Molecular patterns of response and treatment failure after frontline venetoclax combinations in older patients with AML.

The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes divergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.

Structural basis of acquired resistance to selpercatinib and pralsetinib mediated by non-gatekeeper RET mutations.

BACKGROUND: Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are highly potent RET-selective protein tyrosine kinase inhibitors (TKIs) for treating advanced RET-altered thyroid cancers and non-small-cell lung cancer (NSCLC). It is critical to analyze RET mutants resistant to these drugs and unravel the molecular basis to improve patient outcomes. PATIENTS AND METHODS: Cell-free DNAs (cfDNAs) were analyzed in a RET-mutant medullary thyroid cancer (MTC) patient and a CCDC6-RET fusion NSCLC patient who had dramatic response to selpercatinib and later developed resistance. Selpercatinib-resistant RET mutants were identified and cross-profiled with pralsetinib in cell cultures. Crystal structures of RET-selpercatinib and RET-pralsetinib complexes were determined based on high-resolution diffraction data collected with synchrotron radiation. RESULTS: RETG810C/S mutations at the solvent front and RETY806C/N mutation at the hinge region were found in cfDNAs of an MTC patient with RETM918T/V804M/L, who initially responded to selpercatinib and developed resistance. RETG810C mutant was detected in cfDNAs of a CCDC6-RET-fusion NSCLC patient who developed acquired resistance to selpercatinib. Five RET kinase domain mutations at three non-gatekeeper residues were identified from 39 selpercatinib-resistant cell lines. All five selpercatinib-resistant RET mutants were cross-resistant to pralsetinib. X-ray crystal structures of the RET-selpercatinib and RET-pralsetinib complexes reveal that, unlike other TKIs, these two RET TKIs anchor one end in the front cleft and wrap around the gate wall to access the back cleft. CONCLUSIONS: RET mutations at the solvent front and the hinge are resistant to both drugs. Selpercatinib and pralsetinib use an unconventional mode to bind RET that avoids the interference from gatekeeper mutations but is vulnerable to non-gatekeeper mutations.

Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.

BACKGROUND: In a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array. RESULTS: The average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton. CONCLUSIONS: The Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.

Intranasally administered serelaxin abrogates airway remodelling and attenuates airway hyperresponsiveness in allergic airways disease.

BACKGROUND: The peptide hormone relaxin plays a key role in the systemic hemodynamic and renovascular adaptive changes that occur during pregnancy, which is linked to its antiremodelling effects. Serelaxin (a recombinant form of human gene-2 relaxin) has been shown to inhibit lung fibrosis in various disease models and reverse airway remodelling and airway hyperresponsiveness (AHR) in allergic airways disease (AAD). OBJECTIVE: Although continuous systemic delivery of exogenous serelaxin alleviates allergic fibrosis and AHR, more direct routes for administration into the lung have not been investigated. Thus, intranasal administration of serelaxin was evaluated for its ability to reverse airway remodelling and AHR associated with AAD. METHODS: Female Balb/c mice were subjected to a 9-week model of chronic AAD. Subgroups of animals (n = 12/group) were then treated intranasally with serelaxin (0.8 mg/mL) or vehicle once daily for 14 days (from weeks 9-11). Saline-sensitized/challenged mice treated with intranasal saline served as additional controls. Differential bronchoalveolar lavage (BAL) cell counts, ovalbumin (OVA)-specific IgE levels, tissue inflammation, parameters of airway remodelling and AHR were then assessed. RESULTS: Chronic AAD was associated with significant increases in differential BAL cell counts, OVA-specific IgE levels, inflammation, epithelial thickening, goblet cell metaplasia, TGF-ß1 expression, epithelial Smad2 phosphorylation (pSmad2), subepithelial collagen thickness, total lung collagen concentration and AHR (all P < 0.05 vs. respective measurements from saline-treated mice). Daily intranasal delivery of serelaxin significantly diminished AAD-induced epithelial thickening, epithelial pSmad2, subepithelial and total lung collagen content (fibrosis) and AHR (all P < 0.05 vs. vehicle-treated AAD mice). CONCLUSIONS AND CLINICAL RELEVANCE: Intranasal delivery of serelaxin can effectively reduce airway remodelling and AHR, when administered once daily. Respirable preparations of serelaxin may have therapeutic potential for the prevention and/or reversal of established airway remodelling and AHR in asthma.

