Automated Assessment of the DWI-FLAIR Mismatch in Patients with Acute Ischemic Stroke: Added Value to Routine Clinical Practice.

BACKGROUND AND PURPOSE: The DWI-FLAIR mismatch is used to determine thrombolytic eligibility in patients with acute ischemic stroke when the time since stroke onset is unknown. Commercial software packages have been developed for automated DWI-FLAIR classification. We aimed to use e-Stroke software for automated classification of the DWI-FLAIR mismatch in a cohort of patients with acute ischemic stroke and in a comparative analysis with 2 expert neuroradiologists. MATERIALS AND METHODS: In this retrospective study, patients with acute ischemic stroke who had MR imaging and known time since stroke onset were included. The DWI-FLAIR mismatch was evaluated by 2 neuroradiologists blinded to the time since stroke onset and automatically by the e-Stroke software. After 4 weeks, the neuroradiologists re-evaluated the MR images, this time equipped with automated predicted e-Stroke results as a computer-assisted tool. Diagnostic performances of e-Stroke software and the neuroradiologists were evaluated for prediction of DWI-FLAIR mismatch status. RESULTS: A total of 157 patients met the inclusion criteria. A total of 82 patients (52%) had a time since stroke onset of ≤4.5 hours. By means of consensus reads, 81 patients (51.5%) had a DWI-FLAIR mismatch. The diagnostic accuracy (area under the curve/sensitivity/specificity) of e-Stroke software for the determination of the DWI-FLAIR mismatch was 0.72/90.0/53.9. The diagnostic accuracy (area under the curve/sensitivity/specificity) for neuroradiologists 1 and 2 was 0.76/69.1/84.2 and 0.82/91.4/73.7, respectively; both significantly (P < .05) improved to 0.83/79.0/86.8 and 0.89/92.6/85.5, respectively, following the use of e-Stroke predictions as a computer-assisted tool. The interrater agreement (κ) for determination of DWI-FLAIR status was improved from 0.49 to 0.57 following the use of the computer-assisted tool. CONCLUSIONS: This automated quantitative approach for DWI-FLAIR mismatch provides results comparable with those of human experts and can improve the diagnostic accuracies of expert neuroradiologists in the determination of DWI-FLAIR status.

Differential baroreflex control of sympathetic drive by angiotensin II in the nucleus tractus solitarii.

Microinjection of angiotensin II into the nucleus tractus solitarii attenuates the baroreceptor reflex-mediated bradycardia by inhibiting both vagal and cardiac sympathetic components. However, it is not known whether the baroreflex modulation of other sympathetic outputs (i.e., noncardiac) also are inhibited by exogenous angiotensin II (ANG II) in nucleus tractus solitarii (NTS). In this study, we determined whether there was a difference in the baroreflex sensitivity of sympathetic outflows at the thoracic and lumbar levels of the sympathetic chain following exogenous delivery of ANG II into the NTS. Experiments were performed in two types of in situ arterially perfused decerebrate rat preparations. Sympathetic nerve activity was recorded from the inferior cardiac nerve, the midthoracic sympathetic chain, or the lower thoracic-lumbar sympathetic chain. Increases in perfusion pressure produced a reflex bradycardia and sympathoinhibition. Microinjection of ANG II (500 fmol) into the NTS attenuated the reflex bradycardia (57% attenuation, P < 0.01) and sympathoinhibition of both the inferior cardiac nerve (26% attenuation, P < 0.05) and midthoracic sympathetic chain (37% attenuation, P < 0.05) but not the lower thoracic-lumbar chain (P = 0.56). We conclude that ANG II in the nucleus tractus solitarii selectively inhibits baroreflex responses in specific sympathetic outflows, possibly dependent on the target organ innervated.

Clinical outcome and virological characteristics of hepatitis B-related acute liver failure in the United States.

