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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 2  |  Page : 159-165

Effect of dexamethasone versus propranolol on surgically induced endometriosis in an experimental animal model


1 Department of Obstetrics and Gynecology, Faculty of Medicine, Al Azhar University (Girls), Cairo, Egypt
2 Department of Experimental Surgery, Medical Research Centre, Ain Shams University, Cairo, Egypt

Date of Submission03-Feb-2020
Date of Decision09-Feb-2020
Date of Acceptance19-Feb-2020
Date of Web Publication29-Jun-2020

Correspondence Address:
Naglaa M.S Emsalem

Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sjamf.sjamf_17_20

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  Abstract 


Background Endometriosis is a disease of adolescents and reproductive-aged women characterized by the presence of endometrial tissue outside the uterine cavity and commonly associated with chronic pelvic pain and infertility. The mechanisms responsible for its pathogenesis and progression remain poorly understood. It is well established that endometriosis grows and regresses in estrogen-dependent fashion, and the diseases can be effectively cured by definitive surgery. However, prolonged medical therapy may be needed in most of the cases.
Objective The objective of this study is to evaluate the effects of dexamethasone versus propranolol in rats with surgically induced endometriosis on vascular endothelial growth factor (VEGF) as a marker in the implanted tissue.
Materials and methods The study was conducted in the Animal Research Unit of Medical Research Center of Ain Shams University. All procedures were performed from May 2018 to September 2018, in compliance with the international guidelines for care and use of experimental animals, on 100 female Wistar albino rats 90 days old, which were randomly divided into three groups: first group was treated with dexamethasone (n=40), and second group was treated with propranolol (n=40), and third group was a control group (n=20). They were used to create a model for the experimental induction of endometriosis and evaluation of therapeutic effect of dexamethasone versus propranolol.
Results In the first group after treatment with dexamethasone, there was a highly statistically significant decrease in size of implant tissue (P<0.001). Moreover, it was found regarding the histopathological finding that there was massive necrosis associated with hyalinization of stroma and complete absence of the vascularity. In addition, it was found regarding immunochemical assay (VEGF) that there was mild amount of VEGF (25–50) in the peritoneal tissue of the endometriotic implant. In the second group after treatment with propranolol, there was higher statistical decrease in implant size (P<0.001). It was found there was diffuse necrosis of the glandular structure and hyalinization of the stroma with absence of vascularity and mild amount of VEGF (25–50) in the peritoneal tissue of the endometriotic implant and absence inflammatory cells. However, in the control group, there was a higher statistical increase in implant size (P<0.001). It was found there was no change in endometrial gland and stroma, with severe amount of VEGF (75–100) and inflammatory cells.
Conclusion Both propranolol and dexamethasone efficiently reduced the size of endometriotic implant in conditions of surgical-induced endometriosis in rats. This is through restriction of angiogenesis through VEGF, which was utilized as a marker. So, it can be deemed that the drugs could be promoted in conditions of endometriosis in humans, but they require more research studies for assessment in humans.

Keywords: animal model, dexamethasone, endometriosis, experimental, propranolol


How to cite this article:
Emsalem NM, Fahmy S, Effat DM, Abdellah AM. Effect of dexamethasone versus propranolol on surgically induced endometriosis in an experimental animal model. Sci J Al-Azhar Med Fac Girls 2020;4:159-65

How to cite this URL:
Emsalem NM, Fahmy S, Effat DM, Abdellah AM. Effect of dexamethasone versus propranolol on surgically induced endometriosis in an experimental animal model. Sci J Al-Azhar Med Fac Girls [serial online] 2020 [cited 2020 Oct 23];4:159-65. Available from: http://www.sjamf.eg.net/text.asp?2020/4/2/159/288265




  Introduction Top


Endometriosis is an estrogen-dependent disease affecting 8–10% of women in the reproductive age. It is diagnosed in 71–87% of women with chronic pelvic pain and in 30% of women with infertility. It is defined as adherence and growth of the functional layer of the endometrium outside the uterine cavity [1].

Hormonal therapies, such as dienogest and danazol, are widely used to reduce functional endometrial tissue by causing atrophy, thus inducing regression of the symptoms of endometriosis [2].

