Saline Animals Research Articles

Chronic low-dose administration of nicotine facilitates recovery and synaptic change after focal ischemia in rats

The current study examines the effects of chronic administration of nicotine on motor behavior after focal stroke in rats. Animals were trained in a tray-reaching task for 2 weeks and then they were divided into: (1) control + saline (2) control + nicotine (3) stroke + saline, and (4) stroke + nicotine groups. Lesions were produced by devascularization of the surface blood vessels of the sensorimotor cortex contralateral to the forepaw used for reaching. Forty-eight hours after the lesions, and for a total of 12 days, animals received two daily injections of either nicotine (0.3 mg/kg) or saline (0.9%). Animals were tested in a motor battery 1 week after the lesions and every other week for a total of 7 weeks. Pyramidal cells in forelimb and cingulate areas were then examined for dendritic length and branching using a Golgi–Cox procedure. Behavioral results demonstrated that by the end of the testing stroke + nicotine animals showed significant behavioral improvement relative to stroke + saline animals. Stroke + nicotine animals showed an increase in dendritic length and branching in pyramidal cells of the forelimb and cingulate areas. The results suggest that the behavioral enhancement in the stroke + nicotine group might be attributable to the enhanced dendritic growth in residual cortical motor regions.

Transient Disturbance in Physeal Morphology Is Associated With Long-Term Effects of Nitrogen-Containing Bisphosphonates in Growing Rabbits

Bisphosphonates have clinical benefit in children with severe osteogenesis imperfecta or osteoporosis and potential benefit in children with Perthes disease or undergoing distraction osteogenesis. However, there is concern about the effects of bisphosphonates on the physis and bone length. In 44 growing rabbits, zoledronic acid caused a transient disruption of physeal morphology, retention of cartilaginous matrix in trabeculae and cortical bone of the metaphysis, and a minor decrement in tibial bone length at maturity. Data from growing animal models suggest that bisphosphonates cause retention of longitudinal cartilaginous septa at the chondro-osseous junction, extension of trabeculae to the metaphyseal-diaphyseal junction, and varying dose-dependent effects on longitudinal growth. However, there is a lack of data regarding effects of intermittent use of nitrogen-containing bisphosphonates on the physis and on tibial length in models reaching maturity. Contralateral tibias of juvenile rabbits were examined after right tibial distraction osteogenesis from two previous studies. Animals were randomized to receive 0.1 mg/kg zoledronic acid (ZA) IV at 8 weeks of age (ZA*1) or 8 and 10 weeks of age (ZA*2) or saline. Body mass was analyzed from 5 to 44 weeks of age; tibial length and proximal physeal-metaphyseal histology and histomorphometry were analyzed at 8-52 weeks of age. Tibial length was 3% less at 14 weeks of age in the ZA*2-treated versus saline group (p<0.05) in both studies, and this difference persisted at maturity in the long-term study group (26 weeks of age, p<0.05). Total body mass gain from 5 to 26 weeks of age was 14% less in ZA*2-treated than saline animals (p<0.05). Rate of weight gain from 8 to 10 weeks of age was 76% less in ZA*2 compared with saline animals (p<0.05). Radiographs showed radiodense lines in the metaphyses of ZA-treated bones, corresponding to the number of doses. Histologically, lines resulting from the first dose of ZA contained longitudinal cartilaginous matrix cores surrounded by bone, whereas those from the second dose contained spherical cores of matrix caused by transient disruption of physeal morphology after the first dose of ZA. Resorption of these lines at later times was radiographically and histologically evident, but remnants of cartilaginous matrix remained in the cortical bone of ZA-treated animals. ZA treatment within the final 13.5% of the rabbit tibial growth period caused a transient disruption in physeal morphology and resorption associated with retention of cartilaginous matrix and coinciding with a persistent 3% decrement in tibial length. Disruption of physeal morphology and potential loss of bone length should be considered when administering nitrogen-containing bisphosphonates to children before closure of the major physes.

