Comparison of surface markers between human and rabbit mesenchymal stem cells.

This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines.

Characterization and spinal fusion effect of rabbit mesenchymal stem cells.

BACKGROUND: The surface markers of mesenchymal stem cells (MSCs) of rabbits have been reported only sporadically. However, interest in the spinal fusion effect of MSCs has risen recently. The purpose of this research was to study the surface markers and spinal fusion effect of rabbit MSCs. RESULTS: Of our rabbit MSCs, 2% expressed CD14, CD29, and CD45, 1% expressed CD90 and 97% expressed CD44. These results implied the MSCs were negative for CD14, CD29, CD45, and CD90, but positive for CD44. The surgical results showed that satisfactory fusion occurred in 10 rabbits (83%) in the study group and unsatisfactory fusion in 2 (17%). In the control group, satisfactory fusion was found in 3 rabbits (25%) and unsatisfactory fusion in 9 (75%). Statistical analysis showed the study group had significantly better spinal fusion results than the control group. CONCLUSIONS: The surface markers of human and rabbit MSCs are not exactly the same. Rabbit MSCs do not have positive reactivity for CD29 and CD90, which are invariably present on human MSCs. The allogeneic undifferentiated rabbit MSCs were able to promote spinal fusion and did not induce an adverse immune response.

Systemic and local reactions of a water-soluble copolymer bone on a bony defect of rabbit model.

BACKGROUND: Ostene, a synthetic water-soluble bone hemostatic agent, is commercially available. In the current study, we evaluated the systemic and local effects of this copolymer in a rabbit model. METHODS: Eighteen rabbits underwent creation of a bony defect at right iliac crest. These rabbits were then evenly divided into 3 groups. In group 1, the defect surfaces were treated with bone wax; in group 2, the defect surfaces were treated with Ostene; in group 3, the defect surfaces were not treated with anything. Then, the animals underwent blood examinations, including WBC count, CRP, and ESR at 0, 1, 3, and 6 weeks, and were killed at 6 weeks for histologic examination. Another 6 rabbits (group 4) underwent the same surgical treatment of group 2 animals but had blood examinations of BUN and creatinine. RESULTS: The blood examinations showed that the WBC count, CRP, and ESR of all the animals in the first 3 groups were within normal limits in the postoperative periods. Microscopic examinations demonstrated residual bone wax and fibrotic tissue at the defect surfaces in group 1 animals. However, there was no Ostene at the defect surfaces in group 2 animals. The groups 2 and 3 animals showed no fibrotic tissue at the defect surfaces. The group 4 animals showed normal serum levels of BUN and creatinine in the postoperative periods. CONCLUSION: Ostene is absorbable and induces no systemic inflammation (including acute renal damage) and local inflammation in animal bodies.

Biomechanical testing of spinal fusion segments enhanced by extracorporeal shock wave treatment in rabbits.

BACKGROUND: Extracorporeal shock wave treatment (ESWT) has been proven effective in enhancing spinal fusion in a preliminary animal study. However, biomechanical tests were not performed. METHODS: All 12 rabbits in this study underwent decortication at the bilateral L5 and L6 transverse processes. Bone was chipped off and placed onto the intertransverse space. The rabbits were divided into two groups, a study group (n = 6) and a control group (n = 6). In the study group, the bilateral L5 and L6 transverse processes were treated with 1000 impulses of ESWT at 14 kilovolts (KV) (equivalent to 0.18 mJ/mm(2)) at 12 and 18 weeks after surgery. The control group rabbits did not undergo ESWT. A series of radiographic examinations on each rabbit were subsequently performed. All rabbits were killed at 21 weeks, and their spines were harvested for biomechanical tests. RESULTS: Radiographic examination showed 5 of the 6 rabbits in the study group had callus formation in the fusion masses. Biomechanical tests of the fusion segments showed that the mean flexion stiffness (with internal control) in the study group was 2.11 +/- 0.46, while that in the control group was 1.17 +/- 0.19. The mean extension stiffness (with internal control) in the study group was 1.70 +/- 0.39, while that in the control group was 1.23 +/- 0.29. Statistical analysis showed that the fusion segments in the study group had significantly better flexion and extension stiffness than those in the control group (p < 0.05). CONCLUSION: In this animal study, radiographic examinations showed that ESWT stimulated new bone growth. Biomechanical tests showed that ESWT significantly increased the flexion and extension stiffness of spinal fusion segments.

Diabetes mellitus downregulates expression of connexin43 in rat aortic medial smooth muscle cells and can be reversed by simvastatin and losartan therapy.

