Cisplatin is an effective antineoplastic drug. It has been used to treat ovarian, testicular, and bladder carcinoma. However, a serious disadvantage of this drug is the induction of various severe side effects in kidneys, bone marrow and liver. Cisplatin if administrated during pregnancy may cause many malformations including ventriculomegaly, microphthalmous and polyhydraminos. Neural tube defects are a multifactorial disorder, arising from a complex combination of genetic and environmental interactions, and while are only now beginning to understand the etiologies of neural tube defects, significant advances have been made in their prevention and treatment by taking folic acid (FA) during child-bearing years . Based on animal studies, epidemiologic studies and intervention trials, maternal folic acid is known to be protective for neural tube defects, primarily spinabifida and anencephalus.
This study was aimed to assess the prenatal and postnatal effect of cisplatin on spinal cord, dorsal root ganglion &sciatic nerve and to investigate the neuro-protective effect of folic acid against prenatal & postnatal cisplatin neurotoxicity.
Sixty adult female albino rats were used in this study. Animals were placed in quarantine for one week prior to breeding and housed in separate plastic cages in a controlled environment with free access to water and balanced diet. After acclimatization to laboratory conditions, two females were kept overnight with one male to allow mating; the presence of sperms in vaginal smear in the next morning indicates the day zero of pregnancy.
Two drugs were used in this study:-The cisplatin in the form of a vial contains 50 mg liquid cisplatin. . Every rat was injected intraperitoneally by 1 mg cisplatin /kg once every other day and this was equivalent to the human low therapeutic dose. The folic acid is in the form of 800 microgram tablets powdered and dissolved in 10 ml of distilled water, each rat was given 5ml of the solution once daily through a gastric tube.
Sixty pregnant rats were divided into two equal groups:
I- Prenatal group: contained thirty pregnant rats and was subdivided into three equal subgroups.
a. Prenatal control subgroup: received saline from 1st day of pregnancy till the time of sacrifice at 18th day& 20th day of pregnancy.
b. Prenatal cisplatin subgroup: were injected intraperitoneally with the therapeutic dose (l mg/Kg) of Cisplatin on alternate days from 1st. day of pregnancy till the time of sacrifice at 18th day& 20th day of pregnancy.
c. Prenatal cisplatin & folic acid subgroup: were injected intraperitoneally with (I mg/Kg) of Cisplatin (every other day) Plus folic acid in a dose of (400microgram) daily by the nasogastric tube from 1st day of pregnancy till the time of sacrifice at 18th day& 20th day of pregnancy.
The fetuses were obtained by caesarian section then the specimens of the spinal cord, dorsal root ganglion and sciatic nerve were obtained as by dissection then the specimens were then prepared for light and electron microscopic examination
II- Postnatal group: contained thirty pregnant rats and was subdivided into three equal subgroups.
a- Postnatal control subgroup: The pregnant rats were allowed to complete pregnancy. Their pups received saline from 1st day of delivery till being sacrificed on the 7th Day,14th Day & 21st day postnatally
b- Postnatal cisplatin subgroup: The pregnant rats were allowed to complete pregnancy. Their pups were injected intraperitoneally with the therapeutic dose of Cisplatin (l mg/Kg) on alternate days from 1st day of delivery till being sacrificed on the 7th Day, 14th Day & 21st day postnatally.
c- Postnatal cisplatin & folic acid subgroup: The pregnant rats were allowed to complete pregnancy. Their pups were injected intraperitoneally with the therapeutic dose of Cisplatin(I mg/Kg) every other day plus folic acid in a dose of (400microgram) daily given to the pups through a gastric tube from 1st day of delivery till being sacrificed on the7th Day,14th Day & 21st day postnatally .
