Angiogenesis and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle

Prolactin (PRL) is a pituitary hormone involved in a broad spectrum of physiological processes, including lactation, development, and immune function. To further investigate the in vivo roles of PRL, rat PRL cDNA, fused to the cytomegalovirus promoter, was introduced into mouse muscle by direct injection. Prolactin mRNA and protein were detected in the muscle following injection. As a result the number of white blood cells (WBC) increased. When injection was combined with adrenalectomy there was an even greater increase. The augmentation of WBCs persisted for at least 20 days after injection of the rPRL plasmid either on its own and after injection combined with adrenalectomy. The increase in WBCs was accompanied in both cases by an increase in blood cell DNA content. We also observed an increase in heart volume, particularly of the left ventricle. Evidence of marked angiogenesis was found in the testis of rPRL- injected mice. New blood vessels were first found at 8 weeks of age and fully developed blood vessels with complex branching patterns were found after 11 weeks. When PRL fused with EGFP was introduced into mice by intramuscular injection, the EGFP localized to areas of the testis that corresponded to the sites of new blood vessel formation. PRL inhibited this binding. Taken together, our data reveal that intramuscularly expressed PRL augments WBC numbers and induces formation of new blood vessels in the testis, suggesting important roles for PRL in hematopoiesis and angiogenesis. They also indicate that direct intramuscular injection of naked DNA can be used effectively to study the function of secreted proteins, including endocrine signaling molecules.     

Immunohistochemical evidence for the existence of rat cytosolic sialidase in rat skeletal muscles

 Histochemical evidence is required to demonstrate the presence of biochemically defined cytosolic sialidase. To meet this requirement, we examined the immunohistochemical localization of the enzyme in rat skeletal muscles. Sections of chemically fixed tissues were incubated with a polyclonal antibody raised against a synthetic peptide which corresponded to a part of the enzyme protein. After incubation with the primary antibody, cryosections for fluorescence microscopy and resin sections for electron microscopy were incubated with a fluorochrome- and colloidal gold-labeled secondary antibody, respectively. Immunofluorescence was diffusely distributed in the muscle fibers and was also found in the perimysium and blood vessels. Many immunogold particles were scattered over the sarcoplasm, myofibrils, nucleoplasm, and matrix of mitochondria. The immunogold particles were also found in the equivalent compartments of axons, Schwann cells, and cells of endomysium and blood vessels. The specificity of the primary antibody was elucidated by immunoblotting and an immunoprecipitation test. These findings clearly indicate that this type of sialidase is essentially located in the cytosolic compartment. Consequently, the name, cytosolic sialidase, will be appropriate for this enzyme. Additionally it is indicated that this enzyme is also present in cells other than skeletal muscle fibers. Accepted: 29 January 1997     

Split flexor carpi radialis muscle

A detailed anatomic and intramuscular neural staining study in 22 human and 5 monkey upper limbs revealed that the flexor carpi radialis can be raised on its proximal neurovascular pedicle and that the muscle can be split along its tendon into two independently functioning neuromuscular compartments, each with its own nerve and blood supply. A study of the muscle architecture in the human specimens found the radial compartment to have significantly longer fiber length and a larger physiologic cross-sectional area than the ulnar compartment. Independence of function of each compartment was demonstrated in electrical stimulation studies in six monkeys (Macaca fascicularis), but no significant difference was noted in the peak isometric load between the two compartments (p = 0.68) in the monkey. The extra functioning muscle units become important in local transfers for restoring function in multiple nerve palsies as in Hansen's disease, severe traumatic loss of muscle in crush injuries and compartment syndromes, and after wide resection in infective and neoplastic conditions in the forearm and hand.     

