Bone Marrow Of Male Rats Research Articles

Therapeutic effects of bone marrow mesenchymal stem cells via modulation of TLR2 and TLR4 on renal ischemia-reperfusion injury in male Sprague-Dawley rats.

Introduction: Acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) injury is a pro-inflammatory process that activates toll-like receptors (TLRs). Stem cell therapy holds a great promise for kidney repair. Therefore, we investigated the immunomodulatory role of bone marrow stromal cells (BMSCs) on TLR2 and TLR4 expression in AKI in male Sprague-Dawley rats. Methods: BMSCs were isolated from the bone marrow of male rats, cultured in DMEM, and characterized using appropriate markers before transplantation. Renal I/R was induced by 45 minutes bilateral ischemia followed by 24 hours of reperfusion. Rats received intraperitoneal injections of BMSCs (1.5 × 106 cells, i.p, per rat) immediately after termination of renal ischemia. Serum samples were collected pre-and post-stem cells injection for assessment of blood urea nitrogen (BUN) and creatinine (Cr) levels. The kidneys were harvested after 24 hours of reperfusion for structural and molecular analysis. Results: Renal I/R caused severe tissue injuries and increased the level of BUN (166.5 ± 12.9 vs. 18.25 ± 1.75) and Cr (3.7 ± 0.22 vs. 0.87 ± 0.06) compared to the sham group. In addition, mRNA expression of TLR2 and TLR4 elevated in the renal I/R group. Administration of BMSCs improved the functional and structural state of the kidney induced by I/R and down-regulated TLR2 and TLR4 gene expression. Conclusion: The results showed a highly significant renoprotection by BMSCs that indicates their therapeutic potential in I/R injures. These effects are most likely associated with the TLR2/4 signaling pathway via modulation of the inflammatory response cascades.

Dihydrotestosterone and 17-Estradiol Enhancement of In Vitro Osteogenic Differentiation of Castrated Male Rat Bone Marrow Mesenchymal Stem Cells (rBMMSCs)

Background: In vitro impact of dihydrotestosterone (DHT) and 17-estradiol (E2) in osteogenic differentiation of castrated rat bone marrow mesenchymal stem cells (rBMMSC) still need to be clarified. Materials and Methods: The viability, proliferation and density of cultured rBMMSC isolated from sham operated (Sham) and castrated (Cast) male rats were evaluated. rBMMSC were cultured with osteogenic differentiating medium (ODM) in the presence of DHT (5,10 nM) and E2 (10,100 nM). Osteogenesis was evaluated by alizarin red staining and measurement of calcium deposition and bone alkaline phosphatase (B-ALP) activity. Results: Population doubling (PD) of rBMMSC isolated from Cast rats was significantly lower (P<0.05) compared to that isolated from Sham rats. rBMMSC from Cast rats showed low scattered calcified nodule after culturing in ODM and did not cause a significant increase in calcium deposition and B-ALP activity compared to rBMMSCs from Sham rats. Exposure of rBMMSC isolated from Cast rats to DHT (5 nM) or E2 (10 nM) in ODM showed medium scattered calcified nodules with significantly higher (P<0.05) calcium deposition and B-ALP activity. Moreover, exposure of rBMMSC to DHT (10 nM) or E2 (100 nM) showed high scattered calcified nodules with higher (P<0.01) calcium deposition and B-ALP activity Conclusion: These results indicated that the presence of testes might participate in controlling the in vitro proliferation and osteogenic differentiation capacity of rBMMSCs. DHT and E2 can enhance the osteogenic capacity of rBMMSCs in a dose-dependent manner. Based on these observations, optimum usage of DHT and E2 can overcome the limitations of MSCs and advance the therapeutic bone regeneration potential in the future.

