Week-old Animals Research Articles

Age‐ and Inactivity‐Dependent Differences in Glutamate (GluN1) and GABA (GABA Aα1 ) Receptor Subunits in Subregions of the Rat Rostral Ventrolateral Medulla

The rostral ventrolateral medulla (RVLM) is an important brain region that regulates sympathetic vasoconstriction and cardiac function under normal conditions and in cardiovascular disease (CVD) states. Among the common, modifiable risk factors for CVD, a sedentary lifestyle is considered the number one cause of preventable death. Sedentary animals have greater blood pressure, and both greater sympathoexcitatory and sympathoinhibitory responses to in vivo activation and inhibition of the RVLM, respectively. Our recent studies also provide evidence of (in)activity-related neuroplasticity in GABAergic and glutamatergic receptor systems in the RVLM and its rostral extension (RVLMRE) (Mueller et al., 2020; Fyk-Kolodziej et al., 2020). However, to our knowledge, no studies have tested the time course and mechanisms of (in)activity-related neuroplasticity in RVLM subregions. The purpose of this study was to determine expression levels of GABAAα1 and GluN1 receptor subunits in the RVLM and RVLMRE of four week-old rats and following 4 or 12 weeks of sedentary or physically active conditions. We hypothesized that sedentary rats would exhibit subregionally-specific increases in GABAAα1 and GluN1 receptor subunits compared to four week-old rats, and to 8 and 16 week-old active rats. To this end, four week-old, male Sprague-Dawley rats were either sacrificed or provided running wheels (active) or normal caging (sedentary) for 4 (n=3 ea) or 12 weeks (GluN1, n=6 ea; GABAAα1, n=12 ea). Bilateral micropunches from 80 μm brainstem cryosections were obtained from all animals and pooled individually into RVLM and RVLMRE subregions relative to the caudal pole of the facial nucleus (FN0). The RVLM included tissue 480 µms caudal to FN0; whereas, the RVLMRE consisted of tissue 480µms rostral to FN0. Western blotting in 4 week-old animals revealed possibly higher GABA Aα1 expression in the RVLMRE vs. RVLM region (1.15±0.01 vs. 0.93±0.04, p=.07, n=3), with the same pattern of higher GABAAα1 expression in the RVLMRE vs. RVLM in rats after 4 or 12 weeks of sedentary (p<.001 both time points) or active conditions (p=.025 and p=.011, respectively). In sixteen week-old sedentary rats had lower expression of GABAAα1 within the RVLM region only (p=.004; RVLMRE p=.64) vs. active rats. Similar to GABAAα1, GluN1 expression was higher in the RVLMRE vs. RVLM of 4 week-old rats, (1.18±0.06 vs. 0.81±0.06, p=.003; n=3). In contrast, after 4 weeks of sedentary or physically active conditions, the RVLM and RVLMRE showed similar GluN1 expression levels (p=.23 both groups). Sixteen week-old sedentary rats showed higher GluN1 expression in RVLM compared with 4 week-old animals (1.25±0.07 vs. 0.81±0.06, p<.001) and compared to sixteen week-old, active rats (0.93±0.06, p=.001). These results suggest that sedentary conditions produce considerable neuroplasticity in GABAergic and glutamatergic systems in subregions of the RVLM via changes in receptor expression. Our findings are consistent with previous data demonstrating enhanced sympathoinhibitory and sympathoexcitatory responses observed in sedentary rats in vivo and are likely related to the increased incidence of CVD associated with a sedentary lifestyle.

