Series Of Animals Research Articles

Intracerebral Diffusion of Paramagnetic Cationic Liposomes Containing Gd(DTPA)2- Followed by MRI Spectroscopy: Assessment of Patternc Diffusion and Time Steadiness of a Non-Viral Vector Model

Cationic liposomes are generally considered as the non-viral counterparts of the more common viral vectors used in several gene therapy protocols, but their use as delivery vehicles is limited by their efficiency even if they display a lower toxicity. However, cationic liposomes are promising delivery systems in cell biology due to their ability to incorporate small molecules into their inner aqueous spheres and to deliver them into cells. Additionally, on the external surface they can bind therapeutic molecules such as nucleic acids, oligonucleotides, plasmids, etc. through electrostatic interactions. The aim of this work was to study the diffusion properties of such vehicles in vivo with a non-invasive technique and to monitor their tissue migration in order to collect information to be further used in gene therapy procedures. For this purpose, cationic liposomes containing the paramagnetic contrast agent Gd(DTPA)2- (Gd(III)-diethylenetriamine-N,N,N',N",N"-pentaacetic acid) were investigated because of their extended paramagnetic persistency in vivo, compared to the use of the contrast agent alone, and they were used to monitor the diffusion of such vehicles in an animal model (rat model). In particular, these vectors were injected into the rat brain through a stereotactic frame in a preformed cavity mimicking the lesion which had originated after surgical removal of the primary tumor. For the purpose of comparison, the same injection procedure was also applied to a control series of animals without a preformed brain lesion. Pattern diffusion and steadiness of the reported paramagnetic cationic liposomes were studied by means of Magnetic Resonance Imaging (MRI) which allowed us to monitor their diffusion and assess their intracerebral time availability up to 24 hours.

Oral NAC supplementation to a high‐sucrose meal improves postprandial glycemic control via glutathione synthesis in rats

Postprandial hyperglycemia may be detrimental to glucose homeostasis by promoting oxidative stress. We hypothesized that dietary cysteine may favor postprandial glucose control by maintaining glutathione (GSH) status. This work investigates the effects of moderate or high N-acetylcysteine (NAC) supplementation to a high sucrose (HS) meal on postprandial glucose homeostasis in healthy rats. In experiment 1, 3 groups of rats received a 3 g meal containing 72% of sucrose, either enriched with 5.5 g.kg−1 NAC (C1), 16 g.kg−1 NAC (C2) or not enriched (C0). Postprandial parameters were monitored for 3h. after the meal ingestion. The same measurements were performed on a second series of animals (experiment 2) that were injected 4 mmol.kg−1 BSO, a specific inhibitor of glutamate-cysteine ligase, 1 hour before the meal. In experiment 1, postprandial hyperglycemia was 15 ± 3 % and 34 ± 8 % lower (P<0.05) and postprandial hyperinsulinemia was 33 ± 12 % and 66 ± 7 % lower (P<0.05), in C1 and C2 rats, respectively, when compared to C0 rats. Insulin sensitivity, as assessed by HOMA index, was higher in C1 and C2 rats, with a NAC dose effect (P<0.05). Conversely, after BSO injection, postprandial hyperglycemia, hyperinsulinemia and HOMA index did not differ anymore between groups. In both experiment 1 and 2, postprandial GSH redox state was lower in C1 and C2 rats, as compared to C0 rats. Taken together, these data show that meal cysteine favors postprandial glycemic control through a mechanism that involves GSH synthesis and limits adverse effects of sucrose-induced oxidative stress.

Spatial cognition and memory: a reversible lesion with lidocaine into the anteromedial/posterior parietal cortex (AM/PPC) affects differently working and long-term memory on two foraging tasks

