Automated detection of microcalcification clusters for digital breast tomosynthesis using projection data only: a preliminary study.

Digital breast tomosynthesis (DBT) is a promising modality for breast imaging in which an anisotropic volume image of the breast is obtained. We present an algorithm for computerized detection of microcalcification clusters (MCCs) for DBT. This algorithm operates on the projection views only. Therefore it does not depend on reconstruction, and is computationally efficient. The algorithm was developed using a database of 30 image sets with microcalcifications, and a control group of 30 image sets without visible findings. The patient data were acquired on the first DBT prototype at Massachusetts General Hospital. Algorithm sensitivity was estimated to be 0.86 at 1.3 false positive clusters, which is below that of current MCC detection algorithms for full-field digital mammography. Because of the small number of patient cases, algorithm parameters were not optimized and one linear classifier was used. An actual limitation of our approach may be that the signal-to-noise ratio in the projection images is too low for microcalcification detection. Furthermore, the database consisted of predominantly small MCC. This may be related to the image quality obtained with this first prototype.

Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland.

The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized sheep and correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid responses (19 +/- 3 vs. 21 +/- 3 microl x min(-1) x g gland(-1)), whereas vasodilatation was greater during stimulation in bursts (-52 +/- 4 vs. -43 +/- 5%; P < 0.01). Continuous stimulation at 8 Hz induced substantially greater responses (66 +/- 9 microl x min(-1) x g gland(-1) and -77 +/- 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 microg/kg iv) reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz (reduced by 69%). Methoctramine ("M2/M4-selective"; 100 microg/kg iv; n = 5) had no effect on fluid or the vascular responses but increased the protein output at 2 (+90%, P < 0.05) and 8 Hz (+45%, P < 0.05). The immunoblotting showed distinct bands for muscarinic M1, M3, M4, and M5 receptors, and immunohistochemistry showed muscarinic M1 and M3 receptors to occur in the parenchyma. Thus muscarinic M1 receptors contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter release caused by blockade of neuronal inhibitory receptors of the M4 subtype would explain the increase in protein output.

Submandibular responses to stimulation of the parasympathetic innervation in anesthetized sheep.

Submandibular secretory and vascular responses to stimulation of the parasympathetic innervation and the output of vasoactive intestinal peptide (VIP) were investigated in anaesthetized sheep in the presence and absence of atropine (>/=0.5 mg/kg). In the absence of atropine, parasympathetic stimulation caused an increase in the flow of saliva and a decrease in submandibular vascular resistance; the latter response persisted after the administration of atropine and was then significantly reduced at the lowest but not at the higher frequencies tested. The output of VIP from the gland was frequency dependent over the range of 10-20 Hz (continuously) and significantly increased after atropine (P < 0.02). Furthermore, the fall in vascular resistance was linearly related to log VIP output after total muscarinic blockade. Intracarotid infusions of synthetic VIP produced dose-dependent falls in submandibular vascular resistance, together with a corresponding increase in submandibular blood flow. It is concluded that the atropine-resistant vasodilatation that occurs in this gland during parasympathetic stimulation is likely to be due largely, if not entirely, to the release of VIP.

Effects of prolonged reduction in blood flow on submandibular secretory function in anesthetized sheep.

Submandibular vascular and secretory responses to parasympathetic chorda-lingual (C-L) stimulation were investigated in anesthetized sheep before, during, and after an intracarotid (ic) infusion of endothelin-1 (ET-1). Stimulation of the peripheral end of the C-L nerve at 4 and 8 Hz produced a frequency-dependent reduction in submandibular vascular resistance (SVR) associated with a frequency-dependent increase in submandibular blood flow, salivary flow, and Na+, K+, and protein output from the gland. During stimulation at 4 Hz, ic ET-1 significantly increased SVR (P < 0.01), without significantly affecting either the aortic blood pressure or heart rate. Submandibular blood flow (SBF) was reduced by 48 +/- 4% and the flow of saliva by 50 +/- 1%. The effect on blood and salivary flow persisted for at least 30 min after the infusion of ET-1. The reduction in SBF was associated with a diminution in the output of Na+,K+, and protein in the saliva (P < 0.01). These effects persisted for 30 min after the infusion of ET-1 had been discontinued and were linearly related to the flow of plasma throughout.

