External Granular Layer Research Articles

Histomorphometry of the cortical layers and the dentate nucleus of the human fetal cerebellum

ObjectivesThis study was aimed at determining the histomorphometry of the cerebellar cortical laminae and the dentate nucleus of the human fetal cerebellum; the number and shape of the neurons; and the gestational age of appearance of the cerebellar folia, white matter and arbor vitae cerebelli. MethodsMicroscopic sections of the human fetal cerebellum stained with hematoxylin and eosin and Bielschowsky silver stain were studied. ResultsThe thickness of the cortical laminae of the human fetal cerebellum varied among gestational weeks as follows: external granular layer: 36.06 ± 9.36–50.05 ± 34.06 μm, molecular layer: 32.76 ± 17.16–52 ± 28.6 μm, Purkinje cell layer: 9.36 ± 6.8–15.6 ± 4.68 μm and internal granular layer: 66.65 ± 24.42–146.63 ± 47.79 μm. Similarly, the number of neurons per field of view at 1000X under a compound microscope varied among gestational weeks as follows: external granular layer: 89.92 ± 42–142.84 ± 50, molecular layer: 15 ± 12.5–25 ± 8.25, Purkinje cell layer: 3.5 ± 1–5 ± 2.5 and internal granular layer: 98.5 ± 69.75–224 ± 47.White matter in the fetal cerebellum was already present at the age of 12th gestational week, whereas cerebellar folia appeared at 16–20 gestational weeks. Arbor vitae cerebelli and the dentate nucleus became conspicuous after the 20th gestational week. Fetal neurons were round except for Purkinje cells. ConclusionsThe thickness and neuronal counts of the human fetal cerebellar cortical layers and the measurements of the dentate nucleus along with other histomorphological features varied with gestational age from the 12th week of gestation until birth.

Growth Hormone (GH) Crosses the Blood-Brain Barrier (BBB) and Induces Neuroprotective Effects in the Embryonic Chicken Cerebellum after a Hypoxic Injury.

Several motor, sensory, cognitive, and behavioral dysfunctions are associated with neural lesions occurring after a hypoxic injury (HI) in preterm infants. Growth hormone (GH) expression is upregulated in several brain areas when exposed to HI conditions, suggesting actions as a local neurotrophic factor. It is known that GH, either exogenous and/or locally expressed, exerts neuroprotective and regenerative actions in cerebellar neurons in response to HI. However, it is still controversial whether GH can cross the blood–brain barrier (BBB), and if its effects are exerted directly or if they are mediated by other neurotrophic factors. Here, we found that in ovo microinjection of Cy3-labeled chicken GH resulted in a wide distribution of fluorescence within several brain areas in the chicken embryo (choroid plexus, cortex, hypothalamus, periventricular areas, hippocampus, and cerebellum) in both normoxic and hypoxic conditions. In the cerebellum, Cy3-GH and GH receptor (GHR) co-localized in the granular and Purkinje layers and in deep cerebellar nuclei under hypoxic conditions, suggesting direct actions. Histological analysis showed that hypoxia provoked a significant modification in the size and organization of cerebellar layers; however, GH administration restored the width of external granular layer (EGL) and molecular layer (ML) and improved the Purkinje and granular neurons survival. Additionally, GH treatment provoked a significant reduction in apoptosis and lipoperoxidation; decreased the mRNA expression of the inflammatory mediators (TNFα, IL-6, IL-1β, and iNOS); and upregulated the expression of several neurotrophic factors (IGF-1, VEGF, and BDNF). Interestingly, we also found an upregulation of cerebellar GH and GHR mRNA expression, which suggests the existence of an endogenous protective mechanism in response to hypoxia. Overall, the results demonstrate that, in the chicken embryo exposed to hypoxia, GH crosses the BBB and reaches the cerebellum, where it exerts antiapoptotic, antioxidative, anti-inflammatory, neuroprotective, and neuroregenerative actions.

Prenatal effects of alprazolam treatment on the immature cerebellum of rats.

