Stress-like Responses to Common Procedures in Individually and Group-Housed Female Rats . Contemporary Topics 42 (1): 09.
The authors examined cardiovascular parameters (HR and MAP via radiotelemetry) and home-cage activity parameters of individually and group-housed female Sprague-Dawley rats under resting conditions and after common husbandry and experimental procedures. The procedures were routine cage change, restraint and subcutaneous (s.c.) injection, restraint and tail vein injection, simulated cage change, transport to another room and s.c. injection, exposure to the odors of urine and feces from stressed rats, exposure to the odor of dried rat blood, handling and weighing, and handling and vaginal lavage. Three different housing densities were examined -- one, two, or four rats per cage.
The results for each experimental condition are summarized as follows:
Baseline (defined as 1 hour prior to human interaction and during entire 12 h dark period): HR was significantly lower in rats housed 4/cage in the 1 hour period prior to human interaction. In contrast, during the night cycle, the 2/cage rats showed the greatest increase in HR
Routine cage change: In all housing groups, HR increased significantly and remained elevated for 75 min. The rats housed singly showed smallest increases in HR
Restraint and subcutaneous injection: Animals housed alone showed significantly larger responses in HR: 1 rat/cage >2 rats/cage > 4 rats/cage
Restraint and tail vein injection: HR response: 1 rat/cage >2 rats/cage > 4 rats/cage
Simulated cage change (rats returned to original cage): In all groups, HR elevated significantly and remained elevated for 45 min.
Transport to another room and s.c. injection: HR increased equivalently in all housing groups
Exposure to the odors of urine and feces from stressed rats: HR increased - no significant differences between groups
Exposure to the odor of dried rat blood: HR increased - no significant differences between groups however, rats housed alone returned to baseline more quickly
Handling and weighing: HR response: 1 rat/cage >2 rats/cage > 4 rats/cage
Handling and vaginal lavage: HR response: 1 rat/cage >2 rats/cage > 4 rats/cage
Evaluation of Active Behaviors (moving, rearing, grooming): All procedures significantly increased these behaviors for at least 30 minutes and group-housing generally reduced the occurrences of these behaviors
Conclusions:
· Group-housing frequently, but not always, reduced the stress-like responses
· There was little evidence for crowding stress in rats housed 4/cage (even though floor space per rat was one-half of that stipulated in the US federal guidelines); resting HR were less in this group as compared to the other 2 housing conditions
· The routine husbandry procedures (i.e.: cage change, weighing, vaginal lavage) increased HR equivalent to or greater than the procedures that were expected to induce stress (restraint, injections, exposure to blood). Caveat: do not dismiss a procedure as nonstressful just because it is simple or routine
1. T/F In several experimental/procedural settings, group-housing of rats typically reduced stress-like responses, as measured by heart rate.
2. T/F Routine husbandry procedures were found to be as stressful or more stressful than experimental procedures (i.e. restraint, injections) in rats.
3. In historical studies of stress in rats, name 2 physiologic indices that were evaluated.
1. True
2. True
3. Adrenal weight and serum corticosterone
Are "By-stander" Female Sprague Dawley Rats Affected by Experimental Procedures? Contemporary Topics 42 (1): 19.
The paper's objective was two fold. To determine whether female rats are stressed by being in the same room as other rats undergoing routine husbandry (handling, weighing, cage changing) and other experimental procedures (injections, vaginal lavage, simulated and actual decapitation). The other goal was also to ascertain whether housing density impacted that level of stress felt undergoing these procedures. To that end several parameters were observed as indicators of stress. They were mean arterial pressure (MAP) and heart rate (HR) measured by telemetric devices, and also behavioral monitoring. It was determined that decapitation significantly elevated the HR of singly housed rats, and did even more so to female rats housed in groups. HR was increased similarly amongst rats witnessing cage changes, but there was no statistical difference between single or group housed rats. HR did not increase significantly among animals witnessing other rats being restrained or injected, but rats housed alone showed significant increases in HR when observing other rats having vaginal lavages done. When using active behaviors(grooming, rearing etc.) as an indicator of stress in the home cage they were only elevated during a cage change or an injection procedure, and the only group impacted was that where the animals were singly housed. Also, group housing often, but not always reduced stress-like responses that occurred in female by-stander rats Materials and Methods: Female Holtzmann Sprague Dawley Rats were obtained, surgically implanted with telemetric devices (intraabdominal placement with femoral arterial catheterization), and divided into single housed, two or four animal groupings in micro isolator cages. Food and water consumption was monitored daily. The animals were allowed 2 weeks to recover from the surgery during which time they were handled frequently to acclimate them to that process. Three separate experiments