Comparative evaluation of isoflurane and sevoflurane in avian patients.

BACKGROUND AND AIM: Literature comparing the use of isoflurane and sevoflurane inhalation anesthetic agents in birds is scarce. This study aimed to evaluate the comparison of isoflurane and sevoflurane during induction, maintenance, and recovery of anesthesia in avian patients. MATERIALS AND METHODS: In this study, 24 injured avian patients (n=24) were selected randomly and divided into four groups during kite flying festival. In the present study, isoflurane and sevoflurane were used as induction and maintenance anesthetic agents, with and without butorphanol tartrate premedication agent in all the birds. Different physiological parameters were evaluated, namely, cloacal temperature (°F), heart rate (beats/min), respiratory rate (breaths/min), and SpO2 (%) were recorded at 0, 10, 20 min, and at recovery time. The quality of anesthesia was assessed on the basis of induction time, quality of induction, production of analgesia, muscle relaxation, body reflexes, recovery time, quality of recovery, sitting, standing, and complete recovery time (CRT). RESULTS: The mean±standard error value of induction time was 230.00±32.55, 280.00±25.29, 180.00±21.90, and 260.00±36.87 s, respectively, in Groups I, II, III, and IV. The feather plucking, pharyngeal, and toe pinching reflexes were noticed, when the birds were passing through the light plane of anesthesia during induction. Comparison of cloacal temperature at the time of recovery between Group-I versus Group-III revealed a significant difference (p<0.05). Comparison of mean respiratory rates at the time of recovery between Group-II versus Group-IV revealed a significant difference (p<0.05). Excellent quality of recovery was observed in all the groups of anesthetic protocols. Sitting, standing, and CRT were observed shortest in avian patients maintained with sevoflurane as compared to isoflurane. CONCLUSION: The quality of induction of anesthesia was rapid in avian patients when induced with sevoflurane as compared to isoflurane. Rapid onset of induction and recovery of anesthesia were found with sevoflurane followed by isoflurane. Induction and maintenance of anesthesia in avian patients with sevoflurane resulted in the lowest time required for sitting, standing, and CRT.

Combining an epithelial repair factor and anti-fibrotic with a corticosteroid offers optimal treatment for allergic airways disease.

BACKGROUND AND PURPOSE: We evaluated the extent to which individual versus combination treatments that specifically target airway epithelial damage [trefoil factor-2 (TFF2)], airway fibrosis [serelaxin (RLX)] or airway inflammation [dexamethasone (DEX)] reversed the pathogenesis of chronic allergic airways disease (AAD). EXPERIMENTAL APPROACH: Following induction of ovalbumin (OVA)-induced chronic AAD in 6­8 week female Balb/c mice, animals were i.p. administered naphthalene (NA) on day 64 to induce epithelial damage, then received daily intranasal administration of RLX (0.8 mg·mL(−1)), TFF2 (0.5 mg·mL(−1)), DEX (0.5 mg·mL(−1)), RLX + TFF2 or RLX + TFF2 + DEX from days 67­74. On day 75, lung function was assessed by invasive plethysmography, before lung tissue was isolated for analyses of various measures. The control group was treated with saline + corn oil (vehicle for NA). KEY RESULTS: OVA + NA-injured mice demonstrated significantly increased airway inflammation, airway remodelling (AWR) (epithelial damage/thickness; subepithelial myofibroblast differentiation, extracellular matrix accumulation and fibronectin deposition; total lung collagen concentration), and significantly reduced airway dynamic compliance (cDyn). RLX + TFF2 markedly reversed several measures of OVA + NA-induced AWR and normalized the reduction in cDyn. The combined effects of RLX + TFF2 + DEX significantly reversed peribronchial inflammation score, airway epithelial damage, subepithelial extracellular matrix accumulation/fibronectin deposition and total lung collagen concentration (by 50­90%) and also normalized the reduction of cDyn. CONCLUSIONS AND IMPLICATIONS: Combining an epithelial repair factor and anti-fibrotic provides an effective means of treating the AWR and dysfunction associated with AAD/asthma and may act as an effective adjunct therapy to anti-inflammatory corticosteroids

Chronic exercise reduces sympathetic nerve activity in rabbits with pacing-induced heart failure: A role for angiotensin II.

