BrdU Expression Research Articles

Impaired neurogenesis and activated caspase 3‐dependent apoptosis pathway in the rostral ventrolateral medulla cause hypertension in stroke‐prone spontaneously hypertensive rats

Recent studies demonstrated that neural progenitor cells responsible for neurogenesis migrate to the sites of brain injury of epilepsy, stroke and neurodegenerative diseases. It is also well known that apoptosis is involved in regulating synaptic plasticity. It remains unknown the impact of balance between neurogenesis and apoptosis in the rostral ventrolateral medulla (RVLM) in the pathogenesis of hypertension. We examined neurogenesis and caspase 3‐dependent apoptosis initiated by mitochondrial translocation of Bax in the RVLM of stroke‐prone spontaneously hypertensive rats (SHRSP). We identified neuroblasts by staining for doublecortin (Dcx), cell proliferation by absorption of bromodeoxyuridine (BrdU), and apoptosis by staining for caspase‐3, Bax, PKC in membranous, cytosolic and mitochondorial fractions. In hypertensive states, SHRSP had significantly lower Dcx and BrdU expression than WKY (Dcx; −41±4%, BrdU; −33±5%, n=8 for each, P<0.01). In pre‐hypertensive states, the expressions of Dcx and BrdU in the RVLM were significantly decreased compared with WKY. The expression of Bax in mitochondorial fraction and caspase‐3 in cytosolic fraction were significantly increased in SHRSP than in WKY, in either pre‐hypertensive or hypertensive states. In conclusion, the impaired neurogenesis and activated caspase 3‐dependent apoptosis pathway in the RVLM might be involved in hypertension in SHRSP.

Early ventricular restraint after MI: Cautionary results

RESULTS: Relative to sham animals, Plk1 was downregulated 4 weeks after banding. One week after debanding, LVH had functionally regressed and Plk1 expression had normalized. Immunohistochemistry revealed the presence of Plk1 in cardiomyocytes. BrdU expression in debanded hearts was increased compared to banded animals (22.4 / 2.3 vs. 12.1 / 1.7 positive cells/1000). Finally, Plk1 expression was upregulated in human myocardium following mechanical relief with LVAD therapy.

Human Embryonic Stem Cells (HSF-6) Show Greater Proliferation and Apoptoses When Grown on Glioblastoma Cells Than Mouse Embryonic Fibroblasts at Day 19 in Culture: Comparison of Proliferation, Survival, and Neural Differentiation on Two Different Feeder Cell Types

Phenotypic guidance of embryonic stem (ES) cell fate is paramount if these cells are to be used for tissue repair and regeneration. Our objective was to compare two different cell culture feeders and their effect on proliferation, apoptosis, and differentiation of human (h) ES cells. HSF-6 hES cells were grown in Knockout Dulbecco's modified Eagle medium (DMEM) on mouse embryonic fibro-blasts (MEFs) or U87 glioblastoma cells at densities of 50,000, 100,000, and 150,000 cells/well of a six-well plate for 7, 12, and 19 days. Immunocytochemistry was performed for bromodeoxyuridine (BrdU), TUNEL, and neural differentiation markers including class III beta-tubulin, NeuN, nestin, and doublecortin. Slides were examined by laser confocal microscopy with semiquantitative analyses of marker expression. BrdUand TUNEL-positive cells were primarily, but not exclusively, at edges and between established colonies. BrdU expression was higher on U87 feeders at low and intermediate densities at day 19. Both feeders demonstrated higher BrdU expression at day 7 compared to days 12 and 19. U87 produced more TUNEL-positive cells than MEFs with increasing numbers with increasing density and time in culture. Nuclear Oct-4 staining was seen only at day 7. MEFs appeared to promote greater neural differentiation of hES cells than U87. We conclude hES cells grown on U87 feeders demonstrate greater numbers of apoptotic cells and BrdU-positive cells at day 19. Independent of the feeders, proliferation and apoptosis may be positively correlated. We speculate differences in proliferation, apoptosis, and neural differentiation may be due to differential elaboration of specific cytokines by MEFs and U87.

