Design-based Stereology Research Articles

Estimating glomerular number: Why we do it and how

There is currently much interest in determining the number of glomeruli, and thereby nephrons, in the kidney. Researchers have been trying to count glomeruli since the 19th century and currently four general approaches are available: (i) acid maceration; (ii) counting glomerular profiles in histological sections; (iii) model-based stereology; and (iv) design-based stereology. Although design-based stereological methods are generally considered the gold-standard method, all current methods have limitations. A new approach using magnetic resonance imaging has recently been described and may ultimately enable glomerular imaging and quantification in vivo. This report considers the advantages and disadvantages of current methods for counting glomeruli and describes the new magnetic resonance approach. In addition, a method for counting glomeruli in developing kidneys is described.

Why and how we determine nephron number

The total number of glomeruli (nephrons) in a kidney is an important microanatomical parameter for at least three reasons: it provides an index of the success/extent of nephrogenesis and can thereby provide insights into the roles of specific genes and feto-maternal environmental factors in nephrogenesis; low nephron number has been linked to an increased risk of cardiovascular and renal disease in adulthood; and knowledge of quantitative kidney microanatomy can illuminate our understanding of physiological mechanisms in health and disease. A range of methods has been used to count glomeruli in kidneys over the past 100 years, with design-based stereology (the physical disector/fractionator combination) considered the gold standard. However, this approach is labor-intensive and expensive, and therefore is not utilized by most laboratories. A new method for counting and sizing every glomerulus in the kidney has recently been described. This method involves in vivo labeling of glomeruli with cationic ferritin, and then magnetic resonance imaging (MRI) of the ex vivo kidney. Values are obtained in one sixth of the time of disector-based approaches. This new MRI method holds great promise for studies of glomerular number and size ex vivo and in vivo.

Decreased parvalbumin mRNA expression in dorsolateral prefrontal cortex in Parkinson′s disease

It has recently been shown that expression of the rate-limiting GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is decreased in Brodmann area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC) in Parkinson′s disease (PD) compared to control brains (Lanoue, A.C., Dumitriu, A., Myers, R.H., Soghomonian, JJ., 2010. Exp. Neurol. 206 (1), 207–217). A subpopulation of cortical GABAergic interneurons expresses the calcium-binding protein parvalbumin and plays a critical role in the control of pyramidal neuron excitability and the generation of cortical gamma frequency oscillations. In view of its key role in the physiology of the cerebral cortex, we sought to determine whether the expression of parvalbumin and the number of parvalbumin-expressing neurons are altered in BA9 of PD brains. First, isotopic in situ hybridization histochemistry was used to examine mRNA expression of parvalbumin on post-mortem brain sections. Second, immunohistochemistry and design-based stereology were used to determine the density of parvalbumin-positive interneurons in BA9. Quantification of mRNA labeling at the single cell level showed a significant decrease in parvalbumin expression in PD cases. In contrast, neuronal density of parvalbumin-positive neurons was not significantly different between PD and controls. Results confirm that the GABAergic system is altered in the DLPFC in PD and identify the contribution of parvalbumin-expressing neurons in these alterations. We speculate that these effects could contribute to altered cortical excitability and oscillatory activity previously documented in PD.

The efficiency of design-based stereology in estimating spiral ganglion populations in mice

Accurate quantification of cell populations is essential in assessing and evaluating neural survival and degeneration in experimental groups. Estimates obtained through traditional two-dimensional counting methods are heavily biased by the counting parameters in relation to the size and shape of the neurons to be counted, resulting in a large range of inaccurate counts. In contrast, counting every cell in a population can be extremely labor-intensive. The present study hypothesizes that design-based stereology provides estimates of the total number of cochlear spiral ganglion neurons (SGNs) in mice that are comparable to those obtained by other accurate cell-counting methods, such as a serial reconstruction, while being a more efficient method. SGNs are indispensable for relaying auditory information from hair cells to the auditory brainstem, and investigating factors affecting their degeneration provides insight into the physiological basis for the progression of hearing dysfunction. Stereological quantification techniques offer the benefits of efficient sampling that is independent of the size and shape of the SGNs. Population estimates of SGNs in cochleae from young C57 mice with normal-hearing and C57 mice with age-related hearing loss were obtained using the optical fractionator probe and traditional two-dimensional counting methods. The average estimated population of SGNs in normal-hearing mice was 7009, whereas the average estimated population in mice with age-related hearing loss was 5096. The estimated population of SGNs in normal-hearing mice fell within the range of values previously reported in the literature. The reduction in the SGN population in animals with age-related hearing loss was statistically significant. Stereological measurements required less time per section compared to two-dimensional methods while optimizing the amount of cochlear tissue analyzed. These findings demonstrate that design-based stereology provides a practical alternative to other counting methods such as the Abercrombie correction method, which has been shown to notably underestimate cell populations, and labor-intensive protocols that account for every cell individually.

