Effect of the method of blood extraction on plasma levels of renin in the Wistar rat

To investigate the influence of blood extraction conditions on the renin-angiotensin system in rats, plasma renin activity (PRA) and plasma renin concentration (PRC) were measured in blood samples obtained by different methods. PRA and PRC in samples obtained by chronic catheterization, cardiac puncture without anesthesia, and decapitation immediately following light ether anesthesia were not significantly different from those obtained by simple decapitation (control group). In contrast, PRA and PRC in samples obtained by cardiac puncture and cavernous sinus puncture after light ether anesthesia were significantly (p less than 0.01) higher than those obtained in the control group. There was a significant direct correlationship between PRA and PRC in all samples studied (r = 0.87, p less than 0.001). The present results suggest that light ether anesthesia increases renin levels, except when blood samples are taken by decapitation, and that chronic catheterization and cardiac puncture are the choice blood extraction methods to evaluate the renin-angiotensin system in rats.     

The Distance to Which Wound Effects Influence the Structure of Secondary Xylem of Decapitated Pinus pinea

Decapitation of stems of annuals and trees for the study of vascular and fiber differentiation with or without hormonal application is a common procedure. There is controversy about whether wound effects play a role in such experiments, and to what distance from the point of decapitation. To examine this question, the distance from the point of decapitation at which apparent wound effects are obvious developmentally, was studied in decapitated 4-year-old Pinus pinea plants. The wound effects just below the cut included differentiation of many traumatic resin ducts, a parenchyma band instead of tracheids, more tracheid files, and a higher proportion of late wood. The increase in the number of resin ducts was still considerable and statistically significant 10 cm below the point of decapitation compared with the nondecapitated control. These results indicate that in pines, wound effects in the first 5 cm below the decapitation point (a common point for tissue examination) cannot be ignored in experiments on the regulation of vascular differentiation.     

Prolactin response to stress in neonatally estrogenized male rats

Male Wistar rats were injected either 500 micrograms of estradiol benzoate or olive oil on their first day of life. Blood samples were obtained from the adult by decapitation, by decapitation after 15 min of restraint, by decapitation 10 min after a 5 min period of ether exposure or by jugular venipuncture after 60 s of ether exposure. Prolactin (PRL) plasma levels were measured by RIA. The PRL levels in samples obtained by decapitation were similar to control and estrogenized groups. A similar response to restraint was also found in both groups. Sixty s of exposure to ether stress stimulates PRL secretion only in the estrogenized males, this effect being blocked by treatment with Normifensine (5 mg/kg two hours prior to blood sampling). These results suggested that estrogenized male rats show greater sensitivity to acute ether stress than the controls, and that changes in the dopaminergic system could be involved in this response.     

Radioimmunoassay of brain peptides: Evaluation of a methodology for the assay of β-endorphin and enkephalin

The brain levels of β-endorphin, α-endorphin and enkephalin were measured by radioimmunoassay after different methods of sacrifice. Microwave irradiation proved not to be better than decapitation followed by boiling of the intact tissue, the latter procedure giving values of β-endorphin 10 fold higher than decapitation alone. Concurrently when decapitation was followed by boiling, α-endorphin was no longer detected. Evaluation in brain tissue of several extraction media--phosphate buffered saline, 5% TCA, HCl methanol, and 1N HOAc--showed the last to be the most satisfactory for both β-endorphin and enkephalin. Since β-endorphin was found to be readily hydrolized by brain homogenates with consequent appearance of α-endorphin, these results indicate that disruption of tissue modifies the content of opioid peptides in brain.     