However, treatment with these hormonal agents is associated with adverse events, which limits their use, such as estrogen-withdrawal symptoms (e.g. headache, hot flushes, vaginal dryness, decreased libido, and bone demineralization), androgenic effects, adverse effects on lipids, arterial thrombosis, liver dysfunction, increased glucocorticoid activity, and a high incidence of abnormal menstrual bleeding [3]. Therefore, there is a need for alternative treatments that are both effective and safe against endometriosis. Propranolol is a nonselective beta-blocker that demonstrates equal affinity for adrenoreceptors and therefore acts on multiple tissues.

Propranolol has been found to be potent and safe for treatment of hemangiomas by inhibition of angiogenesis [4]. Beta-blockers also decrease the expression of vascular endothelial growth factor (VEGF), thus preventing angiogenesis [5].

Studies have demonstrated that the glucocorticoids, e.g., dexamethasone, inhibit the cellular proliferation, and their effects can be observed in the reproductive functions because they block several induced responses by the estrogen in the uterus [6].

In the estrous cycle of rats, as well as other animals, several phases of estrogen action are observed (pro estrous and estrous) and of progesterone (meta-estrous and di estrous) [6]. This way, several experimental models of endometriosis in rats (induced endometriosis), which histologically and endocrinologically reproduce this pathology, have been elaborated, causing a better understanding about the physiological and biochemical aspect [7].


  Aim Top


The objective of this study is to evaluate the effects of dexamethasone versus propranolol in rat with surgically induced endometriosis on VEGF as a marker in the implanted tissue.


  Materials and methods Top


Design

A randomized controlled experimental study was conducted.

Study setting

The study was conducted in the Animal Research unit of Medical Research Center of Ain Shams University. All procedures were performed in compliance with the international guidelines for care and use of experimental animals.

Animals

A total of 100 female Wistar albino rats 90 days old, nonpregnant, were used to create a model for the experimental induction of endometriosis and evaluation of therapeutic effect of dexamethasone versus propranolol.

Study intervention

A total of 100 female rats were randomly divided into three groups and caged individually in controlled environment with 24-h light/dark cycle. The animals were sexually mature and demonstrated normal estrous cycle change in the uterine histology. All rats were left for 10 days before the experimental procedures to become adapted to the experimental environment. During this period, vaginal smear was done and examined microscopically for determination of the stage of estrous cycle before the operation.

Study procedures

All groups were anesthetized by intraperitoneal administration of ketamine hydrochloride (80 mg/kg body weight) and xylazine (chlorpromazine hydrochloride) (5 mg/kg body weight) ([Figure 1]). Before surgery, the abdominal skin was shaved, and antisepsis was done by 10% povidone–iodine solution. Ventral midline incision about 3 cm long was made to expose the reproductive organs.
Figure 1 Administration anesthesia before surgery.

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First laparotomy (induction of endometriosis)

Endometriosis was induced surgically when the rats were in estrous cycle. A distal segment 1 cm in length was resected from the left uterine horn; the endometrial layer was separated and trimmed into a 5×5mm piece. Next, the trimmed endometrial piece was sutured to inner side of the abdominal wall close to the artery with 4-0 vicryl sutures. Before closure of abdominal wall, 2 ml of saline was administrated into the abdominal cavity to prevent drying and to minimize adhesion formation. Finally, the peritoneal cavity, the abdominal wall, and skin were closed. After the first endometriosis-inducing operation, all groups were observed for 21 days with daily dressing of the wound without any medication ([Figure 2]).
Figure 2 The first operation induction of endometriosis.

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Second laparotomy

All groups underwent a second exploratory laparotomy to observe the growth of endometriotic implants ([Figure 3]). The surface sizes of implants were recorded (length in mm×width in mm). The endometriotic tissues were photographed ([Figure 4] and [Figure 5]), followed by the closure of the peritoneal cavity, abdominal wall, and skin.
Figure 3 Second operation after 21 days to examine the endometrial implants for size and viability.

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Figure 4 Measures of the length and width of implanted tissues.

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Figure 5 Third operation for sample collection.

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After second laparotomy, we started treatment and categorized the groups as follows:
  1. First group (endometriosis model tread with dexamethasone): 40 rats were treated with dexamethasone concentration 8 mg/ml, being administered through intraperitoneal injections in dosage of 0.1 ml/day/animal for 15 successive days.
  2. Second group (endometriosis model treated with propranolol): 40 rats were treated with propranolol 10 mg/kg (Indral; Astrazeneca) for 15 successive days.
  3. Third group (untreated endometriosis, as a control group): 20 rats were given normal saline intraperitoneally for 15 successive days.


