Tail Artery Research Articles

A paradoxical increase of force development in saphenous and tail arteries from heterozygous ANO1 knockout mice.

A Ca2+‐activated Cl− channel protein, ANO1, is expressed in vascular smooth muscle cells where Cl− current is thought to potentiate contraction by contributing to membrane depolarization. However, there is an inconsistency between previous knockout and knockdown studies on ANO1’s role in small arteries. In this study, we assessed cardiovascular function of heterozygous mice with global deletion of exon 7 in the ANO1 gene. We found decreased expression of ANO1 in aorta, saphenous and tail arteries from heterozygous ANO1 knockout mice in comparison with wild type. Accordingly, ANO1 knockdown reduced the Ca2+‐activated Cl− current in smooth muscle cells. Consistent with conventional hypothesis, the contractility of aorta from ANO1 heterozygous mice was reduced. Surprisingly, we found an enhanced contractility of tail and saphenous arteries from ANO1 heterozygous mice when stimulated with noradrenaline, vasopressin, and K+‐induced depolarization. This difference was endothelium‐independent. The increased contractility of ANO1 downregulated small arteries was due to increased Ca2+ influx. The expression of L‐type Ca2+ channels was not affected but expression of the plasma membrane Ca2+ ATPase 1 and the Piezo1 channel was increased. Expressional analysis of tail arteries further suggested changes of ANO1 knockdown smooth muscle cells toward a pro‐contractile phenotype. We did not find any difference between genotypes in blood pressure, heart rate, pressor response, and vasorelaxation in vivo. Our findings in tail and saphenous arteries contrast with the conventional hypothesis and suggest additional roles for ANO1 as a multifunctional protein in the vascular wall that regulates Ca2+ homeostasis and smooth muscle cell phenotype.

Fine tuning by protein kinases of CaV1.2 channel current in rat tail artery myocytes

Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular CaV1.2 channel current (ICa1.2) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate ICa1.2 and disclose any unexpected modulation of CaV1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced ICa1.2 in dialysed cells, while weakly increasing it under the perforated configuration. The β-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased ICa1.2; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced ICa1.2, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased ICa1.2, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated ICa1.2 in a concentration-dependent manner; conversely, the PKCα inhibitor Gö6976 markedly reduced basal ICa1.2 and, similarly to the PKCδ (rottlerin) and PKCε translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited ICa1.2 in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Gö6976 appeared to block directly CaV1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α1C subunit in a region involved in CaV1.2 channel inactivation, forming an interaction network comparable to that of CaV1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.

Biological effects of inhaled hydraulic fracturing sand dust. VI. Cardiovascular effects

Hydraulic fracturing is used to access oil and natural gas reserves. This process involves the high-pressure injection of fluid to fracture shale. Fracking fluid contains approximately 95% water, chemicals and 4.5% fracking sand. Workers may be exposed to fracking sand dust (FSD) during the manipulation of the sand, and negative health consequences could occur if FSD is inhaled. In the absence of any information about its potential toxicity, a comprehensive rat animal model study (see Fedan et al., 2020) was designed to investigate the bioactivities of several FSDs in comparison to MIN-U-SIL® 5, a respirable α-quartz reference dust used in previous animal models of silicosis, in several organ systems. The goal of this study was to assess the effects of inhalation of one FSD, i.e., FSD 8, on factors and tissues that affect cardiovascular function. Male rats were exposed to 10 or 30 mg/m3 FSD (6 h/d for 4 d) by whole body inhalation, with measurements made 1, 7 or 27 d post-exposure. One day following exposure to 10 mg/m3 FSD the sensitivity to phenylephrine-induced vasoconstriction in tail arteries in vitro was increased. FSD exposure at both doses resulted in decreases in heart rate (HR), HR variability, and blood pressure in vivo. FSD induced changes in hydrogen peroxide concentrations and transcript levels for pro-inflammatory factors in heart tissues. In kidney, expression of proteins indicative of injury and remodeling was reduced after FSD exposure. When analyzed using regression analysis, changes in proteins involved in repair and remodeling were correlated. Thus, it appears that inhalation of FSD does have some prolonged effects on cardiovascular, and, possibly, renal function. The findings also provide information regarding potential mechanisms that may lead to these changes, and biomarkers that could be examined to monitor physiological changes that could be indicative of impending cardiovascular dysfunction.

