Implantation Of Osmotic Pump Research Articles

Reversion Of Lung Fibrosis In C57BL6 Mice Using Statins

Objective: In radiation enteropathy models, activation of Rho/ROCK signalling pathway trigger radio-induced fibrosis and constitutes a new therapeutic target as Rho inhibition using anti-fibrotic properties of statins. In this study we investigate whether the pathological activation of the Rho/ROCK pathway was specific to the gut or could represent a more general pathogenic path. Therefore, anti-fibrotic properties of Rho/ROCK pharmacological inhibitors were tested in murine models of lung fibrosis. Materials and methods: Pulmonary fibrosis was induced in pathogen-free 10-wk-old female C57BL/6 mice by 1) IP injection of Bleomycin at 40 mg/kg body weight on Days 0, 2, 4, 6 and 8 and 2) Thorax irradiation (16 Gy, 17 Gy; RX 250 KeV). Mice follow-up was performed twice a week for the overall experimental period and tissue collection was performed at Day 15 and 30 and at week 15 and 26 respectively for bleomycin and irradiation model. Safron stain was used for collagen quantification after slide scanning (Nikon Scan) and analysis by Pixcyt program. Fibrosis development was monitored by HES staining. CTGF and α SM actin expression were studied by Western Blot and Q-RT-PCR. Curative effect of Rho and ROCK inhibition were studied in vivo using respectively pravastatin (40 mg/Kg/d) and simvastatin (20 mg/Kg/d) delivered over 14 days via osmotic pump implantation. Results: histopathological examinations show inflammatory lesions mixed up with fibrotic areas at Day 15 whereas fibrosis is fully established D30. The fibrotic lesions are located in the subpleural zones and within the large peripheral vessels. Quantification of the collagen deposition show progressive increase of collagen after D15. Curative administration of pravastatin increased the body weight of the animals by 25% as compared to the vehicle-treated group and significantly improved survival (83% vs 56%). In the same groups, HES analysis showed an almost complete regression of fibrosis in all animals. These results were less impressive in simvastatin group with a survival gain equal to 10%. Experiments on irradiation-induced lung fibrosis are currently ongoing. Conclusion: This study confirms and extends the anti-fibrotic therapeutic action of statins and shows that bleomycin-induced lung fibrosis can be modulated by Rho/ROCK pathway inhibition. However, further investigation are required to investigate the distinct action elicited by pravastatin versus simvastatin. As the Rho family is composed of several members, one hypothesis is that pravastatin and simvastatin could target different member of the Rho family.

17β-estradiol attenuates hippocampal neuronal loss and cognitive dysfunction induced by chronic restraint stress in ovariectomized rats

Several lines of evidence suggest that hormonal changes after menopause may play an important role in the incidence of cognitive dysfunction, and also in the development of Alzheimer’s disease. In this study, we investigated the effect of estrogen on cognitive function in rats under different stress environment. Female rats were divided into four groups: two groups were ovariectomized (OVX) and two were sham-operated. One group each of OVX and sham rats was kept in a normal environment, and the other groups were assigned to a daily restraint stress (6 h/day) for 21 days from 2 months after the operation. Following the stress period, subjects were tested for performance in novel object recognition test and then used for morphological and neurochemical analyses. The OVX plus stress (OVX/stress) group showed a significant impairment of recognition of novel objects, compared with the other groups. The OVX/stress group also showed a marked decrease in the number of pyramidal cells of the CA3 region and levels of brain-derived neurotrophic factor mRNA in the hippocampus. We further examined the effect of estrogen against cognitive dysfunction and hippocampal changes of OVX/stress rats. Vehicle or 17β-estradiol (E2) at 20 μg/day was s.c. administered to OVX/stress rats from 2 days before the stress period to the end of behavioral analysis through an implantable osmotic pump. Chronic E2 treatment decreased stress response and improved the cognitive and morphological impairments relative to vehicle group. These data have important implications for cognition enhancing effect of estrogen treatment in postmenopausal women.

