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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 46  |  Issue : 1  |  Page : 38-53

Histological and immunohistochemical study of the effect of tramadol on the seminiferous tubules of adult albino rat and the effect of its withdrawal


Department of Histology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission01-Nov-2017
Date of Acceptance23-Jan-2018
Date of Web Publication26-Jul-2018

Correspondence Address:
Amira Ali Abou Elnaga
51 Ali Eldeen Street, El Mehalla El Kobra El Gharbia, 31951
Egypt
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DOI: 10.4103/tmj.tmj_85_17

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  Abstract 


Background Tramadol is a centrally acting opioid analgesic drug that is widely used in the treatment of pain. It is currently the most prescribed opioid in the world, so its abuse, dependence, toxicity, and tramadol-related deaths have increased.
Aim This study aimed to evaluate the histological effect of tramadol on the seminiferous tubules of testis of adult albino rat and assess the effect of its withdrawal.
Materials and methods A total of 52 adult male albino rats were used and divided into two main groups: the first served as control, and the second was treated with tramadol and subdivided into four subgroups. The first and third subgroups received tramadol once daily by gastric tube in a dosage of 40 mg/kg for 4 and 6 weeks, respectively, whereas the second and the fourth (recovery subgroups) received tramadol in a dosage of 40 mg/kg once daily for 4 and 6 weeks, respectively, and then kept without treatment for another 4 weeks. Specimens from the testes were processed for light and electron microscopic examination. Immunohistochemical study was carried out. Morphometric study was also done, and statistical results were analysis.
Results Specimens from tramadol-treated animals showed obvious structural changes. Some spermatogenic cells appeared disorganized with wide intercellular spaces. Many spermatogonia showed vacuolated cytoplasm and deeply stained nuclei. Ultrastructurally, Sertoli cells showed cytoplasmic vacuoles. Some primary spermatocytes appeared with concentric lamellar formations. The immunohistochemical study showed a highly significant decrease in Bcl-2 immunoreaction. In contrast, minimal changes were observed in the recovery groups, with a nonsignificant change in the immunoreaction.
Conclusion Tramadol-induced histological and immunohistochemical effects on the seminiferous tubules of rat testis, so its abuse should be avoided except with medical prescription.

Keywords: Bcl-2, recovery, seminiferous tubules, tramadol


How to cite this article:
Abou Elnaga AA, Kassab AA, Soliman GM, El Shal AO. Histological and immunohistochemical study of the effect of tramadol on the seminiferous tubules of adult albino rat and the effect of its withdrawal. Tanta Med J 2018;46:38-53

How to cite this URL:
Abou Elnaga AA, Kassab AA, Soliman GM, El Shal AO. Histological and immunohistochemical study of the effect of tramadol on the seminiferous tubules of adult albino rat and the effect of its withdrawal. Tanta Med J [serial online] 2018 [cited 2018 Dec 16];46:38-53. Available from: http://www.tdj.eg.net/text.asp?2018/46/1/38/237628




  Introduction Top


Opioids are used as analgesics because they are effective for treatment of cancer and noncancer pain. Tramadol is one of the synthetic opioid analgesics that are centrally acting drugs, and its potency ranges between weak opioids and morphines [1],[2],[3]. It is used for treatment of various degrees and types of pain and is also considered as an antidepressant drug. Its mechanism for analgesia is not only mediated by being opioid but also through inhibition of norepinephrine and serotonin reuptake [4],[5], so its efficacy is improved by this dual action.

Being opioid, it has many adverse effects such as dizziness, headache, nausea, constipation, and central nervous system stimulation [6] but tramadol is devoid of many serious effects of traditional opioids. Recently, toxicity, abuse, dependence, and tramadol-related deaths have been increased [7],[8] as young addicts substitute tramadol for heroin.

So, many research studies were performed to study biochemical and histological changes owing to long-term use of tramadol on liver, kidney, and brain [9]. It seemed important to study its histological effect on testis. So, our research was carried out to study histological and immunohistochemical changes induced by tramadol on the testes and the effect of its withdrawal.


  Materials and methods Top


Materials

Chemicals

The drug used in this study was tramal capsules produced by Minapharm, Egypt; each capsule contains 50 mg tramadol hydrochloride. The doses of tramadol were calculated according to the formula adapted by Paget and Barnes [10]. The dose of tramadol was 40 mg/kg, which was equivalent to human effective therapeutic dose. Tramadol was dissolved in normal saline 0.9%, and the calculated doses were given orally using gastric tube [11].

