Stathmokinetic Technique Research Articles

Effect of defunction on cytokinetics and cancer at colonic suture lines

An intestinal suture line potentiates experimental carcinogenesis in its vicinity, probably due to adaptive hyperplasia. By contrast, a defunctioning colostomy causes distal hypoplasia, and fewer tumours develop. Male Sprague-Dawley rats (n = 160) were used to study adaptation (assessed by a stathmokinetic technique) and carcinogenesis (induced by azoxymethane) at an end-to-end anastomosis that was raised in either functioning or defunctioned left colon. Controls had no procedure and other rats had proximal colostomy alone. Defunction had a profound antitropic effect on the colon, reducing bowel length and weight and crypt cell production rate (CCPR) by 22-56 per cent. Anastomotic CCPR was increased by a factor of 2.6 over controls: mean(s.e.m.) values of 12.71(2.85) versus 4.87(0.41) cells/crypt/h (P less than 0.01), but defunction reduced this by 76 per cent to a mean(s.e.m.) value of 3.00(0.52) cells/crypt/h below that in the intact colon. In the left colon, 39-50 per cent of tumours were sited at the anastomosis. Compared with controls there were 77 per cent fewer tumours in defunctioned colon, but they still favoured the site of anastomosis. Neoplasms at the colostomy site accounted for 74-77 per cent of all right-sided tumours. Anastomosis and defunction have powerful but contrasting effects on colonic adaptation and carcinogenesis; when combined they tend to cancel each other out.

Measurement of cell production rates in human gastro-intestinal cancer--a guide to treatment?

Cell production rates were measured in 19 cases of rectal carcinoma and in 11 cases of gastric carcinoma using a stathmokinetic technique. These measurements were compared with results from normal rectal and gastric mucosa. It was found that most of the cases of rectal cancer were proliferating more slowly than morphologically normal rectal mucosa. This was true for the majority of gastric cancers, but a small proportion appeared to be proliferating more rapidly. These results are discussed with their relevance to appropriate therapy.

Colorectal cell kinetics.

The assessment of cell proliferation in colorectal tissue may provide information with both prognostic and therapeutic implications. A variety of methods are available, including flow cytometric estimations of S phase fraction, immunohistochemical and autoradiographic visualization of exogenous and endogenous proliferation proteins, and morphological and stathmokinetic techniques. There is some correlation between Dukes stage and proliferation state features, and there is increased proliferative activity throughout the adenoma-carcinoma sequence. Data on cell proliferation rates are difficult to obtain. When correctly applied, the metaphase arrest technique remains the 'gold standard' of measuring proliferation, but its usefulness in clinical practice is limited. Recent studies have employed dual measurement flow cytometry and double labelling techniques to produce rate data.

Hypoplasia of defunctioned rectum.

The adaptive response of the large bowel to surgical defunction in man is essentially unknown, although in the rat there is progressive hypoplasia and a reduced propensity to experimental carcinogenesis. Mucosal biopsies were taken from the upper rectum completely defunctioned by a proximal stoma from 2 months to 5 years earlier in 11 patients and from 14 controls without abdominal operations or disease. Samples were established in organ culture and, after 16 h, crypt cell production rate (CCPR) was determined by a stathmokinetic technique. Crypt morphometry was also undertaken. CCPR in defunctioned large bowel was less than half that of controls: (mean (s.d.)-1.96 (0.68) versus 4.65 (0.54) cells crypt-1 h-1, P less than 0.0001). Likewise, crypt length was 24 per cent lower (0.34 (0.05) versus 0.44 (0.04) mm, P less than 0.0001) and crypt width was 38 per cent lower (0.04 (0.01) versus 0.07 (0.01) mm, P less than 0.0001). Rectal defunction causes profound and persistent hypoplasia in man.

The effect of pinealectomy on the crypts of defunctioned rat colon.

