Evaluation of endothelial cell migration with a novel in vitro assay system

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349–354). An aliquot of 15–20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor (TNF). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm² and 5.3 mm² for TNF-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater – 6.4 mm² and 6.5 mm² with and without TNF treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30–40% on vWF/collagen, and 60–80% on FN/collagen, with increased proliferation observed with TNF- treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.     

Extracellular potassium concentration and membrane potential in rabbit gastrocnemius muscle during tourniquet ischemia

The relationship between the extracellular potassium concentration (K⁺ e.c.) and the resting membrane potential (MP) of the gastrocnemius muscle was studied in the anesthetized rabbit during a 3-h period of hindleg ischemia and a subsequent 1.5-h period of resumed flow. The K⁺ e.c. was measured on the skeletal muscle surface, using a potassium selective electrode. The MP was recorded with conventional microelectrodes. Small biopsies were taken and analysed for content of ATP and lactate.The lactate content increased fourfold during the ischemic period, but returned to normal values after reflow. No significant changes occurred in the ATP content during the period of the experiment. K⁺ e.c. increased from 3.6±0.2 to 16.1±0.7 mmol/l, and the MP decreased from –90.2±0.6 to –58.5±1.9 mV during the 3-h period of ischemia. After release of the tourniquet there was an initial rapid decrease in K⁺ e.c. to 7.8±0.9 mmol/l during the first 15 min of resumed flow, followed by a slower decrease. The MP increased linearly during the first hour of resumed flow and both variables returned to near normal values 1.5 h after releasing the tourniquet.This work was supported by grants from the Swedish Medical Research Council (project No. 17X-05416), from the Medical Faculty, University of Göteborg and from the Göteborg Medical SocietyPresented in part at the meeting of the Scandinavian Physiological Society, Tammerfors, Finland, May 1981     

The effects of collagen concentration and crosslink density on the biological,structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering

In this study, we examined the effects of varying collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. Three different collagen contents (0.25%, 0.5% and 1% collagen) and two different dehydrothermal (DHT) crosslinking processes [1] 105 ^°C for 24 h and [2] 150 ^°C for 48 h were investigated. These scaffolds were assessed for (1) pore size, (2) permeability (3) compressive strength and (4) cell viability. The largest pore size, permeability rate, compressive modulus, cell number and cell metabolic activity was all found to occur on the 1% collagen scaffold due to its increased collagen composition and the DHT treatment at 150 ^°C was found to significantly improve the mechanical properties and not to affect cellular number or metabolic activity. These results indicate that doubling the collagen content to 1% and dehydrothermally crosslinking the scaffold at 150 ^°C for 48 h has enhanced mechanical and biological properties of the scaffold making it highly attractive for use in bone tissue engineering.     

Effect of the protease inhibitor contrycal on granulation tissue metabolism

Investigation of the effect of contrycal on granulation tissue metabolism in rats showed that the inhibitor stimulated incorporation of glycine-C¹⁴ into collagen proteins and modified the lactate dehydrogenase isozyme spectrum; meanwhile maturation of the granulation tissue was delayed despite its well-marked growth.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Kraevskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 6, pp. 677–679, June, 1976.     

The association of photo-induced collagen degeneration and the development of telangiectasias in rosacea

Rosacea is a chronic, often progressive disorder characterized by facial erythema, telangiectasias, papules, pustules, and/or rhinophyma. In this study, we investigated the tissue structure in rosacea compared to controls. We performed a case–control study between five patients with mild-to-moderate erythematotelangiectatic rosacea (ETR) and five matched controls. Facial biopsy samples from rosacea patients and controls were stained with picrosirius red for collagen and CD31 for microvessel identification. Mean collagen content was significantly greater in control samples (19.603% ±8.821%) compared to rosacea samples (16.812% ± 7.787%, p = 0.030). In contrast, mean microvessel density was significantly higher in rosacea patients (4.775 E-5 ± 1.493 E-5 µm⁻³) compared to controls (2.559 E-5 ± 8.732 E-6 µm⁻³, p = 0.004). Mean microvessel lumen area was also significantly higher in rosacea patients (491.710 ± 610.188 µm²) compared to controls (347.879 ± 539.624 µm², p = 0.003). We identified a correlation between decreased collagen content and increased microvessel size and density in rosacea patients that was not observed in controls. These structural changes to the dermal matrix may contribute to the characteristic vessel growth and dilation in rosacea.     

