CGRP,but not substance P,induces an increased negativity of the interstitial fluid pressure in rat trachea

Neurogenic inflammation is mediated by neuropeptides released from sensory nerves following electrical stimulation of the vagal nerve or by capsaicin. The released neuropeptides are, among others, calcitonin gene-related peptide and substance P, which both induce vasodilation, while only substance P induces plasma extravasation. Electrical stimulation of the vagal nerve induces increased negativity of interstitial fluid pressure (P_if), which will contribute to enhance oedema formation. P_if was measured, on the abluminal side of the surgically exposed trachea, with sharpened glass capillaries (4–10 μm) connected to a servo-controlled counterpressure system. Measurements were performed after circulatory arrest, since the oedema formation associated with acute inflammation will increase P_if in a positive direction, which may potentially underestimate the increased negativity of P_if. Experiments were carried out in pentobarbital anaesthetized (50 mg kg^?1) Wistar–Møller rats. P_if in control rats averaged ?1.2 ± 0.9 (SD) mmHg (n=9). Intravenous injection of capsaicin (65.0 nmol) and calcitonin gene-related peptide (1.3 nmol) increased the negativity of P_if to ?4.0 ± 1.2 mmHg (n=8)(P < 0.01) and ?4.7 ± 2.0 mmHg (n=9) (P < 0.01), respectively. Intravenous injection of substance P (7.4 nmol, n=9; and 37.0 nmol, n=8) did not affect P_if compared to control (P > 0.05). Similarly, potentiation of the available substance P with thiorphan or captopril did not increase the negative P_if, nor did injection of stable substance P analogues. Thus, the present study seems to support the theory that, in rat trachea, the increased negativity of P_if after intravenous injection of capsaicin and after vagal stimulation is caused by calcitonin gene-related peptide.     

Lowering of interstitial fluid pressure in rat trachea after substance P alone and in combination with calcitonin gene‐related peptide

Neurogenic inflammation is mediated following a release of sensory neuropeptides including calcitonin gene‐related peptide (CGRP) and substance P (SP). The release of peptides can be mediated chemically with capsaicin, or electrically by stimulation of the vagal nerve, both inducing vasodilation, plasma protein extravasation and lowering of interstitial fluid pressure (P_if) which will contribute to the enhancement of oedema formation. Aim: Lowering of P_if has previously been demonstrated following intravenous (i.v.) treatment with CGRP, but it was not possible to demonstrate that SP had this effect under the same condition. Methods: Micropuncture measurements of P_if in the submucosa, without opening of the trachea, was conducted on rats anaesthetized with pentobarbital sodium (50 mg kg^?1) and cardiac arrest was induced with i.v. KCl. Results: P _if in vehicle‐treated animal averaged ?1.7 ± 0.4 (SD) mmHg (n = 9). Intravenous injection of SP induced significant lowering of P_if compared with control, both at low dose (0.47 nmol kg^?1 body weight) with 1 min distribution time (P < 0.007, ?4.2 ± 2.3 mmHg) and at high dose with seconds of distribution time (P < 0.03, ?4.2 ± 1.6 mmHg). The same response was observed after treatment with SP co‐injected with CGRP. Conclusions: Substance P alone or in combination with CGRP is able to induce a rapid lowering of P_if showing that this peptide is a potent agent in increasing the hydrostatic driving pressure initially transporting fluid into the tissue during an acute inflammatory reaction.     