Autonomic control of protein production by the parotid gland of the sheep.

The role of adrenoceptors in the control of parotid salivary function has been investigated in anaesthetized sheep. The enhancement of parotid protein output that occurs when the parasympathetic and sympathetic innervations to the gland are stimulated simultaneously in bursts at a low frequency (20 Hz for 1 s at 10-s intervals) was effectively abolished by pretreatment with propranolol (> or = 1.0 mg kg(-1), i.v., P < 0.001), without a comparable reduction in the flow of saliva or in the output of sodium or potassium. Secretion of protein was similarly augmented by simultaneous stimulation of the sympathetic innervation and an intracarotid infusion of acetylcholine (0.4-0.6 microg min(-1) g gland(-1)). This effect was also abolished by pretreatment with propranolol. Pretreatment with phentolamine (>1.0 mg kg(-1), i.v.) had no effect on the output of protein that occurred during combined stimulation of the parasympathetic and sympathetic innervations but increased the flow of saliva and the output of electrolytes. Stimulation of the parasympathetic innervation to the parotid gland caused a substantial fall in vascular resistance, which was reduced by the administration of atropine (0.5 mg kg(-1)). Stimulation of the sympathetic innervation caused a substantial rise in parotid vascular resistance in atropinized sheep. This effect was greater during continuous stimulation than during intermittent stimulation and enhanced by pretreatment with propranolol. It was virtually eliminated by pretreatment with phentolamine. It is concluded that the enhancement of protein output from the ovine parotid gland, that occurs during combined stimulation of the parasympathetic and sympathetic innervations at relatively low frequencies, depends upon interaction between cholinergic muscarinic and beta-adrenergic receptors. The vasoconstriction that occurs during sympathetic stimulation alone can be accounted for by activation of alpha-adrenoceptors.

The effect of parasympathetic postganglionic denervation on parotid salivary protein secretion in anaesthetized sheep.

Effects of unilateral parasympathetic denervation of ovine parotid glands were examined in anaesthetized sheep 21-28 days after nerve section. Parasympathetic denervation reduced the mass of the ipsilateral gland while increasing that of the contralateral gland to the extent that total gland mass was greater than in sheep with normally innervated glands. The spontaneous secretion (8.8 +/- 1.1 microl min(-1) g gland(-1)) was significantly less from denervated than from innervated glands of normal control animals (26.0 +/- 2.7 microl min(-1) g gland(-1); P< 0.01) and contained more protein. Rates of flow, and the outputs of sodium and potassium, in response to sympathetic stimulation, were similar from normally innervated and chronically denervated glands, when allowance was made for the discrepancy in weights, whereas the output of protein was significantly enhanced following parasympathetic denervation (innervated--31.4 +/- 7.3 microg g gland(-1), denervated--83.4 +/- 26.6 microg g gland(-1); P< 0.05). Intra-arterial infusions of acetylcholine (130 pmol min(-1) kg(-1)) elicited a flow of parotid saliva, the protein content of which was significantly enhanced by prior parasympathetic denervation. Intra-arterial infusions of vasoactive intestinal peptide (VIP; 2.5 pmol min(-1) kg(-1)) produced a small but statistically significant (P< 0.05) increase in the flow of parotid saliva from the contralateral, innervated but not from denervated glands. It also caused a small increase in protein output, which was significantly enhanced by prior denervation. VIP had no synergistic effect on the parotid responses to acetylcholine. The results show that the parasympathetic innervation to the parotid gland of the sheep exerts important trophic effects on the gland. Interaction of adrenergic and cholinergic receptors makes an important contribution to stimulation of the secretion of protein and prior denervation potentiates the protein responses to both acetylcholine and VIP.

Effects of endothelin on submandibular salivary responses to parasympathetic stimulation in anaesthetized sheep.

