Coronal Sections Research Articles

Cytoarchitectonic and connection stripes in the dysgranular insular cortex in the macaque monkey.

The insula has been classically divided into broad granular, dysgranular, and agranular architectonic sectors. We previously proposed a novel partition, dividing each sector into four to seven sharply delimited architectonic areas, with the dysgranular areas being possibly further subdivided into subtle horizontal partitions or "stripes." In architectonics, discrete subparcellations are prone to subjective variability and need being supported with additional neuroanatomical methods. Here, using a secondary analysis of indirect connectional data in the rhesus macaque monkey, we examined the spatial relationship between the dysgranular architectonic stripes and tract-tracing labeling patterns produced in the insula with injections of neuronal tracers in other cortical regions. The injections consistently produced sharply delimited patches of anterograde and/or retrograde labeling, which formed stripes across consecutive coronal sections of the insula. While the overall pattern of labeling on individual coronal sections varied with the injection site, the boundaries of the patches consistently coincided with architectonic boundaries on an adjacent cyto- (Nissl) and/or myelo- (Gallyas) architectonic section. This overlap supports the existence of a fine dysgranular stripe-like partition of the primate insula, with possibly major implications for interoceptive processing in primates including humans. The modular organization of the insula could underlie a serial stream of integration from a dorsal primary interoceptive cortex toward progressively more ventral egocentric "self-agency" and allocentric "social" dysgranular processing units.

Immunohistochemical detection of amyloid beta and tau proteins in the Monodelphis domestica brain

AbstractBackgroundAnimal models have been important for the understanding of Alzheimer’s disease and related dementias (ADRD). However, most of the research involving these diseases has been with rodents. Researchers have raised awareness about the need for alternative and feasible animal models in order to understand these types of diseases. To increase the knowledge of alternative animal models for the study of ADRD, the current study proposed the use of the Monodelphis domestica animal model to detect the presence of amyloid beta and tau proteins.MethodFixed brains from young adult Monodelphis were used in the present study. A standard immunohistochemistry (ABC‐DAB) staining protocol was used to detect amyloid beta and tau proteins. To detect the presence of amyloid beta, a 1:100 anti‐amyloid primary dilution was used; for tau protein, a 1:1000 anti‐tau primary dilution was utilized. Only the hippocampal region was selected for description and quantification. An Axioscan Z.1 microscope system was utilized to capture images, and image analysis was performed using ImageJ software.ResultThe area of the hippocampus of the first coronal section was 30262602.934 um2. The results from an amyloid‐beta dilution of 1:100 detected amyloid beta in a number of neurons in the hippocampus. Amyloid beta was also detected in the hippocampus of another brain section (total area: 7048008 um2). The negative control tissue did not reveal positive signal in neurons in the hippocampal area, suggesting low levels of non‐specific binding. The results for tau antibody are pending.ConclusionOur study revealed that in Monodelphis domestica animal model brain tissue, a 1:100 dilution resulted in identification of positive amyloid beta protein. However, no abnormal amyloid beta plaque formation was found with this animal model. A possible explanation was that a natural, non‐induced animal model was used in the study.

CircRNAs mediate the effect of chronic lysosomal dysfunction on Alzheimer’s disease pathology

AbstractBackgroundCircular RNAs (circRNAs) and microRNAs (miRNAs) are non‐coding RNAs. CircRNAs modulate miRNA levels by sequestration. MiRNAs regulate amyloidogenic pathways, disrupting transcripts including APP, BACE1, and ADAM10. CircRNAs regulate key endolysosomal transcripts. Chronic lysosomal dysfunction (CLD) is linked to Alzheimer’s Disease (AD). However, the role circRNAs downstream of CLD and AD is unclear. Here, we tested whether CLD affects the amyloidogenic pathway in 5xFAD mice through circRNAs.MethodCortical circRNA expression was obtained of 5xFAD, PPT1+/−, 5xFAD: PPT1+/− (P5X), Naglu+/−, 5xFAD: Naglu+/− (N5X) mice. TruSeq Stranded total RNA was extracted with Ribo‐Zero to deplete rRNA and generate stranded‐RNA‐seq. Libraries were sequenced on a NextSeq 500 platform of Illumina and analysis was done with the DESeq2 package with an FRD <0.05. In silico microRNA binging was done using circAtlas 2.0 browser. The insoluble Aβ‐40 and Aβ‐42 were quantified by ELISA in the hippocampal fraction. The Aβ plaque load was quantified in coronal brain sections using immunohistochemistry.ResultThe P5X and N5X mice exhibit increased amyloid plaque load, hippocampal Aβ‐40, and Aβ‐42 levels, and reduced lifespan compared to 5xFAD mice. We detected 1286 cicRNAs in all the groups. 38 from P5X and N5X groups, 37 from PPT1+/−, and 29 from Naglu+/− passed FRD correction compared with 5xFAD mice. Both linear and circRNAs of 4933406I18Rik, Zfp609, Zfp532, and Nnt changed between PPT1+/− and P5X compared to 5xFAD. The levels of circAdam10, circPan3, circMbtd1, circ4930402H24Rik, circSt6gal2, and circCdc14b were reduced, while circZranb1 and circMyo9a were increased in P5X and N5X compared with 5xFAD. CircNlgn1 levels changed only downstream of PPT1+/−. In silico analyses show amyloidogenic‐related microRNAs exhibit circRNAs binding sites. MiR‐361‐3p has binding sites for circZranb1 and circADAM10, as well as miR‐298‐3p, can bind circNlgn1. Both miR‐361‐3p and miR‐298‐3p trap and inhibit BACE1 function and Aβ production, interfering in the amyloidogenic pathway. CircPan3 regulates the autophagy‐related miR‐421. Experimental evidence shows that overexpression of circPan3 suppresses autophagy through a miR‐421/Pink1 pathway.ConclusionCLD (PPT1+/− and Naglu+/−) boosted AD pathology and induced differential changes in circRNAs levels affecting amyloidogenic and autophagy pathways. There is a positive feedback loop between CLD and circRNAs in AD mouse models.

