Regulation of testicular LH/hCG receptors in golden hamsters (Mesocricetus auratus) during development

During prepubertal development in the golden hamster, there are major age-related changes in the number of testicular LH/hCG receptors. Between 22 and 35 days of age, there was greater than 10-fold increase in testicular LH/hCG receptors, followed by a decrease at Day 37. Concomitant with, but preceding slightly, the changes in receptors, were increases in plasma LH and FSH and most noticeably prolactin concentrations, between Days 10 and 20 of age. Inhibition of the increases in plasma levels of prolactin by daily injections of bromocriptine, between 14 and 31 days of age, resulted in suppressed testicular and seminal vesicle weights, and decreased content and concentration of testicular LH/hCG receptors. Similarly, the premature increase in plasma prolactin concentrations in prepubertal hamsters between 6 and 20 days of age, by means of ectopic pituitary transplants, resulted in increased testicular and seminal vesicle weights, as well as an increase in the concentration of testicular LH/hCG receptors. These results strongly suggest that increases in plasma prolactin values during development are important in enhancement of the development of testicular LH/hCG receptors.     

Effects of pinealectomy and melatonin or 5-methoxytryptamine on testicular LH and PRL receptors in Syrian hamsters (Mesocricetus auratus)

The pineal has been previously shown to be an important factor in the regulation of testicular function in photoperiodic mammals. The effects of lack or increase in pineal hormones on testicular hormonal receptors has, therefore, been examined. Pinealectomy decreased the concentration of testicular LH receptors in hamsters exposed to either a long or short photoperiod but had no effect on the concentration of testicular PRL receptors. In animals exposed to a short photoperiod, pinealectomy prevented testicular regression and the concomitant decreases in total LH and PRL receptor contents. Treatment for 12 weeks with either melatonin or 5-methoxytryptamine caused a decrease in testicular PRL receptor levels, whereas the only changes in LH receptor levels were due to melatonin-induced testicular regression. The present results indicate that some of the effects of pineal hormones on the testes are independent of the pineal-induced changes in testes mass and are the consequence of long-term action. Furthermore, testicular function appears to be affected by both the lack or the increase in pineal hormones.     

Effects of long-term treatment with melatonin or melatonin plus ectopic pituitary transplants on testicular LH/hCG and prolactin receptors in juvenile Syrian hamsters (Mesocricetus auratus)

Juvenile hamsters were injected daily with melatonin and some were also given transplants of 2 pituitaries under the kidney capsule. Weights of the testes and the accessory reproductive glands were reduced after 8 and after 12 weeks of melatonin treatment, but remained unaltered in animals treated with ectopic pituitary transplants. Levels of testicular LH/hCG receptors were significantly reduced by daily melatonin injections for 8 and 12 weeks. The presence of pituitary transplants in melatonin-injected hamsters prevented these reductions, and increased LH/hCG receptors above control levels. These changes in testicular LH/hCG receptors were closely related to alterations in serum prolactin concentration induced by melatonin and pituitary transplants. After 8, but not after 12 weeks of treatment, testicular prolactin receptor levels were reduced by melatonin and maintained by the presence of pituitary transplants. We conclude that: juvenile male hamsters become sensitive to the effects of daily melatonin injections when they reach maturity; daily melatonin injections can reduce the levels of testicular LH/hCG and prolactin receptors; and the effects of melatonin on LH/hCG and prolactin receptors are probably due to suppression of endogenous prolactin release.     

Effects of different numbers of ectopic pituitary transplants on regulation of testicular LH/hCG and prolactin receptors in the hamster (Mesocricetus auratus)

Adult male hamsters were given transplants of 1/2, 1, 2, 3 or 4 pituitaries under the kidney capsule and were killed 4 weeks later. Pituitary transplants produced a significant, dose-related increase in plasma prolactin levels, no changes in plasma LH and an increase in plasma FSH. Concentration of LH/hCG receptors in the testes was significantly increased in animals with 2 or 3 transplants and concentration of testicular prolactin receptors was significantly increased in those given 2 transplants. The apparent stimulatory effects of 1/2, 1 or 4 transplants on testicular LH/hCG and prolactin binding were not statistically significant. Some of the animals were injected with 0.3 i.u. hCG/g body weight 24 h before being killed. This produced a significant reduction in the levels of prolactin receptors and an apparent reduction in the levels of LH/hCG receptors in the testes. Elevation of plasma testosterone concentrations in response to hCG was significantly greater in animals given 3 or 4 pituitary transplants than in the remaining groups. These results provide further evidence that prolactin increases the number of LH/hCG and prolactin receptors in the hamster testis and suggest that changing the number of ectopic pituitary transplants may result in biphasic effects on the testis, with 2 or 3 transplants being maximally stimulatory.     