Ginsenoside Rg1 protects rat bone marrow mesenchymal stem cells against ischemia induced apoptosis through miR-494-3p and ROCK-1

This study aimed to verify the cytoprotective effect of ginsenoside Rg1 in vivo, and to elucidate the mechanism of Rg1 in the ischemic microenvironment. Male rat bone marrow mesenchymal stem cells (rBMSCs) or rBMSCs treated with Rg1 were injected into ischemic region of the arterial embolism hind limb in female rats. Behavioral and histological data, obtained one-week post injection, showed that rBMSCs with Rg1 could improve the survival rate of BMSCs and enhance the therapeutic effects. rBMSCs treated with hypoxia and serum deprivation for 24h (H/SD-rBMSCs) showed the up-regulated expression of ras homolog family member A (RhoA), Rho associated coiled-coil containing protein kinase 1 (ROCK-1), myosin light chain 2 (MLC-2), Bcl2 associated agonist of cell death (Bad) and Bcl2 associated X, apoptosis regulator (Bax); while the expression of miR-148b-3p, miR-148b-5p and miR-494-3p was down-regulated. H/SD with Rg1 treatment (H/SD+Rg1-rBMSCs) inhibited the expression of ROCK-1, MLC-2, Bad and Bax, increased the expression of Bcl-2, miR-494-3p. After ROCK-1 knockout, the expression of Bad and Bax were downregulated and Bcl-2 upregulated, but Rg1 no longer altered their expression. Mir-494-3p functional study established that miR-494-3 mimic downregulated and miR-494-3 inhibitor upregulated ROCK-1 gene expression, Rg1 did not have the ability to change the ROCK gene expression after loss of function of miR-494-3p. Also, the function loss of mir-494-3p promoted apoptosis; otherwise reduced apoptosis. The anti-apoptotic effect of Rg1 disappeared after mir-494-3p loss or gain function. In conclusion, Ginsenoside Rg1 has shown to have protective effects on ischemic-induced rBMSCs apoptosis through mir-494-3p→ROCK-1→Bcl-2 signaling pathway.

A Memory of Early Life Physical Activity Is Retained in Bone Marrow of Male Rats Fed a High-Fat Diet.

Studies have reported opposing effects of high-fat (HF) diet and mechanical stimulation on lineage commitment of the bone marrow stem cells. Yet, how bone marrow modulates its gene expression in response to the combined effects of mechanical loading and a HF diet has not been addressed. We investigated whether early-life (before onset of sexual maturity at 6 weeks of age) voluntary physical activity can modulate the effects of a HF diet on male Sprague Dawley rats. In the bone marrow, early-life HF diet resulted in adipocyte hypertrophy and a pro-inflammatory and pro-adipogenic gene expression profile. The bone marrow of the rats that undertook wheel exercise while on a HF diet retained a memory of the early-life exercise. This memory lasted at least 60 days after the cessation of the voluntary exercise. Our results are consistent with the marrow adipose tissue having a unique response to HF feeding in the presence or absence of exercise.

PHYTOHORMONE INDUCED CHANGES ON BONE HISTOLOGY IN NORMAL AND DIABETIC MALE RAT

&lt;p&gt;&lt;strong&gt;Objective: &lt;/strong&gt;To study the effect of brasslinolide keto isoform 28-homocastasterone in diabetic male rat bone marrow cells, bone histological changes, electrolytes and alkaline phosphatase activity in rat blood.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;Diabetes was induced in the group (n=6) of rats with a single peritoneal injection of streptozotocin (60 mg/kg bwt). With a treatment schedule of 15 consecutive days, control (n=6) and diabetic rats received 666µg/kg bwt of 28-homocastasterone. Circulating blood cell count, cell indices, bone marrow cells, bone histology, electrolytes Na&lt;sup&gt;+&lt;/sup&gt;, K&lt;sup&gt;+&lt;/sup&gt;, Cl&lt;sup&gt;-&lt;/sup&gt;, P, Ca&lt;sup&gt;2+&lt;/sup&gt;and alkaline phosphatase activity was assessed.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;Cytological examination showed an increased erythrocyte progenitor and megakaryocyte cell lineage along with improved osteoblastic and bone histomorphology in the treated group was noted. A significant reduction in electrolytes level (p&amp;lt;0.05) and increased in alkaline phosphatase activity (p&amp;lt;0.05) was noted in treated groups.&lt;/p&gt;&lt;strong&gt;Conclusion: &lt;/strong&gt;It is suggested that brassinosteroid keto isoform 28-homocastasterone exhibits a hematopoietic effect in diabetic rat and improves bone histology while reducing hyperglycemic damage in bone.&lt;p&gt; &lt;/p&gt;