Place memory is relevant for exploration and forage behaviour. When food supply is dispersed, a win-shift has advantage over a win-stay strategy. In the Olton Octagonal Maze, the rat follows a win-shift strategy using working memory. However, in the Olton 4x4 version, the rat follows a win-stay strategy, using both working and long-term memories. It has been suggested that the neocortex is required for the resolution of tasks demanding long-term, but not for that demanding working memory alone. The role of anteromedial/posterior parietal cortex (AM/PPC) was investigated here, using a reversible lesion induced by intracerebral lidocaine infusion. Long-Evans rats were implanted with guide cannulae into the AM/PPC and trained in an Olton 4x4 maze, counting working and long-term memory errors after a delay. Then, the animals were infused with lidocaine or saline during the delay phase and tested for three days. Another series of animals, treated as before, was tested in an Olton Octagonal Maze and subjected to the same injection schedule. In the Olton 4x4 Maze, lidocaine produced a significant increase in working and long-term memory errors, compared to saline and post-lidocaine conditions. In contrast, in the Olton Octagonal Maze, lidocaine did not induce any effect on working memory errors. Thus, AM/PPC is required when both working with previous information and long-term memories are needed, but not when only working memory is required, as it happens under ethological conditions. Whenever food supply is dispersed, a win-shift strategy is preferable.

Percutaneous In Situ Coronary Venous Arterialization: A Catheter‐Based Procedure for Coronary Artery Bypass

Diffuse coronary artery disease is frequently untreatable by coronary artery bypass or angioplasty. Open chest surgical arterialization of the coronary venous system was successfully performed before the widespread use of coronary artery bypass grafting and has been applied in ungraftable patients thereafter. Percutaneous in situ coronary venous arterialization (PICVA) follows this surgical experience, using a series of unique catheter-based devices to arterialize isolated segments of coronary vein, forcing retroperfusion of severely ischemic myocardium. In a series of animals, this procedure had been shown to reduce ischemia and myocardial damage in an infarction model. In the PICVA European Safety trial, the first 11 percutaneous applications of catheter-based coronary bypass in humans revealed feasibility of the technique in highly symptomatic "no-option" patients. Despite two deaths and six failures to create the bypass, we continue to believe that on further development, the catheter-based coronary bypass can become a broad-based interventional application.

Structure and size of bronchopulmonary anastomoses in sheep lung

The distribution and drainage of bronchial arterial blood flow are complex. We used two different methods to study the bronchial-pulmonary anastomoses in sheep lung. Initially, we injected two different sizes of fluorescent microspheres (15 and 100 microm diameter) into the bronchial artery and histologically determined where the different-size microspheres were entrapped in the lung. In a second series of animals, we injected Microfil into the bronchial artery to observe the anastomotic vessels. The microsphere data confirmed the existence of bronchial-to-pulmonary anastomoses. No microspheres were found in the systemic organs (heart and kidney), confirming the absence of large bronchial artery-to-pulmonary vein anastomoses. Unexpectedly, proportionately more large microspheres (100 microm) lodged in the alveolar parenchyma when compared to 15 microm microspheres. This suggests that there are many more small bronchial (< 100 microm) arterioles feeding the airway mucosa than the larger anastomotic vessels feeding into the parenchyma. In the Microfil cast lungs, we observed four types of anastomotic vessels: bronchial arteries/arterioles that anastomose with pulmonary arteries/arterioles that accompany airways; bronchial arterioles that anastomose directly with parenchymal (and eventually alveolar) vessels; bronchial arterioles that anastomose with blood vessels that do not accompany airways; and bronchial arterioles that anastomose with bronchial veins. Based on our in vivo microsphere data, the vessels that do not accompany the airways are most likely bronchial venules, not pulmonary venules.

Brain Energetics and Tolerance to Anoxia in Deep Hypothermia

The remarkable time-resolution enhancement by deep lethargic hypothermia (15 degrees C rectal temperature, "cold narcosis," "anesthesia by internal cold") of metabolic events in the rat brain after oxygen deprivation has been exploited to monitor metabolic changes by in vivo (31)P-NMR. A correlation was established between the bioenergetic status of the brain and physiological descriptors of tolerance (survival and revival times) determined in parallel experiments with large series of animals. Spectral peak integrals were transformed into absolute concentrations by comparison to biochemically determined time series of data obtained in freeze-trapping experiments conducted under identical conditions. Serial spectra were used to reconstruct the time-course kinetics of intracellular brain pH and of concentration changes of inorganic phosphate, phosphocreatine, ATP, and ADP. Both the biochemical and NMR time series of data were simultaneously fitted by a set of exponential kinetic equations accounting for relationships imposed by the Lohmann and adenylate kinase reactions. Depletion profiles were then computed for a number of descriptors of brain energy status (energy charge, phosphorylation potential, total adenylate, and primary energy stores expressed as the sum of high-energy phosphate-bond equivalents). The results contribute to the understanding of the role of brain energetics in tolerance to oxygen deprivation.

