الفهرس 
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Abstract
The first objective of our study was conducted to evaluate the therapeutic effect of curcumin on MnCl2 induced neuropathlogical lesions and toxic lesions in other organs (testes, kidney and liver) by histopathological examination of selected organs and neuromorphometrical analysis associated with brain lesions of different group.
To fulfill our goals, seventy–four male albino rats were used for this study. The rats were apparently healthy, weighting about 200-250gms and randomly assigned to three groups according to the experimental design.
In the first group (MnCl2 toxicity group), 32 rats were given 10 mg MnCl2/kg b.w daily orally for 8 weeks and 8 rats scarified 2, 4, 6 and 8 weeks post dosing (subgroup 1, 2, 3 and 4). The effect of MnCl2 toxicity was assessed histopathologically and neuromorphometrically. The tissue samples from prominent lesions of brain parts (cerebrum, cerebellum, midbrain), testes, kidney and liver were preserved in neutral buffered formaline for histopathological examination.
The neuropathlogical lesions of MnCl2 toxicity group demonstrated at different parts of brain (cerebrum, cerebellum and midbrain). The neuronal changes of cerebrum appeared in 2 rats out of 8 rats after 2 and 4 weeks post toxicity which expressed by mild pyknotic degeneration of neurons with perineuronal space, complete karyolysis in few neurons associated with proliferation of astroglia cells. Moreover, after 6 weeks the neuronal changes became more sever and appeared in all 8 examined rats which expressed as pyknotic degeneration of large number of neurons surrounded with perineuronal space, appearance of neurophagia around damaged neurons as well as focal proliferation of glia cells were seen. Finally, at the end of the experiment, the neuronal changes of the cerebrum were more aggressive as pyknotic degeneration of large numbers of neurons surrounded with perineuronal spaces and focal proliferation of glia cells forming nodules in all examined rats.
The angiopathic alterations of cerebral blood vessels, after 2 weeks post toxicity were found in 6 rats out of 8 rats which expressed by congestion of blood vessel, swelling of endothelial cells of some blood vessel surrounded with perivascular edema. The vascular changes became more sever after 4, 6 and 8 weeks post toxicity expressed as sever congestion of blood vessel, thrombotic blood vessels, swollen endothelial cells of some blood vessels surrounded with perivascular edema and submeningal perivascular hemorrhages in all 8 examined rats.
In the cerebellum, the neurons of molecular and granular layer were still healthy and the neuronal changes involved mainly the Purkinje cells layer. After 2 weeks post toxicity, the neuronal changes of Purkinje cells appeared in 4 rats out of 8 rats and expressed as pyknotic degeneration of Purkinje cells, chromatolysis of some Purkinje cells and karyolysis of other Purkinje cells as well as reduction in numbers of Purkinje cells. The neuronal changes after 4 weeks became sever and appeared in all 8 rats which expressed by pyknotic degeneration of large number of Purkinje cells with proliferation of Bergman cells, chromatolysis of Purkinje cells and karyolysis of other Purkinje cells. Moreover, after 6 and 8 weeks, the neuronal changes became more aggressive in all rats which expressed as pyknotic degeneration of majority of Purkinje cells, chromatolysis of some Purkinje cells surrounded with increased number of Bergman cells associated with marked loss of Purkinje cells. Disarrangement and disorganization of Purkinje cells appeared after 6 weeks and progressed to status spongosis after 8 weeks. Mild demylination of nerve fibers was seen in medulla of cerebellum in form of tiny small clear vacuoles in 2 rats after 2 weeks and appeared in all rats after 4, 6 and 8 weeks post toxicity.
Neuromorphometrical analysis of Purkinje cells of MnCl2 toxicity group showed that after 2 weeks, the mean of Purkinje cells number was (32.25 ± 1.80B) compared to that of the control group (40.00 ± 2.08A) indicating significance loss while after 8 weeks Purkinje cells number showed highly significant loss (17.25 ± 1.25B) versus the control values (40.00 ± 2.08A).
After 2weeks, the vascular changes of cerebellar blood vessels were mild and increased in severity along different time intervals in all examined 8 rats. These changes were expressed by sever congestion of blood vessels, perivascular hemorrhage, thrombosis of blood vessel and hemorrhage at cerebellar medulla.