Scientific Opinion on Flavouring Group Evaluation 208 Revision 1 (FGE.208Rev1): Consideration of genotoxicity data on representatives for 10 alicyclic aldehydes with the α,β‐unsaturation in ring / side‐chain and precursors from chemical subgroup 2.2 of FGE.19

The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids was requested to evaluate the genotoxic potential of flavouring substances from subgroup 2.2 of FGE.19 in the Flavouring Group Evaluation 208 Revision 1 (FGE.208Rev1). The Flavour Industry has provided additional genotoxicity studies on p-mentha-1,8-dien-7-al [FL-no: 05.117], the representative substance for FGE.19 subgroup 2.2. This substance was tested in vivo in a combined micronucleus assay in bone marrow and Comet assay in liver and duodenum. It did not induce any increase in micronucleated polychromatic erythrocytes of the bone marrow of male rats in the micronucleus test and it did not induce DNA damage in duodenum of the same animals as analysed by the Comet assay. The Comet assay performed in liver shows a positive result and therefore the Panel concluded that p-mentha-1,8-dien-7-al [FL-no: 05.117] is genotoxic in vivo and that, accordingly, there is a safety concern for its use as flavouring substance. Since p-mentha-1,8-dien-7-al [FL-no: 05.117] is representative for the nine remaining substances of this subgroup 2.2 (p-mentha-1,8-dien-7-ol [FL-no: 02.060], myrtenol [FL-no: 02.091], myrtenal [FL-no: 05.106], 2,6,6-trimethyl-1-cyclohexen-1-carboxaldehyde [FL-no: 05.121], myrtenyl formate [FL-no: 09.272], p-mentha-1,8-dien-7-yl acetate [FL-no: 09.278], myrtenyl acetate [FL-no: 09.302], myrtenyl-2-methylbutyrate [FL-no: 09.899] and myrtenyl-3-methylbutyrate [FL-no: 09.900]), there is a potential safety concern for these substances.

Paracrine Effect of Wnt11-Overexpressing Mesenchymal Stem Cells on Ischemic Injury

Our previous studies have suggested that transduction of Wnt11 directly increases bone marrow-derived mesenchymal stem cells (MSCs) differentiation into cardiac phenotypes. In this study, we investigated whether Wnt11 enhances MSC-mediated cardioprotection via paracrine fashion after acute ischemia. MSCs were harvested from male rat bone marrow and transduced with Wnt11 (MSC(Wnt11)). An acute myocardial infarction model in rats was developed by ligation of the left anterior descending coronary artery. MSC(Wnt11) were transplanted into the peri-infarct region after acute myocardial infarction. To mimic ischemic injury, cultured cardiomyocytes (CMs) isolated from neonatal ventricles were exposed to hypoxia. ELISA studies indicated that the release of Wnt11 (3.45-fold) as well as transforming growth factor-β2 (TGFβ2) (1.5-fold) was significantly increased from MSC(Wnt11) compared with transduced control MSC (MSC(Null)). Hypoxia-induced apoptosis and cell death was significantly reduced when CM were co-cultured with MSC(Wnt11) in a dual chamber system. The cell protection mediated by MSC(Wnt11) was mimicked by treating CM with conditioned medium obtained from MSC(Wnt11) and abrogated by Wnt11- and TGFβ2 neutralizing antibodies. Further, animals receiving MSC(Wnt11) showed a significant improvement in cardiac contractile function as assessed by echocardiography. Masson trichrome and TUNEL staining showed a significant reduction in infarct size and apoptosis of CM in MSC(Wnt11)-treated animals. Transplantation of MSC(Wnt11) improved cardiac function. The release of Wnt11 and other factors from transplanted MSC(Wnt11) is more likely responsible for protection of native CM at risk.

