Female Brown Norway Rats Research Articles

The establishment of chronic rejection model of rat's aortic transplantation

Objective To establish an aortic transplantation model in the rat. Methods A thoracic aorta to abdominal aorta interposition transplant model in the rat was established. The male Lewis rats were used as donors and the female Brown Norway(BN) rats were used as recipients.Intercepted the thoracic aorta of lewis rats about one and a half centimeter. Transplanted it on the abdominal aorta of BN rats. The activity and weight change of BN rats were observed everyday until 1 week after transplantation. Then observed these index one time a week. The transplanted aortas were harvested at 8 weeks after transplantation. The intimal thickening were observed by pathological. The expression of CD3,CD68 and α-actin on transplanted aorta were observed by immunohistochemistry. Resuts All operations were finished within 2 hours. No recipients died during operation. All recipients could survival more than 8 weeks. The symptoms of bowing back, hair shedding and weight loss was appeared after transplantation. The weight of the rats were upturn at 4 or 5 days after transplantation. About 1 week after transplantation, all situations of the rats were recovery to the lever before transplantation. 8 weeks after transplantation the intimal thickening of the transplanted aortas were observed obviously by pathological. And the expressions of CD3, CD68 and α-actin on aortic allograft were observed. Conclusion The intimal thickening and the expressions of CD3, CD68 and α-actin on aortic allograft were typical expressions of chronic rejection. So this aortic transplantation model is feasible and reliable for studying chronic rejection. Key words: Aorta; Vessel transplantation; Allograft rejection; Animal model

RNA profiles of rat olfactory epithelia: individual and age related variations

BackgroundMammalian genomes contain a large number (~1000) of olfactory receptor (OR) genes, many of which (20 to 50%) are pseudogenes. OR gene transcription is not restricted to the olfactory epithelium, but is found in numerous tissues. Using microarray hybridization and RTqPCR, we analyzed the mRNA profiles of the olfactory epithelium of male and female Brown Norway rats of different origins and ages (newborn, adult and old).Results(1) We observed very little difference between males and females and between rats from two different suppliers. (2) Different OR genes were expressed at varying levels, rather than uniformly across the four endoturbinates. (3) A large proportion of the gene transcripts (2/3 of all probes) were detected in all three age groups. Adult and older rats expressed similar numbers of OR genes, both expressing more OR genes than newborns. (4) Comparisons of whole transcriptomes or transcription profiles of expressed OR genes only showed a clear clustering of the samples as a function of age. (5) Most OR genes were expressed at lower levels at birth than in older animals, but a small number of OR genes were expressed specifically or were overexpressed in newborns.ConclusionNot all OR genes are expressed at a detectable level. Pups expressed fewer OR genes than adult rats, and generally at a lower level; however, a small subset of OR genes were more strongly expressed in these newborn rats. The reasons for these differences are not understood. However, the specific expression of some OR genes in newborn olfactory epithelia may be related to the blindness and deafness of pups at birth, when these pups are heavily reliant on olfaction and their mother.

Diazepam Metabolism in the Kidneys of Male and Female Rats of Various Strains

Previously, we have reported drastic strain differences of diazepam metabolism in the livers of a variety of rat strain. In this study, to characterize strain and sex differences of diazepam metabolism in the kidney, renal microsomal diazepam metabolic activities were determined in the Dark Agouti (DA), Sprague-Dawley (SD), Brown Norway (BN) and Wistar (WS) strains of rat. We found that the major pathway of diazepam metabolism in the kidney was diazepam N-demethylation, which is different from that in the liver, 3-hydroxylation. A Dose-course (12.5-200 muM of diazepam) study revealed that the DA and WS male rats had higher diazepam N-demethylation activity than the SD and BN rats. In contrast to the males, a lower activity of diazepam N-demethylation was observed in female BN rats. By Western blot analysis, constitutive protein expressions of cytochrome P450 (CYP) 2C11, which is responsible for diazepam N-demethylation, were detected in the 4 strain in both the male and female rats, and the BN rats had lower expression levels of CYP2C11 protein. However, we did not observe significant differences in the kinetic parameters of diazepam N-demethylation. Our results suggested that there was a strain difference in CYP-dependent diazepam N-demethylation in the rat kidney, which is different from the finding in liver microsomes.