Prolonged daily exposure to IV cocaine results in tolerance to its stimulant effects

We have previously shown that 1 h, but not 6 h, of daily access to IV cocaine induces a sensitized response to IV cocaine challenge after 14 days of withdrawal. Here we tried to replicate these results using an IP cocaine challenge and adding a group of animals that had 1 h daily access to cocaine, but maintained levels of administration comparable to that of saline animals (i.e. a Coc group). Since addiction-associated neuroadaptations are particularly long lasting, we also tested the response to cocaine challenge after a longer withdrawal period of 60 days. Rats had daily access to IV self-administered saline or cocaine for 1 h (Coc1h), or to cocaine for 6 h (Coc6h) over 8 days. Subsequently, after 14 days of withdrawal only Coc animals showed a sensitized locomotor response to cocaine challenge administered IP. After 60 days of withdrawal, IP cocaine failed to produce a sensitized response in Coc1h animals and produced a tolerant response in Coc6h animals. The present data support the notion that 6 h of daily access to cocaine leads to different neuroadaptations than those resulting from 1 h of daily access to the drug. In addition, these data further demonstrate a dissociation between sensitization and addiction to cocaine.

FK506 reduces tissue damage and prevents functional deficit after spinal cord injury in the rat

We examined the efficacy of FK 506 in reducing tissue damage after spinal cord injury in comparison to methylprednisolone (MP) treatment. Rats were subjected to a photochemical injury (T8) and were given a bolus of MP (30 mg/kg), FK 506 (2 mg/kg), or saline. An additional group received an initial bolus of FK 506 (2 mg/kg) followed by daily injections (0.2 mg/kg intraperitoneally). Functional recovery was evaluated using open-field walking, inclined plane tests, motor evoked potentials (MEPs), and the H-reflex response during 14 days postoperation (dpo). Tissue sparing and glial fibrillary acidic protein (GFAP), biotinylated tomato lectin LEC, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and interleukin 1 beta (IL-1 beta) immunoreactivity were quantified in the injured spinal cord. FK 506-treated animals demonstrated significantly better neurologic outcome, higher MEP amplitudes, and lower H-wave amplitude compared to that of saline-treated rats. In contrast, administration of MP did not result in significant differences with respect to the saline-treated group. Histologic examination revealed that tissue sparing was largest in FK 506-treated compared to saline and MP-treated animals. GFAP and COX-2 reactivity was decreased in animals treated with FK 506 compared to that in animals given MP or saline, whereas IL-1 beta expression was similarly reduced in both FK 506- and MP-treated groups. Microglia/macrophage response was reduced in FK 506 and MP-injected animals at 3 dpo, but only in MP-treated animals at 7 dpo with respect to saline-injected rats. Repeated administrations of FK 506 improved functional and histologic results to a greater degree than did a single bolus of FK 506. The results indicate that FK 506 administration protects the damaged spinal cord and should be considered as potential therapy for treating spinal cord injuries.

Delayed Menarche Causes A Transient Reduction In Cancellous Bone Area In Female Rats

Optimizing peak bone mass during late adolescence may help reduce the effects of osteoporosis. Multiple factors affect the structural development of the skeleton; including estrogen levels during growth which are an important factor in the pathogenesis of bone fragility. The delay of menarche decreases estrogen levels during adolescence and subsequent peak bone mass. Therefore, low estrogen levels during bone development decrease peak bone mass accrual in young adults and may lead to an increased risk of fracture later in life. PURPOSE The investigative hypothesis predicts that administration of a GnRH antagonist initiated prior to the onset of the first estrus cycle will reduce estrogen levels and impede the development of cancellous bone area in the proximal tibial metaphysis of female rats (6 weeks) and the peak bone area at maturity (6 months). METHODS Twenty-five day old female Sprague Dawley rats were randomly assigned into short-term control (C-ST) (n = 12), long-term control (C-LT) (n = 12), short-term GnRH antagonist (GnRH-a ST) (n = 12) and long-term GnRH antagonist groups (GnRH-a LT) (n = 12). Injections (0.2 ml) were given intraperitoneally for 18 days of either saline or GnRH antagonist (100 ug/day) and animals were monitored daily for vaginal opening. Both short-term and long-term groups received the GnRH antagonist for 18 days. However, the short-term groups were sacrificed after the last injection (day 42) and the long-term groups were sacrificed at 6 months of age. Uterine weights were measured. Serum Estradiol levels were assayed. Bone area (%) was measured at the proximal tibial metaphysis. Differences were detected using Students t-test. The Institutional Animal Care and Use Committee at Brooklyn College approved the study. RESULTS Day of vaginal opening was delayed 22% in the short term GnRH-a group and 30% in the long-term group. The delay in pubertal development was also confirmed by decreased uterine weights (74 %) in the GnRH-a ST group, uterine weights were normal in the GnRH-a LT group. GnRH-a ST estrogen levels were suppressed after the injection period (38.1 (13.6) pg/mL v 26.8 (16.6) pg/mL). Bone area was decreased 16.5% (p = 0.05) in the short-term groups but was not significantly different at 6 months of age. CONCLUSION GnRH antagonist given to animals prior to the first estrus cycle significantly decreased the bone area in the proximal tibial metaphysis at 6 weeks but not at 6 months of age. Delayed vaginal openings and decreased uterine weights in the GnRH-a ST group resulted in a reduction in the level of available estrogen. Supported by NIH R15 AG19654-01A1and PSC-CUNY 64293-00 33