Knock down of caveolin‐1 affects morphological and functional hallmarks of human endothelial cells

Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.

Application of design‐based stereology for estimation of absolute volume and surface area of the articular and calcified cartilage compartments of undecalcified human femoral heads

Design-based stereological methods using systematic uniform random sampling, the Cavalieri estimator and vertical sections are used to investigate undecalcified human femoral heads. Ten entire human femoral heads, obtained from normal women and normal men, were systematically sampled and thin undecalcified vertical sections were obtained. Absolute volumes and surface areas of the entire femoral head, the articular cartilage and the calcified cartilage compartments were estimated. In addition, the average thickness of the articular cartilage and the calcified cartilage were calculated. The stereological procedures applied to the human femoral heads resulted in average coefficient of errors, which were 0.03-0.06 for the volume estimates and 0.03-0.04 for the surface area estimates. We conclude that design-based stereology using the Cavalieri estimator and vertical sections can successfully be used in large undecalcified tissue specimens, like the human femoral head, to estimate the absolute volume and surface area of macroscopic as well as of microscopic tissue compartments. The application of well-known design-based stereological methods carries potential advantage for investigating the pathology in inflammatory and degenerative joint diseases.

SAT0337 Growth of the Calcified Cartilage in Human Osteoarthritis Femoral Heads in Comparison to Normal Aging

BackgroundOsteoarthritis (OA) is the most common rheumatic disorder world-wide. The disease affects multiple structures inside the joint, including the articular cartilage, the calcified cartilage, and the subchondral bone. The calcified...

Quantitative microscopy of the lung: a problem-based approach. Part 2: stereological parameters and study designs in various diseases of the respiratory tract

Design-based stereology provides efficient methods to obtain valuable quantitative information of the respiratory tract in various diseases. However, the choice of the most relevant parameters in a specific disease setting has to be deduced from the present pathobiological knowledge. Often it is difficult to express the pathological alterations by interpretable parameters in terms of volume, surface area, length, or number. In the second part of this companion review article, we analyze the present pathophysiological knowledge about acute lung injury, diffuse parenchymal lung diseases, emphysema, pulmonary hypertension, and asthma to come up with recommendations for the disease-specific application of stereological principles for obtaining relevant parameters. Worked examples with illustrative images are used to demonstrate the work flow, estimation procedure, and calculation and to facilitate the practical performance of equivalent analyses.

Quantitative microscopy of the lung: a problem-based approach. Part 1: basic principles of lung stereology

The growing awareness of the importance of accurate morphometry in lung research has recently motivated the publication of guidelines set forth by a combined task force of the American Thoracic Society and the European Respiratory Society (20). This official ATS/ERS Research Policy Statement provides general recommendations on which stereological methods are to be used in quantitative microscopy of the lung. However, to integrate stereology into a particular experimental study design, investigators are left with the problem of how to implement this in practice. Specifically, different animal models of human lung disease require the use of different stereological techniques and may determine the mode of lung fixation, tissue processing, preparation of sections, and other things. Therefore, the present companion articles were designed to allow a short practically oriented introduction into the concepts of design-based stereology (Part 1) and to provide recommendations for choosing the most appropriate methods to investigate a number of important disease models (Part 2). Worked examples with illustrative images will facilitate the practical performance of equivalent analyses. Study algorithms provide comprehensive surveys to ensure that no essential step gets lost during the multistage workflow. Thus, with this review, we hope to close the gap between theory and practice and enhance the use of stereological techniques in pulmonary research.

