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PubMed1997

Effects of magnesium cardioplegia on regulation of the porcine coronary circulation.

We compared the effect of hypermagnesium and hyperkalemic crystalloid cardioplegia on beta-adrenoceptor-mediated and endothelium-dependent relaxation and myogenic responses of coronary arterioles. Pigs were placed on cardiopulmonary bypass. Hearts were arrested with cold hypermagnesium (25 mM Mg2+, hyper-Mg, n = 12) or hyperkalemic (25 mM K+, hyper-K, n = 12) crystalloid cardioplegia for 1 hr. Hearts of selected pigs (n = 6 in each group) were then reperfused for 1 hr. In vitro relaxation responses of acetylcholine-pre-contracted arterioles were studied in a pressurized no-flow state with video-microscopy. Relaxation of pre-contracted coronary microvessels (70-150 microns) to isoproterenol (beta-adrenergic agonist) and forskolin (adenylate cyclase activator) was preserved after cardioplegia using a hyper-Mg solution. In contrast, responses were impaired to isoproterenol [P < 0.01 (two-factor ANOVA) vs. controls, n = 6] and forskolin (P < 0.01) after hyper-K cardioplegia. After 1 hr of reperfusion, relaxation responses to isoproterenol and forskolin were partially recovered. Hyper-Mg cardioplegia and post-cardioplegic reperfusion did not affect receptor-mediated endothelium-dependent relaxation to ADP, non-receptor-mediated endothelium-dependent relaxation to A23187, and endothelium-independent relaxation to nitroprusside. However, responses to ADP (P < 0.01) and A23187 (P < 0.05) were selectively impaired after hyper-K cardioplegia. Myogenic contraction was impaired after either hyper-Mg or hyper-K cardioplegia. Left ventricular systolic pressure, coronary blood flow, and +dP/dt were similar after hyper-Mg or hyper-K cardioplegia. These results suggest that hyper-Mg cardioplegia is superior to hyper-K cardioplegia in terms of preserving beta-adrenoceptor-mediated and endothelium-dependent regulation of the coronary microcirculation, yet it has minimal if any additional beneficial effect on preserving myogenic responses or myocardial contractile function.

Adrenergic beta-Agonists↗
PubMed1997

Decreased myogenic reactivity in skeletal muscle arterioles after hypothermic cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is associated with a generalized defect in the intrinsic control of vascular smooth muscle. To determine if myogenic reactivity of skeletal muscle arterioles was altered by CPB, sheep (n = 7) were placed on hypothermic CPB (27 degrees C) for 90 min and hearts were arrested by cold blood cardioplegia ([K+] = 25 mM) for 60 min. Arterioles (70-180 microns) were isolated from the gracilis muscle before (control) and 15 min after CPB. In vitro arteriolar responses were studied with video-microscopy. Myogenic reactivity was examined to stepwise increases in intraluminal pressure from 10 to 100 mm Hg. Mean arterial pressure was decreased from 80 +/- 15 prior to CPB to 55 +/- 4 mm Hg (P < 0.01) 15 min after CPB. Myogenic contraction was observed in control vessels and was markedly attenuated by the protein kinase C inhibitor staurosporine (P < 0.01). CPB decreased myogenic contraction and shifted the pressure-diameter relation upward, suggesting a decrease in the intrinsic tone (both P < 0.05 vs control). CPB reduced contractile responses to the alpha 1-adrenoceptor agonist phenylephrine from -43 +/- 7% to -23 +/- 5% (P < 0.01) and the protein kinase C activator 12-deoxyphorbol 13-isobutyrate 20-acetate (phorbol ester) from -64 +/- 6% to -38 +/- 16% (P < 0.01). CPB-associated decrease in myogenic reactivity of skeletal muscle arterioles is likely due to alterations in protein kinase C and/or downstream signal transduction. This may account in part for reduction in systemic vascular resistance and hypotension associated with CPB.

Adrenergic alpha-Agonists↗
PubMed1998

Clotrimazole is a potent vasodilator of the rat coronary microcirculation.

