Assessment of Histopathological Changes in the Liver and Spleen of Mice Infected with Salmonella typhi Following Treatment with Ciprofloxacin

This study evaluated the toxicity of ciprofloxacin to spleen and liver when used for the treatment of mice infected with S. typhi for seven days. The dose concentration used in these experiments was 100mg/kg. Mice were divided into two groups . The first group (negative control) was not given ciprofloxacin, but rather a sterile phosphate buffer solution (PBS) as an alternative. Ciprofloxacin was administered to the second group. After seven days , the animals were sacrificed and organs (liver and spleen) were collected . The histopathological examination showed normal hepatocytes in the liver and normal structure of spleen cells in animals of control group . However, the treated group showed dilated and congested blood vessels with perivascular inflammatory cell cuffing and acute cell swelling in the liver, as well as white pulp activation with an increased number of megakaryocyte cells in the spleen. Therefore, the current study suggests that the concentration of 100 mg/kg of ciprofloxacin is considered to be toxic to hepatocytes and splenocytes of mice during the treatment period.


Introduction
The use of antibiotics is considered as a general cause of DILI (drug-induced liver injury). Ciprofloxacin is a fluoroquinolones with a broad spectrum of anti-bacterial efficacy. However, ciprofloxacin has excellent bio-availability and safety following the oral administration [1]. Ciprofloxacin is active towards Gram positive and negative organisms. The mode of action of ciprofloxacin involves the blocking of the synthesis of bacterial DNA throughout inhibiting bacterial topoisomerase IV and DNA gyrase (topoisomerase II) [2]. Inhibiting topoisomerase IV interferes with the separation of the replicated chromosomal DNA in special daughter cells by cell division [3].The liver is considered to be the main organ that is responsible for the metabolism and excretion of chemotherapeutic agents, xenobiotics, and environmental pollutants; these substances, together with oxidative stress, are responsible for hepatotoxicity (liver injury) which is a main clinical concern [4]. Liver toxicity is related to cell necrosis, depletion of glutathione levels, and increased lipid peroxidation [5]. Salmonenella typhi is known to cause typhoid fever and, subsequently, liver injury. Delay in the diagnosis of infection, as well as the development of DILI, may result in acute liver failure and cirrhosis [6]. The spleen is a secondary lymphoid organ that is highly sensitive to various chemical materials. It consists of lymphoid and vascular elements and is the location of hematopoiesis. The spleen is also partially involved in the removal of the ageing and degenerating red blood cells, in addition to particulate materials and circulating bacteria from the blood circulation [7]. This organ is considered to be the location of indirect and direct toxicity, the location of malignant neoplasms arising in other locations, and the target for several carcinogens. The immune system undergoes continuous differentiation and self-renewal to maintain immunecompetence and , therefore, can be affected by xenobiotics that change the current cellular balance [7,8].Materials and Methods

Bacterial strains and growth conditions
Salmonella typhi was isolated from human patients suffering from typhoid fever and then cultured in XLD and MacConkey agar. The isolate was incubated at 37°C for 18-24 hr [9]. The next day, prior to oral inoculation, it was confirmed that all animals were pathogen-free by using bacteriological examinations which showed that the mice had a negative culture for S. typhi . At the beginning of the study, the mice were deprived of food overnight and of water for 3 hrs. They were then fed with 0.2 ml of bacteria suspended in LB (Luria broth). The actual dosage was confirmed using the plating of serial dilutions of the inoculum, and prepared the LD50 dose of mic. The mice were gavaged with 0.2ml of Salmonella typhi suspension according to the LD50 dose, while the control group was gavaged with the same amount of sterile saline [10,11]. Colonies of bacteria were recovered from the intestines of mice. The mice were sacrificed and their intestines were extracted, homogenized in sterile phosphate-buffered saline (PBS) , plated on MacConkey and XLD agar plates, and incubated overnight at 37°C to obtain the results [10].

