Rapid Freezing Method Research Articles

Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxiophyceae)

Autosporulation is a common mode of propagation for unicellular algae. Autospore-forming species of Chlorellaceae, Chlorella vulgaris Beijerinck, C. sorokiniana Shihira et Krauss, C. lobophora Andreyeva, and Parachlorella kessleri (Fott et Nováková) Krienitz et al. have glucosamine as the main constituent of their rigid cell wall. Recent phylogenetic analyses have showed that the Chlorellaceae divided into two sister groups: the Chlorella-clade and the Parachlorella-clade. We compared the cell wall structure and synthesis of the daughter cell wall in the four species by electron microscopy using rapid freezing and freeze substitution methods. The cell wall of C. vulgaris, C. sorokiniana, and C. lobophora consisted of an electron-dense thin layer with an average thickness of 17-20, 22, and 19 nm, respectively. In these three species, daughter cell wall synthesis occurred on the outer surface of the plasma membrane in the early cell-growth phase. The cell wall of P. kessleri, however, was electron-transparent and 54-59 nm in thickness. Ruthenium red staining of P. kessleri indicated that ruthenium-red-specific polysaccharides accumulated over the outer surface of the plasma membrane. Immunoelectron microscopic observation with an anti-beta-1, 3-glucan antibody and staining with wheat germ agglutinin (WGA) indicated that the cell wall contained beta-1, 3-glucan and WGA specific N-acetyl-beta-D-glucosamine. In P. kessleri, daughter cell wall synthesis began after successive protoplast division. The daughter cell wall synthesis during autosporulation in the four species of Chlorellaceae can be classified into two types-the early and the late types.

Increased Constriction of the Ductus Arteriosus by Dexamethasone, Indomethacin, and Rofecoxib in Fetal Rats

To find a better treatment for patent ductus arteriosus (PDA) in premature infants, the present study investigated the synergism of clinical doses of dexamethasone, indomethacin, and rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on the fetal ductus arteriosus (DA) in rats. Dexamethasone (0.3 mg/kg), indomethacin (0.3 mg/kg), and rofecoxib (0.3 mg/kg), alone or in combination, were administered to preterm (d19) and near-term (d21) fetal rats. The ratio of the inner diameter of the DA to that of the main pulmonary artery (PA) (DA/PA) was studied at 2, 4 and 8 h after drug administration, using a rapid whole-body freezing method. In near-term rats, the combined administration of dexamethasone and indomethacin caused severe constriction, with a DA/PA ratio of 0.52+/-0.08 at 8 h, whereas the DA/PA ratios were 0.83+/-0.03 and 0.90+/-0.02 with dexamethasone and indomethacin, respectively. Combined administration of dexamethasone and rofecoxib also caused severe constriction, with a DA/PA ratio of 0.64+/-0.07 at 8 h, compared with the DA/PA ratio of 0.92+/-0.03 with rofecoxib alone. Combined therapy may be an option in the medical management of PDA in premature infants before considering surgical treatment.

Measurement of density distributions for colloidal β-FeOOH rods in suspensions exhibiting phase separation: The role of long-range forces in smectic ordering

We prepared monodisperse colloidal beta-FeOOH rods with length-to-width ratios L/W of 3.6-7.0 (L=210-330 nm and W=40-58 nm). Density gradients of the rods occurred in the suspensions by gravity, inducing a phase separation. The denser phase showed smectic (Sm) liquid crystalline structures exhibiting iridescent colors in a wide range of pH from 1.2 (at which the rods interact attractively) to 4.7 (repulsively). The lower density phase was disordered, but frequently emitted diffuse colors locally (at pH>2.6), implying the occurrence of short-range order. The nematic phase was not observed in the beta-FeOOH systems, being consistent with theoretical predictions. The particle density distributions were measured over the whole region of the suspensions (separated into two phases) at various pH values using a rapid freezing method. A phase diagram was determined thereby, where the critical (minimal) packing fraction of the particles for the Sm phase showed a nonlinear decrease from 0.43 to 0.12 with increasing pH. Rod-rod spacings in the Sm phase estimated experimentally at various pH were well explained using Derjaguin-Landau-Verwey-Overbeek (DLVO) type pair potentials. It is suggested that Sm ordering can be induced by attractive minima at pH<2.2, while driven by soft repulsions at pH>2.6. The former Sm ordering is expected to be the condensation-type phase transition and the latter the disorder-order transition.