were done. Each one had a varying home cage density (single, 2 rat and 4 rats). For each experiment. Instrumented animals were divided into a "manipulated group" (subjected to common husbandry procedures like cage changing, tail vein and sub cut. injections, handling and vaginal lavage), and a "bystander group" (witnessed the "manipulated group undergoing their procedures and husbandry). The two groups were combined for the decapitation and simulated decapitation studies. The radiotelemetry data was collected by computer (data Sciences Corp.), and imported into MS Excel spreadsheets. Data was collected starting one hour and concluding 3 hours after each scheduled experiment. Behavioral assessment was carried out beginning 15 min before and up to 45 min after each experiment and subsequently scored. Results: 1. Animals showed small but statistically significant increases in HR when other animals were decapitated. In context, however, these increases were smaller than those shown by female rats when research staff entered the room. HR's in all groups returned to normal within 30 minutes of witnessing the procedure. Smaller increases in HR were also seen in just the 4 animals groups of female rats after witnessing simulated decapitation. 2. HR was increased when animals witnessed actual cage changing, but were not impacted by simulated cage changing. 3. Restraint and injection of female rats either in the room or on rats removed to another room for injection had no major impact on HR. 4. Witnessing handling, weighing and vaginal lavage had little impact on the group housed female rats, but caused significant elevation of HR in singly housed female rats. Discussion/conclusion: Rats witnessing decapitation did experience some increased stress levels (increased HR), but it was either of lesser or similar magnitude than the stress observed in animals observing routine procedures. The results may suggest that the AVMA Panel on Euthanasia's recommendation that animals be removed for euthanasia may bear some reassessment at least in the case of rats (just this strain?).
1. T or F Female rats displayed greater elevations in HR to cage changing than they did to decapitation?
2. T or F Group housing always reduces stress like responses that occur in female by-stander rats.
1. T, 2. F
Evaluation of Postoperative analgesia in a rat model of incisional pain. Contemporary Topics 42 (1): 28.
Four commonly used analgesics in laboratory animal medicine were identified to administer to mice (Day 0, 1, and 2 SID) to compare their effectiveness after a surgical procedure. An incision ws made on the left rear foot pad of all mice and sutured with silk. Mechanical sensitivity was measured by applying Semmes-Weinstein von Frey filiments (nylon monofiliments) at varying callibrated forces at the incision site until the foot was withdrawn. Baseline pressure measurements were taken on both rear feet prior to the surgical intervention. Additionally weight gain was monitored. Buprenorphine, a partial mu opioid receptor agonist, was administered SC at 3 dosages (0.025, 0.05, and 0.1mg/kg) following surgery. Fentanyl, a potent relatively short-acting synthetic opioid agonist, was injected IP at doses of 0.01 and 0.1mg/kg after surgery. Flunixon meglumine, a potent nonsteroidal anti- inflammatory which exhibits analgesic, anti-inflammatory, and antipyretic activity, was given SC at 1.1 and 2.5mg/kg post operatively. The last treatment was acetominophen which produces analgesia and antipyresis by inhibiting cyclooxygenases. This was given PO before surgery at 100 and 300mg/kg. Controls were given saline SC, IP, and PO. Buprenorphine provided the most profound analgesia at all doses but, once discontinued, led to significant decreased mechanical thresholds (heightened mechanical sensitivity). Interestingly, decreased mechanical thresholds were also detected in the back foot with no incision. Mice receiving fentenyl at both doses exhibited significant decreases in mechanical thresholds, however, mice receiving the high dose had a significant increase in their mechanical threshold when compared to the control group receiving saline IP. The mice receiving the high dose also exhibited heightened sensitivity following cessation of the drug, as with buprenorphine. This condition is known as opioid-induce hyperalgesia and has been reported in other studies. Animal receiving buprenorphine (0.05 and 0.1mg/kg) or fentanyl (0.1mg/kg) displayed other signs: sedation, shallow respiration, exophthalmia, lack of blink response, and postural rigidity. When recovering from these signs, they also exhibited rhythmic swaying of head, licking, chewing, and repeated grooming and rearing behavior. Flunixin did not decrease mechanical thresholds in either dose but did decrease mechanical allodynia when compared with the control receiving saline SC. Weight gain was unaffected by the lower dose but dropped when given the higher dose. Animals receiving acetominophen exhibited decreased mechanical thresholds during the treatment period and also showed signs of pain when exposed to innocuous stimuli. The low dose of acetominiphen caused siginificantly lower weight gains than the control group and the higher dose produced higher weight gains than the control group. While opioid based drugs produce more profound analgesia, they also produce some side effects that may be deleterious to particular research projects. In light of this, nonsteriodal anti-inflammatory agents may provide analgesia and avoid these side effects.
