الفهرس 
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Abstract
Toxoplasma gondii is an intracellular protozoan parasite of worldwide distribution that can infect nearly all worm-blooded hosts including human. It is characterized by an acute phase of rapidly replicating tachyzoites with nearly no or little symptoms in immunocompetent hosts followed by a chronic phase with cysts in tissues especially in the immune-privileged organs as the brain. Although it was believed that chronic toxoplasmosis has no clinical impact on the hosts, in the last decades a lot of neuropsychiatric problems have been linked with chronic toxoplasmosis. Schizophrenia, manic-depressive disorder, obsessive–compulsive disorder, Alzheimer disease, cryptogenic epilepsy, increased suicide attempts and risk of traffic accidents are all among the neuropsychiatric problems that were associated with latent toxoplasmosis (Dalimi and Abdoli 2012). Till now, the dormant Toxoplasma tissue cyst has no available treatment as it is highly resistant to most of the known anti-Toxoplasma therapeutic modalities. Although the treatment of choice in toxoplasmosis is the combination therapy of pyrimethamine and sulfadiazine, it is still showing no satisfactory results against tissue cysts in chronic toxoplasmosis in addition to its severe side effects and the emergence of pyrimethamine and sulfadiazine resistant strains (Konstantinovic et al., 2019). Thus, an urgent need for a safe effective drug that can cross the blood brain barrier and affect the cysts in the brain has been emerged. So, the aim of the present work was to test the efficacy of guanabenz alone and guanabenz-loaded PEG-PLGA nanoparticles against chronic experimental toxoplasmosis. In order to fulfill the aim of the study, the following steps were performed: One hundred and sixty laboratory-bred male Swiss albino mice were divided into two main groups as follows: group I: Control group This group included 100 mice that were subdivided equally into five subgroups (each of 20 mice): Subgroup Ia: non-infected non-treated mice as a control healthy group. Subgroup Ib: infected non-treated mice as a control diseased group. Subgroup Ic: non-infected and injected with nanoparticles alone. Subgroup Id: infected and injected with nanoparticles in the same dose of subgroup Ic. Subgroup Ie: infected and treated with pyrimethamine (4 mg/kg) and sulfadiazine (100 mg/kg) daily. group II: Infected treated group This group included 60 chronically-infected mice that were subdivided into three subgroups (each of 20). Subgroup IIa: infected and treated with guanabenz in a dose of 5mg/kg/day for 19 successive days. Subgroup IIb: infected and treated with guanabenz-loaded nanoparticles (5mg/kg/day for 19 successive days). Subgroup IIc: infected and treated with guanabenz-loaded nanoparticles (2.5 mg/kg/day for 19 successive days). The Mice were infected intraesophageally with a cystogenic strain of Toxoplasma gondii (ME49) in a dose of 10 cysts/mouse according to El- Zawawy et al. (2015b). The treatment started on day 25 post-infection for 19 successive days according to Benmerzouga et al. (2015). On the 20th day, the mice were anesthetized and scarified. Blood samples were collected, sera were separated, divided into aliquots, and stored at -20 ° C until used for detection of interferon gamma (IFN-γ) and estimating the level of urea, creatinine, SGPT and SGOT. The Brains were isolated and used for the histopathological, immunohistochemical and scanning electron microscopy examinations. Parasitological evaluation Estimation of the mortality rate (MR) The mortality rate was calculated in each subgroup according to the following equation: MR = Number of dead mice at the sacrifice time/ Number of mice at the beginning of the experiment × 100 according to El-Zawawy et al. (2015b). Histopathological evaluation The brain specimens were fixed in 10% formaline, and prepared for both histopathological haematoxylin and eosin stainning and immunohistochemical staining to detect the Toxoplasma cyst and assess the degree of inflammation. Estimation of the parasite burden: It was performed by counting the mean number of tissue cysts in the brain sections of the mice. This was done using immunohistochemical staining according to Benevides et al. (2008). Toxoplasma gondii cyst counting