Received Placebo Pellets Research Articles

Male Offspring of Hyperandrogenemic Female (HAF) Rats Develop Hypertension Beginning at 16 Weeks of Age

Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in women. It is characterized by hyperandrogenemia, oligo/anovulation, and polycystic ovaries. Children born to women with PCOS often have intrauterine growth restriction (IUGR) or are small for gestational age. This present study was conducted to test the hypothesis that maternal hyperandrogenemia will affect blood pressure (mean arterial pressure (MAP)) in male offspring.Hyperandrogenemia was produced by implantation of 5α‐dihydrotestosterone pellets (DHT; 7.5 mg/90 days, sc) in SD female rats at 4 wks of age (hyperandrogenemic females (HAF)). Control females received placebo pellets. Females were mated with SD males at 10–12 wks of age. Approximately 60% of HAF rats became pregnant, compared to 100% of controls. At 48 hrs, offspring were weighed and measured, and litters were culled to 4 males and 4 females. DHT and control dams were allowed to nurse offspring for 3 wks. Offspring (F1) were left untreated and studied at 4–6 mos (adult) and ≥16 mos (aging).Despite similar litter sizes (n=10–12±2 for HAF and control dams; p=NS), male offspring from HAF dams (F1HAF) weighed significantly less than offspring from control dams (F1contr.): (male F1HAF vs. F1contr: (PN 12 hrs :6.4±0.1 vs. 7.0±0.2g; n=13–15; p<0.05; PN 48 hrs: 7.4±0.3 vs. 8.1±0.1 g; n=13–15; p<0.05). Male F1HAF were also significantly shorter in length (6.2±0.6 vs. 6.8±0.2 cm; n= 15–18, p<0.05) and had smaller head circumference (4.4±0.1 vs. 4.6±0.1 cm; n=12–15) at 48hrs than male F1contr. At 16 wks, fat mass (normalized to BW) was significantly higher in male F1HAF than F1contr (0.07±0.01 vs. 0.06±0.01; p<0.05). MAP (radiotelemetry) was significantly elevated in adult male F1HAF compared to F1contr (121± 3 vs. 113 ± 3 mmHg; n=4–5/grp, p<0.05). DHT levels were significantly lower in male F1HAF than F1contr (57.4±34.9 vs 136.4±55.7 ng/ml; n=7–11/grp; p<0.01). With aging, male F1HAF had higher levels of proinflammatory cytokines than did male F1contr (by Bioplex; n=3–7/grp; all p<0.05): IL‐17a (149.5±39 vs. 32.1±5 pg/ml; p<0.05), IL‐1α (426±159 vs. 138±33 pg/ml), IL‐1β (122.6±36 vs. 36±6 pg/ml), and PAI‐1 (1690±386 vs. 590±63 pg/ml).Male offspring of HAF dams are born with IUGR compared to offspring of control females. As adults, male offspring of HAF dams have lower levels of androgens, increased fat mass, and develop hypertension. With aging, HAF male offspring have higher levels of inflammation than control offspring. These data are important since physicians rarely ask their middle‐aged, hypertensive, overweight male patients with androgen deficiencies if their mothers had PCOS during their pregnancy. With this knowledge, physicians may not only be able to predict which men will suffer from these complications, but may also provide the best therapeutic options for these men.Support or Funding InformationStudies were supported by NIH R01HL135089, R01HL66072, P01HL05971, P20GM121334

Proteomic Analysis of Focal Lesions in a Rat Model of Progenitor Marker‐Positive Hepatocellular Carcinoma

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR), markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt‐Farber) model of HCC in the rat. In the present study, we characterized the proteome of persistent, pre‐neoplastic focal lesions in rats subjected to the Solt‐Farber protocol. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and sacrificed 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to animals that underwent a protocol to activate hepatic progenitor cells. Normal rat liver was used as an additional control. Regions of formalin‐fixed, paraffin‐embedded tissue were obtained by laser capture microdissection. Peptides were generated and analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), which yielded 11,070 unique peptides representing 2,227 proteins. Peptides that showed increased abundance in placebo foci represented known HCC markers (glutathione S‐transferase‐P, epoxide hydrolase, 6 others) and potential markers (aflatoxin aldehyde reductase, carbonic anhydrase 2, 11 others). Peptides derived from cytochrome P450 enzymes were generally reduced. Neither placebo nor rapamycin samples showed similarity with the progenitor cell samples, indicating that the foci in this model, while considered to be progenitor marker‐positive, do not show evidence of persistent progenitor cells. Pathway analysis, based on non‐parametric identification of affected peptides, was consistent with an effect of rapamycin to reduce dedifferentiation in foci. Finally, we have demonstrated that the combination of LCM and LC‐MS/MS is an effective approach to characterize focal liver lesions.Support or Funding InformationNIH R01DK100301 NIH R01HD024455Rhode Island Hospital Department of Pediatrics

