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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 4  |  Page : 1125-1134

The effect of tramadol on seminiferous epithelium of albino rats and the protective effect of vitamin C


Medical Histology Department, Menoufia University, Menoufia, Egypt

Date of Submission29-Dec-2016
Date of Acceptance19-Mar-2017
Date of Web Publication04-Apr-2018

Correspondence Address:
Reham A Mahmoud
Department of Medical Histology, Menoufia University, Shebin Elkom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_716_16

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  Abstract 


Objective
The aim of the study was to assess the effect of tramadol on the testes of adult male albino rats and to elucidate the possible protective effect of vitamin C.
Background
Tramadol is an opioid pain medication used to treat mild to severe pain, both acute and chronic, and premature ejaculation and as an antidepressant.
Materials and methods
Sixty adult male albino rats were used in the present study. Animals were divided into four groups: group I, the control group; group II, which received 500 mg/kg/day vitamin C orally for 4 weeks; group III, which was divided into two subgroups, subgroup IIIa and subgroup IIIb (receiving 50 mg/kg/day tramadol orally for 4 weeks) and subgroup IIIb, which was left without treatment for another 4 weeks; and group IV, which received tramadol and vitamin C in the doses same as the previous ones. At the end of the experiment, the testes were excised and specimens were processed for light, electron microscopic, morphometric studies and statistical analyses.
Results
Tramadol-treated rats showed degeneration, irregularity of seminiferous tubules with disorganization and exfoliation of spermatogenic cells in the lumen, which showed absence of sperms. Vascular dilatation and congestion was present. In addition, there was collagen fiber deposition in the interstitium and around the blood vessels with thickening of tunica albuginea. Cessation of tramadol administration showed reversibility of the previous changes but not complete recovery. However, coadministration of vitamin C with tramadol showed improvement.
Conclusion
Tramadol has harmful effect on the testes of rats. This effect is reversible when tramadol administration is stopped but complete recovery is not achieved. Vitamin C and tramadol in combination revealed improvement.

Keywords: testes, tramadol, vitamin C


How to cite this article:
Soliman ME, Atteya SE, Ghobashy HA, Noya DA, Mahmoud RA. The effect of tramadol on seminiferous epithelium of albino rats and the protective effect of vitamin C. Menoufia Med J 2017;30:1125-34

How to cite this URL:
Soliman ME, Atteya SE, Ghobashy HA, Noya DA, Mahmoud RA. The effect of tramadol on seminiferous epithelium of albino rats and the protective effect of vitamin C. Menoufia Med J [serial online] 2017 [cited 2018 Jul 21];30:1125-34. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1125/229239




  Introduction Top


Tramadol hydrochloride is a synthetic opioid analgesic that is centrally acting drug widely used in the treatment of acute and chronic pain of moderate to severe intensity associated with a variety of diseases including diabetic neuropathy, neuropathic pain, and perioperative pain[1]. It is used also for premature ejaculation[2] and as an antidepressant[3]. In addition, it is used in the treatment of diabetic neuropathy[4] and postherpatic neuralgia[5].

Long-term administration of tramadol has negative impacts on many organs like the liver, kidney, thyroid gland[6], and testis[7].

Vitamin C is a powerful reducing agent capable of rapidly scavenging a number of reactive oxygen species, for example, oxygen, nitrogen, and sulfur radicals. Vitamin C can protect against plasma lipid peroxidation and protect biomembranes against peroxidative damage. Vitamin C enhances the activity of tocopherol (vitamin E), which is the principle lipid soluble antioxidant. It reacts with the tocopheroxyl radicals that arise in cell membranes as a result of vitamin E antioxidant activity and regenerates tocopherols[8].


  Materials and Methods Top


Materials

Animals

This study was carried on 60 adult male albino rats. Strict care and hygiene were maintained to keep them in normal and healthy conditions. Food and water were given ad libitum. Animals were treated in accordance to the Animal Ethical Committee of the Faculty of Medicine, Menoufia University.