Modulation of Erbb2 signaling during development: a threshold level of Erbb2 signaling is required for development

We have generated a series of Erbb2 cDNA knock-in animals to explore the role of signaling pathways coupled to Erbb2 during development. Although this knock-in allele was hypomorphic, expressing tenfold less Erbb2 protein than wild type, the knock-in animals were healthy. However, a further twofold reduction in Erbb2 levels in hemizygous knock-in animals resulted in perinatal lethality with defects in the innervation of the diaphragm. Genetic rescue of this hypomorph was accomplished by expression of the Erbb2-Y1028F mutant in a comparable knock-in allele. Interestingly, hemizygous Y1028F animals were viable with normal innervation of the diaphragm. Molecular analyses revealed that the Y1028F allele expressed higher levels of Erbb2 and that Y1028 promoted the turnover of the receptor. In addition, ablation of the Shc-binding site in Erbb2 (Y1227) resulted in subtle defects in the sensory nerves not observed in the other mutant erbb2 strains. Thus, we have established how Erbb2 levels may be modulated through development and that a minimum threshold level of Erbb2 is required.

Uncoupling of vasopressin signaling in collecting ducts from rats with CBL-induced liver cirrhosis.

Vasopressin (AVP) stimulates collecting duct water reabsorption through cAMP-mediated membrane targeting and increased expression of the aquaporin-2 (AQP2) water channel. Rats with liver cirrhosis induced by common bile duct ligation (CBL) show decreased protein expression of AQP2 despite increased plasma concentrations of AVP. The present study was conducted to investigate possible mechanisms behind this uncoupling of AVP signaling. The rats were examined 4 wk after CBL or sham operation. The CBL rats had increased plasma AVP concentrations (CBL: 3.2 +/- 0.2 vs. sham: 1.4 +/- 0.4 pg/ml, P < 0.05) and reduced AQP2 (0.62 +/- 0.11) and phosphorylated AQP2 (0.50 +/- 0.06) protein expression compared with sham-operated rats. However, examination of subcellular AQP2 localization by immunohistochemistry showed unchanged plasma membrane targeting in CBL rats, indicating a sustained ability of AQP2 short-term regulation. In a separate series of animals, thirsting was found to normalize AQP2 expression, indicating that AVP uncoupling in CBL rats is a physiological compensatory mechanism aimed at avoiding dilutional hyponatremia. Studies on microdissected collecting ducts from CBL rats showed decreased cAMP accumulation in response to AVP stimulation. The presence of the nonspecific phosphodiesterase inhibitor IBMX normalized the cAMP accumulation, indicating that cAMP-phosphodiesterase activity is increased in CBL rats. However, in contrast to this, Western blotting showed a decreased expression of several phosphodiesterase splice variants. We conclude that CBL rats develop an escape from AVP to prevent the formation of dilutional hyponatremia in response to increased plasma AVP concentrations. The mechanism behind AVP escape seems to involve decreased collecting duct sensitivity to AVP as a result of increased cAMP-phosphodiesterase activity.