The role of hepatitis B virus (HBV) genotypes in the outcome of acute HBV infection is unclear. In this study, we aimed to evaluate the clinical and virological features of patients with hepatitis B-related acute liver failure (HBV-ALF) in the US. Clinical and laboratory features of consecutive patients with HBV-ALF from the US ALF Study Group were analysed. Prevalence of HBV genotypes, precore stop (G1896A) and core promoter dual (T1762A, A1764T) variants among patients with HBV-ALF were compared with a cohort of 530 patients with chronic HBV infection. Thirty-four HBV-ALF patients were studied: mean age 41 years, 56% men, 25 had detectable HBV-DNA. HBV genotypes A, B, C and D were found in 36, 24, 8 and 32% patients, respectively. Precore stop and core promoter dual variants were detected in 32 and 44% of patients, respectively. Twenty-three (68%) patients survived: 14 after liver transplant, nine without transplant. Older age was the only independent factor associated with poor outcome. Compared with patients with chronic HBV infection, patients with ALF were more likely to be non-Asians (88% vs 44%, P = 0.005) and to have genotype D (32% vs 10%, P < 0.01). A higher prevalence of HBV genotype D persisted even after matching for race and HBeAg status (32% vs 16%, P = 0.007). We concluded that HBV genotype D was more frequently found in patients with HBV-ALF than those with chronic HBV infection in the US. Further studies are needed to determine if HBV genotypes play a role in the outcome of acute HBV infection.

Medullary and supramedullary mechanisms regulating sympathetic vasomotor tone.

AIM: Neurons in the rostral ventrolateral medulla (RVLM) that project directly to sympathetic preganglionic neurons in the spinal cord play a critical role in maintaining tonic activity in sympathetic vasomotor nerves. Intracellular recordings in vivo from putative RVLM presympathetic neurons have demonstrated that under resting conditions these neurons display an irregular tonic firing rate, and also receive both excitatory and inhibitory synaptic inputs. This paper will briefly review some recent findings on the role of glutamate, GABA and angiotensin II (Ang II) receptors in maintaining the tonic activity of RVLM presympathetic neurons. RESULTS: Based on these findings, the following hypotheses will be discussed: (1) RVLM neurons receive tonic glutamatergic excitatory inputs, which originate from both medullary and supramedullary sources; (2) at least some neurons that project to and tonically inhibit RVLM presympathetic neurons are themselves tonically inhibited by GABAergic inputs originating from neurons in the caudalmost part of the ventrolateral medulla (caudal pressor area); (3) under normal conditions, Ang II receptors in the RVLM do not contribute significantly to the tonic activity of RVLM presympathetic neurons, but may do so in abnormal conditions such as heart failure or neurogenic hypertension; (4) RVLM presympathetic neurons maintain a significant level of tonic resting activity even when glutamate, GABA and Ang II receptors on the neurons are completely blocked. Under these conditions, the tonic activity is a consequence either of the intrinsic membrane properties of the neurons (autoactivity) or of synaptic inputs mediated by receptors other than glutamate, GABA or Ang II receptors. CONCLUSION: The current evidence indicates that the resting activity of RVLM presympathetic neurons is determined by the balance of powerful tonic excitatory and inhibitory synaptic inputs. Ang II receptors also contribute to the raised resting activity of these neurons in some pathological conditions.

Central mechanisms underlying short- and long-term regulation of the cardiovascular system.

1. Sympathetic vasomotor nerves play a major role in determining the level of arterial blood pressure and the distribution of cardiac output. The present review will discuss briefly the central regulatory mechanisms that control the sympathetic outflow to the cardiovascular system in the short and long term. 2. In the short term, the sympathetic vasomotor outflow is regulated by: (i) homeostatic feedback mechanisms, such as the baroreceptor or chemoreceptor reflexes; or (ii) feed-forward mechanisms that evoke cardiovascular changes as part of more complex behavioural responses. 3. The essential central pathways that subserve the baroreceptor reflex and, to a lesser extent, other cardiovascular reflexes, have been identified by studies in both anaesthetized and conscious animals. A critical component of these pathways is a group of neurons in the rostral ventrolateral medulla that project directly to the spinal sympathetic outflow and that receive inputs from both peripheral receptors and higher centres in the brain. 4. Much less is known about the central pathways subserving feed-forward or 'central command' responses, such as the cardiovascular changes that occur during exercise or that are evoked by a threatening or alerting stimulus. However, recent evidence indicates that the dorsomedial hypothalamic nucleus is a critical component of the pathways mediating the cardiovascular response to an acute alerting stimulus. 5. Long-term sustained changes in sympathetic vasomotor activity occur under both physiological conditions (e.g. a change in salt intake) and pathophysiological conditions (e.g. heart failure). There is evidence that the paraventricular nucleus in the hypothalamus is a critical component of the pathways mediating these changes. 6. Understanding the central mechanisms involved in the long-term regulation of sympathetic activity and blood pressure is a major challenge for the future. As a working hypothesis, a model is presented of the postulated central mechanisms that result in sustained changes in sympathetic vasomotor activity that are evoked by different types of chronic stimulation.

