Adolescent alcohol exposure produces sex differences in negative affect-like behavior and group I mGluR BNST plasticity.

Adolescent alcohol exposure increases the risk of developing alcohol use disorders (AUDs), yet the mechanisms responsible for this vulnerability remain largely unknown. One potential target for alcohol-induced changes is the circuitry that modulates negative affect and stress, two sexually dependent drivers of alcohol relapse. The bed nucleus of the stria terminalis (BNST) is a sexually dimorphic region that critically regulates negative affective- and stress-induced relapse. Group I metabotropic glutamate receptors (mGluR) are a target of interest due to their regulation of stress, anxiety behaviors, and BNST plasticity. The current studies investigate sex-dependent sensitivity to the effects of adolescent intermittent ethanol vapor exposure (AIE) on negative affect during acute and protracted alcohol withdrawal and following stress in adulthood. This work also assessed whether BNST group I mGluR-mediated long-term depression (LTD) was disrupted at these timepoints. During acute withdrawal, AIE altered LTD induced by the group I mGluR antagonist DHPG in females, but not males. During adulthood, stress unmasked persistent changes in DHPG-induced LTD and behavior that were not present under basal conditions. Females with an AIE history demonstrated enhanced negative affective-like behavior in the novelty-induced hypophagia test following restraint stress-a phenotype that could be blocked with systemic mGluR5 allosteric antagonism via MTEP. Conversely, males with an AIE history demonstrated elevated freezing in a contextual fear conditioning paradigm. These studies demonstrate long-lasting, sex-dependent phenotypes produced by AIE and suggest pharmaceutical interventions for alcohol use and comorbid disorders may be more effective if designed with sex differences in mind.

Dried plum prevents bone loss in a male osteoporosis model via IGF-I and the RANK pathway.

Previously, dietary supplementation with dried plums, a rich source of polyphenolic compounds with antioxidant and anti-inflammatory properties, has been shown to improve bone density, microstructure and biomechanics in female animal models of osteopenia. We designed this study to determine the extent to which dried plum prevents skeletal deterioration in gonadal hormone deficient male animals and to begin to understand its mechanism of action. Sixty 6-month-old male Sprague-Dawley rats were either sham-operated (Sham = 1 group) or orchidectomized (ORX = 4 groups) and randomly assigned to dietary treatments: standard semi-purified diet (Control) with either LD = 5%, MD = 15%, or HD = 25% (w/w) dried plum for 90 days. At the end of the treatment period, both the MD and HD dried plum completely prevented the ORX-induced decrease in whole body, femur, and lumbar vertebra bone mineral density (BMD). Biomechanical testing indicated that the MD and HD of dried plum prevented the ORX-induced decrease in ultimate load of the cortical bone as well as the compressive force and stiffness of trabecular bone within the vertebrae. Analyses of trabecular microarchitecture of the distal femur metaphysis and vertebral body revealed that HD dried plum protected against the decrease in trabecular bone volume (BV/TV) induced by ORX. In the distal femur, all doses of dried plum improved trabecular number (TbN) and separation (TbSp) compared to the ORX-control group, while MD and HD dried plum prevented the ORX-induced changes in vertebral TbN and TbSp. At the end of the 90-day treatment, no remarkable changes in serum osteocalcin or alkaline phosphatase in any of the treatment groups were observed, while serum insulin-like growth factor (IGF)-I was increased by dried plum. The ORX-induced increase in urinary deoxypyridinoline (DPD) excretion was completely prevented by all doses of dried plum coinciding with down-regulation of gene expression for receptor activator of NFkappa-B ligand (RANKL) and osteoprotegerin (OPG) in the bone. We conclude that dried plum prevents osteopenia in androgen deficient male rats, and these beneficial effects may be attributed in part to a decrease in osteoclastogenesis via down-regulation of RANKL and stimulation of bone formation mediated by IGF-I.

Systemic bone loss and induction of coronary vessel disease in a rat model of chronic inflammation.