Semi-interpenetrating networks of hyaluronic acid in degradable PEG hydrogels for cartilage tissue engineering

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9 × 10⁴ and 2 × 10⁶ Da) and concentration (0.5 and 5 mg g⁻¹) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4 weeks. A steady release of HA was observed over the course of the study with 90% released by 4 weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8 days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4 weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.     

Changes in somatotrophic and lactotrophic functions of the adenohypophysis in rats with acute alloxan diabetes

By electrophoresis followed by colorimetric study of the stained gels an increased content of growth hormone and prolactin was found in the pituitary of rats during the development of alloxan diabetes. The STH and prolactin levels 4–5 days after injection of alloxan were higher by 45–58 and 38–43% respectively than in intact animals. Experiments on primary cell cultures using [¹⁴C]-L-leucine as labeled precursor revealed increased secretory activity of the somatotrophs and lactotrophs of rats with alloxan diabetes.Laboratory of Biological Standardization of Hormones, Institute of Experimental Endocrinology and Hormone Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Yudaev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 11, pp. 536–538, November, 1979.     

Ca2+-calcineurin signaling is involved in norepinephrine-induced cardiac fibroblasts activation

Cardiac fibroblasts (CFs) activation plays a vital role in cardiac fibrosis. There are some studies demonstrate that norepinephrine (NE, an α₁-adrenoceptor agonist) induced CFs proliferation. But whether Ca²⁺-calcineurin, a signaling concerned with growth and differentiation in various cell types, is participated in NE-induced CFs activation is unclear. In present study, we determined NE-induced CFs proliferation and differentiation, synthesis of collagen, and calcineurin (CaN) activity, and the effects of phentolamine (Phen, an α₁-adrenoceptor antagonist), verapamil (Ver, a calcium channel blocker) and cyclosporine A (CsA, an inhibitor of CaN) on NE-induced CFs activation. The results showed that NE induced CFs proliferation and differentiation, increased α-SMA protein expression, increased collagen I, collagen III and fibronectin production, promoted ECM expression, activated CaN and increased CaN protein expression, which were inhibited by Phen, Ver and CsA. In vivo, more collagen deposition could be observed and total collagen volume fraction (CVF) was significantly increased in NE group. Phen, Ver and CsA decreased NE-induced collagen deposition, reduced cardiac fibrosis. Thus, our results demonstrate that Ca²⁺/CaN is involved in NE-induced CFs proliferation and collagen synthesis.     

Effect of cholinergic agents on survival of mice after acute microwave irradiation

Administration of cholinomimetics (pilocarpine, neostigmine, cytisine) immediately after microwave irradiation (62±5 mW/cm², =12.5 cm, 15–16 min) increased the survival rate of the irradiated mice by 1.2–1.3 times; after administration of cholinolytics fewer of the irradiated mice survived.Vladivostok Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 285–286, March, 1976.     

Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy

Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (～200 mg ml^–1). Young’s modulus (E) measurements from acellular collagen gels (range 0.5–12 kPa) exhibited a power-law concentration dependence (range 3–9 mg ml^–1) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5–27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ～5–200 mg ml^–1). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R² = 0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R² = 0.83, exponents of ?0.7 and ?1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.     