Experimental iron deficiency in Syrian hamsters (Mesocricetus auratus)

An animal model was developed in which the effect of iron deficiency on the oral mucosa could be studied. Iron deficiency was induced by feeding hamsters a low-iron powdered diet together with withdrawal of 0.5 ml of blood weekly, for a period of 9 weeks. At the end of this period the mean haematological values for control animals were, Hb 15.9 g/dl, plasma iron 40.3 mumol/l, TIBC 90.5 mumol/l and transferrin saturation 44.5%, compared with 7.4, 7.2, 111.4 and 6.5 respectively for experimental animals. These results were reproducible in successive groups of animals and indicate that this is a useful model for the study of iron deficiency anaemia.     

Age affects hibernation in Syrian hamsters (Mesocricetus auratus)

In this study, we aimed to show how age affects hibernation in the Syrian hamster. Experimentally, we used 30 male animals differing in age. The old animals were 20 months of age and the adults were 8 months of age at the end of the test. The young animals were 3 weeks old at the start of testing and 5 months old at the end of the testing period. The torpor observation started October 15, 1996, and ended March 11, 1997, in the laboratory colony maintained under natural photoperiod and outdoor air. Observations were performed around noon daily. Three measures (i.e., prehibernation period [hibernation latency], proportion of hibernation spent in torpor, and proportion of animals in torpor), all of which reflect the strength of occurrence of hibernation, indicated that the older hamsters (1) started hibernation earlier, (2) spent more time in torpor, and (3) had a higher chance of being in torpor than the younger ones during the hibernation season. (Chronobiology International, 17(5), 623-630, 2000)     

Effects of a circadian mutation on seasonality in Syrian hamsters (Mesocricetus auratus).

In Syrian hamsters, exposure to short photoperiods or constant darkness induces a decrease in gonadotrophin secretion and gonadal regression. After 10-12 weeks, animals undergo spontaneous gonadal reactivation, gonadotrophin concentrations rise, and in males, testes size increases and spermatogenesis resumes. The tau mutation shortens the period of circadian wheel-running activity by 4 h in the homozygote. Here, we examine the impact of this mutation on the reproductive response to photoperiod change. Seventeen adult tau mutant and nine adult wild-type males were housed in complete darkness for 25 weeks and testes size determined at weekly intervals. Gonadal regression and subsequent recrudescence occurred in both groups of animals. Regression occurred more rapidly in tau mutants, with a nadir significantly earlier than wild-types but after a similar number of circadian cycles. Rates of testicular recrudescence were similar in both groups. Our data suggest that an acceleration of the circadian period increases the rate of reproductive inhibition in animals exposed to inhibitory photoperiods. Once initiated, the rate of spontaneous reactivation may be independent of the circadian axis.     

Routine chromosome screening in Syrian hamsters (Mesocricetus auratus) using orbital sinus blood

Blood from the posterior orbital sinus of Syrian hamsters, obtained under halothane anaesthesia, can be cultured to give large numbers of metaphase chromosome spreads for analysis. The procedure has been used for rapid routine screening of the karyotypes of all offspring in a breeding colony where translocations are present. Results can be obtained within 3 days of collecting the blood sample.     