Clinically, osteopenia or low bone mass has been observed in a variety of chronic inflammatory diseases, and elevated proinflammatory mediators have implicated this process. The purpose of this study was to develop an in vivo model of bone loss induced by chronic systemic inflammation. Time-release pellets designed to deliver one of three doses of LPS: Low (3.3 microg/day), High (33.3 microg/day), or Placebo over 90 days, were implanted subcutaneously in 3-month-old male Sprague-Dawley rats (n = 8/group). Neutrophil counts, indicative of ongoing inflammation, were elevated (P < 0.05) in both LPS groups at 30 days post-implant and remained significantly elevated in the High dose throughout the 90-day study period. At the end of the study, bone loss occurred in the femur as indicated by decreased bone mineral density (BMD) in both LPS-treated groups, but vertebral BMD was reduced in the High dose animals only. Microcomputed tomography revealed that trabecular bone volume (BV/TV) of the proximal tibial metaphysis tended to be reduced in the High dose LPS group. Deleterious effects on trabecular number (TbN) and trabecular separation (TbSp) were observed in both LPS-treated groups, but only the High dose group reached statistical significance. These alterations in trabecular microarchitecture resulted in compromised biomechanical properties. No changes in cortical thickness, porosity, or area of the tibia midshaft were evident at either dose of LPS. Up-regulation of the proinflammatory mediators, cyclooxygenase (COX)-2, interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha was demonstrated in the metaphyseal region where the deleterious effects of LPS were observed. In addition to these alterations in bone, trichrome staining indicated changes in the coronary arterioles, consistent with vascular disease. Utilization of a LPS time-release pellet appears to provide an in vivo model of chronic inflammation-induced bone loss and a potentially novel system to study concurrent development of osteopenia and vascular disease.

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel.

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 microl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IkappaBalpha (inhibitor of NF-kappaB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IkappaBalpha were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).

Vitamin E provides protection for bone in mature hindlimb unloaded male rats.

The deleterious effects of skeletal unloading on bone mass and strength may, in part, result from increased production of oxygen-derived free radicals and proinflammatory cytokines. This study was designed to evaluate the ability of vitamin E (alpha-tocopherol), a free-radical scavenger with antiinflammatory properties, to protect against bone loss caused by skeletal unloading in mature male Sprague-Dawley rats. A 2 x 3 factorial design was used with either hindlimb unloading (HU) or normal loading (ambulatory; AMB), and low-dose (LD; 15 IU/kg diet), adequate-dose (AD; 75 IU/kg diet), or high-dose (HD; 500 IU/kg diet) vitamin E (DL-alpha-tocopherol acetate). To optimize the effects of vitamin E on bone, dietary treatments were initiated 9 weeks prior to unloading and continued during the 4-week unloading period, at which time animals were euthanized and blood and tissue samples were collected. Serum vitamin E was dose-dependently increased, confirming the vitamin E status of animals. The HD treatment improved oxidation parameters, as indicated by elevated serum ferric-reducing ability and a trend toward reducing tissue lipid peroxidation. Histomorphometric analysis of the distal femur revealed significant reductions in trabecular thickness (TbTh), double-labeled surface (dLS/BS), and rate of bone formation to bone volume (BFR/BV) due by HU. AMB animals on the HD diet and HU animals on the LD diet had reduced bone surface normalized to tissue volume (BS/TV) and trabecular number (TbN); however, the HD vitamin E protected against these changes in the HU animals. Our findings suggest that vitamin E supplementation provides modest bone protective effects during skeletal unloading.

A rat model of syngeneic bone marrow transplantation during breast cancer therapy.

The purpose of this study was to develop a breast cancer model in rats, in which myeloablative chemotherapy and syngeneic bone marrow transplantation (SBMT) could be evaluated systematically for therapeutic effect. The Wistar-Furth (WF) DMBA-4 breast cancer cell line transplanted into naive WF rats produced rapidly growing tumors that were lethal within 2 months. SBMT was performed following preparation with a regimen (Bu-Cy), consisting of busulfan 16 mg/kg by gastric gavage on days -3 and -2 followed by 250 mg/kg of cyclophosphamide i.p. on day -1. Marrow was prepared from the femurs of donors and infused i.v. into the recipient on day 0. In all, 15 rats treated with Bu-Cy without marrow died, while 22 of 25 transplanted rats survived. In total, 16 rats with measurable tumors showed tumor responses following transplantation, but tumors recurred and survival was minimally prolonged. Of nine rats transplanted before clinical tumors were detected, five became long-term survivors that resisted further tumor challenge. It was concluded that the DMBA-4 breast cancer in WF rats could serve to evaluate SBMT following myeloablative doses of chemotherapy at various tumor loads. At large tumor loads therapy was not curative, but at low tumor burdens cures were possible and resistance to subsequent tumor challenge was demonstrated. The model may be useful for further studies of stem cell infusion in rodent tumor systems.

