Hardening Of Tissue Research Articles

Determining the Proportions of Bone and Cartilage Growth in the Crucian Carp (carassius auratus) Using the Modified Simultaneous Differential Staining Technique

The modified simultaneous differential staining technique, which enables double staining of cartilage and bones, needs to be improved to prevent soft tissues from being damaged during the staining process. Key factors influencing the extent to which soft tissues are damaged include the fixative used, macerating time, potassium hydroxide concentration, incubation temperature and the removal of skin from specimens. Here we describe a protocol that enables the hardening of tissues during bleaching and maceration. We also describe a method for objectively measuring rates of cartilage and bone growth. The use of formalin as a fixative rendered soft tissues more rigid due to the resulting chemical bonds formed between proteins. Blotted specimens were immersed in 1% potassium hydroxide (KOH) and incubated at <TEX>$37^{\circ}C$</TEX> for 1 day (smaller specimens) or 2-3 days (larger specimens). The 1% KOH solution was also used as the diluent solution for the subsequent immersion in a graded series of 30%, 50%, 70%, 90%, 100% glycerol solutions, a procedure that made soft tissues even more transparent and hardened. It was not necessary to remove the skin of specimens shorter than 2 cm, since the macerating solution could easily penetrate their thin skin layer and continuously remove those pigments hindering visibility. Since excessive osmosis is another factor that can damage soft tissues in the macerating process by causing the rupture of those cells not able to withstand the osmotic pressure, here it was minimized by balancing the salt concentration between the interior and exterior of cells with the addition of 0.9% sodium chloride (NaCl) in the macerating solution. Finally, to determine the proportions of cartilage and bone growth, photographs of the stained specimens were taken with a dissecting microscope and sections corresponding to the cartilage and bones were cut out from the printed pictures and weighed. Our results show that this method is suitable for the objective evaluation of bone and cartilage growth.

Effect of Heparin Oligomer Chain Length on the Activation of Valvular Interstitial Cells

A key event in connective tissue remodeling involves the transformation of fibroblasts to myofibroblasts, also revealed by expression of α-smooth muscle actin (α-SMA). However, misregulation of this transition can lead to fibrosis, an overgrowth and hardening of tissue due to excess extracellular matrix deposition, a process that is linked to heart valve disease and many others. Both disease treatment and regenerative strategies would benefit from strategies for the controlled delivery and presentation of bioactive factors that can promote or suppress this transformation. In this regard, the ability of heparin to complex a plethora of growth factors offers a broad range of possibilities for this purpose. Here, the effects of heparin chain length and structure on valvular interstitial cell (VIC) phenotypic expression were explored. Heparin from porcine intestinal mucosa was depolymerized with heparinase and fractionated to obtain oligosaccharides of different sizes. VICs cultured with octasaccharides and decasaccharides exhibited higher expression of a-SMA when compared to other saccharides and full-length heparin. No activation of VICs was observed in response to full-length heparin presence in media.

RISK FACTORS FOR SCLEREMA NEONATORUM IN PRETERM NEONATES IN BANGLADESH

This study presents a retrospective analysis of risk factors for sclerema neonatorum in preterm neonates in Bangladesh. Preterm neonates admitted to Dhaka Shishu Hospital in Bangladesh were enrolled in a clinical trial to evaluate the effects of topical treatment with skin barrier-enhancing emollients on prevention of sepsis and mortality. Four hundred ninety-seven neonates were enrolled in the study and 51 (10.3%) developed sclerema neonatorum. We explored risk factors for sclerema neonatorum by comparing patients with and without sclerema neonatorum. Diagnosis of sclerema neonatorum was based on the presence of uniform hardening of skin and subcutaneous tissues to the extent that the skin could not be pitted nor picked up and pinched into a fold. Cultures of blood and cerebrospinal fluid were obtained in all neonates with clinical suspicion of sepsis. In multivariate analysis, lower maternal education (OR: 1.94; 95% CI: 1.02-3.69; P = 0.043), and signs of jaundice (OR: 2.82; 95% CI: 1.19-6.69; P = 0.018) and poor feeding (OR: 4.71; 95% CI: 1.02-21.74; P = 0.047) on admission were risk factors for developing sclerema neonatorum. The incidence rate ratio of sepsis in neonates who developed sclerema neonatorum was 1.81 (95% CI: 1.16-2.73; P = 0.004), primarily due to Gram-negative pathogens, and risk of death in infants with sclerema neonatorum was 46.5-fold higher (P < 0.001, 95% CI: 6.37-339.81) than for those without sclerema neonatorum. Sclerema neonatorum was a relatively common, grave condition in this setting, heralded by poor feeding, jaundice, and bacteremia, and signaling the need for prompt antibiotic treatment.