The best site of transplantation of neural stem cells into brain in treatment of hypoxic-ischemic damage: experiment with newborn rats

To investigate the best site of transplantation of neural stem cells (NSCs) into the brain to treat hypoxic-ischemic damage (HIBD). Forty-eight 7-day-old Spraque-Dawley rats underwent ligation of the left common carotid artery and exposure to 8% oxygen at 37 degrees C for 2 hours to establish HIBD models and then were randomly divided into 4 equal groups 3 days later: HIBD control group, HIBD + cortex transplantation group (CT group) undergoing NSC transplantation into the sensorimotor cortex, HIBD + hippocampus transplantation group (HT group) undergoing NSC transplantation into the hippocampus, and HIBD + ventricle transplantation group (VT group), undergoing NSC transplantation into the lateral ventricle. Since the rats were 40-day-old, they underwent radial maze water-seeking test and 4 sensorimotor tests. Then the brains of the rats were taken out to undergo histological examination by Nissl staining and immunohistochemistry to observer the 5-bromodeoxyuridine (BrdU) expression. The times the rats took to find the water in all 3 arms of the maze were arranged form short to long in the order HT group < VT group < CT group < HIBD group with significant differences between the HT, VT, and CT groups and HIBD group (All P < 0.05) and between the groups HT and VT and the group CT (both P < 0.05). The results of the 4 sensorimotor tests were arranged from the best to the worst in the order CT group > VT group > HT group > HIBD group with significant differences between the groups CT and VT and the group HIBD (both P < 0.05). Nissl staining showed that the number of normal neurons in the cortex area from more to less were arranged in the order CT group > VT group > HT group > HIBD group; and the number of normal neurons in the CAI area from more to less were arranged in the order HT group > VT group > CT group > HIBD group. Transplantation of NSC attenuates the brain damage and the cognitive and sensorimotor dysfunction after HIBD. Transplantation into the lateral ventricle is the most effective.

Stimulation of ES-cell-derived cardiomyogenesis and neonatal cardiac cell proliferation by reactive oxygen species and NADPH oxidase

After birth the proliferation of cardiac cells declines, and further growth of the heart occurs by hypertrophic cell growth. In the present study the cell proliferation capacity of mouse embryonic stem (ES) cells versus neonatal cardiomyocytes and the effects of reactive oxygen species (ROS) on cardiomyogenesis and cardiac cell proliferation of ES cells was investigated. Low levels of hydrogen peroxide stimulated cardiomyogenesis of ES cells and induced proliferation of cardiomyocytes derived from ES cells and neonatal mice, as investigated by nuclear translocation of cyclin D1, downregulation of p27(Kip1), phosphorylation of retinoblastoma (Rb), increase of Ki-67 expression and incorporation of BrdU. The observed effects were blunted by the free radical scavengers vitamin E and 2-mercaptoglycin (NMPG). In ES cells ROS induced expression of the cardiac-specific genes encoding alpha-actin, beta-MHC, MLC2a, MLC2v and ANP as well as the transcription factors GATA-4, Nkx-2.5, MEF2C, DTEF-1 and the growth factor BMP-10. During differentiation ES cells expressed the NADPH oxidase isoforms Nox-1, Nox-2 and Nox-4. Treatment of cardiac cells with ROS increased Nox-1, Nox-4, p22-phox, p47-phox and p67-phox proteins as well as Nox-1 and Nox-4 mRNA, indicating feed-forward regulation of ROS generation. Inhibition of NADPH oxidase with diphenylen iodonium chloride (DPI) and apocynin abolished ROS-induced cardiomyogenesis of ES cells. Our data suggest that proliferation of neonatal and ES-cell-derived cardiac cells involves ROS-mediated signalling cascades and point towards an involvement of NADPH oxidase in cardiovascular differentiation of ES cells.

Neurogenesis decreases during brain maturation from adolescence to adulthood

Adolescence is an important stage of brain development. Recent studies have indicated that neurogenesis in the brain occurs throughout life prompting comparisons of adolescent and adult neurogenesis. Since insulin-like growth factor 1 (IGF-1) has been implicated in promoting neurogenesis we investigated the levels of neurogenesis in adolescents (PND30) and adults (PND120) using IGF-1 over-expressing mice and IGFBP-1 (IGF binding protein-1) over-expressing mice. Proliferation and differentiation of neuroprogenitors were determined using bromodeoxyuridine (BrdU)- and doublecortin (DCX)-labeling. High levels of neurogenesis were found in both the hippocampal dentate gyrus (DG) and the forebrain subventricular zone (SVZ) of the adolescents as compared with the adults. Both adolescent IGF-1 and IGFBP-1 transgenic mice as well as their wildtype controls have significantly higher expression of BrdU and DCX in the hippocampus and SVZ when compared with their adult counterparts. However, no significant differences on BrdU-labeling were found when either of transgenic mice were compared with their wildtype littermates in both age groups. These studies indicate that adolescent mice have high levels of neurogenesis compared to adults suggesting a dramatic loss of neurogenesis during the transition from adolescence to adulthood. However, the role of IGF-1 during adolescent development is still unclear.