The relationship between interstitial fluid pressure and volume in rat trachea

A change of interstitial fluid volume (IFV) will normally change the interstitial fluid pressure (P_if) so as to counteract further fluid movement across the capillaries and changes in IFV. Contrary to this, several acute inflammatory reactions in the trachea are associated with increased negativity of P_if, which will interstitial fluid balance in the trachea, interstitial compliance (ΔIFV/ΔP_if) was measured in pentobarbital anaesthetized rats. IFV was measured as the plasma equivalent extravascular distribution space of [⁵¹Cr]EDTA. P_if was measured in the same animal with sharpened glass pipettes (diameter 3–6 μm) connected to a servocontrolled counterpressure system. In dehydration (30 mL saline i.v., n=10) interstitial compliance was 0.083 mL g dry wt^-1 mmHg^-1. Since control IFV was 1.046 mL g dry wt^-1 (n=10) the interstitial compliance is 8% of IFV per mmHg. In overhydration (30 mL NaCl, n=10) and dextran anaphylaxis (1 mL dextran 70, n=10) compliance remained the same for the first 15% increase in IFV and then increased several-fold since P_if did not increase more than 2 mmHg above control level. The increased negativity of P_if by -10 mmHg associated with acute inflammation will require a reduction of IFV by 80% when interstitial compliance is 8% per mmHg. A more likely explanation is therefore that structural rearrangements are responsible for the events leading to increased negativity of P_if in acute inflammation.     

Increased negativity of interstitial fluid pressure contributes to development of oedema in rat skin following application of xylene

Intradermal interstitial fluid pressure (p₁) has been studied in rat skin during formation of inflammatory oedema caused by application of xylene. P, was measured with sharpened micropipettes connected to a servocontrolled counter-pressure system. Control p₁ averaged –1.3 + 0.6 (SD) mmHg. Following xylene application p₁ decreased to – 5.0 mmHg after 5 min and then increased to stabilize at about 0 mmHg at 45–60 min and later. When the transvascular fluid shifts accompanying the inflammatory reaction were prevented by inducing circulatory arrest prior to xylene application, p₁ fell to –7.5 mmHg within 5 min and remained at this level throughout the observation period of 90 min. Aprotinin in large doses (80,000 KIE kg^-1) before xylene application reduced the fall in P_i, whereas indomethacin had no effect. The increased negativity in P₁ will add directly to a normal transcapillary net filtration pressure of about 0.5 mmHg, resulting in a 10– to 20-fold increase in this pressure. The present experiments therefore suggest that the interstitium plays an active role in oedema formation in the initial phase of xylene-induced inflammation in rat skin through the development of an increased negativity of P_i.     

Effects of lactoferrin on rat dermal interstitial fluid pressure (Pif) and in vitro endothelial barrier function

We recently demonstrated that intravenous (i.v.) injection of the iron‐binding protein lactoferrin (Lf) followed by antilactoferrin (aLf) antibodies or iron‐saturated Lf alone increased albumin extravasation in vivo in several tissues including skin. Increased driving pressure for blood‐tissue exchange or direct effects of Lf on the endothelial barrier are possible mechanisms. We therefore, firstly, measured interstitial fluid pressure (P_if) in dermis of rats given 1 mg Lf i.v. followed 30 min later by aLf or saline and circulatory arrest 1 or 5 min thereafter and compared with controls. Secondly, transmonolayer passage of Evans blue labelled albumin (EB‐albumin) was evaluated in porcine pulmonary artery endothelial cells exposed to iron‐free or iron‐saturated Lf (both 100 μg mL^–1) in the absence and presence of 0.5 mM hydrogen peroxide. P_if increased significantly at 11–30 min following Lf to +2.1 ± 0.3 and +1.7 ± 0.2 mmHg at 11–20 and 21–30 min, respectively, compared with +0.1 ± 0.2 mmHg before Lf (P < 0.05, n=25). Endothelial transmonolayer passage of EB‐albumin during 3 h was not affected by iron‐free or iron‐saturated Lf neither in the absence nor presence of hydrogen peroxide that increased passage 3.5 times compared with controls. In conclusion, Lf‐induced increase in albumin extravasation in rat skin is not explained by changes in P_if (because Lf raised P_if significantly) or direct effects of Lf on the endothelial barrier.     