Digital Oedema, Adhesion Formation and Resistance to Digital Motion after Primary Flexor Tendon Repair

The development of digital oedema, adhesion formation, and resistance to digital motion at days 0, 3, 5, 7, 9 and 14 after primary flexor tendon repairs using 102 long toes of 51 Leghorn chickens was studied. Oedema presented as tissue swelling from days 3 to 7, which peaked at day 3. After day 7, oedema was manifest as hardening of subcutaneous tissue. The degree of digital swelling correlated with the resistance to tendon motion between days 3 and 7. At day 9, granulation tissues were observed around the tendon and loose adhesions were observed at day 14. Resistance to digital motion increased significantly from day 0 to day 3, but did not increase between days 3 and 9. The early postoperative changes appear to have three stages: initial (days 0-3, increasing resistance with development of oedema), delayed (days 4-7, higher resistance with continuing oedema) and late (after day 7-9, hardening of subcutaneous tissue with development of adhesions).

Cellular and molecular mechanisms of fibrosis

Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components including collagen. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Although current treatments for fibrotic diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis typically target the inflammatory response, there is accumulating evidence that the mechanisms driving fibrogenesis are distinct from those regulating inflammation. In fact, some studies have suggested that ongoing inflammation is needed to reverse established and progressive fibrosis. The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell. Myofibroblasts are generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells in processes termed epithelial/endothelial-mesenchymal (EMT/EndMT) transition, as well as from circulating fibroblast-like cells called fibrocytes that are derived from bone-marrow stem cells. Myofibroblasts are activated by a variety of mechanisms, including paracrine signals derived from lymphocytes and macrophages, autocrine factors secreted by myofibroblasts, and pathogen-associated molecular patterns (PAMPS) produced by pathogenic organisms that interact with pattern recognition receptors (i.e. TLRs) on fibroblasts. Cytokines (IL-13, IL-21, TGF-beta1), chemokines (MCP-1, MIP-1beta), angiogenic factors (VEGF), growth factors (PDGF), peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, and components of the renin-angiotensin-aldosterone system (ANG II) have been identified as important regulators of fibrosis and are being investigated as potential targets of antifibrotic drugs. This review explores our current understanding of the cellular and molecular mechanisms of fibrogenesis.

The Effectiveness of Honey as a Substitute for Formalin in the Histological Fixation of Tissue

The preservative and antimicrobial powers of honey have been known for centuries. In addition, studies have shown that honey has acidic and dehydrative properties. The influence of these powers on tissue fixation was determined in this work to ascertain whether honey could prove to be a safe and acceptable substitute for formalin in the histological fixation of tissue. In this study, rat liver and kidney tissues were fixed at 37°C and at room temperature with and without agitation in concentrations of honey ranging from 10% to 100% diluted with distilled water. Tissues were fixed for 24 h, processed to paraffin wax, sectioned and stained by hematoxylin and eosin, and examined microscopically. Initial results obtained from this work concluded that tissues fixed in honey concentrations of 10% and 20% at room temperature with and without agitation gave comparable results with those obtained by formalin-fixed control tissues. Higher concentrations of honey were less successful and produced slower penetration rates, hardening of tissues, and difficulty in sectioning. Nonetheless, the results using low concentrations of honey for tissue fixation were encouraging and make further research using this and other brands of honey with a wider range of tissues, fixation times, and special stains of great interest. (The J Histotechnol 29:173, 2006)Submitted June 27, 2006; accepted with revisions July 25, 2006

704 生体由来組織の脱細胞化のための超臨界流体抽出(G02-3 バイオマテリアル,G02 バイオエンジニアリング)