Developing an anatomically valid segmentation protocol for early tau regions: the anterior medial temporal lobe cortices

AbstractBackgroundThe anterior portion of the medial temporal lobe (MTL) is one of the earliest regions showing tau deposition in Alzheimer’s disease (AD) and hence a key focus area for AD imaging biomarkers. However, the anatomical variability of the anterior MTL presents a challenge for segmentation protocols. Leveraging a novel postmortem dataset with histology and MRI, we aimed to develop a histologically‐informed segmentation protocol for the anterior entorhinal cortex (ERC), Brodmann Area (BA) 35 (approximates transentorhinal cortex), and BA36 for in vivo 3 tesla (T) MRI.MethodDigitized 50‐µm thick MTL Nissl‐stained coronal histology sections from 20 cases (Table 1) were annotated by expert neuroanatomists. Cases with and without neurodegenerative diseases were included to ensure broad generalizability of the protocol. The histology sections were registered to same‐subject 0.2×0.2×0.2‐mm3 9.4 T postmortem MRI and were analyzed together to determine the location of the histological borders of the MTL cortices in relation to anatomical landmarks observable on MRI.ResultThe distance between the anterior histological border of ERC, BA35 and BA36 was assessed in relation to different anatomical landmarks observable on MRI. The distance relative to the hippocampus was chosen, as there was relatively low between‐subject variability in distance (low SD in Table 2) and this region is easily identifiable on MRI. The ERC starts 4.75 mm (median) anterior to the hippocampus, BA35 9.25 mm and BA36 10.25 mm. There was no significant difference in distances between patients with and without neurodegenerative disease. Next, we analyzed the location of the ERC on histology sections. We determined that regardless of the depth of the collateral sulcus (CS) (contrary to more posterior regions), the inferior border of the ERC is on average at the edge of the CS and the superior border at the superior edge of the parahippocampal gyrus (Figure 1a), but at the halfway point for the first section with ERC. These segmentation rules for ERC can be applied to T1‐MRI (Figure 1b).ConclusionSegmentation rules for anterior BA35 and BA36 are under development. This histologically‐informed segmentation protocol is expected to improve the efficacy of imaging biomarkers for tau deposition in AD.

A CLU/APOJ GWAS AD risk variant suppresses the astrocytic response to plaques and reduces axonal and neuritic damage in 5xFAD mice

AbstractBackgroundGenome‐Wide Association Studies (GWAS) implicate clusterin (CLU, also known as apolipoprotein J) as a risk factor for late‐onset Alzheimer’s disease (LOAD), with elevated expression of CLU being associated with increased risk of LOAD. The region surrounding the SNP risk allele rs2279590 is poorly conserved within the mouse genome. Consequently, to model the effect of the rs2279590 risk allele on development of AD‐related pathology in mice we developed mice with a partially humanized allele of Clu.MethodCRISPR was used to substitute a 2kb region of human CLU (intron 7 to exon 9) containing the enhancer region and the rs2279590 SNP variant for the orthologous region of Clu within the C57BL/6J mouse genome. Clu‐rs2279590_h2kb mice were crossed with 5xFAD mice and aged to 4 and 12. Coronal brain sections were immunolabeled to visualize dense‐core plaques (ThioS), microglia (IBA1), astrocytes (S100β and GFAP), axonal (NfL) and neuritic (LAMP1) damage, confocal images of subiculum and cortical regions were taken and analyzed using Imaris software. In addition, brain Aβ and plasma NfL levels were quantified using meso scale discovery technology.Result5xFAD;Clu‐rs2279590_h2kb showed significantly reduced microglial density compared to 5xFAD mice in both brain areas at 4 months. By 12 months of age, microglial density was comparable between genotypes in cortex while in subiculum, elevated IBA1 cell density was observed in 5xFAD;Clu‐rs2279590_h2kb mice. Clu‐rs2279590_h2kb variant in 5xFAD background increased the density of S100β astrocytes in subiculum at 4 months and decreased the total S100β volume at 12 months. Reduced GFAP (reactive astrocytes) levels were found in cortex but not subiculum at 12 months. Despite comparable plaque load and insoluble Aβ42 levels, neuritic and axonal damage was decreased in the brain of 5xFAD;Clu‐rs2279590_h2kb compared to 5xFAD mice at 12 months. Plasma NfL was also significantly decreased.ConclusionDespite the minimal impact on Aβ plaque deposition, humanization of 2kb of Clu allele and introduction of rs2279590 risk SNP suppresses astrocytic response to plaques and is protective against axonal and neuritic damage which is mirrored by the reduced levels of plasma NfL.