Building a More Perfect Beast: APP Transgenic Mice with Neuronal Loss

While there have been a number of successful attempts to develop transgenic mice that express mutant amyloid precursor protein (APP) and develop amyloid deposits that have features similar to those in aging and Alzheimer’s disease (AD),1 until now there has been either no neuronal loss or less than convincing evidence in favor of neuronal loss. In this issue of The American Journal of Pathology, Schmitz and colleagues2 provide compelling evidence for age-dependent neuronal loss in the hippocampus of bigenic transgenic mice expressing a double mutation in APP (Swedish APP mutation APPK670N, M671L (APPsw) and APPV717I, under the mouse Thy1 promoter) and mutant presenilin-1 (PS-1 M146L under the pHMG promoter). The neuronal loss was ascertained by unbiased stereologic methods on Nissl-stained sections. The animals showed significant (30 to 35%) neuronal loss in the hippocampal pyramidal layer compared to controls, which was disproportionate to the degree of amyloid deposition. No neuronal loss was detected in the dentate fascia of the hippocampus, the only other region assessed for neuronal loss. While the regions analyzed are of interest, if the model is to have relevance to AD, it will be important in future studies to extend neuronal counts to limbic and association cortical regions, which are especially vulnerable to amyloid deposition in humans. The behavioral consequences, if any, of neuronal loss in this model are not described in this report or other descriptions of this model3 and are left to future work.

Effects of sildenafil on myocardial infarct size, microvascular function, and acute ischemic left ventricular dilation.

Adverse cardiac events in patients treated with the phosphodiesterase-5 inhibitor sildenafil for erectile dysfunction raised concerns about its safety in ischemic heart disease. In anesthetized open-chest rabbits, receiving 1.45 mg/kg sildenafil intravenously or saline 30 min prior to ischemia (n=12, each), infarct size (IS, triphenyltetrazolium), the area of no-reflow (ANR, thioflavin S) (% of the risk area, RA, blue dye), and regional myocardial blood flow (RMBF, radioactive microspheres) were measured after 30 min of coronary occlusion and 180 min of reperfusion. Left ventricular hemodynamics and dimensions (echocardiography) were determined in a separate series of animals (n=5, each). Sildenafil significantly lowered arterial blood pressure before occlusion (-17 to -19 mmHg over 30 min), but during ischemia and reperfusion hemodynamics were comparable to controls. IS in treated animals (51+/-4%) did not significantly differ from control animals (47+/-4%). No major arrhythmias or lengthening of QT/QTc occurred. While sildenafil slightly increased RMBF and significantly reduced specific vascular resistance in the RA during reperfusion (51+/-7 versus 73+/-10 mmHg g min/ml, P<0.05), the ANR (46+/-3%) was similar to control animals (44+/-4%). Sildenafil reduced left ventricular dP/dt(max) (P<0.05) and dP/dt(min) (P<0.01) in non-ischemic conditions, and slightly during ischemia, along with a pronounced decrease in ischemic left ventricular end-diastolic pressure (9+/-2 versus 15+/-2 mmHg after saline, P<0.05), but did not attenuate acute ischemic left ventricular dilation. Sildenafil reduced cardiac pre- and afterload, and parameters of left ventricular contractility. Myocardial necrosis and microvascular dysfunction were neither exacerbated nor attenuated.

Ecological series of soil animals in darlidai mountain

The ecological series of soil animals under the broad-leaved and pine mixed forest in Darlidai Mountain was studied. Seven sample plots were selected according to different altitude gradients, which belong to different vegetation types. By investigating and analyzing soil animals in every sample plot it is found that there are 45 groups and 1956 individuals, which are involved in 3 phylums, 7 classes, 16 orders, respectively. The altitude is a key factor which affects ecological series of soil animals. Both the groups and individuals of soil animals increase with altitude increasing under certain conditions, which contrastes with ordinary cases, resulting from special micro-climate in studied area. The groups and individuls of soil animals are the most under the broad-leaved and pine forest on the top of the mountain, and the least under Picea-Abies forest in the foot of the mountain.

Examination of OLETF-derived non-insulin-dependent diabetes mellitus QTL by construction of a series of congenic rats.

The Otuska Long-Evans Tokushima Fatty (OLETF) rat is one of the well-characterized animal models for the study of type 2 diabetes. Our previous QTL mapping identified 11 loci responsible for non-insulin-dependent diabetes mellitus (NIDDM) susceptibility in the OLETF rat. Here we generated a series of congenic animals by individually introgressing all 11 OLETF-derived NIDDM loci into a normoglycemic F344 background. Subsequent oral glucose tolerance test revealed that the congenic strains for Nidd1/of, Nidd2/of, Nidd3/of Nidd4/of, Nidd7/of, and Nidd10/of showed significantly higher levels of blood glucose in comparison with parental host strain F344. Furthermore, simultaneously made heterozygote animals for Nidd1/of and Nidd2/of did not increase blood glucose levels, indicating that these loci are recessively inherited as predicted by the QTL analysis. Congenic strains for the other five loci-Nidd5/of, Nidd6/of, Nidd8/of, Nidd9/of, and Nidd11/of-were apparently normoglycemic, presumably owing to heterosis or because the effect of these loci may not be detected unless interactions with other OLETF genes exist. We believe that these congenic strains should provide useful agents for decomposing complex diabetic traits and for positional cloning.