Involvement of brainstem noradrenergic system in cutaneous heat loss during exercise

The involvement of brainstem noradrenergic system in thermoregulation during exercise was evaluated by assessing the neuronal activation of A1, A2, locus coeruleus (LC) during exercise. Male Wistar rats weighing 280−330 g were used in the present study. Ninety minutes after exercise bout until fatigue, animals were anaesthesiated and brain removed and processed immunohistochemically for Fos protein and tyrosine hydroxylase in A1, A2 and LC and for Fos in POA subregions. Core and tail temperature were recorded during all running period by telemetry system. Heat storage rate (HSR, cal.min−1), maximum tail vasoconstriction (°C) and vasodilatation threshold (°C) were calculated and correlated with Fos expression in all nuclei studied. Fos expression in LC correlated inversely with maximum tail skin vasoconstriction (r = −0.787, p < 0.03) and HSR (r = −0.834, p < 0.02) and positively to time to fatigue (r = 0.862, p < 0.01). A1 nucleus showed an inverse correlation with tail skin vasodilatation threshold (r = −0.861, p < 0.01). Fos expression in LC correlated inversely with Fos expression in the median (MnPO, r = −0.909, p < 0.01) and medial preoptic nucleus (MPOM, r = −0.942, p < 0.05). Our results bring further evidences that noradrenergic neurons from LC and A1 nuclei are involved in cutaneous heat loss mechanisms during exercise. LC nucleus probably modulates the sympathetic tonus of tail artery and integrates the central network LC / POA that could represent an important circuitry of temperature regulation during exercise. Also, noradrenergic neurons from A1 nucleus could be involved in cutaneous heat loss during exercise by modulating of vasodilatation threshold.

Pharmacological Effects of a Novel Bradykinin-Related Peptide (RR-18) from the Skin Secretion of the Hejiang Frog (Ordorrana hejiangensis) on Smooth Muscle.

Bradykinin (BK) and bradykinin-related peptides (BRPs), which were identified from a diversity of amphibian skin secretions, exerted contractile and relaxing effects on non-vascular and vascular smooth muscle, respectively. Here, we report a novel bradykinin-related peptide with a molecular mass of 1890.2 Da, RVAGPDKPARISGLSPLR, which was isolated and identified from Ordorrana hejiangensis skin secretions, followed by a C-terminal extension sequence VAPQIV. The biosynthetic precursor-encoding cDNA was cloned by the “shotgun” cloning method, and the novel RR-18 was identified and structurally confirmed by high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the myotropic activity of the synthetic replicate of RR-18 was investigated on the rat bladder, uterus, tail artery and ileum smooth muscle. The peptide was named RR-18 in accordance (R = N-terminal arginine, R = C-terminal arginine, 18 = number of residues). In this study, the synthetic replicates of RR-18 showed no agonist/antagonism of BK-induced rat bladder and uterus smooth muscle contraction. However, it displayed an antagonism of bradykinin-induced rat ileum contraction and arterial smooth muscle relaxation. The EC50 values of BK for ileum and artery, were 214.7 nM and 18.3 nM, respectively. When the tissue was pretreated with the novel peptide, RR-18, at the maximally effective concentration of bradykinin (1 × 10−6 M), bradykinin-induced contraction of the ileum and relaxation of the arterial smooth muscle was reduced by 50–60% and 30–40%, respectively. In conclusion, RR-18 represents novel bradykinin antagonising peptide from amphibian skin secretions. It may provide new insight into possible treatment options for chronic pain and chronic inflammation.

Neuroprotective effects of quercetin on cerebral vasospasm following experimental subarachnoid haemorrhage in rats

Background/aim We examined the protective effects of the natural flavonoid, quercetin, against cerebral vasospasm in an experimental rat subarachnoid haemorrhage (SAH) model. Materials and methods Thirty-eight albino Wistar rats were divided into five groups as follows: group 1 (G1, n=8), no experimental intervention; group 2 (G2, n=8), subarachnoid physiological saline; group 3 (G3, n=8), SAH; group 4 (G4, n=7) SAH and low-dose (10 mg/kg) quercetin treatment; group 5 (G5, n=7), SAH and high-dose (50 mg/kg) quercetin treatment. Subarachnoid haemorrhage was induced by injection of 0.15 cc of autologous blood taken from the tail artery into the cisterna magna from the craniocervical junction and basilar arteries and blood samples were taken for biochemical and histopathological analyses. Results Malondialdehyde (MDA) levels were significantly higher in G2 and G3 than in G1 (P < 0.05). Significant decreases in MDA were observed in G4 and G5 compared with G2 (P < 0.05, G4–G2; P < 0.05, G5–G2). There were no significant differences between G2 and G3 or among G1, G4, and G5. No statistically significant differences were found in total antioxidant capacity between the groups (P > 0.05). There were no significant differences in basilar artery (BA) wall thickness between G3 and G4 or between G3 and G5, but G4 and G5 showed greater luminal diameters than G3 (P < 0.05). There were no significant differences in BA thickness or luminal diameter between G4 and G5. Conclusion Our results suggested that quercetin may be beneficial in SAH therapy by preventing vasospasm.