Animals

This work was carried out on 52 adult male albino rats. Their weight ranged between 150 and 200 g for each animal. All animals were housed in suitable clean properly ventilated cages under similar conditions and were fed on a similar commercial laboratory diet with free access to food and water. They were acclimatized to their environment for 1 week before starting the experiment, which was approved by the local ethical committee, Faculty of Medicine, Tanta University.

Grouping of animals

The rats were divided into two main groups.

Control group (group I)

It included 24 rats that were further subdivided into two subgroups:
  1. Group IA: it included 12 rats for histological and ultrastructural study of the normal testis. They were kept without treatment for the same periods as experimental animals (three rats for each period).
  2. Group IB: it included 12 rats that were given 1 ml of normal saline 0.9% by a gastric tube daily (three rats for each period).


Experimental group (group II)

It included 28 rats that were further subdivided into four subgroups:
  1. Group IIA: it included seven rats that were orally given tramadol in a dose of 40 mg/kg body weight dissolved in saline daily for 4 successive weeks.
  2. Group IIB: it included seven rats that were orally given tramadol in a dose of 40 mg/kg body weight dissolved in saline daily for 4 successive weeks and then kept without treatment for 4 weeks.
  3. Group IIC: it included seven rats that were orally given tramadol in a dose of 40 mg/kg body weight dissolved in saline daily for 6 successive weeks.
  4. Group IID: it included seven rats that were orally given tramadol in a dose of 40 mg/kg body weight dissolved in saline daily for 6 successive weeks and then kept without treatment for another 4 weeks.


After 24 h from the last administration, the animals were anesthetized by an intraperitoneal injection of sodium thiopental (30 mg/kg) [12],[13]. The testes were dissected, and specimens from the testes were obtained and processed for histological and immunohistochemical examination.

Methods

Histological examination

The testicular specimens were immediately fixed in Bouin’s solution, dehydrated, cleared, and embedded in paraffin. Sections of 5 μm thickness were stained with hematoxylin and eosin stain [14],[15] and periodic acid Schiff (PAS) reagent [16].

Immunohistochemical examination

Sections of 5 µm thickness were dewaxed, rehydrated, and washed with PBS. The sections were then incubated overnight in a humid chamber with the primary antibody (Rabbit monoclonal Bcl-2 antibody)) Sigma Aldrich, Cairo, Egypt) diluted in PBS at 4°C. Washing in PBS buffer and co-incubation with biotinylated secondary antibody for 1 h at room temperature were carried out. Streptavidin peroxidase was then added for 10 min and rinsed again three times in PBS. Immunoreactivity was visualized using 3,3′-diaminobenzidine–hydrogen peroxide as a chromogen. Sections were counterstained with Mayer’s hematoxylin. The negative control sections were prepared by excluding the primary antibodies [17],[18].

Electron microscopic examination

The specimens were fixed by immersion in 2.5% phosphate buffered glutaraldehyde, processed, and embedded in epoxy resin by routine protocol. Semithin sections (1 µm thick) were obtained and stained with 1% toluidine blue and examined by light microscope. Ultrathin sections (80–90 nm) were then stained with uranyl acetate and lead citrate to be examined by JEOL electron microscope at 80 kV in Electron Microscopy Unit, Faculty of Medicine, Tanta University [19],[20].

Morphometric study

Image analysis system (Leica Qwin 500C Image analyzer computer system, Leica Imaging System Ltd, Cambridge, England) at Central Research Laboratory was used to measure the color intensity of PAS and Bcl-2 immunoreactivity (in 3,3′-diaminobenzidine stained sections) in all groups. A total of 10 different nonoverlapping randomly selected fields at magnification of 400 were examined for each slide for each animal of each group.

Statistical analysis

The data obtained were analyzed using SPSS software version 13 (SPSS Inc., Chicago, Illinois, USA), and then compared by one-way analysis of variance test. The results were expressed as mean±SD. The differences were considered statistically significant if probability value P value of less than 0.05 and highly significant if P value of less than 0.001.


