Neonatal Rabbit Research Articles

Intrauterine Endotoxin Administration Leads to White Matter Diffusivity Changes in Newborn Rabbits

Maternal intrauterine inflammation has been implicated in the development of periventricular leukomalacia and white matter injury in the neonate. We hypothesized that intrauterine endotoxin administration would lead to microstructural changes in the neonatal rabbit white matter in vivo that could be detected at birth using diffusion tensor magnetic resonance imaging (MRI). Term newborn rabbit kits (gestational age 31 days) born to dams exposed to saline or endotoxin in utero on gestational day 28 underwent diffusion tensor imaging, and brain sections were stained for microglia. Comparison between normal and endotoxin groups showed significant decreases in both fractional anisotropy and eigenvalue (e(1)) in all periventricular white matter regions that showed an increase in the number of activated microglial cells, indicating that after maternal inflammation, microglial infiltration may predominantly explain this change in diffusivity in the immediate neonatal period. Diffusion tensor imaging may be a clinically useful tool for detecting neuroinflammation induced by maternal infection in neonatal white matter.

Probiotic fortified diet reduces bacterial colonization and translocation in a long-term neonatal rabbit model

Background Probiotic fortified diet reduces bacterial colonization and translocation in a short-term neonatal rabbit model when continuously challenged with pathogen. The purpose of this study was to determine if live probiotic diet could remain effective at decreasing colonization/translocation of pathogens in a long-term neonatal rabbit model without ill effects of the probiotic outside the gastrointestinal (GI) tract. Methods Rabbit pups were born via cesarean delivery 1 day preterm and assigned to 2 diets: a newly formulated diet (controls) vs the same diet fortified with the live probiotic Lactoccocus lactis. Enterobacter cloacae was added to both preparations before each feed. Pups were gavage fed twice daily, and weights were recorded. Rabbits were sacrificed on day 7, and organs were harvested and cultured for target organism growth. Results The probiotic fortified diet resulted in a significant decrease in Enterobacter translocation to the liver and decreased colonization in the stomach and lungs. There was no evidence of Lactococccus translocation or colonization outside of the GI tract. Conclusion This probiotic fortified diet was effective at decreasing pathogenic bacteria colonization and translocation in a long-term neonatal model. The addition of L lactis to the diet resulted in appropriate growth without any colonization or translocation of the probiotic outside of the GI tract.

Inactivation of pulmonary surfactant by silicone oil in vitro and in ventilated immature rabbits

Surface activity of pulmonary surfactant is impaired by exposure to syringes lubricated with silicone oil (SO). These syringes are used daily in clinical practice. In vitro experiments were used for detection of SO, determination of surface activity, and semiquantitative measurement of surfactant protein (SP)-B and -C in SO/surfactant mixtures. Randomized, controlled animal studies were applied for determination of in vivo activity. University research laboratory. Mass spectrometry of SO originating from syringes with and without surfactant was performed. The surface activity of SO plus surfactant phospholipids (PLs) or modified natural surfactant (Curosurf) was measured. SO/Curosurf preparations were further analyzed for changes in the content of SP-B and SP-C using immunoblotting. Neonatal rabbits received mixtures of SO/Curosurf (ratio 0-1.3 mg/mg PL) intratracheally and were then ventilated with a standardized sequence of peak insufflation pressures. Tidal volume curves were recorded, gas volumes of excised lungs were measured, and histologic analysis was performed. Dissolved SO was found after rinsing syringes with organic solvents or Curosurf. Surface activity of Curosurf was significantly reduced after addition of 0.13-1.3 mg SO/mg PL. Immunoblotting revealed interference of SO with SP-B, but not with SP-C. With increasing SO/Curosurf ratios, patchy alveolar air expansion was observed, lung gas volumes were reduced, and time to inflate the lungs was increased, whereas compliance and tidal volumes remained unimpaired. In vitro SO impairs surface activity of Curosurf and leads to interference with SP-B. SO contamination of exogenous surfactant impairs lung function in animal studies and should be avoided.

