PROLIFERATION KINETICS OF THE MOUSE BLADDER AFTER IRRADIATION

The proliferative response of the mouse bladder was investigated, using continuous labelling with tritiated thymidine, at various times after a single dose of radiation. Bladder epithelial and vascular endothelial cells were studied. The cell turnover rate in unirradiated epithelium and endothelium was found to be extremely slow (in excess of 1 year). Irradiation with a single dose of 25 Gy resulted in compensatory proliferation of the epithelium but the response was not initiated for many months. At 3 months after irradiation there was little difference from the control proliferation rate, but from 6 to 22 months after irradiation (the end of the study) there was a period of sustained rapid proliferation with the cell turnover time reduced to approximately 1 week. The increase in proliferative activity observed at 22 months was found to be dose—dependent. Endothelial cells in the blood vessels of the submucosa also showed an increased turnover rate after irradiation and the timing of this reponse was found to be similar to that of the epithelium. The onset of compensatory proliferation in both cell types was found to coincide with marked histological and functional changes in the bladder. In this slowly proliferating tissue, the onset of rapid compensatory proliferation after irradiation is delayed and occurs at the time that functional impairment is observed. This supports the postulate that proliferation is unlikely to contribute much to the sparing effect of prolonged fractionated radiotherapy in slowly dividing tissues.     

CHANGES IN THE PROLIFERATION RATE OF MOUSE EPIDERMIS AFTER IRRADIATION: CONTINUOUS LABELLING STUDIES

The proliferative response of mouse skin to damage caused by X-irradiation has been tested by giving repeated injections of tritiated thymidine and scoring the percentage of labelled cells in high resolution autoradiographs. Four, nine and fourteen daily fractions of 300 rads of X-rays were used and labelling commenced 4 days after the last fraction. The epidermis of the upper surface and the sole of the foot were scored separately and were compared with the skin of unirradiated feet. In unirradiated skin the proliferation rate of the basal layer cells is more rapid on the sole than on the upper surface. The cell cycle times deduced from continuous labelling curves were 81 hr and 111 hr respectively and the growth fractions were 97% and 85%. After irradiation with small daily doses the homeostatic response to cell killing was slow. More rapid proliferation occurred after nine fractions in the sole, but was not apparent in the skin of the upper surface until fourteen fractions had been given. After fourteen fractions the cell cycle time was about 24 hr on both surfaces and the growth fraction was about 90%. The initial labelling index after a single thymidine injection was a poor measure of proliferation rate. The delay in the time of onset of faster proliferation is similar, both qualitatively and quantitatively, to that measured previously from the additional dose increments needed if large doses were given at different times after the multifraction treatments (Denekamp, 1973).     

PROLIFERATION STUDIES OF THE ENDOTHELIAL AND SMOOTH MUSCLE CELLS OF THE MOUSE MESENTERY AFTER IRRADIATION

A continuous labelling technique was employed to study the effects of external β-radiation on the proliferation of endothelial cells and smooth muscle cells in the mesenteric arterioles of mice. Labelled and non-labelled cells of either type were determined by autoradiographic techniques in control animals and at different times (3, 12 and 48 weeks) after single doses of 20 and 45 Gy (2000 and 4500 rads). The fraction of cells labelled, even after 7 days of repeated injections was very low in all instances. Calculations showed very long turnover times for the two cell populations in control animals (>2 years for endothelium and >3 years for smooth muscle). After 20 and 45 Gy, no significant increase in endothelial proliferation was seen except at 3 weeks. No significant increase in labelling was observed in smooth muscle at any time after irradiation. These labelling data have been compared with the pattern of cell depletion of the irradiated endothelium. It was concluded that the depletion was much earlier than expected for a slowly proliferating tissue, if all the cells were cycling very slowly. Such an early depletion is, however, consistent with cell death resulting from a small proportion of the cells having a short cell cycle. The recovery of the endothelial cell numbers between 9 and 12 months was not accompanied by a rise in the fraction of labelled cells. It is suggested that repopulation may occur from outside the treated area.     

