The ultrastructural identification of cells was based on published criteria.6,9,31–37 Myofibroblasts were identified by the presence of fibronexi that are regarded as being crucial to the identification of myofibroblasts, as emphasized by Eyden37 (Fig 2
). Fibroblasts were identified by their bipolar morphology, rough endoplasmic reticulum (RER), and lack of other features such as cytoplasmic filaments or fibronexi (Fig 3
, top, A). In addition to fibronexi, myofibroblasts also showed other typical features, including spindle-like cytoplasmic projections, dilated RER, crenated nuclear membranes, patchy basal lamina, intermediate or gap junctions, and pinocytotic vesicles with or without bundles of cytoplasmic microfilaments,6,32,34,37–38 (Fig 3, top, B; center left, C; and center right, D). When the microfilaments were present, they tended to be scanty, to run parallel to the long axis of the cell, and were located peripherally. We have called these cells type 1 myofibroblasts. A minority of cells with fibronexi (and therefore classified as myofibroblasts) had an unusual morphology with numerous, prominent bundles of microfilaments distributed throughout the cytoplasm and absent or scanty RER (Fig 3, bottom left, E). In addition, a thick basal lamina around the perimeter of the cells was easily identified, although it was not completely continuous as is the case with smooth-muscle cells (Fig 3, bottom right, F). These cells were labeled as type 2 myofibroblasts. Eosinophils were characterized by a segmented nucleus and distinctive granules containing a crystalline core, with activated eosinophils undergoing cytolysis,39 as well as clusters of free eosinophil granules (CFEGs) also being identified39 (Fig 4
, top left, A; top right, B). Mast cells, basophils, and neutrophils were distinguished by their nuclei, characteristic granules, and presence/absence of cytoplasmic glycogen particles (Fig 4, center left, C; center right, D; Fig 5
). Neutrophils were classified as cells with an intact cytoplasmic membrane and granules (Fig 5, top left, A) and those undergoing lysis with a degenerating nucleus and cytoplasmic membrane, and granules spilling out into extracellular matrix (Fig 5, center, C). Lymphocytes and monocytes were not always easy to distinguish, both having scanty cytoplasm and a paucity of organelles with occasional mitochondria; therefore, they were classified as lymphomononuclear cells (Fig 4, bottom, E). Some cells could not be identified due to their lack of characteristic features or excessive degeneration and were labeled difficult to classify. Cells were only counted if the nucleus was identified.