Previously it has been found that 6 months after pinealectomy hyperplasia occurred in the crypt cells of the rat small bowel and colon. It is also known that defunctioning a loop of colon, using a colostomy, results in crypt cell hypoplasia, emphasizing the prime importance of luminal factors in the control of crypt cell proliferation. To determine if the effects of pinealectomy on the colon could be modified by the absence of colonic luminal contents, the crypt cell kinetic effects of combined pinealectomy and defunctioning of a colonic loop by colostomy for 6 months were examined by using a stathmokinetic technique. It was found that the hypoproliferative effect of defunctioning a loop of colon was largely but not completely overridden by the hyperproliferative effect of pinealectomy. However, previously it has been found that in the rat small bowel, the hypoproliferative effects of defunctioning a loop were completely overridden by the effect of pinealectomy. This and other evidence suggests that the role of the pineal in the control of crypt cell proliferation in the colon may possibly be different from its role in the small bowel. There is other evidence of possible involvement of the pineal in carcinoma of the colon and it is possible that its role in the colon may be to prevent excessive mitotic activity, which is known to be present in the early stages of carcinoma. The Pineal gland may have a role in modulating the usual mechanisms of crypt cell mitotic control.

Germinal‐center cell proliferation in response to T‐independent antigens: A stathmokinetic, morphometric and immunohistochemical study in vivo

Although the nature of the germinal center reaction during responses to T-dependent antigens has been well documented, much less is known regarding the relationship between germinal centers and T-independent antigens. In this study, germinal-center cell proliferation was determined at specific time points in spleens of C3H/HeN mice following immunization with either the type-1, T-independent antigen dinitrophenol-lipopolysaccharide (DNP-LPS), or the type-2, T-independent antigen DNP-Ficoll. A stathmokinetic technique was employed to assess proliferation in terms of germinal center cell birth rate and morphometry was used to measure actual growth and regression of the germinal center cell population. An estimate of the absolute rate of germinal-center (GC) cell proliferation was derived from these two values. In addition, immunohistochemistry was performed to correlate changes in GC cell proliferation with the presence or absence of antigen within GC. Following immunization with both antigens, there was an initial reduction of proliferation within pre-existing germinal centers which manifested as either GC dissociation (DNP-LPS) or a suppression of birth rates (DNP-Ficoll). This was followed by a period of increased GC cell proliferation in animals immunized with DNP-LPS, but not in those exposed to DNP-Ficoll. GC cell proliferation was then measured in mice treated with cyclosporin A from 1 day before to 2 days after immunization with DNP-LPS. In these animals, the expected increase in GC cell birth rates did not take place. Immunohistochemistry showed that DNP-Ficoll and DNP-LPS were present in GC from 1 day after immunization until the end of the experiment on day 7. Treatment with cyclosporin A did not affect the deposition of DNP-LPS in GC. These results show that only some T-independent antigens are able to stimulate GC cell proliferation, and we propose that this is related to their ability to recruit precursors of GC B cells into the GC reaction. In addition, the results indicate that GC proliferation seen in response to a so-called T-independent antigen is at least partly driven by T cell-derived cytokines.

The Trophic Action of Human Growth Hormone on Human Duodenal Mucosa Cultured In Vitro

SummaryThe influence of human growth hormone (hGH) on crypt cell proliferation in cultured explants of human duodenal mucosa has been studied, using a stathmokinetic technique with crypt microdissection. The addition of hGH (0.004 μ/ml) to paired duodenal explants from eight patients significantly increased epithelial crypt cell proliferation (p < 0.001), showing that hGH has a trophic action on the human duodenal mucosa in vitro.

The trophic action of human growth hormone on human duodenal mucosa cultured in vitro.

The influence of human growth hormone (hGH) on crypt cell proliferation in cultured explants of human duodenal mucosa has been studied, using a stathmokinetic technique with crypt microdissection. The addition of hGH (0.004 IU/ml) to paired duodenal explants from eight patients significantly increased epithelial crypt cell proliferation (p < 0.001), showing that hGH has a trophic action on the human duodenal mucosa in vitro.

The long-term effect of pinealectomy on the crypts of the rat gastrointestinal tract.

