Automated rodent in situ muscle contraction assay and myofiber organization analysis in sarcopenia animal models.

Age-related sarcopenia results in frailty and decreased mobility, which are associated with increased falls and long-term disability in the elderly. Given the global increase in lifespan, sarcopenia is a growing, unmet medical need. This report aims to systematically characterize muscle aging in preclinical models, which may facilitate the development of sarcopenia therapies. Naïve rats and mice were subjected to noninvasive micro X-ray computed tomography (micro-CT) imaging, terminal in situ muscle function characterizations, and ATPase-based myofiber analysis. We developed a Definiens (Parsippany, NJ)-based algorithm to automate micro-CT image analysis, which facilitates longitudinal in vivo muscle mass analysis. We report development and characterization of translational in situ skeletal muscle performance assay systems in rat and mouse. The systems incorporate a custom-designed animal assay stage, resulting in enhanced force measurement precision, and LabVIEW (National Instruments, Austin, TX)-based algorithms to support automated data acquisition and data analysis. We used ATPase-staining techniques for myofibers to characterize fiber subtypes and distribution. Major parameters contributing to muscle performance were identified using data mining and integration, enabled by Labmatrix (BioFortis, Columbia, MD). These technologies enabled the systemic and accurate monitoring of muscle aging from a large number of animals. The data indicated that longitudinal muscle cross-sectional area measurement effectively monitors change of muscle mass and function during aging. Furthermore, the data showed that muscle performance during aging is also modulated by myofiber remodeling factors, such as changes in myofiber distribution patterns and changes in fiber shape, which affect myofiber interaction. This in vivo muscle assay platform has been applied to support identification and validation of novel targets for the treatment of sarcopenia.

Differential effects of inhibitors of cellular function on inflammatory mediator-stimulated increases in vascular permeability.

The suffused noneverted cheek pouch of pentobarbital anesthetized hamsters was used to study the effects of various inhibitors of receptor/cellular function on inflammatory mediator stimulated increases in vascular permeability. Fluorescein isothiocynate dextran (FITC-D, 70,000 Da) was utilized as a tracer, and intra-vital light microscopy was employed to monitor the formation of vascular leakage sites while direct measurement of plasma and suffusate tracer concentrations were used to monitor tracer clearance. Vascular permeability increases were triggered by suffusing the cheek pouch with histamine, bradykinin, or Compound 48/80 which stimulated the formation of focal FITC-D leakage sites in the postcapillary venules resulting in marked increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. Saline, calmidazolium, and papaverine lacked intrinsic permeability increasing activity, and failed to alter histamine, bradykinin, and compound 48/80 stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. In contrast, treatment with cytochalasin B, DDAVP, diphenhydramine, tubulazole C, or verapamil inhibited histamine and Compound 48/80 stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. Bradykinin stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance were not affected by treatment with calmidazolium, cytochalasin B, DDAVP, diphenhydramine, tubulazole C, or verapamil. These findings demonstrate that inflammatory mediator stimulated increases in vascular permeability may be differentially affected by inhibitors of receptor/cellular function.

Effects of local mast cell degranulation on vascular permeability to macromolecules.

The suffused noneverted cheek pouch of pentobarbital anesthetized hamsters was used to study the effects of localized, selective mast cell degranulation on vascular permeability. Fluorescein isothiocynate dextran (FITC-D, 70,000 Da) was utilized as a tracer, and intra-vital light microscopy was employed to monitor the formation of vascular leakage sites while direct measurement of plasma and suffusate tracer concentrations were used to monitor tracer clearance. Varying the time at which the FITC-D tracer was injected i.v. relative to the start of the Compound 48/80 suffusion permitted direct determination of the duration of any observed increase in vascular permeability. Selective, local mast cell degranulation was triggered by suffusing the cheek pouch with Compound 48/80 for 10 minutes which stimulated the formation of focal FITC-D leakage sites in the postcapillary venules resulting in increases in [FITC-D]S, [FITC-D]S/[FITC-D]P. 10(-6), and FITC-D clearance. In contrast, suffusion of the cheek pouches with saline failed to trigger the formation of venular FITC-D leakage sites or to promote increases in [FITC-D]S, [FITC-D]S/[FITC-D]P. 10(-6), and FITC-D clearance. The increase in permeability produced by Compound 48/80 was marked but transient (duration less than 20 minutes), and subject to inhibition by treatment with either the H1 receptor antagonist diphenhydramine or the endothelial cell stabilizer isoproterenol. There was no evidence for for a non-histamine mediated or delayed-onset increase in vascular permeability to macromolecules during the course of these experiments.