Parasitological detection and associated risk factors of Cryptosporidium in bovines of Karnataka state

The prevalence of Cryptosporidium in cattle and buffalo calves of less than one month age was studied in Karnataka state using modified Ziehl-Neelsen staining method. Associated risk factors viz., age, sex, breed, consistency of dung, rearing systems and season were evaluated for their influence on the prevalence of Cryptosporidium infection. An overall prevalence of 12.20% (40/328) of Cryptosporidium infection was observed with a prevalence rate of 12.84% in cattle calves and 10.91% in buffalo calves. Age wise, three week-old animals were infected more (16.95%) followed by the four week (13.27%), one week (6.90%) and two week (4.41%) old animals. Female animals (13.14%) showed more prevalence than male animals (11.11%). Among the breeds, crossbred animals (15.08%) were infected more compared to non-descript animals (7.75%). Higher prevalence of infection was noticed in diarrhoeic samples (16.41%) followed by formed (10.31%) and semisolid dung samples (7.77%). Rearing system was correlated for its effect on its prevalence rate, in which the free-range animals (12.61%) had a slightly higher prevalence than that of intensively reared animals (11.32%). Season-wise, the higher prevalence of infection was noted in monsoon (14.74%) followed by winter (11.48%), autumn (11.38%) and summer (10.20%).

Age-related changes of wall composition and collagen cross-linking in the rat carotid artery – In relation with arterial mechanics

In association with age-related changes in arterial wall mechanics, the composition of connective tissues, the fraction and size of vascular smooth muscle cells (SMCs), and the degree of collagen cross-linking were studied with common carotid arteries harvested from 8, 16, 32, and 64 week-old Wistar rats. For histomorphometric studies, each arterial segment was fixed under in vivo operating force condition, and then sequentially sliced into thin specimens, followed by selective staining for the observation of collagen, elastin, and SMCs. Then, the fraction of each component, and the number and size of SMCs were determined with an image analyzer. The content of collagen and elastin, their ratio, and the number and the area fraction of SMCs showed no significant correlations with age, while the density and the size of SMCs were significantly smaller and larger, respectively, in 64 week-old animals than in the others. The results of collagen and elastin cannot explain the biomechanical data obtained in our previous study using the same animal model, which showed that the elastic modulus and wall stiffness were significantly larger in 64 week-old animals compared to younger ones. To investigate the reason for the discrepancy between the histological and the biomechanical results, a hydrothermal isometric tension method was applied to the analysis of the cross-linking of collagen, and we found that the amount of cross-links was significantly greater in 64 week-old arteries than in the others. This result corresponded well with the biomechanical results, and therefore the higher wall stiffness and elastic modulus in older arteries might be ascribed to their larger amount of collagen cross-links.

Abstract 827: Analysis of p63 protein expression in rat ventral prostate submitted to intrauterine undernutrition associated to hormonal exposure in adult life

Abstract Basal cells expressing p63 contain multipotent stem cells that can efficiently differentiate during postnatal prostate morphogenesis, adult prostate epithelial homeostasis and carcinogenesis. Our project aim is to investigate p63 expression by focusing on the rates of basal epithelial cell proliferation/differentiation in the ventral prostate after intrauterine undernutrition (IUN) associated with or without hormonal exposure in adult life. Briefly, 20 Sprague Dawley dams were distributed equally into two groups during gestation: control diet (NP; fed a normal diet containing 17% protein) and restricted protein diet (RP, fed a diet containing 6% protein). After birth, all animals were maintained on the control diet. Experiment 1: To analyze p63 expression during prostate development, NP and RP animals were euthanized at postnatal day (PND) 3, 10, 21 and 35. The ventral prostate (VP) was dissected, weighed and processed for IHC (Ki67, p63 and AR) and Western blot (WB) analysis (PCNA, p63 and AR). Experiment 2: To analyze p63 expression in adults after hormonal exposure, 14 week-old NP and RP animals were subjected to 17-beta estradiol+testosterone administration (subcutaneous implant) for 16 weeks. The animals were killed at 35 weeks old and the VP was excised, weighed and processed for IHC and WB analysis. RP treated animals showed lower body weight compared to NP control treated animals. Reductions of testosterone plasma levels as well as the absolute and relative values of VP weights from the RP animals compared to the NP animals were observed. Normal glandular architecture was observed in both groups at PND 3, 10, 21 and 35. There was a reduction of AR expression in VP from RP animals at all ages analyzed. However, the proliferation rate and p63+ cells were higher in the RP group indicating that the low androgenic stimulation induced by IUN promoted a delay in VP development. As a consequence, the proliferation/differentiation cell dynamic was impaired, leading to an increase in basal cell number and damaging their differentiation into secretory luminal cells. We observed in hormone exposed adults that prostatitis aggressiveness and prostatic intraepithelial neoplasia (PIN) incidence were higher in the RP compared to NP animals. These results were followed by a decrease in apoptosis index and increases in proliferation rate and p63+ cells. It is clear that IUN alters adult prostate response to androgen/estrogen exposure and interferes in adult prostate susceptibility to diseases. In conclusion, IUN-induced fetal programming impairs ventral prostate growth, maturation and homeostasis highlighting the importance of adequate nutritional status during embryonic/fetal development. Note: This abstract was not presented at the meeting. Citation Format: Jaqueline C. Rinaldi, Caroline N. Barquilha, Sergio AA Santos, Ana C. Camargo, Ketlin T. Colombelli, Sergio L. Felisbin, Luis A. Justulin. Analysis of p63 protein expression in rat ventral prostate submitted to intrauterine undernutrition associated to hormonal exposure in adult life. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 827. doi:10.1158/1538-7445.AM2015-827

