Freeze-fracture studies of nexuses between smooth muscle cells. Close relationship to sarcoplasmic reticulum.

The freeze-fracture appearance of the nexus was compared in the smooth muscle of guinea pig sphincter pupillac, portal vein, pulmonary artery, taenia coli, uretzr, and vas diferens, mouse vas deferens, chicken gizzard and anterior mesenteric artery, and toad stomach. Nexuses are particularly numerous in the guinea pig sphincter pupillae; they are usually oval and their average area is 0.15 mum2, although some as large as 0.6 mum2 were seen. Small aggregations of particles were observed which would not be recognizable as nexuses in thin section. What constitutes the minimum size of a nexus is discussed. It is estimated that the number of nexuses per cell in this preparation is of the order of tens rather than hundreds. All nexuses examined had 6-9-nm particles in the PF face, with corresponding 3-4-nm pits on the EF face forming a polygonal tending towards a hexagonal lattice. The nexuses are arranged in rows parallel to the main axis of the cell, usually alternating with longitudinal rows of plasmalemmal vesicles. Many nexuses in the guinea pig sphincter pupillae, chicken gizzard, and toad stomach show a close relationship with sarcoplasmic reticulum. The possibility that this may have some role in current flow across this specialized junction is discussed.

Thick filaments in vascular smooth muscle.

Two sets of myofilaments were demonstrated after incubation of strips of rabbit portal-anterior mesenteric vein under moderate stretch in a physiological salt solution. Thick filaments had a mean diameter of 18 nm and reached a maximum length of 1.4 microm with a mean length of 0.61 microm. In transverse sections, 2.5-5 nm particles were resolved as subunits of the thick filaments. Thin filaments had an average diameter of 8.4 nm and generally conformed to the structure believed to represent actin filaments in smooth and striated muscles. In the areas of maximum concentration there were 160-328 thick filaments/microm(2) and the lowest ratio of thin to thick filaments was 12:1. Thick filaments were present in approximately equal numbers in vascular smooth muscle relaxed by theophylline, in Ca(++)-free solution, or contracted by norepinephrine. The same preparatory procedures used with vascular smooth muscle also enabled us to visualize thick filaments in guinea pig and rabbit taenia coli and vas deferens.

Localization of tritiated norepinephrine in vascular sympathetic axons of the rat intestine and mesentery by electron microscope radioautography.

The distribution of infused tritiated norepinephrine (NE-(3)H) in small mesenteric arteries and intestinal arterioles in rats was investigated with electron microscopic radioautography. Silver grains, indicating the presence of the tritium label on the sections, were found lying mainly over axon bundles, but some were present over collagen and smooth muscle cells. Axons with the highest concentrations of silver grains had been sectioned at points where they were naked of Schwann cell sheath, were dilated into varicosities, and contained small granular vesicles. This finding was taken as confirmatory circumstantial evidence that the small granular vesicles were the sites of uptake and storage of NE. The short interval between the start of infusion and the fixation of the tissue appeared to rule out any process other than a direct uptake of NE by the peripheral axons. If axonal sites of uptake of NE-(3)H correspond to sites of release of NE, then the evidence suggests that such sites of release are widespread over the terminal part of the axon and are not confined to those parts of the axon which are in close contact with smooth muscle cells. Since the fixation and embedding procedures will remove NE which is not strongly bound to tissues, the localization of NE-(3)H in the radioautographs does not necessarily correspond to the distribution of all the NE present in vivo.

Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles.