Methylprednisolone Inhibits Production of Interleukin-1?? and Interleukin-6 in the Spinal Cord Following Compression Injury in Rats

Interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are major inflammatory cytokines produced after spinal cord injury (SCI). This study sought to evaluate the effects of methylprednisolone (MP) on IL-1beta and IL-6 protein in spinal cord tissue following SCI. Halothane-anesthetized, female Sprague-Dawley rats weighing (280-320 g) underwent laminectomy at T7-T8. No lesions were produced in animals in the saline control and MP control groups. SCI was induced by temporary placement of an aneurysm clip at T7-T8, with a closing pressure of 55 g at the spinal level of T7-T8, resulting in spinal cord compression for one minute. Animals with SCI were treated with MP (30 mg/kg sc) or an equal volume of saline. IL-1beta and IL-6 spinal cord protein were measured by enzyme-linked immunosorbent assays (ELISA). Data were summarized as mean +/- SD and compared by two-way analysis of variance (ANOVA). IL-1beta and IL-6 levels were elevated in the SCI + Saline animals (P < 0.01) compared with saline control, MP control, and SCI + MP-treated animals. The rise in IL-1beta and IL-6 levels after SCI was blunted after administration of MP, suggesting an interaction between glucocorticosteroids and the cytokine cascade after spinal cord trauma. Further evaluation of the effects of MP on the cytokine cascade may be important in assessing whether or not the anti-inflammatory effects of glucocorticosteroids confer neuroprotection after SCI.

Monocyte Chemoattractant Protein 1-Induced Monocyte Infiltration Produces Angiogenesis but Not Arteriogenesis in Chronically Infarcted Myocardium

Monocyte chemoattractant protein 1 (MCP-1) stimulates the invasion of monocytes into ischemic tissue with concomitant adhesion to endothelial cells. Monocyte stimulation has been shown to be involved in the induction of arteriogenesis, which is the development of functional arterioles resulting in improvement of perfusion. However, angiogenesis (newly developed capillaries contribute to improved tissue perfusion) in several models has not resulted in any improvement in blood flow. The effects of MCP-1 on potential angiogenesis and arteriogenesis as well as changes in left ventricular function were tested in a chronic infarct model in rat hearts. Anesthetized rats were subjected to open-chest ligation of the left coronary artery with subsequent myocardial infarction. After 6 weeks, animals were randomized to receive either MCP-1 (3 microL in 0.15 mL NaCl, group 1, n = 9) or saline (0.15 mL, group 2, n = 9), which was injected into the myocardium at the border zones of the infarcts. For assessment of left ventricular dimensions and global cardiac function, transthoracic two-dimensional echocardiography was performed at baseline, 6 weeks after myocardial infarction, and 4 weeks after MCP-1 or saline injection, by use of a 12-MHz pediatric transducer. For light microscopic analysis, myocardial tissue was stained with Elastica-van-Giesson and von Willebrand factor for blood vessels and endothelial cells, respectively. In a subset of animals, hearts were excised 24 hours after MCP-1 administration (n = 4) or saline administration (n = 4) for assessment of monocyte infiltration by immunohistologic staining of the CD31 antigen. Left ventricular dimensions and ejection fraction changed after coronary occlusion (from 60.4% +/- 2.85% to 24.8% +/- 5.01% ejection fraction in group 1, and from 58.4% +/-2.06% to 26.3% +/- 4.3% ejection fraction in group 2 at 6 weeks, P < .005) without any further change 4 weeks after treatment (ejection fraction in group 1, 26.3% +/- 2.7%, ejection fraction in group 2, 25.0% +/- 5.18%). The MCP-1 group resulted in 390.6 +/- 10.36 endothelial cells compared with 285.2 +/- 13.56 in group 2 (P < .005) at the injection site. Monocyte infiltration was observed at the MCP-1 injection site with an increase in capillary growth (angiogenesis). However, there was no difference in the number of arteriolar structures between animals treated with MCP-1 and saline animals (group 1, 19.0 +/- 1.52 vs group 2, 16.4 +/- 0.68, P > .05). A single intramyocardial injection of MCP-1 into the infarct border zone resulted in neo-angiogenesis and monocyte infiltration but not arteriogenesis in the rat heart. There was no functional change of chronically infarcted myocardium in the present model.

