Upright tilt resets dynamic transfer function of baroreflex neural arc to minify the pressure disturbance in total baroreflex control

Maintenance of arterial pressure (AP) under orthostatic stress against gravitational fluid shift and pressure disturbance is of great importance. One of the mechanisms is that upright tilt resets steady-state baroreflex control to a higher sympathetic nerve activity (SNA). However, the dynamic feedback characteristics of the baroreflex system, a hallmark of fast-acting neural control, remain to be elucidated. In the present study, we tested the hypothesis that upright tilt resets the dynamic transfer function of the baroreflex neural arc to minify the pressure disturbance in total baroreflex control. Renal SNA and AP were recorded in ten anesthetized, vagotomized and aortic-denervated rabbits. Under baroreflex open-loop condition, isolated intracarotid sinus pressure (CSP) was changed according to a binary white noise sequence at operating pressure +/- 20 mmHg, while the animal was placed supine and at 60 degrees upright tilt. Regardless of the postures, the baroreflex neural (CSP to SNA) and peripheral (SNA to AP) arcs showed dynamic high-pass and low-pass characteristics, respectively. Upright tilt increased the transfer gain of the neural arc (resetting), decreased that of the peripheral arc, and consequently maintained the transfer characteristics of total baroreflex feedback system. A simulation study suggests that postural resetting of the neural arc would significantly increase the transfer gain of the total arc in upright position, and that in closed-loop baroreflex the resetting increases the stability of AP against pressure disturbance under orthostatic stress. In conclusion, upright tilt resets the dynamic transfer function of the baroreflex neural arc to minify the pressure disturbance in total baroreflex control.     

Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells

Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70 degrees C or in liquid nitrogen in a rate-controlled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)- and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70 degrees C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells.     

Gastric or coeliac vagotomy decreases drinking after peripheral angiotensin II

After bilateral subdiaphragmatic vagotomy, rats drank later and less in response to peripherally administered angiotensin II [13]. We attempted to localize this deficit neurologically by performing selective gastric, hepatic or coeliac vagotomies. The drinking responses of such selectively lesioned rats to 0.1 and 1.0 mg·kg^?1 angiotensin II (SC) were compared to those of total bilateral vagotomized rats and sham vagotomized rats. Gastric or coeliac vagotomy produced drinking deficits that were similar to those produced by total abdominal vagotomy, but hepatic vagotomy did not. These results demonstrate the importance of abdominal vagal mechanisms in the drinking response to circulating angiotensin II.     

Cocaine or delta 9-tetrahydrocannabinol does not affect cellular cytotoxicity in vitro

Cocaine or delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has no effect on in vitro cytotoxicity mediated by natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) at concentrations similar to those observed in vivo.     

Genetic modification does not affect the stemness of neural stem cells in nestin promoter-GFP transgenic mice

Because nestin promoter-GFP mice have frequently been used in neural stem cell (NSC) research, it is essential to prove that there is no alteration in the stemness of NSCs derived from this transgenic model for the interpretation and validity of the data. We compared the stemness of NSCs derived from transgenic mice expressing GFP driven by the nestin enhancer with those from wild-type (C57BL/6) mice with respect to the general gene expression profile, expression of neural stem cell markers as nestin and Sox2, and responsiveness to neurotrophins (BDNF, PDGF-BB, and NT-3). The gene expression profile analysis showed that the coefficient of correlation between the two groups was very high (r = 0.9865) in the total genes. We found that 23 genes were either up- or down-regulated more than two-fold in the NSCs from the transgenic mice (p < 0.05), without any obvious functional relatedness among them. Likewise, there was no difference between the two mouse groups in the expression of nestin or Sox2, the ability to form neurospheres and the neuronal differentiation of NSCs by neurotrophins. Taken together, the self-renewal and neuronal differentiation ability of NSCs from the transgenic mice showed the great similarity to those from wild-type mice. Such information will be useful when the properties of NSCs are evaluated following genetic modification in such a nestin-GFP Tg model.     

