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Biomedical subjects

S R Milligan

Publications and source records attributed to S R Milligan.

At least 37 records · Page 2Linked to original sources

Sound levels in rooms housing laboratory animals: an uncontrolled daily variable.

High sound levels are known to have adverse effects on the behaviour and physiology of laboratory animals, yet their acoustic environment is rarely monitored. In particular, high-frequency sounds that are above the limit of human hearing, but are well within the limits of many laboratory species (i.e., ultrasounds), are usually ignored. In this study, the acoustic environment of laboratory animals was investigated in a variety of different animal facilities. Sound pressure levels (dB SPL) were monitored for periods up to 24 h over two frequency ranges: a relatively low range (0.01-12.5 kHz), and a high range (12.5-70 kHz). While background sound levels in undisturbed situations were generally low (i.e., below 50 dB SPL), marked increases in sound levels often occurred during the working day, producing characteristic daily variations in the sound profile. Peak SPLs commonly reached values of 80-95 dB in the low-frequency range and 50-75 dB in the higher range. In most cases, sound levels were low over weekends. The results suggested that human activities were a very important source of sound in most animal facilities. In a few situations (e.g., rabbits, marmosets, dogs), the animals themselves provided a significant contribution to the acoustic environment. It is clear that the acoustic environment of laboratory animals is a daily variable that is usually uncontrolled and that may have important implications for behavioural and physiological experiments and for animal welfare.

Animal Welfare↗

Silastic implants for delivery of oestradiol to mice.

Silastic implants containing oestradiol were developed for delivering a range of physiological concentrations of oestradiol to mice over long periods. The implants consisted of discrete lengths of Silastic tubing containing oestradiol in arachis oil, with a small reservoir of the oestradiol solution at either end of the implant. Studies showed that the release of oestradiol in vitro was proportional to the concentration of steroid within the implant. Implants containing oestradiol at concentrations from 1 to 100 micrograms ml-1 could induce biological responses in ovariectomized mice, ranging from minimal effects on uterine weight and vaginal smears to supraphysiological increases in uterine weight and rapid vaginal cornification. Studies of uterine vascular permeability indicated that significant effects occurred within a few hours of initial placement of the implant. These results suggest that the design of the Silastic implants described in this study provides a useful method for delivering controlled and easily manipulated physiological doses of oestradiol to mice.

Animals↗

Timing of the window of uterine sensitivity to decidual stimuli in mice.

The refractory period that follows the period of sensitivity to a decidual stimulus in ovariectomized hormone treated mice was investigated. Medroxyprogesterone acetate provided constant progestin concentrations and silastic implants containing oestradiol maintained constant nidatory oestrogen concentrations. The nidatory stimulus was provided by crushing the uterus with a haemostat or by the intrauterine instillation of arachis oil. The decidual response was assessed by measuring changes in uterine weight or by examining the stroma for the presence of alkaline phosphatase. Sensitivity to the oil was first observed approximately 14 h after the insertion of the oestradiol implant but this sensitivity had waned by 32 h and was absent at 40 h. Crushing the uterus initiated a decidual response in mice treated with progestin alone but if the oestradiol implant was inserted then the uterus was responsive to crushing 24 h after insertion but not at 45 h. The traumatic decidual cell reaction (crushing), although not requiring nidatory oestradiol for its successful initiation, was nevertheless subject to the refractoriness that followed oestradiol sensitivity.

Alkaline Phosphatase↗

Failure of platelet-activating factor (PAF-acether) to induce decidualization in mice and failure of antagonists of PAF to inhibit implantation.

The possible role of platelet-activating factor (PAF) in the uterine responses associated with implantation was investigated. Attempts to trigger a decidual cell response in the uteri of hormonally sensitized, ovariectomized mice by instilling PAF-acether (1-1000 ng) intraluminally were unsuccessful. The effect of PAF antagonists on implantation was investigated in females ovariectomized on Day 3 of pregnancy and treated with progesterone. Implantation was induced in these females by injection of 10 ng oestradiol-17 beta on Day 8. Hourly intraperitoneal injections of three PAF antagonists (WEB 2086, CV 3988 and BN 52021 at doses of 1.2-1.4 mg/kg) given over a 24-h period starting 1 h before the injection of oestradiol-17 beta had no significant effect on the occurrence of implantation sites. Intraluminal injection of WEB 2086 (15 micrograms) or BN 52021 (5 micrograms) either 3 h before or 6 h after the nidatory oestradiol also had no significant inhibitory effect on implantation. SRI 63-441 given once daily over the first 4 days of pregnancy at a dose of 40 micrograms/30 g body weight had no inhibitory effect on the establishment of pregnancy. These results are not consistent with a critical role for PAF in implantation in mice.