1. What is allodynia?
2. How do buprenorphine, fentanyl, flunixin, and acetominophen cause analgesia?
1. Inflammation induces a state in which pain behavior is evoked by previously innocuous stimuli.
2. Buprenorphine-partial mu opioid receptor agonist Fentanyl-relatively short-acting synthetic opioid agonist Flunixin-nonsteroidal anti-inflammatory Acetominophen-nonsteroidal anti-inflammatory by inhibiting cyclooxygenases
The Effects of Confinement Environment on Reproductive Efficiency in the Baboon. Contemporary Topics 42 (1): 35.
The authors report on their observation of increased reproductive efficiency in a population of research baboons when moved from indoor housing meeting animal welfare act space requirements to a much larger indoor-outdoor facility providing significantly more space per animal and exposure to natural sunlight. Factors evaluated included photoperiodicity, stress from nutritional, spatial, and social causes. The mean number of days post-partum to first estrus was 165 days for indoor animals (Annex) and 69 days for the new facility (El Reno). The length of time from first estrus to conception was 61 in the Annex and 47 for El Reno (not significantly different), and the mean time between conceptions was 403 days for Annex housed animals and 296 for El Reno animals. Some improvement in reproduction had been anticipated, but this was greater than expected. Research animals studied were from a breeding colony at the Oklahoma University Health Sciences Center. The animals had been housed in the "Annex" which was an indoor facility and reproductive efficiency data collected over a period of 45 months. Approx. 80% of these animals were moved to the new El Reno facility and data collected over a 7 month period, at which time significant improvement in reproductive efficiency was noted to have occurred. Gang housing was used in both facilities, but the size of the gangs was larger in the new facility. Indoor gangs were composed of one male, 12-13 adult females, and 3-8 infants/juveniles. Indoor-outdoor gangs were composed of 2-4 adult males, 23-30 adult females, and 5-18 infants/juveniles. Reasons for the improvement in reproductive efficiency were believed to include photoperiodicity and stress due to nutritional, social, or spatial causes. Decreased length of lactational anestrus was believed to be due to influences on the hypothalamic-pituitary-ovarian axis and production of prolactin, decreased stress with more space and places to play and hide (thus less social stress), and less competition for food in the El Reno facility vs. the Annex resulting in improved nutrition even though the diet was not changed.
1. Give the taxonomic name (Family, Genus, Species, and Sub-species) of the Olive and Yellow baboons.
2. What is the gestation period in baboons?
3. Which reproductive parameter was NOT significantly different in the two housing situations?
a. days post-partum to first estrus
b. days from first estrus to conception
c. days from one conception to the next
d. all of the above were significantly different
4. Estrus in the female baboon is indicated by _______ and ovulation usually occurs ____________ .
a. Vaginal bleeding, 5 days after bleeding stops
b. Perineal swelling or turgescence, 1-2 days prior to detumescence
c. Turgescence, 5 days prior to detumescence
d. No visible change, mid-cycle
5. Adult baboons usually require cages in which groups, based on The Guide for the Care and Use of Laboratory Animals?
a. Females 4,5,& 6. Males 5,6,& 7.
b. Females 6 &7. Males 5 & 6.
c. Females and Males, 4 & 5.
1. Cercopithecidae, Papio, hamadryas anubis for olive and cynocephalus for yellow.
2. 180 days
3. B
4. B
5. A
Development of a quantitative method for evaluation of the electroencephalogram of rats by using radiotelemetry. Contemporary Topics 42 (1): 40.
