Dentate gyrus neurons that are born at the peak of development, but not before or after, die in adulthood

In the dentate gyrus of the rodent hippocampus, neurogenesis begins prenatally and continues to the end of life. Adult-born neurons often die in the first few weeks after mitosis, but then survive indefinitely. In contrast, neurons born at the peak of development are initially stable but can die later in adulthood. Physiological and pathological changes in hippocampal structure may therefore result from both the addition of new neurons and the loss of older neurons. However, it is unknown whether neurons born at other stages of development also undergo delayed cell death. Here, we used BrdU to label dentate granule cells that were born in rats on embryonic day 19 (E19; before the developmental peak), postnatal day 6 (P6; peak) and P21 (after the peak). We quantified BrdU+ neurons in separate groups of rats at 2 and 6 months post-BrdU injection. Consistent with previous work, there was a 15% loss of P6-born neurons between 2 and 6 months of age. In contrast, E19- or P21-born neurons were stable throughout young adulthood. Delayed death of P6-born neurons suggests they may play a unique role in hippocampal plasticity and pathology in adulthood.

Ineffective Erythropoiesis in β-Thalassemia Is Characterized by Enhanced Survival Mechanisms That Reduce P21-Mediated Cell Death.

Ineffective erythropoiesis (IE) in β-thalassemia is described as increased expansion of erythroid progenitor cells in combination with accelerated apoptosis and intramedullary hemolysis. However, evidence for this assumption is not particularly strong. In this study we evaluated the kinetics of red blood cell proliferation and survival in thalassemic mice that exhibit levels of anemia consistent with thalassemia intermedia (th3/+) and major (th3/th3), as we described previously. Th3/+ mice show anemia and increased reticulocyte counts (8.9±1.1 g/dL and 17.7±2.6x105/ul compared to 14.9±1.1 g/dL and 2.6±0.4x105/ul in +/+), whereas th3/th3 mice show more severe anemia than th3/+ mice and reduced production of reticulocytes (3.0±1.2 g/dL and 1.8±0.7x105/ul). As soon as two months of age, EPO levels in thalassemic mice are significantly increase over normal mice, 10 times in th3/+ and up to three orders of magnitude in th3/th3 mice. The total number of nucleated erythroid cells (spleen + bone marrow) increased from 3.3±0.9x108 in +/+ to 1.2±0.5x109 (or 3.6 folds) in th3/+ and 1.6±0.6x109 (or 4.8 folds) in th3/th3 mice (N=5 per group), whereas the level of apoptotic cells increased only 2 to 3 folds in percentage in th3/+ and th3/th3, respectively, as observed using the apoptotic Annexin-V and erythroid specific Ter119 markers (9.9±5.0, 14.3±2.5, 24.2±6.7 in BM and 10.6±3.5, 14.8±6.5, 25.4±6.2 in spleen of +/+, th3/+ and th3/th3, respectively; n=3 per genotype). This data was confirmed by TUNEL, cleaved caspase-3/7 and by immunostaining assays. Bilirubin and LDH levels were not different between thalassemic and +/+ mice. Altogether these observations indicated that in thalassemia there is a disproportion between number of proliferating and dying cells with a net increase of erythroid cells. Furthermore, microarray analysis on erythroid cells indicated increased expression of cell cycle promoting genes such as Ki67, Mcm3, Cyclin-A, CDK2 and BclXL (2 to 6 folds compared to +/+ mice, n=5 per genotype). This data was confirmed by Q-PCR, Western blot, immunostaining, clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after in vivo BrdU injection (22, 30 and 44% BrdU+ in +/+, th3/+ and th3/th3, respectively). On the other hand, in th3/th3, which show more apoptosis than th3/+ mice, the cyclin-dependent kinase inhibitor p21 was upregulated both at the RNA (50-folds) and protein level. P53 was also analyzed in th3/th3 mice, showing no expression. In order to investigate the function of p21 in thalassemic erythroid cells, its expression was analyzed on purified erythroid cells isolated from th3/th3 that were transfused (10.4±0.4 g/dL of Hb) or thalassemic mice showing different levels of anemia (6.2±0.2 and 2.1±0.8 g/dL of Hb, respectively; N=3 per each group). We observed that the level of p21 increased with anemia and the severity of the pathology. However, in th3/th3 mice that were injected with BrdU, immunostaining analysis indicated that a large amount of p21+ erythroid cells were also BrdU+. In conclusion, we propose that erythropoiesis in β-thalassemia is characterized by enhanced expression of cell cycle promoting and survival factors that are able to overcome or mitigate p21 cell cycle block and, probably, apoptosis.

