Carisoprodol-Induced Histological and Immunohistochemical Changes in Spleen of Pregnant Rats

Background: Carisoprodol (Somadril®) is a centrally acting skeletal muscle relaxant that is commonly used to treat serious musculoskeletal disorders. Carisoprodol's mechanism of action is unknown and it is often attributed to its active metabolite, meprobamate. Objective: This study aimed to detect histopathological and immunohistochemical changes in the tissues of the splenic pregnant rats treated orally with Carisoprodol (Soma). Method: Thirty pregnant female albino rats were randomly categorized into three groups. The first group was a negative control and administered oral doses of distilled water. The other two groups were administered oral doses of carisoprodol in the distilled water equivalent to 10.8 mg and 21.6 mg/100g body weight/day respectively for 15 days from the 6th day to the 20th day of gestation. Results: Histological and immunohistochemical studies have been performed on the tissue of the spleen to detect histopathological and immunohistochemical changes. Compared to untreated (negative control) and pregnant rats treated with carisoprodol, the treated rats showed numerous histopathological and immunochemical changes. Conclusion: Treatment of pregnant rats with carisoprodol has induced many atrophic changes in the tissue of the spleen in pregnant rats, so the use of this drug during pregnancy should be under strict protection.


INTRODUCTION
Muscle relaxants, also known as myorelaxants, are a class of chemical compounds that relax the skeletal muscle and are used to minimize the need for anaesthesia and make intubation easier (Vardanyan and Hruby, 2006).

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It has been on the market for more than 40 years and is widely used with over 10 million prescriptions filled per year (Rall, 1985). Because of their role in the US opioid crisis, highrisk combinations of controlled drugs, such as those containing opioid analgesics, are being scrutinised more closely. The triple-drug categoryopioids, benzodiazepines, and skeletal muscle relaxants, especially carisoprodol -should not be prescribed, according to prescribing guidelines (Wang et al., 2019). Soma, Carisoma, Sodol, Somadril, and Vanadom are trade names for carisoprodol, which comes in 250 and 350 mg generic formulations (Simon et al., 2010). Due to the risk of dependency and abuse, the recommended adult dose is 250 to 350 mg three to four times daily for just two to three weeks (Zacny et al., 2011). It is metabolised by the cytochrome P450 oxidase isozyme CYP2C19 in the liver and excreted by the kidneys, with a half-life of approximately 8 hours (Bramness et al., 2007). Dizziness, drowsiness, nausea, and psychomotor weakness are all side effects of carisoprodol (Raffel et al., 1969). Drug addiction, drug dependency, drug overdose, extreme withdrawal, progressive obtundation, coma, neuromuscular rigidity, myoclonus, and seizures can occur at high doses or with long-term use (National Institutes of Health 2017). Reeves and Carter (1999) discovered that carisoprodol causes recurrent withdrawal symptoms and toxic effects, which are consistent with previous case reports and series. Carisoprodol, on the other hand, is a pregnancy category C drug; animal tests show that it affects foetal growth and postpartum survival.
Carisoprodol's mechanism of action is unknown, although it is often attributed to that of its active metabolite, meprobamate (Carbonaro et al., 2020). Carisoprodol is metabolised to meprobamate (Olsen et al., 1995), which has been shown to have an addiction risk comparable to benzodiazepines, which are widely abused drugs (Rho et al., 1997). Meprobamate is known to produce phenomena that lead to physical and psychological dependence (Jackson et al., 2014).
Carisoprodol, on the other hand, is now known to control and specifically gate -aminobutyric acid types A receptors (GABA A Rs), the inhibitory neurotransmitter receptors found in mammalian brains. This may be attributable to carisoprodol's potential to improve the sedative effects of central nervous system (CNS) depressants, raising the risk of violence (Kumar and Dillon, 2015;Gonzalez et al., 2009). Carisoprodol was classified as Schedule IV in the United States due to its increased risk of adverse effects. Carisoprodol was listed as schedule A in Norway and was taken off the market, although it can still be used by patients who have been specially approved. Carisoprodol comes in the form of somadril in Egypt, which contains 200 mg carisoprodol, 160 mg paracetamol, and 32 mg caffeine. Mina Pharm has a prescription medication for pharmaceutical regulations as a result of Minister of Health andPopulation Decision No.172 of 2011 (Abouel-Magd, 2018). The analgesic and antipyretic effects of paracetamol (acetaminophen) are commonly used over-the-counter (Bernal et al., 2010). Hepatic dysfunction was exacerbated by paracetamol, which could lead to liver transplantation (Reuben et al., 2010). Caffeine is the most commonly consumed CNS stimulant, and it is absorbed quickly and almost entirely (up to 90%) by the stomach, with peak plasma concentrations occurring in 20 to 40 minutes. As a result, toxic levels can be reached easily and last for a long time after caffeine's 3-to 10-hour half-life has passed (Holstege et al., Immunohistochemical and histological alterations in the spleen of pregnant rats induced by carisoprodol 23 1993). Drugs may induce spleen enlargement through a direct effect on spleen cells or as a side effect of disorders in other organs or systems, such as the liver or the haematoimmunological system. Some medications cause serious haemolysis and splenomegaly (Petroianu, 2007). There have been no studies available regarding changes in the splenic tissue after administration of carisoprodol to pregnant. Therefore, the aim of this study was to investigate the effect of carisoprodol on splenic tissues with two different doses.

