Remote Dilation Research Articles

Hysteroscopic Removal of Foreign Body

<h3>Study Objective</h3> To present a unique case of hysteroscopic removal of retained fetal bone <h3>Design</h3> This is a unique video case report of a patient whose infertility workup revealed evidence of suspected retained fetal bone from a remote second trimester abortion. She was counseled and consented for hysteroscopic removal of suspected foreign body. <h3>Setting</h3> Operating room. <h3>Patients or Participants</h3> There is one patient in this video. She presented for infertility workup with past medical history significant for surgical abortion at 20 weeks gestation, 17 years prior. She was unable to conceive following this procedure. <h3>Interventions</h3> Hysteroscopy to remove foreign object. <h3>Measurements and Main Results</h3> Ultrasound showed irregular echogenic structure 1.7 by 0.4 cm with dense posterior shadowing in the cervix of uncertain etiology. Differential included retained foreign body such as a fragmented IUD or retained fetal bone from her remote abortion, which was the leading diagnosis. Hysteroscopy was performed for direct visualization and removal of this foreign body. Pathology report confirmed it to be degenerated mature bone. <h3>Conclusion</h3> Multiple calcified bony remnants, likely from remote dilation and curettage for termination, were extending through the length of the endocervical canal. Hysteroscopy was utilized to directly visualize and remove these bony remnants to clear the endocervical canal and entrance to uterine cavity.

Downstream Exposure to Growth Factors Causes Elevated Velocity and Dilation in Arteriolar Networks

Our goal was to characterize changes in flow and diameter with vascular endothelial cell growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Observations were made in arteriolar networks of the cheek pouch tissue in anesthetized hamsters (pentobarbital 70 mg/kg, i.p., n = 45). Local and remote dilation responses to micropipette-applied VEGF or FGF2 yielded similar EC₅₀ values. The role of gap junctions in the remote response was tested by applying sucrose, halothane or 18αGA to the feed arteriole midway between the remote stimulation and upstream observation sites; all remote dilation to FGF2 was prevented, while only the early dilation to VEGF was blocked. The remote dilation to VEGF displayed a second rheologic mechanism. The second mechanism involved an abrupt increase in upstream velocity and shear rate, followed by nitro-arginine sensitive dilation. To test whether the abrupt increase in shear could be caused by other agents known to cause edema, remote responses to histamine and thrombin were tested. Each caused an abrupt increase in velocity followed by nitro-arginine-sensitive dilation. This study shows that VEGF or agents that increase permeability can initiate an upstream velocity increase with dilation that recruits flow to the network; this is in addition to simultaneous gap junction-mediated dilation.

Different pathways with distinct properties conduct dilations in the microcirculation in vivo

Conduction of vasomotor signals along the vessel coordinates the behaviour of vascular cells and is attributed to the spread of hyperpolarizations through gap junctions. Intriguingly, conducted dilations encompass larger distances than can be expected by passive electrotonic spread. Because distances are quite distinct for different dilators, we hypothesized that separate pathways with distinct properties are involved. We characterized local and conducted responses elicited by acetylcholine (ACh) and adenosine (Ado) in the murine microcirculation in vivo. Local (and remote) ACh dilations were nearly abrogated by blockade of K(Ca) channels (charybdotoxin), but dilations to Ado were abolished by the K(ATP) blocker glibenclamide. Bupivacaine, a blocker of Na+ and K+ channels, and similarly the blockade of inwardly rectifying K+ channels (barium) revealed different conduction mechanisms, as the remote dilation to Ado, but not ACh, was abrogated. Surprisingly, expression of connexin37 (Cx37) was not detected in Cx40-deficient arterioles, although abundantly expressed in endothelium of wild-type arterioles. In contrast to the wild-type mice, the amplitude of conducted ACh and Ado dilations decreased similarly with distance in Cx40-deficient mice. Recordings of membrane potential in vivo showed endothelial hyperpolarization by approximately 10 mV in response to ACh, whereas Ado did not alter endothelial membrane potential. Distinct pathways conduct responses along the vessel wall which involve dissimilar K+ channels and connexins in initiation and spreading. Most likely, the endothelium is the preferential conduction pathway activated by ACh, whereas in the case of Ado the smooth muscle serves as the signalling pathway. However, in arterioles nearly devoid of Cx40 and Cx37, ACh responses can likewise be conducted along the smooth muscle.