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to unmanipulated control animals and to animals that underwent an incomplete Solt-Farber protocol to activate hepatic progenitor cells. Regions of formalin-fixed, paraffin-embedded tissue were obtained by laser capture microdissection (LCM). Proteomic analysis yielded 11,070 unique peptides representing 2,227 proteins. Quantitation of the peptides showed increased abundance of known HCC markers (e.g., glutathione S-transferase-P, epoxide hydrolase, 6 others) and potential markers (e.g., aflatoxin aldehyde reductase, glucose 6-phosphate dehydrogenase, 10 others) in foci from placebo-treated and rapamycin-treated rats. Peptides derived from cytochrome P450 enzymes were generally reduced. Comparisons of the rapamycin samples to normal liver and to the progenitor cell model indicated that rapamycin attenuated a loss of differentiation relative to placebo. We conclude that early administration of rapamycin in the Solt-Farber model not only inhibits the growth of pre-neoplastic foci but also attenuates the loss of differentiated function. In addition, we have demonstrated that the combination of LCM and mass spectrometry-based proteomics is an effective approach to characterize focal liver lesions.

Effects of Homeopathic Medicines on Mood of Adults with Histories of Coffee-Related Insomnia

Background/Aims: The purpose of this within-subjects feasibility study was to determine whether two different homeopathic remedies, Nux Vomica (NV) and Coffea Cruda (CC), exert effects on subjective mood ratings in healthy adults with a history of coffee-induced insomnia. The impact of individual personality traits, anxiety sensitivity or Type A cynical hostility, and homeopathic constitutional type (HTYPE-NV, HTYPE-CC), on remedy effects was examined to evaluate differential responsivity, in accord with clinical claims. Subjects and Methods: Young adults of both sexes (ages 18–31) with above-average scores on standardized personality scales for either cynical hostility or anxiety sensitivity, and a history of coffee-induced insomnia, participated in the month-long study. At-home polysomnographic recordings were obtained on successive pairs of nights once per week for a total of 8 recordings (nights 1, 2, 8, 9, 15, 16, 22, 23). Subjects (N = 59) received placebo pellets on night 8 (single-blind) and verum pellets in 30c doses of one of two homeopathic remedies, NV or CC, on night 22 (doubleblind). Subjects completed the Profile of Mood States Scales at bedtime. Results: The remedies produced differential effects on anger and overall mood, with improved mood following CC administration. A similar trend for depression was observed. Anxiety sensitive subjects experienced less tension following CC, whereas hostile subjects receiving CC became more tense. The high HTYPE-CC receiving CC experienced less vigor. The high HTYPE-CC receiving NV experienced more vigor.

Effects of homeopathic medicines on polysomnographic sleep of young adults with histories of coffee-related insomnia

Background Homeopathy, a common form of alternative medicine worldwide, relies on subjective patient reports for diagnosis and treatment. Polysomnography offers a modern methodology for evaluating the objective effects of taking homeopathic remedies that clinicians claim exert effects on sleep quality in susceptible individuals. Animal studies have previously shown changes in non rapid eye movement sleep with certain homeopathic remedies. Methods Young adults of both sexes (ages 18–31) with above-average scores on standardized personality scales for either cynical hostility or anxiety sensitivity (but not both) and a history of coffee-induced insomnia participated in the month-long study. At-home polysomnographic recordings were obtained on successive pairs of nights once per week for a total of eight recordings (nights 1, 2, 8, 9, 15, 16, 22, 23). Subjects ( N = 54) received placebo pellets on night 8 (single-blind) and verum pellets on night 22 (double-blind) in 30c doses of one of two homeopathic remedies, Nux Vomica or Coffea Cruda. Subjects completed daily morning sleep diaries and weekly Pittsburgh sleep quality index scales, as well as profile of mood states scales at bedtime on polysomnography nights. Results Verum remedies significantly increased PSG total sleep time and NREM, as well as awakenings and stage changes. Changes in actigraphic and self-rated scale effects were not significant. Conclusions The study demonstrated the feasibility of using in-home, all-night sleep recordings to study homeopathic remedy effects. Findings are similar though not identical to those reported in animals with the same remedies. Possible mechanisms include initial disruption of the nonlinear dynamics of sleep patterns by the verum remedies.