Pathophysiological studies of trinitrobenzene sulfonic acid-induced colitis in Syrian hamsters (Mesocricetus auratus)

We developed a colitis model in Syrian hamsters (Mesocricetus auratus) to investigate the relationship between colitis and neutrophil elastase (NE). Colitis was induced by a single intracolonic dose of trinitrobenzene sulfonic acid (TNBS; 90 mg/ml) dissolved in 15% (vol/vol) ethanol. The ulcer area, tissue myeloperoxidase (MPO) activity, and luminal NE activity all were increased on Days 1 and 5, corresponding with the acute inflammatory histopathological changes. These acute inflammatory parameters subsequently decreased by Day 14, and chronic inflammatory histopathological changes became evident. Recurrence of inflammation was not observed during the period up to Day 28. To evaluate our colitis model, the effects of prednisolone were examined. Prednisolone was administered orally once on the day before induction of colitis, and animals were treated twice daily thereafter. Although prednisolone had little effect on the tissue MPO activity, prednisolone inhibited the ulcer area and NE activity. In addition, the effects of an NE-specific inhibitor (ONO-6818) on our TNBS-induced colitis model were examined. In the subcutaneous treatment study, ONO-6818 was administered once before the induction of colitis. Although ONO-6818 had little effect on the tissue MPO activity, the ulcer area and NE activity were decreased in the ONO-6818-treated group. The inhibitory effects on the ulcer area and NE activity were confirmed after oral treatment with ONO-6818 after induction of colitis. We conclude that our colitis model is useful for investigating the relationship between colitis and NE, and inhibition of NE activity can prevent the progression of ulceration.     

Enhanced longevity in tau mutant Syrian hamsters,Mesocricetus auratus

The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span of male and female hamsters from three genotypes (wild-type, heterozygous, and homozygous tau mutants, all derived from heterozygote crosses to randomize the genetic background) was recorded in constant darkness. Male hamsters lived significantly longer than females: the overall average life span was 96.9 weeks (SE = 2.5, n = 118) for males and 82.0 weeks (SE = 2.1, n = 99) for females. To our surprise, male and female homozygous mutant hamsters lived significantly longer rather than shorter compared to wild-types. For males, the difference between the two genotypes was on average 14%; for females, the difference was 16%. The mortality rate of wild-type males was significantly different from that of homozygous tau males but not different from that of heterozygotes. Overall, survival of wild-type females was statistically distinguishable from both heterozygous and homozygous mutant females. Male and female wild-type hamsters were heavier than homozygote mutants throughout the entire life span, and heterozygous mutants had intermediate weights. There was no correlation between body mass and life span, and the causes of the extended life span in tau mutant hamsters remain unresolved.     

Isolation of Corynebacterium kutscheri from aged Syrian hamsters (Mesocricetus auratus).

Corynebacterium kutscheri was isolated from the oral cavities of 12 male Syrian hamsters (Mesocricetus auratus) which were about 12 months old. At 1, 5, and 9 months after initial isolation of C. kutscheri from the oral cavity, hamsters were euthanatized, and attempts were made to culture C. kutscheri from 13 additional sites. Corynebacterium kutscheri was isolated from nine hamsters, and regardless of the hamsters' ages, the organisms were most frequently isolated from the oral cavity (100%), esophagus (100%), cecal content (100%), and colon and rectum (88.9%). Isolation rates in the nasal cavity were 66.7%, followed by 55.5% in the trachea and 33.3% in the submaxillary lymph nodes. The number of the organisms found in the submaxillary lymph nodes and esophagus was 10(3) to 10(4) CFU/g. The number found in the cecal content and in the colon and rectum was 10(2) to 10(5) CFU/g. The organisms were not isolated from lung, stomach, kidney, spleen, and mesenteric lymph node tissues. The hamsters had neither clinical signs nor lesions. However, 7 of 12 animals had low agglutinating antibody titers. The Syrian hamster can therefore be an asymptomatic carrier of C. kutscheri.     

Short days and exogenous melatonin increase aggression of male Syrian hamsters (Mesocricetus auratus)

