[Change of Rho kinase activity in brain tissue in high altitude condition and its relationship with high altitude cerebral edema: experiment with rats].

OBJECTIVE: To observe the change of Rho kinase activity in brain tissue in the high altitude condition and its relationship with blood brain barrier permeability and high altitude cerebral edema (HACE), and to explore the pathological mechanism of HACE. METHODS: 30 Wistar rats were divided randomly into 3 equal groups, high altitude (HA) group put in low pressure cabin mimicking high altitude of 7000 m for 24 hr and then gradually exposed to higher pressure until the normal pressure, fasudil group undergoing intraperitoneal injection of fasudil hydrochloride 30 mg/kg, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and then treated as the HA group, and normal control group (HC group). Five rats from each were decapitated with their brains taken out. The ratio of dry brain weight and wet brain weight was calculated. Western blotting was used to detect the expression of ROCK. Another 5 rats from each groups underwent injection of sodium fluorescein into the caudal vein and then the rats were decapitated to examine the natrium (sodium) fluorescence index (NaFI) of the central brain slice so as to observe the blood brain barrier permeability. RESULTS: The ROCK activity in brain tissue, ratio of dry and wet brain weight and NaFI of the HA group were significantly higher than those of the HC group (all P<0.001), and ROCK activity in brain tissue, ratio of dry and wet brain weight and NaFI of the fasudil group were all significantly lower than those of the HA group (all P<0.001) and not significantly different from those of the HC group (all P>0.05). CONCLUSIONS: ROCK in brain tissue is activated in high altitude condition, which may play a key role in the development of HACE.

Theophylline attenuates microwave-induced impairment of memory acquisition.

Numerous studies have shown that acute microwave exposure causes cognitive deficits in animals, possibly via hyperthermia, but the biological effect of microwave exposure on memory processing is still unknown. The release of adenosine is demonstrated to be a general way for the cells to respond to metabolically stressful conditions such as hypoxia and ischemia. The present study aimed to examine whether adenosine mediates biological effects of microwave exposure on memory processing using a continuous multiple-trial inhibitory avoidance task. Results demonstrated that microwave exposure for 20 min before training impaired memory acquisition and retention performance in mice, assessed by the number of training trials and by latency to enter the dark compartment. The mice exposed to microwave radiation showed a dose-dependent hyperthermia. Moreover, the cell numbers of hippocampus were decreased in the mice receiving microwave exposure at an average power density of 50 mW/cm(2), indicating the anatomical correlation to hippocampal-amygdaloid structures corresponding with the memory disrupt of the mice. Administration of theophylline, a nonspecific adenosine receptor antagonist, 30 min before microwave exposure, completely antagonized the impairment of inhibitory avoidance acquisition but not retention. These results suggest that the adenosine regulation pathway was partially involved in microwave-induced impairment of inhibitory avoidance memory.

Protective effects of inosine on mice subjected to lethal total-body ionizing irradiation.

Mammals can barely survive total-body ionizing irradiation greater than 10 Gy. To date, there are few drugs available for radioprotective therapy under such circumstances. Inosine, a natural derivative of adenosine, has been known to provide powerful protection for many kinds of cells and tissues against various insults both in vitro and in vivo. In the present study, we examined whether inosine was also beneficial for mammals subjected to an absolutely lethal total-body ionizing irradiation. Immediately after adult Balb/c mice were exposed to (60)Co gamma-rays at a single dose of 12 Gy, a moiety of them were administered daily with inosine or adenosine, either at doses of 375 or 750 micromol/kg up to death, and their body weight and survival time were recorded. Some irradiated mice were administered inosine or adenosine daily at doses of 750 micromol/kg and assessed for spatial memory abilities using the Morris water maze. The results demonstrated that, although inosine could not prevent body weight loss in irradiated mice, it was able to significantly prolong their survival time at doses of 750 micromol/kg. Moreover, inosine but not adenosine could suppress spatial memory deficit in irradiated mice. The data suggested that inosine had protective effects on mammals suffering from total-body ionizing irradiation at a single lethal dose.

[The study of the effect of the administration of mannitol on flash visual evoked potentials].

