EFFECT OF OSELTAMIVIR PHOSPHATE (TAMIFLU H) ON THE PRE AND POSTNATAL DEVELOPMENT OF THE ALBINO RAT CEREBELLAR CORTEX (Histological and Morphometric studies)

EFFECT OF OSELTAMIVIR PHOSPHATE (TAMIFLU H) ON THE PRE AND POSTNATAL DEVELOPMENT OF THE ALBINO RAT CEREBELLAR CORTEX

(Histological and Morphometric studies)

By

Amany F. Mohamed

Department of Anatomy & Embryology, Faculty of Medicine (Girls), Al-Azhar University, Cairo, Egypt

Mail: fekryamany@yahoo.com or amanyfekry.medg@azhar.edu.eg

ABSTRACT

Background: Oseltamivir phosphate (OP) is preferred treatment for influenza with activity against influenza A and B. It is commonly recommended for the treatment and chemoprophylaxis in pregnancy and lactation. Serious neuropsychiatric adverse reactions were observed in patient treated with OP.

Objective: This study aims to evaluate the effects of OP on prenatal and post-natal development of the albino rat cerebellar cortex.

Material and methods: This study was carried out on 40 pregnant albino rats and 120 of their offspring of both sex, 3 from each mother. The pregnant rats were divided equally into 2 main groups A and B. Group A (control): it was subdivided equally into 2 subgroups A1 and A2. Group B (OP treated): it was subdivided equally into 2 subgroups B1 and B2. In group A, each rat mother was given 0.9 ml distilled water twice daily by oral gavage for successive 5 days. In group B, each rat mother was given 0.9 ml distilled water containing 1.35 mg OP twice daily by oral gavage for successive 5 days. Dosing started from the 15^th to 19 ^th of pregnancy in subgroups A1 and B1 while it started from the 1^st to 5^th day after delivery in subgroups A2 and B2. Cerebelli were collected, on the 1^st day of birth in subgroups A1 and B1, on the 7 ^th day of birth in subgroups A2 and B2, and on the 14 ^th day of birth in subgroups A1, A2, B1 and B2. The cerebelli of all subgroups were subjected to light microscopic study and the cerebelli of 14 days old offspring were subjected also, to transmission electron microscopic and morphometric studies and statistical analysis.

Results:  OP induced delayed development and neurotoxic effects on the cerebellar cortex of albino rat offspring whose mother dosed OP during pregnancy or during lactation.

Conclusion: Oseltamivir phosphate induced pre and post-natal delayed development and neurotoxic insults on the cerebellar cortex of albino rat.

Key words: influenza, oseltamivir phosphate, rat, pregnancy, lactation, cerebellum.

INTRODUCTION

     Antiviral use in pregnancy presents challenges because of the paucity of clinical and safety data for many agents in this class. While vaccination is important for prevention, many viral diseases lack this option, making antiviral prophylaxis or treatment necessary (Cottreau, and Barr, 2016).

     Oseltamivir is a preferred treatment of influenza with activity against influenza A and B, it is commonly recommended for the treatment and chemoprophylaxis for influenza in pregnancy (Committee for Medicinal Products for Human Use     ‟CHMPˮ, 2012). It is administrated orally as oseltamivir phosphate (OP) which dissociates in the gastrointestinal tract and absorbed.  It is metabolized by the liver to its active metabolite, oseltamivir carboxylate (OC) by hepatic carboxylesterase (Worley et al., 2008 and Fiore et al., 2011).   

     The metabolite has excellent penetration into tissues. The peak plasma concentrations are lower and the half-life is shorter for OP than for OC respectively.                  The placenta of a pregnant woman would presumably be exposed to higher sustained levels of OC than the prodrug (Worley et al., 2008). Oseltamivir inhibits viral neuraminidase by binding to neuraminidase sialic acid receptors and preventing neuraminidase from cleaving host-cell receptors and releasing newly synthesized virus so it inhibits the propagation of influenza virus. Almost, all OC is secreted in urine (Dalvi et al., 2011; Cottreau Bennet, Barr, 2016 and Hama 2017).

     Serious neuropsychiatric adverse reactions were observed in patient treated with OP, including sudden death and abnormal behaviors leading to accidental death (Hama, 2008). More data is needed to conclusively rule out effect of OP use on psychiatric events (Doll et al., 2017).

     Limited safety data exist for OP therapy in pregnancy and lactation. So, this study aimed to evaluate the effect of OP on the pre and postnatal development of the cerebellar cortex of albino rat.

MATERIALS AND METHODS

Drug:

     Oseltamivir phosphate (Trade name ‘Tamiflu’) was purchased from F. Hoffmann-La Roche, Basel, Switzerland. It was available in the form of capsules. Each capsule contained 75 mg oseltamivir phosphate. The recommended human therapeutic dose is 75 mg twice daily for 5 days (Brunton et al. 2013). According to Paget and Barnes, (1964), the equivalent dose of OP per adult rat, weighing about 180±20 gram, was 2.7 mg/rat/day. OP is highly water soluble (Tanaka et al., 2009). So, the powder of capsule was dissolved in 50 ml of distilled water and each rat was given 0.9 ml distilled water containing 1.35 mg OP by oral gavage twice daily for successive 5 days.

Animals:

     Fifty six local strain adult albino rats weighing about 180± 20 gm were used (42 females and 14 males) in this experimental study. They were obtained and provided by veterinary care in the Animal House of Faculty of Medicine (Girls), Al- Azhar University according to the guidelines for animal research approved by the Animal Ethics Committee.  The adult female albino rats were isolated from the adult male rats. All rats were left to acclimate for one week prior to the experiment and every 3 or 2 rats were housed in stainless steel cages, (40x27.5x19.5 cm) and maintained at controlled room temperature (22-24 ᵒC) with normal day and night cycle. Throughout the experiment, all animals were observed and maintained on standard diet pellets (El-Nasr- Company, Abou- Zaabal- Egypt) and water ad libitum.

     Each male was kept overnight with three females in a separate cage. In the next morning, females showed a vaginal plug was considered to be in the 1^st day of pregnancy (Barcellona et al., 1977).The pregnant albino rats were kept in separate cages till the end of the experiment.

Experimental design:

     This study was carried out on the offspring of 40 pregnant albino rats (120 offspring of both sex, 3 from each mother). Every rat mother and their offspring were kept in a cage. The pregnant rats and their offspring were divided into 2 groups:

Group A (control group): consisted of 20 pregnant rats which was subdivided equally into 2 subgroups:

A1: Each pregnant rat in this subgroup was given 0.9 ml distilled water twice daily by oral gavage for successive 5 days staring from the 15^th   to 19 ^th day of pregnancy. After delivery, fifteen offspring were sacrificed on the 1^st day and other fifteen on the 14 ^th day of birth.