Comparison of mRNA Expression of P2X Receptor Subtypes in Different Arterial Tissues of Rats.

This study aims to compare the expression of P2X receptor subtype mRNA in different arterial tissues of rats. After the rats were sacrificed, the internal carotid, pulmonary, thoracic aorta, mesenteric and caudal arteries were dissected out. Then, the P2X receptor mRNA expression in different blood vessels was detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction. The P2X1, P2X4 and P2X7 receptor mRNA amplification products revealed specific bands of the same size as the amplified target fragment in their respective lanes, while the P2X2, P2X3, P2X5 and P2X6 receptor mRNA amplification products did not reveal significant specific bands in their respective lanes by RT-PCR. Based on the P2X1 receptor mRNA expression of the mesenteric artery, there were no significant differences in the internal carotid, pulmonary and thoracic aorta (0.64 ± 0.07, 0.17 ± 0.11 and 1.49 ± 0.65, respectively). However, the P2X1 receptor mRNA expression level in the caudal artery significantly increased (11.06 ± 1.99, P < 0.01). Furthermore, there was no difference in P2X4 receptor mRNA expression among these five blood vessels (P > 0.05). The P2X7 receptor mRNA expression level was significantly different: pulmonary artery < tail artery = thoracic aorta < internal carotid artery < mesenteric artery. The relative P2X1 receptor mRNA expression in the caudal artery was observed to be elevated when compared to that of the internal carotid, pulmonary and thoracic aorta as well as the mesenteric arteries. The P2X7 receptor mRNA expression level is pulmonary artery < caudal artery = thoracic aorta < internal carotid artery < mesenteric artery. P2X4 receptor mRNA expression was not significantly different among these five blood vessels.

Efficiency of Minimized Circuits of a Heart Roller Pump on Systemic Inflammatory Response Syndrome and Multiorgan Effects in a Rat Model.

The aim of this study is to compare the effects of tubing length on systemic inflammatory response syndrome and myocardial protection in a rat model of cardiopulmonary bypass (CPB) from a histological standpoint. Twelve adult male Wistar Albino rats weighing >180 g were randomly selected and divided into 2 groups. In 1 group, the pump lines were kept 1 m shorter than standard. The right jugular vein and tail artery were cannulated using a 16-gauge catheter. Animals received 500 IU/kg intravenous heparin. Cardiac index and rectal temperature were set at 2.4 mL and 36°C, respectively. Total line volume was maintained at 8 mL. A roller pump was adjusted to supply a blood flow of 6 to 28 mL/min (mean 10 mL/min), similar to the typical cardiac output of rats. CPB duration was 15 minutes throughout the experiment. After sacrifice, tissue samples were collected from heart, liver, and kidney for histomorphologic examination. All histochemical and histomorphologic analyses, performed by 2 blinded researchers, revealed band loss in cardiomyocytes, mononuclear (MNL) cell infiltration, and impaired fibrillar organization in the standard-line group. Additionally in that group, sinusoidal dilatation in the liver, low-level congestion, focal necrosis, and periportal MNL infiltration were noted. In the shorter-line group, on the other hand, MNL cell infiltration, band loss in myofibrils, and cardiomyocyte degeneration were rarely observed. Higher liver congestion and lower MNL cell infiltration were observed in the shorter-line group. No significant differences were found in kidney samples. In a shorter-line roller pump test model, less multiorgan damage and fewer systemic inflammatory responses were observed. It may be applicable to keep CPB lines as close to the table as possible, especially in pediatric cardiac surgery cases.

