Cerebellar Toxicity Induced by Maternal Exposure to Topical Betamethasone Prior to Fertilization in Newborn Rabbits

Topical glucocorticoids are used for treatment of psoriasis, mainly due to their immunosuppressive, anti-inflammatory and antiproliferative properties (Castela et al., 2012) as endogenous biosynthesis of glucocorticoids is suppressed in psoriatic skin (Sarkar et al., 2017).
       
Endogenous and synthetic glucocorticoids regulate epidermal development and combat skin inflammatory diseases (Bigas et al., 2018). So, glucocorticoids are considered as the first line treatment for all grades of psoriasis (Laws and Roung, 2010). Betamethasone is more effective than any other steroids (Ni et al., 20l8). It is well known that the highest level of glucocorticoid receptors is present in the cerebellum, so cerebellum is an important area of study (Pavlik et al., 1984).
       
Glucocorticoids (GC) act throughout the body via the widely expressed glucocorticoid receptor and the mineralocorticoid receptor (Pujols et al., 2002). GC binding to its receptors results in a wide array of genomic and non-genomic signaling changes at the cellular level (Kadmiel and Cidlowski, 2013).
       
Corticosteroids have widespread pharmacological effects mediated via gene transcription and protein expression effects in many organ systems. This leads to both desired fetal effects such as increased protein production in the lungs with resulting phospholipid biosynthesis and the appearance of surfactant (Ballard and Ballard, 1995), potentially less desirable effects such as decreased cerebral blood flow that could be linked to adverse neurodevelopment (Schwab et al., 2000).
       
In this regard, some animal and human-based studies could confirm the beneficial effects of corticosteroids, but some others did not recommend such regimens due to their related adverse consequences (Arimi et al., 2021). There is considerable evidence from experimental animals that corticosteroids can have an adverse effect on the growth and development of the immature brain (Whitelawa and Thoresen, 2000).
       
Prenatal glucocorticoids increase the susceptibility of cerebellar neurons to oxidative cell death (Fuentes-Pardo et al., 1990) and glucocorticoids therapy may disrupt cerebellar development through the rapid induction of apoptosis (Noguchi, 2014). Hypercorticosteronemia induced brain region-specific changes that might include aberrant myelination and a degree of white matter damage (Amya et al., 2021).
       
Glucocorticoids play a pivotal role in fetal programming (Czamara et al., 2021). Although, there are no enough studies about the risk or potential alterations due to accumulation of Betamethasone before fertilization. In addition, previous studies paid attention to intramuscular injection during pregnancy neglecting the long-term impact of the use of topical solution. So, the goal of the present study was to determine these adverse side effects topical administration of betamethasone solution on liver and kidney tissues.
       
Therefore, the goal of the present study was to establish whether maternal topical exposure to betamethasone, 2 months before fertilization, would induce alterations in new born rabbits.

Experimental animals
 
Twenty-four adult healthy sexual mature, White New Zeeland rabbits Oryctolagus cuniculus of both sexes (6 males and 18 females) of average weight of 2.5±0.5 kg, 6-8 months obtained from research station in king Faisal University. The animals were housed in steel cages under controlled condition like temperature 25±2°C and 12 hrs. light /dark cycle with free access to water, labium and pellet food diets. They were left for two weeks for acclimatization before starting the experiment. All experimental procedures were reviewed and approved by the research ethics committee at King Faisal University (Ref. No. KFU-REC/2021-03-05). They were housed in the animal house of College of Science, Building 9, King Faisal University.
 
Experimental design
 
The experiment involved three experimental groups as follows:
 
G I (Control group)
 
In this group, female rabbits (n=6) were topically exposed-on the shaved area of back skin -to the saline solution for 2 months and allowed to mate with males and then kept until delivery.
 
G II
 
In this group, female rabbits (n=6) were topically treated with betamethasone solution at a dose of 0.02 mg/kg body weight on the shaved area of back skin for 2 months, allowed to mate with males and then kept until delivery.
 
G III
 
In this group, female rabbits (n=6) were treated with betamethasone solution at a dose of 0.20 mg/kg body weight on the shaved area of back skin for 2 months, allowed to mate with males and then kept until delivery. 
 
Mating
 
Mating process was done according to the method of Wangikar et al., (2005).
 
Application of medication
 
Betnovate solution - Scalp application 30 ml -Betamethasone 0.1% w/w (as Betamethasone Valerate) was topically applied on shaving area of back skin (5 ´ 5), for 2 months according to Vose et al. (2014).
       