Previously it has been found that rat small bowel crypt cell hyperplasia occurred several weeks after pinealectomy. To determine if this effect was longer-lasting (because of the possible role of the pineal in bowel malignancy) the crypt cell proliferation rate was determined in rat small bowel and colon 6 months after pinealectomy, using a stathmokinetic technique. Although the hyperproliferative effect of pinealectomy was well maintained in the small bowel crypts after 6 months, the hyperproliferative effect in the colonic crypts was much less marked. There is no obvious explanation for these findings, although it is possible that regional differences in levels of gut neuropeptides or melatonin are involved. The mechanism of the effect of pinealectomy on the crypts remains unexplained--in particular, why the effect is so prolonged.

Regulation of murine germinal centre cell proliferation in vivo: a stathmokinetic study examining the effect of differently timed doses of cyclosporin A.

Although studies have identified factors which affect germinal centre cell proliferation in vitro, their relative contributions in vivo remain largely undetermined. In this study, the proliferative rate of germinal centre cells was measured in sheep red blood cell-immunized C3H/HeN mice exposed to variously timed doses of cyclosporin A. Germinal centre (GC) cell proliferation was measured by a stathmokinetic technique to determine GC cell birth rates at specific time points after immunization. Changes in total GC volume were determined by morphometry in order to assess actual growth and regression of the GC cell population. An estimate of the absolute rate of GC cell proliferation was derived from these two values. Following exposure to antigen, there was an initial inhibition of proliferation within pre-existing germinal centres, followed by a rapid rise, then a sustained phase of increased GC cell proliferation. By comparing the effects of the different cyclosporin A treatment regimes, it was possible to deduce that the initial inhibition of proliferation was mediated by a T-cell-derived cytokine, as was the final sustained phase of the proliferative response. The intervening rise in GC cell proliferation, however, was attributable to a contact-dependent signalling mechanism.

Enhancement of ethanol induced rectal mucosal hyper regeneration with age in F344 rats.

Experimental studies in rats have shown an independent stimulation of rectal cell turnover by either chronic ethanol consumption or age. In this study the combined effect of these two factors on colorectal cell regeneration has been investigated. Ninety male F344 rats aged 2, 12, and 22 months were pair fed nutritionally adequate liquid diets containing 36% of total energy either as ethanol or isoenergetic carbohydrates. After four weeks of feeding, colorectal crypt cell production rates were measured using a stathmokinetic technique with vincristine. While age by itself did not affect colorectal cell renewal, chronic ethanol consumption stimulated rectal, but not colonic crypt cell production rate in an age dependent manner. While no significant effect of ethanol was noted in young animals, cell proliferation was significantly enhanced in middle aged animals by 81% (4.1 (2.7-5.5) v 7.4 (6.0-8.7) cells/crypt/hour, p < 0.001) and in old animals by 138% (4.5 (3.3-5.6) v 10.7 (8.9-12.4) cells/crypt/hour, p < 0.001) after ethanol ingestion. Because acetaldehyde, the first and most toxic metabolite of ethanol, has been detected in the colorectal mucosa and may lead to tissue injury influencing cell regeneration, acetaldehyde concentrations have been measured in the colons of 15 male F344 rats of various ages after an acute intraperitoneal dose of ethanol (2.5 g/kg bodyweight). There was a significant positive correlation between crypt cell production rate and acetaldehyde concentrations measured in the distal and proximal colon after an acute dose of ethanol (r = 0.5955, p < 0.005). These data clearly show that the ethanol mediated stimulation of cell regeneration in the rectum is age dependent. As reported earlier, there was found indirect evidence that acetaldehyde participates in the pathogenesis of rectal hyperregeneration after chronic alcohol consumption. This hyperregeneration of the rectal mucosa after alcohol drinking could by itself favour carcinogenesis, which is especially relevant in old age.

Differential cell kinetics in the ileum and colon of germfree rats.