Fire in the operating room: a case report and laboratory study.

UNLABELLED: In July, 1998 a fire occurred in an operating room (OR) at the University Medical Center in Tucson, AZ. A patient was burned on the face, neck, and shoulders by the fire, which started during cranial burr-hole placement under monitored anesthesia care. This paper describes the actual case in some detail. The incident was simulated as accurately as possible in a laboratory experiment, in an attempt to determine specific risk factors for this event. The experiment found that a specific combination of factors was required to produce a fire similar in appearance to the one in the OR. The risk factors determined in these experiments are discussed in the context of previous reports of OR fires. Although other reports demonstrate some common characteristics of these events, the fire at the University Medical Center appears to be unique within the literature regarding the specific chain of events that led up to it. IMPLICATIONS: A patient was seriously burned in a fire that occurred during surgery. We performed laboratory experiments to re-create the fire, and found some of the key factors that led to this event.

Modulation of ACh-induced currents in rat adrenal chromaffin cells by ligands of alpha2 adrenergic and imidazoline receptors.

The aim of this study was to investigate the expression of the alpha2-adrenergic receptors in the adrenal medulla, and to examine the mechanism by which clonidine and related drugs inhibit acetylcholine (ACh)-induced whole-cell currents in adrenal chromaffin cells. Reverse transcription-polymerase chain reaction (RT-PCR) performed on punches of rat adrenal medulla demonstrated expression of mRNA for the 2A-, alpha2B- and alpha2C-adrenergic receptors. Similar experiments conducted with tissue punches obtained from the adrenal cortex did not reveal expression of these receptor subtypes. Whole-cell currents were recorded in isolated chromaffin cells using the perforated-patch configuration. ACh (50 microM) evoked inward currents with a peak amplitude of 117.8+/-9.3 pA (n = 45; Vhol = -60 mV). The currents were inhibited in a dose-dependent manner (0.5-50 microM) by clonidine, UK 14,304 and rilmenidine (agonists of alpha2/imidazoline receptors), as well as by SKF 86466 and efaroxan (antagonists). Adrenaline and noradrenaline (50-100 microM) had no significant effect. Thus, although the adrenal medulla expresses mRNA for the alpha2-adrenergic receptors, the lack of agonist-antagonist specificity observed in our whole-cell recordings (in the absence of intracellular dialysis) provides additional evidence against the possibility that these inhibitory effects are mediated by classical alpha2 or imidazoline receptor interactions.

Descending pathways mediating cardiovascular response from dorsomedial hypothalamic nucleus.

Physiological and anatomic methods were used to determine whether neurons in the rostral ventrolateral medulla (RVLM), nucleus tractus solitarius (NTS), or hypothalamic paraventricular nucleus (PVN) mediate the cardiovascular response evoked from the dorsomedial hypothalamic nucleus (DMH), which is believed to play a key role in mediating responses to stress. In urethane-anesthetized rats, activation of neurons in the DMH by microinjection of bicuculline resulted in a large increase in arterial pressure, heart rate, and renal sympathetic nerve activity. The pressor and sympathoexcitatory responses, but not the tachycardic response, were greatly reduced after bilateral muscimol injections into the RVLM even when baseline arterial pressure was maintained at a constant level. These responses were not reduced by muscimol injections into the PVN or NTS. Retrograde tracing experiments identified many neurons in the DMH that projected directly to the RVLM. The results indicate that the vasomotor and cardiac components of the response evoked from the DMH are mediated by pathways that are dependent and independent, respectively, of neurons in the RVLM.

What drives the tonic activity of presympathetic neurons in the rostral ventrolateral medulla?

1. The present review discusses the mechanisms that maintain the tonic activity of presympathetic cardiovascular neurons in the rostral part of the ventrolateral medulla. 2. Experimental evidence is reviewed that indicates that these neurons receive both tonic excitatory and tonic inhibitory synaptic inputs. The former appear to be mediated, at least in part, by glutamate receptors and the latter appear to be mediated by GABA receptors. 3. There is also evidence that these neurons have the capacity to generate action potentials in the absence of synaptic inputs. However, at present, there is not clear evidence that such an intrinsic pacemaker-like mechanism contributes to the tonic activity of these neurons under normal resting conditions. 4. These neurons are also chemosensitive and this may contribute to their tonic activation under conditions of hypoxia or hypercapnia.