OBJECTIVE: To study the relationship between the change of N2 wave in flash visual evoked potentials (fVEP) and the intracranial pressure after the administration of mannitol. METHODS: Fifty-two patients with elevated intracranial pressure were chosen and are divided into two groups: the former (n = 32) were treated with mannitol, the latter (n = 20) were treated with mannitol and glycerol and sodium chloride injection. The latency and amplitude of N2 wave of fVEP was measured by NIP-200 noninvasive intracranial pressure apparatus before and after the 1st, 4th, 10th administration of mannitol. RESULTS: The latency of N2 wave began to reduce at 30 min after the injection of mannitol and was lowest at 2 h. At 4 h after the injection of mannitol, the latency of N2 wave increased but was still shorter than that before the injection of mannitol. The amplitude of N2 wave did not change significantly. The reduction of the latency of N2 wave after the administration of mannitol decreased with the multiple mannitol injection. The latency of N2 wave did not change significantly after the administration of mannitol when combined with glycerol and sodium chloride injection. CONCLUSION: The latency of N2 wave of fVEP changes after the administration of mannitol, which suggests that the change of the latency of N2 wave of fVEP can reflect the change of intracranial pressure.

Progesterone alleviates neural behavioral deficits and demyelination with reduced degeneration of oligodendroglial cells in cuprizone-induced mice.

Demyelination occurs widely in neurodegenerative diseases. Progesterone has neuroprotective effects, is known to reduce the clinical scores and the inflammatory response. Progesterone also promotes remyelination in experimental autoimmune encephalomyelitis and cuprizone-induced demyelinating brain. However, it still remains unclear whether progesterone can alleviate neural behavioral deficits and demyelination with degeneration of oligodendroglial cells in cuprizone-induced mice. In this study, mice were fed with 0.2% cuprizone to induce demyelination, and treated with progesterone to test its potential protective effect on neural behavioral deficits, demyelination and degeneration of oligodendroglial cells. Our results showed noticeable alleviation of neural behavioral deficits following progesterone treatment as assessed by changes in average body weight, and activity during the open field and Rota-rod tests when compared with the vehicle treated cuprizone group. Progesterone treatment alleviated demyelination as shown by Luxol fast blue staining, MBP immunohistochemical staining, and electron microscopy. There was an obvious decrease in TUNEL and Caspase-3-positive apoptotic cells, and an increase in the number of oligodendroglial cells staining positive for PDGFRα, Olig2, Sox10 and CC-1 antibody in the brains of cuprizone-induced mice after progesterone administration. These results indicate that progesterone can alleviate neural behavioral deficits and demyelination against oligodendroglial cell degeneration in cuprizone-induced mice.

[Effects of mesenchymal stem cells on cell cycle and apoptosis of hematopoietic tissue cells in irradiated mice].

The aim of this study was to investigate the effect of mesenchymal stem cells (MSCs) on cell cycle and apoptosis of thymus, spleen and bone marrow cells in mice totally irradiated with sublethal dose, and to explore its mechanisms. BALB/c mice irradiated with 5.5 Gy 60Co gamma-ray were randomly divided into control group and MSC group. Mice in MSC group were infused with 0.4 ml containing 2.5x10(7)/kg of MSCs through tail vein at 1 hour after irradiation. Mice in control group were infused with 0.4 ml normal saline. The cell apoptosis and cell cycle of thymus, spleen and bone marrow cells were detected by flow cytometry at 6, 12, 24 and 72 hours after irradiation and the P53 protein expressions in thymus and bone marrow cells were assayed by immunohistochemistry at 12 hours after irradiation. The results showed that the arrest of cells in G0/G1 and G2/M phase, and decrease of cells in S phase appeared at 6 hours after irradiation, those reached peak respectively at 12 hours in thymus cells, 6 hours in spleen and 24 hours in bone marrow, then the cell counts in G0/G1 phase decreased and the cell counts in S and G2/M phases increased. At 72 hours the cell counts in G0/G1 phase were less than the normal level and the cell counts in S phase were more than the normal level. The above changes of cell cycle in thymus and spleen were more rapid in spleen and more obvious in amplitude than that in bone marrow, the change of cell cycle in MSC group was more rapid and obvious than those in control group. After irradiation the apoptosis cells increased from 6 hours, reached the highest level at 12 hours and decreased to the normal level gradually after 24 hours in two groups; the apoptosis rates in spleen and thymus cells were higher than that in bone marrow cells. In comparison with the control group, the apoptosis rate in thymus cells at 12 hours, in spleen cells at 12 and 24 hours, and in bone marrow cells at 24 hours were fewer in MSC group. The cells expressing P53 protein in control group were more than that in MSC group. It is concluded that the MSCs accelerate the running of cell cycle in these hematopoietic tissue cells of irradiated mice, reduce the cell apoptosis and promote the recovery from injuries in hematopietic and immunological organs, thus protect the irradiated mice at early stage.