Memory interference in APPswe/PS1dE9 mice: Role of astrocytic β‐adrenoreceptors

AbstractBackgroundPrevious studies have reported that long‐term β‐adrenoceptor (β‐AR) agonist, isoproterenol hydrochloride (IPE), treatment brings about beneficial effects in an induced model of Alzheimer’s disease (AD) by neutralizing microglial inflammatory morphology against soluble amyloid β oligomers (PMID: 30093491). However, we do not have a clear understanding of how such treatment impacts astrocyte morphology, and whether that afford beneficial effects. Introducing a proactive memory interference step to the classical novel object recognition (NOR) paradigm impairs long‐term memory formation in 2‐month‐old familial model of AD ‐ APPswe/PS1dE9 (APP/PS1) mice. Long‐term IPE treatment rescued long‐term memory. In this study we attempt to understand the impact of long‐term IPE treatment on astrocyte morphology, and how they bring about the reversal in cognitive deficits.Method(1) Mice were taken through a proactive interference step in which they were exposed to objects of varied shape, size and colour before taking them through NOR paradigm. (2) Mice were treated with β‐AR agonist IPE at P26 (0.05g/litre) in drinking water for 21 days till P47. (3) They were sacrificed at 2‐months of age by transcardial perfusion and 100µm coronal sections of hippocampus were stained with astrocytic markers GFAP and S100β by immunohistochemistry. The number and morphology of astrocytes were analysed using ImageJ, SMorph (PMID: 34137444) and Imaris software.ResultOur observations suggest that NOR memory is intact in 2‐month‐old APP/PS1. However, introducing an object‐based short‐term interference before NOR leads to recognition memory deficits in APP/PS1 but not in wild‐type mice. Treatment with IPE restored long‐term memory of APP/PS1 mice to normal levels, thereby protecting them from the effects of interference during the NOR task. Our preliminary findings indicate that IPE impacts the morphology of astrocytes and thus contribute to the reversal of recognition deficits in APP/PS1 at 2 months of age.Conclusionβ‐AR activation can bring about morphological changes in astrocytes that in turn can modulate memory representations.

Developing Topics.

Limbic-predominant age-related TDP-43 encephalopathy neuropathological change (LATE-NC) is found in over half of pathologically confirmed Alzheimer's disease (AD) dementia cases. However, the region-specific relationship between AD neuropathologic change (ADNC) and LATE-NC remains unclear. Here, we investigated the intraregional and interregional associations between ADNC and LATE-NC. We analyzed multi-regional postmortem neuropathology data from the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP). Quantitative ADNC burden was assessed with amyloid-β (Aβ) and tau immunohistochemistry in 8 brain regions. The Aβ burden was averaged across all regions, given strong inter-regional correlations. Semi-quantitative LATE-NC burden was assessed with TDP-43 immunohistochemistry in 7 brain regions, scored in 6 grades. Hippocampal sclerosis (HS) was defined as a severe neuronal loss in a mid-hippocampal coronal section, irrespective of ADNC or LATE-NC burden. We assessed the association between regional LATE-NC and global Aβ burden, adjusting for APOE ε4, age at death, and sex. Then, we tested the intra- and inter-regional associations between LATE-NC and tau, adjusting for Aβ, APOE ε4, age at death, and sex. We performed a post-hoc subgroup analysis in participants without HS. We analyzed the data from n = 1264 deceased ROSMAP participants (Table 1). Global Aβ was positively associated with LATE-NC in most regions (Fig.1A). Interestingly, even after accounting for Aβ and APOE ε4, tau and LATE-NC were positively associated within the entorhinal (t = 7.29, p = 5.4×10-13) and midfrontal cortex (t = 4.33, p = 1.6×10-5) but not within the hippocampus (Fig.1B, bold boxes). Tau and LATE-NC were positively correlated across most interregional pairs (Fig.1B) except for negative associations between hippocampal tau (CA1/subiculum) and frontal LATE-NC. There was no association between hippocampal tau and frontal LATE-NC in the HS(-) subgroup (n = 1143; inferior frontal, p = 0.67; midfrontal, p = 0.15), suggesting that extensive hippocampal neuronal loss in HS(+) cases could have artifactually driven the negative association between hippocampal tau and frontal LATE-NC. Aβ was positively associated with LATE-NC in most regions. Even after adjusting for Aβ and APOE ε4, tau was positively associated with LATE-NC across most regions, except for hippocampal tau and frontal LATE-NC. Further studies are required to determine the underlying mechanisms linking ADNC and LATE-NC.