Determination of arginine-vasopressin in small volumes of rat plasma

A radioimmunoassay to determine arginine-vasopressin concentration in rat plasma is described. This method was a modification from a previously described technique from which the incubation volume was reduced. It allows us to use only 300 microliter of plasma, 1/4 of the original volume, without any impairment to the sensitivity. The intra- and inter-assay coefficients of variation are 11% and 13% respectively. The accuracy ranges between 90 and 99%. To investigate the influence of sampling blood conditions on arginine-vasopressin levels, five groups of Sprague-Dawley rats are analyzed: Group I, decapitation; Group II, ether anaesthesia and cardiac puncture; Group III, ether anaesthesia and decapitation; Group IV, ketamine anaesthesia and cardiac puncture; Group V, ketamine anaesthesia and decapitation. Arginine-vasopressin levels in Group V (2.2 +/- 0.8 pg/ml) are significantly lower than those from the other four groups and similar to those obtained by other authors using a chronic catheter.     

A temporal profile of the endocrine control of trypsin synthesis in the yellow fever mosquito,Aedes aegypti

Trypsin synthesis and secretion is induced after the female mosquito takes a blood meal. Its peak activity has been shown to be proportional to the amount and quality of food uptake. Further regulatory elements, hormones from the brain and the ovary, increase the synthethic rate of trypsin in the midgut by a factor of two. We investigated the temporal effect of removing the humoral factors by decapitation and ovariectomy. Trypsin synthesis was reduced to less than half its normal output when the operations were performed prior, or immediately after the blood meal. Postponing decapitation resulted in an increased activity. However, the dependence on hormones extended up to 14-16hrs after a meal, when maximal synthethic rates are assumed. Similarly, ovariectomy had a prolonged effect on trypsin synthesis. Finally, the lack of hormones reduced the synthetic capacity of the midgut even when small blood meals were given. We conclude that for continued efficient trypsin synthesis, humoral stimulation is necessary but is not part of the feedback mechanism that links the presence of food with the amount of trypsin secreted.     

Comparison of effects of decapitation and anesthesia on metabolic and hormonal parameters in Sprague-Dawley rats.

The modes of euthanasia by either anesthesia or by decapitation were compared by assessing several metabolic and hormonal parameters from plasma and hormone receptors from liver plasma membranes. Two different anesthetics were used. Compared to decapitation, euthanasia by anesthesia significantly increased plasma glucose and triglyceride levels but not plasma cholesterol. Plasma insulin was also significantly increased by anesthetics. No significant differences were observed in plasma glucagon levels or insulin and glucagon receptors from liver plasma membranes between rats euthanized by decapitation and anesthesia. Glucagon receptors were however, affected by dietary carbohydrates. It is concluded that in studies involving measurements of metabolic and hormonal parameters, the use of anesthesia is to be avoided for euthanasia and that decapitation should be the method of choice.     

Corpus allatum control of ovarian development in Aedes aegypti

Ovarian follicles of Aedes aegypti normally pass through a potential stage of arrest 1 day after adult ecdysis and after blood feeding. Removal of the corpora allata or decapitation within an hour of these events interrupts normal development, and development is restored by reimplantation of active allata or by administration of synthetic analogues of juvenile hormone. Only follicles in the normal stage of arrest deposit yolk when stimulated by exogenous ecdysone or after the female takes a meal of blood. Follicles that ceased development as a result of allatectomy or decapitation respond to these oögenic stimuli, but degenerate without depositing yolk.     

Role of water relations and photosynthesis in the release of buds from apical dominance and the early reinvigoration of decapitated poplars

A decrease in xylem pressure potential starting 1 h after decapitation of young hybrid poplars ( Populus deltoides Bartr. × Populus nigra L. cv. DN22) reduced stomatal conductance and transpiration rates for the first 3 days after decapitation. This early moisture stress was alleviated 4 to 5 days after decapitation, resulting in substantial increases in stomatal aperture, transpiration and net photosynthetic rates which continued for the remainder of the one week measurement period. The results suggest the following sequence of events in the decapitated plant: After a brief moisture stress, decapitation increases moisture availability by increasing the root/shoot ratio and by reducing shoot competition for moisture. Improvement in hydration releases buds from apical dominance and increases stomatal conductance and rates of net photosynthesis. This, in turn, leads to the acceleration of growth observed when plants are reinvigorated by decapitation.     