Molecular cloning and functional characterization of murine cysteinyl-leukotriene 1 (CysLT(1)) receptors.

We sought to clone and characterize the murine cysteinyl-leukotriene D(4) receptor (mCysLT(1)R) to complement our studies with leukotriene-deficient mice. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase chain reaction, has two potential initiator ATG codons that would encode for polypeptides of 352 and 339 amino acids, respectively. These two potential forms, predicted to be seven transmembrane-spanning domain proteins, have 87% amino acid identity with the human CysLT(1) receptor (hCysLT(1)R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT(1)R demonstrated high affinity and specific binding for leukotriene D(4) (LTD(4), K(d) = 0.25 +/- 0.04 nM). In competition binding experiments, LTD(4) was the most potent competitor (K(i) = 0.8 +/- 0.2 nM) followed by LTE(4) and LTC(4) (K(i) = 86.6 +/- 24.5 and 100.1 +/- 17.1 nM, respectively) and LTB(4) (K(i) > 1.5 microM). Binding of LTD(4) was competitively inhibited by the specific CysLT(1) receptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlukast (Onon), and zafirlukast (Accolate), while the CysLT(1)/CysLT(2) receptor antagonist BAY-u9773 [6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD(4). In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT(1)R, LTD(4) induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isoform, LTD(4) induced the dispersion of pigment granules, consistent with the activation by LTD(4) of a G(alphaq) (calcium) pathway. Functional elucidation of mCysLT(1)R properties as described here will enable further experiments to clarify the selective role of LTD(4) in murine models of inflammation and asthma.

Efficient, long-term transgene expression in Xenopus laevis dermal melanophores.

Xenopus laevis dermal melanophores provide an excellent model system for the investigation of complex cellular processes. Specifically, the expression of exogenous genes in Xenopus melanophores is the basis of recombinant bioassays for the study of receptor-ligand interactions. However, due to their slow rate of cell division and to the relatively low efficiency of current transfection protocols, long-term expression of exogenous genes and the generation of stable melanophore cell lines remains problematic. In this report we demonstrate the efficient, long-term expression of two exogenous proteins, the enhanced green fluorescent protein (EGFP) and the human CD4 (hCD4) cell surface receptor, following stable introduction into Xenopus melanophores via an HIV-1 based vector. Transduction of melanophores with the EGFP expression vector resulted in up to 80% EGFP+ cells. After 1 year in continuous culture in the absence of antibiotic selection, more than 60% of the cells remained EGFP+. Furthermore, we demonstrate the expression of hCD4 melanophores for over 9 months in continuous culture in the absence of antibiotic selection. Our results indicate that lentivirus vectors provide an efficient means of introducing genetic information into Xenopus melanophores, resulting in sustained levels of gene expression. The significance of this gene transfer system for the study of cellular signal transduction pathways is discussed.

Melatonin-induced organelle movement in melanophores is coupled to tyrosine phosphorylation of a high molecular weight protein.

Melanophores, brown to black pigment cells from, for example, Xenopus laevis, contain mobile melanin filled organelles, and are well suited for studies on organelle movement. The intracellular regulation of the movement seems to be controlled by serine and threonine phosphorylations and dephosphorylations. Melatonin induces aggregation of the melanosomes to the cell centre through a G(i/o)-protein-coupled receptor, Mel1c, which leads to an inhibition of PKA and a stimulation of PP2A. However, this study shows that the melatonin-induced aggregation of melanosomes is also accompanied by tyrosine phosphorylation of a protein with a molecular weight of approximately 280 kDa. Cells pre-incubated with genistein, an inhibitor of tyrosine phosphorylations, showed inhibited melanosome movement after melatonin stimulation, and a lower degree of tyrosine phosphorylation of the approximately 280 kDa protein. The adenylyl cyclase activator forskolin, and the G(i/o) protein inhibitor pertussis toxin, also inhibited tyrosine phosphorylation of the approximately 280 kDa protein. The results indicate that melatonin stimulation generates tyrosine phosphorylation of a high molecular weight protein, an event that seems to be essential for melanosome aggregation.

