Tips for Studies with Quantitative Morphology (Morphometry and Stereology)

Mandarim-de-Lacerda, Carlos Alberto; Sol, Mariano del

doi:10.4067/s0717-95022017000401482

Cited by 46 publications

(28 citation statements)

References 27 publications

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“…Images were morphometried by ImageJ software (Madison, WI, USA). The Vv of mesangium, glomerular beclin-1, and LAMP-1 positive areas were calculated using previously described techniques [66].…”

Section: Light Microscopy and Ihcmentioning

confidence: 99%

“…Single-membrane compartments with electron dense cytoplasmic material and/or organelles at various stages of degradation were recognized as autolysosomes ( Figure 8b) [27,68]. The Vv of autophagosomes, autolysosomes and lysosomes in the podocyte cytoplasm was assessed on magnification ×40,000 or ×60,000 by previously described methods [66,71] by ImageJ software (Madison, WI, USA). cytoplasm was assessed on magnification ×40,000 or ×60,000 by previously described methods [66,71] by ImageJ software (Madison, WI, USA).…”

Section: Temmentioning

confidence: 99%

“…The Vv of autophagosomes, autolysosomes and lysosomes in the podocyte cytoplasm was assessed on magnification ×40,000 or ×60,000 by previously described methods [66,71] by ImageJ software (Madison, WI, USA). cytoplasm was assessed on magnification ×40,000 or ×60,000 by previously described methods [66,71] by ImageJ software (Madison, WI, USA).…”

Section: Temmentioning

confidence: 99%

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SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes

Korbut

Taskaeva

Bgatova

et al. 2020

IJMS

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Recent data have indicated the emerging role of glomerular autophagy in diabetic kidney disease. We aimed to assess the effect of the SGLT2 inhibitor empagliflozin, the DPP4 inhibitor linagliptin, and their combination, on glomerular autophagy in a model of type 2 diabetes. Eight-week-old male db/db mice were randomly assigned to treatment with empagliflozin, linagliptin, empagliflozin-linagliptin or vehicle for 8 weeks. Age-matched non-diabetic db/+ mice acted as controls. To estimate glomerular autophagy, immunohistochemistry for beclin-1 and LAMP-1 was performed. Podocyte autophagy was assessed by counting the volume density (Vv) of autophagosomes, lysosomes and autolysosomes by transmission electron microscopy. LC3B and LAMP-1, autophagy markers, and caspase-3 and Bcl-2, apoptotic markers, were evaluated in renal cortex by western blot. Vehicle-treated db/db mice had weak glomerular staining for beclin-1 and LAMP-1 and reduced Vv of autophagosomes, autolysosomes and lysosomes in podocytes. Empagliflozin and linagliptin, both as monotherapy and in combination, enhanced the areas of glomerular staining for beclin-1 and LAMP-1 and increased Vv of autophagosomes and autolysosomes in podocytes. Renal LC3B and Bcl-2 were restored in actively treated animals. LAMP-1 expression was enhanced in the empagliflozin group; caspase-3 expression decreased in the empagliflozin-linagliptin group only. Mesangial expansion, podocyte foot process effacement and urinary albumin excretion were mitigated by both agents. The data provide further explanation for the mechanism of the renoprotective effect of SGLT2 inhibitors and DPP4 inhibitors in diabetes.Int. J. Mol. Sci. 2020, 21, 2987 2 of 22 dedifferentiation. In its turn, the loss or damage of podocytes causes dysfunction of the filtration barrier and increases albuminuria [2]. Some recent studies have indicated an emerging role of autophagy downregulation in diabetic podocytopathy [4][5][6]. Autophagy is a cellular recycling process involving self-degradation and reconstruction of damaged organelles and proteins [6]. The process is vital for highly differentiated post-mitotic cells, such as neurons and podocytes [7]. A growing body of evidence indicates a critical role of autophagy in maintaining podocyte integrity and renal function [6]. Mice with podocyte-specific deletion of autophagic regulators, such as class III PI3K vacuolar protein sorting 34 (Vps34) and the Atg5 gene, develop early proteinuria, progressive glomerulosclerosis, and renal failure [6]. Accordingly, autophagy is considered a potential therapeutic target for renal protection [8,9].Sodium-glucose cotransporter-2 (SGLT2) inhibitors and dipeptidylpeptidase-4 (DPP4) inhibitors are promising antidiabetic agents introduced into clinical practice in the last decade. The antihyperglycemic effect of SGLT2 inhibitors is mediated by increment of glucosuria, while DPP4 inhibitors realize their activity through an increase in the half-life of incretin hormones. Both SGLT2 and DPP4 inhibitors demonstrated renal protective...

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Section: Light Microscopy and Ihcmentioning

confidence: 99%

Section: Temmentioning

confidence: 99%

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SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes

Korbut

Taskaeva

Bgatova

et al. 2020

IJMS

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“…Here we propose to estimate some parameters that we find useful for studying quantitative changes in mitochondria: volume density, numerical density per area, cross-sectional area, and surface density (for cristae and inner and outer membranes). Before proceeding, it is essential for the reader to be familiar with the stereology (jargon and principles) that are easily found in review articles (Gundersen et al, 1988;Cruz-Orive & Weibel, 1990;Mandarim-de-Lacerda, 2003;Mandarim-de-Lacerda & Del-Sol, 2017), and textbooks (Weibel, 1979;Elias et al, 1983;Russ & Dehoff, 2000;Howard & Reed, 2005;Mouton, 2011).…”

Section: Estimating Mitochondrial Stereologymentioning

confidence: 99%

Commentary on Mitochondrial Stereology in Transmission Electron Microscopy

Reis-Barbosa¹,

de-Carvalho²,

Sol³

et al. 2020

Int. J. Morphol.