ELECTRON MICROSCOPIC AUTORADIOGRAPHY OF GERMINAL CENTER CELLS IN MOUSE SPLEEN

The fine structure of tritiated thymidine-labeled cells in antigen-stimulated mouse spleen germinal centers is described. In studies on the ultrastructural level, two labeled cell types found in germinal centers are observed. Large lymphocytes are characterized by their very numerous free ribosomes, a paucity of endoplasmic reticulum, relatively few mitochondria, and a poorly developed Golgi region. The nuclei are large and vesicular, and large nucleoli are present. A second labeled cell type appears to contain more mitochondria and has a higher development of the Golgi area. The nucleus contains large, numerous blocks of chromatin, indicative of a more differentiated cell type. Reticular cells, both phagocytic and non-phagocytic, were not observed to be labeled in the germinal centers.     

ELECTRON MICROSCOPE OBSERVATIONS ON TINGIBLE BODY MACROPHAGES IN MOUSE SPLEEN

The structure of the ciliary epithelium of the adult albino rabbit has been studied by electron microscopy. Material was fixed in osmium tetroxide and embedded in epoxy resins. Two hitherto unappreciated features of the non-pigmented epithelial layer are described. First, the "infolded plasma membranes" described by previous workers are shown by serial sections to be projections or interdigitations from adjacent cells. Second, the "rows of vesicles" described by previous workers are shown by serial sections to be part of an unusual form of smooth-surfaced tubular endoplasmic reticulum. The tubules are highly convoluted and extensively interconnected. They are arranged in sheets, so that a cross-section through a sheet gives the appearance of a row of vesicles. The other structural features of the ciliary epithelium are also described. Previous workers have reported that Diamox, which inhibits the secretory activity of the epithelium, causes profound structural changes. An effort has been made to confirm these reports under carefully controlled experimental conditions. It was found that secretion could be inhibited by a maximally effective dose of Diamox without the occurrence of any detectable structural changes. The physiological significance of these findings is discussed.     

REGIONAL CHANGES IN BRAIN CATECHOLAMINES AFTER PROTON IRRADIATION OF THE STRIATE CORTEX IN THE SQUIRREL MONKEY

To establish possible functional relationships between anatomically distinct cortical centres in primates, we compared changes in visual acuity, nucleic acids, protein, enzymes and catecholamines in cortical and subcortical areas six months after 10,000 and 20,000 rd of proton irradiation of striate area 17 of the visual cortex of the squirrel monkey. Minimum separable acuity was reduced from 1 minute of arc in controls to 4 minutes in the 20,000-rd group. DNA, RNA and protein contents were not altered, but the activity of acetylcholinesterase was increased significantly in area 17. A decrease of norepinephrine occurred in the hypothalamus, hippocampus, caudate nucleus, putamen and brain stem of the irradiated brains. Since no ultrastructural basis has been found to account for these changes, we assume that a sustained chemical change occurred at the irradiated site and that the effect was transmitted to non-irradiated regions of the brain.     

SPERMATOGONIAL STEM CELL RENEWAL IN THE MOUSE: II AFTER CELL LOSS

The repair of the mouse seminiferous epithelium after cell loss has been studied in seminiferous tubules mounted in toto . Cell loss was inflicted by injection of Myleran in a dose of 10 mg/kg body weight. In stages 7–8, in which we mainly counted, the numbers of A_isolated (A_is), A_paired (A_pr), A_aligned (A_al) and A₁ spermatogonia and resting primary spermatocytes decreased after injection. After about 24 days normal numbers of A₁ spermatogonia were found again. Thereafter a substantial overshoot in the number of A₁ spermatogonia was found.
While normally most of the A_pr and A_al cells differentiate into A₁ spermatogonia in stages 3 and 4 and do not divide until stage 9, during repair they pass through one more division during stages 6 and 7. Normally, during these stages divisions of these spermatogonia are rare. Owing to this extra division the transformation of A_pr and A_al into A₁ spermatogonia is delayed from stage 3 or 4 to stage 8, i.e. still before stage 9, in which A₁ spermatogonia divide. From 16 days after the injection onwards the extra division takes place less generally and more and more cells transform into A₁ spermatogonia at the normal time.     