The International Conference on Harmonization Guidelines for safety pharmacology includes a core battery of in vitro tests to investigate the effects of new substances on vital functions. These tests evaluate cardiovascular, respiratory, and CNS. The EEG is used in supplemental safety pharmacology evaluations. The EEG provides a continuous tracing of regional electrical activity in the brain, usually recorded from the skin surface. MV is recorded on the y-axis and time on the x-axis. Amplitudes vary from 0 to +/- 300mV(0.3mV). Brain waves on the EEG appear very "chaotic" and are highly complex. The character of the cortical EEG is dependent on the degree of activity of the cortex and brain waves change between states of wakefulness and sleep. Delta waves(<4Hz) occur during deep sleep, infancy, and pronounced organic brain disease. Theta (4-7Hz) are associated with parietal and temporal regions in children and during frustration in adults and with many brain disorders. Alpha (8-13Hz) are associated with a relaxed state, and beta (>14Hz) are activated during intense activation of the CNS. Further signal processing techniques provide a clearer picture of the character of a tracing. Digital sampling and filters provides a spectrum display that has phase and amplitude information. Voltage across a range of frequencies can be quantified. The result is a logarithmic display of signal power (dBV) versus a linear display of frequency (Hz) termed a fast Fourier transformation(FFT). Computerized frequency analysis by FFT provides and objective and quantitative method for evaluation of the EEG. This method is faster, easier, more consistent, and more objective than scoring of raw tracings. FFT analysis may provide information regarding the effects of some compounds on brain activity. Recording the EEG by telemetry has the potential advantage of eliminating behavioral artifacts because animals are undisturbed. Mechanical and electrical artifacts can be avoided because there is no tether. The objective in this study was to develop and evaluate a method for the quantitative analysis of EEG changes caused by compounds known to effect unrestrained, undisturbed rats by using averaged FFTs on EEG data acquired from rats by radiotelemetry. Methods and materials: Electrodes were stereotactically placed and transmitter was secured subcutaneously to fascia of the dorsal lumbar muscles and leads were tunneled to the neck area. The compounds evaluated are given in the below.
Discussion: The data did not show strong statistical significance in changes in EEG spectral patterns due to drug administration. However, there was consistency between all four animals, and the changes were consistent with previous reports. For atropine, caffine, and pentobarbital, the overall direction of changes was the same as that reported previously. For atropine, caffine, and ketamine, changes over specific frequency ranges (relative peaks in power) were consistent with those in previous reports. Differences could be attributed to differences in dose etc. Radiotelemetry has advantages for recording of the EEG. First, behavior effects of a tether apparatus are eliminated, along with the possible associated EEG changes. Telemetry generally is accepted as a more humane alternative to exteriorized leads and tethers. FFT provides an effective way to measure subtle drug-induced changes in EEGs. Placement of leads is important. "Theta walking", a vigilance state in the rat was not detected in the frontal cortex, but was in the occipital cortex. Electrode placement in the present study was selected to detect limbic activity. Electrode placement designed for detection of seizures is a good choice for drug safety studies. Diurnal variations in the EEG power spectrum have been shown for the rat.
1. What is fast Fourier transformation (fFT)?
2. What are the four types of waves described in the paper?
3. What are the advantages of using radiotelemetry for EEG recordings?
1. FFT is the logarithmic display of signal power (dBV) versus a linear display of frequency (Hz).
2. Delta waves (<4Hz), Theta (4-7Hz), Alpha (8-13Hz), Beta (>14Hz).
3. Behavioral artifacts are avoided because animals are undisturbed. Mechanical and electrical artifacts are avoided because there is no tether.
Aspiration Method to Collect Epithelial Cells from Mouse, Rat, and Monkey Oviducts. Contemporary Topics 42 (1): 46.
Oviduct epithelial cells are often used, both in vitro and in vivo, in the research area involving reproductive biology. Conventional method for epithelial cell collection may not be practical for collecting sufficient numbers of cells from small laboratory animals. For example, in larger species, enzymatic method involving protease (e.g. trypsin) sometimes damages the oviduct epithelial cells; there is a potential for contamination by other cell types due to protease activity; mechanical squeezing of oviducts is not desirable in small animals. The objective of this study is to develop a simple, nonenzymatic technique for collection of oviduct epithelial cells from rodents which can also be applied to non-human primates. The method involves using a glass micropipette to aspirate epithelial cells from the oviduct. Glass micropipette diameter (stretched after heating): 100 um for mice/rats and 300 um for cynomolgus monkey. For specific description of actual collection protocol, please refer to the article. 1) The oviduct is first flushed out with D-MEM; 2) glass micropipette is inserted into the oviduct via the fimbriae; 3) aspirate as the pipette tip slides down the oviduct; 4) transfer recovered cells into a petri dish with culture medium. Total number of cells collected from each oviduct was 1x10^4 to 1x10^5 from mice and rats, and 1x10^6 to 1x10^7 from the cynomolgus monkeys. The collected epithelial cells were able to adhere to petri dishes and grow in culture. Active movement of the cilia of the cells collected using the aspiration method was observed, although movement was weaker from the NHP when compared to those collected from rodents.
