BrdU Labeling Index Research Articles

Involvement of Vasoactive Intestinal Peptide on Insulin-Like Growth Factor I-Induced Proliferation of Rat Pituitary Lactotropes in Primary Culture: Evidence for an Autocrine and/or Paracrine Regulatory System

In previous studies we demonstrated that insulin-like growth factor I (IGF-I) induces pituitary vasoactive intestinal peptide (VIP) gene expression and secretion, and that IGF-I-induced prolactin (PRL) release is mediated by VIP. In this study, we investigate the mitotropic action of IGF-I and VIP on pituitary lactotropes, and their possible interplay in this effect. Cultured male rat pituitary cells were treated with rhIGF-I (10^–7M) and/or VIP (10^–7M) for 48 h. 5-Bromo-2′-deoxyuridine (BrdU) (10 µM) was added for labeling proliferation of pituitary cells. BrdU-labeling indices indicative of the proliferation rate of lactotropes were determined by double-labeling immunofluorescence staining for PRL and BrdU. Treatment with either IGF-I or VIP increased BrdU-labeling indices of lactotropes, but there was no further increase upon combined incubation with both factors, suggesting an interaction between the signal transduction pathways of IGF-I and VIP. VIP antiserum partially suppressed IGF-I-induced BrdU-labeling indices of lactotropes. We also investigated the intracellular signal transduction pathways in the action of IGF-I and VIP on the proliferation of lactotropes. Treatment of pituitary cells with an inhibitor of the mitogen-activated protein kinase (MAPK) pathway completely abolished IGF-I-induced lactotrope proliferation, whereas it partially suppressed VIP-induced BrdU-labeling indices. The protein kinase A (PKA) inhibitor, which abolished the mitogenic action of VIP, markedly suppressed IGF-I-induced lactotrope proliferation. These results indicate that both IGF-I and VIP stimulate lactotrope proliferation, and that IGF-I-induced lactotrope proliferation is partially mediated by VIP produced locally. Also, this study suggests that interactions between MAPK and cyclic adenosine 3′,5′-monophosphate-PKA signaling pathways are implicated in the lactotrope proliferation induced by IGF-I and VIP.

Inhibition of STAT3 prevents neointima formation by inhibiting proliferation and promoting apoptosis of neointimal smooth muscle cells.

In cultured vascular smooth muscle cells (SMCs), STAT3 mediates proliferation signal by directly activating transcription of early growth response genes. Recently, we have found that balloon injury transiently induces JAK2 and STAT3 expressions and activations with a peak at day 7 in rat carotid artery. However, the specific role of STAT3 in neointima formation remains unknown. Adenoviral vector encoding a dominant negative STAT3 (AxCAdnSTAT3) or beta-galactosidase (control) was overexpressed in a balloon-injured artery to inhibit endogenous STAT3 activation selectively. In controls, neointima became evident after day 4, and reached a maximum at day 14. The number of bromodeoxyuridine (BrdU)-positive proliferating or TUNEL-positive apoptotic neointimal SMCs peaked at day 7, decreasing to lower levels by day 14. AxCAdnSTAT3 not only abrogated STAT3 phosphorylation but also decreased BrdU labeling index by 60% and increased TUNEL index by 35% at day 7 versus controls, resulting in the 40% reduction in the intima/media area ratio at day 14. At day 7, in controls, vascular injury upregulated antiapoptotic mediator Mcl-i and Bcl-xL expression by 8-fold to 5-fold, respectively, versus sham, whereas proapoptotic Bax slightly increased by 1.5-fold versus sham. AxCAdnSTAT3 reversed the upregulated Mcl-1 and Bcl-xL levels by 70% and 37%,respectively, while having no affect on Bax expression. In conclusion, the STAT3-mediated pathway plays an important role in neointima formation through enhanced vascular SMC accumulation by promoting cell proliferation and survival.

Suppression of proliferative cholangitis in a rat model by local delivery of paclitaxel.