Paracrine Effect of Wnt11 Overexpressing Mesenchymal Stem Cells on Ischemic Injury

In this study, we investigated whether Wnt11 enhances bone marrow‐derived mesenchymal stem cell (MSC) mediated cardioprotection via paracrine fashion following acute ischemia.MethodsMSCs were harvested from male rat bone marrow (BM) and transduced with Wnt11 (MSCWnt11). An acute myocardial infarction (MI) model in rats was developed by ligation of the left anterior descending coronary artery (LAD). MSCWnt11 were transplanted into the peri‐infarct region following acute MI. To mimic ischemic injury, cultured cardiomyocytes (CM) isolated from neonatal ventricles were exposed to hypoxia.ResultsELISA studies indicated that the release of Wnt11 (3.45‐fold) as well as transforming growth factor‐β2 (TGFβ2) (1.5‐fold) was significantly increased from MSCWnt11 compared to transduced control MSC (MSCNull). Hypoxia induced apoptosis and cell death was significantly reduced when CM were co‐cultured with MSCWnt11 in a dual chamber system. The cell protection mediated by MSCWnt11 was mimicked by treating CM with conditioned medium (CdM) from MSCWnt11 and abrogated by Wnt11‐ and TGFβ2‐neutralizing antibodies. Furthermore, animals receiving MSCWnt11 showed a significant improvement in cardiac contractile function as assessed by echocardiography. Infarct size measured from infarcted hearts was reduced from 44.6 ± 4.1% in control to 30.1 ± 3.0% in treated hearts (p &lt; 0.05). Similarly, apoptosis of cardiomyocytes evaluated by TUNEL staining was reduced from 9.9 ± 0.71% in control group to 5.30 ± 0.35% in treated group (p &lt; 0.05).ConclusionsTransplantation of MSCWnt11 improved cardiac function. The release of Wnt11 and other factors from MSCWnt11 transplanted in the infarcted left ventricle is more likely responsible for protection of CM at risk. This work was supported by National Institutes of Health grants: HL083236, HL105176 (M.X.) and HL087246 (M.A.).

Paracrine factors released by GATA-4 overexpressed mesenchymal stem cells increase angiogenesis and cell survival.

Transplanted mesenchymal stem cells (MSC) release soluble factors that contribute to cardiac repair and vascular regeneration. We hypothesized that overexpression of GATA-4 enhances the MSC secretome, thereby increasing cell survival and promoting postinfarction cardiac angiogenesis. MSCs harvested from male rat bone marrow were transduced with GATA-4 (MSC(GATA-4)) using the murine stem cell virus retroviral expression system; control cells were either nontransduced (MSC(bas)) or transduced with empty vector (MSC(Null)). Compared with these control cells, MSC(GATA-4) were shown by immunofluorescence, real-time PCR, and Western blotting to have higher expression of GATA-4. An increased expression of angiogenic factors in MSC(GATA-4) and higher MSC resistance against hypoxia were observed. Human umbilical vein endothelial cells (HUVEC) treated with MSC(GATA-4) conditioned medium exhibited increased formation of capillary-like structures and promoted migration, compared with HUVECs treated with MSC(Null) conditioned medium. MSC(GATA-4) were injected into the peri-infarct region in an acute myocardial infarction model in Sprague-Dawley rats developed by ligation of the left anterior descending coronary artery. Survival of MSC(GATA-4), determined by Sry expression, was increased at 4 days postengraftment. MSC(GATA-4)-treated animals showed significantly improved cardiac function as assessed by echocardiography. Furthermore, fluorescent microsphere and histological studies revealed increased blood flow and blood vessel density and reduced infarction size in MSC(GATA-4)-treated animals. We conclude that GATA-4 overexpression in MSCs increased both MSC survival and angiogenic potential in ischemic myocardium and may therefore represent a novel and efficient therapeutic approach for postinfarct remodeling.