Preclinical analysis of treosulfan in combination with total body irradiation as conditioning regimen prior to bone marrow transplantation in rats

Treosulfan (Treo) and total body irradiation (TBI) demonstrate a high therapeutic activity in treatment of acute leukemia and lymphoma. We investigated the combination of Treo and TBI prior to bone marrow transplantation (BMT) in rats. Female Lewis rats were treated with Treo on 3 consecutive days followed by TBI with either 5 Gy (n = 28) or 7.5 Gy (n = 48). After conditioning animals received 4 × 10E7 bone marrow cells (BC) from female Lewis rats. Additional 16 rats were transplanted with 4 × 10E7 BC and 1.5 × 10E7 spleen T-cells from female Brown-Norway (BN) rats. Animals were examined daily for clinical signs and toxicity was investigated by necropsy and histology in all animals. Gastrointestinal toxicity was the dose-limiting factor of Treo in combination with TBI. The highest tolerable dose of Treo in combination with 7.5 Gy TBI was 3 × 0.5 g/kg and the highest tolerable dose of Treo in combination with 5 Gy TBI was 3 × 0.6 g/kg. Allogeneic BMT from BN donors resulted in engraftment and survival of 12 out of 16 animals. Gastrointestinal toxicity is the dose-limiting factor in the treatment with Treo and TBI. Furthermore, Treo possesses certain characteristics of a radiosensitizer.

Inhibitory Effect of YC-1, 3-(5′-Hydroxymethyl-2′-Furyl)-1-Benzylindazole, on Experimental Choroidal Neovascularization in Rat

Purpose: It was the aim of this study to evaluate the effects of YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole, one of the hypoxia-inducible factor 1 (HIF-1) inhibitors, on laser-induced choroidal neovascularization (CNV) in rats. Methods: Thirty female Brown Norway rats underwent laser photocoagulation to induce CNV. Twenty of them (treatment group) were treated with a single intravitreal injection of 5 µg YC-1, and the remaining 10 (control) were sham-treated with a single intravitreal injection of 2.5 mg/ml dimethyl sulfoxide 2 weeks after laser photocoagulation. The expression of HIF-1α and vascular endothelial growth factor (VEGF) in CNV was evaluated by immunofluorescence staining. Fluorescein angiography was performed 2 weeks before and 2 weeks after single intravitreal YC-1 (5 µg) and dimethyl sulfoxide (2.5 mg/ml) injection, grading fluorescein leakage from 0 to 3. The size of the CNV was measured on histologic sections. Results: Both HIF-1α and VEGF were expressed in CNV lesions in the control group 4 weeks after laser photocoagulation, whereas the expressions of HIF-1α and VEGF were not observed in the intravitreally YC-1-treated group. The mean fluorescein leakage score decreased from 2.56 ± 0.49 to 0.79 ± 0.71 in the intravitreally YC-1-injected group and from 2.62 ± 0.49 to 1.58 ± 0.60 in the control group. Sixty-eight (71.6%) out of 95 CNV lesions of intravitreally YC-1-treated eyes (71.4%) and 12 (21.8%) out of 55 lesions in DMSO-treated eyes showed a decreased fluorescein leakage score of 2 or more. The mean difference of fluorescein leakage scores between the intravitreally YC-1-treated group and the control group was significant (p = 0.004). The mean thickness of the CNV lesions in the intravitreally YC-1-treated group (27.30 ± 6.47 µm) was smaller than that of the control group (64.36 ± 8.26 µm, p < 0.001). There was no ocular inflammation, retina hemorrhage or systemic toxicity induced by YC-1 treatment. Conclusion: These results suggest that YC-1 inhibits the HIF-1 expression after photocoagulation and suppresses the development of laser-induced CNV formation.