Proliferative cholangitis (PC) leads to biliary stricture, which is the main cause of hepatolithiasis, recurrent cholangitis, and biliary cirrhosis. The aim of this study was to determine whether local delivery of paclitaxel, which inhibits cell proliferation by overstabilization of microtubules, prevents PC in a rat model. PC was induced by introducing a fine nylon thread into the bile duct in a rat. Paclitaxel (100 microl of 10, 100, and 1000 micromol/l) or solvent vehicle was administered into the bile duct for 15 min. One week after treatment, histopathologic examination and 5-bromodeoxyuridine (BrdU) labeling of the bile duct were performed. In comparison with the control, the mean thickness of the bile duct was reduced by 29% in the 1000 micromol/l paclitaxel-treated group (2.61 +/- 0.31 microm vs 3.67 +/- 0.25 micro m, P << 0.05). The luminal area increased ( P << 0.0001) and the grade of epithelial-glandular proliferation was decreased ( P << 0.01) as the dose of paclitaxel increased. Ductal fibrosis and inflammatory cell infiltration were similar in both groups. The BrdU labeling index was significantly lower in the paclitaxel-treated group ( P << 0.05). Local delivery of paclitaxel suppressed PC in a rat model by the inhibition of epithelial-glandular proliferation and may offer an effective therapeutic option for biliary stricture.

The effects of ad libitum overfeeding and moderate and marked dietary restriction on age-related spontaneous pituitary gland pathology in Sprague-Dawley rats.

This study compared the effects of ad libitum (AL) overfeeding and moderate or marked dietary restriction (DR) on the pathogenesis of aged-related pituitary gland changes in Sprague-Dawley (SD) rats. SD rats were fed Purina Certified Rodent Diet AL (group 1), DR at 72-79% of AL (group 2), DR at 68-72% of AL (group 3) or DR at 47-48% of AL (group 4) for 106 weeks. Interim necropsies were performed at 13, 26, and 53 weeks, after a 7-day 5-bromo-2-deoxyuridine (BrdU)-filled minipump implantation. Body weights, organ weights and insulin-like growth factor 1 (IGF-1) serum levels were measured at interim and final necropsies. Serum levels of prolactin (PRL), progesterone, estradiol, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured at 53 and/or 106 weeks. In addition to the routine histopathologic examination, determination of 7 stereologic parameters after pituitary immunohistochemistry of PRL, growth hormone (GH) and BrdU was done in both sexes at 13, 26, and 53 weeks. Body and pituitary weights were proportional to the food intake. In AL-fed rats, hyperplastic and neoplastic changes developed early and progressed with age, affecting almost all animals by 106 weeks. These changes were associated with high PRL serum levels. Pituitary adenomas were the most common cause of death in both sexes. In DR rats, a delayed onset and a decreased incidence of pituitary tumors were observed in association with decreased serum IGF-1, PRL, estradiol, and LH levels. The results of the stereological analysis demonstrated that, compared to AL-fed rats, pituitary glands from DR rats contained lower PRL and GH secreting cell volumes, and a lower epithelial cell BrdU labeling index, which correlated with a lower incidence of pituitary tumors at study termination. Moderate and marked degrees of DR delayed the onset of pituitary tumors in a temporal- and dose-related manner. In contrast to marked DR, which dramatically reduced the incidence of hyperplastic and neoplastic pituitary gland changes, moderate DR delayed the onset but did not prevent the development of pituitary tumors.

Effects of 3,3′,4,4′,5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis

3,3′,4,4′,5-Pentachlorobiphenyl (PCB126), a congener with a planar configuration, has been established to have relatively strong toxicities similar to those of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) via aryl hydrocarbon receptors. We investigated the effects of this coplanar PCB on mammalian early spermatogenesis and steroidogenesis in a mouse neonatal testicular organ culture system. Testes collected from newborn mice were subjected to organ culture in medium containing 0, 10, 100 or 1000 n m PCB126. Histochemical analysis revealed that the BrdU-labeling indices of both spermatogenic cells and Sertoli cells were unchanged in all testis specimens exposed to the coplanar PCB. CYP1A1 and steroidogenic enzymes (P450scc, P450c17, 3β-HSD and 17β-HSD) mRNA levels were determined by semiquantitative RT-PCR. The CYP1A1 mRNA level in cultured testis was significantly increased by PCB126 in a dose-dependent manner. Although mRNA levels of 3β-HSD and 17β-HSD were unchanged, the P450scc mRNA level was significantly down-regulated by PCB126 in a dose-dependent manner. In contrast, the P450c17 mRNA level was significantly higher in 1000 n m PCB126-exposed testis than in control testis. These results suggest that the coplanar PCB does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, but that it directly affects the expression of steroidogenic enzyme genes.