Submandibular responses to stimulation of the parasympathetic chorda tympani nerve have been investigated in anaesthetized sheep before, during and after an intracarotid infusion of endothelin, which reduced the blood flow through the gland by 56+/-5%. Stimulation of the peripheral end of the chorda tympani nerve produced a frequency-dependent increase in the flow of submandibular saliva, and in sodium, potassium and protein output. The reduction in submandibular blood flow, which occurred in response to endothelin, was associated with a decrease in the flow of saliva at all frequencies tested amounting on average to 44+/-6% (P<0.01). The flow of saliva was linearly related to the blood flow before and after endothelin. Both parameters were also linearly related during the infusion of endothelin and the regression lines were parallel. Salivary sodium concentration was significantly increased at the lower frequencies (1 and 2 Hz). Protein output was generally reduced but the difference only achieved statistical significance during stimulation at 1 Hz (P<0.01). Thus, submandibular secretory responses to parasympathetic stimulation are significantly attenuated by reducing the blood flow through the gland in this way.

A GATA-6 gene heart-region-specific enhancer provides a novel means to mark and probe a discrete component of the mouse cardiac conduction system.

The transcriptional programs that specify the distinct components of the cardiac conduction system are poorly understood, in part due to a paucity of definitive molecular markers. In the present study we show that a cGATA-6 gene enhancer can be used to selectively express transgenes in the atrioventricular (AV) conduction system as it becomes manifest in the developing multichambered mouse heart. Furthermore, our analysis of staged cGATA-6/lacZ embryos revealed that the activity of this heart-region-specific enhancer can be traced back essentially to the outset of the cardiogenic program. We provide evidence that this enhancer reads medial/lateral and anterior/posterior positional information before the heart tube forms and we show that the activity of this enhancer becomes restricted at the heart looping stage to AV myocardial cells that induce endocardial cushion formation. We infer that a deeply-rooted heart-region-specific transcriptional program serves to coordinate AV valve placement and AV conduction system formation. Lastly, we show that cGATA-6/Cre mice can be used to delete floxed genes in the respective subsets of specialized heart cells.

Pancreatic exocrine responses to parasympathetic stimulation in anaesthetized pigs.

Pancreatic exocrine responses to stimulation of the peripheral ends of the vagus nerves intermittently have been investigated in anaesthetized pigs and compared with the effects of continuous stimulation at corresponding frequencies. At relatively low frequencies < or =20 Hz in bursts or 2 Hz continuously) both the flow of pancreatic juice and the output of protein therein were potentiated by stimulating in bursts. Thus stimulation at 20 Hz in bursts produced a significantly greater flow of pancreatic juice than stimulation at 2 Hz continuously (10.9+/-0.9 compared to 4.8+/-0.7 microl min(-1) (g gland)-1 , respectively; P<0.01). Likewise the output of protein during intermittent stimulation at 20 Hz (144+/-23 microg min(-1) (g gland)-1) far exceeded that produced during continuous stimulation at 2 Hz (49+/-9 microg min(-1) (g gland)-1; P<0.01). Both differences were abolished by atropine (0.5 mg kg(-1) i.v.), which augmented the flow during continuous stimulation (to 8.7 +/- 1.5 microl min(-1) (g gland)-1; P<0.05 at 2 Hz) and substantially reduced the output of protein during intermittent stimulation (to 27+/-7 ng min(-1) (g gland)-1; P<0.01 at 20 Hz in bursts). These results show that a variety of pancreatic exocrine responses can be enhanced by stimulating the parasympathetic innervation in bursts. They are also consistent with the contention that the secretion of protein from the gland, in response to parasympathetic stimulation, is dependent mainly on activation of muscarinic receptors. They confirm that the flow of pancreatic juice is due mainly to the release of VIP and show that, in the absence of atropine, this is restricted by muscarinic inhibition which may be presynaptic as elsewhere.

Submandibular secretory and vascular responses to stimulation of the parasympathetic innervation in anesthetized cats.

