Histopathologic evaluation of the peritoneum exposed to heat shock: experimental study in rats

Avaliação histopatológica do peritônio exposto a choque térmico: estudo experimental em ratos

ORIGINAL ARTICLE

PERITONEUM

Histopathologic evaluation of the peritoneum exposed to heat shock. Experimental study in rats¹

Avaliação histopatológica do peritônio exposto a choque térmico. Estudo experimental em ratos

João Vieira Lopes^I; Paulo Gonçalves de Oliveira^II; João Batista de Sousa^II; Sonia Nair Báo^III; Gustavo Henrique Soares Takano^IV; Isabel Irene Rama Leal^IV

^IMaster, Affiliate Professor, Medical School, UnB, Distrito Federal, Brazil

^IIAssociate Professor, Medical School, UnB, Distrito Federal, Brazil

^IIIAssociate Professor, Department of Cellular Biology, Biological Science Institute, UnB, Distrito Federal, Brazil

^IVMD, Pathologist, Pathologic Anatomy Center, UnB, Distrito Federal, Brazil

^{Correspondence}

ABSTRACT

PURPOSE: To evaluate histopathologic alterations of the peritoneum exposed to heat shock.

METHODS: Sixty rats were randomly distributed into 6 groups: Heat Shock (HS), High Temperature (HT), Body Temperature (BT), Temperature 0oC (TZ), Sham (SH) and Control (CG) with 10 animals each. The peritoneal cavity of animals from groups HS, HT, BT and TZ was irrigated with NaCl solution 0.9% at temperatures 50ºC, 0ºC, 50ºC, 37ºC and 0ºC, respectively. For animals from group SH, the procedures were simulated and those from group CG, laparotomy and biopsies were conducted. Twenty-four hours later, biopsies of the peritoneum for exams under light and electronic microscopy were performed.

RESULTS: Edema was found in groups HS 80%, HT 60%, BT 30% TZ 70%, SH 40% and CG 30%. Vascular congestion was found in groups HS 20%, HT 30%, BT 10% and TZ 20%. Erythrocyte extravasation was found in groups HT 60% and SH 10%. Mesothelium destruction was found in 100% of specimens from groups HS, HT, BT, TZ, SH and CG 90%. Necrosis was found in groups HS 30%, HT 20% and BT 10%. The mean peritoneal thickness ranged from 42.26 µm (TZ) to 26.42 µm (CG).

CONCLUSION: The heat shock caused no deaths, but promoted significant peritoneal edema without affecting the other histopathologic indicatives.

Key words: Heat-Shock Response. Peritoneal Lavage. Peritonitis. Rats.

RESUMO

OBJETIVO: Avaliar alterações histopatológicas do peritônio exposto a choque térmico.

MÉTODOS: Sessenta ratos foram distribuídos aleatoriamente em seis grupos: Choque Térmico (CT), Temperatura Elevada (TE), Temperatura 0ºC (TZ) Sham (SH) e Controle (GC) com 10 animais. A cavidade peritoneal dos animais dos grupos CT, TE, TC e TZ foi irrigada com solução de NaCl 0,9% nas temperaturas, 50ºC e 0ºC, 50ºC, 37ºC e 0ºC, respectivamente. Nos animais do grupo SH foram simulados os procedimentos e nos do GC laparotomia e biópsias. Depois de 24 horas foram realizadas biópsias do peritônio para exames sob microscopia de luz e eletrônica.

RESULTADOS: Edema foi encontrado nos grupos CT 80%, TE 60%, TC 30%, TZ 70%, SH 40% e GC 30%. Congestão vascular foi encontrada nos grupos CT 20%, TE 30%, TC 10% e TZ 20%. Extravasamento de hemácias foi encontrado nos grupos TE 60% e SH 10%. Destruição de mesotélio foi encontrada em 100% dos espécimes dos grupos CT, TE, TC, TZ, SH e no grupo GC 90%. Necrose foi encontrada nos grupos CT 30%, TE 20% e TC 10%. A espessura média do peritônio variou de 42,26 µm (TZ) a 26,42 µm (GC).

