الفهرس 
List of contentsOnly 14 pages are availabe for public view
 |  | from | 314 |  |  |
| | | from | 314 | | |
Abstract
The cerebellum is an important part of the circuitry that links sensory to motor areas of the brain, and it functions to coordinate movement. It provides corrections during movement, which are the basis for precision and accuracy, and it is critically involved in motor learning and reflex modification.
Cisplatin, (a platinum-based antineoplastic agent), is one of the most effective chemotherapeutic drugs used in the treatment of solid tumors, However, nephrotoxicity, hepatotoxicity, neurotoxicity and ototoxicity have been reported to be some of the adverse side effects upon the use of cisplatin. Cisplatin appears to affect the axons, myelin sheath, neuronal cell body and the glial structures of the neurons, interferes with DNA replication and metabolic function of the neurons.
Alpha-lipoic acid (ALA) is a naturally occurring dithiol compound that functions as an essential cofactor for mitochondrial bioenergetic enzymes. It has also been recognized as a powerful antioxidant capable of prevention or treatment of many diseases associated with oxidative stress, such as diabetes, chronic liver diseases, and neurodegenerative processes. Alpha-lipoic acid (ALA) is a naturally occurring substance that
acts as an essential cofactor for various enzymes required for oxidative
metabolism.
In this study two experiments was done:
1-Experiment (I): It was performed to study the development of the cerebellum and the effect of cisplatin, and its combination with alpha lipoic acid on its development. In this study 50(40 female and 10 male) adult( three months old) albino rat (Wister strain) were used in the study. The animals were left to mate with each other and we observed the presence of vaginal plug and its time of presence is considered the first day of pregnancy. Then , the animals were equally divided into 4 groups (10 animals each):
2- The first group : (control group)
2-The second group :(cisplatin treated group): animals were treated with cisplatin at a dose of 4 mg/ kg body weight once weekly by intra peritoneal injection started from the sixth day of pregnancy and continued till the end of lactation period of rat (20 days ).
3-The third group :( alpha lipoic acid treated group): animals were treated with α lipoic acid at a dose of 100 mg/ kg /day by intra peritoneal injection started from the beginning of pregnancy till end of lactation (20 days).
4-The fourth group: This group was treated with both cisplatin and alpha lipoic acid using the same doses.
At the end of pregnancy some of the offspring was sacrificed at the following ages, new born (18 rat), tenth day(18 rat) and 20 days old rats(18 rat).
-Experiment (II): was done to study the aging changes of the cerebellum and the effect of cisplatin, and its combination with alpha lipoic acid on the aged cerebellum.
This study was done on :
B- Adult group: 18 adult Albino rats (3 months old Wister strain). They considered as a control for the aged rats. They received no treatment.
B- A total number of 60 rats (18 months old) albino rat (Wester strain) were equally divided into:
1-Control aged group :in which the animals received no treatment.
2-Cisplatin treated aged group :in which animals were treated with cisplatin at a dose of 4 mg/ kg body weight once weekly by intra peritoneal injection for 2 month till reaching 20 month old.
4- Both cisplatin and alpha lipoic acid treated aged group: in which animals were received both cisplatin as previous group and alpha lipoic acid in which animals will be treated with α lipoic acid at a dose of 100 mg/ kg /day by intra peritoneal injection for 2 month.
In both developmental groups and aged group, 6 rats in all studied age groups were processed for Gallocyanine chrom –alum staining technique to study the cytoarchitecture of the cerebellum. Transmission electron microscopic study was done in which 6 animals were used in all age groups to show the ultra structure of the cerebellar cortex. Furthermore, immunohistochemical study using anti- Glial fibrillary acidic protein was performed. Morphometric study was done to study the thickness of cerebellar cortex and volume proportion in all age groups. In addition, nuclear diameter of pyrkinje cell in the cerebellar cortex and cell number of pyrkinje and granule cells were measured. The morphometric study was done on paraffin sections using 6 animals in each age group. The nuclear diameter was done on semithin sections. The parameters were statistically analyzed.
In the experiment (I) the present study revealed that the cerebellum of new born rat of control group is composed of cerebellar cortex and white matter containing deep cerebellar nuclei. The external granular layer was observed as the most superficial layer of cerebellar cortex just beneath the pia matter. It is formed of 2 zones which are the multiplying zone which located just beneath the pia matter and the other layer is the premigratory zone which located more deeply and its cells migrate to the internal granular layer. The pyrkinje cell layer appeared to be formed of several rows of pyrkinje cells.
The fourth layer was the internal granular layer whose cells are small and rounded with well defined nuclei. At the 10 days old control group, the developing cerebellar cortex still shows the presence of external granular layer. There was apparent increase in the size of the molecular layer. The pyrkinje cell layer at this age is characterized by the presence of pyrkinje cell somata which still arranged in several rows. The pyrkinje cell body is pear shape containing apical cytoplasmic cone with euchromatic nucleus. The presynaptic terminal making synaptic contact with the pyrkinje cell is characterized by the presence of many synaptic vesicles and mitochondria. The ultrastructure of the granule cells shows that the cells have oval nuclei with peripheral chromatin condensation.
The present study at the age of 20 days old of control group reveals that the cerebellar cortex is differentiated in to molecular layer, pyrkinje cell layer and granular layer . The external granular layer is nearly disappeared with the exception of few cells can be identified. The molecular layer has few scattered cells. In this age, the pyrkinje cells are arranged in one row which is characteristic of its maturity. Its nucleus is oval in shape with uniformly distributed chromatin. The granule cells in the internal granular layer appears to be arranged in clusters. The cells have well defined nuclei. Immunohistochemical examination using anti GFAP of control group at 10 days and 20 days old rat, shows the presence of weak expression of anti GFAP in all layers of the cerebellum.