Pharmacokinetic studies of moracin C in mice using various blood sampling methods

Mice have been increasingly used in pharmacokinetic evaluation due to using less amount of compound and availability of various disease models and blood sampling methods in mice have produced the distinct pharmacokinetic profiles even in the same compounds. In this study, three blood sampling methods [i.e., automated dosing/blood sampling (ABS), tail artery collection (TA) and heart puncture (HP)] were applied to investigate the pharmacokinetics of moracin C in mice. Depending on blood sampling sites, significant differences were observed in plasma concentrations as well as pharmacokinetic parameters such as AUC (area under the plasma‐time concentration curve). The AUC values in ABS and TA groups were smaller than that in HP group. Considering that moracin C was greatly distributed to liver and gastrointestinal tracts (e.g., stomach, small intestine, and large intestine) and was extensively metabolized via glucuronidation in liver and small intestines, the AUC differences could be mainly caused by the stronger stress from blood sampling via heart puncture, which might interfere the elimination (i.e., hepatic and/or intestinal first‐pass effects). The remaining amounts of moracin C in gastrointestinal tract at 24 h after its oral administration were similar among three groups with different blood sampling methods, indicating that the absorption of moracin C was not affected during the blood sampling stress of HP. These results indicate that the ABS or TA method can be utilized to obtain better reliable and accurate pharmacokinetic parameters in mice.Support or Funding InformationThis study was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF‐2016R1C1B2010849, NRF‐2019R1A2C2009053 and NRF‐2018R15A2023217).

Abstract No. 438 Tail artery access for transarterial experiments in rats: feasibility study in a survival model

The rat is an increasingly valuable model to study interventional therapeutic procedures. Suitable arterial access is essential for vascular intervention. In rats, transtail artery access has been recently reported as a feasible technique compared to conventional carotid or femoral access. The aim of this study was to evaluate repeat accessibility and complications of transtail angiography in a survival model. Seventeen male Sprague-Dawley rats were used (460.2 ± 45.0g), one rat died before the procedure, hence 16 rats were included in this study. 1-cm longitudinal ventral incisions were created 5 cm distal to the base of the tail to dissect the tail artery (ventral caudal artery). A 1.5–1.7 Fr microcatheter was advanced over a 0.010–0.014 inch microguidewire through a partial arteriotomy. Selective renal angiography was then performed. Intraarterial antibiotics injection was performed in 11 of 16 rats. The accessed segment of the tail artery was ligated and the incision closed with sutures. Repeat renal angiography via the tail artery was performed 48 hours after the initial procedure using a separate tail incision 1 cm proximal to the initial incision. Procedure time from skin incision to catheterization was recorded. Complications including tail ischemia, infection and bleeding were evaluated during the 2-day follow-up period. All initial and repeat procedures were successfully performed. The average time for the 32 tail artery cannulations was 11.0 ± 4.5 min. Transient focal skin color change in the tail was observed in one rat immediately after the initial procedure, which resolved within 90 mins. No tail necrosis, major bleeding or signs of infection were observed. Dehiscence of the incision was observed in 3 of 16 rats from self-grooming, with no bleeding or infection. Repeat tail artery access for angiography in rats was successful without major complications in a survival model. This reproducible technique can provide a new experimental platform to test transarterial procedures.

Mechanisms underlying the improvement of preeclampsia through salvianolic acid B-regulated miRNA-155/CXCR4.

The aim of this study was to evaluate the effects and mechanisms of salvianolic acid B (Sal B) in preeclampsia treatment by in vivo and in vitro study. Rats were randomly divided into 5 groups. In order to establish the model of preeclampsia, endotoxin was administered to the rats in the Sal B intervention and model groups. The systolic blood pressure (SBP) of the tail artery and urine protein concentration were observed at different points, the miRNA-155 and CXCR4 gene expression levels by RT-PCR and the CXCR4 and p-AKT protein expression by WB assay. Using HTR8/SVneo to explain the mechanisms; evaluating the miRNA-155 and CXCR4 mRNA expression by RT-PCR assay, measuring the cell invasion and migration by transwell and wound healing assay in different groups; evaluating the CXCR4 and p-AKT protein expression by WB assay and p-AKT nucleation volume by cellular immunofluorescence were evaluated. Compared with the normal group, the systolic blood pressure and urine protein were significantly increased in the model group (p < 0.05), serum NO concentration was significantly down-regulated (all p < 0.05), CXCR4 and miRNA-155 mRNA expression was significantly different and CXCR4 and p-AKT protein expression was significantly suppressed (all p < 0.05). With Sal B supplement, the SBP, urine protein and NO concentration were significantly improved with dose-dependent (all p < 0.05). In the cell experiment, the cell invasion and migration ability were significantly improved with Sal B supplement (both p < 0.05). However, with miRNA-155 transfection, the cell invasion and migration ability were suppressed with Sal B treatment (both p < 0.05). Sal B improved preeclampsia via regulation of miRNA-155/CXCR4 in the in vitro and vivo study.

