الفهرس 
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Abstract
Tramadol, a broadly used opioid in recent years, is an effective analgesic agent for the treatment of moderately severe acute or chronic pain. The liver and kidneys are responsible for the metabolism and excretion of opioids, which may cause hepatotoxicity and nephrotoxicity. The present study was designed to evaluate the toxic effects of tramadol on the liver and kidney of experimental rats. Tramadol is becoming abused among teens; especially between males. The study aimed to investigate the histopathological and biochemical profiles of acute and chronic toxic effects of tramadol on testicular functions. Tramadol, a potent immunosuppressive agent, has several adverse effects on diverse organs including the spleen.So the present study aimed to investigate the adverse effects of tramadol on the spleen of rats. Tramadol administration to rats caused some histological deproduced changes in lymphocytes, plasma cells and neutrophils as well as it caused a reduction in CD3+ T cell expression in the spleen and in IL-2, IL6-, IFN-c, IgM and IgG levels in the plasma.
The potential for respiratory depression is commonly associated with opioid analgesics and occurs due to a reduction in the sensitivity of the respiratory centre to carbon dioxide. This results in a reductiond tidal volume and respiratory rate. Tramadol reduce the sensitivity of the respiratory centre to carbon dioxide. This may result in reductiond tidal volume and reductiond respiratory rate. Because of the μ-opioid agonist activity of O-desmethyltramadol, tramadol may lower the respiratory rate and potentially lead to severe respiratory depression. This has incidentally been observed in overdose cases of tramadol. This study aims to investigate pulmonary toxic effect of tramadol in rats by assessment of pulmonary histopathological changes using light and electron microscope examination. A total of 160 male adult rats (Rattus norvegicus) weighing approximately 180-200g were used in the present study.This research divided into two experimental studies acute and chronic.
*Acute mode of administration:
Included oral administration of tramadol tabs suspended in saline by using gavage daily.It also included injected rat intra-peritoneal daily for a month, rats sacrificed after 10,20,thirty days.
*Chronic mode of administration:
Included oral administration of tramadol tabs suspended in saline by using gavage twice weeks for three months. It also included injected rat intra-peritoneal twice weeks for three months, rat sacrificed after three months.
First acute experimental study:
Animal were divided randomly into seven group’s fifteen rats in each groups and mediated as follows:
group 1: Comprised thirty rat, served as control groups. It is divided into 6 groups .Three groups each one consists of 5 rats administered oral dose of saline solution ten, twenty, and thirty days by using gavage. Others 15 rats, also divided into three groups and injected intra-peritoneal with saline for ten, twenty, and thirty days .Rats sacrificed after ten, twenty, ,and thirty days.
group 2: Comprised 15 rats, five rats was daily administered oral dose of tramadol Hcl suspended in saline solution equal to 20mg/kilogramb.wt./day for ten days, other five rats was daily administered oral dose 40mg/kilogramb.wt./day for ten days, other five rats was daily administered oral dose 80mg/kilogramb.wt./day for ten days . This group sacrificed after ten days.
group 3: Comprised 15 rats, five rats was daily administered oral dose of tramadol Hcl suspended in saline solution equal to 20mg/kilogramb.wt./day for twenty days, other five rats was daily administered oral dose 40mg/kilogramb.wt./day for twenty days, other five rats was daily administered oral dose 80mg/kilogramb.wt./day for twenty days. This group sacrificed after twenty days.
group 4: comprised 15 rats, five rat was daily administered oral dose of tramadol Hcl suspended in saline solution equal to 20mg/kilogramb.wt.day for thirty days, other five rats was daily administered oral dose 40mg/kilogramb.wt./day for thirty days, other five rats was daily administered oral dose 80mg/kilogramb.wt./day for thirty days. This group sacrificed after thirty days.
group 5: comprised 15 rats, five rats was daily injected intra peritoneal with tramadol Hcl ampoule equal to 20mg/kilogramb.wt./day for ten days , other five rats was daily injected dose 40mg/kilogramb.wt./day for ten days,other five rats was daily injected dose 80mg/kilogramb.wt./day for ten days. This group sacrificed after ten days.