Denervation does not affect the growth of rat vibrissae

This study examines the hypothesis that neural factors influence the growth of rat vibrissae. We divided the vibrissae in rows alpha-delta, 1 and 2 and examined their regrowth during the first complete growth period in normal and nerve-lesioned rats. The lesions used were denervation through neonatal capsaicin treatment, surgical sympathecomy in adult rats, neurectomy of the mandibular and buccal branches of the facial nerve in adult rats or division of the infraorbital nerve in adult rats. Normal vibrissae developed a length of 51.1 mm and a diameter of 178 microm (row alpha-delta), 44.1 mm and 181 microm (row 1) and 33.2 mm and 165 microm (row 2). In all experimental groups the examined vibrissae developed a normal final length and proximal diameter. This indicates that local nerves do not influence vibrissal growth to any major extent.     

Endothelin does not affect aggregation of human platelets

Endothelin (ET-1) is a recently discovered endothelial-derived peptide with pronounced vasoconstrictor activity. The present study addressed whether ET-1, in analogy with several other vasoactive agents, can induce or modulate aggregation of human platelets in vitro. Venous blood from healthy donors was collected in citrate or heparin and platelet-rich plasma (PRP) was prepared. Portions of the PRP were added to drugs, and platelet aggregation was recorded according to Born & Cross (1963). ET-1 added to the PRP (final concentrations 1-100 nM) did not induce aggregation of platelets, either in citrate- or heparin-containing plasma. Adenosine-diphosphate (0.5-2 microM) or thrombin (0.1-0.4 NIH units ml-1) induced dose-dependent aggregation of platelets in citrate- or heparin-containing PRP; such aggregation was, however, not affected by ET-1 (1-100 microM) either. We conclude that ET-1, in contrast to other endothelial-derived vasoactive agents, lacks direct effect on platelet aggregation in vitro.     

Interleukin-4 does not influence development of hypercholesterolemia or angiotensin II-induced atherosclerotic lesions in mice

Interleukin-4 (IL-4) has been detected in both human and mouse atherosclerotic lesions, although its effects on the development of the disease are undefined. We determined the role of IL-4 in the most commonly used murine models of atherosclerosis by defining the effects of exogenous delivery and genetic deficiency of this cytokine on both hypercholesterolemia and AngII-induced atherosclerosis in apolipoprotein E (apoE)(-/-) mice and different dietary stimuli in low-density lipoprotein (LDL) receptor(-/-) mice. Exogenous administration of IL-4 (1.1 ng g(-1) day(-1) i.p. for 30 days) into female apoE(-/-) mice had no effect on lesion size or composition in mice fed normal or saturated fat diets. Also, IL-4 deficiency had no significant effect on the size or composition of atherosclerotic lesions in two vascular areas of male and female apoE(-/-) mice fed either a normal or saturated fat diet. IL-4 deficiency was also studied in age-matched male mice infused with AngII (1000 ng kg(-1) min(-1)) for 28 days. Whereas AngII infusion augmented atherosclerotic lesion formation, IL-4 deficiency did not influence atherosclerotic lesion size or composition. Finally, different dietary stimuli also had no effect on atherosclerotic lesion size in female LDL receptor(-/-) mice. These data demonstrate that IL-4 does not significantly influence the development of atherosclerotic lesions in apoE(-/-) mice of either gender or in female LDL receptor(-/-) mice, irrespective of the mode of induction of atherosclerosis.     

Treatment of human peripheral blood leukocytes with proteases does not affect Sendai virus-induced interferon production.

Trypsinization of human peripheral blood leukocytes was found to have no effect on Sendai virus adsorption or on interferon induction by the virus. alpha-Chymotrypsin and papain also did not affect interferon induction, although the three proteases did remove part of the leukocyte surface material. In contrast, treatment of leukocytes with neuraminidase reduced virus adsorption and thoroughly abolished interferon induction. We conclude that protease-resistant structures on leukocyte surfaces serve as the receptor of Sendai virus for the induction of interferon.     