Influence of Hydroxyurea On Imatinib Mesylate (Gleevec) Transport at the Mouse Blood-Brain Barrier

The combination of imatinib mesylate and hydroxyurea provides a therapeutic benefit in patients with glioblastoma, although each drug is not effective when used alone. The increase of brain delivery of one or both drugs has been suggested to be a potential cause of this therapeutic benefit. The cross-influence of hydroxyurea and imatinib on their respective brain distribution was examined in mice and rats. We used in situ brain perfusion in mice to determine whether these two drugs have an influence on their respective initial transport across the blood-brain barrier. The brain penetration of hydroxyurea, assessed by its brain uptake clearance, Knet, was low in mice (approximately 0.10 microl/g/s) and not modified by coperfusion of imatinib (0.5-500 microM). Likewise, the brain penetration of imatinib was low (Knet, 1.39 +/- 0.17 microl/g/s) and not modified by direct coperfusion of hydroxyurea (0.2-1000 microM) or by intravenous pretreatment with 15 or 1000 mg/kg hydroxyurea. We also examined a potential time-dependent influence of hydroxyurea on imatinib brain distribution after sustained subcutaneous administration in rats using an implantable osmotic pump. The brain penetration of imatinib in rats increased with time, approximately 1.6-fold (p < 0.01) after 7 and 14 days' infusion of imatinib (3 mg/day) with or without hydroxyurea (15 mg/day), and was not influenced by hydroxyurea. The results of these two sets of experiments indicate that hydroxyurea has no significant influence on the brain distribution of imatinib in mice and rats.

In vivo characterization of a novel GnRH (gonadotropin-releasing hormone) antagonist, LXT-101, in normal male rats

LXT-101 is a newly developed GnRH (gonadotropin-releasing hormone) analogue. In this study, the in vivo pharmacological profile in intact male rats and binding characters of LXT-101 were illustrated, and regulation of mRNA of hormone receptors related to the pituitary–gonadal axis during and after administration was observed to reveal its molecular mechanism of potent effect and reversibility. After single subcutaneous injections, LXT-101 produced a dose- and time-dependent suppression of serum testosterone level. Multiple administrations and osmotic pump implantation revealed that the time of onset and dose needed to maintain the effect of chemical castration decreased as the frequency of injection increased and gave direct proof that depot formulation could significantly improve the duration of antagonist delivery and pharmacological activities compared to the injectable formulation. And LXT-101 showed excellent character of regulating the pituitary–gonadal axis quickly and reversibly. Competitive binding assay showed that LXT-101 could specifically bind a pituitary GnRH receptor with high affinity. These results indicated that LXT-101 is fit for sustained-release formulation and it might possibly be developed as an ideal candidate for treating sex hormone-sensitive tumors and other disorders.

Duration and Intensity of NF-κB Activity Determine the Severity of Endotoxin-Induced Acute Lung Injury

Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-kappaB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-kappaB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-kappaB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-kappaB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IkappaB kinase inhibitor (BMS-345541) to down-regulate NF-kappaB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-kappaB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-kappaB activation correlates with severity of lung injury, and interdiction in the NF-kappaB pathway is beneficial even after the onset of lung inflammation.

A potent and selective soluble epoxide hydrolase inhibitor improves angiotensin II‐induced hypertension

Soluble epoxide hydrolase (sEH) participates in the metabolism of epoxyeicosatrienoic acids (EETs), and pharmacological inhibition of the enzyme is considered as a novel approach to improve hypertension. In vitro assay with cytosol fraction prepared from human liver, we have discovered sEH inhibitors, Anilide Derivative (AD) and Benzimidazole Derivative (BD), from our chemical library. These compounds completely inhibited sEH-catalyzed the conversion of trans-stilbene oxide (AD, IC50=4.5 nM; BD, IC50=28 nM) and 14,15-EET (AD, IC50=121 nM; BD, IC50=50 nM) to their corresponding diols, but had little inhibition for cycloxygenase-1 and 5-lipoxygenase. BD also showed a strong inhibition on recombinant human sEH activity with the IC50 value of 19 nM. To clarify the potency of BD in vivo, we investigated the hypotensive effect of BD in angiotensin II-induced hypertensive rats. Subcutaneous implantation of osmotic pump filled with angiotensin II exhibited the elevation in systolic blood pressure up to 170 mmHg, whereas, given BD to these rats (60mg/kg/day, po) reduced the rise in blood pressure to 140 mmHg. In conclusion, we have discovered potent and selective inhibitors of sEH that may be useful for treating hypertension.