The cells of the deep cerebellar nuclei at the newborn rat of the control group appears to be non differentiated.At 10 days old rat of the control group, the nuclei are differentiated into 2 groups which are the caudal group which formed mainly of fastigial nucleus and the rostral group which is formed mainly of the dentate nucleus. At 20 days old rat of control group, the cells of the cerebellar nuclei are completely differentiated in which the fastigial nucleus is formed of small rounded cells with well defined nuclei and large cells with oval shaped nuclei. The cells of dentate nucleus are divided into magnocellular part in which the cells are oval in shape with prominent nucleus and parvocellular part in which the neurons are rounded in shape .
In cisplatin received group at the age of new born, the external granular layer appears to be thicker in comparison with control group. Both pyrkinje cell layer and internal granular layer show degenerative changes. The nucleus of pyrkinje cell has irregular nuclear membrane. Its cytoplasm contained dense bodies and many vacuoles. The cells of internal granular layer in this group appear to have vacuolated cytoplasm and darkly stained nuclei.
The age of ten days old of cisplatin group , shows increase in the thickness of external granular layer in comparison with the control. Most of cells of pyrkinje layer are faintly stained . Also, many cells of the internal granular layer have degenerative changes. The examination of pyrkinje cell with electron microscope reveals that the nucleus shows chromatin condensation with irregularity in the nuclear membrane.
In cisplatin received group at the age of 20 days, there is marked increase in the thickness of the external granular layer is still present and it appears to be formed of several rows. Many of pyrkinje cells had darkly stained nuclei. Examination of the presynaptic terminal at 10 and 20 days rat whose mother received cisplatin , reveales marked decrease in the synaptic vesicles and the presence of damaged mitochondria.The granule cells in the internal granular layer had condensed chromatin. All layers of the cerebellar cortex at 10 and 20 days of cisplatin received group show intense immunostaining with extensive network of fibers and many glial cells in pyrkinje cell layer and granular cell layer using anti- GFAP immunostaining.
In new born rat of cisplatin received group, the neurons of deep cerebellar nuclei appear to have darkly stained nuclei. At the age 10 and 20 days of cisplatin received group, cells of fastigial nucleus appear to have darkly stained nuclei. Also, neurons of dentate nucleus are widely dispersed and lightly stained due to reduction of Nissl granules.
At the developmental group which received both cisplatin and alpha lipoic acid , The ultrastructure of the pyrkinje cell at the age of newborn, 10 days and 20 days old reveals that the cells have oval nuclei with uniform chromatin distribution and the cytoplasm contains free ribosomes and mitochondria. However, it also shows the presence of some vacuoles and dense bodies. The presynaptic terminal making synaptic contact with pyrkinje cell at age of new born , 10 days and 20 days old shows a lot of synaptic vesicles and mitochondria which is comparable to that of the control. Examination of the granule cells at the age of new born , 10 days and 20 days old shows rounded nuclei and prominent nucleoli .Some of the cells appear to have darkly stained nuclei.
At new born ,10 days ,20 days old rat of both alpha lipoic acid and cisplatin received group, the cells of cerebellar nuclei have normal appearance with few cells which are darkly stained. There is reduction in the immunoreactivity in all layers of the cortex as compared with cisplatin received group at 10 and 20 days old rat.
In the experiment (II), the cerebellar cortex of the adult control group was differentiated into the molecular layer, pyrkinje cell layer and granular layer. The nucleus of the pykinje cell is oval in shape with uniformly distributed chromatin .The granule cells at this age group are small and rounded with well defined nuclei and spaces between cells. The cells of the fastigial and dentate nuclei have rounded nuclei with prominent nucleolus and lot of Nissl granules in the cytoplasm.
The nucleus of the pyrkinje cells of the control aged group have uniformly distributed chromatin with irregularity in the nuclear membrane. The cytoplasm also contains vacuoles. The granule cells are arranged in groups and some cells had darkly stained nuclei and lightly stained cytoplasm.The cells of the fastigial and dentate nuclei had prominent nucleus and nucleolus .Some cells have darkly stained nuclei.
Examination of pyrkinje cell nucleus of cisplatin treated group shows marked irregularity of nuclear membrane. The cytoplasm contains vacuoles and some lipofuscin pigment. The granule cells had darkly stained nuclei and vacuolated cytoplasm. The cytoplasm of granule cells appears to be rarified with the presence of lipofuscin pigment. The fastigial and dentate nuclei have darkly stained nuclei and vacuolated cytoplasm. There are extensive immunoreactive cells and processes in all layers of the cerebellar cortex as compared with the control aged group.
In cisplatin and alpha lipoic acid received group, most of the pyrkinje cells have prominent nucleus and nucleolus .Some cells are lightly stained and have vacuolated cytoplasm. Most of the granule cells have normal appearance. The ultrastucture of both pyrkinje and granule cells appear nearly similar to that of the aged control rat.The presynaptic terminal contains mitochondria and synaptic vesicles. Some cells have darkly stained nuclei. Most of the cells of fastigial and dentate nucleus have normal appearance with some cells are lightly stained with vacuolated cytoplasm. There is apparent decrease in the immunoreactivity in comparison with group which received cisplatin only.
In conclusion, the cisplatin has neurodegenerative effect on the cerebellum of both developmental and aged groups. The alpha lipoic acid has antioxidant effect against the degenerating effect of cisplatin when it is used in combination with cisplatin.
So , it is recommended to combine the alpha lipoic acid with cisplatin to reduce the neurotoxic effect of cisplatin.