  Results Top


Histological results

Hematoxylin and eosin-stained sections

Group I (control group)

Sections of the testes obtained from control rats were similar, showing normal histological structure of the testes. The seminiferous tubules were cut transversely and appeared rounded to oval in shape with regular basement membrane ([Figure 1]a), lined by stratified epithelium ([Figure 1]b) and ensheathed by single layer of flattened myoid cells. Leydig cells were identified by their central nuclei and acidophilic vacuolated cytoplasm ([Figure 1]c).
Figure 1 Photomicrographs of hematoxylin and eosin-stained testis section. (a) Control group showing oval seminiferous tubules (ST) lined with different spermatogenic cells. Interstitium can be seen (It) (×200). (b) Control group showing a normal seminiferous tubule (ST) and normal blood vessel (→) (×400). (c) Control group showing  Sertoli cells More Details (St), spermatogonia, (↷) primary spermatocyte (zigzag), early spermatids (Sd), Leydig cells (→), and myoid cells (►) (×1000). (d) group IIA showing wide interstitium (*) and increased spaces between spermatogenic cells (→) (×200).

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Group II (experimental group)

Group IIA animals received 40 mg/kg tramadol daily for 4 successive weeks: many changes occurred in this subgroup. Marked widening of the intercellular spaces and separation of spermatogenic cells were detected in many tubules ([Figure 1]d and [Figure 2]a); moreover, deeply stained nuclei of the basal cell layer, and diminished germ cell layers as well as atrophy of some tubules (absent spermatids and sperms) were also seen. On the contrary, the interstitial tissue between the seminiferous tubules showed markedly congested blood vessels and vacuolated homogenous acidophilic material as well as Leydig cells with dark stained nuclei were also detected ([Figure 2]b–[Figure 2]d and [Figure 3]a [Figure 2] and [Figure 3]).
Figure 2 Photomicrographs of hematoxylin and eosin-stained testis sections of group IIA. (a) Wide intercellular spaces (zigzag) and deeply stained nuclei of the basal cells (→). (b) A deeply stained nuclei of spermatogonia (zigzag) and spermatocytes (→), halo spermatids (tailed arrows), and detached cells from the basal layer (*). (c) A seminiferous tubule having one layer of spermatogonia (→). Another tubule shows vacuolated spermatogonia (►). (d) Dilated congested blood vessel (→), vacuolated acidophilic material (zigzag), vacuolated spermatogonia (►), and wide intercellular spaces (*) (×400).

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Group IIB animals received 40 mg/kg tramadol daily for 4 successive weeks and then kept without treatment for another 4 weeks: examination of the testis sections of this subgroup revealed less prominent structural changes when compared with the previous group. Most of the seminiferous tubules appeared with increased spermatogenic population and increase in the number of sperms. The space between the tubules was reduced in size, but homogenous acidophilic material was still present within the interstitium ([Figure 3]b).
Figure 3 A photomicrograph of hematoxylin and eosin-stained testis sections. (a) Group IIA showing dark nuclei of Leydig cells (►), vacuolated spermatogonia (→), and spermatocytes (zigzag) (×1000). (b) Group IIB showing seminiferous tubules with sperms (Sz) and vacuolated acidophilic material (→) (×200). (c) Group IIC showing shrunken tubule (→), others with separated basement membrane (►), congested blood vessel (۩), and wide interstitium (*) (×200). (d) Group IIC showing tubule with absent spermatids and sperms (*), another tubule shows vacuoles (→), dark nuclei of spermatogonia (►), and spermatocytes (۩) (×400).

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Group IIC animals received 40 mg/kg tramadol daily for 6 successive weeks: severe structural changes in all specimens were observed. Concerning the seminiferous tubules, focal disorganization, irregularity, increased spaces between the tubules and detached basement membrane of some tubules as well as shrunken seminiferous tubules were also detected. Marked depletion of spermatogenic population was observed in most tubules ([Figure 3]c). Many spermatogenic cells were replaced by vacuoles, whereas other seminiferous tubules lack sperms and spermatids. Darkly stained nuclei of spermatogonia and spermatocytes were also detected ([Figure 3]d). Vacuolated homogenous acidophilic material of the interstitium, Pyknotic nuclei, and binucleated cells were observed, and vacuolated spermatogonia and spermatocytes as well as deeply stained nuclei of Leydig cells were evident ([Figure 4]a–c).
Figure 4 A photomicrograph of hematoxylin and eosin-stained testis sections. (a) Group IIC showing displaced spermatocytes (→), vacuolated cells (►) with vacuolated acidophilic material (*) (×400). (b) Group IIC showing a binucleated cell (→) and large vacuole replacing the cells (×1000). (c) Group IIC showing deeply stained nuclei of Leydig cells (→), vacuolated spermatogonia (►), and spermatocytes(zigzag arrows) (×1000). (d) Group IID showing one tubule with sperms in its lumen (Sz) and the other with wide separation of its germ cells (→) (×200).