Angiotensin‐II receptors regulate ACE2 expression in the brain

We previously reported the presence of ACE2 in brain nuclei involved in the central regulation of blood pressure (BP). Using real-time PCR, we tested the hypothesis that angiotensin-II receptors blockade would modulate ACE2 expression in these brain regions. Non-transgenic (NT) and transgenic mice (n=6 per group) with brain-selective overexpression of AT1a receptors (NSE-AT1) were instrumented with radiotelemeters for continuous BP monitoring. Baseline BP (mmHg) was recorded for 1 wk before subcutaneous implantation of osmotic pumps containing saline, an AT1 blocker (losartan, 10 mg/Kg/day) or an AT2 antagonist (PD123319, 10 mg/Kg/day). Mice were infused and BP recorded for 2 wks. In losartan-treated mice, mean BP decreased significantly (NT:−10±3; NSE-AT1:−12±3; P<0.05) by 2 wks but remained unchanged in the saline groups (NT:−1±2; NSE-AT1:-2±3). On the other hand, AT2 blockade increased BP only in NSE-AT1 (12±5; P<0.05) mice. Basal ACE2 mRNA expression was increased in the nucleus of tractus solitarius (NT:1±0.1; NSE-AT1:1.3±0.2; P<0.05) and decreased in the ventrolateral medulla (NT:1±0.1; NSE-AT1:0.7±0.1; P<0.05) of NSE-AT1 mice but was similarly decreased by losartan (NT:0.5±0.1; NSE-AT1:0.7±0.1 and NT:0.6±0.1; NSE-AT1:0.4±0.1, respectively, P<0.05) in both genotypes. On the contrary, AT2 blockade increased ACE2 expression in these regions (NT:1.9±0.4; NSE-AT1:1.5±0.2 and NT:1.9±0.3, respectively, P<0.05). These data suggest that both AT1 and AT2 receptors regulate ACE2 expression in brain regions controlling BP, confirming the involvement of brain ACE2 in the central regulation of cardiovascular function. (NS52479)

Relaxin and β-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by β-estradiol. We postulated that relaxin and β-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, β-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered β-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 μg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. β-estradiol, relaxin, or β-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or β-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by β-estradiol, relaxin, or β-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.

EFFECTS OF α 1-ADRENERGIC RECEPTOR SUBTYPE SELECTIVE ANTAGONISTS ON LOWER URINARY TRACT FUNCTION IN RATS WITH BLADDER OUTLET OBSTRUCTION

Antagonists of alpha 1-adrenergic receptors (alpha 1ARs) relieve obstructive and irritative symptoms in patients with bladder outlet obstruction. However, to our knowledge mechanisms underlying the relief of irritative symptoms remain unknown. Because bladder alpha 1dARs are up-regulated in some rats with bladder outlet obstruction, we investigated the effect of the alpha 1aAR antagonist 5-methyl urapidil (5MU) vs the alpha 1a/alpha 1dAR antagonist tamsulosin on urinary frequency in obstructed rats. Baseline frequency was measured using a chronic micturition recording system and then obstruction (40 rats) or sham obstruction surgery (11 rats) was performed. After 6 weeks frequency was reassessed, followed by subcutaneous implantation of osmotic pumps to deliver 5MU, tamsulosin or vehicle for 1 week. Upon the completion of drug treatment urinary frequency was again measured and the pressor response to the alpha 1AR agonist phenylephrine was documented. Obstructed bladder mass was an average of 4.9 times greater than bladder mass in sham operated rats (p <0.001). Urinary frequency was elevated in obstructed rats with a bladder mass of greater than 500 mg vs all rats with a bladder mass of under 255 mg (p = 0.01). Of rats with a bladder mass of greater than 500 mg frequency was decreased in those treated with tamsulosin (p = 0.03) but not in those treated with 5MU. Tamsulosin and 5MU inhibited the pressor response to phenylephrine. Urinary frequency is increased in rats with a bladder mass of greater than 500 mg. The combined alpha 1a/alpha 1dAR antagonist tamsulosin decreases urinary frequency more than the alpha 1aAR selective antagonist 5MU. This finding supports the hypothesis that the alpha 1dAR is important for mediating irritative symptoms.

