A Simple Method to Estimate the Number of Autophagic Elements by Electron Microscopic Morphometry in Real Cellular Dimensions

Autophagic elements typically appear as spherical bodies. During their life they undergo a series of changes (e.g., fusion, degradation of content, and swelling) which influence their size in a way that may be characteristic for cell type, stage of maturation, or various experimentally manipulated parameters. A simple and time efficient method is suggested here to use exactly calculated specific surface values and estimate average diameter and number of autophagic elements in real cellular dimensions. The method is based on the easiest morphometric determination of relative surface (surface density) and volume (volume density) data by electron microscopy. A series of data from real experimental samples of liver and exocrine pancreatic cells are offered to illustrate the potential of these measurements and calculations.

Content of intramyocellular lipids derived by electron microscopy, biochemical assays, and 1H-MR spectroscopy

Three different methods to determine intramyocellular lipid (IMCL) contents in human skeletal muscle have been compared. 1H-magnetic resonance spectroscopy (MRS) was evaluated against electron microscopic morphometry and biochemical assays of biopsy samples from m. tibialis anterior of 10 healthy subjects. The results of 1H-MRS and morphometry were strongly correlated, proving the validity of the1H-MRS results for the noninvasive determination of IMCL. Biochemical assays yielded results that did not significantly correlate with the results of the other methods. When IMCL levels obtained from the three methods are expressed in common units, it was found that1H-MRS yielded IMCL average levels that were 1.8 times lower than those found by morphometry. Potential reasons for the discrepancy are discussed. It is expected that 1H-MRS will be suitable to replace invasive techniques for IMCL determination, whenever noninvasiveness is crucial, e.g., for repeated investigations in studies of substrate recruitment and recovery in exercise.

Molecular analysis of spontaneous glomerulosclerosis in Os/+ mice, a model with reduced nephron mass

Oligosyndactyly mice (ROP Os/+) are a radiation-induced mutant strain with reduced glomerular number and increased glomerular size. We found that they develop glomerulosclerosis. At 3 mo, ROP Os/+ mice had diffuse mesangial expansion by light microscopy, whereas their +/+ littermates did not. Electron microscopic morphometry revealed a twofold increase in mesangial areas but no changes in the thickness of glomerular basal laminae. Mean glomerular volume was increased 1.8-fold. Cell number and thymidine labeling index were increased 1.3- and 2.4-fold, respectively. The amount of glomerular type IV collagen and tenascin but not laminin was increased by immunofluorescence microscopy. mRNA levels in microdissected glomeruli were measured by competetive reverse transcription-polymerase chain reaction and corrected for cell number. alpha 1-Chain type IV collagen and tenascin mRNAs were increased 3.2-fold and 1.8-fold, whereas laminin B1 mRNA levels were not. The levels of 72-kDa collagenase mRNA were increased 1.6-fold. Transforming growth factor-beta 1 mRNA levels were elevated 1.8-fold, but platelet-derived growth factor-B mRNA levels remained normal. This is the first analysis of glomerular molecular and cellular changes in a model of congenital nephron reduction.