Contribution of kininase II to the waning of vascular actions of bradykinin.

The mechanism(s) of the waning of the vasodilation and increase in vascular permeability during prolonged local intraarterial infusions of bradykinin (BK) was investigated in this study. Treatment with phentolamine or saralasin failed to prevent the waning of the vasodilation during the prolonged infusion of BK into forelimbs perfused at constant flow. In contrast, BK produced a sustained vasodilator response after treatment with captopril. Forelimb weight and lymph analysis were used to quantitate edema formation and to determine the duration of the increase in vascular permeability during prolonged local intra-arterial infusions of BK into forelimbs perfused at constant flow. The lymph-to-plasma ratios (L/P) for protein and FITC-Dextrans (fluorescein isothiocyanate dextrans, 70,000 Da) were determined, and clearances for protein and FITC-D were calculated. BK markedly increased fluid filtration, the protein L/P, and protein clearance resulting in edema formation. The protein L/P remained markedly elevated throughout the experimental period. The FITC-D L/P was markedly increased in the groups of animals in which the tracer was injected intravenously at the start or 8 min after the start of the prolonged BK infusion. In the groups of animals in which the tracer was injected intravenously 15-60 min after the start of the prolonged BK infusion, the FITC-D L/P failed to exceed the FITC-D L/P in control animals, although the protein L/P remained elevated. Pretreatment with both captopril and propranolol dramatically potentiated the magnitude of the increase in protein clearance, the filtration rate, and edema formation produced by BK but failed to affect the duration of the transient increase in vascular permeability.

The role of venular endothelial cells in the regulation of macromolecular permeability.

It is well documented that inflammatory mediators produce increases in fluid filtration promoting massive edema formation in limb skin and skeletal muscle. The edema is attributable to an increase in the transmural hydrostatic pressure gradient subsequent to an increase in microvascular pressure (Pmv) and, more importantly, to a decrease in the transmural colloid osmotic pressure gradient subsequent to an increase in vascular permeability to macromolecules. The mediators produce increases in net fluid filtration, protein clearance, the L/P total protein ratio, and edema formation in the absence of increases in blood flow and Pmv demonstrating that increases in vascular permeability are not dependent on changes in blood flow, Pmv, or surface area per se. Since the inflammatory mediators produce increases in the L/P protein ratio and edema formation in vascular beds perfused with cell-free, artificial perfusates, the increase in permeability likely results from an interaction between the mediator and the vascular endothelium. The findings demonstrating that mediator-stimulated increases in net fluid filtration, protein clearance, the L/P ratio, and edema formation are subject to inhibition by selective mediator receptor antagonists suggests that the increase in vascular permeability is dependent on the activation of a receptor mediated physiological mechanism which regulates the porosity of the vascular endothelium to macromolecules. Findings from microscopic studies demonstrate that the inflammatory mediators trigger the extravasation of macromolecules exclusively from the postcapillary venules, and that increases in the flux of macromolecules between the vascular and interstitial fluid compartments is dependent on the formation of leakage sites in the postcapillary venules. Electron microscopic studies of the vascular leakage sites reveals the formation of large gaps between adjacent endothelial cells, and changes in cell shape including a wrinkling of the nucleus suggestive of endothelial cell contraction. Contractile proteins and receptors for many substances including inflammatory mediators are found in endothelial cells, and receptor modulated changes in endothelial cell shape associated with active changes in the contractile proteins have been demonstrated in endothelial cells in culture. The inflammatory mediators appear to trigger active endothelial cell contraction inducing the transient, reversible formation of large junctional gaps between adjacent cells in the postcapillary venules.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of macromolecular permeability by immune-complexes and a beta-adrenoceptor stimulant.