Distribution of neurons projecting to the rostral ventrolateral medullary pressor region that are activated by sustained hypotension.

Hypotension produces a reflex increase in the activity of sympathetic vasomotor and cardiac nerves. It is believed that the reflex sympathoexcitation is due largely to disinhibition of sympathoexcitatory neurons in the rostral ventrolateral medulla, but it is possible that it may also be mediated by excitatory inputs from interneurons that are activated by a fall in blood pressure. The aim of this study in conscious rabbits was to identify and map neurons with properties that are characteristic of interneurons conveying excitatory inputs to the rostral ventrolateral medullary pressor region in response to hypotension. In a preliminary operation, a retrogradely-transported tracer, fluorescent-labelled microspheres, was injected into the functionally-identified pressor region in the rostral ventrolateral medulla. After a waiting period of at least one week, a moderate hypotension (decrease in arterial pressure of approximately 20 mmHg) was induced in conscious rabbits for 60 min by the continuous infusion of sodium nitroprusside. In confirmation of a previous study from our laboratory, [Li and Dampney (1994) Neuroscience 61, 613634] hypotension resulted in the expression of Fos (the protein product of c-fos, a marker of neuronal activation) in many neurons in several distinct regions in the brainstem and hypothalamus. Some of these regions (nucleus tractus solitarius, area postrema, caudal and intermediate ventrolateral medulla, parabrachial complex in the pons, and paraventricular nucleus in the hypothalamus) also contained large numbers of retrogradely-labelled cells. Approximately 10% of the Fos-positive neurons in the nucleus tractus solitarius, and 15-20% of Fos-positive neurons in the caudal and intermediate ventrolateral medulla were also retrogradely-labelled from the rostral ventrolateral medullary pressor region. In other brain regions, very few double-labelled neurons were found. In previous studies from our laboratory, we have determined the distribution of neurons in the brainstem that project to the rostral ventrolateral medullary pressor region and that are also activated by hypertension [Polson et al. (1995) Neuroscience 67, 107-123] or by hypoxia. [Hirooka et al. (1997) Neuroscience 80, 1209-1224] Comparison of the present results with those from these previous studies indicate that although hypotension and hypoxia both elicit powerful reflex sympathoexcitatory responses, the central pathways subserving these effects in conscious animals are fundamentally different. Hypoxia activates rostral ventrolateral medullary sympathoexcitatory neurons mainly via a major direct excitatory projection from the nucleus tractus solitarius, as well as from the Kölliker-Fuse nucleus in the pons, while in contrast the activation of these neurons in response to hypotension appears to be due mainly to disinhibition, mediated via inhibitory interneurons. In addition, however, inputs originating from excitatory interneurons in the nucleus tractus solitarius and caudal and intermediate parts of the ventrolateral medulla appear to contribute to the hypotension-evoked activation of sympathoexcitatory neurons in the rostral ventrolateral medulla.

Entropic collapse transition of a polymer in a solvent with a nonadditive potential.

We use molecular dynamics simulations to study an entropy-driven collapse transition of a flexible polymer in a solvent. Monomers and solvent particles interact with a steeply repulsive soft-sphere potential. We consider a nonadditive potential system in which the effective diameter describing the solvent-monomer interaction is greater than or equal to the diameters corresponding to the solvent-solvent and monomer-monomer interactions, which are set equal. We examine the effects of nonadditivity of the solvent-monomer potential and solvent density on the collapse transition. We find that a small degree of nonadditivity will drive the transition at sufficiently high solvent density. Increasing the density leads to a collapse transition at lower values of nonadditivity.

Effect of CI-930 [3-(2H)-pyridazinone-4,5-dihydro-6-[4-(1H-imidazolyl) phenyl]-5-methyl-monohydrochloride] and rolipram on human coronary artery smooth muscle cell proliferation.