Genetic control of estrogen action in the rat: mapping of QTLs that impact pituitary lactotroph hyperplasia in a BN × ACI intercross

Estrogens are important regulators of growth and development and contribute to the etiology of several types of cancer. Different inbred rat strains exhibit marked, cell-type-specific differences in responsiveness to estrogens as well as differences in susceptibility to estrogen-induced tumorigenesis. Regulation of pituitary lactotroph homeostasis is one estrogen-regulated response that differs dramatically between different inbred rat strains. In this article we demonstrate that the growth response of the anterior pituitary gland of female ACI rats to 17beta-estradiol (E2) markedly exceeds that of identically treated female Brown Norway (BN) rats. We further demonstrate that pituitary mass, a surrogate indicator of absolute lactotroph number, behaves as a quantitative trait in E2-treated F(2) progeny generated in a genetic cross originating with BN females and ACI males. Composite interval mapping analyses of the (BNxACI)F(2) population revealed quantitative trait loci (QTLs) that exert significant effects on E2-induced pituitary growth on rat chromosome 4 (RNO4) (Ept5) and RNO7 (Ept7). Continuous treatment with E2 rapidly induces mammary cancer in female ACI rats but not BN rats, and QTLs that impact susceptibility to E2-induced mammary cancer in the (BNxACI)F(2) population described here have been mapped to RNO3 (Emca5), RNO4 (Emca6), RNO5 (Emca8), RNO6 (Emca7), and RNO7 (Emca4). Ept5 and Emca6 map to distinct regions of RNO4. However, Ept7 and Emca4 map to the same region of RNO7. No correlation between pituitary mass and mammary cancer number at necropsy was observed within the (BNxACI)F(2) population. This observation, together with the QTL mapping data, indicate that with the exception of the Ept7/Emca4 locus on RNO7, the genetic determinants of E2-induced pituitary growth differ from the genetic determinants of susceptibility to E2-induced mammary cancer.

Alveolar macrophages suppress non-specific inflammation caused by inhalation challenge with trimellitic anhydride conjugated to albumin

Occupational exposure to low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter these inhaled compounds and were previously shown to affect TMA-induced asthma-like symptoms in the Brown Norway rat (Valstar et al., Toxicol. Appl. Pharmacology 211:20-29, 2006). TMA is a hapten that will bind to endogenous proteins upon entrance of the body. Therefore, in the present study we determined if TMA conjugated to albumin is able to induce asthma-like symptoms and if these are affected by AM depletion. Female Brown Norway rats were sensitized by dermal application of TMA or received vehicle alone on days 0 and 7. One day prior to the inhalation challenge the rats were treated intratracheally with either empty liposomes or liposomes containing clodronate (dichloromethylene diphosphonate) to specifically deplete the lungs of AMs. On day 21, all groups of rats were challenged by inhalation of TMA-BSA. Breathing frequency, tidal volume, and minute ventilation were measured before, during, within 1 h, and 24 h after challenge and the gross respiratory rate score was determined during challenge. Total and TMA-specific IgE levels were determined in serum and lung lavage fluid and parameters of inflammation and tissue damage were assessed in lung lavage fluid and/or lung tissue 24 h after challenge. Sensitization with TMA had no effect on the lung function before challenge, but TMA-BSA challenge resulted in an early asthmatic response as compared to the non-sensitized rats, irrespective of AM depletion. AM depletion had no effect on the sensitization-induced serum and lung lavage fluid IgE levels. TMA-BSA inhalation did not induce airway inflammation and tissue damage irrespective of sensitization, unless AM were depleted. Data indicate that AMs inhibit immunologically non-specific damage and inflammatory cell influx into the lungs as caused by TMA-BSA inhalation. Since effects of inhalation challenge with TMA-BSA are partly different from those of TMA, challenge with the latter is to be preferred for hazard identification.

Alveolar macrophages have a dual role in a rat model for trimellitic anhydride-induced occupational asthma

Occupational exposure to low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter inhaled compounds. These cells can produce many different mediators that have a putative role in asthma. In this study, we examined the role of AMs in lung function and airway inflammation of rats exposed to TMA. Female Brown Norway rats were sensitized by dermal application of TMA or received vehicle alone on days 0 and 7. One day before challenge, rats received intratracheally either empty or clodronate-containing liposomes to deplete the lungs of AMs. On day 21, all rats were challenged by inhalation of TMA in air. Lung function parameters were measured before, during, within 1 h after, and 24 h after challenge. IgE levels and parameters of inflammation and tissue damage were assessed 24 h after challenge. Sensitization with TMA led to decreased lung function parameters during and within 1 h after challenge as compared to non-sensitized rats. AM depletion alleviated the TMA-induced drop in lung function parameters and induced a faster recovery compared to sham-depleted TMA-sensitized rats. It also decreased the levels of serum IgE 24 h after challenge, but did not affect the sensitization-dependent increase in lung lavage fluid IL-6 and tissue TNF-alpha levels. In contrast, AM depletion augmented the TMA-induced tissue damage and inflammation 24 h after challenge. AMs seem to have a dual role in this model for TMA-induced occupational asthma since they potentiate the immediate TMA-induced decrease in lung function but tended to dampen the TMA-induced inflammatory reaction 24 h later.