The sarcoplasmic reticulum (SR) was studied in the smooth muscles of rabbit main pulmonary artery, mesenteric vein, aorta, mesenteric artery, taenia coli, guinea pig mesenteric artery, and human uterus, and correlated with contractions of the smooth muscles in Ca-free media. SR volumes were determined in main pulmonary artery (5.1%), aorta (5%), portal-anterior mesenteric vein (2.2%), taenia coli (2%), and mesenteric artery (1.8%): because of tangentially sectioned membranes these estimates are subject to a correction factor of up to +50% of the values measured. Smooth muscles that contained a relatively large volume of SR maintained significant contractile responses to drugs in the virtual absence of extracellular calcium at room temperatures, while smooth muscles that had less SR did not. The unequal maximal contractions of main pulmonary artery elicited by different drugs were also observed in Ca-free, high potassium-depolarizing solution, indicating that they were secondary to some mechanism independent of changes in membrane potential or calcium influx. Longitudinal tubules of SR run between and are fenestrated about groups of surface vesicles separated from each other by intervening dense bodies. Extracellular markers (ferritin and lanthanum) entered the surface vesicles, but not the SR. The peripheral SR formed couplings with the surface membrane: the two membranes were separated by gaps of approximately 10 nm traversed by electron-opaque connections suggestive of a periodicity of approximately 20-25 nm. These couplings are considered to be the probable sites of electromechanical coupling in twitch smooth muscles. Close contacts between the SR and the surface vesicles may have a similar function, or represent sites of calcium extrusion. The presence of both thick and thin myofilaments and of rough SR in smooth muscles supports the dual, contractile and morphogenetic, function of smooth muscle.

Characterization of a unique muscle cell line.

A clonal cell line derived from a mouse neoplasm is described which shares many properties with smooth muscle. The cells have electrically excitable membranes capable of generating overshooting action potentials, and they contract both spontaneously and with electrical stimulation. They respond to the iontophoretic application of acetylcholine with a depolarizing response, and to norepinephrine with a hyperpolarizing response. Electron microscopy reveals that the cells have a morphology similar in many, but not all, respects to that of smooth muscle cells in vivo. The cells secrete soluble collagen-like molecules in addition to several proteins of undefined function. Finally, there is an increase in the specific activities of creatine phosphokinase and myokinase associated with increased cell density and the cessation of cell division.

Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calcium-free solutions.

The contractile response of turtle oviduct smooth muscle to acetylcholine after 30 min of incubation of muscles in Ca-free, 4 mM ethylene (bis) oxyethylenenitrilotetraacetic acid (EGTA) solutions at room temperature was greater than the contractile response after 30 min of incubation in the Ca-free medium at 37 degrees C. Incubation in Ca-free solution at 37 degrees C before stimulation with acetylcholine in Ca-free solutions at room temperature also reduced the contractile response, suggesting that activator calcium was lost from the fibers at a faster rate at higher temperatures. Electron micrographs of turtle oviduct smooth muscle revealed a sarcoplasmic reticulum (SR) occupying approximately 4% of the nucleus- and mitochondria-free cell volume. Incubation of oviduct smooth muscle with ferritin confirmed that the predominantly longitudinally oriented structures described as the SR did not communicate with the extracellular space. The SR formed fenestrations about the surface vesicles, and formed close contacts (couplings) with the surface membrane and surface vesicles in oviduct and vena caval smooth muscle; it is suggested that these are sites of electromechanical coupling. Calculation of the calcium requirements for smooth muscle contraction suggest that the amount of SR observed in the oviduct smooth muscle could supply the activator calcium for the contractions observed in Ca-free solutions. Incubation of oviduct smooth muscle in hypertonic solutions increased the electron opacity of the fibers. A new feature of some of the surface vesicles observed in oviduct, vena caval, and aortic smooth muscle was the presence of approximately 10 nm striations running approximately parallel to the openings of the vesicles to the extracellular space. Thick, thin, and intermediate filaments were observed in turtle oviduct smooth muscle, although the number of thick filaments seen in the present study appeared less than that previously found in mammalian smooth muscles.

Electron microscopy and electron probe analysis of mitochondrial cation accumulation in smooth muscle.