Drugs and chemicals

Tramadol: the drug was in the form of tramadol hydrochloride tablets (200 mg/tablet). It was provided by Hikma Pharmaceutical Co. (Giza, Egypt). Each tablet is suspended in 20 ml distilled water to produce the desired concentration.

Vitamin C: the drug was available as 500 mg capsules manufactured by Hikma Pharmaceutical Co. The content of each capsule is dissolved in 10 ml distilled water to produce the desired concentration.

Experimental protocol

The animals were divided into four groups as follows:

  • Group I (control group): it included 10 animals. They received 0.1 ml of physiological saline solution by oral route for 4 weeks.
  • Group II (vitamin C-treated group): it included 10 animals. They received vitamin C at a dose of 500 mg/kg body weight daily orally by intragastric gavage needle for 4 weeks.
  • Group III (tramadol-treated group): it included 20 animals subdivided into two subgroups:


    1. Subgroup IIIa: it included 15 animals. They received tramadol at a dose of 50 mg/kg body weightdaily (nearly 10 mg/rat) orally by intragastric gavage needle for 4 weeks.
    2. Subgroup IIIb: it included five animals. They received tramadol at a dose of 50 mg/kg body weightdaily orally for 4 weeks, then left without treatment for another 4 weeks.


  • Group IV (tramadol and vitamin C-treated group): it included 20 animals. They received tramadol at a dose of 50 mg/kg body weightdaily orally and vitamin C at a dose of 500 mg/kg body weightdaily orally for 4 weeks.


Methods

At the end of the determined period for each group, animals from were anesthetized using ether inhalation, then were killed. Both testes of each animal were excised.

Light microscopic study

Half of the testes were fixed in 10% formol saline for light microscopy using hematoxylin and eosin (H and E) stain for routine histological examination, Masson's trichrome stain for detection of collagen fibers[9], and Periodic Acid-Schiff (PAS) stain for detection of mucopolysaccharides[10].

Electron microscopic study

The other half of the testes were fixed in 1% glutaraldehyde in phosphate buffer for electron microscopic study[11].

Morphometric study

Using H and E-stained sections, morphometric measurements in the form of seminiferous tubular diameter and germinal epithelial height measuring were taken (under light microscopy using an objective lens of magnification 40, that is, of total magnification 400). From each rat, 10 microscopic fields were used. This was carried out using the interactive measuring menu of image analyzer (Lecia Qwin 500 image analyzer computer system, UK) (Ltd., Cambridge, England) in the faculty of medicine for girls, Al-Azhar University.

Statistical analysis

The data (seminiferous tubular diameter and germinal epithelial height) were expressed as mean ± SD. P value was used to test the significant change in each parameter in the experimental animals (treated groups in comparison with the control group). The data collected were tabulated and analyzed using the statistical package for the social science software (version 17.0 on an IBM compatible computer; SPSS Inc., Chicago, Illinois, USA). P value was set at 0.05, P values more than 0.05 was considered NS, less than 0.05 was significant, and less than 0.01 was highly significant[12].


  Results Top


Light microscopic results

Group I (control)

H and E stain of this group showed the well-known histological picture of rat testis. The testis consists of multiple rounded to oval seminiferous tubules. Each tubule is bounded by a basal lamina and lined by spermatogenic and Sertoli cells [Figure 1]a. The spermatogenic cells formed of spermatogonia, which appeared as small rounded cells resting on a basement membrane, primary spermatocytes with rounded dark nuclei, which were the largest in size, and early-rounded and late-elongated spermatids. Sertoli cells with triangle nuclei were observed in between the germ cells. The lumen of the tubules appeared filled with sperms. In between the tubules, there was a testicular interstitium containing blood vessels together with clumps of Leydig cells [Figure 1]b.
Figure 1: Photomicrographs of sections of testes of the control group showing rounded to oval seminiferous tubules (ST) bounded by a basal lamina (arrow) with normal spermatogenic cells and in between there is the interstitial tissue (tailed arrow) (a; H and E, ×100). Parts of adjacent seminiferous tubules lined by normal spermatogenic cells: spermatogonia (arrow), primary spermatocytes (tailed arrow), spermatids (curved arrow), and sperms (asterisks). Sertoli cells with their triangular nuclei (zigzag arrow) are seen between the spermatogenic cells. Notice the interstitial tissue between the tubules containing blood vessels (BV) and Leydig cells (L) (b; H and E, ×400). Intense positive PAS reaction in well circumscribed basal lamina (arrow) and a strong reaction in the interstitium (curved arrow), spermatogenic cells (tailed arrow), and sperms (asterisk) is noticed (c; PAS, ×200). Collagen fibers (blue color) in tunica albuginea (arrowhead) surrounding the testis and minimal amount of collagen fibers in the interstitium (arrow) (d; Masson's trichrome, ×200). H and E, hematoxylin and eosin.

