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PubMed1994

Protection of the rat retina from ischemic injury by brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor.

PURPOSE: The protective effects of three survival-promoting agents on ischemia-induced retinal injury in the rat were investigated. The agents included brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and basic fibroblast growth factor (bFGF). METHODS: Retinal ischemia was induced in Lewis albino rats by increasing intraocular pressure to 160 mm Hg for 90 minutes. The agents or buffer controls were injected intravitreally at different times, either before or after the ischemic insult, and the postischemic survival time was either 7 or 14 days. The degree of retinal damage was assessed from plastic-embedded sections by cytologic analysis, measurement of the thickness of several layers, and neuronal counts of the ganglion cell layer. RESULTS: Retinal ischemia thinned and reduced cell numbers in the inner retinal layers, but not in the photoreceptor nuclear layer. Each agent transiently ameliorated the degenerative changes when it was injected 2 days before ischemia. At 7 days postischemia, the inner retinal layers were far less damaged, and more ganglion cells were present than in buffer-injected or uninjected eyes. The protective effect was no longer evident at 14 days postischemia, except in the inner nuclear layer of the BDNF-treated eyes. If a second injection of BDNF was made 5 days after the ischemic insult, then the inner retinal layers were more preserved than buffer controls at 14 days postischemia, but the survival of ganglion cells was not enhanced. A single injection of BDNF at either 1 or 3 days postischemia reduced the degree of inner retinal damage and increased the number of surviving ganglion cells over that in buffer-injected controls. (CNTF and bFGF were not studied with postischemic injections.) CONCLUSIONS: BDNF, CNTF, and bFGF transiently protect the retina from pressure-induced ischemic injury when given 2 days before ischemia, and a second injection of BDNF given postischemically can prolong the protective effect. Moreover, protection afforded by BDNF can be seen even when applied only 1 or 3 days after the ischemic insult, although the protective effect is greater at 1 day than at 3 days postischemia.

Animals↗
PubMed1993

[Acquired aplastic anemia: a stereological analysis of bone marrow fatty tissue and its clinical correlations].

BACKGROUND: To analyze the changes of the number and size of bone marrow adipocytes in aplastic anaemia. To discover possible clinical and haematological factors which may be associated with predominant expansion of the number or size of adipocytes. To analyze the prognostic value of the adipocyte number and size. METHODS: The adipose tissue of 25 plastic-embedded bone marrow specimens corresponding to patients with aplastic anaemia was studied stereologically. The correlation study between the results obtained and clinical and haematological data was carried out. RESULTS: The expansion of the adipose tissue is due to the predominant increase of the adipocyte number in some cases and of the adipocyte size in others. Among different clinical and haematological factors, only a lower age is significantly associated with a predominant increase of adipocyte number. In the prognostic stratification performed according to three different systems, neither the adipocyte number nor the size showed a statistically significant association. CONCLUSIONS: In the aplastic anaemia, the younger patients expand their bone marrow adipose tissue by increasing predominantly the adipocyte number, whereas in the older patients this occurs by predominantly increasing the adipocyte size (may be due to the decrease in proliferative potential). On the other hand, both the adipocyte number and size lack prognostic significance.

Adipocytes↗
PubMed1995

Cryptococcus neoformans survive and replicate in human microglia.