Animals↗

Tissue spaces during development and regression of the decidual cell reaction in ovariectomized, steroid-treated mice.

Changes in the extracellular and blood spaces of the uterus were assessed from the distribution volumes of 51Cr-EDTA and 51Cr-labelled red blood cells during the development and regression of the artificially induced decidual cell reaction in ovariectomized, steroid-treated mice. The normally high values for uterine extracellular space (0.35-0.40 microliter/mg) fell to less than 0.20 microliter/mg in association with decidual growth. Uterine blood space increased from around 0.02 microliter/mg to 0.03-0.05 microliter/mg with decidual development. Induction of decidual regression by removal of s.c. progesterone implants caused a rapid decline in tissue blood volume to reach control values (0.01-0.02 microliter/mg) within 24 h and preceded any reduction in uterine weight. Uterine vascular permeability, as determined from the tissue accumulation of 125I-labelled human serum albumin, fell with a similar time course. Tissue extracellular space returned to the higher control values within 48 h of initiating decidual regression.

Animals↗

The emission and elicitation of mouse ultrasonic vocalizations: the effects of age, sex and gonadal status.

Subject' mice of varying gonadal status (castrate males; intact or neonatally gonadectomized females and males) were paired for 3-min with intact 'stimulus' females and ultrasonic vocalizations were monitored. Vocalization patterns from home cages differed from the test pairings. The results suggested that the age, experience and gonadal status of the subject influenced the vocalizations from the pair. As the source of ultrasonic calls from these vocally intact pairs could not be individually identified, the 'subjects' were paired with a range of ultrasonically silent (inferior laryngeal nerve-transected) stimulus animals. Vocalizations were detected from all combinations of animals. Gonadally intact females were most effective in eliciting ultrasonic vocalizations from the subjects and gonadally intact males were least effective. The responses of castrate males were lower than from intact males. Anesthetized adults of either sex elicited only poor vocalization responses from other adults. Ultrasonic calls have often previously been studied using vocally intact 'subject' and 'stimulus' animals: the present results confirm the difficulty of establishing who is who in such situations.

Aging↗

A study of the early morphological changes initiated in the uterine luminal epithelium by substances (oil and carrageenan) which induce the decidual cell reaction in mice.

Oil, carrageenan or saline were injected into the uteri of ovariectomized mice treated with hormones on schedules which would sensitize, partly sensitize or not sensitize the uterus to an intraluminal decidual stimulus. The uterine epithelium was examined histologically at various times over the succeeding 5 h. Saline did not produce any morphological change whereas almost immediately after the injection of oil or carrageenan epithelial cell death was apparent in the uterus, regardless of hormone treatment. Within 45 min the dead cells had been removed and the epithelium was re-established. Oil droplets were still present in the uterus after 5 h and these were able to stimulate a decidual reaction in partly sensitized animals when oestrogen was administered 18-44 h after the oil instillation, well after the re-establishment of the epithelium. It is suggested that the early transient cell death in the uterine epithelium is not responsible for triggering the decidual reaction but that it is the contact of the oil droplet with an intact epithelium which triggers the response when the hormonal conditions so allow.

Animals↗

Long-term monitoring of mouse ultrasonic vocalizations.

The temporal pattern of ultrasonic vocalizations by mice in an undisturbed 'home' environment can now be assessed using a system based on amplitude discrimination. Within a chosen frequency band, vocalizations of sufficient intensity are detected by an amplitude discriminator. The output from a pulse generator is sent to a microcomputer which records the time of the incoming event. The system has been validated for monitoring ultrasonic vocalizations in the mouse.

Animals↗

The effects of different anaesthetic agents on the estimation of uterine vascular permeability in mice.