Utilization of the novel adeno‐associated virus PHP.eB serotype to modulate Intersectin 1 expression in the Ts65Dn mouse model of Down syndrome

AbstractBackgroundDown Syndrome (DS) and Alzheimer’s disease (AD) include cholinergic neurodegeneration which parallels cognitive decline. Triplicated genes in DS are thought to play a role in basal forebrain cholinergic neuron (BFCN) pathology; one avenue to assess triplicated genes’ role is to normalize their expression in DS mouse models by stereotaxic injection of adeno‐associated viruses (AAVs) carrying silencing constructs such as single hairpin mRNA (shRNA). However, stereotaxic surgeries have drawbacks particularly when targeting areas lying deep within the brain. The introduction of the AAV‐PHP.eB serotype, which can cross the blood‐brain‐barrier when introduced into peripheral vasculature, has been shown to vary in efficiency depending on mouse background. Here, we investigated tropism of the AAV‐PHP.eB serotype in the Ts65Dn mouse, the most commonly used DS mouse model. We then used AAV‐PHP.eB to assess our ability to globally modulate expression of intersectin 1 (ITSN1), a protein triplicated in DS that may relate to BFCN dysfunction and memory impairments.MethodsWe retro‐orbitally injected N = 16 4‐4.5 month‐old male mice, (C57Bl, Ts65Dn trisomic and normosomic) with AAV‐PHP.eB‐CamKII‐GFP‐scrmble‐shRNA, to assess tropism and optimize virus concentration (2, 5, and 10µL). Four weeks post injection we imaged for GFP expression in 50µm coronal sections. To knock down ITSN1 globally, we injected N = 58 Ts65Dn mice (3 months, balanced by sex, genotype, virus) with either 5μl AAV‐PHP.eB‐U6‐scrmble‐GFP (control) or AAV‐PHP.eB‐U6‐shITSN1‐GFP (ITSN1); mice were behaviorally tested at 7 months and euthanized at 8 months followed by tissue collection.ResultsMice injected with AAV‐PHP.eB‐CamKII‐GFP‐scrmble‐shRNA showed GFP expression 4 weeks following injection, with robust expression visible with 5µL virus. Mice subsequently injected with control and ITSN1 AAVs showed significant effects of ITSN1 knockdown, including increased tremors, increased latency in the radial arm water maze – suggesting impaired spatial reference learning and memory – decreased brain weight, and altered weight change.ConclusionThe AAV‐PHP.eB serotype shows successful CNS tropism in the Ts65Dn mouse model, with 5µL of AAV providing the best efficiency throughout key brain regions affected in DS and AD. Furthermore, the AAV‐PHP.eB serotype can be used to modulate expression of genes triplicated in DS to investigate their mechanistic role in neurodegeneration both molecularly and behaviorally.

The Human Brain Nucleus Incertus: A New Target in Dementia?

AbstractBackgroundThe brainstem nucleus incertus (NI) or ‘uncertain nucleus’, was originally described by Streeter in 1903 as a midline region in the floor of the fourth ventricle of the human brain with an ‘unknown’ function. A century later, the NI has been shown to play a key role in memory formation in rodents, through direct and indirect inhibition of the hippocampus; and the anatomy of the NI and its projections throughout the brain have been systematically mapped in rats, mice, and primates, but not in humans. In these novel studies, we mapped the human NI using the neuropeptide, relaxin‐3 (RLN3), as neurochemical marker. In rodents, RLN3 is produced mostly in NI neurons with some subsidiary positive cells in the periaqueductal gray and an area dorsal to the substantia nigra.MethodsSerial, free‐floating coronal sections of formalin‐fixed postmortem tissue were prepared from the pons of an adult female (aged 96 years) without a history of dementia or other neurological disorders, provided by the Banner Brain and Body Donation Program, Sun City, Arizona, USA. Using immunohistochemistry, we first identified the NI by mapping the distribution of microtubule‐associated protein‐2 (MAP2) and RLN3. The specificity of the RLN3 antibody used was verified in a dot‐blot using native RLN3 peptide. We also investigated the presence of markers detected in the NI of rats ‐ glutamic acid dehydrogenase‐65/67 (GAD65/67) and corticotropin‐releasing factor type 1 receptor (CRF1); and used a phosphorylated‐tau (AT8) to determine if the NI displayed tau accumulation.ResultsDetection of MAP2‐immunoreactivity revealed the neuronal distribution throughout the pons. RLN3‐immunoreactivity was detected in neurons in the dorsal, anterior‐medial region, revealing the broad anatomy of the human NI. Neurons within this same area were also positive for GAD65/67‐ and CRF1‐immunoreactivity, in line with previous studies; and AT8‐immunoreactivity was also detected.ConclusionsRLN3 is present in neurons of the human NI and aspects of its anatomy are shared across species, including the macaque. Accumulation of phosphorylated‐tau in the NI suggests its possible involvement in the pathology of Alzheimer’s disease and related dementias. Further comparative studies are required to better understand the neurochemical anatomy of the human NI.