The suffused, noneverted cheek pouch of ovalbumin (OA)-immunized hamsters was employed to study changes in vascular permeability utilizing intravital light microscopy and direct measurements of plasma to suffusate tracer efflux. Suffusion of the cheek pouches of immunized animals with OA for 10 min stimulated the formation of focal venular fluorescein isothiocyanate dextran (FITC-D, 70,000 Da) leakage sites and produced increases in the plasma to suffusate FITC-D efflux resulting in marked increases in concentration of FITC-D in suffusate ([FITC-D]s). The intravenous injection of the FITC-D 15 to 60 min after the start of the OA suffusion failed to reveal the formation of vascular FITC-D leakage sites or increases in [FITC-D]s. In contrast, the intravenous injection of the tracer at the start or 10 min after the start of the OA suffusion revealed the formation of venular FITC-D leakage sites and marked increases in [FITC-D]s. Treatment with diphenhydramine or isoproterenol completely inhibited the antigen-stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]s. It is concluded that antigen-antibody reactions resulting in the production of immune complexes trigger immediate increases in the plasma to suffusate FITC-D clearance via the formation of venular FITC-D leakage sites subsequent to the release of endogenous histamine. The increase in venular permeability is marked but transient, lasting approximately 10 min, and is subject to inhibition by either diphenhydramine or isoproterenol.

Patterns of constriction produced by vasoactive agents.

The patterns of vasoconstriction produced by local infusions of constrictor agents and neurogenic stimuli are unique and varied. Although vasoconstrictors or neurogenic stimuli may produce similar increases in total resistance to blood flow, the effects on consecutive vascular segments may differ dramatically. Vasoconstrictors may affect primarily small vessels, large vessels, or a combination of both. The constrictor response may be restricted to precapillary vessels or may recruit both pre- and postcapillary vessels. The baroreceptors elicit a pattern of vasoconstriction distinct from that produced by electrical stimulation of a vasomotor nerve. Prearteriolar and venous resistance may contribute more than arterioles to increases in total vascular resistance produced by local infusions of vasoconstrictor agents or nerve stimulation. The constriction of large vessels also affects fluid filtration, vascular capacity, and the distribution of blood flow between shunt and exchange vessels. The waning of the resistance increase that occurs during prolonged infusions of vasoconstrictors varies, in part, as a function of the vessel segments that participate in the vasoconstrictor response. Large vessels participate in vasoconstrictor responses triggered by stimuli that impose a severe stress on the circulation. In contrast, small vessels participate primarily in normal vascular adjustments required to maintain blood pressure at the set point.

Evidence that prolonged histamine suffusions produce transient increases in vascular permeability subsequent to the formation of venular macromolecular leakage sites. Proof of the Majno-Palade hypothesis.

The aim of this study was to determine whether histamine-stimulated increases in macromolecular efflux are dependent on the formation of specific vascular leakage sites, or whether other mechanisms need to be invoked to explain the increase in macromolecular efflux produced by this inflammatory mediator. Intravital light microscopy was used to localize and quantitate vascular macromolecular leakage sites in the noneverted hamster cheek pouch. Fluorimetric measurements of plasma and suffusate tracer (FITC-D 70,000 mol wt) concentrations were utilized to quantitate changes in macromolecular efflux. In some experiments, the FITC-D was injected intravenously either at the start of or after the start of a prolonged histamine suffusion for estimation of the duration of the vascular FITC-D leakage response. In saline control cheek pouches there were few, if any, visible FITC-D vascular leakage sites and only small increases in the [FITC-D]s. The arteriolar vasodilators papaverine (1 X 10(-5) M) and isoproterenol (1 X 10(-5) M) failed to increase the formation of vascular FITC-D leakage sites, and the magnitude of the increase in [FITC-D]s produced by these agents was similar to that observed in saline controls. Histamine (1 X 10(-5) M) suffused for either 15, 60, or 120 minutes produced marked increases in [FITC-D]s and in the number of venular FITC-D leakage sites. The venular FITC-D leakage sites began to fade after 10-20 minutes, eventually disappearing altogether. In contrast, the [FITC-D]s was markedly increased throughout the 120-minute observation period. Treatment with papaverine prior to and during the 60-minute histamine suffusion failed to prevent the mediator-stimulated vascular leakage response. In contrast, similar treatment with isoproterenol inhibited the histamine-stimulated increases in [FITC-D]s and the formation of venular FITC-D leakage sites. When the tracer was injected intravenously at the start of the 60-minute histamine suffusion (1 X 10(-5) M), the [FITC-D]s and the number of vascular leakage sites were markedly increased. However, when the tracer was injected intravenously 30 minutes after the start of the 60-minute histamine suffusion, there were only minimal increases in [FITC-D]s and the formation of venular leakage sites. These findings suggest that prolonged suffusions of histamine produce transient increases in macromolecular efflux which are dependent on the formation of discrete venular macromolecular leakage sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Physiological and pharmacological evidence for the regulation of permeability.