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Histochemical results

PAS reaction was intense in the basal lamina and strong in the interstitium, spermatogenic cells and sperms [Figure 1]c.

Collagen fibers were normally distributed in the tunica albuginea surrounding the testis while minimal amount of collagen fibers was present in the interstitium and around blood vessels by Masson's trichrome stain [Figure 1]d.

Group II (vitamin C-treated group)

Sections of the testes of this group revealed the same normal histological and histochemical architecture as the control group [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d.
Figure 2: Photomicrographs of sections of testis of the vitamin C-treated group showing the normal architecture of testis formed of normal seminiferous tubules (ST) surrounded by basal lamina (arrow), lined by normal spermatogenic cells (tailed arrow), and separated by interstitium (I) (a; H and E, ×100). Notice parts of adjacent seminiferous tubules lined with spermatogenic cells (arrow) and Sertoli cells with triangular nucleus (zigzag arrow). Notice also that the lumen is full of sperms (sper). In between the tubules there is interstitial tissue containing Leydig cells (asterisk) and blood vessels (BV) (b; H and E, ×400). Notice the intense positive PAS reaction in the basement membrane (tailed arrow), strong positive reaction in spermatogenic cells (sp), sperms (sper), and interstitium (arrow) (c; PAS, ×200). Collagen fibers (blue color) in tunica albuginea (arrowhead). Notice the small amount of collagen fibers in the form of tinge of blue color around the BV in the interstitium (arrow) (d; Masson's trichrome, ×200). H and E, hematoxylin and eosin.

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Group III (tramadol-treated group)

Subgroup IIIa: H and E stain of this group revealed irregularity of the seminiferous tubules with disorganization and degeneration of spermatogenic cells. The damaged spermatogenic cells were exfoliated in the lumens, which showed absence of sperms [Figure 3]a. Vacuoles appeared between the degenerated spermatogenic cells, replacing them. Multinucleated giant cells appeared in the lumen of some tubules [Figure 3]b. Some seminiferous tubules were ruptured [Figure 3]c. Widening of the interstitium with appearance of vacuoles and congestion of blood vessels were also noticed [Figure 3]d.
Figure 3: Photomicrographs of sections of testis of the tramadol-treated group showing irregular degenerated seminiferous tubules (arrow) with no sperms in the lumen (asterisk). There are exfoliative cells in the lumen (arrowhead) (a; H and E, ×100). Degenerated spermatogenic cells with pyknotic nuclei (arrow) with multinucleated giant cells (curved arrow) are also seen in the lumen. Vacuoles are seen in between the spermatogenic cells (V) (b; H and E, ×400). Rupture of a seminiferous tubule (arrow) with large vacuoles between the spermatogenic cells and in the interstitium (V) can be noticed (c; H and E, ×400). Dilated congested blood vessel (BV) and wide interstitium (arrowhead) with vacuoles (v) are noticed. There is also disorganization of spermatogenic cells (arrow) with no sperms in the lumen (asterisk) (d; H and E, ×400). Mild PAS reaction in degenerated basement membrane (arrows) and interstitium (asterisk) and weak reaction in degenerated spermatogenic cells (arrowhead) is observed (e; PAS, ×200). Abundant collagen fibers around a congested BV (arrowhead) are noticed with extensive thickening of tunica albuginea (arrow) (f; Masson's trichrome, ×200). H and E, hematoxylin and eosin.