BACKGROUND: Chronic exercise (EX) improves the quality of life and increases the survival of patients with chronic heart failure (CHF). Because sympathetic nerve activity is elevated in the CHF state, it is possible that EX is beneficial in this disease due to a decrease in sympathetic outflow. METHODS AND RESULTS: We evaluated arterial baroreflex function and resting renal sympathetic nerve activity (RSNA) in EX normal and CHF rabbits before and after angiotensin II type 1 (AT(1)) receptor blockade. Four groups of rabbits were studied: a normal non-EX group, a normal EX group, a CHF non-EX group, and a CHF EX group. EX lowered resting RSNA in rabbits with CHF but not in normal rabbits. In addition, EX increased arterial baroreflex sensitivity in the CHF group (heart rate slope: CHF 1. 7+/-0.3 bpm/mm Hg, EX CHF 4.9+/-0.3 bpm/mm Hg; P:<0.01; RSNA slope: CHF 2.2+/-0.2%max/mm Hg, EX CHF 5.7+/-0.4%max/mm Hg; P:<0.01. AT(1) receptor blockade enhanced baroreflex sensitivity in the non-EX CHF rabbits but had no effect in EX CHF rabbits. Concomitant with this effect, EX lowered the elevated plasma angiotensin II concentration in the CHF group. A significant positive correlation was observed between sympathetic nerve activity and plasma angiotensin II. CONCLUSIONS: These data strongly suggest that EX reduces the sympathoexcitatory state in the setting of CHF. Enhanced arterial baroreflex sensitivity may contribute to this reduction. In addition, EX lowers plasma angiotensin II concentration in CHF. These data further suggest that the lowering of angiotensin II may contribute to the decrease in sympathetic nerve activity after EX in the CHF state.

Blunted diuretic and natriuretic responses to central administration of clonidine in streptozocin-induced diabetic rats.

The purpose of this study was to determine whether diuretic and natriuretic effects are altered in response to intracerebroventricular (ICV) infusion of clonidine in diabetic rats. Diabetes was induced in male Sprague-Dawley rats by 65 mg/kg i.p. injection of streptozocin, and control rats were injected with vehicle 2 wk before the experiment. Blood glucose levels were significantly elevated in the diabetic group (26.3 +/- 1.3 mM) compared with the control group (8.4 +/- 1.6 mM). Before and during ICV infusion of clonidine (2 micrograms.kg-1.min-1 for 45 min), urine flow and sodium excretion were measured from intact and denervated kidneys in anesthetized diabetic and control rats. The ICV infusion of clonidine significantly increased urine flow in both innervated and denervated kidneys from control rats but not from diabetic rats. There was a significant increase in sodium excretion during ICV infusion of clonidine from innervated kidneys of control rats, and denervation abolished this effect. In diabetic rats, clonidine failed to promote natriuresis from intact kidneys, and similar to control rats, did not promote natriuresis in denervated kidneys. This study demonstrates that 1) the diuretic response to the ICV infusion of clonidine is blunted in diabetic rats, and 2) a natriuretic response to the ICV infusion of clonidine is blunted in innervated kidneys of diabetic rats.

Volume reflex in diabetes.

After examining various components of the volume reflex in different models of diabetic rats, it is clear that the neural component of the volume reflex is altered in the early stage of diabetes. Nevertheless, the mechanisms involved in the neural component need to be examined further in diabetes. In terms of the humoral component of the effector limb of the volume reflex, the actions of ANF within the kidney appear to contribute to the altered volume reflex in diabetes. Results of our preliminary studies suggest that intrarenal factors may also contribute to the blunted renal excretory responses to acute VE in early diabetes. Finally, the present review has outlined the major abnormalities in the volume reflex in diabetes and the gaps in our knowledge of the various components of the volume reflex in diabetes.