Raloxifene: its ossification-promoting effect on female mesenchymal stem cells

BackgroundRaloxifene acts like estrogen in preventing bone loss in postmenopausal women, but it selectively activates biological responses in bone tissue. It has a direct effect on osteoblasts’ differentiation and bone formation in bone marrow culture. However, the point at which raloxifene has an effect on bone marrow-derived mesenchymal stem cells (MSCs), regardless of sex difference, is not known. The purpose of this study was to examine the osteogenic effect of raloxifene on MSCs derived from female and male rats and to assess the sex difference of raloxifene with or without osteogenic supplements (OSs) in the regulation of bone formation. MethodsFemale and male rat bone marrow cells were cultured with or without OSs. In each experimental group, 106 M or 10-8 M raloxifene was added. As a control, cells were cultured without raloxifene. Histologically, mineralization was assessed by alizarin red S staining. Biochemically, alkaline phosphatase (ALP) activity, calcium content, and osteocalcin content were assessed. ResultsOn histological analysis, mineralized nodules were seen on alizarin red S staining in the groups treated with OS. On the biochemical analysis, OS increased ALP activity, calcium content, and osteocalcin content. Among female groups with OSs, 10-6 M raloxifene significantly increased ALP activity, calcium content, and osteocalcin content compared with the controls. Among male groups, raloxifene had negligible effects. Conclusions10-6 M Raloxifene had no ossification-inducing effect on female MSCs, but it had an ossification-promoting effect; it had no osteogenic effect on male MSCs. Therefore, raloxifene has a sex difference with regard to its osteogenic effect on MSCs. Moreover, combined treatment with raloxifene plus OS has an effect on female MSCs. These results provide a useful insight into the possible influence of raloxifene after MSC transplantation in clinical practice.

Assessment of Cadmium Genotoxicity in Peripheral Blood and Bone Marrow Tissues of Male Wistar Rats

In this study, Cadmium (Cd) genotoxicity was investigated in both bone marrow and peripheral blood treatment using rat micronucleus technique as genotoxicity test at acute and chronic treatment in the same animals. This study evaluated the frequency of micronuclei in the peripheral blood and bone marrow of male rats treated with unique cadmium dose (15 mg/kg. body w/day) by gavage for 60 days and acute treatment for 24 h, respectively. Mitomycin C (MMC) 2 mg/kg body wt was used as a positive control. This study shows that cadmium chloride treatment significantly induced the frequency of micronucleus in polychromatic erythrocytes in both tibia bone marrow and peripheral blood (p < 0.001, p < 0.01, respectively). This increase in micronucleus frequency shows that cadmium has a genotoxic effect on bone marrow and peripheral blood at this level. Also, in order to determine cytotoxicity in bone marrow and peripheral blood, the ratio of polychromatic erythrocytes to normochromatic erythrocytes was calculated in bone marrow and peripheral blood. Cd treatment decreased this ratio in only bone marrow. The results of this study demonstrate that Cd has both toxic and genotoxic potential in bone marrow and only genotoxic potential in peripheral blood. There is a significant difference between the control group and exposed group, including acute and chronic treatment for blood Cd level (p < 0.001). No significant difference was found between acute and chronic exposure group (p > 0.05).