Neutrophil influx into guinea-pig airway lumen during cholinergic and non-cholinergic bronchoconstriction

Sensory nerve activation will produce adherence of neutrophils to tracheobronchial microvessels. The aim of the present study was to investigate whether this adherence would lead to an influx of neutrophils into the airway lumen. To do this, we studied the effects of 20 minutes of vagal stimulation (1 Hz, 5 ms, 5 V) in anaesthetized and tracheostomized guinea-pigs on both lung resistance, and the cell picture in bronchoalveolar lavage. Any changes were compared to those of intravenous methacholine influsion, producing similar changes in lung resistance. Since high pressure ventilation could produce lung damage, we also studied the effects of ventilation through an extracorporeal resistor, producing a similar change in transpulmonary pressure (45 ± 2 cmH₂O) as vagal stimulation (42 ± 4 cmH₂O). The total number of cells recovered in the lavage was not increased by vagal stimulation, methacholine influsion or the extracorporeal resistor. However, both vagal stimulation and methacholine influsion significantly increased the relative number of neutrophils in the lavage compared to sham stimulated animals (21 ± 11%, 13 ± 4% and 4±1% respectively), but the extracorporeal resistor had no effect (4 ± 2%). Our data suggests that prolonged bronchoconstriction per se may induce an influx of neutrophils into the airway lumen of the guinea-pig.     

Platelet activating factor (PAF) increases plasma protein extravasation and induces lowering of interstitial fluid pressure (Pif) in rat skin

Aim: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). Methods: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. Results: Platelet activating factor (50 μg mL^?1) administered locally via the fibre, increased extravasation of radiolabelled ¹²⁵I‐HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 Å, 56 Å and 39 Å after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P_if) pressure after subcutaneous administration (50 μg mL^?1). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 μg kg^?1) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma‐to‐tissue clearance (E‐alb) showed a significant increase after PAF (n = 7, P < 0.05). Conclusions: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P_if, indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.     

Quantitative MRI in murine radiation‐induced rectocolitis: comparison with histopathological inflammation score

Murine radiation‐induced rectocolitis is considered to be a relevant animal model of gastrointestinal inflammation. The purpose of our study was to compare quantitative MRI and histopathological features in this gastrointestinal inflammation model. Radiation rectocolitis was induced by localized single‐dose radiation (27 Gy) in Sprague‐Dawley rats. T₂‐weighted, T₁‐weighted and diffusion‐weighted MRI was performed at 7 T in 16 rats between 2 and 4 weeks after irradiation and in 10 control rats. Rats were sacrificed and the histopathological inflammation score of the colorectal samples was assessed. The irradiated rats showed significant increase in colorectal wall thickness (2.1 ± 0.3 mm versus 0.8 ± 0.3 mm in control rats, P < 0.0001), normalized T₂ signal intensity (4 ± 0.8 versus 2 ± 0.4 AU, P < 0.0001), normalized T₁ signal intensity (1.4 ± 0.1 versus 1.1 ± 0.2 AU, P = 0.0009) and apparent and pure diffusion coefficients (ADC and D) (2.06 × 10^?3 ± 0.34 versus 1.51 × 10^?3 ± 0.23 mm²/s, P = 0.0004, and 1.97 × 10^?3 ± 0.43 mm²/s versus 1.48 × 10^?3 ± 0.29 mm²/s, P = 0.008, respectively). Colorectal wall thickness (r = 0.84, P < 0.0001), normalized T₂ signal intensity (r = 0.85, P < 0.0001) and ADC (r = 0.80, P < 0.0001) were strongly correlated with the histopathological inflammation score, whereas normalized T₁ signal intensity and D were moderately correlated (r = 0.64, P = 0.0006, and r = 0.65, P = 0.0003, respectively). High‐field MRI features of single‐dose radiation‐induced rectocolitis in rats differ significantly from those of control rats. Quantitative MRI characteristics, especially wall thickness, normalized T₂ signal intensity, ADC and D, are potential markers of the histopathological inflammation score.     