Increased susceptibility of the lungs of hyperthyroid rats to oxidant injury: Specificity of effects

Results from previous studies indicate that hyperthyroidism increases the risk of ozone-induced lung toxicity. This observation raised the possibility that pulmonary damage from other oxidant substances might be greater in a hyperthyroid state. To address this hypothesis, pulmonary responses to crystalline silica, a particulate with oxidant properties, were evaluated in normal or hyperthyroid adult male rats. To induce a hyperthyroid condition, time-release pellets containing thyroxine were implanted subcutaneously; control rats received placebo pellets. After 7 days, the animals were exposed to saline or silica (0.1 mg/100 g BW or 1.0 mg/100 g BW) by intratracheal instillation. Following silica treatment, there was a dose-related increase in bronchoalveolar lavage (BAL) albumin levels and neutrophil numbers. However, the effects of silica were similar in both normal and hyperthyroid rats. These findings were confirmed and contrasted with those regarding ozone (1 ppm, 4 h inhalation) in a subsequent experiment. The results indicated that, although exposure to either ozone or silica resulted in increases in BAL albumin levels and neutrophil numbers, only responses to ozone were enhanced in hyperthyroid rats. These findings suggest that specificity exists in regards to the modulation of oxidant-induced lung damage and inflammation by thyroid hormones.

Increased Susceptibility of Hyperthyroid Rats to Ozone: Early Events and Mechanisms

Previous studies demonstrated that ozone-induced lung damage and inflammation are much greater in hyperthyroid rats, compared to normal rats, at 18 h postexposure. The purpose of the present investigation was to study early events and mechanisms underlying the increased sensitivity to ozone in a hyperthyroid state. Specifically, the degree of lung epithelial cell barrier disruption, the antioxidant status of the extracellular lining fluid, and the release of inflammatory mediators were examined. To induce a hyperthyroid state, mature male Sprague-Dawley rats were implanted with time-release pellets containing thyroxine; control rats received placebo pellets. After 7 d, the animals were exposed to air or ozone (2 ppm, 3 h). Immediately following the end of the exposure, bronchoalveolar lavage (BAL) fluid and cells were harvested. BAL fluid albumin levels and total antioxidant status were examined. In addition, levels of prostaglandin E2 (PGE2), macrophage inflammatory protein (MIP)-2, MCP-1, and tumor necrosis factor (TNF)-α were determined in BAL fluid and in media samples following ex vivo culture of BAL cells harvested after in vivo inhalation exposures. The results of this study are consistent with the following hypotheses: (1) A marked increase in the permeability of the alveolar–capillary barrier is an early event following ozone exposure in a hyperthyroid state; however this does not appear to be due to overall changes in BAL fluid antioxidant potential. (2) Early increases in MIP-2, but not PGE2, are involved in the enhanced lung response to ozone in a hyperthyroid state. (3) Inflammatory mediator production (i.e., PGE2, MIP-2, MCP-1, and TNF-α) by alveolar macrophages plays a minimal role in the initial responses to ozone in a hyperthyroid state. We thank D. J. Prugh, M. Donlin, A. Frazer, and D. Shahan for expert technical assistance.

Norgestimate and medroxyprogesterone acetate do not attenuate the atheroprotective effects of 17β-estradiol in ovariectomized, apolipoprotein E–deficient mice

To determine whether progestins counteract the cardioprotective effects of estrogen. Controlled animal study. Academic laboratory environment. Female apolipoprotein E-deficient mice. Mice were randomly assigned to groups receiving a sham operation plus placebo pellet, bilateral gonadectomy plus placebo pellet, or gonadectomy plus one of nine combinations of estrogen/progestin SC pellets. Total plasma cholesterol, body weight, fat depot weight, uterine weight and size, and the cross-sectional area of fatty streaks in the aortic sinus were measured in each animal. After 8 weeks of treatment, plasma cholesterol levels were significantly higher only in the ovariectomized and sham-operated animals that received placebo pellets. No differences in plasma cholesterol were observed relative to the type or amount of progestin administered. There was a reduction in fatty streaks in all of the hormone treatment groups as compared with both the ovariectomized and sham-operated animals that received placebo pellets. There were no significant differences in lesion area in response to estrogen alone or to estrogen plus the different types and doses of progestins.