Experiments were conducted to determine how selective inhibitors of certain cyclic nucleotide phosphodiesterase (PDE) families, namely CI-930 (PDE3 inhibitor; 3-(2H)-pyridazinone-4,5-dihydro-6-[4-(1H-imidazolyl) phenyl]-5-methyl monohydro chloride) and rolipram (PDE4 inhibitor), may affect human coronary artery smooth muscle cell (HCASMC) proliferation. CI-930- and rolipram-inhibitable PDEs accounted for most of the cyclic AMP hydrolyzing activity in HCASMC. Twenty micromolar CI-930 and 20 microM rolipram used individually attenuated proliferation of HCASMC from some, but not all donors, as measured by flow cytometry. The simultaneous addition of 10 microM CI-930 plus 10 microM rolipram caused greater attenuation. This attenuation represented a reduction of the number of cells entering the S phase of the cell cycle and not merely a delay in cell cycle traverse. No statistically significant elevation of cyclic AMP was detected following the addition of either PDE inhibitor individually, but the combination produced significant elevations. It is concluded that CI-930- and rolipram-inhibitable PDE isozymes are expressed in HCASMC and that selective inhibitors of these isozymes can attenuate HCASMC proliferation. The data suggest that selective PDE inhibitors may prevent restenosis in patients following percutaneous transluminal coronary angioplasty because of their effect on HCASMC proliferation, and they may also be useful in retarding the progression of atherosclerosis in individuals at risk. PDE3 and PDE4 inhibitors in combination are more effective than the inhibitors used individually.

Hypoxia-induced Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla.

Previous studies in anaesthetized animals have shown that the hypoxia-induced increase in sympathetic vasomotor activity is largely dependent on synaptic excitation of sympathoexcitatory pressor neurons in the rostral part of the ventrolateral medulla. The primary aim of this study was to determine, in conscious rabbits, the distribution of neurons within the brain that have properties characteristic of interneurons conveying excitatory inputs to the rostral ventrolateral medullary pressor region in response to systemic hypoxia. In a preliminary operation, a retrogradely-transported tracer, fluorescent-labelled microspheres, was injected into the physiologically-identified pressor region in the rostral ventrolateral medulla. After a waiting period of one to two weeks, the conscious rabbits were subjected to moderate hypoxia (induced by breathing 10% O2 in N2) for a period of 60 min. Control groups of animals were exposed to room air or to mild hypoxia (12% O2 in N2). Moderate hypoxia resulted in a modest hypertension of approximately 15 mmHg, and in the expression of Fos (a marker of neuronal activation) in many neurons in the nucleus tractus solitarius, the rostral, intermediate and caudal parts of the ventrolateral medulla, the Kölliker-Fuse nucleus, locus coeruleus, subcoeruleus and A5 area in the pons as well as in several midbrain and forebrain regions, including the periaqueductal grey in the midbrain and the paraventricular, supraoptic and arcuate nuclei in the hypothalamus. Fos expression was also observed in these regions in rabbits subjected to mild hypoxia or normoxia, but it was much reduced compared to rabbits subjected to moderate hypoxia. Approximately half of the neurons in the ventrolateral medulla, 27% of neurons in the nucleus tractus solitarius, and 49-81% of neurons in the locus coeruleus, sub-coeruleus and A5 area that expressed Fos following moderate hypoxia were also immunoreactive for tyrosine hydroxylase, and were therefore catecholamine cells. Approximately half of the neurons in the nucleus tractus solitarius and two-thirds of neurons in the Kölliker-Fuse nucleus that expressed Fos following moderate hypoxia were retrogradely labelled from the rostral ventrolateral medullary pressor region. Similarly, approximately one quarter of Fos-positive cells in the caudal and intermediate ventrolateral medulla were retrogradely labelled, but very few Fos-positive/retrogradely-labelled cells were found in other pontomedullary or suprapontine brain regions. The results indicate that systemic hypoxia results in activation of neurons in several discrete nuclei in the brainstem and forebrain, including neurons in all the major pontomedullary catecholamine cell groups. However, neurons that are activated by systemic hypoxia and that also project to the rostral ventrolateral medullary pressor region are virtually confined to the lower brainstem, primarily in the nucleus tractus solitarius and Kölliker-Fuse nucleus and to a lesser extent the caudal/intermediate ventrolateral medulla. In a previous study from our laboratory, we determined the distribution of neurons in the brainstem that are activated by hypertension and that also project to the rostral ventrolateral medullary pressor region. [Polson et al. (1995) Neuroscience 67, 107-123]. Comparison of the present results with those from this previous study indicates that the hypoxia-activated neurons in the nucleus tractus solitarius and Kölliker-Fuse nucleus that project to the rostral ventrolateral medulla are likely to be interneurons conveying excitatory chemoreceptor signals, while those in the caudal/intermediate ventrolateral medulla are likely to be mainly interneurons conveying inhibitory baroreceptor signals, activated by the rise in arterial blood pressure associated with the hypoxia-induced hypertension.