An oral Brown Norway rat model for food allergy: comparison of age, sex, dosing volume, and allergen preparation.

The purpose of the presented experiments was to study the possibility of using the Brown Norway rat as a model for food allergy in our laboratory. Specific serum IgE against ovalbumin (OVA) was induced after dosing male and female Brown Norway rats daily by gavage for 35 days. The influence of various preparations of allergen: OVA grade II, OVA grade V, and fresh egg white, age (4 versus 8 weeks), dosing volumes, and animal suppliers was studied. A general finding was that females had statistically significantly higher specific IgE and IgG titres and number of responders than males. Egg white preparation, age, dosing volume, and animal supplier did not statistically significantly influence the median IgE and IgG titres and number of responders. The difference between immune responses in males and females could not be attributed to variations in daily intake of OVA or exposure via the lung. In our hands, the oral Brown rat food allergy model gives rise to a moderate number of IgE responders, 13-38 and 38-75% in males and females, respectively. For further experiments with this model in our laboratory, females seem the sex of choice.

Age- and sex-related resistance to chronic experimental autoimmune myasthenia gravis (EAMG) in Brown Norway rats.

The influence of age and sex on the induction of chronic EAMG was analysed. Aged male rats, immunized with Torpedo acetylcholine receptor (tAChR), showed no clinical signs of disease or AChR loss. Immunization of young male Brown Norway (BN) rats resulted in both clinical signs of disease and 65% AChR loss. In contrast, both young and aged female BN rats showed comparable AChR loss (58% and 50%, respectively), although aged female rats did not develop clinical signs of disease. Differences in antibody titres, isotype distribution, fine specificity or complement activation could not account for the observed resistance. These results suggest that resistance against EAMG in aged rats is due to resistance of the AChR against antibody-mediated degradation, or to mechanisms able to compensate for AChR loss.

Characterization of a potential animal model of an idiosyncratic drug reaction: nevirapine-induced skin rash in the rat.

Idiosyncratic drug reactions are difficult to study in humans due to their unpredictability. Unfortunately, this characteristic also hinders the development of animal models needed for mechanistic studies. Nevirapine, used to treat human immunodeficiency virus (HIV) infections, results in a severe idiosyncratic skin rash in some patients. We found that nevirapine can also cause a significant rash in some strains of rats. At a dose of 150 mg/kg/day, the incidence in female Sprague-Dawley rats was 6/28 (21%), in female Brown Norway rats 32/32 (100%), and in female Lewis rats 0/6 (0%) while no male Sprague-Dawley or Brown Norway rats developed a rash. Female SJL mice 0/7 also did not develop nevirapine-induced skin lesions. The first sign of a reaction in Brown Norway rats was red ears at days 7-10 followed by a rash with scabbing mainly on the back; this was a shorter time to onset than in Sprague-Dawley rats. Light microscopy of the skin revealed a primarily mononuclear inflammatory infiltrate and lesions typical of self-trauma. Immunohistochemistry results suggest that the infiltrate was composed of CD4 and CD8 T cells as well as macrophages. A lower dose of either 40 or 75 mg/kg/day did not lead to a rash and, in fact, 2 weeks of the lower doses induced tolerance to the 150 mg/kg/day dose in female Brown Norway rats. A dose of 100 mg/kg/day resulted in rash in 2/4 (50%) of female Brown Norway rats. Rechallenge of Brown Norway rats that had been allowed to recuperate after a nevirapine-induced rash led to red ears in less than 24 h followed by hair loss and occasional skin lesions. Although the skin rash was less evident on rechallenge, microscopically, the cellular infiltrate was more prominent, especially surrounding the hair follicles. Moreover, there were lesions of interface dermatitis with apoptosis and satellitosis, indicative of a cell-mediated immune attack on the epidermis. While systemic signs of illness did not accompany the rash on primary exposure, on rechallenge, the animals appeared generally unwell and this forced sacrifice after 2 weeks or less of treatment. Importantly, splenocytes isolated from rechallenged animals were able to transfer susceptibility to nevirapine-induced skin rash to naïve female Brown Norway recipients, which was illustrated by a faster time to onset of rash in the recipients. The characteristics of this adverse reaction are similar to that seen in humans; that is, it is idiosyncratic in that it only occurs in some strains of animals, is delayed in onset, is more common in females, is dose-dependent, and appears to be immune-mediated. Therefore, it may represent a good animal model for the study of idiosyncratic drug reactions.