Proliferation of neural precursors in the subventricular zone after chemical lesions of the nigrostriatal pathway in rat brain

The proliferative activity of neural precursors from the subventricular zone (SVZ) was investigated after a unilateral lesion was formed in the nigrostriatal pathway in adult rats. The lesion was formed by unilateral injection of 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway, and then bromodeoxyuridine (BrdU) was injected (ip) for 4 days or 2 weeks 10 days after the lesion was formed. The rats were killed, and the brain sections were immunohistochemically stained to detect the expression of BrdU, polysialylated neural-cell-adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP) and tyrosine hydroxylase (TH) in the SVZ and the striatum (STR). The results showed that the BrdU+ cells increased significantly in the SVZ, ipsilateral to the lesion at 2 weeks after the lesion. The PSA-NCAM+ and GFAP+ cells were also increased in the SVZ at this time. Some BrdU-labeled cells were seen in the same side of the STR and were double-labeled with PSA-NCAM. These cells had a tendency to migrate from the SVZ to the STR. The number of positive cells decreased at 4 weeks after the lesion was formed. The number of nigrostriatal projections with TH+ decreased significantly in the STR on the lesion side, and the level of decrease was related to the quantity of BrdU-labeled cells at 2 weeks. These results indicate that the neural precursors in the SVZ of adult rats may increase after a lesion has been formed in the nigrostriatal pathway, and these cells might migrate into the STR on the same side.

Stem cells marked with bromodeoxyuridine and telomerase reverse transcriptase in hyperoxia lung injury in neonatal rats

目的 探讨5溴脱氧尿苷(bromodeoxyuridine,Brdu)和端粒酶逆转录酶(tolomerase reversetranscriptase,TERT)标记肺干细胞特点及其在肺发育和损伤修复中的作用.方法95%左右氧气7 d制备新生鼠高氧肺损伤模型;将Brdu自腹腔注入模型鼠,免疫组化法检测Brdu和TERT阳性显色细胞,并和表面蛋白C(surfactant protein C,SPC)抗体免疫双染.结果(1)Brdu阳性显色部位主要在各级支气管黏膜下和肺间隔,支气管黏膜上皮及肺泡壁有散在分布,阳性细胞数量较少,多呈立方形、核大;TERT阳性显色在外周肺组织,肺间隔和肺泡壁部位,数量明显少于Brdu,且阳性显色呈不对称性,即多集中在某一肺叶;(2)SPC阳性显色细胞在肺间隔和肺泡壁,分别和Brdu、TERT双染,可见少量阳性细胞;(3)Brdu、TERT和SPC表达积分在高氧组与正常氧组差异无统计学意义(P>0.05).Brdu表达积分无论在高氧组(1.61±0.83)还是正常氧组(1.43±0.85),均高于TERT(分别为0.83±0.84和0.62±0.55,P<0.05).结论Brdu和TERT作为肺干细胞标记,两者各有其特征;肺损伤时Brdu和TERT标记阳性细胞量可提示肺干细胞增殖分化及肺泡Ⅱ型上皮细胞损伤修复情况.至于Brdu和TERT哪一个指标更具特异性,是否Brdu同时标记了已从干细胞增殖分化但尚保留干细胞特征的短暂扩增细胞,或TERT更能反映干细胞特征尚待深入研究。

The Effects of BR003 on Memory and Cell Proliferation in the Dentate Gyrus of Rat Hippocampus

BR003 is a multi-herbal formula that contains twelve medicinal herbs. We investigated the effects of oral administration of BR003 to Wistar rats on (a) learning and memory using a passive avoidance test and (b) cell proliferation in the dentate gyrus (DG) of the hippocampus using immunohistochemical analysis of 5-bromo-2-deoxyuridine (BrdU) expression. In the passive avoidance test, the retention time of the BR003-treated group was significantly longer than that of the control group (182.64+/-39.88 vs. 73.08+/-29.30 s, respectively; n=11; p<0.05). There were significantly more BrdU-immunoreactive cells in the DG in the BR003-treated group than in the control group (1281.07+/-151.16 vs. 818.01+/-132.98 cells per DG, respectively; n=11; p<0.05). These results suggest that the administration of BR003 not only improves learning and memory but also increases cell proliferation in the DG of the rat hippocampus.

