saline (PBS); washed in 0% fat free powder milk to decrease the background and incubated in wet chamber for 30 minutes; incubated with 300 µL of primary antibody PCNA solution diluted 1:200 in wet chamber for two hours; washed with PBS; incubated in wet chamber with eight drops of secondary antibody (Histofine ® -Nichirei -cod.414191f) in room temperature for 30 minutes; washed in PBS; incubated with 3mL of peroxidase inhibitor (H 2 O 2 3%) at room temperature for five minutes; washed with PBS; incubated in wet chamber with diaminobenzidine solution (DAB) at room temperature for five minutes; washed with distilled water; stained with 1 mL Harris' hematoxylin for two minutes; washed with distilled water; immersed in 5% ammonium hydroxide in order to obtain blue nucleus and brown cytoplasm; washed in distilled water; dehydrated in alcohol and xylol and mounted with coverslips using Canada Balsam.

FIGURE 1 -
FIGURE 1 -(A) Negative Control -a section of the spleen without brown staining indicating the absence of PCNA (x100) marker.(B) Positive Control -a section of human amygdala with positive PCNA marker, made evident by the brown staining (x100).

TABLE 2 -FIGURE 2
FIGURE 2 -(A)There is a greater number of PCNA-positive cells (indicated by the arrow) in a section of a treated animal spleen -B15 (x100).(B) There is a small number of PCNA-positive cells in a section of a non-treated animal spleen -A15 (x100).

TABLE 1 -
Distribution of rats in two groups according to the use of hyperbaric oxygenation and in four subgroups according to the postoperative day when the collection of specimens of the spleen inferior pole (n=20) was done.