Submandibular secretory responses to stimulation of the parasympathetic chorda-lingual nerve in anaesthetized cats have been investigated before, during, and after intracarotid infusion of endothelin-1 (ET-1), which reduced blood flow through the gland by 64+/-7%. Stimulation at different frequencies (2, 4, 8, and 16 Hz) evoked a frequency-dependent increase in the flow of submandibular saliva, sodium concentration and output, and output of both potassium and protein. The reduction in submandibular blood flow, which occurred in response to the infusion of ET-1, was associated with a decreased flow of saliva and a diminished output of both sodium and protein. The flow of saliva was linearly related to submandibular blood flow both in the presence and absence of ET-1. It is concluded that submandibular secretory responses to electrical stimulation of the parasympathetic innervation can be significantly attenuated by reducing the blood flow through the gland by ET-1 infusion, just as it is when the blood flow is reduced by hypotension.

The role of nitric oxide in mediating pancreatic endocrine responses to insulin-induced hypoglycaemia in the conscious calf.

The role of nitric oxide (NO) in mediating pancreatic endocrine responses to moderate hypoglycaemia has been investigated in conscious unrestrained calves. The synthesis of endogenous NO was inhibited by the administration of N-nitro-L-arginine methyl ester (L-NAME; 100 mg kg-1 I.A.), while sodium nitroprusside was infused continuously (2-4 microg min-1 kg-1 I.V.) to mimic the tonic production of NO. This effectively abolished the rise in plasma pancreatic polypeptide (PP) concentration during moderate hypoglycaemia (0.7 nmol kg-1 insulin I.V.) and significantly reduced the response to more intense hypoglycaemia (2.0 nmol kg-1 insulin I. V.). In contrast, the glucagon response was not significantly affected in either group, although consistently higher plasma glucagon values were obtained in response to the higher dose of insulin following the administration of L-NAME. It is concluded that, in the absence of L-NAME, production of NO contributes to the PP response, but not the glucagon response to hypoglycaemia in this species under physiological conditions.

Effects of reducing submandibular blood flow on secretory responses to parasympathetic stimulation in anaesthetized cats.

Submandibular secretory responses to stimulation of the parasympathetic chorda-lingual nerve were investigated in five anaesthetized cats before, during and after withdrawal of blood (ca 20 ml kg-1) in order to investigate the consequences of a reduced blood flow through the gland. Stimulation at different frequencies (2, 4, 6 and 8 Hz) evoked a frequency-dependent increase in the flow of submandibular saliva, sodium concentration, electrolyte and protein output. When the blood pressure was reduced (by about 50%) there was a significant reduction in submandibular blood flow and the secretion of both saliva and protein during stimulation. Under each set of conditions the flow of saliva was linearly related to the blood flow through the gland. It is concluded that submandibular secretory responses to electrical stimulation of the parasympathetic innervation can be significantly attenuated when the blood flow through the gland is reduced under the conditions employed in this study.

The role of nitric oxide in the control of protein secretion in the parotid gland of anaesthetized sheep.

Parotid secretion has been investigated in anaesthetized lambs in the presence and absence of N omega-nitro-L-arginine methyl ester (L-NAME) and sodium nitroprusside to block de novo synthesis of nitric oxide (NO). Following administration of L-NAME the basal rate of flow was unaffected and changes in electrolyte secretion failed to achieve statistical significance but there was a significant fall in the basal rate of protein secretion. The flow of parotid saliva which occurred in response to stimulation of the parasympathetic innervation was reduced by 34% and sodium output was reduced in approximately the same proportion. L-NAME had no significant effect on these parameters during stimulation of the sympathetic innervation. During combined stimulation of the parasympathetic and sympathetic innervations L-NAME caused a reduction in parotid salivary flow and sodium output which was roughly the same as that observed during parasympathetic stimulation alone. However, L-NAME caused a much greater reduction in protein output during each of these experimental protocols: -92% during parasympathetic stimulation, =63% during sympathetic stimulation, and -60% during combined stimulation. Whereas the absolute amount of protein secreted was reduced after L-NAME in each instance, the extent of potentiation of protein output recorded during combined stimulation was increased roughly fivefold. It is concluded that the output of protein in response to autonomic stimulation exhibits a greater No dependence than either the flow of saliva or secretion of electrolytes in this gland.

