Study of the Effect of Prenatal Administration of Pregabalin on Cerebellar Cortex of Albino Rat's Offspring and the Possible Protective Role of Folic Acid

INTRODUCTION

The incidence of congenital malformations has increased because of the use of traditional anti-epileptic drugs during gestation (e.g., valproate, phenytoin, carbamazepine, and phenobarbital). Thus, it is necessary to find new safer drugs for the mother and the fetus to lower the incidence of these complications. This may need adequate clinical and experimental studies.¹

Pregabalin is the latest compound that joins the list of approved new AEDs. The European Commission granted Pfizer Company the approval for pregabalin in July 2004 for the treatment of variable types of peripheral neuropathic pain and in the same year, it was approved as adjunctive therapy for epilepsy by the American Food and Drug Administration (FDA).²

It has been shown that pregabalin can cross easily the placenta and the blood-brain barrier in rats, mice, and

monkeys. Therefore, pregabalin is an important drug that can influence the development of the nervous system after birth.³

The cerebellum of mammals has very interesting histological events during the nervous system development. Therefore, the cerebellum is an ideal model for studying many aspects of neural development, because each stage of its development has a distinct morphology and special histological features with different types of cells. As well as the process of cerebellar development is not complete during gestation only, but also, continues after birth.⁴

Pregabalin has been shown to have harmful effects on the developing cerebellum when given in higher doses. It causes loss of cellular components and distortion of the cerebellar cortical layers that may affect the physiological functions of the cerebellum.⁵

Investigations showed that the incidence of malformations of the nervous system and musculoskeletal system has decreased with supplementation of folic acid during gestation, while folic acid deficiency during gestation was associated with a wide spectrum of congenital malformations.⁶

Therefore, this study was carried out to investigate the folic acid’s protective potential against the postnatal histological changes in the cerebellar cortex of albino rat’s offspring due to pregabalin administration during gestation.

MATERIAL AND METHODS

Pregabalin capsules 300 mg were supplied from Pfizer-Egypt Company. In 37.5 ml of distilled water, 10 capsules of pregabalin 300 mg were dispersed to produce a suspension containing 80 mg of pregabalin/ ml of distilled water. Folic acid tablets 500 μg were supplied from MEPACO-MEDIFOOD Company, Egypt, Cairo. In 25 ml of distilled water, 5 tablets of folic acid 500 μg were dissolved to produce a suspension containing 100 μg of folic acid / 1 ml of distilled water.

Sixty-six adult albino rats (forty-four female and twenty-two male albino rats) were selected for this study with their weight ranged between 200gm and 250 gm and their age ranged between 4 and 5 months old. After acclimatization to the laboratory conditions for one week, every two female rats were kept with one male rat and left overnight in a separate cage to allow mating. Early in the next morning, copulation was confirmed by vaginal smear. The copulation was confirmed in thirty female albino rats. The gestational day zero was defined when spermatozoa were observed in a smear of the vaginal contents.

Twenty-four pregnant rats were selected for the present study and divided into (group I, group II and group III). Each group comprised of eight pregnant rats. Pregabalin was given to group II. Pregabalin and folic acid were given to group III. No medications were given to group I. The Pregabalin dose was 80 mg/kg B.w /day (about 1ml of the produced suspension),⁷ and the Folic acid dose was 400 μg/kg B.w /day (about 4 ml of the produced suspension).⁸ The medications were given by oral gavage, once daily, from gestational day one till birth.

30 offspring born to the three experimental groups, were sacrificed at two and four weeks after birth (5 offspring from each group for each age), therefore two subgroups for each group were formed as follow: Subgroup I-A of 5 rats; sacrificed at 2 weeks old,  Subgroup I-B  of 5 rats; sacrificed at 4 weeks old, Subgroup II-A of 5 rats; sacrificed at 2 weeks old, Subgroup II-B  of 5 rats; sacrificed at 4 weeks old, Subgroup III-A of 5 rats; sacrificed at 2 weeks old and Subgroup III-B  of 5 rats; sacrificed at 4 weeks old. The cerebellum was carefully dissected out from the skull of each rat and prepared for examination by the light and electron microscopes.