TREATMENT OF TRAUMATIC BRAIN INJURY WITH A COMBINATION THERAPY OF MARROW STROMAL CELLS AND ATORVASTATIN IN RATS

This study investigated the effects of a combination therapy of marrow stromal cells (MSCs) and statins (atorvastatin) after traumatic brain injury in rats. Thirty-two female Wistar rats were injured by controlled cortical impact and divided into four groups. Group I was injected with MSCs (1 x 10(6)) intravenously 24 hrs after traumatic brain injury. Group II was administered atorvastatin (0.5 mg/kg) orally for 14 days starting 24 hours after traumatic brain injury. Group III received MSCs (1 x 10(6)) combined with atorvastatin (0.5 mg/kg). Group IV (control) was injected with saline. MSCs were harvested from the bone marrow of male rats to identify male donor cells within female recipient animals by localization of Y chromosomes. Functional analysis was performed using modified neurological severity scores and the Morris water maze test. Animals were sacrificed 35 days after injury and brain sections stained with immunohistochemistry. No functional improvement was seen in animals treated with MSCs or atorvastatin alone (Groups I and II). However, functional improvement was seen with both testing modalities (modified neurological severity scores and Morris water maze) in animals receiving combination therapy (Group III). Microscopic analysis showed that significantly more MSCs were present in animals receiving combination therapy than in those receiving MSCs alone. Also, significantly more endogenous cellular proliferation was seen in the hippocampus and injury boundary zone of the combination therapy group than in the monotherapy or control groups. When administered in combination with MSCs, atorvastatin increases MSC access and/or survival within the injured brain and enhances functional recovery compared with monotherapy.

Homing and differentiation of mesenchymal stem cells delivered intravenously to ischemic myocardium in vivo: a time-series study

Mesenchymal stem cells (MSCs) are potential sources of cells for tissue repair. However, little information is available about the time course of homing and differentiation of systemically delivered MSCs after acute myocardial ischemia (MI). In the present study, MSCs were isolated from male rat bone marrow and expanded in vitro. Female rats were divided randomly into three groups. Three hours after coronary ligation, the transplanted group received an infusion of MSCs through the tail vein; at the same time, a coronary-ligated control group was injected with culture medium, and a normal (unligated) group received MSCs. Homing of MSCs to the heart was assessed by expression of the Y chromosome sry gene using fluorescence in situ hybridization (FISH) at 3 days, 1, 4, and 8 weeks after transplantation. Immunofluorescent staining was used to examine markers for cardiomyocytes, endothelial cells, and smooth muscle cells. Hemodynamics in the hearts was also measured to assess cardiac function. At each time point, sry-positive cells were present in the cardiac tissue in transplanted group but not in the hearts of normal and control group animals. The number of sry-positive cells was significantly higher at 1 week compared to 3 days after transplantation. No significant difference was found in the number of sry-positive cells among those of 1, 4, and 8 weeks after transplantation. At 3 days and 1 week after transplantation, the sry-positive cells in the transplanted group lacked troponin, desmin, smooth muscle alpha-actin, and CD31. At the later time points, cardiomyocytes, smooth muscle cells, and endothelial cells bearing sry were identified in the transplanted group. The cardiac function in transplanted group showed higher improvement at 4 and 8 weeks compared to 1 week after transplantation. Our data suggest that intravenously delivered MSCs are capable of homing toward the ischemic myocardium, and the fastigium of homing appeared around 1 week after MI. The differentiation of MSCs to cardiomyocytes, smooth muscle cells, and endothelial cells shows to be time dependent and arises at 1 to 4 weeks after transplantation.

Intravenous transplantation of mesenchymal stem cells improves cardiac performance after acute myocardial ischemia in female rats