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Group IID animals received 40 mg/kg tramadol daily for 6 successive weeks and then kept without treatment for additional 4 weeks: improvement in the histological structure of some seminiferous tubules was manifested by restoration of the spermatogenic epithelium and appearance of spermatozoa in the lumen, whereas the lining epithelium of other tubules failed to regenerate, so the lumen was seen empty of sperms ([Figure 4]d).

Periodic acid Schiff’s reaction

Control groups (group I)

Sections of the testes obtained from control rats showed PAS-positive reaction in the basement membrane which appeared in close relation with the germinal epithelium ([Figure 5]a).
Figure 5 A photomicrograph of periodic acid Schiff (PAS)-stained testis sections. (a) Control group showing PAS-positive reaction of the basement membrane (→) (×200). (b) Group IIA showing separated basement membrane (→) and apparent decrease in the reaction (×200). (c) Group IIA showing vacuolated PAS-positive material in Leydig cells (→) with detachment of the basement membrane (►) (×400). (d) Group IIB showing obvious PAS-positive reaction as compared with group IIA with wrinkling of the basement membrane (→) and PAS-positive material in the interstitium (►) (×200).

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Experimental groups (group II)

Group IIA animals received 40 mg/kg tramadol daily for 4 weeks: sections of the testes obtained from this subgroup showed decrease PAS-positive reaction in the separated parts of the basement membrane surrounding the seminiferous tubules ([Figure 5]b) with presence of homogenous PAS-positive material between these tubules ([Figure 5]c).

Group IIB animals received 40 mg/kg tramadol daily for 4 weeks then kept without treatment for another 4 weeks): sections obtained from this subgroup showed marked improvement of the tubules and increase in PAS-positive reaction, but still wrinkling of the basement membrane with presence of homogenous acidophilic material from the interstitium was evident ([Figure 5]d).

Group IIC animals received 40 mg/kg tramadol daily for 6 weeks: sections of the testes obtained from this subgroup showed decrease in PAS-positive reaction as compared with the control group, with detachment of basement membrane of many seminiferous tubules ([Figure 6]a). Vacuolated PAS-positive material in-between the tubules was detected ([Figure 6]b).
Figure 6 A photomicrograph of periodic acid Schiff (PAS)-stained testis sections. (a) Group IIC showing detachment of the basement membrane of many seminiferous tubules (→) (×200). (b) Group IIC showing vacuolated PAS-positive material in the interstitium (►) (×200). (c) Group IID showing apparent increase in PAS-positive reaction of the basement membrane but PAS-positive material in the interstitium still present (→) with focal separation of the basement membrane (►) (×100).

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Group IID animals received 40 mg/kg tramadol daily for 6 weeks and then kept without treatment for additional 4 weeks): some seminiferous tubules showed improvement by increasing PAS-positive reaction. Other tubules still have separation of the basement membrane and homogenous acidophilic material in the interstitium ([Figure 6]c).

Immunohistochemical results

Bcl-2 immunostaining (antiapoptotic marker)

Control group (group I)

Examination of the testis of control rats revealed apparently strong positive immunoreactivity for Bcl-2 in the cytoplasm of the cells of the seminiferous tubules when examined in high magnification ([Figure 7]a).
Figure 7 A photomicrograph of Bcl-2-stained testis sections of different groups. The reaction was as follows: (a) Control group showing apparently strong positive cytoplasmic reaction (→). (b) Group IIA showing mild positive cytoplasmic reaction (→). (c) Group IIB showing moderate positive cytoplasmic reaction (→). (d) Group IIC showing apparently weak positive cytoplasmic reaction (→). (e) Group IID showing apparently mild positive cytoplasmic reaction (→) (Bcl-2 immunostaining, ×1000).

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Experimental groups (group II)

Group IIA: examination of this subgroup showed apparently mild positive immunostaining reaction of Bcl-2 in the cytoplasm of the cells of the seminiferous tubules ([Figure 7]b).

Group IIB: examination of this subgroup showed improvement of the immunostaining reaction of Bcl-2 in the cytoplasm of the cells of the tubules ([Figure 7]c).

Group IIC: examination of this subgroup showed apparently weak positive immunostaining reaction of cytoplasm of the cells of the seminiferous tubules for Bcl-2 ([Figure 7]d).