Systemic administration of Bordetella pertussis enhances pulmonary sensitization to house dust mite in juvenile rats.

The incidence of allergies and asthma has increased significantly in the past few decades. The objectives of this study were to establish an allergy model in weanling rats to more closely reflect the developing immune system of children, and to determine whether systemic administration of inactivated Bordetella pertussis could enhance pulmonary and systemic immune responses to locally administered house dust mite antigen (HDM). Three-week old female Brown Norway rats were sensitized with 10 micro g HDM intratracheally or intraperitoneally, with or without a simultaneous injection of 10(8) whole killed B. pertussis organisms. Ten days later, all the rats were challenged with 5 micro g HDM via the trachea. Bronchial lymph nodes and bronchoalveolar lavage fluid (BAL) were collected 0, 2, and 4 days post-challenge. Coadministration of pertussis and intratracheal instillation of HDM enhanced HDM-specific lymphoproliferative responses and increased BAL levels of total protein, lactate dehydrogenase, HDM-specific IgE and IgG antibodies, and the number of eosinophils in BAL to the same extent as had occurred in the systemically immunized animals. The data show that intratracheal instillation of HDM induces a mild allergic sensitization in juvenile rats, and that ip injection with B. pertussis enhances this sensitization process to levels seen in animals injected with antigen and B pertussis together. These results suggest that simultaneous exposure to Th2-inducing vaccine components and allergenic proteins may be a risk factor for allergic sensitization and the development of asthma in susceptible individuals.

Mast cells in ongoing acute rejection: increase in number and expression of a different phenotype in rat heart transplants.

Mast cells (MC) are resident in healthy hearts and play important physiological and pathophysiological roles. In the transplanted heart, correlations have been found between MC number and the severity of rejection episodes, the intensity of chronic inflammation, and allograft arteriosclerotic changes. However, not much emphasis has been placed on the fact that resident donor MC, and infiltrating recipient MC do not forcedly need to share the same properties and function. To gain insight in the role of cardiac MC during acute, and ongoing acute rejection of heart transplants, we investigated MC kinetics and MC phenotype in a rat heart transplantation model. Donor hearts from female Brown-Norway rats were transplanted to male Lewis rats. Immunosuppression was started at day 5 using ciclosporin and prednisolone. Connective tissue type MC (CTMC) were distinguished from mucosa type MC (MMC) by immunohistochemistry for rat MC protease (RMCP) -1 and -2. Expression of RMCP-1 and -2 mRNA was quantified by real time reverse transcription-polymerase chain reaction. Infiltrating Y chromosome positive MC were detected by fluorescence in situ hybridization. mRNA expression of interleukin-3 (IL-3) and of the two differentially spliced isoforms of kit ligand (KL, stem cell factor) was quantified using reverse transcription-polymerase chain reaction. Resident cardiac donor MC are almost exclusively CTMC and decrease in number during acute rejection. MC increase in number, and recipient MC invade the cardiac allograft during ongoing acute rejection. The phenotype of the invading MC is characterized by the expression of RMCP-2, or both RMCP-1 and RMCP-2, and thus resemble a MMC type. IL-3 mRNA is highly expressed, and the ratio of the differentially spliced mRNAs for KL-1 and KL-2 rises up to 2-fold during ongoing acute rejection. Our data show that MC in posttransplant hearts during ongoing acute rejection differ from MC in healthy hearts and isografts by expressing a different phenotype. Changes in IL-3 and KL expression might be responsible for the predominance of MMC over CTMC. The notion is of importance that MC in cardiac allografts may have properties and functions that differ from those in nontransplanted healthy hearts.