[Effect of bone marrow mesenchymal stem cells on immunoregulation in H-2 haploidentical bone marrow transplantation mice].

OBJECTIVE: To explore immunoregulatory mechanism of mesenchymal stem cells (MSCs) in H-2 haploidentical bone marrow cells transplantation mice. METHODS: BALB/c female mice irradiated with 8Gy 60Co gamma-rays were divided into two groups: MSCs group, infused cm-DiI labeled MSCs from female CB6F1 mice and monocytes from the bone marrow and spleen of male CB6F1; Control group, only infused monocytes from the bone marrow and spleen of male CB6F1. T-lymphocyte subpopulation of peripheral blood cells, T and B cells proliferation stimulated by ConA and LPS, mixed lymphocyte reaction between donor and recipient and third part, the sry-gene chimerism of bone marrow, spleen and thymus of the recipient, the distribution of MSCs in the recipient, the incidence rate of GVHD and survival were observed. RESULTS: The CD3 at +90 d the percent of CD3+ CD4+ cells, and CD4/CD8 at +30 d in the MSCs group were higher than that in control post-transplantation, respectively (P < 0.05). The proliferation activity of B cells recovered more rapidly and that of T cells recovered comparably in MSCs group as compared with that in control group. The result of MLR between donor and recipient was lower in MSCs group than that in the control; and that between recipient and the third part had no difference. The sry-gene chimerism of bone marrow and spleen of the recipient was higher in MSCs group than in control at +30 d. The MSCs mainly distributed in intestine, thymus, bone marrow, liver, heart of the recipient after transplantation. The incidence of acute GVHD was higher and the survival rate was lower in MSCs group than that in control group (P < 0.05). Chronic GVHD occurred in the control group at +90 d, while in the MSCs group at +120 d. CONCLUSIONS: MSCs might improve stem cell engraftment, promote lymphocyte and humoral immunity recovery, decrease incidence of GVHD and increase survival by inducing specific immunologic tolerance and repairing organs injuries.

[Expression profiling of UBF, a novel member of UBC family].

UNLABELLED: From large-scale sequence of human fetal liver cDNA library, we have obtained a full-length cDNA from an EST after further sequencing. It has been demonstrated by the alignment comparison with data base available that it is a novel member of Ubc family and got the number from GeneBank: UBF-F1 AF 294842. AIM AND METHODS: To demonstrate its authenticity, UBF was amplified from the total RNA of human fetal liver and HL-60 cell line using RT-PCR, and the PCR products were further sequenced and compared with the original UBF sequence. To evaluate the expression level and subcellular location of UBF in human multiple tissues, in situ hybridization was carried out on the frozen section of human fetal multiple tissues and HL-60 cell line with DIG-labeled UBF cDNA probes. RESULTS: The experimental results of RT-PCR and sequencing showed that the sequence of RT-PCR products were the same as the original UBF. The experimental results of in situ hybridization showed that UBF was expressed widely by human multiple fetal tissues and the expression level were very high in HL-60 cells. CONCLUSION: It is suggested that the special structure of UBF is authentic, and the expression profiling research of UBF shows that UBF is expressed widely by human multiple fetal tissues and the expression level is very high in HL-60 cells, implying that UBF plays the important function in the developing tissues and leukemia cells. It is also suggested that UBF may be functionally related with the nucleic-involving cellular activities based on the results of sub-cellular localizations.

