Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats.

PURPOSE: Local delivery of therapeutic molecules encapsulated within liposomes is a promising method to treat ocular inflammation. The purpose of the present study was to define the biodistribution of rhodamine-conjugated liposomes loaded with vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, following their intravitreal (IVT) injection in normal rats. METHODS: Healthy seven- to eight-week-old Lewis male rats were injected into the vitreous with empty rhodamine-conjugated liposomes (Rh-Lip) or with VIP-loaded Rh-Lip (VIP-Rh-Lip; 50 mM of lipids with an encapsulation efficiency of 3.0+/-0.4 mmol VIP/mol lipids). Twenty-four h after IVT injection, the eyes, the cervical, mesenteric, and inguinal lymph nodes (LN), and spleen were collected. The phenotype and distribution of cells internalizing Rh-Lip and VIP-Rh-Lip were studied. Determination of VIP expression in ocular tissues and lymphoid organs and interactions with T cells in cervical LN was performed on whole mounted tissues and frozen tissue sections by immunofluorescence and confocal microscopy. RESULTS: In the eye, 24 h following IVT injection, fluorescent liposomes (Rh-Lip and VIP-Rh-Lip) were detected mainly in the posterior segment of the eye (vitreous, inner layer of the retina) and to a lesser extent at the level of the iris root and ciliary body. Liposomes were internalized by activated retinal Müller glial cells, ocular tissue resident macrophages, and rare infiltrating activated macrophages. In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected. In lymphoid organs, Rh-Lip and VIP-Rh-Lip were distributed almost exclusively in the cervical lymph nodes (LN) with only a few Rh-Lip-positive cells detected in the spleen and mesenteric LN and none in the inguinal LN. In the cervical LN, Rh-Lip were internalized by resident ED3-positive macrophages adjacent to CD4 and CD8-positive T lymphocytes. Some of these T lymphocytes in close contact with macrophages containing VIP-Rh-Lip expressed VIP. CONCLUSIONS: Liposomes are specifically internalized by retinal Müller glial cells and resident macrophages in the eye. A limited passage of fluorescent liposomes from the vitreous to the spleen via the conventional outflow pathway and the venous circulation was detected. The majority of fluorescent liposomes deposited in the conjunctiva following IVT injection reached the subcapsular sinus of the cervical LN via conjuntival lymphatics. In the cervical LN, Rh-Lip were internalized by resident subcapsular sinus macrophages adjacent to T lymphocytes. Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment. In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

Dose and time effects of treatment with low doses of a LRH agonist on testicular axis and accessory sex organs in rats.

LRH and its agonists have been shown to exert both stimulatory and inhibitory effects on testicular function. In the present study, the dose and length of treatment were tested to determine the appearance of the stimulatory and inhibitory effects of LRH agonist on testicular axis including the three levels. Two doses of an agonist of LRH, 40 and 100 ng/100 g body weight (buserelin, 'agonist'), were administered daily for 1 to 15 days to adult male rats. Control rats received the vehicle only. On day 1, 2, 4, 8 and 15 of treatment, the pituitary, testicular and peripheral levels (weight of accessory sex organs and androgen receptors in ventral prostate) were tested 6 h after the last injection. For the 15 days of treatment with both doses, a stimulatory effect of the 'agonist' was observed on LH and FSH release. A short exposure (1-2 days) to the low dose of the 'agonist' had a stimulatory effect on the density of LH/hCG testicular receptors (326 +/- 49 vs control 185 +/- 21 fmol/mg protein, mean +/- SEM), on the weights of seminal vesicles and ventral prostate and exposure to both doses led to high plasma testosterone levels (13.8 +/- 0.5 and 13.7 +/- 0.7 ng/ml, respectively, vs control 2.6 +/- 0.3 ng/ml), and to an increased density of nuclear androgen receptors in the ventral prostate (142 +/- 9 and 144 +/- 15 fmol/mg protein respectively vs control 97 +/- 12 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

Dietary calcium restriction enhances cadmium-induced metallothionein synthesis in rats.