Morphology and ultrastructure of the somatic cells in Astacus leptodactylus ovary

We defined the somatic environment in which female germinal cells develop, and performed ultrastructural analyses of various somatic cell types, with particular reference to muscle cells and follicle cells, that reside within the ovary at different stages of oogenesis. Our findings show that ovarian wall of the crayfish is composed of long muscle cells, blood cells, blood vessels and hemal sinuses. The follicle and germinal cells lie within a common compartment of ovarian follicles that is defined by a continuous basal matrix. The follicle cells form branching cords and migrate to surround the developing oocytes. A thick basal matrix separates the ovarian interstitium from ovarian follicles compartment. Transmission electron microscopy shows that inner layer of basal matrix invaginates deeply into the ovarian compartment. Our results suggest that before being surrounded by follicle cells to form follicles, oogonia and early previtellogenic oocytes reside within a niche surrounded by a basal matrix that separates them from ovarian interstitium. We found coated pits and coated vesicles in the cortical cytoplasm of previtellogenic and vitellogenic oocytes, suggesting the receptor mediated endocytosis for transfer of material from the outside of the oocytes, via follicle cells. The interstitial compartment between the inner muscular layer of the ovarian wall and the basal matrix of the ovarian follicle compartment contains muscle cells, hemal sinuses, blood vessels and blood cells. Granular hemocytes, within and outside the vessels, were the most abundant cell population in the ovarian interstitium of crayfish after spawning and in the immature ovary. J. Morphol. 277:118–127, 2016. © 2015 Wiley Periodicals, Inc.     

Hypertrophic smooth muscle

Summary In adult guinea-pigs, oral to a partial obstruction to the flow of ingesta in the ileum there is a marked increase in the diameter of the intestine and a hypertrophy of the muscle coat. The features of the intramuscular blood vessels and of the extracellular materials were studied by electron microscopy. There is a small increase in the amount of intercellular space measured morphometrically. The basal lamina surrounding the hypertrophic muscle cells is more prominent than in controls. In the intercellular space between muscle cells, in addition to collagen fibrils, there is abundant amorphous material of medium electron density and streak-like, electron-dense material often similar to thickened basal laminae. The total amount of stroma (intercellular materials) present in a unit length of intestine is greatly increased in hypertrophy; a role of the muscle cells in the production of new collagen and other extracellular elements is suggested by the present observations. Many new intramuscular blood vessels (mainly capillaries, some of which are fenestrated) are formed during hypertrophy of the intestinal wall, so that the circular muscle layer remains as well vascularized in the hypertrophic intestine as in the controls. Blood vessels are not formed within the longitudinal muscle layer.     

Intramuscular pressure and muscle blood flow in supraspinatus

Intramuscular pressure and muscle blood flow was measured in the supraspinatus muscle in 6 healthy subjects. The recordings were performed at rest, during isometric exercise, during an isometric muscle contraction of 5.6 kPa (42 mm Hg) and 10.4 kPa (78 mm Hg) and at rest after the contraction. Intramuscular pressure was measured by the microcapillary infusion technique, and muscle blood flow by the Xenon-133 washout technique. Intramuscular pressure was 38.2 kPa (SD 12.0) (287 mm Hg) during maximal voluntary contraction. A muscle contraction pressure of 5.6 kPa (42 mm Hg), which is 16% of maximal voluntary contraction, reduces local muscle blood flow significantly. It is concluded that the high intramuscular pressures found in supraspinatus during work with the arms elevated impedes local muscle blood flow.     

Muscle soreness and intramuscular fluid pressure: comparison between eccentric and concentric load

This study investigates the dynamic and resting intramuscular pressures associated with eccentric and concentric exercise of muscles in a low-compliance compartment. The left and righ leg anterior compartments of eight healthy males (ages 22-32 yr) were exercised by either concentric or eccentric contractions of the same load (400 submaximal contractions at constant rate, 20/min for 20 min at a load corresponding to 15% of individual maximal dorsiflexion torque). Tissue fluid pressures were measured with the slit-catheter technique before, during, and after the exercise. Average peak intramuscular pressure generated during eccentric exercise (236 mmHg) was significantly greater than during concentric exercise (157 mmHg, P less than 0.001). Peak isometric contraction pressure in the eccentrically exercised compartment was significantly higher both within 20 min postexercise and on the second postexercise day (P less than 0.001). Resting pressure 2 days postexercise was significantly higher on the eccentrically exercised side (10.5 mmHg) compared with the concentrically exercised (4.4 mmHg, P less than 0.05). The ability to sustain tension during postexercise isometric contractions was impaired on the "eccentric" side. Soreness was exclusively experienced in the eccentrically exercised muscles. We conclude that eccentric exercise causes significant intramuscular pressure elevation in the anterior compartment, not seen following concentric exercise, and that this may be one of the factors associated with development of delayed muscle soreness in a tight compartment.     