The use of benzodiazepines (BZDs) during pregnancy, especially alprazolam, is common and its impact on the fetal neural tissue is not known. In this sense, the present study aimed to investigate the effects of prenatal treatment with alprazolam on the cerebellum of Wistar rat pups. Thirty animals (24 females and six males, CEUA protocol 014/17) were separated into pairs for copulation. Females were divided into three groups: Control (CT), treatment 1 (T1, 1.25 mg per animal), and treatment 2, which is an overdose (T2, 30 mg per animal). Alprazolam was administered 10 days before copulation and throughout pregnancy. We evaluated the number and weight of pups and the macroscopic changes in the brain. Eight neonates (n = 8) from each group were used in the following analyses: Cellular and chromatin density, gliosis, synaptic density, inflammation, and oxidative stress. The results showed no significant differences regarding the number of pups, body weight, and macroscopic changes. The morphological study focused on the external granular layer (EGL) that is presented only in the immature cerebellum. Here, we detected more cells after alprazolam treatment; the T2 group showed large nuclei and some pyknotic nuclei; also, both treated groups presented an increase in the euchromatin density compared with the control. The molecular and biochemical analyses used the total protein extract of the entire cerebellum and showed an increased expression of Iba-1 and NF-κBp65 but without indication of inflammation or degeneration in the T1 group. Overdose of alprazolam presented an increased level of oxidative degradation of lipids. The treatment with alprazolam during pregnancy involved cellular and molecular changes in the immature cerebellum.

Emerging signs of autism spectrum disorder in infancy: Putative neural substrate.

Autism spectrum disorder (ASD) is characterized by altered development of the social brain with prominent atypical features in the fronto-temporo-parietal cortex and cerebellum. Early signs of ASD emerge between 6 and 12 months: reduced social communication, slightly less advanced motor development, and repetitive behaviour. The fronto-temporo-parietal cortex and cerebellum play a prominent role in the development of social communication, whereas fronto-parietal-cerebellar networks are involved in the planning of movements, that is, movement selection. Atypical sensory responsivity, a core feature of ASD, may result in impaired development of social communication and motor skills and/or selection of atypical repetitive behaviour. In the first postnatal year, the brain areas involved are characterized by gradual dissolution of temporary structures: the fronto-temporo-parietal cortical subplate and cerebellar external granular layer. It is hypothesized that altered dissolution of the transient structures opens the window for the expression of early signs of ASD arising in the impaired developing permanent networks. WHAT THIS PAPER ADDS: The early social and motor signs of autism spectrum disorder emerge between the ages of 6 and 12 months. Altered dissolution of transient brain structures in the fronto-temporo-parietal cortex and cerebellum may underlie the emergence of these early signs.

Expression Analyses of Cep152, a Responsible Gene Product for Autosomal Recessive Primary Microcephaly, during Mouse Brain Development

Centrosomal protein 152 (Cep152) regulates centriole duplication as a molecular scaffold during the cell cycle. Its gene abnormalities are responsible for autosomal recessive primary microcephaly 9 and Seckel syndrome. In this study, we prepared an antibody against mouse Cep152, anti-Cep152, and performed expression analyses focusing on mouse brain development. Western blotting analyses revealed that Cep152 with a molecular mass of ∼150 kDa was expressed strongly at embryonic day (E)13 and then gradually decreased during the brain development process. Instead, protein bands of ∼80 kDa and ∼60 kDa came to be recognized after postnatal day (P)15 and P30, respectively. In immunohistochemical analyses, Cep152 was enriched in the centrosome of neuronal progenitors in the ventricular zone at E14, whereas it was diffusely distributed mainly in the cytoplasm of cortical neurons at P18. In developing cerebellum at P7, Cep152 was localized at the centrosome in the external granular layer, where neurogenesis takes place. Notably, biochemical analysis revealed that Cep152 was also present in the postsynaptic density fraction. Subsequent immunofluorescent analyses showed co-localization of Cep152 with excitatory synaptic markers, PSD95 and synaptophysin, but not with an inhibitory synaptic marker gephyrin in differentiated primary cultured hippocampal neurons. The obtained results suggest that Cep152 takes part not only in neurogenesis during corticogenesis but also in the regulation of synaptic function in differentiated neurons.