Decreased parvalbumin mRNA expression in dorsolateral prefrontal cortex in Parkinson's disease

Gene expression of the 67 kilodalton isoform of the GABA synthesizing enzyme, glutamic acid decarboxylase, is decreased in the dorsolateral prefrontal cortex (DLPFC) in Parkinson's disease (PD) (Lanoue et al., 2010, Experimental Neurology). Parvalbumin (PV) is a calcium‐binding protein expressed by a subset of GABAergic interneurons in the cerebral cortex. Computational models show that decreased cortical GABA and PV levels reduce gamma oscillatory activity (Volman et al., 2011, J. Neuroscience) and cortical oscillations are altered in PD. To test the hypothesis that PV levels are decreased in the cerebral cortex in PD, PV was analyzed on post‐mortem tissue from DLPFC from PD and control cases. Radioisotopic in situ hybridization histochemistry was used to examine PV mRNA expression. Quantitative image analysis of emulsion radioautographs showed a significant decrease in mRNA labeling per neuron in PD compared to control brains. Immunohistochemistry was used to label PV‐positive neurons in fixed tissue sections. Design‐based stereology indicated that the average density of PV‐labeled neurons was not significantly different between groups. In conclusion, PD is paralleled by a decrease in PV gene expression without loss of PV neurons in the DLPFC. Decreased PV expression in cortical GABAergic interneurons may be contributing to altered oscillations in PD. American Parkinson's Disease Association

Left Ventricular Morphology of the Giraffe Heart Examined by Stereological Methods

The giraffe heart has a relative mass similar to other mammals, but generates twice the blood pressure to overcome the gravitational challenge of perfusing the cerebral circulation. To provide insight as to how the giraffe left ventricle (LV) is structurally adapted to tackle such a high afterload, we performed a quantitative structural study of the LV myocardium in young and adult giraffe hearts. Tissue samples were collected from young and adult giraffe LV. Design-based stereology was used to obtain unbiased estimates of numbers and sizes of cardiomyocytes, nuclei and capillaries. The numerical density of myocyte nuclei was 120 × 10(3) mm(-3) in the adult and 504 × 10(3) mm(-3) in the young LV. The total number (N) of myocyte nuclei was 1.3 × 10(11) in the adult LV and 4.9 × 10(10) in the young LV. In the adult LV the volume per myocyte was 39.5 × 10(3) µm(3) and the number of nuclei per myocyte was 4.2. The numerical density of myocytes was 24.1 × 10(6) cm(-3) and the capillary volume fraction of the adult giraffe ventricle was 0.054. The significantly higher total number of myocyte nuclei in the adult LV, the high density of myocyte nuclei in the LV, and the number of nuclei per myocyte (which was unusually high compared to other mammalian, including human data), all suggest the presence of myocyte proliferation during growth of the animal to increase wall thickness and normalize LV wall tension as the neck lengthens and the need for higher blood pressure ensues.

Neurons Activated During Fear Memory Consolidation and Reconsolidation are Mapped to a Common and New Topography in the Lateral Amygdala

A key question in neuroscience is how memory is selectively allocated to neural networks in the brain. This question remains a significant research challenge, in both rodent models and humans alike, because of the inherent difficulty in tracking and deciphering large, highly dimensional neuronal ensembles that support memory (i.e., the engram). In a previous study we showed that consolidation of a new fear memory is allocated to a common topography of amygdala neurons. When a consolidated memory is retrieved, it may enter a labile state, requiring reconsolidation for it to persist. What is not known is whether the original spatial allocation of a consolidated memory changes during reconsolidation. Knowledge about the spatial allocation of a memory, during consolidation and reconsolidation, provides fundamental insight into its core physical structure (i.e., the engram). Using design-based stereology, we operationally define reconsolidation by showing a nearly identical quantity of neurons in the dorsolateral amygdala (LAd) that expressed a plasticity-related protein, phosphorylated mitogen-activated protein kinase, following both memory acquisition and retrieval. Next, we confirm that Pavlovian fear conditioning recruits a stable, topographically organized population of activated neurons in the LAd. When the stored fear memory was briefly reactivated in the presence of the relevant conditioned stimulus, a similar topography of activated neurons was uncovered. In addition, we found evidence for activated neurons allocated to new regions of the LAd. These findings provide the first insight into the spatial allocation of a fear engram in the LAd, during its consolidation and reconsolidation phase.