CONCLUSÃO: O choque térmico não causou óbitos, mas promoveu edema peritoneal significante sem alterar os demais indicadores histopatológicos.

Descritores: Resposta a Choque Térmico. Lavagem Peritoneal. Peritonite. Rats.

Introduction

Heat shock may be stimulated in the peritoneal cavity (PC) through irrigation with NaCl solution 0.9% (saline) at different temperatures employed in peritoneal lavage (PL). PL is used by abdominal surgeons since 1905 when Price1 proposed its use to remove purulent secretions and other impurities from the PC with peritonitis^2,6,10. Although the incorporation of new surgical techniques, the better comprehension about the disease pathophysiology, the use of antibiotics and employment of intensive care in prevention and treatment of the diffuse peritonitis have contributed for the reduction on the mortality rate, this disease is still a fearful complication and its mortality rate has decreased only from 40% to 30% in the last 30 years³. In the localized form of the secondary peritonitis, the mortality rate ranges from 1% to 3% while in the diffuse peritonitis, it ranges from 30% to 85%^4,16. On the other hand, tertiary peritonitis associated infection due to Candida albican may present mortality rate of up to 75%⁵. The PL was initially discussed as routine procedure for the reduction of the contamination level in peritonitis by authors who supported the PC irrigation as contamination propagation factor⁶. Maingot⁷ is among these authors and proposed: "In my opinion, irrigation of the peritoneal cavity with cleanness purposes is never justified even in the presence of gross fecal contamination". However, although the concept of contamination propagation remains, it has not been clinically verified⁶ and cannot not applied to diffuse peritonitis when the contamination already involved the entire peritoneum. The parietal peritoneum consists of a single layer of mesothelial cells and a conjunctive tissue layer containing cells, blood vessels, lymphatic vessels and nervous fibers^8,9, what may be physically and biologically similar to the dermis that once injured and contaminated, needs the purulent secretion, strange bodies, dead tissues and other impurities to be washed off³. Once recognized that PL was vital for the treatment of the secondary peritonitis^3,4,6,23, researchers added to the saline solution substances such as antibiotics^2,11,24, antimicrobial agents¹², chemotherapic agents¹³, hypertonic glucose 10%¹⁴ and immunoglobulin¹⁵ with the objective of increasing the procedure efficiency. Although there are clinical and experimental researches on PL with saline solution associated or not to different substances, references on histopathologic alterations that the temperature of these solutions could cause in the peritoneum are scarce^16-18. In practice, the temperature of the saline solution used in the PL does not receive adequate attention¹⁹ and is commonly referred as "lukewarm serum", according to the surgeon's tactile sensitivity. However, the temperature and/or its sudden variation, the heat shock, may cause stress and cell death^16-19,, and produce protective effects in the sepsis with protein expression of the heat shock²⁰, in malignant neoplasia²¹ and in the food deterioration prevention^22,25. In 1864, Pasteur apud Debré²² studied the wine manufacturing process and observed that deterioration occurred due to the action of microorganisms present in the medium and later found that heating at controlled temperature conditions from 55ºC to 60ºC eliminated microorganisms without affecting the wine organoleptic properties, which remained salutary if kept cooled in closed recipient with no contact with oxygen²². This partial sterilization process with temperature raise at controlled conditions followed by cooling widely used for food conservation was called as pasteurization²². The employment of saline solution for PL at high temperature followed by lavage with solutions at low temperature, the heat shock, could also produce damages to microorganisms found in the peritoneum, similarly to the milk pasteurization process²², but it could also cause histopathologic alterations in the peritoneum, muscles or in the intestine wall as those observed in response to the exposition of these tissues to biological agents⁹. In this context, in an experimental research, Silva, et al.¹⁹ reported that when the saline solution at temperature of 60ºC used for PL in rats remained within the PC for 1 min could cause histopathologic alterations in the peritoneum and in the muscular fibers of the PC posterior wall perceived at light microscopy with mortality rate of 66% at the first 24 hours. However, the solution caused no histopathologic alteration when used at temperature of 45ºC during equal period of time. In 1991, the employment of peritoneal irrigation with saline solution at different temperatures for the peritonitis treatment in human beings with encouraging results was reported. The process described as heat shock consists of the irrigation of the peritoneal cavity with saline solution at temperature between 48ºC and 50ºC during 20 minutes shortly followed by peritoneal lavage with other solution at temperature between 0ºC and 2ºC during 5 to 10 min¹⁷. The objective of the present study was to evaluate possible histopathologic alterations in the peritoneum of rats exposed to heat shock due to PL with saline solution at temperature of 50ºC shortly followed by another PL with saline solution at temperature between 0ºC and 2ºC.