Effects of sinoaortic denervation on Fos expression in the brain evoked by hypertension and hypotension in conscious rabbits.

We have previously shown [Li and Dampney (1994) Neuroscience 61, 613-634] that periods of sustained hypertension and hypotension each induces a distinctive and reproducible pattern of neuronal expression of Fos (a marker of neuronal activation) in specific regions of the brainstem and forebrain of conscious rabbits. The aim of this study was to determine the contribution of afferent inputs from arterial baroreceptors to the activation of neurons in these various brain regions that is caused by a sustained change in arterial pressure. Experiments were carried out on rabbits in which the carotid sinus and aortic depressor nerves were cut in a preliminary operation. Following a recovery period of seven to 10 days, a moderate hypertension or hypotension (increase or decrease in arterial pressure of 20-30 mmHg) was induced in conscious barodenervated rabbits for 60 min by the continuous infusion of phenylephrine or sodium nitroprusside, respectively. In control experiments, barodenervated rabbits were subjected to the identical procedures except that they were infused with the vehicle solution alone. Compared with the effects seen in barointact rabbits, [Li and Dampney (1994) Neuroscience 61, 613-634] the number of neurons that expressed Fos in response to hypertension was reduced by approximately 90% in the nucleus of the solitary tract and in the caudal and intermediate parts of the ventrolateral medulla. In supramedullary regions, baroreceptor denervation resulted in a reduction of approximately 60% in hypertension-induced Fos expression in the central nucleus of the amygdala and in the bed nucleus of the stria terminalis, but no significant reduction in the parabrachial complex in the pons. Following hypotension, the number of neurons that expressed Fos in barodenervated rabbits, compared with barointact rabbits, [Li and Dampney (1994) Neuroscience 61, 613-634] was reduced by approximately 90% in the nucleus of the solitary tract, area postrema, and caudal, intermediate and rostral parts of the ventrolateral medulla. Baroreceptor denervation also resulted in a similar large reduction in hypotension-induced Fos expression in many supramedullary regions (locus coeruleus, midbrain periaqueductal grey, hypothalamic paraventricular nucleus, and in the central nucleus of the amygdala and the bed nucleus of the stria terminalis in the basal forebrain). In the supraoptic nucleus, hypotension-induced Fos expression in barodenervated rabbits was reduced by 75% compared to barointact animals, but was still significantly greater than in control animals. There was also a high level of Fos expression, much greater than in control animals, in the circumventricular organs surrounding the third ventricle (subfornical organ and organum vasculosum lamina terminalis). The results indicate that in conscious rabbits the activation of neurons that occurs in several discrete regions at all levels of the brain following a sustained change in arterial pressure is largely dependent upon inputs from arterial baroreceptors, with the exception of neurons in the circumventricular organs surrounding the third ventricle that are activated by sustained hypotension. The latter group of neurons are known to project to vasopressin-secreting neurons in the supraoptic nucleus, and may therefore via this pathway trigger the hypotension-induced release of vasopressin that occurs in the absence of baroreceptor inputs.

Pressor and sympathoexcitatory effects of nitric oxide in the rostral ventrolateral medulla.

