Effect of Nectaroscordum koelzi Methanolic Extract on Acute and Chronic Inflammation in Male Mice.

INTRODUCTION: The present study deals with the effect of Nectaroscordum koelzi fruit extract on acute and chronic inflammation. METHODS: A total of 84 NMRI mice were used in this study. The effect of the extract on acute inflammation was analyzed by increasing vascular permeability via acetic acid and xylene induced ear edema among mice. The extract was evaluated in terms of effects on chronic inflammation by means of the cotton pellet test among mice. For the assessment of inflammation degree, the mice paw edema volume was measured by the plethysmometric test. RESULTS: The findings showed that the extract was effective on acute inflammation induced by acetic acid in mice. In the xylene ear edema, N. koelzi extract indicated a significant activity in mice. In the cotton pellet method, the methanol extract produced a significant reduction in comparison with the control and dexamethasone. Mice paw edema volume decreased with the extract. CONCLUSION: In general, the data from the experiments indicated that the methanol extract of N. koelzi has an anti-inflammatory effect on acute and chronic inflammation. However, the exact contributing mechanisms have not been investigated for the pharmacological effects.

High-precision, non-invasive anti-microvascular approach via concurrent ultrasound and laser irradiation.

Antivascular therapy represents a proven strategy to treat angiogenesis. By applying synchronized ultrasound bursts and nanosecond laser irradiation, we developed a novel, selective, non-invasive, localized antivascular method, termed photo-mediated ultrasound therapy (PUT). PUT takes advantage of the high native optical contrast among biological tissues and can treat microvessels without causing collateral damage to the surrounding tissue. In a chicken yolk sac membrane model, under the same ultrasound parameters (1 MHz at 0.45 MPa and 10 Hz with 10% duty cycle), PUT with 4 mJ/cm2 and 6 mJ/cm2 laser fluence induced 51% (p = 0.001) and 37% (p = 0.018) vessel diameter reductions respectively. With 8 mJ/cm2 laser fluence, PUT would yield vessel disruption (90%, p < 0.01). Selectivity of PUT was demonstrated by utilizing laser wavelengths at 578 nm or 650 nm, where PUT selectively shrank veins or occluded arteries. In a rabbit ear model, PUT induced a 68.5% reduction in blood perfusion after 7 days (p < 0.001) without damaging the surrounding cells. In vitro experiments in human blood suggested that cavitation may play a role in PUT. In conclusion, PUT holds significant promise as a novel non-invasive antivascular method with the capability to precisely target blood vessels.

Observation of vasculature alternation by intense pulsed light combined with physicochemical methods.

Intense pulsed light (IPL) with low energy insufficient to completely destroy a vasculature was applied to rabbit ears to investigate vasculature alteration. Glycerol was combined with IPL to enhance the transfer efficacy of IPL energy. Both trans-illumination and laser speckle contrast images were obtained and analyzed after treatment. The application of IPL and glycerol combination induced vasodilation and improvement in blood flow. Moreover, such phenomenon was maintained over time. IPL may be applied to treat blood circulatory diseases by inducing vasodilation and to improve blood flow.

Long-circulating lipoprotein-mimic nanoparticles for smart intravenous delivery of a practically-insoluble antineoplastic drug: Development, preliminary safety evaluations and preclinical pharmacokinetic studies.