Experiments were conducted with adult male rats to investigate the effects of dietary calcium (Ca) restriction upon intake and tissue distribution of cadmium (Cd), and Cd-metallothionein (Mt) synthesis. Four groups of animals were fed either a low-Ca, semisynthetic diet (0.1% Ca) or the same diet supplemented with 0.8% Ca (normal diet). The caloric intake was similar in all groups. Two groups (low-Ca and normal diet) were used as controls, and two groups (low-Ca and normal diet) received 100 mg/l Cd (as CdCl2) in drinking water. Cd levels in liver, kidney, spleen and red cells were measured in all animals after 8 weeks of treatment. Concomitantly, Mt levels in plasma, liver and kidney were evaluated by radioimmunoassay. Ca deficiency entailed marked and significant increases in accumulation of Cd and synthesis of Mt in all assayed tissues. It is concluded that dietary Ca restriction, independent of caloric intake, enhances Cd intestinal absorption and tissue accumulation, which is followed by increased tissue Mt synthesis.

Liver regeneration with l-glutamine supplemented diet: experimental study in rats

OBJECTIVE: To assess liver regeneration in rats after 60% hepatectomy with and without supplementation of L-glutamine through liver weight changes, laboratory parameters and histological study.METHODS: 36 male rats were divided into two groups: glutamine group and control group. Each group was subdivided into three subgroups, with death in 24h, 72h and seven days. The glutamine group received water and standard diet supplemented with L-glutamine, and the control recieved 0.9% saline. In all subgroups analysis of liver regeneration was made by the Kwon formula, study of liver function (AST, ALT, GGT, total bilirubin, indirect and indirect bilirubin and albumin) and analysis of cell mitosis by hematoxylin-eosin.RESULTS: In both groups there was liver regeneration by weight gain. Gamma-GT increased significantly in the control group (p < 0.05); albumin increased in the glutamine group. The other indicators of liver function showed no significant differences. Histological analysis at 72h showed a higher number of mitoses in the glutamine group, with no differences in other subgroups.CONCLUSION: Diet supplementation with L glutamine is beneficial for liver regeneration.

The effects of supraphysiological supplementation of b-carotene in spontaneously hypertensive rats (SHR and SHR-sp)

Objective: to investigate the effect of administration of supraphysiological âcaroteno on biological parameters (ectoscopy and blood pressure), laboratory (malondialdehyde) and histological (liver and carotid arteries) of spontaneously hypertensive rats prone to stroke (SHR-sp).Methods: we used 36 male rats were divided into three groups, each containing 12 rats Wistar, SHR and SHR-sp, subdivided into six control animals and six animals treated with supraphysiological doses of âcaroteno for two periods of ten weeks interspersed with one week interruption. In the experiment were assessed daily physical examination and blood pressure (plethysmography). At sacrifice, blood was collected for measurement of serum malondialdehyde, liver and carotid arteries for histological examination.Results: temporary change in color of the fur, decreased significantly (p<0.0001) blood pressure (20mg supplementation âcaroteno) and serum levels of malondialdehyde (p<0.05) and increased amount of elastic fibers in the carotid wall of SHR and SHR-sp.Conclusion: supplementation of supraphysiological âcaroteno caused no toxic effects, showed positive response in the modulation of blood pressure and lower serum malondialdehyde. No significant morphological changes were found in both groups, except an increase in the number of elastic fibers in the muscle layer carotid suggesting elastosis in SHR and SHR-sp.

Correlations between ANP concentrations in atria, plasma and cerebral structures and sodium chloride preference in Wistar rats

We determined whether ANP (atrial natriuretic peptide) concentrations, measured by radioimmunoassay, in the ANPergic cerebral regions involved in regulation of sodium intake and excretion and pituitary gland correlated with differences in sodium preference among 40 Wistar male rats (180-220 g). Sodium preference was measured as mean spontaneous ingestion of 1.5% NaCl solution during a test period of 12 days. The relevant tissues included the olfactory bulb (OB), the posterior and anterior lobes of the pituitary gland (PP and AP, respectively), the median eminence (ME), the medial basal hypothalamus (MBH), and the region anteroventral to the third ventricle (AV3V). We also measured ANP content in the right (RA) and left atrium (LA) and plasma. The concentrations of ANP in the OB and the AP were correlated with sodium ingestion during the preceding 24 h, since an increase of ANP in these structures was associated with a reduced ingestion and vice-versa (OB: r = -0.3649, P<0.05; AP: r = -0.3291, P<0.05). Moreover, the AP exhibited a correlation between ANP concentration and mean NaCl intake (r = -0.4165, P<0.05), but this was not the case for the OB (r = 0.2422). This suggests that differences in sodium preference among individual male rats can be related to variations of AP ANP level. Earlier studies indicated that the OB is involved in the control of NaCl ingestion. Our data suggest that the OB ANP level may play a role mainly in day-to-day variations of sodium ingestion in the individual rat