A2: Each delivered rat in this subgroup was given 0.9 ml distilled water twice daily by oral gavage for successive 5 days staring from the 1^st to 5^th day after delivery. Fifteen offspring were sacrificed on the 7 ^th day and other fifteen on the 14 ^th day of birth.

Group B (OP treated group): consisted of 20 pregnant rats which was subdivided equally into 2 subgroups B1 and B2.  They received OP as follow: 

Subgroup B1: Each pregnant rat in this subgroup was given 0.9 ml distilled water containing 1.35 mg OP twice daily by oral gavage for successive 5 days staring from the 15 ^th to 19 ^th of pregnancy. After delivery, fifteen offspring were sacrificed on the 1^st day and other fifteen on the 14 ^th day of birth.

Subgroup B2: Each delivered rat in this subgroup was given 0.9 ml distilled water containing 1.35 mg OP twice daily by oral gavage for successive 5 days staring from the 1^st to 5^th day after delivery. Fifteen offspring were sacrificed on the 7 ^th day and other fifteen on the14 ^th day of birth.

Collection and preparation of the specimens:

     The cerebelli of the offspring of all subgroups were collected. All offspring were anesthetized lightly by isoflurene inhalation.  Each offspring was decapitated with straight, thin and sharp scissors. The cap and side walls of the cranium were carefully removed. The dura mater was carefully incised all around then raised up. The falx cerebelli was removed and tentorium cerebelli was cut out. Cerebellum was gently removed. Half of cerebellum was used for light microscopic examination and morphometric study. The other half was used for transmission electron microscopic (TEM) examination. Cerebelli of one and seven days old offspring were examined by light microscope and the cerebelli of fourteen days old offspring all subgroups were examined by also, by transmission electron microscope and morphometric study.

Light microscopy: The cerebelli which were used for light microscopic examination were fixed by immersion in Bouin’s solution for 3days(Suvarna et al., 2013). The specimens were dehydrated in ascending grades of ethyl alcohol and cleared in benzene. They were impregnated for three changes in paraffin and were finally embedded in paraffin wax. The paraffin blocks were cut into serial sagittal sections at 5 μm thick with a rotatory microtome. Successive sagittal paraffin sections were attached to an albumenized glass slides. The Hematoxylin and Eosin stain  was used to study the cerebellar architectures. (Bancroft and Gamble, 2008) Also, the cerebellar semi-thin sections which were stained with toluidine blue were examined by the light microscope. The images were taken by a microscope (Leica) DM750 connected to a digital camera in Anatomy Department, Faculty of Medicine for Girls, Al-Azhar University, Cairo. Egypt.

Transmission electron microscopy (TEM):

     The cerebelli used for electron microscopic examination were cut into small pieces.  The specimens were immediately fixed in cold 5% glutaraldehyde and washed in 0.1 ml phosphate buffer (PH 7.2). Then, post fixed with 1% osmium tetraoxide (OsO4), dehydrated and embedded in epoxy resin. The semi thin sections (1μm thick) were cut on an LKB ultratome and stained with toluidine blue. Ultrathin sections (60 nm thick) were cut, mounted on copper grids, and then stained with uranyl acetate and lead citrate (Bancroft and Gamble, 2008). The ultrathin sections were examined using a transmission electron microscope (JEOL1010 EX II, Japan) at the Regional Mycology and Biotechnology Center, Al-Azhar University, Cairo, Egypt.

Morphometric study and its statistical analysis:

     The image analyzer computer system Leica Qwin 500 (England) at the Regional Mycology and Biotechnology center, Al-Azhar University, Cairo, Egypt was used to evaluate the number of Purkinje cells  and the thickness of the molecular layer of 14 days old offspring of  all subgroups  using Haematoxylin and Eosin -stained sections. The cerebellar sections of subgroups were examined; the central five folia were selected for measurements. Counting the number of Purkinje cells was done in special fixed squares. Measuring the thickness was done by taking 5 different areas in each folium at magnification X 200 using Image J software version 1.48. The data were subjected to statistical analysis. The means and the cumulative means of the numbers of Purkinje cells of folia and the thickness of the molecular layer for each subgroup were calculated. Statistical analysis of the data obtained was expressed as mean values and standard deviations using one-way analysis of variance (ANOVA F test) (Mould, 1989). When the ANOVA F test results were significant, Post- hoc Tukey's test was performed for pair-wise subgroups comparisons. If P value was ≤0.05, the results was considered significant (Knapp et al., 2014).

     Statistical calculations of morphometric study were performed using Microsoft Excel 2007 and Statistical Package for the Social Science (SPSS) (SPSS Inc., IBM Corp., Armonk, USA) v. 15 for Microsoft Windows.

RESULTS

Histopathological results:

Group A (control group)

     The light microscopic examination of the sagittal section of the cerebellar cortex stained with haematoxylin and eosin of one day old albino rat of subgroup A1 showed that the cerebellar cortex was covered by the pia matter and consisted of four layers; the external granular layer, the molecular layer, Purkinje cell layer and the internal granular layer (fig.1AI). The external granular layer was present just beneath the pia mater and it was formed of granular cells which were packed together and arranged perpendicular to the pia matter. It consisted of 4 to 8 rows. The granular cells were rounded or oval in shape of variable size and had rounded or oval deeply stained basophilic nuclei with small prominent nucleoli, these nuclei were surrounded by a rim of eosinophilic cytoplasm (figs.1AI and 2AI). The molecular layer appeared as a narrow pale zone beneath the external granular layer and its cells could be hardly recognized as small oval cells with lightly stained basophilic nuclei and eosinophilic cytoplasm (figs.2AI and 2AI). Purkinje cell layer appeared superficial to the internal granular layer and consisted of 2 to 3 irregular rows. They were nearly rounded in shape and had large rounded vesicular basophilic nuclei with one or two prominent nucleoli, these nuclei were surrounded by huge eosinophilic cytoplasm (figs.1AI and 2AI). The internal granular layer was consisted of numerous granular cells which were widely separated from each other by eosinophilic areas. They were nearly rounded or oval in shape with darkly stained basophilic nuclei containing one or two prominent nucleoli, the nuclei were surrounded by a narrow rim of eosinophilic cytoplasm (figs. 1AI and 2AI).