Abstract 253: Restoring GDF11 to Normal Levels in Old Mice Has no Effect on Cardiac Structure and Function

Rationale: GDF11 (Growth Differentiation Factor 11) is a member of the TGFβ super family of secreted factors, which play an important role in the regulation of cell processes including proliferation, differentiation, death, adhesion, and migration. Recently it was shown that circulating GDF11 levels fall with aging and this change is associated with pathological cardiac hypertrophy (PCH). Restoring GDF11 to normal levels was shown to rescue PCH. Objective: To determine if we can confirm the hypothesis that correcting the levels of a single factor, GDF11, determines aging related PCH. Methods and Results: We used the study design described by Loffredo et al, 2013. Investigators were blinded to treatment group. 24 month old C57BL/6 male mice were given a daily injection of recombinant GDF11 at 0 .1mg/kg or vehicle for 28 days. GDF11 bioactivity was confirmed in-vitro. After treatment, GDF11 levels were significantly increased but there was no difference in heart weight (HW) to body weight (BW) ratio (4.74 vs 4.70) between GDF11 and vehicle treated old mice. HW/BW ratios of old mice were not different from12 week-old animals (4.56) and the PCH markers ANP and BNP were not different in young verses old mice. Before GDF11 treatment there were no significant differences in ejection fraction (46 versus 46 %), internal ventricular dimensions (4.33 vs 4.28 mm), and septal wall thickness (0.72 versus 0.78 mm) between GDF11 and vehicle treated groups. All structural and functional parameters remained unchanged at 1, 2 and 4 weeks of treatment. Strain analysis showed no significant difference in radial or longitudinal strain. Invasive hemodynamics performed at time of sacrifice also showed no significant differences in max dP/dt (6499.17 vs 6011.95 mmHg/s) or min dP/dt (-6551.73 vs -6214.33 mmHg/s) between GDF11 and vehicle treated animals respectively. Conclusions: There is no significant age-related PCH in C57Bl6 mice. GDF11 injections had no effects on cardiac structure or function in old male C57BL/6 mice. Our results question the idea that there is age-related PCH in disease free C57BL6 mice and question the findings that restoring GDF11 in old mice has any effect on cardiac structure or function.

Sexual maturation in common vole (Microtus arvalis) males raised under laboratory conditions.

The common vole is one ofthe most numerous rodents in Europe and Asia but its reproductive biology is not fully described. It is thought that females reach reproductive abilities at a very young age, however, there is no data concerning male sexual maturation. The aim of the present study was to determine the rate of sexual maturation of males of the common vole. Research was carried out on 4, 6, 8 and 10 week-old animals. Body, testes and accessory sex glands weights were compared to evaluate morphological development. Epididymal sperm quality was assessed by a motility test, sperm tail membrane integrity, viability, maturity and sperm head morphology. Moreover, the number of sperm cells in 1 mm3 of semen was evaluated. The largest body weights were observed in 8 and 10 week-old males, which also possessed the highest relative weights of gonads and accessory sex glands. These groups of males produce about 2 times more gametes than 6 week-old individuals. The highest seamen quality was noted in 8 and 10 week-old males. Based on the obtained results, it is concluded that among the investigated age groups the most appropriate male age for fertile copulation is between 8 and 10 weeks.

