Cerebellar Layer Research Articles

Probiotic microorganisms affect the reproductive and nervous systems of male rats treated with acrylamide

Objective: To evaluate the protective effects of probiotic microorganisms on the reproductive and nervous systems of male rats treated with acrylamide. Methods: Thirty-two rats were randomly divided into 4 groups and received normal saline through gavage (control), acrylamide 20 mg/kg body weight, acrylamide plus probiotic microorganisms (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Bifidobacterium breve, Bifidobacterium infantis, Streptococcus thermophilus and fructooligosaccharides, all mixed in sachets) 20 or 200 mg/kg body weight, respectively. After 30 days, the testis, prostate, seminal vesicle and cerebellum were removed, fixed and stained with hematoxylin-eosin (H & E). The Johnsen score was used to classify spermatogenesis. Cavalieri's principle method was used to evaluate the total volume (in mm3) of the testes. The number of each intratubular cell type as well as intertubular Leydig cells in whole samples was measured using the physical dissector counting techniques. Stereological analysis and the grids were used to determine the volume of cerebellar layers as well as the Purkinje cell number. Results: The testis weight decreased significantly in the acrylamide- treated group compared to the other groups (P<0.001). The number of spermatogonia, spermatocytes, spermatids and Leydig cells in the acrylamide-treated group were significantly less compared to the control group (P<0.05), while they were increased significantly in the acrylamide+200 mg/kg probiotic group (P<0.05; P<0.01). The mean Johnsen score in the acrylamide-treated group was lower than in the control group (P<0.001). Acrylamide-induced changes including congestion, vacuolization in the secretory epithelial cells, and epithelial rupture were observed in the prostate and seminal vesicle. The volumes of cerebellar layers were decreased in the acrylamide group compared to the control group while recovered in both probiotic treated groups. Conclusions: Probiotic microorganisms alleviate acrylamide- induced toxicities against the reproductive and cerebellar tissues in rats.

Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

AbstractThe prognosis for many pediatric brain tumors, including cerebellar medulloblastoma (MB), remains dismal but there is promise in new therapies. We have previously generated a mouse model developing spontaneous MB at high frequency, Ptch1+/−/Tis21−/−. In this model, reproducing human tumorigenesis, we identified the decline of the Cxcl3 chemokine in cerebellar granule cell precursors (GCPs) as responsible for a migration defect, which causes GCPs to stay longer in the proliferative area rather than differentiate and migrate internally, making them targets of transforming insults. We demonstrated that 4‐week Cxcl3 infusion in cerebella of 1‐month‐old mice, at the initial stage of MB formation, forces preneoplastic GCPs (pGCPs) to leave lesions and differentiate, with a complete suppression of MB development. In this study, we sought to verify the effect of 4‐week Cxcl3 treatment in 3‐month‐old Ptch1+/−/Tis21−/− mice, when MB lesions are at an advanced, irreversible stage. We found that Cxcl3 treatment reduces tumor volumes by sevenfold and stimulates the migration and differentiation of pGCPs from the lesion to the internal cerebellar layers. We also tested whether the pro‐migratory action of Cxcl3 favors metastases formation, by xenografting DAOY human MB cells in the cerebellum of immunosuppressed mice. We showed that DAOY cells express the Cxcl3 receptor, Cxcr2, and that Cxcl3 triggers their migration. However, Cxcl3 did not significantly affect the frequency of metastases or the growth of DAOY‐generated MBs. Finally, we mapped the expression of the Cxcr2 receptor in human MBs, by evaluating a well‐characterized series of 52 human MBs belonging to different MB molecular subgroups. We found that Cxcr2 was variably expressed in all MB subgroups, suggesting that Cxcl3 could be used for therapy of different MBs.

Zonisamide Ameliorated the Apoptosis and Inflammation in Cerebellar Tissue of Induced Alcohol Addiction Animal Model.