Clotrimazole (CLT), used in the treatment of patients with sickle cell disease, directly blocks Ca2+-activated K+ (K+ Ca) channels in red cells and in portal vein smooth muscle cells by a cytochrome P450(cyt P450)-independent mechanism. Therefore, we examined the effects of CLT on vasomotor tone of coronary arterioles. Rat coronary arterioles (80-180 micro(m) in diameter) were studied in vitro in a pressurized no-flow state with a video microscopy. CLT (0.1 micromol/L) elicited in nonprecontracted vessels a small contraction (<10% baseline diameter, P < 0.05 vs time control), consistent with blockade of a hyperpolarizing K+ channel. However, similar contraction was produced by the cyt P450 blocker 17-octadecynoic acid (17-ODYA, 100 micromol/L), suggesting possible involvement of arachidonate metabolites of cyt P450. In contrast, microvessels precontracted with the thromboxane A2 analog U46619 dilated in response to CLT [>90% relaxation of the U46619-induced precontraction at 100 micromol/L (P < 0.01 vs time control)] and its structural analogs flutrimazole (FLT), UR-4055, UR4057, UR-4058, and UR-4059. This relaxation was cyt P450-independent, since the in vivo CLT metabolite (CLT-carbinol) was equipotent with CLT, and 17-ODYA did not promote relaxation. CLT-induced dilation was not inhibited by the nitric oxide synthase inhibitor NGnitro-l-arginine (100 micromol/L, P > 0.5) or affected by endothelial denudation (P > 0.5). Thus, CLT at concentrations >1 micromol/L is a potent vasodilator of rat coronary arterioles. This dilation is likely mediated through a vascular smooth muscle mechanism independent of cyt P450 and is not modulated by nitric oxide or by the endothelium. This effect may arise from CLT's reported ability to inhibit voltage-gated Ca2+ channels or to inhibit, in some tissues, Ca2+ release from intracellular stores. The CLT- and FLT-induced relaxation may be a property common to this class of drugs and have clinical applicability.

Animals↗
PubMed1999

The role of ischemic preconditioning in the recruitment of rolling and adherent leukocytes in hepatic venules after ischemia/reperfusion.

BACKGROUND: We have recently shown that hepatic ischemia/reperfusion (I/R) results in rolling and adherence of leukocytes in terminal hepatic venules (THV) followed by hepatic enzyme elevation and tissue destruction. The objective of this study was to determine the effect of ischemic preconditioning on the recruitment of leukocytes in THV after liver I/R. METHODS: Left hepatic lobe ischemia was induced for 5 min (preconditioning) in anesthetized C57B1/6 mice followed by reperfusion for 10 min and then prolonged ischemia for 30 min. The number of rolling, saltating, and adherent leukocytes in THV was measured at 0.5, 2, 5, 12, and 24 h after reperfusion using intravital video microscopy. Matching sham groups were evaluated after 30 min of ischemia. RESULTS: Hepatic I/R elicited significant increases in the number of rolling, saltating, and adherent leukocytes, with peak values observed at 30 min and 5 h after reperfusion. All of these responses were significantly attenuated in mice undergoing ischemic preconditioning. Rolling leukocytes in THV following I/R without preconditioning reached peak levels of 25.2 +/- 1.4 leuk/2 min (leukocytes/2 min) at 30 min reperfusion and 31.4 +/- 1.5 leuk/2 min at 5 h reperfusion. With ischemic preconditioning these values fell to 12.3 +/- 0.9 leuk/2 min and 14.4 +/- 1.0 leuk/2 min, respectively (P < 0.001). Similarly, adherent leukocytes in nonpreconditioned mice reached peak values of 4.8 +/- 1.3 leuk/2 min at 30 min reperfusion and 8.3 +/- 1.2 leuk/2 min at 5 h reperfusion compared with 2.0 +/- 1.5 leuk/2 min and 1.6 +/- 1.1 leuk/2 min in preconditioned mice, respectively (P < 0.001). CONCLUSION: Ischemic preconditioning attenuates the initial events leading to leukocyte-mediated hepatic destruction following I/R injury. Delineating these mechanisms may play an important role in hepatic transplantation, resection, shock, and sepsis.

Animals↗
PubMed2001

N-cadherin is involved in axon-oligodendrocyte contact and myelination.

