BRIEF REPORT |
Correspondence to: Eduardo Rocha, Laboratory of Histology & Embryology, Dept. of Microscopy, Inst. of Biomedical Sciences Abel Salazar, Largo Prof. Abel Salazar no. 2, 4099-003 Porto, Portugal. E-mail: histo@icbas.up.pt
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Summary |
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Evaluation of activation and proliferation of hepatic stellate cells (HSCs) must be grounded on solid quantitative data under normal conditions. The HSC index (HSCI), number of HSCs per 1000 hepatocytes (HEP), is often used in hepatology but has been never determined using stereology. Systematically sampled sections were immunostained against glial fibrillary acidic protein and carcinoembryonic antigen, allowing unequivocal distinction of HSC and mononuclear/binuclear HEP. With the optical disector the HSCI was estimated as 109 (coefficient of error = 0.04). This work provides a sound technical basis for experiments in which the estimation of HSCI and/or simultaneous quantification of HSC and HEP are relevant. (J Histochem Cytochem 51:11011104, 2003)
Key Words: hepatic stellate cell index, double immunohistochemistry, disector
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Introduction |
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WHEN HEPATIC STELLATE CELLS (HSCs) were first described 125 years ago, no one could imagine that this research would be pivotal for understanding liver fibrosis. HSCs produce extracellular matrix and cell mediators, store vitamin A, and regulate sinusoidal blood flow (
Currently, the unbiased disector principle is the most adequate baseline procedure to estimate cell number in microscopy (
We aimed to obtain a precise and unbiased estimation of the HSCI in healthy conditions. For this, we combined the optical disector with a double IHC against (a) glial fibrillary acidic protein (GFAP), a specific marker for quiescent HSCs in the rat liver (
Five male Wistar rats (Gulbenkian Institute of Science; Oeiras, Portugal), 3 months old and weighing about 336 g (CV = 0.12) were used. Liver fixation was achieved by controlled flux perfusion of 10% formalin with a peristaltic pump. The liver was removed, weighed, and a fractionator sampling scheme was applied (
A double IHC procedure against GFAP and CEA followed. After deparaffinization, slides were placed in buffered citrate (pH 6.0) and microwave treatment was carried out for antigen retrieval (600 W, 12 min). After rinsing in PBS, the endogenous peroxidase was blocked with 0.3% H2O2 for 30 min. The first streptavidinbiotin protocol then followed (Histostain Plus; Zymed, San Francisco, CA). After rinsing in PBS, 10% non-immune goat serum was applied for 90 minutes, followed by a long incubation with 1:3000 rabbit polyclonal antibody against GFAP (Dako; Glostrup, Denmark) at 4C for 4 days to ensure immunomarking through the full section thickness. After rinsing in PBS, the secondary antibody and the streptavidinperoxidase complex were applied for 60 min each. Then, after rinsing in PBS and Tris-buffered saline (TBS), the slides were developed in 0.05% diaminobenzidine (DAB) in TBS with 0.03% H2O2. After rinsing in tapwater, sections were dipped in 50 mM glycine buffer (pH 2.2) for 120 min to strip the antibodies used for the first staining. The second streptavidinbiotin protocol then followed. This was similar to that described above, except that sections were incubated with 1:1600 rabbit polyclonal antibody against CEA (Dako), at 4C for 4 days. For this reaction, slides were developed in 0.05% DAB in TBS containing 0.003% H2O2 and 0.0025% CoCl2. After rinsing, sections were stained in Gill's hematoxylin and mounted in DPX. Sections in which the antibodies were either omitted or replaced by rabbit non-immune serum showed no immunomarking (negative controls).
The HSCI was estimated with the optical disector, using a workstation made of a microscope (Olympus BX-50; Tokyo, Japan), a microcator (Heidenhain MT-12; Traunreut, Germany) to control the movements in the z-axis (accuracy 0.5 µm), a motorized stage (Prior; Fulbourn, UK) for stepwise displacement in the xy axis (accuracy 1 µm), and a CCD video camera (Sony; Tokyo, Japan) connected to a 17'' PC monitor (Sony). The entire system was controlled by the software Olympus CAST-Grid version 1.5 (Albertslung, Denmark). A x100 oil immersion lens (Olympus Uplan; NA 1.35) provided a high magnification at the monitor (x4750), allowing unambiguous recognition of HSC and HEP borders. The first field was selected randomly. Thereafter, fields were sampled systematically by stage stepwise movements (stepx,y=1250 µm). Through the disector height (20 µm), software-generated counting frames with defined areas (1673 µm2 for HSCs and 418 µm2 for HEPs) were superimposed.
The HSCs and HEPs were counted when two conditions simultaneously met: (a) the rim of the nucleus was in focus at a plane below 4 µm and above or equal to 24 µm (the upper and lower surfaces were avoided with guard heights of 4 and 7 µm, respectively); (b) at the plane of focus, the nucleus was within the counting frame or touching the inclusion lines but did not touch the forbidden lines or their extensions. In every disector it was confirmed that the immunomarking extended beyond 24 µm, ensuring that all HSC and HEP borders could be unambiguously recognized. At the same time, in every fifth field, section thickness was measured with the microcator. The same author (R. Marcos) performed the countings.
The HSCI was estimated via the NV, obtained from the optical disector, by the formula
HSCI = 1000 x Nv(HSC)/Nv(HEP) = 1000 x Q-HSC x (aHEP/
Q-HEP x aHSC)
where NV (HSC) and NV (HEP) are the numerical density of HSC and HEP, Q-HSC and
Q-HEP are the total number of cells counted, and
HSC and
HEP are the counting frame area for HSCs and HEPs. To determine the coefficient of error (CE) of the index estimate, a two-step procedure was used. The CE of the number of cells counted, HSC and HEP, was estimated in each rat by the formula:
Since the CE of the index represents a ratio, another formula was then used: (
A consistent and reliable marking of HSCs and biliary canaliculi was achieved with GFAP and CEA. The brown staining of HSCs, visualized at high magnification, allowed an accurate identification of these cells at every section depth and in all sections. In contrast, biliary canaliculi were black stained. This outlined HEP borders, permitting clear distinction and counting of mononucleated and binucleated cells (Fig 1).
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A mean of 1340 disectors was analyzed per rat, with an average of 907 HSCs and 2099 HEPs counted (1540 MnHEP and 559 BnHEP). The HSCI was 109 HSCs/1000 HEPs (CV = 0.11), with CE = 0.05 only.
IHC has been often used in liver research, but this is the first time a double immunotagging is combined with an unbiased stereological tool to determine the HSCI. In recent years, crosstalking between liver cells became a focus of research. Respecting HSCs and HEPs, different modes of communication, either by membrane contact or soluble mediators, have been identified (
Despite its introduction 30 years ago (
In quantitative studies of HSCs involving IHC, routine cryostat and paraffin sections have been used (e.g.,
The marking of HSCs is still controversial. Concerning GFAP, it was established that not all HSCs express this protein (-smooth muscle actin, should be used, whereas in humans the use of a neurotrophin receptor, such as Trk-C, is a better option (
In conclusion, the use of stereology and double IHC against GFAP and CEA is presented as an innovative approach to estimating the HSCI and obtaining an unbiased simultaneous quantification of HSCs and HEPs. By using stereology, the CE could be estimated, allowing a critical judgment about the sampling and precision. In view of the high numbers of cells counted and the low CE, we can regard our sampling as efficient and the estimate as precise.
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Footnotes |
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1 This work was performed at the Institute of Biomedical Sciences Abel Salazar (ICBAS), Porto, Portugal.
Received for publication October 11, 2002; accepted February 12, 2003.
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