BACKGROUND: Cryptococcus neoformans (CN) is an opportunistic pathogen that can cause fatal meningoencephalitis in patients with immune deficiency. Among the central nervous system (CNS) cells that are infected by CN are perivascular microglia and macrophages. Little is known about the interaction of CN and CNS phagocytes at the cellular level. EXPERIMENTAL DESIGN: To better understand the fate of CN in microglia, we followed Ab-opsonized CN in human fetal microglial culture by phase-contrast microscopy, combined lighted microscopy and transmission electron microscopy of plastic-embedded monolayers, and immunocytochemistry for localization of capsular Ag. RESULTS: Phase-contrast microscopy revealed that microglia initially internalized and contained Ab-opsonized yeast cells within phagolysosomes. However, CN escaped from microglia and resumed extracellular growth 16 to 24 hours after being phagocytosed. Transmission electron microscopy/1-mu epoxy sections revealed that intracellular CN were localized in two types of phagosomes in microglia: spacious phagosomes (SP) and close-fitting phagosomes (CP). Three lines of evidence indicate that SP are the primary sites for intracellular CN survival and replication: (a) SP contained multiple, budding yeast cells, whereas CP contained only single yeast cells within a tightly bound phagosomal membrane; (b) the number of SP and the number of CN within SP increased considerably at 24 hours compared with at 2 hours; and (c) microglial cultures challenged with heat- or amphotericin B-treated CN had significantly fewer SP than those challenged with live CN. Both SP and CP phagosomes fused with lysosomes, suggesting that CN survival in SP was not due to failure of phagolysosomal fusion. In SP, there was attenuation and diffusion of capsular polysaccharide within the phagosome, whereas in CP the fungal capsules remained compact and homogeneous. Immunocytochemistry with an mAb directed to capsular glucoronoxylomannan supported continued synthesis of polysaccharide within SP. CONCLUSION: The results suggest that, in human microglia, CN survive and replicate within SP. Modification of CN capsular polysaccharide within SP may be a contributing factor to this aberrant microglial-CN interaction.

Animals↗
PubMedDate not supplied

Two in situ labeling techniques reveal different patterns of DNA fragmentation during spontaneous apoptosis in vivo and induced apoptosis in vitro.

BACKGROUND: Two new enzymatic reactions were described recently to detect apoptotic cell death in situ viz in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) of fragmented DNA. A comparative study was conducted to detect in vivo and in vitro apoptotic death using these two techniques. Experimental design; Spontaneous apoptous cell death was detected in plastic embedded tumor biopsies from patients with non-Hodgkin's lymphoma (NHL), head and neck squamous cell carcinomas (HNSCC), and breast cancer using these two in situ methods. Uninvolved normal tissues adjacent to breast tumors and a lymph node metastasis of breast tumor were also studied. Furthermore, apoptotic death induced by different doses of etoposide (VP16) was also studied in HL60 cells by in situ methods and by agarose gel electrophoresis. RESULTS: Interestingly, whereas NH1 and HNSCC biopsies showed comparable levels of detectability with the two techniques, the breast tissues be it neoplastic, normal or metastatic, revealed apoptosis detectable only by TUNEL and not by ISEL. Similarly in HL60 cells, the percentage of apoptotic cells or apoptotic index (AI) determined by TUNEL was significantly higher than that determined by ISEL. A double labelling of these HL60 cells for ISEL and TUNEL also revealed a higher proportion of cells labeled positively for TUNEL as compared to those labeled for ISEL. Agarose gel electrophoresis revealed characteristic DNA laddering only at 35 microM dose of VP 16. No smearing of DNA was found in any group ruling out the necrotic death. In vivo, in one HNSCC specimen apoptosis and necrosis could be differentiated by the difference in staining intensity. Both methods stained necrotic chromatin fragments very lightly. The DNA fragments generated during apoptosis could be of unique lengths (ie 180-200 bp or multiples) but have differently staggered ends. These fragments may be 3' recessed, 5' recessed or blunt ended. While TUNEL can label all three types, ISEL labels only those with 3' recessed ends. CONCLUSIONS: Thus our data show that the DNA fragments formed during spontaneous apoptosis in breast tissues and preferentially during VP16 induced apoptosis in HL60 cells are either 5' recessed or blunt ended, being distinctly different from 3' recessed fragments seen in NHL and HNSCC or with a lesser frequency in VP 16 treated HL60 cells. Specific fragmentation patterns could be a result of activation of different endonucleases which as indicated by our data could be tissue specific and may be differentially activated by different chemotherapeutic agents. Therefore, screening for the presence of specific endonucleases in different tissues and for agents specifically activating them would have major clinical implications.

Apoptosis↗
PubMed1996

Indication of an involvement of interleukin-1 beta converting enzyme-like protease in intramedullary apoptotic cell death in the bone marrow of patients with myelodysplastic syndromes.