Drug:
The carisoprodol drug was obtained in the form of tablets of a combination of some products containing carisoprodol 200mg, paracetamol 160mg and caffeine 32mg. The drug was purchased from Mina Pharm for Pharmaceuticals and Chemical Industries, Cairo, Egypt.

Animals:
Adult male and female albino rats were used in the current work, with an average weight of 150-180g and put in cages alone for two weeks under a standard laboratory environment of light and temperature. They were taken from the animal house of El-Nasr Pharmaceutical Chemicals Company. All rats were kept under standard breeding conditions of dry food diet supply, temperature, tap water and light cycle. Gestation Course: The estrous phase was performed according to Taylor (1986). Daily, the vaginal signs were calm to check the estrus phase. The female rats were caged with one male overnight. The presence of sperm and a vaginal plug meant that reproduction was established and this was reversed on the zero-day of pregnancy.

Study Design:
Thirty rats were randomly categorized into three groups (10 pregnant female rats in each). The first was administered oral doses of distilled water and was served as negative control (NC). The other two groups (S1 and S2) were administered oral doses of carisoprodol in the distilled water equivalent to 10.8 mg and 21.6 mg/100g body weight/day respectively for 15 days from day 6 to day 20 of gestation. The equivalent dose for rats was adjusted according to the method Paget and Barnes (1964) formula on the basis of the human dose. All groups were sacrificed after 4 hours from the last drug administration. Histological and Immunohistochemical Studies: All rats were sacrificed and small pieces of spleen were picked out, rinsed in formalin (10%) and Bouin's solution for histological and immunohistochemical purposes. Sections of the spleen were prepared and stained with hematoxylin and eosin stain according to the method reported by Bancroft and Gamble (2008). Another set of slides were processed for detection of Caspase 3, CD163 and CD68 as immune markers according to Tan et al. (1986).

RESULTS Histopathological
Changes in Splenic Tissue: The spleen is a large lymphoid organ that performs two main functions: immune reaction to blood-borne foreign antigens and filtration of the blood of particulate substances/debris. Each of these two functions is performed by specific compartments known as the "white pulp" and the "red Pulp" respectively. The white pulp is essentially a collection of both B and Tlymphocytes, whereas the red pulp is the intervening splenic tissue that consists of sinuses and cords (Fig. 1A). Histologically, treatment of pregnant rats with carisoprodol at current doses S1 and S2 caused many pathological changes in splenic tissues, as opposed to negative control rats. Splenic tissue of group S1 showed numerous changes include thickened central arteries with narrow lumens, congested blood sinuses in the red pulps and congested veins. There was a proliferation of lymphocytes in some white pulps. Atrophic white pulp and thickened central arteries were observed (Figs. 1 B1&2). S2 group showed connected white pulps with increased proliferation of lymphocytes. Central arteries were congested and with a narrow lumen. There were congested blood sinuses in the red pulps (Figs. 1 C1&2).