In the midbrain, the neuronal changes were found in all 8 examined rats along different time intervals. After 2 weeks, the examined rats showed mild neuronal changes expressed by necrosis of focal area of motor neurons with perineuronal space, karyolysis of motor neurons and diffuse gliaosis. After 4 weeks, the neuronal changes were moderate and appeared as degeneration of motor neurons, karyolysis of other motor neurons and chromatolysis of some motor neurons as well as proliferation of astrocyte cells. The neuronal changes became more aggressive after 6 and 8 weeks which demonstrated as pyknotic degeneration of large number of motor neurons, chromatolysis of motor neurons. In addition, activation of glia cells and neuronophagia around damaged neurons were also observed. Mild demylination started from 2 weeks till the end of experiment.
The toxic effect of MnCl2 on the testes revealed marked changes in seminiferous tubules and interstitial tissue throughout the experiment periods. After 2 and 4 weeks, the changes expressed by moderate degeneration of seminiferous tubules, massive necrosis of whole germinal epithelial layers of some seminiferous tubules with formation of spermatid giant cells in seminiferous lumina in 6 rats out of 8 examined rats. The changes became more sever in all 8 examined expressed as diffuse necrosis of most seminiferous tubules with formation of spermatid giant cells in lumina of damaged seminiferous tubules.
The vascular damage was obvious in 4 rats after 2 weeks, increased in severity after 4 and 6 weeks in 6 rats while appeared in all rats after 8 weeks. The angiopathic alteration of testicular blood vessels expressed as sever congestion of blood vessels of tunica albugina, thrombosis of blood vessels, vacuolar degeneration of endothelial cells as well as edema of interstitial tissue. However, the intensity and severity of vascular damage varied affecting 4 rats after 2 weeks, 6 rats after 4 and 6 weeks and all rats after 8 weeks post toxicity.
The nephropathological changes of MnCl2 toxicity group appeared as moderate lesions in 6 rats sacrificed after 2 and 4 weeks which expressed as congestion of glomerular capillaries, swelling and proliferation of mesangial cells obliterating bowman’s space, periglomerular mononuclear cellular infiltrations, vacuolar degeneration of renal tubules and focal area of coagulative necrosis of renal tubules. Moreover, 6 rats examined after 6 weeks post toxicity demonstrated progressive changes as congestion of glomerular capillaries, glomerularsclerosis, periglomerular mononuclear cell infiltration, vacuolar degeneration of renal tubules and hyaline cast in renal tubules lumina. At the end of the experiment, 8 weeks post toxicity, the same changes became more pronounced in all examined rats beside evidence of focal tubulointerstitial nephritis.
The angiopathic changes expressed by congestion of interstitium blood vessel, thrombosis of blood vessels, damaged endothelial cells, hyalinization of the wall of some blood vessels as well as intertubular hemorrhages. However, the intensity and severity of vascular damage were moderate in 6 rats scarified after 2 weeks and severe in all examined rats at other time intervals.
The hepatopathological changes of MnCl2 toxicity demonstrated as mild vacuolar degeneration and centralobular necrosis in 4 rats after 2 weeks post toxicity and became moderate in 6 rats after 4 weeks. After 6 and 8 weeks, all 8 rats showed progressive hepatocellular coagulative necrosis with loss of hepatic architecture, kupffer cells proliferation and infiltration of mononuclear inflammatory cells in the portal triad.
The angiopathic alterations of hepatic blood vessels were expressed by congestion of central vein, thrombotic blood vessels, damage of endothelial cells and congestion of hepatic sinusoids. Moderate changes appeared at 2 weeks in 4 rats and became more severe at 4, 6 and 8 weeks in all 8 rats.
In the second group (curcumin-treated group), 32 rats received curcumin co-administrated with MnCl2 at a dose of 100 mg curcumin/kg b.w daily orally for 8 weeks. Eight rats were sacrificed after 2, 4, 6 and 8 weeks post curcumin treatment (subgroup 1, 2, 3 and 4). The histopathological examinations of the selected tissue samples and neuromorphometrical analysis of brain lesions were performed.