This study investigated the effects of zonisamide treatment on cerebellar tissues in an experimental alcohol addiction (AA) model and its potential mechanisms of action, particularly regarding apoptotic protease activating factor-1 (APAF-1) and tumor necrosis factor-alpha (TNF-α) expression. Thirty rats were divided into three groups: sham, ethanol (EtOH), and EtOH + zonisamide. AA was induced by administering 6 cc of EtOH orally every 8 h for 4 days. Zonisamide (100 mg/kg) was given to rats once daily before EtOH administration. Motor defects were evaluated using an open field maze. Serum TNF-α levels were measured from blood samples. Cerebellar sections were processed for histological examination and immunostained for APAF-1 and TNF-α. Protein interaction networks were constructed using Cytoscape, and functional annotations were performed with ShinyGO (version 0.80) software. The traveled area in the EtOH group was significantly reduced compared to the sham group (p = 0.0005). Rats in the EtOH + zonisamide group covered a larger area, with zonisamide treatment significantly improving locomotor ability compared to the EtOH group (p = 0.0463). Serum TNF-α levels were significantly elevated in the EtOH group compared to the sham group (p < 0.0001) and were significantly decreased in the EtOH + zonisamide group compared to the EtOH group (p = 0.0309). Regular cerebellar histological layers were observed in the sham group, while EtOH induction caused loss of cerebellar tissue integrity, neuronal degeneration, vascular dilatation and congestion, reduced myelin density, and neuropils in the EtOH group. Zonisamide treatment improved these pathologies, enhancing myelination and neuropil formation. Negative APAF-1 and TNF-α expressions were observed across cerebellar layers in the sham group. Due to EtOH toxicity, APAF-1 and TNF-α expression were upregulated in the EtOH group compared to the sham group (p < 0.001 for both). Zonisamide treatment downregulated these protein expressions in the EtOH + zonisamide group compared to the EtOH group (p < 0.001 and p = 0.0087, respectively). APAF-1 was primarily associated with AA through antifolate resistance, endopeptidases, and the interleukin-1 pathway, while TNF-α was predominantly enriched in infections and choline-binding, indicating zonisamide's impact on immune and inflammatory pathways. In conclusion, zonisamide treatment significantly mitigated ethanol-induced cerebellar damage and inflammation in an AA model. Zonisamide improved locomotor function and reduced serum TNF-α levels, as well as APAF-1 and TNF-α expression in cerebellar tissues. These findings suggest that zonisamide exerts its protective effects by modulating immune and inflammatory pathways, thereby preserving cerebellar integrity and function.

An unbiased approach of individual tissue-specific autoantibody identification using Immunoprecipitation coupled to Mass-Spectrometry (IP-MS)

Abstract Background and aim Autoreactive antibodies have gained increasing recognition in autoimmune diseases of the central nervous system (CNS) serving as disease drivers and / or biomarkers. Identification of novel autoantibodies therefore is crucial to optimize diagnostic work and therapy of these diseases as well as understanding their pathogenesis. Hence, we aimed to develop an unbiased strategy to identify patients’ individual main autoantibodies directed against brain epitopes. Methods We used murine tissue-based assays to screen patients’ sera and cerebrospinal fluids (CSFs) for autoreactive IgGs with known and unknown antibody targets. IgGs from autoreactive patients’ samples were coupled to magnetic beads and used as a bait to pull down potential targets from homogenized brain tissue of wildtype mice. After precipitation and several washes to reduce unspecific binding, beads were subjected to on-bead tryptic digestion followed by mass spectrometry based proteomic analyses using liquid chromatography tandem mass-spectrometry (LC-MS/MS). For antibody targets identification the fold change enrichment compared to a negative control or to all other pulldown samples, as well as the protein abundance was used. Antibody target validation was done via recombinant expression of the target protein in HEK-293 cells and neutralization with the respective antigen in tissue-based immunofluorescence assays. Results As proof of principle, we were able to identify Grin1 Caspr2, Trim46 and Nf-h [MOU1] as main targets of patients’ sera or CSF with autoimmune encephalopathy. All four antigens showed highest enrichment and/or highest protein abundance in the respective samples and have been confirmed in cell-based assays. In addition, we identified one novel anti-neuronal antibody target in serum and CSF of a patient showing a speckled staining of the granular cerebellar layer. Preabsorbance of the serum and CSF with the recombinant protein fully neutralized the signal of the tissue-based immunofluorescence. Conclusions Our IP-MS approach is suitable to identify the main autoantibody targets in patients’ serum and CSF within murine CNS tissue thereby providing an unbiased and scalable technique allowing for tissue-specific autoantibody determination.