Chlorambucil (CHL) is a water-insoluble antineoplastic drug having a short elimination half-life. It suffers from remarkable differences in pharmacokinetics following oral administration. The current work aimed to assess safety and pharmacokinetics of CHL-loaded, lipoprotein-mimic, nanoparticles (NPs) following intravenous administration. The design of NPs was based on complexation between egg yolk lecithin (EYL) and bovine serum albumin (BSA). The NPs were preliminary evaluated via FT-IR, DSC and P-XRD. The NPs were characterized for particle size, zeta potential, morphology and drug entrapment efficiency (EE%). The best achieved NP dispersion (LP6) and CHL solution were challenged for in vitro hemolytic potential, in vivo vascular irritation studies in rabbits and in vivo pharmacokinetics following intravenous administration in rats. The results confirmed that NPs were stabilized by hydrophobic-attractions and hydrogen-bondings between CHL, BSA and EYL. The amorphous dispersion of CHL within NPs was revealed. LP6 dispersion displayed monodispersed nano-spherical particles (144.33 ± 2.17 nm). It possessed the highest negative zeta potential (-30.55 ± 0.24 mV) and the largest EE% (86.35 ± 2.33%). The significantly (P < 0.05) prolonged MRT(0-∞), longer elimination t50% and reduced plasma clearance highlighted the long-circulating characteristics of LP6. The preliminary safety evaluations and the seven-fold increase in bioavailability elucidated potentiality for smart intravenous delivery of CHL.

[Acupuncture combined with auricle cutting method for blood stasis-type psoriasis: a randomized controlled trial].

OBJECTIVE: To verify the clinical efficacy of acupuncture combined with auricle cutting method for treatment of blood stasis-type psoriasis. METHODS: Fifty-six cases of blood stasis-type psoriasis were randomly divided into a combined therapy group, a auricle cutting group, an acupuncture group and a control group, 14 cases in each one. Based on regular treatment of TCM decoction in four groups, the combined therapy group was treated with acupuncture and auricle cutting method, and the auricle cutting group was treated with sham-acupuncture and auricle cutting, and the acupuncture group was treated with acupuncture and sham auricle cutting, and the control group was treated with sham-acupuncture and sham auricle cutting. The acupuncture was applied at Dazhui (GV 14), Feishu (BL 13), Ganshu (BL 18) and Geshu (BL 17), etc., and manipulated with routine technique; in the sham acupuncture, the needle was inserted into dermis layer so that the needles could be swung without being dropped out. In the auricle cutting, erbeixin (P1) of unilateral auricle was selected and cut by Chan needle to perform bloodletting; in the sham auricle cutting, the neighborhood approximately 0.5 cm next to erbeixin (P) of auricle was selected as cutting area. The treatment was given once a day, seven days as a treatment session for totally two sessions. Psoriasis area and severity index (PASI) before and after treatment was observed and efficacy of each group was compared. RESULTS: The effective rate was 57.1% (8/14) in the combined therapy group, which was superior to 14.3% (2/14) in the auricle cutting group, 7.1% (1/14) in the acupuncture group and 0.0% (0/14) in the control group (all P < 0.05). The scores of PASI were all decreased in each group after the treatment (all P < 0.05), which was the most significant in the combined therapy group (all P < 0.05). After factorial analysis, the main effect was P < 0.05 in the auricle cutting, P < 0.05 in the acupuncture and P < 0.05 in interaction effect of combined therapy. CONCLUSION: The scores of PASI of blood stasis-type psoriasis could be effectively reduced by acupuncture, auricle cutting method and TCM decoction, among which the interaction effect of auricle cutting and acupuncture combined with TCM decoction is the most significant.

Microvascular dysfunction with increased vascular leakage response in mice systemically exposed to arsenic.

The mechanisms underlying cardiovascular disease induced by arsenic exposure are not completely understood. The objectives of this study were to investigate whether arsenic-fed mice have an increased vascular leakage response to vasoactive agents and whether enhanced type-2 protein phosphatase (PP2A) activity is involved in mustard oil-induced leakage. ICR mice were fed water or sodium arsenite (20 mg/kg) for 4 or 8 weeks. The leakage response to vasoactive agents was quantified using the Evans blue (EB) technique or vascular labeling with carbon particles. Increased EB leakage and high density of carbon-labeled microvessels were detected in arsenic-fed mice treated with mustard oil. Histamine induced significantly higher vascular leakage in arsenic-fed mice than in water-fed mice. Pretreatment with the PP2A inhibitor okadaic acid or the neurokinin 1 receptor (NK1R) blocker RP67580 significantly reduced mustard oil-induced vascular leakage in arsenic-fed mice. The protein levels of PP2Ac and NK1R were similar in both groups. PP2A activity was significantly higher in the arsenic-fed mice compared with the control group. These findings indicate that microvessels generally respond to vasoactive agents, and that the increased PP2A activity is involved in mustard oil-induced vascular leakage in arsenic-fed mice. Arsenic may initiate endothelial dysfunction, resulting in vascular leakage in response to vasoactive agents.