Targeted Gene Transfer of Hepatocyte Growth Factor to Alveolar Type II Epithelial Cells Reduces Lung Fibrosis in Rats

Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-β(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

Stereological assessment of mouse lung parenchyma via nondestructive, multiscale micro-CT imaging validated by light microscopic histology

Quantitative assessment of the lung microstructure using standard stereological methods such as volume fractions of tissue, alveolar surface area, or number of alveoli, are essential for understanding the state of normal and diseased lung. These measures are traditionally obtained from histological sections of the lung tissue, a process that ultimately destroys the three-dimensional (3-D) anatomy of the tissue. In comparison, a novel X-ray-based imaging method that allows nondestructive sectioning and imaging of fixed lungs at multiple resolutions can overcome this limitation. Scanning of the whole lung at high resolution and subsequent regional sampling at ultrahigh resolution without physically dissecting the organ allows the application of design-based stereology for assessment of the whole lung structure. Here we validate multiple stereological estimates performed on micro-computed tomography (μCT) images by comparing them with those obtained via conventional histology on the same mouse lungs. We explore and discuss the potentials and limitations of the two approaches. Histological examination offers higher resolution and the qualitative differentiation of tissues by staining, but ultimately loses 3-D tissue relationships, whereas μCT allows for the integration of morphometric data with the spatial complexity of lung structure. However, μCT has limited resolution satisfactory for the sterological estimates presented in this study but not for differentiation of tissues. We conclude that introducing stereological methods in μCT studies adds value by providing quantitative information on internal structures while not curtailing more complex approaches to the study of lung architecture in the context of physiological or pathological studies.

The effect of simvastatin treatment on behavioral parameters, cognitive performance, and hippocampal morphology in rats fed a standard or a high-fat diet

The aim of this study was to examine the effect of simvastatin (SMV), a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, in rats fed with a standard or a high-fat diet (HFD) throughout the prenatal and the postnatal periods. The emotional status of the animals was evaluated by elevated plus-maze and modified forced swimming tests, whereas cognitive performance was assessed by a Morris water maze task. Morphological changes in the hippocampus were examined by design-based stereology. HFD exposure significantly increased blood serum triglycerides without altering cholesterol levels relative to the controls. After four weeks of oral SMV (5 mg/kg) administration, serum triglycerides reverted to the control level. SMV caused significant anxiolytic, antidepressant-like, and nootropic effects in animals fed the standard diet. In the HFD group, enhanced anxiety and depression, and reduced cognitive performances of animals were reversed by SMV treatment. Although a total volume estimation of the hippocampal subfields indicated no significant change among the groups, the total number of pyramidal neurons decreased significantly in animals fed the HFD; following SMV treatment, this detrimental effect was reversed. In conclusion, chronic SMV administration has significant therapeutic potential for the treatment of affective and cognitive disorders with or without altering the serum lipid profiles.

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology even in complex tissue sections. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

A stereological study of the numbers of neurons and glia in the primary visual cortex across the lifespan of male and female rhesus monkeys

Mild age-related declines in visual function occur in humans and monkeys, independent of ocular pathology, suggesting involvement of central visual pathways (Spear [1993] Vision Res 33:2589-2609). Although many factors might account for this decline, a loss of neurons in primary visual cortex (V1) could be a contributing factor. Previous studies of neuron numbers in V1 reported stability across age, but were limited in the ages and genders studied and sampled only limited parts of V1 or limited cell types, allowing for the possibility of a subtle loss of neurons. We pursued this question in 26 behaviorally tested adult male and female rhesus monkeys ranging from 7.4 to 31.0 years of age by using design-based stereology to estimate numbers of NeuN-labeled neurons and thionin-stained glia within three laminar zones, supragranular (layers II-IVB), granular (IVC), and infragranular (V-VI), across the entirety of V1. There were no significant differences between males and females on any measures, except for total brain weight (P = 0.0038). There was an average of 416,000,000 neurons in V1, but no effect of age on this total or numbers within any laminar zone. Similarly, there was an average of 184,000,000 glia in V1 (44% of the number of neurons), but no effect of age on this total. However, there was a significant age-related increase in numbers of glia in the infragranular zone, perhaps reflecting a glial response to pathology in myelinated projection fibers. This study provides further evidence that in normal aging neurons are not lost and hence cannot account for age-related dysfunction.