We have analyzed the influence of the calcium-dependent cell adhesion molecule, N-cadherin, on events leading to CNS myelination. Interactions between axons and oligodendrocyte progenitor (OP) cells and the CG4 OP cell line were examined by video-microscopy. OPs cocultured with dorsal root ganglia explants migrated around the culture and formed numerous contacts with axons. The duration of these contacts depended on the morphology of the OP, with OPs containing four or more processes forming long-lasting contacts and OPs with three or fewer processes forming short-termed contacts. Treatment with N-cadherin function blocking peptides approximately halved the duration of contacts made by cells with four or more processes but contact times for cells with three or less processes were unaffected. The L7 cadherin-blocking antibody and calcium withdrawal had similar effects. Contacts with axons regenerating from explants of adult retina, which do not have N-cadherin on their surface was examined. The contact duration of OPs to adult retina axons was short and similar in length to those formed between OPs and dorsal root ganglion axons in the presence of cadherin blocking reagents. Oligodendrocyte myelination was examined in organotypic rat cerebellar slice cultures, taken before myelination at postnatal day 10 and then allowed to myelinate in vitro over 7 days. When incubated with an N-cadherin function-blocking peptide, myelination of Purkinje cell axons was reduced to about half of control levels, while control peptides were without effect. Cadherin-blockade did not prevent maturation of OPs, since oligodendrocytes showing myelin basic protein immunostaining were still found in these cultures. However, many of the cell processes did not colocalize with calbindin-positive axons. From these data we conclude that N-cadherin is important for the initial contact between a myelinating oligodendrocyte and axons and significantly contributes to the success of myelination.

Animals↗
PubMed1994

Delayed recovery of endothelium-dependent vasodilation in regenerating arterioles of skeletal muscle autografts.

This study investigated the responses of regenerating arterioles in grafted skeletal muscle to the endothelium-dependent dilator acetylcholine (ACh); the substrate for endothelium-derived relaxing factor (EDRF), L-arginine (L-Arg); and sodium nitroprusside (SNP). Additionally, responses of graft arterioles to the endothelium-independent substances adenosine (Ado) and norepinephrine (NE) were measured. The retractor muscle of hamsters was removed, placed in a myotoxic solution, and grafted into its original site. The graft revascularized spontaneously by sprouting of vessels in surrounding tissue. Quantification of changes in arteriolar luminal diameter was accomplished using in vivo video microscopy at 30, 45, and 60 days postgrafting. In 30-day grafts, there was little or no response to topically applied ACh or L-Arg. By 45 days, arteriole response to 10(-6), 10(-5), and 10(-4) M ACh and 10(-4) L-Arg was 0, 4, 20, and 17% of the control response, respectively. SNP (10(-6)-10(-4) M) produced approximately 50% of the control response at both 30 and 45 days. By 60 days the response of graft arterioles to ACh, L-Arg, and SNP was not different from controls. Arteriolar response in 30-day grafts to Ado (10(-6)-10(-4) M) was significantly attenuated, but was not different from control by 45 days. Responsiveness of arterioles to NE had recovered to control levels by 30 days postgrafting. These data indicate that the restoration of endothelium-dependent regulation of arteriolar dilation lags behind endothelium-independent, receptor-mediated mechanisms. The diminished response of regenerating arterioles may be related to dysfunctions in both the synthetic pathway for EDRF and the guanylate cyclase/cGMP mechanism for vascular smooth muscle cell relaxation and/or flow-dependent mechanisms of blood flow regulation.

Acetylcholine↗
PubMed1995

Quantitative angiogenesis in a syngeneic tumor spheroid model.

The aim of this work was to develop a system for noninvasive, in vivo, and in situ study of tumor angiogenesis in awake mice. Tumor spheroids were prepared from Lewis lung carcinoma cells prelabeled with methylrhodamine. A transparent chamber consisting of two titanium frames was implanted into the dorsal skin of CB6 mice. One layer of the skin was removed in a 15-mm area and covered with a coverslip. A few days later, the coverslip was removed and one to three tumor spheroids (diameters, 500-900 microns) were placed over the upper tissue layer. The selected fields were recorded under trans- and epi-illumination using video microscopy. Separate fluorescence filter sets were used to visualize the FITC-labeled plasma and the rhodamine-labeled tumor spheroids. The dual labeling technique allowed precise identification of the tumors and the study of tumor and microvessel growth for up to 14 days. The tumor area and morphometric parameters of tumor vessels were measured from recorded images. After implantation, tumor cells formed well-defined tumor foci. Venular and capillary dilation and tortuosity were observed in the surrounding tissue 1-2 days after implantation, followed by the appearance of buds and sprouts. After that, vascular networks developed around and within the spheroid. During the first week, angiogenesis was very intense: at Day 6, vascular density and tumor area reached 81 and 19% of their respective maximum values. Vascular densities at Days 3, 6, 10, and 14 were 106 +/- 59, 147 +/- 62, 183 +/- 108, and 173 +/- 38 cm-1, respectively. Tumor volume increased exponentially, with a doubling time of 2 days. Similar results were obtained in nude mice. The model allows detailed repeated observations of angiogenesis and permits quantitative evaluation of tumor growth and angiogenesis in vivo. It is applicable for mechanistic studies as well as therapeutic and pharmacokinetic studies of angiostatic and cytotoxic anti-tumor agents.