Cardiovascular and behavioral effects produced by administration of liposome-entrapped GABA into the rat central nervous system.

Liposomes are nanosystems that allow a sustained release of entrapped substances. Gamma-aminobutyric acid (GABA) is the most prevalent inhibitory neurotransmitter of the central nervous system (CNS). We developed a liposomal formulation of GABA for application in long-term CNS functional studies. Two days after liposome-entrapped GABA was injected intracerebroventricularly (ICV), Wistar rats were submitted to the following evaluations: (1) changes in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) to ICV injection of bicuculline methiodide (BMI) in anesthetized rats; (2) changes in cardiovascular reactivity to air jet stress in conscious rats; and (3) anxiety-like behavior in conscious rats. GABA and saline-containing pegylated liposomes were prepared with a mean diameter of 200 nm. Rats with implanted cannulas targeted to lateral cerebral ventricle (n = 5-8/group) received either GABA solution (GS), empty liposomes (EL) or GABA-containing liposomes (GL). Following (48 h) central microinjection (2 µL, 0.09 M and 99 g/L) of liposomes, animals were submitted to the different protocols. Animals that received GL demonstrated attenuated response of RSNA to BMI microinjection (GS 48 ± 9, EL 43 ± 9, GL 11 ± 8%; P < 0.05), blunted tachycardia in the stress trial (ΔHR: GS 115 ± 14, EL 117 ± 10, GL 74 ± 9 bpm; P<0.05) and spent more time in the open arms of elevated plus maze (EL 6 ± 2 vs. GL 18 ± 5%; P = 0.028) compared with GS and EL groups. These results indicate that liposome-entrapped GABA can be a potential tool for exploring the chronic effects of GABA in specific regions and pathways of the central nervous system.

Noradrenergic mechanisms in the brain and peripheral organs of normotensive and spontaneously hypertensive rats at various ages.

Previous studies of noradrenergic mechanisms in spontaneously hypertensive rats (SHR) have yielded conflicting results, as many have used: 1) rats of only one age; 2) a single organ such as heart or brain; or 3) either Wistar-Kyoto (WKY) or an outbred normotensive control rat. We have studied the turnover of norepinephrine (NE) in three brain areas (cortex, hypothalamus, brain stem) and three peripheral organs (duodenum, skeletal muscle, kidney) of SHR, WKY, and Wistar rats at 5, 9, and 18 weeks of age. The rate of decline of norepinephrine [NE] in tissue was determined with a fluorescence assay at 0, 2, 4, and 8 hours after inhibition of tyrosine hydroxylase with alpha-methyltyrosine. Differences in NE turnover were inferred by comparing slopes of regression lines calculated for the plot of log [NE] (expressed as a percent of the initial concentration) vs time. Systolic arterial pressure of SHR was similar to that of WKY and Wistar rats at 5 weeks of age, but increased to 150 mm Hg by 9 weeks and reached an average of 190 mm Hg by 18 weeks. The turnover of NE in 5-week-old SHR compared to two normotensive strains was significantly lower in the cortex and significantly higher in the kidney and skeletal muscle. By 9 weeks, in SHR, NE turnover had increased significantly in the hypothalamus and brain stem, while decreasing significantly in the kidney and duodenum. No such changes were seen in these organs of WKY or Wistar rats when comparing turnover of NE at 5 and 9 weeks. At 18 weeks, there were no further differences in the organs of SHR when compared to values obtained at 9 weeks. These data support the hypothesis that the turnover of NE may be altered in central and peripheral organs of young SHR, and may initiate or contribute to the development of hypertension. Changes in turnover of NE in the brain and peripheral organs between 5 and 9 weeks in SHR suggest compensatory responses to increasing arterial pressure; however, similar changes in turnover were not seen between 9 and 18 weeks, although arterial pressure continued to increase.

Effect of renal denervation on arterial pressure in rats with aortic nerve transection.