Which Connexins Connect?

See related article, pages 1292–1299 Electrical signaling via gap junctions modulates vascular function via 2 physiologically interrelated mechanisms, namely dilations/constrictions that propagate longitudinally along the vessel wall,1–4 and the EDHF phenomenon, in which relaxation is facilitated by the transmission of endothelial hyperpolarization to smooth muscle via a radial myoendothelial pathway.5 Gap junctions are composed from 2 hemichannels, each constructed from 6 connexin (Cx) protein subunits, whose alignment across the extracellular space permits intercellular diffusion of ions and small molecules <1 kDa in size and confers electrical continuity. Coupling is enhanced by the clustering of up to several hundred individual gap junction channels to form plaques that can be visualized by immunostaining at points of cell–cell contact. Vascular cells variably express 4 connexin subtypes (Cxs 37, 40, 43, and 45) with protein expression generally being more abundant in the endothelium than in smooth muscle cells. Most commonly, endothelial plaques contain Cx37 and Cx40 and medial plaques contain Cx43. This distribution is seen, for example, in the rabbit iliac artery in which synthetic peptides selectively targeted against Cx37 and Cx40 attenuate endothelium-dependent subintimal smooth muscle hyperpolarization, whereas peptides targeted against Cx43 attenuate the spread of medial hyperpolarization.6 In muscular arteries electrical signaling pathways thus appear to correlate closely with immunostaining findings. In this issue of Circulation Research Wolfle and colleagues1 have analyzed longitudinal conduction in mice with replacement of Cx40 by “knock in” of Cx45 (denoted as Cx40KI45 mice). Responses were initiated by localized application of the endothelium-dependent dilators acetylcholine (ACh) and bradykinin (BK) to cremasteric arterioles in wild-type (WT), Cx40 knockout (Cx40KO), and Cx40KI45 animals, and diameter changes were monitored dynamically by intravital microscopy. Responses were found to propagate for more than 1 mm within 1 sec in WT mice without significant decay, whereas remote dilation …

Connexin45 Cannot Replace the Function of Connexin40 in Conducting Endothelium-Dependent Dilations Along Arterioles

Intercellular communication through gap junctions coordinates vascular tone by the conduction of vasomotor responses along the vessel wall. Gap junctions in arterioles are composed of different connexins (Cxs) (Cx40, Cx37, Cx45, Cx43), but it is unknown whether Cxs are interchangeable. We used mice with a targeted replacement of Cx40 by Cx45 (Cx40KI45) to explore whether Cx45 can functionally replace Cx40 in arterioles. Arterioles were locally stimulated using acetylcholine, bradykinin, adenosine, and K(+) in the cremaster of Cx40KI45, Cx40-deficient (Cx40ko), and wild-type mice, and diameter changes were assessed by intravital microscopy. Additionally, arterial pressure was measured by telemetry and Cx expression verified by immunofluorescence. Acetylcholine initiated a local dilation of a similar amplitude in all genotypes ( approximately 50%), which was rapidly conducted to upstream sites (1200 mum distance) without attenuation in wild type. In marked contrast, the remote dilation was significantly reduced in Cx40ko (25+/-3%) and Cx40KI45 (24+/-2%). Likewise, dilations initiated by bradykinin application were conducted without attenuation up to 1200 mum in wild type but not in Cx40ko and Cx40KI45. Adenosine-induced dilations and K(+)-induced constrictions were conducted similarly with decaying amplitude in all genotypes. Arterial pressure was strongly elevated in Cx40ko (161+/-1 versus 116+/-2 mm Hg) but only moderately in Cx40KI45 (133+/-8 mm Hg). This demonstrates that Cx40 function is critical for the conduction of acetylcholine and bradykinin dilations and cannot be substituted by Cx45. Therefore, unique properties of Cx40 are required for endothelial signal conduction, whereas nonspecific restoration of communication maintains additional functions related to blood pressure control.