group 6: comprised 15 rats, each 5 rats was daily injected intra peritoneal with tramadol Hcl ampoule equal to 20mg/kilogramb.wt./day for twenty days, other five rats was daily injected dose 40mg/kilogramb.wt./day for twenty days,other five rats was daily injected dose 80mg/kilogramb.wt./day for twenty days. This group sacrificed after twenty days.
group 7: comprised 15 rats, each rat was daily injected intra peritoneal with tramadol Hcl ampoule equal to 20mg/kilogramb.wt./day for thirty days, other five rats was daily injected dose 40mg/kilogramb.wt./day for thirty days ,other five rats was daily injected dose 80mg/kilogramb.wt./day for thirty days. This group sacrificed after thirty days.
Second chronic experimental study:
group 1: Comprised 5 rats, served as control group and administered oral dose of saline solution twice week by using gavage for three months. Others 5 rats, injected intra-peritoneal with saline twice a week for three months .Rats sacrificed after three months.
group 2: comprised 15 rats, five rat was administered oral dose of tramadol Hcl suspended in saline solution equal to 20mg/kilogramb.wt.day for twice a week, other five rats was administered oral dose 40mg/kilogramb.wt./day twice a week, other five rats was administered oral dose 80mg/kilogramb.wt./day twice a week. This group sacrificed after three months.
group 3: comprised 15 rats, each 5 rats was injected intra peritoneal with tramadol Hcl ampoule equal to 20mg/kilogramb.wt./day twice week, other five rats injected with dose 40mg/kilogramb.wt./day twice week, other five rats injected with dose 80mg/kilogramb.wt./day twice week. This group sacrificed after three months.
Collection of samples
Following decapitation, the rat liver, kidney, lungs, testis, and spleen of each rat were freed from the surrounding connective tissue and organs, then excised. They were immediately immersed in saline solution (.9%Nacl) for blood removal. days for recovery. Control group injected with distilled water. Formalin-fixed liver, kidney,spleen,testies and lungs were processed by the standard paraffin wax technique,segemented in (4–5μm) thicknesses, stained with Harris’s alum heamatoxylin and eosin for histopathological investigation. For the histochemical study,segements were stained with periodic acid-Schiff’s method to demonstrate total carbohydrates and with mercury bromophenol blue method to demonstrate total proteins. Olso Mallory triable stain used for illustrated changes in collagenous fibrous.
For transmission electron microscopy, small pieces of liver, kidney, spleen, lungs, and testis were fixed in 2.5% glutaraldehyde buffered with .1M cacodylate (pH7.2) at room temperature for 2 hours.
For Immunohistochemical Studies ,Segements were taken on positive slides and immunostained using avidin-biotin technique.
Biochemical indices:
Blood samples were collected in dry centrifuge tubes for serum preparation, sera were separated and preserved at -20°C till used for biochemical analysis to detect liver, kidney sexual hormonal levels urea and create.
Results:
Several alterations were observed. The changes in the liver histological structure include congestion in the central vein, diffuse of Kupffer cells, karyolysis and complete pyknosis of many cells were noticed after treatment with tramadol for 10, 20 and thirty days and congestion in sinusoids after treatment for 10 days in both oral and injected groups. Incrementd of inflammatory cells infiltration, piecemeal necrosis and fibroblastic cells proliferation was observed in groups mediated for thirty day. Bile duct hyperplasia appeared in acute and chronic highly doses groups. Vesicular fatty change is often associated with metabolic disturbances and is generally readily reversible, whereas micro vesicular fatty change is more likely a reflection of toxicity, possibly involving mitochondrial disturbances also appeared in acute and chronic studied with highly doses.