Down-regulation of renal endothelial nitric oxide synthase expression in experimental glomerular thrombotic microangiopathy.

Infection with certain strains of Escherichia coli and endotoxemia results in renal glomerular thrombotic microangiopathy (TMA) characterized by endothelial swelling and prominent glomerular microthrombus formation. Nitric oxide (NO) is an endogenous biologic modulator with diverse physiologic functions including vasodilation and inhibition of platelet adhesion and aggregation. NO is synthesized from conversion of L-arginine to L-citrulline by a family of NO synthases (NOS), which include constitutive and inducible isoforms. Indirect evidence supports the hypothesis that TMA is associated with depressed intrarenal NO production. However, the effect of TMA on renal tissue NOS expression has not been fully elucidated. We studied rats with TMA induced by iv bolus injection of high dose (20 mg/kg) E. coli endotoxin. Subgroups of six animals each were sacrificed before or at 30, 90, 180, 360, and 720 minutes after the administration of endotoxin. Renal histology and tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) were examined. Additionally, we examined the effect of endotoxin on glomerular NO production, and eNOS and iNOS protein expression in vitro. Glomerular capillary thrombosis developed by 180 minutes after endotoxin administration in approximately half of the animals. The glomeruli without thrombotic lesions apparent by light microscopy disclosed early signs of TMA characterized by endothelial swelling, platelet accumulation/adhesion, and patchy fibrinogen deposition. These morphologic changes were associated with a marked reduction of renal tissue eNOS expression beyond 180 minutes after the endotoxin administration. The fall in eNOS expression was coupled with a significant rise in iNOS protein abundance, which was expressed largely by glomerular circulating neutrophils and endothelial cells, peritubular vascular endothelium, and collecting ducts of cortex and medulla. In vitro incubation of isolated glomeruli with endotoxin also resulted in a marked reduction in eNOS expression and a significant rise in iNOS content. Administration of E. coli endotoxin leads to a sustained fall in renal eNOS expression both in vivo and in vitro. The associated decline in intrarenal endothelial NO production/availability may result in renal vasoconstriction and a hypercoagulative state, which may contribute to the pathogenesis of endotoxin-induced TMA.

Smoothened activates Galphai-mediated signaling in frog melanophores.

The 7-pass transmembrane protein Smoothened was investigated for its ability to act as a G-protein-coupled receptor in Xenopus laevis melanophores. A plasmid containing the human Smoothened cDNA insert was transfected into immortalized frog pigment cells. Cells expressing the protein showed a phenotype of persistent pigment aggregation, a hallmark of constitutive Galpha(i) activation. Smoothened-mediated pigment aggregation was reversed by treatment with pertussis toxin or by co-expression with dominant negative Galpha(i). The ability of melanophores to express functional Smoothened was also determined by its co-expression with the twelve-pass transmembrane protein, Patched. Patched blocked Smoothened-mediated melanosome aggregation in a dose-dependent manner, consistent with its physiological role as an inhibitor of Smoothened. That the reconstituted Patched-Smoothened receptor complex functions normally in pigment cells was demonstrated by co-transfection with the activating ligand, Sonic hedgehog, as well as by direct application of the recombinant Sonic hedgehog protein. Sonic hedgehog reversed Patched-mediated inhibition of Smoothened and induced pigment aggregation. The findings demonstrate that the human Sonic hedgehog receptor complex can be functionally reconstituted in melanophores and that it is capable of transmembrane signaling by utilizing endogenous Galpha(i).

Cocaine-induced cardiovascular responses in rats during acute ethanol withdrawal.

The effects of cocaine administration during acute ethanol withdrawal on both the cardiovascular system and cocaine pharmacokinetics are unclear. This study demonstrated differences in the cardiovascular effects of i.v.-administered cocaine during acute ethanol withdrawal in awake, freely moving rats. The altered responses to cocaine while in acute ethanol withdrawal compared to control animals included: enhanced increases in mean arterial pressure and systemic vascular resistance, attenuated heart rate decreases, and enhanced cardiac index and stroke volume decreases. These results may suggest that acute ethanol withdrawal disrupts myocardial contractility when the myocardium is subjected to a large increase in blood pressure. Serial arterial blood sampling in additional groups of rats were done to assess plasma cocaine concentrations and to confirm the absence of ethanol in the blood. Plasma cocaine concentrations were not effected by acute ethanol withdrawal. These results indicate that the altered cardiovascular responses to cocaine during acute ethanol withdrawal were not a result of differences in cocaine plasma concentrations.