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Histochemical results

PAS reaction was mild in degenerated basement membrane and interstitium. Weak reaction was observed in degenerated spermatogenic cells [Figure 3]e.

There was increase in connective tissue fibers in the form of extensive thickening of tunica albuginea, fibrosis of interstitium, and fibrosis around the blood vessels, as noticed by Masson's trichrome stain [Figure 3]f.

Subgroup IIIb (recovery subgroup)

H and E stain of this group revealed signs of improvement after cessation of tramadol administration for 4 weeks. The signs of improvement were in the form of increased spermatogenic cell layer with increased sperm concentration in the lumen of the seminiferous tubules. About 85% of seminiferous tubules were nearly normal but nearly 15% of seminiferous tubules were still affected after tramadol withdrawal. The affected tubules showed absence of sperms from the lumen with presence of some vacuoles between the spermatogenic cells [Figure 4]a and [Figure 4]b.
Figure 4: Photomicrographs of sections of testis of the recovery group showing signs of improvement in the form of increased spermatogenic cell layer (arrow) with increased sperm bundles in the lumen (asterisk). Some tubules are still affected (arrowhead) (a; H and E, ×100). Adjacent seminiferous tubules with signs of regeneration in the form of increased spermatogenic cell layers (arrow) but vacuoles are still present between the spermatogenic cell (V) with absence of sperms in the lumen (asterisk) (b; H and E, ×400). Moderate positive PAS reaction in the basement membrane (arrow) and in the interstitium and mild reaction in spermatogenic cells (arrowhead) (c; PAS, ×200). Large amount of collagen fibers in the interstitium (arrow) with mild thickening of tunica albuginea (arrowhead) (d; Masson's trichrome, ×200). H and E, hematoxylin and eosin.

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Histochemical results

PAS reaction was moderate in the basement membrane and the interstitium. Mild reaction was observed in spermatogenic cells [Figure 4]c.

There was moderate amount of collagen fibers in the interstitium with mild thickening of tunica albuginea, as noticed by Masson's trichrome stain [Figure 4]d.

Group IV (tramadol and vitamin C-treated group)

The histological study of the H and E stain of the testis of tramadol and vitamin C-treated group showed about 92% of seminiferous tubules appearing normal with normal spermatogenic cells and a lumen full of sperms. Only 8% of seminiferous tubules were affected with no sperms in the lumen [Figure 5]a and [Figure 5]b.
Figure 5: Photomicrographs of sections of testis of the tramadol and vitamin C-treated group showing that most of seminiferous tubules appear normal (arrow) while few tubules are affected (arrowhead) (a; H and E, ×100). More or less normal seminiferous tubules with normal spermatogenic cells (arrow) and a lumen full of sperms (asterisk) (b; H and E, ×400). Mild to moderate positive PAS reaction in the basement membrane (arrow), spermatogenic cells (sp), sperms (sper), and interstitium (tailed arrow) is noticed (c; PAS, ×200). Some collagen fibers (blue color) around the blood vessel (arrow) and mild thickening of tunica albuginea (arrowhead) can be noticed (d; Masson's trichrome, ×200). H and E, hematoxylin and eosin.

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Histochemical results

Mild to moderate positive PAS reaction in the basement membrane of seminiferous tubules, spermatogenic cells, sperms, and interstitium was observed [Figure 5]c. Some increases in collagen fibers in tunica albuginea, in the interstitium, and around the blood vessels were present, as noticed by Masson's trichrome stain [Figure 5]d.