     The light microscopic examination of the sagittal sections of cerebellar cortex   stained with haematoxylin and eosin of 7 days old albino rat of subgroup A2 showed that the cerebellar cortex appeared more developed than previous age. It was covered by the pia matter and was still consisted of four layers (fig. 1AII). The external granular layer became consisted of 8 to 9 rows of granular cells. The molecular layer apparently increased in thickness and its cells were easily recognized (figs. 1AII and 2AII). Also, Purkinje cell layer became consisting of 2 irregular rows and some Purkinje cells exhibited their characteristic flask shape. The internal granular cells appeared aggregated in clusters and these clusters were separated by eosinophilic areas (figs. 1AII and 2AII).

     The results of histological examination of cerebellar cortex of 14 days old offspring of subgroups A1 and A2 were similar, so they pooled together. 

     The light microscopic examination of the sagittal sections of the cerebellar cortex stained with haematoxylin & eosin and toluidine blue of 14 days old albino rat  of  group A showed that the cerebellar cortex (fig.1AIII) was more developed than the previous ages (figs.1AI and 1AII), it was still consisted of the four layers (fig.1AIII). The external granular layer showed marked reduction in its thickness, it became formed of 2 to 3 rows of granular cells (figs.2AIII and 3AIII). The molecular cell layer was more developed as its thickness was increased (fig.1AIII) compared to those of the previous ages (figs. 1AI and 1AII). The molecular layer contained 2 types of cells; stellate and basket cells. The stellate cells were present in the outer part of the molecular layer while the basket cells were present in the deep part and also, between Purkinje cells and both of them were also present in the middle part. They were oval or rounded in shape, of variable size and had lightly stained nuclei with prominent nucleoli. The migrating granular cells could also be seen in the molecular layer (figs.2AIII, 3AIII, 4AIII and 5AIII). Purkinje cell layer (figs.4AIII and 5AIII) was more developed than the previous age (figs. 2AI and 2AII). Its cells were arranged in an irregular single row superficial to the internal granular layer. Most of Purkinje cells had a characteristic flask shaped and some of them still oval in shape. They had large rounded vesicular nuclei, with prominent nucleoli, and huge cytoplasm. Their cytoplasmic processes appeared directed towards the molecular layer (figs. 1AIII, 4AIII and 5AIII). The granular cells of the internal granular layer were apparently increased in number and arranged in clusters and separated by small eosinophilic areas (figs. 4AIII and 5AIII). Electron microscopic examination of cerebellar cortex of 14 days old offspring of group A showed that the external granular cells lied close together beneath the pia mater. They were oval in shape with large heterochromatic oval nuclei and eccentric nucleoli. Their nuclear envelopes contained many nuclear pores. The nuclei were surrounded by rim of cytoplasm and plasma membrane. The cytoplasm contained rough endoplasmic reticulum, rounded or oval mitochondria and free ribosomes (fig. 6AIII). The molecular layer composed of a complex network of neuronal cells and neuronal fibers. The neuronal cells were stellate and basket cells. The stellate and basket cells appeared rounded or oval in shape with well-defined plasma membrane. They had euchromatic nuclei with nuclear envelope containing many nuclear pores. The nuclei contained small clumps of heterochromatin and eccentric nucleoli. The cytoplasm contained rough endoplasmic reticulum, rounded or elongated mitochondria, Golgi apparatus and free ribosomes (figs. 7AIII and 8AIII). The neuropil of the molecular layer was formed of dendrites and axons. The dendrites contained mitochondria and parallel microtubules and neurofilaments. The dendritic spine could be detected. The axons contained vesicles of varying size. There were symmetrical and asymmetrical types of synapse (figs. 7AIII, 8AIII and 9AIII). Purkinje cells had large rounded euchromatic nuclei with eccentric nucleoli. The nuclei had well-developed nuclear envelope having invaginations and many nuclear pores. The cell had huge cytoplasm surrounded by plasma membrane. The cytoplasm contained rough endoplasmic reticulum, Golgi apparatus, rounded or oval mitochondria and free ribosomes (fig. 10AIII). The internal granular cells lied close together. They appeared nearly oval in shape with oval heterochromatic nuclei containing eccentric nucleoli and clumps of heterochromatin. The nuclear envelope contained many nuclear pores and was surrounded by a rim of cytoplasm and a plasma membrane. The cytoplasm contained few rough endoplasmic reticulum, and small rounded or oval mitochondria and free ribosomes (fig. 11AIII).

Group B

Subgroup B1

     Light microscopic examination of the sagittal sections of cerebellar cortex stained with haematoxylin and eosin of one day old albino rat of subgroup B1 showed that the cerebellar cortex was consisted of four layers; the external granular layer, the molecular layer, Purkinje cell layer and the internal granular layer (fig.1BI). The external granular layer was poorly developed. It consisted of 2-3 rows of granular cells which were not packed together and most of them were apparently not perpendicular to the pia matter which contained congested blood vessels.  Also, most of the external granular cells had vacuolated cytoplasm and small deeply stained nuclei (figs.1BI and 2BI). The molecular layer appeared vacuolated and contained cells with vacuolated cytoplasm and small deeply stained nuclei (figs. 1BI and 2BI). Purkinje cell layer consisted of irregularly arranged Purkinje cells superficial to the internal granular layer. Some Purkinje cells appeared small irregular in shape with small deeply stained nuclei and they were surrounded by vacuolated areas (figs. 1BI and 2BI). The cells of the internal granular layer were widely separated from each other by vacuolated eosinophilic areas. Some internal granular cells had small deeply stained nuclei and vacuolated cytoplasm (figs.1BI and 2BI).