Animals↗
PubMed1995

Interaction of activated natural killer cells with normal and tumor vessels in cranial windows in mice.

A mammary carcinoma, MCa IV, was grown in syngeneic C3H mice in a cranial window preparation which permitted the in vivo observation of the growth and microcirculation of the tumors. Fluorescently labeled activated natural killer (A-NK) cells were injected into the external carotid artery and their interactions with normal and tumor vessels were quantified by video microscopy. Cells which entered the tumor vessels adhered heterogeneously to these vessels, regardless of vessel size or blood flow rates and bound with an efficiency ranging from 0 to 82% of the incoming cell flux. Normal brain tissue showed significantly fewer binding cells per microscopic field (9 +/- 5 vs 85 +/- 27 cells/1.3 mm2) and the few cells which were retained by the normal tissue were highly deformed, suggesting mechanical rather than adhesive entrapment. These studies indicate that A-NK cells bind in high numbers to segments of the vessels of mammary tumors growing in an intracranial site when administered through an arterial route; however, some tumor vessels may escape recognition by these cells. These findings suggest that A-NK cells may be used as carriers of genes for anti-cancer agents.

Animals↗
PubMed1996

Implementation of intussusceptive microvascular growth in the chicken chorioallantoic membrane (CAM): 1. pillar formation by folding of the capillary wall.

Intussusceptive microvascular growth is a new mode of capillary network growth originally described in the lungs of rabbits and rats. It constitutes an alternative to endothelial sprouting. The capillary network grows by insertion of new intercapillary meshes with dimensions around 1.5 microns called tissue pillars or posts. In a recent investigation, growth by intussusception was demonstrated in the chicken chorioallantoic membrane (CAM). In the present study the first of several modes of its implementation can now be presented in the CAM by in vivo video microscopy and analyses of light and electron microscopic serial sections: Cores of tissue pillars containing collagen fibrils ensheathed by extensions of endothelial-like cells will form within the tips of vertically running tissue folds that project into the capillary lumen. Due to retraction of tissue toward the intercapillary space the fold is thinning. Finally, the pillar's core is connected to its fold by a very slender extension of a single endothelial cell. Cell membrane fusion within that slender membrane-like structure causes subsequent separation of the pillar from its fold throughout an increasing vertical distance. This mechanism allows for expansion of the capillary network into the surrounding tissue, leaving behind tissue pillars as remnants of folds.

Allantois↗
PubMed1997

Effects of platelet-activating factor on arteriolar and venular tone in rat trachea.

Platelet-activating factor (PAF) causes altered vascular tone in the mesenteric, pulmonary, and skeletal muscle vascular beds, enhanced macromolecular leak in postcapillary venules, and bronchoconstriction. In spontaneously breathing, anesthetized male Sprague-Dawley rats, we examined responses of individual tracheal microvessels using intravital video microscopy with epi-illumination using a polarizing filter and a long working distance lens (10x). Topical application of PAF (0.1 ml; 10(-9), 10(-7), or 10(-5) M) caused venules (diameter, 51 +/- 5 microm, mean +/- SEM) to constrict in a concentration-dependent fashion. Venoconstriction began within 0-3 min and was maximal within 10 min. In contrast, PAF (10(-5) M) applied to arterioles (diameters, 37 +/- 1 microm) ultimately caused constriction by 21 +/- 7%, but in three dilation (38 +/- 9%) occurred first. PAF (10(-7) and 10(-9) M) applied to arterioles (diameter 36 +/- 3 and 38 +/- 3 microm, respectively) caused no significant change in diameter. Infusion of BN52021, a PAF receptor antagonist, completely blocked constriction of venules and arterioles to PAF (10(-5) M), but dilation (17 +/- 0.2%) still occurred in three of five arterioles. Infusion of Nomega-nitro-l-arginine methyl ester (L-NAME; 1 mg/kg/min), a nitric oxide (NO) synthase inhibitor, potentiated venoconstriction by 34 +/- 18% in response to PAF (10(-7) M) and blocked arteriolar dilation to PAF (10(-5) M). Arteriolar constriction was unaffected by inhibiting NO release. Topical application of L-NAME (10(-3) M) constricted venules (n = 6) by 13 +/- 2% and arterioles (n = 5) by 26 +/- 4%. Venules constricted 33 +/- 15% less to PAF (10(-7) M, n = 5) following topical L-NAME (33 +/- 15%) than with infused l-NAME. Arterioles (n = 5) constricted 27 +/- 4% to PAF (10(-5) M) after topical l-NAME, no different from the group receiving infused L-NAME. We conclude that (1) PAF has a greater vasoconstrictive effect on tracheal venules than arterioles; (2) arterioles have a biphasic response to PAF at some concentrations; (3) PAF-induced vasoconstriction, but not dilation, is receptor mediated; (4) nitric oxide attenuates tracheal venoconstriction and may cause arteriolar dilation in response to PAF; and (5) endogenous release of NO appears to modulate the basal tone of tracheal arterioles more than venules.

