BACKGROUND: Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap. METHODS: A fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks. RESULTS: Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal. CONCLUSIONS Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.
Animals ; Bioreactors ; Groin ; Male ; Perfusion ; RNA ; analysis ; Rats ; Rats, Inbred Lew ; Surgical Flaps ; Tissue Engineering

PURPOSE: This study was performed to investigate the availability of the serum HSP72 and HSP27 as serologic markers of cardiac allograft rejection through rat heterotopic heart transplatation model. METHODS: Inbred Lewis rats were randomly divided into three groups: the allograft heart transplant group, the isograft heart transplant group, and the sham-operated group. Six animals were studied in each group. In allograft heart tranplant group, the Brown Norway rats were used as donors and in isograft heart tranplant group, the Lewis rats were used as donors. The sera of the allograft heart transplanted rats, isograft heart transplanted rats, and sham- operated rats were collected at preoperative time, 3 days after operation and 6 days after operation, and analyzed for HSP72 and HSP27 by Western blots. Quantifications of band densities were carried out by laser densitometer and the results were expressed as % preoperative densities. RESULTS: The levels of serum HSP72 of 3 days and 6 days after heart transplantation significantly increased in the allograft heart transplant group than in the isograft heart transplant group, respectively (160.2+/-44.8% vs. 109.0+/-34.7%, 276.0+/-72.1% vs. 175.0+/-44.2%, P<0.05). The levels of seum HSP27 of 3 days and 6 days after heart transplantation significantly increased in the allograft heart transplant group than in the isograft heart transplant group, respectively (162.3+/-62.7% vs. 118.4+/-37.0%, 235.7+/-67.1% vs. 127.9+/-40.8%, P<0.05). CONCLUSION: It is concluded that serum HSP72 and HSP27 are useful markers to detect the cardiac allograft rejection.
Allografts ; Animals ; Blotting, Western ; Heart Transplantation ; Heart* ; Heat-Shock Proteins ; Humans ; Isografts ; Norway ; Rats* ; Rats, Inbred Lew ; Tissue Donors ; Transplantation

This study was purposed to investigate the effects of rat marrow mesenchymal stem cell (rMSC) transplantation on left ventricular (LV) function in a rat myocardial infarction model. Myocardial infarction was performed in male Lewis rats by ligating the proximal left coronary artery. Rats were randomly divided into 3 groups: sham operation group (only thoracotomy, n = 8), AMI group (DF12 injection, n = 10), rMSC group (Dil-Labeled rMSC transplantation). At 8 weeks later, the cardiac functions including left ventricular ejection fraction (LVEF), left ventricular end systolic pressure (LVESP), left ventricular end diastolic pressure (LVEDP), +dp/dtmax and -dp/dtmax were evaluated by echocardiography and cardiac catheterization. The presence and differentiation of engrafted cells were assessed. CD31 was detected by immunohistochemical staining to demonstrate neovascular formation. The results indicated that the cultured in vitro rMSC expressed CD90, CD44, CD105, CD54; did not express CD34, CD45, CD31, as compared with AMI group, rMSC group showed a significant increase of LVEF, LVESP, +dp/dtmax, -dp/dtmax and a significant decrease of LVEDP. Immunofluorescence demonstrated that some transplanted rMSCs were positive for myosin, suggesting that small number of transplanted rMSCs differentiated into cardiac-like cells. Immunostaining showed marked augmentation of capillary density in the rMSC group than that of AMI group. It is concluded that transplanted rMSCs can differentiate into cardiac-like cells and rMSC transplantation can improve LV function after myocardial infarction in rats.
Animals ; Bone Marrow Transplantation ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardial Infarction ; physiopathology ; surgery ; Rats ; Rats, Inbred Lew ; Ventricular Function, Left

BACKGROUNDWhether plasma can transfer the protective effect(s) of remote ischemic preconditioning (RIPC) between animals remains unresolved. We therefore investigated the effects of plasma collected 48 hours after transient limb ischemia on blood pressure recovery during myocardial ischemia reperfusion (IR) in homogenic rats.

METHODSPlasma was collected from Lewis rats, and the donor rats were randomly assigned to 2 groups: transient limb ischemia and control (n = 8 each). Transient limb ischemia was achieved by four cycles of 5-minute ischemia and 5-minute reperfusion by noninvasive ligation and deligation of the both legs using elastic rubber bands after anesthesia. In the control group, no ligation was performed. Forty-eight hours later, whole blood was collected, and the plasma spun off. Study Lewis rats underwent 30-minute left anterior descending coronary artery occlusion followed by 180-minute reperfusion, and were randomly assigned to 2 groups (group A and group B, n = 24 each), each further subdivided into 3 subgroups (n = 8 each). The subgroups of group A received normal saline (group A1) , plasma of control rats (group A2), plasma of transient limb ischemia rats (group A3) respectively at 1 hour before IR; the subgroups of group B received normal saline (group B1), plasma of control rats (group B2), plasma of transient limb ischemia rats (group B3) respectively at 24 hours before IR. BIOPAC systems were used to measure hemodynamics of rats during myocardial ischemiareperfusion.