Determination of urinary lithogenic parameters in murine models orthologous to autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), a genetic disease caused by mutations in PKD1 or PKD2 genes, is associated with a high prevalence of nephrolithiasis. The underlying mechanisms may encompass structural abnormalities resulting from cyst growth, urinary metabolic abnormalities or both. An increased frequency of hypocitraturia has been described in ADPKD even in the absence of nephrolithiasis, suggesting that metabolic alterations may be associated with ADPKD per se. We aimed to investigate whether non-cystic Pkd1-haploinsufficient (Pkd1(+/-)) and/or nestin-Cre Pkd1-targeted cystic (Pkd1(cond/cond):Nestin(cre)) mouse models develop urinary metabolic abnormalities potentially related to nephrolithiasis in ADPKD. 24-h urine samples were collected during three non-consecutive days from 10-12 and 18-20 week-old animals. At 10-12 weeks of age, urinary oxalate, calcium, magnesium, citrate and uric acid did not differ between test and their respective control groups. At 18-20 weeks, Pkd1(+/-) showed slightly but significantly higher urinary uric acid vs. controls while cystic animals did not. The absence of hypocitraturia, hyperoxaluria and hyperuricosuria in the cystic model at both ages and the finding of hyperuricosuria in the 18-20 week-old animals suggest that anatomic cystic distortions per se do not generate the metabolic disturbances described in human ADPKD-related nephrolithiasis, while Pkd1 haploinsufficiency may contribute to this phenotype in this animal model.

Premature aging-related peripheral neuropathy in a mouse model of progeria

Peripheral neuropathy is a common aging-related degenerative disorder that interferes with daily activities and leads to increased risk of falls and injury in the elderly. The etiology of most aging-related peripheral neuropathy is unknown. Inherited defects in several genome maintenance mechanisms cause tissue-specific accelerated aging, including neurodegeneration. We tested the hypothesis that a murine model of XFE progeroid syndrome, caused by reduced expression of ERCC1-XPF DNA repair endonuclease, develops peripheral neuropathy. Nerve conduction studies revealed normal nerve function in young adult (8 week) Ercc1 −/Δ mice, but significant abnormalities in 20 week-old animals. Morphologic and ultrastructural analysis of the sciatic nerve from mutant mice revealed significant alterations at 20 but not 8 weeks of age. We conclude that Ercc1 −/Δ mice have accelerated spontaneous peripheral neurodegeneration that mimics aging-related disease. This provides strong evidence that DNA damage can drive peripheral neuropathy and offers a rapid and novel model to test therapies.

INFLUENCE OF AGE, STIMULATION BY PMSG OR FLUSHING ON THE OVARIAN RESPONSE TO LHRHa IN YOUNG RABBIT FEMALES.

Fifty young Californian rabbit females have been used to study the effect of age (14 or 17 weeks) and ovarian stimulation (nutritional flushing or PMSG) on the ovarian response to LHRHa injection. Stimulation by nutritional flushing showed, in the 14 and 17 week-old animals, a positive effect on live weight after 8 days, ovulation rate was satisfactory in females aged 17 weeks but not in the 14 week-old group, nevertheless 14 weeks old does showed an increase in the number of follicles larger than 0.6 mm (P&lt;0.05). Stimulation by PMSG (100 IU) provoked a six folds increase in the number of follicles larger than 0.6 mm (90.6 vs 15.1; P&lt;0.001), but failed to in crease the proportion of ovulating females, thus suggesting that a too high dose had been used. These results indicate that introduction of rabbit females to reproduction can be advanced to 17 week-old, provided that previous ovarían stimulation by nutritional flushing is done.