Decellularization of biological tissue has been investigated using supercritical fluid extraction method. Carbon dioxide and fluoroform which have gentle critical conditions were applied as extraction media under various experimental conditions. Decellularization was evaluated by means of HE-stain and chemical quantitative analysis of DNA and phospholipids. Supercritical carbon dioxide that contained small amount of entrainer had a potential to extract phospholipids and nucleus without losing mechanical strength of tissue. In addition, decellularization with carbon dioxide could be attained within 30 minutes treatments. On the other hand, treatments with supercritical fluoroform alone caused slight hardening of tissue.

Revisiting the surgical creation of volume load by aorto-caval shunt in rats.

Cardiac hypertrophy is an early landmark during the clinical course of heart failure, and is an important risk factor for subsequent morbidity and mortality. The hypertrophy response to different types of cardiac overload is distinguished both at the molecular and cellular levels. These changes have been extensively characterized for pressure load hypertrophy; however, similar information for volume load hypertrophy is still needed. This study was undertaken to improve the existing method of producing experimental cardiac volume load. Previous investigators have employed surgical aorto-caval shunt (ACS) as a model for volume load hypertrophy (VO) in rats. The procedure is relatively simple and involves glue to seal the aortic hole after ACS. However, it has several limitations mostly related to the use of glue e.g. poor visualization due to hardening of tissues, imperfect sealing of the puncture site and glue seeping through the aortic hole resulting in shunt occlusion. We have modified the procedure using aortic adventitial suture instead of glue and 18G angiocatheter instead of 16G needle, which eliminated the technical difficulties from the former method. The ACS was visually confirmed at sacrifice, and the VO demonstrated by time-related changes in the heart weight/body weight ratio which increased from 78% at 4 weeks to 87% at 10 weeks and increased liver/body weight ratio by 22% at 10 weeks of post aorto-caval shunt. Cardiac expression of atrial natriuretic peptide (ANF) also demonstrated time-related increase in ANF mRNA (+275% increase at 4 weeks, p < 0.05, and +370% increase at 10 weeks, p < 0.001). This modified technique of aorto-caval shunt offers simpler, reproducible and consistent model for VO hypertrophy in rats.

Serial notching of the platysma bands

Background: Traditional procedures for the reduction of platysma bands can result in scarring and hardening of tissue in the submental region. Objective: A technique for serial notching of the platysma bands is described that is more effective and causes fewer complications than the traditional submental and midline approaches. Methods: The platysma bands are marked for incision; optimal results require multiple incisions with a distance of 3 to 4 cm between each incision. The muscle band is dissected and undermined, and notching is performed with an electrocautery. Suturing is necessary only if severe skin laxity is present; otherwise, Steri-strips for 3 to 4 days are sufficient. In most cases, submental lipoplasty is also necessary. Results: A series of 51 procedures performed over 14 months produced satisfactory results with no major complications. Conclusions: Serial notching is a good adjuvant to a neck lift or lower-face procedures, particularly in older patients. (Aesthetic Surg J 2001;21:412-417.)

Allomonal and hepatotoxic effects following methyl eugenol consumption in Bactrocera papayae male against Gekko monarchus.

Methyl eugenol (ME), is converted into two major phenylpropanoids, 2-allyl-4,5-dimethoxyphenol and trans-coniferyl alcohol, following consumption by the male fruit fly Bactrocera papayae. Chemical analysis of wild male B. papayae rectal glands, where the compounds are sequestered, revealed the presence of ME metabolites in varying quantities. These phenylpropanoids are shown to be involved in the fruit fly defense both in no-choice and choice feeding tests against the Malayan spiny gecko, Gekko monarchus. After being acclimatized to feeding on fruit flies, geckos consumed significantly fewer ME-fed male flies than controls that consumed all the ME-deprived male flies offered throughout a two-week period. Diagnosis of dissected livers from geckos that consumed ME-fed male flies revealed various abnormalities. These included discoloration and hardening of liver tissue, whitening of the gallbladder, or presence of tumor-like growths in all geckos that consumed ME-fed male flies. Control geckos fed on ME-deprived male flies had healthy livers. When given an alternative prey, geckos preferred to eat untreated house flies, Musca domestica to avoid preying on ME-fed fruit flies.