Immunohistochemical Changes:
Immune reactions evaluated in splenic tissues of all pregnant rats and caspase 3 was immunopositive in all treated groups with a varying positivity level (Fig. 2). Caspase 3 in splenic tissues of negative control rats showed a negative (−) caspase 3 stain ( Fig. 2A). There was a moderate positive (+3) for caspase 3 in both S1 (Fig. 2B) and S2 groups (Fig. 2C). CD163 was positive in all treated pregnant rat splenic tissues when compared to negative control rats (Fig.   3). CD163 immunohistochemical staining of splenic tissues of negative control rats showed negative (−) CD163 stain (Fig. 3A) while was Mild positive for CD163 in both S1 (Fig.  3B) and S2 groups ( Figure 3C). CD68 staining was positive in all treated pregnant rat splenic tissues when compared to negative control rats (Fig.  4). Negative control rats showed negative (−) CD68 stain ( Figure 4A) but the splenic tissues of S1 was mild CD68 positivity in S1 ( Figure 4B) and S2 groups (Fig. 4C).     (Spence et al., 2013). Patients who take carisoprodol for a long time or who take it with other drugs that are affected may be at a higher risk of carisoprodol side effects (Abdelhafez and Abd Rabou, 2020).
In the present study, carisoprodol treatment in pregnant rats with the current doses S1 and S2 induced several pathological changes in splenic tissues compared to negative control rats. These changes include an increase in the number of connected white pulp with increased proliferation of lymphocytes and a highly thickened wall of central arteries with a narrow lumen. Emam (2020) also mentioned these changes in his report, stating that carisoprodol treatment in pregnant rats induced many dystrophic changes in lung tissue that worsened in a dosedependent manner. Extensive lung tissue injury, multiple necrotic areas, and narrow alveolar cysts with thick alveolar septa are among the pathological changes. There was also alveolar hemorrhage, severe damage to the bronchi, very thick walls of blood vessels, most of which were congested with dissolved blood cells and cytological infiltration in lung tissue. Abd Rabou (2019) discovered that giving carisoprodol to pregnant rats caused dystrophic changes in the mother and foetal esophageal tissue as compared to the control group, implying that its use during pregnancy should be strictly controlled. The current findings were consistent with Abouel-Majd (2018) findings, which showed that carisoprodol-treated mother and foetal liver tissues were seriously affected. These findings were in line with those of Ali et al. (2017), who found testicular vacuole, degeneration, and diminution in spermatozoa within the lumen, as well as loss of normal structure of the seminiferous tubules in Somadriltreated rats. These findings matched those of Awadalla and Salah-Eldin (2016), who found that tramadol, like other opioids, caused severe pathological changes in rat lung tissue and resulted in lower antioxidant plasma levels, suggesting a failure of the antioxidant protection mechanism. Lung depression, moderate sinus tachycardia, annexations, and ataxia were among the toxic effects of carisoprodol in a dog case study (Lane and Mazzaferro, 2005). Carisoprodol can cause congenital malformations in the first trimester of pregnancy (Robertson and Marinetti, 2003). Gomaa (2018) research has also shown that diclofenac, ibuprofen, and paracetamol have negative effects on immune-related organs (bone marrow, thymus, spleen, and lymph nodes). Both tapentadol and tramadol (CNS depressants) were found to cause significant cellular damage, inflammatory cellular infiltrations, and cell death in the pulmonary tissues, according to Faria et al. (2017), but tapentadol caused more damage. These findings were consistent with those of Liu et al. (2006), who found that infiltration of inflammatory cells into the liver in cases of direct drug-induced hepatotoxicity depletes neutrophils from the periphery, implying that the innate immune system (e.g., neutrophils and macrophages) plays a role in drug hepatotoxicity defence. Furthermore, paracetamol is metabolised into a toxic reactive metabolite that damages hepatocytes and releases risk signals, triggering an innate immune response that results in inflammation and collateral tissue damage to the liver, potentially lowering the number of leukocytes in the blood (Rainsford and Velo, 2012). Treatments with paracetamol resulted in a substantial reduction in the relative weight of the spleen, according to Gomaa (2017). Changes in the organ-body weight ratio may indicate cell constriction or inflammation, with the latter occurring as a result of a loss of fluid from the organ associated with damage; however, an improvement in the organbody weight ratio may indicate inflammation (Moore et al., 1999). Furthermore, several medications are thought to have immunotoxic effects in humans and animals, resulting in immune system disturbances manifested by changes in the weight of immune-related organs (spleen and thymus) (Descotes, 1986). According to Bessone (2010) and Kishida et al. (2012), nonsteroidal anti-inflammatory drugs (NSAIDs) such as paracetamol cause liver injury due to their acidic moiety or reactive metabolites that bind to host proteins and cause cellular injury. Moreover, paracetamol is metabolized into a toxic reactive metabolite that binds to glutathione and depletes it in the hepatocytes resulting in impaired mitochondrial function causing hepatocyte damage and necrosis. This damage causes cellular leakage and loss of functional integrity of the cell membrane in the liver, resulting in the release of high levels of serum markers into the blood, particularly AST and ALT that which account for 90% of the total enzyme and are a better predictor of liver injury (Watkins and Seef, 2006;Ansari and Asif, 2012). McIntyre et al. (2012) demonstrated that carisoprodol was concentrated in the liver with the organism dying before being distributed to the rest of the body. Besides this, because of its central nervous system depressant activity, the active metabolite, meprobamate, can contribute significantly to the overall toxic effects of carisoprodol in many cases. Concerning splenic sections, the experimental groups of Abbasi et al.