Increased Proteolysis of Collagen in an <Emphasis Type="Italic">In Vitro</Emphasis> Tensile Overload Tendon Model

Presently, there is a lack of fundamental understanding regarding changes in collagen’s molecular state due to mechanical damage. The bovine tail tendon (BTT; steers approximately 30 months) was characterized and used as an in vitro model for investigating the effect of tensile mechanical overload on collagen susceptibility to proteolysis by acetyltrypsin and α-chymotrypsin. Two strain rates with a 1000-fold difference (0.01 and 10 s⁻¹) were used, since molecular mechanisms that determine mechanical behavior were presumed to be strain rate dependent. First, it was determined that the BTTs were normal but immature tendons. Water content and collagen content (approx. 60% of wet weight and 80% of dry weight, respectively) and mechanical properties were all within the expected range. The collagen crosslinking was dominated by the intermediate crosslink hydroxylysinonorleucine. Second, tensile overload damage significantly enhanced proteolysis by acetyltrypsin and, to a lesser degree, by α-chymotrypsin. Interestingly, proteolysis by acetyltrypsin was greatest for specimens ruptured at 0.01 s⁻¹ and seemed to occur throughout the specimen. Understanding damage is important for insight into injuries (as in sports and trauma) and for better understanding of collagen fiber stability, durability, and damage mechanisms, aiding in the development of durable tissue-based products for mechanically demanding surgical applications.     

High density type I collagen gels for tissue engineering of whole menisci

This study investigates the potential of high density type I collagen gels as an injectable scaffold for tissue engineering of whole menisci, and compares these results with previous strategies using alginate as an injectable scaffold. Bovine meniscal fibrochondrocytes were mixed with collagen and injected into micro-computed tomography-based molds to create 10 and 20 mg ml^?1 menisci that were cultured for up to 4 weeks and compared with cultured alginate menisci. Contraction, histological, confocal microscopy, biochemical and mechanical analysis were performed to determine tissue development. After 4 weeks culture, collagen menisci had preserved their shape and significantly improved their biochemical and mechanical properties. Both 10 and 20 mg ml^?1 menisci maintained their DNA content while significantly improving the glycosaminoglycan and collagen content, at values significantly higher than the alginate controls. Collagen menisci matched the alginate control in terms of the equilibrium modulus, and developed a 3- to 6-fold higher tensile modulus than alginate by 4 weeks. Further fibrochondrocytes were able to reorganize the collagen gels into a more fibrous appearance similar to native menisci.     

Estrogen Induces Different Responses in Dermal and Lung Fibroblasts

Fibroblast-like cells derived from dorsal skin and lung of AKR strain mouse embryos were cultured with estrogen for 4 days. The labeled hydroxyproline content was measured as newly synthesized collagenous protein in the cell layer and media. Although collagen synthesis by both cell lines was increased in both fractions under the influence of β-estradiol-3-benzoate, at physiological concentrations of 10^-3-10^-1μ g/ml, the rate of increase differed. Fibroblasts derived from skin showed increased collagen synthesis of approximately 76%, while those from lung showed an increase of approximately 25%. Total protein synthesis by both cell lines also increased. In lung fibroblast cultures the synthesis of total protein was increased more than the synthesis of collagen; on the other hand in skin fibroblast cultures the synthesis of collagen was increased more than the synthesis of total protein. DNA synthesis by both cell lines was not affected by estrogen at the concentrations used. These findings suggest that fibroblasts develop in a different manner in each organ.     

Antithrombogenic effect of urokinase bound to collagen substrate with bifunctional antibodies

In vitro experiments simulating collagen-induced thrombogenesis in arteries damaged during angioplasty demonstrate a marked inhibitory effect of surface-bound urokinase. In contrast to free urokinase, bound enzyme is more effective in preventing platelet aggregation on the collagen surface. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 5, pp. 504–507, May, 1994 Presented by V. N. Smirnov, Member of the Russian Academy of Medical Sciences     

Targeted Deletion of Collagen V in Tendons and Ligaments Results in a Classic Ehlers-Danlos Syndrome Joint Phenotype