Many nontropical rodent species rely on photoperiod as a primary cue to coordinate seasonally appropriate changes in physiology and behavior. Among these changes, some species of rodents demonstrate increased aggression in short, "winter-like" compared with long "summer-like" day lengths. The precise neuroendocrine mechanisms mediating changes in aggression, however, remain largely unknown. The goal of the present study was to examine the effects of photoperiod and exogenous melatonin on resident-intruder aggression in male Syrian hamsters (Mesocricetus auratus). In Experiment 1, male Syrian hamsters were housed in long (LD 14:10) or short (LD 10:14) days for 10 weeks. In Experiment 2, hamsters were housed in long days and half of the animals were given daily subcutaneous melatonin injections (15 microg/day in 0.1 ml saline) 2 h before lights out for 10 consecutive days to simulate a short-day pattern of melatonin secretion, while the remaining animals received injections of the vehicle alone. Animals in both experiments were then tested using a resident-intruder model of aggression and the number of attacks, duration of attacks, and latency to initial attack were recorded. In Experiment 1, short-day hamsters underwent gonadal regression and displayed increased aggression compared with long-day animals. In Experiment 2, melatonin treatment also increased aggression compared with control hamsters without affecting circulating testosterone. Collectively, the results of the present study demonstrate that exposure to short days or short day-like patterns of melatonin increase aggression in male Syrian hamsters. In addition, these results suggest that photoperiodic changes in aggression provide an important, ecologically relevant model with which to study the neuroendocrine mechanisms underlying aggression in rodents.     

Volitional ethanol consumption affects overall serotonin metabolism in Syrian golden hamsters (Mesocricetus auratus)

Methods were established for the determination of serotonin (5-HT)(1) metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL) in the urine of Syrian golden hamsters (Mesocricetus auratus) and used to study the effect of volitional ethanol consumption on overall 5-HT metabolism in this ethanol-preferring rodent. The basal levels of 5-HIAA and 5-HTOL in 24-h urine of ethanol-naive hamsters were 300 +/- 101 and 4.96 +/- 1. 06 nmol (n = 8), respectively. Given free choice between water and a 15% ethanol solution, these hamsters chose to consume increasing amounts of ethanol. The increase was accompanied by a concomitant decrease in urine 5-HIAA and increase in urine 5-HTOL, indicating that volitional ethanol intake diverted part of the 5-HT metabolic flux from an oxidative into a reductive pathway. In a separate experiment, the amounts of ethanol consumed by and blood ethanol concentrations attained in ethanol-drinking golden hamsters were determined at 5 different time intervals between 6 PM and 7 AM when most feeding activities occurred. Except in the first hour after lights were turned off, ethanol was consumed at a relatively even pace throughout the night (2-3 g/kg/3 h) and blood ethanol levels were maintained at the low mM range which rarely exceeded 2 mM. These results suggest that the biochemical pathway that catalyzes 5-HT metabolism is extremely sensitive to ethanol and can play an important role in mediating the reported clinically beneficial action of a low concentration of ethanol during alcohol detoxification.     

Suprachiasmatic nuclei may regulate the rhythm of leptin hormone release in Syrian hamsters (Mesocricetus auratus)

The suprachiasmatic nuclei (SCN) generate the circadian rhythm of many hormones. The hormone leptin is a metabolic signal that informs the brain about fat and energy stores of the body. We investigated whether the rhythm of leptin hormone release in Syrian hamsters is directly controlled by the SCN. Three experiments were performed: in the first, hamsters were SCN-lesioned; in the second, hamsters were exposed to different feeding regimes; and in the third, hamsters were adrenalectomized and implanted with cortisol capsules to maintain constant glucocorticoid release. Blood samples were collected before and after the experiments at different clock times and examined for leptin levels by enzyme-linked immunosorbant assay (ELISA). Different feeding regimes and constant glucocorticoid release did not alter the rhythm of leptin release; whereas, SCN lesions abolished the rhythm. The results of the present study suggest the rhythm in leptin release in Syrian hamsters may be controlled by the SCN.     

Auditory brainstem responses in Golden Syrian hamsters (Mesocricetus auratus) affected with the Wh gene.

BACKGROUND AND PURPOSE: The anophthalmic white (Wh) gene in Golden Syrian hamsters (Mesocricetus auratus) is autosomal semi-dominant and causes several developmental defects, including hearing loss. The Wh mutation is thought to be homologous to Waardenburg syndrome in humans, apparently affecting similar developmental processes. The purpose of this study was to assess the hearing of hamsters in the AN/As-Wh strain. METHODS: Using auditory brainstem responses, electrophysiologic activity was determined in 20 hamsters of the AN/As-Wh strain, with the aim of elucidating hearing status. Hamsters were classified into five genotypes and were evaluated by use of click stimuli. RESULTS AND CONCLUSION: Hamsters assigned to the genotypes differed in their hearing sensitivity and could be classified into categories of normal hearing, moderate hearing loss, and profound hearing loss.     