Mechanisms initiating integrin-stimulated flow recruitment in arteriolar networks

Our purpose was to investigate the local mechanisms involved in network-wide flow and diameter changes observed with localized downstream vitronectin receptor ligation; we tested specific K or Cl channels known to be involved in either dilation or elevated permeability following vitronectin receptor activation and tested integrin-linked pathway elements of tyrosine phosphorylation and protein kinase C (PKC). Arteriolar networks were observed in the cheek pouch tissue of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n=86) using intravital microscopy. Terminal arteriolar branches of the networks were stimulated with micropipette LM609 (0.5-10 microg/ml, 60 s) alone or with inhibitors (separate micropipette). Hemodynamic changes (diameter, red blood cell flux, velocity) were observed at the upstream entrance to the network. LM609 alone stimulated first an increase in wall shear stress (WSS), followed by a dilation that recovered WSS to baseline or below. K channel inhibition (glybenclamide, 4-AP) had no effect on the initial peak in WSS, but decreased remote vasodilation. Cl channel inhibition (DIDS, IAA-94, niflumic acid) or inhibition of PKC (chelerythrine) prevented the initial peak in WSS and decreased remote vasodilation. Inhibition of tyrosine phosphorylation (genistein) prevented both. With the use of nitro-arginine at the observation site, the initial peak in WSS was not affected, but remote vasodilation was decreased. We conclude the remote response consists of an initial peak in WSS that relies on both PKC activity and depolarization downstream, leading to an upstream flow mediated dilation and a secondary remote dilation that relies on hyperpolarization downstream at the stimulus site; both components require tyrosine phosphorylation downstream.

Summation through gap junctions spreads microvascular preconditioning

Brief remote exposure to key agonists preconditions (PC) upstream arterioles, altering local responses (remote microvascular preconditioning, RMP; AJP HC in press 8/26/05). We tested whether RMP could summate to nearby regions. The cheek pouch of anesthetized (Nembutal 70mg/kg) male hamsters (n=31) was observed. The terminal branches of an arteriolar network (NET) were stimulated with nitroprusside (60 s micropipette, NP 10-4M), initiating both remote dilation, and RMP (op cit). RMP is noted 1000μm away at the entrance to the NET (~12μm dia.) shifting the response to L-arg (10-4M) from constrict (−8±5% baseline diameter) to dilate (43±8%). RMP is blocked within NETS by inhibiting gap junctions (micropipette) 18aGA (10–5M n=6, −1±5%), or gap27 (10–4M n=11, 17±15%). With 1 stimulus, neighboring NETS arising along the same arcade arteriole are not PC (−14±1%). To test whether 2 stimuli could summate to spread RMP to a 3rd NET, we selected sequential NETS arising in alternating R – L branching patterns from an arcade (n=14). Two NETS branching R were stimulated with NP, resulting in the expected RMP of those NETS (41±6%). One NET arising L between the stimulated NETS was PC (24±8%). Two NETS arising to the L before and after the stimulated NETS were also PC (16±11%). NETS that arose sequentially next along the arcade, i.e., 2 away, were not PC (−16±6%). With dual stimulation, blocking gap junctions along the arcade between stimulated and un-stimulated NETS, prevented spread of RMP (high OsM sucrose, 2±12%; 10–4M halothane, -20±1%). We conclude that an important means for amplifying a microvascular preconditioning signal is through active gap junctional activity. (NIH HL55492)