Methods

The experimental procedures were conducted according to ethic principles of the Brazilian Bureau of Animal Experimentation (COBEA) and were approved by the Ethics Committee for Animal Use (CEUA) from the Biological Sciences Institute (ICB) – University of Brasilia. Sixty healthy male Wistar rats (Rattus norvergicus) weighting between 340 and 400 g, from 100 to 120 days old were distributed by simple allotment with no reposition into 6 groups, namely: Group 1, heat Shock (HS); Group 2, High Temperature (HT); Group 3, Body Temperature (BT); Group 4, Temperature 0ºC (TZ); Group 5, Sham (SH) and Group 6, Control (CG). The animals were anesthetized with intramuscular cetamine chloridrate at dose of 90 mg/kg associated to xylazin at dose of 5 mg/kg and submitted to median laparotomy of 4 cm with the caudal extreme of the incision 2.0 cm from the external genitalia.

Experimental protocol

The PC was exposed with the aid of an aluminum frame (Figure 1, A and ^B), filled and emptied with saline solution according to protocol established for each group of animals, except for those from the control group, which were submitted to laparotomy and biopsies and those from the Sham group, which were submitted to experimental simulation (Table 1). Twenty-four hours after laparotomy, all animals were anesthetized and re-operated for the performance of biopsies, except for animals from the control group that had already been submitted to biopsies at the first procedure. During re-operation, samples from the anterolateral wall of the left side of the peritoneal cavity were collected for histopathologic examination under light microscopy (LM) and in the fifth animal from each group, biopsy using the biopsy punch technique of the right side was also performed for analysis under transmission electronic microscopy (TEM). After these procedures, the animals received intracardiac lethal dose of sodium thiopental 2.5%.

Light Microscopy (LM)