[Randomized controlled clinical trials for treatment of external sty with ear-apex blood-letting].

OBJECTIVE: To observe the clinical therapeutic effect of ear-apex blood-letting for external sty. METHODS: A total 102 sty patients were randomized into ear-apex blood-letting group (n = 51) and routine treatment (medication) group (n = 51) according to computer-aided randomization procedure. Ear-apex-bloodletting (5-6 blood drops/time) was performed once daily for 3 times for the patients of blood-letting group. Patients of the medication group were treated by local application of hydrochloric levofloxacin and erycin ointment to the affected eyelid lining. Additionally, local warm compress of the affected eyelid was given to patients of both groups. The therapeutic effect was assessed by measuring the size of the sty swell and visual analogue scale (VAS) was determined for evaluating pain severity changes. The outcomes were analyzed by researchers who did not know the grouping. RESULTS: Comparison between patients of the two groups showed that the difference vahees of the styrize and VAS score between pre- and post-treatment in the ear-apex bloodletting group were significantly bigger than those of the medication group on day 3, 5 and 7 after treatment (P<0.05). The cure rates of the blood-letting group and medication group were 64.7% and 41.2%, 90.2% and 62.7%, 94. 1% and 80.4%, respectively on day 3, 5 and 7 after the treatment. The therapeutic effects of blood-letting were significantly superior to those of the medication group in relieving external sty (P<0.05). CONCLUSION: Ear-apex blood-letting therapy for external sty is effective in relieving pain, reducing the size and shortening the duration of disease.

Development of intravenous lipid emulsion of α-asarone with significantly improved safety and enhanced efficacy.

Severe adverse events have been frequently associated with taking the commercially available formulation of α-asarone injection (α-asarone-I). Hence, we sought to develop an intravenous lipid emulsion of α-asarone (α-asarone-LE), where we hypothesized that these adverse events could be prevented. Using a central composite design-response surface methodology, we developed and optimized an emulsion formulation of α-asarone-LE that composed of 10.0% (w/v) soybean oil, 0.4% (w/v) α-asarone, 1.2% (w/v) soybean lecithin, 0.3% (w/v) F68, and 2.2% (w/v) glycerol. The mean particle size of α-asarone-LE was 226±11 nm, the ζ-potential was -25.6±1.2 mV, the encapsulation efficiency was 99.2±0.1% and the drug loading efficiency was 3.45%. Stability, safety, and efficacy studies of α-asarone-LE were systematically investigated and compared to those of α-asarone-I. The α-asarone-LE not only showed a desired stability, but also exhibited excellent safety and improved efficacy in vivo, indicating its great potential for clinical application in the future.

A selective cysteinyl leukotriene receptor 2 antagonist blocks myocardial ischemia/reperfusion injury and vascular permeability in mice.

Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed ß-galactosidase-ß-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be ∼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 µM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.

[Cellular reaction of the skin and underlying tissues on prolonged acupuncture needle introduction].

The changes of a skin and hypodermic are studied at the prolonged introduction (till 3 months) original acupuncture needles under a skin of an auricle and neck at rats by the technique Muhina M.M. In early terms in an introduction place develops an aseptic (or septic) inflammation reaching of peak in 1-2 weeks. On a measure reduction of inflammatory reaction activation of epydermis (keratinocites) which acquire from periphery an acupuncture needle is marked, isolating it from surrounding tissues and forming the implant channel (acustract). Formation process of acustract comes to the end through 2 - 2,5 months.