OBJECTIVE: It has been shown that nitric oxide (NO) plays an important role in the central control of arterial pressure and sympathetic nerve activity. The aim of this study was to determine whether NO can regulate sympathetic nerve activity by an action on pressor neurons within the rostral part of the ventrolateral medulla (VLM). DESIGN AND METHODS: Experiments were performed on anaesthetized rabbits with denervated arterial and cardiopulmonary baroreceptors. The mean arterial pressure (MAP), heart rate and renal sympathetic nerve activity were measured. Microinjections of the NO donors sodium nitroprusside (SNP, 4-50 nmol) and S-nitroso-glutathione (10 nmol), the NO precursor L-arginine (50 nmol) and the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 50 nmol), were made into the functionally identified pressor region in the rostral VLM. The effects of SNP were also determined before and after injection of 5 nmol methylene blue into the same area. In control experiments, injections of D-arginine (50 nmol) and D-NAME (50 nmol), which are the inactive isomers of L-arginine and L-NAME, respectively, were also made into the functionally identified pressor region in the rostral VLM. RESULTS: Microinjections of SNP into the rostral VLM pressor region produced a dose-dependent increase in mean arterial pressure and renal sympathetic nerve activity. At the highest dose of 50 nmol, the increase in MAP was 26 +/- 5 mmHg (P < 0.001) and the sympathetic nerve activity was 53 +/- 5% (P < 0.001). These effects were abolished following methylene blue injection into the same region. Injection of 10 nmol S-nitroso-glutathione also produced increases in MAP (15 +/- 2 mmHg, P < 0.001) and in renal sympathetic nerve activity (28 +/- 2%, P < 0.001). Microinjections of L- or D-arginine resulted in very small depressor responses, but had no significant effect on renal sympathetic nerve activity. Microinjections of L-NAME, but not of D-NAME, caused significant decreases in MAP (19 +/- 1 mmHg, P < 0.001) and in sympathetic nerve activity (30 +/- 3%, P < 0.001). CONCLUSIONS: The results indicate that, in the anaesthetized rabbit with denervated baroreceptors, NO has a pressor and sympathoexcitatory action in the rostral VLM, which is mediated by a cyclic GMP-dependent mechanism. Second, endogenous NO may modulate sympathetic activity tonically, by a direct or indirect action on sympathoexcitatory neurons within the rostral VLM.

Cyclic nucleotide phosphodiesterases and vascular smooth muscle.

At least 30 different phosphodiesterase (PDE) enzymes have now been identified in mammalian tissues and cells, many of which are products of separate genes. These different isoenzyme forms can be subdivided into seven families based on their genetic and functional characteristics. Relatively specific inhibitors are available for at least five of these PDE families. A functional classification based on substrate specificity, regulatory properties, and sensitivity to inhibition by isozyme- and tissue-selective inhibitors can be used in describing the PDEs of vascular smooth muscle. Inhibition of these PDEs, especially with inhibitors of the PDE3 isoform, promotes vascular relaxation, particularly if the preparation of smooth muscle has been preconracted. For the most part, the drugs appear to act directly on smooth muscle; their effects are usually observed in endothelium-denuded preparations. In addition to their cardiotonic properties, many PDE3 inhibitors possess antiplatelet and thrombolytic activities, thereby suggesting the potential benefit of these drugs in treating diseases of the cardiovascular system. Isozyme- and cell-specific drugs have been shown to alter the synthetic state (i.e. proliferative phenotype) of smooth muscle cultures toward the appearance of the contractile phenotype. This suggests the possible use of selective PDE inhibitors to minimize the problem of restenosis seen after angioplasty. The development of novel methods to deliver more potent and selective PDE inhibitors to individual cell types and subcellular locales will lead to new therapeutic uses for this class of drugs in diseases of the cardiovascular system.

Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla after sustained hypertension in conscious rabbits.

Previous studies in anaesthetized animals have shown that the baroreflex control of sympathetic vasomotor activity is mediated to a large extent by inhibitory inputs to sympathoexcitatory pressor neurons in the rostral part of the ventrolateral medulla. The aim of this study was to determine, in conscious rabbits, the distribution of neurons within the brain that have two properties characteristic of interneurons conveying baroreceptor signals to the rostral ventrolateral medulla: (i) they are activated by an increase in arterial pressure; and (ii) they project specifically to the rostral ventrolateral medulla pressor region. In a preliminary operation, an injection of the retrogradely transported tracer, fluorescent-labelled microspheres, was made into the physiologically identified pressor region in the rostral ventrolateral medulla. After a waiting period of one to eight weeks, hypertension was produced in the conscious rabbit by continuous intravenous infusion of phenylephrine at a rate sufficient to increase arterial pressure by approximately 20 mmHg, maintained for a period of 60 min. A control group of animals was infused with the vehicle solution alone. In confirmation of our previous study, hypertension produced by phenylephrine resulted in the neuronal expression of Fos (a marker of neuronal activation) in the nucleus of the solitary tract, area postrema, the intermediate and caudal parts of the ventrolateral medulla parabrachial complex, and in the central nucleus of the amygdala. Approximately 50% of the Fos-immunoreactive neurons in both the caudal and intermediate parts of the ventrolateral medulla were also retrogradely labelled from the rostral ventrolateral medulla pressor region; such double-labelled neurons were confined to a discrete longitudinal column located just ventrolateral to the nucleus ambiguus. Significant numbers of double-labelled neurons were also found in the nucleus of the solitary tract and area postrema, although these represented a much lower proportion (13-16%) of the total number of Fos-immunoreactive neurons in these regions. In the parabrachial complex, Fos-immunoreactive and retrogradely labelled neurons were largely separate populations, while in the amygdala they were entirely separate populations. In the control group of rabbits, virtually no double-labelled neurons were found in any of these regions. The results indicate that putative baroreceptor interneurons that project to the pressor region of the rostral ventrolateral medulla are virtually confined to the lower brainstem. In particular, they support the results of previous studies in anaesthetized animals indicating that neurons in the intermediate and caudal ventrolateral medulla convey baroreceptor signals to the rostral ventrolateral medulla pressor region, and extend them by demonstrating the precise anatomical distribution of these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of c-fos functional mapping to identify the central baroreceptor reflex pathway: advantages and limitations.