Irradiated Third Party Peripheral Blood Mononuclear Cells Enhance Umbilical Cord Blood Engraftment.

Abstract 3533Poster Board III-470Umbilical Cord Blood (UCB) is a readily available, alternate donor source for allogeneic Hematopoietic Stem Cell (HSC) transplantation and can be used with lower stringency HLA-matching between donor and host. However, only limited numbers of cells are available and non-engraftment or delayed engraftment occurs often when recipients receive single UCB units that contain < 2.5 × 107 TNC/kg recipient body weight. This excludes single UCB as a donor source for many adult patients who might otherwise benefit from an UCB transplant. Both immunologic and non-immunologic mechanisms may contribute to UCB HSC engraftment failure. UCB T-cells are phenotypically and functionally immature with a high percentage of CD45RA+ T cells and T cells are known to facilitate engraftment in human HSC transplantation. Co-transplantation of irradiated T-cells supported engraftment in an immunocompetent mouse transplant model without increasing GvHD (Waller et al. Blood94: 3222,1999).The working hypothesis for his study is that the irradiated third party peripheral blood mononuclear cells (PBMNCs) induce a cytokine or cellular response that facilitates hematopoietic engraftment. Long-term culture initiating cell (LTC-IC) frequencies determined with limiting dilution assays were statistically higher when either purified UCB derived CD34+ cells or CB MNCs cells were plated with irradiated PBMNCs (Figure 1). [Display omitted] To assess the impact of irradiated third party PBMCs on UCB engraftment we utilized sub-lethally irradiated NOD/SCID-IL2Rgnull mice in the presence and absence of irradiated third-party human PBMNCs. After sublethal irradiation (240 cGy) female 8 week-old animals were transplanted with either UCB MNCs corresponding to 8×103 (n=5) or 2.66×103 (n=9), or 8.88×102 CD34+ cells (n=9) with or without co-transplantation of 1×106 irradiated (2500 cGy) PBMNCs, and 4 animals received only irradiated PBMNCs as the control group. Engraftment levels are compared by calculating SCID repopulating cell (SRC) frequency. Co-transplantation of PB MNCs with UCB MNCs provided a steady, statistically significant increase in both short-term and long term hematopoietic engraftment (Figure 2). [Display omitted] In these pre-clinical studies, we have confirmed in vitro effects on LTIC formation and in vivo UCB graft-enhancing effects of irradiated PBMNCs. If applied in clinic, this cellular therapy option might overcome a major barrier to successful allogeneic UCB HSC transplantation: engraftment failure, making the procedure more applicable and safer. Disclosures:No relevant conflicts of interest to declare.

Tumor Induction by Monoenergetic Neutrons in B6C3F1 mice

This study was undertaken to investigate induction of tumors by monoenergetic neutrons in B6C3F1 mice. Individual groups of 6 week-old animals of both sexes (about 30 mice/group) were exposed to 0.5 Gy of various monoenergetic neutrons (dose rate 0.5 cGy/min) and then observed for 13 months. The incidences of tumors (mainly liver neoplasms) in non-irradiated male and female controls were 11% and 0%, respectively. In the irradiated animals, the incidences were 53%, 50%, 60% and 43% in males, and 75%, 81%, 71%, and 85% in females, after 0.18, 0.32, 0.6 and 1.0 MeV neutron exposure, respectively. There were no significant differences in the tumor induction rate among the different energy groups.