(2018) search not only demonstrate shrinkage as well as a cellular distortion of white pulp at each time point compared to the control sections. Furthermore, in the white pulp, a decrease in lymphocyte population, follicle activation, and cellular disruption were observed. These changes may be attributed to toxic metabolites of the dose of paracetamol.
The current study produced a quantity of Caspases-3 in the splenic tissue of treated rats indicating apoptosis compared to negative control rats. These results were identical with Emam (2020) who indicated that when comparing treated pregnant rats to the control group, the amount of caspase-3 indicated by the dense brown stain increased.
These findings were consistent with those of other studies of CNS depressants or analgesic drugs as Gloor et al. (2019), who found that acetaminophen causes apoptosis, necrosis, and mitochondrial harm. Jaeschke (2015) agreed that overdosing on paracetamol, which is considered a safe medication at therapeutic doses, can result in serious liver damage, acute liver failure, and cell death. Moreno et al. (2016) examined the main pathways for apoptosis following ethanol (CNS depressants) exposure and concluded that ethanol exposure has a substantial impact on the degree of apoptosis in the CNS. They revealed widespread proantiapoptotic responses, suggesting that while apoptosis increased cellular attempts to compensate and resolve exposure also increased. Gomaa (2018) concluded that consuming diclofenac, ibuprofen, or paracetamol can cause immunological and biochemical changes, as well as an increase in toxicity depending on the dosage. Oxidative stress and subsequent overproduction of reactive oxygen species (ROS), which have been proposed as the most significant mechanism of toxicity, may be the cause of such extreme histopathological changes with carisoprodol therapy (Rajapakse et al., 2007). Ostrovsky et Immunohistochemical and histological alterations in the spleen of pregnant rats induced by carisoprodol 29 al. (2009) demonstrated that oxidative stress and ROS cause cellular damage, including apoptosis and DNA fragmentation. NSAIDs (such as paracetamol) can impair thymocyte development and induce bone marrow suppression by reducing the production of cells that provide immunity (leukocytes). It can also have various immunomodulatory effects on activated macrophages, granulocytes, and extrathymic T cells, which may become cytotoxic effectors against self-tissues or self-cells when they are overactivated. The aetiology of the tissue impairment caused by NSAIDs may be linked to these activated leukocytes (Cho, 2007;Aronson, 2009).
In the current study, CD163 was positive in all treated rats when compared to negative control rats. Splenic immune cells, especially macrophages, play a key role in a variety of pathological processes, and mesencephalic astrocyte-derived neurotrophic factor (MANF) has been shown to have anti-inflammatory and immunoregulatory properties in inflammation (Hou et al., 2019). The upregulation of CD163, a macrophagespecific protein, is one of the main changes in the macrophage switch into alternative active phenotypes in inflammation. As a result, tissues that lead to inflammation have increased CD163 expression in macrophages. CD163 indirectly contributes to the anti-inflammatory response by scavenging oxidative and proinflammatory haemoglobin (Hb), which leads to activation of hemeoxygenase-1 and the development of anti-inflammatory heme metabolites. CD163 is a potential biomarker of inflammation and a therapeutic target, in addition to its biological function in inflammation (Etzerodt and Moestrup, 2013). Bacterial sepsis/infection is an acute inflammatory disease (Møller et al., 2006), while rheumatoid arthritis is a chronic inflammatory disease (Greisen et al., 2011).
Our study found that immunohistochemical staining of splenic sections for the detection of CD68 was positive in all treated rats compared to negative control rats. CD68 expression can be significantly upregulated in rat resident peritoneal macrophages in response to inflammatory stimuli (Rabinowitz and Gordon, 1991). CD68 is a typical histochemical/cytochemical marker of inflammation in the presence of monocytes/macrophages (Ferenbach and Hughes, 2008). Cells of the macrophage lineage, such as tissue histiocytes, multinucleated giant cells, kupffer cells, and osteoclasts, have also been identified using CD68 (Brooks et al., 2009). CD68 immunostaining can be seen in a number of cell types, including fibroblasts and inflammatory cells (Chen et al., 2017). Increased CD68 staining was characterised by activated kupffer cells in a previous study (Lee et al., 2016), and appeared to be co-stimulated by increased secretion of the cytokine macrophage colonystimulating factor (M-CSF). Mossanen et al. (2016) determined that acetaminophen (paracetamol) toxicity is a leading cause of acute liver failure (ALF) in humans, resulting in hepatocyte necrosis and innate immune system activation. Overdosage of depleted glutathione (GSH) with acetaminophen (paracetamol) resulted in liver damage and thus increased hepatotoxicity and CD68 expressions (Fadda et al., 2017).

Conclusion
Despite the fact that carisoprodol is believed to be the most powerful pain reliever, our findings showed that taking it during pregnancy induced numerous histopathological and immunohistochemical changes in the spleen tissues of pregnant rats. As a result, the toxicity of carisoprodol must be noted, and extreme care should be used when using it in therapies.