Letters to the Editor

Applying stereology to measure thickness of the basement membrane zone in bronchial biopsy specimens

Diabetes is associated with lower limb co-morbidities, including ulceration and subsequent amputation. As a systemic disease, diabetes affects the microstructure of soft tissues, and material microstructural changes are known to affect the macroscale mechanics. However, the associations between diabetes-related disruptions to essential microstructural components and mechanical changes in plantar skin with diabetes has not been thoroughly characterized. Plantar skin specimens were collected from four diabetic and eight non-diabetic donors at six plantar locations (hallux; first, third, and fifth metatarsals; lateral midfoot; calcaneus) from matched pairs. Mechanical testing was performed on fresh frozen specimens from one foot, and histomorphological measurement and biochemical quantification were performed on specimens from the other foot. Mechanical (compressive and shear moduli and viscoelastic slopes) and biochemical/histological (total quantity of collagen and elastin; dermal and epidermal thickness) parameters were correlated using linear mixed effects regression. There were no significant differences by disease state. Skin thicknesses were positively correlated with initial compression modulus and all three shear moduli. The final compressive modulus was significantly lower at the third metatarsal than the fifth metatarsal, lateral midfoot, and calcaneus, while the final shear modulus was significantly higher at the calcaneus than at the hallux, first, and third metatarsals. Epidermal thickness was significantly higher at the calcaneus compared to all other locations. While differences were not significant by disease state, the strong differences by locations and significant but weak correlations between skin thickness and mechanics can inform future research to understand the mechanism of ulcer formation in the diabetic foot.

Many studies have reported that inflammation and oxidative stress are involved in the pathogenesis of polycystic ovary syndrome (PCOS). Bone marrow mesenchymal stromal cells (BM-MSCs) have anti-oxidant and anti-inflammation properties. In this study, we investigate the beneficial effect of stem cell therapy on folliculogenesis in mice with induced PCOS

Mouse model of PCOS was performed through daily injection of testosterone enanthate (1 mg/100 g/body weight subcutaneous (s.c).) for a period of 5 weeks. Naval Medical Research Institute (NMRI) mice (21 days old) were divided into three groups: control, PCOS and PCOS + BM-MSCs. BM-MSCs were labeled with Hoechst 33342 (0.5 µg/mL) and then injected into the mice (10⁶/animal, via the tail vein) at 1 and 14 days after PCOS confirmation. Mice were humanely killed at 2 weeks after last transplantation. Ovarian stereological studies were done. Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), testosterone, interleukin (IL)-6 and tumor necrosis factor (TNF)-α serum levels were measured. The levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in serum were analyzed. Apoptotic index for ovarian follicles was assessed using Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). CD31 expression in ovarian vessels was assessed with the immunohistochemistry.

There was a significant increase in the total volume of ovary, cortex, number of antral follicles, volume of oocyte and zona pellucida thickness, and there was a significant decrease in the primary and preantral follicles number in the PCOS + BM-MSCs group compared with the PCOS group. There was a significant increase in the serum level of FSH and TAC and a significant decrease in the serum level of testosterone, LH, MDA and percentage of TUNEL-positive apoptotic cells in the PCOS + BM-MSCs group in comparison with the PCOS group.

BM-MSC transplantation improves folliculogenesis in mice with induced PCOS. BM-MSC therapy can be an operative treatment for PCOS via anti-inflammatory, anti-oxidant and anti-apoptotic properties.

Diabetes results in pathophysiological changes, leading to tissue that is unable to withstand and adapt to the same loads, resulting in breakdown. Certain locations are more susceptible to breakdown, yet differences between locations are largely not well understood. The authors performed a histological and biochemical analysis of isolated plantar adipose tissue at six relevant locations.

Tissue from six plantar locations (hallux, first, third and fifth metatarsal heads, lateral midfoot and calcaneus) was taken from fresh cadaveric feet of older diabetic and older non-diabetic intact donors. Histomorphological and biochemical analysis of isolated plantar tissue from both diabetic and non-diabetic feet at six relevant locations was performed.

The main differences found between diabetic and non-diabetic tissue were in the thickness of the septal walls and the elastin content. Diabetic tissue had significantly thicker septal walls and an increased elastin concentration. When comparing the calcaneus to other locations, although there were no differences found in the thickness of the septal walls of diabetic tissue, elastin content was lower in the calcaneous tissue compared to the non-calcaneus sites.