Changes in 5-Fluorouracil-induced external granular cell damage during the time-course of the developing cerebellum of infant rats.

5-Fluorouracil (5-FU) is widely used as a chemotherapeutic agent that blocks DNA synthesis and replication by inhibiting thymidylate synthetase. This study aimed to elucidate 5-FU-induced changes in the external granular cells (EGCs) in the cerebellum of infant rats and the possible underlying mechanism. Six-day-old infant rats were injected subcutaneously with 40 mg/kg of 5-FU, and their cerebellums were examined at 6, 9, 12, and 24 h after treatment (HAT), and 2, 4, and 10 d after treatment (DAT). The width of the external granular layer (EGL) decreased from 24 HAT to 4 DAT in the 5-FU group compared to that in the control group. However, the width in the 5-FU group was comparable to that of the control group at 10 DAT. The number of apoptotic cells, cleaved caspase 3-labeling index (LI%), p21cip1-LI%, and expression levels of p53, p21cip1, and Fas mRNAs increased at 24 HAT. However, no changes were detected in the expression levels of Puma and Bax mRNAs at any time point. BrdU-LI% increased at 6 and 12 HAT but decreased at 24 HAT. The phospho-histone H3-LI% decreased from 6 HAT to 2 DAT. The width of the molecular layer decreased compared to that of the control group at 10 DAT. No differences were observed in Purkinje cell development. These results indicate that 5-FU inhibited cell proliferation by inducing apoptosis of EGCs via activation of Fas and caspase-3 without the involvement of the mitochondrial pathway and induced p53-dependent G1-S and G2-M phase arrest.

Aquoeus Extracts of Daucus Carota (Linn) Protected the Postnatal Developing Cerebellum of Wistar Rats Against Arsenic-Induced Oxidative Stress.

The neuroprotective effects of the aqueous extract of Daucus carota (Dc) tuber against arsenic-induced oxidative damage on the developing cerebellum of Wistar rats were studied. Twenty-five pregnant rats (110-200g) were divided into five groups (n=5) - control received distilled water; Arsenic (As); Dc (200mg/kg); Dc (200mg/kg) +As; Vitamin C (Vc) (100mg/kg) +As. The pregnant rats in all the groups were treated orally from the first day of pregnancy to postnatal day 21. The Dc extract and Vc were administered one hour before the administration of As. Body weight of the pups on days 1, 7, 14, 21 and 28 were recorded, while neurobehavioural (forelimb grip strength and negative geotaxis) tests were done on day 21 pups. The rats were sacrificed and cerebellar tissues were collected for oxidative stress, histological (H and E), and immunohistochemical studies.Decreased forelimb grip strength, increased lipid peroxidation and decreased glutathione, glutathione peroxidase, catalase and superoxide dismutase was observed in the As group compared with the control and other treated groups. Histologically, the cerebellar cortex of the As pups showed persistent external granular layer (EGL) on postnatal day 21, reduced thickness of the molecular layer (ML) on postnatal day 28, pyknotic and depleted Purkinje cells compared with the control and other treated rats. Immunohistochemical evaluations of the cerebellar cortex showed astroliosis in the As-treated group on day 21 pups compared with the control and other treated groups. Aqueous extracts of Daucus carota and Vitamin C reversed the toxicity caused by arsenic. From the results of the study, arsenic-induced oxidative stress with morphological alterations in the perinatal developing rat cerebellum. Extracts of Daucus carota exhibited antioxidant activity as such may be a potential neuroprotective agent.

Aquoeus Extracts of Daucus Carota (Linn) Protected the Postnatal Developing Cerebellum of Wistar Rats Against Arsenic-Induced Oxidative Stress