Investigating brain alterations in the Dp1Tyb mouse model of Down syndrome

Down syndrome (DS) is one of the most common birth defects and the most prevalent genetic form of intellectual disability. DS arises from trisomy of chromosome 21, but its molecular and pathological consequences are not fully understood. In this study, we compared Dp1Tyb mice, a DS model, against their wild-type (WT) littermates of both sexes to investigate the impact of DS-related genetic abnormalities on the brain phenotype.We performed in vivo whole brain magnetic resonance imaging (MRI) and hippocampal 1H magnetic resonance spectroscopy (MRS) on the animals at 3 months of age. Subsequently, ex vivo MRI scans and histological analyses were conducted post-mortem. Our findings unveiled the following neuroanatomical and biochemical alterations in the Dp1Tyb brains: a smaller surface area and a rounder shape compared to WT brains, with DS males also presenting smaller global brain volume compared with the counterpart WT. Regional volumetric analysis revealed significant changes in 26 out of 72 examined brain regions, including the medial prefrontal cortex and dorsal hippocampus. These alterations were consistently observed in both in vivo and ex vivo imaging data. Additionally, high-resolution ex vivo imaging enabled us to investigate cerebellar layers and hippocampal subregions, revealing selective areas of decrease and remodelling in these structures.An analysis of hippocampal metabolites revealed an elevation in glutamine and the glutamine/glutamate ratio in the Dp1Tyb mice compared to controls, suggesting a possible imbalance in the excitation/inhibition ratio. This was accompanied by the decreased levels of taurine. Histological analysis revealed fewer neurons in the hippocampal CA3 and DG layers, along with an increase in astrocytes and microglia. These findings recapitulate multiple neuroanatomical and biochemical features associated with DS, enriching our understanding of the potential connection between chromosome 21 trisomy and the resultant phenotype.

Reduction of Microvessel Number and Length in the Cerebellum of Purkinje Cell Degeneration Mice.

Degenerative effects of nerve tissues are often accompanied by changes in vascularization. In this regard, knowledge about hereditary cerebellar degeneration is limited. In this study, we compared the vascularity of the individual cerebellar components of 3-month-old wild-type mice (n = 8) and Purkinje cell degeneration (pcd) mutant mice, which represent a model of hereditary cerebellar degeneration (n = 8). Systematic random samples of tissue sections were processed, and laminin was immunostained to visualize microvessels. A computer-assisted stereology system was used to quantify microvessel parameters including total number, total length, and associated densities in cerebellar layers. Our results in pcd mice revealed a 45% (p < 0.01) reduction in the total volume of the cerebellum, a 28% (p < 0.05) reduction in the total number of vessels and a lower total length, approaching 50% (p < 0.001), compared to the control mice. In pcd mutants, cerebellar degeneration is accompanied by significant reduction in the microvascular network that is proportional to the cerebellar volume reduction therefore does not change density of in the cerebellar gray matter of pcd mice.

High-Resolution Motion-corrected 7.0-T MRI to Derive Morphologic Measures from the Human Cerebellum in Vivo.