Abstract TP280: A Bland-new Rat Model of Embolic Cerebral Ischemia Using Micro Catheter Under Fluoroscopic Guide

Background: The failure of clinical trials on neuroprotective treatment may be partially caused by unestablished animal models. To verify a trans-arterial regeneration therapy, our project is aimed at developing a brand-new focal stroke model using micro catheter. Methods: Using Sprague-Dawley rats (n=8), a micro catheter (ID 0.26mm, OD 0.35mm) was navigated from the caudal ventral artery to the middle cerebral artery in order to establish local occlusion under digital subtraction angiography apparatus. Results: We succeeded in brain angiography by percutaneous tail artery puncture and occlusion of the middle cerebral artery by a radiopaque micro bead with 6 rats (75%). Ischemic stroke lesion volume is 480.3 mm3±84.2 mm3 on sustained 1% TTC solution after 24 hours occlusion. Conclusion: We present a new rat model for focal stroke using micro catheter under fluoroscopic control. The model is capable of repeated super selective administration of therapeutics directly to cerebral artery, and practice the 3Rs principles in experimental animals because of minimal invasive.Figure Legend- A: A sheath is inserted through the ventral midline, approximately 5 cm from the root of the tail of the rat, with a sharp angle. B: Cerebral angiography of rats. Arrowhead pointe middle cerebral artery. C: Occlusion of the middle cerebral artery by a radiopaque micro bead (arrowhead). D: Brain is sliced and stained 1% TTC solution after 24 hours occlusion.

Vasodilation of rat skeletal muscle arteries by the novel BK channel opener GoSlo is mediated by the simultaneous activation of BK and Kv 7 channels.

Background and PurposeBK channels play important roles in various physiological and pathophysiological processes and thus have been the target of several drug development programmes focused on creating new efficacious BK channel openers, such as the GoSlo‐SR compounds. However, the effect of GoSlo‐SR compounds on vascular smooth muscle has not been studied. Therefore, we tested the hypothesis that GoSlo‐SR compounds dilate arteries exclusively by activating BK channels.Experimental ApproachExperiments were performed on rat Gracilis muscle, saphenous, mesenteric and tail arteries using isobaric and isometric myography, sharp microelectrodes, digital droplet PCR and the patch‐clamp technique.Key ResultsGoSlo‐SR compounds dilated isobaric and relaxed and hyperpolarised isometric vessel preparations and their effects were abolished after (a) functionally eliminating K+ channels by pre‐constriction with 50 mM KCl or (b) blocking all K+ channels known to be expressed in vascular smooth muscle. However, these effects were not blocked when BK channels were inhibited. Surprisingly, the Kv7 channel inhibitor XE991 reduced their effects considerably, but neither Kv1 nor Kv2 channel blockers altered the inhibitory effects of GoSlo‐SR. However, the combined blockade of BK and Kv7 channels abolished the GoSlo‐SR‐induced relaxation. GoSlo‐SR compounds also activated Kv7.4 and Kv7.5 channels expressed in HEK 293 cells.Conclusion and ImplicationsThis study shows that GoSlo‐SR compounds are effective relaxants in vascular smooth muscle and mediate their effects by a combined activation of BK and Kv7.4/Kv7.5 channels. Activation of Kv1, Kv2 or Kv7.1 channels or other vasodilator pathways seems not to be involved.

The Dorsomedial Hypothalamus Is Involved in the Mediation of Autonomic and Neuroendocrine Responses to Restraint Stress.