The neuroprotective effects of the aqueous extract of Daucus carota (Dc) tuber against arsenic-induced oxidative damage on the developing cerebellum of Wistar rats were studied. Twenty-five pregnant rats (110-200g) were divided into five groups (n=5) – control received distilled water; Arsenic (As); Dc (200mg/kg); Dc (200mg/kg) +As; Vitamin C (Vc) (100mg/kg) +As. The pregnant rats in all the groups were treated orally from the first day of pregnancy to postnatal day 21. The Dc extract and Vc were administered one hour before the administration of As. Body weight of the pups on days 1, 7, 14, 21 and 28 were recorded, while neurobehavioural (forelimb grip strength and negative geotaxis) tests were done on day 21 pups. The rats were sacrificed and cerebellar tissues were collected for oxidative stress, histological (H and E), and immunohistochemical studies. Decreased forelimb grip strength, increased lipid peroxidation and decreased glutathione, glutathione peroxidase, catalase and superoxide dismutase was observed in the As group compared with the control and other treated groups. Histologically, the cerebellar cortex of the As pups showed persistent external granular layer (EGL) on postnatal day 21, reduced thickness of the molecular layer (ML) on postnatal day 28, pyknotic and depleted Purkinje cells compared with the control and other treated rats. Immunohistochemical evaluations of the cerebellar cortex showed astroliosis in the As-treated group on day 21 pups compared with the control and other treated groups. Aqueous extracts of Daucus carota and Vitamin C reversed the toxicity caused by arsenic. From the results of the study, arsenic-induced oxidative stress with morphological alterations in the perinatal developing rat cerebellum. Extracts of Daucus carota exhibited antioxidant activity as such may be a potential neuroprotective agent.

Cerebellar Patterning Defects in Mutant Mice.

The cerebellar cortex is highly compartmentalized and serves as a remarkable model for pattern formation throughout the brain. In brief, the adult cerebellar cortex is subdivided into five anteroposterior units—transverse zones—and subsequently, each zone is divided into ∼20 parasagittal stripes. Zone-and-stripe pattern formation involves the interplay of two parallel developmental pathways—one for inhibitory neurons, the second for excitatory. In the inhibitory pathway, progenitor cells of the 4th ventricle generate the Purkinje cells and inhibitory interneurons. In the excitatory pathway, progenitor cells in the upper rhombic lip give rise to the external granular layer, and subsequently to the granular layer of the adult. Both the excitatory and inhibitory developmental pathways are spatially patterned and the interactions of the two generate the complex topography of the adult. This review briefly describes the cellular and molecular mechanisms that underly zone-and-stripe development with a particular focus on mutations known to interfere with normal cerebellar development and the light they cast on the mechanisms of pattern formation.

Anti-NMDA-Receptor Encephalitis in a Patient with Ovarian Teratoma, Harboring Brain Histology of Varying Developmental Stages and Regions with Chronic Inflammation

Abstract Introduction/Objective Anti-NMDA-receptor encephalitis is a subacute, autoimmune disorder thought to be caused by autoantibodies directed against the N-methyl-D-aspartate (NMDA) receptor. Clinical symptoms of anti-NMDAR encephalitis may mimic schizophrenia and psychotic spectrum disorders or substance-induced psychosis. Although initially described in association with ovarian teratomas in women, anti-NMDAR encephalitis has been reported in individuals without paraneoplastic association, as well as in males. Herein, we report a case of a 29-year-old woman with suicidal ideation and other neuropsychiatric manifestations who was found to have a right ovarian cystic mass by imaging study. Microscopically, the resected ovarian mass is composed of mature skin, fat, cartilage and neural tissues. Nerve, ganglions and multiple brain tissues are present. Cerebellum including external granular cell layer (normally only seen in infants), cerebrum-like, choroid plexus and other neural elements are present. There is peripheral lymphoplasmacytic infiltrates around and within the neuroglial matrix. Cerebral spinal fluid was concurrently tested positive for Anti-NMDAR. The combined clinical, histological, and laboratory findings confirmed the above diagnosis. Although Anti-NMDAR encephalitis is a familiar entity to many clinical psychiatrist and neurologists, it is less commonly reported in the pathology literature. Its resultant relationship to cystic teratoma warrants awareness of this condition by pathologists. Methods/Case Report Case Report Results (if a Case Study enter NA) NA Conclusion Anti-NMDA-receptor encephalitis is related to cystic teratoma, therefore pathologists need to be aware of this condition.