Overhydroxylation of Lysyl Residues is the Initial Step for Altered Collagen Cross-Links and Fibril Architecture in Fibrotic Skin

In fibrotic skin of lipodermatosclerosis a substantial increase of the cross-link hydroxylysylpyridinoline is observed. Hydroxylysylpyridinoline is a typical cross-link of skeletal tissue and is thought to play a major part in the hardening of sclerotic tissue. We investigated whether the increase in hydroxylysylpyridinoline is due to overhydroxylation of lysyl residues in the collagen molecule, which may also be associated with an increase of glycosylated hydroxylysine residues. Furthermore, we determined whether the collagen fibrils in lipodermatosclerosis showed a decrease of the diameter in the tissue as well as in vitro after fibrillogenesis of pepsin-solubilized collagens. Isolated alpha-chains of pepsin solubilized collagen I showed an increase in lysyl hydroxylation (hyl/(hyl + lys)) as compared with normal control [alpha1(I): lipodermatosclerosis 0.18 +/- 0.01; control 0.12 +/- 0.01; alpha2(I): lipodermatosclerosis 0.36 +/- 0.02; control 0. 25 +/- 0.03, p < 0.001]. Furthermore, the content of enzymatic glycosylated hydroxlysine residues increased. This increase is associated with a decrease of fibril diameter of both tissue and fibrils formed in vitro of pepsin-solubilized collagens. In the same pool of collagens an increase in collagen III content was observed as compared with controls (lipodermatosclerosis 14.5% +/- 1.6, control 10.3% +/- 1.6, p < 0.001). Our results showed that the overhydroxylation of lysyl residues, which is required for the generation of hydroxylysylpyridinoline, is not only restricted to the telopeptides but also affects the helical part of the molecule. This process is further associated with an increase of glycosylated hydroxylysyl residues. These changes along with the increase in collagen III content seem to be responsible for the observed alteration in the architecture of collagen fibrils in sclerotic skin.

Histopathological and enzymological studies on the effects of chronic lead nitrate intoxication in the digestive system of a freshwater teleost, Channa punctatus

The effect of lead nitrate on the digestive system of a teleost fish, Channa punctatus has been studied after 125 days of exposure to a sublethal concentration (6.8 mg/liter). The results show that considerable degenerative changes are produced in the histological structure of liver, intestine, and pyloric caeca. In the liver, the damage is in the form of liver cord disarray, necrosis, inflammation of portal areas, hardening of connective tissue, shrinkage of nuclei, and septa formation around blood vessels. No fatty infiltration or glycogen depletion has been observed. Lipofuscin granules accumulated in the cytoplasm of hepatocytes. In the intestine and pyloric caeca flattening of villi at a number of places, inflammation, and necrosis are the most conspicuous changes. The activities of alkaline phosphatase and aminotripeptidase are inhibited in the liver. In stomach, alkaline phosphatase is inhibited but an elevation in amylase activity was noted. Acid phosphatase showed an increase in the intestine and pyloric caeca while aminotripeptidase and glycylglycine dipeptidase were inhibited.

Early frost damage on Sitka spruce seedlings and the influence of phosphorus nutrition

A frost on 8 September 1972 caused widespread damage to young Sitka spruce ( Picea sitchensis (Bong.) Carr.) plantations. In an experiment designed to examine the interaction of climate and P nutrition on the growth of young transplants in south Scotland damage was greater on plants growing at low elevation and with high P content. This effect was attributed to their extended growth period compared to those with low P supply. The lack of low temperatures prior to the frost resulted in insufficient hardening of tissues to avoid damage from relatively high temperatures. Damage and recovery were assessed by the reduction in height and dry weight increase compared to undamaged plants.

Cold panniculitis in children.