Post-oral and Perioral Stimulations during Nursing Enhance Appetitive Olfactory Memory in Neonatal Rabbits

Nursing-suckling interactions facilitate olfactory learning in newborns as long as suckling and the olfactory stimulus temporally overlap. We tested the hypothesis that olfactory preferences would develop even with a long delay between odor presentation and nursing. Thyme was presented to 2-day-old rabbit pups by placing an odorized plate 2 cm above their nest box. Duration and time of nursing were controlled and occurred before, during, or after odor presentation. Controls were not nursed. When exposed to the odor for 15 min, control pups preferred thyme to a novel odor in a 2-choice test immediately after exposure but not 3 and 22 h later. When pups were nursed immediately before thyme exposure or during exposure, they preferred the familiar odorant until 22 h later. Identically, when nursing occurred 30 min before odor exposure, a preference for thyme was maintained up to 22 h. This was not observed when nursing occurred 60 min before odor presentation. We concluded that enhancement of olfactory memory occurs in neonates during nursing but also after post-oral stimulation by postprandial internal state.

Potential of stem/progenitor cell cultures within polyester fleeces to regenerate renal tubules

The cell biological mechanism controlling the regeneration of renal tubules in renal failure after application of stem/progenitor cells is subject of actual research. Unsolved issues are the integration of stem/progenitor cells in a diseased organ environment, the differentiation into epithelial tissue and the formation of tubules in a spatial environment. Following this therapeutic strategy new biomaterials have to be found promoting spatial development of tubules. To obtain new information about the growth of tubules renal stem/progenitor cells from neonatal rabbit kidney were isolated and mounted in a tissue carrier between a selection of commercially available polyester fleeces. This procedure replaces coating by extracellular matrix proteins and creates an artificial interstitium supporting development of tubules. Perfusion culture was performed with chemically defined IMDM containing aldosterone as tubulogenic factor. Polyester fleeces were investigated by scanning electron microscopy. The spatial development of tubules was registered on whole-mount specimens and on cryosections labeled with SBA and antibodies indicating tubule differentiation. It is found that some polyester fleeces promote the spatial development of tubules between the fibers, whereat each of them produces its individual growth pattern.

Onset of Antibody Repertoire Diversification in the Rabbit Appendix (81.17)

Abstract Unlike mice and humans, rabbits generate their primary antibody repertoire by somatically diversifying V(D)J genes in gut-associated lymphoid tissues (GALT). Rabbit V(D)J genes are diversified by somatic hyper-point mutation and somatic gene conversion, both of which require activation-induced cytidine deaminase (AID). To determine when repertoire diversification begins, we examined AID expression in neonatal rabbit appendix by in situ hybridization. We first detected AID mRNA expression at one week of age, in B cells in the basolateral region of appendix follicles. We asked whether AID+ B cells were undergoing V(D)J gene diversification by using laser capture microdissection (LCM) to isolate AID+ and AID- B cells from 1-week-old appendix follicles. We RT-PCR-amplified and cloned VDJ genes from the two B cell populations, and searched for evidence of somatic diversification by comparing their nucleotide sequences to those of the appropriate germline genes. We found somatic gene conversion events in the VDJ genes from AID+ B cells, but not in those from AID- B cells. These results demonstrate that diversification of the rabbit primary antibody repertoire begins around 1 week of age, in B cells residing at the basolateral region of appendix follicles. This work was supported by NIH Grant 1 RO1 AI49458-01A1.

Mammary olfactory signalisation in females and odor processing in neonates: Ways evolved by rabbits and humans

Mammalian females have long been known to release olfactory attraction in their offspring. Mammary odor cues control infant state, attention and directional responses, delay distress responses, stimulate breathing and positive oral actions, and finally can boost learning. Here, we survey female–offspring odor communication in two mammalian species – European rabbits and humans – taken as representatives of evolutionary extremes in terms of structure and dynamics of mother–infant relations, and level of neonatal autonomy. Despite these early psychobiological differences, females in both species have evolved mammary structures combining multiple sources of endogenous and exogenous odorants, and of greasy fixatives, conferring on them a chemocommunicative function. To process these mammary chemosignals, neonates have co-evolved multiple perceptual mechanisms. Their behaviour appears to be driven by plastic mechanism(s) calibrated by circumstantial odor experience in preceding and current environments (fetal and postnatal induction of sensory processes and learning), and by predisposed mechanisms supported by pathways that may be hard-wired to detect species-specific signals. In rabbit neonates, predisposed and plastic mechanisms are working inclusively. In human neonates, only plastic mechanisms could be demonstrated so far. These mammary signals and cues confer success in offspring's approach and exploration of maternal body surface, and ensuing effective initial feeds and rapid learning of maternal identity. Although the duration of the impact of these mammary signals is variable in newborns of species exposed to contrasting life-history patterns, their functional role in setting on infant–mother interaction in the context of milk transfer can be crucial.