The contractile responses to barium and the ultrastructure and ionic composition of mitochondria were studied in vascular smooth muscle. In normal rabbit portal anterior mesenteric vein (PAMV) and main pulmonary artery (MPA) smooth muscle mitochondria were frequently associated with the surface vesicles. The average distance between the outer mitochondrial and inner surface vesicle membrane was 4-5 nm. Ba contractures of MPA were tonic and of PAMV were phasic. Incubation of MPA and PAMV with Ba resulted in the accumulation of mitochondrial granules, followed in the MPA by massive mitochondrial swelling. Oligomycin and anoxia inhibited the appearance of mitochondrial electron-opaque granules and prevented the Ba-induced mitochondrial swelling in the MPA. Electron probe analysis of mitochondria in PAMV incubated with Ba and containing granules showed characteristic Ba signals over the mitochondria. Electron probe X-ray microanalysis also showed a highly significant (P < 0.001) correlation of P with mitochondrial Ba, in an estimated elemental ratio of approximately 3 Ba/4 P. Mitochondrial granules were still prominent after block staining of the osmium-fixed, Ba-loaded PAMV, but electron probe microanalysis showed no Ba, but only U, emissions. Tissues incubated with strontium had electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum. X-ray microanalysis of mitochondria containing granules showed the presence of characteristic Sr and Ca emissions. The presence of Sr was similarly verified in the sarcoplasmic reticulum. These findings indicate the energy dependent uptake of divalent cations, in association with phosphate, by mitochondria in vascular smooth muscle in situ and the possibility that mitochondria may contribute to the regulation of intracellular divalent cation levels in smooth muscle.

Imaging and Staging of Endometrial Cancer.

Endometrial carcinoma is the most common female pelvic malignancy in the United States. Although endometrial cancer is staged according to the International Federation of Gynecology and Obstetrics surgical system, early and accurate diagnostic assessment of disease status of gynecologic malignancies is important for optimal treatment planning and outcome prediction. Preoperative imaging may assist in evaluation of local extent and detection of distant metastatic disease guiding the optimal course of treatment. Several imaging techniques such as transvaginal ultrasound, computed tomography, and magnetic resonance imaging have been used as tools for preoperative staging of endometrial cancer. Positron emission tomography/computed tomography and more recently, positron emission tomography/magnetic resonance imaging have also been used in the management of endometrial cancer. Cross-sectional imaging, especially MRI, may detect gross myometrial invasion or extension of tumor to the cervical stroma which can alter management. Imaging studies can also evaluate the presence of lymph nodal involvement, and detect local and distant metastatic disease at diagnosis. Additionally, imaging also plays a role in the monitoring of treatment and surveillance of the patients for detection of early recurrent disease. In this article, we will review the imaging and staging of endometrial cancer.

Uterine Carcinosarcomas.

Uterine carcinosarcoma (UCS) is a rare and aggressive variant of endometrial cancer, distinguished by its containment of both epithelial and sarcomatous elements. This article reviews the epidemiology, pathologic classification and staging of UCS, along with the typical findings seen on different imaging modalities. Prognosis and therapies will also be discussed.

Pre-slaughter stress arising from on-farm handling and its interactions with electrical stimulation on tenderness of lambs.

The effect of electrical stimulation of lamb carcasses (n=269) or its absence (n=257) on shear force of m. longissimus thoracis et lumborum (LT) was monitored during ageing in pasture-fed merino lambs (n=526). The lambs were slaughtered on four different days allowing durations of between one to 10 days of recovery from pre-slaughter handling (yarding, weighing and crutching) that affected ultimate pH (pH(u)). The right LT was removed 20-40min post-slaughter, tightly-wrapped in cling film (prevents the muscle cross-section increasing and thus minimising shortening) and rapidly cooled to 15°C to enter rigor mortis and age. At 0, 4, 24 and 72h post-slaughter, pH measurements and samples for shear force measurement were taken. Pre-slaughter handling had a significant negative effect on pH(u) and several days recovery were required for pH(u) to reach values associated with optimal meat quality as reflected by pH(u). Lambs with one and three days recovery (no significant difference between them) had a pH(u)>5.7 in 50% of the muscles and 19.4%>pH(u) 5.8. Whereas, in lambs with 8-10 days recovery (no significant difference between them), only 8% had a pH(u)>5.7 and 3.1%>pH(u) 5.8. Within each slaughter day electrically stimulated lambs were always more tender than non-stimulated lambs. For non-stimulated muscles at 72h, shear force values >40N occurred for 11.2% of the muscles: for electrically stimulated muscles at 72h, shear force values >40N occurred for 1.9% of the muscles. The rates of tenderisation were slower for intermediate pH(u) values resulting in higher shear force values at all ageing durations. With ageing at 72h for intermediate pH(u), non-stimulated muscles (n=38) 17.64% were >40N and for stimulated muscles (n=34), 7.9% were >40N.