Our previous studies using in situ end labeling (ISEL) of fragmented DNA revealed extensive apoptotic cell death in the bone marrows (BM) of patients with myelodysplastic syndromes (MDS) involving both stromal and hematopoietic cells. In the present report we show greater synthesis of interleukin-1 beta (IL-1 beta) in 4 hour cultures of density separated BM aspirate mononuclear (BMAM) cells from MDS patients as compared to the cultures of normal BM from healthy donors or lymphoma patients (1.7 +/- 0.37 pg/10(5) cells, n = 29 v 0.42 +/- 0.24 pg/10(5) cells, n = 11, respectively, P = .049). Further, these amounts of IL-1 beta in MDS showed a significant correlation with the extent of apoptosis detected by ISEL in corresponding plastic embedded BM biopsies (r = .480, n = 30, P = .007). In contrast normal BMs did not show any correlation between the two (r = .091, n = 12, P = .779). No significant correlation was found between the amounts of IL-1 beta and % S-phase cells (labeling index; LI%) in MDS determined in BM biopsies using immunohistochemistry following in vivo infusions of iodo- and/or bromodeoxyuridine. Neither anti-IL-1 beta antibody nor IL-1 receptor antagonist blocked the apoptotic death of BMAM cells in 4 hour cultures (n = 5) determined by ISEL (apoptotic index; AI%), although the latter led to a dose-dependent accumulation of active IL-1 beta in the culture supernatants. On the other hand, a specific tetrapetide-aldehyde inhibitor of ICE significantly retarded the apoptotic death of BMAM cells at 1 mumol/L in 5/6 MDS cases studied (AI% = 2.99 +/- 0.30 in controls v 1.58 +/- 0.40 with ICE-inhibitor, P = .05) and also reduced the levels of active IL-1 beta synthesized (5.59 +/- 2.63 v 2.24 +/- 0.93 pg/10(6) cells, respectively). In normal cells, neither IL-1 blockers nor the ICE inhibitor showed any effect on the marginal increase in apoptosis observed in 4 hour cultures. Our data thus suggest a possible involvement of an ICE-like protease in the intramedullary apoptotic cell death in the BMs of MDS patients.

Antibodies, Monoclonal↗
PubMedDate not supplied

Pituitary control of proliferation and differentiation of Leydig cells and their putative precursors in immature hypophysectomized rat testis.

The objective of this study was to determine the effects of pituitary hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], growth hormone [GH], and prolactin [PRL]) on interstitial cell proliferation and differentiation in the testis of immature hypophysectomized rats. Macrophages, Leydig cells, precursor mesenchymal cells, endothelial lymphatic cells, and myoid cells were studied. Our experimental approach was aimed at determining whether changes in a cellular subpopulation observed after pituitary hormone treatments were the result of division of existing cells in the population, of differentiation of interstitial precursor cells, or both. In this context, it must be stressed that our data reflected the effects of hormones to prevent the decline of cells due to hypophysectomy rather than their recovery. Macrophage proliferation was taken into account because macrophages closely resemble Leydig cells and are known to proliferate after hormonal treatment. A double-labeling procedure (acid phosphatase and anti-bromodeoxyuridine [anti-BUdR]) revealed that LH, FSH, and PRL increased the number of testicular macrophages 105-, 104-, and 103-fold, respectively, in hypophysectomized rats compared to hypophysectomized control animals. BUdR incorporation in testicular macrophages was greater after PRL treatment than after LH and FSH supplementation. In contrast, we were unable to demonstrate any effect of rat GH on the macrophage population. Light microscopic analysis of plastic embedded sections of treated rat testis revealed that LH increased the numbers of Leydig, precursor mesenchymal, and myoid cells 6-, 4-, and 1.3-fold, respectively. LH also stimulated BUdR incorporation into all interstitial cell types. PRL administration increased both the number of Leydig and precursor mesenchymal cells (each 3-fold) but decreased the number of endothelial lymphatic cells (1.5-fold) when compared to the control animals. In contrast, FSH did not increase the number and proliferation of Leydig cells but exerted a slight proliferative effect on the other interstitial cell populations. In GH-treated rats, the number of precursor mesenchymal cells increased two fold above the control rats. GH also exerted slight proliferative effects on both precursor mesenchymal and myoid cells. Immunohistochemical studies of steroidogenic enzymes in the testicular interstitium of treated rats demonstrated the presence of steroidogenic enzymes, not only in Leydig and precursor mesenchymal cells, but also in some (1%-2%) endothelial lymphatic cells and myoid cells. This may indicate that both of these cell types are also constitutively equipped to perform steroidogenesis or that they are precursor cells undergoing differentiation. Taken together, changes in the number of Leydig cells in our animal model appeared more likely to be dependent on the transformation of precursor cells than on division of preexisting mature Leydig cells.