Samples of 10 x 5 mm composed of peritoneum and a muscular layer of the PC anterolateral wall were fixed in formaldehyde 10%, dehydrated in ethanol, paraffin included, cut in microtome Leica RM2125RT into sections of 6 µm thickness, assembled in microscope slides and stained with hematoxilin/eosin (HE). The examination of the microscope slides was performed using microscope Zeiss oxicop 2 with lens of 40X and ocular of 10x23 mm to investigate the presence of edema, vascular congestion, erythrocyte extravasation, mesothelium destruction and focal necrosis classified from 1 to 4, corresponding to the following levels: 1 – absent; 2 – slight; 3 – moderate and 4 – intense. Five different fields from each slide were photographed and two peritoneal thickness evaluations were performed in two different sites of each one of the five fields with the aid of a digital camera (lens of 20x) using the Zeiss Axio Vision® software version 3.0 calibrated with the digitalized image of the Neubauer chamber photographed with the same parameters adopted in microscope slides photography. The evaluation of the peritoneal thickness in the digitalized images was performed considering the mesothelium as surface limit and the first muscular cell as deep limit of the segment that included the mesothelium and the submesothelial zone in two different sites of each one of the five photographed fields in the slides. Transmission Electronic Microscopy (TEM) . For the analysis under transmission electronic microscopy, fragments of 1 mm³ were obtained and included in fixing solution composed of 2% paraformaldehyde, 2% glutaraldehyde in sodium cacodylate buffer 0.1 M, pH. 7.2. The fixing procedure occurred for a period of 24 h. After fixation, the tissue was washed three times with the same buffer for a period of 1 h. The post-fixation procedure was performed in osmium tetroxide solution and potassium ferricyanide for 1 h in the absence of light. The dehydration was performed in progressive concentrations of acetone and inclusion in Spurr resin. The cuts were obtained in ultramicrotome Leica AG™ Type 705001 in sections of 70 to 90 nm with the use of diamond cutting blade. The sections were collected in copper web of 200 mesh and contrasted in uranile acetate and lead citrate solutions. The analyses were performed in transmission electronic microscope (Jeol JEM-1011C™ Electronic Microscopy) operated at 80 Kv in the Electronic Microscopy Laboratory of the Department of Cellular Biology – University of Brasília by evaluating morphological alterations and the ultrastructure of the mesothelial cells. In the TEM analysis, although the mesothelial cell was the main focus, other cells found at the submesothelial zone, conjunctive tissue and muscular tissue of the PC anterolateral wall were also evaluated.

Statistical analysis

The statistical calculations were performed with the use of the Statistical Package Social for Sciences software (SPSS.13) applying the Levene Proof, Analysis of Variance and the Mann-Whitney test. Probabilities of p<0.05 were considered as significant.

Results

The experimental model allowed performing the study with the use of aluminum frame for PC exposition, which facilitated the opening and uniform exposition of the surgical wound. No inter-group and intra-group significant differences in relation to the weight of animals were verified, which was performed at the operation and at the re-operation days: p> 0.05, (Table 2). In relation to weight, the sample seemed to be homogeneous, with p = 0.151 at the operation day and p = 0.481 at the re-operation day and in agreement with the Levene test for homoscedasticity.

Clinical evolution

No death was verified. Two animals presented diarrhea, one from group HS and another from group HT. One animal from group SH developed with abdominal distension and dehiscence of the surgical wound suture; however, after reestablished from anesthesia, all animals remained active until the re-operation moment.

Light microscopy

The examination of the microscope slides showed normal mesothelium, edema, vascular congestion, erythrocyte extravasation, mesothelium destruction, focal necrosis and thickening of the peritoneum, mesothelium and submesothelial zone (Figure 2).

Edema

Slight-degree edema was found in 80% of samples of animals from group HS, 60% from group HT, 20% from group BT, 50% from group TZ, 40% from group SH and 30% from group CG. Moderate-degree edema was found in 10% of samples of animals from group HS and in 20% from group TZ. No edema that could be classified as intense was found. A significant difference (P = 0.028) was observed in the edema incidence when samples of animals from group HS were compared to those from group CG (Table 3).

Vascular congestion

Slight-degree vascular congestion was found in 10% of samples of animals from group HS, 20% from group HT, 10% from group BT, and 20% from group TZ. Moderate-degree vascular congestion was found in 10% of samples of animals from groups HS and HT. No intense vascular congestion was verified. No significant difference (P < 0.05) was observed in the vascular congestion incidence when samples of animals from group HS were compared to those from groups CG and SH (Table 3).

Erythrocyte extravasation

Slight-degree erythrocyte extravasation was found in 60% of samples of animals from group HS and in 10% from group HT. No moderate or intense erythrocyte extravasation was verified. No significant difference (P < 0.05) was observed in the erythrocyte extravasation incidence when samples of animals from group HS were compared to those from groups CG and SH (Table 3).