Effect of the cholinesterase inhibitor donepezil on cardiac remodeling and autonomic balance in rats with heart failure

In an earlier study we demonstrated the beneficial effect of direct vagal electrical stimulation on cardiac remodeling and survival. In the study reported here, we attempted to reproduce the effect of vagal enhancement through the administration of an acetylcholinesterase inhibitor, donepezil. A rat model of heart failure following extensive healed myocardial infarction was used. Compared to their nontreated counterparts, rats given donepezil (5 mg/kg/day) in their drinking water had a smaller biventricular weight (3.40 ± 0.13 vs. 3.02 ± 0.21 g/kg body weight, P < 0.05), and maximal rate of rise (3256 ± 955 vs. 3822 ± 389 mmHg/s, P < 0.05) and the end-diastolic value (30.1 ± 5.6 vs. 23.2 ± 5.7 mmHg, P < 0.05) of left ventricular pressure were improved. Neurohumoral factors were suppressed in donepezil-treated rats (norepinephrine 1885 ± 1423 vs. 316 ± 248 pg/ml, P < 0.01; brain natriuretic peptide 457 ± 68 vs. 362 ± 80 ng/ml, P < 0.05), and the high-frequency component of heart rate variability showed a nocturnal increase. These findings indicated that donepezil reproduced the anti-remodeling effect of electrical vagal stimulation. Further studies are warranted to evaluate the clinical usefulness of donepezil in heart failure.     

4‐Thiouridine induces dose‐dependent reduction of oedema,leucocyte influx and tumour necrosis factor in lung inflammation

Recent reports demonstrate a role for nucleotides as inflammatory modulators. Uridine, for example, reduces oedema formation and leucocyte infiltration in a Sephadex‐induced lung inflammation model. Tumour necrosis factor (TNF) concentration was also reduced. Previous in vivo observations indicated that 4‐thiouridine might have similar effects on leucocyte infiltration and TNF release. The aim of this study was thus to investigate the effects of 4‐thiouridine in greater detail. We used a Sephadex‐induced acute lung inflammation model in Sprague–Dawley rats. The dextran beads were instilled intratracheally into the lungs, which were excised and examined after 24 h. Sephadex alone led to massive oedema formation and infiltration of macrophages, neutrophils and eosinophils. Microgranulomas with giant cell formations were clearly visible around the partially degraded beads. A significant increase in bronchoalveolar lavage fluid (BALF) content of TNF and leukotrienes was also seen. 4‐Thiouridine co‐administration affected all variables investigated in this model, i.e. oedema, microscopic and macroscopic appearance of lung tissue, total leucocyte and differential leucocyte counts in BALF, TNF and leukotrienes C₄ (LTC₄), LTD₄ and LTE₄ in BALF, indicating a reproducible anti‐inflammatory effect. In conclusion, we have demonstrated that 4‐thiouridine has anti‐inflammatory effects similar to those of uridine. To our knowledge, this is the first demonstration of pharmacological 4‐thiouridine effects in vivo. The results suggest nucleoside/nucleotide involvement in inflammatory processes, warranting further studies on nucleoside analogues as attractive new alternatives in the treatment of inflammatory diseases.     

Fractional ventilation mapping using inert fluorinated gas MRI in rat models of inflammation and fibrosis