Nulliparous CCAAT/enhancer binding proteindelta (C/EBPdelta) knockout mice exhibit mammary gland ductal hyperlasia.

CCAAT/Enhancer binding proteins (C/EBPs) are a family of nuclear proteins that function in the control of cell growth, death, and differentiation. We previously reported that C/EBPdelta plays a key role in mammary epithelial cell G(0) growth arrest. In this report, we investigated the role of C/EBPdelta in mammary gland development and function using female mice homozygous for a targeted deletion of C/EBPdelta (C/EBPdelta -/-). C/EBPdelta -/- females develop normally and exhibit normal reproductive and lactational performance. Adult nulliparous C/EBPdelta -/- females, however, exhibit mammary epithelial cell growth control defects. The mean number of mammary ductal branches is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ (wild-type control) females (66.8 +/- 5.2 vs 42.9 +/- 6.3 branch points/field, P < 0.01). In addition, the mean total mammary gland cellular volume occupied by epithelium is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls (29.0 +/- 1.4 vs 20.4 +/- 1.3, P < 0.001). Our results showed that the BrdU labeling index was significantly higher in mammary epithelial cells from nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls during the proestrus/estrus (4.55 +/- 0.70 vs 2.14 +/- 0.43, P < 0.01) and metestrus/diestrus (6.92 +/- 0.75 vs 3.98 +/- 0.43 P < 0.01) phases of the estrus cycle. In contrast, the percentage of mammary epithelial cells undergoing apoptosis during both phases of the estrus cycle did not differ between C/EBPdelta -/- and C/EBPdelta +/+ females. The increased epithelial cell content and proliferative capacity was restricted to the nulliparous C/EBPdelta -/- females as no differences in mammary gland morphology, ductal branching or total epithelial content were observed between multiparous C/EBPdelta -/- and C/EBPdelta +/+ females. These results demonstrate that C/EBPdelta plays a novel role in mammary epithelial cell growth control that appears to be restricted to the nulliparous mammary gland.

Liver regeneration in heparin-binding EGF-like growth factor transgenic mice after partial hepatectomy

Background & Aims: Heparin-binding epidermal growth factor–like growth factor (HB-EGF), a member of the EGF family, is synthesized in the form of a membrane-anchored precursor (proHB-EGF), which subsequently is processed proteolytically to mature HB-EGF. This study describes the effects of HB-EGF on liver regeneration after 70% partial hepatectomy in proHB-EGF transgenic mice with liver-specific expression. Methods & Results: No significant differences in liver/body weight ratios and in bromodeoxyuridine (BrdU)-labeling index (the ratios of BrdU-positive hepatocyte nuclei) were found between adult transgenic and wild-type mice. However, in regenerating liver after partial hepatectomy, transgenic mice had higher liver/body weight ratios than wild-type mice and at 120 hours reached a level equal to that before partial hepatectomy. The BrdU-labeling index was about 5 times higher in the livers of transgenic mice compared with the wild type (51.5% vs. 10.2%, respectively; P < 0.01) at 48 hours after partial hepatectomy. Activation of microtubule-associated protein kinase after partial hepatectomy was higher and earlier in the transgenic mice as compared with the wild-type mice. Soluble HB-EGF was increased in the liver (at 8 min) after partial hepatectomy, indicating that the shedding of proHB-EGF occurred after partial hepatectomy. Conclusions: The transgenic expression of HB-EGF accelerates the proliferation of hepatocytes after partial hepatectomy, suggesting that HB-EGF functions as a hepatotrophic factor in vivo.GASTROENTEROLOGY 2003;124:701-707

Lateral functional shift of the mandible: Part I. Effects on condylar cartilage thickness and proliferation