309 THE SPERMATOGENIC CYCLE IN MAMMALIAN TESTIS XENOGRAFTS

Grafting of immature testis tissue from different mammalian donor species into mouse hosts results in production of spermatozoa from the donor species. Xenografting of testis tissue from rhesus monkeys, pigs, and sheep accelerates sperm production. To determine whether this shortened time to sperm production is due to the reduced spermatogenic cycle length, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Testes from 1-2-week-old Yorkshire cross pigs and 1-week-old Suffolk sheep were cut into small fragments (approximately 1 � 1 � 2 mm) and eight fragments were grafted under the back skin of each castrated male immunodeficient NCR nude recipient mouse (n = 7 for pig, n = 5 for sheep). Mice were given BrdU (100 mg/kg i.p.) at 7 months (porcine tissue) or 6 months (ovine tissue) post-transplantation. Mice carrying porcine tissue were sacrificed 1 h, 9 days or 18 days after BrdU injection. Mice with ovine testicular tissue were sacrificed 1 h, 11 days or 22 days after BrdU injection. Analysis time points were chosen based on the reported length of the spermatogenic cycle in pigs and sheep (approximately 9 days and 11 days, respectively). All eight stages of the spermatogenic cycle were analyzed to identify the most advanced germ cells labeled in each time period after BrdU injection. All seminiferous tubules containing full spermatogenesis were analyzed. Histologically, 51.8% (range 7 to 98%, n = 2040 tubules) of seminiferous tubules from porcine grafts, and 64.4% (range 2 to 92%, n = 2903 tubules) of seminiferous tubules from ovine grafts presented complete spermatogenesis. In porcine grafts, the most advanced germ cells labeled 1 h after BrdU injection were primary spermatocytes in pre-leptotene/leptotene at stage I of the spermatogenic cycle. At 9 days and 18 days after injection, the most advanced labeled germ cells were primary spermatocytes at pachytene at stage I and elongating spermatids at late stage II, respectively. In ovine grafts, the most advanced labeled germ cells at 1 h, 11 days and 22 days were pre-leptotene/leptotene at stage II, primary spermatocytes at the pachytene at stage I and elongating spermatids at stage II, respectively. These results indicate that each spermatogenic cycle in porcine and ovine testis xenografts lasts around 9 days and 11 days, respectively. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts and shortened time to sperm production is likely due to accelerated maturation of the testicular somatic components, such as Sertoli cells. This work was supported by NIH R01 RR17359-01.

272. Progesterone stimulates endothelial cell proliferation, but not stromal cell proliferation, in mouse endometrium

Previous studies have suggested that progesterone stimulates stromal cell (SC) proliferation in the mouse endometrium1. However, these studies have not differentiated endothelial cells (EC) from other SC. In this study, we investigated the effects of progesterone on cellular proliferation in ovariectomised mouse endometrium. We hypothesised that progesterone would stimulate both SC and EC proliferation. One group of CBA × C57 mice (n = 6) were treated with a single injection of 100 ng of estradiol on day eight following ovariectomy, followed by a day with no treatment and three consecutive daily injections of 1 mg progesterone. Other groups were treated with either the vehicle (n = 5), estradiol (n = 4) or progesterone (n = 5) injections only. All groups were dissected on day 13 after ovariectomy, 4 h following a BrdU injection. CD31/BrdU double staining immunohistochemistry allowed proliferating EC to be differentiated from proliferating SC. Mice treated with progesterone only had significantly higher EC proliferation in comparison to females treated with progesterone following oestrogen priming (P = 0.05) or vehicle only (P = 0.01) (progesterone only: median=97.3 proliferating EC (PEC)/mm2 [range = 60.8–203.4]; oestrogen plus progesterone: 41.0 PEC/mm2 [8.9–86.9]; vehicle only: 0.0 PEC/mm2 [0.0–3.1]). Unexpectedly, there was no significant difference in SC proliferation among the treatment groups (progesterone only: 50.1 PSC/mm2 [39.2–102.6]; oestrogen plus progesterone: 46.1 PSC/mm2 [12.6–120.8]; vehicle only: 44.8 PSC/mm2 [17.3–68.4]). To determine if VEGF had a role in the progesterone-induced EC proliferation, the previous experiment was repeated with the inclusion of mice treated with VEGF anti-serum. The addition of VEGF anti-serum significantly inhibited progesterone-induced EC proliferation (46.8 PEC/mm2 [38.9–128.0]; P = 0.04], but had no effect on SC proliferation (P = 0.3). These results demonstrate that progesterone stimulates endometrial EC proliferation, but not SC proliferation as reported by earlier studies1. Studies are currently underway to further investigate the role of VEGF in mediating progesterone effects on endometrial EC. (1)Clarke, C.L. and Sutherland, R.L. (1990) Endocrine Reviews 11, 266–301.