We hypothesized that dorsomedial hypothalamus (DMH) modulates autonomic and neuroendocrine responses in rats at rest and when subjected to restraint stress (RS). Male Wistar rats were used, and guide cannulas were bilaterally implanted in the DMH for microinjection of vehicle or the nonspecific synaptic blocker CoCl2 (1 mM/100 nl). A polyethylene catheter was inserted into the femoral artery for the recording of arterial pressure and heart rate (HR). Tail temperature was measured using a thermal camera. The session of RS started 10 min after DMH treatment with vehicle or CoCl2. Under home-cage condition, the pretreatment of DMH with CoCl2 increased baseline blood pressure (BP), and heart rate (HR) without affecting the tail temperature. In addition, it decreased plasma vasopressin levels without affecting plasma corticosterone and oxytocin contents. When rats pretreated with CoCl2 were exposed to RS, the RS-evoked cardiovascular were similar to those observed in vehicle-treated animals; however, because cobalt pretreatment of the DMH increased baseline BP and HR values, and the RS-evoked cardiovascular responses did not exceed those observed in vehicle-treated animals, suggesting a possible celling limit, the possibility that DMH is involved in the modulation of RS-evoked cardiovascular responses cannot be certainly excluded. Nonetheless, the pretreatment of DMH with CoCl2 blocked the reduction in tail temperature caused by RS. The DMH pretreatment with CoCl2 did not modify the RS-evoked increase in plasma corticosterone and oxytocin contents. In conclusion, the present data suggest the involvement of DMH in the maintenance of BP, HR, and vasopressin release under the rest conditions at the home-cage. Furthermore, indicate that DMH is an important thermoregulatory center during exposure to RS, regulating tail artery vasoconstriction.

Frequency-dependent changes in mitochondrial number and generation of reactive oxygen species in a rat model of vibration-induced injury

ABSTRACTRegular use of vibrating hand tools results in cold-induced vasoconstriction, finger blanching, and a reduction in tactile sensitivity and manual dexterity. Depending upon the length and frequency, vibration induces regeneration, or dysfunction and apoptosis, inflammation and an increase in reactive oxygen species (ROS) levels. These changes may be associated with mitochondria, this study examined the effects of vibration on total and functional mitochondria number. Male rats were exposed to restraint or tail vibration at 62.5, 125, or 250 Hz. The frequency-dependent effects of vibration on mitochondrial number and generation of oxidative stress were examined. After 10 days of exposure at 125 Hz, ventral tail arteries (VTA) were constricted and there was an increase in mitochondrial number and intensity of ROS staining. In the skin, the influence of vibration on arterioles displayed a similar but insignificant response in VTA. There was also a reduction in the number of small nerves with exposure to vibration at 250 Hz, and a reduction in mitochondrial number in nerves in restrained and all vibrated conditions. There was a significant rise in the size of the sensory receptors with vibration at 125 Hz, and an elevation in ROS levels. Based upon these results, mitochondria number and activity are affected by vibration, especially at frequencies at or near resonance. The influence of vibration on the vascular system may either be adaptive or maladaptive. However, the effects on cutaneous nerves might be a precursor to loss of innervation and sensory function noted in workers exposed to vibration.

Endothelium-Independent Vasodilatory Effect of Sailuotong (SLT) on Rat Isolated Tail Artery.

Background Sailuotong (SLT) is a standardized three-herb formulation consisting of extracts of Panax ginseng, Ginkgo biloba, and Crocus sativus for the treatment of vascular dementia (VaD). Although SLT has been shown to increase cerebral blood flow, the direct effects of SLT on vascular reactivity have not been explored. This study aims to examine the vasodilatory effects of SLT and the underlying mechanisms in rat isolated tail artery. Methods Male (250–300 g) Wistar Kyoto (WKY) rat tail artery was isolated for isometric tension measurement. The effects of SLT on the influx of calcium through the cell membrane calcium channels were determined in Ca2+-free solution experiments. Results SLT (0.1–5,000 μg/ml) caused a concentration-dependent relaxation in rat isolated tail artery precontracted by phenylephrine. In the contraction experiments, SLT (500, 1,000, and 5,000 μg/mL) significantly inhibited phenylephrine (0.001 to 10 μM)- and KCl (10–80 mM)-induced contraction, in a concentration-dependent manner. In Ca2+-free solution, SLT (500, 1,000, and 5,000 μg/mL) markedly suppressed Ca2+-induced (0.001–3 mM) vasoconstriction in a concentration-dependent manner in both phenylephrine (10 μM) or KCl (80 mM) stimulated tail arteries. L-type calcium channel blocker nifedipine (10 μM) inhibited PE-induced contraction. Furthermore, SLT significantly reduced phenylephrine-induced transient vasoconstriction in the rat isolated tail artery. Conclusion SLT induces relaxation of rat isolated tail artery through endothelium-independent mechanisms. The SLT-induced vasodilatation appeared to be jointly meditated by blockages of extracellular Ca2+ influx via receptor-gated and voltage-gated Ca2+ channels and inhibition of the release of Ca2+ from the sarcoplasmic reticulum.