Lateral functional shift of the mandible is characterized by transverse rotation of the entire mandible about a vertical axis toward 1 side of the head. The effect of this shift is that the condyle on the side opposite the direction of the shift is displaced anteriorly, or protruded, while the condyle on the side toward the shift is more stable positionally and is likely to be slightly retruded. According to the view that growth of the mandibular condylar cartilage (MCC) adapts to its local functional-biomechanical environment, differential changes in metabolic activity of the MCC would be expected on the nonprotruded and the protruded sides. To evaluate this hypothesis, 21 rats (28 days old) were fitted with intraoral positioners designed to shift the mandibular postural position asymmetrically. Cartilage thickness and BrdU labeling index in the MCC 3, 7, and 14 days after placement of the positioners were compared with those in age-matched controls that received no positioners. Cartilage thickness in the MCC on the protruded side was significantly greater than that in the controls at each time interval, with the difference increasing slightly with time. The labeling index for the protruded MCC was significantly greater than the controls at both 7 and 14 days after positioner placement. Trends on the nonprotruded side were generally opposite, culminating in reduced thickness and proliferation after 14 days. Thickness and labeling index were greater on the protruded side at every time interval except 3 days (labeling index). These trends in cartilage thickness and proliferation are consistent with previous studies of MCC changes after bilateral functional protrusion or retrusion. These data suggest that changes in MCC thickness and proliferative activity might accompany a lateral functional shift of the mandible in growing persons. (Am J Orthod Dentofacial Orthop 2003;123:153-9)

Betacellulin Promotes Growth of the Gastrointestinal Organs and Effects a Diuresis in Normal Rats

Betacellulin is a relatively new member of the epidermal growth factor peptide family, however, its function remains poorly defined. We investigated its physiological effects in rats implanted with pumps to deliver vehicle or recombinant rat betacellulin [46 7 g/day] for 7 days. At kill, blood and gastrointestinal tissues were collected for determinations of betacellulin levels, proliferation (bromodeoxyuridine-BrdU incorporation) and growth. Plasma betacellulin levels were increased 8-fold compared to vehicle, whilst serum insulin, body weight and food intake were decreased by 32, 15 and 9%, respectively. Water intake, urine and faecal output and small intestinal weight were respectively increased by 36, 78, 47 and 24%. Ileal and proximal colonic crypt depths were increased by 25 and 51% although the BrdU labelling index was unaffected. Betacellulin stimulated gastrointestinal growth, the increased responsiveness of the terminal ileum and colon suggesting therapeutic potential in disease conditions in which ileal or colonic re-growth is desirable. Betacellulin further stimulated a diuresis suggesting an additional role in fluid homeostasis.

Effect of Juvenile Aging on Hepatocellular Proliferation in Male Han Wistar(GALAS) Rats.

In order to investigate the age group which shows constant hepatocellular proliferation rates in normal male Wistar Hannover GALAS juvenile rats, cell proliferation in the left lateral, the right lateral, and the right medial lobes of the liver was evaluated by BrdU labeling index (LI%) at 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of age. LI% was significantly increased from 4 to 7 weeks of age and lower rate values were shown constantly from 8 to 16 weeks of age. In addition, there was no clear interlobular difference in LI% of the liver in rats of any age.

Immunohistochemical proliferation markers may overestimate the growth potential after ionizing radiation: in vivo study in the rat anterior pituitary gland.

The effect of ionizing radiation on the expression of immunohistochemical proliferation markers was examined in the rat pituitary gland. Rats were irradiated in the pituitary region with a dose of 40 Gy, or were sham-irradiated as controls. Bromodeoxyuridine (BrdU) was given to the rats after one week, either one hour (Br-1 group) or 17 hours (Br-17 group) before perfusion fixation. Immunohistochemical staining for BrdU, topoisomerase II-alpha (TopoII), Ki-67 (MIB-5), p21WAF1/Cip1 (p21), and p27Kip1 (p27) was performed. Apoptotic cells were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling method. The mean BrdU labeling index (LI) and MIB-5 LI were significantly higher in the irradiated rats than in the sham rats in the Br-1 group. TopoII LI was higher in the irradiated rats than in the sham rats, although not significantly. p27-positive cells decreased in irradiated rats, but p21-positive cells increased more than in the sham rats. The number of apoptotic cells increased significantly after radiation. BrdU LIs were lower in the irradiated rats than in the sham rats in the Br-17 group. A few small BrdU-positive fragments with apoptotic features were phagocytosed in the anterior lobe cells. These results indicate that some "immunohistochemically proliferating cells" subsequently undergo apoptosis in the irradiated pituitary gland. The values of proliferative indices should be cautiously interpreted after irradiation of tissue.