Cardiovascular responses to microinjection of trans-(±)-ACPD into the NTS were similar in conscious and chloralose-anesthetized rats

The changes in mean arterial pressure (MAP) and heart rate (HR) in response to the activation of metabotropic receptors in the nucleus tractus solitarii (NTS) with trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-(±)-ACPD) were evaluated in conscious and anesthetized Wistar, male rats weighing 240-260 g (N = 8). The responses obtained with trans-(±)-ACPD were compared with the responses to L-glutamate (1 nmol/100 nl), since in a previous study we showed that anesthesia converted a pressor response to L-glutamate microinjected into the NTS of conscious rats to a depressor response in the same rats under urethane or chloralose anesthesia. Microinjection of 3 doses of trans-(±)-ACPD (100, 500 and 1000 pmol/100 nl) produced a dose-dependent fall in MAP (range, -20 to -50 mmHg) and HR (range, -30 to -170 bpm) under both conscious and chloralose anesthesia conditions. These data indicate that the cardiovascular responses to the activation of metabotropic receptors by trans-(±)-ACPD are not affected by chloralose anesthesia while the cardiovascular responses to the activation of excitatory amino acid (EAA) receptors by L-glutamate are significantly altered

The hyperinsulinemia produced by concanavalin A in rats is opioid-dependent and hormonally regulated

The present study examines the effect of concanavalin A (Con A) on the blood insulin and glucose levels of rats. Male and female rats treated with Con A (62.5-500 µg/kg) for three days showed a dose- and time-dependent hyperinsulinemia that lasted more than 48 h. Male rats were more sensitive to Con A. Thus, 6 h after treatment with Con A the circulating insulin levels in male rats had increased by 85% (control: 10.2 ± 0.9 mU/l and Con A-treated: 18.8 ± 1 mU/l) compared to only 38% (control: 7.5 ± 0.2 mU/l; Con A-treated: 10.3 ± 0.9 mU/l) in females. An identical response was seen after 12 h. Con A (250 µg/kg) produced time-dependent hypoglycemia in both sexes but more pronounced in males. There was no correlation between the hypoglycemia and hyperinsulinemia described above. The Con A-induced hyperinsulinemia in rats of both sexes was abolished in gonadectomized animals (intact males: +101 ± 17% vs orchiectomized males: -5 ± 3%; intact females: +86 ± 23% vs ovariectomized females: -18 ± 7.2%). Pretreating intact male and female rats with human chorionic gonadotropin also significantly inhibited the Con A-induced hyperinsulinemia. Estradiol (10 µg/kg, im) significantly blocked the Con A-induced increase in circulating insulin in male rats (101 ± 17% for controls vs 32 ± 5.3% for estradiol-treated animals, P<0.05) while testosterone (10 mg/kg, im) had no similar effect on intact female rats. Pretreating Con A-injected rats with opioid antagonists such as naloxone (1 mg/kg, sc) and naltrexone (5 mg/kg, sc) blocked the hyperinsulinemia produced by the lectin in males (control: +101 ± 17% vs naloxone-treated: +5 ± 14%, or naltrexone-treated: -23 ± 4.5%) and females (control: +86 ± 23% vs naloxone-treated: +21 ± 20%, or naltrexone-treated: -18 ± 11%). These results demonstrate that Con A increases the levels of circulating insulin in rats and that this response is opioid-dependent and hormonally regulated.