Early postnatal growth of skeletal muscle blood vessels of the rat

The development of blood vessels during the first three postnatal weeks was studied in the ventral stripe of the spinotrapezius muscle of the rat by use of India ink-gelatine injections, and electron microscopy. The number of terminal arterioles and collecting venules remained unchanged postnatally in the observed area. A remarkable proximodistal gradient of vascular development was apparent: while the basic structure of the hilar vessels remained unchanged in the time studied, the intramuscular arteries and veins matured gradually. More peripherally, gradual maturation of terminal and precapillary arterioles was observed. The capillary endothelium and the pericytes showed immature features, and remained unchanged during the time studied. An intense rebuilding activity was found in the endothelial cells of the growing venules, expressed by various forms of gaps, covered by an intact basal lamina and pericytes. Numerous mast cells and macrophages were found along all vessels. Intramuscular lymphatics were not present prior to the first postnatal week.     

The blood supply of muscle spindles in some mammals and the pigeon.

The blood supply of muscle spindles was studied in serial cross sections in macaque, cat, rabbit, guinea pig, mouse and pigeon muscles which had been incubated in a medium containing 3,3' diaminobenzidine. Lumina of blood vessels were recognized by the reaction product that was localized within erythrocytes. The outer capsule was well vascularized, but few or no capillaries were seen in the periaxial space. The inner spindle capsule, which closely invests the axial bundle, was rarely contacted by periaxial capillaries at the equator and juxtequator. Capillaries occurred more frequently adjacent to intrafusal fibers at the polar region and beyond the end of the outer capsule. Shorter diffusion distances and, usually, higher capillary densities were found at the polar region than at the spindle midsection. This suggests that transcapillary exchange at the polar segment is nearer to conditions prevalent in extrafusal muscle than elsewhere in the spindle, provided the inner and outer capsules are not less permeable at the poles than at the midsection. Differences in blood supply among mammalian species appear to be related to receptor size.     

The organogenesis of murine striated muscle: a cytoarchitectural study

The ultrastructure and the three-dimensional cytoarchitecture of the developing murine extensor digitorum longus muscle has been studied in spaced, serial, transverse and longitudinal ultrathin sections of the muscles of 12-, 14-, 16-, and 18-day in utero, newborn, and 5-day-old 129 ReJ mice. Despite the fact that in vivo myogenesis is asynchronous (i.e., during most of the fetal period, multiple stages of myogenesis can be seen in a single developing muscle mass), a distinct temporal pattern of development can be seen across the entire width and length of the developing muscle. At 12 days in utero, the developing extensor digitorum longus muscle consists of primary myotubes surrounded by a pleomorphic population of mononucleated cells devoid of myofilaments. At this stage, blood vessels and nerves are found peripheral to but not within the developing muscle mass. A delay of 2 days occurs between the time of formation of the primary and secondary myotubes. Clusters (consisting of one primary myotube and secondary myotubes), axon bundles, capillaries, and primitive motor endplates are found in the muscle by 16 days in utero. Evidence is presented consistent with the hypothesis that cluster formation and cluster dispersal occur simultaneously in the developing muscle, beginning as early as 16-days in utero. By 18 days in utero, many of the primary myotubes of the cluster and the independent myotubes (i.e., single myotubes enclosed in their own basal lamina) have begun to acquire the polygonal shape, fascicular arrangement, and ultrastructure characteristic of more mature myofibers. At birth, clusters are infrequently encountered, and intramuscular axons have begun to undergo myelination. At this time, the only undifferentiated, mononucleated cells present in the muscle are myosatellite cells. The first week postnatal was characterized by further maturation of the myofibers.     