Curcumin treatment resulted in improvement of the histopathological changes in brain parts. In the cerebrum, after 2 weeks, the neuronal changes in all examined rats were similar to those described in MnCl2 toxicity group. Gradual improvement of neurons detected in all rats examined after 4 weeks expressed by evidence of sporadic healthy neurons beside damaged neurons. After 6 and 8 weeks post treatment, highly significant improvement of neuronal damage was noticed in all examined rats indicated by predominance of healthy neurons and presence of few damaged neurons.
In the cerebellum, curcumin ameliorated the changes in the Purkinje cells as mild improvement at 2 and 4 weeks post curcumin treatment evidenced by presence of sporadic healthy Purkinje cells beside damaged cells. Moderate improvement at 6 weeks post treatment, expressed as 4 rats out of examined 8 rats demonstrated by healthy Purkinje cells beside damaged cells while after 8 weeks, majority of Purkinje cell population appeared healthy in 6 rats represented remarkable neuroprotection by curcumin.
Neuromorphometrical analysis of Purkinje cells of the curcumin- treated group showed that after 4 weeks, the mean number of Purkinje cells was (35.50 ± 1.76AB) compared to that of the toxicity group indicating significant increase while after 8 weeks, the mean of Purkinje cells number showed highly significant increase (36.75 ± 1.31A) versus toxicity group.
In the midbrain, after 2 weeks post treatment the neuronal changes in all rats began to improve and expressed by few healthy motor neurons beside damaged neurons. Moderate improvement recorded after 4 weeks and expressed as moderate number of healthy neurons beside damaged neurons. After 6 and 8 weeks post treatment, well marked improvement of neuronal damage was noticed in all examined rats indicated by predominance of healthy motor neurons and presence of few damaged neurons.
The vascular changes of brain blood vessels were persistent after 2 weeks post treatment in all examined 8 rats, improved gradually after 4 and 6 weeks (lesser intensity and severity) and at end of experiment after 8 weeks completely disappeared. The glia cellular reactions gradually decreased and disappeared at 6 and 8 weeks.
Regarding the protective effect of curcumin on the testicular lesions, the histopathological changes of the testes in rats sacrificed after 2 weeks post curcumin treatment did not vary from those described in the toxicity group. Gradual improvement of the testicular lesions could be demonstrated after 4 weeks post treatment in 4 examined rats, which prevailed sporadic healthy seminiferous tubules beside other damaged seminiferous tubules. However, after 6 and 8 weeks post curcumin treatment, highly significant improvement was obvious in all examined rats indicated by predominance of healthy seminiferous tubules and presence of few damaged seminiferous tubules.
The vascular changes were persistent at 2 weeks post treatment in all 8 rats, improved gradually at 4 and 6 weeks (lesser intensity and severity) and completely disappeared at end of experiment after 8 weeks.
In our work, curcumin did not significantly improve the histopathological changes of renal tissues in rats sacrificed 2, 4, and 6 weeks post treatment. The nephropathic and angiopathic changes were persistent and resembled those seen in the toxicity group. Significant improvement of the renal and vascular lesions could be demonstrated after 8 weeks post treatment in all examined rats which expressed by slight congestion of glomerular capillaries with appearance of bowman’s space, slight tubular degeneration, periglomerular proliferation of few mononuclear inflammatory cells and slight congestion of blood vessels.
The hepatoprotective effect of curcumin throughout the experimental period was not significantly expressed since coagulative necrosis either affecting sporadic hepatocytes cells or involving the whole lobular structure was prominent. . However, the vascular changes subsided starting from the 4th weeks post curcumin treatment till the 8th weeks and kupffer cell proliferation also persisted till the end of the experiment.
In the present study, our second objective was to evalute the therapeutic effect of curcumin on MnCl2 induced hematological alterations. Blood samples were collected from the experimental groups to prepare blood smears for routine hematological examination and to assess the whole blood picture for each group.
In the first group (MnCl2 toxicity group), the number of total white blood cells count (WBCs) started to increase from 2 weeks post toxicity (18.13 ± 0.43) and reached the maxmium peak (22.65± 0.55) at the end of experiment. In the second group (curcumin- treated group), the number of total white blood cells count (WBCs) statred to decrese from 4 weeks (16.63± 1.44) and reached to the lowest number (14.03 ± 2.55) at end of experiment.