Collagen V mutations underlie classic Ehlers-Danlos syndrome, and joint hypermobility is an important clinical manifestation. We define the function of collagen V in tendons and ligaments, as well as the role of alterations in collagen V expression in the pathobiology in classic Ehlers-Danlos syndrome. A conditional Col5a1^flox/flox mouse model was bred with Scleraxis-Cre mice to create a targeted tendon and ligament Col5a1-null mouse model, Col5a1^Δten/Δten. Targeting was specific, resulting in collagen V–null tendons and ligaments. Col5a1^Δten/Δten mice demonstrated decreased body size, grip weakness, abnormal gait, joint laxity, and early-onset osteoarthritis. These gross changes were associated with abnormal fiber organization, as well as altered collagen fibril structure with increased fibril diameters and decreased fibril number that was more severe in a major joint stabilizing ligament, the anterior cruciate ligament (ACL), than in the flexor digitorum longus tendon. The ACL also had a higher collagen V content than did the flexor digitorum longus tendon. The collagen V–null ACL and flexor digitorum longus tendon both had significant alterations in mechanical properties, with ACL exhibiting more severe changes. The data demonstrate critical differential regulatory roles for collagen V in tendon and ligament structure and function and suggest that collagen V regulatory dysfunction is associated with an abnormal joint phenotype, similar to the hypermobility phenotype in classic Ehlers-Danlos syndrome.Ehlers-Danlos syndrome (EDS) is a hereditary connective tissue disorder characterized by joint hypermobility, skin extensibility, and connective tissue fragility.^1–5 The combined prevalence of all types of EDS is approximately 1 in 5000. More than half of these cases are characterized by joint hypermobility,⁶ and hypermobility is one of the major diagnostic criteria for the classic subtype of EDS. Joint hypermobility can cause chronic joint and limb pain, recurring joint dislocation, and sports injuries with potential degenerative complications, including muscle weakness, precocious osteoarthritis, spondylosis, and lower bone mass.^6–9In more than 90% of patients with classic EDS, collagen V mutations have been identified,^3,10 and approximately half are null-allele mutations resulting in COL5A1 haploinsufficiency.^10–12 Besides the null-allele mutations, mutations scattered throughout COL5A1 and COL5A2 genes and some mRNA splicing mutations in COL5A1 also have been identified.¹⁰ This finding has led to the proposal that classic EDS is a collagen V disease resulting from altered collagen V expression.¹⁰Collagen V is a quantitatively minor, regulatory component in collagen I–rich connective tissues, including dermis, tendons and ligaments, bones, blood vessels, and cornea.¹³ Collagen V content relative to collagen I varies from a high of 10% to 20% in cornea to 2% to 5% of the total fibril-forming collagens in most other tissues.^14,15 Collagen V regulates collagen fibrillogenesis by nucleating fibril assembly in in vitro self-assembly assays, cell culture analyses, and mouse models.^14,16–19 The data support a model whereby the collagen V:I ratio in different tissues determines the initial diameter and number of fibrils assembled. By varying the number of collagen V nucleation sites for a given collagen I concentration, the fibroblast can regulate fibril number and diameter in a tissue-specific manner, ie, more sites result in increased fibrils assembled with smaller diameters.^13,17Several mouse models have been established to elucidate the function of collagen V tissue-specific fibril assembly. A traditional homozygous deletion in the Col5a1 gene is embryonic lethal because of a virtual lack of fibril formation at the beginning of organogenesis in the early embryo, although the Col5a1^−/− mice synthesize and secrete collagen I at a level comparable with that of wild-type controls.¹⁸ This model demonstrates that collagen V is essential for the assembly of collagen I–containing fibrils in the low-collagen-concentration environment of the embryo and is consistent with a critical role in nucleation of fibril assembly. The heterozygous Col5a1^+/− mice demonstrate haploinsufficiency, with approximately 50% of wild-type collagen V expression. Col5a1^+/− mice are excellent models of classic EDS.²⁰ There were two subpopulations of fibrils in the mutant dermis. One had increased diameters and normal fibril structure, and these were immunoreactive for collagen V; the second group had very large diameters with aberrant fibril structure and were negative for collagen V reactivity. This suggests relatively normal assembly when nucleated with collagen, but the expression level of collagen V was insufficient to nucleate all available collagen I and, therefore, dysfunctional fibril assembly in the high-collagen-content environment. This abnormal fibril growth recapitulated that seen clinically in the dermis of EDS patients. In addition, the Col5a1^+/− flexor digitorum longus tendon (FDL) also demonstrates decreased cross-sectional area and stiffness compared with that in the wild-type controls,²¹ consistent with the joint hypermobility and dislocations seen in EDS patients.To overcome the embryonic lethal phenotype and permit analyses of the roles of collagen V in the development and maturation of a tissue-specific extracellular matrix, our group created a conditional collagen V–null mouse model by using a Cre/loxP approach.²² When targeted to the corneal stroma, a severe dysfunctional regulation of fibrillogenesis and corneal opacity was observed in the targeted collagen V–null mice. Unlike cornea, the tendon has a low collagen V content. The mature tendon contains uniaxial fibrils with a very heterogeneous population of fibril diameters, and the mechanical properties of the tendon depend on the increases in fibril diameter seen with development.²³Our aim was to explore specific regulatory roles for collagen V in tendons and ligaments, as well as its roles in the pathophysiology associated with joint hypermobility in classic EDS. Our conditional Col5a1^flox/flox mouse model was targeted to tendons and ligaments using Cre driven by a Scleraxis (Scx) promoter (Scx-Cre) to produce the Col5a1^Δten/Δten mouse model. Scx-Cre targets the deletion to tendons and ligaments. The data demonstrate that the absence of collagen V results in a disruption in tendon and ligament structure and function. In addition, there were consistent differences between the FDL tendon and the anterior cruciate ligament (ACL), indicating tissue-specific regulatory properties. The absence of collagen V also resulted in alterations in the joint consistent with the hypermobility seen in classic EDS.     