Mesothelium destruction

Slight-degree mesothelium destruction was found in 40% of samples of animals from group HS, 60% from group HT, 30% from group BT, 60% from group TZ, 40% from group SH and 20% from group CG. Moderate-degree mesothelium destruction was found in 60% of samples of animals from group HS, 40% from group HT, 70% from group BT, 40% from group TZ, 60% from group SH and 70% from group CG. No intense mesothelium destruction was found. No significant difference (P < 0.05) was observed in the mesothelium destruction incidence when samples of animals from group HS were compared to those from groups CG and SH (Table 3).

Focal necrosis

Slight-degree focal necrosis was found in 30% of samples of animals from group HS, 20% from group HT and 10% from group BT. No moderate or intense focal necrosis was found. No significant difference (P < 0.05) was observed in the focal necrosis incidence when samples of animals from group HS were compared to those from groups CG and SH (Table 3).

Peritoneal thickness

The peritoneal thickness ranged from 14.01µm to 93.37µm, but no significant difference in the peritoneal thickness was found in inter-group and intra-group samples according to the analysis of variance p = 0.146 (Table 4). The means of the peritoneal thickness measurements of animals from each group grouped as quartiles were similar for groups HE and CG, with higher incidence in the 3^rd quartile, while for group SH, the highest concentration occurred in the 1^st quartile. A discrepant value for group BT in relation to the other groups was found ^93,37, concerning the animal number 31, which was excluded from the nalysis (Figure 3).

Electronic microscopy

The analysis was limited to the morphology of mesothelial cells found in the conjunctive tissue, although samples presented muscular tissue, collagen, capillaries, erythrocytes and inflammatory cells. Specimens collected from groups SH and CG presented mesothelial cells that adhered more easily to the subjacent conjunctive tissue than those from groups BT and HT, where mesothelial cells were free or partially detached from the conjunctive tissue (Figure 4). On the other hand, cells with morphological alterations such as membrane rupture with cytoplasm extravasation and abundant vacuoles, erythrocytes outside capillaries, were found in samples from groups HS, HT and TZ, but these alterations were more frequent in groups HS and HT.