was calculated by counting the total number of cysts per sagittal section in the brain of the infected mice. Two histological sections of each mouse from 3-5 randomly chosen mice per group were examined. Immunological study The stored collected serum samples at -20 ° C were used for estimation of interferon gamma level by enzyme-linked immunosorbent assay (ELISA) according to Hamad et al. (2018). Morphological study: The brains of mice in different infected subgroups were collected on the 20th day post treatment. Homogenization of the brains was done and examined by light microscopy then was fixed in buffered glutraldehyde and paraformaldehyde and processed for scanning electron microscopy (SEM) examination according to Karnovsky (1965) for detection of the morphological changes of Toxoplasma gondii cyst. Biochemical study Urea, creatinine and liver enzymes; serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic transaminase (SGOT) were estimated to assess the toxicity of these nanoparticles in subgroup Ic (non-infected nanoparticles-treated control group) on the liver and kidney, in comparison to subgroup Ia according to Sharma et al., (2003) and Pissinate et al. (2014). from the present study, the following results were obtained: 1-Estimation of the mortality rate (MR) The mortality rate between different subgroups was not statistically significant (P= 0. 0.741), however, the mortality rate in the subgroups IIa, IIb and IIc was 15% which is less than that of subgroup Ie. 2- Histopathological evaluation: Histopathological examination of brain sections of the infected nontreated mice (subgroup Ib) showed necrosis, perivascular cuffing by a large number of mononuclear cells, neuronal degeneration and many Toxoplasma cysts. Marked improvement in the histopathological changes was detected in the guanabenz treated subgroups either alone or in combination with PEGPLGA polymeric nanoparticles by full or half dose. No perivascular cuffing or mononuclear cellular infiltration was found in subgroup IIb. In subgroup IIa there was only an area of focal mononuclear cellular infiltrate with no perivascular cuffing, while mild perivascular cuffing was noticed in subgroup IIc. 3-Estimation of the parasite burden: No statistically significant difference was detected in cyst count between subgroup Ib and Id (a mean of 13±1.58 and 11.4± 1.14) respectively. A highly significant reduction in the number of brain cysts was obtained in subgroups treated with guanabenz or guanabenz-loaded nanoparticles in full or half doses (64.61%, 77% and 67.69% respectively). However, there was no statistically significant difference between the three subgroups. In subgroup Ie, there was a highly significant reduction (53.85 %) in the cyst count compared to the infected non-treated subgroup Ib. However, the lowest number of brain cysts was observed in subgroup IIb with statistically significant difference in comparison with subgroup Ie (P= 0.01). 4-Immunological study: There was statistically significant difference between all subgroups (P= 0.021) regarding the mean IFN-γ level. On comparing each two subgroups, the mean IFN-γ level in subgroup IIb was statistically significant higher in comparison to subgroups Ic, Ie, IIa and IIc with (P value < 0.05), while the lowest level of IFN-γ was observed in subgroup IIa with statistically significant difference in comparison to subgroup Ia (P value = 0.037). 5-Morphological study: In subgroup Ib, Toxoplasma cyst was spherical, having a regular surface with some fine granulation. In subgroup Id, Toxoplasma cyst also had a spherical regular shape with either smooth surface or some protrusions in the cyst wall. In subgroup Ie, there was multiple grooves and holes in the wall of Toxoplasma cyst. Infected subgroups that were treated by either guanabenz or guanabenz loaded nanoparticles in full or half dose in subgroups IIa, IIb and IIc respectively, showed similar changes of depression and compression of the cyst wall. A marked compression and distortion of the wall with release of crescent shaped bradyzoite outside the cyst were observed in subgroup IIb. 6-Biochemical study There was no statistically significant difference between subgroup Ia (healthy control) and Ic (healthy control injected with nanoparticles) (P> 0.05) regarding urea, creatinine and liver enzymes.