Local intraarterial infusions of histamine-type mediators produce increases in microvascular pressure (Pmv), protein efflux, and net fluid filtration that promote edema formation. The rise in Pmv is not the primary determinant of edema formation inasmuch as mediator-stimulated edema formation develops without an increase in Pmv. The inflammatory mediators increase the hydraulic conductivity of the microvascular membrane as evidenced by a large increase in the capillary filtration coefficient (CFC) subsequent to an increase in permeability. The development of inflammatory edema is primarily attributable to the increase in protein efflux, which decreases the lymph-to-plasma total-protein ratio (L/P ratio), virtually eliminating the transmural colloid osmotic pressure gradient. Hence, fluid filtration is increased at almost any level of Pmv. Noninflammatory vasodilators and venous occlusion produce increases in Pmv and protein clearance, but fail to increase the L/P ratio. The increase in protein efflux and L/P ratio is attributable to a nonhemodynamic action of the inflammatory mediators, an increase in microvascular permeability to macromolecules. The increase in protein efflux, CFC, and net fluid filtration produced by various inflammatory mediators is largely inhibited by cooling, treatment with endothelial cell stabilizers, or perfusion with blood from hemorrhaged animals. This inhibition is independent of changes in hemodynamics and must be ascribed to a direct effect on the microvascular membrane, providing evidence for a variable macromolecular transport pathway. In contrast, increases in protein clearance produced by increasing Pmv are not inhibited by these maneuvers, which provides evidence for a static macromolecular transport pathway. These findings correlate well with those from microscopic studies supporting the concept that macromolecular permeability may be directly regulated at the level of the venular endothelial cell subsequent to the modulation of interendothelial cell junction gap size.

Is there evidence for venular large junctional gap formation in inflammation?

Inflammatory edema is associated with vascular macromolecular leakage. Various patterns of vascular macromolecular leakage may be produced depending on the severity and nature of the inflammatory stimulus resulting in transient and/or sustained increases in macromolecular permeability. Inflammatory stimuli which cause endothelial cell damage or destruction induces non-specific increases in macromolecular permeability in all injured microvessels. In the absence of endothelial cell injury, macromolecular permeability is increased in inflammation subsequent to the formation of inter-endothelial cell gaps in capillaries and venules. Various inflammatory mediators including histamine-type agents, immune complexes, and activated leukocytes induce venular large junctional gap formation. Individual, simultaneous, or sequential mediator effects could explain the various patterns of venular macromolecular leakage found in inflammation. The formation of endothelial cell junctional gaps in capillaries cannot be attributed to any known inflammatory mediator. The classical static small pore/large pore model of the microvascular membrane cannot explain the increased extravasation of macromolecules in inflammation. A dual static/variable large pore system would best describe macromolecular transport under normal and inflammatory conditions.

Effects of AVP and DDAVP on histamine-induced increases in macromolecular permeability in the hamster cheek pouch.