Discussion

The efficiency obtained in the use of the saline solution in PL associated or not to other substances to reduce the peritonitis contamination level is frequently reported; however, reports on the effects of the heat shock that the temperature of these solutions may produce on the peritoneum and even on the muscular fibers of the abdominal wall are scarce. The heat shock caused by PL with heated saline solution followed by another PL with cooled solution could produce damages to microorganisms with no death or irreversible injuries to host tissue similarly to what occurs in pasteurization, what makes HS an additional resource in the peritonitis treatment. Thus, this study evaluated histopathologic alterations of peritoneum exposed to heat shock so that a subsequent study would evaluate its effect on the peritonitis treatment. In a pilot study, 60% of rats exposed to peritoneal lavage with saline solution at temperature of 60ºC died in the first 24 hours and no deaths were observed when irrigations were conducted with solution at temperature lower than or equal to 50ºC. This result is similar to that obtained by Silva et al.¹⁹, who found mortality rate of 66% when saline solution at 60ºC was used for PL during 1 min and no death among animals was observed when the solution temperature was 45ºC. Therefore, in order to avoid high mortality rates and to reproduce the process, saline solution at maximum temperature of 50ºC and minimum between 0ºC and 2ºC was used, unlike methods used by authors who do not support the immediate cooling through irrigation of the PC with saline solution at temperature between 0ºC and 2ºC and consequent heat shock ^16,18,19. During the surgical procedure, the PC exposition through aluminum frame promoted the opening and immobilization of the surgical wound, thus allowing safe peritoneal irrigation and avoiding different positions in relation to operated animals. This technique avoided overflow of saline solution like when wound edges are fixed with homeostatic nippers and presented by the assistant that hardly ever keep stable while the surgeon pours the saline solution inside the PC and especially during exposition periods, thus causing a bias that could distort results and cause complications such as tissue rupture due to excessive tension. When the PC was filled up with saline solution at temperature of 50ºC, a dilatation of the subserous vessels was observed and when the solution temperature was between 0ºC and 2ºC, a contraction of the intestinal vessels was otherwise observed, which presented smaller caliber when compared to the macroscopic aspect of the subserous vascularization before irrigations. Similar results were reported by Silva et al.¹⁶ when an intraperitoneal infusion of 20 ml of saline solution at 38ºC was used. Histopathologic alterations such as edema, vascular congestion, erythrocyte extravasation, mesothelium destruction and focal necrosis of muscular cells observed at light microscopy that characterized cellular aggression occurred; however, the intensity of these alterations was not sufficient to produce death. When histopathologic alterations found for group HS were compared to those observed for group CG, significant differences in relation to variable edema and differences statistically not significant in relation to variables congestion, erythrocyte extravasation, mesothelium destruction and focal necrosis of muscular cells were found. When these histopathologic indicatives found for the other groups HT, BT and TZ were compared to those observed for groups SH and CG, no significant differences were found, except for the erythrocyte extravasation incidence for group HT, which was significant in relation to SH and CG, thus justifying the necessity to control the temperature of saline solutions employed, as proposed by Silva et al.¹⁹. Mesothelium destruction occurred to 100% of samples from all groups, except for group CG, which presented 90%, similarly to what was reported by Silva et al.¹⁹, it cannot be exclusively attributed to PC irrigations with saline solution, but also to the technical difficulty found in maintaining the peritoneal mesothelial cells. In general, the peritoneum is quite thin and easily detaches from the subjacent conjunctive tissue. Histopathologic alterations observed in muscular cells of the PC anterolateral wall of animals from groups HS, HT (20%) and TZ suggestive of degeneration and necrosis such as loss of striations, cytoplasm vacuolization and shapeless aspect of the tissue, as reported by Silva et al.¹⁹ were not found in samples of animals from group CG. The thickness of the peritoneum, mesothelium and submesothelial zone may exceed 300 µm after secondary peritonitis, peritoneal dialyses and in rats when the PC is exposed to biological agents ⁹. Although in this study a physical agent has been used to stimulate the peritoneum, the heat shock, an increase on the average peritoneal thickness was also observed especially in groups HT and TZ in relation to the group CG. This increase was mainly caused by edema and was smaller than that observed when the peritoneum is exposed to biological agent that produces increases on the cellularity, as reported by Margetts et al. ¹⁶, who injected intraperitoneal AdIL-70 (Adenovirus Interleukin Innocuous – control) and AdIL-1ß (Adenovirus Interleukin ß) and observed increase on the peritoneal thickness 9. The fact that the average peritoneal thickness of group HS, x = 29.26 µm was smaller in relation to groups HT and TZ leads us to infer that the exposition of the PC to saline solution at temperature between 0ºC and 2ºC shortly after irrigation with saline solution at 50ºC had no accumulative effect on the average thickness (HT+TZ), but rather, the average thickness was smaller than that verified when irrigation was conducted with saline solution at 50ºC, group HT, or between 0ºC and 2ºC, group TZ. On the other hand, morphological alterations and cellular ultra-structure observed under TEM such as rupture of the cell membrane, cytoplasm extravasation, vacuolization and degeneration of cell nucleuses in samples from different groups were analyzed and based on histopathologic alterations that suggest that cellular architecture of the peritoneum was more preserved in the Control Group than in the Heat Shock Group, corroborating results obtained through light microscopy.

Conclusion

The heat shock under conditions established in the present study did not cause any death, but promoted significant peritoneal edema without altering the other histopathologic indicatives.

Received: April 09, 2007

Review: June 14, 2007

Accepted: July 16, 2007

Conflict of interest: none

Financial support: none

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1

Research performed at Experimental Surgery Laboratory, Medical School, University of Brasília (UnB), Distrito Federal, Brazil.

Publication Dates

• Publication in this collection
  27 Sept 2007
• Date of issue
  Oct 2007

History

• Accepted
  16 July 2007
• Received
  09 Apr 2007
• Reviewed
  14 June 2007