RESULTSSystolic blood pressure (SBP) after IR in group B3 was different from that in groups B1 and B2 (B3 vs. B1, P = 0.007; B3 vs. B2, P = 0.039) at the beginning of reperfusion and 30 minutes after reperfusion. SBP was higher in group B3 than in groups B1 and B2 at the beginning of perfusion (B3 vs. B1, P = 0.010; B3 vs. B2, P = 0.002) and 30 minutes after reperfusion (B3 vs. B1, P = 0.001; B3 vs. B2, P = 0.001). SBP did not differ among subgroups A1, A2 and A3. Diastolic blood pressure and heart rate did not change in group A or group B.

CONCLUSIONSThe transfusion of plasma collected 48 hours after transient limb ischemia into homogenic rats 24 hours before IR can improve the SBP recovery during reperfusion. This may suggest that cardioprotective effect of late phase of RIPC is transferable via plasma.

Animals ; Blood Pressure ; physiology ; Extremities ; blood supply ; Ischemia ; Ischemic Preconditioning ; Male ; Plasma ; Rats ; Rats, Inbred Lew ; Time Factors

OBJECTIVEKupffer cells (KCs) are resident macrophages in the liver. Because the densities and sizes of KCs show a significant overlap with other sinusoidal cells, it is difficult to separate and purify them. We aim to find an improved procedure that could optimize the method for isolation of highly purified rat Kupffer cells.

METHODSIn ex vivo rat liver perfusion with pronase E and collagenase IV, density gradient centrifugation by Histodenz and selective attachment of Kupffer cells were used to isolate them. Cell proliferation and morphological characterization were studied under light phase-contrast microscopes; KCs were also studied with transmission electron microscopy and scanning electron microscopy using standard techniques. Immunocytochemistry was used to detect the expression of ED2 CD163 and lysosome associated membrane protein 2 (LAMP2). Phagocytosis of latex-beads by KCs was also studied.

RESULTSThe yield rate of KCs was 5 x 10(7) and the KCs viability exceeded 98%. The purity of KCs identified by ED2 was higher than 98%, and over 99% of the collected KCs were LAMP2 positive.

CONCLUSIONIn the future this simple, stable and effective method of collecting highly purified Kupffer cells is expected to help in further studies.

Animals ; Cell Culture Techniques ; Cell Separation ; methods ; Cells, Cultured ; Kupffer Cells ; cytology ; Male ; Rats ; Rats, Inbred Lew

The aim of this study was to investigate the immune regulatory effect of dendritic cells phagocytosing photochemotherapy-treated allogeneic spleen lymphocytes on syngeneic T cells. DA rat spleen lymphocytes were treated with 8-methoxypsoralen plus UVA irradiation (PUVA). LEW rat bone marrow-derived DCs were co-cultured with PUVA-treated DA spleen lymphocytes (PUVA-SP), and the surface markers (MHC-II, CD86 and CD40) of treated DC were detected by flow cytometry. CFSE-labeled PUVA SP were incubated with LEW DCs and the phagocytosis of DCs on PUVA-SP was observed by using fluorescent microscope. The ability of DC phagocytosing allogeneic PUVA-SP (PUVA-SP DC) to stimulate the proliferation of LEW T cells was analyzed by mixed leukocyte reactions (MLR). The production of IL-4, IL-10, IL-2, IFN-gamma in MLR culture supernatant was determined by luminex method. The results indicated that the PUVA treatment effectively induced early apoptosis of DA rat spleen lymphocytes. After co-culture, DC efficiently phagocytosed allogeneic PUVA-SP and still maintained an immature phenotype with low levels of MHC II, CD40 and CD86. PUVA-SP DC induced LEW T cell hyporesponsiveness to DA rat antigen, and led to skewing of T cell cytokine expression toward Th2 (IL-10 and IL-4). It is concluded that the PUVA-SP DC effectively down-regulate T cell response to alloantigen and induce Th2 immune deviation in vitro.
Animals ; Dendritic Cells ; cytology ; immunology ; physiology ; Flow Cytometry ; Isoantigens ; Phagocytosis ; immunology ; Photochemistry ; Rats ; Rats, Inbred Lew ; T-Lymphocytes ; immunology

OBJECTIVETo observe the effects of iodine/selenium on the function of antigen presentation of peritoneal macrophages in rats and explore the immunological mechanisms of iodine/ selenium's role in pathogenesis of autoimmune thyroid diseases (AITD).