Group IID: examination of this subgroup showed apparent mild positive immunostaining reaction of the cytoplasm of the cells for Bcl-2 ([Figure 7]e).

Electron microscopic results

Group I (control group)

Electron microscopic examination of the ultrathin sections of the testes from control group showed the basal compartment of the seminiferous tubules formed of cells resting on the basement membrane formed of Sertoli cells and spermatogonia. Sertoli cell appeared as a large pyramidal cell having large indented nucleus with prominent nucleolus ([Figure 8]a). A spermatogonia appeared with oval nuclei containing peripheral clumps of heterochromatin ([Figure 8]b). The adluminal compartment showed primary spermatocytes, spermatids, and spermatozoa. The primary spermatocytes revealed rounded to oval nuclei ([Figure 8]c), and secondary spermatocytes were rarely seen as they have short lifespan. The early rounded spermatids were identified by their characteristic acrosomal caps ([Figure 8]d). Moreover, sections in the tail of the sperms at different levels were observed in the lumen of the seminiferous tubules ([Figure 9]a). The interstitial cells of Leydig showed rounded to oval nucleus with heterochromatin and prominent nucleolus. The cytoplasm was full of well-developed smooth endoplasmic reticulum (sER), mitochondria, and lipid droplets ([Figure 9]b).
Figure 8 An electron micrograph of a control rat testis (a) A Sertoli cell having a nucleus (N) with a prominent nucleolus (Nu), mitochondria (m), and smooth endoplasmic reticulum (S) (×2500). (b) A type A dark spermatogonium on the basal lamina with oval nucleus (N), mitochondria (m), and dense ribosomal granules (→) (×2000). (c) A primary spermatocyte with oval nucleus (N) and mitochondria (m) (×2500). (d) An early rounded spermatid has acrosomal cap (→) over the nucleus (N) with fine granular chromatin and mitochondria (m) (×2000).

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Figure 9 An electron micrograph of rat testis showing (A) control group with transverse sections of sperm tails: the middle piece (MP), the principal piece (PP), and the end piece (EP) (×5000). (b) Control Leydig cell reveals nucleus (N), prominent nucleolus (Nu), mitochondria (m), smooth endoplasmic reticulum (s), and fat droplet (L) (×3000). (c) Group IIA showing Sertoli cell with nucleus (N), cytoplasmic vacuoles (V), and destroyed mitochondria (m). Leydig cell has nucleus (N2), dilated perinuclear space (→) and cytoplasmic vacuoles (►) (×1500).

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Experimental groups (group II)

Electron microscopic examination confirmed the results that were found by light microscopic examination. The degree of cell damage was associated with increased duration of drug administration.

Group IIA animals received 40 mg/kg of tramadol daily for 4 successive weeks): ultrathin sections of this subgroup revealed damaging effects of tramadol on the cells. Sertoli cells observed resting on the basement membrane with multiple cytoplasmic vacuoles. Regarding Leydig cells, they showed dilated perinuclear space and cytoplasmic vacuoles ([Figure 9]c). Some primary spermatocytes showed nuclei with clumped chromatin and dilated perinuclear space ([Figure 10]a). Spermatogonia contained nuclei with discontinuous nuclear membrane and separated from the basal lamina ([Figure 10]b). Primary spermatocytes showed lamellar bodies and secondary lysosome; in addition, widening of the intercellular space between cells and axoneme was abnormally present between spermatocytes ([Figure 10]c). Spermatids appeared with increased intercellular spaces between them ([Figure 11]a). Regarding Leydig cells, they showed dilated perinuclear space and their cytoplasm revealed vacuolated mitochondria, dilated sER, and vacuoles ([Figure 11]b).
Figure 10 An electron micrograph of a rat testis of group IIA. (a) Sertoli cell has nucleus (N1) and cytoplasmic vacuoles (V). Primary spermatocytes have nuclei (N2 and N3), dilated perinuclear space (→), and destroyed mitochondria (m) (×2500). (b) Spermatogonium contains nucleus (N), area of separation (*), and discontinuous nuclear membrane (→) (×3000). (c) Primary spermatocytes reveal nuclei (N1 and N2), mitochondria (m), lysosome (L), and concentric lamellar formation (→). Principal piece of sperm tail abnormally present (►) and wide intercellular spaces (*) (×2000).