Animals used:
31-32 week old SPF ICR (n=3), BDF1 (n=3), and C57BL/6 (n=2) mice
36-37 week old SPF Wister rats (n=8)
Cynomolgus monkeys (n=3; 6mo., 12 years, and 18 years old)
1. T or F - Collection of oviduct epithelial cells using aspiration method from mice, rats, and cynomolgus monkey was done using glass micropipette connected to a mouth piece via a silicon tube.
2. T or F The collected oviduct epithelial cells were able to adhere and grow in cell culture.
1. FALSE - Due to biohazard/zoonosis risk working with NHP tissue, a glass syringe (instead of a mouth piece) was used for controlling the glass micropipette for cell collection.
2. TRUE
Coinfection of laboratory rats with Mycoplasma pulmonis. Contemporary Topics 42 (1): 52.
Natural coinfection of Chlamydia pneumoniae and Mycoplasma pulmonis can occur in laboratory rats and is associated with multiple organ compromise.
Introduction: Mammals and birds are natural hosts for bacteria of the Chlamydia genus. They are obligate intracellular parasites and multiply in intracytoplasmic vacuoles, primarily in macrophages and monocytes. These vacuoles are resistant to lysosomal hydrolases because they do not merge with lysosomes. The infective forms, called "elementary bodies", are released when the infected cell is lysed.
Chlamydia pneumoniae causes acute subclinical bronchitis and pnuemonia in humans worldwide. Natural infection of this organism has not been described in rats.
Mycoplasma pulmonis usually causes subclinical upper respiratory disease in rats. In situations where the host immune status is compromised, pneumonia or otitis may develop.
Purpose of Study: To describe the histological findings caused by M. pulmonis and C. pneumoniae coinfection.
Materials and Methods: Outbred Wistar rats housed under conventional conditions were used. Wistar specific pathogen-free rats were used as control animals. Blood was collected by cardiac puncture under k/x anesthesia and was submitted to a laboratory for analysis of alanine aminotranferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and urea. Five rats were euthanized for isolation of bacteria, and eight rats were euthanized for histopathology and electron microscopy.
Results: Serum levels of ALT, AST, ALP, and urea in the conventionally housed, outbred Wistar rats differed significantly from the SPF control WIstar rats. Histopathological exam of heart, lung, liver, kidney, and spleen from conventionally housed rats revealed that the lungs were the most severely and frequently affected. Lungs had characteristics of chronic bronchitis, hyperplasia of respiratory epithelial cells, preribronchial lymph node enlargement and severe interstitial pneumonia. The most seriously affected animals had microscopic lesions consistent with chronic obstructive pulmonary disease (COPD). M. pulmonis was identified in interstitial tissue using immunoperoxidase staining. Immunohistochemical and in situ hybridization assays demonstrated C. pneumoniae in mesothelial cells, macrophages, lymphocytes, and plasma cells in the lungs.
In the liver, hepatocytes contained C. pneumoniae organisms. M. pulmonis antigens were present in hepatocytes and in the interstitial space. Electron microscopy identified Mycoplasma organisms in hepatocytes, Kupffer cells, and endothelial cells. Hepatocytes exhibited marked intracellular edema and mitochondrial lysis.
The heart had foci of chornic inflammatory infiltrates in the endocardium, valves and myocardium. Immunohistochemical staining demonstrated the presence of C. pneumoniae. Kidneys and spleen were also positive for C. pneumoniae.
Discussion: The presence of M. pulmonis and C. pneumoniae was identified in lungs, liver, spleen, kidneys, and heart, and were associated with chronic inflammatory infiltrate and parenchymal lesions. No other pathogenic organisms were identified in these tissues. The authors found no other reports describing an association by its morphological appearance during electron microscopy. It has a characteristic pear-shaped form due to the expansion of its external membrane.
In this study, what organ was most severely affected?
Lungs
Open-Field Assessment of Spontaneous Activity. Contemporary Topics 42 (1): 57.
SUMMARY: There are a number of behavioral tests that have been developed to assess motor function in rodents (i.e. rotarod, balance beam, hanging wire/grip test, vertical pole test, walking pattern analysis, and open field testing). The latter has been shown to be best used to assess traits such as anxiety, response to novel situations, stimulant activity of drugs and experimental compounds, and the effect of induced brain injury. This paper describes both non-automated and automated open field assessment in rodents. Due to the labor intensive non-automated method, new automated activity boxes have been developed. These automated systems utilize infrared beams and photocells to act as motion detectors. Coupled with the computer system, all of this data can then be collected and analyzed.
1. What part of the brain initiates movement?
2. What part of the brain coordinates movement?
3. What part of the brain provides input on location of the animal, relative to the head & body?
1. cerebrum
2. cerebellum
3. vestibular system