Histological and Mucin Histochemical Study of Colon Ulcers Induced in Rats by a Rapid Freezing Method

The purpose of this study was to make a new experimental colon ulcer model that is independent on chemicals and to assess the ulcerations and subsequent changes at various time points. Histological and mucin histochemical assessment of colon ulcers and associated regeneration induced in Wistar rats using a rapid freezing method was performed. Male rats were fasted 48 hours prior to surgery. A lower midline incision of the abdominal wall was made and a stainless steel rod, kept in dry ice-acetone, was applied to the serosal surface of the colon for 4 seconds, twice, with a 5 seconds interval. The rats were sacrificed at various time points thereafter. Histological examination showed the ulcers to be accompanied by acute inflammation, and healing was complete in almost all cases at day 24. In the ascending colon, sulfomucin was found in goblet cells in the upper half of the normal crypts, and sialomucin was found in the lower half. In the descending colon, sulfomucin was apparent almost throughout normal crypts remote from ulcers. Sialomucin dominant regenerative crypts were found near areas of ulceration in both the ascending and descending colon. Thus, the simple freezing method can produce colon ulcers, and the sialomucin that was predominant in regenerative crypts may have an important role for regeneration.

Randomized comparison of murine blastocyst cryopreservation methods

Objective: To evaluate vitrification and controlled-rate freezing of murine blastocysts. For optimal survival and implantation potential, human blastocysts at varying developmental stages from the same cohort may need to be frozen at different times. Multiple freezing procedures/patient can be costly, disruptive to workflow, and impractical with standard controlled-rate freezing, since each freeze requires considerable time to perform. Rapid freezing methods including vitrification could overcome these problems.

Are there gap junctions between chief (glomus, type I) cells in the carotid body chemoreceptor? A review.

Since the dye- and electronic couplings between the carotid body chief cells have been demonstrated, the detection and localization of the gap junctions in the carotid body is crucial to understanding the functional mechanism of chemoreception. However, conventional electron microscopy has been unsuccessful in unquestionably detecting ultrastructural features equivalent to the gap junctions, such as close (2 nm in width) membrane appositions in ultrathin sections and aggregations of intramembranous particles in freeze-fracture replicas of the carotid body. We previously reported using a modified electron microscopic study by chemically fixed and subsequent rapid freezing and freeze-substitution method a number of close membrane appositions comparable to the gap junctions. However, we later found that the freeze-substitution also induces numerous close apposition of the membrane in sites where the gap junctions are not known to occur, indicating that the modified electron microscopy by freeze-substitution is not always confirmative in the detection of the gap junction. With regard to the molecular evidence for the gap junction in the carotid body, there have so far been few data on the immunohistochemical demonstration on connexin 32 and 43 in cultured chief cells, but not in the in situ cells.

Electron microscopy of pathogenic yeasts Cryptococcus neoformans and Exophiala dermatitidis by high-pressure freezing.

A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.

Evaluation of human sperm function after repeated freezing and thawing.

Sperm storage via freezing has been useful for men who have difficulty masturbating during assisted reproductive technology (ART) programs and before impotency caused by chemotherapy, vasectomy, and other procedures. Studies were undertaken to evaluate the extent of cryoinjury to sperm after repeated freezing and thawing. The results showed that normozoospermic and oligozoospermic sperm survived after 3 repeated freeze-thaw cycles. The inclusion of seminal plasma did not seem to protect human sperm during freezing and thawing. There were no significant differences in recovery percentages for motile, vital, and morphologically normal sperm between slow and rapid freezing methods in thaws 1, 2, and 3 of normozoospermic and oligozoospermic unwashed (u), washed (w), and washed + seminal plasma (ws) samples. However, there were significant percentage drops in the recovery of motile and vital sperm between each thaw (ie, first to second thaw, and second to third thaw) using both slow and rapid freezing for u, w, and ws samples (P < .01). There were also no significant differences in percentage recovery of motile, vital, and morphologically normal sperm between u, w, and ws samples during thaws 1 to 3 in the normozoospermic and oligozoospermic groups. Sperm were capable of fertilizing hamster oocytes microinjected with single sperms after 3 freeze-thaw cycles as evidenced by the formation of 2 distinct pronuclei and 2 polar bodies in 22.2% and 17.2% of normozoospermic and oligozoospermic samples, respectively. The numbers of normal vital motile sperm after 3 serial freeze-thaw cycles are adequate for bringing about fertilization via intracytoplasmic sperm injection in ART programs. Thus, leftover washed sperm in laboratories that perform in vitro fertilization can be frozen, thawed, and refrozen several times without loss of the sperms' ability to fertilize. This approach has tremendous benefits for men who have difficulty producing sperm and for those with low and declining sperm counts.