[Comparative study of differentially expressed genes in mouse bone marrow and fetal liver cells].

Hematopoiesis undergoes several migrations from yolk sac to liver and spleen, and finally bone marrow until the end of life. A number of investigations have demonstrated that the hematopoietic microenvironment plays very important role in this process. However, the exact mechanisms remain unknown. In order to systematically analyze and understand the role of hematopoietic microenvironment in the regulation and control of hematopoiesis, a microarray containing 588 complementary DNAs was used to compare the gene expressions between those in murine fetal liver and bone marrow cells. The results obtained from array hybridization were analyzed and reconfirmed by using bioinformatics and RT-PCR as well as Northern blot. The results showed that 65 and 131 genes were relatively high expressed in bone marrow and fetal liver cells respectively among 588 known genes in the array-membrane. According to the survey in the PubMed, 39 out of bone-marrow-expressed genes and 71 in fetal-liver-expressed genes were closely related to the hematopoiesis. Further reconfirmation by RT-PCR or Northern blot has demonstrated that CD18, CD44 an d PSGL-1 genes chosen for analysis were highly expressed in adult bone marrow, but unexpressed or lower expressed in fetal liver cells, resulting in high similarity to the array results. Moreover, the expressions of CD18 and CD44 in fetal liver were down-regulated with the increment of gestational age. In conclusion, the gene expressions in bone marrow and fetal liver cells are obviously different, some of the genes are down-regulated at the different stages of ontogeny. The different gene expression levels between bone marrow and fetal liver, especially those genes closely related to the hematopoiesis, may be the molecular basis for the explanation of why hematopoietic stem cells derived from different tissues have different characterizations as well as the differences from the beginning and terminating of fetal liver hematopoiesis, and why hematopoietic stem cells derived from fetal liver is tremendously difficult to be grafted in bone marrow.

[Screening of differentially expressed genes in the mouse hematopoietic stromal cells after long-term culture].

Hematopoietic stromal cells, being the essential ingredient of the hematopoietic microenvironment, play very important roles in the control and regulation of self-renewal, proliferation and differentiation of hematopoietic stem cells (HSC) via complex interactions of cell-cell, cell-humoral and cell-extracellular matrix. Evidence from in vivo experiment has proved that HSC derived from normal mice could reconstitute hematopoiesis of mice with HSC defects but failed to reconstitute hematopoiesis of those mice with microenvironment defects, showing the importance of hematopoietic microenvironment in the maintenance of hematopoiesis in vivo. A well-known long-term culture (LTC) system established by Dexter demonstrated in another way that stromal cell layer in the system could support ex vivo hematopoiesis for several months, even more than one year under the optimal conditions. It, however, has not been demonstrated that what is the key elements and in which way the ex vivo hematopoiesis could be maintained for so long time. As the inventions for the large-scale screening methodologies the suppression subtractive hybridization (SSH) was chosen for the screening differentially expressed genes expressed by LTC cultured stromal cells but not by the uncultured bone marrow cells (BMC). mRNA extracted from both cultured adherent cells (tester) and BMC (driver) were hybridized according to the protocol provided by CLONTECH. Total of 130 clones differentially expressed by cultured cells were randomly picked up and 106 ESTs were obtained after sequencing. They represent 26 identical or similar genes and 7 novel genes after the bioinformatics analysis. 5 of the novel genes with the entire open reading frame, without functional clues, have been cloned into the mammalian expression vectors and the functions of them in the control of proliferation and differentiation of HSC will be further exploring. The most interesting discovery is that 3 novel genes have signal peptides, implying the potential discovery of novel growth factors as 80% known growth factors have signal peptides. Our experimental results suggest that: (a) based on the results of subtractive efficiency, the SSH could be a reliable method to screen differentially expressed genes; (b) gene expression may be regulated by multiple factors, even conditioning-dependent, in this experiment the genes expressed by bone marrow stromal cells are LTC-cultivation inducible; (c) it is possible to find interesting genes or special gene after relatively large-scale screen.