Connexin45 (Cx45) cannot replace the function of Cx40 in arterioles

Intercellular communication via gap junction channels (GJC) contributes to the coordination of arteriolar tone which is reflected by conducted vasomotor responses. GJC in arterioles are composed of Cx40, 37, 45 and 43. It is unknown if Cx are interchangeable or exhibit specific properties to support cellular function. We investigated if Cx45, which exhibits a smaller conductance than Cx40, can functionally replace Cx40 using mice in which Cx45 is expressed in place of Cx40 (Cx40KI45). Propagation of dilations and constrictions initiated by acetylcholine (ACh, 1mM) or KCl (3M) were studied in arterioles (≈33μm) of the cremaster muscle by intravital microscopy in Cx40-deficient (Cx40KO), Cx40KI45, and wildtype (wt) mice. ACh and KCl were applied with micropipettes in a confined manner. ACh induced a similar dilation in all genotypes at the site of stimulation (58-69%). This dilation was conducted to distant sites and the amplitude was 54±8% at 1200μm upstream in wt. However, in Cx40KO this remote dilation was significantly reduced to 29±4%. In Cx40KI45 remote dilations were impaired to a similar degree (21±4%). In contrast, the propagation of KCl-induced constrictions was comparable in all genotypes (local: −52 to −65%, 600μm:−21 to −25%). In summary, Cx40 function is critical for the conduction of dilations and cannot be compensated for by Cx45 substitution. Therefore, it appears that specific Cx-properties are necessary to support conduction along the endothelium.

Remote arteriolar dilations caused by methacholine: a role for CGRP sensory nerves?

Remote vasodilation caused by arteriolar microapplication of acetylcholine cannot be completely attributed to passive cell-cell communication of a hyperpolarizing signal. The present study was undertaken to ascertain whether a neural component may be involved in the remote response. In the cheek pouch of anesthetized hamsters, methacholine (100 microM) was applied to the arteriole by micropipette for 5 s, and the arteriolar responses were measured at the site of application and at remote locations: 500 and 1,000 microm upstream from the application site. Superfusion with the local anesthetic bupivacaine attenuated a local dilatory response and abolished the conducted dilation response to methacholine. Localized micropipette application of bupivacaine 300 microm from the methacholine application site also attenuated the remote dilation but did not inhibit the local dilation. Blockade of neuromuscular transmission with botulinum neurotoxin A (1 U, 3 days), micropipette application of calcitonin gene-related peptide (CGRP) receptor inhibitor CGRP-(8-37) (10 microM) 300 microm upstream from the methacholine application site, and denervation of the CGRP sensory nerve by 2 days of capsaicin treatment reduced the conducted dilation response to methacholine but did not affect the local dilatory response. Together, these data support involvement of a TTX-insensitive nerve, specifically the CGRP containing nerve, in vascular communication. Understanding the effect of regulation of a novel neural network system on the vascular network may lead to a new insight into regulation of blood flow and intraorgan blood distribution.

Localized adenovirus-mediated gene transfer into vascular smooth muscle in the hamster cheek pouch.

Our purpose was to develop a method for adenovirus delivery to the hamster cheek pouch to experimentally target gene transfer in tissue used for microvascular studies. Separate constructs were tested with transgenes for lacZ or green fluorescent protein (GFP) driven by three promoters: RSV, CMV, and SM22. With university approval, adenovirus was delivered in anesthetized (pentobarbital, 70 mg/kg) hamsters (n = 28) by using either a vascular systemic injection or tissue infiltration (interstitial space behind the pouch). During 3 days, animals receiving infiltration gained the expected weight, whereas those receiving vascular injection lost weight; no other behavior changes were noted. On day 3 postadenoviral delivery (infiltration), expression of lacZ (histology, beta-galactosidase) or GFP (fluorescence microscopy) was confirmed across the tissue (CMV and RSV promoters) and exclusively in vascular smooth muscle cells (specific SM22 promoter), without evidence of tissue inflammation. In vitro microvascular experiments verified normal responses in the cheek pouch of day 3 postadenoviral delivery animals. We tested local dilation to methacholine, adenosine, remote dilation to methacholine, adenosine, nitroprusside, and LM609 (alpha(v)beta3 integrin agonist), flow-dependent dilation, and flow recruitment. Thus, this method enables targeted, cell-specific gene transfer to one tissue important for microvascular studies, without significant systemic exposure and without adverse inflammation.