Autonomic control of submandibular protein secretion in the anaesthetized calf.

The autonomic control of submandibular secretion has been investigated in fully weaned, anaesthetized calves 7 weeks after birth. Stimulation of the parasympathetic (chorda-lingual) innervation invariably produced a flow of saliva, the rate of which was frequency dependent over the range 2-8 Hz continuously. Neither the rate of flow nor the output of protein was enhanced by stimulating in bursts at relatively high frequencies. Stimulation of the sympathetic innervation (20 Hz for 1 s at 10 s intervals) alone produced a much slower flow of saliva but with a considerably higher protein content. Stimulation of both together produced no greater flow of saliva than occurred with either alone at the lower frequencies (2 and 4 Hz) but there was a pronounced synergy in respect of the secretion of protein. Following pre-treatment with propranolol (1.0 mg kg-1 i.v.), during on-going chorda-lingual stimulation at 4 Hz, intra-arterial injections of 1 nmol of either vasoactive intestinal peptide (VIP) or pituitary adenylate cyclase activating peptide (PACAP) elicited an increase in the flow and protein output of about the same order of magnitude. Calcitonin gene-related peptide (CGRP) also produced these same effects with roughly half the efficacy of VIP and PACAP but substance P had no detectable effect. It is concluded that VIP, PACAP and possibly CGRP are candidates for neurotransmitters with a role in the control of secretion in this gland.

Nitric oxide in the control of submandibular gland function in the anaesthetized ferret.

Stimulation of parasympathetic innervation of the submandibular gland (2 or 20 Hz continuously or 20 Hz for 1 s at 10 s intervals), in the ferret, produced secretion of fluid and protein and a fall in vascular resistance. The responses following the administration of N omega-nitro-L-arginine methyl ester (L-NAME; 2 mmol kg-1 i.a.) to block the synthesis of nitric oxide (NO) were reduced, and the persisting responses were abolished (at 2 Hz continuously and 20 Hz intermittently) or further reduced (at 20 Hz continuously) by the additional administration of atropine. The output of vasoactive intestinal peptide (VIP) from the gland was not affected. Neither the secretory nor the vascular response to intra-arterial infusions of acetylcholine (1.25 nmol kg-1) was affected by L-NAME, whereas the vascular responses to both VIP (10 pmol kg-1) and pituitary adenylate cyclase-activating peptide (1-38) (PACAP) (0.5 pmol kg-1) were reduced thereby. Neither peptide evoked a fluid secretion per se. However, when infused during parasympathetic stimulation of saliva secretion, VIP increased both flow rate and the output of protein. These effects of VIP were abolished by L-NAME. The experiments were performed in the presence of sodium nitroprusside at doses (4-8 nmol min-1 kg-1 i.v.) aimed to counterbalance the systemic effects of L-NAME. The results show that, in the ferret, parasympathetic nerve activity increases submandibular blood flow, and elicits the flow of saliva and output of protein by mechanisms that involve in situ generation of NO, upon which the effects of VIP and PACAP but not acetylcholine are largely dependent.

Cardiovascular and pancreatic endocrine responses to glucagon-like peptide-1(7-36) amide in the conscious calf.

Intravenous infusions of glucagon-like peptide-1(7-36) amide (GLP-1; 35 pmol min-1 kg-1 for 10 min) produced a significant rise in mean heart rate, without significant change in mean aortic blood pressure, together with a significant rise in mean arterial plasma insulin, but not in plasma pancreatic glucagon or pancreatic polypeptide concentration, in conscious calves given exogenous glucose (30-60 micromol min-1 kg-1 i.v.). The insulinotropic effect was eliminated in the presence of exogenous amino acids (0.03 mmol min-1 kg-1 i.v.). It was not affected predictably by blocking the synthesis of nitric oxide or by the simultaneous administration of the established incretin factor gastrin-releasing peptide (GRP). Whereas GLP-1 produced a statistically significant rise in plasma insulin concentration in these animals, it was much less effective than GRP in this respect, when given by continuous i.v. infusion.