Pharmacokinetics of an Implanted Osmotic Pump Delivering Sufentanil for the Treatment of Chronic Pain

A matchstick-sized implanted osmotic pump (Chronogesic) that delivers sufentanil subcutaneously for more than 90 days is being developed to treat chronic pain. This study evaluates pharmacokinetic characteristics related to the absorption of sufentanil using a prototype 60-day system. Twelve opioid-naive volunteers were given naltrexone to prevent opioid effects. Sufentanil, 60 microg, was infused intravenously over 6 h, then 48 h later, the pump was implanted subcutaneously in the upper arm under local anesthesia. Pumps were removed 9 days later. In six volunteers, fever (1.6-3.3 degrees C) was induced with interleukin-2. Plasma was sampled and population pharmacokinetic modeling was performed to estimate in vivo release rate and absorption half-life. Bioavailability was calculated by comparing in vivo to in vitro release rates. The impact of perturbations in release rate on sufentanil plasma concentration (Cp) was simulated. Fever had no systematic effect on Cp. Release rate estimated in vivo was similar to that measured in vitro; bioavailability did not differ from 100%. Absorption half-life was 16.2 h. Simulation demonstrated that supplemental release of sufentanil from the implant (as might occur with local heating) increases Cp an average of 2.5-2.8% per hours supplemental dose. An implantable osmotic pump delivered sufentanil in vivo at the rate predicted from in vitro experiments. The rate at which sufentanil was absorbed from the subcutaneous space (half-life > 16 h) was markedly slower than reported with subcutaneous or intramuscular administration of large volumes of dilute opioids; this slow absorption dampens potential changes in Cp if release rate is perturbed.

Inhibition of central amylin signaling increases food intake and body adiposity in rats.

Amylin is a 37-amino acid peptide hormone that is co-secreted with insulin by pancreatic beta cells in response to feeding. We recently reported that amylin potently reduces food intake, body weight, and adiposity when delivered into the 3rd cerebral ventricle (i3vt) of rats. We have now infused i3vt a specific antagonist (AC187) to ascertain the physiological relevance of central amylin in the control of energy balance. After establishing the ability of i3vt AC187 to block the anorexic effect of i3vt amylin, we performed an experiment to examine the impact of acute inhibition of central amylin signaling on feeding. Separate groups (n = 7/group) of ad lib-fed male Long Evans rats were given one bolus i3vt infusion of synthetic cerebrospinal fluid vehicle (CSF) or AC187 (250 or 1000 pmol). Acute infusion of AC187 tended to increase 1-h food intake and significantly elevated 4-h intake. Both the 250 and 1000 pmol doses produced significant increases as compared to CSF. In another experiment designed to tonically inhibit central amylin signaling over an extended period, two other groups of rats (n = 6/group) received continuous i3vt infusion of CSF or 100 pmol/h AC187 over 14 days via implantable osmotic pumps. Rats receiving AC187 ate significantly more food over the 14-day infusion period relative to controls (CSF = 322 +/- 6 g, AC187 = 360 +/- 12 g). Although body weight was not significantly affected, body fat was increased by about 30% in the AC187 rats, with no difference in lean tissue between the groups. Additionally, although fasting plasma glucose did not differ between the CSF and AC187 groups after 14 days of infusion, plasma insulin was significantly elevated in the AC187 rats. In summary, the present results document significant increases of food intake and body adiposity resulting from inhibition of central amylin signaling. They are consistent with our hypothesis that CNS actions of endogenous amylin contribute to the long-term regulation of energy balance.

Pharmacodynamic and receptor binding changes during chronic lorazepam administration

To assess pharmacodynamic and neurochemical aspects of tolerance, lorazepam (2 mg/kg/day), or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice via implantable osmotic pump. Open-field behavior, benzodiazepine receptor binding in vitro, receptor autoradiography, and muscimol-stimulated chloride uptake were examined at both 1 and 14 days. Open-field activity was depressed in lorazepam-treated animals on Day 1. On Day 14, open-field parameters were indistinguishable from those of vehicle-treated animals, indicating behavioral tolerance. Benzodiazepine binding, as determined by the specific binding of [ 125I]diazepam, was also decreased in cortex on Day 14. Hippocampal binding was unchanged following chronic lorazepam exposure. Apparent affinity in cortical membrane preparations was unchanged, indicating that altered ligand uptake was due to decreased receptor number. Muscimol-stimulated chloride uptake into cortical synaptoneurosomes from lorazepam-treated animals was not significantly different on Day 1 or Day 14 compared to vehicle-treated animals. These results confirm that down-regulation of benzodiazepine receptor binding is closely associated with behavioral tolerance to benzodiazepines. These observed changes in binding are not necessarily associated with robust changes in receptor function.