This study was carried out in accordance with the NIH recommendations and guidelines for the use and care of laboratory animals with approval from the ethics committee of the Faculty of Medicine, Al-Azhar University, Cairo, Egypt.

For examination by the light microscope, samples from the cerebellum were taken from each hemisphere and cut into small pieces (about 2-4 mm³), put in Bouin’s solution for fixation, and then proceeded to be embedded in the paraffin wax. Sections of 5-micron thickness were obtained for staining with hematoxylin and eosin (Hx & E), ⁹ also; some of these sections were used for glial fibrillary acidic protein (GFAP) immune-reaction.¹⁰

For examination by the electron microscope, small pieces (about 1 mm³) were obtained from the cortices of each cerebellar hemisphere, put in 2.5% glutaraldehyde for fixation, and then prepared for embedding in epon resin. Semithin sections of 1μm thickness were obtained and stained with toluidine blue. The ultrathin sections of (50 nm) thickness were obtained and mounted on grids of copper to be ready for staining with lead citrate and uranyl acetate.¹⁰ the examination of the sections was carried out by using a transmission electron microscope. Photomicrographs were taken under different magnifications to detect the ultrastructure of the cerebellar cortex.

Histomorphometric measurements were conducted on Hx & E and GFAP stained sections in different random fields with a fixed area (about 4 X 104 μm²). The density of Purkinje cells and the percentage of the area of GFAP immune reaction were measured in magnification X400. The measurements were obtained in non-overlapping 10 fields for each specimen by using an image analyzer computer system, V4.8, Leica LAS, Switzerland.

The collected data by the histomorphometric measurements, from the three experimental groups, was first represented as means with standard deviations (±SD), and then statistically analyzed by using SPSS 22 program. A comparison between the experimental groups was carried out by using ANOVA test. A comparison of significance was made between the subgroups of group I and the other subgroups. P-value was considered significant when its value ≤ 0.05 and was considered highly significant when its value ≤ 0.001.

RESULTS

Results of the light microscope examination: Histological evaluation of Hx & E stained sections of subgroups I-A and I-B showed the normal cerebellar cortex morphology. The layer of Purkinje cells was observed containing large oval cells arranged in on row between the molecular and the internal granular layers. Their nuclei appeared large, vesicular in the pale cytoplasm. The molecular layer was observed with a small number of the basket and stellate cells. The internal granular layer was observed containing granule cells. These cells were small and deeply stained. In subgroup I-A, the external granular layer comprised many layers of small darkly stained cells. These cells were closely packed. In subgroup I-B, the external granular layer comprised one or two layers of cells (Figures 1a, 1b). Degenerative changes were observed in the cerebellar cortices of the offspring of group II (subgroups II-A, II-B). These changes included depletion of Purkinje cells that appeared deformed, shrunk, and surrounded by vacuolated spaces. The external granular layer displayed deeply stained pyknotic cells. Closely packed cells with small size and pyknotic changes were seen in the internal granular layer. Some spaces between these cells were seen congested. Basket and stellate cells were observed within the molecular layer with pyknotic changes; also, some vacuolated areas were

seen in this layer. There was a hemorrhage between the folia of the cerebellum (Figures 1c, 1d).

Improvement in the morphology of the cerebellar cortices of subgroups III-A and III-B was noticed. The oval-shaped Purkinje cells were observed again as in group I with vesicular nuclei in granular cytoplasm. The molecular layer was observed with a scattered basket and stellate cells, and with minimum vacuolated areas. Besides, normal granule cells were observed in the internal granular layer. (Figures 1e, 1f).

Fig. 1: Photomicrographs of Hematoxylin and eosin-stained sections of the cerebellar cortex of albino rat. a = 2weeks old rat (subgroup I-A), b = 4weeks old rat (subgroup I-B), c = 2 weeks old rat (subgroup II-A), d = 4 weeks old rat (subgroup II-B), e = 2 weeks old rat (subgroup III-A), f = 4 weeks old rat (subgroup III-B) (all photomicrographs with magnification X 400).