Pancreatic endocrine responses to the peptides VIP and PACAP in the conscious calf.

Pancreatic endocrine responses to vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP) and acetylcholine have been investigated in conscious calves, while continuously monitoring aortic blood pressure and heart rate, in order to assess the effects of these neuropeptides under normal physiological conditions. VIP was found to modulate the release of pancreatic polypeptide (PP) in response to acetylcholine at a dose which had no significant effect on the release of either pancreatic glucagon or insulin. PACAP stimulated the release of insulin and the effect was potentiated during infusions of exogenous glucose. PACAP, when infused together with acetylcholine, also stimulated the release of pancreatic glucagon in the absence of exogenous glucose but not in its presence. Release of PP was stimulated during infusions of PACAP, whether or not exogenous glucose was given, and this effect was found to be additive with that of acetylcholine.

Secretory interactions between the sympathetic and parasympathetic innervations of the submandibular gland in the anaesthetized cat.

Interactions between the sympathetic and parasympathetic innervations of the submandibular gland have been investigated in the anaesthetized cat. At low frequencies of chorda lingual (parasympathetic) stimulation, simultaneous stimulation of the ascending cervical sympathetic nerve in bursts (20 Hz for 1 s at 10 s intervals) increased the flow of submandibular saliva, but the effect was never more than additive. The output of protein was consistently reduced by simultaneous stimulation of both the sympathetic and parasympathetic innervations, below that evoked by stimulation of either alone. Sympathetic stimulation was more effective than parasympathetic stimulation in promoting the secretion of tissue kallikrein and peroxidase in the submandibular saliva. The output of the latter enzyme, in response to sympathetic stimulation, was significantly reduced by simultaneous stimulation of the parasympathetic innervation at frequencies greater than 1 Hz, but nevertheless exceeded the amount secreted during chorda stimulation alone. Thus, this protocol provided no evidence of synergy between the two divisions of the autonomic nervous system with respect to any submandibular secretory function that was recorded. However, following the administration of a small dose of atropine (2-15 micrograms kg-1 i.v.), sufficient to block secretion during chorda stimulation alone, the flow of saliva, in response to sympathetic stimulation, was potentiated when superimposed on a background of parasympathetic stimulation at all frequencies that were employed. This effect was abolished by larger doses of atropine, indicating that it was dependent upon activation of muscarinic receptors, only some of which could have been blocked by the initial dose.

Aspects of autonomic and neuroendocrine function.

This article provides a brief review of aspects of autonomic and neuroendocrine function studied initially in collaboration with the late Marian Silver. The importance of the sympathetic innervation to the liver in the control of glycogenolysis was established in anaesthetised animals of various species. Otherwise the work has been carried out mainly in conscious animals under strictly physiological conditions and below behavioural threshold. Investigations of the role of the autonomic innervation to the endocrine pancreas in controlling the release of pancreatic hormones, led to the realisation that the parasympathetic innervation mediates responses to glycaemic stimuli while the sympathetic innervation mediates responses to any form of stress. Studies of adrenal medullary function have confirmed that its threshold for many forms of stress is much higher than that of other components of the sympathetic system and revealed the importance of the pattern of electrical stimulation in determining the rates of release of catecholamines, enkephalins, corticotrophin-releasing factor (CRF) and adrendocorticotrophin (ACTH). The splanchnic sympathetic innervation to the adrenal cortex also plays an important role in determining glucocorticoid output by sensitising the cells to ACTH, probably mainly by the release of vasoactive intestinal peptide (VIP) from cortical nerve terminals. Finally studies of feeding in milk-fed calves have shown that suckling is associated with a remarkable hypertension and tachycardia. These cardiovascular effects are due to a selective sympathetic discharge, which does not involve the adrenal medullae, or the release of neuropeptide Y (NPY) and, at least in the calf, can be attributed to activation of adrenoceptors.