The purpose of this study was to extend established methods for fractional ventilation mapping using ¹⁹F MRI of inert fluorinated gases to rat models of pulmonary inflammation and fibrosis. In this study, five rats were instilled with lipopolysaccharide (LPS) in the lungs two days prior to imaging, six rats were instilled with bleomycin in the lungs two weeks prior to imaging and an additional four rats were used as controls. ¹⁹F MR lung imaging was performed at 3 T with rats continuously breathing a mixture of sulfur hexafluoride and O₂. Fractional ventilation maps were obtained using a wash‐out approach, by switching the breathing mixture to pure O₂, and acquiring images following each successive wash‐out breath. The mean fractional ventilation (r) was 0.29 ± 0.05 for control rats, 0.23 ± 0.10 for LPS‐instilled rats and 0.19 ± 0.03 for bleomycin‐instilled rats. Bleomycin‐instilled rats had a significantly decreased mean r value compared with controls (P = 0.010). Although LPS‐instilled rats had a slightly reduced mean r value, this trend was not statistically significant (P = 0.556). Fractional ventilation gradients were calculated in the anterior/posterior (A/P) direction, and the mean A/P gradient was ?0.005 ± 0.008 cm^?1 for control rats, 0.013 ± 0.005 cm^?1 for LPS‐instilled rats and 0.009 ± 0.018 cm^?1 for bleomycin‐instilled rats. Fractional ventilation gradients were significantly different for control rats compared with LPS‐instilled rats only (P = 0.016). The ventilation gradients calculated from control rats showed the expected gravitational relationship, while ventilation gradients calculated from LPS‐ and bleomycin‐instilled rats showed the opposite trend. Histology confirmed that LPS‐instilled rats had a significantly elevated alveolar wall thickness, while bleomycin‐instilled rats showed signs of substantial fibrosis. Overall, ¹⁹F MRI may be able to detect the effects of pulmonary inflammation and fibrosis using a simple and inexpensive imaging approach that can potentially be translated to humans. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of alveolar hypoxia on segmental pulmonary vascular resistance and lung fluid balance in dogs

In a biventricular bypass preparation with constant-flow perfusion, pulmonary arterial pressure (P_pa), average pulmonary capillary pressure (P_pc), venous pressure (P_v), extravascular lung water volume (EVW_d) and capillary permeability-surface area product for urea (PS) were determined in control animals and in animals subjected to alveolar hypoxia. During hypoxia, P_pa increased in a biphasic manner, the site of hypoxic pulmonary vasoconstriction being located in the arterial upstream segment. At baseline, P_pc values were identical in control and experimental animals (3.4 ± 0.4 vs. 3.6 ± 0.2 mmHg). During 150 min of airway hypoxia, the rise in P_pc (5.1 ± 0.3mmHg) did not exceed the rise in P_pc (4.9 ± 0.5mmHg) recorded in control animals at same time interval during normoxic ventilation. EVW_d increased during hypoxia to values significantly higher than those obtained in control animals (0.559 ± 0.036 vs. 0.466 ± 0.027 mL water g^?1 lung). PS remained unchanged at baseline level throughout experiments in both groups of animals. Present data suggest that lung oedema formation during alveolar hypoxia may be caused by increased transcapillary fluid loss preferentially through transcellular hydraulic pathways in capillary endothelial cells.     

Muscle microvascular oxygenation in chronic heart failure: role of nitric oxide availability

Aim: To test the hypothesis that diminished vascular nitric oxide availability might explain the inability of individuals with chronic heart failure (CHF) to maintain the microvascular PO₂’s (PO_2mv ∝ O₂ delivery‐to‐uptake ratio) seen in healthy animals. Methods: We superfused sodium nitroprusside (SNP; 300 μm ), Krebs–Henseleit (control, CON) and l ‐nitro arginine methyl ester (l ‐NAME; 1.5 mm ) onto the spinotrapezius muscle and measured PO_2mv by phosphorescence quenching in female Sprague–Dawley rats (n = 26) at rest and during twitch contractions (1 Hz). Seven rats served as controls (Sham) while CHF was induced by myocardial infarction. CHF rats were grouped as moderate (MOD; n = 15) and severe CHF (SEV; n = 4) according to morphological data and baseline PO_2mv. Results: In contrast to Sham and MOD,l ‐NAME did not affect the PO_2mv response (dynamics and steady‐state) of SEV when compared with CON. SNP restored the PO_2mv profile of SEV to that seen in Sham animals during CON. Specifically, the effect of l ‐NAME expressed as Δ(l ‐NAME – CON) were: Baseline PO_2mv [in mmHg, ΔSham = ?7.0 ± 1.6 (P < 0.05); ΔSEV =?1.2 ± 2.1], end‐contractions PO_2mv [in mmHg, ΔSham = ?5.0 ± 1.0 (P < 0.05); ΔSEV = ?2.5 ± 0.5] and time constant of PO_2mv decrease [in s, ΔSham = ?6.5 ± 3.0 (P < 0.05); ΔSEV = ?3.2 ± 1.8]. Conclusion: These data provide the first direct evidence that the pathological profiles of PO_2mv associated with severe CHF can be explained, in part, by a diminished vascular NO availability.     