Evaluation of the Postnatal Effects Induced by Diazinon on the Growth of the Mice Offspring and the Development of Their Cerebellar Cortex

Diazinon (DZN) is a commonly used organophosphorus pesticide. Exposure to the residuals of DZN may lead to toxic effects. The current work was performed to clarify the possible physical variations and cellular changes in the developing cerebellar cortex of mice offspring after administration of DZN to their pregnant mothers. 27 adult males and 54 adult females were housed for mating. In the morning, vaginal smears were done to detect vaginal plug. Pregnant mice were divided into 3 groups: GI (control), GII (DZN), and GIII (sham control). Their offspring were subdivided into 3 subgroups (a) at birth, (b) on postnatal day 7 (PD7), and (c) on postnatal day 14 (PD14). At the end of the experiment, fetal crown-rump length and weight were measured. The cerebellar cortex was extracted, and samples were prepared for tissue homogenate, light and electron microscopic examination, morphometric and statistical studies. DZN treatment induced a statistically significant difference in pups’ crown-rump length and weight associated with a highly statistically significant decrease in acetylcholinesterase enzyme level. A histopathological degenerative change was observed in the cerebellum of GII. Postnatally, a separation between cerebellar layers occurred along with shrunken cells leading to multiple enfolding and vacuolated matrix. At PD14, pericellular halos and hemorrhage between the pia matter and external granular layer were noticed. Ultrastructural examination revealed dilated rough endoplasmic reticulum, swollen mitochondria, and shrunken hyperchromatic nuclei. Moreover, morphometric studies detected a statistically significant increase in external granular layer thickness and a statistical decrease in Purkinje cell numbers. These findings demonstrated that prenatal administration of DZN to pregnant mice adversely influenced the developing cerebellum of the offspring and leads to neurodegenerative changes.

Kola nut from Cola nitida vent. Schott administered to pregnant rats induces histological alterations in pups' cerebellum.

Kola nut (from Cola nitida) is popular in Nigeria and West Africa and is commonly consumed by pregnant women during the first trimester to alleviate morning sickness and dizziness. There is, however, a dearth of information on its effects on the developing brain. This study, therefore, investigated the potential effects of kola nut on the structure of the developing neonatal and juvenile cerebellum in the rat. Pregnant Wistar rats were administered water (as control) or crude (aqueous) kola nut extract at 400, 600, and 800 mg/kg body weight orally, from pregnancy to day 21 after birth. On postnatal days 1, 7, 14, 21 and 28, the pups were weighed, anaesthetised, sacrificed and perfused with neutral buffered formalin. Their brains were dissected out, weighed and the cerebellum preserved in 10% buffered formalin. Paraffin sections of the cerebellum were stained with haematoxylin and eosin for cerebellar cytoarchitecture, cresyl violet stain for Purkinje cell count, Glial Fibrillary Acidic Protein (GFAP) immunohistochemistry (IHC) for estimation of gliosis, and B-cell lymphoma 2 (Bcl-2) IHC for apoptosis induction. The kola nut-treated rats exhibited initial reduction in body and brain weights, persistent external granular layer, increased molecular layer thickness, and loss of Bergmann glia. Their Purkinje cells showed reduction in density, loss of dendrites and multiple layering, and their white matter showed neurodegeneration (spongiosis) and GFAP and Bcl-2 over-expression, with evidence of reactive astrogliosis. This study, therefore, demonstrates that kola nut, administered repeatedly at certain doses to pregnant dams, could disrupt normal postnatal cerebellar development in their pups. The findings suggest potential deleterious effects of excessive kola nut consumption on human brain and thus warrant further studies to understand the wider implications for human brain development.

Early Diagnostics and Early Intervention in Neurodevelopmental Disorders-Age-Dependent Challenges and Opportunities.

This review discusses early diagnostics and early intervention in developmental disorders in the light of brain development. The best instruments for early detection of cerebral palsy (CP) with or without intellectual disability are neonatal magnetic resonance imaging, general movements assessment at 2–4 months and from 2–4 months onwards, the Hammersmith Infant Neurological Examination and Standardized Infant NeuroDevelopmental Assessment. Early detection of autism spectrum disorders (ASD) is difficult; its first signs emerge at the end of the first year. Prediction with the Modified Checklist for Autism in Toddlers and Infant Toddler Checklist is possible to some extent and improves during the second year, especially in children at familial risk of ASD. Thus, prediction improves substantially when transient brain structures have been replaced by permanent circuitries. At around 3 months the cortical subplate has dissolved in primary motor and sensory cortices; around 12 months the cortical subplate in prefrontal and parieto-temporal cortices and cerebellar external granular layer have disappeared. This review stresses that families are pivotal in early intervention. It summarizes evidence on the effectiveness of early intervention in medically fragile neonates, infants at low to moderate risk, infants with or at high risk of CP and with or at high risk of ASD.