Kinetic analysis of root growth and georeaction

The kinetics of growth and georeaction of horizontal or vertical maize (cv. ORLA 264) apical root segments were analysed. Elongation and curvature data were fitted to a mathematical function and the effect of both light and decapitation reported. Elongation of horizontal segments was found to be more sensitive to light than to decapitation. A light treatment or the decapitation presented several effects on the shape of the growth curves. Growth of vertical segments was affected much more by decapitation than by light treatment. The shape of the curves was clearly different for decapitated and intact segments.
Curvature is affected both by light and decapitation but the shape of the bending curves is modified principally by decapitation.
Under the present conditions used, growth and georeaction of root segments do not seem to be strictly correlated.     

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel “repeated” blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be ≤0.4% of body weight every week or ≤1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.     

Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [¹³C₆]-IAA or [²H₆]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

The increase of cerebellar cAMP level after decapitation: the effect of propranolol

The increase of cAMP level of the rat cerebellum induced by decapitation was studied. Administration of 5 mg/100 g propranolol 1 hour before decapitation completely prevented this increase. Neither the depletion of catecholamine pools, inhibition of their synthesis, nor barbiturate treatment influenced the increase of cAMP level evoked by decapitation. It has been concluded that noradrenergic neurotransmission is involved in the cerebellar cAMP level increase after decapitation.     

Differences in the stress response of prolactin in young and aged female rats

The drawing of blood by orbital sinus puncture (OSP) under ether anesthesia is known to produce a marked increase in serum prolactin (PRL levels in young cycling female rats. The effect of this stressful procedure on PRL release was compared in young and aged female rats. Nonstressed PRL levels were obtained from blood drawn by decapitation. Whereas OSP with a one-minute ether exposure induced a marked increase in PRL levels in young rats on all days of the estrus cycle, older cycling female rats on the day of diestrus -1 and aged rats exhibiting prolonged diestrus (PD) showed virtually no increase above nonstressed levels. However, increasing the ether exposure time to five minutes did produce a rise in PRL levels. Old cycling female rats on the day of estrus and aged rats exhibiting constant estrus (CE) did show a PRL increase comparable to that seen in young animals. Ovariectomy (OVX) completely abolished the stress response seen in aged CE rats. The response, though markedly decreased, was still present in young ovariectomized rats. These experiments show that the stress-induced rise in PRL promoted by OSP under either anesthesia is markedly diminished in aged rats exhibiting a diestrus state. The attenuated response seen in these rats is believed due to factors characteristic of the diestrous state of aging.     

Influence of blood sampling conditions upon histamine concentrations in rat plasma: a study of a complex relationship with plasma epinephrine

The concentrations of histamine reported vary considerably from species to species. The present studies sought to determine if blood sampling techniques were at least in part responsible for this large variability. Since plasma catecholamines are influenced by the stress associated with blood sampling, these biogenic amines also were measured. Finally, we explored the possible existence of a relationship between plasma histamine and plasma catecholamine concentrations.The present study confirms that concentrations of histamine in rat plasma are particularly large and establishes that the manner (e.g. awake, anesthetized) and site (e.g. intravenous, decapitation) of blood removal influence the concentrations obtained. The lowest histamine values were seen in samples taken from blood vessels in anesthetized rats. Blood obtained after decapitation showed increasing concentrations of plasma histamine in sequentially obtained samples.An inverse relationship appeared to exist between plasma histamine and plasma catecholamines (predominantly epinephrine). An inhibitory role of epinephrine upon decapitation-associated histamine release was suggested by the observation that both adrenalectomy and catecholamine depletion (alpha-methyl-para-tyrosine) elevated histamine concentrations. Our studies with propranolol, as well as work by other investigators, establish an inhibitory role of β-receptor stimulation on the release of histamine. On the other hand, histamine injected into the perfused rat adrenal caused a marked release of adrenomedullary catecholamines.In summary our study suggests the presence of a complex interaction between catecholamines and histamine in the regulation of the release of the individual amines. Our findings point to the existence of a histamine-adrenal axis in which the release of histamine may facilitate the release of epinephrine which in turn may restrict further release of histamine.     

An assessment of auxin-promoted transport in decapitated stems and whole shoots of Phaseolus vulgaris L.