Impaired liver regeneration after partial hepatectomy in db/db mice

Fatty liver is the most common hepatic disorder in humans and supposed to be a cause of poor prognosis after liver transplantation and hepatic resection which could be resulted from impaired liver regeneration. This study was carried out to analyze the process of liver regeneration in db/db mice which show severe steatosis because of abnormal leptin receptor. We performed 70% partial hepatectomy (PH) on db/db mice and normal +m/+m mice, and then sacrificed the animals 1, 2, 3, 5, 7 and 10 days later. The liver samples were weighed and examined histologically or immunohistochemically. As a result, the liver mass restitution was significantly inhibited in db/db mice compared with +m/+m mice. The BrdU labelling index peaked at 2 days after PH in both strains, although the value was lower in db/db mice. After that, interestingly, it decreased to the control level at 5 days in +m/+m mice while the recovery was delayed in db/db mice. Similar sequence was also observed in the PCNA labelling index. In addition, the peak time of the mitosis index was 2 days and 5 days after PH in +m/+m mice and in db/db mice, respectively. Thus, although not significant, the proliferative response of hepatocytes to PH occurred somewhat more transient and sharply in +m/+m mice while it lasted somewhat longer in db/db mice. This suggests that db/db mice may be valuable as one of the animal models for the investigation of the effects of steatosis on the liver regeneration.

Proliferative Activity of Canine Mast Cell Tumours Evaluated by Bromodeoxyuridine Incorporation and Ki-67 Expression

The proliferative activity of 91 canine mast cell tumours was assessed on the basis of the Ki-67 positive index (Ki-67 PI) and mitotic index (MI) and, in 15 cases, also by the labelling index of bromodeoxyuridine (BrdU; an analogue of tritiated thymidine) incorporated in vivo into S-phase cells. BrdU and Ki-67 were detected immunohistochemically. The tumours were graded histologically (I, II or III). The BrdU labelling index (BrdU LI) tended to increase as the grade became higher. In terms of the mean values of Ki-67 PI, significant differences were found between histological tumour grades I and II (P<0·01) and between grades II and III (P<0·01). In terms of mean MI, grades I and II were found to differ significantly (P<0·05). With Spearman rank correlation coefficient and linear regression analysis, the BrdU LI and Ki-67 PI showed a highly significant correlation. This strong correlation indicated that Ki-67 was, like BrdU, a useful marker for proliferative potential in canine mast cell tumours; moreover, its use did not require the prior administration of any reagent to the live animal.

Decreased beta-cell proliferation impairs the adaptation to pregnancy in rats malnourished during perinatal life.

We investigated the cellular mechanisms responsible for the inability of 8-month-old previously malnourished (PM) females to adapt their beta-cell mass during pregnancy. The evolution during pregnancy of beta-cell fraction, size and proliferation was studied. At day 21 of pregnancy beta-cell fraction increased less in PM than in control females, compared with their non-pregnant values. A slight beta-cell hypertrophy was observed during pregnancy in both groups. In control females, beta-cell 5-bromo-2'-deoxyuridine (BrdU) labelling index (LI) increased from 0.07+/-0.04% before pregnancy to 1.13+/-0.20% at day 12 and decreased thereafter to reach again basal levels at day 21. In PM females, beta-cell proliferation rate was decreased at day 12 (0.74+/-0.15%, P<0.05) but similar to controls at all other stages studied. Separate analysis of the head and tail parts of the pancreas in control animals revealed that the beta-cell fraction during pregnancy increased more in the head than in the tail; similarly, BrdU LI increased 20-fold in the head and 10-fold in the tail, compared with non-pregnant values. In PM females, no adaptation of beta-cell fraction could be observed in the head, where BrdU LI was decreased by half at day 12 of pregnancy. In PM females the lactogenic activity was twice that of controls at day 12 whereas all beta-cells expressed the prolactin receptor. In conclusion, perinatal malnutrition impairs subsequent adaptation to pregnancy by decreasing beta-cell proliferation in the head of the pancreas at a critical time during pregnancy.