20-Hydroxysteroid Dehydrogenases↗
PubMed1997

Immunoelectron microscopic demonstration of regulated pathway for calcitonin and constitutive pathway for carcinoembryonic antigen in the same cells of human medullary carcinomas of thyroid glands.

The human medullary carcinomas are well known to secrete calcitonin (CT) as a neuroendocrine peptide and carcinoembryonic antigen (CEA) as a serum protein that is integrated into the cell membrane. This ultrastructural study is designed to elucidate whether CT and CEA are secreted via two different intracellular secretory pathways, a regulated pathway and a constitutive pathway. The immunoelectron microscopic postembedding method, performed on four cases of the human medullary carcinomas of the thyroid using plastic embedding material, disclosed two distinct different localization patterns for CT and CEA. CT was localized exclusively in dense cored secretory granules. CEA was present in the cell membrane and in the secretory vesicles. The secretory granules were completely negative for CEA. The trans-Golgi networks were also positive for CT and CEA. Electron microscopic double staining confirmed these localization in the same carcinoma cells. These observations suggest the presence of two distinct pathways in the endocrine cancer cells, i.e., the regulated pathway for CT and the constitutive pathway for CEA.

Biological Transport↗
PubMed1977

Ultrastructural changes in tracheal epithelial cells exposed to oxygen.

White albino rats were sacrificed after 24, 36, 48, 72, and 96 h of exposure to 100% O2 at 1 atm. Tissue was prepared for the scanning electron microscope (SEM) by Critical Point Drying and for the transmission electron microscope (TEM) by plastic embedding. Scanning microscopy showed a loss of microvilli after 48 h of exposure. Cilia appeared relatively normal with SEM, but TEM revealed changes in the outer membrane. In TEM, nonciliated cells appeared swollen and often encroached on the ciliated cells. A heavy mucous blanket remained even after processing. All the changes observed that are induced by oxygen exposure contribute to mucostasis, reducing and/or halting mucociliary clearance.

Animals↗
PubMed1996

A comparison of methods for the estimation of retroviral burden.

The presence of retroviral contamination is of vital concern in the manufacture of cell culture-derived biopharmaceuticals. These cell lines usually have A- or C-type retrovirus-like particles which are visible by transmission electron microscopy (TEM) even when infectivity (IF) or reverse transcriptase activity (RTA) cannot be demonstrated. The supernatant of the post-production cell cultures, therefore, also needs to be evaluated by TEM for viral burden. A major question, however, is how to establish a quantitative viral load estimate for the evaluation of a purification process. The FDA recommends that a purification process for viral contaminants remove or inactivate 3-5 logs over the estimated viral burden. Viral particles are difficult to identify and quantify, however, by conventional negative staining. We present a comparison of infectivity assay, reverse transcriptase assay, negative staining, and thin sectioned TEM. These assays were performed on four samples. Ultracentrifuged sediments of cleared cell-culture media were measured, fixed and processed. Thin sections were evaluated by TEM and the number of viral particles estimated by morphometric derived quantification. Retrovirus particles were easily identified and quantified when examined by TEM as compared to negative staining and correlated with the other viral assays (IF, RTA). These results demonstrate that the TEM thin section method was a superior technique to negative staining for estimating viral particle load in cell-culture supernatant. To validate further the plastic embedding with thin sectioning, we evaluated cell culture supernatants (pellets) for retroviral burden at various dilutions, from two cell lines. Morphometric determinations were made of the number of viral particles present per unit volume and compared to results obtained by infectivity assay. Since the morphometric calculation for viral density assumes even distribution of viral particles, we also evaluated and calculated viral counts on multiple thin sections taken throughout selected pellets.