METHODSFemale Lewis rats were randomly divided into four groups including (1) low selenium and normal iodine group (L(sE)N(I)) (2) low selenium and high iodine group (L(Se)H(I)) (3) normal selenium and normal iodine group (N(Se)N(I) ) (4) normal selenium and high iodine group (N(Se)H(I)). All rats were fed by a special diet with lower selenium and iodine in it and drunk ion-free water containing different levels of iodine and selenium for 3 months. Peritoneal macrophages of each group and OVA allergized T cells were prepared and cultured together. Then the function of antigen presentation were estimated by detecting the levels of IL-2 in the culture supernatant. The levels of the expression of co-stimulator CD86 in the spleen of each group were determined by RT-PCR.

RESULTSThe level of IL-2 in the supernatant in N(Se)H(I) (43.22 +/- 3.27) pg/ml was much stronger than N(Se)N(I) [the level of IL-2 was (25.74 +/- 2.45) pg/ml, P < 0.05]. The level of IL-2 in L(Se)N(I) (15.79 +/- 2.13) pg/ml was significantly lower than N(Se)N(I) (P < 0.05). The expression of CD86 mRNA in N(Se)H(I) (CD86/beta-actin: 0.52 +/- 0.10) were higher than N(Se)N(I) (CD86/beta-actin: 0.35 +/- 0.04), P < 0.05.

CONCLUSIONSHigh iodine could promote the presentation function of macrophages to a higher state than normal. Therefore, high iodine intake might become an importantly inducing factor in thyroid autoimmunity. Low selenium could weaken the ability of recognizing and presenting OVA antigen of peritoneal macrophages which might destroy immunological homeostasis and thus the low selenium intake might also become an inducer of AITD.

Animals ; Antigen Presentation ; drug effects ; immunology ; Female ; Iodine ; pharmacology ; Macrophages, Peritoneal ; drug effects ; immunology ; Rats ; Rats, Inbred Lew ; Selenium ; pharmacology

Animals ; Female ; Follicle Stimulating Hormone ; blood ; Iliac Vein ; Luteinizing Hormone ; blood ; Male ; Rats ; Rats, Inbred Lew ; Spermatogenesis ; Testis ; pathology ; transplantation

OBJECTIVETo evaluate the effect of emodin on hepatocellular apoptosis following orthotopic liver transplantation (OLT) in rats.

METHODThe LEW --> BN OLT models were established. A total of 24 rats were divided randomly and equally into 4 groups. Group A was treated with normal saline at dose of 0.5 mL x d(-1) intraperitoneally from 1st day to 8 th day after operation. Group B, CsA at dose of 10.0 mg x kg(-1) x d(-1). Group C, emodin at dose of 50.0 mg x kg(-1) x d(-1). Group D, CsA at dose of 10.0 mg x kg(-1) x d(-1) and emodin at dose of 50.0 mg x kg(-1) x d(-1). Fifteen days after operation, rejection active index (RAI) and hepatocellular apoptosis index (AI) was confirmed after observing the pathologic change of transplanted liver in recipients.

RESULTRespectively, the RAI of group A, B, C, D was 7.67 +/- 0.98, 5.17 +/- 0.40, 5.83 +/- 0.75, 3.83 +/- 0.75 and the AI of group A, B, C, D was 35.83 +/- 2.320, 15.83 +/- 1.33, 16.50 +/- 2.35, 11.50 +/- 1.05. The RAI and AI of group B, C, D was significantly lower than group A (P < 0.01) and group D was significantly lower than group B, C too (P < 0.05). There was no significant distinction between group B and C in RAI and AI.

CONCLUSIONEmodin has the effect of reduce the hepatocellular apoptosis following OLT in rats and the effect can stronger by CsA.

Animals ; Apoptosis ; drug effects ; Emodin ; pharmacology ; Graft Rejection ; Hepatocytes ; cytology ; drug effects ; immunology ; Liver Transplantation ; Male ; Rats ; Rats, Inbred Lew

PURPOSE: The purpose of this study is to determine the effect of platelet-rich plasma(PRP) on rat sciatic nerve regeneration in a 10mm silicone chamber. METHODS: A total of 6 inbred Lewis rats were used in this study. Bilateral sciatic neurectomy was performed on each rat. On one side, silicone chambers containing PRP solutions were implanted; on the contralateral side, the chambers without PRP were implanted as a control. In 12 weeks post-implantation, chambers were retrieved and both gastrocnemius muscles were excised. Nerves biopsy samples were examined under a light microscope after Masson trichrome staining. RESULTS: Cross sections of the midpoints of PRP treated nerves were significantly larger and appeared more mature than those of controls. CONCLUSION: Based on morphological evidence, PRP has a positive effect on neural regeneration, and it may therefore be useful for treating peripheral nerve injuries.
Animals ; Biopsy ; Light ; Muscles ; Nerve Regeneration ; Peripheral Nerve Injuries ; Platelet-Rich Plasma ; Rats ; Rats, Inbred Lew ; Regeneration ; Sciatic Nerve ; Silicones