Cancer cachexia alters intracellular surfactant metabolism but not total alveolar surface area

Dyspnoea is frequently observed in cancer cachectic patients. Little is known whether this is accompanied by structural or functional alterations of the lung. We hypothesized that in analogy to calorie restriction cancer cachexia leads to loss of alveolar surface area and surfactant. Mice were subjected to subcutaneous injection of Lewis lung carcinoma cells (tumour group, TG) or saline (control group, CG). Twenty-one days later blood samples and the lungs were taken. Using design-based stereology, the alveolar surface area and the lamellar body (Lb) content were quantified. Messenger RNA expression of surfactant proteins, ABCA3 and various growth factors was investigated by quantitative RT-PCR. Intraalveolar surfactant subtype composition was analyzed by differential centrifugation. TG mice showed reduced body weight and anaemia but no reduction of lung volume or alveolar surface area. The volume of Lb was significantly reduced and mRNA levels of ABCA3 transporter tended to be lower in TG versus CG. Surfactant protein expression and the ratio between active and inactive intraalveolar surfactant subtypes were not altered in TG. Growth factor mRNA levels were not different between CG and TG lungs but the tumour expressed growth factor mRNA. Vascular endothelial growth factor was significantly enhanced in blood plasma. The present study demonstrates structural alterations of the lung associated with cancer cachexia. These include reduction of Lb content despite normal intraalveolar surfactant and alveolar surface area. The pulmonary phenotype of the cancer cachectic mouse differs from the calorie restricted mouse possibly due to growth factors released from the tumour tissue.

NOS2 mediates lung structure and function changes in the SP-D model of emphysema without improving surfactant homeostasis

The collectin Surfactant protein D (SP-D) is an innate host defense protein with well-established immuno-modulatory properties. Mice deficient in SP-D (SP-D−/−) spontaneously develop typical emphysematous and inflammatory alterations: a significant decrease in alveolar number, accumulation of enlarged alveolar macrophages overproduction of the inducible nitric oxide synthase (iNOS), hypertrophy and hyperplasia of alveolar type II cells and disturbances of surfactant homoeostasis. Using design-based stereology, wild type, iNOS(−/−), SP-D(−/−) and double knockout (DiNOS) mice at 12 weeks of age were measured for a series of structural parameters. Lung resistance and elastance as functions of frequency at positive end-expiratory pressures in the range of 0–9 cm H2O were measured using Flexivent. The additional ablation of the iNOS gene improved emphysematous alterations in the SP-D(−/−) mouse resulting in a significant increase in alveolar number and a reduction in mean alveolar volume. DiNOS mice exhibited reduced amount of enlarged alveolar macrophages and altered their inflammatory phenotype relative to SP-D(−/−) mice. Neither disturbances of intracellular surfactant homeostasis nor the hyperplasia or hypertrophy of alveolar type II cells were corrected by iNOS ablation. However, despite a lack of effect on surfactant homeostasis, iNOS ablation did normalize lung function. The increased iNOS-activity in SP-D(−/−) mice is critical to the development of inflammation and consequent emphysema but is not important in altered surfactant homeostasis, implying that emphysema and alterations of type II cells are mediated via different pathways. The structural remodeling appears to dominate in terms of lung function.

Cell-specific expression of human HGF by alveolar type II cells induces remodeling of septal wall tissue in the lung: a morphometric study

Hepatocyte growth factor (HGF) is involved in development and regeneration of the lungs. Human HGF, which was expressed specifically by alveolar epithelial type II cells after gene transfer, attenuated the bleomycin-induced pulmonary fibrosis in an animal model. As there are also regions that appear morphologically unaffected in fibrosis, the effects of this gene transfer to normal lungs is of interest. In vitro studies showed that HGF inhibits the formation of the basal lamina by cultured alveolar epithelial cells. Thus we hypothesized that, in the healthy lung, cell-specific expression of HGF induces a remodeling within septal walls. Electroporation of a plasmid of human HGF gene controlled by the surfactant protein C promoter was applied for targeted gene transfer. Using design-based stereology at light and electron microscopic level, structural alterations were analyzed and compared with a control group. HGF gene transfer increased the volume of distal air spaces, as well as the surface area of the alveolar epithelium. The volume of septal walls, as well as the number of alveoli, was unchanged. Volumes per lung of collagen and elastic fibers were unaltered, but a marked reduction of the volume of residual extracellular matrix (all components other than collagen and elastic fibers) and interstitial cells was found. A correlation between the volumes of residual extracellular matrix and distal air spaces, as well as total surface area of alveolar epithelium, could be established. Cell-specific expression of HGF leads to a remodeling of the connective tissue within the septal walls in the healthy lung, which is associated with more pronounced stretching of distal air spaces at a given hydrostatic pressure during instillation fixation.