Origins, Development, and Compartmentation of the Granule Cells of the Cerebellum.

Granule cells (GCs) are the most numerous cell type in the cerebellum and indeed, in the brain: at least 99% of all cerebellar neurons are granule cells. In this review article, we first consider the formation of the upper rhombic lip, from which all granule cell precursors arise, and the way by which the upper rhombic lip generates the external granular layer, a secondary germinal epithelium that serves to amplify the upper rhombic lip precursors. Next, we review the mechanisms by which postmitotic granule cells are generated in the external granular layer and migrate radially to settle in the granular layer. In addition, we review the evidence that far from being a homogeneous population, granule cells come in multiple phenotypes with distinct topographical distributions and consider ways in which the heterogeneity of granule cells might arise during development.

The Possible Effect of pregabalin on the postnatal development of the cerebellar cortex in albino rats (Histological &Ultrastrucural study).

Background: Pregabalin is an oral medication that is used to treat neuropathic pain, generalized anxiety disorder and epilepsy; as a therapy for partial seizures. Cerebellar development is not complete during gestation only; but continues after birth to maturation of the cerebellum (postnatal development).Aim of the Work: To study the possible effect of Pregabalin on the postnatal development of the cerebellar cortex of albino rat.Materialandmethods:60 pregnant albino rats were allocated into three groups; Group A: Included 20offspring of 20 control mothers, Group B: Included 20 offspring of 20 mothers treated orally with pregabalin at a dose of 150mg/kg, and Group C:included 25 offspring of 20 mothers treated orally with pregabalin at a dose of 600mg/kg. In the three groups rats were sacrificed at postnatal (age 1, 2, 3 weeks), in addition to adult age (2.5 months).The skulls were opened and the cerebella were removed and processed for light and transmission electron microscope. Results: Pregabalin administration during pregnancy and lactation caused its marked effect during early postnatal life and this effect extended till the adult stage; in the form of increase in the external granular layer (mainly in the 2nd and 3rd weeks); less thickness of the molecular layer in all treated groups; shrunken and abnormal shaped Purkinje cells which appeared with ill-defined cell membranes and destructed nuclei; mainly at higher dose. There were also delayed differentiation of the internal granular layer and granule cells appeared with destructed cytoplasmic membrane and organelles which appeared prominent at higher doses. Conclusion: Pre and postnatal administration of pregabalin in both low and high doses caused loss of cellular components, distortions of cerebellar cortical cells in a dose dependent manner.

Neuroprotective potentials of Lycopersicon esculentum fruit extract on cadmium-induced toxicity in postnatal developing cerebellum of rats

The neuroprotective potemtials of Lycopersicon esculentum fruit extract on cadmium-induced oxidative stress in postnatal developing cerebellum of rats. Twenty-five pregnant Wistar rats (110-200g) were divided into five groups (n=5) and used for the experiment. Group I served as control and received water, Group II received 0.5mg/kg of Cadmium only, Group III received 50mg/kg aqueous extract of Lycopersicon esculentum only, Group IV received 0.5mg/kg body weight of cadmium+50mg/kg aqueous extract of Lycopersicon esculentum and Group V received 0.5mg/kg of cadmium +200mg/kg of vitamin C, orally from the first day of gestation to postnatal day 21. Lycopersicon esculentum was administered one hour before the administration of cadmium. Neurobehavioural tests was done on the pups of day 21. Then, pups of day 1, 7, 14, 21, and 28 were weighed, sacrificed and their brains dissected out, weighed with the cerebellum preserved for biochemical and histomorphological evaluations. Data were analysed using ANOVA at p < 0.05. Cadmium-treated rats showed significant decreased in body and brain weight, decreased time spent on the forelimb grip, increased negative geotaxis, increased lipid peroxidation (LPO), and decreased glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the developing cerebellum. Histologically, there was persistent external granular layer (EGL), reduced molecular layer (ML) thickness and increased astrocytes population in the cerebellar cortex of cadmium-treated day 21pups. Lycopersicon esculentum extract and vitamin C reversed the behavioural and morphological alterations induced by cadmium toxicity. Cadmium-induced oxidative stress, behavioural deficit and morphological alterations in the postnatal developing cerebellum of rat was ameliorated by Lycopersicon esculentum fruit extracts as such may be a potential neuroprotective agent.
 Key words: Cadmium - oxidative stress - Lycopersicon esculentum – Neurobehaviour - Developing cerebellar cortex