Our aim was to study the cell kinetics and epithelial structure in the ileum and colon of conventional and germfree AGUS rats by means of a classic stathmokinetic technique. A slight hyperplasia was observed in the villi of germfree animals (p < 0.05), associated with a comparatively short cell cycle time. The crypt cell production rate was reduced in the colon of germfree rats (p < 0.05), and their crypts contained fewer cells than those of conventional animals (p < 0.05). It is concluded that intraluminal bacteria influence cell proliferation in the colon. The absence of microflora prolongs the cell cycle time and reduces the proliferative activity in the colonic crypts, which contributes to a steady state that is different from that of conventional animals.

The identification of high and low risk groups for colorectal cancer using rectal mucosal crypt cell production rate (CCPR)

Rectal mucosal proliferation was measured in 116 individuals using the metaphase arrest technique crypt cell production rate (CCPR). CCPR was found to be significantly elevated in individuals with adenomas (n = 42, CCPR = 13 cc c-1h-1, range 7-25 Cl 10-15) compared with normals (n = 21, CCPR = 10 cc c-1h-1 range 5-24 Cl 7-11, Mann-Whitney P = 0.001 z = 3.2). Mucosal proliferation was increased among individuals who were undergoing adenoma follow up but in whom no further adenomas were found (n = 37 CCPR = 12 range 5-26 cc c-1h-1 Cl 10-14) compared to controls (Mann-Whitney P = 0.01 z = 2.4) Proliferation in vegetarians i.e. low risk (n = 16) was similar to controls. Measurement of proliferative indices in rectal mucosa by the stathmokinetic technique CCPR can discriminate between high and low risk groups for colorectal cancer.

Cellular proliferation at sutured and sutureless colonic anastomoses.

Elevated cellular proliferation in the vicinity of an anastomosis may explain the enhanced susceptibility to carcinogens. The aim of this study was to determine whether anastomotic cellular proliferation was altered by different suture materials and whether a rise in cell turnover also occurred after a "sutureless" closure. A transverse descending colon enterotomy was repaired with interrupted sutures of 5/0 silk (n = 20), stainless steel (n = 20), or Vicryl (Ethicon, Inc., Somerville, NJ) (n = 20) or by a sutureless technique (n = 20). Using a stathmokinetic technique, crypt cell production rates (CCPR) were calculated at the anastomosis and in the adjacent colon at varying intervals between one week and six months after treatment. Overall colonic cellular proliferation appeared to be elevated at a sutured colotomy for at least three months (P < 0.05). In contrast, no significant elevation in cellular proliferation was observed at sutureless anastomoses. The duration of elevated proliferative response varied among the sutures.

Effect of systematic interleukin-3 administration on epithelial cell proliferation in mouse intestine

The effect of interleukin-3 (IL-3) on the crypt cell production rate (CCPR) in the intestine of mice was studied using a stathmokinetic technique combined with crypt microdissection. Interleukin-3 (0.71 micrograms/injection) was administered subcutaneously (s.c.) as two injections per day for 7 successive days and small mucosal pieces (duodenum, jejunum, ileum and colon) removed at necropsy were organ cultured in the presence of the metaphase arrest agent vincristine sulfate for two hours. The number of metaphases was enumerated in dissected crypts and CCPR calculated. The results demonstrated that the CCPR was significantly increased in all mucosal segments in the IL-3 treated animals compared to saline injected controls. These results suggest that the growth promoting properties of IL-3 are not restricted to hematopoietic cells when used in vivo and may directly or indirectly increase epithelial cell turnover in gut mucosa.