The pups were anaesthetized by ether inhalation and the specimens were obtained by dissection. The specimens of the spinal cord, dorsal root ganglion and sciatic nerve were prepared for light and electron microscopic examination
In the present study, cisplatin has a toxic effect on the developing spinal cord. This toxicity is represented as degeneration in the grey and white matter. The shape of the spinal cord was severely affected and this effect ranges from disfigurement of the grey matter in fetuses aged 18 days to shrinkage of the spinal cord in pubs aged 14 days to end in complete degeneration of the cord which is replaced by large vacuoles and spots of hemorrhage in rats aged 21 days. Ultrastructure examination confirms this toxicity as the sections of the spinal cord show distorted neurons with eccentric piknotic nuclei, these nuclei consists mainly of heterochromatin and surrounded by irregular nuclear membrane, the cytoplasm is full of vacuoles and dilated rough endoplasmic reticulum and swollen mitochondria. In the current study, the folic acid can alter the cisplatin toxicity of the spinal cord as the specimens received folic acid during treatment with cisplatin show signs of improvement as, the vaculations in the grey and white matter decreased and the size of the spinal cord increased and its outlines are regular. The ultrastructure confirms that as, the amount of euchromatin increased in the nuclei, these nuclei have regular nuclear membrane. The cytoplasm of theses neurons is full of healthy mitochondria but some of them are swollen. The myelinated axons have no vacuoles in the myelin sheath or shrinkage of the axoplasm.
Regarding the dorsal root ganglia, in this study, the cisplatin has a toxic effect on the dorsal root ganglia. This toxicity appears in a mild form in the prenatal groups which show that the ganglia have dark neurons with small nuclei; there are wide spaces separate the neurons and the nerve fibers. Then the toxicity became sever on the older ages as ganglia of rats aged fourteen and twenty one days show that some neurons are piknotic with dark nuclei and others are degenerated with chromatolytic nuclei and the neuroglia are accumulated on one side as residual bodies the nerve fibers show large vacuoles. The ultrastructure of the ganglia confirmed the cisplatin toxic effect as the neurons are shrunken with pale nucleus and irregular nuclear membrane, the cytoplasm has large vacuoles and the mitochondria are the most subjected organ for the toxicity as these mitochondria are swollen and vacuolated. In this study, the folic acid has a minor protective role on dorsal root ganglia against cisplatin toxicity, as the specimens of the dorsal root ganglia that received folic acid during cisplatin therapy show that some neurons have large pale nucleus and prominent nucleolus. The ultrastructure of the dorsal root ganglia confirmed that the groups received folic acid show little improvement as the nuclear membrane of the neurons in these groups is more regular than that of the cisplatin group but the cytoplasm has many vacuoles these vacuoles decreased in older ages.
Regarding the sciatic nerve, the cisplatin has neurotoxic effects on the sciatic nerve; these toxic effects appear in the prenatal groups as multiple vacuoles and spots of hemorrhage in the nerve fibers. The signs of toxicity also appear in the postnatal groups that show swollen and degenerated axons with few Schwann cells and fibrosis in the nerve fibers with excess fibrous tissue in the perineurium. The ultrastructure confirms the cisplatin neurotoxicity as the sciatic nerve of the prenatal groups treated by cisplatin show unmyelinated fibers with vacuolated axoplasm, the axoplasm of these fibers contains few mitochondria. In this study the postnatal groups show shrinkage of axoplasm of the myelinated axons with few mitochondria and neurofibrils and vacuoles in the myelin sheath.
In this study the folic acid cannot protect the sciatic nerve against cisplatin toxicity as the prenatal groups received folic acid during cisplatin therapy show spaces in the nerve fibers. The postnatal groups show multiple vacuoles in the nerve fibers with areas of fibrosis some nerve fibers are degenerated. The ultrastructure confirmed that the folic acid cannot protect sciatic nerve against toxicity as the groups treated with folic acid during cisplatin therapy show shrinkage of the axoplasm with few mitochondria, neurofibrils and vacuoles in the axoplasm the surrounding myelin sheath is thin.
Cisplatin has neurotoxic effects on the developing spinal cord, dorsal root ganglia and sciatic nerve. Folic acid can protect the spinal cord and dorsal root ganglia against these toxicities but cannot protect the toxicity of the sciatic nerve. So the pregnant female should be supplied by adequate folic acid doses during cisplatin therapy.
We recommend further studies concerning other drugs that may prevent cisplatin neurotoxicity.