A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis.

The plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, gamma-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong fluorescence. To confirm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1-3 microm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them 'bulbs'. Three-dimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The fluorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give fluorescent signals of bulbs in transgenic plants, but the existence of bulbs was still confirmed by electron microscopy. These results suggest that bulbs define a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.

Stability of the meiotic spindle following rapid cryopreservation and thawing of Syrian hamster eggs.

Objective: Cryopreservation of unfertilized eggs can result in damage to the meiotic spindle. This study used a simple method of rapid freezing and analysis of meiotic spindles of thawed eggs to assess such damage.

Review article fine structure of the exocrine cells of rat sublingual gland revealed by rapid freezing and freeze substitution method.

The ultrastructure of mucous cells of rat sublingual gland processed by rapid freezing, followed by freeze substitution, was compared with that obtained by the standard chemical fixation technique. The rapid freezing method gave a very good preservation of membrane structure with round and discrete mucous droplets (granules) not showing any sign of coalescence. The cisterns of the Golgi apparatus and the trans Golgi network also were well preserved. Upon secretory stimulation by pilocarpine, mucous droplets were discharged by the usual mechanism of exocytosis. From all these findings it emerged that mucous cells had the same structural characteristics as serous cells. In the endpieces of rat sublingual gland prepared by the rapid freezing method, serous cells aligned with mucous cells around the central lumen, and no cap-like arrangement of serous cells (demilunes) was observed. Furthermore, computer reconstruction of stereo images from serial section light micrographs prepared by the rapid freezing method showed that, within a given endpiece, all serous cells had direct access to the lumen and that they were disseminated throughout it and not only in its fundus. From our observations it seems very likely that, at least in rat sublingual gland, serous demilunes are an artificial product caused by the compression exerted on serous cells by the mucous cells distended during the conventional fixation procedure.

Fine Structure of the Exocrine Cells of Rat Sublingual Gland Revealed by Rapid Freezing and Freeze Substitution Method

Fine Structure of the Exocrine Cells of Rat Sublingual Gland Revealed by Rapid Freezing and Freeze Substitution Method

Fine structure of transforming-type granules in mucous cells in the early postnatal rat parotid gland when processed by rapid freezing followed by freeze-substitution fixation.

The present study was designed to clarify the more precise ultrastructural feature of granules, especially mucous granules in the early postnatal rat parotid gland by using rapid freezing followed by freeze-substitution fixation. The parotid gland of Wistar rats (aged 0-10 days) was removed under anesthesia and immediately underwent cryofixation followed by substitution with osmium tetroxide. After fixation, the samples were embedded in Epon-Araldite, cut into ultrathin section, and then examined by transmission electron microscopy. Electron microscopy showed that the mucous granules of samples treated by freeze-substitution fixation had low electron density and were almost spherical in shape with a clear limiting membrane. By Day 5, granules that were a little more electron dense than mucous granules, granules with a more electron dense portion at their periphery, and heterogeneous granules with an internal highly electron dense portion were found. Moreover, cells containing both homogeneous granules with a high electron density similar to that of mature serous granules and heterogeneous granules were observed. These findings demonstrated that the morphology of the transforming-type mucous granules by chemical fixation in the previous study was an artifact and, as a result, strongly suggested that on the sequential morphological changes of transitional mucous/serous granules by rapid freezing method in the present study, the mucous cells in the early postnatal rat parotid gland undergo transformation to serous cells.

Criopreservação de embriões de suínos adicionando trealose aos crioprotetores etilenoglicol ou glicerol