The role of renal nerves in influencing the control of arterial pressure was studied in Wistar rats with aortic depressor nerve (ADN) transection. Renal denervation prevented or reversed the normal increase in arterial pressure seen after ADN transection. This effect was not due to an effect on the renin-angiotensin system, as the elevated arterial pressure after ADN section in rats with renal nerves intact was shown to be due to increased alpha-adrenergic activity. Food and water intake and urine output decreased significantly in both renal-denervated and sham-denervated rats after ADN section, suggesting that a pressure diuresis mechanism was not responsible for preventing the rise in pressure in renal-denervated rats. In another study, the concentration of norepinephrine in skeletal muscle and hypothalamus at 0 and 8 hours after inhibition of tyrosine hydroxylase with alpha-methyltyrosine was used as an index of norepinephrine turnover. Norepinephrine turnover in skeletal muscle was increased significantly over control values by ADN transection in sham renal-denervated rats, but was not significantly different from controls in renal-denervated rats with ADN section. In the hypothalamus, there was a significant difference between the turnover of norepinephrine in the two groups of ADN-sectioned rats. The results taken together suggest that renal denervation prevents the arterial pressure response to ADN transection by altering the central mechanisms governing sympathetic outflow. It is suggested that this effect may be due to elimination of information carried by afferent renal fibers.

L-Arginine does not restore dilatation of the basilar artery during diabetes mellitus.

The goal of this study was to test the hypothesis that administration of L-arginine, a substrate for the synthesis of nitric oxide, restores endothelium-dependent dilatation of the basilar artery during diabetes mellitus. We measured the diameter, of the basilar artery in vivo in nondiabetic and diabetic (streptozotocin; 50-60 mg/kg i.p.) rats in response to endothelium-dependent agonists (acetylcholine and bradykinin) and an endothelium-independent agonist (nitroglycerin) before and during application of L-arginine. Topical application of acetylcholine (1.0 and 10 muM) and bradykinin (1.0 and 10 microM) produced dilatation in nondiabetic rats of the basilar artery which was impaired in diabetic rats. Topical application of nitroglycerin (0.1 and 1.0 microM) produced similar dilatation of the basilar artery in nondiabetic and diabetic rats. Topical application of L-arginine (0.1 and 3 mM) did not enhance dilatation of the basilar artery in response to acetylcholine and bradykinin in diabetic rats. Thus, impairment of dilatation of the basilar artery in diabetic rats in response to acetylcholine and bradykinin appears to be related to a mechanism unrelated to the availability of L-arginine for nitric oxide synthase.

The regulation of sympathetic outflow in heart failure. The roles of angiotensin II, nitric oxide, and exercise training.

Sympatho-excitation is a hallmark of the chronic heart failure (CHF) state. It has long been assumed that this sympatho-excitation is mediated by a reduction in sensory input from cardiopulmonary and arterial baroreceptors. However, recent data suggest that these reflexes may only be important in the initiation of the sympatho-excitatory state and may not be necessary for the sustained increase in sympathetic nerve activity (SNA) in CHF. Two humoral factors that can influence SNA are nitric oxide (NO) and angiotensin II (AngII). Animals with CHF exhibit a downregulation in central gene expression for the neuronal isoform of nitric oxide synthase (nNOS). In addition, blockade of AngII receptors in combination with NO donation reduces SNA in animals with CHF, while NO donation alone has no effect on SNA. Chronic exercise training (EX) reduces both plasma AngII and SNA in rabbits with CHF while improving baroreflex function. Blockade of AT1 receptors enhances baroreflex function in non-EX CHF rabbits, but has little effect in EX CHF rabbits. These data suggest that the sympatho-excitatory state that is typical of CHF is, in part, due to changes in AngII and NO. Depressed baroreflex function and the elevated SNA can be improved by EX in animals with CHF.

Hemodynamic and norepinephrine responses to pacing-induced heart failure in conscious sinoaortic-denervated dogs.