     The light microscopic examination of the sagittal sections of cerebellar cortex of 14 days old rat cerebellar cortex of subgroup B1 stained with haematoxylin & eosin and toluidine blue, showed that the cerebellar cortex was still consisted of four layers (fig.1BII). The external granular layer (figs.1BII, 2BII and 3BII) was apparently thick compared to the control one of the same age (figs. 1AIII, 2AIII and 3AIII). It consisted of 5 to 7 rows of irregularly arranged granular cells which appeared not packed, nor perpendicular to the pia matter. Most of the granular cells were irregular in shape with vacuolated cytoplasm. Also,  some  of them had  small, irregular and deeply stained nuclei (figs.2BII and 3BII).The thickness of the molecular layer (fig.1BII) appeared markedly decreased compared to that of the control one of the same age (fig. 1AII). Moreover, some stellate and basket cells had vacuolated cytoplasm and others had small deeply stained nuclei (figs. 2BII, 4BII, and 5BII). Purkinje cells appeared arranged in a single row and widely separated from each other. Some cells appeared irregular in shape with deeply stained and ill-defined nuclei (figs. 4BII and 5BII). Most internal granular cells appeared irregular in shape and had vacuolated cytoplasm. Few had irregular deeply stained nuclei (figs. 4BII and 5BII). Electron microscopic examination of cerebellar cortex of 14 days old albino rat of subgroup B1 showed that most external granular cells had ill- defined plasma membrane. There were multiple areas of ill-defined structures. Some nuclei appeared small and irregular in shape with condensed heterochromatin. There were small and electron dense masses surrounded a nucleus. The cytoplasm of granular cells contained few dilated rough endoplasmic reticulum and few vacuolated mitochondria with destructed cristae .Other cells had normal structure (fig.6BII). The molecular layer showed also multiple areas of ill-defined structures, some stellate cells appeared small and irregular with ill-defined plasma membrane in some areas, some nuclei were small and contained electron dense clumps of heterochromatin. Their cytoplasm contained swollen and vacuolated mitochondria with destructed cristae, few dilated rough endoplasmic reticulum and free scattered ribosomes (fig.7BII). Some basket cells had ill-defined plasma membrane, nuclei with less heterochromatin. Their cytoplasm contained swollen and vacuolated mitochondria with destructed cristae, fragmented and dilated rough endoplasmic reticulum and numerous vacuoles (fig. 8BII). The neuropil of the molecular layer showed areas of ill-defined structures. Some dendritic profiles had swollen and vacuolated mitochondria and few ill- defined organelles. The others had microtubules and neurofilaments. Also, there were nearly rounded areas of ill-defined structures which might be axons. The synapses could be detected (fig.9BII). Some Purkinje cells had irregular electron dense nucleus with more electron dense nucleolus, the cytoplasmic organelles could not be differentiated easily. However, small electron dense mitochondria and numerous dilated rough endoplasmic reticulum could only be differentiated.  Others had euchromatic nuclei and their cytoplasm had vacuolated mitochondria and dilated and fragmented rough endoplasmic reticulum (figs. 9BII and 10BII). Some internal granular cells had ill-defined plasma membrane in some areas and irregular hyperchromatic nuclei with condensed clumps of heterochromatin and dilated nuclear envelope.  The cytoplasm contained dilated rough endoplasmic reticulum, and swollen mitochondria with destructed cristae. Some cells had electron dense nucleus and cytoplasm and their cytoplasm organelles could not be recognized. Areas of ill-defined structures were detected. Some myelinated nerve fibers were present between the cells (fig. 11BII).

Subgroup B2

     The light microscopic examination of the sagittal sections of cerebellar cortex stained with haematoxylin & eosin of 7 days old rat of subgroup B2 showed that the cerebellar cortex was consisted of four layers; the external granular layer, molecular layer, Purkinje cell layer and the internal granular layer (fig. 1CI). The external granular layer was formed nearly from 3 to 5 rows of cells that were irregularly arranged and separated from each other. Some cells had small irregular deeply stained nuclei and vacuolated cytoplasm (figs. 1CI and 2CI). The molecular layer appeared as narrow zone beneath the external granular layer. Some molecular cells had small deeply stained nuclei. Areas of vacuolation could be detected (fig. 2CI). Purkinje cell layer appeared as large cells scattered in between the internal granular cells and some of them were irregular in shape with deeply stained nuclei (fig. 2CI). The internal granular cells appeared widely separated from each other (fig. 2CI) compared to those of the control of the same age (fig. 2AII). Some cells had irregular and deeply stained nuclei (fig.2CI).

     The light microscopic examination of the sagittal sections of cerebellar cortex stained with haematoxylin & eosin and toluidine blue of 14 days old albino rat of subgroup B2 showed that the cerebellar cortex was still consisted of the four layers (fig.1CII). The external granular layer (figs.1CII, 2CII and 3CII) was apparently thick compared to the control one of the same age (figs.1AIII, 2AIII and 3AIII). It consisted of nearly 5-6 rows of granular cells. The granular cells were irregular in shape, not packed together and had vacuolated cytoplasm. Some had small deeply stained nuclei (figs.2CII and 3CII).The thickness of the molecular layer (fig. 1CII) was apparently decreased compared to that of the control one of the same age (fig. 1AIII). Some stellate and basket cells had vacuolated cytoplasm, some had deeply stained nuclei (figs. 2CII, 3CII, 3CII and 4CII). Purkinje cells were   arranged in a single row and were widely separated from each other. Some Purkinje cells appeared irregular in shape with ill-defined nuclei (figs. 1CII, 4CII and 5CII). Some internal granular cells had vacuolated cytoplasm and few cells had deeply stained nuclei (figs. 4CII and 5CII). Electron microscopic examination of 14 days old albino rat cerebellar cortex of subgroup B2 showed that some external granular cells appeared irregular in shape with large or small irregular heterochromatic nuclei, the plasma membrane could hardly be recognized in some areas. There were areas of ill-defined structures. The cytoplasm of the granular cells contained few dilated rough endoplasmic reticulum and few mitochondria with destructed cristae (fig.6CII). The molecular layer showed also some areas of ill-defined structures, the cytoplasm of some stellate cells contained vacuolated mitochondria with destructed cristae and dilated rough endoplasmic reticulum and free ribosomes (fig.7CII). Some basket cells had dilated nuclear envelop. Their cytoplasm contained areas of ill-defined structures, small electron dense mitochondria, fragmented and dilated rough endoplasmic reticulum (fig. 8CII). The neuropil of the molecular layer showed areas of ill-defined structures. Some dendritic profiles had dark elongated mitochondria and few ill- defined organelles. The others had microtubules and neurofilaments. The axons and synapse were hardly identified. Myelinated nerve fibers were seen (figs.8CII and 9CII). Some Purkinje cells had ill-defined nuclei. Their cytoplasm appear more electron dense and contained small electron dense mitochondria and dilated rough endoplasmic reticulum (fig.10CII). Some internal granular cells had irregular heterochromatic nuclei with condensed clumps of heterochromatin and dilated nuclear envelope. They had electron dense cytoplasm and their cytoplasm organelles could not be recognized. Other cells appeared normal (fig. 11CII).