Electron microscopic results

Group I (control)

Electron microscopic study for this group showed the seminiferous tubule surrounded by basal lamina, sertoli cell with its characteristic intended nucleus resting on the basal lamina, and the cytoplasm containing mitochondria and lipid droplets. Spermatogonial cell appeared near the basal lamina with pale-stained nucleus, which showed peripheral chromatin clumps and the cytoplasm that contained few mitochondria [Figure 6]a. The primary spermatocyte had a rounded euchromatic nucleus with prominent nucleolus and peripherally arranged mitochondria [Figure 6]b. The spermatids had a rounded euchromatic nucleus covered on the proximal part of its circumference with acrosomal vesicle and acrosomal granule. The cytoplasm showed peripherally arranged mitochondria. Notice the presence of the head of mature sperm and transverse sections in the middle piece of the sperm. Notice that a stage of spermiogenesis is present (tailed arrow) [Figure 6]c.
Figure 6: Electron micrographs of ultrathin section of testis of the control group showing the basal lamina of a seminiferous tubule (BL), Sertoli cell with its intended nucleus (S), and the cytoplasm contain mitochondria (M) and lipid droplets (L). Notice the spermatogonial cells with their rounded vesicular pale stained nuclei (N) with peripheral chromatin clumps and cytoplasm containing mitochondria (m) (a; TEM, ×5000). A primary spermatocyte with a rounded euchromatic nucleus (N) and prominent nucleolus. Notice the peripherally arranged mitochondria (m) in the cytoplasm (b; TEM, ×8000). A spermatid (SD) with rounded euchromatic nucleus (N) covered on the proximal part of its circumference with acrosomal vesicle (arrow) and acrosomal granule (Ag). The cytoplasm shows peripherally arranged mitochondria (arrowheads). Notice the presence of the head of mature sperm (S) and transverse sections in the middle piece of the sperm. Notice that a stage of spermiogenesis is present (tailed arrow) (c; TEM, ×8000).

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Group II (vitamin C-treated group)

Electron micrographs of ultrathin section of this group showed the normal histological architecture as the control group [Figure 7]a and [Figure 7]b.
Figure 7: Electron micrographs of ultrathin section of testis of the vitamin C-treated group showing a normal primary spermatocyte with its euchromatic rounded nucleus (N) and a cytoplasm full of mitochondria (m) (a; TEM, ×8000). A normal spermatid with a rounded euchromatic nucleus (N) covered with acrosomal vesicle (arrow) and acrosomal granule (Ag). The cytoplasm contains peripherally arranged mitochondria (arrowhead). Notice the transverse sections in the middle piece (curved arrow) (b; TEM, ×6000).

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Group III (tramadol-treated group)

Subgroup IIIa: electron micrographs for this group showed degeneration of spermatogonia. Their cytoplasm contained vacuoles and they appeared with irregular cell membranes [Figure 8]a. The primary spermatocytes showed shrunken hyperchromatic nuclei with irregular heterochromatin clumps. The cell outlines appeared irregular. The cytoplasm appeared vacuolated, containing few degenerated mitochondria [Figure 8]c. Some of them showed ill-defined nuclear membranes with loss of intercellular bridges [Figure 8]a. Sertoli cells showed accumulation of electron-dense bodies in their cytoplasm, which contained electron-dense mitochondria and lipid droplets. Intracytoplasmic vacuoles and dilated smooth endoplasmic reticulum were observed. Tight junctions between adjacent Sertoli cells were lost [Figure 8]b. Ultrastructural adverse effects of tramadol on the spermatids were in the form of irregular outlines and vacuolated cytoplasm with few damaged mitochondria. Intracellular vacuolations were observed between the degenerated spermatogenic cells [Figure 8]d.
Figure 8: Electron micrographs of ultrathin section of testis of the tramadol treated group showing vacoules (V) in the cytoplasm of spermatogonia (Sg). The primary spermatocytes (Ps) have ill-defined nuclear envelopes (arrowheads) and disturbed intercellular bridges (arrow). Notice the presence of damaged mitochondria (m) (a; TEM, ×6000). Two affected Sertoli cells with their characteristic nuclei (N1, N2) having a transverse fold. Their nuclei show clumps of heterochromatin (H). Their cytoplasm show vacuoles (V) and many electron-dense bodies (L). Notice the dilated smooth endoplasmic reticulum (S) in one of the two cells. Notice also the loss of the intercellular bridges (arrow) between the two adjacent cells (b; TEM, ×8000). Notice a primary spermatocyte with irregular outlines (arrowhead) and intracytoplasmic vacuoles (V). The nucleus (N) is heterochromatic and the cytoplasm contains degenerated mitochondria (m) (c; TEM, ×8000). A spermatid with acrosomal cap covering its nucleus (arrowhead). The cytoplasm appear vacuolated (V) with few peripheral damaged mitochondria (m). Notice that the outlines of the cell are irregular (arrow) (d; TEM, ×6000).