Animals↗
PubMed1997

Structural neurotoxicologic investigation of the glycine antagonist 5-nitro-6,7-dichloroquinoxalinedione (ACEA-1021).

NMDA antagonists of glutamate have psychotomimetic side effects and structural side effects which have been shown to be lethal to CNS neurons in the cingulate and retrosplenial cortex of rodents, yet these compounds may reduce focal ischemic brain damage. This investigation used 38 Wistar rats to determine whether the structural toxicologic profile of a newly developed halogenated quinoxalinedione derivative, a pharmacologic antagonist of the glycine site on the NMDA receptor complex, is identical to that seen with MK-801. In the cingulate and retrosplenial cortex, examination of glutaraldehyde perfusion-fixed, plastic-embedded tissue 4 to 6 hours after intravenous administration of 10 mg/kg of the glycine antagonist 5-nitro-6,7-dichloroquinoxalinedione (ACEA-1021), no changes were seen by light or electron microscopy. At a dose of 30 mg/kg, neurons were seen containing 1 to 2 microns granules in perikarya and axons. Vacuolated neurons, as described in NMDA-antagonist neurotoxicity, were exceedingly rare, comprising only 4 in the entire study. Electron microscopy of the granulated profiles showed intracytoplasmic areas containing grouped mitochondria and lysosomes, located in neuronal perikarya, and rarely in myelinated axons. Neuronal necrosis was evaluated in formaldehyde perfusion-fixed, paraffin-embedded tissue at one week survival, and was absent. MK-801 5 mg/kg, in contrast, caused irreversible (necrotizing) neuronal changes. The results demonstrate that this glycine antagonist is devoid of lethal neurotoxicity, but causes a reversible alteration in a small proportion of cingulate and retrosplenial cortical neurons. Since previous studies have shown anti-ischemic efficacy of this compound in focal, but not global ischemia, it appears that the therapeutic profile of this antagonist of the strychnine-insensitive glycine site is similar, but the toxicologic structural profile is different, from NMDA receptor antagonists.

Animals↗
PubMed1977

How useful is a biopsy in a case of suspected fungal infection?

In suspected cases of superficial fungal infections, biopsies are not routinely performed. This paper has analyzed some diagnostic features of histopathologic examination in dermatomycosis that are not usually emphasized. The usefulness of light microscopic observation of PAS-stained and plastic-embedded specimens has also been shown.

Biopsy↗
PubMedDate not supplied

[A method for the re-embedding of tissues from paraffin blocks into epoxy resins for the study of stained semithin sections by electron microscopy].

The proposed method or re-embedding tissue specimens from paraffin in epoxy resins (epon, araldite, etc.) permits study of the same material at histo-, cyto-, and ultrastructural levels by modern morphological methods and thus detecting a number of additional morphological signs important for thanatogenesis. The proposed method provides objective results and improves the quality of forensic medical studies in general.

Forensic Medicine↗
PubMedDate not supplied

Early ultrastructural changes in the dorsal mucosa of rat tongue after irradiation, with special reference to the microvasculature.