Changes in β1 integrin in renal tubular epithelial cells after intrauterine asphyxia of rabbit pups

We investigated the role of beta(1) integrin in acute renal tubular injury caused by intrauterine asphyxia of neonatal rabbits by exploring the distribution and expression changes in beta(1) integrin and its mRNA in renal tubular epithelial cells. A catheter was used to temporarily block the abdominal aortas of New Zealand pregnant rabbits in order to set up the intrauterine asphyxia animal model. The rabbit pups were randomly divided into control, asphyxia, and calpain inhibitor intervention groups and their renal tubular tissues were examined at 2 h after asphyxia. Immunofluorescence and in situ hybridization were used to examine the expression of beta(1) integrin and its mRNA, respectively. Western blot analysis was used to show the proteolysis of beta(1) integrin. Calpain inhibitor I was used to show the protective effect of keeping beta(1) integrin from being hydrolyzed after asphyxia. (1) Normally, beta(1) integrin was located exclusively at the basal surface of renal tubular epithelial cells. After asphyxia a large amount of beta(1) integrin shifted from the basal surface to the cytoplasma and the lateral and apical surfaces and its expression decreased significantly, with simultaneous damage to renal tubular integrity and structure, many exfoliated cells and cell fragments obstructed the tubular lumen. (2) The mRNA of beta(1) integrin was mainly expressed in the cytoplasma. After asphyxia its expression increased significantly. (3) Proteolysis of beta(1) integrin was evident after asphyxia, but was significantly reduced in the calpain inhibitor intervention group. Calpain inhibitor I prevented the decrease and dislocation of beta(1) integrin and protected renal tubular integrity and structure. Intrauterine asphyxia caused proteolysis of beta(1) integrin, with reduced expression and depolarized distribution, leading to tubular lumen obstruction and renal tubule destruction. Damage to beta(1) integrin and the renal tubule was related to the activation of calpain, and calpain inhibitor curtailed these effects.

Abstract 2600: Mitochondria Dysfunction Precedes Cardiomyocyte Apoptosis in the Pressure-Overloaded Neonatal Left Ventricle

Objective: In pressure overload LV hypertrophy (LVH) cardiomyocyte apoptosis has been recognized as a major contributor to the progression from compensated hypertrophy to failure. We hypothesize that the mitochondrial apoptotic pathway is the likely source via electron transport chain (ETC) dysfunction and oxidative stress with resultant activation of the intrinsic mitochondrial pathway. Methods: Neonatal rabbits underwent thoracic aortic banding at 10 days of age. Compensated hypertrophy (4wks), decompensated hypertrophy (6wks), and age-matched controls (n=4/group) were studied by serial echocardiography measuring LV mass/volume ratio (M/V) and fractional shortening (FS). ETC Complex I activities were determined by spectophotometry in isolated mitochondria (nmolesNADH/mg/min). Cardiomyocyte apoptosis (TUNEL staining) and Bcl-2/Bax ratios (immunoblotting) were measured. Data are expressed as median ± SEM with p≤.05 being significant by t-test. Results: At 4wks M/V ratio was significantly increased (control 1.47±0.11 vs. banded 2.01±0.18, p=.009) and FS was preserved. Complex I activity was significant decreased (control 1,355±123 vs banded 949±25, p=.04), however no significant loss of cardiomyocytes had yet occurred. By 6wks M/V ratio was significantly decreased (LV dilation; control 1.67±0.13 vs banded 1.26±0.09, p=.01) and FS was impaired (39.2±1.3 vs 27.7±4.3, p=.016). Complex I activity was further decreased (Control 940±47 vs Banded 773±21, p=.03). Bcl-2/Bax ratios were significantly decreased (control 0.97±.002 vs banded 0.9±.003, p=.028) with a resultant significant increase in cardiomyotcyte apoptosis compared to 4wk banded animals (3.93±0.86 vs 13.03±4.19 TUNEL pos nuclei/1000 nuclei, p=.05). Conclusion: In pressure overload hypertrophy, the transition from compensated LVH to failure and cardiomyocyte apoptosis is preceded by mitochondrial ETC dysfunction followed by a decrease in Bcl-2/Bax ratios. The likely mechanism is through increased reactive oxygen species generation and increased oxidative stress. Interventions to mitigate oxidative stress during compensated hypertrophy may prevent the onset of failure.