Effects of regulators of G protein-signaling proteins on the functional response of the mu-opioid receptor in a melanophore-based assay.

The goal of the present study was to investigate a possible role for regulators of G protein-signaling (RGS) proteins in opioid receptor (OR) desensitization using cultured Xenopus laevis dermal melanophores. Morphine-induced pigment aggregation in a melanophore cell line stably expressing the murine mu OR (muOR) was quantified over time. Responses of the muOR (a G(i)-linked receptor) exhibited a time-dependent desensitization, which varied with the concentration of morphine used. In contrast, much less desensitization was observed in response to melatonin, effects mediated through the cells' endogenous melatonin receptor (which is also G(i)-linked). To further study OR desensitization, melanophores lacking a muOR were transiently transfected with plasmids encoding the muOR alone or in combination with plasmids encoding one of several RGS subtypes (RGS1, RGS2, RGS3, or RGS4). Overexpression of RGS2, but not the other RGS subtypes, produced a rightward shift in the morphine concentration-response curve. RGS protein overexpression also decreased the magnitude of morphine-induced responses. Finally, the effect of a mutant form of Galpha(i1), which is insensitive to RGS action, was investigated with respect to its ability to alter the response of the muOR to morphine. Expression of the mutant Galpha(i1) prolonged morphine-induced pigment aggregation and produced leftward shifts in concentration-response curves, compared with expression of wild-type Galpha(i1). These results demonstrate that specific RGS proteins can dampen signals initiated by agonist activation of the muOR, and support a possible role for RGS proteins in OR desensitization.

Expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumor suppressor in dysplastic progression and adenocarcinoma in Barrett esophagus.

BACKGROUND: Barrett esophagus predisposes individuals to esophageal carcinoma, which develops from intermediate stages of tissue dysplasia primarily in the vicinity of the gastroesophageal junction. Understanding the cellular and molecular events in the progression of Barrett esophagus to adenocarcinoma may contribute to its early diagnosis and treatment. Mutation and overexpression of the tumor suppressor p53 have previously been observed in Barrett high grade dysplasia and adenocarcinoma. The expression of the cyclin-dependent kinase (CdK) inhibitor p21 can be up-regulated by p53, resulting in the down-regulation of cell division at the G(1)/S-phase transition. The current study examined the correlation between the expression of p21 and p53 by quantifying their levels during the progression of dysplasia and adenocarcinoma in Barrett esophageal tissues. METHODS: Barrett esophageal tissue samples that were negative or indefinite for dysplasia, contained dysplasia, and contained adenocarcinoma were examined by immunohistochemistry. Paraffin embedded sections of lining and glandular epithelia were adsorbed with primary murine antibodies against human p21 or p53 followed by horseradish peroxidase secondary antibody. An immunoreactivity score for each primary antibody and section was obtained by multiplying a staining intensity factor by the percent of positively stained cells. RESULTS: Nuclear p21 expression was detectable immunohistochemically in Barrett esophagus that was negative for dysplasia, but it was significantly elevated (P

Shock after blast wave injury is caused by a vagally mediated reflex.

OBJECTIVE: Bomb blast survivors occasionally suffer from profound shock and hypoxemia without signs of external injury. We hypothesize that a vagally mediated reflex such as the pulmonary defensive reflex is the cause of shock from blast wave injury. This study was a prospectively randomized, controlled animal study. METHODS: By using a previously described model of blast wave injury, we randomized rats to one of four groups: control, blast-only, bilateral cervical vagotomy plus atropine 200 microg/kg i.p. only, and bilateral cervical vagotomy plus atropine 200 microg/kg i.p. before blast injury. Cardiopulmonary parameters were recorded for 90 minutes after the blast or until death. RESULTS: Bradycardia, hypotension, and absence of compensatory peripheral vasoconstriction, typically seen in animals subjected to a blast pressure injury, were prevented by bilateral cervical vagotomy and intraperitoneal injection of atropine methyl-bromide. Hypoxia and lung injury were not statistically significant between the blasted groups, suggesting equivalent injury. CONCLUSION: Our data implicate a vagally mediated reflex such as the pulmonary defensive reflex as the cause of shock seen immediately after a blast pressure wave injury.

