Male DA Rats Research Articles

Olive Leaf Polyphenols Attenuate the Clinical Course of Experimental Autoimmune Encephalomyelitis and Provide Neuroprotection by Reducing Oxidative Stress, Regulating Microglia and SIRT1, and Preserving Myelin Integrity.

Numerous evidences suggest that plant polyphenols may have therapeutic benefits in regulating oxidative stress and providing neuroprotection in many neurodegenerative diseases, including multiple sclerosis (MS). However, these mechanisms are not yet completely understood. In this study, we investigated the effect of olive leaf polyphenols on oxidative stress through oxidation marker level and activity (TBARS, SOD, and GPX) and their protein expression (SOD1, SOD2, and GPX1), as well as the protein expression of Sirtuin 1 (SIRT1) and microglia markers (Iba-1, CD206, and iNOS) and myelin integrity (proteolipid protein expression) in the brain of rats with induced experimental autoimmune encephalomyelitis (EAE) and subjected to olive leaf therapy. Experiments were performed in male EAE DA rats, which were randomly divided into 2 main groups: EAE groups treated with the therapy of olive leaf (EAE+TOL) and untreated EAE control groups. The EAE treated groups consumed olive leaf tea instead of drinking water (ad libitum) from the beginning to the end of the experiment. In addition, olive leaf extract was injected intraperitoneally (i.p.) for the 10 continuous days and started on the 8th day after EAE induction. The clinical course was monitored in both groups until the 30th day after EAE induction. Our results demonstrated that TOL attenuated the clinical course of EAE; reduced the oxidative stress (by decreasing the concentration of MDA); upregulated antioxidant enzymes (SOD1, SOD2, and GPX1), SIRT1 (overall and microglial), and anti-inflammatory M2 microglia; downregulated proinflammatory M1 type; and preserved myelin integrity. These data support the idea that TOL may be an effective therapeutic approach for treating MS and other neurodegenerative diseases.

Chronic iron overload induces gender-dependent changes in iron homeostasis, lipid peroxidation and clinical course of experimental autoimmune encephalomyelitis

To analyze iron- and gender-dependent mechanisms possibly involved in pathogenesis of multiple sclerosis (MS) in this study we evaluated the effects of iron overload (IO) on iron status and lipid peroxidation processes (LPO) in tissues of female and male DA rats during chronic relapsing experimental autoimmune encephalomyelitis, a well-established MS animal model.Rats were treated by iron sucrose (75mg/kg bw/day) or with saline solution during two weeks before the sensitization with bovine brain homogenate in complete Freund's adjuvant. Clinical signs of EAE were monitored during 29 days. Serum and tissues of CNS and liver were sampled before immunization and at day 13th post immunization (during acute phase of EAE). The determination of ferritin, iron, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and evaluation of histopathology were performed by ELISA, ICP spectrometry and immunohistochemistry.Results showed that IO in female EAE rats accelerated the onset of disease. In contrast, in male rats it accelerated the progression of disease and increased the mortality rate. During acute phase of EAE female IO rats sequestered more Fe in the liver, spinal cord and in the brain and produced more ferritin than male EAE rats. Male rats, however, reacted on IO by higher production of MDA or 4-HNE in the neural tissues and showed greater signs of plaque formation and gliosis in spinal cord.The data point to sexual dimorphism in mechanisms that regulate peripheral and brain iron homeostasis and imply that men and women during MS might be differentially vulnerable to exogenous iron overload.

Chronic central serotonin depletion attenuates ventilation and body temperature in young but not adult Tph2 knockout rats.