Mineralocorticoid Receptor, Heat Shock Proteins and Immunophilins Participate in the Transmission of the Tubulogenic Signal of Aldosterone

Aldosterone exhibits a tubulogenic activity on renal stem/progenitor cells. Little information is available about the signaling between the mineralocorticoid receptor (MR) and the proteins in the cytoplasm. To obtain information, renal stem/progenitor cells from neonatal rabbit were cultured at the interface of an artificial polyester interstitium in a perfu- sion container for 13 days. Development was registered with Soybean Agglutinin (SBA). Microscopy revealed that treat- ment with aldosterone (1 x 10 -7 M) resulted in numerous SBA-labeled tubules. Interference with mineralocorticoid recep- tor (MR) and heat shock protein (hsp) 90 using geldanamycin (3,6 x 10 -6 M) or radicicol (1 x 10 -6 M) showed a lack of tu- bule development. Disturbing MR and hsp 70 with quercetin (2 x 10 -4 M) and KNK 437 (1 x 10 -4 M) generated SBA- labeled cell cluster but not structured tubules. Interference with MR and immunophilins by cyclosporin A (2 x 10 -6 M) and rapamycin (2,2 x 10 -9 M) did not influence the generation of numerous SBA-labeled tubules. In contrast, administration of FK 506 (2 x 10 -8 M) resulted in a lack of tubule development. Thus, the signal for development of tubules induced by al- dosterone is mediated by interaction of MR, hsp 90, hsp 70 and immunophilin FKBP 12.

Remote ischemic preconditioning elaborates a transferable blood-borne effector that protects mitochondrial structure and function and preserves myocardial performance after neonatal cardioplegic arrest

Remote ischemic preconditioning is known to elicit production of a blood-borne cardioprotective factor that is infarct sparing in models of ischemia-reperfusion injury and myocardial damage reducing after cardiopulmonary bypass in human subjects. The mechanism of protection remains incompletely understood. In this study, we examined effects on mitochondrial structure and function in a noninfarct model of cardioplegic arrest. Explanted neonatal rabbit hearts were mounted in a Langendorff preparation and perfused with dialysate of blood taken from sham-treated or remotely preconditioned rabbits. Each heart was subsequently subjected to 1-hour cardioplegic arrest and 30-minute reperfusion periods, during which hemodynamic responses were measured. Mitochondria were isolated for structural and functional measurements. Relative to hearts with sham-treated dialysate, myocardial performance (systolic pressure, maximum positive and negative first derivatives of left ventricular pressure, and left ventricular end-diastolic pressure) was better preserved with dialysate from preconditioned rabbits. Similarly, mitochondria isolated from hearts with dialysate from preconditioned rabbits showed preserved respiration at complex I and IV in the electron transport chain (P < .01 and P < .05, respectively). Mitochondrial outer membrane integrity was also preserved, with diminished sensitivity of mitochondrial respiration to exogenous cytochrome c (P < .01) and less cytosolic diffusion of cytochrome c (P < .01). Mitochondrial resistance to calcium-mediated mitochondrial permeability transition pore opening was not affected. The cardioprotective factor in plasma dialysate after remote preconditioning preserves mitochondrial structure and function in a noninfarct cardioplegic arrest model. This protection is associated with preservation of global myocardial performance.

Degradation of electrodeposited calcium phosphate and calcium phosphate-chitosan coatings in vitro

To investigate the degradation of electrodeposited calcium phosphate (ECP) coating and calcium phosphate/chitosan (ECPC) coating in vitro. Osteoclasts were isolated from neonatal rabbit long bone cavities and incubated with ECP and ECPC coatings. Calcium ion concentrations in the culture medium were analyzed at 3 days and 6 days. The osteoclastic resorption was observed with scanning electron microscope. Both coatings demonstrated osteoclastic resorption lacunae. The calcium ion concentrations of the culture mediums were decreased when incubated with calcium phosphate coatings (P < 0.05). Compared with coatings cultured with osteoclasts, the calcium ion concentrations of those cultured without osteoclasts were higher on day 3 (P > 0.05) but lower on day 6 (P < 0.05). Both ECP and ECPC coatings can be resorbed by osteoclasts in vitro and can dissolve in the culture medium.