Prolonged stimulation of many neurons results in the expression of the immediate early gene c-fos, which in turn cause the production of the protein Fos, whose presence in a cell can be detected by immunocytochemistry. This method has been used in both conscious and anaesthetized animals to identify central neurons involved in the baroreceptor reflex. In this paper we review the factors that can influence c-fos expression, with particular emphasis on the effects of different anaesthetic agents. We conclude that the c-fos method of functional mapping, when applied carefully and critically, is a very useful method of identifying central neurons that are activated by cardiovascular stimuli in conscious animals. Anaesthetic agents can significantly alter c-fos expression, and this effect differs greatly according to the type of anaesthetic used.

Synergistic interactions between selective pharmacological inhibitors of phosphodiesterase isozyme families PDE III and PDE IV to attenuate proliferation of rat vascular smooth muscle cells.

The interaction between selective inhibitors of 3',5'-cyclic-nucleotide phosphodiesterase (PDE) III (cyclic GMP inhibited phosphodiesterase) and selective inhibitors of PDE IV (Ro 20-1724 inhibited phosphodiesterase) to attenuate fetal bovine serum-stimulated incorporation of [3H]thymidine into DNA and cell proliferation was studied in a line (A10) of vascular smooth muscle cells (VSMC). The nonselective PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and papaverine attenuated DNA synthesis with EC50 values (16 and 18 microM, respectively) in the same range as their published IC50 values (2-50 and 2-25 microM, respectively) as PDE inhibitors. The selective PDE III inhibitors CI-930 and cilostamide used alone attenuated DNA synthesis with EC50 values (> 300 and 5.3 microM, respectively) that were much higher than published IC50 values (0.15-0.46 and 0.005-0.064 microM, respectively) for inhibition of PDE III. In the presence of the PDE IV inhibitor rolipram (10 microM), their EC50 values were shifted (0.66 and 0.16 microM, respectively) much closer to their respective IC50 values. When the selective PDE IV inhibitors rolipram and Ro 20-1724 were used alone, they attenuated DNA synthesis with EC50 values (111 and > 100 microM, respectively) much higher than their IC50 values (0.6-2.6 and 2-13 microM, respectively) as inhibitors of PDE IV, but 10 microM CI-930 (PDE III inhibitor) shifted their EC50 values (0.56 and 1.5 microM, respectively) much closer to their IC50 values. In experiments that assessed VSMC proliferation using the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] method, IBMX and papaverine attenuated proliferation with EC50 values (27 and 58 microM, respectively) close to their IC50 values. CI-930 and cilostamide used alone did not cause 50% attenuation of proliferation at the highest concentrations tested (100 and 10 microM, respectively). In the presence of 5 microM rolipram, however, their effects were enhanced greatly with EC50 values (0.86 and 0.23 microM, respectively) that were close to their IC50 values as PDE III inhibitors. Similarly, rolipram and Ro 20-1724 attenuated VSMC proliferation with EC50 values close to their IC50 values in the presence (2.1 and 4.6 microM, respectively) but not in the absence (> 100 and > 10 microM, respectively) of 2 microM CI-930. The interactions between PDE III inhibitors and PDE IV inhibitors to attenuate DNA synthesis and VSMC proliferation were synergistic as determined by the combination index. The data demonstrate that the synergistic interactions that attenuate incorporation of [3H]thymidine into DNA are accompanied by synergistic attenuations of VSMC division.(ABSTRACT TRUNCATED AT 400 WORDS)