Application of modern endocrine methods to conservation biology

Ishii, S. 1999. Application of modem endocrine methods to conservation biology. In: Adams, N.J. & Slotow, R.H. (eds) Proc. 22 Int. Ornithol. Congr., Durban. Ostrich 70 (1): 33–38. I report here on novel endocrinological methods originally developed for the artificial breeding of the Japanese Crested Ibis, Nipponia nippon. According to the most recent information. the only wild population in the world is one of about 60 individuals in Yang Xian, China. Captive populations of 54 and 17 individuals are located in Yang Xian and at Beijing Zoo, respectively and a single female of this species survives in Japan. Females of many birds stop breeding when they are brought to captivity. Hormone administration is considered to be effective in stimulating the breeding activity of such females. If the ovary of a captive female contains well-developed ova but does not ovulate, the problem is not serious. Ovulation can be induced by injection of gonadotropic hormone. However, if the ovary is regressed and contains no developed ova, the problem is serious. Until recently, no avian endocrinologist had been able to induce ovulation in hens with a regressed ovary. We developed a non-invasive method to assess the ovarian and testicular condition of birds by analysing gonadal sex steroid hormones in droppings. By using this method, we can decide whether the ovary contains developed ova or not and whether a male is sexually active or not. To reactivate a regressed ovary, we employed gonadotropin administration by means of the osmotic pump implantation in female Japanese Quail Coturnix japonica kept under short-days and then injected with the gonadotropin preparation once a day for several days. About 50% of the females laid an egg or eggs in response to the injection. Seven of the eggs were artificially incubated and two of them hatched. One of the two was revealed to be a male and fertile. The faecal sex steroid assays have been successfully applied to males of the Japanese Crested Ibis and Kakapo Strigops habroptilus in New Zealand. Although too late to apply the hormone administration method to the Japanese Crested Ibis for breeding, application of the method to the Kakapo is now being examined in New Zealand.

Obstructive Jaundice Alters Kupffer Cell Function Independent of Bacterial Translocation

Background.There is a high incidence of perioperative morbidity and mortality in patients with obstructive jaundice. The absence of bile in the gastrointestinal tract promotes bacterial overgrowth and the increased translocation of bacteria and endotoxin to the liver which has been postulated to inhibit Kupffer cell function in these patients. But, biliary tract obstruction can directly damage liver cells and thus alter their function. Thus, we hypothesized that obstructive jaundice alone alters Kupffer cell function independent of the effects of bacterial translocation. This study was designed to evaluate the contribution of bacterial translocation to the altered Kupffer cell function observed in patients with obstructive jaundice.Methods.Sprague–Dawley rats were randomized to three groups of six animals each. Group 1 underwent common bile duct ligation with intestinal bile salt replacement (sodium taurocholate 100 mg/kg/day) via gastrostomy and an implantable osmotic pump (CBDL + bile salts), Group 2 underwent common bile duct ligation with normal saline replacement (CBDL + saline), and Group 3 underwent a sham operation (sham control). After 7 days, tissue and blood were collected for bacterial translocation and biochemical analyses. Examination of cultured Kupffer cell function included measuring the phagocytosis of heat-killedCandida albicansand endotoxin (LPS)-induced TNFα and nitric oxide production.Results.While bacterial translocation and cecal bacterial counts were significantly increased in the CBDL + saline group, these parameters were both reduced to control levels following intestinal bile salt replacement (CBDL + bile salts). Altered Kupffer cell function, as measured by the increased phagocytosis ofC. albicansand LPS-induced NO production, and decreased LPS-induced TNFα production were observed in all animals with obstructive jaundice regardless of bile salt replacement.Conclusion.Kupffer cell function appears to be differentially affected by obstructive jaundice and these altered functions can occur independent of bacterial translocation.

Characterization of a prolonged regenerative attempt by diffusely injured axons following traumatic brain injury in adult cat: a light and electron microscopic immunocytochemical study.