In vivo effect of hyperbaric oxygen on wound angiogenesis and epithelialization.

Hyperbaric oxygen (HBO) therapy is increasingly being used in different areas of medical practice. While demonstrated to be effective in several settings, its mechanism of action is not well understood. In the present study, we determined the effects of HBO on wound epithelialization and neovascularization in an in vivo hairless mouse ear "impaired" wound model. To impair wound healing, macrophages were depleted by pretreatment with iota-carrageenan. Wound epithelialization and neovascularization were measured using intravital microscopy and computerized planimetry. Metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and tumor necrosis factor-alpha (TNF-alpha) were measured on days 2 and 7 using immunohistochemistry. In nonimpaired healing wounds, the rate of epithelialization and neovascularization was significantly accelerated in the groups treated with HBO. Time to wound closure was significantly delayed in impaired compared with nonimpaired healing wounds and HBO treatment completely reversed this delay. Neither HBO treatment nor macrophage depletion caused significant alterations in MMP-2 expression in wounds. In contrast, TNF-alpha, MMP-9, and TIMP-1 were significantly up-regulated in the impaired healing group receiving HBO treatment. These results show that HBO therapy effectively reversed the negative effect exerted by macrophage reduction on wound epithelialization and neovascularization. This beneficial effect could be due to stimulation of TNF-alpha production and, to a lesser degree due to release of metalloproteinases.

A lipid microsphere vehicle for vinorelbine: Stability, safety and pharmacokinetics.

A lipid microsphere vehicle for vinorelbine (VRL) was designed to reduce the severe venous irritation caused by the aqueous intravenous formulation of VRL. Lipid microspheres (LMs) were prepared by high pressure homogenization. The physical stability was monitored by the appearance, particle size and zeta potential changes while the chemical stability was achieved by using effective antioxidants and monitored by long-term investigations. Safety tests were performed by testing rabbit ear vein irritation and a guinea pig hypersensitivity reaction. A pharmacokinetic study was performed by determining the drug levels in plasma up to 24h after intravenous administration of VRL-loaded LMs and conventional VRL aqueous injection separately. The VRL-loaded LMs had a particle size of 180.5+/-35.2nm with a 90% cumulative distribution less than 244.1nm, while the drug entrapment efficiency was 96.8%, and it remained stable for 12 months at 6+/-2 degrees C. The VRL-loaded LMs were less irritating and toxic than the conventional VRL aqueous injection. The pharmacokinetic profiles were similar and the values of AUC(0-t) were very close for the two formulations. A stable and easily mass-produced VRL-loaded LM preparation has been developed. It produces less venous irritation and is less toxic but has similar pharmacokinetics in vivo to the VRL aqueous injection currently commercially available.

Protective effect of policosanol on endothelium and intimal thickness induced by forceps in rabbits.

Policosanol is a cholesterol-lowering drug isolated from sugar cane wax with concomitant antiplatelet effects that prevents lipofundin-induced atherosclerotic lesions in rabbits and rats, including foam cell formation, and also reduces foam cell formation in carrageenan-induced granulomas in rats, while it inhibits proliferation of smooth muscle cells induced in rabbit cuffed artery. This study was undertaken to determine whether policosanol prevents endothelium damage and increase in arterial wall thickness in rabbits with arterial walls damaged with a forceps. Artery forceps were placed over the central artery of the right ear of all rabbits, and each artery was injured eight times. Animals were randomly distributed into four groups: a positive control group treated with Tween 20/H2O vehicle, two groups treated with policosanol (5 and 25 mg/kg, respectively), and a group treated with aspirin (8 mg/kg). Treatments were given for 30 days. Damaged arteries were examined by light and electron (transmission and scanning) microscopy. To evaluate intimal thickening, areas of intima were measured, and a significant reduction in policosanol-treated animals was observed. The endothelial surface, studied with scanning electron microscopy, revealed several types of damage. In control group, the endothelial surface was severely damaged. De-endothelialized areas were reduced in policosanol-treated animals. Platelet adhesion to subendothelium was seen in all animals of the control group, whereas policosanol-treated groups exhibited significantly reduced platelet adhesion. Policosanol also reduced, dose-dependently, the platelet sequestration induced in the damaged vessel wall, partially preventing the reduction in platelet count. It is concluded that policosanol prevents endothelium injury and reduces significantly intimal thickness of rabbit arteries damaged with forceps.