At the end of the experimental period (after 3 weeks of delivery), six newborn rabbits of both sexes (1 newborn rabbit per mother) were dissected under light anesthesia. The animals were euthanized anesthetic exsanguination using the combination of 10 mg/kg xylazine and 100 mg/kg ketamine HCl. The blood samples (5 ml) were collected aseptically through vein puncture from lateral cephalic vein of each rabbit. Cerebellar tissues were collected and kept frozen at -80°C before assays for 24 hours. Collected cerebellar tissue samples were used for biochemical and histological evaluations. All the measurements were performed in one assay.
 
Determination of bioaccumulation of betametathone in sera of rabbits mothers treated with betamethasone (2 monthes before fertilization)
 
Evaluation of betamethasone concentrations in the blood of female rabbits, treated with betamethasone for 2 months before fertilization, were done every 7 days during the period of treatment using commercial REAGEN betamethasone ELISA Test Kit Manual, which is a competitive enzyme immunoassay. The concentration of betamethasone was determined as ng/ml.
 
Determination of antioxidant markers
 
Activities of catalase, superoxide dismutase and glutathione peroxidase were determined in chosen tissues using commercial kits.
 
Determination of acetylcholinesterase activity
 
The activity of acetylcholinesterase enzyme was performed using AmpliteTM colorimetric acetylcholinesterase assay kit according to the instructions of the supplier.
 
Estimation of GABA level
 
The level of GABA was determined as described in the manual of Rat Gamma-aminobutyric acid, GABA ELISA Kit. GABA level was calculated as mg/ml/g Tissue.
 
Determination of serotonin and dopamine levels
 
Serotonin and Dopamine levels were measured using competitive ELISA kits. They were calculated as ng/mg protein.
 
Light microscopic study
 
Cerebellar specimens were collected from all experimental groups and fixed in 10% neutral buffered formalin and processed routinely to be stained with Haematoxylin and Eosin stain (H&E). Tissue blocks were cut into thin section 5 microns and stained with HandE stain (Bancroft and Gamble, 2002). The stained sections were examined using light microscopy.
 
Statistical analysis
 
Results were presented as mean±standard error of the mean (SE). SPSS program was used for the statistical analysis of data with one-way ANOVA to compare the experimental groups. A difference was considered significant when P£0.05.

Bioaccumulation of betametathone in sera of rabbits mothers treated with betamethasone (2 monthes  before fertilization)
 
In the present study (Table 1) the exposure to betamethasone, for 2 months before fertilization during 8 weeks, resulted in bioaccumulation of betamethasone in sera of treated rabbits. It resulted to approximately 16-fold increase in low dose betamethasone treated group (from 0.63 to 3.85 ng/ml) and high dose betamethasone treated group (from 0.78 to 4.87).
 

 
Effect of maternal betamethasone exposure before fertilization on oxidative stress markers in newborn rabbits
 
In the present study (Table 2), new born rabbits of G II and G III, showed significant decrease in studied antioxidant enzymes activities and increase in the MDA levels in cerebellum tissues as (G I). These alterations were more pronounced in rabbits of GIII than those of G II. These can be considered as indicators of oxidative stress. It was reported that prenatal or postnatal glucocorticoid therapy induced oxidative stress in cerebellar granular cells (Ahlbom et al., 2000) or in developing brain in rats (Camm et al., 2011) respectively.
 

 
Effect of maternal betamethasone exposure before fertilization on some neurotransmitter levels and acetylcholinesterase and caspase 3 activities in cerebella of  newborn rabbits
 
In this study, there was a significant increase in acetylcholinesterase and caspace-3 activities and GABA level but a significant decrease in the concentrations of serotonin and dopamine (Table 3) in newborn rabbits, whose mothers were exposed to betamethasone before fertilization for 2 months (G II and G III) relative to control group (G I). This result is in agreement with the finding of Yamate et al. (2010) who reported that administration of betamethasone caused higher level of acetylcholinesterase activity in cerebellum of chick embryos (19 days of age) than control. Serotonin acts as a differentiation factor in early neurogenesis. So, change in serotonin level during brain development altered neuronal differentiation (Buznikoy, 1984). A hypothesis is that glucocorticoids regulate the development of mechanisms which couple neuronal depolarization with release of neurotransmitter (Puro, 1983). The results of Kawamura et al. (1984) suggest that adrenal glucocorticoids may directly act on brain serotonin metabolism and there may be a feed-back relation between adrenal glucocorticoid and brain serotonin.
 