The present study was undertaken to determine the effects of chronic sinoaortic (baroreceptor) denervation (SAD) on the hemodynamic and sympathetic alterations that occur in the pacing-induced model of congestive heart failure. Two groups of dogs were examined: intact (n = 9) and SAD (n = 9). Both groups of dogs were studied in the control (prepace) state and each week after the initiation of ventricular pacing at 250 beats/min. After the pacemaker was turned off, hemodynamic and plasma norepinephrine levels returned toward control levels in the prepaced state and after 1 and 2 wk of pacing. However, by 3 wk all hemodynamic and norepinephrine levels remained relatively constant over the 10-min observation period with the pacemaker off. With the pacemaker off, left ventricular end-diastolic pressure went from 2.7 +/- 1.4 (SE) mmHg during the prepace state to 23.2 +/- 2.9 mmHg in the heart failure state in intact dogs (P < 0.01). Left ventricular end-diastolic pressure increased to 27.1 +/- 2.2 mmHg from a control level of 4.2 +/- 1.9 mmHg i SAD dogs (P < 0.0003). Mean arterial pressure significantly decreased in intact and SAD dogs. Resting heart rate was significantly higher in SAD dogs and increased to 135.8 +/- 8.9 beats/min in intact dogs and 136.1 +/- 6.5 beats/min in SAD dogs. There were no significant differences in the hemodynamic parameters between intact and SAD dogs after pacing. Plasma norepinephrine was significantly lower in intact than in SAD dogs before pacing (197.7 +/- 21.6 vs. 320.6 +/- 26.6 pg/ml; P < 0.005). In the heart failure state, plasma norepinephrine increased significantly in both intact (598.3 +/- 44.2 pg/ml) and SAD (644.0 +/- 64.6 pg/ml) groups. There were no differences in the severity or the magnitude of the developed heart failure state in SAD vs. intact dogs. We conclude from these date that the arterial baroreflex is not the sole mechanism for the increase in sympathetic drive in heart failure.

Amelioration of depressed cardiopulmonary reflex control of sympathetic nerve activity by short-term exercise training in male rabbits with heart failure.

The reflex regulation of sympathetic nerve activity has been demonstrated to be impaired in the chronic heart failure (CHF) state compared with the normal condition (Liu JL, Murakami H, and Zucker IH. Circ Res 82: 496-502, 1998). Exercise training (Ex) appears to be beneficial to patients with CHF and has been shown to reduce sympathetic outflow in this disease state (Hambrecht R, Hilbrich L, Erbs S, Gielen S, Fiehn E, Schoene N, and Schuler G. J Am Coll Cardiol 35: 706-713, 2000). We tested the hypothesis that Ex corrects the reduced cardiopulmonary (CP) reflex response to volume expansion in the CHF state. Normal, normal with Ex, CHF, and CHF with Ex (CHF-Ex) groups (n = 10-21) of male New Zealand White rabbits were studied. CHF was induced by chronic ventricular pacing. Rabbits were instrumented to record left ventricular end-diastolic pressure (LVEDP), left ventricular end-diastolic diameter (LVEDD), and renal sympathetic nerve activity (RSNA). Experiments were carried out with the animals in the conscious state. Volume expansion was performed with 6% dextran in normal saline at a rate of 5 ml/min to approximately 20% of estimated plasma volume without any significant effect on mean arterial pressure being exhibited. The relationships between RSNA and LVEDP and between RSNA and LVEDD were determined by linear regression; the slopes served as an index of CP reflex sensitivity. Normal rabbits exhibited a CP reflex sensitivity of -8.4 +/- 1.5%delta RSNA/mmHg. This value fell to 0.0 +/- 1.3%delta RSNA/mmHg in CHF rabbits (P < 0.001). Ex increased CP reflex sensitivity to -5.0 +/- 0.7%delta RSNA/mmHg in CHF-Ex rabbits (P < 0.05 compared with CHF). A similar trend was seen when related to the change in LVEDD. Furthermore, resting RSNA expressed as a percentage of maximum RSNA in response to cigarette smoke was also normalized by Ex in rabbits with CHF. Ex had no effect on these parameters in normal rabbits. These data confirm an impairment of CP reflex sensitivity and sympathoexcitation in CHF vs. normal animals. Ex substantially restored both CP reflex sensitivity and baseline RSNA in CHF animals. Thus Ex beneficially affects reflex regulation in CHF, thereby lowering resting sympathetic nerve activity.