METABOLISM OF POLYAMINES IN NORMAL AND SCRAPIE-AFFECTED MOUSE BRAIN AND SPLEEN

Abstract— Following intracerebral inoculation of mouse adapted scrapie agent into mice, polyamine concentration in the brain decreases to about 75 per cent of the normal level during the first 2 months after intracerebral inoculation of the agent. Between 2 and 4 months after infection thelevel of spermidine and spermineincreased by 80 and 40 percent respectively to reach concentrations of 25 and 20 per cent higher than controls of the same age. During the same period the rate of incorporation of [¹⁴C]putrescine into spermidine is increased four-fold as compared with controls. The changes in polyamine levels correlate well with the pattern of astrocyte hypertrophy and are similar to those reported for human brain tumours. The concentration of polyamines in spleen increases soon after inoculation. Whilst changes in brain polyamines might be referred to the hypertrophic growth of astrocytes those in spleen are perhaps due to an increased metabolic activity of spleen cells associated with the replication of the agent. These results are derived from experimental mouse scrapie and not naturally occurring disease in sheep.     

INFLUENCE OF AGE AND ANTIGENIC STIMULATION ON GRANULOCYTE AND MACROPHAGE PROGENITOR CELLS IN THE MOUSE SPLEEN

The frequency and proliferative activity of granulocytic and macrophage progenitor cells were determined in the spleens of C₅₇BL, BALD/c, NZB and CBA mice. These cells were detected by their capacity to form granulocytic and/or macrophage colonies ( in vitro colony-forming cells, CFC) in agar culture. In vitro CFCs were low in frequency in the adult spleen (4–28/10⁵ cells) compared with the bone marrow (180–280/10⁵ cells). However, the neonatal spleen, both in germfree and conventional mice, contained high levels of in vitro CFCs. From the low suiciding index with tritiated thymidine and the small numbers of cluster-forming cells in relation to colony numbers, many in vitro CFCs in the adult C₅₇BL spleen appear to be in a non-cycling state. The level and activity of in vitro CFCs were extremely low in the spleen of adult germfree CBA mice but were greatly increased in conventional mice following the injection of a bacterial antigen.     

实验性脾虚小鼠脾脏淋巴细胞增殖周期和免疫细胞化学的研究

本文用流式细胞术（ＦＣＭ）和免疫细胞化学方法,检测了实验性小鼠脾的淋巴细胞增殖周期和５－溴脱氧尿苷阳性细胞（ＢｒｄＵ￣＋）在脾脏内的分布．以及脾小结中ＩｇＭ￣＋细胞的反应。结果显示由利血平诱发的脾虚小鼠Ｓ期细胞数最少（占５％）,Ｇ＿１和Ｇ＿２＋Ｍ期细胞增加。经过健脾汤治疗后Ｓ期细胞增至１０％,与对照组比较无显著性差异．但高于自然恢复组（Ｐ＜０．００１）。经免疫细胞化学显示：脾虚组小鼠的脾中５－溴脱氧尿苷阳性细胞（ＢｒｄＵ￣＋）的数量远低于其它组。脾小结缩小,帽状区ＩｇＭ￣＋Ｂ细胞基本消失,经健脾汤治疗后,复健组的脾小结与自然恢复组比较,体积明显增大,生发中心的ＢｒｄＵ￣＋和帽状区ＩｇＭ￣＋Ｂ淋巴细胞均相应增多。提示健脾汤有促进ＤＮＡ和ＩｇＭ合成及细胞增殖作用。     