264.The effects of progesterone on endometrial angiogenesis in pregnant and ovariectomised mice

In mice, early pregnancy is associated with an increase in endometrial angiogenesis in preparation for the implanting embryo. The aims of this study were to quantify endometrial angiogenesis in pregnant mice and to investigate the role of progesterone in promoting endothelial cell (EC) proliferation in ovariectomised mice; we hypothesised that EC proliferation would increase with increasing plasma progesterone concentrations in pregnant mice and that progesterone would stimulate EC proliferation in ovariectomised mice, but only following oestrogen priming. Uterine tissue from CBA x C57 mice was collected on Days 1–4 of pregnancy (n�=�4–5/day) when circulating progesterone concentrations are increasing but before implantation occurs. Prior to dissection, mice were injected with BrdU enabling proliferating EC to be quantified and localised within blood vessels by CD31/BrdU double staining immunohistochemistry. There was a significant increase in proliferating EC (Kruskal-Wallis statistic (KW) = 17.1, P = 0.002) on Day 3 of pregnancy (Days 1 and 2, no proliferation; Day 3,126.6 � 45.6 proliferating EC/mm2 (mean � s.e.)), when plasma progesterone also began to increase (as measured by radioimmunoassay). To determine if the EC proliferation was due to progesterone, a second experiment was performed on ovariectomised mice. One group of mice (n = 6) were treated with a single injection of 100 ng of estradiol on day eight after ovariectomy, followed by a day with no treatment and three consecutive daily injections of 1 mg progesterone. Other groups were treated with either the vehicle (n = 5), estradiol (n = 4) or progesterone (n = 5) injections only. All groups were dissected following BrdU injection on Day 13 following ovariectomy. Unexpectedly, mice treated with progesterone only had the highest amount of EC proliferation (114.7 � 30.9 proliferating EC/mm2); oestrogen priming was not required and actually significantly reduced progesterone induced EC proliferation (44.8 � 15.5 proliferating EC/mm2; KW = 13.8, P = 0.008). We are currently investigating the interaction between progesterone and VEGF using immunohistochemistry and inhibition studies.

Regulation of parietal cell migration by gastrin in the mouse

Recent studies suggest that gastrin regulates parietal cell maturation. We asked whether it also regulates parietal cell life span and migration along the gland. Dividing cells were labeled with 5′-bromo-2′-deoxyuridine (BrdU), and parietal cells were identified by staining with Dolichos biflorus lectin. Cells positive for D. biflorus lectin and BrdU were reliably identified 10–30 days after BrdU injection in mice in which the gastrin gene had been deleted by homologous recombination (Gas-KO) and wild-type (C57BL/6) mice. The time course of labeling was similar in the two groups. The distribution of BrdU-labeled parietal cells in wild-type mice was consistent with migration to the base of the gland, but in Gas-KO mice, a higher proportion of BrdU-labeled cells was found more superficially 20 and 30 days after BrdU injection. Conversely, in transgenic mice overexpressing gastrin, BrdU-labeled parietal cells accounted for a higher proportion of the labeled pool in the base of the gland 10 days after BrdU injection. Gastrin, therefore, stimulates movement of parietal cells along the gland axis but does not influence their life span.