Effects of intrauterine inflammation on developing rat brain.

Damage to the white matter in the brain during development can lead to cerebral palsy (CP), a heterogeneous group of clinical syndromes that results in life-long disorders of movement and posture. Periventricular leukomalacia (PVL) is a pathological process within the white matter characterized by oligodendrocyte loss and is associated with the development of CP. Clinically, CP and PVL are associated with intrauterine infection and inflammation, but mechanisms involved are not well understood. We developed a model of intrauterine inflammation in Lewis and Fischer 344 rats to study the effects of intrauterine inflammation on developing glia. Pregnant rats were intracervically injected with lipopolysaccharide (LPS) at 15 days of gestation (E15) and a dose of LPS that caused low fetal mortality was determined. At E20, treated fetuses had increased TUNEL(+) nuclei and tumor necrosis factor (TNF)-alpha-immunoreactive areas within the brains. In a second series of animals allowed to survive until postnatal day 21 (PND 21), immunostaining was performed against several glial markers. Staining for the oligodendrocyte-specific proteins 2', 3'-cyclic nucleotide phosphodiesterase (CNP) and myelin proteolipid protein (PLP) was decreased in treated pups compared to shams within the corpus callosum, a white matter structure used as a representative area of developing white matter. Treated pups had activated astrocytes lining cerebral blood vessels, as observed by glial fibrillary acidic protein (GFAP) staining, while sham pups did not. Activated microglia were not detected using OX42 as a cell marker. Our model of intrauterine inflammation causes increased TUNEL and TNF-alpha staining early after injury, suggesting increased apoptotic cell death, possibly by cytokine-related mechanisms.

Effect of melatonin and nifedipine on some antioxidant enzymes and different energy fuels in the blood and brain of global ischemic rats.

The brain normally derives most of its energy from the aerobic oxidation of glucose and therefore it must be nourished with a rich supply of both glucose and oxygen. Interference with the blood supply, such as in ischemia, could shift the brain to search for another source of energy and to spare its own glucose. Ischemia results not only in energy fuel disturbance, but also in free radical formation and Ca(2+) homeostasis disruption. Therefore, our investigations studied the influence of ischemia on energy fuels, on some natural free radical scavengers, and the relationship between the changes of these parameters in brain and blood. Each of these was also studied under the influence of melatonin, a well-known free radical scavenger, and nifedipine, a Ca(2+)-channel blocker and antioxidant, during ischemia followed by reperfusion (I/R). Adult male Wistar rats were subjected to global ischemia by occlusion of the two carotid arteries for 1 hr (group I), followed by reperfusion for another hour in group II. Drugs were injected after ischemia (group I), and before or after reperfusion onset in the second group. Two series of animals were used. In the first series the effect of the two drugs on the activity of superoxide dismutase (SOD), glutathione reductase (GR), and lactate dehydrogenase (LDH) was investigated in the cytosolic fraction of four brain areas, viz., cortex (CC), thalamus/hypothalamus (T/TH), midbrain (MB) and medulla, pons and cerebellum (MPC). Moreover, the level of both SOD and GR in the erythocytes of these rats was also estimated. In the second series, we studied the effect of each drug on the content of glucose and beta-hydroxybutyrate (beta-HB) in whole brain, in addition to the plasma levels of glucose, beta-HB and lactate. The results showed that (i), ischemia elevated the brain levels of LDH and beta-HB, as well as the plasma level of glucose, beta-HB, lactate and erythocytic GR. Conversely, it lowered glucose, SOD and GR levels in the brain; (ii), reperfusion reversed the ischemic effect on all the previously altered parameters except for plasma levels of lactate and glucose; (iii), melatonin (10 mg/kg, i.p) and nifedipine (1.5 mg/kg, i.p), restored the energy fuel levels in the brain of ischemic and I/R rats, as well as the ischemic effect on the erythocyte activities of SOD and GR. Furthermore, both drugs reversed I/R effect on the cytosolic activities of the antioxidant enzymes. We conclude that melatonin and nifedipine are both neuroprotective with improvement in the antioxidant system and energy fuels.