Traumatic brain injury in animals and humans is well known to cause axonal damage diffusely scattered throughout the brain without evidence of other brain parenchymal change. This observation has prompted some to posit that such damaged axons are well positioned to mount a regenerative attempt. The present study uses an immunocytochemical marker specific for regenerating neurites to explore this issue. Further, in an attempt to expedite and enhance any potential regenerative effort, this study evaluates the efficacy of intrathecally applied nerve growth factor. Three sets of experiments were performed in adult cats. One group of animals was subjected to moderate fluid percussion brain injury and followed for 7 or 14 days post injury, with the continuous intraventricular infusion of nerve growth factor delivered by implanted osmotic pumps. These animals were compared to a second group of time-matched, sham-operated animals receiving artificial cerebrospinal fluid infusion. To assess axonal damage immunohistochemical staining for the low molecular weight neurofilament subunit (NF-L) was carried out using an NR4 monoclonal antibody. To localize axons exhibiting a regenerative response immunohistochemical staining for the growth associated protein GAP43 was employed. In sham controls, at the light microscopic level NF-L-immunoreactive axonal swellings were numerous at 7 days, but by 14 days post injury their frequency declined markedly. In contrast, GAP43-immunoreactive, disconnected reactive axonal swellings were rarely observed at 7 days but were numerous at 14 days. Ultrastructural analysis at 14 days post injury of carefully matched sections revealed reactive axons demonstrating sprouting consistent with a regenerative effort. Analysis of tissue from animals of 14 days of survival indicated that supplementation with nerve growth factor did not appear to enhance the capacity of damaged brain axons to mount a regenerative attempt. Rather, it appears that regenerative efforts seen reflect a spontaneous response. A third group of adult cats, subjected to the same injury but not subjected to osmotic pump implantation, was allowed to survive for 22-28 days. Animals in this group also demonstrated GAP43 immunoreactivity in reactive axonal swellings in the brain stem. This study demonstrates that diffusely injured axons can mount a sustained regenerative attempt that is associated with a reorganization of their cytoskeleton and accompanied by an up-regulation of growth-associated proteins.

Differential Effects of Nonselective Nitric Oxide Synthase (NOS) and Selective Inducible NOS Inhibition on Hepatic Necrosis, Apoptosis, ICAM-1 Expression, and Neutrophil Accumulation during Endotoxemia

The roles of nitric oxide derived from either the constitutive endothelial NO synthase (eNOS or NOS3) or the inducible NOS (iNOS or NOS2) in hepatic injury during endotoxemia remain controversial. To investigate this further, rats received a bolus of lipopolysaccharide (LPS) following implantation of osmotic pumps containing one of two nonselective NOS inhibitors (NMA or NAME), one of two inducible NOS inhibitors (NIL or AG), or saline. The inhibitors were infused continuously into the liver via the portal vein. Treatment of LPS-injected rats with NMA and NAME resulted in 106 and 227% increases, respectively, in circulating hepatic enzyme levels compared to LPS-treated control rats. In contrast, infusion of the iNOS-selective inhibitors had no effect on the LPS-induced hepatic necrosis. In rats receiving NAME, LPS induced greater neutrophil infiltration and ICAM-1 expression than in the LPS + saline group, whereas NIL infusion did not. The increased hepatic necrosis and PMN infiltration in the LPS + NAME group was partially prevented by a simultaneous infusion of a liver-selective NO donor. Inhibition of PMN accumulation using an anti-ICAM-1 antibody or by PMN depletion using vinblastine pretreatment, however, did not reverse the increased necrosis with NAME infusion during endotoxemia. In contrast to the assessment for necrosis, increased apoptosis was observed in the livers of LPS-treated rats receiving infusions of either NAME or NIL, but not with LPS alone. These data indicate that NO produced by eNOS may be adequate to prevent necrosis by a mechanism independent of PMN, while induced NO appears to prevent apoptosis.

Concomitant infusion of ovine corticotropin-releasing hormone does not prevent suppression of the hypothalamus-pituitary-adrenal axis by dexamethasone in male rats.

Glucocorticoids (GCs) suppress the hypothalamus-pituitary-adrenal (HPA) axis at various sites including hypothalamus, pituitary and extrahypothalamic brain. Previous studies have shown that corticotropin-releasing hormone (CRH) and vasopressin facilitate the recovery of HPA axis suppressed by GCs. In this study, we investigate whether the concomitant continuous infusion of CRH may prevent the suppression of HPA axis by GCs. Groups of male Wistar rats weighing 140 to 160 g were implanted subcutaneously with Alzet osmotic pump for delivery of dexamethasone (DEX), 2 micrograms/h and/or CRH, 0.66 microgram/h. Control rats were implanted with sialistic tube of similar size. Rats were decapitated 3 or 7 days after osmotic pump implantation. In spite of the suppression of plasma corticosterone, the body weight (BW), adrenal weight (AW), plasma corticotropin (ACTH) and pituitary ACTH content of rats treated with DEX for 3 days were not significantly different from those of control rats. Concomitant infusion of ovine CRH (oCRH) and DEX for 3 days caused impaired BW gain, adrenal atrophy in addition to further reduction of plasma corticosterone. Treatment with DEX and/or oCRH for 7 days caused further suppression of HPA axis as shown by reduced pituitary ACTH content. In conclusion, simultaneous infusion of oCRH and DEX does not prevent and may even worsen HPA axis suppression by DEX.