Decellularization of submillimeter-diameter vascular scaffolds using peracetic acid

Various decellularization methods for allogenic and xenogenic bioscaffolds have been previously reported; however, decellularization methods for very thin (submillimeter-diameter) vascular tissues have not been discussed well. In this study, rat tail arteries (inner diameter, 0.6mm) were decellularized with peracetic acid (PAA) and DNase I. PAA treatment is expected not only to disrupt cell membranes which improves the decellularization efficiency in the subsequent DNase treatment, but also to sterilize vascular scaffolds. We succeeded in adequate cell removal by immersing in 0.3% isotonic PAA solution and subsequent washing with DNase solution. For the DNase washing process, the perfusion method was superior in terms of cell removal to the static immersion method. Graft lumen was modified with a peptide composed of a collagen binding sequence and endothelial progenitor cell-binding sequence, (Pro-Hyp-Gly)7-Gly-Gly-Gly-Arg-Glu-Asp-Val, as previously reported. They were patent in rat allogeneic transplantation model for 2weeks, but unexpectedly resulted in graft rupture or tear formation, thereafter, suggesting reduced mechanical strength of the decellularized scaffolds. Histology showed that the thickness of the extracellular matrix (ECM) was decreased by the perfusion of the DNase solution. The method of combination of PAA and DNase was not necessarily optimal for the decellularization of very thin vascular tissues. The decellularization method is a compromise between effective cell removal and maintenance of the ECM nature. Since the acceptability of ECM denaturation by the host tissue highly depends on individual cases, decellularization methods should be carefully selected according to the type of target tissue and its intended use.

Effect of electroacupuncture on renal fibrosis in spontaneously hypertension rats and its related mechanisms

To observe the effect of electroacupuncture (EA) on blood pressure, renal fibrosis and expression of tissue inhibitors of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor 1 (PAI-1), and alpha smooth muscle actin (α-SMA) in spontaneous hypertension rats (SHR), so as to explore its mechanisms underlying improving hypertensive renal damage. Forty male SHR (15 weeks in age) were randomly divided into 5 groups: model, medication (Losartan), Shenshu, Geshu, and Shenshu＋Geshu groups(n=8 rats in each group), and the same age-old male 8 Wistar-Kyoto (WKY) rats were used as the normal control group. Rats of the medication group were treated by gavage of Losartan potassium solution (3 mg/mL, 30 mg·kg-1·d-1, once a day for 12 weeks), and those of the 3 EA groups treated by EA stimulation of bilateral "Shenshu" (BL23), "Geshu"(BL17) or both BL23 and BL17 (2 Hz/100 Hz, 1 mA, 15 min each time, once every other day for 12 weeks). The systolic blood pressure of the tail artery was measured before, and 4, 8 and 12 weeks after the intervention. The expression of TIMP-1, PAI-1 and α-SMA proteins of the right kidney tissue was measured by immunohistochemistry. Histopathological changes of the right renal tissue were observed under light microscope after H.E. stain. The blood pressure was significantly higher in the mo-del group than those in the normal control group (P<0.01), and considerably decreased at the 4th , 8th, and 12th week of the interventions in the medication and 3 EA groups (P<0.01). The expression levels of renal TIMP-1, PAI-1 and α-SMA proteins were notably higher in the model group than those in the normal control group and considerably decreased at the 12th week of the interventions in the medication and 3 EA groups than in the model group (P<0.01). H.E. staining of the renal tissue showed disordered arrangement of the renal cells, congestion and dilation of capillaries with thickened vascular wall, renal tubule atrophy and lumen stenosis with some necrosis of renal tubules, protein tubule and cell tubules, increase of some glomerular mesangial matrix and hyperplasia of fibrous tissue in the model group, which was re-latively milder in the medication and 3 EA groups. EA of BL23 and BL17 can reduce the blood pressure in SHR, which may be related to its function in down-regulating expression of TIMP-1, PAI-1 and α-SMA proteins.

