الفهرس 
Introduction and Aim of the workOnly 14 pages are availabe for public view
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Abstract
The present work on male adult Sprague Dawley rats investigated the possible neuroprotective effect of sea urchin extracts (shell and spine) against the neurotoxicity induced by cisplatin.
The current study used various techniques for investigation such as, chemical studies, MTT assay for In vitro studies, behavioral studies, oxidative stress assays, histological, immunohistochemical and transmission electron microscope studies.
The rats were divided into 6 groups, 10 rats in each group, as following: group I: normal, group II: Cisplatin 2mg/kg twice a week for 4 weeks, group III: Urchin shell high (100 mg/kg) twice a week for 4 weeks then intoxicated with cisplatin as in group II. group IV: Urchin shell low (50 mg/kg) twice a week for 4 weeks then intoxicated with cisplatin as in group II, group V: Urchin spine high (100 mg/kg) twice a week for 4 weeks then intoxicated with cisplatin as in group II, group VΙ: Urchin spine low (50 mg/kg) twice a week for 4 weeks then intoxicated with cisplatin as in group II.
Rats in the above-mentioned groups were tested for the chosen behavioral parameters then sacrificed after 48 hours from the last dose, and brain was dissected out and cut into pieces for the oxidative stress, histological, immuno-histochemical studies, and transmission electron microscope.
Analysis of the mass spectra of the constituents of both urchin extracts with reference to NIST and WILEY standard library identified 15 (in spine) and 32 (in shell) compounds, respectively. Most of the separated compounds are phenolic compounds. Results of spine extract revealed that bisabol oxide (27.88%) and oleic acid (12.56%) were found to be major components, followed by hexadecenoic acid, ethyl ester (10.12%), and cholesterol (9.47%). While in shell extract eugenol (15.1%), mint lactone (8.09%), and levomenthol (7.36) were the major components.
The total antioxidant capacities of the shell and spine extracts were significantly high and increased in a concentration dependent manner from 50 µg/ml to 500 µg/ml.
The scavenging activity of the shell and spine extracts was tested at four different concentrations of 12.5, 50, 100, and 300 µg/mL using % DPPH inhibition method which gave 73-75 % inhibition percentages of DPPH• radical scavenging activity.
To determine the safety and toxicity of the sea urchin shell and spine extracts, they were tested on human neuroblastoma cells (SH-SY5Y) and fibroblasts (L929 cells). The results showed no toxic effects for the tested concentrations (25, 50, 100, and 200 µg/mL). The two extracts are safe and did not cause cell death. Both the spine and shell extracts showed a significantly high effect on cell viability and proliferation on the two cell lines.
Administration of cisplatin resulted in significant behavioral changes. A decrease in withdrawal latencies in heat tests from 15 seconds to 5 seconds was observed indicating the development of hyperalgesia. Treatment with both urchin extracts caused a significant improvement in withdrawal time, with best effect when using shell extract at the dose 100 mg/kg. The cold sensitivity test showed a dramatic decrease in the latency from approximately 30 seconds to about 5 seconds after injection with cisplatin. The administration of both urchin extracts shell doses (50-100 mg/kg) and spine dose (50 mg/kg) reversed the cisplatin effect and brought the withdrawal latencies back to normal levels. But the spine dose (100 mg/kg) showed a slightly lower effect.
Oxidative stress markers, MDA and NO, significantly increased in cisplatin-treated rats compared to the normal group. However, treatment with 50 and 100 mg/kg of spine and shell extracts restored the levels to the normal.
Catalase activity (CAT) and reduced glutathione levels (GSH) significantly decreased in cisplatin-treated rats compared to the normal group. However, there was no significant difference in CAT and GSH activities between the normal, spine (50-100 mg/kg), and shell (50-100 mg/kg) groups.
Histological investigation of normal group showed all types of neurons occupying the 6 layers of cerebral cortex appeared with normal histological distribution and homogeneous neuropil. Administration of cisplatin caused marked histological alterations in the tissue of the frontal cortex like congestion of the blood vessels of the pia mater, as well as cavitation of the neuropil and extracellular vacuolar degeneration. Most pyramidal cells were shrunken and had pyknotic nuclei, severe pericellular edema in addition to deeply stained neuroglial cells with pericellular halo.
Injection of cisplatin-treated rats with a low dose 50 mg/kg of shell extracts resulted in notable protection in the tissues of the brain. Also, with the dose 100 mg/kg of shell extract neuroprotection was apparent among cortical layers which were intact, embedded within intact neuropil.