Carcinogenicity of dimethylarsinic acid in male F344 rats and genetic alterations in induced urinary bladder tumors.

Arsenic is a well-documented human carcinogen, and contamination with this heavy metal is of global concern, presenting a major issue in environmental health. However, the mechanism by which arsenic induces cancer is unknown, in large part due to the lack of an appropriate animal model. In the present set of experiments, we focused on dimethylarsinic acid (DMA), a major metabolite of arsenic in most mammals including humans. We provide, for the first time, the full data, including detailed pathology, of the carcinogenicity of DMA in male F344 rats in a 2-year bioassay, along with the first assessment of the genetic alteration patterns in the induced rat urinary bladder tumors. Additionally, to test the hypothesis that reactive oxygen species (ROS) may play a role in DMA carcinogenesis, 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in urinary bladder was examined. In experiment 1, a total of 144 male F344 rats at 10 weeks of age were randomly divided into four groups that received DMA at concentrations of 0, 12.5, 50 and 200 p.p.m. in the drinking water, respectively, for 104 weeks. From weeks 97-104, urinary bladder tumors were observed in 8 of 31 and 12 of 31 rats in groups treated with 50 and 200 p.p.m. DMA, respectively, and the preneoplastic lesion, papillary or nodular hyperplasias (PN hyperplasia), was noted in 12 and 14 rats, respectively. DMA treatment did not cause tumors in other organs and no urinary bladder tumors or preneoplastic lesions were evident in the 0 and 12.5 p.p.m.-treated groups. Urinary levels of arsenicals increased significantly in a dose-responsive manner except for arsenobetaine (AsBe). DMA and trimethylarsine oxide (TMAO) were the major compounds detected in the urine, with small amounts of monomethylarsonic acid (MMA) and tetramethylarsonium (TeMa) also detected. Significantly increased 5-bromo-2'-deoxyuridine (BrdU) labeling indices were observed in the morphologically normal epithelium of the groups treated with 50 and 200 p.p.m. DMA. Mutation analysis showed that DMA-induced rat urinary bladder tumors had a low rate of H-ras mutations (2 of 20, 10%). No alterations of the p53, K-ras or beta-catenin genes were detected. Only one TCC (6%) demonstrated nuclear accumulation of p53 protein by immunohistochemistry. In 16 of 18 (89%) of the TTCs and 3 of 4 (75%) of the papillomas, decreased p27(kip1) expression could be demonstrated. Cyclin D1 overexpression was observed in 26 of 47 (55%) PN hyperplasias, 3 of 4 (75%) papillomas, and 10 of 18 (56%) TCCs. As a molecular marker of oxidative stress, increased COX-2 expression was noted in 17 of 18 (94%) TCCs, 4 of 4 (100%) papillomas, and 39 of 47 (83%) PN hyperplasias. In experiment 2, 8-OHdG formation in urinary bladder was significantly increased after treatment with 200 p.p.m. DMA in the drinking water for 2 weeks compared with the controls. The studies demonstrated DMA to be a carcinogen for the rat urinary bladder and suggested that DMA exposure may be relevant to the carcinogenic risk of inorganic arsenic in humans. Diverse genetic alterations observed in DMA-induced urinary bladder tumors imply that multiple genes are involved in stages of DMA-induced tumor development. Furthermore, generation of ROS is likely to play an important role in the early stages of DMA carcinogenesis.

Human limbal progenitor cells expanded on intact amniotic membrane ex vivo.