The effects of topical and injected mitomycin c on wound healing

Objectives: To determine the effect of topical and injected Mitomycin C (MMC) on skin wound healing by examining wound strength and histology. Methods: Paramedian incisions were placed on the back of each of 18 adult male Sprague-Dawley rats. The wounds were treated with topical MMC, intradermal MMC (0.5 mg/mL), or normal saline and closed with staples. Two animals in each study arm were sacrificed at 1 and 2 weeks, and 1 and 6 months. After wound harvest 1 section was reserved histologic examination (blinded) with H & E staining and TFG-B1-specific immunohistochemical staining. Using a tensiometer, the remaining wound sections were used to evaluate the force needed to cause wound dehiscence. Kruskal-Wallis and Mann-Whitney tests were used for statistical comparison. Results: Application of topical MMC resulted in decreased wound integrity compared with saline-treated animals at 1 week ( P < 0.001), 2 weeks ( P < 0.001), and 1 month ( P < 0.001). Intradermal MMC showed decreased wound integrity 1 week ( P < 0.001), 2 weeks ( P > 0.001), and 1 month ( P > 0.001). (Six month data pending.) Small areas of skin necrosis occurred in 7 of 8 animals receiving intradermal MMC, and no animals in the other groups. There was no consistent difference in fibroblast proliferation or concentration of TGF-B1 between saline and MMC-treated animals. Conclusions: Intradermal MMC can cause skin necrosis. MMC-treated wounds had decreased wound strength compared to controls. Despite the wound strength changes, no consistently identifiable histological changes in fibroblast proliferation or TGF-B1 expression was noted. This data suggest cautious use of MCC in clinical situations when wound breaking strength is critical.

Immune cell activity during the initial stages of withdrawal from chronic exposure to cocaine or morphine

The immunosuppression accompanying illicit drug use has been shown to contribute to a decreased resistance to a variety of pathogens; however, there is relatively little information on how long these effects persist following withdrawal from chronic drug exposure. To begin to address this question, Sprague–Dawley male rats were administered either cocaine (10 mg/kg, i.p., b.i.d.) for 7 days or morphine (escalating doses up to 40 mg/kg, s.c., b.i.d.) for a 10-day period. Control groups of animals received similar saline injections for equivalent time periods. Drug administration was abruptly discontinued and animals were sacrificed at 2, 24, 72 or 96 h following the last dose. At these time points, proliferation responses of peripheral blood T-lymphocytes stimulated by concanavalin A (Con A) and plasma levels of corticosterone were measured. Plasma corticosterone levels of cocaine- or morphine-treated animals were found to be significantly elevated 24 h following drug cessation as compared to saline animals. At this time, proliferation responses were significantly decreased and were further suppressed during cocaine and morphine withdrawal at 96 and 72 h, respectively. These results suggest that abrupt cessation of cocaine or morphine administration leads to activation of stress-related pathways that may contribute to an increased susceptibility of infection during the initial withdrawal phase.

Effects of adrenocorticotropin stimulation on cortisol dynamics of pregnant gilts and their fetuses: implications for prenatal stress studies

The effect of adrenocorticotropic hormone (ACTH) administration on plasma cortisol concentrations was determined in pregnant gilts and their fetuses. In a first experiment, 100 IU ACTH (Synacthen ® Depot) was administered intramuscularly to the gilts every second day from Days 49 to 75 of gestation. ACTH injections were carried out at 08:00 h and, thereafter, 10 blood samples were taken within the following 8 h via jugular catheters. Blood samples were analysed for plasma cortisol concentrations, and results were compared with values from animals which were treated with physiological saline and untreated animals (blood sampling only). The values for plasma cortisol concentrations increased until 3 h after ACTH applications to a mean maximum level of 276.5±17.2 nmol/l in the whole 4-week stimulation period. Plasma cortisol levels did not return to pre-treatment values within the 8 h post-injection. No differences in cortisol levels were found between the physiological saline and untreated control, and no habituation of the adrenocortical response to ACTH was found during the 4-week stimulation period. In a second experiment, 100 IU ACTH were administered to pregnant gilts at gestation Day 65. After 3 h, fetuses were recovered under general anaesthesia and blood samples were taken from the umbilical vein, artery, and, after decapitation, from periphery. Application of ACTH to the sows significantly increased their plasma cortisol concentrations ( P<0.001), and also increased plasma cortisol concentrations in peripheral blood samples from the fetuses ( P=0.09) and in the umbilical vein ( P<0.001) and artery ( P<0.01), respectively. Plasma ACTH concentrations did not differ in fetuses from ACTH-treated or control sows. The results show that in gilts the adrenocortical response to an exogenous application of Synacthen ® Depot is consistent over time during mid-gestation. Furthermore, cortisol but not ACTH levels were increased in fetuses from ACTH-treated sows, indicating that maternal cortisol can cross the placenta during mid-gestation. The stimulation of maternal cortisol release through exogenous ACTH with subsequent elevation of fetal cortisol levels is, therefore, a useful approach for studying effects of elevated maternal glucocorticoids in prenatal stress studies in pigs.