Histoimmunological Evaluation for the Efficacy of Entero Nutrient Containing n-3 Fatty Acids in TNBS Rat Colitis Model

Inflammatory bowel disease (IBD) is a chronic disease of the digestive tract, with complicated and multifactoral etiology. An exaggerated intestinal immune response to otherwise innocuous stimuli plays a key role in the pathophysiology of this intestinal disorder. Nutritional intervention is an important therapy for patients with IBD. But the ratio of n-3 and n-6 fatty acids on the modulation of intestinal inflammation is not clear. Interleukin-16 (IL-16) is a pleiotrophic cytokine secreted mainly by CD8+ T cells. The properties of IL-16 suggest that it may be involved in pathophysiological process of chronic inflammatory diseases. The aim was to determine the effect of an n-3 fatty acid-rich diet (RACOL®) on the expressions of cytokines and mucosal integrity in trinitrobenzene surufonic acid (TNBS) induced rat colitis. 7 week-male Wistar rats were divided into 4 groups. 1. Normal laboratory diet (n = 10), 2. RACOL® diet only (n = 10), 3. TNBS induced colitis with normal laboratory diet (n = 10), 4. TNBS induced colitis with RACOL® diet (n = 10). TNBS induced colitis rats were given an intracolonic injection of 50 mg of TNBS dissolved in 50% ethanol. From day 2, rats were fed 80 kcal/day of RACOL® and/or normal laboratory diet, and sacrified on the 14th and the 28th days. One hour before sacrifice, 0.2 mg/g/rat of BrdU was injected. The expressions of BrdU labeled cells, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-16 were studied by the ABC staining method and DAB staining lesions in colonic mucosa were estimated by an image analyzer system. Apoptosis was assessed by the TUNEL method. Macroscopic and histological findings were classified by the Morris and Vilaseca method. TNBS application increased colonic epithelial cell proliferation, but nutrition by RACOL® reduced this stimulation. Number of apoptosis cells was also increased by TNBS application, but nutrition of RACOL® reduced the number of apoptosis cells, which had been increased by TNBS treatment. The expressions of cytokines had been increased in TNBS-colitis rats, but were reduced by the RACOL®. In conclusion, the effect of nutrition by n-3 fatty acid-rich diet, RACOL®, is an important treatment through the inhibition of inflammatory mediators, and colonic hyper-proliferation.

Hepatocyte Growth Factor/Scatter Factor and MET Are Involved in Arterial Repair and Atherogenesis

Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE(-/-) mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.

Analgesic effect of intrathecal transplant of immortalized rat astrocyte strain genetically modified by human preproenkephalin gene on rat chronic neuropathic pain

To observe the analgesic effect of intrathecal transplant of immortalized rat astrocyte genetically modified by human preproenkephalin gene (IAST/hPPE) on chronic neuropathic pain. 40 adult male Sprague-Dawley rats were randomly divided into four groups, 10 rats for each group. Naive group, SNI group, SNI + IAST group and SNI + IAST/hPPE group. The immortalized rat astrocyte (IAST) or IAST/hPPE co-incubated with bromodeoxyuridine (BrdU) in vitro were transplanted in the lumbar 4 to 6 subarachnoid space near the spinal cord 1 week after right side spared nerve injury (SNI). All animals were tested for bilateral 50% hindpaw withdrawal threshold (PWT) to a graded series of Von Frey hairs stimulation once a week from one week before SNI to six weeks after transplant, the difference value for right 50% PWT minus left 50% PWT was calculated and the effect of intraperitoneal naloxone on the analgesic efficacies was also observed. The content of L-EK in the spinal cord of L4 - 6 and was determined using immunohistochemistry and radioimmunoassay, and the expression of BrdU in grafts was determined using immunohistochemistry. Allodynia-like behaviour after 1 week following SNI was observed. As compared with Naive group, the difference value for the right 50% PWT minus left 50% PWT in the other three groups was higher significantly (P < 0.01). The tactile allodynia induced by SNI was significantly alleviated during the 1 to 6 week period after transplantation of IAST/hPPE cells, but transplants of IAST cells had no effect on the allodynia-like behaviour. The difference value for the right 50% PWT minus left 50% PWT in SNI + IAST/hPPE group was lower significantly than that in the SNI and SNI +I AST group (P < 0.01), but there was no significant difference between SNI and SNI + IAST group (P > 0.05). The efficacies were reversed by intraperitoneal naloxone in SNI + IAST/hPPE group. The content of L-EK in the lumbar spinal cord in IAST/hPPE group (108.1 pg/mg +/- 12.5 pg/mg) was significantly higher than that in other three groups (P < 0.01), but there was no significant difference between SNI and SNI + IAST group (25.4 pg/mg +/- 1.9 pg/mg vs 28.0 pg/mg +/- 2.1 pg/mg, P > 0.05). Furthermore, The grafts in the surface of dorsal horn were still stained positively for BrdU, they survived greater than 6 weeks on the pia mater around the spinal cord. Intrathecal transplant of IAST/hPPE cells could alleviate the allodynia-like behaviour after chronic neuropathic pain, which is associated with enkephalin secreted continuously from the grafts and conducted via opiate receptors.