Modifications in the structural and biochemical properties could translate to changes in the mechanical properties. This information could lead to an understanding of how the structural and biochemical changes result in an increase in susceptibility of tissue to breakdown with load at the different locations of the foot.

Ophidian paramyxovirus (ferlavirus) is a global threat to reptilian sauropsids in herpetological collections, with occasional but fatal effects. This study characterizes the effects of three different genetic strains of ferlavirus on the dynamic changes of histology and morphometry of the lung of corn snakes (Pantherophis guttatus). Lungs from 42 corn snakes were either sham-infected or infected experimentally under standardized conditions. From 4 to 49 days after intratracheal inoculation, the lungs were examined qualitatively and quantitatively. Progressive microscopical changes were seen in the lung. Initially, increased numbers of heterophils were observed in the interstitium followed by proliferation and vacuolation of epithelial cells lining faveoli. Electron microscopy revealed loss of type-I pneumocytes, hyperplasia of type-II pneumocytes, and interstitial infiltrates of heterophils and mononuclear cells. With progression of disease the respiratory epithelium was initially overgrown by transformed type-II pneumocytes and later became multilayered. The results of the study suggest that the respiratory capacity of the lungs declines with disease development. The dynamics of disease development and histopathology differed in snakes infected with different ferlavirus genogroups. Animals infected with virus genogroup B developed histopathological changes and morphometric changes more rapidly and of greater intensity than snakes infected with viruses from genogroups A or C.

Diabetes, and the subsequent complication of lower limb ulcers leading to potential amputation, remains an important health care problem in United States, even with declining amputation rates. It has been well documented that diabetes can alter the mechanical properties (i.e., increased stiffness) of the plantar soft tissue, although this finding is not universal. Similarly, biochemical, and histological changes have been found in the plantar soft tissue, but, as with the mechanical changes, these findings are not consistent across all studies. Our group׳s work has demonstrated that diabetes increases plantar soft tissue modulus and increases elastic septal thickness. The purpose of the current study was to explore the association between mechanical, biochemical and histological properties. Using previously collected data, a linear mixed effects regression was conducted. The correlations were weak; of the 32 that were tested, only 3 (modulus to septal thickness when location was accounted for, energy loss to total collagen, and energy loss to collagen/elastin ratio) were statistically significant, none with an R² greater than 0.10. The main differences in the means were increased tissue stiffness and increased septal wall thickness, both trends were supported in the literature. However, as the correlations were weak, it is likely that another unexamined biochemical factor (perhaps collagen crosslinking) is associated with the mechanical tissue changes.

A qualitative and quantitative morphological study of the pulmonary exchange capacity of healthy and diseased Burmese pythons (Python molurus) was carried out in order to test the hypothesis that the high morphological excess capacity for oxygen exchange in the lungs of these snakes is one of the reasons why pathological processes extend throughout the lung parenchyma and impair major parts of the lungs before clinical signs of respiratory disease become apparent. Twenty-four Burmese pythons (12 healthy and 12 diseased) were included in the study. A stereology-based approach was used to quantify the lung parenchyma using computed tomography. Light microscopy was used to quantify tissue compartments and the respiratory exchange surface, and transmission electron microscopy was used to measure the thickness of the diffusion barrier. The morphological diffusion capacity for oxygen of the lungs and the anatomical diffusion factor were calculated. The calculated anatomical diffusion capacity was compared with published values for oxygen consumption of healthy snakes, and the degree to which the exchange capacity can be obstructed before normal physiological function is impaired was estimated. Heterogeneous pulmonary infections result in graded morphological transformations of pulmonary parenchyma involving lymphocyte migration into the connective tissue and thickening of the septal connective tissue, increasing thickness of the diffusion barrier and increasing transformation of the pulmonary epithelium into a columnar pseudostratified or stratified epithelium. The transformed epithelium developed by hyperplasia of ciliated cells arising from the tip of the faveolar septa and by hyperplasia of type II pneumocytes. These results support the idea that the lungs have a remarkable overcapacity for oxygen consumption and that the development of pulmonary disease continuously reduces the capacity for oxygen consumption. However, due to the overcapacity of the lungs, this reduction does not result in clinical signs and disease can progress unrecognized for an extended period.