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Figure 11 An electron micrograph of a rat testis of group IIA showing (a) multiple rounded spermatids with nuclei (N1 and N2), but nuclei (N3, N4, and N5) are apparently smaller with increased intercellular spaces (*) (×1500). (b) A Leydig cell having rounded nucleus (N), dilated perinuclear space (→), vacuoles (►), vacuolated mitochondria (m), and dilated sER (S) (×2500).

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Group IIB animals received 40 mg/kg of tramadol for 4 successive weeks then kept without treatment for another 4 weeks): electron microscopic examination of this subgroup revealed marked improvement in the structure and arrangement of the cells of the seminiferous tubules. Sertoli cells appeared with normal indented nuclei, and the cytoplasm contained normal mitochondria and lipid droplets ([Figure 12]a). Primary spermatocytes appeared with normal euchromatic nuclei with large marginated nucleolus and increased number of peripheral mitochondria ([Figure 12]b). Leydig cells had large nuclei with peripheral clumps of heterochromatin, and their cytoplasm showed large mitochondria and sER ([Figure 12]c).
Figure 12 An electron micrograph of a rat testis of group IIB. (a) An apparently normal Sertoli cell containing nucleus (N), normal mitochondria (m), and lipid droplets (L) (×2500). (b) Primary spermatocyte with rounded nucleus (N) with large marginated nucleolus (Nu), and mitochondria (m) (×3000). (c) Group IIB showing a Leydig cell with ovoid nucleus (N) with heterochromatin arranged at the periphery. The cytoplasm contains multiple mitochondria (m) and sER (s) (×2000).

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Group IIC animals received 40 mg/kg of tramadol daily for 6 successive weeks: in this subgroup, more damaging effects had occurred to most of cells. Some seminiferous tubules showed very thick and wavy basal lamina with marked deposition of collagen fibers in it ([Figure 13]a). Most primary spermatocytes appeared with ruptured nuclear membrane and rarified cytoplasm ([Figure 13]b). The normal arrangement of cells in the tubules was lost, as some spermatids located near the basement membrane or even resting on it ([Figure 13]c). Abnormal shape of sperms was detected within the tubules or even in-between spermatogenic cells ([Figure 13]d). Dilated perinuclear space of Leydig cells was detected, as well as dilated sER and destroyed mitochondria were also observed ([Figure 14]a).
Figure 13 An electron micrograph of a rat testis of group IIC. (a Sertoli cell has cytoplasmic vacuoles (v) and resting on thick wrinkled basal lamina (BL) collagen (C) in it. (b) Primary spermatocyte has ruptured nuclear membrane (N) and rarified cytoplasm (*). (c) Spermatid with rounded nucleus (N) acrosome (►) directed to the basal lamina (BL). (d) Spermatid has nucleus (N) and lysosome (L). Abnormal shapes (→) of sperms (Sp) are present. Vacuolated mitochondria (►) and wide intercellular space (*) are present (×1500).

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Figure 14 An electron micrograph of a rat testis. (a) Group IIC showing congested blood vessel (BV) and Leydig cells with nuclei (N1, N2, and N3), dilatation of perinuclear space (►), dilated sER(S), and destroyed mitochondria (→) (×1500). (b) Group IID showing Sertoli cell with euchromatic nucleus (N), nucleolus (Nu), normal mitochondria (m), sER (s), and lipid droplets (L). Thick basal lamina (BL) and collagen (C) are present (×2500). (c) Group IID shows spermatogonia containing normal nuclei (N1 and N2), but wide intercellular space is present (*) (×2000).

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Group IID received 40 mg/kg of tramadol for 6 successive weeks and then kept for 4 weeks without any treatment: electron microscopic examination of this subgroup revealed an improvement in the structure and arrangement of the cells of the seminiferous tubules with decrease vacuolation and absence of exfoliated and apoptotic cells. Sertoli cells contained normal-shaped mitochondria, sER, and lipid droplets. The basement membrane remained thickened with persistence of collagen fibers ([Figure 14]b). Spermatogonia contain normal nuclei, but wide intercellular space is noticed between cells ([Figure 14]c). Spermatogenic cells appeared normal, and the sperms appeared within the lumen. The interstitial cells of Leydig contained normal nucleus and organelles ([Figure 15]a–d).
Figure 15 An electron micrograph of a rat testis of group IID showing (a) primary spermatocyte having nucleus (N), rarified chromatin (*), some normal mitochondria (m), and others with discontinuous membranes (→) (×2000). (b) Two spermatids have nuclei (N1 and N2), acrosomal caps (►) and mitochondria arranged at the periphery, but some mitochondria are abnormal in position (→) (×2000). (c) Transverse sections in sperms appear normal (×5000). (d) Leydig cell has nucleus (N), mitochondria (m), and sER (s) (×2000).