Hippocampal neurogenesis and PSA-NCAM expression following exposure to 56Fe particles mimics that seen during aging in rats

Exposure to particles of high energy and charge can disrupt the neuronal systems as well as the motor and cognitive behaviors mediated by these systems in a similar fashion to that seen during the aging process. In the hippocampus, adult neurogenesis is affected both by aging and irradiation with ionizing particles. Likewise, the maturation of newly formed cells in this region as measured by PSA-NCAM expression is also altered by the aging process. The present study was designed to investigate the effects of 2.5 Gy of 1 GeV/n 56Fe particles on neurogenesis using the nuclear proliferation marker 5-bromodeoxyuridine (BrdU and PSA-NCAM expression in the dentate gyrus of rats exposed to whole-body irradiation or simply placed in the chamber without being irradiated. All subjects ( n=10) were sacrificed 28 days after the last BrdU injection (50 mg/kg X 3 days) and their brains were processed for immunohistochemistry. Results illustrate a decrease in the number of BrdU-positive cells as well as different distribution of these cells in the dentate gyrus of irradiated animals. Additionally, irradiated subjects show decreased levels of PSA-NCAM expression. These changes are consistent with those found in aged subjects indicating that heavy-particle irradiation is an adequate model for the study of aging.

Efficacy of doublecortin as a marker to analyse the absolute number and dendritic growth of newly generated neurons in the adult dentate gyrus.

Doublecortin (DCX), a microtubule-associated phosphoprotein, has been recently utilized as a marker of newly born neurons in the adult dentate gyrus (DG). Nonetheless, it is unknown whether DCX exclusively labels newly formed neurons, as certain granule cells with the phenotype of differentiated neurons express DCX. We addressed the authenticity of DCX as a marker of new neurons in the adult DG by quantifying cells that are positive for 5'-bromodeoxyuridine (BrdU), DCX and both BrdU and DCX in hippocampal tissues of adult rats treated with daily injections of BrdU for 12 consecutive days. We provide new evidence that neurons visualized with DCX immunostaining in the adult rat DG are new neurons that are predominantly born during the 12 days before euthanasia. This is confirmed by the robust expression of BrdU in 90% of DCX-positive neurons in the DG of animals injected with BrdU for 12 days. Furthermore, DCX expression is specific to newly generated healthy neurons, as virtually all DCX-positive cells express early neuronal antigens but lack antigens specific to glia, undifferentiated cells or apoptotic cells. As DCX expression is also robust in the dendrites, DCX immunocytochemistry of thicker sections facilitates quantification of the dendritic growth in newly born neurons. Thus, both absolute number and dendritic growth of new neurons that are generated in the adult DG over a 12-day period can be quantified reliably with DCX immunostaining. This could be particularly useful for analysing changes in dentate neurogenesis in human hippocampal tissues as a function of ageing or neurodegenerative diseases.