Protein Kinase A Transgenes

The β-adrenergic receptor/adenylyl cyclase/protein kinase A (PKA) axis is the central signaling pathway that serves to stimulate cardiac function. It is classically perceived as a linear signaling cascade (Figure), and cAMP is thought to be the second messenger responsible for the positive inotropic, chronotropic, and lusitropic effects of catecholamines. New data gained from a series of transgenic animals overexpressing various elements of this axis show that this system has a far more complex makeup than previously thought. Transgenic models of the cardiac β-adrenergic receptor/PKA system. The signaling cascade is represented as a simple linear chain. Proteins that are detrimental when overexpressed are shown in black; those causing beneficial effects upon overexpression are depicted in white. PLB indicates phospholamban. It has been known for about two decades that the β-adrenergic receptor system is dysfunctional in heart failure.1 The loss of receptor responsiveness has been attributed to a reduced β-receptor number, particularly of the β1-subtype, plus a functional desensitization of the remaining receptors—presumably mediated via increased activity of G protein–coupled receptor kinases.2 The extent of the receptor downregulation and the functional loss correlate with the severity of the disease.3,4 This phenomenon has long been regarded as detrimental to the compromised function of the failing heart, and it has been proposed that restoration of β-adrenergic receptor responsiveness might be a strategy to improve cardiac function. However, recent clinical studies and observations on a panel of transgenic animals suggest that the opposite is the case. Sustained stimulation or overexpression of cardiac β1-adrenergic receptors leads to hypertrophy, fibrosis, and heart failure,5–7 and blockade of β-adrenergic receptors with several antagonists—bisoprolol, carvedilol, and metoprolol—improves survival in heart failure patients.8 Thus, the loss of β-adrenergic receptor responsiveness in heart failure is more likely to represent a protective mechanism than …

Iulius Casserius (1552-1616): the self-made anatomist of Padua's golden age.

Giulio Cesare Casseri (1552– 1616), whose name was Latinized into Iulius Casserius, was born in Piacenza; therefore, the nickname Piacentino (Placentinus) was often used. According to Sterzi (1910), who based his claim on a statement contained in Casserius’s will, his date of birth was around 1552. It should be noted, however, that although most modern authors accept this date (Roberts and Tomlinson, 1992; Premuda, 1993), some (Singer, 1957) still report 1561 as his birth date on the basis of the inscription (Fig. 1) appearing on the portrait published in Casserius’s work De Vocis Auditusque organis that ascribes to the author the age of 39 years. According to most of his early biographers (Tomasini, 1630; Ghilini, 1647; Papadopoli, 1726), Casserius’s family was very poor and the young Iulius, perhaps as the servant of some student (Sterzi, 1910), moved to Padua, the city that shared with Bologna the reputation of being the seat of Italy’s most illustrious university. He soon assumed the job of servant in the house of the famed Gerolamo Fabrici d’Acquapendente (Fabricius, 1533– 1619) (Fig. 2), Public Lecturer of Anatomy and Surgery (Tomasini, 1630; Ghilini, 1647). As Tomasini (1630) reports, “from a servant he became first Fabricius’s auditor, then instructor and brilliant disciple.” We do not know when Casserius matriculated in the School of Medicine of the Universita Artista. As mentioned several times in his works, Casserius, in addition to Fabricius, had the well-known physician Gerolamo Mercuriale (Mercurialis) as a teacher and mentor, who held the chair of Clinical Medicine of Padua in the years 1569–1587. The precise date he obtained his degree in medicine and philosophy is unknown, because the official records are missing from 1580 to 1587; a likely date is ca.1580 (Sterzi, 1910; De Ferrari, 1978; Premuda, 1993). After the award of his degree, Casserius, in addition to giving private lectures on anatomy to the students of the Universita Artista and working as Fabricius’s preparator, started a practice as physician and surgeon in Padua that was very successful (Tomasini, 1630). In 1584, Casserius’s reputation was already so great that he took Fabricius’s place as member of the board of examiners for the finals in surgery. These examinations, held in private houses in the presence of the Rector of the University, conferred the license to practice surgery. The profession of surgery was considered in those days as a minor branch of Medicine and the surgeon had to take an oath that he would not involve himself in the treatment of serious diseases, but would call a licensed physician whenever the patient was in real danger.