Perinatal exposure to nonylphenol promotes proliferation of granule cell precursors in offspring cerebellum: Involvement of the activation of Notch2 signaling

Nonylphenol (NP), a widely diffused persistent organic pollutant (POP), has been shown to impair cerebellar development and cause cerebellum-dependent behavioral and motor deficits. The precise proliferation of granule cell precursors (GCPs), the source of granular cells (GCs), is required for normal development of cerebellum. Thus, we established an animal model of perinatal exposure to NP, investigated the effect of NP exposure on the cerebellar GCPs proliferation, and explored the potential mechanism involved. Our results showed that perinatal exposure to NP increased cerebellar weight, area, and internal granular cell layer (IGL) thickness in offspring rats. Perinatal exposure to NP also resulted in the GCPs hyperproliferation in the external granular layer (EGL) of the developing cerebellum, which may underlie the above-mentioned cerebellar alterations. However, our results suggested that perinatal exposure to NP had no effects on the length of GCPs proliferation. Meanwhile, perinatal exposure to NP also increased the activation of Notch2 signaling, the regulator of GCPs proliferation. In conclusion, our results supported the idea that exposure to NP caused the hyperproliferation of GCPs in the developing cerebellum. Furthermore, our study also provided the evidence that the activation of Notch2 signaling may be involved in the GCPs hyperproliferation.

The Effect of Induction of Maternal Hypothyroidism on Postnatal Cerebellar Cortex Development in Albino Rat Offspring and the Role of Thyroxin Replacement Therapy Histological, Immunohistochemical and Genetic Study

Background: Maternal thyroid hormones are necessary for the growth of the central nervous system before birth and their shortage can delay cerebellum development.Aim: This study aimed to evaluate the effect of maternal hypothyroidism induction on the development of the cerebellar cortex postnatally in offspring and to compare thyroxin replacement to mothers and postnatally to offspring.Material and Methods: Rat offspring were divided into 3 groups; group I (control), group II (hypothyroid); 15 offspring whose mothers received carbimazole (20 mg/kg/day orally) from the 1st gestational day to the 21st day of lactation. Group III (thyroid hormone replacement) included subgroup IIIa (15 rats) their mothers received carbimazole as group II and Levothyroxine (20 μg/kg/day subcutaneously) from the 10th day of gestation to 21st day of lactation, and subgroup IIIb (15 rats), their mothers received carbimazole as group II and offspring received Levothyroxine (20μg/kg/day subcutaneously) from day 1 postnatally. At the end of 1st, 2nd, and 3rd postnatal weeks, serum Thyroid-stimulating hormone, Free triiodothyronine, and thyroxin were estimated. Cerebellar cortex sections were stained with hematoxylin and eosin, Neurofilament, Myelin basic protein, and Bcl2 immunohistochemical stains. The real-time polymerase chain reaction was done for the reelin gene.Results: Group II showed a significantly reduced Free triiodothyronine, thyroxin, and increased Thyroid-stimulating hormone. Vacuolation in the external granular layer and delayed its disappearance and degeneration of Purkinje cells that increased with age were observed. Reduced myelination, neurofilament content, and Reelin gene expression in the offspring were also detected. Replacement therapy (group III) especially to the mothers (subgroup IIIa) revealed amelioration of these changes.Conclusion: Maternal hypothyroidism impaired development of the offspring cerebellar cortex. However, thyroxin replacement for mothers was more effective than the treatment of offspring. Therefore, treatment of hypothyroid mothers during pregnancy is essential to ensure adequate cerebellar cortex development.