The effects of histamine alone and in the presence of AVP or DDAVP on microvascular permeability to macromolecules was evaluated in the superfused hamster cheek pouch. FITC-Dextran (MW 70,000) was employed as a macromolecular tracer to quantitate the increase in macromolecular permeability produced by the topical application of histamine. Intra-vital light microscopy was utilized to quantitate and localize FITC-D extravasation sites along the vascular tree, and fluorimetric measurement of the FITC-D concentration in the suffusate (S) and plasma (P) was used to calculate the FITC-D S/P ratio to quantitate the increase in macromolecular permeability. The infusion of histamine for 5 minutes at a rate which produced a suffusate histamine concentration of 1 X 10(-5) M produced a marked increase in the number of venular FITC-D leakage sites, the [FITC-D]s, and the FITC-D S/P ratio. These effects of histamine were prevented by treatment with either AVP or DDAVP which was infused at a rate sufficient to produce a suffusate concentration of 1 X 10(-8) M. AVP produced profound vasoconstriction whereas DDAVP prevented the histamine-induced increase in the formation of venular FITC-D leakage sites, the [FITC-D]s, and FITC-D S/P ratio without producing vasoconstriction. These data suggest that the antagonism of the histamine-induced increase in macromolecular permeability by AVP and DDAVP is not dependent on vasoconstriction per se, but rather is attributable to the stimulation of a vasopressin receptor on the venular endothelial cell which is identical to or similar to the vasopressin receptor mediating the anti-diuretic effects of these agents.

Effects of dopamine and vasopressin on histamine-induced increases in macromolecular permeability in the canine forelimb.

Local intra-arterial infusions of histamine, 16 micrograms base/min, for 60 minutes produced increases in lymph flow, lymph total protein concentration, the lymph/plasma total protein ratio, and weight in canine forelimbs perfused at constant flow. The weight gains were far greater than can be accounted for by an increase in vascular volume, and must, therefore, be attributed to edema formation. Treatment with dopamine (2, 4, or 8 micrograms base/min) or arginine vasopressin (AVP) initially produced an increase in perfusion pressure reflecting constriction of the forelimb vasculature. AVP prevented the histamine-induced increase in lymph flow, lymph total protein concentration, the L/P total protein ratio, and weight gain. In contrast, treatment with DA at three different dose levels failed to affect the histamine response. The increases in lymph flow, lymph total protein concentration, lymph/plasma total protein ratio, and weight were comparable to that produced by histamine in the absence of DA. These data fail to provide evidence for DA mediated regulation of macromolecular permeability, and suggest that endothelial cells either do not contain DA receptors, or that their activation does not affect macromolecular permeability.

Effects of dopamine (DA) and SKF 82526, a selective DA1-receptor agonist, on vascular resistances in the canine forelimb.

This study was performed to determine the presence of vascular dopamine (DA1) receptors in the canine forelimb. Local i.a. infusions of DA (2, 4 or 8 micrograms base/min) produced cutaneous and skeletal muscle vasoconstriction in the forelimb comparable to that produced by local i.a. infusions of norepinephrine (0.5, 1 or 4 micrograms base/min). In the cutaneous vasculature, DA produced large artery, small vessel and large vein constrictions. The small vessels and large veins constricted proportionately more than the large arteries. The pattern of constriction produced by DA along the cutaneous vascular tree was similar to that produced by norepinephrine. The forelimb vasoconstriction produced by both DA and norepinephrine was abolished completely by treatment with phentolamine, indicating that both agents produce vasoconstriction mediated by stimulation of alpha adrenoceptors. The failure of DA to produce vasodilation after phentolamine suggests that there are no vascular or DA1-subtype DA receptors in the canine forelimb vasculature. The local i.a. infusion of SKF 82526, a selective DA1-receptor agonist, for 3 min produced cutaneous and skeletal muscle dilation which could be antagonized by either sulpiride or phentolamine. This neurogenic dopaminergic vasodilation produced by SKF 82526 (25, 50 or 100 micrograms base/min) was converted into vasoconstriction after ganglionic blockade. Inasmuch as SKF 82526 does not activate presynaptic or DA2-subtype DA receptors, the neurogenic vasodilation caused by SKF 82526 may be due to a dopaminergic inhibition of ganglionic transmission. Whereas the results of our study fail to provide any evidence for the presence of vascular DA (DA1) receptors in the canine forelimb, they show that SKF 82526, a DA1-receptor agonist, produces neurogenic vasodilation probably by activating ganglionic DA receptors.