Os objetivos deste trabalho foram: comparar (a) o efeito da adição de trealose ao crioprotetor etilenoglicol e, (b) o efeito dos crioprotetores etilenoglicol ou glicerol associados à trealose sobre a viabilidade de embriões congelados de suínos . A trealose foi adicionada à solução de congelamento de etilenoglicol 1.5 M nas concentrações de 0.0 M (tratamento 1) e 0.25 M (tratamento 2), e na concentração de 0.25 M (tratamento 3) à solução de congelamento de glicerol 1.5 M. Os embriões foram congelados no estágio de blastocisto expandido. O método de congelamento utilizado foi o rápido, sendo que desde a temperatura ambiente (± 25°C) até a temperatura do "seeding" (-7°C), a velocidade de resfriamento foi de 1°C/minuto. Após o "seeding", foi estabelecido um período de equilíbrio de 10 minutos à -7°C, sendo, a partir de então, adotada a velocidade de resfriamento de 0.3°C/minuto até a temperatura de -35°C. A seguir, as palhetas foram colocadas no nitrogênio líquido (-196°C). O descongelamento foi realizado mediante exposição das palhetas ao ar durante 30 segundos e, posteriormente, em banho-maria à 35°C, durante 30 segundos. A remoção dos crioprotetores foi realizada pelo método em etapas. O critério de viabilidade adotado consistiu na observação da reexpansão da cavidade blastocélica dos embriões, após período de cultivo in vitro de 18-24h. O cultivo foi feito em meio TCM 199 (tissue culture medium), acrescido de 20% de soro fetal bovino, em microgotas de 60 mil, sob óleo mineral em estufa com 6% CO2, 95% de umidade e 37°C. A viabilidade embrionária logo após o descongelamento (0h) foi de 17.2%, 37.5% e 42.8%, para os tratamentos 1, 2 e 3, respectivamente. Após o período de cultivo de 18-24h, a viabilidade embrionária foi de 6.9%, 28.1% e 28.5%, para os tratamentos 1, 2 e 3, respectivamente. Os resultados indicaram que não houve diferença (p&gt;0.05) significativa entre os tratamentos ao descongelamento. Após o cultivo in vitro, verificou-se que a porcentagem de embriões viáveis foi superior (p&lt;0.05) pela adição de trealose na concentração de 0.25 M à solução de congelamento, contendo etilenoglicol. No entanto, não foi detectada diferença significativa entre os crioprotetores etilenoglicol ou glicerol, associados à trealose.

Effect of freezing and grinding method on near-infrared reflectance (NIR) spectra variation and chemical composition of fresh silage

In order to evaluate possible effects of rapid freezing and grinding methods on near-infrared reflectance (NIR) spectra variability and chemical composition, samples from 18 pasture silages, previously pre-homogenized, were frozen either with liquid nitrogen (LN) or dry ice (DI) and, subsequently, ground with a Tecator ® homogenizer (Tec) or a Moulinex ® food processor (Mou). NIR spectra were taken (monochromator NIRSystems 6500) on four fresh subsamples per silage and root mean squares (RMS) computed on derivatized spectra (software NIRS 3, ISI, 1992) as a means of estimating spectra dissimilarities among subsamples. Principal components were computed and samples plotted according to the first three scores to visualize potential effects of treatments. Seven silages from the four treatments plus a control (pre-homogenized only) were freeze-dried and analyzed for toluene dry matter (DM Tol), crude protein (CP) and acid detergent fiber (ADF). Average RMS value for silages was 423.97 and no effect of freezing or grinding method, nor any interaction between them, was found ( P > 0.05), although DI-Mou and DI-Tec treatments showed lower (402.8) and higher (459.4) mean RMS values, respectively. Plotting of samples according to the main principal component scores of spectra did not show any effect of treatments, as samples tended to cluster by silage instead as by treatment. Chemical composition of treated, and control samples, was not different ( P > 0.05). On average, composition was as follows: DM Tol, 269.2 g kg −1; CP and ADF (oven DM basis), 126.0 and 403.2 g kg −1 DM, respectively. It is concluded that any sample handling method could be selected for calibration and subsequent routine analysis, but if a method is chosen, it is suggested to consistently adhere to that method.

The serous demilune of rat sublingual gland is an artificial structure produced by conventional fixation.

The ultrastructure of the secretory end-piece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the terminal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells distributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation.

Anomalies of the aortic arch and arterial duct induced by small doses of bis-diamine in fetal rats

AbstractDeletion of chromosome 22 and DiGeorge syndrome are associated with inappropriate development of the neural crest and abnormalities of the ventricular outflow tracts. Bis-diamine induces comparable cardiac anomalies by inhibiting the normal development of the neural crest. In order to clarify the effect of damage on the development of the neural crest and its relation to the cardiac anomalies, the effects of giving a small dose of bis-diamine were compared with the results of a large dose of bis-diamine. A small dose of bis-diamine (20 mg), therefore, was administered to 40 pregnant rats on the 10th day of pregnancy, and 330 fetal hearts were studied on the 21st day with the rapid whole-body freezing method. Intracardiac anomalies were rare, being found in only 55 fetuses. The anomalies discovered were: aberrant origin of the subclavian artery in 88 fetuses, right aortic arch in 26 fetuses, interruption of the aortic arch in 23 fetuses, and high location of the aortic arch in 8 fetuses. Anomalies of the arterial duct were also common; with a vascular ring formed by the aortic arch and contra-lateral duct being seen in 21 fetuses, isolation of the right subclavian artery associated with either bilateral ducts (9 fetuses) or a right duct and absent left duct (9 fetuses), absent duct with tetralogy of Fallot in 7 fetuses, and with common arterial trunk in one fetus. In conclusion, a small dose of bis-diamine induced extra cardiac anomalies of the aortic arch and arterial duct, indicating a greater susceptibility of the aortic arch and the duct to inappropriate formation of the neural crest cells in the early developing embryo. Clinically, these anomalies of the aortic arch and the duct should be searched for in all patients suspected of having deletion of their 22nd chromosome.