Mesenchymal stem cells (MSCs) are potential sources of cells for tissue repairing. However, little information is available regarding the therapeutic potency of intravenously transplanted MSCs for myocardial ischemia (MI). In the present study, MSCs were isolated from bone marrow of male rats and expanded in vitro. Three hours after ligation of left anterior descending artery, the transplanted group received an infusion of MSCs through the tail vein. At the same time, a coronary-ligated control group was injected with culture medium. Homing of MSCs to the heart was assessed by expression of the Y chromosome sry gene using fluorescent in situ hybridization (FISH). At 1 week or 8 weeks after transplantation, sry positive cells were present in cardiac tissue in the transplanted group, but not in the hearts of control group. Cardiomyocytes, smooth muscle cells, and endothelial cells that bore sry gene were identified in transplanted group at 8 weeks after transplantation. Ultrastructural observation revealed that a large number of capillary and some immature myocytes were found to survive in the ischemia region. MSCs transplantation also decreased LVEDP pressure and -dP/dt, but increased LVSP and +dP/dt. The cardiac infarct size was significantly smaller in transplanted group than in control group. Our data suggest that intravenously transplanted MSCs improve cardiac performance and promote the regeneration of blood vessels and cardiomyocytes.

Central autonomic control of the bone marrow: Multisynaptic tract tracing by recombinant pseudorabies virus

Bone marrow is the primary place of hematopoiesis, where the development, survival and release of multipotent stem cells, progenitors, precursors and mature cells are under continuous humoral and neural control. Dense network of nerve fibers, containing various neurotransmitters is found in the bone marrow, however, the central neuronal circuit that regulates the activities of the bone marrow through these fibers remained unexplored. Transsynaptically connected neurons were mapped by virus-based transneuronal tracing technique using two isogenic, genetically engineered pseudorabies viruses, Bartha-DupGreen and Ba-DupLac expressing green fluorescent protein and β-galactosidase, respectively. Bartha-DupGreen was injected into the femoral bone marrow of male rats and the progression of infection was followed 4–7 days post-inoculation. Virus-labeled cells were revealed in ganglia of the paravertebral chain and in the intermediolateral cell column of the lower thoracic spinal cord. Neurons were retrogradely labeled in the C1, A5, A7 catecholaminergic cell groups and several other nuclei of the ventrolateral and ventromedial medulla, the periaqueductal gray matter, the paraventricular and other hypothalamic nuclei, and in the insular and piriform cortex. Nerve transections and double-virus tracing from the bone marrow and the surrounding muscles were used to confirm the specific spreading of the virus. These results provide anatomical evidence for the CNS control of the bone marrow and identify putative brain areas, which are involved in autonomic regulation of the hematopoiesis, the release of progenitor cells, the blood supply and the immune cell function in the bone marrow.

Strong and Rapid Induction of Osteoblast Differentiation by Cbfa1/Til-1 Overexpression for Bone Regeneration

Core binding factor alpha-1 (Cbfa1), known as an essential transcription factor for osteogenic lineage, has two major N-terminal isoforms: Pebp2alphaA and Til-1. To study the roles of these isoforms in bone regeneration, we applied an adenoviral vector carrying their genes to transduce primary osteoprogenitor cells in vitro and in vivo. Overexpression of the two isoforms induced rapid and marked osteoblast differentiation, with Til-1 being more effective in vitro, by examination of the alkaline phosphatase activity, calcium content, and Alizarin red staining. Til-1 overexpressing cells/porous ceramic composites were transplanted into subcutaneous and bone defect sites in Fischer rats (cultured bone transplantation model) and markedly affected in vivo bone formation and osteoblast markers. The results demonstrated that the reconstitution of bone tissues, such as cortical bone and trabecular bone was accelerated by implantation of Til-1 overexpressing cells/porous ceramic composites. Moreover, the new bone formation by Til-1 overexpression appeared to reflect replacement of new bone within the implant boundaries. To ascertain whether implanted Cbfa1 overexpressing cells could differentiate into osteogenic cells to create bone or whether it stimulated the surrounding recipient tissue to regenerate bone, implanted male donor cells were visualized by fluorescent in situ hybridization analysis. The proportion of implanted cells in the presumptive bone forming region was over 80% and did not change throughout from 3 days to 8 weeks after implantation. These findings suggested that the newly formed bone in the porous area of the scaffold is mostly produced by the implanted donor cells or their derived cells, effectively by Til-1 overexpression.

DNA damage in tissues of rat treated with potassium canrenoate

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463–472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50.