Microscopic examination of the cerebral cortex from the group treated with low dose 50 mg/kg of the spine extract showed significant neuroprotection in the form of intact well-organized neurons among cortical layers with only few sporadic occasional records of neuronal injury. On the other hand, cerebral cortex in groups treated with high dose 100 mg/kg of spine extract demonstrated mild vacuolization and edema in the neuropil.
Electron microscopic examination of frontal cortex in normal brain tissues showed that pyramidal cells with large, rounded, central euchromatic nuclei surrounded by cytoplasm and large axons with the wide axon hillock containing many Golgi fields and mitochondria. The surrounding neuropil contains glial cell processes, and numerous cross sections of myelinated axons. In the cisplatin-treated group, a marked karyorrhexis of the nucleus surrounded with degenerated cytoplasm was noted associated with darkly stained coagulated mitochondria and highly dilated endoplasmic reticulum. The surrounding neuropil contained capillaries with irregular wall, irregular degenerated myelin sheaths which lost laminated pattern. Axons contained vacuoles while the whole neuropil suffered from cavitation.
Cisplatin-treated rats given low dose of shell extract showed nerve cells with a centrally located nucleus encircled by cytoplasm which contains scattered mitochondria, Golgi bodies, lysosomes, and intact rough endoplasmic reticulum. Only a few cisternae of the RER were mildly dilated, in addition to cavitation of neuropil. Rats given high doses of shell extract showed pyramidal cells with large, rounded, central euchromatic nucleus surrounded by cytoplasm and containing mitochondria lysosomes and intact RER. Also, a mild dilation in rough endoplasmic reticulum can be noticed. The neuropil appeared homogeneous and normal.
Frontal cortex of cisplatin-treated rats given the low and high doses of spine extract showed pyramidal cells with large, oval, central euchromatic nucleus surrounded by cytoplasm and having a large axon with RER, cisternae slightly dilated and Golgi bodies. The surrounding neuropil contained glial cell processes, in addition to longitudinal and cross sections of axons.
The normal healthy group showed very weak expression of Glial fibrillary acidic protein (GFAP) in the form of few intense cytoplasmic staining of astrocytes and other glial cells. While the cisplatin-treated group showed strong positive GFAP expression and hypertrophic astrocytes with numerous elongated processes indicating severe astrogliosis. In brain sections of rats treated with cisplatin followed by low and high doses of shell extracts displayed mild GFAP expression. In cisplatin-treated rats injected with low and high doses of spine extracts, GFAP stained sections revealed moderate expression.
The normal group showed positive expression with the anti-apoptotic marker B-cell lymphoma 2 (Bcl2), in the form of intense cytoplasmic staining of neurons and oligodendrocytes, while examination of cisplatin-treated group showed a negative Bcl2 reaction in most apparent neurons. On the other hand, low and high doses of shell extracts increased the expression of Bcl2 in most apparent neurons. In cisplatin-treated rats injected with low dose of spine extract showed high positive expression of Bcl2, while the high dose of spine extract showed moderate expression of Bcl2.
Conclusions
Results obtained from this study highlight the significant positive effect of the urchin extracts against cisplatin-induced neurotoxicity in rats.
• Administration of adult male rats with 50 and 100 mg/kg of urchin extracts (shell-spine) prohibit and protect the cortical degeneration induced by cisplatin. The neuroprotective potential of the urchin extracts may be due to:
• Presence of biologically active phenolic compounds in the urchin spine and shell extracts.
• The total antioxidant capacity assay of urchin extracts confirmed the good antioxidant capacity of all extracts.
• The tendency of urchin extracts to scavenge DPPH radicals that initiates lipid peroxidation.
• The potential safety of the extracts, proved by the In vitro studies using MTT assay.
o The obtained results highlight the protective effect of sea urchin which was showed by the enhanced behavioral activities.
Supplementation of urchin extract in low and high doses prevented the cisplatin-induced neurotoxicity in rats. The protective effect is due to antioxidant property of urchin extracts which prevents the formation of free radicals.
Histological and immunohistochemical studies of the brain tissues have shown substantial improvement in the histological architecture after treatment with urchin extracts; reversed the immunohistochemical expression of GFAP and Bcl2 to the normal levels.
Ultrastructural studies proved that cisplatin caused some subcellular damage, however both spine and shell extracts exerted noticeable protective effects and disappearance of most of the injuries and organelle damages.
Recommendations
The present results suggest that extracts of sea urchin have effective pharmacological components with properties that candidate them to develop emergent neuroprotective dugs for cancer patients.