Background The human cerebellum has a large, highly folded cortical sheet. Its visualization is important for various disorders, including multiple sclerosis and spinocerebellar ataxias. The derivation of the cerebellar cortical surface in vivo is impeded by its high foliation. Purpose To image the cerebellar cortex, including its foliations and lamination, in less than 20 minutes, reconstruct the cerebellocortical surface, and extract cortical measures with use of motion-corrected, high-spatial-resolution 7.0-T MRI. Materials and Methods In this prospective study, conducted between February 2021 and July 2022, healthy participants underwent an examination with either a 0.19 × 0.19 × 0.5-mm3, motion-corrected fast low-angle shot (FLASH) sequence (14.5 minutes) or a whole-cerebellum 0.4 × 0.4 × 0.4-mm3, motion-corrected magnetization-prepared 2 rapid gradient-echo (MP2RAGE) sequence (18.5 minutes) at 7.0 T. Four participants underwent an additional FLASH sequence without motion correction. FLASH and MP2RAGE sequences were used to visualize the cerebellar cortical layers, derive cerebellar gray and white matter segmentations, and examine their fidelity. Quantitative measures were compared using repeated-measures analyses of variance or paired t tests. Results Nine participants (median age, 36 years [IQR, 25-42 years; range, 21-62 years]; five women) underwent examination with the FLASH sequence. Nine participants (median age, 37 years [IQR, 34-42 years; range, 25-62 years]; five men) underwent examination with the MP2RAGE sequence. A susceptibility difference between the expected location of the granular and molecular cerebellar layers was visually detected in the FLASH data in all participants. The segmentations derived from the whole-cerebellum MP2RAGE sequence showed the characteristic anatomic features of the cerebellum, like the transverse fissures and splitting folds. The cortical surface area (median, 949 cm2 [IQR, 825-1021 cm2]) was 1.8 times larger, and the cortical thickness (median, 0.88 mm [IQR, 0.81-0.93 mm]) was five times thinner than previous in vivo estimates and closer to ex vivo reference data. Conclusion In vivo imaging of the cerebellar cortical layers and surface and derivation of quantitative measures was feasible in a clinically acceptable acquisition time with use of motion-corrected 7.0-T MRI. Published under a CC BY 4.0 license. Supplemental material is available for this article. See also the editorial by Dietrich in this issue.

Pathogenesis of selective damage of granule cell layer in cerebellum of rats exposed to methylmercury.

Granule cell-selective toxicity of methylmercury in the cerebellum is one of the main unresolved issues in the pathogenesis of Minamata disease. Rats were orally administered methylmercury chloride (10 mg/kg/day) for 5 consecutive days, and their brains were harvested on days 1, 7, 14, 21, or 28 after the last administration for histological examination of the cerebellum. It was found that methylmercury caused a marked degenerative change to the granule cell layers but not to the Purkinje cell layers. The generative change of the granule cell layer was due to cell death, including apoptosis, which occurred at day 21 and beyond after the methylmercury administration. Meanwhile, cytotoxic T-lymphocytes and macrophages had infiltrated the granule cell layer. Additionally, granule cells are shown to be a cell type susceptible to TNF-α. Taken together, these results suggest that methylmercury causes small-scale damage to granule cells, triggering the infiltration of cytotoxic T-lymphocytes and macrophages into the granule cell layer, which secrete tumor necrosis factor-α (TNF-α) to induce apoptosis in granule cells. This chain is established based on the susceptibility of granule cells to methylmercury, the ability of cytotoxic T lymphocytes and macrophages to synthesize and secrete TNF-α, and the sensitivity of granule cells to TNF-α and methylmercury. We propose to call the pathology of methylmercury-induced cerebellar damage the "inflammation hypothesis."

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.

Coenzyme Q10 Supplementation Increases Removal of the ATXN3 Polyglutamine Repeat, Reducing Cerebellar Degeneration and Improving Motor Dysfunction in Murine Spinocerebellar Ataxia Type 3.

Coenzyme Q10 (CoQ10), a well-known antioxidant, has been explored as a treatment in several neurodegenerative diseases, but its utility in spinocerebellar ataxia type 3 (SCA3) has not been explored. Herein, the protective effect of CoQ10 was examined using a transgenic mouse model of SCA3 onset. These results demonstrated that a diet supplemented with CoQ10 significantly improved murine locomotion, revealed by rotarod and open-field tests, compared with untreated controls. Additionally, a histological analysis showed the stratification of cerebellar layers indistinguishable from that of wild-type littermates. The increased survival of Purkinje cells was reflected by the reduced abundance of TUNEL-positive nuclei and apoptosis markers of activated p53, as well as lower levels of cleaved caspase 3 and cleaved poly-ADP-ribose polymerase. CoQ10 effects were related to the facilitation of the autophagy-mediated clearance of mutant ataxin-3 protein, as evidenced by the increased expression of heat shock protein 27 and autophagic markers p62, Beclin-1 and LC3II. The expression of antioxidant enzymes heme oxygenase 1 (HO-1), glutathione peroxidase 1 (GPx1) and superoxide dismutase 1 (SOD1) and 2 (SOD2), but not of glutathione peroxidase 2 (GPx2), were restored in 84Q SCA3 mice treated with CoQ10 to levels even higher than those measured in wild-type control mice. Furthermore, CoQ10 treatment also prevented skeletal muscle weight loss and muscle atrophy in diseased mice, revealed by significantly increased muscle fiber area and upregulated muscle protein synthesis pathways. In summary, our results demonstrated biochemical and pharmacological bases for the possible use of CoQ10 in SCA3 therapy.

