The Use of Immunohistochemistry for Evaluating the Liver

SummaryTo better evaluate the activation and proliferative response of hepatic stellate cells (HSC) in hepatic fibrosis, it is essential to have sound quantitative data in non-pathological conditions. Our aim was to obtain the first precise and unbiased estimate of the total number of HSC in the adult rat, by combining the optical fractionator, in a smooth sampling design, with immunocytochemistry against glial fibrillary acidic protein.Moreover, we wanted to verify whether there was sufficiently relevant specimen inhomogeneity that could jeopardize the high expected estimate precision when using the smooth fractionator design for HSC. Finally, we wanted to address the question of what sampling scheme would be advisable a priori for future studies. Microscopical observations and quantitative data provided no evidence for inhomogeneity of tissue distribution of HSC. Under this scenario, we implemented a baseline sampling strategy estimating the number ( N ) of HSC as 207E 06 (CV = 0.17). The coefficient of error [CE( N )] was 0.04, as calculated by two formerly proposed approaches. The biological difference among animals contributed ≅ 95% to the observed variability, whereas methodological variance comprised the remaining 5%. We then carried out a half reduction of sampling effort, at the level of both sections and fields. In either occasion, the CE( N ) values were low ( ≅ 0.05) and the biological variance continued to be far more important than methodological variance. We concluded that our baseline sampling (counting 650 -1000 cells/rat) would be appropriate to assess the lobular distribution and the N of HSC. However, if the latter is the only parameter to be estimated, around half of our baseline sampling (counting 250-600 cells/rat) would still generate precise estimates [CE( N ) < 0.1], being in this case more efficient to reduce the number of sections than to reduce the sampled fields.

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“…The IC approach here used was mandatory given that HSC cannot be adequately recognized in routine sections (Hall & Rojko, 1996). Gard et al .…”

Section: Discussionmentioning

confidence: 99%

Estimation of the number of stellate cells in a liver with the smooth fractionator

Marcos

Monteiro

Rocha

2004

Journal of Microscopy

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“…Ito cells ("Sternzellen") were described over a century ago, but this is the first time that an unbiased stereological tool, combined with immunocytochemistry, is applied to these cells. The immunocytochemical approach here used was mandatory, as IC cannot be distinguished in routine sections (Hall and Rojko, 1996). We tagged IC with GFAP, an intermediate filament (IF) protein recognised as a specific marker for these hepatic cells (Neubauer et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Stereological Estimation of Ito Cells From Rat Liver Using the Optical Fractionator - A Preliminary Report

Marcos

Rocha²,

Monteiro³

2011

Image Anal Stereol

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In the last two decades, much light has been shed on hepatic fibrosis, and the activation / proliferation of Ito cells (IC) emerged to play a central role. Therefore, it is essential to have solid quantitative data in nonpathological statuses; yet, this data is scarce and confined to "number per area" or semiquantitative information. Moreover, the supposed heterogeneous distribution of IC in the hepatic lobule was never analysed with design-based (unbiased) stereology. In the present study, the total number (N) of IC in rat liver was estimated for the first time, by combining immunocytochemistry with the optical fractionator. Quantification was extended to the hepatocytes, to disclose the IC index, an often-used ratio in hepatology. Systematic uniform random liver sections were obtained from male Wistar rats (n = 3), and immunostained against glial fibrillary acidic protein (GFAP), a known specific marker for hepatic IC. For the first time, these were marked against GFAP in thick (30 μm) paraffin sections. The estimated N of IC was 224E06; with a coefficient of error of 0.04 or 0.06, depending on the particular equation used (based on the so-called "quadratic approximation"). The IC index was 91 IC/1000 hepatocytes. Concerning the lobular heterogeneity, it was proved the liver harbours a larger total number of periportal IC and hepatocytes

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“…Liposarcomas are uncommon spontaneous findings in domestic animals and other species. Based on a review of data the frequency of this tumour type in the laboratory rat is generally less than 0.1 % (1).…”

Section: Genta Rm Robason Go Graham Dy (1994)mentioning

confidence: 99%

Gesellschaft für Toxikologische Pathologie

1997

Experimental and Toxicologic Pathology

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The Use of Immunohistochemistry for Evaluating the Liver

Cited by 15 publications

References 69 publications

Estimation of the number of stellate cells in a liver with the smooth fractionator

Estimation of the number of stellate cells in a liver with the smooth fractionator

Stereological Estimation of Ito Cells From Rat Liver Using the Optical Fractionator - A Preliminary Report

Gesellschaft für Toxikologische Pathologie

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