The transplantation of human limbal epithelium on amniotic membrane as a substrate is a new treatment for limbal stem cell deficiency. Limbal epithelial stem cells are characterized by a slow cell cycle and the lack of K3 keratin and connexin 43 (Cx43), a gap junction protein. We investigated Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of limbal epithelium expanded on amniotic membrane. Connexin 43 expression and bromodeoxyuridine (BrdU) incorporation were determined by immunohistology. The GJIC was investigated by a scrape-loading dye transfer assay. Expression of Cx43 and K3 keratin as well as BrdU-retaining nuclei were also analyzed after xenotransplantation in nude mice. Limbal epithelium showed mean +/- SD 12.4% +/- 14.5% positive units of Cx43 expression and a low BrdU labeling index of 2.4% +/- 0.9% (n = 5), of which the latter was due to slow cycling, as proved by its increase to 62.0% +/- 9.5% after continuous BrdU labeling for 5 days. Most of the expanded epithelium did not show GJIC (83%), significantly more than that grown on plastic (6%; P<.002). Basal cells of the stratified epithelium after xenotransplantation did not express Cx43 and K3 keratin, but their nuclei retained BrdU. These results support the hypothesis that intact amniotic membrane preferentially preserves and expands Cx43-negative, keratin K3-negative, and GJIC-deficient limbal epithelium, a phenotype resembling that of stem cell-containing limbal basal epithelial cells in vivo. Intact amniotic membrane is a suitable substrate for bioengineering limbal epithelia for ocular surface reconstruction.

Induction of PDX-1-positive cells in the main duct during regeneration after acute necrotizing pancreatitis in rats.

Pancreatic regeneration involves two pathways; proliferation and differentiation of pancreatic progenitor cells, which probably exist in pancreatic ductal epithelium, and replication of pre-existing differentiated acinar, islet, and ductal epithelial cells. During pancreatic development, differentiated cells arise from the ductal progenitor cells expressing the pancreatic/duodenal homeobox-1 (PDX-1) homeodomain transcription factor. The aims of this study were to characterize cell proliferation and differentiation during regeneration after acute necrotizing pancreatitis and to evaluate the role of PDX-1-positive stem cells. Necrotizing pancreatitis was induced in rats by retrograde intraductal infusion of sodium taurocholate. Cell types were classified into five categories: main, large, and small ductal epithelial cells, tubular complexes and acinar cells. Each category was scored using a 5-bromo-2'-deoxyuridine (BrdU) labelling index (LI) at various time points after induction of pancreatitis. Tissue sections were also immunostained for PDX-1 to determine the source of pancreatic stem cells. Acinar necrosis was observed at 24 h after induction of pancreatitis and most lobules were filled with tubular complexes on day 5. Subsequently, newly formed acinar cells were observed on day 7, but the lobular architecture returned to normal appearance on day 28. Proliferation started in the main and large ducts at 24 h; marked mitotic activity was evident in small ductal epithelial cells and tubular complexes on day 3, and in acinar cells on day 7. At 24 h after induction of pancreatitis, epithelial cells of the main duct with PDX-1-positive nuclei were greatly increased, simultaneously with the peak LI of BrdU. These results suggest that regeneration after necrotizing pancreatitis involves proliferation and differentiation of pancreatic progenitor cells, and that ductal epithelial cells with PDX-1-positive nuclei may contribute to the differentiation of pancreatic stem cells in the main duct.

Epithelial Kinetics in Mouse Heterotopic Tracheal Allografts

Obliterative bronchiolitis (OB) is the most important cause of graft dysfunction post-lung transplantation. It is likely that the small airway epithelium is a target of the alloimmune response, and that epithelial integrity is a crucial determinant of airway patency. Our goals are to elucidate epithelial cell kinetics in the heterotopic mouse trachea model and to determine potential mechanisms of cell death in allografts. Allografts and isografts were obtained by transplanting BALB/c tracheas into C57BL/6 and BALB/c immunosuppressed and non-immunosuppressed hosts, respectively and harvested from day 3-20. Morphometry, BrdU and TUNEL labeling, and EM studies were performed. Columnar epithelium in isografts and allografts sloughs during day 0-3, but regenerates in both sets of grafts by day 10. Subsequently, allografts become inflamed and denuded, while isografts retain an intact epithelium. Prior to airway denudation, allografts exhibited significantly increased epithelial cell density, BrdU labeling index (LI), and TUNEL positive cells. Epithelial apoptosis was confirmed by electron microscopy. Allograft percent ciliated columnar epithelium and lumenal circumference were significantly decreased. Cyclosporin delayed airway fibrosis but did not alter the progression of the allograft through the phases of early ischemic injury, airway epithelial cell regeneration, and eventual cell death. These studies quantitatively demonstrate that the allograft epithelium actively regenerates in the alloimmune environment, but succumbs to increased apoptotic cell death, underscoring the importance of the airway epithelium as a self-renewing source of alloantigen.