Tissue changes in the rabbit periodontal ligament during orthodontic tooth movement

In this (semi) quantitative animal study the reaction of the periodontal ligament (PDL) to experimental tooth movement is described. To this end, rabbit first incisors were moved sideways with helical torsion springs for periods varying from 3-24 hours. The initial force of the springs was 50 gf. The histomorphology of the PDL was studied in 5 microns thick plastic sections. Comparison with control animals and animals wearing passive springs showed that tooth movement leads to an increased trauma in the PDL within only a few hours. This trauma is characterized by hyalinization, tears and ruptures in the fibres and blood vessels, and by the presence of extravascular erythrocytes and pyknosis. Tissue damage significantly increased with time. After 24 hours of tooth movement, the PDL fibers are compressed or stretched in 68% of the sections and the blood vessels in the PDL are compressed or stretched in 62% of the sections. Even in the controls, more than 15% of the sections displayed slightly stretched or compressed fibers, and about 10% showed slightly compressed or stretched blood vessels. This indicates that some damage is regularly present in a normally functioning PDL. Increases in the percentage of sections with blood vessel compression are found in all groups wearing passive springs, especially after 6 hours. A high concordancy in compression and tension patterns of blood vessels and fibers is present in 83% of the sections. Pyknotic cells are practically confined to areas with compressed PDL fibers in rabbits wearing active springs. Extravascular erythrocytes were found in sections with all types of fiber patterns. A significant majority of extravascular erythrocytes, however, was found in areas with compressed fibers.     

Formation of lamellar cross bridges in the annulus fibrosus of the intervertebral disc is a consequence of vascular regression

Cross bridges are radial structures within the highly organized lamellar structure of the annulus fibrosus of the intervertebral disc that connect two or more non-consecutive lamellae. Their origin and function are unknown. During fetal development, blood vessels penetrate deep within the AF and recede during postnatal growth. We hypothesized that cross bridges are the pathways left by these receding blood vessels.Initially, the presence of cross bridges was confirmed in cadaveric human discs aged 25 and 53 years. Next, L1-L2 intervertebral discs (n = 4) from sheep ranging in age from 75 days fetal gestation to adult were processed for paraffin histology. Mid-sagittal sections were immunostained for endothelial cell marker PECAM-1. The anterior and posterior AF were imaged using differential interference contrast microscopy, and the following parameters were quantified: total number of distinct lamellae, total number of cross bridges, percentage of cross bridges staining positive for PECAM-1, cross bridge penetration depth (% total lamellae), and PECAM-1 positive cross bridge penetration depth.Cross bridges were first observed at 100 days fetal gestation. The overall number peaked in neonates then remained relatively unchanged. The percentage of PECAM-1 positive cross bridges declined progressively from almost 100% at 100 days gestation to less than 10% in adults. Cross bridge penetration depth peaked in neonates then remained unchanged at subsequent ages. Depth of PECAM-1 positive cross bridges decreased progressively after birth. Findings were similar for both the anterior and posterior.The AF lamellar architecture is established early in development. It later becomes disrupted as a consequence of vascularization. Blood vessels then recede, perhaps due to increasing mechanical stresses in the surrounding matrix. In this study we present evidence that the pathways left by receding blood vessels remain as lamellar cross bridges. It is unclear whether the presence of cross bridges in the aging and degenerating intervertebral disc would be advantageous or detrimental, and this question should be addressed by future studies.     