Third laparotomy

By the end of the medication period, all groups underwent the third laparotomy procedure. The rats were euthanized under ketamine overdose anesthesia. After opening of the abdominal cavity, the endometriotic implants were measured and photographed. Next, the implants were excised and. trimmed; a portion of each implant was fixed in formalin 10% and then embedded in paraffin for histopathological examination and immunohistochemistry.

The severity of immunohistochemical alteration in the endometriosis of different experimental groups was assessed using VEGF antibodies [8]:
  1. From 75 to 100, it means severe score (+++).
  2. From 50 to 75, it means moderate score (++).
  3. From 25 to 50, it means mild score (+).
  4. From zero to 25, it means nil (˗).


Sample collection

Autopsy samples were taken from the implanted uterine tissue of rats in different group and fixed in 10% formalin for 24 h. Washing was done in tap water, and then serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56° in hot air oven for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 4-μm thickness by a sliding microtome.

Staining methods

Hematoxylin and Eosin staining

The obtained tissue sections were collected on glass slides, deparaffinized, and stained by hematoxylin and eosin stain for routine examination through the light electric microscope [8].

Immunochemistry

Endometriosis sections were immunohistochemically stained for VEGF detection.

Procedure for immunohistological staining

We prepared all solutions according to manufacturer’s recommendations. Optimized antibody detection was accomplished by antigen retrieval screening, titration, and validation according to clinical practice standards. Sections were dewaxed in xylene, rehydrated, and pretreated with 3% hydrogen peroxide for blocking endogenous peroxidase activity. Microwave-assisted antigen retrieval was then performed for 20 min. Sections were incubated overnight at 40°C with the corresponding antibody. After washing with phosphate buffered saline, sections were incubated with biotinylated IgG and then with streptavidin–peroxidase conjugate. Sections were then washed with phosphate buffered saline and incubated with diaminobenzidine for 5 min and counterstained with Mayer’s hematoxylin. Negative control sections were performed by omission of incubation with the primary antibody [9].

Statistical analysis

Data were analyzed using Statistical Program for Social Science (SPSS), version 15.0. Quantitative data were expressed as mean±SD. Qualitative data were expressed as frequency and percentage.

The following tests were done:
  1. Independent samples t test of significance: it was used when comparing between the two means.
  2. A one-way analysis of variance: it was used when comparing between more than two means.
  3. Post-hoc test: it was used for multiple comparisons between different variables.
  4. P value:
    1. P value less than 0.05 was considered significant.
    2. P value less than 0.001 was considered as highly significant.
    3. Pvalue more than 0.05 was considered insignificant.



  Results Top


[Table 1] shows no statistical significant difference (P>0.05) among the studied groups regarding weight.

There was no statistical significant difference among studied groups regarding implant size at second laparotomy and before treatment (P>0.05).

[Table 3] shows higher statistical decrease in implant size after treatment in comparison before treatment in dexamethasone group (P<0.001).

Hematoxylin and eosin stain

In the rats with experimental induction of endometriosis and administration of dexamethasone, there was massive necrosis observed and associated with hyalinization of the stroma and complete absence of vascularity.

Immunochemistry results

In the dexamethasone group, the immunochemical result showing mild amount of VEGF and absence of inflammatory cell ([Figure 6]).
Figure 6 The peritoneum of endometriosis area in the treatment groups showing the mild amount of VEGF indicated by arrows with absence of inflammatory cell. VEGF, vascular endothelial growth factor.

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[Table 4] shows higher statistical decrease in implant size after treatment in comparison before treatment in propranolol group (P<0.001).

Hematoxylin and eosin stain

In the rats with experimental induction of endometriosis and administrated propranolol, there was diffuse necrosis of the glandular structure and hyalinization of the stroma with absence of vascularity.

Immunochemistry results

In the propranolol group, the immunochemical result showing mild amount VEGF and absence of inflammatory cell in [Figure 6].

[Table 5] shows higher statistical increase in implant size in control group (P<0.001).

Hematoxylin and eosin stain result

Group of rats with induction of endometriosis and kept as control showed the endometrial glands and stroma were histological intact associated with well-supplied vascularity as well as focal hemosiderosis.