Phenotypic, metabolic, and biogenesis properties of human stem cell-derived cerebellar spheroids

Human cerebellum consists of high density and complexity of neurons. Thus, it is challenging to differentiate cerebellar-like organoids with similar cellular markers and function to the human brain. Our previous study showed that the combination of retinoic acid (RA), Wingless/integrated (Wnt) activator, and Sonic Hedgehog (SHH) activator promotes cerebellar differentiation from human induced pluripotent stem cells (hiPSCs). This study examined phenotypic, metabolic, and biogenesis in early cerebellar development. Cerebellum spheroids were differentiated from human iPSK3 cells. During day 7–14, RA and Wnt activator CHIR99021 were used and SHH activator purmorphamine (PMR) was added later to promote ventralization. Gene expression for early cerebellar layer markers, metabolism, and extracellular vesicle (EV) biogenesis were characterized. Zinc-induced neurotoxicity was investigated as a proof-of-concept of neurotoxicity study. Flow cytometry results showed that there was no significant difference in NEPH3, PTF1A, OLIG2, and MATH1 protein expression between RCP (RA-CHIR-PMR) versus the control condition. However, the expression of cerebellar genes for the molecular layer (BHLE22), the granule cell layer (GABRB2, PAX6, TMEM266, KCNIP4), the Bergmann glial cells (QK1, DAO), and the Purkinje cell layer (ARHGEF33, KIT, MX1, MYH10, PPP1R17, SCGN) was significantly higher in the RCP condition than the control. The shift in metabolic pathways toward glycolysis was observed for RCP condition. The EV biogenesis marker expression was retained. Mild zinc-induced neurotoxicity may exist when zinc exposure exceeds 1.0 µM. RCP treatment can promote specific cerebellar-like differentiation from hiPSCs indicated by gene expression of early cerebellar markers and regionally enriched genes. The higher cerebellar marker expression is accompanied by the elevated glycolysis with the retained EV biogenesis. This study should advance the understanding of biomarkers during early cerebellar development for cerebellum organoid engineering and neurotoxicity study.

The Selective Loss of Purkinje Cells Induces Specific Peripheral Immune Alterations.

The progression of neurodegenerative diseases is reciprocally associated with impairments in peripheral immune responses. We investigated different contexts of selective neurodegeneration to identify specific alterations of peripheral immune cells and, at the same time, discover potential biomarkers associated to this pathological condition. Consequently, a model of human cerebellar degeneration and ataxia -the Purkinje Cell Degeneration (PCD) mouse- has been employed, as it allows the study of different processes of selective neuronal death in the same animal, i.e., Purkinje cells in the cerebellum and mitral cells in the olfactory bulb. Infiltrated leukocytes were studied in both brain areas and compared with those from other standardized neuroinflammatory models obtained by administering either gamma radiation or lipopolysaccharide. Moreover, both myeloid and lymphoid splenic populations were analyzed by flow cytometry, focusing on markers of functional maturity and antigen presentation. The severity and type of neural damage and inflammation affected immune cell infiltration. Leukocytes were more numerous in the cerebellum of PCD mice, being located predominantly within those cerebellar layers mostly affected by neurodegeneration, in a completely different manner than the typical models of induced neuroinflammation. Furthermore, the milder degeneration of the olfactory bulb did not foster leukocyte attraction. Concerning the splenic analysis, in PCD mice we found: (1) a decreased percentage of several myeloid cell subsets, and (2) a reduced mean fluorescence intensity in those myeloid markers related to both antigen presentation and functional maturity. In conclusion, the selective degeneration of Purkinje cells triggers a specific effect on peripheral immune cells, fostering both attraction and functional changes. This fact endorses the employment of peripheral immune cell populations as concrete biomarkers for monitoring different neuronal death processes.