Vascular-nervous relationships in the valves of the heart]

Method of silver nitrate impregnation was used in order to study 50 preparations of not-changed atrioventricular valves of the heart of domestic bulls and 30 preparations of the same valves of adult humans. It has been shown that in heart valves there are certain relationships between striated muscle fibres, blood vessels and nerve elements. The nerve structures of the valves are represented by nerve bundles of different thickness. In their composition there are comparatively thin non-myelinated and thicker myelinated fibres. Towards the free edge of cusps the nerve bundles become thinner and the nerve trunks give off separate thin nerve fibres disposed along the vessels of a capillary type and in some places getting around them. In certain portions of cusps the nerve bundles, some of which have zigzag sinuosity, cross blood vessels in different directions. In man the major mass of blood vessels and nerve elements are disposed near the base of the valve cusps, accompanying the muscle fibre bundles penetrating from the base side. In the bull heart valves an amount of blood vessels and nerve elements is found in considerable portions of the cusps not connected with muscle fibres.     

In vitro contractility of normal and aneurysmal abdominal aorta muscle coat sections in human and animal material

The objective of the study was to demonstrate spontaneous contractile activity of the smooth muscle coat of the aorta in human and animal material. Spontaneous contractility of smooth muscle tissue, or tonus, is essential for the proper function of many internal organs as observed in the many types of muscle cells which make up the internal structures. The spontaneous contractile activity of the muscle tissue in blood vessels is particularly marked in resistance vessels, regulating circulation within organs or tissues. It can also be observed in large blood vessels such as arteries and veins. The contractile activity of muscular tissue isolated from arteries is the result of a number of factors, including endogenous paracrine substances, neurotransmitters released at postganglionic endings (mostly within the sympathetic system), cells capable of spontaneously generation of functional potentials (pacemaking cells) and the vascular endothelium. Pacemaking cells present in the aortic wall are an important factor in the development of the spontaneous contractility of the muscular coat of the aorta. They are capable of generating functional potentials, resulting in the constant tonus of the smooth muscular coat (comprising the aortic wall) due to tonic contraction. In vitro studies were carried out on abdominal aortic sections collected from 30 New Zealand rabbits with a body mass of 3-4 kilograms each and also on aneurysmal abdominal aortic sections collected during elective aneurysm repair procedures in humans (10 abdominal aortic sections). The 1.5 cm-long sections were mounted in chambers of an automated water bath. The sections were oriented in a transverse and longitudal fashion in order to compare contractility. The incubation medium consisted of Krebs-Henseleit buffer. Spontaneous contractile activity was observed during the study, characterized by rhythmic contractions of the muscular layer of the aorta. The contractile tension within the sections was 0.15 mN in the case of rabbit sections and 0.8 mN in the case of human sections. The average duration of a single contraction was 38.3 +/- 15.05 seconds. The average contraction frequency, i.e. the average number of contractions per minute, was 1.61 +/- 0.54 contractions per minute. The spontaneous contraction is modulated by many factors like endogenous paracrine substances, neurotransmitters or vascular endothelium.     

Biochemical and cytochemical characterization of extracellular proteoglycans in the inner circular smooth muscle layer of dog small intestine

Current literature concerning smooth muscle blood vessels has shown versican as the main proteoglycan (PG) component of the matrix. To show whether smooth muscle matrix has the same PG distribution when present in organs, other than the blood vessels, the inner circular smooth muscle layer of the small intestine was obtained by dissection as a highly purified tissue and analyzed by biochemical and cytochemical methods. The smooth muscle layer PGs were extracted from dog small intestine with 4 M guanidine-HCl in the presence of proteinase inhibitors, purified by charge equilibrium, isolated by equilibrium CsCl density gradients, and analyzed in terms of anion exchange, size, and glycosaminoglycan (GAG) distribution. Proteoheparan sulfate itself represented 91.5% of the PGs present in this tissue. The remainder was proteodermatan sulfate. Cytochemical analyses using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinases ABC and heparitinase III showed the arrangement and identification of PGs in basal lamina and intramuscular connective tissues. The PGs in the basal lamina were proteoheparan sulfate, and those associated with collagen fibrils in the endomysium and perimysium were rich in dermatan sulfate. In contrast to the blood vessels, inner circular muscle smooth tissue in intestine has, as the main PG, perlecan.     