Effect of repeated stress on novelty-induced antinociception in rats

There is extensive evidence that acute stress induces an analgesic response in rats. On the other hand, repeatedly stressed animals may present the opposite effect, i.e., hyperalgesia. Furthermore, exposure to novelty is known to induce antinociception. The effects of repeated restraint stress on nociception after exposure to novelty, as measured by the tail-flick latency (TFL), were studied in adult male rats. The animals were stressed by restraint 1 h daily, 5 days a week for 40 days. The control group was not submitted to restraint. Nociception was assessed with a tail-flick apparatus. After being familiarized with the TFL apparatus, each group was subdivided into two other groups, i.e., with or without novelty. Animals were subjected to the TFL measurement twice. For the animals exposed to novelty, the first TFL measurement was made immediately before, and the second 2 min after a 2-min exposure to a new environment. While the control group presented an increased TFL after exposure to a novel environment, chronically stressed animals did not show this effect. These results suggest that repeated restraint stress induces an alteration in the nociceptive response, perhaps as a result of an alteration in endogenous opioids in these animals.

Metabolic markers following beta-adrenoceptor agonist infusion in footshock-stressed rats

Stress hormones can alter metabolic functions in adipose tissue and liver, as well as the sensitivity of rat white adipocytes and rat atrial responses to ß-adrenergic agonists. In this study, we examined the effects of three daily footshock stress sessions on the plasma corticosterone, glucose, glycerol and triacylglycerol levels of fed, conscious male rats, and on the plasma glucose, glycerol and triacylglycerol levels of the same rats following iv infusions of ß-adrenergic agonists (isoproterenol: 0.4 nmol kg-1 min-1, noradrenaline: 5.0 µg kg-1 day-1, and BRL 37344 ([±]-[4-(2-[(2-[3-chlorophenyl]-2-hydroxyethyl)amino]propyl)phenoxy]acetic acid), a selective ß3-adrenoceptor agonist: 0.4 nmol kg-1 min-1). Plasma corticosterone levels increased significantly after each stress session, while triacylglycerol levels increased after the first session and glucose increased after the second and third sessions. Glycerol levels were unaltered after stress. These results suggest that repeated footshock stress may induce a metabolic shift from triacylglycerol biosynthesis to glucose release by hepatic tissue, with glycerol serving as one of the substrates in both pathways. Stressed rats were more sensitive to infusion of noradrenaline plus prazosin and to infusion of isoproterenol, with elevated plasma glucose, glycerol and triacylglycerol levels. The higher sensitivity of stressed rats to isoproterenol and noradrenaline was probably related to the permissive effect of plasma corticosterone. Only BRL 37344 increased plasma glycerol levels in stressed rats, probably because ß3-adrenoceptors are not involved in hepatic triacylglycerol synthesis, thus allowing glycerol to accumulate in plasma.

Episodes of water deprivation enhance daily hypertonic NaCl intake in rats

Water and 1.8% NaCl intake was recorded daily in adult male rats (N = 6) submitted to four water deprivations plus four sodium appetite tests, each at the end of each 7-day interval, or in controls (non-deprived, N = 6). Water deprivation was achieved by removing water and 1.8% NaCl for 24 h. Water was then offered for 2 h. At the end of this period, 1.8% NaCl was also offered in addition to water (sodium appetite test). Average daily 1.8% NaCl intake was enhanced from 5.2 ± 1.0 to 15.7 ± 2.5 ml from the first to the fifth week in the experimental group and was unchanged in the control group. Daily water intake was not altered in either group. Thus, repeated episodes of water deprivation enhance daily NaCl intake.

Chronic converting enzyme inhibition normalizes QT interval in aging rats

The aim of the present study was to investigate the effects of converting enzyme inhibition by captopril on ECG parameters in aged rats. Four-month-old male rats received captopril dissolved in tap water (0.5 mg/l) or tap water for 2 or 20 months. At the end of treatment, under anesthesia, RR and PR interval, P wave and QRS duration, QT and corrected QT interval were measured in all animals. On the following day, chronic ECG (lead II) recordings were performed to quantify supraventricular (SVPB) or ventricular premature beats (VPB). After sacrifice, the hearts were removed and weighed. RR interval was similar in young and untreated aged rats, but significantly larger in aged rats treated with captopril. P wave and QRS length did not differ among groups. PR interval was significantly larger in old than in young rats and was not affected by captopril. Corrected QT interval was larger in aged than in young rats (117 ± 4 vs 64 ± 6 ms, P<0.05) and was reduced by captopril (71 ± 6 ms, P<0.05). VPB were absent in young rats and highly frequent in untreated old animals (8.4 ± 3.0/30 min). Captopril significantly reduced VPB in old rats (0.3 ± 0.1/30 min, P<0.05). The cardiac hypertrophy found in untreated aged rats was prevented by captopril (3.44 ± 0.14 vs 3.07 ± 0.10 mg/g, P<0.05). The beneficial effects of angiotensin converting enzyme inhibition on the rat heart during the aging process are remarkable.

Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats

The pharmacokinetics of scorpion venom and its toxins has been investigated in experimental models using adult animals, although, severe scorpion accidents are associated more frequently with children. We compared the effect of age on the pharmacokinetics of tityustoxin, one of the most active principles of Tityus serrulatus venom, in young male/female rats (21-22 days old, N = 5-8) and in adult male rats (150-160 days old, N = 5-8). Tityustoxin (6 µg) labeled with 99mTechnetium was administered subcutaneously to young and adult rats. The plasma concentration vs time data were subjected to non-compartmental pharmacokinetic analysis to obtain estimates of various pharmacokinetic parameters such as total body clearance (CL/F), distribution volume (Vd/F), area under the curve (AUC), and mean residence time. The data were analyzed with and without considering body weight. The data without correction for body weight showed a higher Cmax (62.30 ± 7.07 vs 12.71 ± 2.11 ng/ml, P < 0.05) and AUC (296.49 ± 21.09 vs 55.96 ± 5.41 ng h-1 ml-1, P < 0.05) and lower Tmax (0.64 ± 0.19 vs 2.44 ± 0.49 h, P < 0.05) in young rats. Furthermore, Vd/F (0.15 vs 0.42 l/kg) and CL/F (0.02 ± 0.001 vs 0.11 ± 0.01 l h-1 kg-1, P < 0.05) were lower in young rats. However, when the data were reanalyzed taking body weight into consideration, the Cmax (40.43 ± 3.25 vs 78.21 ± 11.23 ng kg-1 ml-1, P < 0.05) and AUC (182.27 ± 11.74 vs 344.62 ± 32.11 ng h-1 ml-1, P < 0.05) were lower in young rats. The clearance (0.03 ± 0.002 vs 0.02 ± 0.002 l h-1 kg-1, P < 0.05) and Vd/F (0.210 vs 0.067 l/kg) were higher in young rats. The raw data (not adjusted for body weight) strongly suggest that age plays a pivotal role in the disposition of tityustoxin. Furthermore, our results also indicate that the differences in the severity of symptoms observed in children and adults after scorpion envenomation can be explained in part by differences in the pharmacokinetics of the toxin.

Motor behavioral abnormalities and histopathological findings of Wistar rats inoculated with HTLV-1-infected MT2 cells

The objective of the present study was to describe motor behavioral changes in association with histopathological and hematological findings in Wistar rats inoculated intravenously with human T-cell lymphotropic virus type 1 (HTLV-1)-infected MT2 cells. Twenty-five 4-month-old male rats were inoculated with HTLV-1-infected MT2 cells and 13 control rats were inoculated with normal human lymphocytes. The behavior of the rats was observed before and 5, 10, 15, and 20 months after inoculation during a 30-min/rat testing time for 5 consecutive days. During each of 4 periods, a subset of rats was randomly chosen to be sacrificed in order to harvest the spinal cord for histopathological analysis and to obtain blood for serological and molecular studies. Behavioral analyses of the HTLV-1-inoculated rats showed a significant decrease of climbing, walking and freezing, and an increase of scratching, sniffing, biting, licking, and resting/sleeping. Two of the 25 HTLV-1-inoculated rats (8%) developed spastic paraparesis as a major behavioral change. The histopathological changes were few and mild, but in some cases there was diffuse lymphocyte infiltration. The minor and major behavioral changes occurred after 10-20 months of evolution. The long-term observation of Wistar rats inoculated with HTLV-1-infected MT2 cells showed major (spastic paraparesis) and minor motor abnormalities in association with the degree of HTLV-1-induced myelopathy.