Alterations in brain hexokinase activity associated with streptozotocin-induced diabetes mellitus in the rat.

The effects of streptozotocin-induced diabetes mellitus on the activity of discrete regions of the brain were studied with histochemical localization and photodensitometric quantification of the metabolic enzyme, hexokinase. Two weeks after a single injection of streptozotocin (65 mg/kg, i.p.), plasma glucose and osmolarity levels were elevated, and plasma sodium concentrations were depressed. These changes were reversed in diabetic rats treated with insulin. Accompanying these symptoms of diabetes were significant increases in hexokinase activity in the magnocellular division of the paraventricular nucleus of the hypothalamus (mPVH, 12.1%), the medial subdivision of the nucleus of the tractus solitarius (mNTS, 15.5%), and the commissural subdivision of the NTS (cNTS, 10.9%). An increase, though just below the level of significance, was also observed in the supraoptic nucleus of the hypothalamus (SON, 11.5%). The increases in hexokinase activity were completely reversed in the cNTS (and SON) and only partly reversed in the mPVH and mNTS of insulin-treated diabetic rats. No changes in hexokinase activity were seen in the subfornical organ, medial preoptic area, parvocellular division of the PVH, locus coeruleus, or dorsal motor nucleus of the vagus of diabetic rats. These results reinforce the idea that the brain is not exempt from changes associated with diabetes mellitus and suggest that metabolic alterations in the mPVH (and SON) and two divisions of the NTS are likely related to changes in vasopressin production and blood volume, respectively.

Altered number of diaphorase (NOS) positive neurons in the hypothalamus of rats with heart failure.

Recently, we have demonstrated a decreased neuronal isoform of nitric oxide synthase (nNOS) message in the hypothalamus of rats with heart failure (HF). The purpose of this study was to determine the changes in NADPH-diaphorase (a commonly used marker for neuronal NOS activity) positive neurons in specific hypothalamic sites of rats with HF. After a standard histochemical protocol, NOS positive neurons were measured in paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MePO), subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and lateral hypothalamus (LH) of rats with coronary artery ligation (HF group; n=8) and sham-operated control rats (n=9). A total of 4 months after coronary ligation, the rats in the HF group displayed infarcts greater than at least 35% of the left ventricular wall (n = 8). Sham-operated rats had no observable damage to the myocardium. Rats with HF had a significantly lower number of NOS positive cells in the PVN (36% less) compared to sham rats. The number of NOS positive cells remained unaltered in the SON, MePO and LH in rats with HF. Conversely there was an increased number of NOS positive cells in the SFO (42% greater) and OVLT (100% greater). These data support the conclusion that the NO system within the hypothalamus involved in controlling autonomic outflow is altered during HF and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in HF.

Increased renal interstitial hydrostatic pressure causes c-fos expression in the rat's spinal cord dorsal horn.

To describe a sympathetic afferent circuit, interstitial hydrostatic pressure in the left kidney was increased in anesthetized rats for 1.5 h to activate renal mechanoreceptor afferents. Following renal afferent stimulation, the number of immunocytochemically stained cells for the immediate early gene c-fos was increased within the dorsal horn of the spinal cord. Relative to the surgical control procedure, increasing renal interstitial hydrostatic pressure produced more immunocytochemically stained cells per tissue section in laminae I and II of the dorsal horn both ipsilateral and contralateral to the stimulated kidney in the three most caudal thoracic spinal segments. Further, the number of c-fos immunocytochemically stained cells per section in the dorsal horn ipsilateral to the stimulated kidney was 28% greater than the number of stained cells contralateral to it. The staining patterns in the dorsal horns of stimulated and control animals were similar with most labeled cells in laminae I and II. These results indicate that (1) c-fos immunocytochemical staining may be useful for tracing specific sympathetic afferent pathways, (2) sensory pathways affected by increased renal interstitial hydrostatic pressure include spinal neurons located at lower thoracic levels, and (3) some of this sympathetic afferent pathway is located contralateral to the stimulated kidney. Neurons in the contralateral dorsal horn activated by renal stimulation may mediate renorenal reflexes.