Chronically infused intrathecal morphine in dogs.

Despite the extensive use of intrathecal morphine infusion for pain, no systematic safety studies exist on its effects in high concentrations. The authors assessed the effects of morphine and clonidine given 28 days intrathecally in dogs. Beagles with lumbar intrathecal catheters received solutions delivered by a vest-mounted infusion pump. Six groups (n = 3 each) received infusions (40 microl/h) of saline or 1.5, 3, 6, 9, or 12 mg/day of morphine for 28 days. Additional groups received morphine at 40 microl/h (1.5 mg/day) plus clonidine (0.25-1.0 mg/day) or clonidine alone at 100 microg/h (4.8 mg/day). In animals receiving 9 or 12 mg/day morphine, allodynia was observed shortly after initiation of infusion. A concentration-dependent increase in hind limb dysfunction evolved over the infusion interval. Necropsy revealed minimal reactions in saline animals. At the higher morphine concentrations (all dogs receiving 12 mg/day), there was a local inflammatory mass at the catheter tip that produced significant local tissue compression. All animals with motor dysfunction displayed masses, although all animals with masses did not show motor dysfunction. The mass, arising from the dura-arachnoid layer, consisted of multifocal accumulations of neutrophils, monocytes, macrophages, and plasma cells. Inflammatory cells and endothelial cells displayed significant IL1beta, TNFalpha, iNOS, and eNOS immunoreactivity. No evidence of bacterial or fungal involvement was detected. There were no other changes in spinal morphologic characteristics. In four other groups of dogs, clonidine alone had no effect and in combination with morphine reduced the morphine reaction. The authors found that high intrathecal morphine concentrations lead to aseptic intrathecal inflammatory masses. The lack of effect of clonidine and the possible suppressive effects of clonidine on the local reaction suggest the utility of such coadministration.

Eicosapentaenoic acid supplementation reduces tumor volume and attenuates cachexia in a rat model of progressive non-metastasizing malignancy.

Eicosapentaenoic acid (EPA) has been shown to have anti-inflammatory and tumor growth inhibitory effects clinically and experimentally; evidence also supports the role of EPA in attenuating cancer-associated weight loss, but the mechanisms of these effects remain to be defined. As the liver plays a central role in modulating nutritional status and the cachexia syndrome, we examined the liver and nutritional parameters indicative of cachexia along with the tumor volume in response to oral EPA supplementation in a rat model of progressive non-metastasizing malignancy. Fischer 344 rats implanted with the methylcholanthrene-induced fibrosarcoma (MCA) were trained to meal-feed with access to food from 8:00 PM to 8:00 AM and water ad libitum. On day 13, rats were randomly divided into 3 study groups: (1) 5.0 g/kg per day EPA plus 10 IU vitamin E/g fat, (2) 5.0 g/kg per day corn oil plus 10 IU vitamin E/g fat, and (3) 5.0 g/kg per day saline plus 10 IU vitamin E/g saline. The treatment was delivered via oral gavage twice daily. The animals were killed on day 29, and serum plus tissues (ie, liver and lung) were collected and frozen for analysis. Parameters evaluated include the following: tumor volume, weight loss, liver weight, total liver protein, liver lipid content, serum albumin content, and macrophage inflammatory protein-2 (MIP-2) levels. EPA-treated rats showed a reduction in tumor volume compared with corn oil (25% reduction, p < .01) and saline (33% reduction, p < .01) animals. EPA rats also demonstrated increased liver weight (p < .01) and total liver protein levels (p < .03) over saline-treated animals. EPA- and corn oil-treated rats received more calories than the saline group because of the dietary fat treatments (p < .01) and had elevated lipid content in their livers (p = .05 and p = .04, respectively) compared with saline rats. Serum albumin (a marker of liver function) and MIP-2 levels (a marker of the hepatic acute phase response) were not different between treatment groups. EPA supplementation resulted in a dramatic reduction of tumor volume and mild improvements in weight maintenance. In addition, EPA-treated animals demonstrated increased total liver protein and serum protein levels. Regression analyses showed that the weight and protein differences between treatment groups were not correlated with individual tumor volumes. The increase in liver and serum protein was not explained by differences in albumin or MIP-2. We conclude that the tumor growth inhibitory effects and anticachexiogenic effects of EPA are independent phenomena, and the effects on cachexia may be related to increased levels of undefined protein(s) in the liver and serum. To our knowledge, this is the first study to demonstrate the effects of EPA in the MCA fibrosarcoma model and is also novel in its evaluation of EPA as an anticachexiogenic therapy in progressive non-metastasizing malignancy. Further studies may identify the protein(s) elevated in the liver and the mechanisms for the development of EPA nutritional therapies for the treatment of progressive malignancies.