Reproductive effects of 6-chloromelatonin implants and/or injections in male and female Syrian hamsters (Mesocricetus auratus)

Adult female hamsters were injected each afternoon for 9 weeks with 2.5, 15 or 25 micrograms of melatonin or 6-chloromelatonin (CM). Each drug resulted in a significant dose-related depression in uterine, ovarian and anterior pituitary gland weights. Additionally, plasma and pituitary concentrations of prolactin fell with increasing dose of either indole whereas pituitary levels of LH and FSH were positively correlated with dose. There was no difference in effectiveness between the two drugs. Adult male hamsters treated for 10 weeks with daily afternoon injections of melatonin and a blank beeswax pellet had depressed testicular and accessory organ weights and plasma and pituitary concentrations of prolactin. Implantation of a 1 mg melatonin or CM beeswax pellet in hamsters concurrently receiving daily afternoon injections of melatonin prevented the organ weight and hormonal changes, except for plasma prolactin. Adult male hamsters treated for 10 weeks with daily afternoon injections of CM and a blank beeswax pellet had depressed reproductive organ weights and pituitary and plasma concentrations of prolactin; this depression in hormonal values and organ weights was totally prevented if the CM-injected hamsters were also bearing a beeswax-melatonin pellet. In conclusion, 6-chloromelatonin is as effective as melatonin with regards to antigonadotrophic and counter-antigonadotrophic effects in male and female Syrian hamsters.     

Intravenous injection of unanesthetized hamsters (Mesocricetus auratus)

A rubber band tourniquet was applied to the proximal portion of the foreleg of unanesthetized male and female golden hamsters followed by intravenous injection in the cephalic vein using a 25 gauge needle. Volumes up to 0.3 ml could be injected easily.     

Ontogeny of oral function in hamsters (Mesocricetus auratus)

The oral apparatus of neonatal and juvenile golden hamsters was investigated by clearing and staining of whole crania, videotaping of behavior, and electromyography of several jaw muscles. Chewing developed during the first postnatal week and matured in the second; however, suckling was still the primary mode of feeding. Micromovements of the jaws occurred early when the osseous skeleton and joints developed. Macromovements correlated well with EMG records and were limited to jaw opening at birth. Muscles of the oral floor generated large bursts of activity during jaw opening and tongue protrusion from 0 days postnatal (dpn), when simple and stereotyped gaping was induced, until 14 dpn, when movements were spontaneous and not stereotyped nor inducible. However, adductor muscle activity was brief, low in amplitude, and primarily involved with jaw stabilization until 4 dpn, when these muscles became active during closing the jaws; closing activity increased in frequency and amplitude until the end of the second week. Development of frequent, coordinated macromovements of chewing was associated with the refinement of joint structure and dental occlusion and with the growth of the craniofacial skeleton. Jaw movements and associated EMG's correlated better with available data on development of neural circuitry than with that for musculoskeletal development.     

Chromosome replication in fibroblasts of the Syrian hamster (Mesocricetus auratus)

The BrdU/Hoechst 33258/Giemsa method for sub-dividing S-phase in asynchronous cell populations has been re-evaluated and modified to give better definition and more even distribution of sub-phases. A reference pattern of early-relicating euchromatic bands is given for all chromosomes at Sk2 in primary cultures of skin fibroblasts. The overall band patterns at each sub-phase have allowed more objective definitions of early and late replication for these cells, and show that in both classes of chromatin light G-bands preceed dark G-bands. Asynchrony between homologous bands is observed at all stages of S, albeit with a variable frequency. The observed in vitro replication patterns and programme for the chromosomes of skin fibroblasts does not appear to be affected by the age or sex of the source.     