BACKGROUND: Diabetes mellitus (DM) plays a crucial role in the pathogenesis of initiation and propagation of atherosclerosis. Although previous studies have suggested that interactions between cells form the framework for understanding the pathogenesis of atherosclerosis, little is known about how DM impacts intercellular communication within arteries, which occurs via connexin43 (Cx43) gap junctions (GJs). This study tested the hypothesis that DM suppresses expression of Cx43 GJs, and that this suppression can be abrogated via simvastatin or losartan treatment. METHODS: An experimental model of DM (induced by streptozocin 60 mg/kg body weight) in adult male rats (n = 24) was utilized to investigate Cx43 expression in the aorta. These rats were divided into group I (insulin therapy only), group II (insulin plus simvastatin 20 mg/kg/day) and group III (insulin plus losartan 20 mg/kg/day). Twenty-four diabetic rats and 8 healthy rats (group IV) were sacrificed 3 weeks after DM induction for Western blot and immunofluorescence analysis. RESULTS: By day 21, the blood sugar level was significantly higher than the respective baseline values in groups I, II and III (all values of p < 0.0001). Additionally, the final blood sugar levels of groups I-III were significantly higher than that of group IV (p < 0.0001). The final body weight in group IV was significantly higher than that in groups I-III (all values of p < 0.0001). Experimental results demonstrated that Cx43 expression in the aortic wall did not differ among groups II-IV (p > 0.1). However, compared with groups II-IV, Cx43 expression in the aortic wall was significantly mitigated in group I (all values of p < 0.05). Western blot results showed that relative density of Cx43 to beta-actin was significantly higher in groups II-IV than in group I (p < 0.01). CONCLUSIONS: DM markedly suppressed expression of Cx43 in rat aortic walls. Both simvastatin and losartan treatment significantly reversed the effects of DM on integrity of Cx43 expression.

Vulnerability of the spinal cord to injury from extracorporeal shock waves in rabbits.

We studied the vulnerability of the spinal cord to extracorporeal shock wave treatment (ESWT). In this experiment, 12 rabbits were divided into three groups (4 in each group). All animals underwent a preceding lumbar laminectomy at L4 1 week before ESWT. In group 1, 2000 impulses of high dose (0.62 mJ/mm2 energy flux density) shockwave energy were applied to the spinal cord at the laminectomy site. In group 2, 2000 impulses of low dose (0.18 mJ/mm2 energy flux density) shockwave energy were applied to the same site as group 1. Group 3 did not receive ESWT and served as a control. None of the rabbits in the study groups (groups 1 and 2) showed weakness or paralysis of the hind limbs throughout the entire post-ESWT period. The spinal cord at the L4 level of all animals was harvested on day 13 after laminectomy. On gross morphology, the cord from the study groups and the control group showed normal surface appearance. On microscopic examination, the cord from the control group was normal, whereas the cords from the study groups showed varying degrees of myelin damage and neuronal loss. These microscopic findings were dose-dependent. For the low-energy group (group 2), neuronal loss was insignificant compared to that in the control group. ESWT produced varying degrees of microscopic changes of the treated cords, but no neurological symptoms. The neuronal injury was dose-dependent and mild in the low-energy group.

Impact of diabetes on cardiomyocyte apoptosis and connexin43 gap junction integrity: role of pharmacological modulation.

The integrity of myocardial structures plays a crucial role in signal transductions and cardiac function. The aim of this study was to test the hypothesis that diabetes mellitus (DM) exerts adverse effects on the integrity of gap junctions (GJs) and induces cellular apoptosis in rat cardiomyocytes that can be abolished by simvastatin or losartan therapy. An experimental model of DM (induced by streptozocin 60 mg/kg body weight) in adult male rats (n = 24) was utilized to investigate the integrity of GJs containing connexin43 (Cx43) and the incidence of cellular apoptosis in the left ventricular myocardium. These rats were divided into 3 groups; group I (insulin therapy only), group II (insulin plus simvastatin 20 mg/kg/day), and group III (insulin plus losartan 20 mg/kg/day). Diabetic rats and 8 healthy rats (group IV) were sacrificed at 3 weeks following DM induction for immunofluorescence analysis. The experimental results demonstrated that the number of intact Cx43 GJs and the integrated area (mum(2)) constituted by clusters of Cx43 spots were significantly higher in groups II and IV than in group III, and in groups II-IV than in group I (all P values < 0.05). Additionally, the number of apoptotic bodies was remarkably higher in group I than in groups II-IV, and notably higher in groups II-III than in group IV (all P values < 0.05). Simvastatin is more effective than losartan at inhibiting the effects of DM on the integrity of myocardial ultrastructures. Both drugs effectively prevent cellular apoptosis in diabetic rat heart.

Effect of glucocorticoid pretreatment on oxidative liver injury and survival in jaundiced rats with endotoxin cholangitis.