Fig. (1AI): A photomicrograph of the sagittal section of cerebellar cortex of one day old rat of subgroup A1 showed the external granular layer (EGL) beneath the pia mater (PM), the molecular layer (ML), Purkinje cell layer (PL) and the internal granular layer (IGL). (H&E X400)

Fig. (2AI): A higher magnification of the figure 1AI showed the external granular cells (EGC) arranged in 4 to 8 rows perpendicular on the pia matter (PM), the molecular cells (MC) could be hardly recognized, Purkinje cells (PC) were formed of 2 to 3 irregular rows of cells and the internal granular cells (IGC) were separated by eosinophilic areas. (H&E X1000)

Fig. (1BI): A photomicrograph of the sagittal section of cerebellar cortex of one day old rat of subgroup B1 showed that external granular layer (EGL) appeared thin and vacuolated beneath the pia mater (PM), molecular layer (ML) appeared vacuolated. Purkinje cell layer (PL) was irregular .The internal granular layer (IGL) contained widely separated granular cells. Notice congested blood vessels of the pia matter.                      (H&E X400)

Fig. (2BI): A higher magnification of the figure 1BI showed the external granular layer consisted of 2-3 rows of granular cells. Most granular cells (EGC) had small deeply stained nuclei and vacuolated cytoplasm. The molecular layer appeared vacuolated (star) and contained vacuolated cells with small deeply stained nuclei (arrow head).  Purkinje cells were irregularly arranged. Some Purkinje cells (PC) appeared small irregular in shape with small deeply stained nuclei and surrounded by vacuolation. Also, some internal granular cells (IGC) had deeply stained nuclei and vacuolated cytoplasm.   (H& E X1000)

Fig. (1AII): A photomicrograph of the sagittal section of cerebellar cortex of 7 days old rat of subgroup A2 showed the external granular layer (EGL) beneath the pia mater (PM), the molecular layer (ML), Purkinje cell layer (PL) and the internal granular layer (IGL). (H & E X400)

Fig. (2AII): A higher magnification of the figure 1AII showed part of external granular layer containing external granular cells (EGCs), the molecular layer containing molecular cells (MC), Purkinje cells (PC) arranged irregularly in two rows and appeared large oval in shape and some were flask shape, the internal granular cells (IGC) were packed under Purkinje cells.                                                                                                 (H & E X1000)

Fig. (1CI): A photomicrograph of the sagittal section of cerebellar cortex of 7 days old albino rat of subgroup B2 showed external granular layer (EGL), the molecular layer (ML), Purkinje cell layer (Pl) and the internal granular layer (IGL).            (H & E X400)

Fig. (2CI): A highermagnification of the figure 1CI showed the external granular layer consisting of 3-5 rows of irregularly arranged granular cells. Most granular cells (EGC) had vacuolated cytoplasm and some had deeply stained nuclei, they are widely separatedby vacuolations (star).The molecular layer contained deeply stained nuclei (arrow).  Some Purkinje cells (PC) appeared irregular in shape with small deeply stained nuclei. The internal granular cells were separated. Some internal granular cells (IGCs) had irregular deeply stained nuclei.                                                                                      (H & E X1000)

Fig.(1AIII): A photomicrograph of the sagittal section of cerebellar cortex of 14 days old albino rat of group A (control group) showed the external granular layer (EGL) under beneath the pia matter (PM), the molecular layer (ML), Purkinje cell layer (PL) which formed of single row of cells,  the internal granular layer (IGL).                      (H&E X400)

 Fig.(1BII)): A photomicrograph of the sagittal section of cerebellar cortex of 14 days old albino rat of subgroup B1 showed the cerebellar cortex consisted of four layers;  external granular layer (EGL) under beneath the pia matter (PM), molecular layer (ML), Purkinje cell layer (PL) which formed of single row of cells and the internal granular layer (IGL). Notice the external granular appeared thick while the molecular layer apparently was thin.  (H& EX400)

Fig. (1CII): A photomicrograph of the sagittal section of cerebellar cortex of 14 days old albino rat of subgroup B2 showed cerebellar cortex consisting of four layers; external granular layer (EGL) under beneath the pia matter (PM), molecular layer (ML), Purkinje cell layer (PL) which formed of single row of cells and the internal granular layer (IGL).Notice the external granular appeared thick while the molecular layer apparently was thin.                                                                                                            (H& E X400)

Fig. (2AIII): A higher magnification of the figure 1AIII showed the external granular layer consisting of 2-3 rows of external granular cells (EGCs). It also showed part of the molecular layer contained oval or rounded stellate cells.                                (H& E X1000)

Fig. (2BII): A highermagnification of the figure 1BII showed the external granular layer consisted of 5 to 7 rows of granular cells (EGCs). Most of the granular cells had vacuolated cytoplasm (V), some granular cells had small deeply stained nuclei (arrow).Notice the stellate cell (SC) with vacuolated cytoplasm.                  (H& EX1000)

Fig. (2CII): A highermagnification of the figure 1CII showed the external granular layer consisting of nearly 5-6 rows of granular cells. Most of granular cells had vacuolated cytoplasm (V) and some had small deeply stained nuclei (arrow). The molecular layer contained vacuolated stellate cells (SC).                                                          (H& EX1000)

Fig.(3AIII): : A photomicrograph of the semi-thin sagittal section of cerebellar cortex of 14 days old  albino rat of group A (control group) showed the upper part of the cerebellar cortex consisting of the external granular layer formed of 2  to 3 rows of external granular cells (EGCs). Also, it showed upper part of the molecular layer containing stellate cells (SC) and migrating external granular cells (MEGC).                     (Toluidine blue X 1000)

Fig. (3BII): A photomicrograph of the semi-thin sagittal section of cerebellar cortex of 14 days old albino rat of subgroup B1 showed the external granular layer consisted of 5 to 7 rows of external granular cells (EGCs). Some granular cells had vacuolated cytoplasm (V) and some cells had small deeply stained nuclei (arrow).                (Toluidine blue X 1000)

Fig. (3CII): A photomicrograph of the semi-thin sagittal section of cerebellar cortex of 14 days old albino rat subgroup B2 showed the external granular layer consisting of nearly 5-6 rows of granular cells. The granular cells had vacuolated cytoplasm (V) and some had small deeply stained nuclei (arrow). Some stellate cells (SC) had vacuolated cytoplasm. (Toluidine blue X 1000)

Fig. (4AIII): A highermagnification of the figure 1AIII showed the basket cells (BC) lied between Purkinje cells (PC). Also, showed the internal granular cells (IGCs) lied deep to Purkinje cells and were aggregated in clusters and separated by small eosinophilic areas. (H&E X1000)