Monocyte chemoattractant protein-1 or macrophage inflammatory protein-1alpha deficiency does not affect angiotensin II-induced intimal hyperplasia in carotid artery ligation model.

BACKGROUND: Angiotensin II (Ang II) promotes atherosclerotic vascular diseases, in which proinflammatory and proliferative effects play a major pathogenic role. Ang II up-regulates chemokines, such as monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha, which are important pro-inflammatory factors mediating infiltration of inflammatory cells into atherosclerotic lesion. The aim of the present study was to determine whether the presence of MCP-1 or MIP-1alpha is essential in Ang II-induced intimal hyperplasia in the carotid artery ligation model. METHODS: Six-month-old male C57BL/6-, MCP-1-, or MIP-1alpha-deficient mice underwent ligation of the common left carotid artery and were randomly assigned to receive either vehicle or Ang II (1.4 mg kg(-1) day(-1)) via a subcutaneously implanted osmotic infusion pump (model 2004, Alzet) for 4 weeks. RESULTS: Ang II not only increased MCP-1 and MIP-1alpha production but also enhanced neo-intimal formation, media thickness, and adventitia development in the ligated carotid arteries in C57BL/6 mice. However, MCP-1 or MIP-1alpha deficiency failed to affect intimal hyperplasia in vascular remodeling. CONCLUSION: These results indicate that MCP-1 or MIP-1alpha may not be essential in mediating the proliferative effects of Ang II, a major pathological changes in intimal hyperplasia in the carotid artery ligation model.     

A wide range of baroreflex stimulation does not alter forearm blood flow

The contribution to the regulation of forearm blood flow (FBF) by different baroreceptor populations has previously only been studied over a limited range of stimuli. Therefore, FBF and R-R interval were recorded during neck suctions and neck pressures ranging from –60 to +40 mmHg. The change in R-R interval (R-R) during neck suction was significantly increased at each stage when compared to the control (P<0.05). R-R did not show any significant change during any of the neck pressure stages (P>0.05). Suction or pressure applied to the neck did not elicit any significant changes in FBF when compared to the control (P>0.05). These data show that widening the range of applied stimuli to carotid sinus baroreceptors does not induce a change in FBF. However, the small transient changes reported previously cannot be discounted.     

Bezold-Jarisch reflex blunts arterial baroreflex via the shift of neural arc toward lower sympathetic nerve activity

Although the Bezold-Jarisch (BJ) reflex is potentially evoked during acute myocardial ischemia or infarction, its effects on the static characteristics of the arterial baroreflex remain to be analyzed in terms of an equilibrium diagram between the neural and peripheral arcs. The neural arc represents the static input-output relationship between baroreceptor pressure input and efferent sympathetic nerve activity (SNA), whereas the peripheral arc represents that between SNA and arterial pressure (AP). In 8 anesthetized rabbits, we increased carotid sinus pressure stepwise from 40 to 160 mmHg in increments of 20 mmHg at one-minute intervals while measuring renal SNA and AP under control conditions and during the activation of the BJ reflex by intravenous administration of phenylbiguanide (PBG, 100 microg.kg(-1).min(-1)). The neural arc approximated a sigmoid curve whereas the peripheral arc approximated a straight line. PBG decreased AP at the operating point from -91.3 +/- 2.4 to -71.7 +/- 3.1 mmHg (P < 0.01), and attenuated the total loop gain at the operating point from -1.31 +/- 0.44 to -0.51 +/- 0.14 (P < 0.05). The equilibrium diagram indicated that PBG caused a parallel shift of the neural arc toward lower SNA such that the maximum SNA was reduced to approximately 60% of control. PBG decreased neural and peripheral arc gains at the operating point to approximately 43% and 77%, respectively. In conclusion, the BJ reflex blunts arterial baroreflex via the shift of the neural arc toward lower SNA.     