Immunochemistry results

Concerning immunochemical staining of abdominal wall biopsy from control group, it revealed significant amount of VEGF and inflammatory cells ([Figure 7]).
Figure 7 The peritoneum of endometriosis area in the control group showing the magnification of to identify the sever amount of VEGF indicated by arrows with perivascular inflammatory cells infiltration. VEGF, vascular endothelial growth factor.

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There was a highly statistical significant difference between treated groups and control group regarding implant size after treatment (P<0.001). However, no statistical significant difference between dexamethasone group and propranolol group as regard implant size after treatment (P>0.05).


  Discussion Top


The present study was done on 100 female Wistar albino rats 90 days old, nonpregnant, which were used to create a model for the experimental induction of endometriosis and evaluation of therapeutic effect of dexamethasone versus propranolol. The rats were divided randomly into three groups.

In the current study, there was no statistical significant difference (P=0.05) among the studied groups regarding weight (200 g) ([Table 1]).
Table 1 Comparison among studied groups regarding weight of rats

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At the first laparotomy, the size of endometriotic tissue which was implanted equally in all groups was ∼5 in 5 mm. After 21 days, the second operation was done to measure the endometriotic implant and calculate the mean size of it in all groups. No statistical difference was found among studied groups regarding implanted tissue (P=0.02) ([Table 2]).
Table 2 Post-hoc test for comparison among studied groups regarding implant size at second laparotomy and before treatment

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Then, the treatment was started in first group with dexamethasone (Fortecortin; Glaxo Smith Kline) up to concentration 8 mg/ml through interperitoneal injection in dosage of 0.1 ml/day for 15 successive days, whereas in second group with propranolol 10 mg/kg for 15 successive days, and normal saline in the control group for 15 successive days.

After 15 days from starting the treatment, a third operation was done to measure the endometriotic implant and calculate the mean size of implant tissue, and sample was taken from all groups for histopathological examination and immunohistochemistry.

In the current study, in the first group (dexamethasone), there was a highly statistical significant decrease in size of implant tissue after treatment (P<0.001) ([Table 3]).
Table 3 Comparison between implant size before and after treatment in dexamethasone group

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It was found regarding the histopathological finding that there was massive necrosis associated with hyalinization of stroma and complete absence of vascularity. It was found regarding immunochemical assay (VEGF) that there was mild amount of VEGF (25–50) in the peritoneal tissue of the endometriotic implant in the first group according to the histological score ([Figure 6]).

This result agrees with the study done by Batista et al. [9]. They found the treatment with dexamethasone for 15 days inhibited angiogenesis and inflammatory process in implant tissue.

In the present study, in the second group (propranolol group), there was a highly statistical significant decrease in size of implant tissue after treatment (P<0.001) ([Table 4]). It was found regarding the histopathological finding that there was diffuse necrosis of the glandular structure and hyalinization of the stroma with absence of vascularity. It was found regarding immunochemical assay (VEGF) that there was mild amount of VEGF (25–50) in the peritoneal tissue of the endometriotic implant in second group according to histological score and absence of inflammatory cells ([Figure 7]).
Table 4 Comparison between implant size before and after treatment in propranolol group

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This result agreed with the study done by Rosa-e-Silva et al. [10]. They did an experimental study on 30 female Wistar rats to assess the anti-angiogenic effect of propranolol on the endometriosis lesions. They found decrease in the size of implanted tissue induced in 15 female rats compared with controls. They concluded that treatment with anti-angiogenic drugs like propranolol offers new prospects for therapeutic approach for patients with endometriosis.

Moreover, a study done by Uzunlar et al. [11] agreed with the present study. They found in the propranolol-treated group, the mean implant volume decreased significantly after treatment (142.5 vs. 32.1 mm3) before and after treatment, respectively, and this was statistically significant (P=0.008).

This result is not far from the study done by Nafiye et al. [12], who found decrease in VEGF amount after propranolol treatment, whereas there was no change in implant size after treatment.

On the contrary, Uzunlar et al. [11] did a study to determine whether propranolol has an inhibitory effect on the angiogenesis of endometriosis in an experimental rat model or not. In their study, they used 24 female Wistar albino rats (200–250 g) to create a model for surgical induction of endometriosis; two rats died during the surgery. The rats were randomly divided into a study and a control group, which were treated with daily intraperitoneal injection propranolol (10 mg/kg) and saline (2 mL), respectively. The study duration was 8 weeks. They found the size of endometriotic implant and histopathological findings, and immunochemistry for VEGF elevated, so there is no significant difference between the studied groups.