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Morphometric results and statistical analysis

Statistical results for the color intensity of periodic acid Schiff reaction

Group IIA and group IIC showed a statistically significant decrease in the mean color intensity as compared with the control group. Concerning group IIB and group IID, there was nonsignificant change in the mean color intensity as compared with the control group. On the contrary, group IIA and group IIC showed a statistically significant decrease in the mean color intensity as compared with groups IIB and IID. All these data were illustrated in [Table 1] and Histogram 1

.
Table 1 The mean color intensity of periodic acid Schiff reaction in different studied groups

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Statistical results for the color intensity of Bcl-2 immunoreaction

There was a statistically significant decrease in the mean color intensity of Bcl-2 immunoreaction in groups IIA and IIC as compared with the control group. Group IIB showed nonsignificant decrease of the mean color intensity as compared with the control group. Concerning group IID, there was a significant decrease in the mean color intensity as compared with the control group. Groups IIA and IIC showed a statistically significant decrease of the mean color intensity as compared with group IIB. All these data were illustrated in [Table 2] and Histogram 2

.
Table 2 The mean color intensity of Bcl-2 immunoreaction in different studied groups

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  Discussion Top


Tramadol is widely used in the treatment of pain owing to its efficacy and potency [21]. It has been suggested that tramadol intake has hazardous effects on several organs as liver and kidney [22]. So, the present work was designed to investigate the effect of tramadol on the histological structure of the seminiferous tubules of adult albino rats by using light and electron microscopes. Moreover, immunohistochemical study was carried out using Bcl-2 as an antiapoptotic marker to detect the possible involvement of apoptosis in these effects.

In the present work, light and electron microscopic examination of sections obtained from the testes of the experimental group revealed many structural changes in the seminiferous tubules as well as the interstitial tissue in-between these tubules. These structural changes were more severe and more prominent in group IIC that received tramadol for 6 weeks than in group IIA that received tramadol for 4 weeks.

El-Gaafarawi [4], who studied the biochemical alteration induced by tramadol administration, reported that tramadol induces oxidative stress in the testicular tissue. This is supported by Costa et al. [23] who studied the hematological changes in propofol anesthetized dogs with tramadol administration. Moreover, Zhang et al. [24] stated that morphine causes a significant decrease of reduced glutathione and the activities of catalase and glutathione. Other investigators reported the risk of increased lipid peroxidation owing to long-term use of opioids [9].

Popovic et al. [25] added that oxidative stress generates reactive oxygen species (ROS) that react with biomolecules such as lipids, nucleic acids, and proteins, which in turn change their structure and function causing cell damage and even death. So, most of the intertubular and intratubular changes that were reported in this study can be explained by lipid peroxidation of the cell and organelles, membranes, with consequent ROS generation. Panchenko et al. [26] found that lipid peroxidation increases in long-term heroin users.

Light microscopic examination of the seminiferous tubules and the interstitium revealed widening of the intertubular spaces. This observation was in agreement with Abdellatief et al. [27] who studied the effects of long-term tramadol administration on testicular tissues in rat, and attributed this to the deposition of homogenous acidophilic PAS-positive material, which was described by El-Sherif and El-Mehi [28] as hyaline material. Salama et al. [29], who studied the changes in testicular vascular permeability during progression of a varicocele, observed similar findings and explained this due to excess lymphatic exudate oozing from degenerated lymphatic vessels as well as increase in vascular permeability that may result from accumulation of free radicals and ROS.

Dilated congested intertubular blood vessels and increase in collagen fibers were detected in the present study. This was in accordance with Awadalla and Salah-Eldin [22] and Hafez et al. [30] who studied the hepatorenal toxicity induced by tramadol in adult male albino rats. ElKhateeb et al. [31] stated that tramadol-induced hepatorenal toxicity and oxidative stress in adult albino rats through significant increase in hepatic and renal malomdialdehyde and a highly significant decrease in glutathione peroxidase.

Wide intercellular spaces between spermatogenic cells were noticed in the present work. Similar findings were observed by Mohamed et al. [32], who studied the effect of cadmium on the testes of adult albino rats and attributed this to exposure of spermatogenic cells to ROS causing disruption of the blood–testis barrier, allowing passage of toxic agents between the cells and widening of intercellular spaces.