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Subgroup IIIb (recovery subgroup): sections of this group showed a picture similar to the control group regardless some primary spermatocytes appearing with shrunken nuclei and vacuolated cytoplasm [Figure 9]a and [Figure 9]b.
Figure 9: Electron micrographs of ultrathin section of testis of the recovery group showing a normal primary spermatocyte with rounded euchromatic nucleus (N1) and another degenerated primary spermatocyte with shrunken nucleus (N2) and cytoplasm containing vacuoles (V) and disrupted mitochondria (m) (a; TEM, ×6000). A normal spermatid with normal nucleus (N1) covered by acrosomal cap (arrowhead) and another spermatogenic with normal nucleus (N2). Notice the more or less normal head (S) and cross-section of the middle piece of mature sperm (arrow) (b; TEM, ×6000).

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Group IV (tramadol and vitamin C-treated group): sections of this group showed more or less normal picture regardless some primary spermatocytes appearing with shrunken nuclei [Figure 10] and [Figure 10]b.
Figure 10: Electron micrographs of ultrathin section of testis of the tramadol and vitamin C-treated group showing a primary spermatocyte with signs of degeneration in the form of shrunken nucleus (N1) and few degenerated mitochondria (m). Notice the presence of another normal spermatocyte with its rounded euchromatic nucleus (N2) and peripherally arranged mitochondria (arrow) (a; TEM, ×8000). Parts of normal spermatogenic cells with normal nuclei (N1, N2, N3). Notice presence of mature sperms (arrow) with more or less normal head (S) and middle piece (curved arrow) (b; TEM, ×6000).

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Morphometric results

Seminiferous tubule diameter

Regarding seminiferous tubule diameter, there was a statistically nonsignificant difference between the control group and vitamin C-treated group (P > 0.05), but a highly statistically significant decrease in seminiferous tubule diameter between the treated group and the control group (P< 0.001). Furthermore, a statistically significant decrease was found between the recovery group compared with the control group (P< 0.05). On the other hand, a statistically nonsignificant difference was found between the tramadol and vitamin C-treated group and the control group (P > 0.05) [Table 1] and [Table 2]. There was a statistically significant decrease (P< 0.05) in the seminiferous tubule diameter in the recovery group when compared with the tramadol and vitamin C-treated group [Table 3].
Table 1: Descriptive statistics for mean (X) and SD of the seminiferous tubular diameter of different studied groups

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Table 2: Comparison between different studied groups as regards seminiferous tubular diameter

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Table 3: Comparison between recovery group and tramadol and vitamin C treated group as regards seminiferous tubular diameter

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Germinal epithelial height

Regarding germinal epithelial height, there was a statistically nonsignificant difference between the control group and the vitamin C-treated group (P > 0.05), but a highly significant decrease in germinal epithelial height was found in the treated group compared with the control group (P< 0.001). Moreover, a statistically significant decrease was found in the recovery group compared with the control group (P< 0.05). On the other hand, a statistically nonsignificant difference was found between the tramadol and vitamin C-treated group compared with the control group (P > 0.05) [Table 4] and [Table 5]. There was a statistically significant decrease (P< 0.05) in the germinal epithelial height in the recovery group when compared with the tramadol and vitamin C-treated group [Table 6].
Table 4: Descriptive statistics for mean (X) and SD of the germinal epithelial height of different studied groups

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Table 5: Comparison between different studied groups as regards germinal epithelial height

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Table 6: Comparison between recovery group and tramadol and vitamin C treated group as regards germinal epithelial height

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  Discussion Top


Tramadol hydrochloride is a synthetic opioid analgesic that is centrally acting drug widely used in the treatment of acute and chronic pain of moderate to severe intensity associated with a variety of diseases including diabetic neuropathy, neuropathic pain, and perioperative pain[1].