Primary vaginal cancer: role of MRI in diagnosis, staging and treatment.

Primary carcinoma of the vagina is rare, accounting for 1-3% of all gynaecological malignancies. MRI has an increasing role in diagnosis, staging, treatment and assessment of complications in gynaecologic malignancy. In this review, we illustrate the utility of MRI in patients with primary vaginal cancer and highlight key aspects of staging, treatment, recurrence and complications.

Social behaviour in sheep relates to behaviour and neurotransmitter responses to nociceptive stimuli.

Sheep in the field display differences in social behaviour. These differences allow a division into three social groups with distinct behavioural occurrences and frequencies. The behavioural and neurotransmission responses of each of these groups to aversive stimuli were compared. Behavioural responses were seen to both forelimb electric shocks and thermal heating of the nose in all groups. These responses changed with stimulus repetition in a group-dependent manner. Microdialysis probe studies of neurotransmitter release in the somatosensory cortex indicated neurotransmitter responses to stimuli in all animals that varied with both animal group and stimulus repetition. Group 1 animals, aggressive and socially active, showed increases in gamma amino-4-butyric acid (GABA) with initial stimulus presentation; this increased with stimulus repetition. Behavioural responses to the stimuli decreased with repetition and nonstimulus-related behaviours, during the course of the experiment, increased. Both of these appeared dependent upon GABA. Group 2 animals, moderately aggressive and socially active, released opioid-like peptides (OLP) upon initial exposure to stimuli but, with repetition, switched to using GABA. Group 3 animals, nonaggressive and socially inactive, released OLP with initial and repeat stimuli. In groups 2 and 3, both GABA and OLP appear to reduce stimulus-related behaviour, but OLP appeared to also reduce nonstimulus-related behaviour and GABA increased these. Changes were independent of animal liveweight. Glutamate was released in response to stimuli in all 3 groups and, with repetition, fell in groups 1 and 2 but increased in group 3. An animal's social behaviour and status may predict its response to a stimulus.

Contribution of amino acid transmitters to epileptiform activity and reflex suppression in electrically head stunned sheep.

In sheep, administration of a combination of zolazepam and tiletamine hydrochloride resulted in a dose dependent reduction in the duration of epileptic activity induced by an electric stun applied to the head. The compound also lengthened the normal period of reflex suppression that occurs after a stun. Excitatory amino acid receptor antagonists (2-amino-7-phosphonoheptanoic and 2-amino-5-phosphonovaleric acids) also reduced the duration of epileptic activity following an electric stun. These drugs did not alter the time of pedal and ear pinch reflex suppression. Administration of bicuculline (a gamma amino-4-butyric acid [GABA] receptor antagonist) reduced the period of stun induced reflex suppression and increased seizure duration. Administration of a GABA receptor agonist, baclofen, increased the duration of reflex suppression. The results suggest that the development of epileptiform-like activity following application of an electric current to the head is dependent upon excitatory amino acid receptors. The reflex suppression that also arises following an electric stun is contributed to by the activation of GABA receptor mechanisms.

Changes in the release of amino acid neurotransmitters in the brains of calves and sheep after head-only electrical stunning and throat cutting.

In calves aged two to five months, throat cutting resulted in an increase in the concentration of the amino acid neurotransmitters glutamate and aspartate in the brain. Electrical head-only stunning by itself also increased the concentrations of these two neurotransmitters. The levels induced by stunning resulted in a seizure state characterised by epileptiform-like activity in the electroencephalograph. Combing head-only stunning with throat cutting within 10 seconds of the stun had a synergistic effect upon glutamate and aspartate, increasing their concentration by a greater amount and more quickly than either procedure on its own. An irreversible loss of brain function also occurred more quickly than after throat cutting alone. The administration of glutamate and aspartate receptor antagonists before the throat cutting lengthened the time to the loss of brain function in a dose dependent manner. Similar changes were observed in sheep but they occurred much more quickly than in cattle.

Onset of insensibility at slaughter in calves: effects of electroplectic seizure and exsanguination on spontaneous electrocortical activity and indices of cerebral metabolism.