Cell kinetics of the germinal center reaction ‐ a stathmokinetic study

The changes in splenic germinal center (GC) cell proliferation were measured during primary and secondary responses to a T-dependent antigen in vivo to examine the regulation of the GC reaction. Adult C3H/HeN mice were immunized with sheep red blood cells and boosted 7 or 21 days later. GC cell proliferation was assessed by measurement of GC cell birth rates using a stathmokinetic technique. Actual GC growth and regression were assessed in terms of total splenic volume and number. Pre-existing GC had a mean cell birth rate of 33 cells/1000 cells/h. The GC reactions following each immunization showed a biphasic pattern of changes in cell birth rate, comprising an initial fall immediately succeeded by a transient, but significant, increase. These fluctuations occurred earlier in secondary compared to primary responses. Significant increases in total GC volumes succeeded the peaks of cell birth rate following both primary and early secondary immunization. However, there was a substantially smaller increase following later secondary immunization. We propose that the initial cell birth rate reduction is due to inhibition of pre-existing GC clones and represents one component of the phenomenon of GC dissociation. The succeeding peak birth rate represents early, massive proliferation of newly activated antigen-specific clones. The different patterns of GC expansion, despite similar proliferative responses, may reflect different pathways of differentiation dependent on the timing of antigenic stimulation.

Effect of dietary calcium on the colonic luminal environment.

Dietary supplementation with calcium may prevent the development of colorectal cancer. This mechanism may be related to fatty acid and bile salt chelation in the small bowel forming non-toxic calcium-soap compounds. Calcium may also act locally or systemically on the colonic mucosa. Faecal concentrations of free fatty acids and free bile acids were measured in 17 Sprague-Dawley rats (weighing 472 (39 g)) whose daily calcium intake had been trebled by enriching the chow and adding calcium lactate (24 g/l) to the drinking water. Mean (SEM) faecal concentrations of free bile acids were 33% less than in 19 controls (1.23 (0.15) v 1.82 (0.20) mg/g; p less than 0.001), whereas free fatty acid concentrations were 117% higher (14.68 (3.59) v 6.76 (2.41) mg/g; p less than 0.02). The 'direct' effect of calcium was assessed by organ culture of rat colonic explants in three different concentrations of calcium. Crypt cell production rate (measured by a stathmokinetic technique), which was (mean (SEM)) 4.80 (0.23) cells/crypt/h in control medium (Ca2+ = 2.14 mmol/l), fell by 43% when calcium concentration was doubled (p less than 0.05) and by a further 43% when the concentration was trebled (p less than 0.02). Calcium binds free fatty acids but not free bile acids intraluminally. Calcium has a direct antitropic action on colonic crypts.

Trophic action of epidermal growth factor on human duodenal mucosa cultured in vitro.

The action of epidermal growth factor on the human duodenal mucosa has been studied by estimating the crypt cell production rate in cultured explants, using a stathmokinetic technique with crypt microdissection. The addition of epidermal growth factor (400 ng/ml) to paired explants from five patients caused an almost fivefold increase in the crypt cell production rate, showing that epidermal growth factor has a trophic action on the human duodenal mucosa in vitro.

Effect of Septal and Fornix Lesions on Rat Small Bowel Crypt Cell Kinetics

The septum and fornix are known to modulate the functional activity of the hypothalamus. Since the hypothalamus has been shown to influence crypt cell mitotic rate, it was considered possible that the septum and fornix might have a similar influence. Using a stathmokinetic technique, bilateral fornix and septal lesions were found to be associated with a marked increase in rat small bowel crypt cell mitotic rate. There was no associated significant change in food intake. It appears possible that the effects of the septal and fornix lesions may have been mediated via their influence over the functions of the hypothalamus.

The effect of bilateral amygdaloid nucleus lesions on rat small bowel crypt cell kinetics

Previous investigators have found that central nervous system lesions, in particular lesions of the hypothalamus, may increase the crypt cell mitotic rate in the rat small bowel. Since the amygdaloid nuclei form part of the limbic system (the "visceral brain") and have functional neural connections with the hypothalamus the effect of bilateral electrocoagulation lesions of the amygdaloid nuclei on crypt cell mitotic rate in the rat small bowel was investigated, using a stathmokinetic technique. Bilateral amygdaloid lesions were found to be associated with a marked increase in crypt cell mitotic rate in the proximal jejunum and distal ileum. Consideration of the neural connections of the amygdaloid nuclei suggests that these effects may possibly be mediated via the hypothalamus and the autonomic nervous system. The effects of lesions of other parts of the limbic system on crypt cell mitotic rate will be published subsequently.