PURPOSE: To clarify the acute effects of irradiation on the ultrastructural conformation of the dorsal mucosa of the rat tongue, with special reference to the changes in microvasculature. METHODS: The proboscis of seventy rats were irradiated. The animals were then perfusion-fixed, followed by India ink-injection or resin casting at 3 to 7 days after irradiation. The bulk, frozen sections, or plastic embedded sections of the treated rat tongues were examined by light and electron microscopy. RESULTS: In the dorsal epithelium of the rat tongue, multi-nucleated cells appeared in the basal layer at 3 days after irradiation. At day 5, the thickness of the epithelial layer and connective tissue papillae decreased dramatically, concomitant with the shortening of the capillary loops. At day 7, lingual papillae and connective tissue papillae disappeared, leaving dissociated epithelial cells and numerous neutrophils migrating throughout the tissue. Subepithelial blood vessels displayed drastic dilation with a number of neutrophils adhering to the endothelial surface, but without ultrastructural abnormalities in its cellular components. CONCLUSION: Early changes in the dorsal mucosa of the irradiated rat tongue were limited to the basal epithelial cells, leading to a total disruption of the epithelial layer. Atrophic changes of the capillary loops is due to the loss of the connective tissue papillae. Dilation and conformational changes of the subepithelial capillaries appear to result from the inflammatory reaction, taking place secondarily to the loss of the epithelial barrier of the irradiated tongue. A difference in radiosensitivity among the epithelial, endothelial, and mesenchymal cell components of the rat tongue in vivo is suggested.

Animals↗
PubMed1998

Suppression of developmental retinal cell death but not of photoreceptor degeneration in Bax-deficient mice.

PURPOSE: Bax, a member of the Bcl2 family of cell death regulators, induces cell death by promoting the induction of apoptosis. Bax-deficient mice were examined in this study to determine whether Bax is required for cell death in the developing retina and for pathologic apoptotic photoreceptor degeneration resulting from the rd mutation. METHODS: Retinas from Bax-deficient mice and their wild-type siblings were harvested at postnatal day (P) 7 and processed for TdT-dUTP terminal nick-end labeling (TUNEL) staining, and the number of nuclei containing fragmented DNA were counted. Adult retinas and optic nerves were processed for plastic-embedded 1-microm sections, and the cross-sectional area was determined. The mutant Bax allele was outbred onto the C3H mouse strain, which carries the rd allele. Retinas from these animals were examined histologically at P21 after most of the photoreceptor cell death had occurred. RESULTS: At P7, around the time of peak cell death in the inner nuclear layer (INL), significantly fewer neurons in INL and ganglion cell layer (GCL) were TUNEL positive in Bax-deficient mice than in their wild-type siblings. In adult Bax-deficient mice, the cross-sectional area of the optic nerve was approximately 50% larger than in wild-type siblings, and the total number of retinal ganglion cell axons was increased to 226%. The INL of Bax-deficient mice was thicker than normal. The Bax genotype did not affect the thickness or histologic appearance of the outer nuclear layer in retinas of mice with wild-type or mutant rd alleles. CONCLUSIONS: In the absence of the expression of the Bax gene, there is a profound increase in the survival of retinal ganglion cells that lasts into adulthood. Similarly, death of INL cells is diminished but not completely abolished. The absence of Bax does not, however, protect photoreceptors from naturally occurring cell death or degeneration induced by the rd mutation. This shows that Bax is involved to a variable degree in the control of developmental cell death in the retina and that not all apoptotic retinal cell deaths are controlled by Bax.

Animals↗
PubMed1994

Effect of embedding methods versus fixative type on karyometric measures.

The influence of fixation and embedding methods in seven urologic tumor samples was studied karyometrically for 12 preparatory techniques. Routine histologic formalin fixation was compared with Carbowax and Kryofix fixatives. Also, histologic material was studied embedded in paraffin and plastic (GMA) and secondarily in plastic (after paraffin embedding). From the cell suspensions, cytocentrifuge preparations were made (10 minutes, 1,500 rpm). The influence on nuclear light microscopic characteristics for two morphometric and five texture features in Feulgen-stained nuclei was evaluated. Nuclear features were influenced by both fixation and embedding method. The most extensive karyometric differences were found between different embedding techniques. More abnormally shaped tumor cell nuclei tended to occur with plastic (GMA) embedding methods. Plastic embedding proved to be superior to paraffin for all three fixation methods. Reembedding of archival formalin-fixed, paraffin-embedded material in plastic greatly influenced nuclear features, nuclear area in particular. We conclude that nuclear features are particularly influenced by the embedding method applied.