Localized Adenovirus‐Mediated Gene Transfer Into Vascular Smooth Muscle in the Hamster Cheek Pouch

ABSTRACTObjective: Our purpose was to develop a method for adenovirus delivery to the hamster cheek pouch to experimentally target gene transfer in tissue used for microvascular studies.Methods: Separate constructs were tested with transgenes for lacZ or green fluorescent protein (GFP) driven by three promoters: RSV, CMV, and SM22. With university approval, adenovirus was delivered in anesthetized (pentobarbital, 70 mg/kg) hamsters (n = 28) by using either a vascular systemic injection or tissue infiltration (interstitial space behind the pouch). During 3 days, animals receiving infiltration gained the expected weight, whereas those receiving vascular injection lost weight; no other behavior changes were noted.Results: On day 3 postadenoviral delivery (infiltration), expression of lacZ (histology, β‐galactosidase) or GFP (fluorescence microscopy) was confirmed across the tissue (CMV and RSV promoters) and exclusively in vascular smooth muscle cells (specific SM22 promoter), without evidence of tissue inflammation. In vitro microvascular experiments verified normal responses in the cheek pouch of day 3 postadenoviral delivery animals. We tested local dilation to methacholine, adenosine, remote dilation to methacholine, adenosine, nitroprusside, and LM609 (αvβ3 integrin agonist), flow‐dependent dilation, and flow recruitment.Conclusions: Thus, this method enables targeted, cell‐specific gene transfer to one tissue important for microvascular studies, without significant systemic exposure and without adverse inflammation.

Local and remote arteriolar dilations initiated by skeletal muscle contraction.

To investigate the relationship between skeletal muscle metabolism and arteriolar dilations in the region local to contracting muscle fibers as well as dilations at remote arteriolar regions upstream, we used a microelectrode on cremaster muscle of anesthetized hamsters to stimulate four to five muscle fibers lying approximately perpendicular to and overlapping a transverse arteriole. Before, during, and after muscle contraction, we measured the diameter of the arteriole at the site of muscle fiber overlap (local) and at a remote site approximately 1,000 microm upstream. Two minutes of 2-, 4-, or 8-Hz stimulation (5-10 V, 0.4-ms duration) produced a significant dilation locally (8.2 +/- 2.0-, 22.5 +/- 2.4-, and 30.9 +/- 2.1-microm increase, respectively) and at the remote site (4.2 +/- 0.8, 11.0 +/- 1.1, and 18.9 +/- 2.7 microm, respectively). Muscle contraction at 4 Hz initiated a remote dilation that was unaffected by 15-min micropipette application of either 2 microM tetrodotoxin, 0.07% halothane, or 40 microM 18-beta-glycyrrhetinic acid between the local and upstream site. Therefore, at the arteriolar level, muscle contraction initiates a robust remote dilation that does not appear to be transmitted via perivascular nerves or gap junctions.

Increased flow precedes remote arteriolar dilations for some microapplied agonists.