Genetic deletion of brain serotonin (5-HT) neurons in mice leads to ventilatory deficits and increased neonatal mortality during development. However, it is unclear if the loss of the 5-HT neurons or the loss of the neurochemical 5-HT led to the observed physiologic deficits. Herein, we generated a mutant rat model with constitutive central nervous system (CNS) 5-HT depletion by mutation of the tryptophan hydroxylase 2 (Tph2) gene in dark agouti (DA(Tph2-/-)) rats. DA(Tph2-/-) rats lacked TPH immunoreactivity and brain 5-HT but retain dopa decarboxylase-expressing raphe neurons. Mutant rats were also smaller, had relatively high mortality (∼50%), and compared with controls had reduced room air ventilation and body temperatures at specific postnatal ages. In adult rats, breathing at rest and hypoxic and hypercapnic chemoreflexes were unaltered in adult male and female DA(Tph2-/-) rats. Body temperature was also maintained in adult DA(Tph2-/-) rats exposed to 4°C, indicating unaltered ventilatory and/or thermoregulatory control mechanisms. Finally, DA(Tph2-/-) rats treated with the 5-HT precursor 5-hydroxytryptophan (5-HTP) partially restored CNS 5-HT and showed increased ventilation (P < 0.05) at a developmental age when it was otherwise attenuated in the mutants. We conclude that constitutive CNS production of 5-HT is critically important to fundamental homeostatic control systems for breathing and temperature during postnatal development in the rat.

Nitric oxide‐dependent long‐term depression but not endocannabinoid‐mediated long‐term potentiation is crucial for visual recognition memory

Synaptic plasticity in perirhinal cortex is essential for recognition memory. Nitric oxide and endocannabinoids (eCBs), which are produced in the postsynaptic cell and act on the presynaptic terminal, are implicated in mechanisms of long-term potentiation (LTP) and long-term depression (LTD) in other brain regions. In this study, we examine these two retrograde signalling cascades in perirhinal cortex synaptic plasticity and in visual recognition memory in the rat. We show that inhibition of NO-dependent signalling prevented both carbachol- and activity (5 Hz)-dependent LTD but not activity (100 Hz theta burst)-dependent LTP in the rat perirhinal cortex in vitro. In contrast, inhibition of the eCB-dependent signalling prevented LTP but not the two forms of LTD in vitro. Local administration into perirhinal cortex of the nitric oxide synthase inhibitor NPA (2 μm) disrupted acquisition of long-term visual recognition memory. In contrast, AM251 (10 μm), a cannabinoid receptor 1 antagonist, did not impair visual recognition memory. The results of this study demonstrate dissociation between putative retrograde signalling mechanisms in LTD and LTP in perirhinal cortex. Thus, LTP relies on cannabinoid but not NO signalling, whilst LTD relies on NO- but not eCB-dependent signalling. Critically, these results also establish, for the first time, that NO- but not eCB-dependent signalling is important in perirhinal cortex-dependent visual recognition memory.

Expression of Receptor Activator of Nuclear Factor-Kappa B Ligand in Leukocytes During Acute Kidney Rejection After Transplantation in Rats

BackgroundAcute cellular rejection of the transplanted kidney is an important cause of impaired graft function. One of the basic characteristics of acute cellular rejection according to the latest Banff classification of renal allograft pathology is the presence of a large number of T lymphocytes in the allografted tissue. Osteoprotegerin, receptor activator of nuclear factor-kappa B (RANK) and RANK ligand (RANKL), three relatively novel members of the tumor necrosis factor superfamily, have crucial roles not only in physiologic and pathologic bone metabolism but also in immunologic processes. The aim of our study was to determine the expression of RANKL and RANK by T lymphocytes and macrophages in acute cellular kidney allograft rejection in rats. MethodsThe study included 15 male Wistar rats of 3 months old and 250–300 g as recipients and 15 male DA rats donors of 3 months old; and weight 250–300 g. When animals were sacrificed at 3 weeks to extract the transplanted kidney for pathohistologic analysis and immunoflorescence. all samples showed acute cellular rejection. Kidney sections were examined by dual-labeled immunofluorescence to detect CD4, CD8, or CD68 (red) and RANK or RANKL (green) with coexpressing cells as orange. ResultsRANKL-positive expression colocalized with CD4+ and CD8+ T lymphocytes in acutely rejected kidney tissue. There was no association between CD4+ and CD8+ T cells with RANK expression, which was evident by infiltrating CD68-positive macrophages in the kidney tissue interstitium. ConclusionRANK and RANKL were expressed by T lymphocytes and macrophages in acute cellular kidney rejection after transplantation in rats.