Activation of postsynaptic 5-HT(1A) receptors by fluoxetine despite the loss of firing-dependent serotonergic input: electrophysiological and neurochemical studies.

Systemic doses of fluoxetine slow dorsal raphe cell firing by blocking the reuptake carrier located in the cell body region with the surplus 5-HT thus generated activating inhibitory autoreceptors. The concurrent actions of fluoxetine on postsynaptic receptors in raphe projection areas has not been as thoroughly investigated, although it is presumed that a reduction in cell firing should curtail these targeted effects. The goal of the present studies was to assess the degree of postsynaptic receptor activation obtained with fluoxetine and relate it to cell body autoreceptor activation and the level of extracellular 5-HT obtained at the nerve endings. Changes in firing rates of CA3 hippocampal neurons following systemic administration of fluoxetine were used as a marker of SSRI-dependent changes in postsynaptic 5-HT receptor activation; monitoring of unit activity of neurons in the dorsal raphe nucleus served to gauge the degree of serotonergic input in a parallel series of animals. Estimates of the corresponding changes in terminal 5-HT release in the CA3 region were analyzed by microdialysis. The results indicate that fluoxetine inhibits hippocampal cell firing in a dose-dependent manner (ED(50) = 4.4 mg/kg i.v.) and one sensitive to pretreatment with the 5-HT(1A) antagonist WAY-100,635. Within the same dose range, increases in hippocampal extracellular 5-HT approaching 300% above basal levels were achieved. Both the changes in hippocampal neuronal activity and extracellular 5-HT are evident at doses of fluoxetine in excess of that required to inhibit dorsal raphe cell firing (ED(50) = 1.1 mg/kg i.v.). Taken together, these data suggest that increases in extracellular levels of 5-HT on the order of that observed are sufficient to alter postsynaptic excitability and that this accumulation of synaptic 5-HT and the subsequent activation of postsynaptic 5-HT(1A) receptors are achievable despite loss of firing-dependent 5-HT release.

An improved clinical model of orthotopic pancreatic cancer in immunocompetent Lewis rats.

The study of pancreatic cancer (PaCa) requires orthotopic, clinically relevant animal models. The aims of this study were to establish an orthotopic model of ductal pancreatic adenocarcinoma in immunocompetent Lewis rats and to develop a scoring system to quantify local tumor infiltration and distant metastasis. Cells (10(7)) of the rat ductal PaCa cell line DSL-6A/C1 were injected s.c. into donor rats. After 8 weeks, either three (IPL-3) or five (IPL-5) fragments (1 mm3) of the resulting s.c. tumors were microsurgically implanted into the pancreas of recipient rats. In another series of animals, 10(7) DSL-6A/C1 cells were directly injected (INJ) into the pancreas. All animals were monitored daily until death or for 16 weeks. At autopsy, volume of primary tumors and ascites, local and systemic tumor spread, and histologic phenotype were assessed. IPL-5 resulted in significantly larger tumors (12,224 +/- 1,933 mm3), more local infiltration and systemic spread (score: 18.3 +/- 2.0 points), severe clinical tumor disease, and lethality (50%) in comparison to the other induction techniques (IPL-3: 283 +/- 115 mm3/3.5 +/- 0.8 points/0; INJ: 752 +/- 207 mm3/4.3 +/- 0.8 points/8%). Histologic examination revealed moderately to well-differentiated ductal tumors, surrounded by dense stroma. Intraperitoneal tumor dissemination in the INJ group occurred simultaneous with primary tumor growth, indicating PaCa cell spread during injection. Orthotopic implantation of five DSL-6A/C1 tumor fragments into the rat pancreas provides a valid clinical model of ductal pancreatic adenocarcinoma in immunocompetent rodents for preclinical treatment studies. The dissemination score we used permitted quantification of local and systemic tumor spread.