A collagen-polypyrrole hybrid: influence of 3-butanesulfonate substitution

Hybrid biomaterials derived from a water-soluble derivative of polypyrrole, poly(sodium 4-(3-pyrrolyl)butanesulfonate), and soluble collagen have been synthesized by chemical polymerization of the 4-(3-pyrrolyl)butanesulfonate monomer in a collagen solution. Interaction between the two polymers was evident in the formation of a precipitated black polymer-collagen gel obtained for a monomer concentration of 0,01 mol/L. The collagen content in the gel was determined to be around 38 wt.-%. When the monomer concentration was increased five-fold, the product was a dispersed precipitate that was polymer rich with a corresponding decrease in collagen (11 wt.-%) content. Fourier transform infrared spectroscopy and thermal analysis of the hybrids provide signs of interaction between the sulfonic acid group on the substituted pyrrole and side groups of amino acids in collagen. The electrical conductivities of these hybrid biomaterials were found to be in the 10^?2 S. cm ^?1 range.     

Rat brain gangliosides during hypoxia

The content and composition of rat brain gangliosides in various forms of hypoxia is studied. The content of lipid-bound sialic acid is found to be considerably elevated in the earliest stages of hypoxia combined with hypercapnia and cooling. The content of gangliosides remains elevated over the hypoxia period and returns to control values 48 hours after its discontinuation. Repeated hypoxia results in the same changes in rat brain gangliosides. Hemic sodium nitrite-induced hypoxia also leads to a reliable elevation of these lipids. Hypoxia also causes changes in the content of individual ganglioside fractions: a decreased content of mono- and an increased content of polysialogangliosides. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 7 pp. 19–21, July, 1994 Presented by A. N. Klimov, Member of the Russian Academy of Medical Sciences     

Effect of benactyzine and arecoline on Na,K-ATPase activity and content of Na+ and K+ ions in the rat brain

Activity of Na,K-ATPase and the content of Na⁺ and K⁺ ions in the rat brain were studied after administration of arecoline and benactyzine. Both drugs increased Na,K-ATPase activity, possibly on account of changes in the redistribution of Na⁺ and K⁺ ions in the nerve cell. Arecoline was shown to cause changes in the distribution of electrolytes characteristic of depolarization, and benactyzine changes characteristic of hyperpolarization of the nerve cell membrane.(Presented by Academician of the Academy of Medical Sciences of the USSR S. N. Golikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 180–183, February, 1977.     