A new model of delayed healing of acetic acid ulcers in rats by indomethacin via osmotic pump

Gastric ulcers were produced in male Donryu rats (8-weeks-old) by subserosal injection of 20 microliters of 20% acetic acid. Five days later, an indomethacin (IND)-filled osmotic pump (OP) was implanted into the dorsal subcutaneous space in the rats and maintained for 2, 3 or 4 weeks. The natural healing of the acetic acid-induced ulcers was significantly (P < 0.05) delayed by administration of IND via OP implanted for 4 weeks. Two, 3 and 4 weeks after OP implantation, the plasma IND levels was 1.87 +/- 0.13, 2.43 +/- 0.16 and 0.74 +/- 0.26 (micrograms/ml), respectively, showing that a steady state IND plasma level was maintained for 3 weeks. The gastric mucosal PGE2 levels in rats with ulcers for 3 weeks after implantation of OP were significantly (P < 0.05) reduced by IND (IND(-):576.6 +/- 83.9 pg/mg, IND(+):355.6 +/- 34.7 pg/mg). Repeated administration of enprostil, famotidine and rabeprazole at 25 micrograms/kg, 100 mg/kg and 10 mg/kg (p.o., b.i.d.), respectively, showed anti-ulcer activity. Teprenone and rebamipide had only slight anti-ulcer activity. These findings indicated that this model of delayed healing of acetic acid ulcers by IND using OP is useful for investigating the anti-ulcer activity of drugs.

Local Extravascular Growth Factor Delivery in Myocardial Ischemia.

A number of angiogenic growth factors have been demonstrated in vivo to promote angiogenesis in ischemic myocardium, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). We used the porcine ameroid constrictor model to simulate chronic ischemia in an effort to study the role of exogenous growth factor delivery in the development of coronary collateral circulation. Heparin alginate microspheres were used to deliver bFGF, while an implantable osmotic pump was used for VEGF delivery. Anti-von Willebrand antibody staining was used to visualize microvessels in the porcine heart sections obtained from both ischemic and non-ischemic myocardium. A significant increase in the number of microvessels in growth factor-treated pigs compared to control animals was noted. Myocardial blood flow was used to determine the physiologic impact of these findings. In bFGF and VEGF treated animals resting collateral flow values significantly exceeded those of the control group. In addition, both bFGF and VEGF administration resulted in improvements in myocardial perfusion sufficient to prevent stress-induced deterioration of myocardial performance. Results of the studies demonstrate that local perivascular drug delivery can be effectively used to induce angiogenesis in chronically ischemic myocardium.

The effect of implantation of osmotic pumps on rat thyroid hormone and testosterone levels in the plasma, an implication for tissue ‘Srs phase studies

In order to monitor the effect of the procedures required to s.c implant osmotic pumps into rats on plasma thyroid and testosterone hormone levels, male Fischer 344 rats (8–10 weeks old) were divided into six groups of 10 rats and the groups treated in the following manner: (1) controls housed 5 per cage; (2) controls housed individually; (3) animals anaesthetised for surgery and individually housed; (4) anaesthetised, sham operated and individually housed; (5) anaesthetised, s.c implanted with osmotic pumps containing saline and individually housed; (6) anaesthetised, s.c implanted with osmotic pumps containing 5-bromo 2-deoxyuridine (BRDU) and individually housed. Four days after performing the surgery the study was terminated and the level of hormones in the plasma determined by radio immuno assay (RIA). Tri-iodothyronine (T 3) and thyroxine (T 4) plasma levels (free and total) were significantly decreased with each additional step in the procedure used for the s.c implantation of an osmotic pump containing BRDU, when compared with the individually housed controls. Similarly, testosterone plasma levels were significantly decreased by the s.c implantation of osmotic pumps, implying a ‘stress’ response might occur following implantation. These observations might need to be considered by investigators when performing texicological research which, as part of the study, uses osmotic pumps for the delivery of the nucleotide precursor required for monitoring cells in ‘S’ phase.