Developmental changes in the response of trigeminal neurons to neurotrophins: influence of birthdate and the ganglion environment

Previous studies have shown that most neurons in cultures established during the early stages of neurogenesis in the embryonic mouse trigeminal ganglion are supported by BDNF whereas most neurons cultured from older ganglia survive with NGF. To ascertain to what extent these developmental changes in neurotrophin responsiveness result from separate phases of generation of BDNF- and NGF-responsive neurons or from a developmental switch in the response of neurons from BDNF to NGF, we administered BrdU to pregnant mice at different stages of gestation to identify neurons born at different times and studied the survival of labelled neurons in dissociated cultures established shortly after BrdU administration. Most early-generated neurons responded to BDNF, neurons generated at intermediate times responded to both factors and late-generated neurons responded to NGF, indicating that there are overlapping phases in the generation of BDNF- and NGF-responsive neurons and that late-generated neurons do not switch responsiveness from BDNF to NGF. To ascertain if early-generated neurons do switch their response to neurotrophins during development, we used repeated BrdU injection to label all neurons generated after an early stage in neurogenesis and studied the neurotrophin responsiveness of the unlabelled neurons in cultures established after neurogenesis had ceased. The response of these early-generated neurons had decreased to BDNF and increased to NGF, indicating that at least a proportion of early-generated neurons switch responsiveness to neurotrophins in vivo. Because early-generated neurons do not switch responsiveness from BDNF to NGF in long-term dissociated cultures, we cultured early trigeminal ganglion explants with and without their targets for 24 hours before establishing dissociated cultures. This period of explant culture was sufficient to enable many early-generated neurons to switch their response from BDNF to NGF and this switch occurred irrespective of presence of target tissue. Our findings conclusively demonstrate for the first time that individual neurons switch their neurotrophin requirements during development and that this switch depends on cell interactions within the ganglion. In addition, we show that there are overlapping phases in the generation of BDNF- and NGF-responsive neurons in the trigeminal ganglion.

Polymorphonuclear leukocyte transit times in bone marrow during streptococcal pneumonia

The release of polymorphonuclear leukocytes (PMN) from the bone marrow (BM) is a hallmark of acute inflammatory conditions. BM stimulation may increase the toxic potential of these newly released PMN and influence their behavior at inflammatory sites. The present study was designed to measure the transit time of PMN in the mitotic and postmitotic pools of the BM in rabbit using 5'-bromo-2'-deoxyuridine (BrdU). Blood samples were obtained at 2- to 24-h intervals from 24 to 192 h after a single BrdU injection, and BrdU-positive PMN (PMNBrdU) was detected as they appear in the circulating blood, using immunohistochemistry. The intensity of nuclear staining for BrdU was used to define a single generation of PMN and graded as either weakly (G1), moderately (G2), or highly (G3) stained. The mean +/- SE transit time of PMNBrdU through the BM was 95.6 +/- 3.6 h, with 51.1 +/- 5.9 h in the mitotic and 65.4 +/- 5.4 h in the postmitotic pool. Streptococcus pneumoniae instillation in the lung (n = 3) shortened the transit time of PMN through the BM to 54.0 +/- 2.6 h with a shorter time in both the mitotic (36.2 +/- 5.7 h) and the postmitotic pool 34.6 +/- 0.8 h). All these values were shorter than the control values (P < 0.05). We conclude that Streptococcus pneumoniae shortens the transit time of PMN in the mitotic and postmitotic pools in the marrow, which may result in the release of immature PMN with higher levels of lysosomal enzymes into the circulation.

Epidermal Cell Migration and Healing of the Tympanic Membrane: An Immunohistochemical Study of Cell Proliferation Using Bromodeoxyuridine Labeling

A monoclonal antibody against bromodeoxyuridine (BrdU) was used to investigate cell proliferation in the tympanic membrane of white rabbits. The BrdU-labeled cells were observed mainly in the epidermis of the annulus, around the malleus handle, and in the anterior and posterior superior quadrants of the normal rabbit tympanic membrane at 2 hours after BrdU injection. At 5 days the localization of the BrdU-labeled cells had changed centrifugally from the malleus handle toward the annulus. This change in the distribution of BrdU-labeled cells suggested that epidermal cell migration is caused by cell proliferation and insertion of newly proliferated epidermal cells at the proliferation center. Immunohistochemical observation of BrdU-labeled cells in the artificially perforated tympanic membrane suggested that the process of healing of the perforation may be as follows. Epidermal cell proliferation in the whole tympanic membrane is accelerated by the perforation stimulus. Then the proliferated epidermal cells migrate to the edge of the perforation. In contrast, proliferation of connective tissue cells and mucosal cells is stimulated only around the perforation, and cooperates with the proliferated epidermal cells to close the perforation.