The total number of lymphocytes (LYM), Monocytes (MONO), Neutrophils (NEUT) and Eosinophils (EOS) started to increase especially lymphocytes from 2 weeks to reach the highest number after 8 weeks post toxicity. The total number of lymphocytes (LYM), Monocytes (MONO), Neutrophils (NEUT) and Eosinophils (EOS) decreased gradually along different time intervals of the experiment of curcumin treatment.
The MnCl2 toxicity group showed reduction in the total RBC count from 2 weeks (8.01 ± 0.48) and reached to the lowest number (6.47 ± 0.00) after 8 weeks post toxicity compared to control value. Also, the hemoglobin concentration (Hb%) started to decrease after 2 weeks post toxicity (13.90 ± 0.60) and progressed until reach (11.05 ± 2.35) at 8 weeks. The hematocrite (Hct) values began to decrease gradually from 2 weeks (37.97 ± 0.49) and reached to maximum effect (35.90 ± 0.00) at 8 weeks after toxicity. The curcumin- treated group showed increase in the total RBC count from 2 weeks (8.27 ± 0.05) and reached to the highest number (8.80 ± 0.40) after 8 weeks post treatment compared to toxicity group. The treatment in the present study slightly corrected hemoglobin after 2 weeks (14.23 ± 0.18) and the maximum improvement was (15.90 ± 1.70) at 8 weeks post treatment. The hematocrite (Hct) values began to increase gradually from 2 weeks (38.83 ± 1.73) and reached to maximum effect (44.05 ± 0.81) at 8 weeks after treatment.
The MnCl2 toxicity group showed reduction in the RBCs distribution width (RDWa) along different time intervals of experiment. Also, the mean corpuscular volume values (MCV) were lower than normal along the whole period of experiment. Curcumin-treated rats showed slight increase in the RBCs distribution width (RDWa) and mean corpuscular volume values (MCV) along the whole period of experiment compared to the toxicity group.
The MnCl2 toxicity group showed increase in total platelets count started from 2 weeks (992.50 ± 125.50) and reached to maximum effect at end of experiment (1470.00 ± 53.00). In the treatment group, the total platelets counts decreased slightly after two weeks (882.30 ± 90.83) and the maximum improvement was (1143.67 ± 92.99) after 8 weeks compared to toxicity group.
In MnCl2 toxicity group, after 8 weeks of experiment the number of Leptocytes (14.57 ± 1.54), Howell jolly bodies (4.43± 1.02) and Basophilic stippling (3.03± 0.9) increased compared to control values. In curcumin- treated group, after 8 weeks the number of Leptocytes (5.03 ± 0.81), Howell jolly bodies (2.06 ± 0.4) and Basophilic stippling (1.06 ± 0.6) decreased compared to toxicity group.
In MnCl2 toxicity group, there was an increase in the number of Echinocytes after 8 weeks (7.33 ± 0.74). In the treatment group, marked correction in the rise of Echinocytes by (2.60 ± 0.52) was observed at the end of experiment.
In the MnCl2 toxicity group, there was increase in the number of Target cells after 8 weeks (11.03 ± 0.94). The treatment became very effective in reducing the number of Target cells after 8 weeks post treatment reaching to (3.23 ± 0.64).
The number of Stomatocytes increased in the MnCl2 toxicity group (1.10 ± 0.24) at the end of experiment. In curcumin- treated group, after 8 weeks the number of Stomatocytes decreased (0.43 ± 0.13) compared to the toxicity group.
In MnCl2 toxicity group, the number of Schistocytes increased after 8 weeks (1.70 ± 0.34), the number of Tear DROP cells increased after 8 weeks (2.57 ± 0.47). In curcumin- treated group, after 8 weeks the number of Schitocytes (0.73 ± 0.16) and Tear DROP cells (1.70 ± 0.42) decreased compared to toxicity group.
In the present study, there was an increase in the number of Polychromaic cells of toxicity group after 8 weeks (2.50 ± 0.47). In curcumin- treated group, after 8 weeks the number of Polychromaic cells (1.37 ± 0.22) decreased compared to toxicity group.