In the present study, in the third group, there was higher statistical increase in implant size after placebo (P<0.001) ([Table 5]). Moreover, regarding the histopathological and immunological analysis of implanted tissue in third group, it showed endometrial gland and stroma histologically were intact and associated with well-supplied vascularity, as well as focal hemosiderosis, with severe amount of VEGF (75–100) and inflammatory cells ([Figure 7]).This result agrees with study done by Uzunlar et al. [11], where implant volume was significantly increased in the control group (141.0 vs. 174.2 mm3, respectively; P<0.0).
Table 5 Comparison between implant size before and after placebo (control group)

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In the current study, there was a highly statistically significant difference between treated groups (dexamethasone and propranolol) and control group regarding the implanted size after treatment (P<0.001). However, there was no statistically significant difference between dexamethasone group and propranolol group regarding implant size after treatment (P>0.05) ([Table 6]).
Table 6 Post-hoc test for comparison among studied groups regarding implant size after treatment

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This result agrees with the study done by Uzunlar et al. [11]. They found a highly statistical significant difference between implant size in propranolol and control group (P<0.001).


  Conclusion Top


Propranolol and dexamethasone efficiently reduced the size of endometriotic implant in conditions of surgically induced endometriosis in rats. This is through restriction of angiogenesis through VEGF, which was used as a marker. So, it can be deemed that the drugs could be promoted in conditions of endometriosis in humans, but they require more research studies for assessment in humans.

Recommendation

New agent such as propranolol and dexamethasone can be used in treatment of endometriosis, but they needed more studies for evaluation in human being.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Acien P, Velasco I. Endometriosis: a disease that remains enigmatic. ISRN Obstet Gynecol 2013; 2013:242149.  Back to cited text no. 1
    
2.
Fischer OM, Kaufmann-Reiche U, Moeller C et al. Effects of dienogest on surgically induced endometriosis in rats after repeated oral administration. Gynecol Obstet Invest 2011; 72:145–151.  Back to cited text no. 2
    
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McCormack PL. Dienogest: a review of its use in the treatment of endometriosis. Drugs 2010; 70:2073–2088.  Back to cited text no. 3
    
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Starkey E, Shahidullah H. Propranolol for infantile haemangiomas : a review. Arch Dis Child 2011; 96:890–893.  Back to cited text no. 4
    
5.
Chen XD, Ma G, Huang JL et al. Serum-level changes of vascular endothelial growth factor in children with infantile hemangioma after oral propranolol therapy. Pediatr Dermatol 2013; 30:549–553.  Back to cited text no. 5
    
6.
Batista Ana P, Ana Paula MD, Álvaro AC, Valéria WT. Histological evaluation of the induced endometriosis in rats, after treatment with dexamethasone. Int J Morphol [online] 2006; 24:565–570.  Back to cited text no. 6
    
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Piva M, Horowitz GM, Sharpe-Timms KL et al. Interleukin-6 differentially stimulates haptoglobin production by peritoneal and endometriotic cells in vitro: a model for endometrial-peritoneal interaction in endometriosis. J Clin Endocrinol Metab 2015; 86:2553–2561.  Back to cited text no. 7
    
8.
Choudhury KR, Yagle KJ, Swanson PE et al. A robust automated measure of average antibody staining in immunohistochemistry images. J Histochem Cytochem 2010; 58:95–107.  Back to cited text no. 8
    
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Batista A, Conceição A, Moraes E et al. Histological evaluation of the induced endometriosis in rats, after treatment with dexamethasone. Int. J Morphol 2016; 24:565–570.  Back to cited text no. 9
    
10.
Rosa-e-Silva JC, Zanardi JVC, Fortunato GG et al. Influence of antiangiogenic agent propranolol on endometriosis experimentally induced in rats. J Minim Invasive Gynecol 2016; 23:175–175.  Back to cited text no. 10
    
11.
Uzunlar O, Ozyer S, Engin-Ustun Y et al. Effects of repeated propranolol administration in a rat model of surgically induced endometriosis. Eur J Obstet Gynecol Reprod Biol 2014; 182:167–171.  Back to cited text no. 11
    
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Nafiye Y, Gulnur O, Raziye K et al. Is montelukast effective in regression of endometrial implants in an experimentally induced endometriosis model in rats? Eur J Obstetr Gynecol aReprod Biol 2014; 184:7–12.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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