天然虫草对小鼠脾脏影响的组织化学和免疫细胞化学的观察

本文用３０只成年雄性小鼠,分为实验和对照两组。实验组每日灌服虫草液一次,共７日。对照组每日灌服生理盐水一次。按时将动物处死,取其脾脏进行组织化学和免疫细胞化学染色。结果显示：（１）虫草组小鼠脾脏白髓成份增加,生发中心明显,嗜派罗宁细胞增多。显微分光光度计检测结果显示虫草组脾脏内ＤＮＡ含量高于对照组,表明虫草能促进小鼠脾脏ＲＮＡ和ＤＮＡ的合成。（２）虫草组脾内ＩｇＧ＋和ＩｇＭ＋浆细胞明显增加,边缘区变宽,可能虫草有促进抗体形成的作用。此外本实验还证明,两组小鼠脾内巨噬细胞数量无明显差别     

在给利血平后的恢复中小鼠颌下腺的去甲肾上腺素含量与神经生长因子水平的关系

给ICR/JCL品系的成年小鼠1次注射利血平(2mg/kg体重)后24小时,其颌下腺(SMG)的去甲肾上腺素(NA)贮存能够被耗竭。在注射后第12天,NA 含量恢复到利血平处理以前的水平。在恢复过程中,雄性腺体的 NA 含量均为雌性的2倍左右。切断颈上神经节(SCG)的节前神经,雌雄腺体的 NA 含量都下降至对照侧的80%左右。消除节前神经的影响以后,在 NA 的恢复过程中,雄性腺体的 NA 含量仍然是雌性的2倍左右。利用神经生长因子(NGF)生物测定法,我们测得本品系成年雄性小鼠 SMG 的 NGF 含量为雌性小鼠的94倍左右。正如外源性 NGF 能选择性地提高交感神经元内的酪氨酸羟化酶活力与 SMG 的 NA 含量一样,在利血平处理后的恢复过程中,雄性腺体比雌性腺体具有较高的 NA 含量可能是由于内源性 NGF 对交感神经元内酪氨酸羟化酶产生效应的结果。     

REPOPULATION OF THE BONE-MARROW STEM-CELL COMPARTMENT AFTER IRRADIATION*

Bone-marrow repopulation has been followed as a function of time after a single whole-body dose of 150 rads X-rays to (C3H × C57B1)F₁ hybrid mice. The number of nucleated cells per bone shaft has been estimated by direct counting and the number of progenitor cells by the exogenous spleen-colony assay method. Vinblastine was also administered under various schedules of time and dose in an endeavour to distinguish between cycling and dormant cells in the progenitor pool. The depopulation of total marrow cells induced by Vinblastine proceeds at a comparable rate in irradiated and in normal mice. On the other hand, the depopulation of the colony-formers is faster in irradiated than in normal animals. The data are interpreted to show that the fraction of cycling progenitor cells is larger in a haemopoietic tissue undergoing numerical expansion.     

去神经后小鼠骨胳肌胞纳的增加和卫星细胞增殖

用生物化学和体外培养法研究了小鼠骨胳肌的胞纳增加和卫星细胞增殖的关系。结果表明：（１）去神经４ｄ或６ｄ的肌肉可引起胞纳的增和卫星细胞的增殖;（２）放线菌素Ｄ抑制正常肌肉的卫星细胞激活和胞纳作用;（３）在去神经的肌肉中,放线菌素Ｄ抑制了卫星细胞增殖的同时还抑制了胞纳的增加,但不能去神经肌肉的萎缩。上述结果：肌肉的卫星细胞增殖和胞纳增加可能发生于去神经后某些因素的出现,或者胞纳的增加即是卫星细胞增殖的     

LOCATION OF THE G0-PHASE IN THE CELL CYCLE OF THE MOUSE HAEMOPOIETIC SPLEEN COLONY FORMING CELLS

Experiments in mice on the fraction of haemopoietic stem cells in S-phase after irradiation indicated that a large fraction of the cells resting in G₀ will enter S-phase after a very short interval of time.
After excluding alternative explanations it must be concluded that cells in G₀ have completed all preparations for going into S-phase or, in other words, that the localization of these G₀ cells in relation to other phases of the cell cycle must be between G₁ and S-phase.