Na +, K + ATPase α-subunit isoform distribution and abundance in guinea-pig longitudinal muscle/myenteric plexus after exposure to morphine

Previous work in the myenteric plexus has shown that the resting membrane potential of morphine-tolerant guinea-pig myenteric S neurons is significantly depolarized relative to placebo-implanted controls, and that this depolarization is associated with reduced electrogenic Na +, K + pumping. Identification of the subunits of the sodium pump which are in the myenteric plexus was undertaken in order to facilitate direct qualitative and quantitative measurements of the abundance of sodium pump isoforms after morphine exposure, thereby confirming and extending the electrophysiological data to the molecular level. Seven days prior to the experiments, tolerance was induced by subcutaneous implantation of morphine pellets (one pellet, 75 mg/100 g body weight) while control guinea pigs received placebo pellets. Using immunohistochemistry and confocal microscopy, the distribution of the α subunit isoforms of the Na +/K +-ATPase in placebo and morphine-tolerant guinea-pig ileum was determined. Only the α 1 and α 3 subunit isoforms were in sufficient abundance to be observed. The α 1 subunit isoform was most highly concentrated in the mucosa and in neurons. In contrast, the α 3 subunit isoform was uniquely localized to neurons. Western and slot blot analyses of longitudinal muscle/myenteric plexus homogenates identified a significant reduction of the α 3 but not the α 1 subunit isoform in tolerant preparations. It is concluded that the reduced electrogenic pumping in the S neurons after morphine exposure is associated with a reduction in the α 3 subunit isoform.

GH gene expression in the submaxillary gland in normal and Ames dwarf mice.

High local GH-releasing hormone (GHRH) levels are capable of inducing transdifferentiation in salivary cells to synthesize GH. However, the factors implicated in this process remain unknown. To study this subject, normal and Ames dwarf mice were implanted in the submaxillary gland with a slow release pellet releasing 21 microgram GHRH (1-29)-NH(2)/day for 2 months. Control animals received placebo pellets at the same site. After 60 days, heart blood was collected and submaxillary glands were removed. Circulating levels of GH and IGF-I were significantly decreased (P<0.05) in dwarf mice in comparison with controls, and GHRH treatment did not modify either of these two parameters. Controls carrying GHRH pellets showed a significantly higher GH content (P<0.05) in the submaxillary gland than the placebo-treated normal mice. There were no differences between the IGF-I concentrations of placebo- and GHRH-treated salivary tissue from normal mice. Analysis of GH mRNA by RT-PCR followed by Southern blot revealed that GH transcripts were present in the salivary gland samples carrying the placebo pellets in both normal and dwarf mice. The expression of GH was significantly (P<0.05) increased by the GHRH pellets in salivary tissue from normal mice, but not in submaxillary glands from dwarf mice. Pit-1 mRNA was not detected in the GHRH-treated glands of normal and dwarf mice by RT-PCR or by Southern blot. Using these highly sensitive methods, we have been able to detect the transcription of both GH and Pit-1 in pituitaries from Pit-1-deficient Ames dwarf mice. The present experiment demonstrates that salivary tissue synthesizes GH when it is exposed to the influence of GHRH. Both basal and GHRH-induced salivary GH expression appear to be independent of Pit-1.