Bearded capuchin monkey as a model for Alzheimer’s disease (AD) research

AbstractBackgroundThe absence of a natural animal model is one of the main challenges to research in AD. AD‐type pathology has been described in chimpanzees and other primates’ brains. The bearded capuchin monkey (Sapajus libidinosus) is a New World primate that attracted attention mainly due to its skill in creating and using tools. We evaluated the brains of three captive capuchins (9, 29 and 33 years‐old) who died from natural causes at the Primate Center of University of Brasilia.MethodWhole brain was fixed in 4% buffered paraformaldehyde within 12 hours of death for three weeks. Structural 7T MRI was acquired in one case before brain slicing. Consecutive coronal sections from the fixed brain were embedded in paraffin, and 5um sections from paraffin blocks were used for staining and immunohistochemistry evaluation. All brain sections were stained with hematoxylin and eosin. Immunohistochemistry with β‐amyloid (4G8), phosphorylated tau (AT8), p62, TDP‐43, α‐synuclein (81A), 68 kDa neurofilament (2F11), GFAP and CD68 antibodies were performed in selected sections to analyze the following areas: frontal cortex, temporal cortex, parietal cortex, occipital cortex, anterior cingulate cortex, hippocampus, amygdala, basal ganglia, thalamus, mesencephalon, pons, medulla oblongata, and cerebellum.ResultMacroscopic evaluation of the brains showed considerable cortical gyrification. In addition, thick cortical ribbon was observed in the MRI. A complete neuropathological evaluation was available for two cases. Widespread β‐amyloid immunoreactivity was observed in the form of diffuse deposits, core‐space‐corona plaques, perivascular plaques, and amyloid angiopathy. A high frequency of classic neuritic plaques was observed. P‐tau antibody failed to label the dystrophic neurites. However, these lesions were revealed with p62 and neurofilament antibodies. Astrocyte hypertrophy surrounding plaques was observed without microglial activation. Very few hyperphosphorylated tau aggregates resembling neurofibrillary tangles and neuropil threads were found in the temporal and frontal cortex, and Lewy‐type and TDP‐43 pathology were not observed.ConclusionIn summary, we found β‐amyloid immunoreactivity, classic neuritic plaques surrounded by astrocyte hypertrophy, but only rare P‐tau aggregates. To our knowledge, this is the first evidence reporting brain AD‐like alterations in capuchin monkeys which suggest that this species can be considered an advantageous animal model for AD research.

On-tissue chemical derivatization-enhanced spatially resolved lipidomics reveals abnormal metabolism in type 2 diabetic rat brain

Neurologic disorders are often accompanied by alterations in lipids and oxylipins in the brain. However, the complexity of the lipidome in the brain and its changes during brain damage caused by diabetes remain poorly understood. Herein, we developed an enhanced spatially resolved lipidomics approach with the assistance of on-tissue chemical derivatization to study lipid metabolism in the rat brain. This method enabled the spatially resolved analysis of 560 lipids and oxylipins in 19 brain microregions in coronal and sagittal sections and remarkably improved the coverage of lipidome detection. We applied this method to lipidomic studies of the diabetic rat brain and found that lipid dysregulation followed a microregion-specific pattern. Carnitines and glycerolipids were mainly elevated in the corpus callosum (midbrain) and pineal gland regions, respectively. In addition, most oxylipins, including fatty aldehydes and oxo fatty acids, were significantly upregulated in nine brain microregions. We produced a spatially resolved analysis of lipids and oxylipins, providing a novel analytical tool for brain metabolism research.

Statistical shape analysis of maxillary palatal morphology in patients with palatally displaced canines

ObjectiveMaxillary morphology has long been a subject of interest due to its possible impact on palatally and labially displaced canines. This study aims to conduct a comparison of the palate morphology between individuals with palatal and labially displaced canines and control subjects using statistical shape analysis on a coronal cross-sectional of CBCT images.Materials and methodsPatients aged between 12 and 43 years with palatally or labially displaced canines referred to Hamadan School of Dentistry between 2014 and 2019 were recruited for this retrospective study. The sample included 29 palatally displaced canines (PDC), 20 labially displaced canines (LDC), and 20 control groups (CG). Initially, the maxillary palate coronal section was acquired and landmarked in the region between the right and the left first molar. Procrustes and principal component analyses were used to identify the primary patterns of palatal shape variation. Statistical tests were then performed to examine both shape and size differences.ResultsAccording to the results of Hotelling’s T2 test, there is a significant difference between the mean shape of palate in PDC and CG (P = 0.009), while the difference between the PDC-LDC and LDC-CG groups is not significant. The longest full Procrustes distance was observed between PDC and CG (distance = 0.043), and the shortest full Procrustes distance was observed between LDC and CG (distance = 0.029). The first two principal components accounted for 84.47% of the total variance. The predictive accuracy of the discriminant analysis model showed that 72.46% of cases were correctly classified into the three study groups.ConclusionsIn terms of centroid size, there was no significant difference in the sectional area between the three groups, but the difference between the mean shape of palate in the PDC and CG groups was significant. The PDC group showed more prominent mid-palatal area in the molar region.