Renal nerve stimulation restores tubuloglomerular feedback after acute renal denervation

Renal nerves play an important role in the setting of the sensitivity of the tubuloglomerular feedback (TGF) mechanism. We recently reported a time-dependent resetting of TGF to a lower sensitivity 3–4 h after acute unilateral renal denervation (aDNX). This effect persisted after 1 week, but was then less pronounced. To determine whether normal TGF sensitivity could be restored in aDNX kidneys by low-frequency renal nerve stimulation (RNS), the following experiments were performed. Rats with aDNX were prepared for micropuncture. In one experimental group proximal tubular free flow (P_t) and stop flow pressures (P_sf) were measured during RNS at frequencies of 2, 4 and 6 Hz. In another series of experiments the TGF sensitivity was evaluated from the P_sf responses at different loop perfusion rates after 20 min of RNS at a frequency of 2 Hz. The maximal drop in P_sf (ΔP_sf) and the tubular flow rate at which half the maximal response in ΔP_sf was observed (turning point, TP), were recorded. At RNS frequencies of 2, 4 and 6 Hz, P_t decreased from the control level of 14.1 ± 0.8–13.1 ± 1.0, 12.4 ± 1.1 and 11.2 ± 0.8 mmHg (decrease 21%, P < 0.05), respectively, while at zero perfusion and during RNS at 2 and 4 Hz P_sf decreased from 42.5 ± 1.6 to 38.2 ± 1.4 and 32.8 ± 4.3 mmHg (decrease 23%, P < 0.05), respectively. The TGF characteristics were found to be reset from the normal sensitivity with TP of 19.0 ± 1.1 nL min^–1 and ΔP_sf of 8.7 ± 0.9 mmHg to TP of 28.3 ± 2.4 nL min^–1 (increase 49%, P < 0.05) and ΔP_sf of 5.8 ± 1.2 mmHg (decrease 33%) after aDNX. After 20 min of RNS at 2 Hz TP was normalized and ΔP_sf was 33% higher. Thus the present findings indicate that the resetting of the TGF sensitivity that occurred 2–3 h after aDNX could be partially restored by 20 min of RNS at a frequency of 2 Hz. These results imply that renal nerves have an important impact on the setting of the sensitivity of the TGF mechanism.     

Glucagon secretory response to hypoglycaemia,adrenaline and carbachol in streptozotocindiabetic rats

Glucagon response to insulin-induced hypoglycaemia is impaired in diabetes, but the mechanism is not established. Pancreatic A cell hyporesponsiveness to adrenergic or cholinergic stimulation could contribute to the impairment. We therefore compared the plasma glucagon responses to intravenous infusion of adrenaline (1200 ng kg^-1 min^-1 for 20 min) or to intravenous injection of the cholinergic agonist carbachol (50 μ kg^-1) in chloral hydrate-anaesthetized rats made diabetic with the use of streptozotocin (80 mg kg^-1 subcutaneously) 6 weeks before and in anaesthetized control rats. Insulin was infused intravenously to reduce plasma glucose levels to below 1.8 mmol L^-1. As expected, the plasma glucagon response was reduced by ～ 45% in streptozotocindiabetic rats compared with controls (P= 0.045). During adrenaline infusion, plasma glucagon levels increased by 277 ± 92 pg mL^-1 in controls (P= 0.009) and by 570 ± 137 pg mL^-1 in the diabetic rats (P= 0.002). Thus, the plasma glucagon response to adrenaline was approximately doubled in the diabetic rats (P= 0.045). Following carbachol injection, plasma glucagon levels were raised by 1211 ± 208 pg mL^-1 (P < 0.001) in controls but only by 555 ± 242 pg mL^-1 in the diabetic rats (P= 0.049). Thus, the plasma glucagon response to carbachol was impaired by ～ 58% in the diabetic rats (P= 0.028). We conclude that carbachol-stimulated glucagon secretion is impaired concomitantly with the impaired glucagon response to hypoglycaemia in streptozotocin-diabetic rats, whereas adrenaline-induced glucagon secretion is exaggerated. We suggest that a reduced pancreatic A cell responsiveness to cholinergic stimulation could contribute to the impairment of the glucagon response to insulininduced hypoglycaemia in diabetes.     