Morphine enhances interleukin-12 and the production of other pro-inflammatory cytokines in mouse peritoneal macrophages

In this study we investigated the capacity of morphine to modulate expression of cytokines in peritoneal macrophages. Mice were implanted subcutaneously with a 75-mg morphine slow-release pellet, and 48 h later resident peritoneal macrophages were harvested. Control groups received placebo pellets, naltrexone pellets, or morphine plus naltrexone pellets. Adherent cells were stimulated with lipopolysaccharide (LPS: 10 microg/mL) plus interferon-gamma (IFN-gamma: 100 units/mL) to induce cytokine production. After 24 h RNA was extracted for analysis of cytokine mRNA levels by reverse transcriptase-polymerase chain reaction, or supernatants were collected after 48 h for determination of cytokine production by enzyme-linked immunosorbent assay (ELISA). Morphine enhanced mRNA expression of interleukin (IL)-12 p40 and tumor necrosis factor alpha (TNF-alpha) compared with controls, whereas IL-10 levels were unchanged by drug treatment. ELISA data showed that both IL-12 p40 and p70 were increased by morphine. The enhancement of IL-12 at both the mRNA and protein levels was antagonized by naltrexone, indicating that the modulation of this cytokine by morphine is via a classic opioid receptor. These results are particularly interesting in light of our previous observation that 48 h after morphine pellet implantation, the peritoneal cavity is colonized with gram-negative and other enteric bacteria. The enhancement of IL-12 by morphine might be related to morphine-induced sepsis.

Insulin and glucocorticoid-dependent suppression of the IGF-I system in diabetic wounds

Background: Growth-promoting polypeptides, including insulin-like growth factor-I (IGF-I), orchestrate different biochemical events that culminate in the restoration of functional integrity of wounded skin. The nonhealing cutaneous wound is a well-documented phenomenon in experimental and clinical diabetes. Accordingly, we undertook this study to ascertain whether diabetes impairs the healing process by suppressing the wound microenvironmental IGF-I system (eg, IGF-I; IGF-I receptor [IGF-I R]; and IGF-I binding protein [IGF-BP3]). Methods: The induction of diabetes was achieved by the intravenous injection of streptozotocin at a dose of 55 mg/kg. Subcutaneously implanted polyvinyl alcohol sponge and stainless steel mesh chamber models were used to study wound healing. Nondiabetic and diabetic animals received, respectively, subcutaneous 30-day time-release pellets of glucocorticoid (200 mg) and mifepristone (RU-486, 25 mg). Corresponding control animals received placebo pellets. Polyvinyl alcohol sponge and wound fluid expression of the IGF-I system were evaluated by using ligand blotting, radioimmunoassay, and reverse transcriptase polymerase chain reaction-based techniques. Results: Polyvinyl alcohol sponge contents of messenger RNA (mRNA) transcripts encoding for IGF-I, IGF-I R, and IGF-BP3 were reduced in diabetic and glucocorticoid-treated control animals. A similar pattern of changes in protein levels of IGF-I and IGF-BP3 occurred in wound fluid collected from these animals. Partial normalization of the associated hyperglycemic and hypercortisolemic states of diabetes with insulin (hyperglycemia) and the glucocorticoid receptor blocker RU-486 (hypercortisolemia) ameliorated the diabetes-related decrease in the IGF-I system during wound healing. Conclusions: The current data, together with data garnered from the literature, support the concept that the state of hypercortisolemia in diabetes mellitus impairs the healing process, at least in part, by suppressing the wound microenvironmental IGF-I system. Confirmation regarding this premise awaits further investigation. (Surgery 2000;127:687-95.)

Heat-shock protein 72/73 and impaired wound healing in diabetic and hypercortisolemic states

Background: Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Emerging evidence favors the involvement of glucocorticoids (GCs) in the pathogenesis of this diabetic complication. Recent data indicated that a heat-shock protein (HSP) with a molecular weight of about 70 kd is expressed in wound healing and it is under the control of the hypothalamic-pituitary-adrenal axis. In view of these findings, the current study was designed to examine the influence of diabetes and the hypercortisolemic state on the expression of HSP 72/73 during wound healing. Methods: Induction of diabetes was achieved by the intravenous injection of streptozotocin at a dose of 55 mg/kg. Subcutaneously implanted polyvinyl alcohol (PVA) sponges were used as a wound healing model. Control and diabetic animals received, respectively, subcutaneous 30-day timed-release pellets of GC (200 mg) and RU 486 (25 mg). Corresponding animals received placebo pellets. Expression of HSP 72/73 within the PVA sponges was assayed with use of Western blotting and immunohistochemical techniques. Results: GCs caused a Cushing-like syndrome with weight loss and adrenal atrophy. A pronounced accumulation of constitutive HSP 72/73 was observed in the cytoplasm of various cell types including fibroblasts, macrophages, and endothelium of nondiabetic controls. The PVA sponge contents of HSP 72/73 were decreased as a function of diabetes. A similar phenomenon was seen in control animals receiving high doses of GCs. Partial normalization of the associated hyperglycemic and hypercortisolemic states of diabetes with insulin (hyperglycemia) and the GC receptor block RU 486 (hypercortisolemia) ameliorated the diabetes-related decrease in PVA sponge contents of HSP 72/73. Conclusions: The current study provides evidence that both diabetes and the hypercortisolemic state are associated with a reduction in PVA sponge content of HSP 72/73. An amelioration of these changes was achieved by the institution of RU 486 therapy. Although our data may point to the possibility that the diabetes-related decrease in HSP 72/73 is mediated at least in part by GCs, a confirmation regarding this premise awaits further investigation. (Surgery 1999;125:594-601.)