Omega-3 fatty acid supplementation reduces tumor growth and vascular endothelial growth factor expression in a model of progressive non-metastasizing malignancy.

Omega-3 fatty acids, the principal component of fish oil, have been demonstrated to have antiinflammatory properties. The role of eicosapentaenoic acid (EPA) supplementation for cancer patients is currently under investigation; however, the mechanisms of EPA activity have not been defined. The purpose of this study was to characterize tumor-specific and treatment-specific effects of supplemental dietary EPA in an animal model of progressive malignancy. Fischer 344 rats (200-250 g) underwent flank implantation of the methycholanthrene (MCA)-induced fibrosarcoma on day 0. Rats were randomly divided into 3 treatment groups on day 13: EPA (1 mL, 5.0 g/kg per day) + 10 IU vitamin E; corn oil (1 mL) + 10 IU vitamin E, and saline (1 mL) + 10 IU vitamin E (vitamin E was used to prevent fatty acid oxidation). On day 14, gavage feeding was started and was continued through day 28. The animals were killed on day 29, and the tumors were removed. The tumors were weighed and divided by the tumor-free carcass weight to obtain percentage of tumor volume, and the livers were flash frozen. Vascular endothelial growth factor-alpha (VEGF-alpha) and cyclo-oxygenase 2 (COX-2) mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR). EPA rats had significant reductions in tumor volume compared with isocaloric corn oil and control saline animals (25%, p < .01 and 33%, p < .01, respectively). Rats receiving EPA demonstrated decreased VEGF-alpha mRNA levels (0.023 +/- 0 0.001) compared with those receiving corn oil (0.129 +/- 0.047) or saline (0.150 +/- 0.026; p < .05). These data demonstrate that EPA supplementation inhibits tumor growth, potentially through alterations in the expression of the pro-angiogenic VEGF-alpha. The mechanism(s) of EPA as an inhibitor of tumor-related angiogenic growth factors may be associated with COX-2 enzyme fatty acid metabolism and merits further study.

A simple procedure for assessing ataxia in rats: Effects of phencyclidine

The present study describes an objective, cost- and time-efficient procedure for characterizing the ataxic effects of psychoactive drugs. Male Sprague–Dawley rats were administered an intraperitoneal injection of either saline or one of three doses (1, 5 or 10 mg/kg) of phencyclidine (PCP) 15 min prior to being placed into an empty standard operant conditioning chamber (all manipulanda were removed). The floor of the test apparatus consisted of parallel rows of metal rods spaced approximately 1.5 cm apart. During a 5-min test, a single observer counted the frequency with which each animal's paws (front or back) slipped between the rows of bars that constituted the cage floor. The data demonstrated that while saline animals exhibited no instability in their ambulation, PCP-treated animals demonstrated a highly reliable dose-dependent increase in the number of “paw slips” in a single trial. Since animals are known to develop tolerance to the ataxic response to PCP, the validity of the test as a measure of drug-induced ataxia was examined in a separate group of animals treated with the middle (5 mg/kg) dose every other day over the course of a 9-day period (i.e., resulting in five injection trials). In this experiment, each subsequent test produced a reliable reduction in the magnitude of the ataxic response, and by the fifth drug challenge, the PCP animals were performing at near-control levels. These results suggest that the “paw slip test” can serve as a simple, reliable, objective and valid measure of drug-induced ataxia. The relevance of the ataxia data for interpreting the locomotor response of animals treated with PCP is also discussed.