Changes in peptidergic nerves in the atrioventricular valves of streptozotocin‐induced diabetic rats: A confocal microscopy study

Several previous studies have described the distribution of neuropeptide Y (NPY)‐like and calcitonin gene related peptide (CGRP)‐like immunoreactive nerve fibres in the atrioventricular valves of humans and various animals. It has been suggested that peptide‐containing nerve fibres might have motor or sensory roles in valvular function. Although there is evidence that diabetic changes occur in the sympathetic (preganglionic and postganglionic), parasympathetic (vagal) and peptidergic nerves of rats, the changes of peptide‐containing nerve fibres in the atrioventricular valves of the diabetic rat have not been studied. The distribution, relative density and staining intensity of NPY‐like and CGRP‐like immunoreactive nerve fibres in the mitral and tricuspid valves were studied in whole mount preparations using confocal microscopy with a computer‐assisted image analysis system. Streptozotocin‐induced diabetic and control rats were sacrificed at 12 and 24 months. The nerve staining intensity within the tricuspid valve was greater than the mitral valve in both control (P < 0.01) and diabetic (P < 0.001) rats. Nerve density in the anterior leaflet was greater than the posterior leaflet of the mitral valve. However, the anterior leaflet of the mitral and tricuspid valves showed a decreased number of nerve fibres, followed by drastic reduction in the staining intensities for both the peptides studied (P < 0.001) in the long‐term diabetic rat. The decrease in the number of nerve fibres that follow the mechanical interruption of nerves raises the possibility that cycles of degeneration may occur. It is suggested that these peptide‐containing nerve fibres in the atrioventricular valves may be involved in valvular dysfunction in the diabetic state. Anat Rec 258:277–285, 2000. © 2000 Wiley‐Liss, Inc.     

Renal response to volume depletion and expansion in Milan hypertensive rats

In previous studies on Milan hypertensive (MHS) rats, we found an impaired tubuloglomerular feedback (TGF) response before, during and after development of hypertension. In the present study MHS rats and rats of the Milan normotensive strain (MNS) were investigated after 24 hours of volume depletion (VD) and subsequently after 5% isotonic volume expansion (VE) with respect to whole kidney function, interstitial hydrostatic (P_int) and oncotic (II_int) pressures, stop-flow pressure characteristics of TGF and changes in early proximal flow rate in response to increased loop of Henle flow. MHS rats had higher mean arterial blood pressure (P_a) than MNS rats (129 vs. 101 mmHg) both after VD and after subsequent VE. No difference in glomerular filtration rate (GFR) was found. Both strains had a low urine flow rate (1.5 μl min^-1) during VD, which increased fourfold after VE. The interstitium was significantly more dehydrated in MHS, as indicated by a more negative net interstitial pressure (P_int–±_int t than in MNS (-1.3 ± 0.3 vs. ± 0.0 ± 0.5 mmHg) after VE. The TGF mechanism was more activated in MHS during volume depletion, as indicated by a larger drop in stop-flow pressure (P_sf) in response to loop of Henle perfusion (7.1 ± 0.7 vs. 4.7 ± 0.2 mmHg, P < 0.05). However, during VD the loop of Henle flow that elicited half maximal response in P_sf, the turning point (TP), was equally low in MHS and MNS (13.5 ± 0.6 and 14.3 ± 0.4, respectively). After VE, however, TP increased significantly more in MNS to (32.6 ± 2.1 nl min^-1) then in MHS (to 21.8 ± 0.9 nl min^-1, P < 0.05). It is concluded that the blunting of the TGF resetting in response to VE in MHS rats may well be of importance in the development of hypertension in the MHS strain.     