Analysis of Variance↗
PubMed1997

Capillary adrenoceptors in rat skeletal muscle.

The purpose of this study was to examine whether functional alpha- and beta-adrenoceptors exist on capillaries of rat skeletal muscle, and further to determine which subtype of these receptors predominates on these capillaries. Using intravital video microscopy, we measured red blood cell velocity (VRBC) responses in capillaries of rat extensor digitorum longus muscle (EDL) following a local application of these agonists: norepinephrine (NE; alpha 1, alpha 2; 10(-7) to 3 x 10(-3) M), phenylephrine (PE; alpha 1; 3 x 10(-4) to 10(-2) M), clonidine (CLO; alpha 2; 3 x 10(-3) to 10(-2) M), UK14304 (alpha 2; 3 x 10(-4) to 10(-2) M), and isoproterenol (IPR; beta 1, beta 2; 10(-7) to 3 x 10(-3) M). Responses to NE (10(-5) M) were also measured after a local pretreatment with prazosin (alpha 1 antagonist; 10(-5) to 10(-3) M) and rauwolscine (alpha 2 antagonist; 3 x 10(-4) to 3 x 10(-2) M), while responses to IPR (10(-5) M) were measured after local atenolol (ATE; beta 1 antagonist; 10(-3) to 10(-2) M) and butoxamine (BUT; beta 2 antagonist; 10(-3) to 10(-2) M) pretreatment. The overall control VRBC was 226 microns/sec. NE, PE, CLO, and UK14304 resulted in concentration-dependent decreases of VRBC (from -12 to -89%) from the control level, while IPR caused concentration-dependent increases (17 to 174%). PE reduced VRBC to a larger degree than CLO and UK14304. NE-induced VRBC responses tended to be attenuated more by prazosin than by rauwolscine. Both ATE (10(-2) M) and BUT (10(-3) and 10(-2) M) alone decreased VRBC. However, only ATE significantly attenuated the IPR-induced VRBC responses. These results suggest that the capillary of rat EDL muscle has alpha- and beta-adrenoceptors. From the two alpha-adrenoceptor subtypes, the capillary may be predominated by the alpha 1-adrenoceptors.

Adrenergic alpha-Agonists↗
PubMed1997

Effects of hypoxia and hypercapnia on capillary flow velocity in the rat cerebral cortex.