Estimation of Lethal Dose 50 (LD50)
The estimated viable count of the bacteria in each diluent was made according to the method of Miles and Misra [12].

Animal groups and ciprofloxacin doses
Twenty (Swiss albino) mice were used for the LD-50 experiment. Animals were aged between 6-8 weeks and weighed 25.4 ± 3.2 g. The animals were stabilized for one week before the commencement of the experiments. Commercial pelleted food and clean drinking water were provided. After stabilization, mice were randomly grouped into two groups of 10 mice each; group one was infected with Salmonella typhi . After 3 days, ciprofloxacin was followed for approximately 7 days. Group two was used as a control. Ciprofloxacin was administered orally, once per day , with a total daily dose of 100 mg \ kg of body weight, At 3 days post infection, a maxim-um dose was used according to previous methods [13,14]. The results were interpreted in relation to animal survival and observed toxicity [15].

Antibiotic solution preparation
Ciprofloxacin powder (Bioanalyse, Turkey) was dissolved in sterile distilled water and the MBC (minimum bactericidal concentration) and the MIC (minimum inhibitory concentration) were calculated according to an earlier work [16]. The solutions were transferred to sterile vials through

Histological examination
Tissue specimens from the liver and spleen were collected for histological examination under the light microscope in accordance with standard protocols. The liver and spleen were removed from the mice used in the experiment. These organs were kept in 10% neutral buffered formalin (pH 7) for fixation . Fixation was achieved after 72 hrs and after that the organs were dehydrated by passing through ascending concentrations of ethyl alcohol-water, cleared in xylene and sequentially embedded in blocks of paraffin wax , which were then sectioned using a rotary microtome. Sections of tissues with three to five μm thickness were sliced, stained with H-E (Haemat-oxylin eosin), and then examined using a light microscope. The results of the histopathological examination were recorded using a digital camera system attached to the microscope [18][19][20].

Results and discussion 1-Estimation of lethal dose (LD50) of S.typhi
In the present study , the lethal dose (LD50) of S. typhi in mice was found to be 3 ×10 8.49 cfu / ml, which is compatible with the previously published results [21][22][23].

2-Evaluation of clinical signs
The medical assessment was performed depending on the clinical responses of each mouse to Salmonella infection. These included less efficacy of mice, appearance of fur, increased feeding, ataxia , hunched position, move abnormally(some time fast and jumps a lot other times), weight loss, tremor, and increased feces in the first week after infection. These responses may be due to infection with Salm-onella that causes diarrhea .The current results are in agreement with those previously published [10].

3-Antibacterial activity in mice
Mice from each group were treated with ciprofloxacin at 3 days post infection. Ciprofloxacin (100 mg/kg, 0.5 µg/ml) was given orally to mice infected with S.typhi for seven days. Histopathological examination of liver and spleen was then carried out [24].

4-Control group
In this study , liver and spleen specimens of the control (negative) group(without treatment) showed normal hepatocyte architectures and normal blood vessels (central vein) in the liver ( Figure-1), as well as normal white and red spleen pulps (Figure-2). These results are in accordance with those of previous studies [25,26] which include polygonal hepatocytes with granular eosinophilic cytoplasm and red blood cell (RBCs)-free central vein in the liver. The spleen showed a red pulp loaded with RBC and a white pulp that contains mixture of lymphocytes and plasma cells.

5-The effects of ciprofloxacin on the liver and spleen of S.typhi infected mice
The effects of ciprofloxacin on the liver and spleen of S.typhi infected mice were reflected by changes in the structure of these organs. In the liver, the results showed dilated and congested blood vessels with perivascular inflammatory cells and acute cell swelling (Figure-3). Acutely swollen hepatocytes could be attributed to mitochondrial injury, endothelial reticulum injury, and organ injury, which can lead to cell death, vascular damage, thrombosis, tissue/organ ischaemia, and necrosis [27]. The recorded hepatotoxic impacts of ciprofloxacin may be due to the