This study asks which occurs first in time for remote responses: a dilation or a remote change in flow. Arteriolar diameter (approximately 20 microm) and fluorescently labeled red blood cell (RBC) velocity were measured in the cremaster muscle of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n = 51). Arterioles were locally stimulated for 60 s with micropipette-applied 10 microg/ml LM-609 (alpha(v)beta(3)-integrin agonist), 10(-3) M adenosine, or 10(-3) M 3-morpholinosydnonimine (SIN-1, nitric oxide donor) as remote response agonists or with 10(-3) M papaverine, which dilates only locally. Observations were made at a remote site 1,200 microm upstream. With LM-609 or adenosine, the RBC velocity increased first (within 5 s), and the remote dilation followed 5-7 s later. N-nitro-L-arginine (100 microM) blocked the LM-609 (100%) and adenosine (60%) remote dilations. SIN-1 induced a concurrent remote dilation and decrease in RBC velocity (approximately 10 s), suggesting the primary signal was to dilate. Papaverine had no remote effects. This study suggests that, although remote responses to some agonists are induced by primary signals to dilate, additionally, network changes in flow can stimulate extensive remote changes in diameter.

Conducted signals within arteriolar networks initiated by bioactive amino acids.

Our purpose was to determine the specificity of L-arginine (L-Arg)-induced conducted signals for intra- vs. extracellular actions of L-Arg. Diameter and red blood cell velocities were measured for arterioles [18 +/- 1.6 (SE) micrometer] in the cremaster muscle of pentobarbital sodium-anesthetized (Nembutal, 70 mg/kg) hamsters (n = 53). Remote (conducted) responses were viewed approximately 1,000 micrometer upstream from the local (micropipette) application. Six amino acids were tested: L-arginine, L-cystine, L-leucine, L-lysine, L-histidine, and L-aspartate (100 microM each). Only L-Arg induced a remote dilation; L-lysine and L-aspartate had no effect, and the others each induced a significant remote constriction. There is a second conducted signal initiated by L-arginine that preconditions the arteriolar network and upregulates a direct response of L-arginine to dilate the remote site. This was blocked by inhibition of L-arginine uptake at the local (preconditioning) site (100 microM L-histidine or 1 mM phenformin). Arginine-glycine-aspartate (100 microM)-induced remote dilations (+3. 2 +/- 0.3 micrometer) were not mimicked by a peptide control and were prevented by anti- integrin alphav monoclonal antibody. Remote dilations were greater in animals with a higher wall shear stress for arginine-glycine-aspartate (r2 = 0.92) but not for L-arginine (r2 = 0.12). Thus L-arginine initiates separate conducted signals related to system y+ transport, integrins, and baseline flow.

Disproportionate epicardial dilation after transmural infarction of the canine left ventricle: Acute and chronic differences

The relation between acute disproportionate infarct dilation and late postinfarct left ventricular remodeling was examined by implanting multiple radiopaque epicardial markers in the left ventricle of eight dogs and determining regional surface deformation after acute and chronic transmural infarction. Transmural injury was produced by combining coronary ligation with distal embolization of a rubber polymer. Dogs were anesthetized and studied before and 1 h, 24 h and 1 week after infarction. Marker positions were recorded by rapid biplane cineradiography, and three-dimensional coordinates were reconstructed by a computer-assisted tracking system. Regional deformation was expressed by a local surface area equal to the sum of multiple (three to four) triangles generated by marker triplets.As early as 1 h after infarction, end-diastolic area in the infarct region increased by 20.3 ± 3.1%, while that in the remote region increased by only 7.9 ± 3.5%. Both changes and the difference between them were significant. At 24 h after infarction, both territories continued to undergo dilation, this time to a similar extent (additional +10.3% in the remote region and +10.1% in the infarct region), thus maintaining the significant disproportionate infarct dilation. At 1 week, however, the infarct territory remained dilated with a mean end-diastolic area 31.4 ± 3.1% above control, while that in the remote region returned to a net mean 8.5 ± 4.7% increase.Thus, the major extent of disproportionate infarct dilation occurs within 1 h after transmural injury and is accompanied by remote dilation as a compensatory response. The extent of further infarct dilation achieved by 24 h is maintained in the chronic infarct, and compensatory mechanisms enable noninjured myocardium to become less dilated. This is an even earlier time course than previously demonstrated, which suggests that attempts to limit infarct dilation should target the acute phase of injury.