Cell Nucleus↗
PubMed2000

Vascular remnants in the rabbit vitreous body. II. Enzyme digestion and immunohistochemical studies.

The purpose of this study was to evaluate the composition of ghost vessels and the newly identified intravitreal structures type 1 and 2 (IVS-1 and 2) observed in the rabbit vitreous body. Rabbit eyes (n = 10, 0.5- approximately 36 months of age) were fixed and embedded in plastic. Post-embedding immuno transmission electron microscopy and enzyme digestion methods specifically directed at vascular extracellular matrix components (collagen IV, elastin and hyaluronan) were used in order to confirm the postulated vascular origin of IVS-1 and 2. In addition, markers of vitreous extracellular matrix components (collagen II, hyaluronan) were used. The postulated vascular nature of ghost vessels and IVS-1 was confirmed by a positive labelling with anti-collagen IV, whereas the demonstration of elastin (by anti-elastin antibodies and elastase digestion) in IVS-1 and 2 confirms their arterial origin. These vascular remnants were also labelled with a hyaluronan marker and with anti-collagen II. The presence of remnants of the hyaloid artery system throughout the vitreous matrix is in conflict with a strict spatial separation between the primary and secondary vitreous during embryonic development as proposed in the literature. It strongly supports an alternative theory which suggests an interactive remodelling of this matrix. The presence of hyaluronan in remnants of the hyaloid system is inconclusive, since hyaluronan is a component both of the adult vitreous matrix and of the vascular extracellular matrix. The presence of collagen II in vascular structures is highly interesting, since it supports another challenging theory, which suggests that lamellae develop alongside tracts formerly occupied by the larger hyaloid vessels.

Animals↗
PubMed1979

Embedding in large plastic blocks. Diagnostic light and potential electron microscopy on the same block.

Specimens destined for light and electron microscopy were fixed in a modified buffered formalin, postosmicated, dehydrated, and embedded in a mixture of epoxy resins (Epon-araldite) in large plastic molds. These blocks were sectioned at 0.5 to 1 micron on a JB-4 microtome and stained with a combined nuclear and cytoplasmic stain (Paragon). The sections were examined by light microscopy for diagnostic evaluation. If ultrastructural examination was also desired, the selected area was isolated using the "mesa" technique. The trimmed block was then sectioned on an ultramicrotome, picked up on grids, stained, and examined in the electron microscope. We think these techniques offer the diagnostic pathologist the potential of viewing 1-micron sections at a light microscopy level with the option of subsequent electron microscopy of the same area of the same block.

Histological Techniques↗
PubMed1983

The effects of embedding in water-soluble plastics on the final dimensions of liver sections.

This paper describes the morphometric changes liver samples undergo in the course of fixation, dehydration, infiltration and embedding in different mixtures of water-soluble plastics. The plastics used were: three different mixtures of glycolmethacrylate (GMA) and the commercially available material JB4. Buffered formaldehyde fixation did not produce significant morphometric changes in the liver specimens. Dehydration obviously affects the volume of the liver specimen (linear shrinkage about 9.3%). The dehydration is followed by an infiltration phase. During this phase a slight swelling (linear, 2-5%) occurs. The final polymerization of the plastic resulted in a further linear shrinkage of 1-2%. The influence of different technical factors on the stretching of sections of pure plastic and of plastic embedded liver appeared to be considerable. The difference in stretching between 2 and 3 micron sections has been studied. A significant influence of temperature upon section stretching was noted. Sections of all plastic mixtures stretched at a temperature of 293 K showed 3% more linear stretch than at a temperature of 333 K. Differences between the four plastic mixtures are discussed. Correction factors must be used in morphometrical and stereological investigations (see for review: Weibel, 1979). It was concluded that in the application of water-soluble plastics as embedding media it is essential to apply a standardized procedure, particularly in the cutting and stretching phase.

Animals↗