How costly is molluscan shell erosion? A comparison of two patellid limpets with contrasting shell structures

The energetic cost of replacing shell loss due to erosion has been overlooked in previous research on molluscan energy budgets, although several studies have shown that shell breakage incurs a significant short-term cost and may reduce growth. We measured the rate of shell erosion for two species of patellid limpets: Patella granatina, which has a shell predominated by calcite, and P. argenvillei, in which aragonite is dominant. Highest rates of erosion of P. granatina shells (36% p.a.) were recorded when the shells were grazed by a co-occurring limpet, P. granularis; in their absence, erosion was 15.2%. P. argenvillei experienced a slightly lower rate of erosion (32.4% p.a.) when its shells were grazed by juvenile conspecifics, whereas ungrazed shells lost 12.5%. P. argenvillei shells are often coated with an encrusting coralline, and infested by a boring lichen, but neither had a significant effect on the rate of erosion. By modelling the energetic costs of shell erosion, we showed that the shell mass produced by P. argenvillei each year is substantially greater than that produced by P. granatina, as is the loss of mass due to erosion. However, the energetic costs of shell production, and the losses due erosion, are higher for P. granatina, reflecting its faster growth and the higher energy content of its shells. For P. argenvillei, the cost of erosion increases with age from about 8–20% of the total energy devoted to “production” (somatic growth, shell production and gonadial output). For P. granatina it is 8–12%. Experimental elimination of erosion failed to yield higher somatic, visceral or gonadial masses. This suggests that shell erosion does not carry a short-term cost equivalent to that experienced when shells are broken. Rather, it seems that compensation for shell erosion is an ongoing process involving a long-term cost. As a more explicit test of this hypothesis, we coated the shells of some individuals to protect them against erosion, left others unprotected, and coated one side of the shells of a third series of animals. Protected shells or sides of shells became thicker than those that were unprotected. Deposition of shell continued whether the shells (or portions of shells) were being eroded or not. Thus, compensation for shell erosion is an process of continual maintenance that is not simply initiated where and when erosion occurs, and will carry long-term costs.

The Slow Induction of Resistant Hepatocytes during Initiation of Hepatocarcinogenesis by the Nongenotoxic Carcinogen Clofibrate

This study was designed to explore whether a well-known nongenotoxic liver carcinogen, clofibrate, would induce rare resistant hepatocytes similar to those seen during initiation of hepatocarcinogenesis with many genotoxic carcinogens. Male young adult F344 rats were exposed to a control diet containing 0.5% (w/w) clofibrate for 3, 6, or 10 months. After 1 month on a diet free of clofibrate, the animals were assayed for resistant hepatocytes by a standardized selection procedure using 2-acetylaminofluorene as the inhibitor and partial hepatectomy as a strong stimulus for cell proliferation. No resistant hepatocytes were found in the animals exposed to clofibrate for 3 months or in any of a series of control animals. However, animals on the clofibrate for 6 and 10 months contained resistant hepatocytes that were clonally expanded to produce hepatocyte nodules. These nodules were indistinguishable on gross and microscopic examination from hepatocyte nodules seen in animals in which nodules are induced with one of many different genotoxic carcinogens. Also, like those nodules, the nodules seen in the animals exposed to clofibrate stained positively for glutathione S-transferase 1-1 and γ-glutamyl transpeptidase and negatively for ATPase. The evidence from this study indicates that the nongenotoxic carcinogen, clofibrate, induces early cellular changes in the liver that are very similar to those induced by many different genotoxic carcinogens. These changes are manifest as a resistance phenotype in a few scattered hepatocytes that now can be clonally expanded selectively to form hepatocyte nodules. However, the resistant hepatocytes are induced by clofibrate much more slowly. Whether this basic similarity pertains to the later steps in the hepatocarcinogenic process remains to be studied.