Giemsa Poststaining of Sections Stained for Acetyl-Cholinesterase Improves Sensitivity and Contrast

During investigations into the cholinergic innervation of blood vessels in skeletal muscle, it was found that poststaining of sections with Giemsa's stain (Gurr, R66) after incubation to reveal acetylcholinesterase activity as copper ferrocyanide (El-Badawi and Schenk 1967) not only produced nuclear and cytoplasmic counter-staining, but also resulted in intensification of the reaction, resulting in deep blue-black nerve endings (Fig. 1). Areas previously distinguishable only by phase contrast were easily recognizable after Giemsa staining. The method described was originally used on 10 μm cryostat sections, pre- or postfixed in formolcalcium and stained in the reaction mixture described by El-Badawi and Schenk (1967) for 30-60 minutes. After rinsing in distilled water (5 min), sections were stained in Giemsa's stain (3 min), washed well in distilled water, rapidly dehydrated, cleared and mounted.     

Improved protocol for processing stented porcine coronary arteries for immunostaining

Percutaneous coronary intervention has resulted in a paradigm shift in the treatment of coronary artery disease and myocardial infarction. However, neither bare-metal stents nor polymer-coated drug-eluting stents represent ideal therapies at this time due to the undesired in-stent stenosis or delayed thrombosis. Hence there is pressing clinical need for greater understanding of the cellular mechanisms involved. It is hoped that this in turn will provide insight into designing and developing the next generation of stents. Although immunohistochemistry and immunofluorescence are appropriate tools in understanding the molecular histology, performing these techniques on stented blood vessels is technically challenging because of poor permeability of antibodies into the stented blood vessels which are embedded in methacrylate-based resins and inadequate image resolution due to autofluorescence. Hence there is a need to develop techniques which can facilitate immunohistochemistry/immunofluorescence procedures on stented blood vessel cross-sections. In this study we describe an improved protocol for processing stented porcine coronary arteries for immunostaining with smooth muscle cell, endothelial cell, monocyte and macrophage markers. We first identified the optimal conditions for resin embedding of stented artery and cross sectioned the vessels using high speed precision wafering diamond blade. The sections were then ground using two levels of water sandpaper on a Metaserve 2000 grinder to achieve the desired thickness. For immunostaining, we developed a novel deplasticization protocol which favors optimal antibody permeabilization. Our protocol not only provides feasibility of improved immunostaining of stented artery sections but also results in high quality images.     

The neuromuscular compartments of the flexor carpi ulnaris

Learning Objectives: After studying this article, the participant should be able to: 1. Report on the vascular supply and innervation pattern of the flexor carpi ulnaris. 2. Describe the muscle architecture of the flexor carpi ulnaris, including the physiological cross-sectional area and fiber length. 3. State the uses of the flexor carpi ulnaris both for resurfacing defects in the vicinity of the elbow and in local functional tendon transfers. 4. Understand the principles of splitting skeletal muscles based on neurovascular supply to enhance its utilization in reconstructive procedures. The aim of this study was to describe the intramuscular innervation and vascular supply of the human flexor carpi ulnaris, with confirmation of findings by a similar study in the primate. Two distinct intramuscular nerve branches running parallel to each other, on either side of a central tendon, from the proximal quarter of the muscle belly to its insertion were found. The muscle could then be split into a humeral and an ulnar compartment, each with its own primary nerve branch. Perfusion studies confirmed the adequacy of circulation to the two compartments. In the primate flexor carpi ulnaris, electrical stimulation of the respective branches revealed independent contraction of each compartment. This study provides useful information for enabling the local transfer of the muscle as a whole, both for resurfacing in the vicinity of the elbow and for functional tendon transfers. It will also enable the transfer of the muscle as one or two separate compartments (for resurfacing, in tendon transfers for muscle paralysis, congenital defects, and muscle defects resulting from trauma, and after resections for neoplasm and infection).