Downregulation of the GABAA receptor β2 subunit in a rat model of autism

Introduction: Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain, and GABA type A receptor (GABAA) activation mediates fast inhibitory actions. Numerous studies have shown that individual with autism spectrum disorder (ASD) exhibit abnormalities in the expression of GABAA receptors in various brain areas. Additionally, animal models of ASD have suggested alterations in GABAergic neurotransmission and a dysregulation in the balance between inhibitory and excitatory systems. Objective: We investigated the immunolabeling of the GABAA receptor β2 subunit (GARB2) in the hippocampus, the amygdala, and thalamus of infant rats prenatally exposed to valproic acid (VPA) as an ASD model. Methods: Pregnant females were injected with VPA (600mg/Kg, i.p.) during the twelfth embryonic day; control rats were injected with saline. On the fourteen-postnatal-day, rats from both experimental groups were anesthetized, transcardially perfused with 0.9% NaCl and 4% paraformaldehyde, and sequential coronal brain sections (40μm thickness) were obtained. Immunohistochemistry was performed to detect GARB2 and the relative optical density (OD) of expression was analyzed. Results: Our data showed a statistically significant downregulation of GARB2 in the lateral amygdaloid nucleus, as well as in the ventral and lateral thalamic nuclei when compared to control rats. No statistically significant differences were detected in the hippocampus. Discussion: Our findings suggest that prenatal exposure to VPA reduces GARB2 expression in limbic brain regions involved in social-emotional behaviors, like previous reports in individuals with ASD. Conclusion These results support for the involvement of the GABAergic system in the pathogenesis of ASD.

Jawbone measurements of edentulous sites related to implant planning using magnetic resonance imaging compared to cone beam computed tomography: An exvivo study.

To compare measurements on images obtained by magnetic resonance imaging (MRI) and cone beam CT (CBCT) for height, width, and area in alveolar bone sites in human jaw specimens. Forty edentulous alveolar posterior sites in human cadaver specimens were imaged using CBCT scanners, and with zero-echo-time MRI (ZTE-MRI). Semi-automatic volume registration was performed to generate representative coronal sections of the sites related to implant planning. ZTE-MRI sections were also presented after grayscale inversion (INV MRI). Three observers measured bone height, bone width 5 mm from the alveolar crest, and bone area stretching from the width measurement to the top of the alveolar crest in the images. Interobserver agreement was assessed by intra-class correlation coefficients (ICC). The measurements were analyzed using two-way repeated measures ANOVA factoring observer and image type. ICC was >0.95 for bone height, width, and bone area. No significant differences among observers (p = 0.14) or image type (p = 0.60) were found for bone height. For bone width, observer (p = 0.14) was not a significant factor, while ZTE-MRI produced width estimates that were significantly different and systematically smaller than CBCT-based estimates (p ≤ 0.001). Observer (p = 0.06) was not a significant factor regarding the bone area measurements, contrary to the imaging type where ZTE-MRI led to significantly smaller area estimates than CBCT (p ≤ 0.001). Bone height measurements were essentially equivalent using CBCT and MRI. This was found regardless of grayscale choice for the MRI. However, ZTE-MRI resulted in smaller estimates of bone width and area.

Assessment of Nasal Septum Change after Le Fort I Osteotomy Using Cone Beam Computed Tomography.

Le Fort I osteotomy (LF-IO) is widely used for the correction of dentofacial deformities, which may cause changes in the postoperative nasal septum (NS). The objective of this study was to evaluate the effects of LF-IO on the NS deviation and to determine whether the NS was affected by differences in the direction of maxilla movement. A retrospective study including 57 patients who underwent LF-IO and received cone beam computed tomography (CBCT) preoperatively and 6-12months postoperative evaluation was performed. The NS angle of each patient was measured both pre- and postoperatively in the two coronal sections (nasion and ostium levels). The patients were divided into five paired groups and calculated. Group 1 to 4 were divided depending on the differences in the direction of maxilla movement (Group 1. Impaction ≥ 5mm vs. Impaction < 5mm; Group 2. Anterior movement vs. Non-anterior movement; Group 3. Impaction symmetry vs. Impaction asymmetry; and Group 4. Impaction vs. Inferior repositioning). Group 5 was divided as One-piece osteotomy vs. Multi-segmental osteotomy. The mean NS angles at both nasion and ostium levels of all patients were significant increased after LF-IO. However, there were no statistical significant differences of the five paired groups. LF-IO osteotomy influenced increasing of the NS deviation at both the nasion and ostium levels. However, differences in the direction of the maxilla movement and maxilla segmentation showed no statistically significant changes.