The optimal equilibration time for mouse embryos frozen by vitrification with trehalose.

To determine the best equilibration time in the cryoprotectant before rapid cooling, 8-cell mouse embryos were exposed to a vitrification solution containing ethylene glycol, Ficoll and trehalose in modified phosphate-buffered saline at 5 degrees C for varying periods of time. They were frozen using an ultra rapid freezing method, thawed in a 20 degrees C water bath and cultured for 24 h with 5-bromo-2-deoxyuridine. Embryo development and the number of sister chromatid exchanges, a sensitive indicator of genetic damage, were observed. The results demonstrated that embryo development after freezing and thawing was similar among the groups exposed for periods of 5-40 min. However, the number of sister chromatid exchanges was significantly smaller in the group exposed for 5 min, indicating that this was the safest equilibration time in the vitrification solution.

Biomechanical and biochemical study of a standardized wound healing model

Standardized protocols were developed for use in a detailed investigation into the biomechanical and biochemical properties of a dermal wound healing model in the rat. The use of a rapid freezing method at − 80°C minimized the detrimental effects of freezing on the biomechanical properties of the tissue and also allowed for convenient inter-laboratory collaboration to be performed. The methodology described allowed for the simultaneous and reproducible measurement of tensile strength, collagen cross-linking and proteolytic enzyme activity. Increases in the tensile properties of the tissue with time were consistent with an active process of remodelling process as indicated by changes in the cross-link and enzyme profiles. Initially the granulation tissue was comparatively rich in the keto-imine cross-link hydroxylysino-keto-norleucine, which was later replaced by the aldimine cross-link dehydro-hydroxylysinorleucine. The mature cross-link histidino-hydroxy-lysinonorleucine was not observed within the granulation tissue at any stage and was also absent in aged control skin. A peak of matrix metalloproteinase-9 activity was observed at early timepoints (48 hr) and then decreased rapidly to normal levels and is consistent with an acute inflammatory response. In contrast matrix metalloproteinase-2 activity peaked later (3 days) and then decreased gradually, consistent with its role as one of the predominant enzymes involved in the remodelling process. The results described validate the animal model used and emphasize its potential for use in combined biomechanical and biochemical studies of acute wound healing.

Expression and ultrastructural localization of HMB-45 antigen

HMB-45 is a monoclonal antibody specific for melanoma cells and premature developing melanocytes. We examined the expression and specific subcellular binding sites of HMB-45 in various types of melanocytes including epidermal melanocytes from fetuses and infants with or without tyrosinase-negative oculocutaneous albinism (type IA), melanin-producing and non-producing melanoma cell lines (G361 and MeWo), and in vivo melanoma cells (melanotic and amelanotic malignant melanoma). Subcellular HMB-45 binding was examined by using post-embedding immunogold electron microscopy with rapid freezing and freeze substitution fixation methods without the use of chemical fixatives to preserve the intracytoplasmic delicate antigen property of HMB-45. HMB-45 antigen was detected not only in in vivo melanoma cells and normal fetal melanocytes, but also in melanocytes in the other conditions. Post-embedding immunogold electron microscopy revealed that HMB-45 antigen was exclusively localized to stages I and II melanosomes in the cytoplasm of neoplastic melanocytes, but was detected mainly on stages II and III melanosomes in the melanocytes from fetuses and infants. In tyrosinase-negative oculocutaneous albinism, only stages I and II melanosomes were detected in the cytoplasm, but both stages of melanosomes were HMB-45 positive. We conclude that HMB-45 appears mainly on the immature melanosomes during melanogenesis in both neoplastic and non-neoplastic melanocytes regardless of their tyrosinase activity, but the intracytoplasmic localization of HMB-45 antigen is different by each condition of melanocytes.