Expression of Receptor Activator of Nuclear Factor-Kappa B Ligand in Leucocytes During Acute Kidney Rejection after Transplantation in Rats

Introduction: Acute cellular rejection of the transplanted kidney is an important cause of impaired graft function. One of the basic characteristics of acute cellular rejection according to the latest Banff classification of renal allograft pathology is the presence of T lymphocytes in a large number in allograft tissue. Recent studies have shown the important role of T lymphocytes and macrophages in acute cellular rejection of kidney allograft. Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B (RANK) and RANK ligand (RANKL) are three relatively novel members of the tumor necrosis factor (TNF) superfamily. They have a crucial role in physiological and pathological bone metabolism but also in immunological processes. The aim of our study was to determine the expression of RANKL and RANK by T lymphocytes and macrophages in acute cellular kidney allograft rejection in rats. Methods: The study included 15 male Wistar rats (3 months old; weight 250-300g) as recipients and 15 male DA rats (3 months old; weight 250-300g) as donors. Animals were sacrificed after 3 weeks and the transplanted kidney was extracted and processed for pathohistological analysis and immunoflorescence. The latest Banff classification of renal allograft pathology was used on all tissue samples by two experienced pathologists. In all tissue samples acute cellular rejection was proven. Acute cellular rejection kidney sections were examined by dual- labeled immunofluorescence to detect CD4, CD8 or CD68 (red) and RANK or RANKL (green) allowing clear definition of cells that co-express both (orange). Results: Cytoplasmic and on surface granular RANKL immunoreaction on fluorescence microscopy of various intensities was detected in all tissue samples. RANKL positive expression was colocalized with CD4+ and CD8+T lymphocytes in acutely rejected kidney tissue. There was no association between CD4+ and CD8+T cells with RANK expression in any obtained sample. RANK but not RANKL, was expressed by infiltrating CD68 positive macrophages in the interstitium of kidney tissue. Conclusion: RANK and RANKL are expressed by T lymphocytes and macrophages in acute cellular kidney rejection after transplantation in rats. They have a possible immunomodulatory role in acute cellular kidney rejection. Serum concentration of RANKL could be a marker for early detection of acute cellular kidney rejection.

Critical Role of VEGF-C/VEGFR-3 Signaling in Innate and Adaptive Immune Responses in Experimental Obliterative Bronchiolitis

Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.

Total body irradiation of donors can alter the course of tolerance and induce acute rejection in a spontaneous tolerance rat liver transplantation model

Liver transplantation is an established therapy for end-stage liver diseases. Graft rejection occurs unless the recipient receives immunosuppression after transplantation. This study aimed to explore the mechanism of acute rejection of liver allografts in rats pre-treated with total body irradiation to eliminate passenger lymphocytes and to define the role of CD4(+)CD25(+) regulatory T cells in the induction of immunotolerance in the recipient. Male Lewis rats were used as donors and male DA rats were recipients. Rats were randomly assigned to the following four groups: control group, homogeneity liver transplantation group, idio-immunotolerance group and acute rejection group. After transplantation, the survival time of each group, serum alanine aminotransferase, total bilirubin levels, number of Foxp3(+)CD4(+)CD25(+) regulatory T cells, expression of glucocorticoid-induced tumor necrosis factor receptor on T cell subgroups, histopathology of the hepatic graft and spleen cytotoxic T lymphocyte lytic activity were measured. In the acute rejection group, where donors were preconditioned with total body irradiation before liver transplantation, all recipients died between day 17 and day 21. On day 14, serum alanine aminotransferase increased significantly to (459.2±76.9) U L(-1), total bilirubin increased to (124.1±33.7) μmol L(-1) (P<0.05) and the ratio of Foxp3(+)CD4(+)CD25(+) regulatory T cells decreased significantly to 1.50%±0.50% (P<0.05) compared with the other groups. Analysis of the T cell subpopulations in the acute rejection group varied from the other groups. Histological analysis showed typical changes of acute rejection in the acute rejection group only. Preconditioning of the donors with total body irradiation eliminated passenger lymphocytes of the liver graft, and thus affected the course of tolerance and induced acute rejection after liver transplantation.

The fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats

The aim of the study was to investigate the fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats. The iliac bone of male inbred DA rats was harvested and used as the larger bone grafts and also prepared as fine particulate (granulated) bone powders (300–500μm size particles) for transplantation into radial defects in female rats. The presence and relative amounts of genes specific to the sex-determining region of the Y-chromosome (Sry) originating from the bone grafts were evaluated by polymerase chain reaction and by in situ hybridization, respectively. Additional samples were evaluated histologically. In the larger bone grafts, the expression of Sry decreased relatively early, disappeared by 1 week, reappeared at 4 weeks and continued to increase with time. In the fine particulate bone powders, Sry was detected all the time and its expression was statistically greater than in the larger bone grafts at each time point. Both bone grafts provided donor cells to repair the defects. The donor cells seemed to function differently between the two groups. The fine particulate bone powders contained more living osteocytes in comparison with the larger bone grafts and may accelerate the healing of bone defects compared with conventional autografts.

Chiral analyses of dextromethorphan/levomethorphan and their metabolites in rat and human samples using LC-MS/MS

In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid-acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out using authentic human samples.

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell–related cytokines.

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter > 150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter < 100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.

Induction of Lymphocyte Apoptosis in Rat Liver Allograft by Adenoviral Gene Transfection of Human Interleukin-10

Background/Aims: Gene therapy can provide a possible avenue in organ transplantation to treat acute allograft rejection. This study was designed to investigate the effect of adenovirus-mediated human IL-10 (hIL-10) gene transfer on the apoptosis of infiltrating lymphocytes and examine the efficacy of hIL-10 gene transfer in combination with subtherapeutic doses of cyclosporine A (CsA) in a rat liver transplantation model. Methods: Inbred male DA and LEW rats were used for liver donors and recipients, respectively. The rats were divided into saline, Ad-lacZ, CsA, Ad-hIL-10 and Ad-hIL-10 + CsA groups. Graft survival, histopathological, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction and flow cytometry were performed in liver specimens obtained from different time points after transplantation in the 5 groups. Results: Ad-hIL-10 pretreatment inhibited allograft rejection, prolonged the survival of hepatic allografts, and downregulated the expression of IFN-γ and IL-2 mRNA, with simultaneous upregulation of IL-4 mRNA. In addition, Ad-hIL-10 pretreatment upregulated the expression of Fas mRNA in the isolated graft-infiltrating lymphocytes and induced graft-infiltrating lymphocyte apoptosis. A single subtherapeutic dose of CsA acted synergistically with it. Conclusion: hIL-10 gene therapy induced alloreactive lymphocyte apoptosis via Fas/FasL pathway. hIL-10 gene transfection in combination with subtherapeutic doses of CsA facilitates the long-term survival of liver grafts.

Total body irradiation of the donor in a spontaneous tolerance rat liver transplantation model and the effects on CD4(+)CD25(+) regulatory T cells content