Increased conditioned fear response and altered balance of dopamine in the shell and core of the nucleus accumbens during amphetamine withdrawal

It has been suggested that neuroadaptations within the nucleus accumbens (NAC) dopaminergic (DA) projection contribute to the negative affect associated with psychostimulant withdrawal. The present study assessed the effects of amphetamine (AMPH) withdrawal on behavioral and NAC DA responses to conditioned fear stress. Animals injected with escalating-dose AMPH (1–5mg/kg, three injections/day, 6 days) or saline (SAL) acquired a tone-shock association on withdrawal day 3 and were tested for extinction of conditioned freezing to the tone on withdrawal day 4. Extracellular levels of NAC shell and core DA were monitored using in vivo microdialysis on both days. AMPH-withdrawn animals exhibited more conditioned freezing than SAL animals during both acquisition and extinction. During acquisition, DA increased more in the shell than the core of the NAC in both AMPH and SAL groups. During extinction to the tone, shell DA increased in SAL- but not AMPH-treated animals, whereas core DA activity was greater in AMPH than SAL animals. These data demonstrate that AMPH withdrawal alters the balance between shell and core DA transmission while increasing the behavioral expression of conditioned fear. Such drug-induced neuroadaptations in the NAC stress response may be involved in the exacerbation of negative emotions associated with drug withdrawal and stimulant-induced psychosis.

Increasing Local Levels of IGF-I mRNA Expression Using Adenoviral Vectors Does Not Alter Oligodendrocyte Remyelination in the CNS of Aged Rats

IGF-I, a growth factor that contributes to developmental myelination, shows increased levels of expression within experimental models of remyelination. The pattern of IGF-I mRNA expression changes with the rate of remyelination, with peak levels of expression occurring earlier during rapid remyelination in young adult rats compared to the slower remyelination in old adult rats. In this study we have attempted to accelerate remyelination in old adult rats by using an IGF-expressing adenoviral vector (IGF-I-Ad) to bring forward the timing of peak level of IGF-I expression. Following injection of IGF-I-Ad into focal areas of lysolecithin-induced demyelination in the spinal white matter of old adult rats we created levels of IGF-I mRNA expression at 10 days that were considerably higher than those normally occurring at this time and more similar to those in young animals. However, despite the elevated levels of IGF-I mRNA expression there was no significant change in the extent of oligodendrocyte remyelination compared to saline controls or animals injected with an adenoviral vector expressing LacZ (NT-LacZ-Ad). There was a small increase in Schwann cell remyelination in IGF-I-Ad- and NT-LacZ-Ad-injected animals compared to saline controls. These results indicate that changing the levels of IGF-I directly within demyelinating lesions undergoing remyelination is not sufficient to alter remyelination and that the proremyelinating effects of systemically delivered IGF-I are unlikely to be due to direct effects on the oligodendrocyte lineage.

Insulin-like growth factor-1 does not ameliorate the age-related decline in presumptive inhibitory synapses in layer 2 of rat sensorimotor cortex

Four old (29 months) Brown Norway×Fischer 344 (BN×F344) rats received intracerebroventricular infusion of insulin-like growth factor-1 (IGF-1) and four middle-aged (18 months) and four old (29 months) rats received infusion of saline for 28 days. Sensorimotor cortex containing Layer 2 was blocked and processed for electron microscopy. Thin (700 Å) and semithin (1 μm) sections were collected from the same anatomical space for quantification of synapses and neurons, respectively, using the physical disector. Numerical density (Nv) of presumptive inhibitory synapses in Layer 2 of sensorimotor cortex has been reported to decline with age Poe et al., 2001, Brunso-Bechtold et al. [Brain Res. 872 (2000) 125]. Infusion of IGF-1 did not affect the density of synapses or neurons when old IGF-1 animals were compared with old saline animals.