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.

Time-dependent diffusion MRI probes cerebellar microstructural alterations in a mouse model of Down syndrome.

The cerebellum is a complex system with distinct cortical laminar organization. Alterations in cerebellar microstructure are common and associated with many factors such as genetics, cancer and ageing. Diffusion MRI (dMRI) provides a non-invasive tool to map the brain structural organization, and the recently proposed diffusion-time (td)-dependent dMRI further improves its capability to probe the cellular and axonal/dendritic microstructures by measuring water diffusion at multiple spatial scales. The td-dependent diffusion profile in the cerebellum and its utility in detecting cerebellar disorders, however, are not yet elucidated. Here, we first deciphered the spatial correspondence between dMRI contrast and cerebellar layers, based on which the cerebellar layer-specific td-dependent dMRI patterns were characterized in both euploid and Ts65Dn mice, a mouse model of Down syndrome. Using oscillating gradient dMRI, which accesses diffusion at short td’s by modulating the oscillating frequency, we detected subtle changes in the apparent diffusivity coefficient of the cerebellar internal granular layer and Purkinje cell layer of Ts65Dn mice that were not detectable by conventional pulsed gradient dMRI. The detection sensitivity of oscillating gradient dMRI increased with the oscillating frequency at both the neonatal and adult stages. The td-dependence, quantified by ΔADC map, was reduced in Ts65Dn mice, likely associated with the reduced granule cell density and abnormal dendritic arborization of Purkinje cells as revealed from histological evidence. Our study demonstrates superior sensitivity of short-td diffusion using oscillating gradient dMRI to detect cerebellar microstructural changes in Down syndrome, suggesting the potential application of this technique in cerebellar disorders.

Cerebellar Differentiation from Human Stem Cells Through Retinoid, Wnt, and Sonic Hedgehog Pathways.

Differentiating cerebellar organoids can be challenging due to complex cell organization and structure in the cerebellum. Different approaches were investigated to recapitulate differentiation process of the cerebellum from human-induced pluripotent stem cells (hiPSCs) without high efficiency. This study was carried out to test the hypothesis that the combination of different signaling factors including retinoic acid (RA), Wnt activator, and sonic hedgehog (SHH) activator promotes the cerebellar differentiation of hiPSCs. Wnt, RA, and SHH pathways were activated by CHIR99021 (CHIR), RA, and purmorphamine (PMR), respectively. Different combinations of the morphogens (RA/CHIR, RA/PMR, CHIR/PMR, and RA/CHIR/PMR) were utilized, and the spheroids (day 35) were characterized for the markers of three cerebellum layers (the molecular layer, the Purkinje cell layer, and the granule cell layer). Of all the combinations tested, RA/CHIR/PMR promoted both the Purkinje cell layer and the granule cell layer differentiation. The cells also exhibited electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. This study should advance the understanding of different signaling pathways during cerebellar development to engineer cerebellum organoids for drug screening and disease modeling. Impact statement This study investigated the synergistic effects of retinoic acid, Wnt activator, and sonic hedgehog activator on cerebellar patterning of human-induced pluripotent stem cell (hiPSC) spheroids and organoids. The results indicate that the combination promotes the differentiation of the Purkinje cell layer and the granule cell layer. The cells also exhibit electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. The findings are significant for understanding the biochemical signaling of three-dimensional microenvironment on neural patterning of hiPSCs for applications in organoid engineering, disease modeling, and drug screening.