Evidence that serotonin receptors mediate the cutaneous vasoconstriction produced by 5-hydroxytryptamine in canine forelimbs.

The effects of local intra-arterial infusions of serotonin (5 or 25 micrograms base/min) or norepinephrine (1 or 5 micrograms base/min) on cutaneous (skin) and skeletal muscle vasculatures were investigated in canine forelimbs perfused at constant flow in dogs anesthetized with pentobarbital. Norepinephrine produced dose-related constriction of the skin and skeletal muscle vasculatures. In the cutaneous vascular circuit, norepinephrine produced large artery, small vessel, and large vein constriction. The increase in cutaneous vascular resistance was primarily due to an increase in small vessel resistance. Serotonin did not increase skeletal muscle vascular resistance but produced marked cutaneous vasoconstriction subsequent to large artery and large vein constriction. The small vessels, if anything, tended to dilate. The skin and skeletal muscle vascular responses to serotonin and norepinephrine were similar in innervated and acutely denervated forelimbs. Phentolamine pretreatment completely blocked all vascular actions of norepinephrine, and largely inhibited the cutaneous vasoconstriction produced by the infusion of the low dose of serotonin. However, the cutaneous large artery and large vein constriction produced by the infusion of the high dose of serotonin was not affected by phentolamine pretreatment. Cyproheptadine pretreatment blocked or largely inhibited the cutaneous vasoconstriction produced by serotonin only in doses which also inhibited norepinephrine and vasopressin cutaneous vasoconstriction. Pretreatment with methysergide blocked or largely inhibited the cutaneous large artery and large vein constriction produced by infusions of serotonin. Norepinephrine and vasopressin produced significant vasoconstriction in the presence of methysergide. These data suggest that the cutaneous large artery and large vein constriction produced by serotonin is not due to the activation of postjunctional alpha-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of captopril and propanolol on bradykinin-induced changes in vascular pressures, lymph total protein concentration, and weight in canine forelimbs.

Sixty-minute local intraarterial infusions of bradykinin (0.8, 5, or 10 micrograms base/min) produced transient forelimb vasodilation and dose-related increases in lymph flow, lymph total protein concentration, total protein transport, and weight in forelimbs perfused at a controlled flow rate. Mean aortic pressure was not affected by these infusion rates of bradykinin. Following pretreatment with captopril, the local intraarterial infusion of these same doses of bradykinin produced sustained systemic hypotension. The increase in protein efflux and edema formation produced by local infusions of bradykinin following pretreatment with captopril was markedly increased during the infusion of the low dose of bradykinin (0.8 micrograms base/min, ia), but was attenuated during the local infusion of the larger dose of bradykinin (5 micrograms base/min, ia). Following pretreatment with both captopril and propranolol, the increase in protein efflux and edema formation produced by this larger dose of bradykinin (5 micrograms base/min) was greater than that produced by infusions of this dose of bradykinin alone or after pretreatment with captopril. Moreover, the increase in protein efflux and edema formation was greater during the infusion of the higher dose of bradykinin than during the infusion of the low dose of this autacoid under these conditions. The 60-min infusion of a massive dose of bradykinin into the left ventricular chamber (280 micrograms base/min) produced sustained decreases in aortic and forelimb perfusion pressure, but little edema formation relative to that produced by local intraarterial infusions of this agent. In contrast, the 60-min intravenous infusion of only 5 micrograms base/min of bradykinin following pretreatment with both captopril and propranolol produced profound systemic hypotension and marked increases in protein efflux and edema formation in forelimbs perfused at a controlled flow rate comparable to that produced by the local intraarterial infusion of this dose of bradykinin alone. These data demonstrate that endogenous kininases and catecholamines may dramatically affect the increase in protein efflux and edema formation produced by either local or systemic infusions of bradykinin by modulating the magnitude of the increase in macromolecular permeability.