The formation of pores in the basal lamina of regenerated renal tubules

Little information is available concerning the generation of renal tubules, but this information is urgently needed in regenerative medicine for the future treatment of acute and chronic renal failures. Of major interests are the integration of stem/progenitor cells, the cellular development and the tubular growth in a spatial environment. In this regard, we investigated the basal aspect of renal tubules generated at the interphase of an artificial interstitium. Stem/progenitor cells derived from neonatal rabbit kidney were mounted inside a specific tissue holder and covered by layers of polyester fleece. The tissue was then kept in a perfusion culture container for 13 days in chemically defined IMDM containing aldosterone (1 × 10 −7 m) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryo-sections labeled with soybean agglutinin (SBA) and tissue-specific antibodies indicating that collecting duct tubules were developed. Scanning electron microscopy (SEM) revealed that the generated tubules were completely covered by a basal lamina. Most interestingly, the matrix was not consistently composed, but exhibited three categories of pores. The most frequently found pore type had an apparent diameter of 133 ± 26 nm followed by a medium-sized pore type of 317 ± 35 nm. Another category of pores with a diameter of 605 ± 101 nm was rather rarely found. All of the pores were evenly distributed and not restricted to particular sites. The newly detected pores are not related to culture artifacts, since they were also detected in collecting duct tubules of the neonatal rabbit kidney. It remains to be evaluated whether these pores support physiological transport functions or if they indicate the site where extracellular matrix proteins are inserted into newly synthesized basal lamina.

Inhibition of mitochondrial remodeling by cyclosporine A preserves myocardial performance in a neonatal rabbit model of cardioplegic arrest

Mitochondrial permeability transition pore opening is associated with apoptotic signaling and alterations in mitochondrial structure and function. We tested whether inhibition of mitochondrial permeability transition pore opening with cyclosporine A preserved mitochondrial structure and function after cardioplegic arrest and whether this preservation is associated with improved myocardial performance. Langendorff-perfused neonatal rabbit hearts were perfused for 30 minutes with Krebs-Henseleit buffer (CCP; n = 6) or Krebs-Henseleit buffer containing 2 mumol/L of cyclosporine A (CCP+CsA; n = 6) followed by 60 minutes of normothermic crystalloid cardioplegia (CCP) and 60 minutes of reperfusion. Control hearts (non-CCP; n = 6) were constantly perfused for 150 minutes without cardioplegic arrest. In comparison with non-CCP, CCP was associated with Bax translocation to the mitochondria, cytochrome c release, and greater frequency of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive myocytes These changes were also associated with deficits in isolated mitochondrial oxygen consumption at complex I. CsA pretreatment minimized or prevented all these findings. Myocardial performance (systolic pressure, maximum positive and negative dP/dt, and elevated left ventricular end-diastolic pressure) at 5, 15, 30, and 60 minutes after reperfusion was diminished in CCP hearts when compared with non-CPB, and these deficits could be minimized with cyclosporine A pretreatment. (P < .05 all comparisons) Cyclosporine A prevents apoptosis-related mitochondrial permeabilization and dysfunction after cardioplegic arrest. This protection is associated with improved myocardial performance. Prevention of mitochondrial permeability transition pore opening is a valuable target for mitochondrial (and myocardial) preservation after neonatal cardioplegic arrest.

Role of BAFF/APRIL in GALT Development

In rabbits, B cells develop in the bone marrow and migrate to GALT where they proliferate, form organized follicles, and develop the primary antibody repertoire by Ig gene diversification. While the molecular events leading to Ig gene rearrangements in the bone marrow are well understood, little is known about the mechanisms regulating B cell proliferation and homeostasis in GALT. We investigated the role(s) played by BAFF and APRIL in stimulating GALT B cells. By in situ hybridization, we found BAFF transcripts in the appendix to be highly expressed in B cell follicles. In striking contrast to human B cells, we detected BAFF expression on both naïve peripheral and GALT B cells. In vitro, purified rabbit BAFF induced splenocytes to undergo proliferation, but not B cells isolated from GALT. However, addition of BAFF to cultures of GALT B cells transiently prolonged their survival. Following in vivo injection of neonatal rabbits with a soluble decoy receptor (TACI‐Ig), we found a marked reduction in both the size and number of B cell follicles and also in Ki‐67 expression, demonstrating that BAFF/APRIL is required for B cell proliferation in GALT. We are currently investigating whether B cells residing in GALT are immature or mature B cells. Since rabbit B cells express BAFF, we propose that B cells in GALT are stimulated in an autocrine fashion.Supported by NIH AI50260