JAK-1/STAT-3 pathway mediated role in aging cerebellar cortex degenerative changes of albino wistar rats

BackgroundGlial fibrillary acidic protein (GFAP) is considered an important component of cerebellar astrocyte cytoskeleton. Previous studies had shown that the amount of GFAP may increase in all aging brain zones reflecting underlying gliosis. JAK-STAT system carry signals to deoxyribonucleic acid (DNA) affecting genes transcription. Different zones of the human brain accumulate Alzheimer's Disease (AD) pathological markers but cerebellum is proved to be the least affected zone. AimThe aim of the present research is to explore the impact of JAK-1/STAT-3 pathway on aging process of rats' cerebellar cortex. MethodSixty albino Wistar rats were divided into 12 M, 24 M and 32 M groups aged twelve, twenty-four and thirty-two months respectively. After one week of acclimatization, cerebella were dissected under general anesthesia and collected for histopathological examination, western blotting and biochemical examination. ResultsDegenerative changes were noticed in the cerebellum in addition to a significant decrease of thickness of all cerebellar layers thickness except granular layer in both 24 M and 32 M groups which also showed moderate and sever GFAP immunoreaction respectively. In 24M-group purkinje cells showed irregular nuclear envelope with disturbed mitochondrial cristae while in 32M-group, purkinje cells showed a greater extent of nuclear envelop irregularity and myelin sheath of granular cells axons appeared splitted. 12 M, 24 M and 32 M groups showed mild, moderate and sever caspase-3 immunoreaction. There was a significant decrease in JAK-1, STAT-3 and GSH expression in 24 M and 32 M groups while SOCS-3, TNF-α and MDA expression increased in the same groups which denotes the occurrence of cerebellar inflammatory pathway linked to aging. ConclusionJAK-1 and STAT-3 activity increase or SOCS-3 activity decrease can protect against cerebellar insult associated with aging cerebellum.

A Prospective Analysis of Lesion-Symptom Relationships in Acute Vestibular and Ocular Motor Stroke.

Background: Diagnosing stroke as a cause of acute vertigo, dizziness, or double vision remains a challenge, because symptom characteristics can be variable. The purpose of this study was to prospectively investigate lesion-symptom relationships in patients with acute vestibular or ocular motor stroke.Methods: Three hundred and fifty one patients with acute and isolated vestibular or ocular motor symptoms of unclear etiology were enrolled in the EMVERT lesion trial. Symptom quality was assessed by the chief complaint (vertigo, dizziness, double vision), symptom intensity by the visual analog scale, functional impairment by EQ-5D-5L, and symptom duration by daily rating. Acute vestibular and ocular motor signs were registered by videooculography. A standardized MRI (DWI-/FLAIR-/T2-/T2*-/3D-T1-weighted sequences) was recorded within 7 days of symptom onset. MRIs with DWI lesions were further processed for voxel-based lesion-symptom mapping (VLSM).Results: In 47 patients, MRI depicted an acute unilateral stroke (13.4%). The chief complaints were dizziness (42.5%), vertigo (40.4%) and double vision (17.0%). Lesions in patients with vertigo or dizziness showed a large overlap in the cerebellar hemisphere. VLSM indicated that strokes in the medial cerebellar layers 7b, 8, 9 were associated with vertigo, strokes in the lateral cerebellar layer 8, crus 1, 2 with dizziness, and pontomesencephalic strokes with double vision. Symptom intensity and duration varied largely between patients. Higher symptom intensity and longer duration were associated with medial cerebellar lesions. Hemispheric lesions of the cortex were rare and presented with milder symptoms of shorter duration.Conclusions: Prospective evaluation of patients with acute vestibular or ocular motor stroke revealed that symptom quality, intensity and duration were not suited to differentiating peripheral from central etiologies. Lesions in the lateral cerebellum, thalamus, or cortex presented with unspecific, mild and transient symptoms prone to being misdiagnosed.