¹⁴C-photosynthate transfer in decapitated stems of P. vulgaris plants, treated with IAA (indol-3yl-acetic acid), appeared, as ascertained by microautoradiography, to be restricted to cells of sieve-element appearance. The IAA-induced promotion of photosynthate transport was found not to depend on any artifacts caused by the decapitation procedure. Rather, decapitation primarily served the purpose of removing photosynthate sources above the point of hormone application which otherwise suppressed the expression of the IAA effect on acropetal photosynthate transport. Furthermore, by manipulating stem levels of endogenous auxins with the inhibitor of polar auxin transport, 1-(2¹-carboxyphenyl)-3-phenylpropane-1,3-dione (ACP1.55), evidence was obtained indicating that photosynthate transfer to the shoot apex depended, at least in part, on endogenous levels of auxins at site(s) remote from the apical sink (i.e. shoot apex).Abbreviations ACP1.55 1-(2¹-carboxyphenyl)-3-phenylpropane-1,3-dione - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - IAA indol-3yl-acetic acid     

Factors affecting the free fatty acids in rat brain cortex

The mode of free fatty acid (FFA) release from rat brain cortex was examined under various treatments prior to decapitation and at various times after decapitation. Brain FFA are comprised mainly of 16:0, 18:0, 18:1 and 20:4 with smaller amounts of 18:2, 22:4 and 22:6. A biphasic mode of FFA release is observed with respect to post-decapitative ischemic treatment. The initial rapid phase involves a 3-fold increase in 18:0 and 20:4 and this process occurs within the first min of decapitation. The second phase involves a less rapid increase of most fatty acids between 2 and 5 min after decapitation. Besides the ischemia-induced increase in brain FFA, the levels of individual fatty acids are also affected by factors such as handling stress and administration of anesthetic agents. Pentobarbital anesthesia, but not ketamine, caused a partial reduction in 18:0 and 20:4 levels in brain in vivo. Pentobarbital treatment also reduced the rapid phase of FFA increase commencing after decapitation. On the other hand, FFA level was higher in animals subjected to ketamine anesthesia, both during the non-ischemic and ischemic phase. Results obtained from this study indicate that the FFA pool in brain is regulated by a complex mechanism contributed by extrinsic and intrinsic factors.     

Insulin content in the pancreas and blood plasma of decapitated and encephalectomized rat fetuses

Insulin content in the pancreas and blood plasma of encephalectomized, decapitated and intact rat fetuses was measured by radioimmunoassay. Encephalectomy and decapitation of 17.5-day-old fetuses did not produce any significant effect on insulin concentration in the pancreas and blood plasma of 21-day-old fetuses. Injection of glucose to 21-day-old operated fetuses raised insulin secretion, which seems to be related to the potentiating action of maternal and (or) fetal humoral factors. The data obtained indicate that synthesis and basal secretion of insulin to the blood are not disturbed by the lack of the hypothalamohypophyseal control in prenatal rats.     

The role of hypophyseo-adrenocortical system in the regulation of enzyme monoamine oxidase in rabbit foetuses

The possible relationships of hypophyseo-adrenocortical axis in the evolution of enzyme monoamine oxidase (MAO) in rabbit foetuses from the age of 20 days was studied. The foetuses were deprived of their hypophysis by decapitation in utero at various ages. MAO was measured radiometrically in adrenals, kidneys, paraganglia, lung, liver and heart. There was a progressive rise in MAO activity determined on the 30th day in all cases in adrenals, kidneys and paraganglia following decapitation on the 20th day to 25th day. The activity in the above three organs remained highly significant from control levels even after decapitation on the 27th day. Lung, liver and heart demonstrated maximum activity after decapitation on the 23rd day. Administration of ACTH and hydrocortisone to the decapited foetuses for only once lowered MAO activity in adrenals, kidneys, heart and liver. The results provide evidence that the hormones of the hypophysis act as a rate limiting factor for MAO activity. Their deprivation upsets this rate limiting control resulting in marked rise in MAO activity.