Heroin self-administration in dependent Wistar rats: increased sensitivity to naloxone.

Non-dependent and dependent opiate users appear to be driven by two distinct motivational factors: the primary reinforcing properties of the drug, and the negative reinforcing effects associated with relieving the negative affective component of opiate withdrawal in the dependent state. To investigate the motivational significance of opioid dependence on heroin self-administration (HSA) in rodents. Rats were trained to self-administer heroin intravenously (0.06 mg/kg per infusion; FRI), and opiate dependence was induced by subcutaneous implantation of two morphine (75 mg base) pellets. Rats in a non-dependent control group received placebo pellets. Three days after pellet implantation, HSA was resumed in daily 3-h sessions until baseline criteria were met and testing was conducted with subcutaneous injections of vehicle or naloxone (0, 0.003, 0.01, 0.03 mg/kg) 115 min into the session. Morphine-dependent rats significantly increased HSA upon 0.01 mg/kg naloxone treatment, but decreased response rates at 0.03 mg/kg. Placebo pellet-implanted rats increased heroin intake at the 0.01 and 0.03 mg/kg doses. In a second experiment, the HSA session was shortened to 1 h and the training dose reduced to 0.03 mg/kg per infusion in new groups of animals. HSA in placebo pellet-implanted rats was increased only following the highest dose of the antagonist, while dependent rats were still affected by naloxone doses of 0.003-0.03 mg/kg. When subjected to a progressive-ratio schedule (experiment 3), breaking point values in dependent animals were 198% above baseline. The present study supports the hypothesis that dependence-induction by morphine-pellet implant in rats resulted in increased sensitivity to very small naloxone doses, as measured by changes in HSA. Taken together, these data suggest that opiate dependence, as measured by changes in sensitivity to naloxone, is a continuum which can contribute to the motivational state of drug-seeking.

Salivary gland is capable of GH synthesis under GHRH stimulation.

Twelve female rats weighing approximately 150 g received in the submaxillary gland a pellet capable of releasing 3.5 microg GHRH/h for 60 days. Another eight sex- and weight-matched animals received placebo pellets in the same place. After two months the animals were killed, heart blood was collected and pituitary and submaxillary glands were carefully dissected. Pituitary GH content in both placebo- and GHRH-treated animals showed similar values, but plasma GH and IGF-I levels were significantly lower in the animals carrying GHRH pellets (P<0.03); these animals also had a significantly higher GH content in the submaxillary gland (19.2+/-8 ng/mg protein) compared with the placebo-treated group (1.1+/-0.3 ng/mg protein). GH mRNA was present only in the submaxillary gland of GHRH-treated rats as determined by PCR-Southern blot and by in situ hybridization methods. It is concluded that high local GHRH levels are capable of inducing transdifferentiation in submaxillary gland cells to synthesize GH.

Mineralocorticoid blockade reduces vascular injury in stroke-prone hypertensive rats.