Comparative study about ageing effect on retina and cerebellum of Columba livia domestica

AbstractThe present research explores the ageing impact of Columba livia domestica on retina and cerebellum. It is an available and a widespread bird species. Three ageing groups (n = 5) of male Columba were collected at about three weeks, six months and five years old. Histological studies were made. Morphometric measurement of both retinal and cerebellar cortex layer thicknesses has been determined. For evaluation of the neuronal synapses, quantitative GFAP biochemically and immunohistochemically staining was performed. Also, biochemical analysis of glucose transporter 1, serotonin and dopamine was carried out to demonstrate their functional activity. Flow cytometric assessments of quantitative cell death were accomplished by using annexin V. The present findings revealed quantitative and qualitative increase of GFAP parallel with decrease of serotonin, dopamine and glucose transporter 1 as well as increase of UR and LR annexin V reflecting cell death in old groups. These were confirmed by damaging of the retina and cerebellar layers. The present findings concluded that retinal and cerebellar cells associated with ageing were determined by sharp rise in both cell death and GFAP levels. Additionally, a decrease of dopamine, serotonin and GluT1 level manifested maybe led to functional alteration of both retina and brain.

Allometric Scaling Rules of the Cerebellum in Galliform Birds

Although the internal circuitry of the cerebellum is highly conserved across vertebrate species, the size and shape of the cerebellum varies considerably. Recent comparative studies have examined the allometric rules between cerebellar mass and number of neurons, but data are lacking on the numbers and sizes of Purkinje and granule cells or scaling of cerebellar foliation. Here, we investigate the allometric rules that govern variation in the volumes of the layers of the cerebellum, the numbers and sizes of Purkinje cells and granule cells and the degree of the cerebellar foliation across 7 species of galliform birds. We selected Galliformes because they vary greatly in body and brain sizes. Our results show that the molecular, granule and white matter layers all increase in volume at the same rate relative to total cerebellum volume. Both numbers and sizes of Purkinje cells increased with cerebellar volume, but numbers of Purkinje cells increased at a much faster rate than size. Granule cell numbers increased with cerebellar volume, but size did not. Sizes and numbers of Purkinje cells as well as numbers of granule cells were positively correlated with the degree of cerebellar foliation, but granule cell size decreased with higher degrees of foliation. The concerted changes among the volumes of cerebellar layers likely reflects the conserved neural circuitry of the cerebellum. Also, our data indicate that the scaling of cell sizes can vary markedly across neuronal populations, suggesting that evolutionary changes in cell sizes might be more complex than what is often assumed.

Mesenchymal stem cells versus curcumin in enhancing the alterations in the cerebellar cortex of streptozocin-induced diabetic albino rats. The role of GFAP, PLC and α-synuclein

BackgroundDiabetes mellitus is the disease, termed either by insulin paucity or resistance and hyperglycemia. The selection of the cerebellum was built on its specific functions. The aim of this study was to investigate a comparison between the possible therapeutic effects of MSCs and curcumin against fluctuations in the cerebellar cortex of STZ-induced diabetic albino rats. Materials and methodsForty rats were divided into five groups: control, sham control, streptozotocin-induced diabetes, diabetes and MSCs administered and diabetes and curcumin administered. Light microscopic (H&E), immune-histochemical; Glial fibrillary acidic protein (GFAP), real-time PCR; phospholipase-C (PLC) and α-synuclein, histomorphometric analysis, oxidative / anti-oxidatants; malondialdehyde (MDA)/ superoxide dismutase (SOD) glutathione (GSH) and were made. ResultsThe histopathological examination of the STZ-induced diabetic rats revealed alterations in the molecular, purkinje and granular layers. Abnormal organizations, vacuolation, patchy loss of purkinje cells were detected. Some purkinje cells migrated into the granular layer.Hemorrhage in pia mater outspreading to cerebellar layers is discerned. Purkinje cells showed karyorrhexis. The mean value of area percentage for GFAP immune- reactivity revealed 360 % significant increase compared to that of the control group. Also, MDA level was significantly increased while the SOD and GSH levels were significantly lower when compared to the control group. Meanwhile, mean values of PLC demonstrated significant decrease, while α-synuclein levels displayed a significant increment in the diabetic group. Administration of curcumin and MSCs extremely ameliorated the previous alterations. Conclusionthe deleterious alterations on the cerebellar cortex induced by diabetes were obviously improved when treated with either MSCs or curcumin.