Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica.

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at -2.5 degrees C, -5.0 degrees C, or -7.5 degrees C. All frogs frozen at -2.5 degrees C, and most frogs (71%) frozen at -5.0 degrees C, recovered within 14 h after thawing began; however, frogs did not survive exposure to -7.5 degrees C. Sciatic nerves isolated from frogs frozen at -7.5 degrees C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to -2.5 degrees C or -5.0 degrees C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at -2.5 degrees C or -5.0 degrees C had little or no effect on tissue and cellular organization, but that (lethal) exposure to -7.5 degrees C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells.

Ultrastructural effects of lethal freezing on brain, muscle and Malpighian tubules from freeze-tolerant larvae of the gall fly, Eurosta solidaginis.

In preparation for winter low temperatures, larvae of the gall fly, Eurosta solidaginis, accumulate the cryoprotectants glycerol, sorbitol, and trehalose. The fat body cells of these freeze-tolerant larvae can survive intracellular freezing to -80 degrees C for 48 h even though no whole larvae survive this treatment. We hypothesized that some other tissue was more susceptible to freezing and therefore may be responsible for larval death. This paper compares the ultrastructure of brain, muscle, and Malpighian tubules between non-lethally frozen and lethally frozen freeze-tolerant larvae. The nuclei of cortical brain cells from lethally frozen larvae exhibited clumped chromatin and nuclear membranes with occasional expansions or 'blebs' of the intermembranous space, while the cytoplasm contained swollen spheres of endoplasmic reticulum. In contrast, non-lethally frozen brain contained nuclei with evenly dispersed chromatin, smooth nuclear membranes and a cytoplasm free of swollen endoplasmic reticulum. Muscle tissue of lethally frozen larvae contained disrupted myofilaments surrounding the Z-line in comparison to non-lethally frozen muscle which had myofilaments extending all the way to the Z-line. Alterations of Malpighian tubule cells from lethally frozen larvae included an extracted cytoplasm with swollen and rounded mitochondria. In contrast, Malpighian tubule cells from non-lethally frozen larvae had a more concentrated cytoplasm with many rod-shaped mitochondria. Results show alterations to all three tissue types due to lethal freezing. The brain tissue contained the most observable alterations and therefore may be the most susceptible to lethal freeze damage.

Visualization of proteoglycans and link protein in embryonic chick limb cartilage via cryofixation, freeze-substitution and immunochemical techniques.

Chick embryo limb bud cartilage contains a family of proteoglycans, a few of which have been identified ultrastructurally by antibody labelling. Limb bud cartilage from stage 30-34 chick embryos was high-pressure frozen, freeze-substituted and embedded in Lowicryl resin. Sections were treated with polyclonal antibodies for core protein and monoclonal antibodies for chondroitin-6-sulphate and link protein. Label for core protein was demonstrated on both structural matrix and free within the compartmental space. Quantitative analysis indicates that core protein is preferentially localized on electron-dense structural matrix, and that this distribution is uniform between stages 30 and 34. The association of protein epitopes on electron-dense lattice is strongly influenced, rather than a chance observation. Significant quantities of core protein are also located in the free compartments of the cartilaginous lattice. Chondroitin-6-sulphate and link protein were localized predominantly within the compartments of the embryonic lattice. Our data provide convincing evidence that the proteoglycans were immobilized within a microcrystalline matrix of the embryonic compartments. A role for core protein as a stabilizer within the lattice and in the free space where it serves to aggregate polymeric proteoglycans is suggested from our results.

Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution.

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Retention and structure of extracellular matrix in early chick embryos after quick-freezing and freeze-substitution.

Early chick embryos, stages 11 to 14, were isolated, quick-frozen by immersion in isopentane/propane cryogen (-185 degrees C) and freeze-substituted for study by scanning electron microscopy. Emphasis was placed on the extracellular matrix (ECM) in the axial region of the segmental plate and developing somites. Ultrarapid freezing, followed by delicate freeze-substitution, immobilizes and retains much more ECM than chemical fixatives that include tannic acid (TA). The matrix on the dorsal surface of the neural tube is preserved as delicate filaments which are expressed bilaterally over the tube in a dorso-ventral orientation. These parallel primary ridges of ECM have a spacing of 1 to 3 micron, forming grooves on the wall of the neural tube. Interrupting this pattern are funnel-shaped ridges about 80 to 100 micron apart along the neural tube. The ridges become decorated with cross-bridges creating a dense lattice in the region of somite development, to the extent that a basal lamina composed of dense fibrillar network and amorphous mats of matrix accumulates on the lateral wall of the neural tube. Heavy strands and fenestrated lamellae of ECM interconnect the neural tube, notochord and somites, and attach the overlying epithelium to the upper surface of the somites. The pattern of ECM is complimentary to the migratory pathways of ventrally migrating neural crest cells and is the basis for suggesting that a physical substratum influencing the direction of neural crest cell migration is an idea that should be revived.