Effects of Stachybotrys chartarum on Surfactant Convertase Activity in Juvenile Mice

We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores, both in vitro and in an in vivo juvenile mouse model. In mice, this sensitivity is manifest in part as a significant increase in the newly secreted, biologically active, heavy aggregate form of alveolar surfactant (H) and the accumulation of the lighter, “metabolically used”, biologically inactive alveolar surfactant forms (Lvivo) in the interalveolar space. Conversion of the heavy, surface-active alveolar surfactant to the light metabolically used, nonsurface active forms is believed to involve the activity of an enzyme, namely convertase, which is thought to be derived from lamellar bodies (LB) in alveolar type II cells. The purpose of this study was to evaluate the effects of S. chartarum spores on mouse H and LB convertase activity by measuring their rates of conversion to Lvivo using the in vitro surface area cycling technique. It was determined whether there were concurrent changes in the protein and phospholipid concentrations of the raw bronchoalveolar lavage fluid (RL) and LB fractions that could be correlated with changes in convertase activity. Conversions of H to Lvivo in untreated control mice and saline-, isosatratoxin F-, and Cladosporium cladosporioides-exposed mice were not significantly different (p > 0.05). However, conversion from H to Lvivo in the mice exposed to S. chartarum spores was significantly higher than all other treatment groups (p < 0.001). LB to Lvivo conversions in untreated and saline-exposed mice were not significantly different, although they were significantly higher than the H to Lvivo conversions in these two animal treatment groups (p < 0.005), which supports the position that LB is a source of convertase activity in animals. LB to Lvivo conversion from C. cladosporioides-, isosatrotoxin F-, and S. chartarum-exposed mice were all significantly depressed (p < 0.003) compared to the LB to Lvivo conversion values obtained from untreated and saline-exposed mice. Protein concentrations in RL, H, Lvivo, and LB from mice exposed to S. chartarum spores were significantly elevated compared to those from the other treatment groups (p < 0.001). Protein concentration in H isolated from C. cladosporioides-exposed mice was also significantly elevated above untreated and saline control animal levels. Phospholipid concentrations in H isolated from S. chartarum-exposed mice were significantly elevated compared to those from other treatment groups, while LB phospholipid concentrations were significantly increased compared to saline and untreated control animal groups. These results show that S. chartarum spores significantly alter convertase activity in both the H and LB surfactant fractions in juvenile mice and that these changes can be related to changes in protein and phospholipid concentrations in alveolar lavage fractions. As surfactant promotes lung stability by reducing the surface tension of the air–alveolar interface, these results further support our position that inhalation exposure to S. chartarum spores in exposed individuals may lead to altered surfactant metabolism, and possibly to lung dysfunction through diminished alveolar surfactant surface tension attributes, and lung stability.

Role of Voltage-Dependent Calcium Channel Long-Term Potentiation (LTP) and NMDA LTP in Spatial Memory

This experiment explores the role of two forms of long-term potentiation (LTP) in behavioral memory. NMDA and/or voltage-dependent calcium channels (VDCCs) were antagonized pharmacologically at levels that block nmdaLTP and vdccLTP, respectively, in rats learning an eight-arm radial maze task. Animals were trained twice a day for 11 d under the systemic influence of MK-801, verapamil, both drugs, or saline. During acquisition, the mixed drug group displayed significantly more working memory errors and reference memory errors than all other groups. The mixed drug group was markedly impaired on the first daily trial but improved dramatically on their second daily trial. After a 7 d delay, saline and MK-801 animals maintained their predelay level of performance. The performance of the verapamil groups declined significantly over the delay. These results demonstrate that: (1) vdccLTP is necessary for the retention of information over a 7 d period, (2) the blockade of both forms of LTP prevents the retention of information over a 21 hr period, and (3) blockade of both forms of LTP does not prevent the storing of information over a short period of time (3 hr).

NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats

Although substance P (SP) has been implicated as a mediator of neurogenic inflammation in the small intestine, little information is available regarding the role of SP in the pathogenesis of chronic ulcerative colitis. In this study, our aim was to investigate whether the intraperitoneal administration of a nonpeptide neurokinin-1 (NK-1) antagonist, CP-96345, which antagonizes the binding of SP to its NK-1 receptor, results in the attenuation of colonic inflammation induced in rats by 5% dextran sodium sulfate (DSS) in drinking water for 10 days compared with an inactive enantiomer, CP-96344. Disease activity was assessed daily for 10 days, after which colonic tissue damage was scored and myeloperoxidase activity and colon and urinary 8-isoprostanes were measured. Animals receiving DSS exhibited marked physical signs of colitis by day 5 compared with controls. Chronic administration of the NK-1 antagonist significantly reduced the disease activity index, mucosal myeloperoxidase activity, colonic tissue damage score, and mucosal and urinary levels of 8-isoprostanes compared with inactive enantiomer- or vehicle-injected (saline) animals receiving DSS alone. These data indicate that the administration of an NK-1 antagonist can attenuate colonic inflammation and oxidative stress and suggest a novel therapeutic approach in the treatment of chronic ulcerative colitis.