Fig. (4BII): A highermagnification of the figure 1BII showed some deeply stained Purkinje cells (PC) which were irregular in shape with ill-defined nuclei. The basket cells (BC) had vacuolated cytoplasm. Some small deeply stained nuclei (arrow) appeared in molecular layer. Most internal granular cells (IGC) had vacuolated cytoplasm (V). (H&E X1000)

Fig. (4CII): A highermagnification of the figure 1CII showed the molecular layer containing vacuolated basket cells (BC). Some Purkinje cells (PC) appeared irregular in shape with ill-defined nuclei. Some internal granular cells (IGC) had vacuolated cytoplasm.                                                                                                         (H&E X1000)

Fig. (5AIII): A photomicrograph of the semi-thin sagittal section of cerebellar cortex of 14 days old albino rat of group A (control group) showed the basket cells (BC) between the Purkinje cells (PC) Purkinje cells arranged in a single row and appeared oval in shape with their cell processes (arrow) directed towards the molecular layer.  Purkinje cells had large rounded vesicular nuclei with prominent nucleoli. The internal granular cells (IGC) had deeply stained nuclei with prominent nucleoli.                               (Toluidine blue X 1000)

Fig. (5BII): A photomicrograph of the semi-thin sagittal section of cerebellar cortex of 14 days old albino rat of subgroup B1 showed lower parts of the cerebellar cortex. The molecular layer contained small deeply stained nuclei (arrow). Some basket cells (BC) had vacuolated cytoplasm.  Some Purkinje cells (PC) appeared small irregular and deeply stained with ill-defined nuclei. Few internal granular cells (IGC) had irregular deeply stained nuclei.                                                                                  (Toluidine blue X 1000)

Fig. (5CII): A photomicrograph of the semi-thin sagittal section of cerebellar cortex of 14 days old albino rat of subgroup B2 showed the lower part of the cerebellar cortex. Purkinje cells (PC) appeared darkly stained with ill- defined nuclei. The basket cells (BC) appeared in the lower part of molecular and between Purkinje cells. Few internal granular cells (IGC) had small deeply stained nuclei.                                   (Toluidine blue X 1000)

Fig. (6AIII): An electron micrograph of the external granular cells of cerebellar cortex of 14 days old offspring of group A (control group) showed that the external granular cells (EGCs) lied beneath the pia mater (PM). They had large oval heterochromatic nuclei (N) with eccentric nucleoli (Nu). The nuclear envelop (NE) contained many nuclear pores (NP). The cytoplasm contained rough endoplasmic reticulum (Re), rounded or oval mitochondria (M) and free ribosomes (R).                                                    (TEM X 10000)

Fig. (6BII): An electron micrograph of the external granular cells of the cerebellar cortex of 14 days old offspring of subgroup B1 showed that the external granular cells (EGCs). Most of the external granular cells had ill- defined plasma membrane. They showed multiple areas of ill-defined structures (star). Some nuclei (N) appeared small and irregular in shape with condensed heterochromatin. Electron dense small masses surrounded a nucleus (arrow). The cytoplasm contained few dilated rough endoplasmic reticulum (Re) and few vacuolated mitochondria (M) with destructed cristae.             (TEM X 10000)

Fig. (6CII): An electron micrograph of the external granular cells of the cerebellar cortex of 14 days old offspring of subgroup B2 showed that the external granular cells (EGCs). Some external granular cells irregular in shape with large or small irregular heterochromatic nuclei (N). Their plasma membrane was ill-defined in some areas. They showed areas of ill-defined structures (star). The cytoplasm contained few dilated rough endoplasmic reticulum (Re) and few vacuolated mitochondria (M) with destructed cristae. (TEM X 10000)

Fig. (7AIII): An electron micrograph of the molecular layer of the cerebellar cortex of 14 days old offspring of group A (control group) containing stellatecell and neuropil. The stellatecell(SC) was surrounded by plasma membrane (Pl). It had euchromatic nucleus (N) with eccentric nucleoli (Nu), the nuclear envelope (NE) contained many nuclear pores (NP). Its cytoplasm contained mitochondria (M), rough endoplasmic reticulum (Re) and free ribosomes (R). Notice the presence of axons (Ax), symmetrical synapse (SS), and dendrites (D) containing mitochondria (m), microtubules (MT) and neurofilaments. Notice thedendritic spine (arrow).                                                                           (TEM X 10000)

Fig. (7BII): : An electron micrograph of the molecular layer of the cerebellar cortex  of 14 days old offspring of  subgroup B1 showed that the stellatecell(SC) appeared small and irregular with ill-defined plasma membrane in some areas. The nucleus (N) small and contained small electron dense clumps of heterochromatin. Its cytoplasm contained swollen and vacuolated mitochondria with destructed cristae (M), rough dilated rough endoplasmic reticulum (Re) and free ribosomes (R). Notice the presence of multiple areas of ill-defined structures(star).                                                                      (TEM X 10000)

Fig. (7CII): An electron micrograph of the molecular layer of the cerebellar cortex of 14 days old offspring of subgroup B2 showed the stellatecell(SC) with large euchromatic nucleus (N).Its cytoplasm contained vacuolated mitochondria (M) with destructed cristae, dilated rough endoplasmic reticulum (Re) and free ribosomes (R). Notice the presence of axons (Ax), dendrites (D) containing mitochondria (m), microtubules (MT) and neurofilaments. Notice the presence of some areas of ill-defined structures(star).      (TEM X 10000)

Fig. (8AIII): An electron micrograph of the cerebellar cortex of 14 days old offspring of group A (control group) showed the basket(BC) cell between Purkinje cells (PC) It had well-defined plasma membrane (Pl) and euchromatic nucleus (N) with  nuclear envelope (NE) containing  many nuclear pores (NP). Its cytoplasm contained mitochondria (M), Golgi apparatus (GA) rough endoplasmic reticulum (Re) and free ribosomes (R). Notice, the presence of asymmetrical synapse (AS).                                                 (TEM X 10000)

Fig. (8BII): An electron micrograph of the cerebellar cortex of 14 days old offspring of subgroup B1 showed that the basket(BC) cell had ill-defined plasma membrane, its nucleus had less euchromatin (N) content. The cytoplasm contained swollen and vacuolated mitochondria with destructed cristae (M), fragmented and dilated rough endoplasmic reticulum (Re), and free ribosomes(R). Notice the presence of part of Purkinje cell (PC) with part of its nucleus (N), its cytoplasm contained vacuolated mitochondria (M) and dilated rough endoplasmic reticulum (Re).               (TEM X 10000)