The velocity of red blood cells (RBC) in individual capillaries of the rat cerebral cortex was assessed using direct, intravital video microscopy under normal conditions and during systemic hypoxia or hypercapnia. The movement of RBC in capillaries within 50-microm depth of the parietal cortex was visualized with the aid of fluorescent labeling of RBC in a closed cranial window preparation in pentobarbital-anesthetized, artificially ventilated adult rats. Hypoxia was produced by lowering the concentration of oxygen in the inspired gas from 30 to 15% for 5 min. Hypercapnia was achieved by increasing the inspired CO2 concentration (FiCO2) from 0 to 5% and then to 10% for 5 min at each level. The mean arterial pressure was maintained constant during both maneuvers. Under control conditions, fast and heterogeneous RBC flow in multioriented, tortuous capillaries was observed. During hypoxia, RBC velocity increased from 0.61 +/- 0.06 to 0.82 +/- 0.10 mm/sec (35% change). During hypercapnia, RBC velocity increased from 0.73 +/- 0.05 to 1.07 +/- 0. 11 mm/sec (46% change) at 5% CO2 and to 1.19 +/- 0.11 mm/sec (63% change) at 10% CO2. Corresponding changes in regional blood flow as assessed by laser-Doppler flowmetry during hypercapnia were 69 +/- 7 and 128 +/- 21%, respectively. The RBC velocity increased in almost all capillaries during hypoxia and during moderate hypercapnia. However, a substantial number of capillaries showed no change or a small decrease in RBC velocity during severe hypercapnia. A significant negative correlation between the velocity change at 10% CO2 and the normocapnic resting velocity was found in a group of capillaries isolated by cluster analysis. These results suggest that the dominant component of cerebral hyperemic response to hypoxia and to moderate hypercapnia is an increase in capillary RBC flow velocity. A more complex change in the velocity distribution occurs during severe hypercapnia and results in increased homogeneity of RBC perfusion in the cerebrocortical capillary network.

Animals↗
PubMed1996

Heat-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing HIV-1 envelope glycoproteins.

It has been shown that human CD4 expressed in nonhuman cells does not support HIV-1 entry into those cells and that components from human cells in addition to CD4 are required to overcome the block. We have used human red blood cells (huRBC) as a source for the accessory components since their membrane composition is less complex than that of nucleated cells and they are well characterized. Components were transferred by fusion of huRBC to nonhuman CD4(+) cells mediated by influenza hemagglutinin or polyethylene glycol. The RBC-modified nonhuman CD4(+) cells were labeled with fluorescent markers and incubated with gp 120-gp41-expressing cells labeled with a different fluorescent probe. Fusion between RBC- modified nonhuman CD4(+) cells and gp 120--gp41-expressing cells was quantified by fluorescence video microscopy. Human erythrocyte components transferred to nonhuman CD4+ cells conferred HIV-1 envelope glycoprotein-mediated fusion susceptibility to those cells. The fusion was enhanced by pretreatment of the erythrocytes for 10 min at 56 degrees. No gp 120--gp41-mediated fusion was observed when components from nonhuman RBC were transferred to nonhuman CD4+ cells. Human cell lines with pre-RBC characteristics (K562-CD4) also supported HIV-1 envelope glycoprotein-mediated fusion.

Animals↗
PubMed2002

Controlled cell killing by a recombinant nonsegmented negative-strand RNA virus.

In most tissue culture cell lines tested, infection with the paramyxovirus simian virus 5 (SV5) results in very little cell death. To determine if SV5 could be used as a vector for controlled killing of tumor cells, a recombinant SV5 (rSV5-TK) was constructed to encode the herpes simplex virus thymidine kinase (TK) gene. MDBK cells infected with rSV5-TK showed a time-dependent loss of viability when infected cells were cultured in the presence of the prodrug acyclovir (ACV) or ganciclovir (GCV) while no significant toxicity was observed in the absence of prodrug. Cells infected with a control rSV5 expressing GFP and cultured with prodrug showed only a slight reduction in growth rate and little cell death. Time-lapse video microscopy of rSV5-TK-infected MDBK cells that were cultured in the presence of ACV showed an accumulation of cells with morphological effects characteristic of apoptotic cell death. An MDBK cell line persistently infected with rSV5-TK retained long-term expression of TK and sensitivity to prodrug-mediated cell killing that were comparable to those found in an acute infection. Titration experiments showed that the rSV5-TK plus GCV combination resulted in cell death for all mouse and human cell lines tested, although the kinetics and efficiency of cell death varied between cell types. Our results demonstrating controlled cell killing by a recombinant paramyxovirus support the use of negative-strand RNA viruses as therapeutic vectors for targeted killing of cancer cells.

Acyclovir↗
PubMed1994

Erythrocyte flow heterogeneity in the cerebrocortical capillary network.

The heterogeneity of erythrocyte flow velocity and erythrocyte flux and their dependence on decreased cerebral perfusion pressure were studied in the rat cerebral cortex using intravital video microscopy. With decreased perfusion pressure, both mean and range of erythrocyte flow velocity of individual capillaries was reduced. Both cell velocity and cell flux decreased more in high flow capillaries than in low flow capillaries. The results are compatible with the hypothesis that redistribution of capillary flow during hypotension may help to maintain the perfusion of arterio-venous capillary flow pathways. Although the data are preliminary, they represent the first direct measurements of capillary flow path length and transit time in the capillary network of the cerebral cortex.