Morphological and immunohistochemical characteristics of the heterogeneous prostate-like glands (paraurethral gland) seen in female Brown-Norway rats.

Morphological and immunohistochemical characteristics of the prostate-like glands (paraurethral gland) seen spontaneously in female Brown-Norway (BN) rats were investigated by gross, and light and electron microscopic examination. At 9- to 10-weeks-old, the paraurethral gland was detected in 50 out of 52 female animals examined (96.2%), and it was observed as single or paired structures located ventrolaterally in the urethra just caudal to neck of the urinary bladder. Microscopically, the glandular acini consisted of flat to cuboidal secretory epithelium surrounded by the smooth muscle. The glands displayed modest secreting activity, and a few secreting materials were observed in the acinar lumens. The main peripheral ducts were located in the urethral wall, and drained into the urethra on both sides. Ultrastructurally, abundant rough endoplasmic reticulum (RER), numerous mitochondria and lysosomes, and secretory granules in the apical portion of the epithelial cells were noted, and basal cells were also observed. These gland epithelial cells showed positive reactions when stained for androgen receptor (AR), prostate specific antigen (PSA), or prostate specific acid phosphatase (PSAP). The nature of this paraurethral gland resembled that of the prostate gland in male rats. Thus, the paraurethral glands seen in the females were considered homologous to the prostate gland in males.

TNF-α ENHANCED ALLERGIC SENSITIZATION TO HOUSE DUST MITE IN BROWN NORWAY RATS

We have recently demonstrated that pulmonary exposure to residual oil fly ash (ROFA) resulted in enhanced sensitization to house dust mite (HDM) and augmented the development of allergic lung disease after allergen challenge. This effect was associated with increased tumor necrosis factor (TNF- α) ,a macrophage- and epithelial cell-derived cytokine that promotes granulocyte migration to the lung. The present study examined whether exogenous administration of TNF- α enhances sensitization to HDM. One day prior to pulmonary sensitization with 10 μ gHDM(5 μ g each on days 1 and 3),female Brown Norway rats were instilled via the trachea with either 2.0 μ g recombinant rat TNF- α, 2.0 μ g bovine serum albumin (BSA),or 1000 μ g ROFA, and were challenged with 10 μ g HDM 14 days later. Antigen-induced immediate bronchoconstriction responses, antigen-specific immunoglobulin E (IgE) titers, lymphocyte proliferation, cytokines (TNF- α and interleukin [IL]-13), and eosinophils were elevated in rats treated with ROFAor TNF- α compared with BSA-treated controls after HDMchallenge. Intratracheal administration of anti-TNF- α monoclonal antibody during ROFA exposure did not reduce ROFA-enhanced lymphocyte proliferation or IgE titers, but had a trend for reduced pulmonary inflammation. This study demonstrates that TNF- α has similar adjuvant activity as ROFA, but other factors may fulfill this function when TNF- α activity is blocked.

RESPIRATORY IRRITATION BY TRIMELLITIC ANHYDRIDE IN BROWN NORWAY AND WISTAR RATS

Several acid anhydrides are known for their sensitizing and irritative properties. Since both irritation and respiratory allergy can cause changes of lung function, proper testing of allergen-dependent effects on the respiratory tract requires knowledge of the respiratory irritant effects. To study the latter effects, groups of female Brown Norway (BN) and Wistar rats were exposed for 30 min to a range of concentrations (10 to 300 mg/m 3) of the well-known respiratory allergen trimellitic anhydride (TMA). Breathing pattern and frequency were monitored before, during, and after exposure. Animals were necropsied and lung weights were determined 1 day after exposure. In BN rats, changes in breathing pattern were seen at levels of 29 mg/m 3 and higher and decreases in frequency at 60 mg/m 3 and higher, whereas in Wistar rats changes in both pattern and frequency (increases followed by decreases) were seen at levels of 34 mg/m 3 and higher. Changes in breathing pattern consisted of a spiked form instead of a wave form of the respiratory cycle, with a pause between breaths at the end of expiration. The length of the pause increased with increasing concentrations of TMA while the duration of the respiratory cycle decreased slightly, implying that breathing frequency was mainly determined by the magnitude of the increase in pause. These reversible changes in breathing pattern and frequency were considered to be suggestive of lower airway irritation, rather than upper airway irritation. No concentration-related changes in lung weights were observed. The highest level at which no acute airway irritation as based on both breathing pattern and frequency was observed in both rat strains was 14 mg/m 3 .