Sevoflurane reduces severity of acute lung injury possibly by impairing formation of alveolar oedema

Pulmonary oedema is a hallmark of acute lung injury (ALI), consisting of various degrees of water and proteins. Physiologically, sodium enters through apical sodium channels (ENaC) and is extruded basolaterally by a sodium–potassium–adenosine–triphosphatase pump (Na⁺/K⁺‐ATPase). Water follows to maintain iso‐osmolar conditions and to keep alveoli dry. We postulated that the volatile anaesthetic sevoflurane would impact oedema resolution positively in an in‐vitro and in‐vivo model of ALI. Alveolar epithelial type II cells (AECII) and mixed alveolar epithelial cells (mAEC) were stimulated with 20 µg/ml lipopolysaccharide (LPS) and co‐exposed to sevoflurane for 8 h. In‐vitro active sodium transport via ENaC and Na⁺/K⁺‐ATPase was determined, assessing ²²sodium and ⁸⁶rubidium influx, respectively. Intratracheally applied LPS (150 µg) was used for the ALI in rats under sevoflurane or propofol anaesthesia (8 h). Oxygenation index (PaO₂/FiO₂) was calculated and lung oedema assessed determining lung wet/dry ratio. In AECII LPS decreased activity of ENaC and Na⁺/K⁺‐ATPase by 17·4% ± 13·3% standard deviation and 16·2% ± 13·1%, respectively. These effects were reversible in the presence of sevoflurane. Significant better oxygenation was observed with an increase of PaO₂/FiO₂ from 189 ± 142 mmHg to 454 ± 25 mmHg after 8 h in the sevoflurane/LPS compared to the propofol/LPS group. The wet/dry ratio in sevoflurane/LPS was reduced by 21·6% ± 2·3% in comparison to propofol/LPS‐treated animals. Sevoflurane has a stimulating effect on ENaC and Na⁺/K⁺‐ATPase in vitro in LPS‐injured AECII. In‐vivo experiments, however, give strong evidence that sevoflurane does not affect water reabsorption and oedema resolution, but possibly oedema formation.     

Effects of altered nitric oxide availability on rat muscle microvascular oxygenation during contractions

Aim: To explore the role of nitric oxide (NO) in controlling microvascular O₂ pressure (Po ₂mv) at rest and during contractions (1 Hz). We hypothesized that at the onset of contractions sodium nitroprusside (SNP) would raise Po ₂mv and slow the kinetics of Po ₂mv change whereas l ‐nitro arginine methyl ester (l ‐NAME) would decrease Po ₂mv and speed its kinetics. Methods: We superfused the spinotrapezius muscle of female Sprague–Dawley rats (n = 7, body mass = 298 ± 10 g) with SNP (300 μM) and l ‐NAME (1.5 mm ) and measured Po ₂mv (phosphorescence quenching) during contractions. Results: SNP decreased mean arterial pressure (92 ± 5 mmHg) below that of control (CON, 124 ± 4 mmHg) and l ‐NAME (120 ± 4 mmHg) conditions. SNP did not raise Po ₂mv at rest but it did elevate the Po ₂mv‐to‐MAP ratio (50% increase, P < 0.05) and slow the kinetics by lengthening the time‐delay (TD, 14.0 ± 5.0 s) and time constant (τ, 24.0 ± 10.0 s) of the response compared with CON (TD, 8.4 ± 3.3 s; τ, 16.0 ± 4.5 s, P < 0.05 vs. SNP). l ‐NAME decreased Po ₂mv at rest and tended to speed τ (10.1 ± 3.8 s, P =0.1), while TD (8.1 ± 1.0 s) was not significantly different. l ‐NAME also caused Po ₂mv to fall transiently below steady‐state contracting values. Conclusions: These results indicate that NO availability can significantly affect Po ₂mv at rest and during contractions and suggests that Po ₂mv derangements in ageing and chronic disease conditions may potentially result from impairments in NO availability.