Liver fibrosis induced by hepatic overexpression of PDGF-B in transgenic mice

In hepatic fibrogenesis, stellate cells are activated leading to production and deposition of extracellular matrix. To clarify the role of PDGF-B in liver fibrogenesis, we overexpressed PDGF-B in the liver of transgenic mice. Transgenic mice for the conditional overexpression of PDGF-B in the liver under control of an albumin promoter were generated utilising the Cre/loxP system. Constitutive PDGF-B expression was achieved after breeding with mice expressing Cre-recombinase under actin promoter control. Tamoxifen inducible expression was achieved after breeding with mice expressing Cre under transthyretin receptor promoter control. Levels of fibrosis were assessed and the expression of regulators of matrix remodelling was measured. PDGF-B expression caused hepatic stellate cell and myofibroblast activation marked by alpha-smooth muscle actin and PDGFR-beta expression. Liver fibrosis was verified macroscopically, histologically and by collagen I mRNA quantification in 4-6 week-old animals. MMP-2, MMP-9 and TIMP-1 were upregulated whereas TGF-beta expression was unchanged. We identified PDGF-B as a proliferative and profibrogenic stimulus and potential inducer of stellate cell transdifferentiation in vivo. PDGF-B overexpression causes liver fibrosis without significantly upregulating TGF-beta1, suggesting a TGF-beta-independent mechanism. The established model provides a tool for testing anti-PDGF-B therapeutic strategies in liver fibrosis in vivo.

Upregulation of Vascular ETB Receptor Gene Expression after Chronic ETA Receptor Blockade in Prediabetic NOD Mice

In the aorta of prediabetic non-obese diabetic mice, a model of human type 1 diabetes, we investigated gene expression of the endothelin receptors and contractility to big endothelin-1 and endothelin-1 at the ages of 10 and 16 weeks. A subgroup of 10- week-old animals was treated with the endothelin ETA receptor antagonist LU461314 (30 mg/kg per day for 6 weeks). Blood glucose levels were normal in all animals. Real-time polymerase chain reaction analysis revealed that vascular ETB receptor expression was higher in 10-week-old non-obese diabetic (NOD) mice compared with controls. In 16-week-old NOD mice, but not in control mice, ETB receptor mRNA was twofold lower (P < 0.05 vs 10-week-old NOD mice). In all groups ETA receptor expression was unaffected by age or treatment. Contractions to big endothelin-1 and endothelin-1 were lower in 10-week-old NOD mice compared with controls. Treatment with LU461314 increased ETB receptor expression in 16-week-old NOD mice, but had no effect on vascular contractility. These data indicate that dysregulation of ETB receptor expression and a decreased contractile response to big endothelin-1 and endothelin-1 are present in the prediabetic state of a model of human type 1 diabetes. These alterations occur independent of glucose levels. Furthermore, ETA receptor blockade is effective in increasing ETB receptor gene expression, suggesting a potential role for endothelin ETA antagonists in the treatment of type 1 diabetes.

Nociception and antinociception during the first week of life in mice: Sex differences and test dependence

Abstract This study demonstrates that reliable sex differences in nociceptive and antinociceptive mechanisms are present in even very young subjects. Sex differences were observed in mice tested either on the day of birth or 1 week later on basal tail-flick latency and morphine analgesic magnitude. Female mice had longer tail-withdrawal latencies; male mice demonstrated stronger analgesic responses to morphine. In addition, basal pain behavior and analgesic responsiveness differed between day-old and week-old animals on the hot plate, with day-old mice showing enhanced pain behavior and reduced morphine antinociception compared to week-old subjects. These findings further support the competence of pain processing circuitry in even very young subjects and highlight the early development of nociceptive and antinociceptive mechanisms. Perspective This study highlights the competence of nociceptive circuitry and the analgesic efficacy of morphine as early as the day of birth in mice, reinforcing the importance of evaluating and treating pain in even the youngest subjects. Sex differences were present, suggesting infant sex as one of several potential factors that predict the experience of procedural or pathological pain and analgesic requirement.

Regulation of estrogen receptor alpha and beta expression by testosterone in the rat prostate gland.

Although ER beta is known to be expressed at high levels in the rat prostate gland, its regulation is not well understood. Here we examined ER mRNA expression and the effects of testosterone administration in male rats at 1, 4 and 9 weeks of age who were castrated and/or treated with testosterone for a week, and then sacrificed. ER alpha was the major type of ER expressed in 2 week-old animals while dominant expression of ER beta mRNA was apparent in older age groups. Interestingly while ER beta expression was diminished and ER alpha mRNA increased in the castrated group, testosterone administration reversed this effect. A time-course study indicated that induction of ER beta mRNA increased within 9 hr and ER alpha decreased in 2 days after an injection (i.p.) of testosterone. Our results suggested that 1) testosterone up-regulates ER beta mRNA expression while ER alpha is down-regulated; and that 2) great changes in ER alpha and beta expression in the prostate gland during development from the newborn to adult may be due to the influence of testosterone.