Fig. (8CII): An electron micrograph of the external granular cells of cerebellar cortex of 14 days old offspring of subgroup B2 showed that the basket(BC) cell had  euchromatic nucleus (N) with  dilated nuclear envelope (NE) containing  many nuclear pores (NP). Its cytoplasm contained areas of ill-defined structures(star). Small electron densemitochondria (M), fragmented and dilated rough endoplasmic reticulum (Re). Notice the presence of myelinated nerve fibers (My) and part of electron dense Purkinje cell (PC).  (TEM X 10000)

Fig. (9AIII): An electron micrograph of the molecular layer of the cerebellar cortex of 14 days old offspring of group A (control group) containing neuropil and stellate cell. The dendrites (D) contained mitochondria (m), microtubules (MT) and neurofilaments. The axons (Ax) contained many smal vesicles. Notice the symmetrical synapse (SS) and stellatecell(SC) with euchromatic nucleus (N) and eccentric nucleoli (Nu).               (TEM X 10000)

Fig. (9BII): An electron micrograph of the molecular layer of the cerebellar cortex of 14 days old offspring of subgroup B1 showed nearly rounded multiple areas of ill-defined structures (star). The dendrites (D) contained vacuolated mitochondria with destructed cristae (m) and some microtubules (MT).Notice the presence of part of stellate cell (SC) and symmetrical synapse (SS).                                                                      (TEM X 10000)

Fig. (9CII): An electron micrograph of the molecular layer of the cerebellar cortex of 14 days old offspring of subgroup B2 showed nearly rounded multiple areas of ill-defined structures (star). The dendrites (D) contained dark elongated mitochondria (m) and some microtubules (MT).Notice the presence of a part of stellate cell (SC).        (TEM X 10000)

Fig. (10AIII):  An electron micrograph of a part of Purkinje cell (PC) of the cerebellar cortex of 14 days old offspring of group A (control group). It had a largeeuchromatic nucleus (N) with a nucleolus (Nu). The nuclear envelope (NE) had many pores (NP) and an invagination (arrow). Its cytoplasm contained free Golgi apparatus (GA), rounded or elongated mitochondria (M), rough endoplasmic reticula (Re) ribosomes (R).             (TEM   X 10000)

Fig. (10 BII): An electron micrograph of a part of Purkinje cell (PC) of the cerebellar cortex of 14 days old offspring of subgroup B1. Ithad irregular electron dense nucleus (N) with more electron dense nucleolus (Nu). Its cytoplasm appeared electron dense. It contained dilated rough endoplasmic reticulum (Re), small electron dense mitochondria (M).  The cell was surrounded by areas of ill-defined structures (star).     (TEM   X 10000)

Fig. (10CII): An electron micrograph of a part of Purkinje cell (PC) of the cerebellar cortex of 14 days old offspring of subgroup B2. Ithad irregular electron dense nucleus (N). Its cytoplasm appeared electron dense. It contained dilated rough endoplasmic reticulum (Re), small electron dense mitochondria (M).  The cell was surrounded by areas of ill-defined structures and internal granular cells (IGC).                         (TEM   X 10000)

Fig. (11AIII):  an electron micrograph of the internal granular cells (IGCs) of 14 days old offspring cerebellar cortex of group A (control group). They had oval heterochromatic nuclei (N) containing clumps of heterochromatin and eccentric nucleoli (Nu). The nuclear envelope (NE) contained many nuclear pores (NP). The cytoplasm contained few rough endoplasmic reticulum (Re), mitochondria (M) and free ribosomes(R) and surrounded by a well-defined plasma membrane (Pl).                                                             (TEM X 10000)

Fig. (11BII): An electron micrograph of the internal granular cells (IGCs) of 14 days old offspring cerebellar cortexof subgroup B1.  Some internal granular cells had ill-defined plasma membrane in some areas and irregular hyperchromatic nuclei (N) with condensed clumps of heterochromatinand dilated nuclear envelope (NE).  The cytoplasm contained dilated rough endoplasmic reticulum (Re) and swollen mitochondria with destructed cristae (M). Some granular cells (arrow) had electron dense nucleus and cytoplasm and their cytoplasm organelles could not be recognized. Notice presence of areas of ill-defined structures (star) and myelinated nerve fibers (My).                                     (TEM X 10000)

Fig. (11CII): An electron micrograph of the internal granular cells (IGCs) of 14 days old offspring cerebellar cortexof subgroup B2.  Some internal granular cells (arrow) had irregular hyperchromatic nuclei (N) with condensed clumps of heterochromatinand dilated nuclear envelope (NE).  They had electron dense cytoplasm and their cytoplasm organelles could not be recognized.Other cells had oval heterochromatic nuclei (N) with eccentric nucleoli (Nu) and nuclear envelope (NE), their cytoplasm contained few rough endoplasmic reticulum (Re), mitochondria (M) and free ribosomes(R).   (TEM X 10000)

Morphometric results: The mean thickness of the molecular layer of  the cerebellar cortex of 14 days old albino rats of subgroups A1, A2, B1 and B2 showed a significant difference (P<0.05). There was a significant decrease in the mean thickness of the molecular layer in subgroups B1 and B2 (P<0.05) compared to subgroups A1 and A2. There was a significant decrease in thickness of the molecular layerin subgroup B1 (P<0.05) compared to B2 (Table 1).

     The mean number of Purkinje cellsof  the cerebellar cortex of 14 days old albino rats ofsubgroups A1, A2, B1 and B2  showed a significant difference (P<0.05). There was a significant decrease in the mean number of Purkinje cellsin subgroups B1 and B2 (P<0.05) compared to subgroups A1 and A2. There was no significant difference (P>0.05) between groups B1 and B2 regarding the mean number of Purkinje cells(Table 2).

Table (1): ANOVA F test and Post- hoc Tukey's tests for thickness of the molecular layer of the cerebellar cortex of 14 days old offspring of A1, A2, B1 and B2

Table (2): ANOVA F test and Post- hoc Tukey's tests for Purkinje cell numbers of the cerebellar cortex of 14 days old offspring of A1, A2, B1 and B2

DISCUSSION

     In this study, oseltamivir phosphate produced delayed development and neurotoxic effects in the cerebellar cortex of one and 14 days old albino offspring whose mothers received OP during the third trimester of pregnancy, also, these effects were seen in 7 and 14 days old albino offspring whose mothers received the drug during the first week after delivery. These findings suggested transplacental and translactional passage of OP.