IN 1902, Carl Hochsinger described a hardening of tissue in the submental region in children, related to cold exposure. 1 His patients were between 4 and 10 years old. After exposure to unusually cold weather, they developed a tender, slightly erythematous, well-demarcated induration of the submental subcutaneous tissue. Most of his patients had an abundance of subcutaneous fat in the area. The induration gradually resolved spontaneously in two to three weeks. Hochsinger compared the lesions to those of subcutaneous fat necrosis of the newborn, and observed a similar lesion after prolonged application of an ice bag to the thigh of an 8-year-old boy. In only one instance was the cheek involved. In 1941, Haxthausen described five additional cases, four in children aged 6 months to 3 years, and one in a 17-year-old girl. 2 His patients all had conspicuous involvement of the cheeks, and the oldest girl had evidence of

RELATIVE AND ABSOLUTE ELECTROLYTIC CONDUCTANCE TESTS FOR FROST HARDINESS OF APPLE VARIETIES

A laboratory method for testing frost hardiness of apple trees is described. Results demonstrated that diffused electrolytes from pre-hardened twigs provided a reliable means of testing their hardiness. There was a definite association between increased diffusion of electrolytes from hardened tissues and increased injury of twigs. This association was found useful in establishing both the relative degrees of hardiness of three named varieties of apples, Antonovka, McIntosh and Northern Spy, and the absolute values for the ultimate frost resistance of their tissues. Thus, although the total conductance of electrolytes of the three varieties was about the same before the artificial freezing treatments, ranging from 900 to 1000 micromhos, the average survival and the conductance of diffused electrolytes were as follows: hardy Antonovka 71 per cent and 296 micromhos; less hardy McIntosh 53 per cent and 445 micromhos; and least hardy Northern Spy 23 per cent and 611 micromhos. Results also established quantitative values for the ultimate frost hardiness of apple trees, namely: no appreciable injury when the conductance from hardened tissues of twigs was within 200 to 250 micromhos or less. Conductance of electrolytes above 350 to 450 micromhos generally signified total killing; whereas intermediary readings indicated partial injury to twigs.The method used also permitted a study of the effects of several factors considered of vital importance in: (a) developing frost resistance of overwintering plants such as seasonal maturity, cold hardening and dehardening of tissues, and (b) causing excessive frost injuries such as increasing intensities and rates of freezing temperatures. The validity of the used laboratory methods is discussed.

Use of Perchloric Acid for Nucleic Acid Histochemistry in Mammalian Nerve and Liver Cells.

Summary(1) PNA was completely extracted from fixed sections of liver by 18 hours of treatment with 10% perchloric acid at 4°C. No significant quantity of DNA was removed by such treatment even after 93 hours of incubation. (2) The above treatment is inadequate to extract PNA completely from nerve cells. However, with formalin-fixed nerve tissue, 12 hours incubation in 10% perchloric acid at 20°C and 15 minutes at 37°C was found to effect complete extraction of PNA with no loss of DNA. (3) At higher temperatures of extraction, the margin of safety between complete removal of FN A and incipient extraction of DNA was much smaller. (4) Prolonged hardening of nervous tissue in 10% formalin rendered PNA resistant to extraction by perchloric acid. (5) Perchloric acid extraction of fixed sections of nervous tissue removed insignificant quantities of nerve cell protein. (6) Cell structure was unaltered by this extraction procedure. (7) This extraction procedure did not alter the E2537A of two tissues without PNA, ...

Formalin as a Hardening Agent for Nerve Tissues

Formol, Formalin, or Formaldehyde was discovered by A. W. Hoffman in 1863 while passing wood spirit and air over a redhot platinum spiral. If the vapor is brought into water to its pzint of saturation, a forty per cent. solution of formaldehyde is obtained, which has long been known under the name of formol. That Formalin possessed antiseptic as well as hardening powers we owe to the investigations of Dr. F. Blum, and these facts induced the elder Blum to make an extended serints of investigations in the hardening of animal and vegetable tissues in the Senckenbergischen Institute at Frankfort, Germany. His preliminary report was published in the Zo6logischer Anzeiger, 1893, No. 434, and a more detailed report in the Berichte fiber die Senckenbergische Naturforscher Gesellschaft, I894, p. I95. Here he details his experience, which in brief is as follows: Several human embryos were finely preserved in formol diluted with ten to twenty parts of water. Small embryos with amnion intact were preserved and the amniotic fluid remained transparent, so