Ultrastructural changes in diaphragm neuromuscular junctions in a severe mouse model for Spinal Muscular Atrophy and their prevention by bifunctional U7 snRNA correcting SMN2 splicing

In Spinal Muscular Atrophy (SMA), the SMN1 gene is deleted or inactivated. Because of a splicing problem, the second copy gene, SMN2, generates insufficient amounts of functional SMN protein, leading to the death of spinal cord motoneurons. For a "severe" mouse SMA model (Smn -/-, hSMN2 +/+; with affected pups dying at 5-7 days), which most closely mimicks the genetic set-up in human SMA patients, we characterise SMA-related ultrastructural changes in neuromuscular junctions (NMJs) of two striated muscles with discrete functions. In the diaphragm, but not the soleus muscle of 4-days old SMA mice, mitochondria on both sides of the NMJs degenerate, and perisynaptic Schwann cells as well as endoneurial fibroblasts show striking changes in morphology. Importantly, NMJs of SMA mice in which a modified U7 snRNA corrects SMN2 splicing and delays or prevents SMA symptoms are normal. This ultrastructural study reveals novel features of NMJ alterations - in particular the involvement of perisynaptic Schwann cells - that may be relevant for human SMA pathogenesis.

Black hair follicular dysplasia in Large Münsterländer dogs: clinical, histological and ultrastructural features

Four Large Münsterländer cross-bred dogs affected with black hair follicular dysplasia (BHFD) and one unaffected control littermate were observed, and skin was sampled weekly over the first 19 weeks of life. Affected dogs were born with silvery grey hair, a consequence of melanin clumping in the hair shafts. Hair bulb melanocytes were densely pigmented, and contained abundant stage IV melanosomes but adjacent matrix keratinocytes lacked melanosomes. Melanin clumping was not prominent in epidermal melanocytes in the haired skin but occurred in the foot pads. Follicular changes progressed from bulbar clumping, clumping in the isthmus/infundibulum and finally to dysplastic hair shafts. Alopecia developed progressively in pigmented areas. Silver-grey hair, melanin clumping, accumulation of stage IV melanosomes within melanocytes and insufficient melanin transfer to adjacent keratinocytes are also classic features of human Griscelli syndrome. The underlying cause in Griscelli syndrome is a defect of melanocytic intracellular transport proteins leading to inadequate and disorganized melanosome transfer to keratinocytes with resultant melanin clumping. In view of the correlation in the phenotype, histology and ultrastructure between both disorders, a defect in intracellular melanosome transport is postulated as the pathogenic mechanism in BHFD.

Ultrastructural changes in otoconia of osteoporotic rats

The etiology of benign paroxysmal positional vertigo (BPPV) remains obscure in many cases and women are affected more often than men. A recent prospective study, performed in women >50 years of age suffering from recurrent BPPV, showed associated osteopenia or osteoporosis in a large percentage of these patients. These results suggested the possible relationship between recurrent BPPV and a decreased fixation of calcium in bone in women >50 years. To test this hypothesis, an experimental study was performed in adult female rats. Utricular otoconia of female rats in which osteopenia/osteoporosis was induced by bilateral ovariectomy (OVX) were compared to those of sham-operated adult females rats (SHAM), as control group. FIRST STUDY: The morphology of theutricles of OVX and SHAM rats was analyzed with scanning electron microscopy. In osteopenic/osteoporotic rats, the density of otoconia (i.e. the number of otoconia per unit area) was decreased (p = 0.036)and their size was increased (p = 0.036) compared to the control group. SECOND STUDY: To test the role of calcium turnover in such morphological changes, utricular otoconia of 2 other groups of OVX and SHAM rats, previously injected with calcein subcutaneously, were examined by conventional and epifluorescence microscopy. In epifluorescence microscopy, labeling with calcein showed no significant fluorescence in either group. This finding was interpreted as a lack of external calcium turnover into otoconia of adult female rats. The ultrastructural modifications of otoconia in osteopenic/osteoporotic female adult rats as well as the role of estrogenic receptors in the inner ear are discussed. The possible pathophysiological mechanisms which support the relationship between recurrent BPPV in women and the disturbance of the calcium metabolism of osteopenia/osteoporosis are debated.

Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue.

Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in ∼50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.

Angiogenesis-related ultrastructural changes to capillaries in human skeletal muscle in response to endurance exercise

The ultrastructure of capillaries in skeletal muscle was morphometrically assessed in vastus lateralis muscle (VL) biopsies taken before and after exercise from 22 participants of two training studies. In study 1 (8 wk of ergometer training), light microscopy revealed capillary-fiber (C/F) ratio (+27%) and capillary density (+16%) to be higher (P ≤ 0.05) in postexercise biopsies than in preexercise biopsies from all 10 participants. In study 2 (6 mo of moderate running), C/F ratio and capillary density were increased (+23% and +20%; respectively, P ≤ 0.05) in VL biopsies from 6 angiogenesis responders (AR) after training, whereas 6 nonangiogenesis responders (NR) showed nonsignificant changes in these structural indicators (-4%/-4%, respectively). Forty capillary profiles per participant were evaluated by point and intersection counting on cross sections after transmission electron microscopy. In study 1, volume density (Vv) and mean arithmetic thickness (T) of endothelial cells (ECs; +19%/+17%, respectively) and pericytes (PCs; +20%/+21%, respectively) were higher (P ≤ 0.05), whereas Vv and T of the pericapillary basement membrane (BM) were -23%/-22% lower (P ≤ 0.05), respectively, in posttraining biopsies. In study 2, exercise-related differences between AR and NR-groups were found for Vv and T of PCs (AR, +26%/+22%, respectively, both P ≤ 0.05; NR, +1%/-3%, respectively, both P > 0.05) and BM (AR, -14%/-13%, respectively, both P ≤ 0.05; NR, -9%/-11%, respectively, P = 0.07/0.10). Vv and T of ECs were higher (AR, +16%/+18%, respectively; NR, +6%/+6%, respectively; all P ≤ 0.05) in both groups. The PC coverage was higher (+13%, P ≤ 0.05) in VL biopsies of individuals in the AR group but nonsignificantly altered (+3%, P > 0.05) in those of the NR group after training. Our study suggests that intensified PC mobilization and BM thinning are related to exercise-induced angiogenesis in human skeletal muscle, whereas training per se induces EC-thickening.

Apoptosis in experimental cerebral malaria: spatial profile of cleaved caspase-3 and ultrastructural alterations in different disease stages

Cerebral malaria (CM) is associated with high mortality and morbidity as a certain percentage of survivors suffers from persistent neurological sequelae. The mechanisms leading to death and functional impairments are yet not fully understood. This study investigated biochemical and morphological markers of apoptosis in the brains of mice infected with Plasmodium berghei ANKA. Cleaved caspase-3 was detected in the brains of animals with clinical signs of CM and immunoreactivity directly correlated with the clinical severity of the disease. Caudal parts of the brain showed more intense immunoreactivity for cleaved caspase-3. Double-labelling experiments revealed processing of caspase-3 primarily in neurons and oligodendrocytes. These cells also exhibited apoptotic-like morphological profiles in ultrastructural analysis. Further, cleavage of caspase-3 was found in endothelial cells. In contrast to neurons and oligodendrocytes, apoptosis of endothelial cells already occurred in early stages of the disease. Our results are the first to demonstrate processing of caspase-3 in different central nervous system cells of animals with CM. Apoptosis of endothelial cells may represent a critical issue for the development of the disease in the mouse model. Neurological signs and symptoms might be attributable, at least in part, to apoptotic degeneration of neurons and glia in advanced stages of murine CM.

Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina

Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death.

Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures

The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, i.e. dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.

Clinical and radiographic changes at implants supporting single-unit crowns (SCs) and fixed dental prostheses (FDPs) with one cantilever extension. A retrospective study

To evaluate the clinical and radiographic changes at implants in posterior maxillary and mandibular areas supporting single-unit crowns (SCs) and fixed dental prostheses (FDPs) with one mesial or distal cantilever extension after an observation period of at least 3 years.