     Transplacental passage was confirmed by results of Koren et al. (2010) who found that OP crosses the placenta in rats and rabbits. Additionally, Lin et al.  (2012) suggested that both OP and its metabolite OC can penetrate the placenta, amniotic fluids and fetus in rats during pregnancy; also, they found that the penetration of OC was much lower than that of OP; however, OC might not be easily metabolized in the fetus due to the lack of carboxylase in the fetus. In an ex vivo human placental model. Also, a human placenta perfusion study by Berveiller et al. (2012) showed that the fetal transfer rate is 8.5% and 6.6% for OP and OC, respectively. Meijer et al. (2012) who measured concentrations of OP and OC metabolites from human blood samples drawn from the mother and from the umbilical cord after taking high-dose OP (150 mg twice/day).They found that fetal transfer rate for OP was 23.5% and OC was 73.4%.  In contrast,  Tanaka et al.  (2009) suggested that oseltamivir was extensively metabolized by the placenta. Also, transplacental transfer of the metabolite was incomplete with minimal accumulation on the fetus.

     Translactional passage is confirmed by a study of Areej et al. (2012) who revealed that OP produced neurotoxic effect in mice pups through indirect administration OP to nursing mothers during lactation period and the level of toxicity was in dose dependent manner. They suggested that OP and/or OC has ability to penetrate blood brain barrier (BBB) which complete maturation   around the third postnatal week. This penetration occurs in time when BBB is still immature during lactation period.  Also, Greer et al. (2011) reported that oseltamivir is excreted in human breast milk.

     In the current study, delayed development was seen in the form of decrease in the rows of external granular cells in one and 7 days old offspring while increase rows in 14 days old albino offspring, also, there was significant decrease in the thickness of molecular layer in 14 days old offspring.  The degenerative changes were observed in the form of irregularity of cells, loss of integrity of their plasma membrane, vacuolation of the cytoplasm, dilation of the rough endoplasmic reticulum, degeneration of mitochondria and nuclear changes in the form of pyknosis. The neuropil showed loss of integrity of their structures with degeneration of mitochondria and dilatation of the rough endoplasmic reticulum. In addition, Purkinje cells were significantly decreased compared to the control. These changes were present in offspring of the mothers received OP during last trimester of pregnancy or during first week of lactation.

     The results of present study were in current with the findings of Yoshino et al.  (2008) in a study on Webester rats, they observed ataxia in these rats after administration of OP to them. Moreover, Areej et al. (2012) observed neurotoxic symptoms in mice pups received OP through milk from dosed nursing mothers. They revealed that is due to the extensive damage involving cerebellum and also cerebrum. These neurotoxic symptoms were weakness, convulsions, incoordination, extended limbs and limbs stiffness. Also, gross and histopathological examination of their bodies demonstrate that the brain was the most affected organ beside extensive lesions in liver, kidney, stomach and small intestine of treated groups in dose dependent manner. The brain showed edema, congestion and hemorrhage. The cerebellum showed edema with degenerative changes and complete dissolution of Purkinje cells. The juvenile compared to adult rats are thought to be more affected due to immature physiological processes such as lower carboxylesterase activity or a lower renal clearance. Also, a prospective cohort studies by Yorifuji et al. (2009) and Fujita et al. (2010) and a systematic review of the prospective cohort studies by Hama et al. (2010) indicate an association between abnormal behaviors and oseltamivir use. Additionally,  Hegazy et al. (2016) hypothesized that neuraminidase inhibited by OC, may participate in vital functions in the central nervous system (CNS), including development of neuronal cells and impulse.  Also, Chen et al. (2019) reported that there have been neuropsychiatric events associated with the drug, mainly in young patients and males.

     In contrast,  Freichel et al. (2009) made a study on 7 days old rats using single dose of oseltamivir phosphate at 300, 500.600,700,850 and 1000 mg/kg, they took samples from blood and brain tissue. Light microscopy revealed no histopathological changes at any dose level indicating wide safety margin of OP.  The difference between our results and results of this study may be due to the difference in the age of the exposed rat and duration of exposure.

     Also, Svensson et al. (2011) made a smaller observational cohort on human infants, and found that human infants exposed to a neuraminidase inhibitor at any point in pregnancy (oseltamivir, n=81; zanamivir, n=2) compared with infants not exposed (n=860), had no differences regarding birth weight, Apgar score, fetal mortality, or preterm birth, so they suggested that oseltamivir does not pose a significant threat to fetal development.

     We suggest that many mechanisms are involved in neurotoxic effect of OP. our suggestion clarified by many authors. Lindemann et al. (2010) suggested the possibility that OP blocks Na+ and Ca2+ channels. Moreover, Kipp et al. (2011) recorded that oseltamivir impaired FABP7 signaling. FABP7 is one of FABPs which are part of the intracellular lipid binding protein group so this could resulted in the malfunction of remyelination and development of clinical disability in the brain. Also, Areej et al  (2012) revealed that OP and OC may be interfering with dopamine and /or another catacholamins and even other CNS neurotransmitters. Hiasa et al. (2013) found that OP but not OC competitively and selectively inhibited human monoamine oxidase-A (MAO-A). This is responsible for the occasionally observed behavioral side effects of oseltamivir. Also, Marois et al. (2014) reported that oseltamivir influences sodium and potassium currents. Additionally, Muraki et al. (2015) showed that oseltamivir blocks nicotinic acetylcholine receptors.

     Additionally, Uchiyama et al. (2015) and Hegazy et al. (2016) showed that oseltamivir significantly altered mitochondrial enzyme complex activities resulting in production of intra-cellular reactive oxygen and nitrogen species which  attack the membrane phospholipids, therefore,  cause mitochondrial impairment.  Mitochondrial impairment may also result in Ca+ dysregulation and activation of nitric oxide synthase (NOS), leading to increased levels of total nitric oxide in the brain which may react with O2 to form peroxynitrite (ONOO–) a potent oxidant species. Also, reactive species liberate apoptosis inducing factors which trigger caspase cascades, causing nuclear condensation and produce secondary ROS, inducing tissue injury.

     Hama and Bennet (2017) added that unchanged oseltamivir inhibits nicotinic acetylcholine receptors, which are closely related to hypothermia, as well as human monoamine oxidase-A (MAO-A), which is closely related to abnormal or excitatory behaviors. Association between oseltamivir use and serious adverse events including sudden death and abnormal behaviors remains controversial.

CONCLUSION

         Oseltamivir phosphate induced pre and post-natal delayed development and neurotoxic insults on the cerebellar cortex of albino rat offspring.

RECOMMENDATION

     Treatment with oseltamivir phosphate during pregnancy should be considered whenever maternal benefit outweighs fetal risk. Also, it is better to postpone lactation during the treatment with OP.

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