The wide abuse of tramadol among young people, especially male population, has reinforced the need to investigate the histopathological effects of chronic use of this drug on testicular tissue in adult male albino rats[13].

Vitamin C is a natural powerful water soluble antioxidant. It acts by scavenging reactive oxygen and nitrogen species[14]. It has a protective effect against lipid peroxidation and can regenerate other small antioxidant molecules such as tocopherol and glutathione[1].

In this study the testes of the rats treated with tramadol showed irregularity of the seminiferous tubules with disorganization and degeneration of spermatogenic cells. The damaged spermatogenic cells were exfoliated in the lumen, which showed absence of sperms. Vacuoles appeared between the degenerated spermatogenic cells, replacing them. There was also irregular degenerated basement membrane of seminiferous tubules. Some seminiferous tubules were ruptured. These findings are in agreement with Abdellatief et al.[15] and Ghoneim et al.[16].

Youssef and Zidan[13] attributed these findings to the oxidative damage and free radical formation by tramadol, especially on testis because testicular tissue and sperms contain abundant amounts of polyunsaturated fatty acids in their plasma membranes.

Ahmed and Kurkar[17] postulated that tramadol causes lipid peroxidation, decreases the antioxidant enzyme activities, and increases the level of the testicular levels of nitric oxide, which facilitate damage of testicular tissue.

Another explanation of the effects of tramadol is reported by Abou El Fatoh et al.[18]. They demonstrated that tramadol decreases the level of sex hormones in male rats compared with controls. There were reductions in plasma levels of luteinizing hormone, follicle stimulating hormone, and testosterone, which may be responsible for bad effects of tramadol on testis.

Abdellatief et al.[15] explained germ cell sloughing on the basis that affected Sertoli cells induced decrease in seminiferous tubule fluid secretion, which further resulted in apical sloughing or shedding and germ cell death.

The tramadol-treated group also showed multinucleated giant cells in the lumen of seminiferous tubules. This was in agreement with the results of the studies conducted by Ghoneim et al.[16] and Abou El Fatoh et al.[18].

Abou El Fatoh et al.[18] reported that the diffuse testicular degeneration caused by tramadol was accompanied by attempts of regeneration and the regenerating spermatocytes and spermatids fuse together to from such giant cells.

The increase in connective tissue fibers noticed by Masson's trichrome stain in our study in the tramadol-treated group in the form of thickening of tunica albuginea, fibrosis around the blood vessels, and fibrosis of interstitium was in agreement with Rabei[19] who detected that tramadol caused thickening of splenic trabeculae and fibrosis around the blood vessels of the spleen. Moreover, Awadalla et al.[20] reported that tramadol caused increase in collagen fibers in the hepatic tissue and around the blood vessels.

These findings can be explained by the results of the study conducted by Elkhateeb et al.[21] who said that increased collagen fibers in liver and kidney tissues treated with tramadol occurred due to decreased collagen metabolism, which may be caused by the oxidative stress caused by tramadol.

In this study, widening of the interstitium was noticed in the tramadol-treated group. This was in agreement with the results of the studies conducted by Abdellatief et al.[15] and El-Ghawet[22].

Rashed et al.[23] attributed the widening of the peritubular tissue to withdrawal of gonadotrophic stimulation, as occurring after hypophysectomy.

In this study, there was congestion and dilatation of testicular blood vessels caused by tramadol, which was in agreement with the results reported by Abou El Fatoh et al.[18].

The tramadol-treated group showed mild PAS reaction in the degenerated basement membrane and interstitium and weak reaction in degenerated spermatogenic cells.

Awadalla and Salah-Eldin[20] reported that there is a considerable glycogen depletion in hepatic and renal tissues after treatment with tramadol and explained these findings by being secondary event to tramadol-induced lipid peroxidation of hepatic and renal tissues. They added that lipid peroxidation of cell membranes leads to loss of membrane fluidity, changes in membrane potential, and an increase in membrane permeability, which may lead to alteration of the chemical composition of the cells.