The influence of pylorus-preserving partial gastrectomy on carcinogenesis of residual gastric mucosa in the rat.

The aim of this study is to investigate a pylorus- and vagus-nerve-preserving partial gastrectomy to discover any correlation to a reduced carcinoma occurrence from residual gastric mucosa. Wistar rats underwent a distal partial gastrectomy (Group D), a pylorus-preserving partial gastrectomy (Group P), or a simple laparotomy (as a control) (Group C) while some also underwent an additional truncal vagotomy (Groups DV, PV, and CV). Continuous per oral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was given every day for 8 weeks before operation. At 3, 6, and 12 months after surgery, the animals in each group were administered an intraperitoneal injection of bromodeoxyuridine (BrdU). At 1 h after injection, the animal was killed, and the residual stomach removed. The histopathological findings showed no intestinal metaplasia and no gastric cancer in any group. No abnormality in the gastric mucosa was found in either Group C or Group CV. However, anastomotic ulcers, cystic glands, and submucosal gland formation were found significantly more in Groups P and in PV than in either Groups D or DV (each P < 0.05). Atypical glands were found in only one rat in each of Groups D and DV, and gastritis cystica polyposa (GCP) was found in only one rat in Group DV. All of these findings occurred in the fundic gland mucosa on the oral side of the anastomosis. The BrdU labeling index in this fundic gland mucosa was significantly higher in Groups D and DV than that in Groups C and CV, at 3, 6, and 12 months postoperatively (P < 0.05). The pH of the gastric juice was higher in all those groups that underwent vagotomy, with a significant difference only between Groups DV and C (P < 0.05). The concentration of bile acids contained in the gastric juice (microM/l) was significantly lower in Groups P and PV than that in Groups D or DV (each P < 0.05). The preservation of the pylorus was considered to be a useful surgical method to prevent the retrograde reflux of the duodenal juice into the residual stomach, and also reduced the proliferative activity of the residual gastric mucosa on the oral side of the anastomosis.

Effects of portal vein arterialization on liver regeneration after partial hepatectomy in the rat.

Although portal venous supply is considered essential to preserve hepatic integrity, in this study, effects of portal arterialization on liver regeneration were evaluated in a rat model of partial hepatectomy (PH). Ninety-six Lewis rats were randomly assigned to four groups of 24 rats each: PH only (group 1), PH with either venous or arterialized portal supply (groups 2 and 3, respectively), and PH without portal supply (group 4). Liver regeneration rate (LRR), 5-bromo-2-deoxyuridine (BrdU) labeling index, and liver biological characteristics were assessed on days 1, 2, 3, and 7. Compared with group 1, all tested rats had a marked body weight loss after surgery, and only rats in group 4 showed no signs of recovery on day 7. With maintained portal inflow (groups 1, 2, and 3), LRRs increased steadily to day-7 values of 89.2% +/- 11.8%, 81.4% +/- 8%, and 77.4% +/- 9.4%, respectively (P = not significant), and 24-hour peak values of BrdU labeling index were 159 +/- 26, 157 +/- 42, and 149 +/- 48, respectively (P = not significant). Conversely, rats deprived of portal supply (group 4) showed profound inhibition of these two parameters (14 +/- 13; P <.01;32.1% +/- 7.7%; P <.001, respectively). These results indicate that proper portal blood supply is essential to initiate and maintain liver regeneration after PH. With an equivalent portal inflow rate of either venous or arterial source, the hepatic regeneration response can be sustained.