Animals↗
PubMed1998

Spontaneous fluctuations in cerebral oxygen supply. An introduction.

Spontaneous, low frequency (4-12 cpm) fluctuations, independent of the cardiac and respiratory cycles, in human and animal brains were first recorded with the O2 polarographic technique in the late 1950s. They were seen in NADH and cytochrome oxidase and associated with spontaneous vasomotion pial and large cerebral arteries. Renewed interest in spontaneous fluctuations was generated by studies with laser-Doppler flowmetry (LDF), reflectance oximetry and functional MRI. Spontaneous fluctuations were consistently produced when cerebral perfusion was challenged by systemic or local manipulations; the fluctuation amplitude reached 30-40% of the mean. The most potent stimuli are hypotension, hyperventilation, cerebral artery occlusion and cerebral vasoconstriction elicited, for example, by a nitric oxide synthase inhibitor but not by indomethacin. The fluctuations are suspended by CO2 and halothane at concentrations that produce hyperemia. Recently, spontaneous fluctuations were recorded by LDF microprobes in areas as small as 130 microns and by video-microscopy in single capillaries. The fluctuations were absent in severe, focally ischemic brain territories. The dependence of spontaneous fluctuations on intravascular pressure argues for the importance of a myogenic mechanism, however, neuronal modulation may also play a role. Coherence of small vessel vasomotion may be required for the emergence of regional flow fluctuations. There is a need to elucidate the spatial and frequency domains in which fluctuations are present under normal physiological conditions and those in which they may reflect brain injury and pathologies of diagnostic or prognostic value.

Aged↗
PubMed2006

Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.

Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and "cycling" of the reaction through interphase and back into metaphase promotes formation of bipolar spindles capable of segregating their duplicated chromosomes. Reactions can be "spun down" onto cover slips for immunofluorescence analysis. High-speed extracts support mitotic chromosome condensation, which can be observed live by fluorescence time-lapse video microscopy. Because of the biochemically accessible nature of the egg extract system, a wide array of biochemical techniques can be combined with spindle and chromosome assembly reactions to evaluate the roles of specific proteins in these processes.

Animals↗
PubMed1992

The impact of liver preservation in HTK and UW solution on microcirculation after liver transplantation.

Severe microcirculatory disturbances due to endothelial cell damage and leukocyte adherence during reperfusion of transplanted livers are considered to contribute to early graft failure. Since the degree of reperfusion injury after liver transplantation depends on the length of preservation time and the solution used for preservation, the aim of our study was to assess three solutions with respect to microvascular perfusion and leukocyte adhesion. Therefore, rat livers were stored up to 24 h in Euro-Collins (EC), University of Wisconsin (UW), or histidin-tryphtophan-ketoglutarate (HTK) solutions prior to orthotopic transplantation. The livers were studied in situ 60 min postoperatively using intravital fluorescence video microscopy. Using simple syringe flushing (10 ml), sinusoidal perfusion decreased below 50% in EC preserved livers after 8 h preservation, in HTK preserved livers after 16 h preservation, and remained higher than 70% in livers preserved in UW up to 24 h. Permanent adhesion of leukocytes was increased more rapidly in organs after 1, 8, 16, and 24 h preservation in HTK (16%, 15%, 34%, and 49.7% +/- 4.7%) compared to those preserved in UW (15%, 18%, 17%; and 32.7% +/- 3.3%; P<0.05). Using a 10-fold volumn of the organ weight of HTK solution during the harvesting procedure, with an 8 min equilibration period, sinusoidal perfusion (39.6 +/- 4.7%) and leukocyte adhesion (42.7 +/- 3.1%) were not improved after 24 h. In contrast, equilibration with a volumn of approximately 40-times the liver weight improved sinusoidal perfusion (70.8% +/- 2.7%; P < 0.01) and leukocyte adhesion (24.9% +/- 3.1%; P < 0.01) significantly. Thus, using HTK solution, simple flushing prior to long-term cold storage resulted in microcirculatory disturbances when compared to UW solution. Larger volumns of HTK solution with an additional equilibration period of 8 min, however, reduced leukocyte adhesion and improved sinusoidal perfusion to a similar degree as UW solution.

Adenosine↗