To study the effect of total body irradiation of the donor in a spontaneous tolerance rat liver transplantation model and the role of CD4(+)CD25(+) regulatory T cells on induction of immunotolerance in the recipient. Liver transplantation was performed using male Lewis rats as donors and male DA rats as recipients. These rats were randomly allocated into the following groups:Control group, Homogeneity Liver Transplantation group, Idio-immunotolerance group and Acute Rejection group. After transplantation, survival time rate of each group were observed. Serum ALT, TB level, Foxp3(+)CD4(+)CD25(+) regulatory T cells, expression of GITR on T cell subgroup, histopathology of the hepatic graft on day 14, spleen CTL lytic activity on day 14 were measured. In the Idio-immunotolerance group, the recipients suffered from transient rejection after surgery but acquired immunotolerance and survived long. In the Acute Rejection group, the donors were preconditioned with total body irradiation before liver transplantation. All recipients died between day 17 to 21. Serum ALT and TB increased significantly and the ratio of Foxp3(+)CD4(+)CD25(+) regulatory T cells decreased significantly compared with the Idio-immunotolerance group, the Homogeneity Liver Transplantation group and the Control group. The expression of GITR on CD3(+)CD4(+)T cells in the peripheral blood decreased, the expression of GITR on CD3(+)CD8(+) T cells and CTL lytic activity of the recipients increased by preconditioning of the donors with total body irradiation. Preconditioning of the donors with total body irradiation eliminated the passenger lymphocytes of the liver graft, decreased the expression of Foxp3(+)CD4(+)CD25(+) regulatory T cells in peripheral blood, and increased the expression of GITR on CD3(+)CD8(+) T cells, thus affected the course of tolerance and induced acute rejection after liver transplantation.

Recognition Memory for Objects, Place, and Temporal Order: A Disconnection Analysis of the Role of the Medial Prefrontal Cortex and Perirhinal Cortex

Recognition memory requires judgments of the previous occurrence of stimuli made on the basis of the relative familiarity of individual objects, or by integrating information concerning objects and location, or by using recency information. The present study examined the role of the medial prefrontal cortex (mPFC) and perirhinal cortex (PRH) in these distinct recognition memory processes using a series of behavioral tests: a novel object preference task, an object-in-place task, and a temporal order memory task. Also, a disconnection procedure was used to test whether these regions form components of an integrated system for recognition memory. Male DA rats received bilateral lesions in the PRH or mPFC or unilateral lesions placed in both cortices in either the same (PRH-mPFC IPSI) or contralateral (PRH-mPFC CONTRA) hemispheres. A fifth group underwent sham surgery (SHAM). In the object-in-place and temporal order memory tasks, the PRH, mPFC, and PRH-mPFC CONTRA groups were significantly impaired. However, performance in the novel object preference task was only impaired in the PRH group. No group was impaired in the object location task. These results demonstrate that the mPFC and PRH are crucial for object-in-place associational and recency discriminations, whereas the PRH but not the mPFC is important for the discrimination of novel and familiar individual objects. Importantly, these results provide direct support for the hypothesis that to make discriminations based on associational or recency information, both cortical regions operate within an integrated neural network for recognition memory.

In Vivo Assessment of Resting Myocardial Function in High and Low Performing Genetic Models of Aerobic Running Capacity

BACKGROUND: We previously identified two inbred rat strains divergent for treadmill aerobic running capacity (ARC); the low performing Buffalo (BUF) and the high performing DA rats. Subsequent cardiac performance assessment using the Langendorff-Neely working heart preparation demonstrated that isolated DA hearts exhibit increased stroke volume and cardiac output compared to the BUF hearts, with no difference in intrinsic heart rate (HR). In the present study, we investigated in vivo resting cardiac function in BUF and DA rats using echocardiography. METHODS: Male DA (n=11) and BUF (n=12) rats (aged 14-17 weeks) were lightly anesthetized with pentobarbital (40mg/kg, ip), the chest was shaved, and the animal was situated in the supine position on a warming pad. Two-dimensional guided M-mode studies were performed from the parasternal long axis for the determination of left ventricular end-diastolic (LVDd) and end-systolic (LVSd) diameters and fractional shortening (FS). The myocardial performance index (MPI), stroke volume (SV) and cardiac output (CO) were calculated using Doppler pulsed-wave traces of mitral and aortic flow measured at the level of the LV outflow tract from the apical four-chamber view. Cardiac index (CI) was calculated by dividing CO by body weight. RESULTS: BUF rats had significantly greater body weight and a larger SV than the DA rats, but had significantly lower HR and CI (Table). No differences in LVDd, LVSd, FS, MPI, or CO were observed (Table).TableCONCLUSION: After correcting for differences in HR and body weight, in vivo assessment of resting cardiac function revealed that DA rats exhibit greater cardiac performance than BUF rats. Our findings contrast with those of the working heart preparation in that in vivo assessment of cardiac function shows DA rats to have a smaller stroke volume and greater HR than the BUF rats, resulting in an identical CO between the two strains. Nevertheless, cardiac index is significantly greater in DA rats, possibly contributing to the superior ARC observed in this rat strain compared to that of the BUF strain.