Cytoplasmic vacuolation of most spermatogenic cells and Sertoli cells was observed in the present study. These vacuoles were most probably derived from dilatation and vesiculation of the ER and mitochondrial swelling [32]. Much larger vacuoles are often phagocytotic vacuoles remaining after the digestion of necrotic germ cells [33]. Marked depletion of spermatogenic population and many spermatogenic cells is replaced by vacuoles. Atrophy of some tubules, focal disorganization, and shrunken seminiferous tubules were also detected. The same changes were found by Caju et al. [34] and Abdellatief et al. [27], who explained these findings by disturbance in the endocrine and paracrine functions through disordered luteinizing hormone (LH), estradiol, somatotropin, somatostatin, prolactin, and gonadotrophin releasing hormone levels acted on the hypothalamus or pituitary gland. Moreover, Ahmed and Kurkar [35] stated that tramadol causes a significant decrease in the plasma levels of LH, follicle-stimulating hormone, testosterone, and total cholesterol, whereas it causes increase in the levels of prolactin and estradiol.

Wavy, thickened, and irregular basement membrane of the seminiferous tubules and sloughed germinal epithelium were observed in the present study. Similar findings were observed by Heidari et al. [36] who studied the effect of administration of methadone and buprenorphine on rat testes. They stated that opioids inhibit release of LH by acting on hypothalamus or pituitary, which leads to reduced levels of testosterone. Collagen deposition into the basement membrane observed in this study might be owing to increase in its production by myoid cells or reduction of proteolysis rate in the extracellular matrix [37]. In addition, Sorge and Stewart [38] added that increased thickness of the basement membrane leads to impairment of the testicular metabolism causing germinal cell hypoplasia and tubular atrophy.

The nuclei of Leydig cells appeared irregular in shape. Their cytoplasm showed dilated sER and many vacuoles. Similar finding was observed by Ghoneim et al. [39]. Moreover, Ahmed and Kurkar [35] stated that tramadol causes malformed Leydig cells and decreases their numbers, and the repeated administration of tramadol to rats for several consecutive weeks produced a progressive increase in testicular levels of nitric oxide. This was in accordance with Abdel-Zaher et al. [40], who reported that treatment with tramadol for several consecutive days resulted in an increase in levels of nitric oxide production in the brain. Halawa [41] explained this is owing to oxidative stress. These findings could suggest hypofunction of Leydig cells and consequent reduction of testosterone secretion [42].

Immunohistochemically, there was strong Bcl-2 positive cytoplasmic reaction in the cells of the seminiferous tubules in the control group. Similar results were observed by Al-Azemi et al. [43] who studied the effect of cadmium on the rat testes and stated that Bcl-2 protein is an antiapoptotic marker. The present study demonstrated that tramadol-induced apoptosis in the spermatogenic cells as indicated by a significant decrease in the expression of Bcl-2 protein. Mao et al. [44] explained that prolonged morphine administration resulted in an upregulation of the proapoptotic Bax protein besides a downregulation of the antiapoptotic Bcl-2 protein. This intracellular imbalance in Bax/Bcl-2 ratio accelerated apoptosis of lymphocytes by morphine with subsequent activation of caspase-3 [45].

At the level of recovery, most of histological findings were subsided. This indicates that apoptotic activity and oxidative stress caused by tramadol administration mostly decreased. They stated that examination of rats after withdrawal recovery period showed marked reduction in cellular damage. Tramadol withdrawal has better histological picture than those under usage, in spite of that it is not returned back to normal, but it is associated with good future for tramadol users [39].


  Conclusion Top


From this study, it can be concluded that tramadol has harmful effects on the testis of adult albino rats. Tramadol abuse may provide a possible explanation for the unexplained delayed fertility associated with its abuse. Tramadol abuse should be avoided, except with medical prescription, owing to its hazardous effects.

Recommendations

Tramadol abuse should be avoided without medical prescription owing to its toxic effects.
  1. Users of tramadol under withdrawal should consult andrologist to minimize its residual undesirable effects.
  2. Further investigations are needed for more comparison and interpretation.
  3. It is recommended that patients on long-term opioid therapy should be screened routinely for symptoms of hypogonadism and laboratory abnormalities in sex hormones.
  4. Future studies on the effect of tramadol on sex hormones and changes in semen parameters are strongly recommended, especially in long-term tramadol users.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflics of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15]
 
 
    Tables

  [Table 1], [Table 2]



 

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