Cerebral venous and femoral arterial blood samples were collected from 21 young calves either during electrical stunning and recovery or electrical stunning and slaughter by carotid severance or slaughter without stunning. The blood samples were analysed for PO2, PCO2, pH, glucose and lactate. The results were compared with simultaneous recordings of spontaneous electrocortical (ECOG) activity. Calves subjected to head-only electrical stunning and slaughter became permanently insensible at the time of the stun. The six calves slaughtered without stunning lost sensibility within 10 seconds. One calf, in which a clot formed in the carotid arteries inhibiting bleeding, maintained some evidence of cortical activity beyond 52 seconds; this was high amplitude low frequency activity and analysis by Fast Fourier Transform showed sensibility was not regained. In the remaining calves the ECOG activity was lost on average within 49 +/- 3.5 (SEM) seconds after slaughter. The cerebral extraction of metabolites increased after carotid severance, indicating inadequacy of cerebral bloodflow after slaughter. No correlations were found between indices of cerebral metabolism and the time of loss of cortical function.

Electrical stimulation, muscle tension and glycolysis in bovine Sternomandibularis.

Electrical parameters previously employed to hasten the onset of rigor mortis in carcasses were derived empirically. Various parameters have been studied to determine their relationship to the two-stage fall of muscle pH resulting from stimulation. At any given stimulation period pulse frequency had a considerable effect on the magnitude of the pH fall that occurs during stimulation, the greatest falls, ∼ 0·7 pH units in a 120 sec period, being achieved with 5 to 16·6 pulses. sec(-1). The same total number of pulses delivered at differing frequencies did not produce pH falls of equal magnitude. Variation in pulse shape and polarity caused minor changes. Increased energy levels per pulse increases the magnitude of the pH fall during stimulation. All stimulation parameters tested resulted in the rate of pH fall after stimulation being one and a half times to twice that of non-stimulated muscle. The tension-time curves obtained with lower frequencies (< 10 pulses . sec(-1)) show incomplete summation of pulses with sustained tension. At higher frequencies a complete tetanus develops, while tension falls rapidly soon after achieving the peak tension. The relationship between the mechanical and biochemical responses and the implications for stimulation as a practical processing method are discussed.

The basal toughness of unaged lamb.

Identification of the commencement of aging in relation to the rigor process was determined on lamb loin muscles by injecting a zinc-TRIS solution, which inhibits meat aging, into the muscle over the period when the pH was falling. Aging was evaluated by measuring the tenderness of cooked meat samples using a MIRINZ tenderometer. When zinc was injected into muscle above pH 6·5, the sarcomeres shortened and the cooked meat had an average shear force of 19 kgF (shear force is the force required to shear through at 1 cm cross-section sample). When sinc was injected into the muscle at pH values below 6·5 the sarcomeres did not shorten, and down to the ultimate pH, the shear force remained at approximately 15 kfF. Aging did not take place pre-rigor. The same relationships occurred for electrically stimulated muscle. The data indicate that the basal shear-force value of lamb loin prior to aging, i.e. the basal toughness level, is 15 kgF.

Electrical stimulation of rats: A model for evaluating low voltage stimulation parameters.

Anaesthetised rats, used either before or after exsanguination, provided a useful preparation with which to measure muscle tension from the M. triceps surae and follow pH changes from the M. longissimus dorsi due to stimulation. With square wave pulses (5 ms duration) the minimum voltage required to elicit a tension response from the M. triceps surae was 0·1 V when applied via the tibial branch of the sciatic nerve and nearly 3 V when applied directly across the muscles. Maximum responses were achieved by 2 V applied to the nerve or 5-8 V across the muscle. The fusion frequency was higher than the ox, occurring at about 30 pulses per second. There was no response (with voltages less than 8 V) from the same preparation when the rats were curarised. Tension responses of curarised rats, approaching those of normal direct stimulation, were achieved around 20-40 V. Stimulation of the whole rat carcass at 20 V resulted in a pH fall in the M. longissimus dorsi and M. triceps surae. most of which occurred in the first 30 s. The rate of fall of pH following stimulation of M. longissimus dorsi was approximately 0·6 pH units per hour, being considerably greater than the 0·4 pH units per hour for unstimulated rats. As the ultimate pH of rats was high (pH 6·1-6·3), short stimulation times had to be used to avoid asymptotic portions of the curve. Although responses of rat muscles are different from those of bovine or ovine muscles, they show many of the principles which can be used to evaluate stimulation characteristics.