Blockage of pituitary-adrenal activity does not affect conditioned suppression

Previous research has shown that exposure to stressful experimental procedures results in increased activity of the pituitary-adrenal system. In the present experiment, although normal animals showed elevated steroid levels during exposure to a conditioned suppression procedure, characteristic behavioral results were also obtained in animals for which pituitary-adrenal activity had been blocked by hypothalamic implants of cortisol.     

Intrarenal administration of angiotensin II does not moderate afferent renal nerve mediated cardiovascular reflexes in the anaesthetized rabbit

Stimulation of the afferent renal nerves in the anaesthetized rabbit by acute reduction in renal perfusion pressure results in a neurally mediated, reflex increase in hindlimb vascular resistance. To determine whether exogenous angiotensin II moderates the reflex, the kidneys of anaesthetized rabbits were vascularly isolated and renal blood flow was occluded acutely, following intrarenal administration of vehicle (0.9% saline) or angiotensin II (0.5 ng), and the hindlimb vascular response was measured. Occlusion of renal blood flow resulted in similar, significant increases in femoral perfusion pressure of 39.7±7.1 mmHg after vehicle and 21.3±8.9 mmHg (P<0.05, n=6) after angiotensin II. The viability of the preparation following repeated episodes of renal blood flow occlusion was tested by a series of three rapid (2–3 min delay) occlusions and three delayed (30 min delay) occlusions. Femoral perfusion pressure rose by 43.1±10.7 mmHg (rapid, P<0.05, n=11) and 64.4±12.3 mmHg (delayed, P<0.05, n = 5) on the first occasion. On the second occasion, the rapid occlusion did not result in a significant increase in femoral perfusion pressure (29.1±8.1 mmHg), but the delayed group did (54.6±22.4 mmHg, P<0.05). On the third occasion, neither group showed a significant change (20.9±16.3 and 30.8±13.5 mmHg). These data suggest that exogenous angiotensin II does not moderate the afferent renal nerve reflex. The decline in hindlimb response following rapid serial occlusion may be attributed to a diminution of an intermediary substance(s) at the nerve receptor site.     

Generation of a specific immunological response to FGF-2 does not affect wound healing or reproduction