Generation of Tubular Superstructures by Piling of Renal Stem/Progenitor Cells

The aim of this study was to assess cell biological processes involved in kidney regeneration. Particular interest was focused on the development of renal stem/progenitor cells into structured tubules. To investigate this process, the availability of a powerful cell culturing system is important. One prerequisite is that embryonic tissue can be maintained for several weeks to observe the progression of tubule development under physiological and under pathological conditions. Additionally, a significant amount of tissue must be harvested to analyze the cellular processes involved in the formation of tubules. In the present experiments, it was demonstrated for the first time that the spatial development of tubules could be modulated by piling stem/progenitor cells derived from neonatal rabbit kidney. Isolated embryonic tissue was mounted inside a specific holder and covered by layers of polyester fleece to create an artificial interstitium. The tissue was cultured in a perfusion container for 13 days in a chemically defined Iscove's modified Dulbecco's medium containing aldosterone (1 x 10(7) M) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryosections labeled with lectins or tissue-specific antibodies. The experiments revealed that culturing renal stem/progenitor cells in one layer resulted in the development of numerous differentiated tubules lining one row. Increasing the layers of renal stem/progenitor cells by piling resulted in the formation of multiple tubules orientated in parallel rows. Consequently, this technique made it possible to pile renal stem/progenitor cells like bricks and experimentally extend the spatial environment for tubular growth. This novel technique is an important step toward the generation of renal superstructures urgently needed for biomedical research and kidney regeneration.

Evolution of Ca2+ signals with the development of nano‐domains in smooth muscle cells of the rabbit inferior vena cava

We have previously shown that phenylephrine(PE)‐induced oscillatory Ca2+ signals underlie excitation‐contraction coupling in adult rabbit inferior vena cava (IVC). These oscillations are caused by waves of regenerative Ca2+ release, which are maintained by sarcoplasmic reticulum (SR) reloading via junctions formed between plasma membrane and SR (PM‐SR). Here we present evidence that these junctions together with oscillatory Ca2+ signaling develop post‐natal. We examined the agonist‐induced Ca2+ signal in in situ vascular smooth muscle cells (VSMC) of the neonatal rabbit IVC in relation to force generation. PE elicits a biphasic Ca2+ signal with an initial Ca2+ transient followed by a steady state plateau averaging 25 % of the peak value. In contrast to the adult, the plateau phase of PE‐induced Ca2+ signal and tonic contraction in the VSMC of neonatal rabbit IVC was abolished by nifedipine. Employing electron microscopy, we found that VSMC in neonatal rabbit IVC posses less junctional SR, 6±1%, as compared with the adult rabbit IVC, 18±1%. Our results suggest that complex structures, defining a nano‐domain (~20 nm) and composition, including clusters of Ca2+ and Na+ transport proteins, of the PM‐SR junctions develops during the neo‐natal period and that this correlates with the appearance of oscillatory Ca2+ signaling in VSMC. This study is supported by funding from the Canadian Institute of Health Research.

Subacute hypoxia suppresses Kv3.4 channel expression and whole-cell K + currents through endogenous 15-hydroxyeicosatetraenoic acid in pulmonary arterial smooth muscle cells