Chronic treatment of saline-drinking stroke-prone spontaneously hypertensive rats (SHRSP) with agents that interfere with the formation or actions of angiotensin II (Ang II) prevents the development of stroke and renal vascular damage. Ang II, in addition to its direct vascular effects, stimulates the synthesis and release of aldosterone. To assess the role of aldosterone in the development of pathologic changes in these rats, we implanted time-release pellets containing 200 mg of the mineralocorticoid receptor antagonist, spironolactone, into 14 SHRSP at 7.5 weeks of age. Eight SHRSP littermates received placebo pellets. Over the period of study (3 to 4 weeks), systolic blood pressure (SBP) was not different between the groups. Spironolactone did not enhance water and electrolyte excretion. All placebo-treated SHRSP developed marked proteinuria (150+/-6 mg/d) whereas in spironolactone-treated SHRSP, urinary protein excretion (UPE) averaged 39+/-9 mg/d (P<.0001). In a second study to assess effects on survival, 6 SHRSP received spironolactone (10 mg/kg/d) and 6 received vehicle. All but one of the control rats displayed signs of stroke and died by 16 weeks of age, while the spironolactone-treated SHRSP remained asymptomatic through 19 weeks of age (P<.03). At 16 weeks of age, spironolactone-treated SHRSP were severely hypertensive (247+/-3 mm Hg), yet UPE remained at baseline levels. In contrast, preterminal UPE averaged 136+/-13 mg/d in control rats (P<.0001). In both studies, histopathologic examination revealed a marked protective effect of spironolactone against the development of malignant nephrosclerotic and cerebrovascular lesions. These observations indicate a vascular and end organ protective effect of spironolactone in the absence of lowered blood pressure in saline-drinking SHRSP and are consistent with a major role for mineralocorticoids as hormonal mediators of vascular injury.

Prevention of chronic cerebral vasospasm in dogs with milrinone.

Delayed cerebral ischemia resulting from vasospasm is a major cause of morbidity and death in patients with aneurysmal subarachnoid hemorrhage. Milrinone, because it inhibits Type IV cyclic adenosine monophosphate-specific phosphodiesterase enzyme in both cardiac and vascular smooth muscle, is a powerful inotrope and vasodilator, but it has little effect on heart rate or blood pressure. Because of these properties, milrinone is an attractive potential therapy after subarachnoid hemorrhage. The purpose of the present study was to investigate the effect of milrinone on chronic experimental cerebral vasospasm. A double-hemorrhage canine model of vasospasm was used to study the efficacy of milrinone. Angiographic vasospasm and systemic hemodynamics were compared in a treatment group of animals that received a loading dose of milrinone (0.05 mg/kg, intravenously) and then slow-release (0.05 microgram/kg/min) milrinone pellets (n = 10) and a control group that received placebo pellets (n = 9), over an 8-day period after the initial subarachnoid hemorrhage. The hemorrhage was created by injection of 4 ml of autologous, nonheparinized, arterial blood into the cisterna magna on Days 1 and 3. Hemodynamic measurements, including cardiac output determinations, were made on Days 0, 1, 3, 6, and 8 with a pulmonary artery catheter, and angiographic vasospasm was assessed on Day 8 by comparison with baseline angiograms. Treatment with milrinone caused no significant changes in systemic hemodynamics. Angiographic vasospasm, however, was significantly reduced in the Day 8 angiograms for the treated group, compared with the control group (98.28 +/- 14.06 and 67.89 +/- 13.06% of original vessel cross-sectional area, respectively; P < 0.001). Milrinone is effective in preventing chronic cerebral vasospasm in a canine model of experimental chronic cerebral vasospasm. This effect is independent of changes in systemic hemodynamics. Milrinone and related drugs warrant further investigation for the treatment of cerebral vasospasm.

Prevention of Chronic Cerebral Vasospasm in Dogs with Milrinone

OBJECTIVE: Delayed cerebral ischemia resulting from vasospasm is a major cause of morbidity and death in patients with aneurysmal subarachnoid hemorrhage. Milrinone, because it inhibits Type IV cyclic adenosine monophosphate-specific phosphodiesterase enzyme in both cardiac and vascular smooth muscle, is a powerful inotrope and vasodilator, but it has little effect on heart rate or blood pressure. Because of these properties, milrinone is an attractive potential therapy after subarachnoid hemorrhage. The purpose of the present study was to investigate the effect of milrinone on chronic experimental cerebral vasospasm. METHODS: A double-hemorrhage canine model of vasospasm was used to study the efficacy of milrinone. Angiographic vasospasm and systemic hemodynamics were compared in a treatment group of animals that received a loading dose of milrinone (0.05 mg/kg, intravenously) and then slow-release (0.05µg/kg/min) milrinone pellets (n = 10) and a control group that received placebo pellets (n = 9), over an 8-day period after the initial subarachnoid hemorrhage. The hemorrhage was created by injection of 4 ml of autologous, nonheparinized, arterial blood into the cisterna magna on Days 1 and 3. Hemodynamic measurements, including cardiac output determinations, were made on Days 0, 1, 3, 6, and 8 with a pulmonary artery catheter, and angiographic vasospasm was assessed on Day 8 by comparison with baseline angiograms. RESULTS: Treatment with milrinone caused no significant changes in systemic hemodynamics. Angiographic vasospasm, however, was significantly reduced in the Day 8 angiograms for the treated group, compared with the control group(98.28 ± 14.06 and 67.89 ± 13.06% of original vessel cross-sectional area, respectively; P < 0.001). CONCLUSION: Milrinone is effective in preventing chronic cerebral vasospasm in a canine model of experimental chronic cerebral vasospasm. This effect is independent of changes in systemic hemodynamics. Milrinone and related drugs warrant further investigation for the treatment of cerebral vasospasm.