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Mitochondria (m) are responsible for the energy availability of cells, and their analysis is indicated for example, in studies related to metabolism and oxidative stress. The direct measurement of mitochondria (morphometry) is biased because of the section obliquity and position relative to the mitochondria length (non-equatorial cut). Therefore, stereology is an appropriate technique to evaluate mitochondria. However, before beginning the study, it is necessary to consider the premises to obtain random and uniform samples to be analyzed stereology. Mitochondria must have the chance to appear in all the possibilities of cut and orientation in the micrographs. The number of micrographs to be analyzed will depend on the distribution and occupation of mitochondria in the cell. After this is resolved, a proposal is the estimation of the following stereological data: volume density (Vv), surface density (Sv), and mean cross-sectional area (A). Overlapping a known test area at each micrograph, the density by area of mitochondria is estimated (N AT). Vv [m] can easily be estimated by point-counting (Vv = Pp/P T ; Pp are the points hitting the structure, P T are the number of points of the test system). Sv is estimated overlaying a test-line (L T) on the micrographs and counting the intersections of the lines (I) with the outer membrane (om), inner membrane (im), and crests (c), thus, Sv [om], Sv [im], Sv [c] (Sv = 2I / L T). A [m] is obtained as the ratio: A = Vv / 2N AT .

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“…Además de arrojar resultados numéricos -absolutos y relativos-, objetivos y reproducibles, permite comparaciones más reales, las cuales son sujeto de ser probadas. Su fortaleza más llamativa es la capacidad de corresponder morfología y función, mediante relaciones bien direccionadas (Mandarim-de-Lacerda & del Sol, 2017). En concordancia con la vigencia de la utilización de ratones -C57BL/6-como modelos animales de experimentación, su empleo en el estudio de las glándulas salivales, y la necesidad de conocer más acerca de sus perspectivas en este tema, se ha realizado el presente trabajo de investigación, con el objetivo de obtener datos morfocuantitativos de la glándula submandibular del ratón C57BL/6, para así comprender, de mejor manera, su morfología y función y sentar las bases para futuros estudios experimentales.…”

Section: Introductionunclassified

Características Morfocuantitativas de la Glándula Submandibular de Ratón (Mus musculus)

et al. 2020

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OCAMPO, J.; VÁSQUEZ, B.; SANDOVAL, C.; NAVARRETE, J.; HAIDAR, Z. S. & OLATE, S. Características morfocuantitativas de la glándula submandibular de ratón (Mus musculus). Int. J. Morphol., 38(3):570-577, 2020. RESUMEN:El ratón (Mus musculus) es un buen modelo animal de experimentación, donde sus glándulas salivales han sido utilizadas en estudios, especialmente de tipo preclínico. Sin embargo, las diferencias en la morfología de estas glándulas y, en particular de las submandibulares, entre humanos y roedores no son de conocimiento común. Por lo anterior, el objetivo de este estudio fue conocer las características morfocuantitativos de la glándula submandibular del ratón macho C57BL/6. Se utilizaron cinco ratones macho, cepa C57BL/ 6, de 11 a 13 semanas de nacidos, con un peso promedio de 30 g. Luego de su ambientación, los ratones fueron eutanasiados. Para el análisis histológico, las glándulas fueron extraídas y fijadas en formalina tamponada por 48 horas, procesada para su inclusión en paraplast y teñidas con hematoxilina y eosina, azul alcián pH 2,5, ácido peryódico de Schiff y Picrosirius Red. Se determinaron las características estereológicas de densidad de longitud (L Vcond ), de volumen (V Vcond ) y de superficie (S Vcond ) de los conductos. En las células acinares se evaluó la densidad de número (N Vcel ) y la densidad de volumen (V Vcel ). Para cada variable se obtuvo la media ± desviación estándar. La glándula submandibular se encontraba organizada de manera lobulada, cuyo parénquima era de tipo túbuloacinar mixto con predominio seroso, que se conectaba a través de conductos. Las células acinares presentaban reacción positiva para azul alcián y ácido peryódico de Schiff. El estroma de la glándula contenía principalmente colágeno tipo 1. Los resultados de las mediciones estereológicas fueron: L Vcond = 220,02 ± 50,51 mm/mm 3 ; V Vcond = 50,48 ± 9,55 %; S Vcond = 26,39 ± 5,45 mm 2 /mm 3 ; N Vcel = 16,85 x 104 ± 9,13 × 10 4 /mm 3 ; V Vcel = 24,98 ± 6,22 %. Con los análisis presentados es posible realizar investigaciones experimentales, considerando las similitudes y diferencias morfocuantitativas que presenta la glándula submandibular de ratón macho, cepa C57BL/6 con la del humano.

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Tips for Studies with Quantitative Morphology (Morphometry and Stereology)

Cited by 46 publications

References 27 publications

SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes

SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes

Commentary on Mitochondrial Stereology in Transmission Electron Microscopy

Características Morfocuantitativas de la Glándula Submandibular de Ratón (Mus musculus)

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