P‐II‐04 Early Phase Regulation of Human Mesenchymal Stem Cells and the Unique Profile of the Proliferation by Bone Morphogenetic Protein‐2

Human mesenchymal stem cells (hMSCs) obtained from a single donor of the iliac crest, was further investigated for the cell proliferation, cell cycle profiles, gene expressions and the ultrastructures by electron microscopy. The hMSCs significantly increased the cell number by day 2 after by treatment of bone morphogenetic protein (BMP)‐2 alone, or basic fibroblast growth factor (bFGF) alone or combination of both in the serum‐free condition (P &lt; 0.01). The hMSCs demonstrated the remarkable proliferation cell nuclear antigen notably at day 1 and pituitary tumor transforming gene all through the experiment, suggesting the cell cycle progression by BMP‐2 treatment as well as the strong cellular nuclear BrdU expression by immunocytochemistry. The fluorescence‐activated cell sorter also demonstrated the similar pattern of the cell cycle progression between BMP‐2 treatment in the serum‐free and 10 % fetal bovine serum treatment. The BMP‐2 treated hMSCs demonstrated the heterochromatin in the nucleus, suggesting the cell differentiation and the well‐developed granular endoplasmic reticulums, indicating the protein production. The hMSCs successfully proliferate, the cell cycle is progressed, and the cell ultrastructure morphology suggest the remarkable nuclear and of granular endoplasmic reticulum induction by BMP‐2 treatment in the serum‐free condition.

Nutrient stimulated glucagon-like peptide-2 and crypt cell production in experimental short bowel syndrome

Glucagon-like peptide-2 (GLP-2) is known to be tmphic to the intestinal mucosa, and its release is stimulated by luminal nutrients; these features suggest it may be important in controlling intestinal adaptation. Aims: To examine: I) Resection induced malabsorption effects on GLP-2 release 2) The relationship over time of GLP-2 release and adaptation 3) I-he correlation of sernm GLP-2 levels with adaptation as measured by crypt cell proliferation rate (CCPR). Methods: Animals undeI~vent either intesuual transection or 50, 75, or 90 % intestinal rasection Animals were lasted overnight, and on day 7 gavaged with 3 ml (4.5 kcal) of a liquid enteral diet; blood samples were dra~m 1 hour post-prandiafiy and analyzed by n-terminal specihc ELISA to quanfifie GLP-2 3 levels A second group underwent intestinal transection or 90 % imestinal resection. Day 0, 4, 7, 14, and 28 blood was drawn at time 0, 15, 3 0 45, 60, and 90 minute intervals to measure post-prandial GLP-2 s3~ levds. Animals were pulsed t)~r 1 hour with BrdU (50 mg/kg, i.p.), tissue harvested and processed for inimunocytochemistry to determine ileal CCPR Results: A post-prandial stimulated rise in sernm GLP-2 levels signfficandy correlated ,aqth the magnitude of intestinal resection (p<0.0002). Resection slguihcantly increased post-prandial serum GLP-2 levels (see day4 GLP-2 table), area under the curve (AUC) and CCPR at all time points tested. Nutrient stimulated G1.P-2 levels assessed at 1 hour and with AUC analysis significantly correlated with BrdU expression (p<O.O002, p<0.O02 respectwely). Conclusion: Basal and postprandial serum GLP-2 levels were sigmhcamly correlated with the magnitude of intestinal resection and adaptation as measured by CCPR. Ongomg increases in GLP-2 levels and CCPR at 28 days follo,~ng resection suggests that GLP-2 is involved in maintaining adaptation. The significant correlation between post-prandial GLP-2 release and CCPR is supportive of a role for GLP-2 in controlling adaptation tbIlownig intestinal resection.

The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis

Adult animals continue to produce new neurons in the dentate gyrus of hippocampus. Until now, the principal method of studying neurogenesis has been to inject either tritiated thymidine or 5′-Bromo-2-deoxyuridine (BrdU) intraperitoneally followed by autoradiographic or immunohistochemical detection methods respectively. However, such exogenous markers may produce toxic effects. Our objective was to determine whether Ki-67, a nuclear protein expressed in all phases of the cell cycle except the resting phase, can be used as an alternative, endogenous marker. Using immunohistochemistry, we examined Ki-67 and BrdU expression pattern in rats. Ki-67 was expressed within the proliferative zone of the dentate gyrus and its expression pattern mimicked that of BrdU when examined soon after exogenous BrdU administration. Quantitative comparison of BrdU and Ki-67-positive cells showed 50% higher numbers of the latter when examined 24 h after the BrdU injection. This was expected, since BrdU can be incorporated into DNA only during the S-phase of the mitotic process, whereas Ki-67 is expressed for its whole duration. Experimental increases (by ischemia) or reductions (by radiation) in the number of mitotic cells produced parallel changes in BrdU and Ki-67 signals. Thus, Ki-67 is an effective mitotic marker and has most of the benefits of BrdU and none of the costs. This study provides evidence for Ki-67 to be used as a marker of proliferation in the initial phase of adult neurogenesis.