Evaluation of a novel nimodipine delivery system in conscious rats that allows sustained release for 24 h

Methodologies that allow prolonged drug administration in animal models, while minimizing surgery and anesthesia, are an important contribution towards studies in awake conditions. Commercially available drug delivery systems like pellets can be customized for the evaluation of experimental therapies with minimal or no discomfort to animals. Our objective was to evaluate pharmacokinetic and physiologic parameters after subcutaneous implantation of rapid 24 h release nimodipine pellets in rats for their potential use as a delivery system for stroke therapeutics. A day prior to the study Sprague-Dawley rats were anesthetized (halothane, N 2O, O 2) for femoral vessel cannulation and later returned to their cages. On the day of the study the rats were briefly anesthetized (identical regimen as before), and assigned to two groups: nimodipine (NP) and placebo (PL). NP rats received either 0.5 ( n = 4), 1 ( n = 3), 2 ( n = 2), 4 ( n = 2), or 15 ( n = 5) mg pellets (Innovative Research of America Inc., Sarasota, FL, USA) and PL rats ( n = 5) received placebo pellets. Nimodipine plasma levels were measured at 1, 3, and 6 h. In addition, the 15 mg NP group was followed at 18 and 24 h. Immediately following decapitation the brain was removed for later determination of nimodipine tissue concentration. The NP 15 mg group showed a significant decline of 10% in MABP from base line to 24 h post implantation ( p < 0.001). All NP animals achieved at least 83% of their highest plasma concentration at 1 h and 94% at 3 h. A high degree of correspondence ( r 2 = 0.95, y = 0.36 + 0.28 x, n = 16) between the plasma and brain concentrations of nimodipine was present. Although a significant drop in MABP was observed the drop was no greater than 10% in 24 h. Plasma nimodipine levels for the 15 mg animals were within the cerebrovascular effective range. This is the first report to show that 24 h release nimodipine pellets subcutaneously implanted in rats are a reliable delivery system that allows rapid rise and constant nimodipine plasma levels. Therefore, 24 h release pellets are a suitable alternative to other delivery systems like osmotic pumps.

T3 treatment does not prevent myocardial dysfunction in chronically diabetic rats.

The isolated working heart preparation was used to investigate the effect of continuous triiodothyronine (T3) administration on cardiac function and metabolism of rats rendered diabetic for a period of 4 wk with streptozocin (STZ). T3 controlled-release pellets were implanted 1 wk after STZ (70 mg/kg) injection. Rats injected with citrate buffer without STZ received T3 pellets 1 and/or 2 wk later. A comparable number of rats received placebo pellets. Untreated diabetic rats exhibited a decrease in spontaneous heart rate and myocardial cytochrome c concentrations concurrent with depressed plasma T3 values compared with untreated controls. T3 treatment did not improve in vitro cardiac performance (assessed as cardiac output times peak systolic pressure per gram dry heart weight) in hearts from diabetic rats perfused with glucose alone. Addition of octanoate reversed this depression and improved cardiac function to a greater extent in treated than in untreated diabetic animals. However, these differences between treated and untreated diabetic animals disappeared when heart rate was controlled by cardiac pacing. Furthermore, T3 treatment of controls and diabetics did not alter the oxidation of octanoate or the cardiac responsiveness to isoproterenol. These results suggest that experimental diabetic cardiomyopathy is partly attributable to a substrate deficiency and is not due entirely to hypothyroidism.