Effect of electrical stunning methods on petechial haemorrhages and on the blood pressure of lambs.

Electrical stunning of lambs to render them unconscious and insensible to pain can result in blood splash (spots of blood in muscles) or speckle (petechial haemorrhages in fat or connective tissue overlying the muscle). Stunning through electrodes applied to the head causes mainly blood splash with some speckle, while currents applied head-to-back or head-to-foreleg can produce speckle without blood splash. To identify the causes of speckle, animal movement and blood pressure changes that occur during stunning were measured. Three different stunning methods were employed: head-only, head-to-back and head-to-foreleg. Head-only stunning, which does not stop the heart and produces a moderate amount of speckling, was accompanied by elevated arterial blood pressures up to 300 mm Hg, and venous pressures up to 100 mm Hg. The carcass became rigid on stunning but reflex kicking occurred when the hind legs were shackled. Head-to-back stunning, which stops the heart, produced a momentary arterial and venous blood pressure rise during the stun, as a result of thoracic pressure changes, followed by a decline in pressure to 40-50 mm Hg. The muscles became markedly rigid due to direct stimulation of the loin and spontaneous kicking was absent following current switch off, although spontaneous coughing persisted. Stillness associated with head-to-back stunning was accompanied by the highest incidence and severity of speckling, whereas head-to-foreleg stunning, which also stops the heart, resulted in less muscle contraction and was accompanied by the least incidence and severity of speckling. Kicking was also eliminated if sufficiently high currents were used. Stopping the heart with KCl before a head-to-back stun lowered arterial blood pressure to 20-30 mm Hg and markedly reduced speckle. Abolition of nervously mediated muscle movement with curare before stunning resulted in no observable speckle in head-only stunned lambs but, in the head-to-back stunned lambs, there was a low incidence of speckle in the loin as a result of the muscle being directly stimulated to contract. The results from this study are consistent with the view that speckle is a result of vigorous movements between muscles during stunning, causing a shearing and rupture of blood vessels in the fat and connective tissues, thus allowing the muscles to squeeze out blood. The incidence of speckle does not appear to be related to blood pressure during stunning.

Electrical stimulation of rats: Part 2-The effect of electrical parameters on muscle tension and post-Mortem glycolysis.

Anaesthetized rats were used as a model to determine the effect of changes in electrical stimulation parameters on the pH fall of the M. longissimus dorsi and M. triceps surae and tension development by the M. triceps surae. Stimulation 5 min before decapitation resulted in a reduced post-stimulation fall in pH. Tension development resulting from stimulation of the sciatic nerve decreased rapidly 5 min after decapitation and ceased 30 min after decapitation, whereas a current pathway through the muscle was still effective. Post-stimulation pH fall was less in the leg not in the stimulation pathway, indicating a lack of crossover effect from one leg to the other; but the difference diminished as stimulation voltage increased. For direct stimulation, 20 V produced a maximal response in the stimulated leg; at least twice that voltage was required for a maximal pH fall in the leg not in the stimulation pathway. The total resistance of the rat with a needle electrode at the nape of the neck and crocodile clips attached to the unskinned legs was 3400 ω, whereas with skinned hindlegs the resistance was 860ω. With this high skin resistance at least 40 V was needed for effective stimulation whereas, for low voltage stimulation of much larger animals, only twice this voltage is used. High contact resistance and unknown current density factors make simple correlations of animal size and voltage effects impossible. The optimal stimulation frequency for rats was 20-30 pulses a second. If the cranial electrode was positive, about 15% more tension was developed than when the cranial electrode was negative. With alternating-polarity pulses, there was a high-low tension response, which was more pronounced at low voltages but evened out as the muscles became exhausted. Alternating and cranial-positive pulses produced similar pH falls but cranial-negative pulses were inferior.