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis.

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.

Synthetic melanocortin receptor agonists and antagonists.

Multiple melanocortin receptor subtypes with distinct cell and tissue distribution patterns have recently been identified, thereby presenting numerous opportunities for biological investigation. Many of these studies could benefit from the availability of subtype selective or specific agonists and antagonists. The purpose of this report is to summarize the state of available melanocortin receptor agonists and antagonists.

Prevention of rat mammary carcinoma utilizing leuprolide as an equivalent to oophorectomy.

A clinical trial is currently under way to examine the effectiveness of leuprolide as a breast cancer chemopreventive agent and contraceptive. This trial, as well as similar proposed studies, is based on the assumption that leuprolide is as effective as surgical castration in preventing the onset of mammary tumors; however, this has not been well documented in the DMBA animal model. We directly compared leuprolide and oophorectomy in this model and examined a combined therapy of leuprolide/bromocriptine. Twenty-seven day old female Sprague-Dawley rats were randomly allocated into one of eight groups. All rats received a 20-mg dose of DMBA at the age of 55 days. Group 1 (n = 10), no treatment; Group 2 (n = 9), leuprolide (100 microg/kg/day) for eight weeks beginning four weeks prior to DMBA; Group 3 (n = 10), oophorectomy four weeks prior to DMBA with replacement estrogen beginning four weeks following DMBA. Estrogen replacement was achieved with a 0.05-mg estradiol tablet releasing 0.833 microg/day over a 60-day period. Group 4 (n = 10), leuprolide (100 microg/kg/day) initiated two weeks prior to DMBA and continuing for two weeks following DMBA; Group 5 (n = 9), oophorectomy two weeks prior to DMBA with 0.05 mg of estradiol in depot form, releasing 0.833 microg/day, beginning four weeks following DMBA and continuing until week 16 of the study; Group 6 (n = 10), leuprolide (100 microg/kg/day) beginning two weeks prior to DMBA and continuing for the duration of the experiment; Group 7 (n = 10), leuprolide (100 microg/kg/day) for eight weeks beginning two weeks prior to DMBA; Group 8 (n = 9), leuprolide (100 microg/kg/day) and bromocriptine (83 microg/day) for eight weeks beginning two weeks prior to DMBA. At nineteen weeks (15 weeks post DMBA), animals were sacrificed and autopsies performed. One hundred percent of untreated animals developed tumors. No animals undergoing oophorectomy four weeks prior to DMBA or receiving leuprolide four weeks prior to and simultaneously with DMBA developed tumors. In animals pretreated two weeks prior to DMBA with leuprolide or oophorectomy, each group had one animal with tumor development. No tumors developed in the animals receiving ongoing injections of leuprolide. However, one tumor developed in those receiving leuprolide for the first eight weeks beginning two weeks prior to DMBA administration. One animal receiving both leuprolide and bromocriptine developed one tumor. We conclude that chemical oophorectomy (with leuprolide) is as effective as surgical oophorectomy in inhibiting DMBA induced carcinogenesis.

Global primary blast injury: a rat model.

Blast wave injury from bombs cause a unique but poorly understood spectrum of injuries. Previous blast wave models involved high energy explosives detonated in an open field without the sophisticated monitoring of laboratory equipment. We characterized a rodent model that produces a global blast injury in a safe laboratory environment. Male rats, prospectively randomized to four groups of ten, were anesthetized and subjected to a blast at 2.0 cm, 2.5 cm, or 3.5 cm from the blast nozzle. The control group received no blast. Intensity of the blast (80-120 psi peak pressure, 1-2 msec duration) was controlled by varying the distance of the blast wave generator to the rat. The rats were monitored for three hours following the blast and then euthanized. Bradycardia was an immediate but transient response to blast injury. Mean arterial pressure was bimodal with severe hypotension occurring immediately after the blast and, again, two to three hours later. The characteristic injuries from a blast wave, such as pulmonary hemorrhage with increased lung weight, intestinal serosal hemorrhage, and hemoperitoneum, were found in the rats subjected to the blast pressure wave. In conclusion, our rodent model accurately reproduces the clinical spectrum of injuries seen in blast victims and will provide a powerful tool for studying the pathophysiology and potential treatments of bomb blast victims.