Results of GFAP immune reaction: Sections of subgroups I-A and I-B showed the weak immune reaction of GFAP in the form of small areas of brown colure (Figures 2a, 2b). Sections of subgroups II-A and II-B showed the strong immune reaction of GFAP indicating large areas of gliosis due to astrocyte proliferation (Figures 2c, 2d). Sections of subgroups III-A and III-B showed the moderate immune reaction of GFAP (Figures 2e, 2f).

Fig. 2:  Photomicrographs of sections of GFAP immune reaction of the cerebellar cortex of albino rat. a = 2 weeks old rat (subgroup I-A), b = 4weeks old rat (subgroup I-B), c = 2 weeks old rat (subgroup II-A), d = 4 weeks old rat subgroup II-B), e = 2 weeks old rat (subgroup III-A), f = 4 weeks old rat (subgroup III B) (all photomicrographs with magnification X 400).

Results of the electron microscope: Examination of the cerebellar cortex of subgroups I-A and I-B, showed normal Purkinje cells with intact cell membranes and normal axon hillocks. The nucleus was seen normal with dispersed chromatin and clear nucleolus. A large number of mitochondria were observed in the cytoplasm. There were a large number of axons, some were myelinated and some were unmyelinated. The axons were seen contained many neurofilaments and mitochondria. The astrocyte nucleus was seen with the intact nuclear membrane and regularly distributed euchromatin. There was a microglial cell with an oval indented nucleus (Figures 3a, 3b). Examination of the cerebellar cortex of subgroups II-A and II-B showed Purkinje cells surrounded by irregular cell membranes and containing shrunken nuclei with small eccentric nucleoli. The nuclear envelopes of these nuclei were irregular and the chromatin material was rarefied. Their cytoplasm contained many vacuolations, many degenerated mitochondria, and rough endoplasmic reticula that were dilated. Also, many degenerated axons were completely vacuolated. A degenerate microglial cell was seen surrounded by degenerated axons. The granule cells appeared degenerated with abnormal nuclei and clumping of their chromatin material (Figures 3c, 3d). Examination of the cerebellar cortex of subgroups III-A and III-B showed intact Purkinje cells. Their nuclei were noticed normal and surrounded by a regular, intact nuclear envelope. The cytoplasm appeared nearly normal containing many mitochondria. A microglial cell was seen containing dense cytoplasm and an irregular nucleus. There were many normal granule cells with normal nuclei and condensed chromatin material. There were many normal axons and few degenerated axons, which were completely vacuolated (Figures 3e, 3f).

Figure 3: Electronmicrographs of the cerebellar cortex of albino rat. a = 2weeks old rat (subgroup I-A) (TEM X 4000), b = 4weeks old rat (subgroup I-B) (TEM X 3000), c = 2 weeks old rat (subgroup II-A) (TEM X 5000), d = 4 weeks old rat (subgroup II-B) (TEM X 4000), e = 2 weeks old rat (subgroup III-A) (TEM X 3000), f = 4 weeks old rat (subgroup III-B) (TEM X 4000).

Histomorphometric results: there was a highly significant decrease in the mean density of Purkinje cells in subgroups II-A and II-B   when compared with the same subgroups of group I, while a non-significant decrease was reported in the mean density of Purkinje cells in subgroups III-A and III-B when compared with the same subgroups of group I (Table I).

 Table I: Showing the mean density of the Purkinje cells of the cerebellar cortices of all experimental subgroups. (**) Indicate a highly significant difference.

A high significant increase in the area percentage of GFAP immune reaction was detected in subgroups III-A and III-B when compared with the same subgroups of group I. Meanwhile, a significant increase in the area percentage of GFAP immune reaction was detected in subgroups III-A and III-B when compared with the same subgroups of group I (Table II).

Table II: The mean area percentage of GFAP immune reaction in the cerebellar cortices of all experimental subgroups. (**) Indicate a high significant difference (*) Indicates a significant difference.