MDMA‐induced neurotoxicity: long‐term effects on 5‐HT biosynthesis and the influence of ambient temperature

1. 3,4-Methylenedioxymethamphetamine (MDMA or 'ecstasy') decreases the 5-HT concentration, [3H]-paroxetine binding and tryptophan hydroxylase activity in rat forebrain, which has been interpreted as indicating 5-HT neurodegeneration. This has been questioned, particularly the 5-HT loss, as MDMA can also inhibit tryptophan hydroxylase. We have now evaluated the validity of these parameters as a reflection of neurotoxicity. 2. Male DA rats were administered MDMA (12.5 mg kg(-1), i.p.) and killed up to 32 weeks later. 5-HT content and [3H]-paroxetine binding were measured in the cortex, hippocampus and striatum. Parallel groups of treated animals were administered NSD-1015 for determination of in vivo tryptophan hydroxylase activity and 5-HT turnover rate constant. 3. Tissue 5-HT content and [3H]-paroxetine binding were reduced in the cortex (26-53%) and hippocampus (25-74%) at all time points (1, 2, 4, 8 and 32 weeks). Hydroxylase activity was similarly reduced up to 8 weeks, but had recovered at 32 weeks. The striatal 5-HT concentration and [3H]-paroxetine binding recovered by week 4 and hydroxylase activity after week 1. In all regions, the reduction in 5-HT concentration did not result in an altered 5-HT synthesis rate constant. 4. Administering MDMA to animals when housed at 4 degrees C prevented the reduction in [3H]-paroxetine binding and hydroxylase activity observed in rats housed at 22 degrees C, but not the reduction in 5-HT concentration. 5. These data indicate that MDMA produces long-term damage to serotoninergic neurones, but this does not produce a compensatory increase in 5-HT synthesis in remaining terminals. It also highlights the fact that measurement of tissue 5-HT concentration may overestimate neurotoxic damage.

The fate of donor bone cells in different diameter bone grafts: an experiment study

To investigate the fate of donor bone cells in bone grafts of different diameters during repairing bone defects. One hundred sixteen female syngeneic inbred DA rats were established as radial defect model of 3 kinds: structural bone grafting group (n = 56), morselized bone grafting group (n = 56), and blank group (without bone grafting, n = 4), to be used as receptors The ilia of 58 male inbred DA rats, as donors, were harvested and made into structural bone grafts 2 mm in diameter and morselized bone grafts 0.3 - 0.5 mm in diameter to be transplanted into the radial defects of the female receptors. One and four days, and 1, 2, 4, 6, and 8 weeks after transplantation DNA was extracted from the grafted bones and polymerase chain reaction (PCR) specific for the sex-determining region of Y-chromosome (Sry) was performed to observe the presence and relative amount of Y-chromosome originating from the bone grafts, expression of the sex-determining gene Sry in the receptors' bones and the histology of the receptors' bones. In structural bone grafting group, the amounts of Sry-specific bands decreased in the early time and disappeared 1 week after transplantation, and re-appeared 4 weeks after transplantation with the amount increasing with the lapse of time. In morselized bone grafting group, Sry-specific bands were detected all the time but their amounts decreased with the lapse of time. At each time point, morselized bone graft provided more living osteocytes with better effect of osteogenesis in comparison with the structural bone graft. Bone grafts of different diameters provide donor cells in repairing bone defects. Having more surviving osteocytes, morselized bone grafts may accelerate the healing pf bone defects, thus providing a new and effective method to repair bone defects and spinal fusion clinically.