Transgluteal Ultrasonography in Spica Cast in the Post-reduction Assessment of Hip Developmental Dysplasia

Concentric circle reduction is one of the outcomes after reduction of developmental dysplasia of the hip (DDH). Radiography and magnetic resonance imaging (MRI) are used to confirm the reduction results. In this study, we evaluated the reduction results of the coronal section of the hip using transgluteal ultrasonography. We enrolled 46 children admitted to our hospital for closed or open reduction plaster fixation of DDH between January 2021 and December 2022. Thirty-eight patients had unilateral DDH, and eight patients had bilateral DDH. Transgluteal ultrasonography, radiography and MRI were done on the patients during the 7 d after reduction, and the results were interpreted by different radiologists. The covering of the femoral head and the distance between the medial margin of the femoral head and the acetabulum (HSD) were measured after calculating the maximum coronal surface of the iliac bone. MRI is regarded as the gold standard for determining hip reduction or dislocation. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of ultrasound and radiography were determined and compared. The sensitivity, specificity, PPV and NPV of transgluteal ultrasonography were 100% (confidence interval [CI]: 69.2%-100%), 97.7% (CI: 88%-99.9%), 90.9% (CI: 59%-98.6%) and 100%, respectively, higher than those of radiography, which were 50% (CI: 18.7%-81.3%), 86.4% (CI: 72.6%-94.8%), 45.5% (CI: 24%-68.7%) and 88.4% (CI: 80.2%-93.5%), respectively. Ultrasonography had a higher sensitivity (100% vs. 50%, p < 0.01) and positive predictive rate (90.9% vs. 45.5%, p < 0.01) than radiography. Ultrasonography revealed that a distance between the head and socket (HSD) >8 mm indicated a strong suspicion for a poor reduction. Transgluteal coronal ultrasound scan with semiquantitative assessment of acetabular femoral head coverage and measurement of HSD can effectively monitor the reduction relationship between the two in children after DDH reduction.

Accuracy and safety of a new robotic arm for both femoral and acetabular side in total hip arthroplasty: a cadaveric study

BackgroundTo investigate the accuracy and safety of a newly constructed robotic arm which can cover the whole process of THA, we performed a series of robot-assisted total hip replacement on the cadaver.MethodsFifteen frozen cadaveric specimens (30 hips) were used for this study. In this investigation, united hip system and Longwell robotic-assisted system were used. The entire lower limb was CT scanned prior to surgery. The 3D model was produced based on CT data; the site of the prosthesis, including acetabular anteversion, inclination angle, and the position of femoral prosthesis, was planned. With the assistance of a robotic arm, the surgeon changed the parameters based on the preoperative plan and the actual condition during surgery, and completed the whole procedure. Following surgery, we measured the acetabular anteversion angle, acetabular inclination angle, femur anteversion angle, combined anteversion angle, stem angulation, and canal fill ratio.ResultsThe parameters proved that the acetabular anteversion angle was 16.85 ± 3.00°, the acetabular inclination angle was 40.38 ± 5.37°, femur anteversion angle was 15.90 ± 9.01°, combined anteversion angle was 32.75 ± 9.03°, stem angulation was 1.84 ± 0.99°, and leg length discrepancy was 2.47 ± 1.43 mm. The canal fill ratio (CFR) of femoral prosthesis of osteotomy line in sagittal section is 99.72 ± 1.54% and in coronal section is 62.94 ± 8.91%; below osteotomy line 2.5 cm in sagittal section is 100.00% and in coronal section is 81.48 ± 12.94%; below osteotomy line 7.5 cm in sagittal section is 59.51 ± 12.94% and in coronal section is 89.79 ± 11.13%; femoral shaft isthmus in sagittal section is 56.41 ± 13.80% and in coronal section is 84.95 ± 15.17%.ConclusionThe accuracy and safety of this novel robotic arm are suitable for preparing both the acetabular and femoral sides, providing evidence for clinical trial.