Simultaneous micronucleus and chromosome aberration assessment in the rat

Using a cellulose column fractionation procedure to eliminate nucleated cells for micronucleus assessment, micronucleus and chromosome aberration endpoints in the same animal were compared in maie and female rats following i.p. injection with cyclophosphamide (CP). Groups of 5 Wistar rats per sex were given single doses of CP at 0, 20, or 40 mg/kg. Two hours prior to sacrifice, animais were given colchicine (4 mg/kg) to arrest cells in metaphase. One femur from each animal was used for micronucleus assessment and the other for chromosome aberration assessment. In the micronucleus assessment, 2000 polychromatic erythrocytes (PCEs) per animal and in the chromosome aberration assessment, 50 metaphase cells per animal were scored. This experiment was repeated once. In both experiments, significant increases in micronucleated PCEs and chromosome aberrations were noted at both doses of CP in both sexes. In general, the clastogenic effects of CP were more pronounced in males than females. Both doses of CP caused a decrease in the proportion of PCEs and in mitotic index in both experiments, indicating toxicity of CP to the bone marrow. These results show the usefulness of this rat model for simultaneous evaluation of two cytogenetic endpoints in the same animal and indicate that assessment of MNPCE frequency in the bone marrow of male rats may be an appropriate model for screening test substances for in vivo clastogenic activity in this species.

電気刺激による骨誘導に関する研究 骨髄内骨形成および電気刺激骨髄移植による筋肉内骨形成について

It has been accepted that bone formation was accelerated by electric stimulation. The present experiments were reported to clarify the nature of the induced bones by electric stimulation from histological, crystallographical and elementally analytical standpoints, as well as clarify induced osteoid into muscle.Femoral bone marrow of male rats (Donryu) was employed. A cathode of electric stimulation wire was inserted in the bone marrow and the anode was placed around the bone. The control dummy wire was used in a similar manner. Periods of the experiment were 3, 5, 7, 9 and 14 days during constant stimulation of 15.5μA.Electrically stimulated bone marrow for 5 days was transplanted into the muscle, and specimens were examined on days 3, 5, 7, 14 and 21 after grafting. For histologic observations, H-E and Van Gieson, and fluorescence microscopy were done. The sections obtained from the same specimen were examined by X-ray diffraction and X-ray microanalyzer to detect crystallization and concentration of Ca, P, Mg.Histologic findings showed that bone formation including developing osteoprogeniter cells, osteoid and calcified bones were enhanced by electric stimulation, whereas crystallization and mineral concentration of induced bones had features similar to those of the control bones.On days 3 and 5 after grafting, new bone formation in the electric stimulation group was increased more than in the control group. On days 14 and 21, histologic pictures of bone formations of both groups were obtained and did not show so many prominent differences.In conclusion, the stimulating effects of bone induction and formation by constant electric stimulation existed in rats femur bone marrow.

Chemical Carcinogenesis in the Rat: Common Mode of Action of Carcinogens at the Chromosome Level&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

The chromosomal distribution of chromosome aberrations (CA) and sister chromatid exchanges (SCE) induced by various chemical carcinogens such as 7,12-dimethylbenz[a]anthracene, 7,8,12-trimethylbenz[a]anthracene, 1-butyl-1-nitrosourea, urethan, 4-nitroquinoline 1-oxide, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, and the antineoplastic compound mitomycin C was studied with the use of noninbred Long-Evans male rat bone marrow cells in vivo and in vitro. The CA and SCE induced by these structurally different chemicals were distributed in a similar pattern among and along chromosomes when the chemicals were given a short time (6 hr) before the metaphase cells were harvested. The specific distribution pattern of chemically induced CA and SCE along chromosomes was attributed to the late DNA replication of the vulnerable chromosome regions. Conversely, X-ray-induced CA were distributed more randomly. Thus the different chemical carcinogens were shown to exert a similar genetic effect at the chromosome level.