Rajab and Turki
Iraqi Journal of Science, 2021, Vol. 62, No. 3, pp: 761-768 267 oxidative stress induced in the liver by the use of ciprofloxacin, through the generation of oxidative radicals leading to protein depletion in the hepatocyte. As a result, a decrease of nucleic acids and DNA damage can occur, leading to remarkable degeneration in mitochondria and reduction in the number of hepatocytes. This result is in agreement with those of previously published reports [28,29].
The extending of the hepatic cells and the congestion of blood vessels in this study may be due to the hepatotoxicity of ciprofloxacin, which could be attributed to its active metabolites [30]. This result agrees with the findings of other studies [31,32]. Ciprofloxacin was shown to inhibit eukaryotic and prokaryotic cell growth and protein synthesis by interfering with the replication of DNA and RNA. An earlier work [28] found a congestion of blood vessels and leukocytic infiltration in the liver of animals treated with ciprofloxacin. The pathogenesis mechanism of Salmonella typhi caused hepatic dysfunction, which was postulated to be either due to the endotoxemia with immune-assisted liver damage or through direct invasion [33]. Histopathological examination of liver tissue of animals infected with S. typhi showed the appearance of typhoid nodules , degeneration, swelling, and mononuclear cell filtratation [34]. In systemic salmonellosis, such as typhoid fever, Salmonella may target specific types of host cells, such as dendritic cells and macrophages that favor spread through the lymphatics and blood stream to the deeper tissues. This then leads to transport to the spleen, bone marrow, liver, and gall bladder and causes damages to cells [35] Following ciprofloxacin treatment of S.typhi infected mice in the present study, the spleen tissues showed megakaryocyte hyperplasia and white pulp activation (Figure-4). Treatment of mice with ciprofloxacin against S. typhi has been changed to extramedullary hematopoiesis in the spleen. Significantly, the megakaryocytes number was increased , which is in line with previously published findings [36,37]. The extra medullary hematopoiesis consisted of erythroid precursors, myeloid precursors, megakaryocytes (the precursors of platelets), or all the three components. The erythroid component, i.e. the erythroid hyperplasia, may lead to secondary hemorrhage or erythrocyte destruction, i.e. hemolytic or autoimmune anemia. Also, the myeloid component may predominate secondary to the inflammatory disorders, i.e. the myeloid or granulocyte hyperplasia. Many histological similarities with granulocytic leukemia were reported in myeloid hyperplasia [38], although some degree of extramedullary hematopoiesis occurred in mice. Extramedullary hematopoiesis was increased due to haematotoxic damage, systemic anemia, and bacterial infections that cause bleeding elsewhere in the body [7].
In particular, spleen was sensitive to enlargement due to the use of erythroid and myeloid hyperplasia, most of which were accompanied by megakaryocyte hyperplasia. The majority of spleens show lymphoid hyperplasia in white pulp in response to foreign antigens expressed by infectious

Rajab and Turki
Iraqi Journal of Science, 2021, Vol. 62, No. 3, pp: 761-768 266 agents or tumors [39,40]. Due to the use ciprofloxacin to treat mice infected with typhoid fever, bone marrow suppression and megakaryocytic hyperplasia were reported [41]. This result is in agreement with those of other studies [42,43]. Ciprofloxacin is a very important antimicrobial against a wide range of aerobic bacteria species. This is due to the association between treatment with and histopathological changes. A previous study reported potent therapeutic effects of ciprofloxacin in the spleen along with the stimulation of the immune system [42]. The spleen is an organ that is present in almost every species of vertebrates. It acts primarily as a blood purifying filter of the destroyed bacteria and red blood cells. This organ is also an important organ in the immune system, producing white blood cells, and infections, and synthesizing antibodies [44][45][46]. The active white pulp shows an increase in the number of lymphocytic cells, indicating an immune response, since splenic and hepatic macrophages are the main cells in which the bacteria reside (40).