THE POTENTIAL ROLE OF ALLOGRAFT INFLAMMATORY FACTOR 1 (AIF-1) IN MACROPHAGE ACTIVATION IN SMALL-FOR-SIZE LIVER TRANSPLANTATION

P967 Aims: Our previous study demonstrated that early macrophage activation was related to inflammatory and acute rejection processes in small-for-size allografts. However, the mechanisms that lead to early macrophage activation in the small-for-size liver grafts remain unclear. The present study aims to identify the potential role of allograft inflammatory factor 1 (AIF-1) in mediating macrophage activation in small-for-size liver transplantation. Methods: An orthotopic liver transplantation model was performed. Male DA (RT1a) and LEW (RT1l) rats, weighing 250-300 g, were used as donors and recipients, respectively. Grafts were either 40% of recipients’ livers or whole grafts. Experimental groups included: 1) whole isograft; 2) 40% isograft; 3) whole allograft; and 4) 40% allograft. No treatment was given to all the groups. Tissue samples were collected at 1, 6, 24, and 72 hr after reperfusion. The expression of AIF-1 was detected at both mRNA (by RT-PCR) and protein (by Western blot) levels. In the in vitro study, the expression of AIF-1 in macrophage cell lines was measured after the animals were treated with pro-inflammatory cytokines IL-1β, TNF-α, and anti-inflammatory drug sodium salicylate. In addition, the macrophage cell lines were transfected with PCDNA3.1/AIF-1 (to enhance AIF-1 expression) or short interference RNA (siRNA to block AIF-1 expression). The expression of AIF-1 and iNOS in macrophages was detected, and cell migration assay was performed. Results: The expression of AIF-1 was significantly increased at both mRNA and protein levels in the small-for-size liver grafts in both isogeneic and allogeneic settings, starting from 1 hr to 72 hr after reperfusion. Immunohistochemical staining revealed that at 1 and 6 hr after reperfusion, the majority of AIF-1 positive cells were Kupffer cells, and at 72 hr after reperfusion, AIF-1 was also expressed in some infiltrating cells in the peri-portal area of liver allografts. Pro-inflammatory cytokines IL-1β and TNF-α could stimulate AIF-1 upregulation, whereas anti-inflammatory treatment by sodium salicylate could downregulate the expression of AIF-1. Transfection of AIF-1 to macrophage cell lines significantly enhanced AIF-1 expression. At the same time, the level of iNOS was also upregulatd and the number of migrated macrophages was increased. Blocking the expression of AIF-1 by siRNA significantly decreased the level of iNOS and inhibited the migration of macrophages. Conclusions: The expression of AIF-1 is upregulated in the small-for-size liver grafts during early phase after reperfusion; the expression of AIF-1 is related to iNOS production and migration activities of macrophages.

EFFECTS OF BETA-ADRENOCEPTOR BLOCKADE ON THE PHENOTYPIC CHARACTERISTICS OF THYMOCYTES AND PERIPHERAL BLOOD LYMPHOCYTES

The study revealed that β-adrenoceptor blockade with propranolol (0.40 mg/100 g/day, s.c.) in adult male DA rats: (i) increased the thymocyte proliferation and apoptosis, (ii) caused disturbances in kinetics of T cell differentiation leading to distinguishable changes in relative proportion of thymocytes at distinct maturational steps and to an expansion of the most mature single positive (CD4+, CD8+) thymocyte pool, (iii) affected the relative proportion of neither CD4+ nor CD8+ peripheral blood lymphocytes (PBL), and (iv) augmented the relative number of CD8+CD25+ cells. Thus, the results suggest the role of β-adrenoceptors in fine-tuning of T cell maturation, and, possibly, distribution and activation of distinct PBL subsets