Neuroprotective Potential of Tetrahydrobiopterin Using a Rat Model

Introduction: Spinal cord ischemia (SCI) is one of the most devastating complications in thoracoabdominal aortic aneurysm surgery and the identification of neuroprotective substances to prevent this complication is of great interest. Tetrahydrobiopterin (BH4) is one of five essential cofactors of nitric oxide synthase (NOS) and is crucial for the conversion of L-arginine and oxygen to L-citrullin and NO. Ischemia reperfusion injury Is associated with intracellular BH4 depletion. This in turn, is known to be associated with the uncoupling of NOS leading to an increased production of hydrogen peroxide and superoxide anion resulting in endothelial dysfunction. Supplementation of BH4 has been shown to play a protective role in ischemia-reperfusion injury following solid organ transplantation. We therefore wanted to test its neuroprotective potential using a SCI model in rats. Methods: SCI has been induced in male Sprague Dawley rats (300 - 350g weight) by balloon occlusion of the descending thoracic aorta via insertion of a 2F Fogarty catheter. Blood pressure monitoring via the left carotid artery and the tail artery was performed to confirm sufficient balloon occlusion. Animals were subdivided into a sham group (n=12), a treatment group (50 mg/kg BH4 intramuscular 15 minutes before SCI; n=11) and a control group (intramuscular saline injection 15 minutes before SCI; n=12). Blinded neurofunctional testing was performed using the Bassio-Beattie-Breshnahan (BBB) locomotor rating scale ranging from 0 (no hind limb movement) to 21 (normal hind limb movement). After an observation period of 7 days, animals were sacrificed and tissue was harvested for further histopathological evaluation using H.E. staining and TUNEL assays. Results: The model was associated with unexpected high perioperative mortality due to hemodynamic and embolic complications. Within the control group, 3 animals (n=12; 25%) and within the treatment group 4 animals (n=11; 36%) survived until day 7. Pre-treatment with BH4 lead to significant better neurofunctional outcome 2 hours after awakening of animals (BBB Score treatment group 18.3 ± 3.4 vs. control group 15.3 ± 2.1; p=0.014). The difference of BBB Scores showed a trend towards a better outcome after pre-treatment with BH4 at day 7 (treatment group 21 ± 0 vs. 18.3 ± 3.8; p=0.205). Histopathological evaluation using H.E. staining revealed no difference between treatment and control group when counting the mean number of neurons in thoracic spinal cords. By using TUNEL assays for detection of vital neurons there was a clear trend towards a benefit of pre-treatment with BH4 (mean number of vital neurons treatment group 13.28 ± 4.77 vs control group 11.89 ± 4.04; p=0.117). Conclusion: We demonstrate a neuroprotective potential of BH4 as immediate postoperative neurofunctional outcome was significantly better in pre-treated animals. Due to the unexpected high postoperative mortality results of neurofunctional and histopathological evaluations at day 7 should be taken with caution. However, the observed trend towards a benefit of BH4 pre-treatment urges to repeat the study with a shorter postoperative observation period. Disclosure: Nothing to disclose

Modulatory effect of laser irradiation on mastoparan-7-induced contraction.

Mastoparan-7 activates guanine nucleotide-binding proteins (G-proteins) and stimulates both apoptosis and increases in cytoplasmic calcium concentration and may induce smooth muscle contraction. The primary aim of the present study was to evaluate the modulatory effect of laser stimulation on vascular smooth muscle contraction induced by direct stimulation of G-protein with mastoparan-7. Experiments were performed on isolated and perfused tail arteries of Wistar rats. Contraction force in the model was measured by increased levels of perfusion pressure with a constant flow. Irradiation treatment was applied directly to the blood vessels. The laser was applied in increasing doses of 10 mW (E=1.8 J), 30 mW (E=5.5 J) and 110 mW (E=19.8 J). Time of exposure was 3 min for each irradiation. In the laser-stimulated arteries, a significant and dose-dependent decrease was observed. The half maximal effective concentration values were 4.43±2.2x10-8, 2.4±0.56x10-7, 3.2±0.72x10-7 and 7.7±0.3x10-7 M/l in the control and 10, 30 and 110 mW laser irradiation groups, respectively. Significant (P<0.001) changes were identified for all laser treatment groups in comparison with the control. When analyzing the function of calcium ion (Ca2+) stores was analyzed, a significant inhibition of influx from both intra- and extracellular Ca2+ stores was observed. The results from the present study suggested that contraction induced by direct activation of G-protein with mastoparan-7 may by effectively inhibited by laser radiation, and that the effect was associated with an inhibition of Ca2+ influx from both intracellular and extracellular Ca2+ stores.