Vascular permeability in the uterus and other tissues of mice was assessed using the accumulation of 125I-human serum albumin 30 min after its intravenous injection. The anaesthetic agent employed for the 125I-albumin injection differentially affected the estimates of vascular permeability: intraperitoneal (i.p.) tribromoethanol of pentobarbitone sodium produced significantly higher values for the uterus and body wall than ether. The i.p. administration of either Saffan or pentobarbitone sodium reduced estimates of vascular permeability in the duodenum. These results emphasize the importance of the choosing a suitable anaesthetic agent in vascular studies of the uterus and other abdominal tissues.

Alfaxalone Alfadolone Mixture↗

Environmental ultrasound in laboratories and animal houses: a possible cause for concern in the welfare and use of laboratory animals.

Many laboratory animals are known to be sensitive to sounds (ultrasounds) beyond the nominal upper limit (20 kHz) of the human hearing range. Sources of sound in laboratories and animal houses were examined to determine the extent of ambient ultrasound. Of 39 sources monitored, 24 were found to emit ultrasonic sounds. Many of these (e.g. cage washers and hoses) also produced sound in the audible range. Running taps, squeaky chairs and rotating glass stoppers created particularly high sound pressure levels and contained frequencies to over 100 kHz. The oscilloscopes and visual display units investigated provided particular cause for concern as they emitted sounds that were entirely ultrasonic and therefore were apparently silent. Ambient ultrasound therefore appears to be common in laboratories and animal houses. It is suggested that its effect on laboratory animals should be investigated and guidelines on acceptable levels be formulated.

Animal Welfare↗

Use of a single-injection tissue-sampling technique to study steroid uptake by the decidualized endometrium of the mouse.

A tissue-sampling paired-tracer method was used to investigate the effect of plasma proteins on uptake by the decidualized endometrium of [3H]progesterone, [3H]oestradiol and [3H]corticosterone. When injected arterially in protein-free Ringer, the extraction of progesterone and oestradiol was 100%, while that of corticosterone was only 60%. The addition of 4% albumin or injection in mouse plasma resulted in significant decreases in progesterone extraction to about 80% and 65% respectively. Injection in pregnant guinea-pig plasma reduced progesterone extraction further (to 33%). While neither 4% albumin nor mouse plasma had any significant effect on the uptake of oestradiol, neonatal rat plasma reduced oestradiol extraction to 40%. These results are consistent with high-affinity binding proteins having a limiting effect on the availability of steroids to target tissues.

Animals↗

The sensitivity of the uterus of the mouse and rat to intraluminal instillation.

The tissue accumulation of 125I-labelled human serum albumin 30 min after intravenous injection was used as an index of uterine vascular permeability. In ovariectomized mice, all sham and experimental instillation procedures produced a 6-10-fold increase in vascular permeability. Some effects were also manifest in the contralateral, control horn. In ovariectomized rats, instillation of saline and arachis oil increased vascular permeability 3-7-fold. After 3 or more days of progesterone treatment following oestradiol priming, fluorocarbon and arachis oil instillation produced marked vascular responses, but these were not restricted to the transient period in which the uterus would respond with decidualization. An IUD prevented the response to arachis oil instillation. These results indicate that the uterus is very sensitive to any manipulation and are consistent with decidualization representing a specialization of a normal uterine inflammatory response.

Animals↗

Uterine blood flow during the development and regression of the decidual cell reaction in ovariectomized, steroid-treated mice.

Uterine blood flow was assessed in mice by measuring organ uptake of intravenously injected [14C]butanol. In ovariectomized mice, injection of 100 ng oestradiol-17 beta increased blood flow 5-fold over that of untreated controls. The injection of oestradiol-17 beta in progesterone-treated mice also increased uterine blood flow at the time of maximal sensitivity to a decidual stimulus, but not 4 days later. Absolute values of blood flow increased during development of the decidual cell reaction in proportion to the increase in uterine weight, reaching maximal values 96 h after decidual induction. When progesterone injections were stopped 72 h after decidual induction, a rapid decrease in absolute and relative blood flow values preceded the decrease in uterine weight. This decrease in uterine blood flow occurred within 12 h of removing a subcutaneous implant containing progesterone. These results are consistent with the view that increased uterine blood flow during decidual development may be necessary to support the rapid increase in uterine weight at implantation and the subsequent decrease in both relative and absolute uterine blood flow on withdrawal of progesterone may promote decidual regression in the mouse.

Animals↗

Intraluminally injected oil induces changes in vascular permeability in the 'sensitized' and 'non-sensitized' uterus of the mouse.