Influence of antiresorptive/antiangiogenic therapy on changes in periodontal and oral tissue structures: a histomorphometrical analysis in rats

ObjectiveThe objective of this study was to investigate the influence of various antiresorptive and antiangiogenic medications on morphological changes in periodontal and oral tissue structures.Materials and methodsFifty-five Wistar rats randomly received dual application (i.e., at baseline and after 12-weeks) one of the following medications: (1) amino-bisphosphonate [zoledronate (Zo)], (2) RANKL inhibitor [denosumab (De)], (3) antiangiogenic [bevacizumab (Be)], (4) Zo + Be, (5) De + Be or (6) no medication [Control (Co)]. Periodontal and oral tissue biopsies were obtained at 17 (n = 21 animals, Phase 1, (De = 3, De + Be = 3, Zo = 5, Be = 3, Zo + Be = 2, Co = 5) and 29 (n = 34 animals, (De = 8, De + Be = 6, Zo = 2, Be = 7, Zo + Be = 4, Co = 7, Phase 2) weeks after the second drug application. The following outcomes were histomorphometrically assessed: periodontal space width in the coronal (PLS-C, mm) and apical sections (PLS- A), number of empty alveolar bone lacunae in the coronal, apical sections and at the apex at respective tooth sites (EL – C, EL- A, EL- Ap), mucosal thickness at edentulous alveolar ridge areas (MT, mm), and, when present, associated areas of inflammatory cell infiltrates (ICI, mm2).ResultsComparable mean PLS-C, PLS-A, ET-A, ET-C, ET-Ap, and MT values were observed in all experimental groups after Phases 1 and 2. The presence of ICI was identified in 3 animals in the Co group (Phase 1: 1, Phase 2: 2), and 17 animals in the test groups (Phase 1: 4; Phase 2: 14). The estimated ICI surface area was significantly higher in the Zo + Be group, followed by the Zo and Be groups compared to that measured in the Co group. The time (i.e., Phases 1 and 2) was not found to be a predictor for the extent of the ICI area. In all groups, the EL-C, EL-A, and EL-Ap values were significantly higher after Phase 2 compared to those assessed after Phase 1. The MT values were significantly reduced in all groups after Phase 2 compared to those measured after Phase 1.ConclusionsThe present evaluation was not able to find any morphological effects of different antiresorptive and antiangiogenic medications on periodontal and oral tissue structures. The presence of inflammatory cell infiltrates was more frequently observed in the animals administered with antiresorptive and antiangiogenic medications as well as combinations thereof.Clinical relevanceAdministration of antiresorptive and antiangiogenic medications may be capable of inducing inflammatory reactions in periodontal tissues.

Changes in the Proliferation of the Neural Progenitor Cells of Adult Mice Chronically Infected with Toxoplasma gondii.

During Toxoplasma gondii chronic infection, certain internal factors that trigger the proliferation of neural progenitor cells (NPCs), such as brain inflammation, cell death, and changes in cytokine levels, are observed. NPCs give rise to neuronal cell types in the adult brain of some mammals. NPCs are capable of dividing and differentiating into a restricted repertoire of neuronal and glial cell types. In this study, the proliferation of NPCs was evaluated in CD-1 adult male mice chronically infected with the T. gondii ME49 strain. Histological brain sections from the infected mice were evaluated in order to observe T. gondii tissue cysts. Sagittal and coronal sections from the subventricular zone of the lateral ventricles and from the subgranular zone of the hippocampal dentate gyrus, as well as sagittal sections from the rostral migratory stream, were obtained from infected and non-infected mice previously injected with bromodeoxyuridine (BrdU). A flotation immunofluorescence technique was used to identify BrdU+ NPC. The scanning of BrdU+ cells was conducted using a confocal microscope, and the counting was performed with ImageJ® software (version 1.48q). In all the evaluated zones from the infected mice, a significant proliferation of the NPCs was observed when compared with that of the control group. We concluded that chronic infection with T. gondii increased the proliferation of NPCs in the three evaluated zones. Regardless of the role these cells are playing, our results could be useful to better understand the pathogenesis of chronic toxoplasmosis.

Retro is the new modern: Contemporary application of gold impregnation staining on brain cryosections for digital image analysis

Background and purpose: Since the times of Golgi and Cajal, impregnation with gold or silver has been used to visualize microscopic details of the nervous tissue. Although immunohistochemistry has largely replaced impregnation techniques, they are still used, and there is a growing interest in combining them with modern image analysis methods for quantitative studies in neuroscience. The aim of this research was to modify the gold chloride impregnation method published by Schmued to improve consistency of staining, to be adequate for digital image analysis. Materials and methods: Brains of 8 six-month-old female Wistar rats were fixed in 4% PFA, cryoprotected in sucrose and flash‑frozen in liquid nitrogen. Neighboring sets of coronal sections were chosen for gold impregnation, Nissl staining and MAP2 immunohistochemistry. Whole-slide images and images of specific regions were taken for analysis. Results: Myelinated fibers were stained dark reddish to brown on goldstained sections, and other tissue was yellowish, which gave an excellent contrast for digital image analysis. Gold staining was consistent in all regions, and no major artifacts were noticed. When compared to Nissl and MAP2, only myelinated structures were stained with gold impregnation. Conclusions: Modified gold impregnation method is an alternative that’s on par with traditional myelin staining methods. The new, modified gold impregnation method gives a consistent and reproducible staining suitable for digital image analysis. It can be useful in morphometric evaluation of nervous tissue and investigation of neuropathological changes in nervous tissue, especially for quantitative studies.