Urethane anesthesia produces selective damage in the piriform cortex of the developing brain

The potential induction of neuronal death by neuroactive drugs at specific stages of embryonic or postnatal development is a serious concern in treating brain disease. Recent evidence indicates that NMDA antagonists, GABA agonists, ethanol and some anesthetics can all produce massive neuronal cell loss at critical times during development. We show here that the anesthetic urethane, once used clinically, produces a selective lesion of the piriform cortex, a region not previously implicated in such toxicity, in the developing brain. Young rats were injected with urethane at 1, 2, 3, and 4 weeks of age and brain damage was measured 1–4 days later. We found that urethane produces a large lesion in subfields of the piriform cortex and that the damage is most severe in 2 week-old animals. These data, together with other recent reports, show that there are multiple neuronal death-inducing pathways in the developing nervous system. It will be important to determine if anesthestics used in pregnant women and young children may have similar effects.

Increased systemic oxygen consumption offsets improved oxygen delivery during dobutamine infusion in newborn lambs.

To determine: 1) if dobutamine elicited a thermogenic response during postnatal development; and 2) if this response impacted on the balance between systemic O(2) delivery (DO(2)) and O(2) consumption (VO(2)), and involved one or a combination of adrenoceptor subtypes. Prospective non-randomized unblinded study. University research laboratory. Thirty-five Border-Leicester cross lambs used in a main study performed at 1-2 days (n=7), 7-10 days (n=7), and 6-8 weeks (n=8), and in a adrenoceptor blockade substudy performed at 1-2 days (n=13). Lambs were instrumented under anaesthesia and dobutamine was infused at incremental rates of 1-40 microg/kg per minute. In separate subgroups of 1-2 day-old lambs, dobutamine was infused after selective or combined alpha1, beta 1, and beta 2-adrenoceptor blockade. Cardiac output, aortic and pulmonary arterial blood gases, and body temperature were measured. DO(2) and VO(2) were calculated. Dobutamine increased DO(2) similarly at all three ages. Dobutamine also increased VO(2) in the absence of muscle shivering, but the average rise in 1-2 day-old lambs was sevenfold to 12-fold greater (P<0.001) than in 7-10 day-old and 6-8 week-old animals, was associated with an increase in systemic O(2) extraction, and accounted for approximately 90% of the rise in DO(2). Body temperature rose by 1.3+/-0.5 degrees C in 1-2 day-old animals (P<0.001), but was unchanged in 7-10 day-old or 6-8 week-old lambs. In 1-2 day-old lambs, rises in DO(2), VO(2), and body temperature induced by dobutamine were not affected by selective alpha1, beta1 or beta2 adrenoceptor blockade, but were markedly attenuated by combined adrenoceptor blockade. A substantial rise in VO(2) which accompanied a pronounced thermogenic effect of dobutamine in newborn lambs utilized most of the associated increase in DO(2) and appeared to be dependent on activation of multiple adrenoceptor subtypes.

Cardiac vagal responsiveness during development in spontaneously hypertensive rats