We have previously reported that subacute hypoxia activates lung 15-lipoxygenase (15-LOX), which catalyzes arachidonic acid to produce 15-HETE, leading to constriction of neonatal rabbit pulmonary arteries. Subacute hypoxia suppresses Kv3.4 channel expression and results in an inhibition of whole-cell K + currents ( I K). Although the Kv channel inhibition is likely to be mediated through 15-HETE, direct evidence is still lacking. To reveal the role of the 15-LOX/15-HETE pathway in the hypoxia-induced down-regulation of Kv3.4 channel expression and inhibition of I K, we performed studies using 15-LOX blockers, whole-cell patch-clamp, semi-quantitative PCR, ELISA and Western blot analysis. We found that Kv3.4 channel expression at the mRNA and protein levels was greatly up-regulated in pulmonary arterial smooth muscle cells after blockade of 15-LOX by CDC or NDGA. The 15-LOX blockade also partially restored I K. In comparison, 15-HETE had a stronger effect than 12-HETE on the expression of Kv3.4 channels. 5-HETE had no noticeable effect on Kv3.4 channel expression. These data indicate that the 15-LOX pathway via its metabolite, 15-HETE, seems to play a role in the down-regulation of Kv3.4 expression and I K inhibition after subacute hypoxia.

Cardioprotection of neonatal heart using normothermic hyperkalaemia: the importance of delivery and terminal cardioplegia

Cardioprotection of immature hearts remains controversial and largely based on the use of hypothermic cardioplegia. Recent clinical trials in pediatric open-heart surgery suggest that normothermic cardioplegic arrest is also cardioprotective. However, the advantages of using normothermic cardioplegia delivered as single- or multi-dose with or without terminal cardioplegia are unknown. This work investigates the efficacy of these techniques and the mechanism(s) underlying their protective effect. Neonatal (7-10 days) rabbit hearts in a working mode were exposed to normothermic global ischemia (60 or 90 min) protected with one of the following cardioplegic (hyperkalaemic buffer) protocols: single-dose, multi-dose infused every 30 min, single-dose or multi-dose with terminal cardioplegia. The extent of functional recovery (e.g., aortic and coronary flow), ischemic stress (e.g., myocardial ATP, lactate) and reperfusion injury (lactate dehydrogenase (LDH) release) were assessed. Recovery following 60 min global ischemia was improved (p < 0.05) by single-dose and multi-dose cardioplegic delivery (from 5% to 60% and 80%, respectively). Improved recovery was augmented by 2 min terminal cardioplegia (to 90% and 97% for single-dose and multi-dose, respectively). Extending ischemia to 90 min with single-dose resulted in 0% recovery that was not improved by 2 min terminal cardioplegia. However, 5 min (not 10 min) terminal cardioplegia significantly improved recovery (32%). Multi-dose followed by 5 min terminal cardioplegia resulted in full recovery. Cardioprotective interventions were associated with a reduction in LDH release and attenuated changes in myocardial metabolites. During normothermic cardioplegic arrest of neonatal heart: (i) multi-dose is superior to single-dose; (ii) terminal cardioplegia confers additional protection to single-dose and multi-dose; and (iii) protection is likely to be due to metabolic preservation.

SR Ca2+ refilling upon depletion and SR Ca2+ uptake rates during development in rabbit ventricular myocytes

While it has been reported that a sparse sarcoplasmic reticulum (SR) and a low SR Ca(2+) pump density exist at birth, we and others have recently shown that significant amounts of Ca(2+) are stored in the neonatal rabbit heart SR. Here we try to determine developmental changes in SR Ca(2+) loading mechanisms and Ca(2+) pump efficacy in rabbit ventricular myocytes. SR Ca(2+) loading (load(SR)) and k(0.5) (Ca(2+) concentration at half-maximal SR Ca(2+) uptake) were higher and lower, respectively, in younger age groups. Inhibition of the L-type calcium current (I(Ca)) with 15 microM nifedipine dramatically reduced load(SR) in older but not in younger age groups. In contrast, subsequent inhibition of the Na(+)/Ca(2+) exchanger (NCX) with 10 microM KB-R7943 strongly reduced load(SR) in the younger but not the older age groups. Accordingly, the time integral of the inward NCX current (tail I(NCX)) elicited on repolarization was highly sensitive to nifedipine in the older groups and sensitive to KB-R7943 in the younger groups. Interestingly, slow SR loading took place in the presence of both nifedipine and KB-R7943 in all age groups, although it was less prominent in the older groups. We conclude that the SR loading capacity at the earliest postnatal stages is at least as large as that of adult myocytes. However, reverse-mode NCX plays a prominent role in SR Ca(2+) loading at early postnatal stages while I(Ca) is the main source of SR Ca(2+) loading at late postnatal and adult stages.