       
Alteration in dopamine level, in this study, may indicate alteration in normal fetuses’ development. This can be supported by the fact that postnatal exposure to glucocorticoids led to preterm cerebellar growth impairment (Tam et al., 2011). In this study, maternal administration of betamethasone (0.02 mg/kg or 0.20 mg/kg) for 2 months before fertilization showed significant increase in GABA level and significant decrease in serotonin and dopamine levels in cerebellum of new born rabbits which may suggest the ability of betamethasone to alter neurotransmitter levels in these newborn rabbits. These results are in agreement with Wyroll and Holmes (2011). Also, it is well known that glucocorticoids facilitate synaptic GABA release (Di et al., 2009).
       
Apoptosis through caspase-mediated mechanisms play a role in brain injury (Gill and Perez-Polo, 2008). This study demonstrated that maternal betamethasone exposure before fertilization increased caspase 3 activity in cerebellum of new born rabbits. This may suggest that Betamethasone can induce apoptosis in cerebellar tissue of these newborn rabbits resulting in neuronal death. The suggestion may be supported by other workers (Vose et al., 2013). This may be due to the physiological role of glucocorticoids in controlling programmed cell death in the mammalian cerebellum (Noguchi et al., 2008). 
       
At the cellular level, endogenous and exogenous glucocorticoids regulate cell proliferation, differentiation and apoptosis and glucocorticoids are powerful mediators of vascular function (Michael and Papageorghiou, 2008). These cellular effects are critical but antenatal glucocorticoids also act on the developing brain. Exogenous glucocorticoids increase cerebral vascular resistance leading to decreased cerebral blood flow (Miller et al., 2007) and impair cerebral oxygen delivery in a region-specific manner (Schwab et al., 2000). These changes in cerebral blood flow are associated with altered electrocortical activity, suggestive of dysfunctional complex neuronal activity and disturbed cerebral metabolism (Schwab et al., 2001). In particular, they induce acute hyperexcitability and sustained alterations in ovine fetal sleep patterns (Davidson et al., 2011). At the cellular level, synthetic glucocorticoids disrupt myelination within the brain of appropriately grown fetal sheep (Antonow-Schlorke et al., 2009) and reduce the neuronal number in fetal primates (Uno et al., 1990).
 
Effect of maternal betamethasone exposure before fertilization on histological structure of cerebellum of new born rabbits
 
In control cerebellar cortex tissues, the Purkinje cell layer appeared in between granular and molecular layers surrounded from outside by meninges (Fig 1a). Densely packed small neurons in granular layer, single row of large neurons with clear extensions in Purkinje layer and primarily glial cells in molecular layer were observed. Neurons in Purkinje layer have single axon extending into the granular layer and multiple dendrites branching in the molecular layer. The axons of the small neurons in granular layer extend into the molecular layer. The axons of granule cells and the dendrites of Purkinje cells are shown in molecular layer (Fig 1b). In cerebellar tissues of G II, there was widespread degenerative changes appeared as spacing and dissociation between Purkinje layer and granular layer with lose molecular layer. Molecular layer showed ore proliferation of glial cells (Fig 1c). The Purkinje cells had cell bodies with marked irregularity in cell boundary, mild eosinophilia with ill-defined arborization into the molecular layer were illustrated (Fig 1d). Cerebellar tissues of GIII demonstrated extremely severe degenerative changes where more spacing and dissociation between Purkinje layer and granular layer were illustrated (Fig 1e). The shrinked Purkinje cells have pyknotic nuclei with no arborization surrounded by cavity were showed. Due to no arborization of the pyramidal cells, molecular layer and granular layer appeared with low content (Fig 1f).
 

       
The present study revealed histological alteration in cerebellum cortex of groups II and III. Similar results were obtained by Noguchi (2014) under glucocorticoid treatment.  The observed nuclear and cytoplasmic changes in studied tissues in betamethasone treatment groups might be indication of oxidative stress and lipid peroxidation damage of DNA and other cytoplasmic macromolecules which may induce damage in membranes and causes degeneration of cells (Badawy et al., 2016).

Accordingly, it was concluded that the treatment of mother with betamethasone, 2 months before fertilization, adversely affect cerebellum of  newborn rabbits by traid  biochemical and histological changes. So, according to the above mentioned, it is recommended to stop betamethasone treatment by mother before fertilization by enough period, more than 2 months, as it accumulates in the blood of  them resulting in serious alterations in the embryos.

The authors acknowledge the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia for financial support under the Project Number: Grant253.  

The experimental protocol of this investigation was approved by Institutional Animal Care and Use Committee (IACUC) at the King Faisal University with Research Ethics Committee number: KFU-REC/2021-03-05.

This work  was financial supported by  Grant from Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Project Number: Grant253].

None.