The mercapturic acid biotransformation pathway of hexachlorobenzene is not involved in the induction of splenomegaly, or skin and lung lesions in the Brown Norway rat.

Involvement of the mercapturic acid pathway in the induction of splenomegaly and skin and lung pathology by hexachlorobenzene (HCB) in the rat was investigated by seeking to determine whether pentachloronitrobenzene (PCNB) has the same inflammatory effects as HCB, since both compounds are directly conjugated to glutathione, and further processed into the same mercapturic acid metabolites which are excreted via the urine. Female Brown Norway (BN/SsNO1aHsd) rats at 3 to 4 weeks of age were orally exposed to diets with or without supplementation with 450 mg HCB or equimolar (467 mg) or higher (934 mg) amounts of PCNB per kilogram of diet over 4 weeks. Gross skin lesion development and body weight gains were assessed during exposure and spleen and liver weights as well as histopathologic changes in skin and lung were assessed after exposure. After 3 weeks of exposure, urinary metabolites of the mercapturic acid and oxidative biotransformation pathways were identified using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Oral exposure of the rats to 450 mg/kg HCB resulted in an increase in relative spleen and liver weights as well as in the development of skin and lung pathology in the absence of overall liver toxicity. Equimolar or higher concentrations of PCNB caused none of these effects. Urinary levels of the mercapturic acid N-acetyl-S-(pentachlorophenyl)-cysteine (PCP-NAC), were comparable in HCB- and PCNB-treated rats. Levels of closely related methylsulfide derivatives of PCP-NAC, also generated via the same mercapturic acid pathway, appeared to be significantly higher in PCNB- than in HCB-treated rats, whereas the reverse was true for the urinary levels of the oxidative metabolite pentachlorophenol (PCP). Thus, results indicate that metabolites of the mercapturic acid pathway are not involved in the induction of splenomegaly and skin and lung pathology caused by HCB exposure in BN rats and that the main urinary metabolite of HCB in these BN rats is PCP. Since PCP itself, as well as other cytochrome P450-derived metabolites from HCB, are not likely to be involved in the induction of splenomegaly and skin and lung pathology, it is suggested that either the parent compound HCB or as-yet-unidentified non-P450-generated metabolites are involved in these inflammatory effects of HCB.

Frequency-dependent effect of nitric oxide donor nitroglycerin on bone.

Recently, we showed that supplementation with nitric oxide (NO) via donor nitroglycerin (NG) alleviated the ovariectomy and corticosteroid-induced bone loss in rats. In humans, high doses or frequent applications of NG (i.e., for angina) lead to rapid loss of its efficacy in relieving angina. To examine whether there is a similar effect on the loss of efficacy of NG on bone, we examined the frequency-dependent effects of NG on bone mineral density (BMD), bone mass, trabecular bone volumes (BV/TV), and blood pressure in rats. Thirty 7-month-old female Brown Norway rats underwent ovariectomy, and an additional six rats were sham-operated. The ovariectomized rats were treated either with vehicle (ovariectomized control), 17beta-estradiol (E2; positive control), or 0.2 mg NG (via dermal application) once, twice, or three times a day. Before and at the end of the 10-week treatment period, BMD of the lumbar spine was measured by dual-energy X-ray absorptiometric (DXA) scanning and expressed as a percentage change. BMD in ovariectomized rats was significantly lower (-2.5 +/- 2.0%) compared with the sham-operated rats (+6.3 +/- 5.3%; p < 0.01). Estrogen therapy completely abolished the ovariectomy-induced potential bone loss (+5.9 +/- 3.4%). Application of NG once daily also completely prevented (+6.2 +/- 2.8%; p < 0.01) the ovariectomy-induced bone loss (i.e., it was as effective as estrogen). However, the beneficial effects of NG on BMD were significantly reduced with increased frequency of application of NG (+1.9 +/- 2.1%, twice a day and -0.2 +/- 3.3% three times a day). Estrogen or once daily administration of NG preserved femur weights, BV/TV, and decreased urinary deoxypyridinoline levels as expected. However, a higher level of serum osteocalcin and bone-specific alkaline phosphatase levels were maintained only with once daily administration of NG. There were no adverse effects of these doses of NG on blood pressure, but a tendency to lower blood pressure was noticed with increased frequency of NG. These results confirmed our previous findings that NO donors counteract the bone loss associated with estrogen deficiency. However, these beneficial effects of maintaining BMD are lost with increased frequency of NG application.