Indicators of Postoperative Pain in Syrian Hamsters (Mesocricetus auratus)

Despite the use of Syrian hamsters (Mesocricetus auratus) in research, little is known about the evaluation of pain in this species. This study investigated whether the frequency of certain behaviors, a grimace scale, the treat-take-test proxy indicator, body weight, water consumption, and coat appearance could be monitored as signs of postoperative pain in hamsters in a research setting. Animals underwent no manipulation, anesthesia only or laparotomy under anesthesia. An ethogram was constructed and used to determine the frequencies of pain, active and passive behaviors by in-person and remote videorecording observation methods. The Syrian Hamster Grimace Scale (SHGS) was developed for evaluation of facial expressions before and after the surgery. The treat-take-test assessed whether surgery would affect the animals’ motivation to take a high-value food item from a handler. The hypothesis was that behavior frequency, grimace scale, treat-take-test score, body weight, water consumption, and coat appearance would change from baseline in the surgery group but not in the no-intervention and anesthesia-only groups. At several time points, pain and passive behaviors were higher than during baseline in the surgery group but not the anesthesia-only and no-intervention groups. The SHGS score increased from baseline scores in 3 of the 9 animals studied after surgery. The frequency of pain behaviors and SHGS scores were highly specific but poorly sensitive tools to identify animals with pain. Behaviors in the pain category were exhibited by chiefly, but not solely, animals that underwent the laparotomy. Also, many animals that underwent laparotomy did not show behaviors in the pain category. Treat-take-test scores, body weight, water consumption, and coat appearance did not change from baseline in any of the 3 groups. Overall, the methods we tested for identifying Syrian hamsters experiencing postoperative pain were not effective. More research is needed regarding clinically relevant strategies to assess pain in Syrian hamsters.

Pain experienced by laboratory animals can affect both animal welfare and research results. Little is known about the evaluation of pain in Syrian hamsters (Mesocricetus auratus) in the laboratory setting. However, various research models using Syrian hamsters involve surgery and are presumed to cause pain.^16,47,49 In 2018 alone, the USDA reported that 35,695 hamsters were used for research studies involving painful procedures.⁴⁸ Previously published behaviors exhibited by hamsters in response to pain include hunched posture with head down, reluctance to move, increased depression or aggression, extended sleep periods, and weight loss.^7,8,10,16,21 How these behaviors are affected by factors such as the type of painful stimulus, anesthetic protocol, handling procedures, and environmental conditions is unclear. The practicality of observing these signs in the research environment is uncertain and likely complicated by the nocturnal nature of Syrian hamsters and an assumed propensity of this species to mask pain, much like other prey species.^8,14,16A significant need exists for published data investigating whether behavioral observations or other clinical indicators can help recognize, quantify, or monitor pain in hamsters in a research setting. Detailed behavioral observations and well-controlled studies are needed to develop a system to assess postoperative pain in laboratory animals.^8,33 Moreover, little information is available on the efficacy of analgesic agents in hamsters.¹ The few studies of analgesics in hamsters rely on the mitigation of evoked pain responses (such as using a hot plate), which has limited relevance to clinical situations such as postoperative pain.^8,32,36,51 To date, no published literature has evaluated the efficacy or safety of analgesics to treat postoperative pain in hamsters. Validated real-time and practical methods for evaluating pain in Syrian hamsters would support the evaluation of analgesic efficacy in this species.Various assessments have been developed to identify signs of pain in other species. Behavioral ethograms have been used to evaluate pain and analgesic efficacy in mice, rats, rabbits, and guinea pigs in the research environment.^{5,6,20,23,25,34,35,39-41,53} Another tool used to evaluate pain in animals is the grimace scale, which has been developed for mice, rats, rabbits, ferrets, cats, sheep, pigs, horses, and even harbor seals.^{3,4,9,11,13,15,19,22,26,30,37,45,50} The use of a proxy indicator, such as burrowing and time-to-integrate-to-nest in mice and time-to-consume in guinea pigs, can be used as an additional tool for the evaluation of pain.^{5,17,18,35,38}Because none of the previously mentioned assessment techniques were specific to hamsters, we here explored using these approaches to detect pain in Syrian hamsters that underwent laparotomy in a laboratory setting. We developed a species-specific ethogram and the Syrian Hamster Grimace Scale (SHGS). We also devised a novel proxy indicator of pain for use in Syrian hamsters, the treat-take-test (TTT), which is based on hamsters’ natural behavior to hoard food.^16,46,49,52 Although water intake, body weight, and coat appearance are non-specific indicators of pain, we also measured these parameters.^5,19,23,33 Furthermore, we analyzed the effects of the presence of an observer and time of day. We hypothesized that behavior frequency, grimace scale, treat-take-test score, body weight, water consumption, and coat appearance would change from baseline in the surgery group but not in the no-intervention and anesthesia-only groups.