Ultrastructurally, testes of tramadol-treated rats showed degeneration of spermatogonia. Their cytoplasm contained vacuoles and few disrupted mitochondria. They appeared with hyperchromatic nuclei and irregular cell membrane. The primary spermatocytes showed shrunken hyperchromatic nuclei with irregular heterochromatin clumps. Some of them showed ill-defined nuclear membranes. The cell outlines appeared irregular with disturbed intercellular bridges. The cytoplasm appeared vacuolated, containing few degenerated mitochondria. Sertoli cells showed accumulation of electron-dense bodies in their cytoplasm, which contained electron dense mitochondria and lipid droplets. Intracytoplasmic vacuoles and dilated smooth endoplasmic reticulum were observed. Tight junctions between adjacent Sertoli cells were lost. Ultrastructural adverse effects of tramadol on the spermatids were in the form of irregular outlines and vacuolated cytoplasm with few damaged mitochondria. Intracellular vacuolations were observed between the degenerated spermatogenic cells. In addition, there was thin degenerated basement membrane. These findings were in agreement with Elsawy and Abdel Malak[7] and Ghoneim et al.[16].

These toxic effects of tramadol at the cellular level could be explained by increasing lipid peroxidation and accumulation of reactive oxygen species, leading to cell damage[24].

Abdellatief et al.[15] explained Sertoli cell vacuolation being secondary to metabolic disturbance. They postulated that Sertoli cells are largely influenced by testosterone, producing androgen-binding protein and other products under the control of follicle stimulating hormone. Tramadol administration disturbed this link and induced morphological alteration due to testosterone deprivation.

In our study, the recovery group showed signs of improvement after cessation of tramadol administration for 4 weeks. The signs of improvement were in the form of increased spermatogenic cell layer with increased sperm concentration in the lumen of the seminiferous tubules. Most of seminiferous tubules appeared nearly normal but few tubules were still affected after tramadol withdrawal. The affected tubules showed absence of sperms from the lumen with presence of some vacuoles between the spermatogenic cells. Mitotic activities were seen indicating regeneration. These findings were confirmed by a previous study done by Ahmed and Kurkar[17] who postulated that testicular and brain tissues showed signs of recovery with marked reduction in cellular damage when compared with tramadol-treated groups after tramadol withdrawal for 4 weeks but not return to completely normal.

Another study, conducted by Azari et al.[1], reported that negative effects of tramadol on sperm quality and testicular tissue were reversible and these toxic effects did not persist after drug cessation for 6 weeks.

In the present study, the histological study of the testis of tramadol and vitamin C-treated group showed that most of seminiferous tubules appeared normal with normal spermatogenic cells and a lumen full of sperms. Only few tubules were affected with no sperms in the lumen. Some congestion of blood vessels and some increase in collagen fibers in tunica albuginea, in the interstitium, and around the blood vessels were present.

The improvement of histopathological findings in this group compared with tramadol-treated group may be due to the antioxidant effect of vitamin C, which ameliorates the oxidative stress caused by tramadol.

Angulo et al.[25] reported that exogenous supplementations of rats with ascorbic acid significantly decreased the level of lipid peroxidation in the testicular tissue and significantly increased epididymis sperm concentration and plasma testosterone level.

The protective effect of vitamin C noticed in our study was in agreement with the results of the study conducted by Muthu and Krishnamoorthy[26], who postulated that vitamin C had a potent protective effect against mercuric chloride-induced testicular damage and oxidative stress in rats and this protective effect is due to the antioxidant properties of vitamin C. In addition,[27] postulated that vitamin C had a protective effect against sulfasalazine-induced oxidative stress in rat testicular tissue.


  Conclusion Top


On the basis of the findings of the present study, we concluded that chronic administration of tramadol has negative effects on the testicular tissue of adult male albino rats and these effects are reversible. Rats supplemented with vitamin C during tramadol administration showed remarkable improvement of the testicular tissue.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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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]
 
 
    Tables

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



 

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