EFFECTS OF PERITRANSPLANT ADMINISTRATION OF HEMATOPOIETIC GROWTH FACTORS ON THE DEVELOPMENT OF CHRONIC REJECTION

13 Purpose: Chronic allograft rejection remains as the major obstacle to successful organ transplantation. We have previously shown that persistent existence of donor hematolymphoid cells after transplantation participated in the resistance to chronic rejection, and hypothesize that augmentation of donor chimerism after transplantation is able to prevent chronic rejection. In this study, we examined effects of perioperative administration of hematopoietic growth factors on the development of chronic rejection using simultaneous heart and bone marrow transplantation model. Results were analyzed with the correlation to levels of chimerism. Methods: Female Brown Norway (BN, RT1n) rats received heterotopic heart allografts (Htx) and simultaneous bone marrow (BM, 2.5 × 108) infusion from male Lewis (LEW, RT11) donors. All recipients received tacrolimus (1.5 mg/kg/day) on days 0 to 13, 20, and 27. Hematopoietic growth factors of variable lineage specificity (table) were injected to recipients on days 0 to 6. All recipients were sacrificed on day 100 and tissues were analyzed for levels of chimerism by PCR and Southern blot hybridization using rat sex determining region-Y (Sry) specific oligonucleotide primers and 32P-labeled probe. Severity of chronic rejection in heart allografts was analyzed by histopathology and scored. Results: At day 100, male DNAs were more easily detected in recipient non-lymphoid organs (skin, native heart) than lymphoid organ (spleen, cervical lymph nodes) or blood. Donor BM infusion significantly augmented chimerism, and Flt-3 ligand and IL-6 were able to further significantly enhance chimerism levels. Histopathological analysis demonstrated the significant amelioration of overall inflammation and obliterative arteriopathy (OA) in recipients treated with BM infusion and GCSF or IL-6 treatment. In addition, a significant regression was found between the levels of overall inflammation and chimerism. Table P values vs Htx: a<0.05, b<0.01, c<0.001, d<0.0001, vs Htx+BM: c<0.05TableConclusions: This study demonstrates that simultaneous infusion of donor BM and the appropriate use of hematopoietic growth factors are effective in enhancing chimerism and improving histopathological abnormalities associated with chronic allograft rejection.

Enhanced allergic responses to house dust mite by oral exposure to carbaryl in rats.

Epidemiological studies have demonstrated an association between use of carbamate insecticides, including carbaryl, and increased incidence of allergic asthma in farmers. In this study the effect of oral carbaryl exposure on the development of allergic responses to house dust mite (HDM) was examined in female Brown Norway rats. Rats were gavaged for 2 weeks with 0, 2, 10, or 50 mg/kg/day of carbaryl. They were sensitized with a subcutaneous injection of HDM in aluminum hydroxide adjuvant 3 days after the beginning of carbaryl exposure and challenged with antigen via the trachea 1 day after the final carbaryl ingestion. Two days after challenge, antigen-specific cell proliferation in pulmonary lymph nodes was significantly higher in the 50 mg/kg group than in controls, while antigen-specific splenocyte proliferation was decreased in groups dosed with 2, 10, and 50 mg/kg carbaryl. Total protein and lymphocyte number in bronchoalveolar lavage (BAL) fluid were also increased in the 50 mg/kg group. By 7 days after challenge, immune-mediated pulmonary inflammation (eosinophils), antigen-specific immunoglobulin (Ig) E level in serum, and antigen-specific IgE and IgA levels in BAL fluid were significantly elevated in the 50 mg/kg group. No apparent change was observed for lactate dehydrogenase and eosinophil peroxidase in BAL fluid, while the number of BAL macrophages were decreased in groups dosed with 10 and 50 mg/kg carbaryl. The results suggest that carbaryl may cause systemic immune suppression, while enhancing pulmonary allergic responses to house dust mite antigen.