In the present study we sought to determine the effect of age, hypertension and endogenous angiotensin on the chronotropic responses to vagal stimulation in urethane anesthetized-normotensive Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHR). After β-adrenoceptor blockade with atenolol, the right and left vagal nerves were stimulated with graded frequencies between 1 and 32 Hz in 5-, 8- and 22-week-old animals. At all ages and in both strains, there was a strong linear relationship between the degree of bradycardia and the log of the stimulation frequency. At the age of 5 weeks, the bradycardia to stimulation of the right vagus was greater in SHR than that observed in WKY ( P<0.05). However, in 8- and 22 week-old animals, no differences were observed between the response to vagal stimulation in WKY and SHR. Thus, there was an age-dependent increase in the response to right vagal stimulation in WKY, but no such trend in SHR. No significant age-dependent changes in left vagal responses were observed in either strain. Left vagal responses were approximately half of the response to right vagal stimulation at all ages in SHR and in 8–22 week WKY, but similar to right vagal responses in 5 week WKY. Administration of the angiotensin converting enzyme inhibitor perindopril, which effectively blocked the formation of endogenous angiotensin, did not affect responses to vagal stimulation at any age, in either strain. These results suggest that the baroreflex vagal deficit observed in adult SHR compared to WKY is not due to a difference in the responsiveness of the cardiac vagal neuroeffector mechanism nor due to an effect of circulating angiotensin II. Furthermore, the enhanced vagal bradycardia observed in very young SHR which was due primarily to the earlier establishment of the adult vagal response pattern may indicate accelerated vagal development in this strain compared to WKY.

Dichotomic effects of IFN-gamma on the development of systemic lupus erythematosus-like syndrome in MRL-lpr / lpr mice.

Systemic lupus erythematosus (SLE)-prone female MRL-lpr / lpr (MRL-lpr) mice were treated with mouse or rat IFN-gamma under different experimental conditions, both prophylactically in 6- to 8 week-old animals and therapeutically in 12- to 18-week-old SLE-affected mice. It was found that IFN-gamma heterogeneously modulated the course of the disease in MRL-lpr mice. When administered prophylactically, IFN-gamma favorably modulated the histological, serological and clinical signs of the disease. Relative to untreated or PBS-treated control animals, the MRL-lpr mice which received IFN gamma were virtually free of inflammatory infiltration of the kidneys and the lungs, had lower levels of azotemia with reduction of both circulating IgG1, IgG2a and IgG3 and anti-double strand (ds) and single strand (ss) DNA antibodies, milder skin vasculitis, significantly reduced enlargement of their lymph nodes and lower weight of the spleens. IFN-gamma also lowered the rate of mortality of MRL-lpr mice. In contrast to these findings, therapeutically administered IFN-gamma worsened the course of the disease in MRL-lpr mice, which exhibited increased proteinuria, higher levels of IgG2a and IgG3 and anti-ds and -ss DNA antibodies, more aggressive nephritis and died at an earlier age than PBS-treated control mice. The dichotomic effect of IFN-gamma on disease manifestation in MRL-lpr mice offers new insights into the complex role of this cytokine in the regulation of systemic autoimmunity such as SLE.

Ah receptor-independent induction of CYP1A2 gene expression in genetically inbred mice

Acenaphthylene is a tricyclic aromatic hydrocarbon which induces hepatic methoxyresorufin O-demethylase (MROD) activity and CYP1A2 mRNA levels in 2 week-old male B6C3F1 mice. In the present study, this induction response was further investigated in genetically-inbred mice which differ in their aryl-hydrocarbon (Ah)-responsiveness. Acenapthylene (300 mg/kg) induced a 5- to 23-fold induction of MROD activity in Ah-nonresponsive (DBA and SJL) and responsive (C3H, C57/BL6, A/J, CBA and B6C3F1) mice. The highest induction response was observed in the DBA strain in which there was a 23- and 15-fold increase in activity in males and females, respectively. Acenaphthylene also caused a 2-fold increase in CYP1A2 mRNA and immunoreactive protein levels in 2 week-old DBA mice; however, this induction response was not observed in 6 week-old animals. For example, MROD activity in 6 week-old DBA mice was induced <2-fold by acenaphthylene, mainly as a consequence of increased basal CYP1A2 expression and MROD activity which, at the age of 6 weeks, approached levels induced by acenaphthylene in the 2 week-old mice. This was also observed by immunohistochemical staining with CYP1A2 antibodies of 2 and 6 week-old hepatic tissue from treated and control mice which also showed that CYP1A2 induction was dependent on the age of the animals.