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Oral and Maxillofacial Surgery Dec 2023The aim of this study is to compare the repair of incisions performed with microdissection electrocautery tip, conventional electrocautery tip, high potency diode laser,...
PURPOSE
The aim of this study is to compare the repair of incisions performed with microdissection electrocautery tip, conventional electrocautery tip, high potency diode laser, and conventional scalpel blade in a in vivo model.
METHODS
Different incisions were performed in adults Holtzman rats using the four types of instruments: microdissection electrocautery tip, conventional electrocautery tip, high potency diode laser, and conventional scalpel blade, in different periods of healing process. Thirty rats were divided into 5 groups, according to the period of euthanasia-24 h, 48 h, 72 h, 7 days, and 14 days. All animals received four incisions, each by a different method. Quantitative histological and histomorphometric analyses were performed using hematoxylin and eosin (HE) and Picrosirius Red staining.
RESULTS
Inflammatory profile and tissue repair presented small statistically significance differences comparing conventional scalpel blade and microdissection tip; moreover, both presented quantitatively superior to the others.
CONCLUSION
It is believed that the microdissection tip can perform a dynamic incision just as a common scalpel blade, but more effective. Furthermore, it can promote a better hemostatic control of the surgical field that is comparable to conventional electrocautery tip without affecting tissue repair.
Topics: Rats; Animals; Surgical Wound Infection; Surgical Instruments; Lasers, Semiconductor; Electrocoagulation; Models, Animal
PubMed: 35915281
DOI: 10.1007/s10006-022-01105-7 -
JAMA Psychiatry Dec 2023Individuals with schizophrenia (SZ) exhibit pronounced deficits in somatostatin (SST) messenger RNA (mRNA) levels in the dorsolateral prefrontal cortex (DLPFC)....
IMPORTANCE
Individuals with schizophrenia (SZ) exhibit pronounced deficits in somatostatin (SST) messenger RNA (mRNA) levels in the dorsolateral prefrontal cortex (DLPFC). Molecularly distinct subtypes of SST neurons, located in the superficial and deep zones of the DLPFC, are thought to contribute to different functional processes of this region; understanding the specificity of SST alterations in SZ across these zones could inform the functional consequences of those alterations, including cognitive impairments characteristic of SZ.
OBJECTIVE
To quantify mRNA levels of SST and related neuropeptides in the DLPFC in individuals with SZ, bipolar disorder (BPD), or major depressive disorder (MDD) and unaffected comparison individuals.
DESIGN, SETTING, AND PARTICIPANTS
This case-control study, conducted from January 20, 2020, to March 30, 2022, used postmortem brain tissue specimens previously obtained from individuals with SZ, MDD, or BPD and unaffected individuals from a community population through 2 medical examiners' offices. Demographic, clinical, and educational information was ascertained through psychological autopsies.
EXPOSURES
Diagnosis of SZ, BPD, or MDD.
MAIN OUTCOME AND MEASURES
The main outcome was levels of SST and related neuropeptide mRNA in 2 DLPFC zones, examined using laser microdissection and quantitative polymerase chain reaction or fluorescent in situ hybridization (FISH). Findings were compared using educational attainment as a proxy measure of premorbid cognition.
RESULTS
A total of 200 postmortem brain specimens were studied, including 65 from unaffected comparison individuals (42 [65%] male; mean [SD] age, 49.2 [14.1] years); 54 from individuals with SZ (37 [69%] male; mean [SD] age, 47.5 [13.3] years); 42 from individuals with MDD (24 [57%] male; mean [SD] age, 45.6 [12.1] years); and 39 from individuals with BPD (23 [59%] male; mean (SD) age, 46.2 [12.5] years). Compared with unaffected individuals, levels of SST mRNA were lower in both superficial (Cohen d, 0.68; 95% CI, 0.23-1.13; P = .004) and deep (Cohen d, 0.60; 95% CI, 0.16-1.04; P = .02) DLPFC zones in individuals with SZ; findings were confirmed using FISH. Levels of SST were lower only in the superficial zone in the group with MDD (Cohen d, 0.58; 95% CI, 0.14-1.02; P = .12), but the difference was not significant; SST levels were not lower in either zone in the BPD group. Levels of neuropeptide Y and tachykinin 1 showed similar patterns. Neuropeptide alterations in the superficial, but not deep, zone were associated with lower educational attainment only in the group with SZ (superficial: adjusted odds ratio, 1.71 [95% CI, 1.11-2.69]; P = .02; deep: adjusted odds ratio, 1.08 [95% CI, 0.64-1.84]; P = .77).
CONCLUSIONS AND RELEVANCE
The findings revealed diagnosis-specific patterns of molecular alterations in SST neurons in the DLPFC, suggesting that distinct disease processes are reflected in the differential vulnerability of SST neurons in individuals with SZ, MDD, and BPD. In SZ, alterations specifically in the superficial zone may be associated with cognitive dysfunction.
Topics: Humans; Male; Middle Aged; Female; Schizophrenia; Depressive Disorder, Major; Case-Control Studies; In Situ Hybridization, Fluorescence; Prefrontal Cortex; Somatostatin; Neurons; Neuropeptides; Cognition; RNA, Messenger
PubMed: 37647039
DOI: 10.1001/jamapsychiatry.2023.2972 -
Genes Jan 2024Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the...
Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the family Bovidae: (KDEXr sequence), (BTAXr sequence) and (ACEXr sequence). The copy numbers of the isolated sequences were assessed using qPCR, and their chromosomal localisations were analysed using FISH in ten bovid tribes and in outgroup species. Besides their localisation on the X chromosome, their presence was also revealed on the Y chromosome and autosomes in several species. The KDEXr sequence abundant in most Bovidae species also occurs in distant taxa (Perissodactyla and Carnivora) and seems to be evolutionarily older than BTAXr and ACEXr. The ACEXr sequence, visible only in several Antilopini species using FISH, is probably the youngest, and arised in an ancestor common to Bovidae and Cervidae. All three repetitive sequences analysed in this study are interspersed among gene-rich regions on the X chromosomes, apparently preventing the crossing-over in their close vicinity. This study demonstrates that repetitive sequences on the X chromosomes have undergone a fast evolution, and their variation among related species can be beneficial for evolutionary studies.
Topics: Cattle; Animals; Humans; Repetitive Sequences, Nucleic Acid; Deer; Y Chromosome; DNA; Antelopes; Chromosomes, Human, X
PubMed: 38397149
DOI: 10.3390/genes15020159 -
Alzheimer's & Dementia : the Journal of... Jan 2024Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and...
INTRODUCTION
Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE-linked differences remain unclear.
METHODS
We performed laser capture microdissection of amyloid beta (Aβ) plaques, the 50 μm halo around them, tangles with the 50 μm halo around them, and areas distant (> 50 μm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing.
RESULTS
Aβ plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aβ plaques had more differentially expressed genes than tangles. We identified a gradient Aβ plaque > peri-plaque > tangle > distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aβ plaques.
DISCUSSION
Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aβ plaques, and are exacerbated by the APOE ε4 allele.
Topics: Humans; Alzheimer Disease; Amyloid beta-Peptides; Neurofibrillary Tangles; Apolipoprotein E4; Neuroinflammatory Diseases; Brain; Transcriptome; Plaque, Amyloid; Gene Expression Profiling
PubMed: 37461318
DOI: 10.1002/alz.13387 -
MethodsX Dec 2023The rat supraoptic nucleus (SON) contains magnocellular neurons that project long axons that terminate in the posterior pituitary gland. To perform molecular...
The rat supraoptic nucleus (SON) contains magnocellular neurons that project long axons that terminate in the posterior pituitary gland. To perform molecular characterization of these regions, such as transcriptome and methylome profiling, it is necessary to obtain large quantities of high-quality RNA and DNA. Prior methods to isolate molecular material from these small regions required fixing or freezing and laser microdissection of whole tissue, which can compromise recovery and integrity. We have established a straight-forward method of dissecting out the SON and posterior pituitary gland from fresh, unfixed tissue that allows for the isolation of RNA or DNA without compromising nucleic acid integrity. Furthermore, this method can be used as a framework for the microdissection of any region of the brain to isolate any sensitive material. In this manuscript, we describe step-by-step instructions from the macro scale dissection, to brain sectioning, and finally the microdissection of the appropriate tissue.•Transcardial perfusion without fixative prevents the shortcomings of nucleic acid cross-linking.•A fast method and the maintenance of tissue in ice-cold HBSS during dissection and sectioning prevents nucleic acid degradation.•A vibratome is used for the sectioning of fresh brain tissue without freezing or gelatin embedding (i.e. cryostat or microtome).
PubMed: 37791008
DOI: 10.1016/j.mex.2023.102388 -
Journal of Genetics and Genomics = Yi... Sep 2023Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context, significantly enhancing our... (Review)
Review
Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context, significantly enhancing our understanding of the intricate and multifaceted biological system. With an increasing focus on spatial heterogeneity, there is a growing need for unbiased, spatially resolved omics technologies. Laser capture microdissection (LCM) is a cutting-edge method for acquiring spatial information that can quickly collect regions of interest (ROIs) from heterogeneous tissues, with resolutions ranging from single cells to cell populations. Thus, LCM has been widely used for studying the cellular and molecular mechanisms of diseases. This review focuses on the differences among four types of commonly used LCM technologies and their applications in omics and disease research. Key attributes of application cases are also highlighted, such as throughput and spatial resolution. In addition, we comprehensively discuss the existing challenges and the great potential of LCM in biomedical research, disease diagnosis, and targeted therapy from the perspective of high-throughput, multi-omics, and single-cell resolution.
Topics: Laser Capture Microdissection; Multiomics; Biomedical Research
PubMed: 37544594
DOI: 10.1016/j.jgg.2023.07.011 -
JCI Insight Jan 2024Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be...
Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be recalcitrant to therapy. Decades ago, John McNeal proposed that BPH originates with the "reawakening" of embryonic inductive activity by adult prostate stroma, which spurs new ductal proliferation and branching morphogenesis. Here, by laser microdissection and transcriptional profiling of the BPH stroma adjacent to hyperplastic branching ducts, we identified secreted factors likely mediating stromal induction of prostate glandular epithelium and coinciding processes. The top stromal factors were insulin-like growth factor 1 (IGF1) and CXC chemokine ligand 13 (CXCL13), which we verified by RNA in situ hybridization to be coexpressed in BPH fibroblasts, along with their cognate receptors (IGF1R and CXCR5) on adjacent epithelium. In contrast, IGF1 but not CXCL13 was expressed in human embryonic prostate stroma. Finally, we demonstrated that IGF1 is necessary for the generation of BPH-1 cell spheroids and patient-derived BPH cell organoids in 3D culture. Our findings partially support historic speculations on the etiology of BPH and provide what we believe to be new molecular targets for rational therapies directed against the underlying processes driving BPH.
Topics: Male; Adult; Humans; Aged; Prostatic Hyperplasia; Prostate; Epithelium; Fibroblasts; Gene Expression Profiling
PubMed: 37971878
DOI: 10.1172/jci.insight.176479 -
Acta Neuropathologica Communications Sep 2023Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our...
Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA.
Topics: Humans; Animals; Mice; Astrocytes; Neuroglia; Central Nervous System; Ependymoglial Cells; Glaucoma
PubMed: 37749651
DOI: 10.1186/s40478-023-01641-7 -
Analytical Chemistry Nov 2023In the era of single-cell biology, spatial proteomics has emerged as an important frontier. However, it still faces several challenges in technology. Formalin-fixed...
In the era of single-cell biology, spatial proteomics has emerged as an important frontier. However, it still faces several challenges in technology. Formalin-fixed paraffin-embedded (FFPE) tissues are an important material in spatial proteomics, in which fixed tissues are excised using laser capture microdissection (LCM), followed by protein identification with mass spectrometry. For a satisfied spatial proteomics upon FFPE tissues, the excision area is expected to be as small as possible, and the identified proteins are countered upon as much as possible. For a general laboratory for spatial proteomics, a routine workflow is required, not relying on any special device, and is easily operating. In view of these challenges in technology, we initiated a technology evaluation throughout the entire procedure of proteomic analysis with micro-FFPE tissues. In contrast to the protocols reported previously, several innovations in technology were proposed and conducted, such as removal of destaining, decross-linking with "hang-down", solution simplification for peptide generation and balancing to excision area, and capture rate of micro-FFPE tissues. After optimization of all the necessary steps, a routine workflow was established, in which the minimized area for protein identification was 0.002 mm, while the excision area for a consistent proteomic analysis was 0.05 mm. Using the developed workflow and collecting the micro-FFPE tissues continuously, for the first time, a spatial proteomic atlas of mouse brain was preliminarily constructed, which exhibited the typical characteristics of spatial-dependent protein abundance and functional enrichment.
Topics: Mice; Animals; Tissue Fixation; Formaldehyde; Proteomics; Paraffin Embedding; Workflow; Proteins
PubMed: 37922386
DOI: 10.1021/acs.analchem.3c03848 -
Fertility and Sterility Oct 2023To explore the dynamic transcriptional regulatory network of primordial follicle fate in obese mice to elucidate the potential mechanism of primordial follicle depletion.
OBJECTIVE
To explore the dynamic transcriptional regulatory network of primordial follicle fate in obese mice to elucidate the potential mechanism of primordial follicle depletion.
DESIGN
Experimental study and transcriptomic analysis.
ANIMALS
Healthy (n=15) and obese (n=15) female mice.
INTERVENTIONS
Six-week-old CD-1 mice were divided into healthy and high-fat diet groups and fed continuously for 12 weeks. The diet of healthy mice contained 10% fat. The diet of high-fat mice contained 60% fat.
MAIN OUTCOME MEASURES
Primordial to primary follicle transition rate, gene expression changes, enriched Kyoto Encyclopedia of Genes and Genomes pathways, and ferroptosis.
RESULTS
Primordial follicle depletion was increased in the ovaries of obese mice. We found that deposited fat around primordial and primary follicles of obese mice was higher than that for healthy mice. The proliferation of granulosa cells around primary follicles was increased in obese mice. In addition, we uncovered specific gene signatures associated with the primordial to primary follicle transition (PPT) in obese mice using laser capture microdissection RNA sequencing analysis. Gene set enrichment analysis indicated that ferroptosis, cell oxidation, vascular endothelial growth factor, and mammalian target of rapamycin signaling were increased significantly in the primordial follicles of obese mice. Notably, the ferritin, acyl CoA synthetase long-chain family member 4, and solid carrier family 7 member 11 associated proteins of the ferroptosis signaling pathway were significantly increased in the PPT phase of obese mice.
CONCLUSION
Our work suggests that ferroptosis is a key pathway activated within immature ovarian follicles in the context of obesity and that the process may be involved in the physiological regulation of the PPT as well.
Topics: Female; Animals; Mice; Transcriptome; Mice, Obese; Vascular Endothelial Growth Factor A; Ovarian Follicle; Granulosa Cells; Mammals
PubMed: 37247688
DOI: 10.1016/j.fertnstert.2023.05.165