After suitable sensitization of ovariectomized mice with progesterone and oestradiol, the intrauterine instillation of oil produces a massive decidual cell reaction. Vascular permeability, as reflected by the extra-vascular accumulation of 125I-labelled human serum albumin, increased after oil instillation and was maintained at 2-3 times control values for at least the next 3 days. Although oil instillation did not produce a decidual response in females treated with progesterone alone, an increase in vascular permeability (about 2 times control levels) still occurred. This response peaked 8 h after oil instillation and was not maintained. These results indicate that the progesterone-dominated uterus which has not been sensitized with oestradiol cannot be viewed as completely unresponsive to the stimulus of oil and demonstrate that a marked increase in vascular permeability is not itself sufficient to induce decidualization of progesterone-dominated uterine stromal cells. The uterine extravascular accumulation of 125I-labelled albumin was increased both in association with tribromoethanol anaesthesia and after oestradiol treatment of progesterone-primed animals. In pregnant mice, the appearance of Pontamine Sky Blue spots provided an earlier indication of implantation than did determination of total uterine extravascular 125I-labelled albumin accumulation.

Animals↗

Time course of the changes in uterine vascular permeability associated with the development of the decidual cell reaction in ovariectomized steroid-treated rats.

Uterine vascular permeability and tissue blood volume during the development of the oil-induced decidual cell reaction (DCR) in ovariectomized steroid-treated rats were assessed by measuring the extravascular accumulation of 125I-labelled human serum albumin and the tissue content of 51Cr-labelled red cells 30 min after intravenous administration. Within 15 min of oil instillation into one uterine horn, the vascular permeability of the horn was significantly elevated. Permeability rose to a sharp peak (10 times control levels) 9 h after oil instillation, but dropped to 5 times control values by 12 h and continued a steady decline over the next 7 days. Although a marked increase in uterine weight was associated with the development of the DCR, there was no significant change in blood volume/g tissue until 4 days after oil instillation.

Animals↗

The resistance of the mouse uterine lumen to flushing and possible contamination of samples by plasma and interstitial fluid.

The hydrostatic pressures generated during controlled flushing of the mouse uterus increased at implantation and under conditions of uterine closure. These pressures may be responsible for inducing tissue damage during flushing. The possibility that samples collected by flushing might be contaminated with interstitial fluid or plasma was studied using intravenously administered 51Cr-labelled EDTA and 125I-labelled human serum albumin as markers. The presence of both tracers was detected in all flushings and was greatest in flushings from uteri with luminal closure and early implantation sites. These observations raise serious doubts about the validity of the flushing technique for analysing uterine luminal constituents in mice.

Animals↗

Changes in prostaglandin levels in the sensitized and non-sensitized uterus of the mouse after the intrauterine instillation of oil or saline.

Intrauterine instillation of oil, but not saline, induced both a decidual cell reaction and a marked elevation in the uterine PGF-alpha content of suitably sensitized ovariectomized mice. Uterine PGF-alpha concentrations were elevated within 5 min of the oil instillation, reached maximal levels within 30-60 min and then declined to near baseline levels again by 3 h. A similar increase in uterine PGF-alpha content in response to oil instillation was seen in non-sensitized females, although no decidual cell reaction developed. No significant changes in PGE or 6-oxo-PGF-1 content were observed. These results suggest that although the increase in uterine PGF-alpha content is not solely due to the distension of the uterus after intrauterine injection, the increase is not necessarily sufficient to induce a decidual cell reaction.

6-Ketoprostaglandin F1 alpha↗

Analysis of the LH surges induced by mating and LH-RH in the vole, Microtus agrestis.

Female voles mated with a male and experiencing a single ejaculatory series exhibited a rapid and marked elevation of plasma LH levels. The profile of the LH surge had a broad peak 20-40 min after mating. Pentobarbitone sodium administered immediately after mating did not affect the magnitude of the surge. The LH response to exogenous LH-RH ws not affected by the state of receptivity of the female or by gonadectomy. There was no evidence of any priming effect of 25 ng LH-RH on the pituitary response to a second injection of LH-RH 20 or 60 min later. These results are consistent with the primary cause of the LH surge in the reflexly ovulating vole being a rapid secretion of LH-RH.

Anesthesia↗