X-ray microanalysis of calcium, potassium, and phosphorus in liver mitochondria stressed by carbon tetrachloride.

Previous measurements of elemental concentrations in liver mitochondria have generally required homogenization and fractionation of liver tissue, a procedure in which it is difficult to rule out ion movement between subcellular units. New techniques involving cryoultramicrotomy of rapidly frozen tissue, high resolution scanning transmission electron microscopy, and X-ray microanalysis were used to measure those elements in rat liver mitochondria reported to have changed following oral administration of carbon tetrachloride (CCl4). Increases in liver mitochondrial calcium were found 24 hr following intoxication by CCl4. Significant early (2 hr) mitochondrial increases in potassium and phosphorus were found following administration of CCl4. The electron microscope technique using quick-frozen samples promises to allow measurement of intracellular ionic concentrations under virtually lifelike conditions.

Affinity of intramitochondrial granules for ruthenium red accompanying induced cell death in chick embryos.

To test the hypothesis that ruthenium red binding of intramitochondrial granules might reflect an altered or pathological state of membranes associated with degeneration, embryos were treated with 6-AN to induce cell death in cartilaginous skeletons of chick embryos. Cervical cartilage from normal, 6-AN-treated and nicotinamide-alleviated 6-AN embryos was examined ultrastructurally for presence of IM RR-positive granules. Mitochondria of normal cervical chondroblasts which undergo normal phenotypic expression acquire RR-positive granules, although few mature cells are observed in young embryos. Necrotic chondroblasts, chondroblasts in various stages of degeneration, and proliferating chondrogenic cells of 6-AN-treated embryos all demonstrated induced RR-positive IM granules. Foci of degenerating chondroblasts, with mitochondria demonstrating RR granules, were observed infrequently in teratogen-alleviated tissue. The cytological features induced by 6-AN confirm its lethal effect and the degenerative effect on membranes presumably "unmasks" mitochondrial Ca-affinity sites which then become RR-positive. Cytochemical observations correspond with the biochemical and structural changes induced by 6-AN and confirm the hypothesis that RR-positive sites are the result of pathological changes.

Histological study of muscular hypoplasia in the crooked neck dwarf mutant (cn/cn) chick embryo.

Tibiotarsal segments of 12-day chick embryos homozygous for the crooked neck dwarf gene (cn/cn) were examined histologically following routine methods of preparation. The myogenic mass fails to divide into separate muscle bundles during the early stages of differentiation. Myoblasts and myotubes are observed, although the proportion favors the mononucleate cell population. Multinucleate myotubes are often wavy in appearance and many contain eosinophilic cytoplasmic inclusions. The entire tissue mass of mutants appears more compacted than in control limbs. Poor organization of muscle appears related to the lack of a suitable connective tissue system. Epimysia, perimysia, and subcutaneous connective tissue fail to develop properly. Tendons are poorly developed or absent. Comparisons between mutant and control embryos show no differences in peripheral innervation. Nerve fascicles penetrate deeply into the developing muscle of both species. The distribution of vascular elements is seemingly normal also. Skeletal muscle of cn/cn embryos is capable of differentiating to the myotube stage, after which it undergoes cellular degeneration without achieving a functional state. Comparisons of this mutant with alleged chemical phenocopies show important differences.

Structural variations during mitosis in the chick embryo.

Selected tissues from chick embryos were fixed in 2% glutaraldehyde and 1% OsO(4), both buffered at pH 7.6 with Veronal-acetate, and were embedded in Maraglas or Araldite. Two types of cell division have been noted. Generally, epithelial cells divide predominantly by a shortening of the chromosome-to-pole distance rather than by spindle elongation; mesenchymal cells undergo extensive spindle elongation. The presence of numerous continuous microtubules in cells that undergo extensive spindle elongation functionally implicates these tubules in the elongation process. In most embryonic epithelia, the cleavage furrow converges to a fixed site forming a mid-body near the anchoring desmosomes at the free surface; symmetrical furrow formation is typical of mesenchymal cells which lack desmosomes. The hypothesis of cleavage furrow formation and the fate of the mid-body that is formed during cytokinesis are discussed.