Lead-induced oxidative stress in postnatal developing cerebellum of Wistar rats: role of aqueous extract of Cucumis sativus Linn and vitamin C

Lead (Pb) is one of the most abundant heavy metal whose toxicity causes environmental and health problems, with the brain being highly sensitive to lead toxicity. There is limited literature regarding the protection of the developing brain against lead-induced toxicity. However, plant-derived products with antioxidant activity have been useful in reducing lead-induced neurotoxicity. This study investigated the role of Cucumis sativus (C. sativus) (Cucumber) and vitamin C in lead-induced oxidative stress in postnatal developing cerebellum of Wistar rats. Twenty-five pregnant Wistar rats weighing between 160 and 180g were divided into five groups (n=5). Group I served as control and received water, group II received 50mg/kg of lead acetate, group III received 200mg/kg aqueous extract of C. sativus, group IV received 200mg/kg of C. sativus and 50mg/kg of lead acetate and group V received 200mg/kg of vitamin C and 50mg/kg of lead acetate. The interventions were administered orally using an oral gavage from the first day of gestation to postnatal day 21. Neurobehavioural assessment (forelimb grip strength and negative geotaxis) was carried out on pups of day 21 and then sacrificed. Some cerebella of pups of days 1, 7, 14, 21 and 28 were fixed in 10% formol-saline for histological and immunohistochemical evaluations, while others (day 21) were preserved in in phosphate buffered saline at 4°C and pH 7.2 for oxidative stress assays. Data were analysed using ANOVA at p<0.05. Decreased body weight of Pb-treated pups on days 14 and 21, decreased forelimb grip and increased negative geotaxis, increased lipid peroxidation (LPO), decreased glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities was seen in the lead-treated day 21 pups compared with the control and other treated groups. Histologically, in the cerebellar cortex, there was persistent external granular layer (EGL) on day 21 in the treated groups compared with the control, decreased molecular layer (ML) thickness and depleted Pc in the Pb-treated day 28 pups, and increased astrocyte population on day 21 pups compared with the control and C. sativus groups. Lead induced oxidative stress which caused behavioural deficit and morphological changes in the postnatal developing cerebellum of rats. Aqueous extracts of Cucumis sativus and vitamin C decreased the rate at which lead induced neurotoxicity.

Immunohistochemical characterization of postnatal changes in cerebellar cortical cytoarchitectures in ferrets.

We immunohistochemically characterized postnatal changes in cerebellar cortical cytoarchitectures in ferrets using markers for cerebellar cortical neurons and glial cells. Although 10 lobules of the vermis were already observed on postnatal day (PD) 4, Purkinje cells were still arrayed into two to three layers. Purkinje cells were aligned in a monolayer by PD 10 and formed mature shapes on PD 42 by developing their dendritic arbors. Parvalbumin immunostaining revealed relatively slower maturation of Purkinje cells in the Lobule X cortex than in other lobules. Basket and stellate cells emerged in the molecular layer on PDs 21 and 42, respectively. Rosette-like arranged glutamate decarboxylase 65 and 67-positive puncta were observed in the inner granular layer (IGL) on PD 21. Proliferating cell nuclear antigen immunostaining appeared in the outer zone of the external granular layer (EGL) containing progenitors of granular neurons on PDs 4-21. Bergmann glial processes extending vertically through the molecular layer and EGL were visible with GFAP immunostaining on PD 10 and thereafter. Their somata, aligned in the Purkinje cell layer, showed immunopositivity to Sox2 already on PD 4 and subsequently to S100 protein on PD 10. Sox2-positive cells were found sparsely in the IGL. Few of them were NeuN positive on PD 90, predicting the possibility of adult neurogenesis. These immunohistochemical results revealed that ferrets underwent cerebellar cortical histogenesis during their postnatal life in sequences. Relatively slow development or maturation of the ferret cerebellum was revealed by the timing of the monolayer alignment and morphological maturation of Purkinje cells.