Angiogenesis, the process of new capillary formation from pre-existing vessels, has been established as an important mechanism involved in pathologic processes, such as cancer, as well as in normal physiology (Ribatti, D.; Vacca, A.; Roncali, L.; Dammacco, F. Angiogenesis under normal and pathological conditions. Haematologica 1991, 76 (4), 311-320). Basic fibroblast growth factor (FGF-2) is a critical mediator of angiogenesis that is important for normal reproduction and wound healing. FGF-2 mediates its pro-angiogenic effects by binding to heparin sulfate proteoglycan in addition to a tyrosine kinase receptor (Baird, A.; Schubert, D.; Ling, N.; Guillemin, R. Receptor and heparin-binding domain of basic fibroblast growth factor. Proc. Natl. Acad. Sci. U. S. A. 1998, 5 (7), 2324-2328; Richard, C.; Roghani, M.; Moscatelli, D. Fibroblast growth factor (FGF)-2 mediates cell attachment through interactions with two FGF receptor-1 isoforms and extracellular matrix or cell-associated heparin sulfate proteoglycans. Biochem. Biophys. Res. Commun. 2000, 276 (2), 399-405; Casu, B.; Guerrini, M.; Naggi, A.; Perez, M.; Torri, G.; Ribatti, D.; Carminati, P.; Giannini, G.; Penco, S.; Pisano, C.; Belleri, M.; Rusnati, M.; Presta, M. Short heparin sequences spaced by glycol-split urinate residues are antagonists of fibroblast growth factor 2 and angiogenesis inhibitors. Biochemistry 2002, 41 (33), 10519-10528; Murphy, P.V.; Pitt, N.; O'Brien, A.; Enright, P.M.; Dunne, A.; Wilson, S.J.; Duane, R.M.; O'Boyle, K.M. Identification of novel inhibitors of fibroblast growth factor (FGF-2) binding to heparin and endothelial cell survival from a structurally diverse carbohybrid library. Bioorg. Med. Chem. Lett. 2002, 12 (22), 3287-3290). We developed a liposomal-based peptide vaccine, L(HBD) that targets the heparin binding domain of the FGF-2 molecule. This vaccine, when inoculated into mice, inhibits angiogenesis in response to FGF-2 in a hepatic sponge model as well as tumor progression in two models of pulmonary metastatic disease. In the present studies, we further characterize the immunological and physiological responses to this vaccine. Vaccinated animals generated a specific anti-FGF-2 antibody (titer of 1:5000) that was able to inhibit FGF-2 binding to heparin sulfate in a dose dependent fashion. Cell mediated immunity was evidenced by a delayed type hypersensitivity response following challenge with the heparin binding domain peptide. Despite an immune response toward FGF-2, vaccination with L(HBD) did not result in alterations in mean time to wound healing when compared to unvaccinated animals or those treated with a liposome control. In reproductive studies, vaccinated females were not impaired in their ability to: 1) become pregnant, 2) support the growth and development of their embryos, and 3) deliver viable offspring. Furthermore, when assessed histologically, these offspring did not demonstrate any alterations in organogenesis when compared to pups born to untreated or liposome control treated females. Thus, while vaccination against FGF-2 induces a specific FGF-2 antibody response, and inhibits angiogenesis and tumor development in a pathological setting, it does not adversely alter normal physiological events dependent on FGF-2.     

Bovine colostrum supplementation does not affect plasma buffer capacity or haemoglobin content in elite female rowers

It has been reported that bovine colostrum (BC) supplementation improves buffer capacity () during exercise, but whether the improvement results from changes in tissue and/or blood buffer systems has not been determined. The purpose of the present study was to examine the effect of supplementation with BC on blood buffer systems. Thirteen elite females rowers were supplemented with 60 g·day^–1 of either BC (n=6) or whey protein (WP, n=7) during 9 weeks of pre-competition training in a randomised, double-blind, placebo-controlled, parallel design. All subjects undertook the study as a group and completed the same training program. Resting haemoglobin (Hb) concentration and plasma buffer capacity (p) (determined by titration with HCl) were measured pre- and post-supplementation. There were no differences in macronutrient intakes (P>0.56) or training volumes (P>0.99) between BC and WP during the study period. There were no differences in Hb [BC 13.28 (0.28) mg·dl^–1, WP 13.70 (0.26) mg·dl^–1; P=0.45] or p [BC 14.8 (1.1) nmol HCl·ml^–1·pH^–1, WP 14.8 (0.5) nmol HCl·ml^–1·pH^–1; P=0.68] between groups at week 0. p increased in both groups during the study period (P<0.001), but the increases were not significantly different between groups (P=0.52). Hb did not change significantly in either group (P=0.35). These data indicate that supplementation with BC does not affect p or Hb. We therefore suggest that adaptations in tissue buffer systems are responsible for the previously reported increases in buffer capacity that result from BC supplementation.     

Absence of homologous restriction factor does not affect CTL-mediated cytolysis

Homologous restriction factor (HRF) is a membrane protein of erythrocytes and leukocytes that inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. HRF has also been reported to inhibit perforin-mediated cytolysis and postulated to play a role in cytotoxic T lymphocyte (CTL) self-protection. We show that paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, lacking HRF, are no more sensitive to Ca2+-dependent human CTL-mediated lysis than normal erythrocytes. Furthermore, mouse and normal human erythrocytes, as well as PNH erythrocytes, are similarly lysed by isolated murine perforin-containing granules. We conclude that HRF does not inhibit perforin-mediated lysis and therefore is not likely to play a role in CTL self-protection.