Formulation and evaluation of nimodipine-loaded lipid microspheres.

The purpose of this study was to develop an alternative, improved and better tolerated formulation and investigate the pharmacokinetic profile of the new formulation of nimodipine (NM) compared with nimodipine ethanol solutions. Lipid microspheres (LMs) prepared using lecithin and vegetable oils have attracted a lot of interest owing to their versatile properties, such as non-immunogenicity, being easily biodegradable and exhibiting high entrapment efficiency. NM incorporated in LMs could reduce irritation by avoiding the use of ethanol as a solubilizer. The solubility of NM was also increased by dissolving it in the oil phase. The particle size distribution, zeta potential, entrapment efficacy and assay of the NM-loaded LMs were found to be 188.2+/-5.4 nm, -31.6 mV, 94.2% and 1.04 mg mL(-1), respectively. The preparation was stable for 1 year at 4-10 degrees C. The formulation and some physicochemical properties of NM-loaded LMs were investigated. The pharmacokinetic and biodistribution studies were performed in rats at a dose of 1.2 mg kg(-1). From the observed data, there is no obvious retention of NM-loaded LMs in plasma. Moreover, incorporation of NM in LMs did not alter the tissue distribution significantly except for the relatively greater drug accumulation in the liver and spleen. The stimulation studies demonstrate that LMs of NM reduce irritation markedly compared with NM solutions. These results suggest that the LM system is a promising option to replace NM ethanol solutions as an intravenous treatment.

Effect of extracts from the Chinese and European mole cricket on wound epithelialization and neovascularization: in vivo studies in the hairless mouse ear wound model.

Until the end of World War II, oily extracts from the European mole cricket, Gryllotalpa gryllotalpa Linné, were used for treating nonhealing wounds and burns. In traditional Chinese medicine, extracts from the Chinese mole cricket, Gryllotalpa africana Beauvois, have been used to treat boils, abscesses, and ulcers successfully for over two centuries and are still being used today. The aim of this study was twofold: first, to measure the effect mole cricket extracts have on wound epithelialization and neovascularization, and second, to identify the active compounds in the Chinese and German mole cricket extracts. For the first aim, the hairless mouse ear wound model was used. The findings showed that wounds treated with the mole cricket extracts epithelialized significantly faster than control wounds 12.7+/-0.9 and 13.2+/-1.4 days vs. 16.3+/-2.2 days (mean+/-SD, p<0.05), respectively. While the rate of wound neovascularization was significantly increased in the first 3 days postwounding from that point on, the rate in treated wounds was the same as in controls. To identify the active compounds in the mole cricket extracts, the extracts were fractionated and tested in a foreskin basal keratinocyte cell culture assay. In this assay, the migration of keratinocytes is similar to skin cell migration or reepithelialization in a healing wound. Using this method, we found the active compound in the mole cricket extracts to be linoleic acid methyl ester. All other fatty acid structures that were isolated were found to be inactive.

Epinephrine-supplemented local anesthetics for ear and nose surgery: clinical use without complications in more than 10,000 surgical procedures.