On the other hand, vascular degeneration in the epithelial cells lining the renal tubules at the cortical zone with pyknotic and karyolysed nuclei were noticed in kidney of rats mediated with tramadol for 10, 20 and thirty days. In addition, swelling in the lining epithelium of the renal tubules and the presence of inflammatory cellular infiltration, expansion of glomerulus, glomerular tuft atrophy, focal tubular necrosis and mild mononuclear cell infiltration were noticed. More excessive necrosis of tubular epithelial cells, dilated tubules and expansion of glomerulus were observed after 30and 3 monthes of treatment. Glomerulosclerosis include segmented of glomerular tuft, degeneration of glomerular capsule , intestinal haemorrhage inside glomerular and proliferation of renal tubules appeared in rat kidney segement group IIIa, rats were mediated with 20mg/kilogram. of tramadol through mouth for thirty days. Renal interstitial fibrosis appeared in chronic groups with highly doses.
Results of biochemical studies, Aspartate amino-transferase (AST), alanine aminotransferase (ALT),creatinin, blood urea nitro-gen (BUN) and testosterone levels were measured in the serum. Serum ALT, AST and BUN levels were significantly higher in acute and chronic mediated group compared to the control group. Serum BUN, creatinin and testosterone levels were none significantly in-creased in the both mediated group compared to the control group.
In spleen mediated groups congestion of red pulp sinuses, incrementd of macrophages, atrophy of red pulp appeared in acute and chronic groups. Fibrosis and edema appeared in highly doses. Atrophy of white pulp with loss of lymphocytes in the T-cell areas appeared in chronic groups. Plasma cell hyperplasia consists of plasma cells and plasma blasts that migrate to the red pulp from the white pulp appeared in highly dose chronic groups.
In testis hyperplasia of interstitial cells, germ cells degeneration and/or apoptosis, testicular amyloid appeared in acute and chronic groups. Dilated blood vessels filled with blood also appeared in both groups.
Incrementd disorganization and degeneration of spermatogenic cells incrementd with highly doses of tramadol administration and injected.
Chronic testicular inflammation is characterized by infiltration of inflammatory cells in response to tissue damage. Fibrosis is usually associated with replacement of testicular parenchyma by fibroblasts and collagen following necrosis and inflammation.
On the other hand, Lungs alveoli infiltration with lymphocytes, emphysema founded between alveoli cells, goblet cells appeared hyperplasia and dilated blood vessels filled with blood appeared in acute and chronic groups. Fibrosis pressed on alviolia cells and caused thickened septa filled with congested capillaries and lymphocytes. Keratinizing cysts found in acute case. Lung - Atypia, cellular should be diagnosed and assigned a severity grade in high doses in both groups.
The liver of rats mediated with tramadol for 10 days preserved the normal contents of glycogen similar to that of control animals, while Specimens mediated for 20 days showed mild glycogen depletion involving many hepatic cells. Rats mediated for thirty days showed marked glycogen depletion in comparison to the rats hepatocytes of the control.
Examination of kidney segements of the rats mediated with tramadol for 10 days revealed a normal PAS response , while kidneys of rats mediated for 20 and thirty days of tramadol showed marked diminution in PAS positive material in the renal corpuscles and tubules.
In chronic groups,marked glycogen deplation incrementd with highly doses in liver and kidney.
The animals received tramadol for 10 days did not manifest obvious changes in the protein contents of their hepatocytes. Hepatocytes of rats mediated with tramadol for 20 and thirty days demonstrated a severe reduction of protein contents in comparison to thehepatocytes of the control rats. Examination of kidney segements of rats injected with tramadol for 20 and30 days manifested obvious changes in the protein contents of their kidney cells. The glomeruli and renal tubules have lost the most protein contents and became slightly less stainable than the control animal.
In chronic groups,marked protein deplation incrementd with highly doses in liver and kidney.
In addiation, glycogen deplation incrementd in spleen, lungs and testies with incrementd trramadol doses.Olso protein deplation incrementd with doses and times in lungs,spleen and testies.
On the other hand, collagenous fibrous incrementd with time and doses in all organs and groups.
Olso the immunoHistochemistry findings ,apoptotic cells incrementd with time and doses in all organs and groups.
Conclusion:
Tramadol can causes histological and histochemical changes in kidney and liver, especially when high or repeated low dose of it are used.