The vulnerability of the human taenia coli to alterations in total collagen within the colon of the elderly

The structure of the colonic wall relies on collagen, distributed within the submucosa and the muscularis externa. A recent analysis of total collagen in human ascending colon (AC) suggests that the muscularis externa is more susceptible to age-related increases in collagen among the elderly. However, it is not clear if this change also occurs in the descending colon (DC) or if the circular and longitudinal muscle layers are similarly affected in either region of colon. The aim of this study is to determine the total collagen content in the DC and its distribution between the circular muscle (CM) and taenia coli (TC) of the AC and DC of adults and compare the same with tissue from the elderly. Masson’s trichrome and Picrosirius red were used to assess total collagen content in the AC and DC; aged 22 – 91 years. Macroscopically normal AC from 22 patients (adults: 22–60 years; 6 male, 6 female; elderly: 70 – 91 years; 6 male, 4 female) and DC from 23 patients (adults: 23–63 years; 6 male, 7 female; elderly: 66 – 88 years; 6 male, 4 female) were obtained following surgery for non-obstructed bowel cancer. The total hydroxyproline content in DC samples was also evaluated. In the DC, tinctorial staining demonstrated an increased occurrence of total collagen fibres in the submucosa of the elderly (159.8 ± 9.6 in elderly vs. 126.9 ± 6.1 in the adults; p 0.05) and in the muscularis externa (respectively 37.4 ± 4.1 vs. 18.8 ± 2.4; p 0.01). In the adult AC and DC, there were no statistically significant differences in the amount of collagen within the CM and TC. In the elderly, the total collagen fibres within the TC was greater in the AC (mean grey intensity: 63.4 ± 3.9% in the elderly vs. 36.6 ± 1.6% in adults; p 0.05) and DC (mean grey intensity: respectively, 59.82 ± 2.4 vs. 40.2 ± 0.9%; p 0.05). In both AC and DC of the elderly samples, several thickened collagen fibrils were microscopically identified within the TC infiltrating to the myenteric plexus. In the TC of the elderly AC, the total collagen fibres were increased by approximately 4% compared to that of the DC. The total collagen concentration in the elderly DC assessed by hydroxyproline assay was increased by approximately 15% compared to the adult. Sex related differences were not found when data combined. We concluded that the total collagen content in the muscularis externa particularly of the TC of human colon increases with age. The subtle change in collagen distribution with age between AC and DC may differentially affect the tensile strength of the colon.     

Effect of histamine on electrical and mechanical activity of the myocardial fibers of the guinea pig heart after blocking of the fast sodium channels

The effect of histamine on electrical (recorded intracellularly) and mechanical (isometric contraction) activity of the auricular myocardial fibers of the guinea pig atrium was investigated after preliminary blocking of the fast sodium channels by prolonged depolarization with K⁺ ions (20 mM) or with tetrodotoxin (3.2×10^–7–2.0×10^–6 g/ml). Slowly increasing gradual responses to short stimuli were found to appear in the depolarized fibers. Histamine (10 g/ml) increased the amplitude and duration of these responses, and at the same time the amplitude of the contractions of the preparation also increased. Compound D-600 (1 mg/liter), which specifically blocks calcium channels, inhibited the slow responses and mechanical activity of the preparation. After administration of tetrodotoxin alone orin combination with KCl, histamine also had a potentiating action of its own. It is concluded that histamine activates the slow sodium-calcium channels of the myocardial excitable membrane.Department of Experimental and Clinical Physiology, A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. A. Vishnevskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 78, No. 11, pp. 23–26, November, 1974.