INTRODUCTION: Biliary tract infection is associated with high mortality. This study investigated the effect of glucocorticoid pretreatment on lipopolysaccharide (LPS)-induced cholangitis. METHODS: Rats undergoing either sham operation or ligation of the extrahepatic bile duct (BDL) for 2 weeks were randomly assigned to receive intravenous injections of dexamethasone (DX) or normal saline (NS) prior to infusing LPS into the biliary tract. The plasma levels of tumor necrosis factor-alpha (TNFalpha), chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) as well as liver mRNA expression of MCP-1 and MIP-2 were determined. Infiltration of monocytes, Kupffer cells, and neutrophils in rat liver were studied with immunohistochemistry. Oxidative liver injury was measured by the malondialdehyde (MDA) content. RESULTS: Dexamethasone pretreatment resulted in significantly decreased plasma levels of TNFalpha at 1 hour, MCP-1 and MIP-2 at 2 and 3 hours, and decreased liver MCP-1 mRNA expression at 3 hours following LPS infusion in BDL-DX rats than in BDL-NS rats. The number of inflammatory cells in the liver was significantly different between sham- and BDL-treated rats but was not affected by DX pretreatment. Pretreatment with DX resulted in significantly decreased liver MDA contents in the BDL-DX group than that in the BDL-NS group. Jaundiced rats pretreated with 5 mg DX prior to infusion of 1 g of LPS were 6.8 times more likely to survive than those that were not pretreated. CONCLUSIONS: Pretreatment of jaundiced, LPS-treated rats with a supraphysiological dose of dexamethasone may rescue their lives by suppression of chemokine expression and alleviation of oxidative liver injury.

Glucocorticoid pretreatment suppresses chemokine expression and inflammatory cell infiltration in cholestatic rats receiving biliary intervention.

AIM: Biliary intervention may augment chemokine expression and inflammatory cell infiltration and aggravates liver injury in cholestatic rats. We tested the efficacy of glucocorticoid pretreatment to prevent the complications. METHODS: A model of biliary intervention was established in rats without (sham) or with bile duct ligation (BDL). Before biliary intervention, rats were randomly assigned to receiving intravenous injection of dexamethasone (DX group) or normal saline (NS group). Plasma levels of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) were measured with enzyme-linked immunosorbent assay, and liver messenger RNA of these chemokines was quantified with real-time quantitative reverse transcriptase-polymerase chain reaction. Monocytes, Kupffer cells, and neutrophils in the rat liver were characterized with antibodies to ectodermal dysplasia 1 (ED1), ED2, and myeloperoxidase, respectively. RESULTS: By 3 hours after biliary intervention, plasma MCP-1 and MIP-2 proteins in BDL-NS rats were significantly higher than in BDL-DX. At 3 hours, liver MCP-1 and MIP-2 messenger RNA levels were significantly upregulated in BDL-NS than in BDL-DX. The amount of ED1-, ED2- and myeloperoxidase-staining cells were significantly greater in BDL-NS than in BDL-DX. Most of the changes returned to baseline levels by 24 hours. CONCLUSION: Glucocorticoid pretreatment suppresses chemokine expression and inflammatory cell infiltration, which may consequently alleviate liver injury in cholestatic rats receiving biliary intervention.

Biliary intervention augments chemotactic reaction and aggravates cholestatic liver injury in rats.

BACKGROUND: Intervention of the biliary system is frequently done in patients with obstructive jaundice and is associated with significant morbidity and mortality. The pathogenesis is unknown. MATERIALS AND METHODS: A rat model of bile duct ligation (BDL) for 2 weeks was established in which biliary intervention was feasible by injection of normal saline through an indwelling catheter in the bile ducts. Plasma levels of C-C chemokine MCP-1 and C-X-C chemokine MIP-2 were measured by using ELISA. Blood monocytes, Kupffer cells, and neutrophils in the liver were characterized with antibodies to ED1, ED2, and myeloperoxidase (MPO). Lipid peroxidation was measured by malondialdehyde contents and apoptosis by TUNEL stain of the liver. RESULTS: Biliary intervention resulted in an increase of plasma MCP-1 and MIP-2 proteins by 1 h, which declined to normal level by 3 h in both sham and BDL rats. The levels in BDL rats were significantly higher than in sham at most points. There was a transient increase of ED1- and ED2-positive cells and MPO-staining cells in sham rat liver by 1 h after intervention. ED2-positive cells increased significantly by 1 h, while ED1- and MPO-positive cells decreased, yet insignificantly after intervention in BDL rats. The cell counts in BDL were constantly higher than in sham. Malondialdehyde increased precipitously in BDL by 3 h and was significantly higher than in sham throughout the study period. Parenchymal liver injury, manifested by elevated ALT, as well as apoptosis and necrosis of liver cells, was significantly increased in BDL rats, but not in sham rats. CONCLUSION: Biliary intervention augments chemokine expression, precipitates lipid peroxidation, and aggravates liver injury in cholestatic rats.