INTRODUCTION: Local anesthetics supplemented with epinephrine are generally regarded as contraindicated for surgical procedures involving the fingers, toes, penis, outer ear and the tip of the nose [1], but epinephrine is essential if automated tumescence local anesthesia (Auto-TLA) is used. MATERIALS AND METHODS: Infiltration anesthesia supplemented with 1:200,000 epinephrine was used from 1985-1997 in our department, while Auto-TLA supplemented with 1:1.000,000 epinephrine was introduced in 1997 for all surgical procedures involving the ear or nose. During this period, 10,201 patients underwent surgery at these locations. In addition, dermal blood flow was analyzed by acral photoplethysmography (APPG) and laser Doppler flowmetry (LDF) in the right ear lobe of five normal volunteers and during epinephrine supplemented Auto-TLA. RESULTS: Epinephrine-induced complications were not observed in a single patient. Cosmetic skin flap surgery was performed in 4,953 of these patients. Even in patients with extended surgical procedures that took up to one to two hours and that included extensive skin flaps or skin grafts, we observed no increase in complications when compared to procedures performed either under general anesthesia or local anesthesia without epinephrine supplementation. Measuring blood perfusion of the earlobe showed a 69% reduction of LDF and a 42% reduction of arterial inflow (APPG) immediately following anesthesia. CONCLUSION: Epinephrine supplementation of local anesthetics does not block blood perfusion in the ear and did not induce organ, tissue or flap necrosis. Local anesthesia with epinephrine supplementation is therefore safe for acral areas such as the ear or nose. Despite the relatively small influence on blood perfusion, epinephrine supplementation results in a relatively bloodless operating field and longer effectiveness of local anesthesia. The relative absence of blood in the operating field of the ear and nose significantly reduces the duration of surgery and increases the healing rate, as less electrocautery is needed.

[Effect of shenfu injection on microcirculation].

This study was aimed to assess the effect of Shenfu injection on different circulation state. Using a microcirculation microscope system, we observed mice's auricle micro-artery diameter, density of capillary, blood velocity in different circulation state (i.e. normal state, epinephrine or endotoxin induced microcirculation disturbance state) after administering Shenfu injection into their caudal vein, and we compared the Shenfu group with Shenmai group and Dexamethasone group. The results showed that Shenfu injection causes the auricle microartery diameter to enlarge and the density of capillary and blood velocity to increase in different microcirculation state, and such effect is especially notable on the epinephrine induced microcirculation disturbance group and endotoxin induced microcirculation disturbance group; the effect of Shenfu injection is stronger than that of Shenmai injection and similar to Dexamethasone injection. In addition, Shenfu injection was shown to have remarkable effect on resisting the lowering of limb temperature when the mice are attacked by endotoxin.

Different cardiovascular neuron groups in the ventral reticular formation of the rostral medulla in rabbits: single neurone studies.

To examine whether the cardiovascular neurons of the ventral medulla consist of functionally different kinds of neurons, single neuronal activity of the ventral medulla, activity of the renal sympathetic nerves (RSNA), blood flow of the ear (EarBF) and arterial pressure (AP) were recorded in urethane-anesthetized, vagotomized and immobilized rabbits during electrical stimulation of the aortic nerve (AN, baroreceptor afferent fibers) and electrical stimulation of the dorsomedial hypothalamus (DMH) that reduced EarBF but less affected on AP and RSNA. The dorsolateral funiculus of the second cervical cord was stimulated to evoke antidromic spikes of medullary neurons. Two kinds of reticulo-spinal neurons were identified. Activities of one kind of neurons were facilitated by stimulation of DMH (latency 48.6+/-27.6 ms, n=11) but they did not respond to stimulation of the AN. Therefore, it was presumed that these neurons controlled vasomotion of the ear through the vasoconstrictor neurons in the spinal cord but did not participate in regulation of systemic AP. Activities of the other neurons were inhibited by stimulation of the AN (latency 47.8+/-8 4 ms, n=16) but they did not respond to the DMH stimulation. These neurons were identical to those reported previously as the RVLM neurons, and they contributed to regulate systemic AP but might not participate in control of cutaneous vascular movement. The former neurons were located medially to the latter in the reticular formation of the rostral ventral medulla. These results provided evidence at the single neuronal level that the cardiovascular neurons in the ventral medulla were consisted of functionally different sympatho-excitatory neurons and they were located at the different sites in the rostral ventral medulla.