Temporal course of intravital and postmortem proliferation of epidermal cells after mechanical injury. An immunohistochemical study using bromodeoxyuridine in rats.

The temporal course of epidermal basal cell proliferation in the wound of the pinna of rats was studied using bromodeoxyuridine (BrdU) immunohistochemistry. Following incisional wounding, the animals were sacrificed at intervals ranging from 0 hours to 32 days. Two biopsies were taken from each animal, one intravitally and one postmortem after 24 hours storage at 8 degrees C. Specimens were incubated in a solution containing BrdU and embedded in paraffin. BrdU expression was demonstrated by a monoclonal antibody against BrdU. In both intravital and postmortem biopsies, the labelling indices increased significantly in the period from 32 to 60 hours post-injury. This suggests that DNS synthesis induced during life continues after death. Applied to forensic practice, the present findings point to the possibility of determining the vitality of a wound in postmortem tissue.

The significance of Ki-67, BrdU and OKT9 expression in determination of myelodysplastic syndromes by use of APAAP technique.

Monoclonal antibodies (McAb) to Ki-67, BrdU and OKT9 were used in APAAP technique for determining the proliferative cells of 46 myelodysplastic syndromes (MDS) with 16 serving as normal controls. According to Ki-67 positive labelling, the proliferative fractions of bone marrow cells can be divided into low (< 25%), intermediate (25%-35%), and high (> 35%) grades. The labelling positivity of Ki-67, BrdU and OKT9 in MDS was all higher than that of the controls. Furthermore, the positive percentages of these three McAbs were significantly correlated. From the results of our experiment, an increase in proliferative fraction was related to clinical classification, prognosis, and therapy of MDS. On the other hand, the positive percentage of Ki-67 > 40% and BrdU > 30% or ratio of BrdU to Ki-67 > 80% may be considered as parameters for unfavourable prognosis.

The teratogenic interaction of hydroxyurea and 5-bromodeoxyuridine examined with the aid of limb culture and image analysis

Pregnant mice were treated on gestation Day 11 (E11) with a single dose of either hydroxyurea (HU, 250 mg/kg) or 5-bromo-2′deoxyuridine (BrdU, 500 mg/kg), or with a combination of the two agents. The dams were either allowed to go to term, when the fetuses were examined for cleft palate (CP) and digital anomalies, or killed 24 hr after the treatment. In the latter case, limbs from the embryos of control and treated dams were excised and cultured for 6 days in submerged culture. At the end of the culture period, the limb explants were stained for cartilage. The various cartilaginous components were subsequently analyzed using image analysis methods. CP was observed in 2.4 and 22.9% of the fetuses, respectively, after a single dose of HU or BrdU on E11. Simultaneous HU + BrdU treatment decreased the incidence of BrdU-induced CP to 3.6%, while treatment with BrdU 3 hr after HU resulted in 33.3% CP. In addition, syndactyly and ectrodactyly, not seen after the treatment with either HU or BrdU alone, were observed when the two agents were administered simultaneously, 1 hr apart or with a 3-hr delay. The teratogenic response was enhanced when limbs were cultured for 6 days. Digital anomalies were observed in the limb explants of BrdU-pretreated embryos; such abnormalities were not observed in vivo. Image analysis of cultured limb explants revealed that with HU-3 hr-BrdU pretreatment, the total limb area occupied by the long bones was increased at the cost of the paw area. However, no consistent changes in the shape or form of individual bones were observed. HU and BrdU given together produced a teratogenic response which was significantly different from that observed following the administration of HU or BrdU alone. This is considered to be due to an interference with the incorporation of BrdU in the DNA, since HU is known to block DNA synthesis. When HU was given before BrdU, the effects of BrdU were enhanced, probably due to the reported synchronizing action of HU on dividing cells. We also found that after a similar in utero exposure, the culture conditions themselves enhanced the frequency of teratogenic expression of BrdU.